{"text":"author: Marie Csete\ndate: 2010\nreferences:\ntitle: Q&A: What can microfluidics do for stem-cell research?\n\n# What can microfluidics do for stem-cell research?\n\nStem-cell biology and microfluidics have both been hotbeds of research activity for the past few years, yet neither field has been able to successfully commercialize a clinical 'killer application'. Stem-cell behavior is exquisitely sensitive to environmental cues, and the important cues are difficult to establish, manipulate and quantify in traditional cell culture. Because the microenvironment can be controlled in microfluidics platforms, microfluidics has a lot to offer stem-cell biology and there are many good reasons for the fields to join forces.\n\n# What exactly is microfluidics?\n\nMicrofluidics is the characterization and manipulation of fluids on the nanoliter or picoliter scale. The behavior and properties of fluids change as amounts decrease from the macroscale (volumes used for everyday applications) to the microscale. This means that microfluidic devices cannot be built by simply scaling down macroscale devices. For instance, at low microliter volumes, fluids act more like solids, and two fluids flowing alongside each other in a microchannel will not mix well (except by diffusion); therefore, a variety of techniques (pumps, valves, electrokinetics) are used in microfluidics platforms to actuate mixing and fluid flows. Most microfluidics applications in research labs concentrate on the 10 to 100 \u03bcm scale, basically the diameter of a single cell.\n\nMicrofluidics lab-on-a-chip devices allow standard laboratory analyses, such as sample purification, labeling, detection and separation, to be carried out automatically as the sample is moved, via microchannels, to different regions of a chip. Various methods have been used to produce microfluidic devices, but inkjet printers offer an easily accessible way of printing channels and other features directly onto the device. This technique has been used to print precise patterns of proteins or protein gradients onto a surface on which cells can subsequently be cultured to investigate or control their behavior. A technically more advanced use of microfluidics is the integration of microchannels with nanoelectrospray emitters for preparing material for mass spectrometry in high-throughput proteomics analyses of biologic samples \\[1\\].\n\n# What background do you need for microfluidics?\n\nPhysics (in particular fluid dynamics), mechanical engineering, or bioengineering backgrounds, the common feature of these being a strong mathematical foundation.\n\n# Why should stem-cell biologists care about miniaturization of cell culture and analysis tools?\n\nOn the one hand, scientists working on the development of pluripotent stem cells for clinical use are encountering a major challenge in scaling up cell cultures for master banks to be used as sources of cell therapies for large numbers of patients. Microfluidics is clearly not the answer to this problem. But on the front end of developing therapies from stem cells, rigorous identification of the starting stem cell and its progeny is a major technical challenge and a regulatory requirement, analogous to the precise chemical identity of a drug. Classically, identification of stem cells is done clonally (at the single-cell level), and it is generally difficult to follow or analyze single cells in mass cell culture. Microfluidics techniques can be used for sensitive discrimination of gene expression (and protein) levels at the single-cell level and they are therefore increasingly useful in stem-cell biology to understand the heterogeneity of stem-cell populations.\n\nSeparation of rare stem cells (or rare cancer cell types) from a mixed population is also not easy using flow cytometers developed for clinical use; harsh conditions imposed on the cells during standard flow cytometry mean that cell recovery is low. Microfluidics-based, benchtop flow cytometry allows separation of small numbers of stem cells under direct visualization, and is less damaging to cells than traditional cell sorters. For both analysis and separation, microfluidics offers the means of controlling the cells' environment rigorously. Several groups have also reported that stem cells (and stem cells committed to a particular lineage) can be separated from mixed cell populations using their dielectric properties (electric and magnetic energy).\n\n# In what ways are microfluidics culture conditions superior to those of traditional mass cell culture?\n\nStem-cell fate (growth, death, differentiation, migration) is highly dependent on environmental cues, but the usual cell culture environment does not mimic the *in vivo* microenvironment in several fundamental ways (20% oxygen is unphysiologically high; physiologic fluid flow and shear stresses are not present; three-dimensional environments cannot be standardized), and overall the environment in conventional cell culture is not controllable. For example, pH inevitably drifts in conventional tissue culture, but in well-designed microfluidics devices, the pH can be held constant by controlling medium inflow and outflow. In other words, engineers can provide steady-state conditions for cells, as well as fast and predictable changes in the environment surrounding the cells. Of particular importance, the best microfluidics devices are supported by mathematical descriptions of the microenvironment, and information from experiments can be fed back into mathematical models to determine optimal design features to promote specific stem-cell behaviors.\n\nGradient cues, so important in embryonic development, can be constructed quite precisely on microfluidics devices, as noted above. For example, migration of stem cells in response to chemotactic gradients is often studied in mass cultures using repeated studies in Boyden chambers (two chambers separated by a filter through which cells migrate), but molecular gradients established with microfluidics tools yield inherently more detailed and precise information because gradient characteristics such as slope and concentration can be quantified and correlated to migration behavior. Overall, flexibility in the configuration of microchips is a major advantage of microfluidics-based cell-culture systems, and the ease with which fluid flows can be controlled over time and space.\n\nHuman embryonic stem cells (hESCs) are particularly sensitive to handling in culture, and automation of hESC growth and differentiation *in vitro* on microfluidics platforms produces more standardized outcomes. Many investigators believe that the stress of manual handling of hESCs is an important factor in their instability over time, and therefore automated techniques for passaging and expansion may be a method for overcoming the problem of karyotypic instability.\n\nThree-dimensional mass culture systems are especially 'noisy' and difficult to control using conventional tissue-culture methods. Embryoid bodies - floating aggregations of undifferentiated cells - are often used as an intermediate stage in differentiation protocols, and are generated from hESCs by passaging the cells onto non-adherent plates. The resulting embryoid bodies are widely heterogeneous in size unless special engineering protocols are used. This size heterogeneity means that diffusion patterns for signaling through the embryoid bodies and cell-cell interactions are also heterogeneous, resulting in lack of control over the differentiation patterns. Printed topographic features of various shapes on microchips or microchannels are a proven method for gaining control over how cells aggregate. The size and development of embryoid bodies can be controlled with microfluidics techniques, providing a more predictable differentiation pattern and organization of the cells into phenotypically distinct layers. In fact, engineers have successfully manipulated parts of embryoid bodies in different ways using microfluidics tools to alter distinct fates for different parts of the cell aggregates.\n\nThe 'micro' in microfluidics plus the configurability of channels can be used to look at simultaneous signals to two parts of a single cell, for example the apical versus basal signals that will be encountered by a polarized cell. In traditional mass culture, cells align in random fashion, and although matrix coatings on tissue-culture plastic can be used to line cells up relative to the matrix, it is impossible to present signals to separate subcellular domains. Epithelial cells are the classical polarized cell in which specific receptors are largely confined to either the apical or basal surface, and signals received at these subcellular domains determine cell function. At the very small scale of microfluidics devices, the apical and basal faces of a cell can be exposed to separate chambers whose composition can be defined and manipulated independently, making it possible to determine the hierarchy of stimuli that determine cell behavior.\n\nAn obvious advantage of microfluidics is that it provides economy in terms of reagent use, especially for high-throughput assays. Of course, this economy will only be realized if device fabrication is also inexpensive.\n\n# What are some of the major limitations of microfluidics-based cell culture systems?\n\nNot surprisingly, from a biologist's perspective, the materials-cell interface is still a problem. Polydimethylsiloxane (PMDS) is commonly used to make microchips because it is cheap, optically transparent, gas permeable, and can be manipulated outside a clean room. Although many groups have reported using PDMS chips for hESC studies, my experience is that PDMS has to be considerably modified (and coated), because it is very toxic to the cells. Other, more biocompatible surfaces are available, but the ideal material for exquisitely sensitive cells such as hESCs has not been developed. Again from the biologist's perspective, cellular debris can occlude small channels, so that optimal washing methods in some applications need improvement.\n\nEngineers have pointed out that the best mathematical framework for handling models, such as differentiation, that start at small scales but result in large-scale processes is still evolving \\[2\\]. So along with the constant improvement in hardware and software needed to make inexpensive devices work optimally, the mathematical tools that make microfluidics approaches so valuable also need continuous refinement. Ultimately, the feedback between biologists using the devices and engineers designing them is the essential key for moving microfluidics-based cell culture forward.\n\nA major issue limiting wide application of microfluidics is that the devices still require experts to operate them, and are not yet biology user-friendly.\n\n# What major problems in translational stem-cell biology can be addressed using microfluidics tools?\n\nHere again, microfluidics techniques afford the ability to define the microenvironment surrounding stem cells. The disease environment into which stem cells will be transplanted is certain to alter their behavior, and is not adequately mimicked in most animal models of disease. Microfluidics-controlled environments can be used to test the tolerance of cells to mechanical and shear forces, gases, oxidants and other extracellular cues that characterize the disease environment. Physical, mechanical and biochemical factors can be tested quantitatively at relatively high throughput on the benchtop using microfluidics to help predict behavior of stem cells *in vivo*.\n\nOverall, microfluidics tools can be used for spatiotemporal control over the stem-cell microenvironment, so that the ideal *ex vivo* niche for cell survival and differentiation can be defined quantitatively and in high throughput. Control over the culture environment also allows investigators to perturb cell fate to generate desired outcomes, and to define the limits of physical, mechanical and biochemical factors that are tolerated by stem cells at different stages of differentiation.\n\n# What have been the important contributions of microfluidics in biology in general?\n\nGeorge Whitesides points out that one of the best developed applications of microfluidics is in protein crystallographic studies, to screen the conditions that encourage growth and protection of crystals \\[3\\]. For cell biologists, the major impact has been in cell separation, single-cell resolution of the dynamics of gene expression, and insights into how mechanical forces applied to individual cells determine their behavior.\n\n# Where can I find out more?\n\nCai L, Friedman N, Xie XS: **Stochastic protein expression in individual cells at the single molecule level**. *Nature* 2006, **440**:358-362.\n\nCimetta E, Figallo E, Cannizzaro C, Elvassore N, Vunjak-Novakovic G: **Micro-bioreactor arrays for controlling cellular environments: Design principles for human embryonic stem cell applications**. *Methods* 2009, **47**:81-89.\n\nMelin J, Quake SR: **Microfluidic large-scale integration: the evolution of design rules for biological automation**. *Annu Rev Biophys Biomol Struct* 2007, **36**:213-231.\n\nStroock AD, Dertlinger SK, Ajdari A, Mezic I, Stone HA, Whitesides GM: **Chaotic mixer for microchannels**. *Science* 2002, **295**:647-651.\n\nTung Y-C, Torisawa Y, Futai N, Takayama S: **Small volume low mechanical stress cytometry using computer-controlled Braille display microfluidics**. *Lab Chip* 2007, **7**:1497-1503.","meta":{"dup_signals":{"dup_doc_count":118,"dup_dump_count":43,"dup_details":{"curated_sources":4,"2019-35":1,"2019-26":1,"2019-13":1,"2019-04":1,"2018-51":1,"2018-43":1,"2018-34":2,"2018-30":1,"2018-13":1,"2018-05":1,"2017-39":2,"2017-30":1,"2017-22":1,"2017-17":1,"2017-09":8,"2016-44":1,"2016-40":1,"2016-36":8,"2016-30":4,"2016-22":1,"2016-18":1,"2015-48":2,"2015-40":3,"2015-35":2,"2015-32":3,"2015-27":3,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":4,"2014-42":9,"2014-41":4,"2014-35":5,"2014-23":5,"2014-15":5,"2019-43":1,"2015-18":3,"2015-11":3,"2015-06":3,"2014-10":3,"2013-48":3,"2013-20":3,"2024-26":1}},"file":"PMC2871530"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  Genomic tumor information, such as identification of amplified oncogenes, can be used to plan treatment. The two sources of a brain tumor that are commonly available include formalin-fixed, paraffin-embedded (FFPE) sections from the small diagnostic biopsy and the ultrasonic surgical aspiration that contains the bulk of the tumor. In research centers, frozen tissue of a brain tumor may also be available. This study compared ultrasonic surgical aspiration and FFPE specimens from the same brain tumors for retrieval of DNA and molecular assessment of amplified oncogenes.\n  .\n  # Methods\n  .\n  Surgical aspirations were centrifuged to separate erythrocytes from the tumor cells that predominantly formed large, overlying buffy coats. These were sampled to harvest nuclear pellets for DNA purification. Four glioblastomas, 2 lung carcinoma metastases, and an ependymoma were tested. An inexpensive PCR technique, multiplex ligation-dependent probe amplification (MLPA), quantified 79 oncogenes using 3 kits. Copy number (CN) results were normalized to DNA from non-neoplastic brain (NB) in calculated ratios, \\[tumor DNA\\]\/\\[NB DNA\\]. Bland-Altman and Spearman rank correlative comparisons were determined. Regression analysis identified outliers.\n  .\n  # Results\n  .\n  Purification of DNA from ultrasonic surgical aspirations was rapid (\\<3\u2009days) versus FFPE (weeks) and yielded greater amounts in 6 of 7 tumors. Gene amplifications up to 15-fold corresponded closely between ultrasonic aspiration and FFPE assays in Bland-Altman analysis. Correlation coefficients ranged from 0.71 to 0.99 using 3 kit assays per tumor. Although normalized CN ratios greater than 2.0 were more numerous in FFPE specimens, some were found only in the ultrasonic surgical aspirations, consistent with tumor heterogeneity. Additionally, CN ratios revealed 9 high-level (\u2265 6.0) gene amplifications in FFPE of which 8 were also detected in the ultrasonic aspirations at increased levels. The ultrasonic aspiration levels of these amplified genes were also greater than 6.0 CN ratio, except in one case (3.53 CN ratio). Ten of 17 mid-level (\u22653.0 & \\<6.0 CN ratio) amplifications detected in FFPE were also detected as being increased (\u2265 2.0 CN ratio) in the aspirations.\n  .\n  # Conclusions\n  .\n  Buffy coats of centrifuged ultrasonic aspirations contained abundant tumor cells whose DNA permitted rapid, multiplex detection of high-level oncogene amplifications that were confirmed in FFPE.\n  .\n  # Virtual slides\n  .\n  <http:\/\/www.diagnosticpathology.diagnomx.eu\/vs\/1883718801686466>\nauthor: Long N Truong; Shashikant Patil; Sherry S Martin; Jay F LeBlanc; Anil Nanda; Mary L Nordberg; Marie E Beckner\ndate: 2012\ninstitute: 1Department of Biological Sciences, Louisiana State University - Shreveport, One University Place, Shreveport, LA 71115, USA; 2Department of Neurosurgery, Baylor College of Medicine, 1327 Lake Point Pkwy, Suite 400, Sugar Land, TX 77478, USA; 3Delta Pathology Group, One Saint Mary Place, Shreveport, LA 71101, USA; 4Department of Emergency Medicine, Long Medical Center, Louisiana State University Health Sciences Center \u2013 New Orleans, 5825 Airline Hwy, Baton Rouge, LA 70805, USA; 5Department of Neurosurgery, Louisiana State University Health Sciences Center \u2013 Shreveport, Rm. 3-215, 1501 Kings Highway, Shreveport, LA 71130, USA; 6Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center \u2013 Shreveport, Rm. B-215, 1501 Kings Highway, Shreveport, LA 71130, USA; 7Departments of Pediatrics & Medicine, Louisiana State University Health Sciences Center-Shreveport, Rm. 2-303, 1501 Kings Highway, Shreveport, LA 71130, USA; 8Delta Pathology Molecular Diagnostics, One Saint Mary Place, Shreveport, LA 71101, USA; 9Department of Neurology, Louisiana State University Health Sciences Center \u2013 Shreveport, Rm. 3-438, 1501 Kings Highway, Shreveport, LA 71130, USA\nreferences:\ntitle: Rapid Detection of high-level oncogene amplifications in ultrasonic surgical aspirations of brain tumors\n\n# Background\n\nOncogenes encode proteins that promote tumor growth, survival under adverse conditions, and invasion. Many studies have detected amplified oncogenes in high grade brain tumors, especially glioblastoma or glioblastoma multiforme (GBM). Therefore, assays to identify amplified genes are proposed to become critical in patient care as monoclonal antibodies and small molecules that inhibit proteins encoded by oncogenes become available. Identification of amplified genes in a rapid, multiplex manner is relevant for stratifying patients to clinical trials and treatments. Brain tumor specimens for molecular studies include ultrasonic surgical aspirations available at the time of surgery and small, diagnostic biopsies that are processed as formalin-fixed, paraffin-embedded (FFPE) samples. Sometimes tumor fragments are resected and are also processed as FFPE samples. Although tissue frozen for storage at the time of surgery can also be released for DNA studies, it is not usually available outside of research settings. Ultrasonic surgical aspirations are not routinely used for diagnostic purposes and represent an untapped source of abundant, fresh tumor DNA. With laboratory support for processing cellular fluids and the expertise for confirming tumor cell content morphologically, pathology departments are well suited for providing cellular or nuclear pellets from ultrasonic surgical aspirations to provide tumor DNA needed in molecular testing. Surgical aspirations can quickly provide DNA to speed up turnaround times and produce higher yields than FFPE sections of small biopsies.\n\nBrain tumors debulked by ultrasonic surgical aspiration yield suspensions of single cells, minute tissue fragments, and blood in saline. Following introduction of this technique in the 1970's, ultrasonic aspiration became routine in microneurosurgery \\[1\\]. Unlike the characteristically small diagnostic biopsies of brain tumors, ultrasonic surgical aspirations contain ample tumor cells in large volumes of fluid. The ultrasonic apparatus consists of a computerized control unit with settings for different amplitudes of ultrasonic waves and speeds of irrigation and aspiration. Tumor tissues are targeted and selectively aspirated. An advantage of the ultrasonic aspiration technique over tissue dissection is the reduced need for retraction of normal brain in the patient. Accordingly, the use of ultrasonic surgical aspiration to debulk intracranial tumors is common and explains the unfortunate paradox of having only small diagnostic tissue biopsies from large brain tumors available for molecular studies outside of research settings. Although ultrasonic aspiration specimens have yielded viable tumor cells for experimental studies \\[2-4\\], their lack of tissue architecture greatly diminishes their usefulness in diagnostic pathology. For example, regions of tumor necrosis that are a useful diagnostic feature in high grade tumors are prone to disintegrate during the aspiration procedure. Although some institutions preserve small portions of ultrasonic aspirations in FFPE blocks, the intact tissue fragments from biopsies are preferred and routinely relied upon for diagnostic evaluation of primary and metastatic brain tumors. Ultrasonic surgical aspirations, that contain the bulk of brain tumor tissue in a dispersed and disaggregated form, seldom provide the complete architectural features of the tumors that aid in determining morphologic diagnoses.\n\nIn this study, ultrasonic aspirations of brain tumors were tested to see if they would yield tumor DNA that is appropriate for studies to detect amplified oncogenes. Testing methods that identify amplified genes as potential treatment targets in individual tumors would ideally be performed as soon after surgery as possible. The genes tested with commercial MLPA kits are known to be amplified in association with malignancy. The amounts of DNA purified from ultrasonic aspirations of GBMs were found to be especially ample. These DNA samples were analyzed for amplifications in seventy-nine oncogenes, with results compared to those from FFPE-derived DNA and with fluorescence *in situ* hybridizaiton (FISH) results for *EGFR* gene amplification in FFPE tissue sections. The MLPA assays on ultrasonic aspirations identified high-level amplified genes within a few days at low cost and served as a preview of the more sensitive FFPE assay results that became available later.\n\n# Methods\n\n## Patient Population, Specimen Retrieval and DNA Purification\n\nPermission from our Institutional Review Board was obtained for retrieval and study of the excess amounts of ultrasonic aspiration and FFPE specimens taken from brain tumors resected in 2008\u20132009 in a pilot study of using these specimens for molecular tests. Ultrasonic aspirations from seven brain tumors, including four glioblastomas (GBM1-4), two lung carcinomas metastases (LCM1-2) and an ependymoma (EP1), were tested. The patients (all male except for one female with a GBM) were 44 to 81\u2009yrs of age. Samples were obtained in saline with a Cavitron Ultrasonic Aspirator Excel (Valleylab-Tyco International, Boulder, CO) set at 23 to 36\u2009MHz. The volume (cc) of each tumor present preoperatively and postoperatively was estimated by the neurosurgeon. The ultrasonic surgical aspirations were not designated to be used for any diagnostic purpose. The specimens in this study were delivered to the laboratory within 24\u2009hr of surgical resection, either fresh or after overnight refrigeration. Portions of ultrasonic aspiration specimens, 15 to 30\u2009ml, were centrifuged at 500\u2009g for 10\u2009min. Tumor cells sedimented as large buffy coats above the erythrocytes and beneath floating necrotic debris. According to the manufacturer's recommendations (Qiagen Midi Kits, Valencia, CA), 500\u2009\u03bcl of each buffy coat were pipetted and combined with purification kit components to obtain a nuclear pellet that was processed immediately or frozen for later purification. All specimens yielded measureable amounts of DNA. For comparisons in each tumor, DNA from routine FFPE sections (10\u2009\u03bcm thick, 10 per tumor) was purified with a Qiagen FFPE DNA purification kit. In each case tumor cells represented at least 90% of nucleated cells in the tissue sections. Proteinase K digestions at 55\u00b0C were extended as needed to achieve digestion of the tissues. Purified DNA was evaluated and quantified with spectrometry using absorbance measurements at 260 and 280\u2009nm (JENWAY Genova, Jenway Limited, Essex, England).\n\n## Histological Review of Tumor Specimens\n\nDiagnostic FFPE tissue sections, available from all tumors, were reviewed microscopically to characterize the pathological features. Portions of ultrasonic aspiration specimens that had also been processed as FFPE specimens were available. Hematoxylin and eosin stained FFPE sections were evaluated. The number of mitotic figures in ten high power fields (HPFs) (400X magnification) was determined for each tumor in duplicate counts. Also, stains for Ki67 reactive cells were performed at the time of diagnosis for some tumors using a pre-diluted antiKi67 antibody (Ventana Medical Systems, Oro Valley, AZ) for direct staining on an automated immunohistochemical stainer (Benchmark XT, Ventana Medical Systems). Pre-diluted, non-reactive mouse monoclonal negative control (Ventana) solutions were applied to separate tissue sections. The percentages of tumor cells positive for Ki67 specific staining were determined with an automated cellular imaging system (Chromavision ACIS, San Juan Capistrano, CA) with verification by manual counts at the time of review. If interference from background staining was present, manual counts were substituted.\n\n## *EGFR* FISH\n\nInclusion of probe sets for *EGFR* in the MLPA kits permitted comparison of MLPA and FISH copy number (CN) data for this gene in each tumor. FISH probes for a control locus, 7p11.1-q11.1, D7Z1, and the *EGFR* band region, 7p11.2-7p12, (Vysis Locus Specific Identifier (LSI) *EGFR* SpectrumOrange\/CEP 7 SpectrumGreen, Abbott Molecular Inc., Des Plaines, IL) were hybridized to interphase nuclei. Paraffin sections of all tumors and cytologic smears prepared from some of the ultrasonic aspiration specimens were examined. Smears from aspirations were fixed, denatured, and hybridized with probes overnight. Un-hybridized probes were washed away. Diamino-phenylindole (DAP1) fluorescent blue (Abbott Molecular) stained the nuclei. Slides were scanned on a fluorescent microscope (Leica DMR, Wetzlar, GM) for analysis with images captured using a digital camera (Applied Imaging, San Jose, CA) and CytoVision v4.02 imaging software (Applied Imaging). Sections from FFPE tissue were cut at a thickness of five microns, deparaffinized (Paraffin Pretreatment Reagent Kit II, Abbott Molecular), processed on a VP2000 Processor (Abbott Molecular) and then FISH probes for *EGFR* and CEP 7 were applied and analyzed as described above. Amplifications of *EGFR* were observed as average *EGFR* to CEP 7 signal ratios greater than two for at least 20 cells (usually many more). The results were determined by averages of all cells that could be evaluated. Proteinaceous debris hindered FISH interpretations in some of the ultrasonic surgical aspiration specimens. Ratios of total *EGFR* and CEP 7 signals per tumor, individual *EGFR*\/CEP 7 signals per cell and medians of the signal ratios per cell were described.\n\n## MLPA\n\nThe PCR-based technique, MLPA, involved multiple steps, including exposure of DNA to gene-specific probe sets, enzymatic ligation, PCR, and capillary electrophoresis (CE) to separate PCR products. Briefly, as explained earlier \\[5\\], 200\u2009\u03bcg of DNA from each tumor sample and normal brain in buffer were denatured at 98\u00b0C for 30\u2009min in a thermocycler (MasterCycler personal Eppendorf, Hamburg, GM) and were then hybridized to MLPA probes (SALSA P171, P172 and P173 kits, MRC-Holland, The Netherlands) according to kit instructions. The manufacturer selected genes according to literature documenting amplification in tumors. In addition to gene specific sequences, the probes also contained universal PCR primers, X and Y, and stuffer sequences for subsequent identification of specific gene amplicons by size using CE. Ligase65 (MRC-Holland) generated ligations specific for perfectly hybridized probe pairs (sets) at ligation junctions in the DNA and the enzyme was then heat inactivated. Ligated probes were PCR amplified with polymerase (MRC-Holland) according to instructions. PCR products were separated on a CEQ 8000 (Beckman-Coulter, Fullerton, CA) and were then identified according to the length of amplicons.\n\nFragment analysis of PCR products produced a series of linear peaks (signals of fluorescence) corresponding to the relative quantities of PCR products that were proportional to initial amounts of DNA (or CN) of the targeted genes. Slightly less efficient amplifications of longer amplicons accounted for minor reductions in their peak heights. All of the genes tested in the three MLPA kits, P171, P172, and P173, are listed as follows: *AKT1, AURKA, BCAR2, BCAS1-2, BCL2, BCL2A1, BCL2L1*, *11* and *13, BCL6, BCLG, BIRC1-5, BRAF, BRMS1, CCNA1, CCND1-2, CCNE1, CDK4* and *6, CENPF, CYP27B1, EGFR, EMS1, ERBB2* and *4, ESR1, EVI1, FGF3* and *4, FGFR1, FLJ20517, GNAS, GSTP1, HMGA1, IGF1R, IGFBP2,4* and *5, IRS2, JAK2, MDM2* and *4, MET, MMP7, MOS, MYCL1, MYBL1* and *2, MYC, MYCN, NFKBIE, NRAS, NTRK1-3, PDGFRA* and *B, PIK3C2B, PIK3CA, PPM1D, PSMB4, PTK2, PTP4A3, PTPN1, RELA, RNF139, RUNX1, SERBINB2, 7* and *9, TERT* and *TOM1L2*.\n\nOccasional failures to see peaks in one of the eight capillary tubes during a CE run were attributed to low current whenever the peak was obtained after repeating CE. Successful concurrent results for NB were required for a CE run of tumor samples to be further analyzed.\n\n## Normalization of CN\n\nFor detection of somatic changes found in highly malignant tissues, there were no known reference probes that could be relied on to maintain their normal CN at the time of this study. Large numbers of genes are lost or amplified in malignant tumors leading to considerable variability in the CN of individual tumors. Also, genetic variations and mutations in tumors, such as nucleotide deletions, insertions, or substitutions near the ligation junctions could potentially impact ligation efficiency. In this study the result of each probe set in each tumor specimen was normalized with CN for the same probe set in DNA from non-neoplastic brain (NB) assayed concurrently. Comparisons of all tumors with the same source of NB controlled for assay to assay variation. Aliquots of NB were from an 82\u2009year old woman's normal occipital lobe (Biochain, Hayward, CA).\n\nThe CN ratios, or fold-differences from normal for each gene, are represented by the ratio, \\[tumor DNA\\] \/ \\[NB DNA\\], derived from measurements of the CE peaks. The CN ratio was calculated after peaks of non-amplified genes on CE graphs, representing relative amounts of DNA, were matched with the average of two NB samples analyzed in the same assay run. Peak heights of non-amplified genes in tumor samples and NB were adjusted to approximately the same scale as graphical printouts from CE were produced. Final, finer adjustments were made in spreadsheets by maximizing alignments of trendlines for non-amplified genes. Scatterplots were evaluated to check the reactions of NB DNA with the MLPA probes compared to expected values provided by the manufacturer. Overlays of NB results obtained in GBM1's assays of FFPE, using P171, P172, and P173 kits, with graphs of normal values provided in the MLPA kits' literature, demonstrated comparable overall detection of the two populations of normal DNA data points (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>).\n\nRegression analysis of NB's scatterplots demonstrated that results obtained using one of the two probe sets for *ERBB4* was an outlier. In P171 data points, *ERBB4*'s probe set result was at the 95% confidence limit for the overall NB sample population. All other probe set results fell above the lower 95% confidence limit for the NB sample. Also, three probe sets were slightly out of range in the opposite direction so that missing a low level amplification was a concern but other probe sets for these genes were within the 95% confidence limits so that missing a significant CN gain after their results were averaged would be unlikely. The probe sets with results above the confidence limits included 1 of 2 for *IGFBP2*, 1 of 2 for *NTRK1*, and 1 of 4 for *IGF1R*.\n\nThe normal CN range was set at\u2009\u2265\u20090.75 to\u2009\u2264\u20091.50, with CN ratios\u2009\u2265\u20092.0 considered to be amplified. High-level gene amplifications in glioblastomas have been previously set at 6-fold greater than diploid or 12 or more copies per nucleus \\[6\\]. In this study CN ratios\u2009\u2265\u20096.0 were also designated as high-level amplifications and lesser amplifications were set at CN ratios of\u2009\u2265\u20093.0 and\u2009\\<\u20096.0 for mid-level and\u2009\u2265\u20092.0 and\u2009\\<\u20093.0 for low-level amplifications. Averaged results for two replicates of amplified and non-amplified genes in ultrasonic aspiration tumor DNA were compared to results for two replicates of genes in NB for each gene's probe set (or ligated pair). Results for multiple probe sets for a gene were averaged. MLPA analysis using the kits, P171, P172, and P173, was performed on FFPE DNA in two replicates from each of 7 tumors in 15 of the 21 assays and in single replicates in 6 due to DNA depletion from the small specimens.\n\n## Statistical methods and correction factors\n\nExcel was used to prepare graphs for images, determine Spearman rank correlation coefficients, regression analysis, etc. Outlier identification was performed with regression analysis to detect data points at or beyond 95% confidence intervals for residuals. A Bland-Altman plot to compare MLPA results of cavitronic ultrasonic surgical aspiration and FFPE assays was generated with R, version 2.10 (The R Foundation for Statistical Computing, http\/\/<http:\/\/www.R-project.org>), statistical software.\n\n# Results\n\n## DNA purification yields\n\nSuccessful DNA purification was achieved for all of the tumors. The greatest yields of DNA (43.0 to 77\u2009\u03bcg) were obtained from buffy coats of glioblastoma ultrasonic aspiration specimens. The FFPE sections from all of the tumors yielded less DNA (6.4 to 20.5\u2009\u03bcg) as seen in Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>.\n\nDNA data according to sample type, cavitronic ultrasonic surgical aspiration (CUSA) or formalin-fixed, paraffin-embedded (FFPE) sections\n\n| **Brain tumors** | **Specimen type** | **260\/280 Absorbance (nm)** | **DNA yield per specimen (\u03bcg)** |\n|:---|:---|:---|:---|\n| GBM1 | CUSA | 1.38 | 49.1 |\n| \u00a0 | FFPE | 1.98 | 6.7 |\n| GBM2 | CUSA | 1.84 | 77.0 |\n| \u00a0 | FFPE | 1.98 | 7.9 |\n| GBM3 | CUSA | 1.84 | 43.3 |\n| \u00a0 | FFPE | 1.86 | 6.4 |\n| GBM4 | CUSA | 1.67 | 44.6 |\n| \u00a0 | FFPE | 1.90 | 7.0 |\n| LCM1 | CUSA | 1.85 | 17.9 |\n| \u00a0 | FFPE | 1.81 | 11.3 |\n| LCM2 | CUSA | 1.16 | 14.6 |\n| \u00a0 | FFPE | 1.63 | 20.5 |\n| EP1 | CUSA | 1.88 | 12.1 |\n| \u00a0 | FFPE | 1.55 | 10.1 |\n\nGBM\u2009=\u2009glioblastoma, LCM\u2009=\u2009Lung carcinoma metastasis, EP1\u2009=\u2009Ependymoma.\n\n## Clinicopathologic features of the tumors\n\nFeatures of the tumors were typical for their diagnostic classification as glioblastomas, metastatic tumors, and an ependymoma. Photomicrographs of the ultrasonic surgical aspirations (100X and 400X magnifications) and FFPE sections (400X magnification) illustrates the similarity of tumor cells in the two types of specimens (Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>). There were too few cases to evaluate whether characteristic morphologic profiles for amplified gene(s) in these tumors were present. Mitotic figures among the glioblastomas were most numerous in GBM1 and mitoses among all tumors were most numerous in LCM2 (Table <a href=\"#T2\" data-ref-type=\"table\">2<\/a>).\n\nSelected clinicopathologic features of the brain tumors in this study\n\n| **Tumor** | **Age & sex** | **Tumor (cc)** |  | **Mitoses per 10 HPF** | **Ki67 positive tumor cells** |\n|:---|:---|:---|:---|:---|:---|\n| \u00a0 | \u00a0 | Pre Op | Post Op | \u00a0 | \u00a0 |\n| GBM1 | 50\u2009yr\u2009M | 80.8 | 19.1 | 46.5\u2009\u00b1\u20096.4 | 61\u2009% |\n| GBM2 | 81\u2009yr\u2009M | 51.4 | 0.0 | 19.5\u2009\u00b1\u20092.1 | 55\u2009% |\n| GBM3 | 52\u2009yr\u2009F | 124.0 | 96.1 | 10\u2009\u00b1\u20092.8 | 38\u2009% |\n| GBM4 | 44\u2009yr\u2009M | 35.6 | 0.0 | 18.5\u2009\u00b1\u20092.1 | Not tested |\n| LCM1 | 55\u2009yr\u2009M | 19.0 | 0.0 | 51.5\u2009\u00b1\u20099.2 | Not tested |\n| LCM2 | 61\u2009yr\u2009M | 6.5 | 0.0 | 64.5\u2009\u00b1\u20095.0 | Not tested |\n| EP1 | 73\u2009yr\u2009M | 20.6 | 1.38 | 0 | 10\u2009% |\n\nThe volumes of tumor were provided as estimates by the neurosurgeon. HPF\u2009=\u2009high power field (400X).\n\n## *EGFR* FISH\n\nAnalysis of *EGFR* with FISH was used to correlate with MLPA assay results. Signals were counted in the FFPE sections. The CN for *EGFR* was increased in the four glioblastomas with ratios of *EGFR* to CEP 7 increased to values of more than 20 in at least some cells of each glioblastoma. In the metastatic tumors averages of CNs for *EGFR* were 2.5 to 2.8 per cell with *EGFR* to CEP 7 ratios remaining below 2.0. Distributions of the *EGFR*\/CEP 7 ratios are shown in Figure <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>A and illustrate \"tailing off\" in the data towards high-level amplifications in individual cells. Representative cells with amplified *EGFR* from the glioblastomas and from one of the metastases lacking amplification are shown in Figure <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>B. The EGFR\/CEP 7 ratios are listed for all tumors in Table <a href=\"#T3\" data-ref-type=\"table\">3<\/a>. The CN of CEP 7 varied and was increased (averages of 2.7 to 3.8) in glioblastomas. The CN of CEP 7 varied in LCM1 and LCM2 with averages of 2.5 and 2.6, respectively.\n\n**FISH versus MLPA results for\u2009*EGFR*\u2009**\n\n| **Tumor** | ***EGFR*\/CEP 7 ratios in FISH assays on FFPE** |  | ***EGFR*'s tumor\/NB DNA ratios (normalized CN) Average of 2 MLPA probe sets \u00b1\u2009SD** |  |\n|:---|:---|:---|:---|:---|\n| \u00a0 | Total *EGFR\/*Total CEP 7 signals | Median ratio | CUSA | FFPE |\n| GBM1 | 7.3 | 7.2 | 8.8\u2009\u00b1\u20090.2 | 21.1\u2009\u00b1\u20092.0 |\n| GBM2 | 8.6 | 9.3 | 22.5\u2009\u00b1\u20097.9 | 19.3\u2009\u00b1\u20097.2 |\n| GBM3 | 10.3 | 9.0 | 10.5\u2009\u00b1\u20093.8 | 33.1\u2009\u00b1\u200917.2 |\n| GBM4 | 11.1 | 11.6 | 19.5\u2009\u00b1\u20099.3 | 26.7\u2009\u00b1\u20096.5 |\n| LCM1 | 1.0 | 1.0 | 1.2\u2009\u00b1\u20090.5 | 1.2\u2009\u00b1\u20090.4 |\n| LCM2 | 1.1 | 1.0 | 2.3\u2009\u00b1\u20090.9 | 2.3\u2009\u00b1\u20091.4 |\n| EP1 | 1.0 | 1.0 | 1.7\u2009\u00b1\u20090.7 | 2.9\u2009\u00b1\u20092.6 |\n\nAlthough amplification of *EGFR* seen in individual cells of GBMs varied greatly and MLPA provided collective results of numerous cells, both techniques demonstrated corresponding large elevations in copy number for this oncogene in GBMs. Polysomy may have accounted for low-level amplifications found with MLPA in LCM2 and EP1. Median ratios were derived from the distributions of signal ratios in individual cells. *EGFR*\u2009=\u2009*Epidermal Growth Factor Receptor*. CEP 7\u2009=\u2009Centromere of chromosome 7, FISH\u2009=\u2009Fluorescence *in situ* hybridization. SD\u2009=\u2009Standard deviation. CUSA\u2009=\u2009Cavitronic ultrasonic surgical aspiration. FFPE\u2009=\u2009Formalin-fixed, paraffin embedded tissue.\n\n## MLPA molecular results\n\nProductive CE runs were obtained for all tumors. Prior to normalization of tumor CN, the output of CE data from all four glioblastomas and one of two metastases revealed obvious amplifications in at least one gene for each tumor. Measurements of the peaks for each gene were connected to create line graphs. The obvious peaks representing high-level and some mid-level gene amplifications in the glioblastomas are shown in Figure <a href=\"#F4\" data-ref-type=\"fig\">4<\/a>. Genes are listed along the *x-*axes according to amplicon size within each kit (P171, 172 and 173). Easily recognized amplifications occurred for *EGFR*, *CDK4*, *MDM2*, *CYP27B1*, and *PDGFRA* in one or more GBMs*,* and also for *CCNE1* in one of the brain metastases (not shown) using DNA from ultrasonic surgical aspirations. Comparable amplifications for the same genes were also detected in corresponding FFPE specimens (not shown). Results for non-amplified genes in the tumors and in normal brain (assayed concurrently) constitute the baselines of graphs in Figure <a href=\"#F4\" data-ref-type=\"fig\">4<\/a>. The *EGFR* data obtained with MLPA is included in Table <a href=\"#T3\" data-ref-type=\"table\">3<\/a> along with FISH data.\n\nFollowing NB normalization to generate CN ratios, Bland-Altman analysis (*n*\u2009=\u2009889, derived from 127 data points\/tumor x 7) found that data in ultrasonic surgical aspiration assays corresponded to data from FFPE assays for the same tumor within 1.96 SD limits, except for some of the amplifications that exceeded CN ratios of 15 (log value\u2009=\u20091.176). A normal CN ratio of 1 corresponds to a log value of 0 (Figure <a href=\"#F5\" data-ref-type=\"fig\">5<\/a>A). Correlation coefficients for comparisons ranged from 0.71 to 0.99 in studies of each tumor according to the kits used. Consistency was high with the exception of LCM2 that showed slightly less correspondence, as seen in Figure <a href=\"#F5\" data-ref-type=\"fig\">5<\/a>. The *n*'s were 42, 42, and 43 for P171, P172, and P173 kits, respectively, in each tumor.\n\nNormalized CN ratios for specific genes in glioblastomas, including those with high-level amplifications (CN ratio\u2009\u2265\u20096.0) and those with no alterations (all CN ratios \u22650.75 and \u22641.5), are shown in Figure <a href=\"#F6\" data-ref-type=\"fig\">6<\/a> (A & B, respectively). Both the ultrasonic surgical aspiration and corresponding FFPE results from the same tumors are shown. The only high-level gene amplification in the brain metastases was *EVI1* (7.02 CN ratio) in LCM2 FFPE but it was not amplified in the corresponding aspiration specimen. The metastases, LCM1 and LCM2, had 2 and 6 mid-level gene amplifications, respectively. The ependymoma (EP1) only had 1 mid-level gene amplification. Among the 10 genes with no alterations in any of the GBMs, including replicates of all probe sets for a specific gene, there were 9 genes that were also unaltered in EP1, and 9 and 6 genes that were unaltered in LCM1 and LCM2, respectively. Totals of all deviations (\\<0.75 or \u22652.0 in CN ratios) from normal values in ultrasonic aspiration and FFPE specimens are shown in Table <a href=\"#T4\" data-ref-type=\"table\">4<\/a>. Alterations in CN were more frequent in FFPE than in ultrasonic aspiration specimens, 85% and 49% of total changes, respectively. However, FFPE did not completely encompass all of the changes. Restriction of some genomic gains and losses to only one type of specimen from the same tumor is consistent with tumor heterogeneity.\n\nCopy number changes in formalin-fixed, paraffin-embedded (FFPE) and cavitronic ultrasonic surgical aspiration (CUSA) specimen DNA\n\n| **Tumors** | **Genes with normalized CN ratios\u2009\u2265\u20092.0 (gains in copy number)** |  |  |  |  | **Genes with normalized CN ratios\u2009\\<\u20090.75 (losses in copy number)** |  |  |  |  |\n|:---|:---|:---|:---|:---|:---|:---|:---|:---|:---|:---|\n| \u00a0 | Total | FFPE | CUSA | Only FFPE | Only CUSA | Total | FFPE | CUSA | Only FFPE | Only CUSA |\n| GBM1 | 18 | 17 | 7 | 11 | 1 | 16 | 12 | 6 | 10 | 4 |\n| GBM2 | 10 | 10 | 5 | 5 | 0 | 21 | 17 | 9 | 12 | 4 |\n| GBM3 | 13 | 13 | 7 | 6 | 0 | 21 | 18 | 8 | 13 | 3 |\n| GBM4 | 10 | 8 | 8 | 2 | 2 | 15 | 13 | 7 | 8 | 2 |\n| LCM1 | 7 | 7 | 5 | 2 | 0 | 15 | 12 | 11 | 4 | 3 |\n| LCM2 | 15 | 13 | 5 | 10 | 2 | 32 | 22 | 18 | 14 | 10 |\n| EP1 | 7 | 7 | 3 | 4 | 0 | 16 | 15 | 6 | 10 | 1 |\n| Totals | 80 | 75 | 40 | 40 | 5 | 136 | 109 | 65 | 71 | 27 |\n\nGBM1-4 had increased CN ratios for 22 genes (*AURKA, BCL2A1, BCL6, BIRC1,2,4, BRAF, CDK4,6, CYP27B1, EGFR, ERBB4, ESR1, EVI1, GNAS, GSTP1, MDM2, NRAS, PDGFRA, PIK3CA, RNF139,* &*SERPINB9*) and decreased CN ratios for 35 (*AKT1, BCAR3, BCAS1, BCL2, BCL2L13, BCLG, BIRC5, BRMS1, CCND1, CCNE1, CDK4, CENPF, EMS, FGF4, FGFR1, FLJ20517, GSTP1, IGF1R, IGFBP2, IRS2, JAK2, MDM4, MYBL2, MYC, MYCL1, MYCN, NFKBIE, NTRK1, PDGFRB, PIK3C2B, PTK2, PTP4A3, PTPN1, TERT,* &*TOM1L2*). LCM1-2 had increased CN ratios for 16 genes that overlapped with those for GBM1-4 plus *CCNE1, MET, PPM1D,* and *PSMB4*, and decreased CN ratios were found for thirty six genes that overlapped with those listed for GBM1-4 plus *BCL2L1, BIRC3, CYP27B1, MMP7, NTRK2, NTRK3, PDGFRA, RELA,* &*SERPINB2*. EP1 had increased CN ratios for genes among those for GBM1-4 and decreased CN ratios for those for GBM1-4 or LCM1-2 plus *CCND3, HMGA1*, &*MOS.*\n\n# Discussion\n\n## Overview of tumor genomics\n\nBrain tumors are known for harbouring genetic abnormalities. Tumor genomes are being evaluated to varying extents by FISH, array comparative genomic hybridization (aCGH), single nucleotide polymorphism (SNP) arrays, specific mutation detection with various PCR methods, methylation studies, targeted sequencing of selected genes, and whole genome sequencing. Therapies are planned accordingly in some institutions. Ideally, molecular testing for potential treatment targets occurs at the time of diagnosis and again in tumor recurrences to indicate appropriate treatment alterations.\n\n## Amplified oncogenes in tumor fluids\n\nThis study demonstrates that high-level amplified oncogenes can be quickly detected by inexpensive multiplex, PCR-based studies of cellular fluids using standard molecular laboratory equipment and techniques. Fluids with significant tumor cellularity offer the opportunity to retrieve fresh tumor DNA in abundant amounts. Advantages in testing the DNA of tumor cells in fluids rather than FFPE tissue sections, include avoidance of exposure to formalin, heat, and organic solvents, and enrichment for tumor via pelleting, filters, and various other methods, such as those developed for isolating tumor cells from blood. Tumor cells in fluids not needed diagnostically constitute as a potential source of DNA for molecular assays. This study demonstrated detection of amplified oncogenes despite concerns regarding ultrasonic, mechanical, and osmotic distortions and mixing of the tumor cells with saline and blood. Endothelial cells were the predominant type of non-tumor cell present but these do not exceed what was originally present in the tumor parenchyma. A tendency for short sections blood vessels to remain intact and sediment differently from tumor cells was noted but was not investigated.\n\n## Adaptive and Co-amplification of Oncogenes\n\nIt is proposed that oncogenes can be adaptively amplified in tumor cells to increase key gene products while circumventing promoters and other traditional methods of gene regulation \\[7,8\\]. Tumor cells are already well known for developing resistance to chemotherapy by being permissive to amplification of a gene that encodes a key protein that aids drug metabolism \\[7\\]. Although genetic instability of brain tumors may account for some genes being amplified by chance, amplification of multiple oncogenes involved in a similar function, such as cellular proliferation or resistance to apoptosis, suggests an adaptive response. Co-amplification of multiple oncogenes that provoke redundant malignant behavior or adaptation to cancer treatments strengthens the rationale for planning multi-agent therapies based on identification of amplified oncogenes with multiplex techniques.\n\nAlthough co-amplifications of several oncogenes have been reported in glioblastomas, the lack of predictable patterns in individual tumors indicates that each tumor needs to be tested. Despite a tendency for genes in close proximity to be amplified together in tumor genomes, the span of amplifications in these regions is variable and frequently interrupted. Amplified expanses of chromosomes include both \"driver\" and \"passenger\" or \"bystander\" genes in regard to their effects on tumor behaviour. The benefits of amplified \"driver\" genes could underlie retention of amplified chromosomal regions in malignant tumor clones.\n\n## Specific oncogenes amplified in glioblastomas\n\nSeveral oncogenes are commonly amplified in brain tumors. Amplification of *EGFR* or one of its variants, *EGFRvIII,* whose encoded protein is constitutively active, is well-known to occur in primary glioblastomas. Amplification of *EGFR* has also been found in anaplastic astrocytomas \\[9\\]. Gains of *EGFR* occurred in 70% of 40 glioblastomas in one study with high levels of gene amplification occurring as double minutes in 42% of the cases. Lower levels of *EGFR* amplification occurred as insertions of extra gene copies distributed along chromosome 7 \\[10\\]. In a previous MLPA study of 104 glioblastomas, 74 (71%) had additional copies of *EGFR*\\[11\\]. Although high levels of *EGFR* most likely underlie some malignant adaptations in glioblastomas, only small subsets of patients have responded to therapies targeted to EGFR in clinical trials \\[12-15\\]. Five moderate to high-level amplified genes in glioblastomas identified in this study, *EGFR, PDGFRA, CDK4, MDM2* and *CYP27B1*, have also been previously reported, sometimes with co-amplification \\[16-20\\]. Although proximity of the locations for *CDK4CYP27B1* and *MDM2* at 12q14, 12q13.1-q13.3 and 12q14.3-q15, respectively, contributes to co-amplification, this cannot be assumed to occur for all three genes in individual tumors. In 20 glioblastomas that harbored at least one of these amplifications, only 7 had amplification of all 3 genes \\[20\\]. In another study when 5 glioblastomas contained amplification of either *MDM2* or *CDK4*, both genes were amplified in only 3 \\[18\\]. In another study, among the 5 glioblastomas that contained amplification of either *CDK4* or *MDM2*, only one tumor had both amplified \\[17\\]. In a survey of 456 glioblastomas, 13.4% had amplifications of *CDK4* but only 9.2% had amplification of *MDM2*\\[16\\]. In our study, 2 of 4 glioblastomas had co-amplifications of *CDK4* and *CYP27B1* and only one also had amplification of *MDM2.* Amplification of *EGFR* was present in all four glioblastomas.\n\n## Amplifications of oncogenes in other brain tumors\n\nBrain metastases from lung primaries in this study also contained amplified oncogenes suggesting that genomic analysis of metastases will detect amplified genes to serve as treatment targets, such as *CCNE1* that encodes cyclin E1 \\[21,22\\]. Additional analyses of brain metastases are indicated to identify the full range of oncogenes that can be amplified. Interestingly, in a previous MLPA study of non-typical meningiomas, 19 oncogenes were found to have amplifications in two or more invasive\/atypical\/anaplastic (mostly Grade II) meningiomas (total of 15) and the sums of copy numbers were inversely correlated with patient age \\[5\\]. Some of those genes were among the high-level amplified genes detected here but none of the amplifications in the non-typical meningiomas were in the high-level range. In this study, sums of CN ratios normalized with NB for the same 19 genes were higher in all 4 glioblastomas and in 1 of the 2 metastases than in the non-typical meningiomas studied previously (not shown).\n\n## Implications of amplified oncogenes\n\nAs products of amplified genes become treatment targets, combinations of specific inhibitors and monoclonal antibodies will need to be tailored for individual patients according to amplifications found in the tumor DNA. Additionally, there is a tendency for amplified oncogenes to undergo mutations. Therefore, testing each brain tumor for oncogene amplifications would be useful for detecting key molecular treatment targets.\n\n## Methods for assessments of oncogenes\n\nWith multiplex PCR methodology, rapid assessment of a relatively large group of oncogenes can be obtained using routine molecular laboratory techniques and equipment. In comparison to FISH assays, numerous genes can be tested simultaneously with comparisons to multiple non-amplified genes, whereas the number of FISH targets is limited to fewer genes. However, in this study, FISH did offer the advantage of detecting centromere duplication as a surrogate of chromosomal duplication so that low levels of *EGFR* amplification attributed to polysomy could be predicted. In comparison to the specificity of other PCR-based assays, amplification occurred with MLPA only if the probes hybridized to the gene targets in pairs (probe sets) and then underwent enzymatic ligation based on perfect sequence correspondence at their junctions to the tumor DNA. Also, the use of only one-fourth of the reaction volume for PCR in MLPA further reduced the chances of non-specific carryover of PCR amplicons from previous reactions. However, our study did not compare MLPA with other multiplex PCR techniques. Quantitative real-time PCR using multiwell plates is among other rapid molecular techniques to consider for testing multiple oncogenes simultaneously at low cost.\n\nThe validity of MLPA has been supported by other techniques. Detection of gene amplification (*ERBB2* or *Her- 2\/neu*) with fluorescent and chromogenic *in situ* hybridization has closely correlated with MLPA results \\[23,24\\]. Results of *EGFR* amplification with MLPA have been comparable to detection of increased protein and RNA expression using immunohistochemistry and non-MLPA PCR, respectively \\[10\\]. In this study, FISH confirmed MLPA results for the presence or absence of mid and high-level *EGFR* amplification. Correlations were not quantified due to the potential for FISH signals to merge in tandem repeats or as aggregates of double minutes and also the heterogeneity of results in individual tumor cells.\n\nThe cost of kit reagents in a single MLPA assay is less than \\$15. Although testing a tumor and normal brain DNA with two replicates each in three reactions to encompass probes for 79 genes increases the cost, testing multiple tumors with the same control DNA could be performed. Also, the number of genes to be screened could be reduced so that only 1 kit would be needed. Although high-level gene amplification results were obtained from the ultrasonic aspirations within a few days and the content of tumor DNA was plentiful, it should be noted that the FFPE assays demonstrated higher sensitivity in detection of low to mid-level gene amplifications. Therefore, ideally both types of specimens would be tested if detection of all levels of oncogene amplifications is desired. Multiplex detection of high-level amplifications using both types of samples strengthens confidence in the results at a relatively low cost and helps to negate concerns about tumor heterogeneity.\n\nAlthough DNA from patients' blood cells has been used as normal controls when searching for somatic genetic alterations in tumors, quantifying high-level amplified oncogenes pose a problem when using blood samples. Very low numbers of circulating tumor cells with high-level amplifications or their free DNA in the blood could bias analyses. Thus this study used normal brain DNA that was comparable to normal values expected with MLPA that were provided by the manufacturer. Pooled DNA from multiple normal donors should be considered for future studies. Based on this pilot study, automation of steps in the MLPA procedure and subsequent data analysis are future goals so that this type of assay can be streamlined, validated, and used clinically.\n\n# Conclusions\n\nIn summary, multiplex detection of high-level amplifications among oncogenes was successful in ultrasonic surgical aspiration DNA obtained from malignant brain tumors when compared with FFPE from the same tumors. The results indicate that morphologic evaluation of ultrasonic surgical aspirations to confirm tumor cell content and retrieval of DNA would aid molecular testing of brain tumors for oncogene amplifications. Many of the oncogenes with copy number gains encode proteins that are potential therapeutic targets. Therefore rapid identification of high-level gene amplifications could stratify patients to clinical trials and treatment plans shortly after surgical resections of brain tumors. Although ultrasonic surgical aspiration specimens are less sensitive than FFPE in detecting low to mid-level amplifications, the bulk of the tumor is present in the aspirations, they are fresh and homogenously mixed, and high-level amplifications can be detected in them. Abundant tumor DNA harvested from cellular fluids could also be used for targeted sequencing of amplified oncogenes to detect activating mutations.\n\n# Abbreviations\n\nCE: Capillary electrophoresis; CGH: Comparative genomic hybridization; CN: Copy number; CUSA: Cavitronic ultrasonic surgical aspiration; DAP1: Diamino-phenylindole; EP1: Ependymoma; FFPE: Formalin-fixed: paraffin-embedded; FISH: Fluorescence in-situ hybridization; GBM: Glioblastoma multiforme; HPF: High power field; MLPA: Multiplex ligation-dependent probe amplification; LCM: Lung carcinoma metastasis; MHz: Mega Hertz; NB: Non-neoplastic (normal) brain; PCR: Polymerase chain reaction; SNP: Single nucleotide polymorphism.\n\n# Competing interests\n\nThe authors declare that they have no competing interests.\n\n# Authors' contributions\n\nMEB, LNT, SSM, and JFL carried out the molecular genetic studies. MEB, LNT, and SP drafted the manuscript. SSM and MLN performed the FISH assays. MEB, SP, MLN and AN participated in the design of the study. SP, JFL, and AN participated in and coordinated specimen and clinical data retrieval and characterization. MEB conceived and coordinated the study. All authors read and approved the final manuscript. This study was conducted with approval from Institutional Review Board (IRB) of LSUHSC-S.\n\n## Acknowledgements\n\nWe acknowledge Lee Ellen Brunson, Dwain D'Souza, JoAnn Dismuke and Neelam Joshi, Department of Pathology, Louisiana State University Health Sciences Center-Shreveport (LSUHSC-S), for advice and technical assistance and also Ashley B. Flowers, Kristopher M. Katira, and Raj B. Patel, while medical students at LSUHSC-S School of Medicine, for technical assistance. We thank the Feist-Weiller Cancer Center and the Department of Pathology, LSUHSC-S, for providing equipment. This study was funded by a LSUHSC-S Grant-in-Aid Award (2008\u20132010) to Marie E. Beckner, MD with the purpose of encouraging high-value intramural research.","meta":{"dup_signals":{"dup_doc_count":122,"dup_dump_count":26,"dup_details":{"curated_sources":2,"2022-27":1,"2021-43":1,"2020-40":1,"2020-24":1,"2019-35":1,"2015-48":5,"2015-40":4,"2015-35":5,"2015-32":5,"2015-27":5,"2015-22":5,"2015-14":4,"2014-52":5,"2014-49":6,"2014-42":13,"2014-41":7,"2014-35":4,"2014-23":10,"2014-15":6,"2023-23":1,"2015-18":5,"2015-11":5,"2015-06":4,"2014-10":6,"2013-48":5,"2013-20":5}},"file":"PMC3475141"},"subset":"pubmed_central"}
{"text":"abstract: Peer review is the foundation quality in science, but now, in the US, the system is broken. What's the best way to fix things.\nauthor: Gregory A Petsko\ndate: 2006\ninstitute: 1Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02454-9110, USA\ntitle: Instructions for repair\n\nLast month I wrote about the sharp decline in the success rate for scientific research proposals submitted to the US National Institutes of Health (NIH) and other agencies. That column provoked numerous responses from both administrators of the funding organizations and life scientists. The administrators, while not denying some of the problems I discussed, argued that things aren't quite as bad as they seem, and that a large part of the difficulty stems from sizeable increases in the number of grant applications and the amounts requested, rather than from poor choices in managing the doubling of the NIH budget that took place not long ago. The scientists, on the other hand, all said that things were even worse than I had claimed.\n\nCare has to be taken in drawing conclusions from either of these sources. I'm sure that people who have experienced difficulty in obtaining funding are more likely to respond to that column than those who've had success. And administrators probably feel the need to defend themselves, and their agencies, from what they might, with some justification, see as an attack by someone who doesn't know the whole story the way they do.\n\nNevertheless, although I think both sets of comments are useful, I also think both largely missed the point. People who wrote to me were all concerned, in one way or another, with the amount of money available for research and how it is being allocated. That's what seems to be on everybody's minds, and it's certainly worth talking about. Whether or not we're allocating the available funding sensibly is something that ought to be engaging officials as well as researchers in an ongoing dialog about priorities in science. (But that dialog isn't taking place. Somehow it just seems easier to keep asking for more money.) Yet, that wasn't the main point of the column. What concerns me is that, whether there really is a crisis in scientific funding or not, the perception that there is - and believe me, that is the perception on the part of just about every researcher I have talked to - has crippled the peer-review system.\n\nPeer review is the foundation of quality in science. It prevents widespread cronyism and slowly weeds out unproductive lines of inquiry. But it requires that reviewers be both fair and wise. When the perception is that there's not nearly enough money to fund even all of the highest-quality proposals, a defensive turf-protection replaces a spirit of curiosity and egalitarianism. When it seems as if the primary job of a reviewer is to eliminate most proposals rather than to fight for the good ones, nit-picking replaces generosity. When the feeling is that every dollar counts so much that no risks dare be taken, conservatism and incremental advances get rewarded at the expense of bold new ideas. And when all of these things happen - and I believe they are happening, now, in the US - then the system is broken.\n\nSocieties based on scarcity tend to become hierarchical, with a well-fed elite and starving masses. As can be seen from publicly available data <http:\/\/grants1.nih.gov\/grants\/financial\/QA_Doubling_Period.doc>, during the recent doubling of the NIH budget over a seven-year period, the number of investigators getting funded changed very little. Where did the money go? Besides a very large increase in the funding for NIH's own intramural research program, it seems to have gone to large increases in funding for established investigators who renewed their grants successfully during this period, or wrote additional ones. Instead of bringing lots of new people into the system, we ended up with more money for roughly the same set of grant holders. Now that funding is tight, those bloated operations are under tremendous pressure to at least maintain their size, which makes it even more difficult for new investigators - or new ideas - to enter the system. The average age at which a scientist receives his or her first NIH grant in the US is currently 42 for PhDs (even older for MDs), and in this time of perceived scarcity a broken peer-review system is not likely to change that.\n\nWhat's the best way to fix things? It could be argued that the problem is temporary, and that when funding loosens up again, as it always has in past boom-bust cycles, peer review will recover along with everything else. After all, that's what happened in the 1970s. No need to tamper with the system. Time will take care of the problem.\n\nI have my doubts. There's one big difference between peer review in 1975 and peer review today: the number of senior investigators participating in the process. Back then most review panels had a preponderance of such scientists, who provided the system with institutional memory of the way things were supposed to work. Nowadays, most established investigators feel they are too busy to put in the considerable time required to deal with the glut of proposals that every panel faces. The result is that less experienced scientists, with no history of a different gestalt, are being fed into a system where fault-finding and conservatism are the norm, so when the funding situation improves, there's no guarantee that the peer-review system will improve with it. (If you doubt this, consider the former Soviet Union. When it collapsed in 1989, newer Soviet-block countries like Poland and Hungary and Czechoslovakia, where there was a generation of people who still had a memory of how a market-based economy should work, did much better than Russia, where no one alive had experienced any system but communism.) In addition, the insistence that the composition of the panels must satisfy a requirement for geographic and institutional balance means that it's hard to have a large number of top scientists on any panel, even if they wanted to serve.\n\nSo my first repair instruction is simple: Do away with the misguided concept of balance, and require that all holders of research grants serve at least one year on a reviewing panel for every five years of funding they receive, regardless of seniority. Renewal of funding would be contingent on fulfillment of this service. If there is a surplus of available talent, then grants administrators could forgive the obligation for any given five-year cycle, but the requirement would kick in again when a grant was renewed. There would need to be a mechanism to deal with people who hold multiple grants - perhaps they would only incur a single one year debt for every five years of total funding, or the length of service could scale with the total budget; these details can be worked out. The important point is to create a pool of the best researchers, and to make sure that they represent the majority on all peer-review panels. As a dyed-in-the-wool advocate of personal freedom, the coercive aspects of this suggestion do trouble me somewhat, but it isn't really all that different from the way things work in the other main form of peer review - the jury system.\n\nMy second idea for how fix things is meant to address the problem of reviewer morale. When someone is given twelve grants to review, and knows that there is only a small probability that even the best one is actually going to be funded, he or she rapidly becomes discouraged. It's even more depressing when some less knowledgeable reviewer nit-picks one's best proposals, and depression is not the best mindset from which to make judgments. I suspect the program officers at the funding agencies must feel equally demoralized: it's no fun having to say \"no\" all the time, and to watch conservative study sections pass over the most exciting new ideas in favor of more of the usual. The solution, I think, is to give the program officers more autonomy in funding decisions. Some NIH institutes and centers claim that they do this, but in practice I have found that program officers rarely go against the recommendations of the reviewing panels. I suggest taking at least 10% of the budget of each institute or center and allowing the program officers to use it to fund grants that they believe to be exciting but that would otherwise miss the payline cut-off. They would need to justify each decision to the council, of course, but this suggestion would empower them to rectify some of the worst mistakes of the panels. In my experience, funding officials tend to be bright, committed individuals with a good broad knowledge of their field; I have no hesitation in giving them more autonomy. This is the way things actually work at the National Science Foundation, a funding agency that many believe has a better long-term history of supporting innovative research than does the NIH.\n\nThere also needs to be a way to improve the judgments coming out of the panels. Having more experienced reviewers would help, but it's hard to deal thoroughly and fairly with each proposal when the number being reviewed has increased so greatly. The way to solve the problem of proposal overload is to reduce proposal size. NIH proposals now are limited to 25 pages for the scientific description (that includes background and significance, progress during the past budget period, and the plan for future research). I think that should be shortened to 15 pages. If you can't describe clearly in 15 pages what you've already done, what you intend to do, and why it's important, you probably can't do it in 50.\n\nBut I think the proposals should be structured differently for different investigators. Scientists submitting their first proposal need to spend more space detailing how they are planning to carry out the work than established investigators should. In fact, I would argue that established investigators shouldn't have to describe their proposed methods in any detail at all, except if these are novel. To ask someone who has demonstrated for years that they can deliver the goods to prove that they know what they're doing is silly and borders on insulting. It also provides the nit-pickers with extra ammunition. People who tout stocks are constantly warning investors that past performance is no guarantee of future returns. But there is one area where it is: scientific research. The best predictor I know of as to whether a project will work is the track record of the principal investigator. Someone who has been consistently successful is not likely to fail, even when doing something risky. We need to stop pretending that isn't true. Most organizations that award pre- and post-doctoral fellowships spend very little time picking over the details of the applicant's research proposal, because they know that these young people haven't had any experience writing proposals and anyway usually end up doing something different, or in a very different way, from what they propose. Instead, fellowship reviewers tend to consider the qualities of the individual to be the most important factor on which to base their judgments. I think that makes sense at all levels of science. We need to be much less concerned with the details of projects, and put our bets on people and ideas.\n\nWhile we're waiting for funding levels to improve, we need additional mechanisms to get young people started. The observation that, while investigator funding went way up during the NIH budget doubling, the number of investigators changed very little, suggests that we should consider putting a cap on the size of each award so as to make more money available for funding new projects and people. This is a serious matter, because it potentially has an impact on current employees, so if we implement a cap we will need to phase it in gradually. I am not proposing that we limit the total amount of funding that an individual can have - I think if someone can justify the need for millions of dollars to do first-rate science they should be able to obtain it. But I do think that we should exercise more scrutiny in such cases, and one way to do that is to force someone who claims to need, say, a million dollars to support a project to submit two or three proposals instead of one. I also think we will do better science, as a community, if we have more individual investigator-initiated projects and fewer mega-sized 'me-too' programs. Most innovation comes from small projects by relatively new people.\n\nTwo final ideas pertain to the machinery of the reviewing process itself. Turf protection is one of the biggest problems in peer review: as fields try to survive in a time of scarce resources, they often fight to fund their own mediocre science at the expense of quality in other areas. This largely stems from the personal and professional relationships that develop among members of a particular discipline. It's less of a problem when there's more money to go around, but right now we need to fight it. Here's a heretical and possibly crazy idea: I think we should consider not allowing people to review grants in their own field. Instead, they should only be allowed to comment on any questions of technical feasibility that come up during the review. This may seem absurd, but I'm not sure it is. If we follow my suggestion to bet on people rather than projects, detailed technical expertise isn't so important. And if we have the best, most experienced people back on our review panels, they usually will have a pretty broad knowledge of genomics, or biology, or whatever the main subject is. That will allow them to assess the importance of the proposed research and the impact of the applicant's previous contributions, which I maintain are the only two criteria that really should matter. Reviewing outside one's primary area of technical expertise happens all the time on fellowship panels, and they usually make pretty good decisions. After all, you don't have to be able to lay an egg in order to tell a good one - or to smell a bad one.\n\nThe second procedural change we should consider is aimed at addressing the issue of possible bias in the system. In times of scarce dollars, reviewers worried about their own chances of obtaining funding have an incentive to prevent others from being funded. Even if we assume such a thing rarely happens, we should want to ensure that proposals are reviewed wisely as well as fairly. I think the best way to guarantee the quality of the peer-review process is to review the reviewers. The data to do so exist, because there is a record of how every member of a panel has voted on every grant. Since most panelists only read their assigned proposals in detail, we need only be concerned with how a reviewer's scoring of such applications compares with the average score awarded to those same applications by the other assigned reviewers. Abnormally high or low scores would not be damning in and of themselves (there's plenty of room for legitimate differences of opinion in science) but a consistent pattern of low or high scores could indicate either poor judgment or bias. You may wonder how to be sure about such an evaluation, but it's actually easy, because we can compare each reviewer with him or herself. Bias or territoriality should be relatively easy to detect by examining how a suspect reviewer treats the same grants when they are resubmitted after revision. Since unsuccessful applicants try hard to answer the criticisms raised by the previous review, the scores of resubmitted grant proposals should improve, on average. If a reviewer's scoring on such resubmissions remains abnormally low compared with other reviewers who are also seeing the proposal for a second time, then there is reason to question the impartiality, or the judgment, of that reviewer, and they can be eased off the panel. There might even be no need to evaluate every reviewer all the time: random checking might be all that is needed to discourage trying to rig the game.\n\nWhen fear and discouragement drive peer-review decisions, then the system is broken. But it's not broken beyond repair. The suggestions I've offered here can help mend it; at the very least, I hope they will start a dialog about what should be done. The worst thing we can do as a community is to throw up our hands in despair or pretend that everything will right itself magically when more money becomes available. Peer review is too important to give up on, or be left to chance. If we're serious about funding the best science and making our profession attractive to the brightest, most creative young minds, then we need to fix the system so that it once again serves those ends. Let's get to work.\n\n## Figures and Tables","meta":{"dup_signals":{"dup_doc_count":101,"dup_dump_count":48,"dup_details":{"curated_sources":2,"2023-14":1,"2022-49":1,"2022-21":1,"2021-43":1,"2020-34":1,"2020-24":1,"2019-47":2,"2019-35":1,"2019-26":1,"2019-13":1,"2019-04":2,"2018-47":2,"2018-39":1,"2018-13":1,"2017-47":1,"2017-34":1,"2017-26":1,"2017-17":2,"2017-09":8,"2016-44":1,"2016-40":1,"2016-36":9,"2016-30":7,"2016-22":1,"2016-18":1,"2016-07":4,"2015-48":2,"2015-40":1,"2015-35":2,"2015-32":1,"2015-27":2,"2015-22":2,"2015-14":2,"2014-52":2,"2014-49":3,"2014-42":3,"2014-41":4,"2014-35":2,"2014-23":3,"2014-15":3,"2023-40":1,"2015-18":2,"2015-11":2,"2015-06":1,"2014-10":2,"2013-48":2,"2013-20":2,"2024-22":1}},"file":"PMC1557998"},"subset":"pubmed_central"}
{"text":"abstract: Lessons in personal genome analysis, social networking or health information?\nauthor: Gregory A Petsko\ndate: 2009\ninstitute: 1Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02454-9110, USA\ntitle: What my genome told me - and what it didn't\n\nWell, it turns out I'm not descended from Genghis Khan. I'm sure that's as surprising to you as it is to me. I mean, according to what we hear from people who use genomics to track human migrations, a huge percentage of the human race is actually descended from Genghis Khan. But not me.\n\nThat's one of the things I learned when I submitted a sample of my DNA for genome-wide single nucleotide polymorphism (SNP) analysis by one of the companies that have sprung up to perform such tests for ordinary individuals for a fee. I was curious to see what sort of information they provide, and wanted to know something about my own genomic makeup, to be honest, so I followed the directions of the company I had selected and spat into a plastic container until I produced the required volume of saliva, mailed it in, and awaited the results. Would I have an allele that doomed me to a rare genetic disorder as I got older? Was I at much higher than normal risk for heart disease, diabetes, or any of the other thousand natural shocks that flesh is heir to? Was I descended from Genghis Khan?\n\nThe company I sent my saliva sample to doesn't actually do any DNA sequencing or hybridization itself; it contracts this out to a specialist laboratory. Once the lab received my sample, DNA was extracted from cheek cells in the saliva and amplified by PCR to produce enough material for the genotyping step. Next, the DNA was cut by restriction digestion into smaller, more manageable pieces. These DNA pieces were then applied to a DNA chip, which in this specific case is a small glass slide with millions of microscopic beads on its surface. Attached to each bead are the probes - bits of DNA complementary to those specific sites in the human genome where important SNPs are located. There is a pair of probes for each SNP, corresponding to the 'normal' and 'non-normal' version of each SNP. Hybridization to the particular probe, detected by fluorescence just as in the case of any other DNA chip experiment, serves to identify the allele.\n\nThe DNA chip that this particular company uses reads 550,000 SNPs that are spread across the entire genome. Although this is still only a fraction of the 10 million SNPs that are estimated to be in the human genome, these 550,000 SNPs are specially selected 'tag SNPs' - because many SNPs are linked to one another, the genotype at many SNPs can often be determined by looking at one SNP that 'tags' its group. This tagging procedure maximizes the information from every SNP analyzed, while keeping the cost of analysis low.\n\nIn addition, all the DNA analysis companies have hand-picked tens of thousands of additional SNPs of particular interest from the scientific literature and added their corresponding probes to the DNA chip. These SNPs include risk factors for common and rare human diseases, genetic traits such as color blindness, and so on.\n\nAccess to the resulting data is through the company's website, which includes the ability to download the entire set of SNP information. Once I was notified that my results were in, I did that, and being a scientist I performed my own bioinformatics on the data, but the website actually does a pretty good job of providing the customer with specific information about alleles for various illnesses, physical traits, and so on.\n\nHere are a few of the things I leaned about myself, physically speaking:\n\nAccording to my genome, my eye color is likely to be brown (good guess). I should be lactose tolerant (I am). My cytochrome P450 data show that I would be quite sensitive to the anti-clotting drug warfarin if I ever had to take it (which I hope I never do - it's a nasty drug). The SNPs in my androgen receptor gene say that I am considerably decreased in risk for male pattern baldness (I have news for them; I'm getting rather thin on top). I have a SNP in a dopamine receptor gene that, in one German study, was found to be associated with reduced efficiency in learning to avoid errors (unless I got the facts wrong). According to a single SNP in one gene associated with insulin metabolism, I have increased odds of living to be 100 (that is, if all the mistakes I don't learn to avoid don't get me first). There are a number of SNPs that have, in some studies, been associated with increased athletic performance (faster running, quicker reaction times, and so on). I don't have any of them, which will come as no surprise to any of my gym teachers.\n\nI am at slightly increased risk, relative to the norm, for rheumatoid arthritis and psoriasis (that last one is interesting, because my father suffered from it). I am at slightly decreased risk for Celiac disease, Crohn's disease, type 1 diabetes, and prostate cancer. In all cases, the change is small - less than two-fold difference, and not enough to cause me to consider any lifestyle changes.\n\nBut one thing did jump out at me when I looked at my data. I have a guanine at rs1799945, which is located in the gene coding for a protein called HFE. HFE is the protein mutated in hereditary hemochromatosis. Hereditary hemochromatosis, the most common form of iron overload disease, is an autosomal recessive genetic disorder that causes the body to absorb and store too much iron. Excess iron is stored throughout the body in organs and tissues, including the pancreas, liver, and skin. Without treatment, the iron deposits can damage these organs and tissues. There are two primary variants that give rise to this disease.\n\nGenetic variant 1 (C282Y\/rs1800562) is in the *HFE* gene. The *HFE* gene makes a membrane protein that is similar structurally to MHC class I-type proteins and associates with \u03b22-microglobulin. It is thought that HFE helps cells in the intestines, liver, and immune system control iron absorption by regulating the interaction of the transferrin receptor with transferrin. The C282Y substitution disrupts the interaction between HFE and its \u03b22-microglobulin light chain and prevents cell-surface expression. Pamela Bjorkman's 2.6 \u00c5 resolution crystal structure of HFE confirms that, as predicted from its sequence, Cys282 (residue 260 in the mature form of the protein) is involved in a disulfide bridge analogous to those found in class I MHC \u03b13 domains. Loss of the disulfide destabilizes the native fold of the protein. The second most common variant is also in the HFE gene. It is a change of histidine 63 to aspartic acid. In the crystal structure of HFE, His63 (41 in the sequence of the mature form) is involved in a crucial salt bridge, which would be destroyed by mutation to a negatively charged residue, thereby also destabilizing the protein. Thus, like so many other hereditary disorders, hemochromatosis is a protein conformational disease.\n\nIn the US, variant 1 is the more frequent. The 'normal' Cys282 allele has guanine in both strands and is found in about 876 out of 1,000 people of European ancestry. The most common form of hereditary hemochromatosis is typically associated with people homozygous for an adenine in both positions; this occurs in about 4 out of 1,000 people of European ancestry (0.4%). However, penetrance is incomplete: only about a third to a half of the homozygotes will show elevated iron levels and perhaps fewer than 10% of the males (and 1 to 2% of the females) will develop the full clinical symptoms of the disease, which include joint pain, fatigue, abdominal pain, liver dysfunction, and heart problems. As Ernest Beutler has pointed out, the hemochromatosis mutation is relatively common; the hemochromatosis disease is rare. Mutation of the HFE gene is a necessary, but not sufficient, condition. The challenge, as in the case of so many diseases in the age of genomics, is to understand what other genetic, epigenetic, and environmental factors determine why a few homozygotes for the C282Y (or H63D) mutations develop severe iron-storage disease, while the majority go through life pretty much unscathed by this genotype.\n\nHeterozygotes for C282Y have an adenine in only one strand and represent about 120 in 1,000 people of European ancestry; they almost never develop clinical symptoms. Heterozygotes for H63D are less common, but also are unlikely to develop clinical symptoms. Like one person in ten in the United States, I am a hemochromatosis carrier. I am heterozygous for H63D.\n\nNow that I know that, what does it do for me? Not much, I guess, but I will remember it, and should I ever develop any of the symptoms of iron overload, I will probably tell my physician to check my iron levels. Maybe that's worth knowing.\n\nBut if you go to the website of the company that did my analysis, you will find that the sort of information I've talked about here is actually not that prominently displayed. What is displayed are all manner of data connecting to ancestry. I spoke to the CEO of the company, and she confirmed that, much to their surprise, people who use their service are much more interested in tracing their roots, genetically speaking, than they are in things related to their health or physical condition. The site offers several tools for connecting yourself with others who share your ancestry, genetically speaking. In other words, for the present, the primary use of personal genome-wide SNP analysis is social networking.\n\nMy maternal haplogroup is T2b2. Haplogroup T originated about 33,000 years ago in the Middle East, as modern humans first expanded out of eastern Africa. Its present-day geographic distribution is strongly influenced by multiple migrations out of the Middle East into Europe, India, and eastern Africa after about 15,000 years ago. T2 is currently widespread in northern Africa and Europe. My mother's family most recently came from Italy, so I guess this makes sense. You can find famous people with your haplotype on the site: for example, if your maternal haplotype is H4a, you have the same type as Warren Buffet, one of the richest men in the world. You'll be delighted - and perhaps not surprised - to know that the only famous person the site lists as sharing my maternal haplotype is the notorious old-west outlaw Jesse James.\n\nMy paternal haplotype is I2. Haplogroup I2 is most abundant in eastern Europe and on the Mediterranean island of Sardinia, where it is found in 40% of the male population. Like its brother haplogroup, I1, I2 expanded northward at the end of the Ice Age about 12,000 to 14,000 years ago. But unlike I1, which expanded from the Iberian peninsula into northwestern Europe, I2 radiated outward from the Balkans and southwestern Russia into the eastern half of the continent. That again makes sense, as my father's family were Cossacks. If my paternal haplotype were the extremely common C3, I would be descended from Genghis Khan. I'm not. If it were type T, I would share paternal lineage with the great American president and founding father Thomas Jefferson. I don't. In fact, the company website doesn't list a single famous person with paternal haplotype I2 (unless you count me, of course).\n\nSo now, thanks to my own personal genome SNP analysis, I know that I'm not likely to be exceptionally athletic and that I'm not a blue-eyed balding blonde, neither of which comes as any surprise whatsoever. But I have also learned that I'm not descended from Genghis Khan. So I've got that going for me. Which is something, I suppose.","meta":{"dup_signals":{"dup_doc_count":102,"dup_dump_count":51,"dup_details":{"curated_sources":2,"2023-23":1,"2022-33":1,"2021-39":1,"2021-21":1,"2020-34":1,"2020-24":1,"2019-47":2,"2019-30":1,"2019-22":1,"2019-09":1,"2019-04":1,"2018-51":1,"2018-47":1,"2018-43":1,"2018-39":1,"2018-34":1,"2018-30":1,"2018-26":1,"2018-13":1,"2018-09":1,"2017-43":2,"2017-22":1,"2017-09":7,"2016-44":1,"2016-36":7,"2016-30":8,"2016-22":1,"2016-18":1,"2016-07":3,"2015-48":2,"2015-40":2,"2015-35":2,"2015-32":2,"2015-27":2,"2015-22":2,"2015-14":2,"2014-52":3,"2014-49":3,"2014-42":5,"2014-41":3,"2014-35":2,"2014-23":3,"2014-15":1,"2023-50":1,"2015-18":2,"2015-11":2,"2015-06":1,"2014-10":3,"2013-48":2,"2013-20":2,"2024-26":1}},"file":"PMC2718485"},"subset":"pubmed_central"}
{"text":"abstract: Aortic valve replacement (AVR) is the gold standard for the treatment of severe symptomatic aortic stenosis. Complications directly related to surgical procedure are relatively infrequent. Coronary ostial stenosis is, generally, referred as late complication. Anecdotal reports concern coronary ostial stenosis as acute complication. A unique fatal case of intraoperative, bilateral coronary ostial obstruction by prosthetic valve leading to an extensive myocardial infarction is reported. Surgeons must have a high level of vigilance regarding the occurrence of acute myocardial ischemia and sudden death soon after AVR.\nauthor: Emanuela Turillazzi; Gabriele Di Giammarco; Margherita Neri; Stefania Bello; Irene Riezzo; Vittorio Fineschi\ndate: 2011\ninstitute: 1Department of Forensic Pathology, University of Foggia, Ospedale Colonnello D'Avanzo, Via degli Aviatori 1, 71100 Foggia, Italy; 2Institute of Cardiac Surgery, University of Chieti, Via dei Vestini, 29, 66100 Chieti, Italy\nreferences:\ntitle: Coronary ostia obstruction after replacement of aortic valve prostesis\n\n# Background\n\nAortic valve replacement (AVR) remains the gold standard for the treatment of severe symptomatic aortic stenosis. Late coronary ostial stenosis is described as late complication of the surgical procedure \\[1\\]. Anecdotal reports concern coronary ostial stenosis as acute complication: right ostial occlusion from aortotomy suture, ostial thrombosis as traumatic consequence from an aortic retractor, coronary artery spasm, calcium debris embolization and partial direct occlusion by the device or edematous reaction have been described \\[2-4\\].\n\nA unique fatal case of intraoperative, bilateral coronary ostial obstruction by prosthetic valve leading to myocardial infarction is reported.\n\n# Case presentation\n\n## Clinical findings\n\nA 50 -year old woman, previously submitted to surgical aortic valve replacement in 2003 for aortic valve stenosis was admitted to a cardiac surgery unit for replacement of dysfunctioning mechanical valve prosthesis. Echocardiographic evaluation documented a prosthetic dysfunction with the evidence of an increased peak gradient up to 105 mmHg and a prosthetic valve area by 0.58 cm^2^ in a normal left ventricular function. Prosthetic valve replacement was then performed in August 2010. Cardiopulmonary bypass (CPB) was instituted between right atrium and ascending aorta and a moderate hypothermia was reached. Myocardial protection was achieved by retrograde injection of blood cardioplegia through coronary sinus as induction; it was completed by antegrade injection directly in both coronary ostia. After ascending aorta had been transversely opened, prosthetic dysfunction was evident as one of the hemidisks appeared to be locked by pannus ingrowth and fresh pivotal thrombi. No perivalvular leaks were described. The prosthesis was then removed; both coronary ostia were described quite close to the aortic annulus. Bovine pericardium bioprosthesis (Edwards Magna Ease 21 mm) was then implanted in supraannular position. Aortotomy was sutured using Teflon felt strips as reinforcement due to an extremely thin aortic wall and patient was uneventfully weaned from CPB. During skin suture patient experienced sudden severe hypotension; an ECG demonstrated signs of transmural myocardial ischemia. CPB was promptly reinstituted. Severe dilatation and hypokinesia of right ventricle and severe hypokinesia of interventricular septum were registered. Intraortic ballon pumping (IABP) was inserted through a femoral artery and a saphenous vein graft to right coronary artery (RCA) immediately added on a beating heart in the hypothesis of RCA ostial obstruction. At the end of the operation cardiopulmonary support by means of Extracorporeal Membrane Oxygenator (ECMO) through femoro-femoral access was initiated and IABP removed. Patient was then transferred to Intensive Care Unit (ICU) for postoperative course. Graft verification was not performed, either intraoperatively and postoperatively. Two days after, following progressive decrease of ECMO support along with a satisfying hemodinamical recovery, she was transferred to the operating theatre to remove mechanical assistance. One hour after the new admission to the ICU, cardiac arrest occurred pushing surgical team to reposition an emergent ECMO at the ICU bed. The day after patient was transferred again to the theatre to remove thrombi from left atrium evident at ecographic inspection. At that moment cardiac activity was silent in a very compromised systemic condition. Two days after the new admission to the ICU under a new femoro-femoral support she was declared dead.\n\n## Pathological findings\n\nA complete post mortem examination was performed. Cerebral and pulmonary edema was detected. Cardiac size was mildly increased (11.5 \u00d7 10 \u00d7 7.7 cm), with conical shape; heart weight was increased (547g). At the inspection, a horizontal aortotomy was evident with prosthetic valve in situ after aorta was opened. Meticulous attention was devoted to observe the coronary ostia: the right one was not found at the inspection of inner surface of sinus of Valsalva. Using a metal probe inserted into right coronary artery distally cut, the entrapment of the ostium in a stitch at the level of prosthetic annulus became evident. Left coronary ostium was identified behind one of the bioprosthesis post that was responsible of its complete obstruction (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>). Coronary vessels were fully patent during their course. Equally patent appeared to be saphenous graft to RCA. The inspection of both ventricles showed many foci of pale myocardium diffused to either free wall and interventricular septum.\n\nHistological investigations showed haemostasis of all organs and mild cerebral and pulmonary edema. Specimens taken from the heart showed diffuse stretching of the myocardium with elongation of the sarcomeres and nuclei. Polymorphonuclear leucocyte infiltration was well evident at the periphery of the necrosis zones (Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>). Numerous foci of hypercontracted myocardial cells with markedly short sarcomeres and anomalous, extreme thickening of Z lines and rexis of the myofibrillar apparatus into cross-fibre, anomalous and irregular, up to a total granular disruption were also observed. Myocells pathological aspects were also studied on confocal laser scanning microscope: stretching of the myocardium in flaccid paralysis, resulting in a very early elongation of sarcomeres and nuclei; polymorphonuclear leucocyte infiltration was well evident.\n\nExtramural coronary arteries showed nodular hyperplasia of smooth muscle cells and elastic tissue with fibrous replacement. Proteoglycan accumulation in the deep intima between tunica media and the fibrous cap was observed too. No critical stenosis was evident.\n\nFinal diagnosis of extensive myocardial infarction due to the intraoperative occlusion of both coronary ostia by bioprosthesis malpositioning was established as the cause of death.\n\n# Conclusions\n\nIn the present case, extensive myocardial infarction due to the intraoperative occlusion of both coronary ostia by prosthetic valve was recorded as the cause of death. Since there are, to date, no reports of similar deaths in patients undergone to AVR, our report provides useful information on this complication of AVR.\n\nCoronary ostial stenosis following AVR is believed to occur in 1% to 5% of AVR procedures. It is a life threatening complication which, generally, becomes evident from 1 to 6 months after the operation \\[5\\]. Several different mechanisms which include the possibility of microinjuries and local hyperplastic reaction related to the infusion pressure and\/or low temperature of cardioplegic solution and overdilation of the vessel by the tip of the cardioplegic catheters are thought to be involved \\[1,5\\]. Other mechanisms were hypothesized such as widespread intimal thickening and fibrous proliferation in proximity of the aortic root, presumably as a reaction to turbulence around aortic ball valve prostheses. An immunological reaction to the heterograft after AVR has been considered in cases of bilateral ostial coronary arteries stenoses revealing several months following the surgical procedure \\[1,2\\]. On the basis of these evidences the exact mechanism underlying late coronary ostial stenosis following AVR is unclear.\n\nAnecdotal reports describe the rare occurrence of acute coronary ostial stenosis; right ostial occlusion from aortotomy sutures and ostial post-traumatic thrombosis due to aortic retractor have been described \\[2\\]. In exceptional cases, embolism from debris, more often calcium related to aortic valve decalcification, or left atrial thrombectomy can be involved \\[1,2\\]. Coronary artery spasm has been recognized as a possible cause of hemodynamic and arrhythmic instability after aortic valve replacement \\[6\\]. Occasionally, secondary fibrosis in the area of suture placement may occur causing ostial stenosis \\[7\\]. Finally, use of surgical glue in aortic surgery, or compression from outside due to the glue used to protect the anastomosis may cause stenosis of one or both coronary ostia \\[8\\].\n\nConclusively, there is a reasonable body of evidence that acute coronary ostial stenoses may occur, even if rarely, after AVR and this complication may be life threatening if not promptly recognized, leading to myocardial ischemia, infarction, or fatal arrhythmia. Consequently, it is important to have an high index of diagnostic suspicion if circulatory collapse and\/or signs of myocardial ischemia occur soon after surgery. Transesophageal echocardiography can be useful in diagnosis of acute complications of cardiac surgery; however urgent coronary angiography remains the gold diagnostic tool \\[8\\].\n\nIn our case, both coronary ostia were iatrogenically occluded by the prosthesis: the right one appeared to be entrapped by a stitch and the left one was occluded by prosthetic post. Neither intraoperative transesophageal echocardiography or postoperative coronary angiography were performed, so impeding to reach a prompt diagnosis.\n\nFrom a clinical point of view, surgeons must have an high level of vigilance regarding the occurrence of acute myocardial ischemia soon after AVR and must be ready to perform either an intraoperative verification of patency or an early coronary angiography during ICU stay since these diagnostic tools may reveal mechanisms underlying ischemia which could make necessary a surgical approach (coronary stenting, device removal and\/or re-replacement with a smaller valve size with or without annular enlargement) \\[3,8\\].\n\n# Consent statement\n\nWritten informed consent was obtained from the Medical Examiner Department, Court of Justice, for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.\n\n# List of abbreviations\n\nAVR: aortic valve replacement; CPB: cardiopulmonary bypass; IABP: intraortic ballon pumping; RCA: right coronary artery; ECMO: Extracorporeal Membrane Oxygenator; ICU: intensive care unit.\n\n# Competing interests\n\nThe authors declare that they have no competing interests.\n\n# Authors' contributions\n\nSB drafted the manuscript. MN carried out the istological analysis. IR performed the microscopic analysis. ET, GDG and VF conceived of the study, and participated in its design and coordination and helped to draft the manuscript.\n\nAll authors read and approved the final manuscript.","meta":{"dup_signals":{"dup_doc_count":186,"dup_dump_count":38,"dup_details":{"curated_sources":4,"2022-49":1,"2022-27":1,"2021-49":1,"2020-29":1,"2019-35":1,"2017-17":1,"2017-09":18,"2017-04":1,"2016-50":1,"2016-44":1,"2016-40":1,"2016-36":1,"2016-30":2,"2016-22":1,"2016-18":1,"2016-07":18,"2015-48":5,"2015-40":4,"2015-35":6,"2015-32":4,"2015-27":6,"2015-22":6,"2015-14":4,"2014-52":6,"2014-49":5,"2014-42":15,"2014-41":8,"2014-35":9,"2014-23":10,"2014-15":7,"2023-40":1,"2015-18":6,"2015-11":6,"2015-06":6,"2014-10":6,"2013-48":6,"2013-20":4,"2024-22":1}},"file":"PMC3162531"},"subset":"pubmed_central"}
{"text":"author: Klaus Kayser\ndate: 2011\ntitle: Diagnostic Pathology in 2011: reflecting on the development of an open access journal during the last five years\n\nToday, electronic communication is involved in all parts of our lives, either in a directed active, communicative, or passive manner. Whether to live with electronic communication or to ignore it, is no longer a question. Instead, the question we have to answer is: How shall we live in our communicative environment? What can we expect? What are we forced to develop in order \"to survive\"?\n\nThis Editorial written at the end of a really successful year of our journal *Diagnostic Pathology* tries to give some answers from different points of view.\n\nLet us start with the publisher's interest which stands on two feet, the commercial success and the scientific reputation. Both feet are linked: The higher the scientific reputation of the journal, the higher the financial benefit, in terms of profit per article, as more articles are likely to be submitted as the reputation increases. The limitations of the required fee are the absolute numbers of published articles: If the publication procedure has not been outsourced the publication expenses are related to a fixed amount that is mandatory for hardware, software, salaries, etc., and upon a relative amount which can be calculated to the number of published articles (manpower required for each article). Thus, publishing more and more articles per year usually will, however may not always, increase the publisher's financial profit. Publishing a low number of articles with a very high reputation might endanger the journal's existence. At present, our journal *Diagnostic Pathology* is in a good shape: in 2011 we will publish more than 100 articles. The citation index has slowly reached 1.39. The percentage of research articles has increased to 60% - 70%, whereas the percentage of case reports has decreased to about 20%. This situation is in favour of a rising citation index despite the negative influence of doubling the published articles.\n\nNaturally, the authors' point of view is somewhat different to the publisher's interest. The publication fee is not small, and can usually only be covered by specific grants. The granting of waivers can only partly solve the \"Tom Sawyer's problem\" which is searching for outstanding articles and afterwards asking for money to cover their publication. This conflict can only be solved by the journal's attraction to its readers, through its formal work and presented content. Only if these parameters can overcome the constraint of the publication fee, will *Diagnostic Pathology* survive. The steep increase of submissions and published articles seems to confirm that our journal *Diagnostic Pathology* is on a successful way.\n\nThe main aim of any Editor-in-Chief is to guide the scientific journal to higher levels of its scientific reputation. Herein both the citation index with all its questionnaires and the geographical distribution of the journal are the main components. The open access journal *Diagnostic Pathology* started six years ago. It possesses more than 1200 registered readers living in all parts of the world. Its scientific reputation lies in the middle of all Thomson Reuter's registered scientific journals. The number of submissions and publications doubled last year and our rejection rate is currently about 50%. So far this data can be compared to other journals, i.e. the more traditional print based, or other open access journals. What else?\n\nWe have been involved in electronic publication since its beginning in the early 1990s, starting with the solely electronically distributed Electronic Journal of Pathology and Histology. Since that time new ideas and trials in scientific publication were also in our focus. The first trials of interactive publications were undertaken as well as publication of articles that included executable programs. The reader could start and execute these programs by selecting specific parameters or functions without influence from the authors. Our philosophy to continue to develop electronic scientific publication has remained unchanged since then. We are proud to offer our authors the possibility to publish whole digitized glass slides (virtual slides, VS) without any additional costs since January 2011. To our knowledge, *Diagnostic Pathology* is the first and until today the only, electronically scientific journal that offers its authors the unique chance to publish whole VS, and not only specific areas of interest, in the form of still images. This opportunity has been accepted by about 50% of authors with suitable articles. It took some time to solve the difficult logistic problems but the gateway is now open, and additional derived innovative offers will follow. One of these is that VS and their corresponding articles will be collected to form a repository that can be used for education, as a basis for additional secondary publications (interactive publication), and for assistance in routine diagnostic work. It will also serve as quality assurance for newly submitted articles.\n\nAn additional approach will be the \"opening\" of published articles for the inclusion of related data submitted by other authors. We are also undertaking tests on how to include the reader into published articles, and some of the results from these tests so far are really promising. Of course, the original authors have to be informed and have to give their permission for such investigations.\n\nOpen access journals can provide the framework that is mandatory for such new publication and communication techniques. Additional trials would be to automatically include published articles into specific social forums, for example facebook or linkedin. Certainly, we are also thinking about how to improve the review process, as one of our main aims is to judge all submitted articles without personal biased methods that are only related to scientific issues.\n\n*Diagnostic Pathology* is an alive scientific journal. All active journals require certain changes and exchanges from time to time. Unfortunately, one of our active Section Editors, Professor Torsten Goldmann, PhD will be leaving, and we want to express our gratitude for all of his efforts in helping to develop our journal *Diagnostic Pathology*. Similarly, some colleagues of our Editorial board will leave in a routine exchange. We would like to thank all of them for being on the board and supporting the journal. We also regret to announce that one of our internationally well known Editorial board members, Professor Dr. Anthony Leong, has passed away. Certainly, we will keep him in good memory.\n\nWe are looking forward to further developing our open access journal *Diagnostic Pathology*, focusing on still not, or only poorly investigated, fields of electronic scientific communication. We will also keep an eye on the progress of molecular and genetic pathology, and on new technologies such as in vivo biopsies, alive imaging publication, or nanotechnologies. For all these promising and growing fields of pathology we do need and appreciate the interest, knowledge, information, and assistance of the scientific community, specifically from our readers and authors.\n\nFor the progress made so far we would like to express our deep gratitude to our authors, readers, reviewers, and our publication team, and we wish them all a Blessed and Merry Christmas and a Happy, Healthy, and Fruitful New Year.\n\nKlaus Kayser\n\nEditor-in-Chief","meta":{"dup_signals":{"dup_doc_count":108,"dup_dump_count":48,"dup_details":{"curated_sources":2,"2022-27":1,"2021-49":1,"2020-40":1,"2020-29":1,"2019-39":1,"2019-26":1,"2019-22":1,"2019-13":1,"2019-04":1,"2018-47":1,"2018-39":1,"2018-30":2,"2018-13":1,"2017-47":1,"2017-34":1,"2017-26":1,"2017-17":1,"2017-09":11,"2017-04":1,"2016-50":1,"2016-44":1,"2016-40":1,"2016-36":1,"2016-30":1,"2016-22":1,"2016-18":1,"2015-48":3,"2015-40":2,"2015-35":3,"2015-32":3,"2015-27":3,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":5,"2014-42":7,"2014-41":4,"2014-35":4,"2014-23":5,"2014-15":3,"2023-50":1,"2015-18":3,"2015-11":3,"2015-06":3,"2014-10":3,"2013-48":3,"2013-20":1,"2024-22":1}},"file":"PMC3284407"},"subset":"pubmed_central"}
{"text":"author: MH Hoffmann; J Tuncel; K Skriner; M Tohidast-Akrad; G Schett; JS Smolen; R Holmdahl; G Steiner\ndate: 2005\ninstitute: 1Center of Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria; 2Department of Rheumatology, Medical University of Vienna, Austria; 3Section for Medical Inflammation Research, Lund University, Sweden; 4Department of Rheumatology, Charit\u00e9 University Hospital, Berlin, Germany; 5Ludwig Boltzmann Institute for Rheumatology, Vienna, Austria\nreferences:\ntitle: Identification of hnRNP-A2 (RA33) as a major B-cell and T-cell autoantigen in pristane\u2013induced arthritis\n\n# Background\n\nPristane-induced arthritis (PIA) in rats is considered an excellent model for rheumatoid arthritis (RA) since it fulfils the criteria for RA including a chronic relapsing disease course and is not dependent on immunization with exogenous antigen. Although the adjuvant pristane is not immunogenic, the disease is MHC associated and dependent on the activation of (autoreactive) T cells. However, so far it has not been possible to link the immune response to joint antigens or other endogenous components. HnRNP A2, the RA33 autoantigen (A2\/RA33), is a multi-functional RNA binding protein involved in splicing and other aspects of post-transcriptional regulation of gene expression. Autoantibodies as well as autoreactive T cells against A2\/RA33 have been found in patients with RA but the pathogenetic role of these autoimmune responses is unresolved \\[1\\]. It was therefore the aim of this study to elucidate a potential involvement of A2\/RA33 in PIA.\n\n# Methods\n\nAutoantibodies against A2\/RA33 were determined by immunoblotting, and MHC association of the anti-A2\/RA33 immune response was specified by the presence of autoantibodies, delayed-type hypersensitivity reactions and T-cell cytokine secretion in DA rats of different haplotypes. Interferon gamma and tumour necrosis factor secretion by T cells isolated from draining lymph nodes 10 days after pristane injection and restimulation with A2\/RA33 *in vitro* was determined. Expression of A2\/RA33 in joints and organs was analysed by immunohistochemistry and western blotting. Nasal vaccinations were performed with A2\/RA33 7 days prior to pristane injection.\n\n# Results\n\nAlthough anti-A2\/RA33 autoantibodies were detected in all four rat strains investigated, the immune response appeared to be particularly linked to the F and U MHC haplotypes. Autoantibodies to A2\/RA33 were found in 60% of DA1.F sera, and T cells of all DA1.F rats tested produced intermediate to high levels of interferon gamma upon stimulation with A2\/RA33. The reaction seemed to be entirely produced by CD4^+^ cells showing a Th1 phenotype. Furthermore, nasal vaccination with A2\/RA33 significantly delayed the onset and decreased severity of arthritis in DA1.F rats. Finally, immunohistochemical and western blot analyses revealed pronounced overexpression of A2\/RA33 in joints of rats suffering from acute PIA, but not in healthy joints or in joints from animals with chronic PIA.\n\n# Conclusion\n\nThe A2\/RA33 autoantigen is targeted by autoantibodies and Th1 cells in rats with PIA shortly after pristane injection. The presence of autoreactive Th1 cells in conjunction with synovial overexpression of A2\/RA33 strongly suggests involvement of this autoantigen in the pathogenesis of PIA. This is further bolstered by the observed alleviation and delay of onset of PIA following nasal vaccination with A2\/RA33. Thus, A2\/RA33 seems to be one of the primary autoantigens in PIA that in conjunction with previous observations on the presence of autoreactive T cells in RA patients may argue for a pathogenetic role also in human RA.\n\n## Acknowledgements\n\nThis work was supported by a grant from the Austrian Academy of Sciences and by Marie Curie Host Fellowship number HPMT-CT-2000-00126 of the European Commission Research Directorate.","meta":{"dup_signals":{"dup_doc_count":111,"dup_dump_count":40,"dup_details":{"curated_sources":2,"2022-21":1,"2021-43":1,"2020-29":1,"2020-05":1,"2019-39":1,"2019-04":1,"2018-43":1,"2018-34":1,"2018-26":1,"2018-17":1,"2018-05":1,"2017-39":1,"2017-22":1,"2017-09":8,"2016-44":1,"2016-40":1,"2016-36":8,"2016-30":8,"2015-48":3,"2015-40":2,"2015-35":3,"2015-32":3,"2015-27":3,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":3,"2014-42":7,"2014-41":4,"2014-35":5,"2014-23":5,"2014-15":4,"2022-40":1,"2015-18":3,"2015-11":3,"2015-06":3,"2014-10":3,"2013-48":2,"2013-20":3,"2024-30":1}},"file":"PMC2834155"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  Recent advances in proteomics technologies such as two-hybrid, phage display and mass spectrometry have enabled us to create a detailed map of biomolecular interaction networks. Initial mapping efforts have already produced a wealth of data. As the size of the interaction set increases, databases and computational methods will be required to store, visualize and analyze the information in order to effectively aid in knowledge discovery.\n  .\n  # Results\n  .\n  This paper describes a novel graph theoretic clustering algorithm, \"Molecular Complex Detection\" (MCODE), that detects densely connected regions in large protein-protein interaction networks that may represent molecular complexes. The method is based on vertex weighting by local neighborhood density and outward traversal from a locally dense seed protein to isolate the dense regions according to given parameters. The algorithm has the advantage over other graph clustering methods of having a directed mode that allows fine-tuning of clusters of interest without considering the rest of the network and allows examination of cluster interconnectivity, which is relevant for protein networks. Protein interaction and complex information from the yeast *Saccharomyces cerevisiae* was used for evaluation.\n  .\n  # Conclusion\n  .\n  Dense regions of protein interaction networks can be found, based solely on connectivity data, many of which correspond to known protein complexes. The algorithm is not affected by a known high rate of false positives in data from high-throughput interaction techniques. The program is available from <ftp:\/\/ftp.mshri.on.ca\/pub\/BIND\/Tools\/MCODE>.\nauthor: Gary D Bader; Christopher WV Hogue\ndate: 2003\ninstitute: 1Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, Toronto ON Canada M5G 1X5, Dept. of Biochemistry, University of Toronto, Toronto ON Canada M5S 1A8; 2Current address: Memorial Sloan-Kettering Cancer Center 1275 York Avenue, Box 460, New York, NY, 10021, USA\nreferences:\ntitle: An automated method for finding molecular complexes in large protein interaction networks\n\n# Background\n\nRecent papers published in *Science* and *Nature* among others describe large-scale proteomics experiments that have generated large data sets of protein-protein interactions and molecular complexes \\[1-7\\]. Protein structure \\[8\\] and gene expression data \\[9\\] is also accumulating at a rapid rate. Bioinformatics systems for storage, management, visualization and analysis of this new wealth of data must keep pace. We previously published a simple graph theory method that identified a functional protein complex around the yeast protein Las17 that is involved in actin cytoskeleton rearrangement \\[10\\]. Here we extend the method to better apply it to the accumulating information in protein networks.\n\nCurrently, most proteomics data is available for the model organism *Saccharomyces cerevisiae*, by virtue of the availability of a defined and relatively stable proteome, full genome clone libraries \\[11\\], established molecular biology experimental techniques and an assortment of well designed genomics databases \\[12-14\\]. Using the Biomolecular Interaction Network Database (BIND \u2013 <http:\/\/www.bind.ca>) \\[15\\] as an integration platform, we have collected 15,143 yeast protein-protein interactions among 4,825 proteins (about 75% of the yeast proteome). Much larger data sets than this will eventually be available for other well studied model organisms as well as for the human proteome. These complex data sets present a formidable challenge for computational biology to develop automated data mining analyses for knowledge discovery.\n\nHere we present the first report that uses a clustering algorithm to identify molecular complexes in a large protein interaction network derived from heterogeneous experimental sources. Based on our previous observation that highly interconnected, or dense, regions of the network may represent complexes \\[10\\], the \"Molecular Complex Detection\" (MCODE) algorithm has been implemented and evaluated on our yeast protein interaction compilation using known molecular complex data from a recent systematic mass spectrometry study of the proteome \\[7\\] and from the MIPS database \\[13\\].\n\nPredicting molecular complexes from protein interaction data is important because it provides another level of functional annotation above other guilt-by-association methods. Since sub-units of a molecular complex generally function towards the same biological goal, prediction of an unknown protein as part of a complex also allows increased confidence in the annotation of that protein.\n\nMCODE also makes the visualization of large networks manageable by extracting the dense regions around a protein of interest. This is important, as it is now obvious that the current visualization tools present on many interaction databases \\[15\\], originally based on the Sun Microsystems embedded spring graph layout Java applet do not scale well to large networks (<http:\/\/java.sun.com\/applets\/jdk\/1.1\/demo\/GraphLayout\/example1.html>).\n\n## Algorithm\n\nThe MCODE algorithm operates in three stages, vertex weighting, complex prediction and optionally post-processing to filter or add proteins in the resulting complexes by certain connectivity criteria.\n\nA network of interacting molecules can be intuitively modeled as a graph, where vertices are molecules and edges are molecular interactions. If temporal pathway or cell signalling information is known, it is possible to create a directed graph with arcs representing direction of chemical action or direction of information flow, otherwise an undirected graph is used. Using this graph representation of a biological system allows graph theoretic methods to be applied to aid in analysis and solve biological problems. This graph theory approach has been used by other biomolecular interaction database projects such as DIP \\[16\\], CSNDB \\[17\\], TRANSPATH \\[18\\], EcoCyc \\[19\\] and WIT \\[20\\] and is discussed by Wagner and Fell \\[21\\].\n\nAlgorithms for finding clusters, or locally dense regions, of a graph are an ongoing research topic in computer science and are often based on network flow\/minimum cut theory \\[22,23\\] or more recently, spectral clustering \\[24\\]. To find locally dense regions of a graph, MCODE instead uses a vertex-weighting scheme based on the clustering coefficient, C~i~, which measures 'cliquishness' of the neighborhood of a vertex \\[25\\]. C~i~ = 2*n*\/*k*~*i*~(*k*~*i*~-1) where *k*~*i*~ is the vertex size of the neighborhood of vertex *i* and *n* is the number of edges in the neighborhood (the immediate neighborhood density of *v* not including *v*). A clique is defined as a maximally connected graph. There is no standard graph theory definition of density, but definitions are normally based on the connectivity level of a graph. Density of a graph, G = (V,E), with number of vertices, \\|V\\|, and number of edges, \\|E\\|, is defined here as \\|E\\|; divided by the theoretical maximum number of edges possible for the graph, \\|E\\|~max~. For a graph with loops (an edge connecting back to its originating vertex), \\|E\\|~max~ = \\|V\\| (\\|V\\|+1)\/2 and for a graph with no loops, \\|E\\|~max~ = \\|V\\| (\\|V\\|-1)\/2. So, density of G, D~G~ = \\|E\\|\/\\|E\\|~max~ and is thus a real number ranging from 0.0 to 1.0.\n\nThe first stage of MCODE, vertex weighting, weights all vertices based on their local network density using the highest *k*-core of the vertex neighborhood. A *k*-core is a graph of minimal degree *k* (graph G, for all *v* in G, deg(*v*) \\>= *k*). The highest *k*-core of a graph is the central most densely connected subgraph. We define here the term core-clustering coefficient of a vertex, *v*, to be the density of the highest *k*-core of the immediate neighborhood of *v* (vertices connected directly to *v*) including *v* (note that C~i~ does not include *v*). The core-clustering coefficient is used here instead of the clustering coefficient because it amplifies the weighting of heavily interconnected graph regions while removing the many less connected vertices that are usually part of a biomolecular interaction network, known to be scale-free \\[6,21,26-29\\]. A scale-free network has a vertex connectivity distribution that follows a power law, with relatively few highly connected vertices (high degree) and many vertices having a low degree. A given highly connected vertex, *v*, in a dense region of a graph may be connected to many vertices of degree one (singly linked vertex). These low degree vertices do not interconnect within the neighborhood of *v* and thus would reduce the clustering coefficient, but not the core-clustering coefficient. The final weight given to a vertex is the product of the vertex core-clustering coefficient and the highest *k*-core level, *k*~max~, of the immediate neighborhood of the vertex. This weighting scheme further boosts the weight of densely connected vertices. This specific weighting function is based on local network density. Many other functions are possible and some may have better performance for this algorithm but these are not evaluated here.\n\nThe second stage, molecular complex prediction, takes as input the vertex weighted graph, seeds a complex with the highest weighted vertex and recursively moves outward from the seed vertex, including vertices in the complex whose weight is above a given threshold, which is a given percentage away from the weight of the seed vertex. This is the vertex weight percentage (VWP) parameter. If a vertex is included, its neighbours are recursively checked in the same manner to see if they are part of the complex. A vertex is not checked more than once, since complexes cannot overlap in this stage of the algorithm (see below for a possible overlap condition). This process stops once no more vertices can be added to the complex based on the given threshold and is repeated for the next highest unseen weighted vertex in the network. In this way, the densest regions of the network are identified. The vertex weight threshold parameter defines the density of the resulting complex. A threshold that is closer to the weight of the seed vertex identifies a smaller, denser network region around the seed vertex.\n\nThe third stage is post-processing. Complexes are filtered if they do not contain at least a 2-core (graph of minimum degree 2). The algorithm may be run with the 'fluff' option, which increases the size of the complex according to a given 'fluff' parameter between 0.0 and 1.0. For every vertex in the complex, *v*, its neighbors are added to the complex if they have not yet been seen and if the neighborhood density (including *v*) is higher than the given fluff parameter. Vertices that are added by the fluff parameter are not marked as seen, so there can be overlap among predicted complexes with the fluff parameter set. If the algorithm is run using the 'haircut' option, the resulting complexes are 2-cored, thereby removing the vertices that are singly connected to the core complex. If both options are specified, fluff is run first, then haircut.\n\nResulting complexes from the algorithm are scored and ranked. The complex score is defined as the product of the complex subgraph, C = (V,E), density and the number of vertices in the complex subgraph (D~C~ \u00d7 \\|V\\|). This ranks larger more dense complexes higher in the results. Other scoring schemes are possible, but are not evaluated here.\n\nMCODE may also be run in a directed mode where a seed vertex is specified as a parameter. In this mode, MCODE only runs once to predict the single complex that the specified seed is a part of. Typically, when analyzing complexes in a given network, one would find all complexes present (undirected mode) and then switch to the directed mode for the complexes of interest. The directed mode allows one to experiment with MCODE parameters to fine tune the size of the resulting complex according to existing biological knowledge of the system. In directed mode, MCODE will first pre-process the input network to ignore all vertices with higher vertex weight than the seed vertex. If this were not done, MCODE would preferentially branch out to denser regions of the graph, if they exist, which could belong to separate, but denser complexes. Thus, a seed vertex for directed mode should always be the highest density vertex among the suspected complex. There is an option to turn this pre-processing step off, which will allow seeded complexes to branch out into denser regions of the graph, if desired.\n\nThe time complexity of the entire algorithm is polynomial O(*nmh*^3^) where *n* is the number of vertices, *m* is the number of edges and *h* is the vertex size of the average vertex neighbourhood in the input graph, G. This comes from the vertex-weighting step. Finding a k-core in a graph proceeds by progressively removing vertices of degree \\< k until all remaining vertices are connected to each other by degree k or more, and is thus O(*n*^2^). The highest k-core is found by trying to find k-cores from one up until all vertices have been found and cannot go beyond a number of steps equal to the highest degree in the graph. Thus, the highest k-core step is O(*n*^3^). Since this k-core step operates only on the neighbourhood of a vertex, the *n* in this case is the number of vertices in the average neighbourhood of a vertex, *h*. The inner loop of the algorithm only operates twice for every edge in the input graph, thus is O(2*mh*^3^). The outer loop operates once on all vertices in the input graph, thus the entire time complexity of the weighting stage is O(*n*2*mh*^3^) = O(*nmh*^3^). The complex prediction stage is O(*n*) and the optional post-processing step can be up to O(*cs*^2^), where *c* is the number of complexes that were found in the previous step and *s* is the number of vertices in the largest complex - O(*cs*^2^) to find the 2-core once for each complex.\n\nEven though the fastest min-cut graph clustering algorithms are faster, at O(*n*^2^log*n*) \\[30\\], MCODE has a number of advantages. Since weighting is done once and comprises most of the time complexity, many algorithm parameters can be tried, in O(*n*), once weighting is complete. This is useful when evaluating many different parameters. MCODE is relatively easy to implement and since it is local density based, has the advantage of a directed mode and a complex connectivity mode. These two modes are generally not useful in typical clustering applications, but are useful for examining molecular interaction networks. Additionally, only those proteins above a given local density threshold are assigned to complexes. This is in contrast to many clustering applications that force all data points to be part of clusters, whether they truly should be part of a cluster or not.\n\n## Pseudocode\n\n### Stage 1: Vertex Weighting\n\n**procedure** MCODE-VERTEX-WEIGHTING\n\n\u00a0\u00a0\u00a0**input**: **graph**: G = (V,E)\n\n\u00a0\u00a0\u00a0**for all** *v* in G **do**\n\n\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0N = find neighbors of *v* to depth 1\n\n\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0K = Get highest *k*-core graph from N\n\n\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0*k* = Get highest *k*-core number from N\n\n\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0*d* = Get density of K\n\n\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0Set weight of *v* = *k* \u00d7 *d*\n\n\u00a0\u00a0\u00a0**end for**\n\nend procedure\n\n### Stage 2: Molecular Complex Prediction\n\n**procedure** MCODE-FIND-COMPLEX\n\n\u00a0\u00a0\u00a0**input**: **graph**: G = (V,E); **vertex weights**: W;\n\n\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0**vertex weight percentage**: *d*; **seed vertex**: *s*\n\n\u00a0\u00a0\u00a0**if** *s* already seen **then return**\n\n\u00a0\u00a0\u00a0**for all** *v* neighbors of *s* **do**\n\n\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0**if** weight of *v* \\> (weight of *s*)(1 - *d*) **then** add *v* to complex C\n\n\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0**call**: MCODE-FIND-COMPLEX (G, W, *d*, *v*)\n\n\u00a0\u00a0\u00a0**end for**\n\nend procedure\n\n**procedure** MCODE-FIND-COMPLEXES\n\n\u00a0\u00a0\u00a0**input**: **graph**: G = (V,E); **vertex weights**: W;\n\n\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0**vertex weight percentage**: *d*\n\n\u00a0\u00a0\u00a0**for all** *v* in G **do**\n\n\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0**if** not already seen v **then call**: MCODE-FIND-COMPLEX(G, W, *d*, *v*)\n\n\u00a0\u00a0\u00a0**end for**\n\nend procedure\n\n### Stage 3: Post-Processing (optional)\n\n**procedure** MCODE-FLUFF-COMPLEX\n\n\u00a0\u00a0\u00a0**input**: **graph**: G = (V,E); **vertex weights**: W;\n\n\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0**fluff density threshold**: *d*; **complex graph**: C = (U,F)\n\n\u00a0\u00a0\u00a0**for all** *u* in C **do**\n\n\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0**if** weight of *u* \\>*d* **then** add *u* to complex C\n\n\u00a0\u00a0\u00a0**end for**\n\nend procedure\n\n**procedure** MCODE-POST-PROCESS\n\n\u00a0\u00a0\u00a0**input**: **graph**: G = (V,E); **vertex weights**: W; **haircut flag**: *h*; **fluff flag**: *f*;\n\n\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0**fluff density threshold**: *t*; **set of predicted complex graphs**: C\n\n\u00a0\u00a0\u00a0**for all** *c* in C **do**\n\n\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0**if** *c* not 2-core **then** filter\n\n\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0**if** *h* is TRUE **then** 2-core complex\n\n\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0**if** *f* is TRUE **then call**: MCODE-FLUFF-COMPLEX(G, W, *t*, *c*)\n\n\u00a0\u00a0\u00a0**end for**\n\nend procedure\n\n### Overall Process\n\n**procedure** MCODE\n\n\u00a0\u00a0\u00a0**input**: **graph**: G = (V,E); **vertex weight percentage**: *d*;\n\n\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0**haircut flag**: *h*; **fluff flag**: *f*; **fluff density threshold**: *t*;\n\n\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0**set of predicted complex graphs**: C\n\n\u00a0\u00a0\u00a0**call**: W = MCODE-VERTEX-WEIGHTING (G)\n\n\u00a0\u00a0\u00a0**call**: C = MCODE-FIND-COMPLEXES (G, W, *d*)\n\n\u00a0\u00a0\u00a0**call**: MCODE-POST-PROCESS (G, W, *h*, *f*, *t*, C)\n\nend procedure\n\n## Implementation\n\nMCODE has been implemented in ANSI C using the cross-platform NCBI Toolkit; <http:\/\/www.ncbi.nlm.nih.gov\/IEB> and the BIND graph library in the SLRI Toolkit; <http:\/\/sourceforge.net\/projects\/slritools>. Both of these source code libraries are freely available. The actual MCODE source code is not yet freely available. The MCODE program has been compiled and tested on UNIX, Mac OS X and Windows. Because a yeast gene name dictionary is used to recognize input and generate output, the MCODE executable currently only works for yeast proteins in a user friendly manner. The algorithm, however is completely general, via the graph theory abstraction, to any graph and thus to any biomolecular interaction network. MCODE binaries are available from <ftp:\/\/ftp.mshri.on.ca\/pub\/BIND\/Tools\/MCODE>.\n\n# Results\n\n## Evaluation of MCODE\n\nThe evaluation of MCODE requires a set of experimentally determined biomolecular interactions and a set of associated experimentally determined molecular complexes. Currently, the largest source for such data is for proteins from the budding yeast, *Saccharomyces cerevisiae*. Recently, a large-scale mass spectrometry study by Gavin et al \\[7\\] provided a large data set of protein interactions with manually annotated molecular complexes. Also available are the protein interaction and complex tables of MIPS \\[13\\] and YPD \\[14\\]. MCODE was used to automatically predict protein complexes in our collected protein-protein interaction data sets. Resulting complexes were then matched to known molecular complexes from Gavin et al. (the Gavin benchmark) and the MIPS benchmark using an overlap score. Parameter optimization was then used to maximize the biological relevance of predicted complexes according to the given benchmarks. YPD was not used as a current version could not be acquired.\n\nTo ensure that MCODE is not unduly affected by the expected high false-positive rate in large-scale interaction data sets, large-scale and literature derived MCODE predictions were compared. MCODE was then used to predict complexes in the entire set of machine readable protein-protein interactions that we could collect for yeast. Complexes of interest were then further examined using the directed mode and complex connectivity mode of MCODE.\n\n## Evaluation of MCODE using the Gavin data set of protein interactions and complexes\n\nIn this study, we wanted to use all forms of protein interaction data available, which requires mixing of different types of experiments, such as yeast two-hybrid and co-immunoprecipitation. Two-hybrid results are inherently pairwise, whereas copurification results are sets of one or more identified proteins. For a copurification result, only a set of size 2 can be directly considered a pairwise interaction, otherwise it must be modeled as a set of hypothetical interactions. Biochemical copurifications can be thought of as populations of complexes with some underlying pairwise protein interaction topology that is unknown from the experiment. In the general case of the purification used by Gavin et al., one affinity tagged protein was used as bait to pull associated proteins out of a yeast cell lysate. The two extreme cases for the topology underlying the population of complexes from a single purification experiment are a minimally connected 'spoke' model, where the data are modeled as direct bait-associated protein pairwise interactions, and a maximally connected 'matrix' model, where the data are modeled as all proteins connected to all others in the set. The real topology of the set of proteins must lie somewhere between these two extremes.\n\nPopulation of complexes: *C* = {*b, c, d, e*} (*b* = bait)\n\nSpoke model hypothetical interactions: *i*~*S*~ = {*b-c, b-d, b-e*}\n\nMatrix model hypothetical interactions; *i*~*M*~ = {*b-b, b-c, b-d, b-e, c-c, c-d, c-e, d-d, d-e, e-e*}\n\nAdvantages of the spoke model are that it is biologically intuitive, biologists often represent their copurification results in this manner, and is about 3 times more accurate than the matrix model \\[31\\]. Disadvantages are that it could misrepresent interactions. The matrix model, alternatively, cannot misrepresent interactions, as all possible interactions are generated, but this is at the cost of generating a large number of false interactions. Matrix topologies are also physically implausible for larger complexes because of increased possibility of steric clash if all subunits are interacting with all others. Ultimately, the spoke model should be reasonable for use in evaluating MCODE.\n\nGavin et al. raw data from 588 biochemical purifications were represented using the spoke model, described above, to get 3,225 hypothetical protein-protein interactions among 1,363 proteins for input to MCODE. A list of 232 manually annotated protein complexes based on the original purification data reported by Gavin et al. was filtered to remove five reported 'complexes' each composed of a single protein and six complexes of two or three proteins that were already in the data set as part of a larger complex. This yielded a filtered set of 221 complexes that were used to evaluate MCODE, although some of these complexes have significant overlap to other complexes in the set.\n\nTo evaluate which parameter choice would allow automatic prediction of protein complexes from the spoke modeled Gavin et al. interaction set that best matched the manually annotated complexes, MCODE was run using all four possible combinations of the two Boolean parameters (haircut: true\/false, fluff: true\/false) over a full range of 20 vertex weight percentage (VWP) and fluff parameters (0 to 0.95 in 0.05 increments). During this parameter optimization process, MCODE was limited to find complexes of size two or higher.\n\nA scoring scheme was developed to determine how effectively an MCODE predicted complex matched a complex from the benchmark set of complexes. In this case, the benchmark complex set was the Gavin et al. hand-annotated complex set. The overlap score was defined as \u03c9 = *i*^2^\/*a*\\**b*, where *i* is the size of the intersection set of a predicted complex with a known complex, *a* is the size of the predicted complex and *b* is the size of the known complex. A protein is part of the intersection set only if it is present in both predicted and known complexes. Thus, a predicted complex that has no proteins in a known complex has \u03c9 = 0 and a predicted complex that perfectly matches a known complex has \u03c9 = 1. Also, predicted complexes that fully overlap, but are much larger or much smaller than any known complexes will get a low \u03c9. The overlap score of a predicted complex vs. a benchmark complex is then a measure of biological significance of the prediction, assuming that the benchmark set of complexes is biologically relevant. The best parameter choice for MCODE on this protein interaction data set is one that predicts the largest set of complexes that match the largest number of benchmark complexes above a threshold \u03c9. Since there is overlap in the Gavin benchmark complex database, a predicted complex may match more than one known complex with a high \u03c9.\n\nTo choose an overlap score that maximizes biological relevance of the predicted complexes without filtering away too many predictions, each of the 840 parameter combinations tested during the parameter optimization stage. The number of MCODE predicted complexes was plotted against the number of matched known complexes over a range of \u03c9 thresholds from 'no threshold' to 0.1 to 0.9 (in 0.1 increments). If no \u03c9 threshold is used, a predicted complex only needs at least one protein in common with a known complex to be considered a match. If predicted and known complexes are only counted as a match when their \u03c9 is above a specific threshold, the number of matched complexes declines with increasing \u03c9 threshold, as shown in Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>. Interestingly, the average and maximum number of matched known complexes drops more quickly from zero until a \u03c9 threshold of 0.2 than from 0.2 to 0.9 indicating that many predicted complexes only have one or a few proteins that overlap with known complexes. A \u03c9 threshold of 0.2 to 0.3 thus seems to filter out most predicted complexes that have insignificant overlap with known complexes.\n\nFigure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a> shows the range of number of complexes predicted and number of known complexes matched for the 0.2 \u03c9 threshold over all tried MCODE parameters. A y = x line is also plotted to show that data points tend to be skewed towards a higher number of matched known complexes than predicted complexes because of the redundancy in the Gavin complex benchmark. Data points closest to the upper right portion of the graph maximize both number of matched known complexes and number of predicted complexes. MCODE parameter combinations that result in these data points therefore optimize MCODE on this data set (according to the overlap score threshold). This result shows that the number of predicted complexes should be similar to the number of matched known complexes for a parameter choice to be reasonable, although the number of matched known complexes may be larger, again, because of some commonality among complexes in the benchmark set. The parameter combination corresponding to the best data point (63,88) at an overlap score threshold of 0.2 is haircut = FALSE, fluff = TRUE, VWP = 0.05 and a fluff density threshold between 0 and 0.1. These parameter optimization results for MCODE over this data set were stable over a range of \u03c9 thresholds up to 0.5. Above 0.5, the result was not stable as there were generally too few predicted complexes with high overlap scores (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>).\n\nA specificity versus sensitivity analysis \\[32\\] was also performed. Defining the number of true positives (TP) as the number of MCODE predicted complexes with \u03c9 over a threshold value and the number of false positives (FP) as the total number of predicted MCODE complexes minus TP. The number of false negatives (FN) equals the number of known benchmark complexes not matched by predicted complexes. Sensitivity was defined as \\[TP\/(TP+FN)\\] and specificity was defined as \\[TP\/(TP+FP)\\]. The MCODE parameter choice that optimizes both specificity and sensitivity is the same as from the above analysis. The optimal sensitivity of this analysis was \\~0.31 and the corresponding specificity was \\~0.79.\n\nThe 63 MCODE predicted complexes only matched 88 of the 221 complexes in the known data set indicating that MCODE could not recapitulate the majority of the Gavin complex benchmark solely using protein connectivity information. As mentioned above, there are more matched complexes than predicted because of some redundancy in the benchmark. This low sensitivity is not surprising, since many of the hand-annotated complexes were created directly from single co-immunoprecipitation results, which are not highly interconnected in the spoke model. For example, Cdc3 was used as a bait to co-immunoprecipitate Cdc10, Cdc11, Cdc12 and Ydl225w. A complex was annotated as containing these five proteins, but only Cdc3 was used as bait. If more elements of a complex are used as baits, the proteins become more interconnected and more readily predicted by MCODE. A good example of this is the Arp2\/3 complex, which is highly conserved in eukaryotes and is involved in actin cytoskeleton rearrangement. The structure of this complex is known by X-ray crystallography \\[33\\] thus actual protein-protein interactions from the structure can be matched up to the co-immunoprecipitation results. MCODE predicted all seven components of the Arp2\/3 complex crystal structure and five extra proteins using the optimized parameters. Six out of the seven Arp2\/3 subunits were used as baits by Gavin et al. and the resulting benchmark complex included the five extra proteins that MCODE also predicted (Nog2, Pfk1, Prt1, Cct8 and Cct5) that are not in the crystal structure. Cct5 and Cct8 are known to be involved in actin assembly, but Nog2, Pfk1 and Prt1 are not. These extra proteins likely represent non-specific binding in the experimental approach. These two cases are shown diagrammatically in Figure <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>. Interestingly, using the haircut parameter would remove all five extra proteins that are not in the crystal structure, leaving only the seven that are present. This shows that while the parameter optimization allows maximum matching of the hand-annotated known complexes, these complexes may not all be physiologically relevant and thus another parameter set may better predict 'real' complexes.\n\nTo explore the effect of certain MCODE parameters on resulting predicted complexes, various features of these complexes were examined while changing specific parameters and keeping all else constant. Linearly increasing the VWP parameter increased the size of the predicted complexes exponentially while reducing the number of complexes predicted in a linear fashion. Figure <a href=\"#F4\" data-ref-type=\"fig\">4<\/a> shows this effect with both fluff and haircut parameters turned off. At high VWP values, very large complexes were predicted and these encompassed most of the data set, thus were not very useful.\n\nBecause using haircut = TRUE would have led MCODE to predict the Arp2\/3 complex perfectly (according to the crystal structure as discussed above), we examined if the haircut parameter has any general effect on the number of matched predicted complexes. Setting haircut = TRUE had no significant effect on the number of complexes predicted at high \u03c9 thresholds, but generally reduced the number of matched known complexes at low \u03c9 thresholds (0 to 0.1) compared to haircut = FALSE. Since the haircut = TRUE option removes less-connected proteins on the fringe of a predicted complex and this reduces the number of predicted complexes with low overlap scores, these fringe proteins likely contribute to low-level overlap (\\<0.2 \u03c9) of the known complexes.\n\nWe also investigated the effect of changing the fluff density threshold when setting fluff = TRUE on the number of matched benchmark complexes. Linearly increasing the fluff density threshold in the MCODE post-processing step linearly decreased the number of matched complexes above an overlap score of 0.2.\n\n## Evaluation of MCODE using MIPS data set of protein interactions and complexes\n\nSince the Gavin et al. data set was developed by only one group using a single experimental method, it may not accurately represent protein complex knowledge for yeast. The MIPS protein complex catalogue <http:\/\/mips.gsf.de\/proj\/yeast\/catalogues\/complexes\/> is a curated set of 260 protein complexes for yeast that was compiled from the literature and is thus a more realistic data set comprised of varied experiments from many labs using different techniques. After filtering away 50 'complexes' each composed of a single protein and 2 highly similar complexes, we were left with 208 complexes for the MIPS known set. This set did not include information from the recent large-scale mass spectrometry studies \\[6,7\\]. While the MIPS complex catalogue may be incomplete, it is currently the best available public resource for yeast protein complexes that we are aware of.\n\nMCODE was run again with a full combination of parameters, this time over a set of 9088 protein-protein interactions among 4379 proteins which did not include the recent large-scale mass spectrometry studies but included all interactions from the MIPS, YPD and PreBIND databases as well as from the majority of large-scale yeast two-hybrid experiments to date \\[2-4,10,34\\]. This interaction set is termed 'Pre HTMS'. All of the interactions in this set were published before the last update specified on the MIPS protein complex catalogue and many are included in the MIPS protein interaction table, thus we assumed that the MIPS complex catalogue took into account the information in the known interaction table. Protein complexes found by MCODE in this set were compared to the MIPS protein complex catalogue to evaluate how well MCODE performed at locating protein complexes *ab initio*.\n\nThe same evaluation of MCODE that was done using the Gavin et al. data set was performed with the MIPS data set. From this analysis, including specificity versus sensitivity plots (optimized sensitivity = \\~0.27 and specificity = \\~0.31), the MIPS complex benchmark optimized parameters were haircut = TRUE, fluff = TRUE, VWP = 0.1 and a fluff density threshold of 0.2. This result was stable up to a \u03c9 threshold of 0.6 after which it was difficult to evaluate the results, as there were generally too few predicted complexes above the high \u03c9 thresholds. This parameter combination led MCODE to predict 166 complexes of which 52 matched 64 MIPS complexes with a \u03c9 of at least 0.2. Examining the \u03c9 distribution for this parameter set reveals that, even though this prediction is optimized, most of the predicted complexes don't show overlap to those in the known MIPS set (Figure <a href=\"#F5\" data-ref-type=\"fig\">5<\/a>). The complexes predicted here are also different from those predicted from the Gavin interaction data. Nine complexes have an overlap score above 0.2 between these two sets, with the highest overlap score being 0.43 and all the rest being below 0.27. This might signify that either the MIPS complex catalogue is not complete, that there is not enough data in the dataset that MCODE was run on, or a human annotated definition of a complex does not perfectly match with a graph density based definition.\n\nThe effect of the VWP parameter on complex size and of the haircut and fluff parameters on number of matched complexes was very similar to that seen when evaluating MCODE on the Gavin complex benchmark.\n\n## Effect of data set properties on MCODE\n\nSince many large-scale protein interaction data sets from yeast are known to contain a high level of false positives \\[35\\], we examined the effect these might have on MCODE predictions. Sensitivity vs. specificity was plotted for MCODE predictions, with parameters chosen to maximize these values at \u03c9 threshold of 0.2 against the MIPS and Gavin complex benchmarks for the various data sets (Figure <a href=\"#F6\" data-ref-type=\"fig\">6<\/a>).\n\nMCODE predictions on the high-throughput data sets, termed 'Gavin Spoke', 'Y2H' and 'HTP only' (see Methods), are about as specific as the literature derived interaction data set, but not as sensitive (Figure <a href=\"#F6\" data-ref-type=\"fig\">6A<\/a>). MCODE predictions on interaction data sets containing the literature derived benchmark, labelled 'Benchmark', 'Pre HTMS' and 'AllYeast', are generally more sensitive and specific than those containing just the large-scale interaction sets. Since the specificity drops from Benchmark to Pre HTMS to AllYeast, with increasing amounts of large-scale data, it could be argued that addition of this data negatively affects MCODE. However, large-scale data is known to contain a high number of false positives, so it should be expected that these false-positives would not randomly contribute to the formation of dense regions, which are highly unlikely to occur by chance (see below). More complexes should be predicted with the addition of the large-scale data, assuming this data explores previously unseen regions of the interactome, but the high number of false-positives should limit the amount of new complexes compared to the amount of added interactions. The MIPS complex benchmark used here is not expected to contain complexes newly found in large-scale studies, explaining the decrease in specificity. This is exactly what occurs in our analysis. In an effort to further test the effect of large-scale data on MCODE prediction performance, the Benchmark interaction data set was augmented with the addition of interactions from large-scale experiments that only connect proteins in the Benchmark set with each other. Over 3100 interactions were added to the Benchmark data set to create a set of over 6400 interactions. MIPS complex benchmark optimised MCODE predicted 52 complexes matching 66 MIPS benchmark complexes, almost exactly the same number of complexes found using the Benchmark set by itself (Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>). These analyses strongly suggest the addition of large-scale experimentally derived interactions does not unduly affect the prediction of complexes by MCODE.\n\nSummary of MCODE Results with Best Parameters on Various Data Sets.\n\n| Data Set | Number of Proteins | Number of Interact-ions | Number of Predicted Complexes | MCODE Complexes Predicted Above \u03c9 = 0.2 | Matched Benchmark Complexes | Complex Benchmark | Best MCODE Parameters |\n|----|----|----|----|----|----|----|----|\n| Gavin Spoke | 1363 | 3225 | 82 | 63 | 88 | Gavin | hFfT\\0.05\\0.05 |\n| Gavin Spoke | 1363 | 3225 | 53 | 20 | 20 | MIPS | hTfT\\0.1\\0.35 |\n| Pre HTMS | 4379 | 9088 | 158 | 21 | 28 | Gavin | hTfT\\0\\0.2\\\\ |\n| Pre HTMS | 4379 | 9088 | 166 | 52 | 64 | MIPS | hTfT\\0.1\\0.2 |\n| AllYeast | 4825 | 15143 | 209 | 52 | 76 | Gavin | hFfT\\0\\0.1 |\n| AllYeast | 4825 | 15143 | 209 | 54 | 63 | MIPS | hTfT\\0\\0.1 |\n| AllYeast | 4825 | 15143 | 203 | 80 | 150 | MIPS+Gavin | hTfT\\0\\0.15\\\\ |\n| Benchmark | 1762 | 3310 | 141 | 23 | 30 | Gavin | hTfT\\0\\0.3 |\n| Benchmark | 1762 | 3310 | 163 | 58 | 67 | MIPS | hTfT\\0.1\\0.05 |\n| HTP Only | 4557 | 12249 | 138 | 46 | 77 | Gavin | hTfT\\0.05\\0.1 |\n| HTP Only | 4557 | 12249 | 122 | 29 | 35 | MIPS | hTfT\\0.05\\0.15 |\n| Y2H | 3847 | 6133 | 73 | 7 | 7 | Gavin | hTfT\\0.2\\0.1 |\n| Y2H | 3847 | 6133 | 78 | 21 | 26 | MIPS | hTfT\\0\\0.1 |\n\nStatistics and a summary of results are shown for the various data sets used to evaluate MCODE. 'Gavin Spoke' is the Gavin et al. data set represented as binary interactions using the spoke model; 'Pre HTMS' is the set of all yeast interaction not including the recent high-throughput mass spectrometry studies \\[6,7\\].; 'AllYeast' is the set of all yeast interactions that we could collect; 'Benchmark' is a set of interactions found in the literature from YPD, MIPS and PreBIND; 'HTP Only' is the combination of all large-scale and high-throughput yeast two-hybrid and mass spectrometry data sets; 'Y2H' is the set of all yeast two-hybrid results from large-scale and literature sources. See Methods for full explanation of data sets. The 'Best MCODE Parameters' are formatted as haircut True of False, fluff True or False\\VWP\\Fluff Density Threshold Parameter.\n\nIt can be seen from Figure <a href=\"#F6\" data-ref-type=\"fig\">6B<\/a> that the Gavin complex benchmark set is biased towards the Gavin et al. spoke modeled interaction data. This is expected and is the main reason why the less biased MIPS complex set is used throughout this work as a benchmark instead of the Gavin set.\n\nSince the result of a co-immunoprecipitation experiment is a set of proteins, which we model as binary interactions using the spoke method, we wished to evaluate whether this affects complex prediction compared to an experimental system that generates purely binary interaction results, such as yeast two-hybrid. As can be seen in Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>, MCODE does find known complexes in the 'Y2H' set of only yeast two-hybrid results, thus this set does contain dense regions that are known protein complexes. This being said, the Y2H set is the least dense of all data sets examined here so is expected to have less dense regions of the network and thus less MCODE predictable complexes per protein present in the set. MCODE predicts a similar amount of complexes as well as finding a similar amount of known complexes in the Y2H and Gavin Spoke data sets indicating that these data sets are not significantly different from each other in the amount of dense network regions that they contain, even though they are different sizes. Taken together, the latter results and those in Figure <a href=\"#F6\" data-ref-type=\"fig\">6B<\/a> show that the spoke model is a reasonable representation of the Gavin et al. tandem affinity purification data.\n\n## Predicting complexes in the Yeast interactome\n\nGiven that MCODE performed reasonably well on test data, we decided to predict complexes in a much larger network. All machine-readable protein-protein interaction data from various data sets \\[2-7,10,13,14\\]. were collected and integrated to form a non-redundant set of 15,143 experimentally determined yeast protein interactions encompassing 4,825 proteins, or approximately three quarters of the proteome. This set was termed 'AllYeast'. MCODE was parameter optimized, as above, using the MIPS benchmark. The best resulting parameter set was haircut = TRUE, fluff = TRUE, VWP = 0 and a fluff density threshold of 0.1. With these parameters, MCODE predicted 209 complexes, of which 54 matched 63 MIPS benchmark complexes above an overlap score of 0.2 (see <a href=\"#S1\" data-ref-type=\"supplementary-material\">Additional file 1<\/a>). Complexes found in this manner should be further studied using MCODE in directed mode by specifying a seed vertex and trying different parameters to examine how large a complex can get before seemingly biologically irrelevant proteins are added (see below).\n\nFigure <a href=\"#F5\" data-ref-type=\"fig\">5<\/a> shows that even when a large set of interactions is used as input to MCODE, most of the MCODE predicted complexes do not match well with known complexes in MIPS. The complex size distribution of MCODE predicted complexes matches the shape of the MIPS set, but the MCODE complexes are on average larger (Average MIPS size = 6.0, Average MCODE Predicted size = 9.7). The average number of YPD and GO functional annotation terms per protein in an MCODE predicted complex is similar to that of MIPS complexes (Table <a href=\"#T2\" data-ref-type=\"table\">2<\/a>). This seems to indicate that MCODE is predicting complexes that are functionally relevant. Also, closer examination of the top, middle and bottom five scoring MCODE complexes shows that MCODE can predict biologically relevant complexes (Table <a href=\"#T3\" data-ref-type=\"table\">3<\/a>).\n\nAverage Number of YPD and GO Annotation Terms in Complex Sets.\n\n| Data Set | YPD Functions | YPD Roles | GO Components | GO Processes |\n|----|----|----|----|----|\n| MCODE on All Yeast Interactions | 0.58 | 0.89 | 0.39 | 0.59 |\n| MIPS Complex Database | 0.50 | 0.75 | 0.39 | 0.48 |\n| MCODE Random Model (100 AllYeast network permutations) | 0.72 | 1.24 | 0.52 | 0.85 |\n\nThe average number of YPD and GO functional annotation terms per protein in an MCODE predicted complex is shown for MCODE predicted complexes on the AllYeast set, the MIPS complex database and the MCODE random model. A lower number indicates that the complexes from a set contain more functionally related proteins (or unannotated proteins). In the cases of multiple annotation, all terms are taken into account. Even though there are multiple annotation terms per protein and a variable amount of unannotated proteins per complex, these numbers should perform well in relative comparisons based on the assumption that the distribution of the latter two factors is similar in each data set.\n\nStatistics for Top, Middle and Bottom Five Scoring Optimized MCODE Predicted Complexes Found in All Known Yeast Protein Interaction Data Set\n\n<table>\n<thead>\n<tr>\n<th style=\"text-align: center;\"><strong>Complex Rank<\/strong><\/th>\n<th style=\"text-align: center;\"><strong>Score<\/strong><\/th>\n<th style=\"text-align: center;\"><strong>Proteins<\/strong><\/th>\n<th style=\"text-align: center;\"><strong>Interactions<\/strong><\/th>\n<th style=\"text-align: center;\"><strong>Density<\/strong><\/th>\n<th style=\"text-align: left;\"><strong>Cell Role<\/strong><\/th>\n<th style=\"text-align: left;\"><strong>Cell Localization<\/strong><\/th>\n<\/tr>\n<\/thead>\n<tbody>\n<tr>\n<td style=\"text-align: center;\">1<\/td>\n<td style=\"text-align: center;\">10.04<\/td>\n<td style=\"text-align: center;\">46<\/td>\n<td style=\"text-align: center;\">236<\/td>\n<td style=\"text-align: center;\">0.22<\/td>\n<td style=\"text-align: left;\">RNA processing\/modification and protein degradation (26S Proteasome)<\/td>\n<td style=\"text-align: left;\">Nuclear<\/td>\n<\/tr>\n<tr>\n<td colspan=\"2\" style=\"text-align: left;\"><strong>Protein names<\/strong><\/td>\n<td colspan=\"5\" style=\"text-align: left;\">Dbf2,Ecm29,Gcn4,Hsm3,Hyp2,Lhs1,Mkt1,Nas6,Pre1,Pre2,Pre4,Pre5,Pre6,<br \/>\nPre7,Pre8,Pre9,Pup3,Rad23,Rad24,Rad50,Rfc3,Rfc4,Rpn1,Rpn10,Rpn11,<br \/>\nRpn12,Rpn13,Rpn3,Rpn4,Rpn5,Rpn6,Rpn7,Rpn8,Rpn9,Rpt1,Rpt2,Rpt3,Rpt4,<br \/>\nRpt5,Rpt6,Scl1,Ubp6,Ura7,Ygl004c,Yku70,Ypl070w<\/td>\n<\/tr>\n<tr>\n<td colspan=\"7\"><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">2<\/td>\n<td style=\"text-align: center;\">9<\/td>\n<td style=\"text-align: center;\">19<\/td>\n<td style=\"text-align: center;\">90<\/td>\n<td style=\"text-align: center;\">0.51<\/td>\n<td style=\"text-align: left;\">RNA processing\/modification<\/td>\n<td style=\"text-align: left;\">Nuclear<\/td>\n<\/tr>\n<tr>\n<td colspan=\"2\" style=\"text-align: left;\"><strong>Protein names<\/strong><\/td>\n<td colspan=\"5\" style=\"text-align: left;\">Cft1,Cft2,Fip1,Fir1,Hca4,Mpe1,Pap1,Pcf11,Pfs2,Pta1,Pti1,Ref2,Rna14,Ssu72,<br \/>\nUba2,Ufd1,Yor179c,Ysh1,Yth1<\/td>\n<\/tr>\n<tr>\n<td colspan=\"7\"><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">3<\/td>\n<td style=\"text-align: center;\">7.72<\/td>\n<td style=\"text-align: center;\">56<\/td>\n<td style=\"text-align: center;\">220<\/td>\n<td style=\"text-align: center;\">0.14<\/td>\n<td style=\"text-align: left;\">Pol II transcription<\/td>\n<td style=\"text-align: left;\">Nuclear<\/td>\n<\/tr>\n<tr>\n<td colspan=\"2\" style=\"text-align: left;\"><strong>Protein names<\/strong><\/td>\n<td colspan=\"5\" style=\"text-align: left;\">Ada2,Adr1,Ahc1,Cdc23,Cdc36,Epl1,Esa1,Fet4,Fun19,Gal4,Gcn5,Hac1,Hfi1,<br \/>\nHhf2,Hht1,Hht2,Ire1,Luc7,Med7,Myo4,Ngg1,Pcf11,Pdr1,Prp40,Rna14,Rpb2,<br \/>\nRpo21,Sap185,Sgf29,Sgf73,Spt15,Spt20,Spt3,Spt7,Spt8,Srb6,Swi5,Taf1,Taf10,<br \/>\nTaf11,Taf12,Taf13,Taf14,Taf2,Taf3,Taf5,Taf6,Taf7,Taf8,Taf9,Tra1,Ubp8,<br \/>\nYap1,Yap6,Ybr270c,Yng2<\/td>\n<\/tr>\n<tr>\n<td colspan=\"7\"><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">4<\/td>\n<td style=\"text-align: center;\">7.58<\/td>\n<td style=\"text-align: center;\">18<\/td>\n<td style=\"text-align: center;\">72<\/td>\n<td style=\"text-align: center;\">0.44<\/td>\n<td style=\"text-align: left;\">Cell cycle control, protein degradation, mitosis (Anaphase Promoting Complex)<\/td>\n<td style=\"text-align: left;\">Nuclear<\/td>\n<\/tr>\n<tr>\n<td colspan=\"2\" style=\"text-align: left;\"><strong>Protein names<\/strong><\/td>\n<td colspan=\"5\" style=\"text-align: left;\">Apc1,Apc11,Apc2,Apc4,Apc5,Apc9,Cdc16,Cdc23,Cdc26,Cdc27,Dmc1,Doc1,<br \/>\nLeu3,Rpt1,Sic1,Spc29,Spt2,Ybr270c<\/td>\n<\/tr>\n<tr>\n<td colspan=\"7\"><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">5<\/td>\n<td style=\"text-align: center;\">7<\/td>\n<td style=\"text-align: center;\">15<\/td>\n<td style=\"text-align: center;\">56<\/td>\n<td style=\"text-align: center;\">0.52<\/td>\n<td style=\"text-align: left;\">Vesicular transport (TRAPP Complex)<\/td>\n<td style=\"text-align: left;\">Golgi<\/td>\n<\/tr>\n<tr>\n<td colspan=\"2\" style=\"text-align: left;\"><strong>Protein names<\/strong><\/td>\n<td colspan=\"5\" style=\"text-align: left;\">Bet1,Bet3,Bet5,Fks1,Gsg1,Gyp6,Kre11,Sec22,Trs120,Trs130,Trs20,Trs23,<br \/>\nTrs31,Trs33,Uso1<\/td>\n<\/tr>\n<tr>\n<td colspan=\"7\"><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">102<\/td>\n<td style=\"text-align: center;\">3<\/td>\n<td style=\"text-align: center;\">3<\/td>\n<td style=\"text-align: center;\">3<\/td>\n<td style=\"text-align: center;\">1<\/td>\n<td style=\"text-align: left;\">RNA splicing<\/td>\n<td style=\"text-align: left;\">Nuclear<\/td>\n<\/tr>\n<tr>\n<td colspan=\"2\" style=\"text-align: left;\"><strong>Protein names<\/strong><\/td>\n<td colspan=\"5\" style=\"text-align: left;\">Msl5,Mud2,Smy2<\/td>\n<\/tr>\n<tr>\n<td colspan=\"7\"><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">103<\/td>\n<td style=\"text-align: center;\">3<\/td>\n<td style=\"text-align: center;\">3<\/td>\n<td style=\"text-align: center;\">3<\/td>\n<td style=\"text-align: center;\">1<\/td>\n<td style=\"text-align: left;\">Signal transduction, Cell cycle control, DNA repair, DNA synthesis<\/td>\n<td style=\"text-align: left;\">Nuclear<\/td>\n<\/tr>\n<tr>\n<td colspan=\"2\" style=\"text-align: left;\"><strong>Protein names<\/strong><\/td>\n<td colspan=\"5\" style=\"text-align: left;\">Ptc2,Rad53,Ydr071c<\/td>\n<\/tr>\n<tr>\n<td colspan=\"7\"><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">104<\/td>\n<td style=\"text-align: center;\">3<\/td>\n<td style=\"text-align: center;\">3<\/td>\n<td style=\"text-align: center;\">3<\/td>\n<td style=\"text-align: center;\">1<\/td>\n<td style=\"text-align: left;\">Cell cycle control, mating response<\/td>\n<td style=\"text-align: left;\">Uknown<\/td>\n<\/tr>\n<tr>\n<td colspan=\"2\" style=\"text-align: left;\"><strong>Protein names<\/strong><\/td>\n<td colspan=\"5\" style=\"text-align: left;\">Far3,Vps64,Ynl127w<\/td>\n<\/tr>\n<tr>\n<td colspan=\"7\"><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">105<\/td>\n<td style=\"text-align: center;\">3<\/td>\n<td style=\"text-align: center;\">3<\/td>\n<td style=\"text-align: center;\">3<\/td>\n<td style=\"text-align: center;\">1<\/td>\n<td style=\"text-align: left;\">Chromatin\/chromosome structure<\/td>\n<td style=\"text-align: left;\">Nuclear<\/td>\n<\/tr>\n<tr>\n<td colspan=\"2\" style=\"text-align: left;\"><strong>Protein names<\/strong><\/td>\n<td colspan=\"5\" style=\"text-align: left;\">Gbp2,Hpr1,Mft1<\/td>\n<\/tr>\n<tr>\n<td colspan=\"7\"><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">106<\/td>\n<td style=\"text-align: center;\">3<\/td>\n<td style=\"text-align: center;\">3<\/td>\n<td style=\"text-align: center;\">3<\/td>\n<td style=\"text-align: center;\">1<\/td>\n<td style=\"text-align: left;\">Pol II transcription<\/td>\n<td style=\"text-align: left;\">Nuclear<\/td>\n<\/tr>\n<tr>\n<td colspan=\"2\" style=\"text-align: left;\"><strong>Protein names<\/strong><\/td>\n<td colspan=\"5\" style=\"text-align: left;\">Ctk1,Ctk2,Ctk3<\/td>\n<\/tr>\n<tr>\n<td colspan=\"7\"><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">205<\/td>\n<td style=\"text-align: center;\">2<\/td>\n<td style=\"text-align: center;\">3<\/td>\n<td style=\"text-align: center;\">4<\/td>\n<td style=\"text-align: center;\">1<\/td>\n<td style=\"text-align: left;\">Vesicular transport<\/td>\n<td style=\"text-align: left;\">ER<\/td>\n<\/tr>\n<tr>\n<td colspan=\"2\" style=\"text-align: left;\"><strong>Protein names<\/strong><\/td>\n<td colspan=\"5\" style=\"text-align: left;\">Rim20,Snf7,Vps4<\/td>\n<\/tr>\n<tr>\n<td colspan=\"7\"><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">206<\/td>\n<td style=\"text-align: center;\">2<\/td>\n<td style=\"text-align: center;\">3<\/td>\n<td style=\"text-align: center;\">4<\/td>\n<td style=\"text-align: center;\">1<\/td>\n<td style=\"text-align: left;\">Protein translocation<\/td>\n<td style=\"text-align: left;\">Cytoplasmic<\/td>\n<\/tr>\n<tr>\n<td colspan=\"2\" style=\"text-align: left;\"><strong>Protein names<\/strong><\/td>\n<td colspan=\"5\" style=\"text-align: left;\">Srp14,Srp21,Srp54<\/td>\n<\/tr>\n<tr>\n<td colspan=\"7\"><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">207<\/td>\n<td style=\"text-align: center;\">2<\/td>\n<td style=\"text-align: center;\">3<\/td>\n<td style=\"text-align: center;\">4<\/td>\n<td style=\"text-align: center;\">1<\/td>\n<td style=\"text-align: left;\">Protein translocation<\/td>\n<td style=\"text-align: left;\">Cytoplasmic<\/td>\n<\/tr>\n<tr>\n<td colspan=\"2\" style=\"text-align: left;\"><strong>Protein names<\/strong><\/td>\n<td colspan=\"5\" style=\"text-align: left;\">Srp54,Srp68,Srp72<\/td>\n<\/tr>\n<tr>\n<td colspan=\"7\"><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">208<\/td>\n<td style=\"text-align: center;\">2<\/td>\n<td style=\"text-align: center;\">3<\/td>\n<td style=\"text-align: center;\">4<\/td>\n<td style=\"text-align: center;\">1<\/td>\n<td style=\"text-align: left;\">Energy generation<\/td>\n<td style=\"text-align: left;\">Mitochondrial<\/td>\n<\/tr>\n<tr>\n<td colspan=\"2\" style=\"text-align: left;\"><strong>Protein names<\/strong><\/td>\n<td colspan=\"5\" style=\"text-align: left;\">Atp1,Atp11,Atp2<\/td>\n<\/tr>\n<tr>\n<td colspan=\"7\"><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">209<\/td>\n<td style=\"text-align: center;\">2<\/td>\n<td style=\"text-align: center;\">4<\/td>\n<td style=\"text-align: center;\">5<\/td>\n<td style=\"text-align: center;\">0.67<\/td>\n<td style=\"text-align: left;\">Nuclear-cytoplasmic and vesicular transport<\/td>\n<td style=\"text-align: left;\">Varied<\/td>\n<\/tr>\n<tr>\n<td colspan=\"2\" style=\"text-align: left;\"><strong>Protein names<\/strong><\/td>\n<td colspan=\"5\" style=\"text-align: left;\">Kap123,Nup145,Sec7,Slc1<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\nScore is defined as the product of the complex subgraph density and the number of vertices (proteins) in the complex subgraph (DC \u00d7 \\|V\\|). This ranks larger more dense complexes higher in the results. Density is calculated using the \"loop\" formula if homodimers exist in the complex, otherwise the \"no loop\" formula is used. The cell role column is a manual combination of annotation terms for the proteins reported in the complex.\n\nMany of the 209 predicted complexes are of size 2 (9 predicted complexes) or 3 (54 predicted complexes). Complexes of this size may not be significant since it is easy to create high density subgraphs of size 2 or 3, but becomes combinatorially more difficult to randomly create high density subgraphs as the size of the subgraph increases. To examine the relevance of these small predicted complexes of size 2 or 3, we calculated the sensitivity and specificity of the optimized MCODE predictions against the MIPS complex benchmark while disregarding the small complexes. First, complexes of size 2, then of size 3, were removed from the optimized MCODE predicted complex set. Removing each of these sets independently resulted in only small sensitivity and specificity changes. Because both sets overlap the MIPS benchmark, small complexes have been reported as predictions. Also, because MCODE found these small complexes in regions of high local density, they may be good cores for further examination with MCODE in directed mode, especially since the haircut option was turned on here to produce them.\n\nComplexes that are larger and denser are ranked higher by MCODE and these generally correspond to known complexes (see below). Interestingly, some MCODE complexes contain unknown proteins that are highly connected to known complex subunits. For example, the second highest ranked MCODE complex is involved in RNA processing\/modification and contains the known polyadenylation factor I complex (Cft1, Cft2, Fip1, Pap1, Pfs2, Pta1, Ysh1, Yth1 and Ykl059c). Seven other proteins involved in mainly RNA processing\/modification (Fir1, Hca4, Pcf11, Pti1, Ref2, Rna14, Ssu72) and protein degradation (Uba2 and Ufd1) are highly connected within this predicted complex. Two unknown proteins Pti1 and Yor179c are highly connected to RNA processing\/modification proteins and are therefore likely involved in the same process (Figure <a href=\"#F7\" data-ref-type=\"fig\">7<\/a>). Pti1 may be an unknown component of the polyadenylation factor I complex. The 23^rd^ highest ranked predicted complex is interesting in that it is involved in cell polarity and cytokinesis and contains two proteins of unknown function, Yhr033w and Yal027w. Yal027w interacts with two kinases, Gin4 and Kcc4, which in turn interact with the components of the Septin complex (Cdc3, Cdc10, Cdc11 and Cdc12) (Figure <a href=\"#F8\" data-ref-type=\"fig\">8<\/a>).\n\n## Significance of MCODE predictions\n\nNa\u00efvely, the chance of randomly picking a known protein complex from a protein interaction network depends on the size of the complex and the network. It is easier to pick out a smaller known complex by chance from a smaller network. For instance, in our network of 15,143 interactions among 4,825 proteins, the chance of picking a specific known complex of size three is about one in 1.9 \u00d7 10^10^ (4,825 choose 3). A more realistic model would assume that the proteins are connected and thus would only consider complex choices of size three where all three proteins are connected. The number of choices now depends on the topology of the network. In our large network, there are 6,799 fully connected subnetworks of size three and 313,057 subnetworks of size three with only two interactions (from the triadic census feature of Pajek). Thus now our chance of picking a more realistic complex is one out of 319,856 (1\/(6,799 + 313,057) = 3.1 \u00d7 10^-6^). As the size of the complex increases, the number of possible complex topologies increases exponentially and, in a connected network of some reasonable density, so does the number of possible subgraphs that could represent a complex. The density of our large protein interaction network is 0.0013 and is mostly connected (4,689 proteins are in one connected component). Thus, it is expected that if a complex is found in a network with MCODE that matches a known complex, that the result would be highly significant. To understand the significance of complex prediction further, the topology of the protein interaction network would have to be understood in general, in order to build a null model to compare against.\n\nRecent research on modeling complex systems \\[21,25,27\\] has found that networks such as the world wide web, metabolic networks \\[26\\] and protein-protein interaction networks \\[36\\] are scale-free. That is, the connectivity distribution of the vertices of the graph follows a power law, with many vertices of low degree and few vertices of high degree. Scale-free networks are known to have large clustering coefficients, or clustered regions of the graph. In biological networks, at least in yeast, these clustered regions seem to correspond to molecular complexes and these subgraphs are what MCODE is designed to find.\n\nTo test the significance of clustered regions in biological networks, 100 random permutations of the large set of all 15,143 yeast interactions were made. If the graph to be randomised is considered as a set of edges between two vertices (*v*~1~, *v*~2~), a network permutation is made by randomly permuting the set of all *v*~2~ vertices. The random networks have the same number of edges and vertices as the original network and follow a power-law connectivity distribution, as do the original data sets \\[37\\]. Running MCODE with the same parameters as the original network (haircut = TRUE, fluff = TRUE, VWP = 0 and a fluff density threshold of 0.1) on the 100 random networks resulted in an average of 27.4 (SD = 4.4) complexes per network. The size distribution of complexes found by MCODE did not match that of the complexes found in the original network, as some complexes found in the random networks were composed of \\>1500 proteins. One random network that had an approximately average number of predicted complexes (27) was parameter optimized using the MIPS benchmark to see how parameter choice affects the size distribution and number of predicted complexes. Parameters of haircut = TRUE, fluff = TRUE, VWP = 0.1 and a fluff density threshold of zero produced the maximal number of 81 complexes for this network, but these complexes were composed of on average 27 proteins (without counting an outlier complex of size 1961), which is much larger than normal (e.g. larger than the MIPS set average of 6.0). None of these predicted complexes matched any MIPS complexes above an overlap score of 0.1. Also, the random network complexes had a much higher average number of YPD and GO annotation terms per protein per complex than for MIPS or MCODE on the original network (Table <a href=\"#T2\" data-ref-type=\"table\">2<\/a>). This indicates, as expected, that the random network complexes are composed of a higher level of unrelated proteins than complexes in the original network. Thus, the number, size and functional composition of complexes that MCODE predicts in the large set of all yeast interactions are highly unlikely to occur by chance.\n\nTo evaluate the effectiveness of our scoring scheme, which scores larger, more dense complexes higher than smaller, more sparse complexes, we examined the accuracy of MCODE predictions at various score thresholds. As the score threshold for inclusion of complexes is increased, less complexes are included, but a higher percentage of the included complexes match complexes in the benchmark. This is at the expense of sensitivity as many benchmark matching complexes are not included at higher score thresholds (Figure <a href=\"#F9\" data-ref-type=\"fig\">9<\/a>). For example, of the ten predicted complexes with MCODE score greater or equal to six, nine match a known complex in either the MIPS or Gavin benchmark above a 0.2 threshold overlap score, yielding an accuracy of 90%. 100% of the five complexes that had an MCODE score better or equal to seven matched known complexes. Thus, complexes that score highly on our simple density based scoring scheme are very likely to be real.\n\n## Directed mode of MCODE\n\nTo simulate an obvious example where the directed mode of MCODE would be useful, MCODE was run with relaxed parameters (haircut = TRUE, fluff = TRUE, VWP = 0.05 and a fluff density threshold of 0.2) compared to the best parameters on the AllYeast network. The resulting fourth highest ranked complex, when visualized, shows two clustered components and represents two protein complexes, the proteasome and an RNA processing complex, both found in the nucleus (Figure <a href=\"#F10\" data-ref-type=\"fig\">10<\/a>). This is an example of where a lower VWP parameter would have been superior since it would have divided this large complex into two more functionally related complexes. The highest weighted vertices in the center of each of the two dense regions in Figure <a href=\"#F10\" data-ref-type=\"fig\">10<\/a> are the Rpt1 and Lsm4 proteins. MCODE was run in directed mode starting with these two proteins over a range of VWP parameters from 0 to 0.2, at 0.05 increments. For Lsm4, the parameter set of haircut = TRUE, fluff = FALSE, VWP = 0 was used to find a core complex, which contained 9 proteins fully connected to each other (Dcp1, Kem1, Lsm2, Lsm3, Lsm4, Lsm5, Lsm6, Lsm7 and Pat1). Above this VWP parameter, the core complex branched out into proteasome subunit proteins, which are not part of the Lsm complex (see Figure <a href=\"#F11\" data-ref-type=\"fig\">11A<\/a>). Using this VWP parameter, combinations of haircut and fluff parameters were used to further expand the core complex. This process was stopped when the predicted complexes began to include proteins of sufficiently different known biological function to the seed vertex. Proteins, such as Vam6 and Yor320c were included in the complex at moderate fluff parameters (0.4\u20130.6), but not at higher fluff parameters, and these are known to be localized in membranes outside of the nucleus, thus are likely not functionally related to the Lsm complex proteins. Therefore, the 9 proteins listed above were decided to be the final complex (Figure <a href=\"#F11\" data-ref-type=\"fig\">11B<\/a>). This is intuitive because of their maximal density (a 9-clique).\n\nUsing this same method of known biological role \"titration\" on Rpt1 found a complex of 34 proteins (Gal4, Gcn4, Hsm3, Lhs1, Nas6, Pre1, Pre2, Pre3, Pre4, Pre5, Pre6, Pre7, Pre9, Pup3, Rpn10, Rpn11, Rpn13, Rpn3, Rpn5, Rpn6, Rpn7, Rpn8, Rpn9, Rpt1, Rpt2, Rpt3, Rpt4, Rpt6, Rri1, Scl1, Sts1, Ubp6, Ydr179c, Ygl004c) and 160 interactions using the parameter set haircut = TRUE, fluff = TRUE, VWP = 0.2 and a fluff density threshold of 0.3. Two regions of density can be seen here corresponding to the two known subunits of the 26S proteasome. The 20S proteolytic subunit of the proteasome is comprised of 15 proteins (Pre1 to Pre10, Pup1, Pup2, Pup3, Scl1 and Ump1) of which Pre7, Pre8, Pre10, Pup1, Pup2 and Ump1 are not found with MCODE. The 19S regulatory subunit of the proteasome is known to have 21 subunits (Nas6, Rpn1 to Rpn13, Rpt1 to Rpt6 and Ubp6) of which Rpn1, Rpn2, Rpn4, Rpn12 and Rpt5 are not found with MCODE. Known complex components not found by MCODE are not present at a high enough local density regions of the interaction network, possibly because not enough experiments involving these proteins are present in our data set. Figure <a href=\"#F11\" data-ref-type=\"fig\">11C<\/a> shows the final Rpt1 seeded complex. Of note, Ygl004c is unknown and binds to almost every Rpt and Rpn protein in the complex although all of these interactions were from a single immunoprecipitation experiment \\[6\\]. As well, Rri1 and Ydr179c have unknown function and both bind to each other and to Rpn5. Thus one would predict that these three unknown proteins function with or as part of the 26S proteasome. The protein Hsm3 binds to eight other 19S subunits and is involved in DNA mismatch repair pathways, but is not known to be part of the proteasome, although all of these Hsm3 interactions are from a particular large-scale experiment \\[7\\]. Interestingly, Gal4, a transcription factor involved in galactose metabolism, is found to be part of the proteasome complex. While this metabolic functionality seems unrelated to protein degradation, it has recently been shown that the binding is physiologically relevant \\[38\\]. These cases illustrate the possible unreliability of both functional annotation and interaction data, but also that seemingly unrelated proteins should not be immediately discounted if found to be part of a complex by MCODE.\n\nOf note, the known topology of the 26S proteasome \\[39\\] compares favourably with the complex visualization of Figure <a href=\"#F11\" data-ref-type=\"fig\">11C<\/a> without considering stoichiometry. Thus, if enough interactions are known, visualizing complexes may reveal the rough structural outline of large complexes. This should be expected when dealing with actual physical protein-protein interactions since there are few allowed topologies for large complexes considering the specific set of defining interactions and steric clashes between protein subunits.\n\n## Complex connectivity\n\nMCODE may also be used to examine the connectivity and relationships between molecular complexes. Once a complex is known using the directed mode, the MCODE parameters can be relaxed to allow branching out into other complexes. The MCODE directed mode preprocessing step must also be turned off to allow MCODE to branch into other connected complexes, which may reside in denser regions of the graph than the seed vertex. As an example, this was done with the Lsm4 seeded complex (Figure <a href=\"#F12\" data-ref-type=\"fig\">12<\/a>). MCODE parameters were relaxed to haircut = TRUE, fluff = FALSE, VWP = 0.2 although they could be further relaxed for greater extension out into the network.\n\n# Discussion\n\nThis method represents an initial step in taking advantage of the protein function data being generated by many large-scale protein interaction studies. As the experimental methods are further developed, an increasing amount of data will be produced which will require computational methods for efficient interpretation. The algorithm described here allows the automated prediction of protein complexes from qualitative protein-protein interaction data and is thus able to help predict the function of unknown proteins and aid in the understanding of the functional connectivity of molecular complexes in the cell. The general nature of this method may allow complex prediction for molecules other than proteins as well, for example metabolic complexes that include small molecules.\n\nMCODE cannot stand alone in this task; it must be combined with a graph visualization system to ease the understanding of the relationships among molecules in the data set. We use the Pajek program for large network analysis \\[40\\] with the Kamada-Kawai graph layout algorithm \\[41\\]. Kamada-Kawai models the edges in the graph as springs, randomly places the vertices in a high energy state and then attempts to minimize the energy of the system over a number of time steps. The result is that the Euclidean distance, here in a plane, is close to the graph-theoretic or path distance between the vertices. The vertices are visually clustered based on connectivity. Biologically, this visualization can allow one to see the rough structural outline of large complexes, if enough interactions are known, as evidenced in the proteasome complex analysis above (Figure <a href=\"#F11\" data-ref-type=\"fig\">11C<\/a>).\n\nIt is important to note and understand the limitations of the current experimental methods (e.g. yeast two-hybrid and co-immunoprecipitation) and the protein interaction networks that these techniques generate when analyzing the resulting data. One common class of false-positive interactions arising from many different kinds of experimental methods is that of indirect interactions. For instance, an interaction may be seen between two proteins using a specific experimental method, but in reality, those proteins do not physically bind each other, and one or more other molecules that are generally part of the same complex mediate the observed interaction. As can be seen for the Arp2\/3 complex shown in Figure <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>, when pairwise interactions between all combinations of proteins in a complex are studied, this creates a very dense graph. Interestingly, this false-positive effect is normally considered a disadvantage, but is an advantage with MCODE as it increases the density in the region of the graph containing a complex, which can then be more easily predicted.\n\nApart from the experimental factors that lead to false-positive and false-negative interactions, representational limitations also exist computationally. Temporal and spatial information is not currently described in interaction networks. A complex found by the MCODE approach may not actually exist even though all of the component proteins bind each other *in vitro*. Those proteins may never be present at the same time and place. For example, molecular complexes that perform different functions sometimes have common subunits as with the three types of eukaryotic RNA polymerases.\n\nComplex stoichiometry, another important aspect of biological data, is not represented either. While it is possible to include full stoichiometry in a graph representation of a biomolecular interaction network, many experimental methods do not provide this information, so a homo-multimeric complex is normally represented as a simple homodimer. When an experiment does provide stoichiometry information, it is not stored in most current databases, such as MIPS and YPD. Thus, one is forced to return to the primary literature to extract the data, an extremely time-consuming task for large data sets.\n\nSome quantitative and statistical information is present when integrating results of large-scale approaches and this is not used in our current graph model. For instance, the number of different types of experiments that find the same interaction, the quality of the experiment, the date the experiment was conducted (newer methods may be superior in certain aspects) and other factors that pertain to the reliability of the interaction could all be considered to determine a reliability index or p-value on edges in the graph. For instance, one may wish to rank results published in high-impact journals above other journals (or vice versa) and rank classical purification methods above high-throughput yeast two-hybrid techniques when determining the quality of the interaction data. It may also be possible to weight vertices on the graph by other quality criteria, such as whether a protein is hypothetical from a gene prediction or not or whether a protein is expressed at a particular time and place in the cell. For example, if one were interested in a certain stage of the cell cycle, proteins that are known to be absent at that stage could be reduced in weight (VWP in the case of MCODE) compared to proteins that are present. It should be noted that any weighting scheme that tries to assess the quality of an interaction might make false assumptions that would prevent the discovery of new and interesting data.\n\nThis paper shows that the structure of a biological network can define complexes, which can be seen as dense regions. This may be attributed to indirect interactions accumulating in the literature. Thus, interaction data taken out of context may be erroneous. For instance, if one has a collection of protein interactions from various different experiments done at different times in different labs from a specific complex that form a clique, and if one chooses an interaction from this clique, then how can one verify if it is indirect or not. We would only begin to know if we had a very detailed description of the experiment from the original papers where we could tell the amount of work and quality of work that went into measuring each interaction. Thus with only a qualitative view of interactions, in reference to Dobzhansky \\[42\\], nothing in the biomolecular interaction network would make sense except in light of molecular complexes and the functional connections between them. If one had a highly detailed representation of each interaction including time, place, experimental condition, number of experiments, binding sites, chemical actions and chemical state information, one would be able to computationally delve into molecular complexes to resolve topology, structure, function and mechanism down to the atomic level. This information would also help to judge the biological relevance of an interaction. Thus, we require databases like BIND \\[15\\] to store this information. The integration of known qualitative and quantitative molecular interaction data in a machine-readable format should allow increasingly accurate protein interaction, molecular complex and pathway prediction, including actual binding site and mechanism information in a sequence and structural context.\n\nBased on our scale-free network analysis, it would seem that real biological networks are organized differently than random models of scale-free networks in that they have higher clustering coefficients around specific regions (complexes) and the vertices in these regions are related to each other, by biological function. Thus, attempts to model biological networks and their evolution in a global way solely using the statistics of scale-free networks may not work, rather modeling should take into account as much extant biological knowledge as possible.\n\nFuture work on MCODE could include researching different, possibly adaptive, vertex scoring functions to take into account, for example, the local density of the network past the immediate neighborhood of a vertex and the inclusion of functional annotation and p-values on edges. Time, space and stoichiometry should also be represented on networks and in visualization systems. The process of 'functional annotation titration' in the directed mode of MCODE could be automated.\n\n# Conclusions\n\nMCODE effectively finds densely connected regions of a molecular interaction network, many of which correspond to known molecular complexes, based solely on connectivity data. Given that this approach to analyzing protein interaction networks performs well using minimal qualitative information implies that large amounts of available knowledge is buried in large protein interaction networks. More accurate data mining algorithms and systems models could be constructed to understand and predict interactions, complexes and pathways by taking into account more existing biological knowledge. Structured molecular interaction data resources such as BIND will be vital in creating these resources.\n\n# Methods\n\n## Data sources\n\nAll protein interaction data sets from MIPS \\[13\\], Gene Ontology \\[43\\] and PreBIND <http:\/\/bioinfo.mshri.on.ca\/prebind\/> were collected as described previously \\[6\\]. The YPD protein interaction data are from March 2001 and were originally requested from Proteome, Inc. <http:\/\/www.proteome.com>. Other interaction data sets are from BIND <http:\/\/www.bind.ca>. A BIND yeast import utility was developed to integrate data from SGD \\[12\\], RefSeq \\[44\\], Gene Registry <http:\/\/genome-www.stanford.edu\/Saccharomyces\/registry.html>, the list of essential genes from the yeast deletion consortium \\[11\\] and GO terms \\[43\\]. This database ensures proper matching of yeast gene names among the multiple data sets that may use different names for the same genes. The yeast proteome used here is defined by SGD and RefSeq and contains 6,334 ORFs including the mitochondrial chromosome. Before performing comparisons, the various interaction data sets were entered into a local instance of BIND as pairwise protein interaction records. The MIPS complex catalogue was downloaded in February 2002.\n\nThe protein interaction data sets used here were composed as follows. 'Gavin Spoke' is the spoke model of the raw purifications from Gavin et al \\[7\\]. 'Y2H' is all known large-scale \\[2-5,10\\] combined with normal yeast two-hybrid results from MIPS. 'HTP Only' is only high-throughput or large-scale data \\[2-7,10\\] The 'Benchmark' set was constructed from MIPS, YPD and PreBIND as previously described \\[6\\]. 'Pre HTMS' was composed of all yeast sets except the recent large-scale mass spectrometry data sets \\[6,7\\]. 'AllYeast' was the combination of all above data sets. All data sets are non-redundant.\n\n## Network visualization\n\nVisualization of networks was performed using the Pajek program for large network analysis \\[40\\]<http:\/\/vlado.fmf.uni-lj.si\/pub\/networks\/pajek\/> as described previously \\[6,10\\]. using the Kamada-Kawai graph layout algorithm followed by manual vertex adjustments and was formatted using CorelDraw 10. Power law analysis was also accomplished as previously described \\[6\\].\n\n# Authors' contributions\n\nGB conceived of the study and carried out all programming and analyses as a Ph.D. student in the lab of CH. CH supervised the study and provided valuable input for the evaluation analyses.\n\n# Supplementary Material\n\n###### Additional File 1\n\nAllYeastPredictedComplexes.zip Zip file containing Pajek .net and annotation files for all 209 complexes found using MCODE on the set of all yeast interactions reported here. Various report files from MCODE are also included as well as basic instructions for using Pajek.\n\nClick here for file\n\n### Acknowledgements\n\nMichel Dumontier, Sherrie Kelly, Katerina Michalickova, Tony Pawson and Mike Tyers provided helpful discussion. This work was supported in part from grants from the Canadian Institutes of Health Research (CIHR), the Ontario Research and Development Challenge Fund and MDS-Sciex to C.H. G.D.B. is supported by an Ontario Graduate Scholarship (OGS).","meta":{"dup_signals":{"dup_doc_count":113,"dup_dump_count":52,"dup_details":{"curated_sources":1,"2023-14":1,"2022-49":2,"2022-27":3,"2022-05":2,"2021-43":1,"2021-21":1,"2021-17":1,"2021-04":1,"2020-50":1,"2020-45":1,"2020-40":1,"2020-24":1,"2020-10":1,"2019-51":1,"2019-39":2,"2019-35":1,"2019-30":2,"2019-26":1,"2019-22":1,"2019-13":1,"2019-09":1,"2018-47":3,"2018-43":1,"2018-34":1,"2018-30":1,"2018-13":1,"2018-09":1,"2017-43":2,"2017-39":1,"2017-30":1,"2017-26":5,"2017-22":8,"2017-17":13,"2017-09":7,"2017-04":1,"2016-44":2,"2016-40":1,"2016-36":7,"2016-30":9,"2016-22":1,"2016-18":1,"2015-40":2,"2015-35":1,"2015-32":2,"2015-22":2,"2014-52":1,"2014-49":1,"2014-42":3,"2014-41":2,"2014-35":1,"2014-23":2,"2023-50":1}},"file":"PMC149346"},"subset":"pubmed_central"}
{"text":"abstract: Pandemics such as the Black Death have altered the course of history. The ravages of the Spanish Flu pandemic were so terrible that at least one culture decided the most humane way of dealing with the aftermath was simply to ignore it\u2014to pretend the disease had not happened. Could a new pandemic possibly be worse than the Black Death or the Spanish Flu? Could it be as bad as the Scarlet Plague of Jack London's story?\nauthor: Stephen Webb\ndate: 2019-01-08\nreferences:\ntitle: Pandemic\n\n# The Scarlet Plague (Jack London)\n\n## I\n\nTHE way led along upon what had once been the embankment of a railroad. But no train had run upon it for many years. The forest on either side swelled up the slopes of the embankment and crested across it in a green wave of trees and bushes. The trail was as narrow as a man's body, and was no more than a wild-animal runway. Occasionally, a piece of rusty iron, showing through the forest-mould, advertised that the rail and the ties still remained. In one place, a ten-inch tree, bursting through at a connection, had lifted the end of a rail clearly into view. The tie had evidently followed the rail, held to it by the spike long enough for its bed to be filled with gravel and rotten leaves, so that now the crumbling, rotten timber thrust itself up at a curious slant. Old as the road was, it was manifest that it had been of the monorail type.\n\nAn old man and a boy travelled along this runway. They moved slowly, for the old man was very old, a touch of palsy made his movements tremulous, and he leaned heavily upon his staff. A rude skullcap of goat-skin protected his head from the sun. From beneath this fell a scant fringe of stained and dirty-white hair. A visor, ingeniously made from a large leaf, shielded his eyes, and from under this he peered at the way of his feet on the trail. His beard, which should have been snow-white but which showed the same weather-wear and camp-stain as his hair, fell nearly to his waist in a great tangled mass. About his chest and shoulders hung a single, mangy garment of goat-skin. His arms and legs, withered and skinny, betokened extreme age, as well as did their sunburn and scars and scratches betoken long years of exposure to the elements.\n\nThe boy, who led the way, checking the eagerness of his muscles to the slow progress of the elder, likewise wore a single garment\u2014a ragged-edged piece of bear-skin, with a hole in the middle through which he had thrust his head. He could not have been more than twelve years old. Tucked coquettishly over one ear was the freshly severed tail of a pig. In one hand he carried a medium-sized bow and an arrow.\n\nOn his back was a quiverful of arrows. From a sheath hanging about his neck on a thong, projected the battered handle of a hunting knife. He was as brown as a berry, and walked softly, with almost a catlike tread. In marked contrast with his sunburned skin were his eyes\u2014blue, deep blue, but keen and sharp as a pair of gimlets. They seemed to bore into aft about him in a way that was habitual. As he went along he smelled things, as well, his distended, quivering nostrils carrying to his brain an endless series of messages from the outside world. Also, his hearing was acute, and had been so trained that it operated automatically. Without conscious effort, he heard all the slight sounds in the apparent quiet\u2014heard, and differentiated, and classified these sounds\u2014whether they were of the wind rustling the leaves, of the humming of bees and gnats, of the distant rumble of the sea that drifted to him only in lulls, or of the gopher, just under his foot, shoving a pouchful of earth into the entrance of his hole.\n\nSuddenly he became alertly tense. Sound, sight, and odor had given him a simultaneous warning. His hand went back to the old man, touching him, and the pair stood still. Ahead, at one side of the top of the embankment, arose a crackling sound, and the boy's gaze was fixed on the tops of the agitated bushes. Then a large bear, a grizzly, crashed into view, and likewise stopped abruptly, at sight of the humans. He did not like them, and growled querulously. Slowly the boy fitted the arrow to the bow, and slowly he pulled the bowstring taut. But he never removed his eyes from the bear.\n\nThe old man peered from under his green leaf at the danger, and stood as quietly as the boy. For a few seconds this mutual scrutinizing went on; then, the bear betraying a growing irritability, the boy, with a movement of his head, indicated that the old man must step aside from the trail and go down the embankment. The boy followed, going backward, still holding the bow taut and ready. They waited till a crashing among the bushes from the opposite side of the embankment told them the bear had gone on. The boy grinned as he led back to the trail.\n\n\"A big un, Granser,\" he chuckled.\n\nThe old man shook his head.\n\n\"They get thicker every day,\" he complained in a thin, undependable falsetto. \"Who'd have thought I'd live to see the time when a man would be afraid of his life on the way to the Cliff House. When I was a boy, Edwin, men and women and little babies used to come out here from San Francisco by tens of thousands on a nice day. And there weren't any bears then. No, sir. They used to pay money to look at them in cages, they were that rare.\"\n\n\"What is money, Granser?\"\n\nBefore the old man could answer, the boy recollected and triumphantly shoved his hand into a pouch under his bear-skin and pulled forth a battered and tarnished silver dollar. The old man's eyes glistened, as he held the coin close to them.\n\n\"I can't see,\" he muttered. \"You look and see if you can make out the date, Edwin.\"\n\nThe boy laughed.\n\n\"You're a great Granser,\" he cried delightedly, \"always making believe them little marks mean something.\"\n\nThe old man manifested an accustomed chagrin as he brought the coin back again close to his own eyes.\n\n\"2012,\" he shrilled, and then fell to cackling grotesquely. \"That was the year Morgan the Fifth was appointed President of the United States by the Board of Magnates. It must have been one of the last coins minted, for the Scarlet Death came in 2013. Lord! Lord!\u2014think of it! Sixty years ago, and I am the only person alive today that lived in those times. Where did you find it, Edwin?\"\n\nThe boy, who had been regarding him with the tolerant curiousness one accords to the prattlings of the feeble-minded, answered promptly.\n\n\"I got it off of Hoo-Hoo. He found it when we was herdin' goats down near San Jos\u00e9 last spring. Hoo-Hoo said it was *money*. Ain't you hungry, Granser?\"\n\nThe ancient caught his staff in a tighter grip and urged along the trail, his old eyes shining greedily.\n\n\"I hope Hare-Lip's found a crab\u2026 or two,\" he mumbled. \"They're good eating, crabs, mighty good eating when you've no more teeth and you've got grandsons that love their old grandsire and make a point of catching crabs for him. When I was a boy\u2014\"\n\nBut Edwin, suddenly stopped by what he saw, was drawing the bowstring on a fitted arrow. He had paused on the brink of a crevasse in the embankment. An ancient culvert had here washed out, and the stream, no longer confined, had cut a passage through the fill. On the opposite side, the end of a rail projected and overhung. It showed rustily through the creeping vines which overran it. Beyond, crouching by a bush, a rabbit looked across at him in trembling hesitancy. Fully fifty feet was the distance, but the arrow flashed true; and the transfixed rabbit, crying out in sudden fright and hurt, struggled painfully away into the brush. The boy himself was a flash of brown skin and flying fur as he bounded down the steep wall of the gap and up the other side. His lean muscles were springs of steel that released into graceful and efficient action. A hundred feet beyond, in a tangle of bushes, he overtook the wounded creature, knocked its head on a convenient tree-trunk, and turned it over to Granser to carry.\n\n\"Rabbit is good, very good,\" the ancient quavered, \"but when it comes to a toothsome delicacy I prefer crab. When I was a boy\u2014\"\n\n\"Why do you say so much that ain't got no sense?\" Edwin impatiently interrupted the other's threatened garrulousness.\n\nThe boy did not exactly utter these words, but something that remotely resembled them and that was more guttural and explosive and economical of qualifying phrases. His speech showed distant kinship with that of the old man, and the latter's speech was approximately an English that had gone through a bath of corrupt usage.\n\n\"What I want to know,\" Edwin continued, \"is why you call crab 'toothsome delicacy'? Crab is crab, ain't it? No one I never heard calls it such funny things.\"\n\nThe old man sighed but did not answer, and they moved on in silence. The surf grew suddenly louder, as they emerged from the forest upon a stretch of sand dunes bordering the sea. A few goats were browsing among the sandy hillocks, and a skin-clad boy, aided by a wolfish-looking dog that was only faintly reminiscent of a collie, was watching them. Mingled with the roar of the surf was a continuous, deep-throated barking or bellowing, which came from a cluster of jagged rocks a hundred yards out from shore. Here huge sea-lions hauled themselves up to lie in the sun or battle with one another. In the immediate foreground arose the smoke of a fire, tended by a third savage-looking boy. Crouched near him were several wolfish dogs similar to the one that guarded the goats.\n\nThe old man accelerated his pace, sniffing eagerly as he neared the fire. \"Mussels!\" he muttered ecstatically. \"Mussels! And ain't that a crab, Hoo-Hoo? Ain't that a crab? My, my, you boys are good to your old grandsire.\" Hoo-Hoo, who was apparently of the same age as Edwin, grinned. \"All you want, Granser. I got four.\"\n\nThe old man's palsied eagerness was pitiful. Sitting down in the sand as quickly as his stiff limbs would let him, he poked a large rock-mussel from out of the coals. The heat had forced its shells apart, and the meat, salmon-colored, was thoroughly cooked. Between thumb and forefinger, in trembling haste, he caught the morsel and carried it to his mouth. But it was too hot, and the next moment was violently ejected. The old man spluttered with the pain, and tears ran out of his eyes and down his cheeks.\n\nThe boys were true savages, possessing only the cruel humor of the savage. To them the incident was excruciatingly funny, and they burst into loud laughter. Hoo-Hoo danced up and down, while Edwin rolled gleefully on the ground. The boy with the goats came running to join in the fun.\n\n\"Set 'em to cool, Edwin, set 'em to cool,\" the old man besought, in the midst of his grief, making no attempt to wipe away the tears that still flowed from his eyes. \"And cool a crab, Edwin, too. You know your grandsire likes crabs.\"\n\nFrom the coals arose a great sizzling, which proceeded from the many mussels bursting open their shells and exuding their moisture. They were large shellfish, running from three to six inches in length. The boys raked them out with sticks and placed them on a large piece of driftwood to cool.\n\n\"When I was a boy, we did not laugh at our elders; we respected them.\"\n\nThe boys took no notice, and Granser continued to babble an incoherent flow of complaint and censure. But this time he was more careful, and did not burn his mouth. All began to eat, using nothing but their hands and making loud mouth-noises and lip-smackings. The third boy, who was called Hare-Lip, slyly deposited a pinch of sand on a mussel the ancient was carrying to his mouth; and when the grit of it bit into the old fellow's mucous membrane and gums, the laughter was again uproarious. He was unaware that a joke had been played on him, and spluttered and spat until Edwin, relenting, gave him a gourd of fresh water with which to wash out his mouth.\n\n\"Where's them crabs, Hoo-Hoo?\" Edwin demanded. \"Granser's set upon having a snack.\"\n\nAgain Granser's eyes burned with greediness as a large crab was handed to him. It was a shell with legs and all complete, but the meat had long since departed. With shaky fingers and babblings of anticipation, the old man broke off a leg and found it filled with emptiness.\n\n\"The crabs, Hoo-Hoo?\" he wailed. \"The crabs?\"\n\n\"I was fooling Granser. They ain't no crabs! I never found one.\"\n\nThe boys were overwhelmed with delight at sight of the tears of senile disappointment that dribbled down the old man's cheeks. Then, unnoticed, Hoo-Hoo replaced the empty shell with a fresh-cooked crab. Already dismembered, from the cracked legs the white meat sent forth a small cloud of savory steam. This attracted the old man's nostrils, and he looked down in amazement.\n\nThe change of his mood to one of joy was immediate. He snuffled and muttered and mumbled, making almost a croon of delight, as he began to eat. Of this the boys took little notice, for it was an accustomed spectacle. Nor did they notice his occasional exclamations and utterances of phrases which meant nothing to them, as, for instance, when he smacked his lips and champed his gums while muttering: \"Mayonnaise! Just think\u2014mayonnaise! And it's sixty years since the last was ever made! Two generations and never a smell of it! Why, in those days it was served in every restaurant with crab.\" When he could eat no more, the old man sighed, wiped his hands on his naked legs, and gazed out over the sea. With the content of a full stomach, he waxed reminiscent.\n\n\"To think of it! I've seen this beach alive with men, women, and children on a pleasant Sunday. And there weren't any bears to eat them up, either. And right up there on the cliff was a big restaurant where you could get anything you wanted to eat. Four million people lived in San Francisco then. And now, in the whole city and county there aren't forty all told. And out there on the sea were ships and ships always to be seen, going in for the Golden Gate or coming out. And airships in the air\u2014dirigibles and flying machines. They could travel two hundred miles an hour. The mail contracts with the New York and San Francisco Limited demanded that for the minimum. There was a chap, a Frenchman, I forget his name, who succeeded in making three hundred; but the thing was risky, too risky for conservative persons. But he was on the right clew, and he would have managed it if it hadn't been for the Great Plague. When I was a boy, there were men alive who remembered the coming of the first aeroplanes, and now I have lived to see the last of them, and that sixty years ago.\"\n\nThe old man babbled on, unheeded by the boys, who were long accustomed to his garrulousness, and whose vocabularies, besides, lacked the greater portion of the words he used. It was noticeable that in these rambling soliloquies his English seemed to recrudesce into better construction and phraseology. But when he talked directly with the boys it lapsed, largely, into their own uncouth and simpler forms.\n\n\"But there weren't many crabs in those days,\" the old man wandered on. \"They were fished out, and they were great delicacies. The open season was only a month long, too. And now crabs are accessible the whole year around. Think of it\u2014catching all the crabs you want, any time you want, in the surf of the Cliff House beach!\"\n\nA sudden commotion among the goats brought the boys to their feet. The dogs about the fire rushed to join their snarling fellow who guarded the goats, while the goats themselves stampeded in the direction of their human protectors. A half dozen forms, lean and gray, glided about on the sand hillocks and faced the bristling dogs. Edwin arched an arrow that fell short. But Hare-Lip, with a sling such as David carried into battle against Goliath, hurled a stone through the air that whistled from the speed of its flight. It fell squarely among the wolves and caused them to slink away toward the dark depths of the eucalyptus forest.\n\nThe boys laughed and lay down again in the sand, while Granser sighed ponderously. He had eaten too much, and, with hands clasped on his paunch, the fingers interlaced, he resumed his maunderings.\n\n\"The fleeting systems lapse like foam,\" he mumbled what was evidently a quotation. \"That's it\u2014foam, and fleeting. All man's toil upon the planet was just so much foam. He domesticated the serviceable animals, destroyed the hostile ones, and cleared the land of its wild vegetation. And then he passed, and the flood of primordial life rolled back again, sweeping his handiwork away\u2014the weeds and the forest inundated his fields, the beasts of prey swept over his flocks, and now there are wolves on the Cliff House beach.\" He was appalled by the thought. \"Where four million people disported themselves, the wild wolves roam today, and the savage progeny of our loins, with prehistoric weapons, defend themselves against the fanged despoilers. Think of it! And all because of the Scarlet Death\u2014\"\n\nThe adjective had caught Hare-Lip's ear.\n\n\"He's always saying that,\" he said to Edwin. \"What is *scarlet?*\"\n\n\"The scarlet of the maples can shake me like the cry of bugles going by,\" the old man quoted.\n\n\"It's red,\" Edwin answered the question. \"And you don't know it because you come from the Chauffeur Tribe. They never did know nothing, none of them. Scarlet is red\u2014I know that.\"\n\n\"Red is red, ain't it?\" Hare-Lip grumbled. \"Then what's the good of gettin' cocky and calling it scarlet?\"\n\n\"Granser, what for do you always say so much what nobody knows?\" he asked. \"Scarlet ain't anything, but red is red. Why don't you say red, then?\"\n\n\"Red is not the right word,\" was the reply. \"The plague was scarlet. The whole face and body turned scarlet in an hour's time. Don't I know? Didn't I see enough of it? And I am telling you it was scarlet because\u2014well, because it *was* scarlet. There is no other word for it.\"\n\n\"Red is good enough for me,\" Hare-Lip muttered obstinately. \"My dad calls red red, and he ought to know. He says everybody died of the Red Death.\"\n\n\"Your dad is a common fellow, descended from a common fellow,\" Granser retorted heatedly. \"Don't I know the beginnings of the Chauffeurs? Your grandsire was a chauffeur, a servant, and without education. He worked for other persons. But your grandmother was of good stock, only the children did not take after her. Don't I remember when I first met them, catching fish at Lake Temescal?\"\n\n\"What is *education?*\" Edwin asked.\n\n\"Calling red scarlet,\" Hare-Lip sneered, then returned to the attack on Granser. \"My dad told me, an' he got it from his dad afore he croaked, that your wife was a Santa Rosan, an' that she was sure no account. He said she was a *hash-slinger* before the Red Death, though I don't know what a *hash-slinger* is. You can tell me, Edwin.\"\n\nBut Edwin shook his head in token of ignorance.\n\n\"It is true, she was a waitress,\" Granser acknowledged. \"But she was a good woman, and your mother was her daughter. Women were very scarce in the days after the Plague. She was the only wife I could find, even if she was a *hash-slinger*, as your father calls it. But it is not nice to talk about our progenitors that way.\"\n\n\"Dad says that the wife of the first Chauffeur was a *lady*\u2014\"\n\n\"What's a *lady?*\" Hoo-Hoo demanded.\n\n\"A *lady*'s a Chauffeur squaw,\" was the quick reply of Hare-Lip.\n\n\"The first Chauffeur was Bill, a common fellow, as I said before,\" the old man expounded; \"but his wife was a lady, a great lady. Before the Scarlet Death she was the wife of Van Worden. He was President of the Board of Industrial Magnates, and was one of the dozen men who ruled America. He was worth one billion, eight hundred millions of dollars\u2014coins like you have there in your pouch, Edwin. And then came the Scarlet Death, and his wife became the wife of Bill, the first Chauffeur. He used to beat her, too. I have seen it myself.\"\n\nHoo-Hoo, lying on his stomach and idly digging his toes in the sand, cried out and investigated, first, his toe-nail, and next, the small hole he had dug. The other two boys joined him, excavating the sand rapidly with their hands till there lay three skeletons exposed. Two were of adults, the third being that of a part-grown child. The old man hudged along on the ground and peered at the find.\n\n\"Plague victims,\" he announced. \"That's the way they died everywhere in the last days. This must have been a family, running away from the contagion and perishing here on the Cliff House beach. They\u2014what are you doing, Edwin?\"\n\nThis question was asked in sudden dismay, as Edwin, using the back of his hunting knife, began to knock out the teeth from the jaws of one of the skulls.\n\n\"Going to string 'em,\" was the response.\n\nThe three boys were now hard at it; and quite a knocking and hammering arose, in which Granser babbled on unnoticed.\n\n\"You are true savages. Already has begun the custom of wearing human teeth. In another generation you will be perforating your noses and ears and wearing ornaments of bone and shell. I know. The human race is doomed to sink back farther and farther into the primitive night ere again it begins its bloody climb upward to civilization. When we increase and feel the lack of room, we will proceed to kill one another. And then I suppose you will wear human scalp-locks at your waist, as well\u2014as you, Edwin, who are the gentlest of my grandsons, have already begun with that vile pigtail. Throw it away, Edwin, boy; throw it away.\"\n\n\"What a gabble the old geezer makes,\" Hare-Lip remarked, when, the teeth all extracted, they began an attempt at equal division.\n\nThey were very quick and abrupt in their actions, and their speech, in moments of hot discussion over the allotment of the choicer teeth, was truly a gabble. They spoke in monosyllables and short jerky sentences that was more a gibberish than a language. And yet, through it ran hints of grammatical construction, and appeared vestiges of the conjugation of some superior culture. Even the speech of Granser was so corrupt that were it put down literally it would be almost so much nonsense to the reader. This, however, was when he talked with the boys.\n\nWhen he got into the full swing of babbling to himself, it slowly purged itself into pure English. The sentences grew longer and were enunciated with a rhythm and ease that was reminiscent of the lecture platform.\n\n\"Tell us about the Red Death, Granser,\" Hare-Lip demanded, when the teeth affair had been satisfactorily concluded.\n\n\"The Scarlet Death,\" Edwin corrected.\n\n\"An' don't work all that funny lingo on us,\" Hare-Lip went on. \"Talk sensible, Granser, like a Santa Rosan ought to talk. Other Santa Rosans don't talk like you.\"\n\n## II\n\nTHE old man showed pleasure in being thus called upon. He cleared his throat and began.\n\n\"Twenty or thirty years ago my story was in great demand. But in these days nobody seems interested\u2014\"\n\n\"There you go!\" Hare-Lip cried hotly. \"Cut out the funny stuff and talk sensible. What's *interested?* You talk like a baby that don't know how.\"\n\n\"Let him alone,\" Edwin urged, \"or he'll get mad and won't talk at all. Skip the funny places. We'll catch on to some of what he tells us.\"\n\n\"Let her go, Granser,\" Hoo-Hoo encouraged; for the old man was already maundering about the disrespect for elders and the reversion to cruelty of all humans that fell from high culture to primitive conditions.\n\nThe tale began.\n\n\"There were very many people in the world in those days. San Francisco alone held four millions\u2014\"\n\n\"What is millions?\" Edwin interrupted. Granser looked at him kindly.\n\n\"I know you cannot count beyond ten, so I will tell you. Hold up your two hands. On both of them you have altogether ten fingers and thumbs. Very well. I now take this grain of sand\u2014you hold it, Hoo-Hoo.\" He dropped the grain of sand into the lad's palm and went on. \"Now that grain of sand stands for the ten fingers of Edwin. I add another grain. That's ten more fingers. And I add another, and another, and another, until I have added as many grains as Edwin has fingers and thumbs. That makes what I call one hundred. Remember that word\u2014one hundred. Now I put this pebble in Hare-Lip's hand. It stands for ten grains of sand, or ten tens of fingers, or one hundred fingers. I put in ten pebbles. They stand for a thousand fingers. I take a mussel-shell, and it stands for ten pebbles, or one hundred grains of sand, or one thousand fingers\u2026.\" And so on, laboriously, and with much reiteration, he strove to build up in their minds a crude conception of numbers. As the quantities increased, he had the boys holding different magnitudes in each of their hands. For still higher sums, he laid the symbols on the log of driftwood; and for symbols he was hard put, being compelled to use the teeth from the skulls for millions, and the crab-shells for billions. It was here that he stopped, for the boys were showing signs of becoming tired.\n\n\"There were four million people in San Francisco\u2014four teeth.\"\n\nThe boys' eyes ranged along from the teeth and from hand to hand, down through the pebbles and sand-grains to Edwin's fingers. And back again they ranged along the ascending series in the effort to grasp such inconceivable numbers.\n\n\"That was a lot of folks, Granser,\" Edwin at last hazarded.\n\n\"Like sand on the beach here, like sand on the beach, each grain of sand a man, or woman, or child. Yes, my boy, all those people lived right here in San Francisco. And at one time or another all those people came out on this very beach\u2014more people than there are grains of sand. More\u2014more\u2014more. And San Francisco was a noble city. And across the bay\u2014where we camped last year, even more people lived, clear from Point Richmond, on the level ground and on the hills, all the way around to San Leandro\u2014one great city of seven million people.\u2014Seven teeth\u2026 there, that's it, seven millions.\"\n\nAgain the boys' eyes ranged up and down from Edwin's fingers to the teeth on the log.\n\n\"The world was full of people. The census of 2010 gave eight billions for the whole world\u2014eight crab-shells, yes, eight billions. It was not like today. Mankind knew a great deal more about getting food. And the more food there was, the more people there were. In the year 1800, there were one hundred and seventy millions in Europe alone. One hundred years later\u2014a grain of sand, Hoo-Hoo\u2014one hundred years later, at 1900, there were five hundred millions in Europe\u2014five grains of sand, Hoo-Hoo, and this one tooth. This shows how easy was the getting of food, and how men increased. And in the year 2000 there were fifteen hundred millions in Europe. And it was the same all over the rest of the world. Eight crab-shells there, yes, eight billion people were alive on the earth when the Scarlet Death began.\n\n\"I was a young man when the Plague came\u2014twenty-seven years old; and I lived on the other side of San Francisco Bay, in Berkeley. You remember those great stone houses, Edwin, when we came down the hills from Contra Costa? That was where I lived, in those stone houses. I was a professor of English literature.\"\n\nMuch of this was over the heads of the boys, but they strove to comprehend dimly this tale of the past.\n\n\"What was them stone houses for?\" Hare-Lip queried.\n\n\"You remember when your dad taught you to swim?\" The boy nodded. \"Well, in the University of California\u2014that is the name we had for the houses\u2014we taught young men and women how to think, just as I have taught you now, by sand and pebbles and shells, to know how many people lived in those days. There was very much to teach. The young men and women we taught were called students. We had large rooms in which we taught. I talked to them, forty or fifty at a time, just as I am talking to you now. I told them about the books other men had written before their time, and even, sometimes, in their time\u2014\"\n\n\"Was that all you did?\u2014just talk, talk, talk?\" Hoo-Hoo demanded. \"Who hunted your meat for you? and milked the goats? and caught the fish?\"\n\n\"A sensible question, Hoo-Hoo, a sensible question. As I have told you, in those days food-getting was easy. We were very wise. A few men got the food for many men. The other men did other things. As you say, I talked. I talked all the time, and for this food was given me\u2014much food, fine food, beautiful food, food that I have not tasted in sixty years and shall never taste again. I sometimes think the most wonderful achievement of our tremendous civilization was food\u2014its inconceivable abundance, its infinite variety, its marvellous delicacy. O my grandsons, life was life in those days, when we had such wonderful things to eat.\"\n\nThis was beyond the boys, and they let it slip by, words and thoughts, as a mere senile wandering in the narrative.\n\n\"Our food-getters were called *freemen*. This was a joke. We of the ruling classes owned all the land, all the machines, everything. These food-getters were our slaves. We took almost all the food they got, and left them a little so that they might eat, and work, and get us more food\u2014\"\n\n\"I'd have gone into the forest and got food for myself,\" Hare-Lip announced; \"and if any man tried to take it away from me, I'd have killed him.\"\n\nThe old man laughed.\n\n\"Did I not tell you that we of the ruling class owned all the land, all the forest, everything? Any food-getter who would not get food for us, him we punished or compelled to starve to death. And very few did that. They preferred to get food for us, and make clothes for us, and prepare and administer to us a thousand\u2014a mussel-shell, Hoo-Hoo\u2014a thousand satisfactions and delights. And I was Professor Smith in those days\u2014Professor James Howard Smith. And my lecture courses were very popular\u2014that is, very many of the young men and women liked to hear me talk about the books other men had written. \"And I was very happy, and I had beautiful things to eat. And my hands were soft, because I did no work with them, and my body was clean all over and dressed in the softest garments\u2014\"\n\nHe surveyed his mangy goat-skin with disgust.\n\n\"We did not wear such things in those days. Even the slaves had better garments. And we were most clean. We washed our faces and hands often every day. You boys never wash unless you fall into the water or go swimming.\"\n\n\"Neither do you Granser,\" Hoo-Hoo retorted.\n\n\"I know, I know, I am a filthy old man, but times have changed. Nobody washes these days, there are no conveniences. It is sixty years since I have seen a piece of soap.\n\n\"You do not know what soap is, and I shall not tell you, for I am telling the story of the Scarlet Death. You know what sickness is. We called it a disease. Very many of the diseases came from what we called germs. Remember that word\u2014germs. A germ is a very small thing. It is like a woodtick, such as you find on the dogs in the spring of the year when they run in the forest. Only the germ is very small. It is so small that you cannot see it\u2014\"\n\nHoo-Hoo began to laugh.\n\n\"You're a queer un, Granser, talking about things you can't see. If you can't see 'em, how do you know they are? That's what I want to know. How do you know anything you can't see?\"\n\n\"A good question, a very good question, Hoo-Hoo. But we did see\u2014some of them. We had what we called microscopes and ultramicroscopes, and we put them to our eyes and looked through them, so that we saw things larger than they really were, and many things we could not see without the microscopes at all. Our best ultramicroscopes could make a germ look forty thousand times larger. A mussel-shell is a thousand fingers like Edwin's. Take forty mussel-shells, and by as many times larger was the germ when we looked at it through a microscope. And after that, we had other ways, by using what we called moving pictures, of making the forty-thousand-times germ many, many thousand times larger still. And thus we saw all these things which our eyes of themselves could not see. Take a grain of sand. Break it into ten pieces. Take one piece and break it into ten. Break one of those pieces into ten, and one of those into ten, and one of those into ten, and one of those into ten, and do it all day, and maybe, by sunset, you will have a piece as small as one of the germs.\" The boys were openly incredulous. Hare-Lip sniffed and sneered and Hoo-Hoo snickered, until Edwin nudged them to be silent.\n\n\"The woodtick sucks the blood of the dog, but the germ, being so very small, goes right into the blood of the body, and there it has many children. In those days there would be as many as a billion\u2014a crab-shell, please\u2014as many as that crab-shell in one man's body. We called germs micro-organisms. When a few million, or a billion, of them were in a man, in all the blood of a man, he was sick. These germs were a disease. There were many different kinds of them\u2014more different kinds than there are grains of sand on this beach. We knew only a few of the kinds. The micro-organic world was an invisible world, a world we could not see, and we knew very little about it. Yet we did know something. There was the *bacillus anthracis*; there was the *micrococcus*; there was the *Bacterium termo*, and the *Bacterium lactis*\u2014that's what turns the goat milk sour even to this day, Hare-Lip; and there were *Schizomycetes* without end. And there were many others\u2026.\"\n\nHere the old man launched into a disquisition on germs and their natures, using words and phrases of such extraordinary length and meaninglessness, that the boys grinned at one another and looked out over the deserted ocean till they forgot the old man was babbling on.\n\n\"But the Scarlet Death, Granser,\" Edwin at last suggested.\n\nGranser recollected himself, and with a start tore himself away from the rostrum of the lecture-hall, where, to another world audience, he had been expounding the latest theory, sixty years gone, of germs and germ-diseases.\n\n\"Yes, yes, Edwin; I had forgotten. Sometimes the memory of the past is very strong upon me, and I forget that I am a dirty old man, clad in goat-skin, wandering with my savage grandsons who are goatherds in the primeval wilderness. 'The fleeting systems lapse like foam,' and so lapsed our glorious, colossal civilization. I am Granser, a tired old man. I belong to the tribe of Santa Rosans. I married into that tribe. My sons and daughters married into the Chauffeurs, the Sacramentos, and the Palo-Altos. You, Hare-Lip, are of the Chauffeurs. You, Edwin, are of the Sacramentos. And you, Hoo-Hoo, are of the Palo-Altos. Your tribe takes its name from a town that was near the seat of another great institution of learning. It was called Stanford University. Yes, I remember now. It is perfectly clear. I was telling you of the Scarlet Death. Where was I in my story?\"\n\n\"You was telling about germs, the things you can't see but which make men sick,\" Edwin prompted.\n\n\"Yes, that's where I was. A man did not notice at first when only a few of these germs got into his body. But each germ broke in half and became two germs, and they kept doing this very rapidly so that in a short time there were many millions of them in the body. Then the man was sick. He had a disease, and the disease was named after the kind of a germ that was in him. It might be measles, it might be influenza, it might be yellow fever; it might be any of thousands and thousands of kinds of diseases.\n\n\"Now this is the strange thing about these germs. There were always new ones coming to live in men's bodies. Long and long and long ago, when there were only a few men in the world, there were few diseases. But as men increased and lived closely together in great cities and civilizations, new diseases arose, new kinds of germs entered their bodies. Thus were countless millions and billions of human beings killed. And the more thickly men packed together, the more terrible were the new diseases that came to be. Long before my time, in the middle ages, there was the Black Plague that swept across Europe. It swept across Europe many times. There was tuberculosis, that entered into men wherever they were thickly packed. A hundred years before my time there was the bubonic plague. And in Africa was the sleeping sickness. The bacteriologists fought all these sicknesses and destroyed them, just as you boys fight the wolves away from your goats, or squash the mosquitoes that light on you. The bacteriologists\u2014\"\n\n\"But, Granser, what is a what-you-call-it?\" Edwin interrupted.\n\n\"You, Edwin, are a goatherd. Your task is to watch the goats. You know a great deal about goats. A bacteriologist watches germs. That's his task, and he knows a great deal about them. So, as I was saying, the bacteriologists fought with the germs and destroyed them\u2014sometimes. There was leprosy, a horrible disease. A hundred years before I was born, the bacteriologists discovered the germ of leprosy. They knew all about it. They made pictures of it. I have seen those pictures. But they never found a way to kill it. But in 1984, there was the Pantoblast Plague, a disease that broke out in a country called Brazil and that killed millions of people. But the bacteriologists found it out, and found the way to kill it, so that the Pantoblast Plague went no farther. They made what they called a serum, which they put into a man's body and which killed the pantoblast germs without killing the man. And in 1910, there was Pellagra, and also the hookworm. These were easily killed by the bacteriologists. But in 1947 there arose a new disease that had never been seen before. It got into the bodies of babies of only ten months old or less, and it made them unable to move their hands and feet, or to eat, or anything; and the bacteriologists were eleven years in discovering how to kill that particular germ and save the babies.\n\n\"In spite of all these diseases, and of all the new ones that continued to arise, there were more and more men in the world. This was because it was easy to get food. The easier it was to get food, the more men there were; the more men there were, the more thickly were they packed together on the earth; and the more thickly they were packed, the more new kinds of germs became diseases. There were warnings. Soldervetzsky, as early as 1929, told the bacteriologists that they had no guaranty against some new disease, a thousand times more deadly than any they knew, arising and killing by the hundreds of millions and even by the billion. You see, the micro-organic world remained a mystery to the end. They knew there was such a world, and that from time to time armies of new germs emerged from it to kill men. \"And that was all they knew about it. For all they knew, in that invisible micro-organic world there might be as many different kinds of germs as there are grains of sand on this beach. And also, in that same invisible world it might well be that new kinds of germs came to be. It might be there that life originated\u2014the 'abysmal fecundity,' Soldervetzsky called it, applying the words of other men who had written before him\u2026.\"\n\nIt was at this point that Hare-Lip rose to his feet, an expression of huge contempt on his face.\n\n\"Granser,\" he announced, \"you make me sick with your gabble. Why don't you tell about the Red Death? If you ain't going to, say so, an' we'll start back for camp.\"\n\nThe old man looked at him and silently began to cry. The weak tears of age rolled down his cheeks and all the feebleness of his eighty-seven years showed in his grief-stricken countenance.\n\n\"Sit down,\" Edwin counselled soothingly. \"Granser's all right. He's just gettin' to the Scarlet Death, ain't you, Granser? He's just goin' to tell us about it right now. Sit down, Hare-Lip. Go ahead, Granser.\"\n\n## III\n\nTHE old man wiped the tears away on his grimy knuckles and took up the tale in a tremulous, piping voice that soon strengthened as he got the swing of the narrative.\n\n\"It was in the summer of 2013 that the Plague came. I was twenty-seven years old, and well do I remember it. Wireless despatches\u2014\"\n\nHare-Lip spat loudly his disgust, and Granser hastened to make amends. \"We talked through the air in those days, thousands and thousands of miles. And the word came of a strange disease that had broken out in New York. There were seventeen millions of people living then in that noblest city of America. Nobody thought anything about the news. It was only a small thing. There had been only a few deaths. It seemed, though, that they had died very quickly, and that one of the first signs of the disease was the turning red of the face and all the body. Within twenty-four hours came the report of the first case in Chicago. And on the same day, it was made public that London, the greatest city in the world, next to Chicago, had been secretly fighting the plague for two weeks and censoring the news despatches\u2014that is, not permitting the word to go forth to the rest of the world that London had the plague.\n\n\"It looked serious, but we in California, like everywhere else, were not alarmed. We were sure that the bacteriologists would find a way to overcome this new germ, just as they had overcome other germs in the past. But the trouble was the astonishing quickness with which this germ destroyed human beings, and the fact that it inevitably killed any human body it entered. No one ever recovered. There was the old Asiatic cholera, when you might eat dinner with a well man in the evening, and the next morning, if you got up early enough, you would see him being hauled by your window in the death-cart. But this new plague was quicker than that\u2014much quicker.\n\n\"From the moment of the first signs of it, a man would be dead in an hour. Some lasted for several hours. Many died within ten or fifteen minutes of the appearance of the first signs.\n\n\"The heart began to beat faster and the heat of the body to increase. Then came the scarlet rash, spreading like wildfire over the face and body. Most persons never noticed the increase in heat and heart-beat, and the first they knew was when the scarlet rash came out. Usually, they had convulsions at the time of the appearance of the rash. But these convulsions did not last long and were not very severe. If one lived through them, he became perfectly quiet, and only did he feel a numbness swiftly creeping up his body from the feet. The heels became numb first, then the legs, and hips, and when the numbness reached as high as his heart he died. They did not rave or sleep. Their minds always remained cool and calm up to the moment their heart numbed and stopped. And another strange thing was the rapidity of decomposition. No sooner was a person dead than the body seemed to fall to pieces, to fly apart, to melt away even as you looked at it. That was one of the reasons the plague spread so rapidly. All the billions of germs in a corpse were so immediately released.\n\n\"And it was because of all this that the bacteriologists had so little chance in fighting the germs. They were killed in their laboratories even as they studied the germ of the Scarlet Death. They were heroes. As fast as they perished, others stepped forth and took their places. It was in London that they first isolated it. The news was telegraphed everywhere. Trask was the name of the man who succeeded in this, but within thirty hours he was dead. Then came the struggle in all the laboratories to find something that would kill the plague germs. All drugs failed. You see, the problem was to get a drug, or serum, that would kill the germs in the body and not kill the body. They tried to fight it with other germs, to put into the body of a sick man germs that were the enemies of the plague germs\u2014\"\n\n\"And you can't see these germ-things, Granser,\" Hare-Lip objected, \"and here you gabble, gabble, gabble about them as if they was anything, when they're nothing at all. Anything you can't see, ain't, that's what. Fighting things that ain't with things that ain't! They must have been all fools in them days. That's why they croaked. I ain't goin' to believe in such rot, I tell you that.\"\n\nGranser promptly began to weep, while Edwin hotly took up his defence. \"Look here, Hare-Lip, you believe in lots of things you can't see.\"\n\nHare-Lip shook his head.\n\n\"You believe in dead men walking about. You never seen one dead man walk about.\"\n\n\"I tell you I seen 'em, last winter, when I was wolf-hunting with dad.\"\n\n\"Well, you always spit when you cross running water,\" Edwin challenged. \"That's to keep off bad luck,\" was Hare-Lip's defence.\n\n\"You believe in bad luck?\"\n\n\"Sure.\"\n\n\"An' you ain't never seen bad luck,\" Edwin concluded triumphantly. \"You're just as bad as Granser and his germs. You believe in what you don't see. Go on, Granser.\"\n\nHare-Lip, crushed by this metaphysical defeat, remained silent, and the old man went on. Often and often, though this narrative must not be clogged by the details, was Granser's tale interrupted while the boys squabbled among themselves. Also, among themselves they kept up a constant, low-voiced exchange of explanation and conjecture, as they strove to follow the old man into his unknown and vanished world.\n\n\"The Scarlet Death broke out in San Francisco. The first death came on a Monday morning. By Thursday they were dying like flies in Oakland and San Francisco. They died everywhere\u2014in their beds, at their work, walking along the street. It was on Tuesday that I saw my first death\u2014Miss Collbran, one of my students, sitting right there before my eyes, in my lecture-room. I noticed her face while I was talking. It had suddenly turned scarlet. I ceased speaking and could only look at her, for the first fear of the plague was already on all of us and we knew that it had come. The young women screamed and ran out of the room. So did the young men run out, all but two. Miss Collbran's convulsions were very mild and lasted less than a minute. One of the young men fetched her a glass of water. She drank only a little of it, and cried out:\n\n\"'My feet! All sensation has left them.'\n\n\"After a minute she said, 'I have no feet. I am unaware that I have any feet. And my knees are cold. I can scarcely feel that I have knees.'\n\n\"She lay on the floor, a bundle of notebooks under her head. And we could do nothing. The coldness and the numbness crept up past her hips to her heart, and when it reached her heart she was dead. In fifteen minutes, by the clock\u2014I timed it\u2014she was dead, there, in my own classroom, dead. And she was a very beautiful, strong, healthy young woman. And from the first sign of the plague to her death only fifteen minutes elapsed. That will show you how swift was the Scarlet Death.\n\n\"Yet in those few minutes I remained with the dying woman in my classroom, the alarm had spread over the university; and the students, by thousands, all of them, had deserted the lecture-room and laboratories. When I emerged, on my way to make report to the President of the Faculty, I found the university deserted. Across the campus were several stragglers hurrying for their homes. Two of them were running.\n\n\"President Hoag, I found in his office, all alone, looking very old and very gray, with a multitude of wrinkles in his face that I had never seen before. At the sight of me, he pulled himself to his feet and tottered away to the inner office, banging the door after him and locking it. You see, he knew I had been exposed, and he was afraid. He shouted to me through the door to go away. I shall never forget my feelings as I walked down the silent corridors and out across that deserted campus. I was not afraid. I had been exposed, and I looked upon myself as already dead. It was not that, but a feeling of awful depression that impressed me. Everything had stopped. It was like the end of the world to me\u2014my world. I had been born within sight and sound of the university. It had been my predestined career. My father had been a professor there before me, and his father before him. For a century and a half had this university, like a splendid machine, been running steadily on. And now, in an instant, it had stopped. It was like seeing the sacred flame die down on some thrice-sacred altar. I was shocked, unutterably shocked.\n\n\"When I arrived home, my housekeeper screamed as I entered, and fled away. And when I rang, I found the housemaid had likewise fled. I investigated. In the kitchen I found the cook on the point of departure. But she screamed, too, and in her haste dropped a suitcase of her personal belongings and ran out of the house and across the grounds, still screaming. I can hear her scream to this day. You see, we did not act in this way when ordinary diseases smote us. We were always calm over such things, and sent for the doctors and nurses who knew just what to do. But this was different. It struck so suddenly, and killed so swiftly, and never missed a stroke. When the scarlet rash appeared on a person's face, that person was marked by death. There was never a known case of a recovery.\n\n\"I was alone in my big house. As I have told you often before, in those days we could talk with one another over wires or through the air. The telephone bell rang, and I found my brother talking to me. He told me that he was not coming home for fear of catching the plague from me, and that he had taken our two sisters to stop at Professor Bacon's home. He advised me to remain where I was, and wait to find out whether or not I had caught the plague.\n\n\"To all of this I agreed, staying in my house and for the first time in my life attempting to cook. And the plague did not come out on me. By means of the telephone I could talk with whomsoever I pleased and get the news. Also, there were the newspapers, and I ordered all of them to be thrown up to my door so that I could know what was happening with the rest of the world.\n\n\"New York City and Chicago were in chaos. And what happened with them was happening in all the large cities. A third of the New York police were dead. Their chief was also dead, likewise the mayor. All law and order had ceased. The bodies were lying in the streets un-buried. All railroads and vessels carrying food and such things into the great city had ceased running and mobs of the hungry poor were pillaging the stores and warehouses. Murder and robbery and drunkenness were everywhere. Already the people had fled from the city by millions\u2014at first the rich, in their private motor-cars and dirigibles, and then the great mass of the population, on foot, carrying the plague with them, themselves starving and pillaging the farmers and all the towns and villages on the way.\n\n\"The man who sent this news, the wireless operator, was alone with his instrument on the top of a lofty building. The people remaining in the city\u2014he estimated them at several hundred thousand\u2014had gone mad from fear and drink, and on all sides of him great fires were raging. He was a hero, that man who staid by his post\u2014an obscure newspaperman, most likely.\n\n\"For twenty-four hours, he said, no transatlantic airships had arrived, and no more messages were coming from England. He did state, though, that a message from Berlin\u2014that's in Germany\u2014announced that Hoffmeyer, a bacteriologist of the Metchnikoff School, had discovered the serum for the plague. That was the last word, to this day, that we of America ever received from Europe. If Hoffmeyer discovered the serum, it was too late, or otherwise, long ere this, explorers from Europe would have come looking for us. We can only conclude that what happened in America happened in Europe, and that, at the best, some several score may have survived the Scarlet Death on that whole continent.\n\n\"For one day longer the despatches continued to come from New York. Then they, too, ceased. The man who had sent them, perched in his lofty building, had either died of the plague or been consumed in the great conflagrations he had described as raging around him. And what had occurred in New York had been duplicated in all the other cities. It was the same in San Francisco, and Oakland, and Berkeley. By Thursday the people were dying so rapidly that their corpses could not be handled, and dead bodies lay everywhere. Thursday night the panic outrush for the country began. Imagine, my grandsons, people, thicker than the salmon-run you have seen on the Sacramento river, pouring out of the cities by millions, madly over the country, in vain attempt to escape the ubiquitous death. You see, they carried the germs with them. Even the airships of the rich, fleeing for mountain and desert fastnesses, carried the germs.\n\n\"Hundreds of these airships escaped to Hawaii, and not only did they bring the plague with them, but they found the plague already there before them. This we learned, by the despatches, until all order in San Francisco vanished, and there were no operators left at their posts to receive or send. It was amazing, astounding, this loss of communication with the world. It was exactly as if the world had ceased, been blotted out. For sixty years that world has no longer existed for me. I know there must be such places as New York, Europe, Asia, and Africa; but not one word has been heard of them\u2014not in sixty years. With the coming of the Scarlet Death the world fell apart, absolutely, irretrievably. Ten thousand years of culture and civilization passed in the twinkling of an eye, 'lapsed like foam.'\n\n\"I was telling about the airships of the rich. They carried the plague with them and no matter where they fled, they died. I never encountered but one survivor of any of them\u2014Mungerson. He was afterwards a Santa Rosan, and he married my eldest daughter. He came into the tribe eight years after the plague. He was then nineteen years old, and he was compelled to wait twelve years more before he could marry. You see, there were no unmarried women, and some of the older daughters of the Santa Rosans were already bespoken. So he was forced to wait until my Mary had grown to sixteen years. It was his son, Gimp-Leg, who was killed last year by the mountain lion.\n\n\"Mungerson was eleven years old at the time of the plague. His father was one of the Industrial Magnates, a very wealthy, powerful man. It was on his airship, the Condor, that they were fleeing, with all the family, for the wilds of British Columbia, which is far to the north of here. But there was some accident, and they were wrecked near Mount Shasta. You have heard of that mountain. It is far to the north. The plague broke out amongst them, and this boy of eleven was the only survivor. For eight years he was alone, wandering over a deserted land and looking vainly for his own kind. And at last, travelling south, he picked up with us, the Santa Rosans.\n\n\"But I am ahead of my story. When the great exodus from the cities around San Francisco Bay began, and while the telephones were still working, I talked with my brother. I told him this flight from the cities was insanity, that there were no symptoms of the plague in me, and that the thing for us to do was to isolate ourselves and our relatives in some safe place. We decided on the Chemistry Building, at the university, and we planned to lay in a supply of provisions, and by force of arms to prevent any other persons from forcing their presence upon us after we had retired to our refuge.\n\n\"All this being arranged, my brother begged me to stay in my own house for at least twenty-four hours more, on the chance of the plague developing in me. To this I agreed, and he promised to come for me next day. We talked on over the details of the provisioning and the defending of the Chemistry Building until the telephone died. It died in the midst of our conversation. That evening there were no electric lights, and I was alone in my house in the darkness. No more newspapers were being printed, so I had no knowledge of what was taking place outside.\n\n\"I heard sounds of rioting and of pistol shots, and from my windows I could see the glare of the sky of some conflagration in the direction of Oakland. It was a night of terror. I did not sleep a wink. A man\u2014why and how I do not know\u2014was killed on the sidewalk in front of the house. I heard the rapid reports of an automatic pistol, and a few minutes later the wounded wretch crawled up to my door, moaning and crying out for help. Arming myself with two automatics, I went to him. By the light of a match I ascertained that while he was dying of the bullet wounds, at the same time the plague was on him. I fled indoors, whence I heard him moan and cry out for half an hour longer.\n\n\"In the morning, my brother came to me. I had gathered into a handbag what things of value I purposed taking, but when I saw his face I knew that he would never accompany me to the Chemistry Building. The plague was on him. He intended shaking my hand, but I went back hurriedly before him.\n\n\"'Look at yourself in the mirror,' I commanded.\n\n\"He did so, and at sight of his scarlet face, the color deepening as he looked at it, he sank down nervelessly in a chair.\n\n\"'My God!' he said. 'I've got it. Don't come near me. I am a dead man.' Then the convulsions seized him. He was two hours in dying, and he was conscious to the last, complaining about the coldness and loss of sensation in his feet, his calves, his thighs, until at last it was his heart and he was dead.\n\n\"That was the way the Scarlet Death slew. I caught up my handbag and fled. The sights in the streets were terrible. One stumbled on bodies everywhere. Some were not yet dead. And even as you looked, you saw men sink down with the death fastened upon them. There were numerous fires burning in Berkeley, while Oakland and San Francisco were apparently being swept by vast conflagrations. The smoke of the burning filled the heavens, so that the midday was as a gloomy twilight, and, in the shifts of wind, sometimes the sun shone through dimly, a dull red orb. Truly, my grandsons, it was like the last days of the end of the world.\n\n\"There were numerous stalled motor cars, showing that the gasoline and the engine supplies of the garages had given out. I remember one such car. A man and a woman lay back dead in the seats, and on the pavement near it were two more women and a child. Strange and terrible sights there were on every hand. People slipped by silently, furtively, like ghosts\u2014white-faced women carrying infants in their arms; fathers leading children by the hand; singly, and in couples, and in families\u2014all fleeing out of the city of death. Some carried supplies of food, others blankets and valuables, and there were many who carried nothing.\n\n\"There was a grocery store\u2014a place where food was sold. The man to whom it belonged\u2014I knew him well\u2014a quiet, sober, but stupid and obstinate fellow, was defending it. The windows and doors had been broken in, but he, inside, hiding behind a counter, was discharging his pistol at a number of men on the sidewalk who were breaking in. In the entrance were several bodies\u2014of men, I decided, whom he had killed earlier in the day. Even as I looked on from a distance, I saw one of the robbers break the windows of the adjoining store, a place where shoes were sold, and deliberately set fire to it. I did not go to the groceryman's assistance. The time for such acts had already passed. Civilization was crumbling, and it was each for himself.\"\n\n## IV\n\n\"I WENT away hastily, down a cross-street, and at the first corner I saw another tragedy. Two men of the working class had caught a man and a woman with two children, and were robbing them. I knew the man by sight, though I had never been introduced to him. He was a poet whose verses I had long admired. Yet I did not go to his help, for at the moment I came upon the scene there was a pistol shot, and I saw him sinking to the ground. The woman screamed, and she was felled with a fist-blow by one of the brutes. I cried out threateningly, whereupon they discharged their pistols at me and I ran away around the corner. Here I was blocked by an advancing conflagration. The buildings on both sides were burning, and the street was filled with smoke and flame. From somewhere in that murk came a woman's voice calling shrilly for help. But I did not go to her. A man's heart turned to iron amid such scenes, and one heard all too many appeals for help.\n\n\"Returning to the corner, I found the two robbers were gone. The poet and his wife lay dead on the pavement. It was a shocking sight. The two children had vanished\u2014whither I could not tell. And I knew, now, why it was that the fleeing persons I encountered slipped along so furtively and with such white faces. In the midst of our civilization, down in our slums and labor-ghettos, we had bred a race of barbarians, of savages; and now, in the time of our calamity, they turned upon us like the wild beasts they were and destroyed us. And they destroyed themselves as well.\n\n\"They inflamed themselves with strong drink and committed a thousand atrocities, quarreling and killing one another in the general madness. One group of workingmen I saw, of the better sort, who had banded together, and, with their women and children in their midst, the sick and aged in litters and being carried, and with a number of horses pulling a truck-load of provisions, they were fighting their way out of the city. They made a fine spectacle as they came down the street through the drifting smoke, though they nearly shot me when I first appeared in their path. As they went by, one of their leaders shouted out to me in apologetic explanation. He said they were killing the robbers and looters on sight, and that they had thus banded together as the only-means by which to escape the prowlers.\n\n\"It was here that I saw for the first time what I was soon to see so often. One of the marching men had suddenly shown the unmistakable mark of the plague. Immediately those about him drew away, and he, without a remonstrance, stepped out of his place to let them pass on. A woman, most probably his wife, attempted to follow him. She was leading a little boy by the hand. But the husband commanded her sternly to go on, while others laid hands on her and restrained her from following him. This I saw, and I saw the man also, with his scarlet blaze of face, step into a doorway on the opposite side of the street. I heard the report of his pistol, and saw him sink lifeless to the ground.\n\n\"After being turned aside twice again by advancing fires, I succeeded in getting through to the university. On the edge of the campus I came upon a party of university folk who were going in the direction of the Chemistry Building. They were all family men, and their families were with them, including the nurses and the servants. Professor Badminton greeted me, I had difficulty in recognizing him. Somewhere he had gone through flames, and his beard was singed off. About his head was a bloody bandage, and his clothes were filthy.\n\n\"He told me he had prowlers, and that his brother had been killed the previous night, in the defence of their dwelling.\n\n\"Midway across the campus, he pointed suddenly to Mrs. Swinton's face. The unmistakable scarlet was there. Immediately all the other women set up a screaming and began to run away from her. Her two children were with a nurse, and these also ran with the women. But her husband, Doctor Swinton, remained with her.\n\n\"'Go on, Smith,' he told me. 'Keep an eye on the children. As for me, I shall stay with my wife. I know she is as already dead, but I can't leave her. Afterwards, if I escape, I shall come to the Chemistry Building, and do you watch for me and let me in.'\n\n\"I left him bending over his wife and soothing her last moments, while I ran to overtake the party. We were the last to be admitted to the Chemistry Building. After that, with our automatic rifles we maintained our isolation. By our plans, we had arranged for a company of sixty to be in this refuge. Instead, every one of the number originally planned had added relatives and friends and whole families until there were over four hundred souls. But the Chemistry Building was large, and, standing by itself, was in no danger of being burned by the great fires that raged everywhere in the city.\n\n\"A large quantity of provisions had been gathered, and a food committee took charge of it, issuing rations daily to the various families and groups that arranged themselves into messes. A number of committees were appointed, and we developed a very efficient organization. I was on the committee of defence, though for the first day no prowlers came near. We could see them in the distance, however, and by the smoke of their fires knew that several camps of them were occupying the far edge of the campus. Drunkenness was rife, and often we heard them singing ribald songs or insanely shouting. While the world crashed to ruin about them and all the air was filled with the smoke of its burning, these low creatures gave rein to their bestiality and fought and drank and died. And after all, what did it matter? Everybody died anyway, the good and the bad, the efficients and the weaklings, those that loved to live and those that scorned to live. They passed. Everything passed. \"When twenty-four hours had gone by and no signs of the plague were apparent, we congratulated ourselves and set about digging a well. You have seen the great iron pipes which in those days carried water to all the city-dwellers. We feared that the fires in the city would burst the pipes and empty the reservoirs. So we tore up the cement floor of the central court of the Chemistry Building and dug a well. There were many young men, undergraduates, with us, and we worked night and day on the well. And our fears were confirmed. Three hours before we reached water, the pipes went dry.\n\n\"A second twenty-four hours passed, and still the plague did not appear among us. We thought we were saved. But we did not know what I afterwards decided to be true, namely, that the period of the incubation of the plague germs in a human's body was a matter of a number of days. It slew so swiftly when once it manifested itself, that we were led to believe that the period of incubation was equally swift. So, when two days had left us unscathed, we were elated with the idea that we were free of the contagion.\n\n\"But the third day disillusioned us. I can never forget the night preceding it. I had charge of the night guards from eight to twelve, and from the roof of the building I watched the passing of all man's glorious works. So terrible were the local conflagrations that all the sky was lighted up. One could read the finest print in the red glare. All the world seemed wrapped in flames. San Francisco spouted smoke and fire from a score of vast conflagrations that were like so many active volcanoes. Oakland, San Leandro, Haywards\u2014all were burning; and to the northward, clear to Point Richmond, other fires were at work. It was an awe-inspiring spectacle. Civilization, my grandsons, civilization was passing in a sheet of flame and a breath of death. At ten o'clock that night, the great powder magazines at Point Pinole exploded in rapid succession. So terrific were the concussions that the strong building rocked as in an earthquake, while every pane of glass was broken. It was then that I left the roof and went down the long corridors, from room to room, quieting the alarmed women and telling them what had happened.\n\n\"An hour later, at a window on the ground floor, I heard pandemonium break out in the camps of the prowlers. There were cries and screams, and shots from many pistols. As we afterward conjectured, this fight had been precipitated by an attempt on the part of those that were well to drive out those that were sick. At any rate, a number of the plague-stricken prowlers escaped across the campus and drifted against our doors. We warned them back, but they cursed us and discharged a fusillade from their pistols. Professor Merryweather, at one of the windows, was instantly killed, the bullet striking him squarely between the eyes. We opened fire in turn, and all the prowlers fled away with the exception of three. One was a woman. The plague was on them and they were reckless. Like foul fiends, there in the red glare from the skies, with faces blazing, they continued to curse us and fire at us. One of the men I shot with my own hand. After that the other man and the woman, still cursing us, lay down under our windows, where we were compelled to watch them die of the plague.\n\n\"The situation was critical. The explosions of the powder magazines had broken all the windows of the Chemistry Building, so that we were exposed to the germs from the corpses. The sanitary committee was called upon to act, and it responded nobly. Two men were required to go out and remove the corpses, and this meant the probable sacrifice of their own lives, for, having performed the task, they were not to be permitted to reenter the building. One of the professors, who was a bachelor, and one of the undergraduates volunteered. They bade good-bye to us and went forth. They were heroes. They gave up their lives that four hundred others might live. After they had performed their work, they stood for a moment, at a distance, looking at us wistfully. Then they waved their hands in farewell and went away slowly across the campus toward the burning city.\n\n\"And yet it was all useless. The next morning the first one of us was smitten with the plague\u2014a little nurse-girl in the family of Professor Stout. It was no time for weak-kneed, sentimental policies. On the chance that she might be the only one, we thrust her forth from the building and commanded her to be gone.\n\n\"She went away slowly across the campus, wringing her hands and crying pitifully. We felt like brutes, but what were we to do? There were four hundred of us, and individuals had to be sacrificed.\n\n\"In one of the laboratories three families had domiciled themselves, and that afternoon we found among them no less than four corpses and seven cases of the plague in all its different stages.\n\n\"Then it was that the horror began. Leaving the dead lie, we forced the living ones to segregate themselves in another room. The plague began to break out among the rest of us, and as fast as the symptoms appeared, we sent the stricken ones to these segregated rooms. We compelled them to walk there by themselves, so as to avoid laying hands on them. It was heartrending. But still the plague raged among us, and room after room was filled with the dead and dying. And so we who were yet clean retreated to the next floor and to the next, before this sea of the dead, that, room by room and floor by floor, inundated the building.\n\n\"The place became a charnel house, and in the middle of the night the survivors fled forth, taking nothing with them except arms and ammunition and a heavy store of tinned foods. We camped on the opposite side of the campus from the prowlers, and, while some stood guard, others of us volunteered to scout into the city in quest of horses, motor cars, carts, and wagons, or anything that would carry our provisions and enable us to emulate the banded workingmen I had seen fighting their way out to the open country.\n\n\"I was one of these scouts; and Doctor Hoyle, remembering that his motor car had been left behind in his home garage, told me to look for it. We scouted in pairs, and Dombey, a young undergraduate, accompanied me. We had to cross half a mile of the residence portion of the city to get to Doctor Hoyle's home. Here the buildings stood apart, in the midst of trees and grassy lawns, and here the fires had played freaks, burning whole blocks, skipping blocks and often skipping a single house in a block. And here, too, the prowlers were still at their work. We carried our automatic pistols openly in our hands, and looked desperate enough, forsooth, to keep them from attacking us. But at Doctor Hoyle's house the thing happened. Untouched by fire, even as we came to it the smoke of flames burst forth.\n\n\"The miscreant who had set fire to it staggered down the steps and out along the driveway. Sticking out of his coat pockets were bottles of whiskey, and he was very drunk. My first impulse was to shoot him, and I have never ceased regretting that I did not. Staggering and maundering to himself, with bloodshot eyes, and a raw and bleeding slash down one side of his bewhiskered face, he was altogether the most nauseating specimen of degradation and filth I had ever encountered. I did not shoot him, and he leaned against a tree on the lawn to let us go by. It was the most absolute, wanton act. Just as we were opposite him, he suddenly drew a pistol and shot Dombey through the head. The next instant I shot him. But it was too late. Dombey expired without a groan, immediately. I doubt if he even knew what had happened to him.\n\n\"Leaving the two corpses, I hurried on past the burning house to the garage, and there found Doctor Hoyle's motor car. The tanks were filled with gasoline, and it was ready for use. And it was in this car that I threaded the streets of the ruined city and came back to the survivors on the campus. The other scouts returned, but none had been so fortunate. Professor Fairmead had found a Shetland pony, but the poor creature, tied in a stable and abandoned for days, was so weak from want of food and water that it could carry no burden at all. Some of the men were for turning it loose, but I insisted that we should lead it along with us, so that, if we got out of food, we would have it to eat.\n\n\"There were forty-seven of us when we started, many being women and children. The President of the Faculty, an old man to begin with, and now hopelessly broken by the awful happenings of the past week, rode in the motor car with several young children and the aged mother of Professor Fairmead. Wathope, a young professor of English, who had a grievous bullet-wound in his leg, drove the car. The rest of us walked, Professor Fairmead leading the pony.\n\n\"It was what should have been a bright summer day, but the smoke from the burning world filled the sky, through which the sun shone murkily, a dull and lifeless orb, blood-red and ominous. But we had grown accustomed to that blood-red sun. With the smoke it was different. It bit into our nostrils and eyes, and there was not one of us whose eyes were not bloodshot. We directed our course to the southeast through the endless miles of suburban residences, travelling along where the first swells of low hills rose from the flat of the central city. It was by this way, only, that we could expect to gain the country.\n\n\"Our progress was painfully slow. The women and children could not walk fast. They did not dream of walking, my grandsons, in the way all people walk today. In truth, none of us knew how to walk. It was not until after the plague that I learned really to walk. So it was that the pace of the slowest was the pace of all, for we dared not separate on account of the prowlers. There were not so many now of these human beasts of prey. The plague had already well diminished their numbers, but enough still lived to be a constant menace to us. Many of the beautiful residences were untouched by fire, yet smoking ruins were everywhere. The prowlers, too, seemed to have got over their insensate desire to burn, and it was more rarely that we saw houses freshly on fire.\n\n\"Several of us scouted among the private garages in search of motor cars and gasoline. But in this we were unsuccessful. The first great flights from the cities had swept all such utilities away. Calgan, a fine young man, was lost in this work. He was shot by prowlers while crossing a lawn. Yet this was our only casualty, though, once, a drunken brute deliberately opened fire on all of us. Luckily, he fired wildly, and we shot him before he had done any hurt.\n\n\"At Fruitvale, still in the heart of the magnificent residence section of the city, the plague again smote us. Professor Fairmead was the victim. Making signs to us that his mother was not to know, he turned aside into the grounds of a beautiful mansion. He sat down forlornly on the steps of the front veranda, and I, having lingered, waved him a last farewell. That night, several miles beyond Fruitvale and still in the city, we made camp. And that night we shifted camp twice to get away from our dead. In the morning there were thirty of us. I shall never forget the President of the Faculty. During the morning's march his wife, who was walking, betrayed the fatal symptoms, and when she drew aside to let us go on, he insisted on leaving the motor car and remaining with her. There was quite a discussion about this, but in the end we gave in. It was just as well, for we knew not which ones of us, if any, might ultimately escape.\n\n\"That night, the second of our march, we camped beyond Haywards in the first stretches of country. And in the morning there were eleven of us that lived. Also, during the night, Wathope, the professor with the wounded leg, deserted us in the motor car. He took with him his sister and his mother and most of our tinned provisions. It was that day, in the afternoon, while resting by the wayside, that I saw the last airship I shall ever see. The smoke was much thinner here in the country, and I first sighted the ship drifting and veering helplessly at an elevation of two thousand feet. What had happened I could not conjecture, but even as we looked we saw her bow dip down lower and lower. Then the bulkheads of the various gas-chambers must have burst, for, quite perpendicular, she fell like a plummet to the earth.\n\n\"And from that day to this I have not seen another airship. Often and often, during the next few years, I scanned the sky for them, hoping against hope that somewhere in the world civilization had survived. But it was not to be. What happened with us in California must have happened with everybody everywhere.\n\n\"Another day, and at Niles there were three of us. Beyond Niles, in the middle of the highway, we found Wathope. The motor car had broken down, and there, on the rugs which they had spread on the ground, lay the bodies of his sister, his mother, and himself.\n\n\"Wearied by the unusual exercise of continual walking, that night I slept heavily. In the morning I was alone in the world. Canfield and Parsons, my last companions, were dead of the plague. Of the four hundred that sought shelter in the Chemistry Building, and of the forty-seven that began the march, I alone remained\u2014I and the Shetland pony. Why this should be so there is no explaining. I did not catch the plague, that is all. I was immune. I was merely the one lucky man in a million\u2014just as every survivor was one in a million, or, rather, in several millions, for the proportion was at least that.\"\n\n## V\n\n\"FOR two days I sheltered in a pleasant grove where there had been no deaths. In those two days, while badly depressed and believing that my turn would come at any moment, nevertheless I rested and recuperated. So did the pony. And on the third day, putting what small store of tinned provisions I possessed on the pony's back, I started on across a very lonely land. Not a live man, woman, or child, did I encounter, though the dead were everywhere. Food, however, was abundant. The land then was not as it is now. It was all cleared of trees and brush, and it was cultivated. The food for millions of mouths was growing, ripening, and going to waste. From the fields and orchards I gathered vegetables, fruits, and berries. Around the deserted farmhouses I got eggs and caught chickens. And frequently I found supplies of tinned provisions in the store-rooms.\n\n\"A strange thing was what was taking place with all the domestic animals. Everywhere they were going wild and preying on one another. The chickens and ducks were the first to be destroyed, while the pigs were the first to go wild, followed by the cats. Nor were the dogs long in adapting themselves to the changed conditions. There was a veritable plague of dogs. They devoured the corpses, barked and howled during the nights, and in the daytime slunk about in the distance. As the time went by, I noticed a change in their behavior. At first they were apart from one another, very suspicious and very prone to fight. But after a not very long while they began to come together and run in packs. The dog, you see, always was a social animal, and this was true before ever he came to be domesticated by man. In the last days of the world before the plague, there were many many very different kinds of dogs\u2014dogs without hair and dogs with warm fur, dogs so small that they would make scarcely a mouthful for other dogs that were as large as mountain lions. Well, all the small dogs, and the weak types, were killed by their fellows. Also, the very large ones were not adapted for the wild life and bred out. As a result, the many different kinds of dogs disappeared, and there remained, running in packs, the medium-sized wolfish dogs that you know today.\"\n\n\"But the cats don't run in packs, Granser,\" Hoo-Hoo objected.\n\n\"The cat was never a social animal. As one writer in the nineteenth century said, the cat walks by himself. He always walked by himself, from before the time he was tamed by man, down through the long ages of domestication, to today when once more he is wild.\n\n\"The horses also went wild, and all the fine breeds we had degenerated into the small mustang horse you know today. The cows likewise went wild, as did the pigeons and the sheep. And that a few of the chickens survived you know yourself. But the wild chicken of today is quite a different thing from the chickens we had in those days.\n\n\"But I must go on with my story. I travelled through a deserted land. As the time went by I began to yearn more and more for human beings. But I never found one, and I grew lonelier and lonelier. I crossed Livermore Valley and the mountains between it and the great valley of the San Joaquin. You have never seen that valley, but it is very large and it is the home of the wild horse. There are great droves there, thousands and tens of thousands. I revisited it thirty years after, so I know. You think there are lots of wild horses down here in the coast valleys, but they are as nothing compared with those of the San Joaquin. Strange to say, the cows, when they went wild, went back into the lower mountains. Evidently they were better able to protect themselves there. \"In the country districts the ghouls and prowlers had been less in evidence, for I found many villages and towns untouched by fire. But they were filled by the pestilential dead, and I passed by without exploring them. It was near Lathrop that, out of my loneliness, I picked up a pair of collie dogs that were so newly free that they were urgently willing to return to their allegiance to man. These collies accompanied me for many years, and the strains of them are in those very dogs there that you boys have today. But in sixty years the collie strain has worked out. These brutes are more like domesticated wolves than anything else.\"\n\nHare-Lip rose to his feet, glanced to see that the goats were safe, and looked at the sun's position in the afternoon sky, advertising impatience at the prolixity of the old man's tale. Urged to hurry by Edwin, Granser went on. \"There is little more to tell. With my two dogs and my pony, and riding a horse I had managed to capture, I crossed the San Joaquin and went on to a wonderful valley in the Sierras called Yosemite. In the great hotel there I found a prodigious supply of tinned provisions. The pasture was abundant, as was the game, and the river that ran through the valley was full of trout. I remained there three years in an utter loneliness that none but a man who has once been highly civilized can understand. Then I could stand it no more. I felt that I was going crazy. Like the dog, I was a social animal and I needed my kind. I reasoned that since I had survived the plague, there was a possibility that others had survived. Also, I reasoned that after three years the plague germs must all be gone and the land be clean again.\n\n\"With my horse and dogs and pony, I set out. Again I crossed the San Joaquin Valley, the mountains beyond, and came down into Livermore Valley. The change in those three years was amazing. All the land had been splendidly tilled, and now I could scarcely recognize it, such was the sea of rank vegetation that had overrun the agricultural handiwork of man. You see, the wheat, the vegetables, and orchard trees had always been cared for and nursed by man, so that they were soft and tender. The weeds and wild bushes and such things, on the contrary, had always been fought by man, so that they were tough and resistant. As a result, when the hand of man was removed, the wild vegetation smothered and destroyed practically all the domesticated vegetation. The coyotes were greatly increased, and it was at this time that I first encountered wolves, straying in twos and threes and small packs down from the regions where they had always persisted.\n\n\"It was at Lake Temescal, not far from the one-time city of Oakland, that I came upon the first live human beings. Oh, my grandsons, how can I describe to you my emotion, when, astride my horse and dropping down the hillside to the lake, I saw the smoke of a campfire rising through the trees. Almost did my heart stop beating. I felt that I was going crazy. Then I heard the cry of a babe\u2014a human babe. And dogs barked, and my dogs answered. I did not know but what I was the one human alive in the whole world. It could not be true that here were others\u2014smoke, and the cry of a babe.\n\n\"Emerging on the lake, there, before my eyes, not a hundred yards away, I saw a man, a large man. He was standing on an outjutting rock and fishing. I was overcome. I stopped my horse. I tried to call out but could not. I waved my hand. It seemed to me that the man looked at me, but he did not appear to wave. Then I laid my head on my arms there in the saddle. I was afraid to look again, for I knew it was an hallucination, and I knew that if I looked the man would be gone. And so precious was the hallucination, that I wanted it to persist yet a little while. I knew, too, that as long as I did not look it would persist.\n\n\"Thus I remained, until I heard my dogs snarling, and a man's voice. What do you think the voice said? I will tell you. It said: '*Where in hell did you come from?*'\n\n\"Those were the words, the exact words. That was what your other grandfather said to me, Hare-Lip, when he greeted me there on the shore of Lake Temescal fifty-seven years ago. And they were the most ineffable words I have ever heard. I opened my eyes, and there he stood before me, a large, dark, hairy man, heavy-jawed, slant-browed, fierce-eyed. How I got off my horse I do not know. But it seemed that the next I knew I was clasping his hand with both of mine and crying. I would have embraced him, but he was ever a narrow-minded, suspicious man, and he drew away from me. Yet did I cling to his hand and cry.\"\n\nGranser's voice faltered and broke at the recollection, and the weak tears streamed down his cheeks while the boys looked on and giggled.\n\n\"Yet did I cry,\" he continued, \"and desire to embrace him, though the Chauffeur was a brute, a perfect brute\u2014the most abhorrent man I have ever known. His name was\u2026 strange, how I have forgotten his name. Everybody called him Chauffeur\u2014it was the name of his occupation, and it stuck. That is how, to this day, the tribe he founded is called the Chauffeur Tribe.\n\n\"He was a violent, unjust man. Why the plague germs spared him I can never understand. It would seem, in spite of our old metaphysical notions about absolute justice, that there is no justice in the universe. Why did he live?\u2014an iniquitous, moral monster, a blot on the face of nature, a cruel, relentless, bestial cheat as well. All he could talk about was motor cars, machinery, gasoline, and garages\u2014and especially, and with huge delight, of his mean pilferings and sordid swindlings of the persons who had employed him in the days before the coming of the plague. And yet he was spared, while hundreds of millions, yea, billions, of better men were destroyed.\n\n\"I went on with him to his camp, and there I saw her, Vesta, the one woman. It was glorious and\u2026 pitiful. There she was, Vesta Van Warden, the young wife of John Van Warden, clad in rags, with marred and scarred and toil-calloused hands, bending over the campfire and doing scullion work\u2014she, Vesta, who had been born to the purple of the greatest baronage of wealth the world had ever known. John Van Warden, her husband, worth one billion, eight hundred millions and President of the Board of Industrial Magnates, had been the ruler of America. Also, sitting on the International Board of Control, he had been one of the seven men who ruled the world. And she herself had come of equally noble stock. Her father, Philip Saxon, had been President of the Board of Industrial Magnates up to the time of his death. This office was in process of becoming hereditary, and had Philip Saxon had a son that son would have succeeded him. But his only child was Vesta, the perfect flower of generations of the highest culture this planet has ever produced. It was not until the engagement between Vesta and Van Warden took place, that Saxon indicated the latter as his successor. It was, I am sure, a political marriage. I have reason to believe that Vesta never really loved her husband in the mad passionate way of which the poets used to sing. It was more like the marriages that obtained among crowned heads in the days before they were displaced by the Magnates.\n\n\"And there she was, boiling fish-chowder in a soot-covered pot, her glorious eyes inflamed by the acrid smoke of the open fire. Hers was a sad story. She was the one survivor in a million, as I had been, as the Chauffeur had been. On a crowning eminence of the Alameda Hills, overlooking San Francisco Bay, Van Warden had built a vast summer palace. It was surrounded by a park of a thousand acres. When the plague broke out, Van Warden sent her there. Armed guards patrolled the boundaries of the park, and nothing entered in the way of provisions or even mail matter that was not first fumigated. And yet did the plague enter, killing the guards at their posts, the servants at their tasks, sweeping away the whole army of retainers\u2014or, at least, all of them who did not flee to die elsewhere. So it was that Vesta found herself the sole living person in the palace that had become a charnel house.\n\n\"Now the Chauffeur had been one of the servants that ran away. Returning, two months afterward, he discovered Vesta in a little summer pavilion where there had been no deaths and where she had established herself. He was a brute. She was afraid, and she ran away and hid among the trees. That night, on foot, she fled into the mountains\u2014she, whose tender feet and delicate body had never known the bruise of stones nor the scratch of briars. He followed, and that night he caught her. He struck her. Do you understand? He beat her with those terrible fists of his and made her his slave. It was she who had to gather the firewood, build the fires, cook, and do all the degrading camp-labor\u2014she, who had never performed a menial act in her life. These things he compelled her to do, while he, a proper savage, elected to lie around camp and look on. He did nothing, absolutely nothing, except on occasion to hunt meat or catch fish.\"\n\n\"Good for Chauffeur,\" Hare-Lip commented in an undertone to the other boys. \"I remember him before he died. He was a corker. But he did things, and he made things go. You know, Dad married his daughter, an' you ought to see the way he knocked the spots outa Dad. The Chauffeur was a son-of-a-gun. He made us kids stand around. Even when he was croaking he reached out for me, once, an' laid my head open with that long stick he kept always beside him.\"\n\nHare-Lip rubbed his bullet head reminiscently, and the boys returned to the old man, who was maundering ecstatically about Vesta, the squaw of the founder of the Chauffeur Tribe.\n\n\"And so I say to you that you cannot understand the awfulness of the situation. The Chauffeur was a servant, understand, a servant. And he cringed, with bowed head, to such as she. She was a lord of life, both by birth and by marriage. The destinies of millions, such as he, she carried in the hollow of her pink-white hand. And, in the days before the plague, the slightest contact with such as he would have been pollution. Oh, I have seen it. Once, I remember, there was Mrs. Goldwin, wife of one of the great magnates. It was on a landing stage, just as she was embarking in her private dirigible, that she dropped her parasol. A servant picked it up and made the mistake of handing it to her\u2014to her, one of the greatest royal ladies of the land! She shrank back, as though he were a leper, and indicated her secretary to receive it. Also, she ordered her secretary to ascertain the creature's name and to see that he was immediately discharged from service. And such a woman was Vesta Van Warden. And her the Chauffeur beat and made his slave.\n\n\"\u2014Bill\u2014that was it; Bill, the Chauffeur. That was his name. He was a wretched, primitive man, wholly devoid of the finer instincts and chivalrous promptings of a cultured soul. No, there is no absolute justice, for to him fell that wonder of womanhood, Vesta Van Warden. The grievousness of this you will never understand, my grandsons; for you are yourselves primitive little savages, unaware of aught else but savagery. Why should Vesta not have been mine? I was a man of culture and refinement, a professor in a great university. Even so, in the time before the plague, such was her exalted position, she would not have deigned to know that I existed. Mark, then, the abysmal degradation to which she fell at the hands of the Chauffeur. Nothing less than the destruction of all mankind had made it possible that I should know her, look in her eyes, converse with her, touch her hand\u2014ay, and love her and know that her feelings toward me were very kindly. I have reason to believe that she, even she, would have loved me, there being no other man in the world except the Chauffeur. Why, when it destroyed eight billions of souls, did not the plague destroy just one more man, and that man the Chauffeur?\n\n\"Once, when the Chauffeur was away fishing, she begged me to kill him. With tears in her eyes she begged me to kill him. But he was a strong and violent man, and I was afraid. Afterwards, I talked with him. I offered him my horse, my pony, my dogs, all that I possessed, if he would give Vesta to me. And he grinned in my face and shook his head. He was very insulting. He said that in the old days he had been a servant, had been dirt under the feet of men like me and of women like Vesta, and that now he had the greatest lady in the land to be servant to him and cook his food and nurse his brats. 'You had your day before the plague,' he said; 'but this is my day, and a damned good day it is. I wouldn't trade back to the old times for anything.' Such words he spoke, but they are not his words. He was a vulgar, low-minded man, and vile oaths fell continually from his lips.\n\n\"Also, he told me that if he caught me making eyes at his woman he'd wring my neck and give her a beating as well. What was I to do? I was afraid. He was a brute. That first night, when I discovered the camp, Vesta and I had great talk about the things of our vanished world. We talked of art, and books, and poetry; and the Chauffeur listened and grinned and sneered. He was bored and angered by our way of speech which he did not comprehend, and finally he spoke up and said: 'And this is Vesta Van Warden, one-time wife of Van Warden the Magnate\u2014a high and stuck-up beauty, who is now my squaw. Eh, Professor Smith, times is changed, times is changed. Here, you, woman, take off my moccasins, and lively about it. I want Professor Smith to see how well I have you trained.'\n\n\"I saw her clench her teeth, and the flame of revolt rise in her face. He drew back his gnarled fist to strike, and I was afraid, and sick at heart. I could do nothing to prevail against him. So I got up to go, and not be witness to such indignity. But the Chauffeur laughed and threatened me with a beating if I did not stay and behold. And I sat there, perforce, by the campfire on the shore of Lake Temescal, and saw Vesta, Vesta Van Warden, kneel and remove the moccasins of that grinning, hairy, apelike human brute.\n\n\"\u2014Oh, you do not understand, my grandsons. You have never known anything else, and you do not understand.\n\n\"'Halter-broke and bridle-wise,' the Chauffeur gloated, while she performed that dreadful, menial task. 'A trifle balky at times, Professor, a trifle balky; but a clout alongside the jaw makes her as meek and gentle as a lamb.'\n\n\"And another time he said: 'We've got to start all over and replenish the earth and multiply. You're handicapped, Professor. You ain't got no wife, and we're up against a regular Garden-of-Eden proposition. But I ain't proud. I'll tell you what, Professor.' He pointed at their little infant, barely a year old. 'There's your wife, though you'll have to wait till she grows up. It's rich, ain't it? We're all equals here, and I'm the biggest toad in the splash. But I ain't stuck up\u2014not I. I do you the honor, Professor Smith, the very great honor of betrothing to you my and Vesta Van Warden's daughter. Ain't it cussed bad that Van Warden ain't here to see?'\"\n\n## VI\n\n\"I LIVED three weeks of infinite torment there in the Chauffeur's camp. And then, one day, tiring of me, or of what to him was my bad effect on Vesta, he told me that the year before, wandering through the Contra Costa Hills to the Straits of Carquinez, across the Straits he had seen a smoke. This meant that there were still other human beings, and that for three weeks he had kept this inestimably precious information from me. I departed at once, with my dogs and horses, and journeyed across the Contra Costa Hills to the Straits. I saw no smoke on the other side, but at Port Costa discovered a small steel barge on which I was able to embark my animals. Old canvas which I found served me for a sail, and a southerly breeze fanned me across the Straits and up to the ruins of Vallejo. Here, on the outskirts of the city, I found evidences of a recently occupied camp.\n\n\"Many clam-shells showed me why these humans had come to the shores of the Bay. This was the Santa Rosa Tribe, and I followed its track along the old railroad right of way across the salt marshes to Sonoma Valley. Here, at the old brickyard at Glen Ellen, I came upon the camp. There were eighteen souls all told. Two were old men, one of whom was Jones, a banker. The other was Harrison, a retired pawnbroker, who had taken for wife the matron of the State Hospital for the Insane at Napa. Of all the persons of the city of Napa, and of all the other towns and villages in that rich and populous valley, she had been the only survivor. Next, there were the three young men\u2014Cardiff and Hale, who had been farmers, and Wainwright, a common day-laborer. All three had found wives. To Hale, a crude, illiterate farmer, had fallen Isadore, the greatest prize, next to Vesta, of the women who came through the plague. She was one of the world's most noted singers, and the plague had caught her at San Francisco. She has talked with me for hours at a time, telling me of her adventures, until, at last, rescued by Hale in the Mendocino Forest Reserve, there had remained nothing for her to do but become his wife. But Hale was a good fellow, in spite of his illiteracy. He had a keen sense of justice and right-dealing, and she was far happier with him than was Vesta with the Chauffeur.\n\n\"The wives of Cardiff and Wainwright were ordinary women, accustomed to toil with strong constitutions\u2014just the type for the wild new life which they were compelled to live. In addition were two adult idiots from the feeble-minded home at Eldredge, and five or six young children and infants born after the formation of the Santa Rosa Tribe. Also, there was Bertha. She was a good woman, Hare-Lip, in spite of the sneers of your father. Her I took for wife. She was the mother of your father, Edwin, and of yours, Hoo-Hoo. And it was our daughter, Vera, who married your father, Hare-Lip\u2014your father, Sandow, who was the oldest son of Vesta Van Warden and the Chauffeur.\n\n\"And so it was that I became the nineteenth member of the Santa Rosa Tribe. There were only two outsiders added after me. One was Mungerson, descended from the Magnates, who wandered alone in the wilds of Northern California for eight years before he came south and joined us. He it was who waited twelve years more before he married my daughter, Mary. The other was Johnson, the man who founded the Utah Tribe. That was where he came from, Utah, a country that lies very far away from here, across the great deserts, to the east. It was not until twenty-seven years after the plague that Johnson reached California. In all that Utah region he reported but three survivors, himself one, and all men. For many years these three men lived and hunted together, until, at last, desperate, fearing that with them the human race would perish utterly from the planet, they headed westward on the possibility of finding women survivors in California. Johnson alone came through the great desert, where his two companions died. He was forty-six years old when he joined us, and he married the fourth daughter of Isadore and Hale, and his eldest son married your aunt, Hare-Lip, who was the third daughter of Vesta and the Chauffeur. Johnson was a strong man, with a will of his own. And it was because of this that he seceded from the Santa Rosans and formed the Utah Tribe at San Jos\u00e9. It is a small tribe\u2014there are only nine in it; but, though he is dead, such was his influence and the strength of his breed, that it will grow into a strong tribe and play a leading part in the recivilization of the planet.\n\n\"There are only two other tribes that we know of\u2014the Los Angelitos and the Carmelitos. The latter started from one man and woman. He was called Lopez, and he was descended from the ancient Mexicans and was very black. He was a cowherd in the ranges beyond Carmel, and his wife was a maidservant in the great Del Monte Hotel. It was seven years before we first got in touch with the Los Angelitos. They have a good country down there, but it is too warm. I estimate the present population of the world at between three hundred and fifty and four hundred\u2014provided, of course, that there are no scattered little tribes elsewhere in the world. If there be such, we have not heard from them. Since Johnson crossed the desert from Utah, no word nor sign has come from the East or anywhere else. The great world which I knew in my boyhood and early manhood is gone. It has ceased to be. I am the last man who was alive in the days of the plague and who knows the wonders of that far-off time. We, who mastered the planet\u2014its earth, and sea, and sky\u2014and who were as very gods, now live in primitive savagery along the water courses of this California country.\n\n\"But we are increasing rapidly\u2014your sister, Hare-Lip, already has four children. We are increasing rapidly and making ready for a new climb toward civilization. In time, pressure of population will compel us to spread out, and a hundred generations from now we may expect our descendants to start across the Sierras, oozing slowly along, generation by generation, over the great continent to the colonization of the East\u2014a new Aryan drift around the world.\n\n\"But it will be slow, very slow; we have so far to climb. We fell so hopelessly far. If only one physicist or one chemist had survived! But it was not to be, and we have forgotten everything. The Chauffeur started working in iron. He made the forge which we use to this day. But he was a lazy man, and when he died he took with him all he knew of metals and machinery. What was I to know of such things? I was a classical scholar, not a chemist. The other men who survived were not educated. Only two things did the Chauffeur accomplish\u2014the brewing of strong drink and the growing of tobacco. It was while he was drunk, once, that he killed Vesta. I firmly believe that he killed Vesta in a fit of drunken cruelty though he always maintained that she fell into the lake and was drowned.\n\n\"And, my grandsons, let me warn you against the medicine-men. They call themselves *doctors*, travestying what was once a noble profession, but in reality they are medicine-men, devil-devil men, and they make for superstition and darkness. They are cheats and liars. But so debased and degraded are we, that we believe their lies. They, too, will increase in numbers as we increase, and they will strive to rule us. Yet are they liars and charlatans. Look at young Cross-Eyes, posing as a doctor, selling charms against sickness, giving good hunting, exchanging promises of fair weather for good meat and skins, sending the death-stick, performing a thousand abominations. Yet I say to you, that when he says he can do these things, he lies. I, Professor Smith, Professor James Howard Smith, say that he lies. I have told him so to his teeth. Why has he not sent me the death-stick? Because he knows that with me it is without avail. But you, Hare-Lip, so deeply are you sunk in black superstition that did you awake this night and find the death-stick beside you, you would surely die. And you would die, not because of any virtues in the stick, but because you are a savage with the dark and clouded mind of a savage.\n\n\"The doctors must be destroyed, and all that was lost must be discovered over again. Wherefore, earnestly, I repeat unto you certain things which you must remember and tell to your children after you. You must tell them that when water is made hot by fire, there resides in it a wonderful thing called steam, which is stronger than ten thousand men and which can do all man's work for him. There are other very useful things. In the lightning flash resides a similarly strong servant of man, which was of old his slave and which some day will be his slave again.\n\n\"Quite a different thing is the alphabet. It is what enables me to know the meaning of fine markings, whereas you boys know only rude picture-writing. In that dry cave on Telegraph Hill, where you see me often go when the tribe is down by the sea, I have stored many books. In them is great wisdom. Also, with them, I have placed a key to the alphabet, so that one who knows picture-writing may also know print. Some day men will read again; and then, if no accident has befallen my cave, they will know that Professor James Howard Smith once lived and saved for them the knowledge of the ancients. \"There is another little device that men inevitably will rediscover. It is called gunpowder. It was what enabled us to kill surely and at long distances. Certain things which are found in the ground, when combined in the right proportions, will make this gunpowder. What these things are, I have forgotten, or else I never knew. But I wish I did know. Then would I make powder, and then would I certainly kill Cross-Eyes and rid the land of superstition\u2014\"\n\n\"After I am man-grown I am going to give Cross-Eyes all the goats, and meat, and skins I can get, so that he'll teach me to be a doctor,\" Hoo-Hoo asserted. \"And when I know, I'll make everybody else sit up and take notice. They'll get down in the dirt to me, you bet.\"\n\nThe old man nodded his head solemnly, and murmured:\n\n\"Strange it is to hear the vestiges and remnants of the complicated Aryan speech falling from the lips of a filthy little skin-clad savage. All the world is topsy-turvy. And it has been topsy-turvy ever since the plague.\"\n\n\"You won't make me sit up,\" Hare-Lip boasted to the would-be medicine-man. \"If I paid you for a sending of the death-stick and it didn't work, I'd bust in your head\u2014understand, you Hoo-Hoo, you?\"\n\n\"I'm going to get Granser to remember this here gunpowder stuff,\" Edwin said softly, \"and then I'll have you all on the run. You, Hare-Lip, will do my fighting for me and get my meat for me, and you, Hoo-Hoo, will send the death-stick for me and make everybody afraid. And if I catch Hare-Lip trying to bust your head, Hoo-Hoo, I'll fix him with that same gunpowder. Granser ain't such a fool as you think, and I'm going to listen to him and some day I'll be boss over the whole bunch of you.\"\n\nThe old man shook his head sadly, and said:\n\n\"The gunpowder will come. Nothing can stop it\u2014the same old story over and over. Man will increase, and men will fight. The gunpowder will enable men to kill millions of men, and in this way only, by fire and blood, will a new civilization, in some remote day, be evolved. And of what profit will it be? Just as the old civilization passed, so will the new. It may take fifty thousand years to build, but it will pass. All things pass. Only remain cosmic force and matter, ever in flux, ever acting and reacting and realizing the eternal types\u2014the priest, the soldier, and the king. Out of the mouths of babes comes the wisdom of all the ages. Some will fight, some will rule, some will pray; and all the rest will toil and suffer sore while on their bleeding carcasses is reared again, and yet again, without end, the amazing beauty and surpassing wonder of the civilized state. It were just as well that I destroyed those cave-stored books\u2014whether they remain or perish, all their old truths will be discovered, their old lies lived and handed down. What is the profit\u2014\"\n\nHare-Lip leaped to his feet, giving a quick glance at the pasturing goats and the afternoon sun.\n\n\"Gee!\" he muttered to Edwin, \"The old geezer gets more long-winded every day. Let's pull for camp.\"\n\nWhile the other two, aided by the dogs, assembled the goats and started them for the trail through the forest, Edwin stayed by the old man and guided him in the same direction. When they reached the old right of way, Edwin stopped suddenly and looked back. Hare-Lip and Hoo-Hoo and the dogs and the goats passed on. Edwin was looking at a small herd of wild horses which had come down on the hard sand. There were at least twenty of them, young colts and yearlings and mares, led by a beautiful stallion which stood in the foam at the edge of the surf, with arched neck and bright wild eyes, sniffing the salt air from off the sea.\n\n\"What is it?\" Granser queried.\n\n\"Horses,\" was the answer. \"First time I ever seen 'em on the beach. It's the mountain lions getting thicker and thicker and driving 'em down.\"\n\nThe low sun shot red shafts of light, fan-shaped, up from a cloud-tumbled horizon. And close at hand, in the white waste of shore-lashed waters, the sea-lions, bellowing their old primeval chant, hauled up out of the sea on the black rocks and fought and loved.\n\n\"Come on, Granser,\" Edwin prompted. And old man and boy, skin-clad and barbaric, turned and went along the right of way into the forest in the wake of the goats.\n\nTHE END\n\n## John Griffith \"Jack\" London\n\n(1876\u20131916) was an American novelist, one of the first fiction writers to get rich from writing fiction. His most famous novels\u2014*The Call of the Wild* (1903) and *White Fang* (1906)\u2014were adventure stories set in the Klondike Gold Rush. A number of London's stories were science fiction, and dealt with topics such as invisibility, germ warfare, and energy weapons.\n\n#### Commentary\n\nIn 1918, just six years after \"The Scarlet Plague\" was published in *London Magazine*, the deadly Spanish flu pandemic struck humanity. (The disease got its name not because it originated in Spain but simply because reporters were free to describe its dread effects there. In order to maintain morale, wartime censors suppressed early reports of the illness in the nations fighting World War I. At the time, therefore, it seemed as if neutral Spain had been particularly badly affected.) The H1N1 influenza virus circulating in the period January 1918 to December 1920 infected half a billion people around the globe and killed as many as 100 million people\u2014five percent of the human population. Flu deaths far exceeded the number killed in four years of battle. The disease struck everywhere, from the Arctic to isolated islands in the Pacific. In some communities the impact was so terrible survivors decided it would be best to never talk about it\u2014to pretend, as it were, that this grim event had simply not happened.\n\nSpanish flu was merely the most recent of large-scale pandemics; infectious disease has attacked humankind throughout history. Bubonic plague has been responsible for the most notable pandemics. The first recorded outbreak was the Plague of Justinian (541\u2013542), which struck the Byzantine Empire. Historians estimate that 25 million people died of the plague\u2014less than the number of fatalities caused by Spanish flu, but the total population was much smaller back in Roman times. The Plague of Justinian killed about 13% of the world's population. The plague returned over the following two centuries, killing a further 25 million people.\n\nThe second major outbreak of plague began with the Black Death in 1347. The disease made numerous returns over the following three centuries. The Black Death was one of the most devastating events in human history: it killed between 75 and 200 million people. The dead were placed in ditches and then isolated; in some towns and villages there weren't enough living to bury the dead. (Figure <a href=\"#Fig1\" data-ref-type=\"fig\">3.1<\/a> shows an example of a plague pit.) The world population did not recover to pre-Black Death levels until the seventeenth century. Many historians argue that the devastation unleashed by this pandemic had a significant impact on the course of European history: the labour shortages it caused accelerated several economic, social, and technical developments, and might even have helped introduce the Renaissance.\n\nSo we know pandemics occur. It's entirely possible\u2014even probable\u2014that a destructive flu pandemic will strike again. And it's not hard to imagine how a mutation in one of the viral hemorrhagic fevers\u2014Ebola, say, or the Marburg virus\u2014could lead to a disease causing widespread suffering. Therefore the basic premise of \"The Scarlet Plague\" is not unrealistic. But Jack London was writing more than a century ago. Although elements of his story were prescient (for example, he guessed a global population of eight billion people by 2010\u2014not bad), science and technology have advanced to a level he could scarcely have imagined. Suppose a disease such as the scarlet plague did break out, and let's assume it was as lethal as, say, Spanish flu. Would our modern civilization collapse in the way London suggests?\n\nThis is not an easy question to answer.\n\nOn the one hand, as I write, the world population is about 7.6 billion. A large fraction of the population is mobile to an extent that would have astonished Jack London. In the past, disease travelled from continent to continent at the speed of sailing boats. Nowadays, if people develop a disease in Beijing, say, they can carry it to Berlin within hours. This combination of a large pool of people in which disease can develop with the rapid, large-scale movement of people mean infections can be transmitted more efficiently than ever before. Furthermore, the threats posed by viruses and bacteria are always evolving. The flu virus, for example, changes constantly. One type of change is a gradual \"drift\" in genetic make-up that leads, over time, to a virus our immune system fails to recognise even if we have previously been exposed to a similar virus. (This is one of the reasons why flu vaccine effectiveness is so hit-and-miss: the vaccine must be reformulated every year, based on an informed guess about which strains are likely to cause most suffering in the coming year.) Another type of change is an abrupt \"shift\" in genetic make-up, caused by a random mutation. When this happens, the possibility of a pandemic occurs: most people will possess no immune protection against the new virus. In short, if the microbial world is our enemy then we face a crafty foe. We might well choose to think of microbes as a threat to our civilisation.\n\nOn the other hand, our understanding of medicine in general and of public health in particular are vastly more advanced than in London's day. Furthermore, information can travel even more quickly than people. These advances help mitigate the threat of pandemic disease. Consider, for example, the case of SARS. Between November 2002 and July 2003, a viral disease causing flu-like symptoms caused 774 deaths in China and neighbouring countries. The disease, which was given the name Severe Acute Respiratory Syndrome (SARS for short) was new and it was dangerous\u2014it had a 9.6% fatality rate. There was no vaccine against SARS; there isn't one now. Nevertheless, the dire threat posed to our civilisation by SARS did not materialise. The rapid response of public health authorities, at national and international level, broke the chain of transmission. Since 2004, no case of SARS has been reported anywhere in the world.\n\nOr consider the case of the swine flu outbreak of 2009. A new strain of the H1N1 virus (see Fig. <a href=\"#Fig2\" data-ref-type=\"fig\">3.2<\/a>) began circulating and, since H1N1 caused the dreadful Spanish flu pandemic, it's no surprise that individuals and organisations were worried. Fortunately, the virus that caused swine flu was about one hundred times less lethal than the 1918 virus. Even if that had not been the case, I suspect the outcome of the 2009 pandemic would have been less severe than what happened in 1918. For one thing, doctors could prescribe antiviral medicines\u2014the antivirals weren't hugely successful, but they were better than nothing. More importantly, the public health response was rapid. My own university was soon covered with posters explaining how to slow the transmission of the disease. Some of those posters are still to be found; they are fading, now, but they still provide basic but effective hygiene advice. Furthermore, although it turned out not to be needed, organisations developed business continuity plans. In my own university, these continuity plans involved teaching online if a pandemic caused students to stay away from lecture theatres. (In 1665, the University of Cambridge closed down because of pandemic. In this case it was a precaution against the Great Plague, the last major outbreak of bubonic plague in England. Cambridge was unable to offer online learning back in 1665, but that turned out not to be a hindrance for Isaac Newton. During enforced private study at his home in Woolsthorpe he developed optics, calculus, and the law of universal gravitation!)\n\nI'm writing this almost exactly one hundred years after the doctors observed the first cases of Spanish flu. For an entire century, humans have managed to avoid a widespread outbreak of contagious disease. So it's tempting to conclude that although technological developments might promote a pandemic they also provide us with tools to help prevent a pandemic. It's comforting to suppose that our science, technology, and medicine will avert the disaster envisaged in \"The Scarlet Plague\".\n\nOur knowledge might well save us. But only a fool would be complacent.\n\nHumanity faces a growing threat from bacteria\u2014a self-inflicted threat we could ward off if people would only act rationally. The menace stems from the misuse of our most effective weapon against bacteria.\n\nPeople are naturally enamoured of antibiotics. Patients demand, and often receive, antibiotics as a treatment for colds, sore throats, earaches\u2026 even in cases where doctors know antibiotics won't work. Antibiotics are used in crop production, as pesticides, or to treat disease in plants. Antibiotics are given to animals as freely as they are given to humans\u2014vets use them to treat disease while farmers use them to promote growth. We live in a world awash with antibiotics. The problem? Well, in order to survive in this antibiotic-filled environment bacteria have evolved resistance. Some bacteria are now resistant to all known antibiotics.\n\nA world without effective antibiotics is a terrifying prospect. Many routine medical interventions we now take for granted\u2014appendix operations, hip replacements, transplant surgery\u2014would be dangerous: patients might survive the knife but succumb to infection. We'd face the same risks people faced before 1928, when Alexander Fleming discovered penicillin. Worse, though, is that possibility of pandemic.\n\nConsider the plague. The disease is caused by the bacterium *Yersinia pestis*. When this pestilence stalked our ancestors the prognosis was poor for anyone infected: chances were high the sufferer would die a horrible death. Antibiotics changed the story. The plague infects people to this day, but nowadays if patients are given streptomycin quickly enough then the chances are high they'll survive. So if a strain of *Yersinia pestis* evolves resistance to all antibiotics then doctors\u2014and, more to the point, patients\u2014will be in trouble. It's a similar story with many other infectious diseases.\n\nScience and medicine might have provided us with tools to help prevent a pandemic, but we're letting one of our best tools get rusty.\n\nBlack death, Spanish flu, or something along the lines of the scarlet plague\u2014another pandemic will surely happen eventually. When it comes, though, we'll at least have the comfort of knowing the pandemic agent won't be *trying* to kill us. Some bacteria and viruses cause us harm, but that's just a byproduct of their life cycle. It's nothing personal. As mentioned in Chapter 10.1007\/978-3-030-03195-4_2, however, advances in biotechnology will soon permit a group or even an individual to *design* a microbial agent that's *intended* to kill. The terrorist, or perhaps merely the misogynist, will have the ability to target disease at specific groups\u2014males, females, the pre-pubescent, those possessing too much or too little skin melanin. And an engineered pandemic could be designed to kill more effectively than the natural variety.\n\nConsider the West African Ebola epidemic of 2013\u20132016. According to official statistics the virus caused 11,310 deaths. This was a shocking outbreak, of course, but in some ways the virus killed too effectively for its own good. Symptoms became obvious between 2 and 21 days after exposure to the virus, and this led to a method of containment and control. Anyone who was in contact with a patient was tracked for 21 days; communities were made aware of risk factors and preventative measures; and quarantines were put in place. Roughly 900 days after the first case was diagnosed, the epidemic was over. But the single-minded bioterrorist could *engineer* an infectious agent in such a way that obvious countermeasures would be ineffective. The agent, for example, might be airborne and easily transmitted through the simple act of breathing. It might establish itself in its host whilst producing no symptoms. After a long latency period it could be \"switched on\"\u2014and death would follow for all those infected.\n\nSF writers have long imagined something along these lines. In a 1982 novel, for example, Frank Herbert's titular \"white plague\" was designed by a molecular biologist. The biologist, driven insane by the death of his wife and children in a car bomb, desires revenge\u2014so he develops a deadly plague, which is carried by men but kills only women. In his 1997 novel *The Cobra Event*, Richard Preston has an antagonist called Archimedes release a genetically engineered virus (called \"Cobra\")\u2014a fusion of the common cold and smallpox viruses\u2014which results in a horrifying disease called brainpox. (Preston is the author of the non-fiction book *The Hot Zone*, which gives a well written account of the viral haemorrhagic fevers.) In Paulo Bacigalupi's award-winning 2009 novel *The Windup Girl*, large corporations release bioengineered plagues that attack crops\u2014a lucrative activity if you possess plague-resistant seeds.\n\nWhite plague, Cobra, bioengineered attack genes\u2026 these nightmares remain science fictional. The knowledge and techniques needed to realise them don't exist yet. But, as described in the commentary to the previous chapter, the rate of progress in biotechnology is astounding. In a few years there'll be research labs possessing the knowledge necessary to create a deadly life form from scratch; a few years later those same techniques will be available to undergraduates.\n\nSuch technology is so dangerous it presents an existential risk. Should we not therefore police it, in the same way we police other existential risks? After all, the world has managed to avoid a nuclear catastrophe by cooperating at the international level to limit the spread of the technology that permits the construction of hydrogen bombs. Unfortunately, nuclear non-proliferation techniques won't work in the case of bioterrorism. The construction of a nuclear arsenal can't easily be hidden from view\u2014the resources of a nation state are required to build a hydrogen bomb\u2014and so the activity can in principle be monitored. But one day soon a small terrorist cell working quietly in a garden shed might be able to engineer a virus using only a few test tubes. How could society possibly police that situation? The human race can survive natural pandemics: there have been many in our past and there'll be more in our future. But could humanity survive an *engineered* pandemic?\n\nIf a pandemic, natural or artificial, did wipe out humanity, what might Earth be like without us? Following London, it's interesting to speculate. Many of our buildings would likely soon vanish under the onslaught of wind, rain, and vegetation; roads would crack and bridges would collapse; a few constructions\u2014the Channel Tunnel, for example\u2014might last much longer. But eventually, most traces of humanity's time on this planet would be erased. Perhaps after a few tens of millions of years\u2014the same sort of timescale separating us from the dinosaurs\u2014evolution might produce another intelligent species. Would all traces of the achievements we so value be dissolved by the passage of time? Or would such a species be able to find evidence that humans once walked the Earth?\n\nIf some future intelligence was indeed able to infer the existence of a bipedal creature, which chose to dig up fossilized carbon and transform it into plastics and a source of power, then we'd surely seem bizarre to them. But would we seem as bizarre as the creatures depicted in Chapter 10.1007\/978-3-030-03195-4_4?\n\n#### Notes and Further Reading\n\n-   *the impact was so terrible*\u2014The Spanish flu is the subject of numerous books; for an entertaining account (if \"entertaining\" is the correct word to use about such a grim event), see Spinney (2017).\n\n-   *attacked humans throughout history*\u2014In *Plagues and Peoples*, William McNeill (1976) explores the effects of disease on human history. He gives an in-depth account of the Justinian Plague, as well as more recent pandemics.\n\n-   *the Black Death in 1347*\u2014One of the best of many accounts of the Black Death, its causes and consequences, is that by John Kelly (2006).\n\n-   *According to official statistics*\u2014For further details of the Ebola epidemic that peaked during 2014\u20132015, see World Health Organisation (2016). For more recent information about this terrible disease, see World Health Organisation (2018).\n\n-   *writers have long imagined something along these lines*\u2014For the books and novels mentioned here, see: Bacigalupi (2009); Herbert (1982); and Preston (1995, 1997).\n\n-   *be able to find evidence that we once walked the Earth?*\u2014This question lies at the core of a fascinating novel by a world-renowned astrophysicist. Jayant Narlikar's (2015) novel *The Return of Vaman* appears in the Springer Science & Fiction series. For non-fiction accounts exploring what the world might be like if humans vanished, see for example Weisman (2007) and Zalasiewicz (2009).\n\n# Bibliography","meta":{"dup_signals":{"dup_doc_count":226,"dup_dump_count":89,"dup_details":{"curated_sources":2,"2023-50":2,"2023-40":1,"2023-23":1,"2023-14":1,"2022-49":1,"2022-40":2,"2022-27":2,"2022-21":1,"2022-05":2,"2021-49":2,"2021-43":2,"2021-31":1,"2021-25":1,"2021-21":1,"2021-17":2,"2021-04":5,"2020-40":2,"2020-29":1,"2020-16":4,"2020-10":1,"2020-05":2,"2019-51":1,"2019-43":1,"2019-39":1,"2019-35":2,"2019-30":1,"2019-26":1,"2019-22":3,"2019-18":4,"2019-13":1,"2019-09":3,"2019-04":3,"2018-51":5,"2018-47":2,"2018-43":3,"2018-39":1,"2018-34":5,"2018-30":1,"2018-26":2,"2018-22":1,"2018-17":3,"2018-13":4,"2018-09":1,"2018-05":3,"2017-51":1,"2017-47":3,"2017-43":5,"2017-39":2,"2017-34":2,"2017-30":3,"2017-26":5,"2017-22":4,"2017-17":5,"2017-09":4,"2017-04":4,"2016-50":4,"2016-44":4,"2016-40":3,"2016-36":1,"2016-30":1,"2016-26":1,"2016-22":1,"2016-18":1,"2016-07":3,"2015-48":4,"2015-40":1,"2015-35":2,"2015-32":3,"2015-27":4,"2015-22":3,"2015-14":3,"2014-52":4,"2014-49":2,"2014-42":3,"2014-41":3,"2014-35":5,"2014-23":4,"2014-15":2,"2024-26":2,"2024-18":1,"2024-10":3,"2017-13":7,"2015-18":2,"2015-11":4,"2015-06":3,"2014-10":2,"2013-48":6,"2013-20":4,"2024-30":1}},"file":"PMC7123587"},"subset":"pubmed_central"}
{"text":"abstract: A report on the 6th annual Future of Genomic Medicine conference, held at the Scripps Seaside Forum, La Jolla, CA, USA, March 7-8, 2013.\nauthor: Konrad J Karczewski\ndate: 2013\ninstitute: 1Biomedical Informatics Training Program, Stanford University School of Medicine, Stanford, CA 94305, USA; 2Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA\ntitle: The future of genomic medicine is here\n\nOn his flight over to San Diego to lead the Future of Genomic Medicine (FoGM) conference, Dr Eric Topol (Scripps Translational Science Institute, USA) used a heart monitor device attached to his smartphone to diagnose a distressed passenger with atrial fibrillation. Already, mobile technologies such as this one are beginning to transform medicine, and genome sequencing, with its rapidly decreasing costs, is no exception. As we get closer to mini-sequencers and what George Church (Harvard Medical School, USA) termed 'wearable sequencing', a future of genomically informed medicine becomes possible. The FoGM conference integrated the patient-oriented perspective of genomic medicine, along with cutting-edge technologies and data integration, and developing methods and models in the aim of clinical utility.\n\n# Patient-oriented genomic medicine\n\nIt is becoming increasingly clear, at this and other conferences, such as the Cold Spring Harbor Personal Genomes meetings, that genomics can have a profound role in guiding diagnoses and treatments. A major theme of this year's conference was the patient perspective and their reaction to having their genome sequenced in a clinical setting. The conference started with a direct perspective from the parents of Lilly Grossman, a patient with a lifelong undiagnosed disease, marked by tremors and sleepless nights. After having her full genome sequenced by the Idiopathic Diseases of Man (IDIOM) study, led by Topol, mutations in *ADCY5* and *DOCK3* were able to putatively explain her phenotype, and suggest a possible treatment, which provided a few weeks of regular sleep. While the result was not a conclusive answer, it provided hope for the patient and her family. Howard Jacob (Medical College of Wisconsin, USA) agreed, stressing that even in the absence of clinical utility (if a diagnosis is not actionable), the personal utility of having a diagnosis is important to the patient and the patient's family. Jacob suggested a consumer-driven economy for personal genomics, and that even though variants and annotations are subject to change as technologies and interpretations improve, involving patients in the process can be an effective way to deal with these changes. Misha Angrist (Duke University, USA) mirrored these sentiments, drawing parallels to open-access publishing: subjects should have the right to their own data and to see results of the studies that use their data. Randy Scott (Invitae, USA) outlined his and Invitae's mission of bringing genetics to the masses by building databases and infrastructure for managing genetic information. The books that were handed out to participants reflected this mission, between AJ Jacob's *Drop Dead Healthy*, a foray into taking control of one's health, as well as the book by myself (Konrad Karczewski, Stanford University, USA) and Joel Dudley (Mount Sinai School of Medicine, USA): *Exploring Personal Genomics*, a handbook to understanding and interpreting personal genetic data.\n\nWhile much of these opinions on patient perspectives were anecdotal, Cinnamon Bloss (Scripps Translational Science Institute, USA) presented hard data on the perceptions of both patients and physicians, and the differences therein, through surveys of families. Parents of patients and their doctor agreed that the doctor was knowledgeable about genetics, but the parents were much less satisfied with the doctor's explanations of the results. However, Bloss noted that the majority of patients, parents and physicians were interested in receiving secondary findings, regardless of age of onset or actionability and desire for these results increased with actionability for all three groups.\n\n# Going beyond SNPs\n\nAnother major scientific theme of FoGM this year involved the expansion past somatic variants (SNPs) to other technologies that could be integrated to inform diagnosis and treatment. In addition to getting his genome sequenced, Michael Snyder (Stanford University, USA) tracked a number of omics technologies over time, including his transcriptome, proteome and metabolome, and used this information to track the onset of diabetes concurrent with infection. Since every patient is unique, an 'N of 1' study, followed longitudinally over time, provided him with interesting observations of altered physiological states (such as infection) compared with his healthy state.\n\nWhile Snyder's analysis was aimed at comprehensive profiling of a healthy individual, Elaine Mardis (Washington University in St Louis, USA) suggested a similar approach for cancers, where sequencing RNAs in addition to DNA would inform predictions of peptides binding with HLA class I. Such an analysis would prioritize antigens that could then be used for personalized immunotherapy. Eric Schadt (Mount Sinai School of Medicine, USA) used similar data types to discover personal cancer drivers and create patient-specific networks that could suggest personalized cancer treatments.\n\nGeorge Weinstock (Washington University in St Louis, USA) and Michael Eisen (University of California, Davis, USA) both brought the microbiome into the mix. Weinstock described efforts to sequence neonatal microbiomes to predict antimicrobial resistance, and discussed a future of fecal transplants and microbiome-based acne treatments. Eisen shared the optimism that the microbiome will become important for human phenotypes, but cautioned against overselling and being careful between correlation and causation, which becomes difficult for large numbers of hypotheses whose outcomes may be linked to their causes. Additionally, we already have a high demand for genetic counselors, but additional data types may bring similar demands, as Eisen called for microbiome counselors.\n\n# Scaling up and towards unified models\n\nFinally, a conference of this nature would not be complete without a call to arms for developing methods and models that will ultimately enable physicians to use genomic information in a clinical setting. Daniel MacArthur (Massachusetts General Hospital, USA) cautioned that consistent calling of exomes and genomes is of utmost importance for variant accuracy. He laid out the challenges for scaling up to variant calling of more than 26,000 exomes, but presented one solution in reduced BAMs, a compressed format, that then can be used for joint variant calling to increase accuracy. From this dataset, MacArthur was able to catalog tolerated protein-coding variation. David Goldstein (Duke University, USA) used such tolerated variants to identify genes that were likely to be functional and focused on these to narrow genetic factors for epileptic encephalopathy.\n\nPeter Visscher (University of Queensland, Australia) described models for predicting complex traits from genotype, which will become increasingly important for sequencing of healthy individuals and prioritization of disease risks. Atul Butte (Stanford University, USA) brought this message back to the clinic, using likelihood ratios, which doctors already use, to combine variant risk information into a unified risk factor. While six gigabytes of genetic data may seem overwhelming at first, Butte reminds us that there is a specialty that routinely analyzes gigabyte scale data: the radiologist. All that are needed are the proper tools.\n\n# Conclusions\n\nThe consensus among speakers at FoGM this year was clear: the genome has an important role in the clinic. With the price of sequencing dropping to costs amenable to regular clinical use, the question now is not if, but how, genomic information will be integrated. There is a coming onslaught of 'big data' that will improve individual healthcare and enable genomic personalized medicine. Challenges still remain, including establishing a patient-centric view of genomic data, which is tied to educating the public and encouraging participation in personal health, as well as standardizing models to most accurately identify causal variation and portray disease risk. As these and new challenges arise, it will take a concerted effort of physicians and scientists to bring forth a future of genomic medicine.\n\n# Abbreviations\n\nBAM: Binary Sequence Alignment Map; FoGM: Future of Genomic Medicine; HLA: human leukocyte antigen; SNP: single nucleotide polymorphism.\n\n# Competing interests\n\nThe author declares that they have no competing interests.","meta":{"dup_signals":{"dup_doc_count":101,"dup_dump_count":34,"dup_details":{"curated_sources":2,"2022-40":1,"2022-21":1,"2021-49":1,"2020-24":1,"2019-47":1,"2017-09":9,"2016-44":1,"2016-40":1,"2016-36":9,"2016-30":9,"2016-22":1,"2016-18":1,"2016-07":4,"2015-48":3,"2015-40":2,"2015-35":3,"2015-32":3,"2015-27":3,"2015-22":3,"2015-14":2,"2014-52":2,"2014-49":3,"2014-42":7,"2014-41":5,"2014-35":4,"2014-23":4,"2014-15":1,"2023-40":1,"2015-18":3,"2015-11":3,"2015-06":2,"2014-10":2,"2013-48":2,"2024-18":1}},"file":"PMC3663104"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  Problems of quality and safety persist in health systems worldwide. We conducted a large research programme to examine culture and behaviour in the English National Health Service (NHS).\n  .\n  # Methods\n  .\n  Mixed-methods study involving collection and triangulation of data from multiple sources, including interviews, surveys, ethnographic case studies, board minutes and publicly available datasets. We narratively synthesised data across the studies to produce a holistic picture and in this paper present a high-level summary.\n  .\n  # Results\n  .\n  We found an almost universal desire to provide the best quality of care. We identified many 'bright spots' of excellent caring and practice and high-quality innovation across the NHS, but also considerable inconsistency. Consistent achievement of high-quality care was challenged by unclear goals, overlapping priorities that distracted attention, and compliance-oriented bureaucratised management. The institutional and regulatory environment was populated by multiple external bodies serving different but overlapping functions. Some organisations found it difficult to obtain valid insights into the quality of the care they provided. Poor organisational and information systems sometimes left staff struggling to deliver care effectively and disempowered them from initiating improvement. Good staff support and management were also highly variable, though they were fundamental to culture and were directly related to patient experience, safety and quality of care.\n  .\n  # Conclusions\n  .\n  Our results highlight the importance of clear, challenging goals for high-quality care. Organisations need to put the patient at the centre of all they do, get smart intelligence, focus on improving organisational systems, and nurture caring cultures by ensuring that staff feel valued, respected, engaged and supported.\nauthor: Mary Dixon-Woods; Richard Baker; Kathryn Charles; Jeremy Dawson; Gabi Jerzembek; Graham Martin; Imelda McCarthy; Lorna McKee; Joel Minion; Piotr Ozieranski; Janet Willars; Patricia Wilkie; Michael WestCorrespondence to Professor Mary Dixon-Woods, Department of Health Sciences, University of Leicester, 22\u201328 Princess Road West, Leicester LE1 6TP, UK; <email@example.com>\ndate: 2014-02\ninstitute: 1Department of Health Sciences, University of Leicester, Leicester, UK; 2Imperial College Centre for Patient Safety and Service Quality (CPSSQ), London, UK; 3Institute of Work Psychology and School of Health and Related Research, University of Sheffield, Sheffield, UK; 4Aston Business School, Aston University, Birmingham, UK; 5Health Services Research Unit, University of Aberdeen, Aberdeen, UK; 6Department of Social and Policy Sciences, University of Bath, Bath, UK; 7National Association for Patient Participation, Surrey, UK; 8Lancaster University Management School, Lancaster, UK\nreferences:\ntitle: Culture and behaviour in the English National Health Service: overview of lessons from a large multimethod study\n\n# Introduction\n\nA commitment to delivering high-quality, safe healthcare has been a policy goal of governments worldwide for more than a decade, but progress in delivering on these aspirations has been modest:1 patients everywhere continue to suffer avoidable harm and substandard care.2 3 England's National Health Service (NHS) has not been immune to these problems. Despite some encouraging evidence of improvement in quality and safety,4 5 large and inexplicable variations in quality of care are evident across multiple domains and sectors of healthcare, from primary through to community and secondary care.6 7 England has also seen a number of high-profile scandals involving egregious failings in the quality and safety of individual providers. These include the case of Mid Staffordshire NHS Foundation Trust,8 the subject of a recently published public inquiry by Sir Robert Francis into how catastrophic failings in the quality and safety of care went undetected and uncorrected.9\n\nFrancis identified the causes of organisational degradation at Mid Staffordshire as systemic; he saw the underlying faults as institutional and cultural in character. He found significant weaknesses in NHS systems for oversight, accountability and influence for patient safety and quality of care. Central to his analysis was evidence of a large-scale failure of control and leadership at multiple levels, from what social scientists term the 'blunt end' of the system where decisions, policies, rules, regulations, resources and incentives are generated,10 through to the 'sharp end', often known as the 'frontline', where care is provided to patients. The distinction between the blunt end and the sharp end is of course a heuristic one; many within healthcare organisations function in hybrid positions as managers and practitioners. Nonetheless, it is useful to recognise how the blunt end, by shaping the environment where care is delivered, may create the 'latent conditions'11 that increase the risks of failure at the sharp end, but may equally generate organisational contexts that are conducive to providing high-quality care. Such contexts include culture: Francis blamed an 'insidious negative culture involving a tolerance of poor standards and a disengagement from managerial and leadership responsibilities'.8 Culture is, of course, a term that is widely used but notoriously escapes consensual definition.12 Many definitions of culture (including Schein's13 influential approach) nonetheless have in common an emphasis on the shared basic assumptions, norms, and values and repeated behaviours of particular groups into which new members are socialised, to the extent that culture becomes 'the way things are done around here'.\n\nThe findings of the Francis inquiry are depressingly familiar. England is not alone in experiencing organisational crises in healthcare; examples of failures in healthcare systems have occurred as far apart as New Zealand, the USA and the Netherlands. Several demonstrate precisely the same features as Mid Staffordshire, including long incubation periods during which warning signs were discounted, poor management systems, failure to respond to patient concerns, cultures of secrecy and protectionism, fragmentation of knowledge about problems and responsibility for addressing them, and cultures of denial of uncomfortable information.14 An important question thus concerns the extent to which the features of the Mid Staffordshire case might be symptoms of more widespread pathologies, given that other organisations in the NHS are exposed to the same institutional and regulatory environment. In this article, we offer lessons from a large multimethod research programme on culture and behaviour related to quality and safety in the NHS.\n\nThe research programme covered a critical period between 2010, following the initial inquiry into Mid Staffordshire15 and the White Paper on the NHS,16 and 2012, when the Health and Social Care Act was passed. The programme involved a large number of substudies using different methods to seek evidence from staff and patients throughout the English NHS, from large subsamples of NHS organisations, strategic level stakeholders, teams, and patient and carer organisations, and from detailed case studies. It was thus able to provide graduated levels of focus and multiple lenses. Each of the individual substudies will be reported separately, but there is considerable value in bringing the learning from them together holistically. In this article, we provide a synthesis across the studies to draw out high-level learning about culture and behaviour in NHS organisations; what influences culture and behaviour; and what needs to change to give effect to the vision of a safe, compassionate service in which patients and their families could have trust and confidence.\n\n# Methods\n\nWe conducted a large, mixed-method research programme involving seven separate substudies (<a href=\"#BMJQS2013001947TB1\" data-ref-type=\"table\">table 1<\/a>). The programme received ethical approval from an NHS Research Ethics Committee. In summary, primary data were drawn from:\n\n-   107 interviews with key, senior level stakeholders from across the NHS and beyond;\n\n-   197 interviews from the 'blunt end' (executive and board level) of NHS primary care and acute organisations through to the 'sharp end' (frontline clinicians) where staff care for patients;\n\n-   over 650\u2005h of ethnographic observation in hospital wards, primary care practices, and accident and emergency units;\n\n-   715 survey responses from patient and carer organisations;\n\n-   two focus groups and 10 interviews with patient and carer organisations;\n\n-   team process and performance data from 621 clinical teams, drawn from the acute, ambulance, mental health, primary care and community trust sectors;\n\n-   793 sets of minutes from the meetings of 71 NHS trust boards from multiple sectors over an 18-month period, including detailed analysis of eight boards' minutes.\n\nSummary of elements of the research programme\n\n<table>\n<thead>\n<tr>\n<th style=\"text-align: left;\">Study element<\/th>\n<th style=\"text-align: left;\">Participants and scheduling<\/th>\n<th style=\"text-align: left;\">Setting<\/th>\n<th style=\"text-align: left;\">Focus of research<\/th>\n<th style=\"text-align: left;\">Analytic approach<\/th>\n<\/tr>\n<\/thead>\n<tbody>\n<tr>\n<td style=\"text-align: left;\">1. Stakeholder interviews<\/td>\n<td style=\"text-align: left;\">107 semi-structured telephone interviews with those closely involved in quality and safety<\/td>\n<td style=\"text-align: left;\">Acute trusts, ambulance trusts, mental health trusts, community trusts, foundation trusts, primary care trusts, strategic health authorities, general practices and healthcare commissioning organisations<\/td>\n<td style=\"text-align: left;\">Understanding of vision of high-quality and safe care; what is required to make it happen; theories of change; plans to implement quality and safety improvement, enhance leadership and promote staff engagement; views on what quality improvement means, how it could best be secured, and obstacles<\/td>\n<td style=\"text-align: left;\">Analysis based on constant comparative method<br \/>\nUse of QSR NVivo 8 software<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">2. Ethnographic case studies: observations and interviews<\/td>\n<td style=\"text-align: left;\">Comparative case studies across seven purposively chosen cases<br \/>\n650\u2005h of observation; 197 semi-structured interviews with executive and board-level staff and frontline staff<\/td>\n<td style=\"text-align: left;\">Four hospital trusts; a quality improvement collaborative; a large-scale quality improvement programme involving dozens of organisations; one primary care provider involving a chain of practices<\/td>\n<td style=\"text-align: left;\">Assessing culture and behaviour in relation to quality, staff engagement with quality, leadership for quality, quality improvement, practical actions for promoting cultures of high-quality care<\/td>\n<td style=\"text-align: left;\">Analysis based on constant comparative method<br \/>\nCoding within and across cases, systematically searching for where clusters of codes formed a pattern<br \/>\nCombining data from interviews across cases and stakeholders to form a single dataset<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">3a. Patient and public involvement: survey<\/td>\n<td style=\"text-align: left;\">715 survey responses<br \/>\nCross-sectional<\/td>\n<td style=\"text-align: left;\">Patient participation groups<\/td>\n<td style=\"text-align: left;\">The survey consisted of 14 statements about patient experience. Open text box provided for each statement<\/td>\n<td style=\"text-align: left;\">Quantitative analysis\u2014largely descriptive<br \/>\nOpen-ended responses subject to content analysis to derive themes inductively<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">3b. Patient and public involvement: focus groups and interviews<\/td>\n<td style=\"text-align: left;\">Two focus groups and 10 interviews<\/td>\n<td style=\"text-align: left;\">Patient and carer organisations<\/td>\n<td style=\"text-align: left;\">Interpret the findings of the survey<br \/>\nAssessing views on obstacles to delivering improved quality and safety and greater accountability in the NHS<\/td>\n<td style=\"text-align: left;\">Qualitative analysis of key themes<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">4a. NHS staff and patient surveys: patient satisfaction survey data<\/td>\n<td style=\"text-align: left;\">165 acute trusts\u2014data from 2007, 2009, 2011<\/td>\n<td style=\"text-align: left;\">Acute trusts<\/td>\n<td style=\"text-align: left;\">Patient satisfaction came from the National Acute Inpatient Survey, using the data on patients' overall ratings of care<\/td>\n<td style=\"text-align: left;\">Descriptive statistics and paired sample t tests<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">4b. NHS staff and patient surveys: national staff survey data<\/td>\n<td style=\"text-align: left;\">309 NHS trusts from 2007, 2009, 2011 national staff survey<\/td>\n<td style=\"text-align: left;\">Primary care, ambulance, acute care and mental health trusts<\/td>\n<td style=\"text-align: left;\">Staff engagement, organisational climate, job satisfaction, manager support, job design, errors and reporting, work pressure, bullying, harassment and abuse, team working, training, appraisal, stress<\/td>\n<td style=\"text-align: left;\">Descriptive statistics and paired sample t tests<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">4c. NHS staff and patient surveys: outcome measures<\/td>\n<td style=\"text-align: left;\">2005\u20132009<\/td>\n<td style=\"text-align: left;\">Primary care, ambulance, acute care and mental health trusts<\/td>\n<td style=\"text-align: left;\">Patient mortality (acute sector only) (hospital standardised mortality ratio); quality of services and use of resources (Annual Health Check ratings by Healthcare Commission between 2005\/2006 and 2008\/2009); infection rates (MRSA) per 10000 bed days; staff absenteeism; staff turnover<\/td>\n<td style=\"text-align: left;\">Detailed correlation analysis between staff survey and inpatient survey; multiple and multilevel regression analysis, using HR practice variables to predict engagement; regression and ordinal logistic regression analysis to predict patient satisfaction, patient mortality, staff absenteeism, staff turnover, infection rates, and Annual Health Check ratings, controlling for trust type, size and location; latent growth curve modelling to predict outcomes<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">5. Clinical teams functioning, effectiveness and innovation<\/td>\n<td style=\"text-align: left;\">621 teams (4604 responses)<br \/>\nAston Team Performance Inventory<br \/>\nCross-sectional data with data on team changes collected from 388 teams (1299 individuals) 3\u2005months later<br \/>\nTeam performance data from team leaders\/external raters<\/td>\n<td style=\"text-align: left;\">51 trusts (13 acute, 17 mental health, 10 ambulance and 11 primary care trusts)<\/td>\n<td style=\"text-align: left;\">Team functioning:<br \/>\ntask design, team effort and skills, organisational support, resources, objectives, participation, creativity, conflict, reflexivity, task focus, leadership, satisfaction, attachment, effectiveness, inter-team relationships, innovation<br \/>\nLeaders'\/external raters' evaluations of effectiveness<br \/>\nInnovations introduced by teams<br \/>\nSources of frustration and resilience<\/td>\n<td style=\"text-align: left;\">Descriptive analysis, ANOVA, regression and relative importance analysis<br \/>\nAnalysis and ratings from domain relevant experts<br \/>\nOpen-ended responses subject to content analysis to derive the themes<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">6a. Objectives and team working of trust boards<\/td>\n<td style=\"text-align: left;\">34 boards (306 individuals) Administered processes section of Aston Team Performance Inventory<br \/>\nDetails of board objectives<\/td>\n<td style=\"text-align: left;\">Primary care, ambulance, secondary care and mental health trusts<\/td>\n<td style=\"text-align: left;\">Team processes and content: objectives, participation, reflexivity, task focus (lack of team) conflict, creativity and innovation<br \/>\nClarity and challenge of board objectives<\/td>\n<td style=\"text-align: left;\">Descriptive analysis, regression and relative importance analysis<br \/>\nAnalysis and ratings from domain relevant experts<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">6b. Trust board innovation<\/td>\n<td style=\"text-align: left;\">71 NHS trust boards<br \/>\n793 sets of minutes<br \/>\nMinutes from 18\u2005months of board meetings<\/td>\n<td style=\"text-align: left;\">Primary care, ambulance, secondary care and mental health trusts<\/td>\n<td style=\"text-align: left;\">Innovations introduced by boards and domain of focus (e.g. productivity, targets, organisational effectiveness, quality, safety, patient complaints, clinical effectiveness)<\/td>\n<td style=\"text-align: left;\">Analysis and ratings from domain relevant experts<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">6c Quality and safety in trust boards<\/td>\n<td style=\"text-align: left;\">Detailed analysis of minutes for eight boards<\/td>\n<td style=\"text-align: left;\">Primary care, ambulance, secondary care and mental health trusts<\/td>\n<td style=\"text-align: left;\">Board discussions of quality and safety<\/td>\n<td style=\"text-align: left;\">Ethnographic content analysis and summative analysis<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\nANOVA, analysis of variance; HR, human resources; MRSA, methicillin-resistant *Staphylococcus aureus*; NHS, National Health Service.\n\nWe did not use a formal protocol for integrating the findings across these studies,17 instead deploying a more interpretive, narrative approach.18 We engaged in extensive discussions as a team, and identified points of convergence and updated our analytic categories as we came closer to agreement. Given the size of our datasets, we are able to provide only very limited primary data in support of our analysis in this article; our focus is on high-level messages. Further details of the methods and the data are available in a longer report.19\n\n# Results\n\nOur synthesis of the findings across the substudies allowed many insights into the challenges of realising a vision of reliably safe, high-quality care across the NHS, and important learning about how improvement can best be secured.\n\n## Goal setting\n\nVirtually all those we interviewed (over 300 in total) were firmly committed to the ideal of a safe, high-quality health service for patients and to good patient experience. Many identified the values of compassion and care as at the heart of the mission for their organisations, and as their most deeply felt personal professional commitment. Our interviews, observations, surveys and documentary analysis were united in suggesting that, for organisations to succeed in delivering high-quality, safe care, they needed to have a clearly articulated vision, including explicit goals for quality and safety and a strategy for achieving them. Interviews and observations repeatedly emphasised the importance of clear goals in establishing and signposting priorities for improvement, motivating staff and ensuring resources were appropriately directed. Our survey evidence from the national patient satisfaction and national staff surveys (NSS) showed that patient satisfaction was highest in trusts that had clear goals at every level. Consistent with the findings of the Francis inquiry, boards of organisations were identified in interviews as particularly influential in setting the overall direction and demonstrating the commitment and organisational priority given to quality and safety.\n\nBut converting laudable aspirations for high-quality, safe and compassionate care into clear goals appeared challenging. Clarity about goals, how they could be achieved, and leadership for delivery were highly variable. Our questionnaire surveys of board members showed that they rarely stated clear board objectives that were challenging and measurable. The 621 frontline clinical teams we studied were generally even less clear about their objectives.\n\nA major challenge in creating unifying visions for patient safety and quality and setting clear objectives was the range, diversity and complexity of external expectations and requirements that NHS organisations faced. Board and executive teams described an institutional and regulatory environment that was populated by external agencies and actors who served different but overlapping functions. They reported that targets, standards, incentives and measures seemed to crowd in from multiple external sources; that the same information was required many times in different formats; and that answering to so many masters and producing data for so many external audiences was costly and distracting. The proliferation of externally set priorities and the number of different agencies and actors created what we termed 'priority thickets'\u2014dense patches of overlapping or disjointed goals that commanded very substantial attention and resources, but did not necessarily provide clear direction or facilitate the development of clear goals, internally coherent visions or strategies linked to local priorities.\n\nFaced with so many competing demands, some organisations tended to revert to a highly bureaucratised form of management, characterised by proliferation of rules, procedures and forms corresponding to externally imposed demands. Many of these seemed to be motivated mostly by a need to make displays of compliance,20 rather than by genuine efforts to make systems safer or of better quality. Much of this activity could be characterised as defensive and reactive. It was a source of frustration throughout organisations; frontline teams complained of 'blanket' policies which were seen as 'very prescriptive and not concentrated on clinical work'.\n\nWe also found considerable variability in how far organisations succeed in making their aspirations for high-quality care real: what we termed 'bright spots' and 'dark spots' were both evident, even within the same organisations. Bright spots included teams and individuals who demonstrated caring, compassion, cooperation and civility, and a commitment to learning and innovation. Direct observations found that in many settings patients were often treated with kindness and respect, systems functioned well, and staff were busy but knew what they were doing and why. Compliance with many standards of good practice, such as hygiene and equipment counting, was observed to be very good in many cases.\n\nThough much care was of such high quality as to be inspiring, substandard care or 'dark spots' were also evident. Dark spots were found where staff were challenged to provide quality care, were harried or distracted, or were preoccupied with bureaucracy. Across our interviews, surveys and observations we found evidence of staff and patient concern about variability in quality of care, and a lack of confidence that care would be reliably good. Interviews and surveys with patient and carer groups suggested that patients and their carers were often concerned about quality and safety. Vulnerable patients, including older patients, young patients or those who lacked the ability to 'speak up', were reported to be at risk of being left to 'fend for themselves' or 'being forgotten'. Our observations in clinical areas and our interviews confirmed that inconsistency was a feature of many settings. For example, many staff spoke to patients politely and with kindness, but some others were brusque, impatient or discourteous. Some senior clinical nursing staff highlighted their concern at what they saw as a tendency towards task-focused rather than person-centred care.\n\nFurther evidence of the challenges of realising a vision of consistently high-quality safe care came from our analysis of the NHS Staff Survey and inpatient survey data over the period 2007\u20132011 (<a href=\"#BMJQS2013001947TB2\" data-ref-type=\"table\">tables 2<\/a> and <a href=\"#BMJQS2013001947TB3\" data-ref-type=\"table\">3<\/a>). This suggested that there had been improvements in scores relating to quality and safety reported by patients and staff nationally between 2007 and 2009, but subsequently these improvements stalled or went into reverse. Some of the plateauing may reflect a natural maximum level being reached. For example, the percentage of staff receiving health and safety training increased from 2007 to 2009, and appears to remain relatively constant in 2011. Likewise, levels of job satisfaction increased from 2007 to a moderately high level in 2009, and then stayed approximately the same in 2011. However, measures on the staff survey relating to error and incident reporting, blame cultures and improvements following incidents, where there was headroom for improvement, appeared to have shown only very modest gains. The number of staff working paid extra hours has decreased consistently, but since 2009 the number working unpaid extra hours has increased sharply. The percentage of staff receiving training in infection control related issues increased from 2007 to 2009, but fell in 2011.\n\nChanges in the National Staff Survey 2007\u20132011 (NHS trusts in England)\n\n|  | 2007 | 2009 | 2011 | Change 2007\u20132009 | Change 2009\u20132011 | Standard deviation (2007) |\n|:---|:---|:---|:---|:---|:---|:---|\n| 'I have adequate materials, supplies and equipment to do my work' | 3.22 | 3.36 | 3.38 | +0.14\\*\\* | +0.02\\* | 0.18 |\n| 'There are enough staff at this Trust for me to do my job properly' | 2.61 | 2.78 | 2.72 | +0.17\\*\\* | \u22120.06\\*\\* | 0.17 |\n| 'I do not have time to carry out all my work' | 3.30 | 3.26 | 3.25 | \u22120.04\\*\\* | \u22120.01 | 0.21 |\n\nResults are based on the 309 NHS trusts in England with data from all 3\u2005years shown. p values are based on paired sample t tests. Responses were on a 1\u20135 scale with 5 indicating greater agreement with statements; an increase of 0.10 is equivalent to 10% of respondents moving up one category of response, for example, from 'neither agree nor disagree' to 'agree'.\n\n\\*p\\<0.05; \\*\\*p\\<0.01.\n\nNHS, National Health Service.\n\nChanges in the National Staff Survey and Acute Inpatient Survey 2007\u20132011 (NHS trusts in England)\n\n|  | 2007 | 2009 | 2011 | Change 2007\u20132009 | Change 2009\u20132011 | Standard deviation (2007) |\n|:---|----|----|----|----|----|----|\n| Staff survey |  |  |  |  |  |  |\n| \u2003'My trust encourages us to report errors, near misses or incidents' | 3.84 | 3.93 | 3.94 | +0.09\\*\\* | +0.01\\* | 0.11 |\n| \u2003'My trust treats reports of errors, near misses or incidents confidentially' | 3.55 | 3.63 | 3.66 | +0.08\\*\\* | +0.03\\*\\* | 0.10 |\n| \u2003'My trust blames or punishes people who are involved in errors, near misses or incidents' | 2.75 | 2.67 | 2.68 | \u22120.08\\*\\* | +0.01 | 0.12 |\n| \u2003'When errors, near misses or incidents are reported, my trust takes action to ensure that they do not happen again' | 3.47 | 3.54 | 3.57 | +0.07\\*\\* | +0.03\\*\\* | 0.13 |\n| Acute inpatient survey |  |  |  |  |  |  |\n| \u2003In your opinion, how clean was the hospital room or ward that you were in? | 3.45 | 3.60 | 3.63 | +0.15\\*\\* | +0.03\\*\\* | 0.13 |\n| \u2003As far as you know, did doctors wash or clean their hands between touching patients? (% of positive responses) | 53 | 59 | 57 | +6\\*\\* | \u22122\\* | 6 |\n\nStaff survey results are based on the 309 NHS trusts in England with data from all 3\u2005years shown. p values are based on paired sample t tests. Staff survey responses were on a 1\u20135 scale with 5 indicating greater agreement with statements; an increase of 0.10 is equivalent to 10% of respondents moving up one category of response, for example, from 'neither agree nor disagree' to 'agree'. Inpatient survey results are based on the 157 NHS acute trusts in England with data from all 3\u2005years shown. Inpatient survey responses were on a 1\u20134 scale, with 4 indicating greater agreement with statements.\n\n\\*p\\<0.05; \\*\\*p\\<0.01.\n\nNHS, National Health Service.\n\n## Variability in intelligence\n\nA major challenge to achieving goals relating to quality and safety was that of ensuring that high-quality intelligence was available to organisations, teams, and individuals about how well they were doing and where the deficits and risks in organisational systems lay. NHS organisations that we studied were putting considerable time, effort and resources into data collection and monitoring systems. They typically used a combination of routinely collected data, specific data collection initiatives, and sporadic sources such as spot checks and audits. To a varying extent they also drew on feedback provided by clinical staff and patients as a means of assessing trends. However, the degree to which data collection efforts translated into actionable knowledge, and then into effective organisational responses, differed markedly between organisations.\n\nSome behaviours in relation to data gathering might be described as 'problem-sensing'; other, less positive behaviours were 'comfort-seeking'. Problem-sensing involved actively seeking out weaknesses in organisational systems, and it made use of multiple sources of data\u2014not just mandated measures, but also softer intelligence. Soft intelligence could be gathered in many ways, including active listening to patients and staff; informal, unannounced visits to clinical areas; and techniques such as 'mystery shoppers', shadowing of staff, and swapping roles for a short period. While sometimes discomfiting, this less routinely gathered knowledge enabled fresh, more penetrating insights to complement quantitative data. Senior teams displaying problem-sensing behaviours tended to be cautious about being self-congratulatory; perhaps more importantly, when they did uncover problems, they often used strategies that went beyond merely sanctioning staff at the sharp end, making more holistic efforts to strengthen their organisations and teams.\n\nComfort-seeking behaviours are defined here as being focused on external impression management and seeking reassurance that all was well; consequently, what was available to organisations was data, but not intelligence. Serious blind spots could arise when organisations used a very limited range of methods for gathering data, were preoccupied with demonstrating compliance with external expectations, failed to listen to negative signals from staff or lacked knowledge of the real issues at the frontline. Comfort-seeking tended to demonstrate preoccupation with positive news and results from staff, and could lead to concerns and critical comments being dismissed as 'whining' or disruptive behaviour. When comfort-seeking was the predominant behaviour, data collection activities were prone to being treated by sharp-end staff as wearisome and fruitless accountability exercises. Some staff reported that they felt the main purpose of much data collection was to allow individuals to be blamed if something did go wrong, not to make the system safer.\n\n## Variability in systems\n\nInterviews, observations and surveys showed that when staff had access to appropriate resources, perceived that staffing levels were adequate with the right skill mix, and had systems that functioned effectively, they felt that they could complete their work successfully, could explore new ways of improving quality and could develop reflective practices. This reinforced their levels of motivation and morale in a virtuous circle. But deficits in systems often obstructed and frustrated well-motivated staff in their mission to provide good care for patients. Our analysis of questionnaire reports from 621 clinical teams showed that many staff felt unable to achieve their goals for patients because of organisational factors outside their control. Observations showed that staff wasted time working with poorly designed IT systems, negotiating clinical pathways with obstructions and gaps, and battling with multiple professional groups and subsystems (e.g. pharmacy, microbiology and imaging, and many others) that did not operate in integrated ways. We also found evidence of problematic handovers between shifts, departments and teams, team conflict and a diffusion of responsibility relating to particular patients. Patient and carer groups reported discontinuities in care between institutional boundaries and even within single organisations. These 'responsibility cordons' left patients variously ill informed, distressed and disappointed, and sometimes in danger.\n\nStaff at the sharp end were very often aware of systems problems but felt powerless to bring about change. Changes within organisations, uncertainty about priorities, poor systems, heavy workloads and staff shortages were all blamed for staff feeling they lacked support, further reducing their motivation and morale. Given that many systems required significant improvement, it was disappointing that we found a clear trend of decreasing levels of board innovation, especially in relation to quality and safety.\n\nWe defined innovation as the intentional introduction of processes and procedures, new to the unit of adoption (team or organisation) and designed to significantly benefit the unit of adoption, staff, patients or the wider public. An analysis of board minutes from 71 NHS Trusts covering an 18-month period between January 2010 and June 2011 identified a total of 144 innovations that were implemented in organisations, representing an average of only 1\u20133 per organisation. More than half were focused on increasing productivity (73), with very few related to safety (14). The largest number of innovations (62) was identified between January and June 2010, followed by 56 innovations between July and December 2010. Only 26 innovations were identified in data covering the time between January and June 2011. Separately, analysis of 4976 responses to open-ended questions in our survey of 486 clinical teams identified 183 innovations over a 6-month period. This also suggested relatively low rates of innovation among frontline teams, though many of the solutions they did devise were ingenious and resourceful. The largest number of frontline staff innovations was focused on enhancing quality of patient care; fewer aimed at improving administrative effectiveness, and the smallest number concerned staff wellbeing.\n\nMany organisations were using specific quality improvement methods to achieve change, including Plan\u2013Do\u2013Study\u2013Act cycles, Collaboratives, Lean, Six Sigma, and Productive Ward (an NHS programme to support ward teams in reviewing the processes and environment used to provide patient care21). Some organisations also used wider techniques to improve quality, including organisation-level campaigns. Great enthusiasm for these approaches was often reported by those leading improvement efforts, but we also sometimes observed a tendency towards uncritical or indiscriminate use, and some evidence of 'magical thinking' ('this initiative will solve many problems easily and quickly'). Frontline staff who had to implement these initiatives were often not consulted or adequately informed about their purpose and implementation, and sometimes initiatives were abandoned or forgotten after a short period of intense activity. In some cases, there was insufficient acknowledgement of the effort, expertise and investment required to make such approaches work, and substantial problems with quality of data collection and interpretation.\n\n## Culture and behaviour\n\nLeadership was important for setting mission, direction and tone. Our observations, interviews and surveys all emphasised the importance of high-quality management in ensuring positive, innovative and caring cultures at the sharp end of care. Some senior teams encouraged and enabled frontline teams to address challenges and to innovate, but recognised that, along with demanding personal accountability from staff, they also needed to fix systems problems that prevented staff from functioning well. A strong focus by executive and board teams on their own role in identifying and addressing systems problems was powerful in supporting cultural change that delivered benefits for patients, and our observations and interviews identified many examples of impressive gains being made by the sharp and blunt ends working together around unifying goals.\n\nNevertheless, an important consequence of the failure to clarify goals, to gather appropriate intelligence or to address systems deficits was the existence of frequent misalignments between the ways the blunt end and the sharp end of organisations conceived of quality and safety problems and their solutions. For sharp-end staff, threats to safety and quality were identified as weaknesses in systems, failures of reliability, suboptimal staffing, inadequate resources and poor leadership. Lack of support, appreciation and respect, and not being consulted and listened to were seen as endemic problems by staff in some organisations. In contrast, some senior managers\u2014particularly those engaged in comfort seeking\u2014tended to see frontline staff behaviour and culture as the cause of quality problems. In consequence, those at the blunt and those at the sharp end often did not agree on the causes of variation in quality and safety and, therefore, on how they should be addressed.\n\nWe also found substantial variation in the quality of management. Our analyses of NSS data showed that hospital standardised mortality ratios were inversely associated with positive and supportive organisational climates. Higher levels of staff engagement and health and wellbeing were associated with lower levels of mortality, as were staff reporting support from line managers, well structured appraisals (e.g. agreeing objectives, ensuring the individual feels valued, respected and supported), and opportunities to influence and contribute to improvements at work. NSS data also showed that staff perceptions of the supportiveness of their immediate managers, the extent of staff positive feeling, staff satisfaction and staff commitment were associated with other important outcomes, including patient satisfaction. In places where staff reported high work pressure, patients on the national surveys also reported too few nurses, insufficient support, and problems with information, privacy and respect. In trusts with poor staff health and wellbeing, high injury rates, and a high level of staff intention to quit their jobs, patients reported that they were generally less satisfied, and Care Quality Commission ratings described poorer care and poorer use of resources. These findings were consistent across trust types (primary care, ambulance, mental health, community and acute).\n\nAlso concerning was evidence that though team-working seemed well established and widespread on the surface, there was a surprising lack of clarity about team purpose, objectives, membership, leadership and performance among many teams. Our survey of 621 clinical teams demonstrated that team inputs and team processes were significantly associated with the effective provision of good-quality care, but senior managers were sometimes unable to identify teams and team leaders. When team leaders were identified, they were often confused about who their team members were. Team members themselves had low agreement about who was part of their team. Factors associated with successful teams were the effort and skills of team members, resources made available and good processes. Clarity and agreement about team objectives were key to clinical team effectiveness, along with a participative approach to decision-making that engaged all team members. Teams who regularly took time out to reflect on their objectives, how they were going about achieving these and how their performance needed to change were particularly likely to be more effective and innovative.\n\n# Discussion\n\nThis large mixed-method study identified many 'bright spots' of excellent caring and practice and high-quality innovation across the NHS, but also considerable inconsistency. Though Mid Staffordshire may have been one particularly 'dark spot' in the NHS, organisations throughout the NHS are likely to have at least some shadows: there was little confidence that care could be relied upon to be good at all times in all parts of organisations, and we found evidence of structural and cultural threats to quality and safety. Our analysis points to how things may improve. First, clear and explicit goals that are coherent from ward to Whitehall are essential. Second, organisations need intelligence: they need to know how well they are really doing as an organisation, and where they need to improve. This means actively seeking uncomfortable and challenging information from patients and staff, rather than relying solely on formal data collection against narrow performance indicators that may not give a fully rounded picture of quality of care. Third, organisations must constantly review, strengthen and improve their systems. System improvement and strengthening may be needed at many different levels, from smoothing clinical pathways to improving communication, teamwork, assuring that clinical areas are adequately staffed with the right kinds of skills, and ensuring personal development, equipment standardisation, and training. Organisations also need to focus on developing cultures that are person centred\u2014not just task focused\u2014by valuing and building on the excellent care and commitment delivered by many staff throughout the NHS. This involves modelling and reinforcing values and behaviours that underpin high-quality care, patient safety and positive patient experience from the blunt end to the sharp end of the whole system.\n\nOur work involving a very large number of organisations confirms that achieving quality and safety in NHS organisations requires a robust strategy and unifying vision. National leadership sets the tone, signals importance, legitimises, and creates accountability mechanisms. Yet the Francis public inquiry showed that a major problem for Mid Staffordshire was the large number of different agencies and bodies with a say in the NHS. This contributed to fragmentation, multiple competing pressures, ambiguity and diffusion of responsibility. Our work similarly demonstrates that proliferation of external agencies and expectations creates conflicts, distraction and confusion from the blunt to the sharp end of organisations about where resources and attention should be directed. Where incentives and external expectations conflict, compete or fail to cohere, the ability of organisations to set themselves clear, internally valued goals for achieving their aspirations is weakened.22\n\nIn a distributed and complex system such as the NHS, failures are least likely when the goals are clear and uniting, and when appropriate, sensitively designed regimes of control and support are found at every level: from policymaking, through the layers of formal regulatory systems, the institutional environment, individual organisations, teams and practitioners, through to patients' experiences.23 Coherence of national direction is therefore essential to avoid dispersing responsibility and accountability, and creating confusing messages and signals.24 As new bodies move forward, including NHS England and the Clinical Commissioning Groups, it is important that they avoid creating further competing priorities, and instead ensure focus and coherence.\n\nNational leadership needs to be matched by high-quality leadership across multiple organisational levels underpinned by clear, patient-focused goals and objectives. The role of organisational boards in securing the quality and safety of health services has become an increasing focus of academic and policy interest,25 26 not least because of evidence of the link between leadership from the top and the priority and resources given to quality25 and clinician engagement.27 But we found worrying evidence of NHS trust boards failing to set clear goals for themselves as boards and for their organisations. Goals do need to be set, and they should be limited in number (to identify priorities while avoiding the creation of priority thickets) and known not only by all board members but (if appropriate) more widely within their trusts. Goals should be shaped by the need to promote quality and safety, to ensure sound financial performance, and to value dignity and respect for patients. They should provide a framework for objectives at all levels of trusts, from senior management to clinical teams at the sharp end. They should be framed to encourage innovation at all levels, and quality and safety of patient care must be overriding. Not all innovations need to be grand and over-arching: fixing (apparently) small problems may result in major gains.28\n\nThe Francis public inquiry showed that discounting of warning signs of deterioration was a key feature of board and executive behaviour at Mid Staffordshire. Those in senior positions appear to have developed 'blindsight'\u2014a way of not seeing what was going wrong. Our work confirms the importance of high-quality intelligence (not just data) and making that intelligence actionable. But we found sobering evidence that NHS organisations are not always smart with intelligence, and need to gear more towards problem-sensing rather than comfort-seeking. At the national level, care needs to be taken to ensure that the number of measures organisations are expected to report externally is well managed,29 and that measures are aligned with local priorities, avoid imposing excessive burdens, generate accurate intelligence,30 and most of all, are useful for informing improvement locally. Thus the right intelligence needs to be gathered, interpreted correctly and fed back clearly to staff at the sharp end of care, so that they consolidate and improve their performance.31\n\nOrganisations need to be especially alert to the possibility of blind spots where they are unaware of problems. They should use multiple strategies to generate intelligence and undertake self-assessment32 of local culture and behaviours\u2014not just rely on mandated measures\u2014and use a range of techniques for hearing the patient's voice and the voice and insights of those at the sharp end of care. Consistent with Francis' findings, good management is as important as good leadership in our analysis: the wellbeing of staff is closely linked to the wellbeing of patients, and staff engagement is a key predictor of a wide range of outcomes in NHS trusts. Achieving high levels of engagement is only possible in cultures that are generally positive, when staff feel valued, respected and supported, and when relationships are good between managers, staff, teams and departments and across institutional boundaries. Staff experience frustration and conflict when asked to work in systems that do not effectively serve them, or the patients they care for; these system defects include staff shortages or inappropriate skill mix to address the needs of specific clinical areas. Our analysis suggests that improving culture, behaviour and systems requires system improvement and better communication between the blunt end and sharp end. This needs to be sustained, intense, mutually respectful and focused on achieving a shared understanding of quality problems and joint working to put them right. Trusts can develop these cultures by using specific strategies (<a href=\"#bx1\" data-ref-type=\"boxed-text\">box 1<\/a>), while recognising the complexities of trying purposefully to engineer culture.12\n\n###### Strategies for creating positive cultures\n\nSenior leaders should:\n\n1.  Continually reinforce an inspiring vision of the work of their organisations\n\n2.  Promote staff health and wellbeing\n\n3.  Listen to staff and encourage them to be involved in decision making, problem solving and innovation at all levels\n\n4.  Provide staff with helpful feedback on how they are doing and celebrate good performance\n\n5.  Take effective, supportive action to address system problems and other challenges when improvement is needed\n\n6.  Develop and model excellent teamwork\n\n7.  Make sure that staff feel safe, supported, respected and valued at work.35\n\nThis article has some limitations. In the available space, we have not been able to provide full details of methods or data from this unusually large research programme. Instead we have sought to provide an overview of the key findings of the component studies. Our synthesis of findings was interpretive and narrative, and did not use a formal protocol. Others might reach somewhat dissimilar conclusions or interpretations of our data.33 However, we believe that our careful scrutiny of the data, extensive discussions, and detailed analysis of themes have enabled us to produce a powerful, robust and rich picture. Future work should assess the generalisability of these findings in other contexts, including the other countries of the UK.\n\n# Conclusions\n\nThis very large-scale research programme suggests that there is room for improvement in the quality and safety of care offered by the NHS, and that this improvement can build on the progress already made. Trusts need continually to refresh, reinforce and model an inspiring vision that keeps the patient at the centre. It is essential to commit to an ethic of learning and honesty,34 to work continually to improve organisational systems, and to nurture the core values of compassion, patient dignity and patient safety through high-quality leadership. This implies equal attention to systems, cultures and behaviours: setting coherent and challenging goals and monitoring progress towards them; empowering staff to provide high-quality care and providing them with the means to achieve this through routine practice and innovation; and exemplifying and encouraging sound behaviours.\n\nWe thank all of the many organisations and individuals who participated in this research programme for their generosity and support. We thank our collaborators and the advisory group for the programme.\n\n# References","meta":{"dup_signals":{"dup_doc_count":137,"dup_dump_count":58,"dup_details":{"curated_sources":2,"2023-50":4,"2023-40":1,"2023-14":3,"2023-06":4,"2022-49":2,"2022-40":2,"2022-33":2,"2022-27":2,"2022-21":4,"2022-05":4,"2021-49":2,"2021-43":3,"2021-39":1,"2021-25":2,"2021-21":1,"2021-17":5,"2021-10":1,"2021-04":2,"2020-50":2,"2020-45":5,"2020-40":2,"2020-34":2,"2020-29":2,"2020-24":2,"2020-10":1,"2020-05":5,"2019-51":1,"2019-47":3,"2019-43":2,"2019-39":3,"2019-35":2,"2019-30":2,"2019-26":2,"2019-22":1,"2019-18":2,"2019-04":3,"2018-47":1,"2018-39":1,"2018-30":3,"2018-26":1,"2018-22":2,"2018-17":2,"2018-13":2,"2018-09":1,"2018-05":4,"2017-47":4,"2017-39":6,"2017-34":1,"2017-30":2,"2017-26":3,"2017-22":3,"2017-17":2,"2017-09":2,"2024-22":2,"2024-18":2,"2024-10":2,"2017-13":1,"2024-30":1}},"file":"PMC3913222"},"subset":"pubmed_central"}
{"text":"abstract: CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci, together with *cas* (CRISPR\u2013associated) genes, form the CRISPR\/Cas adaptive immune system, a primary defense strategy that eubacteria and archaea mobilize against foreign nucleic acids, including phages and conjugative plasmids. Short spacer sequences separated by the repeats are derived from foreign DNA and direct interference to future infections. The availability of hundreds of shotgun metagenomic datasets from the Human Microbiome Project (HMP) enables us to explore the distribution and diversity of known CRISPRs in human-associated microbial communities and to discover new CRISPRs. We propose a targeted assembly strategy to reconstruct CRISPR arrays, which whole-metagenome assemblies fail to identify. For each known CRISPR type (identified from reference genomes), we use its direct repeat consensus sequence to recruit reads from each HMP dataset and then assemble the recruited reads into CRISPR loci; the unique spacer sequences can then be extracted for analysis. We also identified novel CRISPRs or new CRISPR variants in contigs from whole-metagenome assemblies and used targeted assembly to more comprehensively identify these CRISPRs across samples. We observed that the distributions of CRISPRs (including 64 known and 86 novel ones) are largely body-site specific. We provide detailed analysis of several CRISPR loci, including novel CRISPRs. For example, known streptococcal CRISPRs were identified in most oral microbiomes, totaling \u223c8,000 unique spacers: samples resampled from the same individual and oral site shared the most spacers; different oral sites from the same individual shared significantly fewer, while different individuals had almost no common spacers, indicating the impact of subtle niche differences on the evolution of CRISPR defenses. We further demonstrate potential applications of CRISPRs to the tracing of rare species and the virus exposure of individuals. This work indicates the importance of effective identification and characterization of CRISPR loci to the study of the dynamic ecology of microbiomes.\nauthor: Mina Rho; Yu-Wei Wu; Haixu Tang; Thomas G. Doak; Yuzhen Ye\\* E-mail: <firstname.lastname@example.com>[^1]\ndate: 2012-06\ninstitute: 1School of Informatics and Computing, Indiana University, Bloomington, Indiana, United States of America; 2Center for Genomics and Bioinformatics, Indiana University, Bloomington, Indiana, United States of America; 3Department of Biology, Indiana University, Bloomington, Indiana, United States of America; University of Toronto, Canada\nreferences:\ntitle: Diverse CRISPRs Evolving in Human Microbiomes\n\n# Introduction\n\nCRISPRs, together with *cas* genes (CRISPR-associated genes), provide acquired resistance against viruses and conjugative plasmids \\[1\\], \\[2\\], and are found in most archaeal (\u223c90%) and bacterial (\u223c40%) genomes \\[3\\], \\[4\\], \\[5\\]. CRISPR arrays consist of 24\u201347 bp direct repeats, separated by unique sequences (spacers) that are acquired from viral or plasmid genomes \\[6\\]. Even though some CRISPR arrays may contain hundreds of spacers (an extreme case is the CRISPR array in the *Haliangium ochraceum* DSM 14365 genome, which has 588 copies of its repeat), they tend to be much smaller, generally with dozens of spacers. The repeat sequences of some CRISPRs are partially palindromic, and have stable, highly conserved RNA secondary structures, while others lack detectable structures \\[7\\].\n\nCRISPR arrays are usually adjacent to *cas* genes, which encode a large and heterogeneous family of proteins with functional domains typical of nucleases, helicases, polymerases, and polynucleotide-binding proteins. CRISPR\/Cas systems commonly use repeat and spacer-derived short guide CRISPR RNAs (crRNAs) to silence foreign nucleic acids in a sequence-specific manner \\[8\\], . CRISPR\/Cas defense pathways involve several steps, including integration of viral or plasmid DNA-derived spacers into the CRISPR array, expression of short crRNAs consisting of unique single repeat-spacer units, and interference with invading foreign genomes at both the DNA and RNA levels, by mechanisms that are not yet fully understood \\[8\\], \\[10\\]. The diversity of *cas* genes suggests that multiple pathways have arisen to use the basic information contained in the repeat-spacer units in diverse defense mechanisms. The CRISPR components are evolutionarily closely linked and potentially evolve simultaneously as an intact locus\u2014sequence analysis reveals that the direct repeats in CRISPR locus and the linked *cas* genes co-evolve under analogous evolutionary pressure \\[11\\].\n\nPrevious studies have shown that CRISPR loci are very diverse and abundant in the genomes of bacteria and archaea. In addition, it has been shown that CRISPR loci with the same repeat sequence and *cas* gene set can be found in multiple bacterial species, implying horizontal gene transfer (HGT) \\[12\\]. Moreover, CRISPR loci can change their spacer content rapidly, as a result of interactions between viruses (or plasmids) and bacteria: several metagenomic studies investigating host-virus population dynamics have shown that CRISPR loci evolve in response to viral predation and that CRISPR spacer content and sequential order provide both historically and geographically insights \\[13\\], \\[14\\], \\[15\\], \\[16\\]\u2014essentially, epidemiology.\n\nAs a reflection of the infectious dynamics of microbial communities, the study of CRISPRs is an essential compliment to the study of the human microbiome, encompassing both disease ecology and ecological immunology \\[17\\]. Infectious disease works to maintain both species diversity \\[18\\], \\[19\\] and genotypic diversity \\[20\\] within a species, as has recently been shown for marine microbiomes \\[21\\], \\[22\\]. As such, infectious agents may be at least partially responsible for the amazing species diversity and turnover found throughout the human microbiome \\[23\\]. The ability of CRISPR loci to prevent plasmid spread is medically relevant, in that the exchange of conjugative elements is perhaps the dominant mechanism by which antibiotic resistance genes (notably multi-drug resistance) move within a biome, and by which pathogens acquire resistance \\[24\\]; CRISPR activities could be expected to retard this exchange (*e.g.* \\[25\\]).\n\nCRISPR composition in human microbial communities, the relative rate of CRISPR locus change, or how CRISPR loci vary between different body sites and between the microbiota of different individuals are less studied, as compared to other environments. A recent analysis of streptococcal CRISPRs from human saliva, in which CRISPR spacers and repeats were amplified from salivary DNA, using the conserved streptococcal CRISPR repeat sequence for priming, revealed substantial spacer sequence diversity within and between subjects over time \\[26\\], which is imagined to reflect the dynamics of phage and other infectious agents in the human mouth \\[2\\].\n\nThe availability of more than 700 shotgun metagenomic datasets from the Human Microbiome Project (HMP) enables us to explore the distribution and diversity of many more CRISPRs, and to discover new ones, across different body sites, in a systematic manner. We developed a targeted assembly strategy (see <a href=\"#pgen-1002441-g001\" data-ref-type=\"fig\">Figure 1<\/a>) to better identify CRISPRs in shotgun metagenomic sequences, as whole-metagenomic assembly failed to reconstruct many CRISPRs that otherwise could be identified. All of the programs available to date \\[27\\], \\[28\\], \\[29\\], \\[30\\] are designed to find CRISPRS from assembled contigs that are sufficiently long to contain at least partial CRISPR loci; however, it is very difficult to assemble metagenome reads into contigs containing CRISPR loci, because of their repeated structures. We thus needed to collect sequencing reads associated with CRISPRs and assemble them specifically. For known CRISPRs (identified in reference genomes), we identified consensus sequences of CRISPR repeats, collected the reads containing these sequences, and assembled these reads into CRISPR contigs. We also identified CRISPRs from the whole-metagenome assemblies, and for the novel CRISPRs or new CRISPR variants (that are not seen in the reference genomes), applied the same assembly strategy to achieve a more comprehensive identification of the novel CRISPRs across the samples. This approach allows us to study the evolution of CRISPRs in human microbiomes.\n\n# Results\n\nWe identified and selected 64 known CRISPRs\u2014including the streptococcal CRISPR\u2014from complete and draft bacterial genomes and 86 novel CRISPRs from the 751 HMP whole-metagenome assemblies, using metaCRT and CRISPRAlign (see <a href=\"#s4\" data-ref-type=\"sec\">Methods<\/a>). For each selected CRISPR, we then applied the targeted assembly approach (for each CRISPR, first pool the reads that contain the repeat, and then assemble the pooled reads only; see <a href=\"#s4\" data-ref-type=\"sec\">Methods<\/a> for a validation of the targeted assembly approach using simulated datasets) to achieve a more comprehensive characterization of the CRISPR loci in the human microbiome shotgun datasets. Below we provide detailed analysis of the targeted assembly approach, and the resulting CRISPR loci (listed in <a href=\"#pgen-1002441-t001\" data-ref-type=\"table\">Table 1<\/a> and <a href=\"#pgen.1002441.s008\" data-ref-type=\"supplementary-material\">Tables S1<\/a> and <a href=\"#pgen.1002441.s009\" data-ref-type=\"supplementary-material\">S2<\/a>).\n\n10.1371\/journal.pgen.1002441.t001\n\n###### List of selected CRISPRs discussed in the paper.\n\n<img src=\"pgen.1002441.t001\" id=\"pgen-1002441-t001-1\" \/>\n\n| ID<a href=\"#nt101\" data-ref-type=\"table-fn\">a<\/a> | Species (or HMP sample ID)Consensus sequence of the CRISPR repeats |\n|:---|:--:|\n| **Known** |  |\n| AhydrL30 | *Anaerococcus hydrogenalis* DSM 7454 (NZ_ABXA01000037)ATTTCAATACATCTAATGTTATTAATCAAC |\n| AlactL29 | *Anaerococcus lactolyticus* ATCC 51172 (NZ_ABYO01000191)AGGATCATCCCCGCTTGTGCGGGTACAAC |\n| BcoprL32 | *Bacteroides coprophilus* DSM 18228 (NZ_ACBW01000156)GTCGCACCCTGCGTGGGTGCGTGGATTGAAAC |\n| FalocL36 | *Filifactor alocis* ATCC 35896 (NZ_GG745527)TTTGAGAGTAGTGTAATTTCATATGGTAGTCAAAC |\n| GhaemL36 | *Gemella haemolysans* ATCC 10379 (EQ973306)GTTTGAGAGATATGTAAATTTTGAATTCTACAAAAC |\n| LcrisL29 | *Lactobacillus crispatus* ST1 (NC_014106)AGGATCACCTCCACATACGTGGAGAATAC |\n| LjassL36 | *Lactobacillus gasseri* JV-V03 (NZ_ACGO01000006)GTTTTAGATGGTTGTTAGATCAATAAGGTTTAGATC |\n| LjensL36 | *Lactobacillus jensenii* 115-3-CHN (NZ_GG704745)GTTTTAGAAGGTTGTTAAATCAGTAAGTTGAAAAAC |\n| Neis_t014_L28 | *Neisseria* sp. oral taxon 014 str. F0314 (NZ_GL349412)GTTACCTGCCGCACAGGCAGCTTAGAAA |\n| Neis_t014_L36 | *Neisseria* sp. oral taxon 014 str. F0314 (NZ_GL349412)GTTGTAGCTCCCTTTCTCATTTCGCAGTGCTACAAT |\n| PacneL29 | *Propionibacterium acnes* J139 (NZ_ADFS01000004)GTATTCCCCGCCTATGCGGGGGTGAGCCC |\n| PpropL29 | *Pelobacter propionicus* DSM 2379 (NC_008609)CGGTTCATCCCCGCGCATGCGGGGAACAC |\n| SmutaL36 | *Streptococcus mutans* NN2025GTTTTAGAGCTGTGTTGTTTCGAATGGTTCCAAAAC |\n| **Novel** |  |\n| SRS012279L38 | SRS012279 (dataset from a tongue dorsum sample)TATAAAAGAAGAGAATCCAGTAGAATAAGGATTGAAAC |\n| SRS018394L37 | SRS018394L37 (dataset from a supragingival plaque sample)GTATTGAAGGTCATCCATTTATAACAAGGTTTAAAAC |\n| SRS023604L36 | SRS023604 (dataset from a posterior fornix sample)GTTTGAGAGTAGTGTAATTTATGAAGGTACTAAAAC |\n\nThe IDs of the CRISPRs are assigned using the following rules: 1) If a CRISPR (*e.g.*, SmutaL36) is identified from a known complete\/draft genome with species name (for SmutaL36, the genome is *Streptococcus mutans* NN2025), its ID uses five letters from the species name (*i.e.*, Smuta) followed by the length of the repeats (length of 36 is shown as L36); 2) If a CRISPR (Neis_t014_L28) is identified from a known complete\/draft genome that has only general genus information (*e.g.*, *Neisseria sp*. oral taxon 014 str. F0314), then its ID is four letters from the genus name, followed by the taxon ID, and the length of the repeats; and 3) the CRISPRs identified in the HMP datasets are named as the ID of the datasets followed by the length of repeat.\n\n## Targeted assembly improves the characterization of CRISPRs\n\nWe first asked if our targeted assembly strategy helps to identify CRISPR elements from metagenomic datasets, and found that it greatly improved detection (see comparison in <a href=\"#pgen-1002441-t002\" data-ref-type=\"table\">Table 2<\/a>). The improvements are twofold. First, the targeted assembly approach identifies known CRISPRs in more human microbiome datasets, as compared to the annotation of CRISPRs using whole-metagenome assemblies. Second, targeted assembly resulted in longer CRISPR arrays, from which we can extract many more diverse spacers for analyzing the evolution of the CRISPRs and other purposes. Here we use three examples to demonstrate the performance of the targeted assembly.\n\n10.1371\/journal.pgen.1002441.t002\n\n###### Comparison of CRISPR identification using whole-metagenome assembly and targeted assembly.\n\n<img src=\"pgen.1002441.t002\" id=\"pgen-1002441-t002-2\" \/>\n\n|  |  | Whole-metagenome assembly |  | Targeted assembly |  |  |\n|:---|:--:|:--:|:--:|:--:|:--:|:--:|\n| CRISPR | Sample datasets | Spacers (max) | Spacers (total) | Short reads | Spacers (max) | Spacers (total) |\n| SmutaL36 (386<a href=\"#nt102\" data-ref-type=\"table-fn\">a<\/a> vs 38<a href=\"#nt103\" data-ref-type=\"table-fn\">b<\/a>) | SRS017025 (plaque) | 1<a href=\"#nt104\" data-ref-type=\"table-fn\">c<\/a> | 1<a href=\"#nt105\" data-ref-type=\"table-fn\">d<\/a> | 1078<a href=\"#nt106\" data-ref-type=\"table-fn\">e<\/a> | 26 | 76 |\n|  | SRS011086 (tongue) | 1 | 2 | 4018 | 24 | 78 |\n| GhaemL36 (257 versus 9) | SRS019071 (tongue) | 0 | 0 | 1718 | 47 | 21 |\n|  | SRS014124 (tongue) | 3 | 3 | 490 | 21 | 58 |\n| SRS018394L37 (238 versus 39) | SRS049389 (tongue) | 0 | 0 | 5778 | 25 | 492 |\n|  | SRS049318 (plaque) | 1 | 1 | 1463 | 38 | 134 |\n\nthe total number of samples that have streptococcal CRISPRs identified if using targeted assembly, and\n\nif using whole-metagenome assembly;\n\nthe total number of spacers found in the longest CRISPR locus found in the given dataset;\n\nthe total number of spacers found in all contigs assembled from the given dataset;\n\nthe total number of sequences that contain the repeats of a given CRISPR, *i.e.*, the recruited reads used for targeted assembly. See <a href=\"#pgen.1002441.s008\" data-ref-type=\"supplementary-material\">Table S1<\/a> for comparison of all the CRISPRs studied in this paper.\n\nThe first example is the streptococcal CRISPR SmutaL36 (see <a href=\"#pgen-1002441-t001\" data-ref-type=\"table\">Table 1<\/a>), a CRISPR that is conserved in streptococcal species such as *Streptococcus mutans* \\[26\\]. This CRISPR was observed only in a limited number of samples (38 out of 751 datasets) when using contigs from whole-metagenome assembly. But our targeted CRISPR assembly identifies instances of CRISPR SmutaL36 in \u223c10 times more (386) datasets. Consistent with the distribution of *streptococcus* across body sites, most of the 386 datasets are from oral samples: 120 of 128 supragingival plaques (94%), 128 of 135 tongue dorsum samples (95%), and 97 of 121 buccal mucosa samples (80%) (see <a href=\"#pgen-1002441-t003\" data-ref-type=\"table\">Table 3<\/a>). CRISPR SmutaL36 was only found in a small proportion of samples from other body locations, where *streptococcus* rarely exists (*e.g.*, 4 of 148 stool samples, and none of the posterior fornix datasets). <a href=\"#pgen-1002441-t002\" data-ref-type=\"table\">Table 2<\/a> shows the details of targeted assembly of this CRISPR in two datasets.\n\n10.1371\/journal.pgen.1002441.t003\n\n###### Distribution of selected CRISPRs across body sites.\n\n<img src=\"pgen.1002441.t003\" id=\"pgen-1002441-t003-3\" \/>\n\n|  |  |  | Oral |  |  |  | Skin |  |\n|:---|:--:|:--:|:--:|:--:|:--:|:--:|:--:|:--:|\n| CRISPR | Anterior nares (94<a href=\"#nt107\" data-ref-type=\"table-fn\">a<\/a>) | Stool (148) | Buccal mucosa (121) | Supra-gingival plaque (128) | Tongue dorsum (135) | Posterior fornix (61) | L- (9)<a href=\"#nt109\" data-ref-type=\"table-fn\">c<\/a> | R- (18)<a href=\"#nt110\" data-ref-type=\"table-fn\">d<\/a> |\n| SmutaL36 | 11<a href=\"#nt108\" data-ref-type=\"table-fn\">b<\/a> | 4 | 97 | 120 | 128 | 0 | 0 | 1 |\n| AhydrL30 | 0 | 53 | 0 | 0 | 0 | 0 | 0 | 0 |\n| BcoprL32 | 0 | 65 | 0 | 0 | 0 | 0 | 0 | 0 |\n| FalocL36 | 0 | 63 | 1 | 18 | 50 | 0 | 0 | 0 |\n| Neis_t014_L28 | 0 | 51 | 15 | 58 | 15 | 0 | 0 | 0 |\n| Neis_t014_L36 | 0 | 0 | 37 | 66 | 82 | 0 | 0 | 0 |\n| PacneL29 | 1 | 0 | 0 | 0 | 0 | 0 | 4 | 7 |\n\nthe total number of datasets;\n\nthe total number of datasets that have CRISPRs identified;\n\nL-Retroauricular crease;\n\nR-Retroauricular crease. Note not all body sites are listed in this table.\n\nThe other two examples are GhaemL36 and SRS018394L37 (see details in <a href=\"#pgen-1002441-t002\" data-ref-type=\"table\">Table 2<\/a>). CRISPR GhaemL36 was initially identified from the genome of *Gemella haemolysans* ATCC 10379 using metaCRT. Targeted assembly further identified instances of this CRISPR in 258 oral-associated samples. The longest contig\u2014of 3121 bases\u2014was assembled from the SRS019071 dataset. This CRISPR array has even more repeats (48; *i.e.*, 47 spacers) than the CRISPR array in the *Gemella haemolysans* reference genome, which has 29 repeats. CRISPR SRS018394L37 (currently not yet associated with a host genome) was initially identified from the whole-metagenome assembly of SRS018394, but targeted assembly reveals the presence of this CRISPR in 238 oral-associated microbiomes. The contig that was assembled in SRS049389 is the longest one (2014 bps), containing 25 spacers.\n\nIn most cases we have tested, targeted assembly dramatically improves the identification of CRISPRs in the HMP datasets: for 142 CRISPRs (out of 150), targeted assembly resulted in CRISPR identification in more HMP samples as compared to using whole-metagenome assemblies, and for 36 CRISPRs, targeted assembly identified instances of the corresponding CRISPR in at least 10 times more datasets (see <a href=\"#pgen.1002441.s008\" data-ref-type=\"supplementary-material\">Table S1<\/a>). It suggests that specifically designed assembly approaches, such as the targeted assembly approach for CRISPR assembly presented here, are important for the characterization of functionally important repetitive elements that otherwise may be poorly assembled in a whole-metagenome assembly (which tends to be confused by repeats), and such a comprehensive identification is important for deriving an unbiased distribution of these functional elements across different body sites among individuals.\n\n## Novel CRISPRs are found in human microbiome samples\n\nIn order to identify novel CRISPR loci, with which to seed further targeted assemblies, we set out to find loci based simply on the structural patterns of CRISPR loci, using the program metaCRT, which we modified from CRT (see <a href=\"#s4\" data-ref-type=\"sec\">Methods<\/a>). As a result, we found and selected 86 different types of novel CRISPR repeats in metagenomic samples, which could not be found in reference genomes, for further targeted assembly (see <a href=\"#s4\" data-ref-type=\"sec\">Methods<\/a>). <a href=\"#pgen-1002441-t001\" data-ref-type=\"table\">Table 1<\/a> lists selected examples of novel CRISPRs that we identified in HMP datasets (see <a href=\"#pgen-1002441-t001\" data-ref-type=\"table\">Table 1<\/a> for naming conventions). A full list of CRISPRs (including the number of CRISPR contigs assembled in each metagenomic dataset) is available as <a href=\"#pgen.1002441.s008\" data-ref-type=\"supplementary-material\">Table S1<\/a>. In this section, we highlight two examples of novel CRISPRs.\n\nCRISPR SRS012279L38 was identified from a whole-metagenome assembly contig of dataset SRS012279 (derived from a tongue dorsum sample; see <a href=\"#pgen-1002441-g002\" data-ref-type=\"fig\">Figure 2A<\/a>). The identified CRISPR contig has 6 copies of a 38-bp repeat (the last copy is incomplete; see <a href=\"#pgen-1002441-t001\" data-ref-type=\"table\">Table 1<\/a> for the consensus sequence of the repeats). *De novo* gene prediction by FragGeneScan \\[31\\] reveals 10 protein-coding genes in this contig, among which 9 share similarities with *cas* genes from other genomes, including *Leptotrichia buccalis* DSM 1135 (NC_013192, an anaerobic, gram-negative species, which is a constituent of normal oral flora \\[32\\]) and *Fusobacterium mortiferum* ATCC 9817, by BLASTP search using the predicted protein sequences as queries (see <a href=\"#pgen-1002441-g002\" data-ref-type=\"fig\">Figure 2B<\/a>). (By contrast, BLASTX search of this contig against nr database only achieved annotations for 7 genes). In addition, similarity searches revealed a single identical copy of this repeat in the genome of *Leptotrichia buccalis* DSM 1135 (from 1166729 to 1166764; *de novo* CRISPR prediction shows that this genome has several CRISPR arrays, including an array that has 84 copies of a 29-bp repeat, but none of the CRISPRs have the same repeat sequence as SRS012279L38). These two lines of evidence (similar *cas* genes, and an identical region in the genome) suggest that the SRS012279L38 CRISPR we found in the human microbiomes could have evolved from *Leptotrichia buccalis* or a related species.\n\nTargeted assembly of this novel CRISPR (SRS012279L38) in HMP datasets resulted in 278 contigs from 97 datasets, confirming the presence of this CRISPR in human microbiomes. In particular, the CRISPR fragments (407 bps) identified from the whole-metagenome assembly of SRS012279 were assembled into a longer CRISPR contig (890 bps) by targeted assembly. A total of 14 unique but related repeat sequences were identified from 278 CRISPR contigs, and two of them (which differ at 3 positions) are dominant, constituting 71% of the repeats in the CRISPR contigs. Notably, all the repeats could be clustered into a single consensus sequence with an identity threshold of 88%. By contrast, the spacer sequences are very diverse across different samples. For example, we obtained a total of 352 unique spacer sequences, which were clustered into 345 consensus sequences with an identity threshold of 90%. Among 352 unique spacers, 114 spacer sequences were shared by multiple samples\u2014a single spacer was shared by at most eight samples.\n\nThe second example is CRISPR SRS023604L36, initially identified in a whole-metagenome assembly contig of dataset SRS023604 (derived from posterior fornix), which has 5 copies of a 36 bp repeat (see consensus sequence of the CRISPR repeat in <a href=\"#pgen-1002441-t001\" data-ref-type=\"table\">Table 1<\/a>). Targeted assembly of this CRISPR across all HMP metagenomic datasets revealed further instances of this CRISPR in several other datasets, including two from stool, and two from posterior fornix. Moreover, the CRISPR contig was assembled into a longer contig of 778 bps containing 12 copies of the CRISPR repeat. BLAST search of the CRISPR repeat against the nr database did not reveal any significant hits.\n\n## Expanding the CRISPR space by human microbiomes\n\nTo investigate how much the CRISPRs identified in the HMP datasets can expand the CRISPR space (sequence space of the CRISPR repeats), we built a network of CRISPRs, based on the sequence similarity between CRISPR repeats. An edge in the network between two CRISPR repeats, each represented by a node, indicates that the two repeats can be transformed from one to another by at most 10 operations (including mutations, insertions, and deletions). Since it is difficult to determine the direction of CRISPR repeats \\[7\\] (especially for the CRISPR arrays that have incomplete structures), given two repeats, we calculated two edit distances\u2014one is the distance between the two repeats, and the other one is between one repeat and the reverse complement of the other\u2014and used the smaller value as the edit distance between the two repeats. The global network (<a href=\"#pgen-1002441-g003\" data-ref-type=\"fig\">Figure 3A<\/a>; see <a href=\"#pgen.1002441.s001\" data-ref-type=\"supplementary-material\">Figure S1<\/a> with node labels) shows that most of the novel CRISPRs identified in the human microbiomes are remotely related to ones identified in complete (or draft) genomes. Still, there are small clusters that contain only novel HMP CRISPRs, indicating that these CRISPRs are substantially different from ones identified in the reference genomes. In <a href=\"#pgen-1002441-g003\" data-ref-type=\"fig\">Figure 3B<\/a>, we have colored nodes by body site: while specific CRISPR repeats can be highly specific to body site (see below), the larger families of repeats shown in <a href=\"#pgen-1002441-g003\" data-ref-type=\"fig\">Figure 3B<\/a> do not appear to cluster based on body site.\n\nWe further studied the sequence patterns of the repeats for each group and our results show 1) distinct patterns among the groups, and 2) high conservation around the stem and start\/end positions in CRSIPR repeats of each group (see sequence logos\u2014for the large groups\u2014in <a href=\"#pgen.1002441.s002\" data-ref-type=\"supplementary-material\">Figure S2<\/a>). The consensuses revealed by the logos show consistencies with the results in a previous study, which used a similar approach, based on alignments of CRISPR repeats, for classification of CRISPR repeats \\[7\\].\n\n## CRISPRs have diverse distributions across human body sites and individuals\n\nOverall, the distributions of CRISPRs are largely body-site specific (see <a href=\"#pgen-1002441-g004\" data-ref-type=\"fig\">Figure 4<\/a> and <a href=\"#pgen.1002441.s003\" data-ref-type=\"supplementary-material\">Figure S3<\/a>; the name of CRISPR and the number of samples in which the CRISPR was found are listed in <a href=\"#pgen.1002441.s010\" data-ref-type=\"supplementary-material\">Table S3<\/a>). For example, CRISPRs AhydrL30 ad BcoprL32 are only found in stool samples (see <a href=\"#pgen-1002441-t003\" data-ref-type=\"table\">Table 3<\/a>). Exceptions include two CRISPRs that were found from both a significant number of gut- and oral-associated samples: Neis_t014_L28 were found in 51 gut samples and 92 oral-associated samples; FalocL36 identified from *Filifactor alocis* ATCC 35896 were found in 63 gut samples and 72 oral-associated samples, including 50 tongue dorsum samples (see <a href=\"#pgen-1002441-t003\" data-ref-type=\"table\">Table 3<\/a>).\n\nThe first 50 CRISPRs shown in <a href=\"#pgen-1002441-g004\" data-ref-type=\"fig\">Figure 4<\/a> are mainly found in stool samples. AshahL36, which was initially identified from *Alistipes shahii* WAL 8301, was found in more than half of gut-related samples (96 out of 147 samples). On the other hand, 99 CRISPRs are mainly found in oral samples, in particular, tongue dorsum, supragingival plaque, and buccal mucosa. We found 5 CRISPRs that exist in more than half of the oral-associated samples (out of 417): SmutaL36, KoralL32 from *Kingella oralis* ATCC 51147, Veil_sp3_1_44_L36 and Veil_sp3_1_44_L35 from *Veillonella sp.* 3_1_44, and SoralL35 from *Streptococcus oralis* ATTC 35037. 4 CRISPRs are mostly found in vaginal samples (AlactL29, LjensL36, LjassL36, and LcrisL29). 1 CRISPR is skin-specific (PacneL29), found mainly in skin samples. Below we discuss the body-site distributions of a few examples.\n\nNeis_t014_L28 and Neis_t014_L36 are inferred from a single genome, *Neisseria sp.* oral taxon 014 str. F0314, but these two CRISPRs show distinct absence\/presence profiles across body sites (see <a href=\"#pgen-1002441-t003\" data-ref-type=\"table\">Table 3<\/a>). For stool samples, there exists only CRISPR Neis_t014_L28 in 51 datasets, but not Neis_t014_L36. And Neis_t014_L36 is relatively more prevalent in oral-associated samples as compared to Neis_t014_L28. The different body site distributions can be explained by the fact that these two CRISPRs are found in different sets of genomes (although both can exist in a common genome, *Neisseria sp.* oral taxon 014 str. F0314). Neis_t014_L36 has been identified in multiple *Neisseria* genomes, including *Neisseria meningitidis* ATCC 13091, *Neisseria meningitidis* 8013 (so Neis_t014_L36 belongs to the Nmeni subtype among the 8 subtypes defined by Haft et al \\[33\\]), *Neisseria flavescens* SK114, and *Actinobacillus minor* NM305. Neis_t014_L28, however, was only found in *Neisseria sp.* oral taxon 014 str. F0314. On the other hand, even though we could not find any CRISPRs containing the exactly same repeat as Neis_t014_L28 in the complete\/draft genomes other than *Neisseria sp.* oral taxon 014 str. F0314, many CRISPRs, when a few mismatches are allowed, were found in diverse genomes (for example, *Crenothrix polyspora*, *Legionella pneumophila* 2300\/99 Alcoy, and *Thioalkalivibrio sp.* K90mix) from environmental samples.\n\nFour CRISPRs (AlactL29, LjensL36, LjassL36, and LcrisL29) exist mostly in vaginal samples. AlactL29, initially identified from the *Anaerococcus lactolyticus* genome, was found only in 3 vaginal samples. Notably, LjensL36 was found in 28 vaginal samples (which comprise 43% of vaginal samples collected) and 1 skin sample. This observation is consistent with a previous study showing that *Lactobacillus* constitutes over 70% of all bacteria sampled from vaginas of healthy, fertile women, and *Lactobacillus jensenii* is one of the major genomes \\[34\\]. Interestingly, we could find evidence of adaptation in the LjensL36 spacer against *Lactobacillus* phage Lv-1 (NC_011801) (see below). LjassL36 was found in 33 vaginal samples by targeted assembly. We confirmed that it is in different *Lactobacillus* genomes, such as *Lactobacillus gasseri* and *Lactobacillus crispatus*, by BLAST search. CRISPR LcrisL29, which was identified in the *Lactobacillus crispatus* genome, was found in 31 vaginal samples, and we found the same repeat sequence in the *Lactobacillus helveticus* genome.\n\nPacneL29 was the only skin-specific CRISPR we found in the HMP datasets. Interestingly, instances of PacneL29 are found in *Propionibacterium acnes* HL110PA4 and *Propionibacterium acnes* J139, but not other *P. acnes* isolates (including KPA171202, SK137, J165, and SK187). This indicates a potential application of CRISPRs in the characterization of specific stains for a species in human microbiomes.\n\n## CRISPRs have very diverse spacers\n\nThe HMP project enables us to explore the diversity of streptococcal CRISPRs (and others) at a much broader scale (with 751 samples from 104 healthy individuals). The CRISPRs that we identified in human microbiomes exhibited substantial sequence diversity in their spacers among subjects. Targeted assembly of the streptococcal CRISPRs (SmutaL36) in HMP datasets resulted in a total of 15,662 spacers identified from 386 samples, among which 7,815 were unique spacers (clustering of the spacers at 80% identify resulted in a non-redundant collection of 7,436 sequences). See <a href=\"#pgen.1002441.s004\" data-ref-type=\"supplementary-material\">Figure S4<\/a> for the sharing of the spacers in streptococcal CRISPRs among all individuals, which shows several large clusters of spacers that are shared by multiple individuals (for clarity, we only keep spacers that were shared by more than eight samples in this figure). In particular, the most common spacer is shared by 25 individuals (in 32 samples).\n\nMore importantly, we could check the sharing of CRISPR spacers across different body sites and sub-body sites (*e.g.*, multiple oral sites) using HMP datasets (Pride *et al.* examined streptococcal CRISPRs in saliva samples from 4 individuals \\[26\\]). <a href=\"#pgen-1002441-g005\" data-ref-type=\"fig\">Figure 5<\/a> shows the spacer sharing among 6 selected individuals, each of whom has multiple samples with identified streptococcal CRISPRs from multiple body sites (see <a href=\"#pgen.1002441.s005\" data-ref-type=\"supplementary-material\">Figure S5<\/a> for the spacer sharing with spacers clustered at 80% sequence identify). By examining the distribution of the spacers across samples, we observed that samples re-sampled from the same individual and oral site shared the most spacers, different oral sites from the same individual shared significantly fewer, while different individuals had almost no common spacers, indicating the impact of subtle niche differences and histories on the evolution of CRISPRs. Our observation is largely consistent with the conclusion from Pride *et al.* \\[26\\]. But our study showed that different samples from the same oral site of the same person, even samples collected many months apart, could still share a significant number of spacers (*e.g.*, the supragingival plaque samples from individual 1 in visit 1 and visit 2, with 238 days between the two visits, and the tongue dorsum samples from individual 5 in visit 1 and visit 3, with 336 days between the two visits; as shown in <a href=\"#pgen-1002441-g005\" data-ref-type=\"fig\">Figure 5<\/a>). Our study also showed that although the different oral sites of the same individual share similar spacers, this sharing (*e.g.*, between the supragingival plaque sample and the buccal mucosa sample for individual 1) is minimal, as compared to the spacer sharing between samples collected in different visits but from the same oral site (*e.g.*, between the supragingival plaque samples from visit 1 and visit 2 for individual 1). Finally, our study shows that the spacer turnover varies among individuals\u2014for the 6 selected individuals, individual 3 shows significantly higher turnover of the spacers between visits, as compared to other individuals.\n\nWe also checked the spacer diversity for the CRISPR KoralL32, since it and its variants are one of the most abundant CRISPRs. This CRISPR was assembled from 339 samples: 327 from oral sites and 2 from gut. The targeted assembly of KoralL32 found 7282 unique spacers, among which the most commonly shared spacer is shared by 35 individuals (in 58 samples). <a href=\"#pgen.1002441.s006\" data-ref-type=\"supplementary-material\">Figure S6<\/a> shows the sharing of the spacers among the individuals for this CRISPR, which shows similar spacer-sharing patterns as those found in the streptococcal CRISPRs.\n\nThe similarity between spacers from the same individual suggests that we may still be able to trace the evolution of CRISPRs, especially in the same body site of the same individual, even though the CRISPR loci tend to have extremely high turnover of their spacers.\n\n## CRISPR spacer sequences can be used to trace the viral exposure of microbial communities\n\nAs a consequence of CRISPR adaptation, the spacer contents in CRISPR arrays reflect diverse phages and plasmids that have passed through the host genome \\[1\\], \\[35\\], \\[36\\], \\[37\\]. However, previous studies have shown that only 2% of the spacer sequences have matches in GenBank, which is probably due to the fact that bacteriophage and plasmids are still poorly represented in databases \\[13\\], \\[14\\]. Similarity searches of identified spacers against viral genomes enable identification of the viral sources of the spacers (*i.e.*, proto-spacers) captured in each CRISPR locus. For example, similarity searches of the 7,815 unique spacers in the streptococcal CRISPR against viral genomes revealed similarities between streptococcal spacers and 22 viral genomes (species names and accession IDs are listed in <a href=\"#pgen.1002441.s011\" data-ref-type=\"supplementary-material\">Table S4<\/a>), and the two most prevalent viruses are *Streptococcus* phage PH10 (NC_012756) and *Streptococcus* phage Cp-1 (NC_001825) (see <a href=\"#pgen-1002441-g006\" data-ref-type=\"fig\">Figure 6A<\/a>). <a href=\"#pgen-1002441-g006\" data-ref-type=\"fig\">Figure 6B<\/a> suggests that the potential proto-spacers are rather evenly distributed along the phage genomes (except for a few regions, including a region that encodes for an integrase, which is highlighted in red in <a href=\"#pgen-1002441-g006\" data-ref-type=\"fig\">Figure 6B<\/a>). Although the positional distribution of the proto-spacers is close to random, the sequences adjoining the proto-spacers for streptococcal CRISPR we identified in the virus genomes showed conserved short sequence motifs (GG) (see <a href=\"#pgen.1002441.s007\" data-ref-type=\"supplementary-material\">Figure S7<\/a> for the sequence logo), which is also the most common proto-spacer adjacent motif (PAM) shared by several CRISPR groups, as reported in \\[38\\].\n\nAnother example is CRISPR PacneL29, which is mainly found in skin-associated microbiomes. BLAST search of the identified spacers against the virus genome dataset revealed similarity between the spacers and several regions in *Propionibacterium* phage PA6 (NC_009541). We also found evidence of adaptation in LjensL36 against *Lactobacillus* phage Lv-1 (NC_011801): BLAST search shows significant matches to a total of 38 regions in the phage genome. Overall we found 23 CRISPRs that have spacers with high sequence similarities (\u226590% over 30 bps) with virus genomes collected from the NCBI ftp site (<a href=\"#pgen.1002441.s012\" data-ref-type=\"supplementary-material\">Table S5<\/a>).\n\nWe also searched the spacers against plasmid sequences (collected in the IMG database). For example, matches were found between the detected streptococcal CRISPR spacers and more than 10 plasmid sequences (including *Streptococcus thermophilus* plasmid pER35, pER36, pSMQ308, and pSMQ173b; *Bacillus subtilis* plasmid pTA1040; and *Streptococcus pneumoniae* plasmids pSMB1, pDP1 and pSpnP1). See <a href=\"#pgen.1002441.s013\" data-ref-type=\"supplementary-material\">Table S6<\/a> for a summary of the plasmids that share high homology with the CRISPR spacers.\n\nThe CRISPER spacers can also be used to identify viral contigs in metagenome assemblies that contain proto-spacers. As an example, similarity searches of identified streptococcal CRISPR spacers against the HMP assemblies revealed 37 potential viral contigs (of lengths from 2,134 to 56,413 bp): these contigs show high homology (\\>80% sequence similarity) with known viral genomes. The largest contig (of 56,413 bps) is similar to the genome of *Streptococcus* phage Dp-1 (NC_015274), with 88% sequence identify, and covers almost the entire viral genome (of 59,241 bps). A future paper will fully explore this approach.\n\n## Conserved CRISPR repeat sequences can be used to reveal rare species in human microbiome\n\nBecause of the large number of repeats that many CRISPR loci contain, CRISPR repeats of rare species with low sequence coverage in a community can still be found. It was reported that repeat-based classification \\[7\\] corresponds to a *cas* gene-based classification of CRISPRs \\[33\\], which revealed several subtypes of CRISPRs largely constrained within groups of evolutionarily related species (*e.g.*, the Ecoli subtype). As such, we may use the presence of the repeats of a particular CRISPR as a first indication of the presence of related genome(s) in a microbiome, even though CRISPR locus has been found transferred horizontally as a complete package among genomes \\[11\\].\n\nWe use CRISPR PpropL29 as an example to demonstrate this potential application, as PpropL29 was identified in only a small proportion of the HMP samples (11 datasets): including 7 supragingival plaque samples (out of 125) and 4 tongue dorsum samples (out of 138). All the PpropL29-related repeats identified in these samples can be clustered into 7 unique sequences. In order to find the most likely reference genomes for these 7 unique repeat sequences, we blasted these repeat sequences against the human microbiome reference genomes and found 100% identity matches in the *Lautropia mirabills* genome. To investigate the overall coverage of this genome by the reads (not only the CRISPR regions), we mapped the entire collection of reads from four samples: SRS019980 and SRS021477 (both are from supragingival plaque, and have an 100% identity match with the CRISPR repeat in the *Lautropia mirabills* genome); SRS019974 (from tongue dorsum, with a slightly different CRISPR repeat sequence with 3 differences); and SRS019906 (which does not contain any CRISPR repeats similar to PpropL29, used as a control). The mapping results show the reads from two samples SRS019980 and SRS021477 each cover \u223c80% of the *Lautropia mirabills* genome, which is very significant evidence that these two microbiomes include *Lautropia mirabills*. But the other two samples have only a limited number of reads mapped to the genome (*e.g.*, only 3089, reads in SRS019906 were mapped into *Lautropia mirabills*). This contrast suggests that identification of CRISPRs by targeted assembly could provide significant evidence for the existence of certain rare genomes.\n\n# Discussion\n\nWe have applied a targeted assembly approach to CRISPR identification, to characterize CRISPRs across body sites in different individuals. Our studies show that a directed approach\u2014such as our targeted assembly approach\u2014is important for a comprehensive (thus less biased) estimation of the distribution of CRISPRs across body sites and individuals, and their dynamics. Note that in this study, we only focused on CRISPRs identified in eubacterial genomes, since archaea are rare in human microbiomes (we looked for, but did not find, archaeal CRISPRs in the HMP assemblies). Also for the sake of simplicity, we derived a non-redundant list of CRISPRs based on the similarity of the CRISPR repeats (see <a href=\"#s4\" data-ref-type=\"sec\">Methods<\/a>), and detailed targeted assembly was only applied to these CRISPRs.\n\nAlthough many CRISPR arrays may be missed by whole-metagenome assembly, we show that whole-metagenome assemblies are useful for identifying novel CRISPRs (as *de novo* prediction of CRISPRs relies on sequence features of CRISPRs that do not exist in short reads). Once seeding CRISPRs are identified from whole-metagenome assemblies, we can go back to the original short read datasets, and pursue a comprehensive characterization of the CRISPRs, using the targeted assembly approach. Also, we did not fully utilize the presence of *cas* genes for identification of novel CRISPRs in our study, since in many cases we can identify arrays of repeats, but not their associated *cas* genes. A future direction is to combine targeted assembly of CRISPRs and whole-metagenome assembly, aiming to achieve even better assembly of the CRISPR loci with more complete structures, including *cas* genes and the arrays of repeats and spacers. Such an improvement is necessary to achieve a more comprehensive characterization of especially the novel CRISPRs discovered in metagenomes, and the temporal order of spacer addition to arrays.\n\nThe immediate utility of this study is to provide more complete inventories of CRISPR loci in human microbiomes and their distributions in different human body sites, and the spacer content of these loci. The identification of CRISPR spacers opens up several potential applications, including tracing the viral exposure of the hosts, studying the sequence patterns of the regions adjoining the spacer precursors in viral genomes, and discovering viral contigs in metagenome assemblies. It has been shown that short sequence motifs found in the regions adjacent to the spacer precursors in the viral genomes determine the targets of the CRISPR defense system \\[38\\], and we were able to analyze the sequence patterns of regions adjacent to spacer precursors for several CRISPRs with the most spacers identified in the HMP datasets (including SmutaL36, LjensL36, and KoralL32; see sequence logos in <a href=\"#pgen.1002441.s007\" data-ref-type=\"supplementary-material\">Figure S7<\/a>). When more metagenomic datasets become available, we will extend the analysis to more CRISPRs, which may provide insights into the mechanism of the CRISPR defense system (including the turnover patterns of the CRISPR spacers, and the target recognition of the CRISPR defense systems). Our preliminary exploration of viral contigs\u2014by searching CRISPR spacers against whole-metagenome assemblies\u2014suggests that we can identify new virus genomes in metagenome assemblies; further computational and experimental analysis will be needed to confirm these contigs.\n\nWe look forward to being able to utilize CRISPR spacer sequences to understand human and human microbiome biology better, utilizing the metadata associated with the HMP datasets. This awaits a more complete sampling of individuals over time, and of known relationships; and a far better characterization of bacteriophage and other selfish genetic elements in the human biome (our inventory of spacers is a standard against which phage and plasmid collections can be judged).\n\n# Methods\n\n## *De novo* identification of CRISPRs\n\nCRT \\[28\\] is a tool for fast, *de novo* identification of CRISPRs in long DNA sequences. CRT works by first detecting repeats that are separated by a similar distance, and then checking for other CRISPR specific requirements (*e.g.*, the spacers need to be non-repeating and similarly sized). We modified CRT to consider incomplete repeats at the ends of contigs from whole-metagenome assembly, and call the modified program metaCRT.\n\n## Identification of CRISPRs by similarity search\n\nWe implemented CRISPRAlign for identifying CRISPRs in a target sequence (a genome or a contig) that has repeats similar to a given CRISPR (query CRISPR). CRISPRAlign works by first detecting substrings in the target sequence (or its reverse complement) that are similar to the repeat sequence of a query CRISPR, and then checking for other requirements, as in metaCRT. Both metaCRT and CRISPRAlign are available for download at <http:\/\/omics.informatics.indiana.edu\/CRISPR\/>.\n\n## Selection of known and novel CRISPRs for targeted assembly in HMP datasets\n\nUsing metaCRT and CRISPRAlign, we prepared a list of known CRISPRs repeats (identified from complete\/draft bacterial genomes) and a list of potentially novel ones (identified only in the whole-metagenome assemblies from the HMP datasets) for further detailed study of their distributions among the HMP datasets. As we show in Results, the targeted assembly strategy is important for an efficient and comprehensive characterization of these CRISPRs in human microbiome datasets.\n\nKnown CRISPRs were first identified from the bacterial genomes (or drafts) collected in the IMG dataset (version 3.3), using metaCRT. We then selected a subset of the identified CRISPRs that meet the following requirements: direct repeats are of length 24\u201340 bps, there are a minimum of 4 copies of the direct repeats, and the individual repeats each differ by at most one nucleotide from the repeat consensus sequence, on average. The parameters were chosen to minimize false CRISPRs, considering that a CRISPR array typically contains 27 repeats, with an average repeat length of 32 base pairs \\[28\\]. We only kept CRISPRs that can be found in at least one of the whole-metagenome assemblies, using CRISPRAlign. We further reduced the number of candidate CRISPRs by keeping only those that share at most 90% sequence identity along their repeats by CD-HIT \\[39\\], as there are CRISPRs that share very similar repeats, and our targeted assembly strategy can recover the CRISPRs with slight repeat differences. To avoid including a repeat and its reverse complete (metaCRT does not consider the orientation for the repeats) in the non-redundant list, we included reverse complement sequences of the CRISPR repeats in the clustering process. Therefore, a repeat would be classified into two clusters by CD-HIT (the reverse complete of the repeat would be classified into a different cluster), one of which was removed to reduce redundancy.\n\nWe consider that a CRISPR identified in the HMP assemblies is novel if we find no instances of this CRISPR in the IMG bacterial genomes and the HMP reference genomes, with at most 4 mismatches using CRISPRAlign. Similarly, we only kept a non-redundant list of the novel candidates.\n\nIn total, we selected a collection of non-redundant CRISPRs\u2014including 64 known CRISPRs and 86 novel ones\u2014for further targeted assembly from HMP shotgun reads. The detailed information for these CRISPRs (repeat sequences, and their resources, and the references for the CRISPRs already collected in the CRISPRdb database <http:\/\/crispr.u-psud.fr\/> \\[6\\]), is provided in <a href=\"#pgen.1002441.s008\" data-ref-type=\"supplementary-material\">Tables S1<\/a> and <a href=\"#pgen.1002441.s009\" data-ref-type=\"supplementary-material\">S2<\/a>.\n\n## Targeted assembly of CRISPRs\n\nFor the targeted assembly of CRISPRs, we first carried out a BLASTN search with each putative CRISPR repeat sequence as the query, to collect reads that contain the repeat sequence (see <a href=\"#pgen-1002441-g001\" data-ref-type=\"fig\">Figure 1<\/a>). In order to make the similarity search tolerant to sequencing errors and genomic variations that are observed among the multiple copies of a CRISPR repeat (in one CRISPR locus or between different CRISPR loci), we allowed three mismatches over the entire CRISPR repeat sequence: we retained only the reads that are aligned with the entire CRISPR repeat sequence with a maximum of three mismatches. With these reads containing CRISPR repeat sequences, we ran SOAPdenovo \\[40\\] with k-mers of 45 bps, which are sufficiently long to assemble reads with the repetitive sequences found in CRISPRs. In general, whole-metagenome contigs are assembled using shorter k-mers (for example, 21\u201323 bps in MetaHit \\[41\\] and 25 bps in HMP assembly \\[42\\]), as longer k-mers often fragment assemblies into short contigs. After CRISPR contigs were assembled, the exact boundaries of the repeats and spacers were obtained using CRISPRAlign.\n\n## Validation of the targeted assembly approach using simulated datasets\n\nWe simulated short reads from 6 reference genomes (*Azospirillum B510*, *Streptococcus mutans NN2025*, *Deferribacter desulfuricans SSM1*, *Dehalococcoides GT*, *Erwinia amylovora ATCC 49946*, and *Escherichia coli K12 MG1655*), and applied our method to attempt to assemble the 10 known CRISPRs in these genomes. All 54 contigs assembled by our targeted assembly approach match perfectly to known CRISPRs in the reference genomes. We listed the genome names, the CRISPR repeats, the coordinates of the known CRISPRS in the reference genomes, and the coordinates of the contigs aligned on the reference genomes in <a href=\"#pgen.1002441.s014\" data-ref-type=\"supplementary-material\">Table S7<\/a>.\n\n## Datasets\n\nWe used the dataset Human Microbiome Illumina WGS Reads (HMIGWS) Build 1.0 available at <http:\/\/hmpdacc.org\/HMIWGS>, and the whole-metagenome assemblies from the HMP consortium (<http:\/\/www.hmpdacc.org\/>). The bacterial genomes were downloaded from the IMG database (<http:\/\/img.jgi.doe.gov\/cgi-bin\/m\/main.cgi>), NCBI ftp site (<ftp:\/\/ftp.ncbi.nih.gov\/genomes>), and human microbiome project website (<http:\/\/www.hmpdacc.org\/data_genomes.php>). The viral genomes were downloaded from the NCBI ftp site (<http:\/\/www.ncbi.nlm.nih.gov\/genomes\/GenomesGroup.cgi?taxid=10239>). Additional phage genomes were downloaded from the PhAnToMe database site (<http:\/\/www.phantome.org\/Downloads\/DNA\/all_sequences\/>).\n\n# Supporting Information\n\nA network of 150 CRISPRS. The CRISPR names were shown in each node. The CRISPR host species for each known CRIPRS are listed in <a href=\"#pgen.1002441.s009\" data-ref-type=\"supplementary-material\">Table S2<\/a>. Known CRISPRs are shown as blue nodes (except for several CRISPRs highlighted in green), and the novel CRISPRs identified in the HMP datasets are shown as red nodes.\n\n(TIF)\n\nClick here for additional data file.\n\nThe consensus of CRISPR repeats for 6 large clusters. See cluster ID in <a href=\"#pgen.1002441.s001\" data-ref-type=\"supplementary-material\">Figure S1<\/a>. The sequence logo was prepared using weblogo (<http:\/\/weblogo.berkeley.edu\/>).\n\n(TIF)\n\nClick here for additional data file.\n\nDistribution of CRISPRs in different body sites. The x-axis represents 150 CRISPRs (listed in <a href=\"#pgen.1002441.s009\" data-ref-type=\"supplementary-material\">Table S2<\/a>) and y-axis represents the proportion of samples in which instances of each of the CRISPR are found.\n\n(TIF)\n\nClick here for additional data file.\n\nCluster of spacers shared by more than eight samples. In this map, rows are spacers (clustered at 80% identify), and the columns are samples: cluster (a) is shared by 22 samples; cluster (b) is shared by 23 samples; cluster (c) is shared by 12 samples; cluster (d) is shared by 32 samples. The red lines indicate the presence of spacers in each of the samples. Multiple lines in the same row represent a spacer that is shared by multiple samples.\n\n(TIF)\n\nClick here for additional data file.\n\nSharing of streptococcal CRISPR spacers among samples from 6 individuals. In this map, the rows are the 761 spacers (clustered at 80% identify; see <a href=\"#pgen-1002441-g005\" data-ref-type=\"fig\">Figure 5<\/a> for the plot using 98% identify) identified in one or more of these 6 individuals, and the columns are samples (e.g., Stool_v1_p1 means a sample from stool of individual 1, in visit 1; Tongue_v2_p1 indicates dataset from tongue, individual 1, in visit 2). Buccal stands for buccal mucosa, and SupraPlaque stands for supragingival plaque. The red lines indicate the presence of spacers in each of the samples. Multiple lines in the same row represent a spacer that is shared by multiple samples.\n\n(TIF)\n\nClick here for additional data file.\n\nSharing of KoralL32 CRISPR spacers among samples from 6 individuals. In this map, rows are the 598 spacers (clustered at 80% identify), and the columns are samples (e.g., Stool_v1_p1 means a sample from stool of individual 1, in visit 1; tongue_v2_p1 indicates dataset from tongue, individual 1, in visit 2). The red lines indicate the presence of spacers in each of the samples. Multiple lines in the same row represent a spacer that is shared by multiple samples.\n\n(TIF)\n\nClick here for additional data file.\n\nSequence logos showing the short sequence motifs in regions adjacent to proto-spacers in the viral genomes for three CRISPRs.\n\n(TIF)\n\nClick here for additional data file.\n\nList of 150 CRISPRs studied in this manuscript and the targeted assembly results in the HMP datasets.\n\n(DOCX)\n\nClick here for additional data file.\n\nList of CRISPRs that are identified from the reference genomes, and their cross-references in the CRISPRdb.\n\n(DOCX)\n\nClick here for additional data file.\n\nList of numbers of datasets from different body sites that have reads (the first number) or CRISPRs (the second number) identified for each CRISPR.\n\n(XLSX)\n\nClick here for additional data file.\n\nList of viral genomes and their accession IDs plotted in <a href=\"#pgen-1002441-g006\" data-ref-type=\"fig\">Figure 6A<\/a>.\n\n(DOCX)\n\nClick here for additional data file.\n\nList of viral genomes sharing high sequence similarities (\u226590% identify over 30 bps) with CRISPR spacers.\n\n(DOCX)\n\nClick here for additional data file.\n\nList of plasmids sharing high sequence similarities (\u226590%) with CRISPR spacers.\n\n(DOCX)\n\nClick here for additional data file.\n\nTargeted assembly results of 10 CRISPRs using reads simulated from 6 genomes.\n\n(DOCX)\n\nClick here for additional data file.\n\nThe authors thank the Human Microbiome Project (HMP) consortium for providing the sequencing data and the whole-metagenome assemblies of the HMP datasets, and the anonymous reviewers for their helpful suggestions.\n\n# References\n\n[^1]: Conceived and designed the experiments: MR YY. Performed the experiments: MR Y-WW YY. Analyzed the data: MR Y-WW HT TGD YY. Wrote the paper: MR Y-WW HT TGD YY.","meta":{"dup_signals":{"dup_doc_count":122,"dup_dump_count":12,"dup_details":{"curated_sources":2,"2014-52":9,"2014-49":7,"2014-42":16,"2014-41":17,"2014-35":12,"2014-23":13,"2014-15":13,"2017-13":1,"2015-18":9,"2015-11":10,"2014-10":12,"2024-26":1}},"file":"PMC3374615"},"subset":"pubmed_central"}
{"text":"abstract: # Abstract\n  .\n  Hemangioblastoma is a benign and morphologically distinctive tumor that can occur sporadically or in association with von Hippel-Lindau disease in approximately 25% of the cases, and which involves the central nervous system in the majority of the cases. Rare occurrences of hemangioblastoma in peripheral nerves and extraneural tissues have been reported. This report describes one case of sporadic renal hemangioblastoma happened in a 16-year-old Chinese female patient, presenting with hematuria, and low back pain. Histologically, the tumors were circumscribed, and composed of sheets of large polygonal cells traversed by arborizing thin-walled blood vessels. The diagnosis of hemangioblastoma was confirmed by negative immunostaining for cytokeratin, and positive staining for \u03b1-inhibin, S100 and neuron-specific enolase (NSE). This benign neoplasm which can be mistaken for various malignancies such as renal cell carcinoma, epithelioid hemangiopericytoma and epithelioid angiomyolipoma, deserves wider recognition for its occurrence as a primary renal tumor.\n  .\n  # Virtual slides\n  .\n  The virtual slide(s) for this article can be found here: http:\/\/www.diagnosticpathology.diagnomx.eu\/vs\/5445834246942699\nauthor: Yang Liu; Xue-shan Qiu; En-Hua Wang\ndate: 2012\ninstitute: 1Department of Pathology, the First Affiliated Hospital and College of Basic Medical Sciences, China Medical University, Shenyang, 110001, China; 2Institute of Pathology and Pathophysiology, China Medical University, Shenyang, 110001, China\nreferences:\ntitle: Sporadic Hemangioblastoma of the Kidney: a rare renal tumor\n\n# Background\n\nHemangioblastoma, also known as capillary hemangioblastoma, is a benign tumor of uncertain histogenesis, consisting of networks of small blood vessels interspersed with lipid-laden stromal cells \\[1\\]. Although the stromal cells are often bland-looking, they can exhibit significant nuclear pleomorphism, mimicking carcinoma, or other malignancies. Hemangioblastoma occurs sporadically, or in a setting of von Hippel-Lindau disease in approximately one-quarter of the cases \\[2\\]. This tumor typically occurs within the central nervous system, predominantly in the cerebellum, but also occasionally in the meninges, retina, spinal cord, corpus callosum, lateral ventricle, pituitary gland, and the optic nerve. Exceptionally, hemangioblastoma occurs in other sites, such as peripheral nerve, soft tissue, retroperitoneum, skin, liver, pancreas, lung, adrenal, kidney, and urinary bladder, usually in the setting of known von Hippel-Lindau disease. There are only a few reports on sporadic hemangioblastoma occurring outside the central nervous system, including kidney \\[3\\]. We report one such case involving the kidney, which might be mistaken for other renal tumors, in particular clear cell renal cell carcinoma.\n\n# Case presentation\n\n## Clinical History\n\nA 16-year-old Chinese female, presented with gross hematuria and low back pain for 18 months. A homogeneous solid mass in the upper pole of the left kidney was detected on a computed tomographic scan. Nephrectomy was performed, showing a 1.2 cm well-encapsulated brownish-white tumor with some hemorrhagic areas in the upper pole of left kidney. There was no polycythemia or clinical evidence of von Hippel-Lindau disease. At imaging, no other tumor was detected, particularly in the central nervous system (CNS). The patient was alive with no tumor recurrence or metastasis at half a year of follow-up.\n\n## Gross features\n\nThe left kidney was normal in size (12.0\u2009\u00d7\u20095.0\u2009\u00d7\u20094.0 cm). A 1.2 cm well-encapsulated mass was found in the upper pole of left kidney. The cut surface of the tumor was brownish-white, solid and homogeneous.\n\n## Microscopic features\n\nThe tumor was characterized by an alternation of cellular and paucicellular areas surrounded by a thick fibrous capsule and well-demarcated from the surrounding renal parenchyma (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>A-E). The paucicellular areas were mainly composed of fibrous stroma containing reticular vascular channels, hemosiderin pigment, and rare stromal cells (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>D). The cellular areas were composed of a rich capillary network of single-layered flat endothelial cells enclosing stromal cells. These last ones showed a pale or eosinophilic cytoplasm exhibiting occasional lipid droplets but no hyaline globules, ovalnuclei, delicate chromatin, and inconspicuous nucleoli (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>F-G). Pleomorphic, or bizarre tumor cell nuclei was barely seen (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>H), and no necrosis or mitoses was found.\n\n## Immunohistochemistry\n\nThe immunohistochemical study showed that stromal cells were diffusely positive for NSE (Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>A), S-100 protein (strong cytoplasmic and nuclear staining, Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>B), and vimentin (Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>C). Stromal cells expressed more focal \u03b1-inhibin (Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>D). They were strictly negative for epithelial membrane antigen (EMA), AE1\/AE3, CD10, and HMB-45 (Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>E-H). The other markers studied were strictly negative (melan-A, CD56, chromogranin, synaptophysin, calretinin, smooth muscle actin, and desmin). Finally, CD34 and CD31 underlined the rich and delicate vascular channels (Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>I, J). All of the immunohistochemical results were summarized in Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>.\n\nPanel of immunohistochemical stains\n\n| **Immunohistochemical Stain**       | **Result** |\n|:------------------------------------|:-----------|\n| Pan-cytokeratin (AE1\/AE3)           | \\-         |\n| epithelial membrane antigen (EMA)   | \\-         |\n| vimentin                            | \\+         |\n| \u03b1-inhibin                           | \\+         |\n| Neuron-specific enolase (NSE)       | \\+         |\n| S100 protein                        | \\+         |\n| Synaptophysin                       | \\-         |\n| Chromogranin                        | \\-         |\n| HMB-45 (melanoma-associated marker) | \\-         |\n| melan-A                             | \\-         |\n| Calretinin                          | \\-         |\n| smooth muscle actin                 | \\-         |\n| Desmin                              | \\-         |\n| CD10                                | \\-         |\n| CD34                                | \\-         |\n| CD31                                | \\-         |\n\n# Discussion\n\nBecause of the few reports of sporadic primary hemangioblastoma, the progression and prognosis of this tumor was still unclear. To our knowledge, there are five cases of hemangioblastoma involving the kidney which have been reported \\[4,5\\]. Hemangioblastoma is likely to be an underrecognized tumor of the kidney, because it mimics many tumor types morphologically and it is usually not considered in the differential diagnosis. A correct diagnosis is important because hemangioblastoma is benign even if there are highly atypical tumor cells, and the patient should be evaluated for possible von Hippel-Lindau disease. Because of the prominent vasculature and large neoplastic cells with atypical nuclei, renal hemangioblastoma can be mistaken for many renal neoplasms including renal cell carcinoma (RCC) and epithelioid angiomyolipoma, adrenal cortical carcinoma and paraganglioma (pheochromocytoma). Sometimes, it can also be mistaken for other rare tumors in this location as epthelioid hemangiopericytoma or lobular capillary hemangioma.\n\nAlthough renal hemangioblastoma mimics various renal neoplasms, it can be recognized or suspected on morphologic grounds. The clues to the diagnosis are: circumscribed borders, paucity of mitotic figures despite prominence of atypical cells, fine vacuoles in some tumor cells indicating presence of intracytoplasmic lipids, and rich capillary network with focal pericytomatous pattern.\n\nIn this case, all the features described above were observed except for fine cytoplasmic vacuoles, so the first diagnosis came to our mind was epithelioid hemangiopericytoma instead of hemangioblastoma. Because of numerous variants of RCC have been described in recent years which markedly expanding the morphologic spectrum and rendering it difficult to totally rule out RCC on morphologic grounds. Among the variants of RCC, clear cell RCC is the main differential diagnosis as it shares several morphologic features with hemangioblastoma. This tumor can have occasionally a hemangioblastoma-like pattern which makes it nearly impossible to distinguish with hemangioblastoma on morphologic grounds. In such particular case, immunohistochemical staining might be the only solution. In contrast to hemangioblastoma, clear cell RCC is usually negative for \u03b1-inhibin, S100, and NSE and positive for AE1\/AE3, EMA and CD10 \\[5,6\\]. And in this case, the tumor cells show striking morphologic mimicry of epithelioid angiomyolipoma. The epithelioid tumor cells of the latter often shows homogeneous or reticulated (spidery) cytoplasm, sometimes with grumous basophilic material, instead of lipid-containing vacuolated cytoplasm. Fat cells and thick-walled blood vessels with spindly cells radiating from the wall, if present, provide further support to the diagnosis. In addition, melanin marker (HMB45 or melan-A) and muscle marker (smooth muscle actin or desmin) will be helpful to diagnosis.\n\nAdrenal cortical carcinoma may directly invade or metastasize to the kidney. But the tumor cells commonly show lipid cytoplasmic vacuoles; mitotic activity, infiltrative growth and vascular invasion are often identifiable. In addition, immunohistochemical staining will be helpful to further demonstrate. Paraganglioma (pheochromocytoma) often shows a definite nested pattern in at least some foci which was hardly seen in this case.\n\nTo our surprise, the immunophenotype (CD34^-^, melan-A^-^, HMB-45^-^, smooth muscle actin^-^, muscle specific actin^-^ and desmin^-^) overthrows the diagnosis of epithelioid hemangiopericytoma or angiomyolipoma. Negative staining for AE1\/AE3, EMA and CD10 exclude the probability of RCC; Negative staining for synaptophysin, chromogranin and calretinin exclude the probability of adrenal cortical carcinoma and paraganglioma. In addition, S100 was diffuse positive in this case, while in paraganglioma, S100+ sustentacular cells are often found surrounding the nests of tumor cells. All of the diagnosises were overthrowed by immunohistochemical staining, so we searched the similar case on PubMed ( <http:\/\/www.ncbi.nlm.nih.gov>). After we learned that sporadic hemangioblastoma might happen in this location, we reviewed this case carefully and found only a few lipid droplets and bizarre tumor cell nuclei in tumor cytoplasm which was indicated by arrow in Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>G-H. NSE and \u03b1-inhibin were also added to stain. The immunophenotype (NSE^+^, S100^+^ and \u03b1-inhibin^+^) also demonstrated the diagnosis of hemangioblastoma. Furthermore, more cytoplasmic multiple sharply delineated vacuoles were observed in the slides performed for immunohistochemistry (Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>A, B, and D). The morphologic characteristics of this case were strikingly superimposable to those previously described in this location, including the clues to the diagnosis defined by Ip et al. \\[4\\] The immunophenotypic profile (AE1\/AE3-, S100+, NSE+, and \u03b1-inhibin+) reported by Ip et al \\[4\\] was also noted.\n\n# Conclusions\n\nIn this case, the tumor was nearly mistaken for epithelioid hemangiopericytoma which indicates it's hard to distinguish between them in the practical work. Cytoplasmic lipid droplet maybe indicates a diagnosis of hemangioblastoma, but don't exclude the probability when it was hardly seen, especially in the unfamiliar renal tumors with abundant capillary network. The stromal cells may be or may not be atypical with bizarre nuclei. Therefore, hemangioblastoma must be included in the dfferential diagnosis of renal tumors to not underestimate this tumor in this location and so to better evaluate its real frequency and not establish wrongly a diagnosis of malignancy to this benign tumor. Using combination of immunohistochemistry may be helpful to some rare renal tumors.\n\n# Competing interests\n\nThe authors declare that they have no competing interests.\n\n# Authors' contributions\n\nYL analyzed the data and wrote the manuscript as a major contributor. X-SQ and E-HW helped to revise the discussion section of this manuscript. All authors have read and approved the final manuscript.\n\n# Consent\n\nWritten informed consent was obtained from the parents of the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in Chief of this Journal.\n\n## Acknowledgements\n\nThe authors express their sincere appreciation of the language editing assistance given by Liang Wang, MD and Juan-han Yu, MD, Department of Pathology, China Medical University.","meta":{"dup_signals":{"dup_doc_count":159,"dup_dump_count":50,"dup_details":{"curated_sources":4,"2023-23":1,"2022-27":1,"2022-05":1,"2021-39":1,"2021-21":1,"2020-40":1,"2020-29":1,"2019-43":1,"2019-35":1,"2019-13":1,"2019-04":1,"2018-47":1,"2018-39":1,"2018-30":1,"2018-17":1,"2018-05":1,"2017-39":1,"2017-30":1,"2017-22":1,"2017-17":1,"2017-09":15,"2017-04":1,"2016-50":1,"2016-44":1,"2016-40":1,"2016-36":1,"2016-30":1,"2016-22":1,"2016-18":1,"2015-48":5,"2015-40":2,"2015-35":5,"2015-32":4,"2015-27":4,"2015-22":4,"2015-14":5,"2014-52":5,"2014-49":8,"2014-42":14,"2014-41":6,"2014-35":7,"2014-23":7,"2014-15":6,"2023-50":1,"2015-18":4,"2015-11":5,"2015-06":6,"2014-10":5,"2013-48":4,"2013-20":5}},"file":"PMC3488519"},"subset":"pubmed_central"}
{"text":"abstract: The vast majority of all agents used to directly kill cancer cells (ionizing radiation, most chemotherapeutic agents and some targeted therapies) work through either directly or indirectly generating reactive oxygen species that block key steps in the cell cycle. As mesenchymal cancers evolve from their epithelial cell progenitors, they almost inevitably possess much-heightened amounts of antioxidants that effectively block otherwise highly effective oxidant therapies. Also key to better understanding is why and how the anti-diabetic drug metformin (the world's most prescribed pharmaceutical product) preferentially kills oxidant-deficient mesenchymal p53^\u2212\\ \u2212^cells. A much faster timetable should be adopted towards developing more new drugs effective against p53^\u2212\\ \u2212^ cancers.\nauthor: Jim Watsone-mail: <email@example.com>\ndate: 2013-01\ninstitute: Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, NY 11724, USA\nreferences:\ntitle: Oxidants, antioxidants and the current incurability of metastatic cancers\n\nAlthough the mortality from many cancers, particularly those of haematopoietic cells, has been steadily falling, the more important statistic may be that so many epithelial cancers (carcinomas) and effectively all mesenchymal cancers (sarcomas) remain largely incurable. Even though an increasing variety of intelligently designed, gene-targeted drugs now are in clinical use, they generally only temporarily hold back the fatal ravages of major cancers such as those of the lung, colon and breast that have become metastatic and gone beyond the reach of the skilled surgeon or radiotherapist. Even though we will soon have comprehensive views of how most cancers arise and function at the genetic and biochemical level, their 'curing' seems now to many seasoned scientists an even more daunting objective than when the 'War on Cancer' was started by President Nixon in December 1971.\n\nPropelling me then, 40 years ago, to turn the Cold Spring Harbor Laboratory into a major site for unravelling the genetic underpinnings of cancer was the belief that once the gene-induced molecular pathways to cancer became known, medicinal chemists would go on to develop much more effective gene-targeted drugs. Unlike most early proponents of the 'War on Cancer', who thought that DNA-damaging chemotherapeutic agents would bring real victories in one to two decades, I thought three if not four more decades of focused research would need to pass before we would be in a position to go all out for total victory \\[1\\]. In fact, only after the 1988\u20132003 Human Genome Project provided the world with the highly accurate sequences for three billion human DNA letters has it been possible to begin to approach the true genetic complexity of cancer.\n\n# Molecular pathways to cancer as revealed through DNA sequencing\n\nBy now we know that mutations in at least several hundred human genes (out of a total of 21 000 genes) become serious 'drivers' of the abnormal cell growth and division process that generates human cancer \\[2\\]. They do so because they encode the protein components of 'signal transduction pathways' that enable external signals (growth factors) to move from the cell surface receptors to key promoter\u2013enhancer regions along the 24 human chromosomes. There they turn up the expression of genes needed for cell growth and division as well as the evasion of programmed cell death, the latter of which much underlies the ever-growing resistance of late-stage aggressive cancer cells to radio- and chemotherapeutic therapies. Most importantly, there exist multiple molecular pathways that bring about cell growth and proliferation, each with their own specific surface receptors, cytoplasmic transducers, and promoters and enhancers of gene expression \\[3\\].\n\nMuch potential cross talk exists between these pathways, allowing new DNA mutations to create new pathways to cancer when pre-existing ones are blocked. Already we know that the emergence of resistance to the gene *BRAF*-targeted anti-melanoma drug Zelboraf frequently results from driver pathway cross talk, as does resistance to the targeted drugs Iressa and Tarceva when they are deployed against EGFR-driven lung cancers. Given the seemingly almost intrinsic genetic instability of many late-stage cancers, we should not be surprised when key old timers in cancer genetics doubt being able to truly cure most victims of widespread metastatic cancer.\n\nResistance to gene-targeted anti-cancer drugs also comes about as a consequence of the radical changes in underlying patterns of gene expression that accompany the epithelial-to-mesenchymal cell transitions (EMTs) that cancer cells undergo when their surrounding environments become hypoxic \\[4\\]. EMTs generate free-floating mesenchymal cells whose flexible shapes and still high ATP-generating potential give them the capacity for amoeboid cell-like movements that let them metastasize to other body locations (brain, liver, lungs). Only when they have so moved do most cancers become truly life-threatening.\n\n# Epithelial-to-mesenchymal transitions are a consequence of changes in transcriptional regulation\n\nEMTs leave intact the pre-existing order of DNA bases while changing the way they are read into RNA transcripts. Underlying transcriptional regulation are site-specific DNA-binding proteins, and sometimes regulatory RNAs, that recruit to genes the machinery required to read those genes. This includes the general transcription machinery and also enzymes that modify the histones around which chromosomal DNA is wound, and the DNA itself. These enzymes mediate methylation and acetylation of histones, as well as remodelling of the nucleosomes in various ways, and methylation of DNA bases, changes that can influence how a given gene is expressed. Regulation of transcription extends far beyond its role in influencing how cancer cells respond to changes in their environmental surroundings. This regulation underlies all the multiple switches that accompany the transition of fertilized eggs into the differentiated cells (lung, kidney, etc.) of mature organisms.\n\n# IL6-like cytokines drive mesenchymal cells to commence cell proliferation\n\nMuch holding back the creation of effective drugs against mesenchymal cancer cells has long been ignorance of the externally driven signalling pathways propelling them into stem cell growth and subsequent differentiation. Most attention until now has been focused on the Wnt signalling pathway that sends \u03b2-catenin into the cell nucleus to activate the TCF transcription factor for essential roles in EMTs as well as stem cell functioning \\[5,6\\]. An even more important villain may have been virtually staring in our faces for almost two decades\u2014one or more of the cytokine mediators of inflammation and immunity, in particular, the IL6 interleukin. IL6 blood serum levels, for example, steadily go up as incurable cancers become more life-threatening \\[7,8\\]. Autocrine loops probably exist where cytokine binding to their respective cell surface receptors sets into motion downstream gene-activating pathways that not only generate more IL6 molecules but give their respective cancer cells an aura of almost true immortality by blocking the major pathway to programmed cell death (apoptosis). *Pushing by cytokines of otherwise quiescent mesenchymal cancer cells to grow and divide probably explains why anti-inflammatory agents such as aspirin lead to much less cancer in those human beings who regularly take them* \\[9\\].\n\nUnfortunately, the inherently very large number of proteins whose expression goes either up or down as the mesenchymal cancer cells move out of quiescent states into the cell cycle makes it still very tricky to know, beyond the cytokines, what other driver proteins to focus on for drug development. Ideally, we should largely focus first on finding inhibitors of cancer cell proliferation as opposed to inhibitors of cancer cell growth. Inhibiting, say, the synthesis of cellular molecular building blocks will slow down not only the metabolism of cancer cells but also that of our body's normally functioning cells. By contrast, blocking proteins specifically moving through the cell cycle should leave untouched the normal functioning of the vast majority of our body's cells and so generate much less unwanted side effects.\n\n# The gene transcription activator Myc allows cells to move through the cell cycle\n\nLong thought to be a key, if not *the* key, protein against which to develop cell-proliferation-inhibiting drugs is the powerful gene transcription activator Myc. First known for its role in driving cancers of blood-forming lymphocytes (e.g. Burkitt's lymphoma), Myc now also has been found to be a key driver of the rapidly fatal 'small cell' lung cancers as well as the likely driver of many late-stage incurable cancers, including receptor negative and ductal breast cancers. *Lots of* *Myc* *may turn out to be an essential feature of much of the truly incurable cancer*. It simultaneously turns up the synthesis of the more than 1000 different proteins required to move all cells through the cell cycle. Although precisely how this almost 400-amino acid long polypeptide works at the molecular level remains to be worked out, it seems to play a unique role that cannot be handled by any other class of transcription factors. Unlike our first hunch that Myc was somehow an on\u2013off specifier of gene activity, it is a nonlinear amplifier of expression acting universally on active genes except for the immediate early genes that become expressed before *Myc* \\[10,11\\]. Already many serious efforts have been made to develop drugs that block its cell-proliferation-promoting activities. Unfortunately, all such direct efforts have so far failed.\n\nUsing a dominant negative plasmid that blocks all Myc functions, Gerard Evans' laboratory, first at UCSF and now in Cambridge, UK, has used mouse xenograph models of several major human cancers to show Myc's indispensable role in moving through the cell cycle \\[12\\]. Although mouse stem cells in Myc's absence stop growing and dividing, they resume normal functioning when *Myc* is turned back on. By contrast, the turning off of *Myc* in human cancer cells preferentially drives them into programmed cell death (apoptosis) with one important exception: pancreatic adenocarcinoma cells do not enter into apoptosis, quite possibly explaining why pancreatic cancer is so resistant to virtually all cell-killing reagents (G. Evans 2012, personal communication).\n\n# Bromodomain 4 proteins play essential roles in maintaining the Myc levels necessary for leukaemic cell growth and division\n\nAn unanticipated powerful way for lowering Myc levels in haematopoietic cancers has emerged from the discovery that the incurable nature of *MLL-AF9* acute myeloid leukaemia (AML) depends upon the presence of the not yet well understood protein bromodomain 4 (BRD4). When JQ1, developed last year to treat the BRD4-driven rare *NUT* midline carcinoma, was used on human *MLL-AF9* AML cells, they rapidly stopped multiplying and differentiated into macrophages \\[13,14\\]. At the same time, Myc levels rapidly plunged. Most importantly, JQ1 does not block the normal macrophage production, suggesting that Myc levels in macrophage-forming stem cells do not depend upon BRD4. Their formation must depend on a different chromosomal remodeller.\n\n# *Myc* is turned on through multiple molecular pathways\n\nHow *Myc* is turned on not only in other cancers but also during normal human development remains largely to be worked out. Likewise not known is how the BRD4 protein at the molecular level helps turn on Myc synthesis in MLL-AF9-driven leukaemia. Until JQ1 goes into the clinic against leukaemia late this year, we will not moreover know for sure whether resistance to JQ1 will compromise its clinical utility. Unfortunately, the answer is probably yes because artificially turning up *Myc* by means that bypass *BRD4* causes JQ1 resistance. Moreover, there are already known multiple ways to turn on *Myc* expression in normal cells, each starting by signals binding to specific cell surface receptors then moving through one or more layers of signal transducers to the nucleus to turn up the transcription of genes needed for cell growth and division. Myc synthesis is not only downstream of the cytokine Jak\u2013Stat3 signal transduction pathway but also downstream of the HER2\u2013RAS\u2013RAF\u2013SHp2\u2013ERK3 pathway that helps drive the growth of much, if not most, breast cancer \\[15\\]. Whether they in turn feed into BRD protein-dependent gene-activating pathways remains for the future to reveal. A multiplicity of Myc-inhibiting specific drugs may have to be in our arsenal before we can routinely move beyond delaying death from incurable cancers to true lifetime long cures.\n\n# Detecting key cancer cell vulnerabilities through RNAi screens\n\nThat the BRD4 protein is among the major Achilles' heels of incurable AML became known not because of a chance observation but by using a powerful new methodology for detecting molecular weaknesses that are cancer cell-specific. At its heart has been the deployment over the past several years by Greg Hannon at Cold Spring Harbor Laboratory of short hairpin RNA molecules (shRNAs) specifically designed to knock back the functioning of single human genes \\[16\\]. A genome shRNA library containing multiple probes (four to six) for each human gene possesses some 100 000 shRNAs. Testing all of them extensively against just one type of cancer still poses a formidable, logistical challenge likely to require 1- to 2-year long intervals for even 'big science laboratories'.\n\nMuch smaller highly focused libraries, however, now can be deployed by high-quality, university-level science laboratories provided there already exist hints as to what molecular vulnerabilities might be found. Forearmed by knowledge that invariably incurable forms of acute myeloblastic leukaemia (AML) originate from rearrangements of a key gene involved in epigenetic chromosomal remodelling, Chris Vakoc and Johannes Zuber at the Cold Spring Harbor Laboratory found the gene-activating BRD4 as the most pronounced potential molecular weakness of an *MLL-AF9* human AML. They did so by screening libraries of only some 1000 probes designed to knockout 234 genes coding for the key proteins involved in epigenetic-driven gene expression.\n\nMost recently, Vakoc has found three other major protein players (Menin, Ezh1\/2 and Eed) that work together with BRD4 to make *MLL-AF9* AML incurable by currently deployed anti-cancer drugs \\[17\\]. Drugs inhibiting their respective functioning should also provide effective anti-AML agents. *Ezh1\/2* and *Eed* code for polycomb proteins that block specific gene expression, whereas the *Menin* gene, like the *BRD4* gene, activates gene expression. Loss of functional Ezh1\/2 and Eed blocks the expression of the *CdKn2a* gene-encoded p16 and p19 proteins that have widespread cell-cycle-progression-blocking roles. The Menin protein's molecular role probably involves its already known binding to MLL. Like BRD4, it may have a Myc-level-raising role. Finding out how such chromosome remodelling dependencies emerge and evolve during tumour progression will directly impact the clinical implementation of epigenetic-based anti-cancer therapies.\n\n# BRD4 functioning is vital not only for fast-growing leukaemias but also for many, if not most, dangerous lymphomas and myelomas\n\nAs soon as possible, we must find out in more detail how far the drug JQ1's anti-cancer actions extend beyond *MLL-AF9*-specific AMLs. Already we know that in mice it stops equally well the more curable, non-*MLL* rearranged strains of AML as well as all forms of acute lymphocytic leukaemia (ALL). BRD4's capacity to heighten Myc levels thus probably extends over almost all leukaemias. Whether the polycomb proteins of ALL, like those of AML, also turn off the cell-cycle-inhibiting *CdKn2a*-coded proteins p16 and p19 remains to be seen. JQ1 also stops the growth in mice of many fast-growing B-, and T-cell lymphomas, suggesting that their untreated BRD4 protein maintains their high Myc levels necessary to make them fatal. In JQ1-resistant lymphomas (e.g. Jurkat cell), Myc synthesis must be turned on by a different route. Cell lines from most human multiple myeloma victims also frequently show high sensitivity to JQ1 \\[18\\]. There, the twosome cocktail of JQ1 and the now widely deployed proteasome inhibitor Velcade reinforce each other's anti-myeloma actions. When JQ1 becomes broadly available clinically, hopefully by mid-2013, it may considerably lengthen the 3\u20135 more years of additional life provided to most myeloma victims by Velcade administration.\n\nJQ1 also significantly slows down the growth of a small but real number of cell lines derived from many major solid cancers (e.g. prostate and melanoma). BRD4 may have been only called into play late as these cancers evolve to become more aggressive. Of more importance is JQ1's failure to stop the growth of the vast majority of solid tumour cell lines. The heightened Myc levels needed by, say, cancers of the prostate and breast may instead be provided by the intervention of one or more of the some 35 other BRD proteins or other chromatin regulators. Unfortunately, we do not yet know how the vast majority of them function beyond the fact that their BRD pockets, by binding to the acetyl groups, help turn on, not turn off, gene activation. JQ1's unanticipated blocking of sperm functioning most excitingly has led to the recent discovery of a testis-specific bromodomain (BRDT) essential for chromatin remodelling during spermatogenesis. Occupancy of the BRDT acetyl-lysine pocket by JQ1 generates a complete and reversible contraceptive effect \\[19\\]. Early evidence suggests that BRDT does not promote Myc synthesis. There may be out there soon to be found, say, breast-specific or prostate-specific *BRD* gene activators. Most important to learn is whether they also do or do not drive Myc synthesis.\n\n# The circadian rhythm regulator (PER2) by negatively regulating Myc levels functions as an important tumour suppressor\n\nMyc's paramount role in moving cancer cells through the cell cycle has recently been reinforced by two highly independent RNAi screens to find genes whose loss of function selectively kills cancer cells \\[20,21\\]. In sampling largely different sets of genes, they both honed in on the gene *CSNKe* coding for protein kinase casein kinase 2 epsilon. Among its many multiple targets for phosphorylation and subsequent proteosome-mediated degradation is the transcription factor *PER2* gene whose selective binding to DNA turns off the function of many genes including *Myc*. Already long known has been PERIOD 2 (PER2) involvement as a clock protein at the heart of the circadian rhythms of higher animal cells. Later, quite unexpectedly, PER2 was found to function as a tumour suppressor, with the absence of both its copies causing the rate of radiation-induced cancers to rise. It now seems obvious that its anti-cancer action arises from its ability to turn off *Myc*. In PER2's absence, Myc levels greatly rise, thereby explaining why tumours of many types all display higher levels of CSNKe than found in their normal cell equivalents. Common sense suggests that specific CSNKe inhibitors should soon be broadly tested against a large variety of human cancers.\n\n# High-Myc-driven, fast proliferating cells possess cell cycle vulnerabilities\n\nHigh-Myc-level proliferating cells less efficiently proceed through the mitotic cycle than cells driven by lower Myc levels. Why high Myc leads to many more mitotic-generated chromosome abnormalities has recently been explained through a large RNAi screen designed to reveal 'synthetic lethal' genes that only have vital function under conditions of high Myc. Most unexpectedly, they pinpointed key roles for the SUMO-activating genes *SAE1* and *SAE2* involved in proteasome-specific protein degradation \\[22\\]. When they are blocked from functioning, large numbers of Myc-driven genes somehow become switched from on to off. As expected, many function in the formation and breakdown of the mitotic spindle. A much less anticipated second class functions in ubiquitin-based, proteasome-mediated protein degradation. Conceivably, the fast growth rates of high-Myc-level-driven proliferating cells generate more mitosis-involved proteins than their respective proteasomes can timely breakdown. In any case, drugs designed to block *SAE1* and *SAE2* should preferentially kill fast-proliferating cancer cells.\n\nHigh-Myc-level vulnerability is also generated by suboptimal supplies of CD kinase 1 (CDK1) that functions with the A type cyclins during the late S phase of the cell cycle. As long as the Myc levels are those of normal cells, proliferating cells have sufficient CDK1. But when more Myc leads to faster cell cycles, much more CDK1 is required to prevent failed cell divisions. So, it makes a prime candidate for the development of an effective drug against high-Myc-driven cancers \\[23\\].\n\n# Selectively killing cancer cells through exploiting cancer-specific metabolic and oxidative weaknesses\n\nWe must focus much, much more on the wide range of metabolic and oxidative vulnerabilities that arise as consequences of the uncontrolled growth and proliferation capacities of cancer cells. As human cancers become driven to more aggressive glycolytic states, their ever-increasing metabolic stress makes them especially vulnerable to sudden lowering of their vital ATP energy supplies. 3-Bromopyruvate, the powerful dual inhibitor of hexokinase as well as oxidative phosphorylation, kills highly dangerous hepatocellular carcinoma cells more than 10 times faster than the more resilient normal liver cells and so has the capacity to truly cure, at least in rats, an otherwise highly incurable cancer \\[24,25\\]. The structurally very different hexokinase inhibitor 2-deoxyglucose, through its ability to block glycolysis, also has the potential for being an important anti-cancer drug. Not surprisingly, it works even better when combined with inhibitors of ATP-generating oxidative phosphorylation such as the mitochondrial target drug Mito Q \\[26\\].\n\nA key mediator of cellular response to falling ATP levels is the AMP-dependent protein kinase AMPK, which in times of nutritional stress phosphorylates key target proteins to push metabolism away from anabolic growth patterns \\[27\\]. By inhibiting mTOR it slows protein synthesis, and by phosphorylating acetyl-CoA carboxylase it slows down lipid synthesis. The glycolytic pathways that produce the cellular building blocks are indirectly controlled by AMPK through its phosphorylation of the p53 transcription factor. Activated p53 slows down glycolysis during cell cycle arrest through turning on its *TIGAR* gene target. Its respective protein breaks down the key regulator of glycolysis fructose 2,6-bisphosphate as well as blocking further cell cycles through turning on the *p21* gene.\n\n# Preferential cancer cell killing by apoptosis reflects high p53 levels\n\nThe enhanced apoptosis capability of early-stage epithelial cancer cells, in comparison with their normal cell equivalents, reflects their higher content of activated p53 transcription factor. Overexpression and amplification of the p53 repressors MDM2 and MDM4 are common across cancer types. In the case of melanomas, p53 function is commonly shut down by overexpression of MDM4. Already a drug exists that through its inhibition of MDM4 makes melanoma much more treatable \\[28\\]. Knowing more about why p53 activation sometimes leads to cell cycle arrest (senescence) and under different circumstances results in apoptosis remains an important challenge for the immediate future.\n\n# P53 induces apoptosis by turning on the synthesis of genes whose primary function is the synthesis of reactive oxygen species\n\nHow p53 turns on apoptosis was first revealed through elegant gene expression studies carried out in Bert Vogelstein's Johns Hopkins laboratory in 1997 \\[29\\]. Although looking for genes expressed only during apoptosis, they discovered a set of 13 p53-induced genes (*PIG* genes), each of which are likely key players in the cellular synthesis of reactive oxygen species (ROS; H~2~O~2~ hydrogen peroxide, the OH^\u2212^ radiation and O~2~^\u2212^ superoxides). *PIG3*, for example, codes for a quinone oxidoreductase that is a potent generator of ROS \\[30,31\\]. p53 target genes also play major roles in downstream processes through turning on synthesis of some 10 different mitochondrial functioning proteins such as BAX, PUMA and NOXA, as well as death receptors such as DR4 and DR5, that in ways yet to be elucidated help carry out the many successive proteolysis stages in apoptosis \\[32\\].\n\nEqually important, p53 turns on the synthesis of the key proteins involved in the apoptotic (programmed cell death) elimination of cells that have no long-term future, say, through unsustainable metabolic stress or damage to cellular chromosomes brought about by exposure to ultraviolet or ionizing radiation. So, removing such cells are complex sets of largely mitochondrial-sited degradation events. As the successive stages in apoptosis unravel, the respective dying cells lose mitochondrial functioning and release cytochrome c, culminating in DNA-liberating cell dissolution.\n\n# Leakage from drug-impaired mitochondrial electron transport chains raises reactive oxygen species levels\n\nThe mitochondrial electron transport generation of ATP and heat is obligatorily accompanied by the production of ROS (such as the OH^\u2212^ radical, H~2~O~2~ and O~2~^\u2212^ superoxides). Normally, preventing ROS molecules from irreversibly damaging key nucleic acid and protein molecules are potent antioxidative molecules such as glutathione and thioredoxin \\[33\\]. When present in normal amounts, they cannot handle the much larger amount of ROS generated when oxidative phosphorylation becomes inhibited by mitochondrial-specific drugs such as rotenone that block feeding of NADH into the respiratory chain or by 3\u2032-3\u2032 diindolylmethane (DIM), the active component in the long-reputed chemo-preventative *Brassica* vegetables, which inhibits the mitochondrial F1F0 ATP synthesis complex \\[34\\]. Still-remaining ROS molecules through oxidizing intra-mitochondrial targets induce the apoptotic elimination of cells damaged from excessive oxidative stress. Already, DIM is used as an adjuvant therapy for recurrent respiratory papillomatosis in humans. The molecular mechanism(s) through which ROS induce apoptosis remains to be found\u2014hopefully soon. Now, we will be surprised if they do not somehow directly oxidize and so activate one or more of the BAX-like proteins involved in p53-mediated apoptosis.\n\nThat ROS by themselves can mediate apoptosis was recently convincingly shown by the finding that the 'first-in-class' anti-cancer mitochondrial drug elesclomol (discovered by Synta Pharmaceuticals through screening for anti-apoptotic agents) kills cancer cells through promoting ROS generation \\[35\\]. When these resulting ROS molecules are destroyed through the simultaneous administration of the antioxidant molecule *N*-acetylcysteine, preferential killing of cancer cells stops. The failure of elesclomol to generate apoptosis in non-cancerous cells probably arises from the inherently lower ROS level generated by normal mitochondrial electron transport machinery.\n\n# Reactive oxygen species may directly induce most apoptosis\n\nThat elesclomol promotes apoptosis through ROS generation raises the question whether much more, if not most, programmed cell death caused by anti-cancer therapies is also ROS-induced. Long puzzling has been why the highly oxygen sensitive 'hypoxia-inducible transcription factor' HIF1\u03b1 is inactivated by both the, until now thought very differently acting, 'microtubule binding' anti-cancer taxanes such as paclitaxel and the anti-cancer DNA intercalating topoisomerases such as topotecan or doxorubicin, as well as by frame-shifting mutagens such as acriflavine \\[36,37\\]. All these seemingly unrelated facts finally make sense by postulating that not only does ionizing radiation produce apoptosis through ROS but also today's most effective anti-cancer chemotherapeutic agents as well as the most efficient frame-shifting mutagens induce apoptosis through generating the synthesis of ROS \\[38\u201340\\]. That the taxane paclitaxel generates ROS through its binding to DNA became known from experiments showing that its relative effectiveness against cancer cell lines of widely different sensitivity is inversely correlated with their respective antioxidant capacity \\[41,42\\]. A common ROS-mediated way through which almost all anti-cancer agents induce apoptosis explains why cancers that become resistant to chemotherapeutic control become equally resistant to ionizing radiotherapy.\n\nRecent use of a 50 000 member chemical library at MIT's Koch Cancer Center to search out molecules that selectively killed *K-RAS*-transformed human fibroblasts revealed the piperidine derivation lanperisone \\[43\\]. ROS generation underlies its cancer cell killing action. Surprisingly, this already clinically used muscle relaxant induced non-apoptotic cell death in a p53 (++ versus\u2212\u2212) independent manner. When lanperisone was applied in the presence of the ROS-destroying antioxidant scavenger molecules deferoxamine, butylated hydroxylamine or the antioxidant trolox, no activity was observed.\n\n# Blockage of reactive-oxygen-species-driven apoptosis by antioxidants\n\nAlthough we know ROS as a positive force for life through their apoptosis-inducing role, for much longer we have feared them for their ability to irreversibly damage key proteins and nucleic acid molecules. So when not needed, they are constantly being neutralized by antioxidative proteins such as glutathione, superoxide dismutase, catalase and thioredoxin. Controlling their synthesis as well as that of many more minor antioxidants is the Nrf2 transcription factor, which probably came into existence soon after life as we know it started. Most importantly, at Cancer Research UK in Cambridge, David Tuveson's laboratory has recently shown that Nrf2 synthesis is somehow upregulated by the cell growth and division-promoting *RAS*, *RAF* and *MYC* oncogenes \\[44\\]. Biologically, this makes sense because we want antioxidants present when DNA functions to make more of itself.\n\nThe fact that cancer cells largely driven by RAS and Myc are among the most difficult to treat may thus often be due to their high levels of ROS-destroying antioxidants. Whether their high antioxidative level totally explains the effective incurability of pancreatic cancer remains to be shown. The fact that late-stage cancers frequently have multiple copies of *RAS* and *MYC* oncogenes strongly hints that their general incurability more than occasionally arises from high antioxidant levels. Clearly important to learn is what other molecules exist that turn on Nrf2 expression. During the yeast life cycle and probably that of most organisms, oxidative phosphorylation is clearly separated by time from when DNA synthesis occurs. Whether Nrf2 levels also go up and down during the cell cycle remains important to be known soon.\n\n# Enhancing apoptotic killing using pre-existing drugs that lower antioxidant levels\n\nAlready there exist experiments with haematopoietic cells in which the cancer-cell-killing capacity of the ROS generator arsenic trioxide (As~2~O~3~) has been shown to be inversely correlated with the content levels of the major cellular antioxidant glutathione \\[45\\]. As~2~O~3~ also knocks down the reductive power of thioredoxin necessary for several key steps in cellular metabolism. Its capacity to inhibit both thioredoxin and glutathione widens its potential for a successful deployment against many major cancers beyond promyeloblastic leukaemia. Also capable of enhancing the cytotoxic effect of As~2~O~3~ is ascorbic acid, which, though known for its antioxidant role in cells, is converted into its oxidizing form dehydroascorbic acid. Unfortunately, up until now, we do not yet have clinically effective ways to lower glutathione levels. Lowering its level through deployment of the drug buthionine sulphazine that blocks its synthesis leads quickly to upregulation of the Nrf2 transcription factor that in turn upregulates glutathione synthesis \\[46\\]. A more general way to reduce antioxidant levels deploys motexafin gadolinium, a member of a class of porphyrin molecules called texaphyrins. Through a process called futile redox recycling, it transfers hydrogen from antioxidants to produce ROS. Unfortunately, clinical trials designed to show its enhancement of chemo- and radiotherapies have so far shown only modest life extensions as opposed to cures.\n\nThrough selecting for compounds that preferentially induce apoptosis in cancer cells as opposed to normal cells, the natural product piperlongumine from the *Piper longum* plant was recently revealed as a potential anti-cancer drug \\[47\\]. Most exciting, it mediates its action through its binding to the active sites of several key cellular antioxidants (e.g. glutathione *S* transferase and carbonyl reductase 1) known to participate in cellular responses to ROS-induced oxidative stress. That piperlongumine failed to raise ROS levels in non-cancerous cells probably resulted from their inherently lower levels of these antioxidants which, in turn, result from less activation of the Nrf2 transcription factor.\n\n# Anti-angiogenic drugs work only when used in conjunction with reactive oxygen species generators\n\nThe non-toxic anti-angiogenesis protein endostatin (discovered and promoted in the late 1990s in Judah Folkman's Boston laboratory and now resurrected by Yongzhang Luo in Beijing) shows anti-cancer activity only when it is used together with conventional chemotherapeutic agents. This fact, long puzzling to me, may be due to the chemotherapeutic component providing the ROS needed for cancer cell killing \\[48\\]. By itself, the hypoxia resulting from endostatin action may not be sufficient for cancer cell killing. A similar explanation may explain why Genentech's avastin also only works when combined with chemotherapy. By contrast, the killing of mutant *BRAF* melanoma cells by Zelboraf works very well in the absence of any obvious direct source of ROS. Conceivably, the metabolic stress resulting from its turning off the RAS\u2013ERK pathways somehow shuts down the Nrf2 pathways, letting ROS rise to the level needed to kill the drug-weakened melanoma cells.\n\n# Lower reactive oxygen species levels in stem cells reflect higher levels of antioxidants\n\nFor more than a decade, there has existed too long ignored evidence that normal stem cells have lower ROS levels than their differentiated progeny. Just a year ago, even more convincing experimentation showed that breast cancer stem cells also contain lower ROS levels than those found in their cancerous epithelial-like progeny cells \\[49\\]. All stem cells, be they normal or cancerous, probably have lower ROS levels as a result of their corresponding higher levels of prominent antioxidant molecules such as glutathione and thioredoxin. Most likely, these heightened amounts have evolved to protect chromosomal RNA from ROS-induced damage to the more exposed region of chromosomal DNA as it undergoes changes in compaction as it moves through the cell cycle. Whether all dividing cells have higher antioxidant levels remains to be worked out. If so, all stem cells will be inherently much more resistant to ROS-induced apoptotic killing than more differentiated, much less antioxidant-rich progeny cells.\n\n# Metformin selectively targets (kills) mesenchymal cancer stem cells\n\nAlready we have at our disposal a relatively non-toxic, excessively well-tested drug that preferentially kills mesenchymal stem cells. In a still much unappreciated article published three years ago in *Cancer Research*, Kevin Struhl's laboratory at Harvard Medical School first showed that metformin, a blocker of stage 2 oxidative phosphorylation, selectively targets stem cells. When so applied with chemotherapeutic agents to block xenographic tumour growth, it induces prolonged remission if not real cures \\[50,51\\]. But when metformin was left out of these experiments, subsequent multiplication of unkillable mesenchymal stem cells lets these xenographs grow into life-threatening forms, showing that chemotherapy by itself does not kill stem cells. This most widely used anti-diabetic drug's heightened ability to kill late-stage mesenchymal cancer cells probably explains why those humans who use it regularly have reduced incidences of many cancers.\n\nMetformin is presently being added to a number of anti-cancer chemotherapeutic regimes to see whether it magnifies their effectiveness in humans. The fact that metformin works much more effectively against p53**^\u2212\\ \u2212^** cells suggests that it may be most active against late-stage cancers, the vast majority of whose cells have lost both of their *p53* genes. By contrast, the highly chemo-radio-sensitive early-stage cancers against which most of anti-cancer drug development has focused might very well show little metformin effectiveness. By the end of 2013, we should know whether it radically improves any current therapies now in use. Highly focused new drug development should be initiated towards finding compounds beyond metformin that selectively kill stem cells. And the reason why metformin preferentially kills p53**^\u2212\\ \u2212^** stem cells should be even more actively sought out.\n\n# Free-radical-destroying antioxidative nutritional supplements may have caused more cancers than they have prevented\n\nFor as long as I have been focused on the understanding and curing of cancer (I taught a course on Cancer at Harvard in the autumn of 1959), well-intentioned individuals have been consuming antioxidative nutritional supplements as cancer preventatives if not actual therapies. The past, most prominent scientific proponent of their value was the great Caltech chemist, Linus Pauling, who near the end of his illustrious career wrote a book with Ewan Cameron in 1979, *Cancer and Vitamin C*, about vitamin C's great potential as an anti-cancer agent \\[52\\]. At the time of his death from prostate cancer in 1994, at the age of 93, Linus was taking 12 g of vitamin C every day. In light of the recent data strongly hinting that much of late-stage cancer's untreatability may arise from its possession of too many antioxidants, the time has come to seriously ask whether antioxidant use much more likely causes than prevents cancer.\n\nAll in all, the by now vast number of nutritional intervention trials using the antioxidants \u03b2-carotene, vitamin A, vitamin C, vitamin E and selenium have shown no obvious effectiveness in preventing gastrointestinal cancer nor in lengthening mortality \\[53\\]. In fact, they seem to slightly shorten the lives of those who take them. Future data may, in fact, show that antioxidant use, particularly that of vitamin E, leads to a small number of cancers that would not have come into existence but for antioxidant supplementation. Blueberries best be eaten because they taste good, not because their consumption will lead to less cancer.\n\n# A much faster timetable for developing anti-metastatic drugs\n\nThe world of Physics already knew 20 years ago that it had no choice but to go very big for the Higgs boson. To the civilized world's great relief, they now finally have it. Biology and Medicine must likewise now again aim big\u2014as when we first promised the world in 1988 that the still to be found human genome would later prove indispensable for the curing of most cancers and so went for it big. If, however, we continue to move forward at today's never frantic, largely five-day working week, the never receding 10\u201320 year away final victory that our cancer world now feels safe to project will continue to sink the stomachs of informed cancer victims and their families. That we now have no General of influence, much less power, say an Eisenhower or even better a Patton, leading our country's War on Cancer says everything. Needed soon is a leader that has our cancer drug development world working every day and all through the night.\n\nThe now much-touted genome-based personal cancer therapies may turn out to be much less important tools for future medicine than the newspapers of today lead us to hope \\[54\\]. Sending more government cancer monies towards innovative, anti-metastatic drug development to appropriate high-quality academic institutions would better use National Cancer Institute's (NCI) monies than the large sums spent now testing drugs for which we have little hope of true breakthroughs. The biggest obstacle today to moving forward effectively towards a true *war against cancer* may, in fact, come from the inherently conservative nature of today's cancer research establishments. They still are too closely wedded to moving forward with cocktails of drugs targeted against the growth promoting molecules (such as HER2, RAS, RAF, MEK, ERK, PI3K, AKT and mTOR) of signal transduction pathways instead of against Myc molecules that specifically promote the cell cycle.\n\nMost needed now are many new anti-Myc drugs beyond the exciting new BRD4 inhibitors, such as JQ1, as well as multiple drugs that inhibit the antioxidative molecules that likely make, say, pancreatic cancer so incurable. They should much enhance the effectiveness of all current radio- and chemotherapeutic regimes. As such, they will likely cure many more now incurable cancers. How they will interact as cocktail partners with the newer targeted therapies that do not directly generate ROS remains to be seen. Equally important may be an expanded search for drugs that prevent p53 breakdown.\n\n# A billion dollars should suffice to identify all the remaining proteins needed for curing most metastatic cancer\n\nThe total sum of money required for RNAi methodologies to reveal the remaining major molecular targets for future anti-cancer drug development need not be more than 500\u20131000 million dollars. Unfortunately, the NCI now is unlikely to take on still one more big science project when it is so hard-pressed to fund currently funded cancer programmes. Still dominating NCI's big science budget is The Cancer Genome Atlas (TCGA) project, which by its very nature finds only cancer cell drivers as opposed to vulnerabilities (synthetic lethals). While I initially supported TCGA getting big monies, I no longer do so. Further 100 million dollar annual injections so spent are not likely to produce the truly breakthrough drugs that we now so desperately need.\n\nHappily, the first RNAi whole genome big screen backed by a 'big pharma' firm has just started with Pfizer working with the Cold Spring Harbor Laboratory. Even, however, if several more giants working separately join in, collectively they will naturally focus on major cancers such as those of the breast, colon and lung. I doubt they will soon go big against, say, either melanoma or oesophageal cancer. Greg Hannon here at Cold Spring Harbor will probably be the first academic scientist to come to grips with the non-trivial experimental challenges provided by whole genome, 100 000 RNAi probe screens, through both his collaboration with Pfizer and through using monies separately provided by the Long Island-based Lustgarten Foundation's support for a comprehensive pancreatic cancer target screen and by Hollywood's 'stand up against cancer' support for breast cancer drug target identification. Although our enthusiasm for big RNAi screens remains far from universally shared, lack of money should not now keep us from soon seeing whether whole genome methodologies live up to their much-touted expectations \\[55\\]. The Cold Spring Harbor Laboratory happily has the means to move forward almost as if it were in a true war.\n\nFurther financial backing, allowing many more cancer-focused academic institutions to also go big using RNAi-based target discovery as well as to let them go on to the early stages of subsequent drug discovery, is not beyond the might of the world's major government research funding bodies nor that of our world's many, many super billionaires. The main factor holding us back from overcoming most of metastatic cancer over the next decade may soon no longer be lack of knowledge but our world's increasing failure to intelligently direct its 'monetary might' towards more human-society-benefiting directions.\n\n![](rsob-3-120144-g1.gif)\n\n**Jim Watson's** (JDW's) interest in cancer first publicly expressed itself through his teaching on tumour viruses after he joined the Harvard University Biology Department in the fall of 1956. Later, for the new Introductory Biology II, his last of 10 lectures focused on how cancer might be induced by DNA tumour viruses, the smallest of which probably only had DNA sufficient to code for 3\u20135 proteins. In his 1965 textbook, *The Molecular Biology of the Gene*, the last chapter ('A geneticist's view of cancer') raised the question of how a virus might have the capacity to turn on the cell cycle. Upon becoming director of the Cold Spring Harbor Laboratory in 1968, he changed its major research emphasis from microbial genetics to cancer (through recruiting Joe Sambrook from Renato Dulbecco's lab at the Salk Institute). Major among its early Cold Spring Harbor Laboratory eukaryotic accomplishments was the 1977 co-discovery of RNA splicing by Richard Roberts and Phil Sharp (MIT). JDW then necessarily devoted much of his time on scientific politics, first toward gaining National Institutes of Health (NIH) acceptance of the safety of recombinant DNA procedures (1973\u20131978), and second arguing for and then leading NIH's role in the Human Genome Project (1986\u20131992). In 2008, JDW's main interest moved to the curing of cancer focusing on the biochemistry of cancer cells as opposed to their genetic origins.\n\n# References","meta":{"dup_signals":{"dup_doc_count":129,"dup_dump_count":44,"dup_details":{"curated_sources":2,"2018-47":3,"2018-43":1,"2018-39":1,"2018-34":2,"2018-30":2,"2018-22":2,"2018-13":3,"2018-09":1,"2018-05":2,"2017-51":2,"2017-47":1,"2017-43":1,"2017-39":1,"2017-34":1,"2017-30":1,"2017-26":2,"2017-22":1,"2017-17":2,"2017-09":3,"2017-04":1,"2016-50":1,"2016-44":2,"2016-40":2,"2016-36":4,"2016-30":4,"2016-26":1,"2016-22":2,"2016-18":2,"2016-07":4,"2015-48":4,"2015-40":5,"2015-35":4,"2015-32":4,"2015-27":5,"2015-22":5,"2015-14":4,"2014-52":4,"2014-49":5,"2014-42":12,"2014-41":7,"2017-13":2,"2015-18":4,"2015-11":3,"2015-06":4}},"file":"PMC3603456"},"subset":"pubmed_central"}
{"text":"abstract: The thermal properties of nanofluids are an especially interesting research topic because of the variety of potential applications, which range from bio-utilities to next-generation heat-transfer fluids. In this study, photopyroelectric calorimetry for measuring the thermal diffusivity of urchin-like colloidal gold nanofluids as a function of particle size, concentration and shape in water, ethanol and ethylene glycol is reported. Urchin-like gold nanoparticles were synthesised in the presence of hydroquinone through seed-mediated growth with homogeneous shape and size ranging from 55 to 115 nm. The optical response, size and morphology of these nanoparticles were characterised using UV-visible spectroscopy and transmission electron microscopy. The thermal diffusivity of these nanofluids decreased as the size of the nanoparticles increased, and the enhancement depended on the thermal diffusivity of the solvent. The opposite effect (increase in thermal diffusivity) was observed when the nanoparticle concentration was increased. These effects were more evident for urchin-like gold nanofluids than for the corresponding spherical gold nanofluids.\nauthor: Gerardo A L\u00f3pez-Mu\u00f1oz; Jos\u00e9 Abraham Balderas-L\u00f3pez; Jaime Ortega-Lopez; Jos\u00e9 A Pescador-Rojas; Jaime Santoyo Salazar\ndate: 2012\ninstitute: 1Qu\u00edmica Arom\u00e1tica SA, R\u00edo Grande S\/N, Col. Santa Catarina, Acolman, Mexico State, CP 55875, Mexico; 2UPIBI-IPN, Av. Acueducto S\/N, Col. Barrio la Laguna Ticom\u00e1n, Mexico City, CP, 07340, Mexico; 3Biotechnology and Bioengineering Department, CINVESTAV, Av. IPN 2508, Col. San Pedro Zacatenco, Mexico City, CP, 07360, Mexico; 4ICGDE-BUAP, 4 Sur 104 Edificio Carolino, Col. Centro Hist\u00f3rico, Puebla, CP, 72000, Mexico; 5Physics Department, CINVESTAV, Av. IPN 2508, Col. San Pedro Zacatenco, Mexico City, CP, 07360, Mexico\nreferences:\ntitle: Thermal diffusivity measurement for urchin-like gold nanofluids with different solvents, sizes and concentrations\/shapes\n\n# Background\n\nCurrently, thermal properties of nanofluids, i.e. mixtures of nanomaterials suspended in an organic or inorganic base fluid, are intriguing because of their various uses, which range from bio-applications to the next generation of heat-transfer fluids. For example, gold nanofluids are a promising material for bio-applications because of their biocompatibility and thermo-optical properties. In particular, urchin-like gold fluids have a higher surface area that endows them with specific catalytic and thermo-optical qualities \\[1\\]. The surface plasmon resonance of urchin-like gold nanofluids can be tuned to the near-infrared region by increasing the particle size, which makes the particles potentially effective in photothermal therapy and as contrast agents for diagnosis \\[2\\]. In addition, local electromagnetic field enhancement at the tips of branched particles and thermal conductivity enhancement make these materials a candidate for application in electromagnetic hyperthermia therapy \\[3,4\\]. Thermal studies of nanofluids have mainly focused on thermal conductivity measurements, but recently, other techniques have been developed for thermal diffusivity measurements in nanofluids, such as the hot-wire technique and thermal lens spectrometry \\[5-7\\].\n\nIn the present study, the aqueous synthesis of urchin-like gold nanoparticles in the presence of hydroquinone through a seed-mediated growth process is presented. The thermal diffusivity in urchin-like gold nanofluids as a function of particle size (55 to 115 nm) and concentration in different solvents was investigated by photopyroelectric calorimetry. A comparative study of thermal diffusivity in urchin-like and spherical gold nanofluids with approximately equal particle sizes as a function of concentration is also reported. These results will open new horizons for thermal studies of nanofluids for bio-applications and heat-transfer applications.\n\n# Methods\n\n## Hydroquinone synthesis of urchin-like gold nanoparticles\n\nUrchin-like gold nanoparticles are commonly synthesised through a self-seeding growth process, where larger particles grow through the deposition of smaller seeds that form from the epitaxial deposition of atoms. The reduction of gold chloride on the nanoparticle seeds (gold nanoparticles, \\<20 nm) paired with hydroquinone as a selective reducing agent improves the homogeneous size and shape distribution of the urchin-like particles through physicochemical effects \\[8,9\\].\n\nGold nanoparticle seeds were synthesised by sodium citrate reduction. For this process, a solution of gold chloride is brought to boil, whereupon a solution of sodium citrate is immediately added. The solution is then removed from the heat source once nanoparticle maturation is completed, as indicated by the colour transition.\n\nUrchin-like gold nanoparticles were synthesised by hydroquinone method using consistent concentrations of gold chloride, sodium citrate and hydroquinone; however, the number of seeds gradually decreased, which resulted in the formation of larger urchin-like gold nanoparticles. Nanofluids with varying particle size were centrifuged at 6,000 rpm for 30 min and re-dispersed in high-performance liquid chromatography (HPLC) water, ethanol and ethylene glycol (EG) at a final concentration of 0.1 mg\/ml.\n\nTo provide nanofluids with different shapes and concentrations, stock solutions of urchin-like gold nanoparticles and spherical gold nanofluids synthesised by hydroquinone method \\[9,10\\] were centrifuged at 6,000 rpm for 30 min. The concentrated solutions were re-dispersed in HPLC water, ethanol and ethylene glycol to obtain different nanoparticle concentrations.\n\nAll chemicals used were of analytical grade as obtained from Sigma-Aldrich Corporation (St. Louis, MO, USA) and were used as received; HPLC water was used for the synthesis of all the urchin-like and spherical gold nanoparticles.\n\n## Basic photopyroelectric theoretical scheme\n\nAs shown in the literature \\[11\\], the photopyroelectric signal in the transmission configuration, assuming that the sample thickness *L* is the only variable (with fixed modulation frequency *f*) in the thermally thick regime of the liquid sample, can be expressed as follows:\n\n$${\\delta P} = E\\exp\\left( {- \\sigma_{s}L} \\right),$$\n\nwhere *E* is a complex constant and *\u03c3*~s~\u2009=\u2009(1\u2009+\u2009*i*)(*\u03c0f*\/*\u03b1*~s~)^1\/2^, with *\u03b1*~s~ is the thermal diffusivity of the sample. The amplitude \u2502\u03b4\u03a1\u2502 and phase \u0424 of this equation can be calculated as follows:\n\n$$|{\\delta P}| = |E|\\exp\\left( {- \\sqrt{\\frac{\\pi f}{\\alpha_{s}}}L} \\right),$$\n\n$$\\Phi = \\Phi_{0} - \\sqrt{\\frac{\\mathit{\\pi f}}{\\alpha_{s}}}L.$$\n\nThe signal phase \u0424 is a linear function of the sample thickness, and slope *B* is given by the following:\n\n$$B = \\left( \\frac{\\mathit{\\pi f}}{a_{s}} \\right)^{\\frac{1}{2}},$$\n\nfrom which the thermal diffusivity of the sample can be determined.\n\n## Experimental setup\n\nA transversal section of the photopyroelectric experimental setup for thermal diffusivity measurements is shown in Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>. The photopyroelectric sensor consisted of a PVDF film (25-\u03bcm thick) with metal electrodes (Ni-Al) on both sides of a stainless steel body. A silicon foil was attached to the top surface of the photopyroelectric sensor to prevent any possible damage that could result from exposure to the liquid environment. The resultant pyroelectric signals were processed by a lock-in amplifier (model SR830; Stanford Research, Menlo Park, CA, USA) for amplification and de-modulation; the transistor-transistor logic output of the lock-in was used for the modulation control of a 660-nm model laser diode system (IFLEX-2000; Qioptiq Photonics Ltd., Hamble, Hampshire, UK) at a fixed modulation frequency of 1 Hz.\n\nThe pyroelectric signal was recorded as a function of the sample thickness by measuring 20 experimental points from a relative sample thickness *l*~*0*~, at 10-\u03bcm intervals using a micro-linear stage (model T-LSM025A; Zaber Technologies, Inc., Vancouver, Canada). Linear fits were performed for the pyroelectric phase to obtain the *B* parameter, as defined in the 'Basic photopyroelectric theoretical scheme' section, from which the thermal diffusivity of the sample was obtained by means of the relationship *\u03b1*~s~\u2009=\u2009*\u03c0*\/*B*^2^. Measurements were performed at room temperature, or 22 \u00b1 2\u00b0C.\n\n# Results and discussion\n\n## Nanoparticle characterization\n\nThe particle size, shape\/morphology and nanoparticle distribution in the base medium were determined by transmission electron microscopy (TEM) using a JEOL JEM2010 (JEOL Ltd., Akishima, Tokyo, Japan) operating at 200 kV with a beam current of 105 \u03bcA. The TEM specimens were prepared by adding 50 \u03bcl of the stock nanoparticle solution onto 200-mesh carbon-coated copper grids. In Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>, the TEM images show the larger urchin-like gold nanoparticles that were synthesised by decreasing the number of nanoparticle seeds, as was expected. The dispersion of the urchin-like gold nanoparticles had a median size distribution of nearly 6% for all of the samples, including the large-diameter nanoparticles. This technique is advantageous over single-step methods that use silver ions to increase the facet selectivity during synthesis of the urchin-like nanoparticles; these methods are also less capable of continuously controlling the diameter of branched particles and spherical particles, which is another important method used to tailor the plasmon resonance absorption \\[8\\].\n\nThe absorption spectra of the synthesised nanoparticles were measured after the HPLC water background was established using a UV-visible (vis) spectrophotometer (8453 Agilent Technologies, Inc., Santa Clara, CA, USA). Figure <a href=\"#F3\" data-ref-type=\"fig\">3A<\/a> shows the UV-vis absorption spectra of the different-sized urchin-like gold nanofluids. As expected, increases in the diameter of the gold nanoparticles resulted in larger absorption (*\u03bb*~max~) values (Figure <a href=\"#F3\" data-ref-type=\"fig\">3B<\/a>) according to Mie theory \\[12,13\\]; these results provide further evidence that the hydroquinone growth method allows superior tuning of surface plasmon resonance for urchin-like gold nanoparticles.\n\n## Thermal diffusivity measurement\n\nThe plots in Figure <a href=\"#F4\" data-ref-type=\"fig\">4<\/a> show the signal phase as a function of sample thickness for the solvents used. The recorded thermal diffusivity values were 14.29 \u00d7 10^\u22124^ \u00b1 0.03 \u00d7 10^\u22124^ cm^2^\u00b7s^\u22121^, 9.25 \u00d7 10^\u22124^ \u00b1 0.03 \u00d7 10^\u22124^ cm^2^\u00b7s^\u22121^ and 8.32 \u00d7 10^\u22124^ \u00b1 0.03 \u00d7 10^\u22124^ cm^2^\u00b7s^\u22121^ for HPLC water (Sigma-Aldrich Corporation), EG and ethanol (Sigma-Aldrich Corporation), respectively. The measurements were obtained by utilising the photopyroelectric system as a reference sample for system characterisation with a coefficient of variation within 1% of similar values reported in the literature. The resulting thermal diffusivity values for colloidal urchin-like gold nanoparticles with different sizes in water, ethanol and ethylene glycol are summarised in Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>. The thermal diffusivity values of colloidal urchin-like and spherical gold nanofluids of two different particle sizes at different concentrations in water, ethanol and ethylene glycol are summarised in Tables <a href=\"#T2\" data-ref-type=\"table\">2<\/a> and <a href=\"#T3\" data-ref-type=\"table\">3<\/a>. All of the values reported are averaged from ten measurements for each sample, and the standard deviation was calculated as an estimation of uncertainty.\n\nThermal diffusivity of urchin-like gold nanofluids for different nanoparticle diameters and solvents\n\n| **Particle size (nm)** | **Water**^**a**^ | **EG**^**a**^ | **Ethanol**^**a**^ |\n|:----------------------:|:----------------:|:-------------:|:------------------:|\n|           55           |      14.76       |     9.83      |        9.05        |\n|           67           |      14.71       |     9.78      |        8.93        |\n|           70           |      14.68       |     9.73      |        8.84        |\n|           79           |      14.48       |     9.49      |        8.61        |\n|          115           |      14.34       |     9.33      |        8.44        |\n\nAt a constant nanoparticle concentration of 0.1 mg\/ml. ^a^Values are presented as (*\u03b1* (10^\u22124^ cm^2^ s^\u22121^ ) \u00b1 0.03).\n\nThermal diffusivity of urchin-like gold nanofluids at different nanoparticle concentrations and in different solvents\n\n| **Concentration (mg\/ml)** | **Water**^**a**^ | **EG**^**a**^ | **Ethanol**^**a**^ | **Water**^**b**^ | **EG**^**b**^ | **Ethanol**^**b**^ |\n|:--:|:--:|:--:|:--:|:--:|:--:|:--:|\n| 0.2 | 14.79 | 9.87 | 9.09 | 14.36 | 9.36 | 8.47 |\n| 0.4 | 14.83 | 9.92 | 9.15 | 14.37 | 9.39 | 8.51 |\n| 0.6 | 14.91 | 10.01 | 9.28 | 14.42 | 9.45 | 8.56 |\n| 0.8 | 14.98 | 10.13 | 9.39 | 14.46 | 9.49 | 8.62 |\n| 1.0 | 15.11 | 10.25 | 9.56 | 14.53 | 9.56 | 8.71 |\n\n^a^Values are presented as (*\u03b1*~55nm~ (10^\u22124^ cm^2^ s^\u22121^) \u00b1 0.03). ^b^Values are presented as (*\u03b1*~115nm~ (10^\u22124^ cm^2^ s^\u22121^) \u00b1 0.03).\n\nThermal diffusivity of spherical gold nanofluids at different nanoparticle concentrations and in different solvents\n\n| **Concentration (mg\/ml)** | **Water**^**a**^ | **EG**^**a**^ | **Ethanol**^**a**^ | **Water**^**b**^ | **EG**^**b**^ | **Ethanol**^**b**^ |\n|:--:|:--:|:--:|:--:|:--:|:--:|:--:|\n| 0.2 | 14.74 | 9.83 | 9.05 | 14.33 | 9.32 | 8.44 |\n| 0.4 | 14.79 | 9.87 | 9.10 | 14.34 | 9.34 | 8.47 |\n| 0.6 | 14.84 | 9.91 | 9.22 | 14.37 | 9.39 | 8.51 |\n| 0.8 | 14.91 | 10.02 | 9.32 | 14.41 | 9.44 | 8.57 |\n| 1.0 | 14.99 | 10.13 | 9.43 | 14.46 | 9.50 | 8.64 |\n\n^a^Values are presented as (\u03b1~55nm~ (10^\u22124^ cm^2^ s^\u22121^) \u00b1 0.03). ^b^Values are presented as (\u03b1~120nm~ (10^\u22124^ cm^2^ s^\u22121^ ) \u00b1 0.03).\n\n### Nanoparticle size\n\nFigure <a href=\"#F5\" data-ref-type=\"fig\">5<\/a> shows the thermal diffusivity values of urchin-like gold colloid nanofluids as a function of nanoparticle size and in different solvents; the plot also reveals that a constant nanoparticle concentration (0.1 mg\/ml) effectively increased the thermal diffusivity ratio (*\u03b1*~sample~\/*\u03b1*~base\\ fluid~) of the nanofluids as the nanoparticle diameter and as the thermal diffusivity of the solvent were reduced. This behaviour can be explained as follows: as the particle size decreases, the surface area-to-volume effective ratio of the particle increases; therefore, the thermal diffusivity and thermal conductivity of the nanofluids are increased. However, for the large-diameter nanoparticles, the surface area-to-volume ratio decreases, and there is consequently no enhancement of thermal diffusivity and thermal conductivity \\[10,14\\]. Moreover, ethanol-based nanofluids were observed to have a higher thermal diffusivity ratio than EG- and water-based nanofluids because of their lower thermal diffusivity.\n\n### Concentration\/shape\n\nFigure <a href=\"#F6\" data-ref-type=\"fig\">6<\/a> shows the dependency of the nanofluid thermal diffusivity on nanoparticle concentration for 55- and 115-nm urchin-like gold nanoparticles; as the nanoparticle concentration increases, the thermal diffusivity ratio (*\u03b1*~sample~\/*\u03b1*~base\\ fluid~) of the nanofluids also increases. Similar results have been reported in the literature for metal nanofluids measured by hot wire method \\[14,15\\]. This dependency can be attributed to the volumetric increase of the nanoparticles that effectively decreases the specific heat of the nanofluids; consequently, the thermal diffusivity of the colloidal suspension increases. The enhancement of the surface area-to-volume ratio paired with the decrease in specific heat resulted in an increase in the thermal diffusivity ratio of 55-nm concentrated nanofluids when compared with 115-nm concentrated nanofluids.\n\nTEM images of spherical gold nanoparticles with sizes similar to those of urchin-like gold nanoparticles are shown in Figure <a href=\"#F7\" data-ref-type=\"fig\">7<\/a>. The thermal diffusivity ratio (*\u03b1*~sample~\/*\u03b1*~base\\ fluid~) for 55- and 120-nm spherical gold nanofluids increased as a function of concentration, as shown in Figure <a href=\"#F8\" data-ref-type=\"fig\">8<\/a>. However, the thermal diffusivity ratio enhancement was smaller when compared with that of urchin-like gold nanofluids. This dependency can be ascribed to the high surface area of the urchin-like gold nanoparticles when compared with the spherical gold nanoparticles, which exhibit an increase in the surface area-to-volume ratio and decrease in the specific heat. These parameters are more strongly affected by the thermal diffusivity ratio of nanofluids containing urchin-like nanoparticles than by the thermal diffusivity ratio of nanofluids containing spherical nanoparticles.\n\n# Conclusions\n\nThe present study investigated the effects of concentration, size and solvent on the thermal diffusivity of urchin-like gold nanofluids prepared by hydroquinone method through seed-mediated growth. The low size dispersion of urchin-like gold nanoparticles synthesised by hydroquinone method is advantageous when compared with single-step methods that are less capable of continuously controlling the diameters of branched particles; such control is critical as it is directly related to the tunable surface plasmon resonance of the urchin-like nanoparticles. The thermal diffusivity ratio changed inversely with nanoparticle size, which varied from 55 to 115 nm for urchin-like gold nanofluids. The thermal diffusivity ratio has been found to increase with nanoparticle concentration and was thus investigated within the range of 1 to 0.2 mg\/ml for the gold nanofluids. The thermal diffusivity ratio increased inversely with the thermal diffusivity of the nanofluid solvent. Moreover, the particle shape was found to have an effect on the thermal diffusivity of nanofluids. Experimental data for the thermal diffusivity ratio as a function of concentration and size for gold nanofluids depict similar behaviours in the enhancement ratio when compared with the thermal diffusivity using other techniques reported in the literature with high accuracy. Because of the small amount of sample required (600 \u03bcl) and the possibility to provide additional information such as the thermal and optical parameters of the sample with the developed sensor, photopyroelectric calorimetry is a promising alternative to classical techniques for measuring the thermal diffusivity of nanofluids.\n\n# Abbreviations\n\nEG: Ethylene glycol; HPLC: High-performance liquid chromatography; TEM: Transmission electron microscopy; Vis: Visible.\n\n# Competing interests\n\nThe authors declare that they have no competing interests.\n\n# Authors' contributions\n\nGALM prepared all the samples, set up the photopyroelectric method and measured and analysed the photopyroelectric data. JAPR and JSS measured and analysed the UV-vis and TEM data. JOL and JABL designed the experiments and wrote the manuscript. All authors read and approved the final manuscript.\n\n## Acknowledgements\n\nThe authors acknowledge Zaber Technologies, Inc. for providing a micro-linear stage and Qu\u00edmica Arom\u00e1tica SA, COFAA-IPN and CONACyT for the partial support of this work.","meta":{"dup_signals":{"dup_doc_count":129,"dup_dump_count":24,"dup_details":{"curated_sources":2,"2022-27":1,"2019-51":1,"2019-35":1,"2015-48":6,"2015-40":2,"2015-35":6,"2015-32":4,"2015-27":5,"2015-22":5,"2015-14":5,"2014-52":6,"2014-49":9,"2014-42":11,"2014-41":6,"2014-35":8,"2014-23":10,"2014-15":6,"2022-49":1,"2015-18":6,"2015-11":5,"2015-06":6,"2014-10":6,"2013-48":5,"2013-20":6}},"file":"PMC3524782"},"subset":"pubmed_central"}
{"text":"abstract: We have developed software for fully automated tracking of vibrissae (whiskers) in high-speed videos (\\>500 Hz) of head-fixed, behaving rodents trimmed to a single row of whiskers. Performance was assessed against a manually curated dataset consisting of 1.32 million video frames comprising 4.5 million whisker traces. The current implementation detects whiskers with a recall of 99.998% and identifies individual whiskers with 99.997% accuracy. The average processing rate for these images was 8 Mpx\/s\/cpu (2.6 GHz Intel Core2, 2 GB RAM). This translates to 35 processed frames per second for a 640 px\u00d7352 px video of 4 whiskers. The speed and accuracy achieved enables quantitative behavioral studies where the analysis of millions of video frames is required. We used the software to analyze the evolving whisking strategies as mice learned a whisker-based detection task over the course of 6 days (8148 trials, 25 million frames) and measure the forces at the sensory follicle that most underlie haptic perception.\nauthor: Nathan G. Clack; Daniel H. O'Connor; Daniel Huber; Leopoldo Petreanu; Andrew Hires; Simon Peron; Karel Svoboda; Eugene W. Myers\\* E-mail: <firstname.lastname@example.com>[^1]\ndate: 2012-07\ninstitute: Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, United States of America; University of California, San Diego, United States of America\nreferences:\ntitle: Automated Tracking of Whiskers in Videos of Head Fixed Rodents\n\n> \"This is a *PLoS Computational Biology* Software article.\"\n\n# Introduction\n\nRats and mice move their large whiskers (vibrissae), typically in a rhythmic pattern, to locate object features \\[1\\], \\[2\\], or to identify textures and objects \\[3\\], \\[4\\]. Whisker movements in turn are influenced by touch \\[1\\], \\[5\\]. The whisker system is a powerful model for studying the principles underlying sensorimotor integration and active somatosensation \\[6\\]. Critical to any mechanistic study at the level of neurons is the quantitative analysis of behavior. Whisker movements have been measured in a variety of ways. Electromyograms, recorded from the facial muscles, correlate with whisker movements \\[7\\]. This invasive method does not report whisker position and shape per se and is complimentary to whisker tracking. Imaging individual labeled whiskers, for example by gluing a high-contrast particle on the whisker \\[8\\], provides the position of the marked whisker, but does not reveal whisker shape. In addition, the particle will change the whisker mass and stiffness and thereby perturb whisker dynamics. Monitoring a single point along the whisker using linescan imaging \\[9\\], or a linear light sheet \\[10\\], can provide high-speed information about whisker position, but only at a single line of intersection.\n\nHigh-speed (\\>500 Hz) videography is a non-invasive method for measuring whisker movements and forces acting on whiskers and yields nearly complete information about whiskers during behavior \\[1\\], \\[11\\]\u2013\\[15\\]. The position of the whisker base with respect to the face reveals the motor programs underlying behavior. The deformation of whisker shape by touch can be used to extract the forces felt by the mouse sensory follicles \\[16\\], \\[17\\]. However, high-speed videography brings its own technical challenges. The large number of images required makes manual analysis impossible for more than a few seconds of video. Comprehensive studies require fully automated analysis. In addition, extraction of motions and touch forces demands accurate measurement of whisker shape, often with sub-pixel precision, and identification of rapidly moving whiskers across time. Finally, the large volume of video data potentially places severe demands even on advanced computational infrastructures, making efficient algorithms necessary.\n\nTracking whiskers is challenging. Whiskers can move at high speeds \\[12\\] and in complex patterns \\[1\\], \\[18\\]. Adjacent whiskers can have distinct trajectories. Moreover, whiskers are thin hairs (e.g. mouse whiskers taper to a thickness of a few micrometers) and thus provide only limited contrast in imaging experiments.\n\nTo address these challenges, we have developed software for tracking a single row of whiskers in a fully automated fashion. Over a manually curated database of 400 video sequences of head fixed mice (1.32\u00d710^6^ cumulative images, 4.5\u00d710^6^ traced whiskers, 8 mice), whiskers were correctly detected and identified with an accuracy of 99.997% (1 error per 3\u00d710^4^ traced whiskers). In other whisker tracking systems, models constraining possible motions and whisker shapes have been used to aid tracking. In contrast, our approach uses statistics gleaned from the video itself to estimate the most likely identity of each traced object that maintains the expected order of whiskers along the face. As a result, the shapes of highly strained whiskers (curvature\\>0.25\/mm) can be traced with sub-pixel accuracy, and tracking is faithful despite occasional fast motion (deflections \\>10,000 degrees\/s).\n\nOur method consists of two steps performed in succession: tracing and linking. Tracing produces a set of piecewise linear curves that represent whisker-like objects for each individual image in a video. Each image is analyzed independently. The linking algorithm is then applied to determine the identity of each traced curve. The trajectory of a whisker is described by collecting curves with the same identity throughout the video. Importantly, once a faithful tracing is completed, the original voluminous image data set is no longer required for downstream processing.\n\nOnce tracing is complete, a small set of features is tabulated for each curve. Based on these features, a heuristic is used to make an initial guess as to the identity of curves in a set of images where it works well. These initial identifications are then used to get a statistical description of the shapes and motions of whiskers. These statistics are then used to compute a final optimal labeling for each traced curve subject to the constraint that whiskers are ordered along the face.\n\nWe validated this approach using manually curated video acquired from a head-fixed, whisker-dependant object localization task \\[1\\]. In these experiments the field of view contains a single row of partially and wholly imaged whiskers as well as the stimulus object (a thin pole, normal to the imaging plane) (<a href=\"#pcbi-1002591-g001\" data-ref-type=\"fig\">Figure 1A<\/a>). The camera is positioned below the whiskers and illuminated from above (<a href=\"#pcbi-1002591-g001\" data-ref-type=\"fig\">Figure 1B<\/a>) producing silhouetted views of the whiskers (<a href=\"#pcbi-1002591-g001\" data-ref-type=\"fig\">Figure 1C\u2013D<\/a>). Automated tracing is robust to occlusions introduced by the stimulus and to whisker crossings. Tracing yields curves for both whiskers and facial hairs (<a href=\"#pcbi-1002591-g001\" data-ref-type=\"fig\">Figure 1C\u2013D<\/a>). Linking is used to uniquely identify each whisker so features such as angle of deflection (<a href=\"#pcbi-1002591-g001\" data-ref-type=\"fig\">Figure 1F<\/a>) and curvature (<a href=\"#pcbi-1002591-g001\" data-ref-type=\"fig\">Figure 1G<\/a>) can be extracted as time-series.\n\n# Design and Implementation\n\n## Tracing\n\nThe process of tracing the backbone of a whisker consists of three phases: initiation, extension, and termination. Tracing starts by using the angle and position estimated at the highest scoring candidate initiation site. As a curve is extended, initiation sites that fall under the traced curve are removed from consideration. When none remain, potentially duplicate curves are resolved.\n\nBefore any analysis, images were preprocessed to remove imaging artifacts specific to particular cameras (<a href=\"#pcbi.1002591.s003\" data-ref-type=\"supplementary-material\">Text S1<\/a>, <a href=\"#pcbi.1002591.s002\" data-ref-type=\"supplementary-material\">Figure S1<\/a>). Tracing begins by searching the image for candidate initiation sites. These are found by performing a pixel-level segmentation isolating locally line-like features in the image. With each segmented pixel, a score and an angle are computed. This is an optimization step; it is possible to initiate tracing at any pixel in an image. However, tracing is relatively expensive, and, by filtering out unproductive initiation sites, computation can be focused on the small number of relevant pixels. On the other hand, the filtration threshold should be set conservatively so that every whisker is sure to have at least one initiation site. Parameters were set by examining results over a small set of 10 images but worked well over the entire data set of over 1 million images. Typically, 50\u2013100 candidate sites were found per whisker with 10\u201320 false positives per image.\n\nA variety of methods have been developed to detect interest points in natural images \\[19\\], and the Hough transform is conventionally used to find linear features. However, given the high contrast and controlled imaging conditions used to acquire data (<a href=\"#pcbi.1002591.s003\" data-ref-type=\"supplementary-material\">Text S1<\/a>), we developed a linear time algorithm, summarized in <a href=\"#pcbi-1002591-g002\" data-ref-type=\"fig\">Figure 2<\/a>, that requires only local image information and yields an estimate of salience and line orientation in a single pass.\n\nFirst, a 7\u00d77 square box is centered on a pixel of interest (<a href=\"#pcbi-1002591-g002\" data-ref-type=\"fig\">Figure 2A<\/a>). The box is divided into two partitions and the position of the intensity minima found in each subset of the two partitions is found. These are designed to ensure that when a whisker passes through the box, one partition will preferentially collect minima along the whisker backbone, and, as a result, the position of those minima will be linearly correlated. For each partition, principle components are computed over the covariance of the collected positions. The principle components describe the major and minor axis of an ellipse. The partition with the highest eccentricity is chosen, and the eccentricity is used to score how line-like the image is at the queried point (<a href=\"#pcbi-1002591-g002\" data-ref-type=\"fig\">Figure 2B<\/a>). The orientation of the major axis is used to estimate the direction of the line (<a href=\"#pcbi-1002591-g002\" data-ref-type=\"fig\">Figure 2C<\/a>, <a href=\"#pcbi.1002591.s004\" data-ref-type=\"supplementary-material\">Video S1<\/a>).\n\nOne advantage of this approach is that the entire image need not be queried for line-like objects. For example, in the data analyzed here, whiskers span hundreds of pixels. A grid of lines spaced appropriately (50 px; this is an adjustable parameter) will cross each whisker at least once. Restricting the search for line-like features to this grid greatly reduces the amount of time required for this step. Additionally, since the algorithm relies on local minima, demands on background subtraction and other preprocessing is minimal. The structure of the partitions ensures that lines in any orientation are detected. Greater than 80% of the pixels that could be used to start tracing a whisker (typically 1000 per whisker over the full image) are detected using an eccentricity threshold of 0.95; only 1 is required to fully trace a whisker. This detector systematically avoids potential starting locations near the face, near occlusions or where whiskers cross or nearly touch.\n\nCurves are bidirectionally extended from candidate initiation sites by performing a small step (1 px) along the measured direction, and repeating until one of several criteria is met. The sub-pixel position and tangent angle are estimated by optimizing the correlation between an oriented line detector that is parameterized as a function of sub-pixel position, angle and width (<a href=\"#pcbi-1002591-g003\" data-ref-type=\"fig\">Figure 3<\/a>).\n\nThe line detector is designed based on modeling the intensity profile of a whisker as a rectangular valley in the image with variable position, width and angle. The center of the whisker is estimated with sub-pixel precision by finding a position that minimizes the Laplacian of the correlation between the model and the image \\[20\\]. The line detector approximates the Laplacian of the model. It consists of two rectangular parallel step-edge detectors (20\u00d71 px) spaced by the detector width (<a href=\"#pcbi-1002591-g003\" data-ref-type=\"fig\">Figure 3A<\/a>). The length was chosen to match the distance over which highly curved whiskers remain approximately linear. The correlation at a given point is evaluated using a pixel representation of the detector that is computed by evaluating the area integral of the detector over the square domain of each pixel (<a href=\"#pcbi-1002591-g003\" data-ref-type=\"fig\">Figure 3B<\/a>). The value of the correlation at that position is then the dot product between pixels in the image and pixels in the evaluated detector. For efficiency, pixel representations of the detector are pre-tabulated for discrete values of the sub-pixel position (0.1 px), width (0.2 px), and angle (2.5 degree precision). Optimizing the width of the detector was important for reliable tracing of whiskers of different width and for reliable tracing of the very tips of whiskers.\n\nTracing is stopped if any one of several criteria indicates that the optimization procedure cannot be trusted to yield accurate results. This is necessary to handle cases where whiskers were partially occluded, such as when whiskers crossed or contacted an obstructing object, such as the stimulus pole in an object localization experiment (<a href=\"#pcbi-1002591-g004\" data-ref-type=\"fig\">Figure 4<\/a>). The tests were for low correlation with the line detector, large left-right asymmetry in the image intensity about the detector, low mean intensity about the detector, and large angular change between steps. A corresponding user-adjustable threshold parameterizes each test. The tests are applied at each step as the curve is traced from the initiation point (<a href=\"#pcbi-1002591-g004\" data-ref-type=\"fig\">Figure 4A,B<\/a>). If one of these fails (<a href=\"#pcbi-1002591-g004\" data-ref-type=\"fig\">Figure 4C,D<\/a>), a number of single pixel steps are taken along the last trusted direction up to a user-settable maximum distance in an attempt to jump past the distrusted region (<a href=\"#pcbi-1002591-g004\" data-ref-type=\"fig\">Figure 4E,F<\/a>). Linear extrapolation is usually a good guess. If all tests are satisfied at one of the points, normal tracing is resumed (<a href=\"#pcbi-1002591-g004\" data-ref-type=\"fig\">Figure 4G,H<\/a>). Otherwise, the trace is terminated at the last trusted point (<a href=\"#pcbi-1002591-g004\" data-ref-type=\"fig\">Figure 4G<\/a>).\n\nOccasionally, overlapping curves are generated during the tracing step. These need to be resolved before tracking so that individual whiskers have exactly one associated curve per frame. Searching for curves that cross the same pixel identifies pairs of potentially redundant curves. If all the points within an interval of one of the curves is within a small distance (typically 2 px) of the other curve and that interval consists of over 50% of the total length, then it is considered redundant. The shorter of the two curves is discarded; the shapes of shorter curves were not reliable enough to average. This procedure is repeated until there are no redundant curves left.\n\n## Linking\n\nIdentifying individual whiskers is challenging. First, the tracing algorithm generates curves (false positives) for facial hairs, trimmed whiskers, or other artifacts in the scene (<a href=\"#pcbi-1002591-g001\" data-ref-type=\"fig\">Figure 1C<\/a>). The linking algorithm must correctly identify true whiskers against this noise. Second, the motion of whiskers can be difficult to reliably model \\[8\\], \\[12\\]. A typical trial may consist of slow, predictable motions interrupted by rapid transient motions that are occasionally too fast even for high speed (1000 fps) cameras to adequately sample \\[1\\]. Although difficult to track, these unpredictable motions may correspond to some of the most interesting data.\n\nOur approach is based on the observation that whiskers are relatively long and maintain a consistent ordering along the face. We use a statistical model to describe the shapes and motions that are characteristic of whiskers. The model is then used to assign each traced whisker the most probable identity subject to the constraint that whiskers are ordered along the face.\n\nFor the majority of images, a simple length threshold can be used to remove false positives and leave exactly one traced curve per whisker. The linking algorithm is either supplied the number, *N*, of whiskers on the subject mouse, or estimates it as follows: For a given length threshold , let be the set of frames that have *n* curves longer than . If *N* is not supplied, then we find and such that is maximal over all possible choices. In most of the data sets we have tested, , i.e. this heuristic is spot on. But when one or more whiskers frequently leave the field of view it may fail. If *N* is supplied, then we let be the value for which is maximal because the set of long curves in each frame in are always true whiskers. For the data sets tested here, represented 99.8% of a video on average (min: 90.1%, max: 100%).\n\nThe curves in the frames and their heuristic classification into whisker and non-whisker based on supplies a training data set for the specific video. From these, we can learn various characteristics to estimate the probability of a curve being one or the other. Specifically, we build normalized histograms, *n~f~*,*~W~* and *n~f~*,*~FP~* (<a href=\"#pcbi-1002591-g005\" data-ref-type=\"fig\">Figure 5C<\/a>), one for whiskers (*W*) and one for false positives (*FP*), for each of six features, *f*, using the training set. The features used are the angle near the face, average curvature, average tracing score, length, and endpoints of a curve. The angle and curvature are computed from a parametric third degree polynomial fit to each curve. These histograms are then used to estimate, , the probability that a curve *c* is of kind *k*, either *W* or *FP*, as the product,\n\n![](pcbi.1002591.e015.jpg)\n\nTo ensure no feature has zero likelihood, a count of one is added to each bin in a histogram before normalizing.\n\nIn (1) above, the features are assumed to be conditionally independent in order to simplify estimating feature distributions, even though this not strictly true in practice. Moreover, errors in the heuristic may introduce a systematic sampling bias. For example, at high deflection angles near the edge of the field of view, only a small segment of a whisker is imaged (<a href=\"#pcbi-1002591-g001\" data-ref-type=\"fig\">Figure 1E<\/a>, purple whisker). The traced curves from such segments might systematically fall below and be excluded from whisker feature distributions. As a result, estimated probabilities will be biased towards longer whiskers at milder deflection angles. Despite these caveats, the use of these feature distributions leads to a highly accurate result.\n\nAppearance alone is not sufficient to uniquely identify individual whiskers in some cases. To address this, we designed a naive Bayes' classifier to determine the most probable identity of each traced curve subject to ordering constraints. The traced curves are ordered into a sequence, *C*, according to their time and relative anterior-posterior position. Identifying a curve, *c*, involves assigning a particular label, *l*, from the following set of *2N+1* labels. There is a label for each whisker, *W~1~* to *W~N~*, and labels, *F~0~* to *F~N~*, where *F~i~* identifies all false positives between whisker *W~i~* and *W~i+1~* (<a href=\"#pcbi-1002591-g005\" data-ref-type=\"fig\">Figure 5A<\/a>). The kind of a label, *K(l)*, is *W* if *l* is a whisker label, and *FP* if *l* is a false positive label. A curve labeled *W~i~* or *F~i~* must be posterior to curves labeled *W~j~* or *F~j~* for all *i\\<j*. For a given *i*, only one curve per frame may be labeled *W~i~*, but several curves may be labeled *F~i~*.\n\nApplying Bayes' theorem, the most probable sequence of labels, , is given by\n\n![](pcbi.1002591.e018.jpg)\n\nwhere is the *a priori* probability of a labeling sequence, and is the likelihood that the curves in *C* are generated from the sequence of items labeled by *L*. By design, certain labeling sequences are ruled out *a priori*. Conceptually drawing a directed edge between one identity and the possible identities of the next curve yields a directed graph (<a href=\"#pcbi-1002591-g005\" data-ref-type=\"fig\">Figure 5B<\/a>). For a video with *T* frames,\n\n![](pcbi.1002591.e021.jpg)\n\nwhere *N^t^* is the number of curves traced in frame *t*, and is the label of the *i*'th curve found in frame *t*. Values for and are estimated by computing the frequency of observed label pairs that occur in the a training set, . This describes a hidden Markov model for labeling curves in a single video frame. The optimal labeling can be computed efficiently with well-known dynamic programming techniques \\[21\\].\n\nThe likelihood, , is computed under the simplifying assumption that the likelihood of a single curve depends only on the curves in the previous or following frame. Using this and applying Bayes' theorem,\n\n![](pcbi.1002591.e027.jpg)\n\nwhere is the label *L* assigns to the curve *c*, *C^t^* is the (possibly empty) set of curves found in frame *t*, and are the labels assigned to the curves in *C^t^*. The first component, , is the likelihood that *c* is an object of the kind denoted by label *l* which we estimate with formula (1).\n\nThe second component of (4), , is interpreted as the likelihood that a curve is part of the same trajectory as corresponding curves in the previous (or following) frame. Similar to the approach used in equation (1) for estimating , we need normalized histograms and of the changes of whiskers and false positives between successive frames for each feature *f* over a \"training\" data set in which the corresponding curves in successive frames is known. While we could use the implied assignment over , we first estimate a hopefully better assignment by restricting the model in (4) to use shape features alone. That is, is treated as a constant and thus the assignment to labels in a frame can be computed independently of other frames. We then use this preliminary labeling over the frames of as the training set over which to build and .\n\nGiven these change histograms, one can estimate the correspondence likelihood according to the formula,\n\n![](pcbi.1002591.e040.jpg)\n\nNote that when evaluating this likelihood, a unique corresponding curve is not always present in the previous (or following) frame. There may be zero or many false positive curves in the previous frame with the same label. Similarly a whisker may be missing because it exited the field of view.\n\nDirectly solving for the most probable labeling (2) is complicated by the fact that the likelihood of a labeling in one frame depends on the labeling in neighboring frames. Our approach is to initially score each frame by computing , where is obtained using shape features alone. In decreasing order of this score, we 'visit' the next best frame, say *t*, and update the label assignment for each of the two adjacent frames that maximizes the full version of (4), provided the adjacent frame has not already been visited. The new assignment replaces the current assignment and the frame's visitation order is updated according to the score of this new assignment (under the full model of (4)). In this way, we let the most confident frames influence their neighbors in a transitive fashion until every frame has been visited. This was critical for achieving high accuracy. Previous whisker tracking algorithms have relied on propagating the identity of a whisker frame-wise from the beginning of the video to the end, and as a result, an error in one frame is propagated throughout the rest of the video \\[11\\]\u2013\\[13\\].\n\n# Results\/Discussion\n\nAverage processing time was 8 Mpx\/s\/cpu (35 fps for 640\u00d7352 pixel video) measured on a 2.6 GHz Intel Core2 Duo Macbook Pro with 2 GB of RAM, and was dominated by the CPU time required to trace curves; linking time was 0.5 ms per frame. This is faster than published speeds of similar whisker analysis packages (typically, 1\u20135 fps.) \\[11\\], \\[13\\], \\[15\\]. However, performance can be difficult to compare as implementations may improve over time. For example, our software is equally fast as the current version of the only other publically available whisker tracker\\[13\\]. More importantly, our software can readily be run on inexpensive cluster nodes to process videos in parallel. This is not the case for whisker trackers that require supervision \\[13\\] or that depend on software with expensive licensing requirements (such as Matlab) \\[11\\]\u2013\\[15\\].\n\nTracing was accurate to within 0.2 px as estimated by analyzing 39 hand-traced whiskers. Individual hand-tracings had an accuracy of 0.23\u00b10.2 pixels when compared against consensus curves. Mouse whiskers (row C) were automatically traced in images with resolutions from 5 \u00b5m\/px to 320 \u00b5m\/px using default settings and in noisy, low-contrast images (<a href=\"#pcbi.1002591.s003\" data-ref-type=\"supplementary-material\">Text S1<\/a>). For the best results, it is important to minimize motion blur and uniformly illuminate the scene, although illumination inhomogeneities can be partially corrected with background subtraction. In contrast with methods that use Kalman filtering \\[13\\], traced curves are not biased by whisker dynamics. Additionally, tracing is faithful even when curves are not well approximated by low order polynomials, in contrast to published tracing methods \\[13\\]\u2013\\[15\\]. Whiskers could be detected and traced in videos of freely behaving rats (<a href=\"#pcbi.1002591.s005\" data-ref-type=\"supplementary-material\">Video S2<\/a>) and mice (<a href=\"#pcbi.1002591.s006\" data-ref-type=\"supplementary-material\">Video S3<\/a>) with all whiskers intact.\n\nLinking accuracy was measured against a hand-annotated set of videos selected by choosing 100 random trials from behavioral sessions of 4 mice (1.32 million frames) \\[1\\], \\[17\\]. The curated videos captured a range of behavior including protracted bouts (\\>1 s) of whisking (<a href=\"#pcbi.1002591.s007\" data-ref-type=\"supplementary-material\">Video S4<\/a>), multiple whiskers simultaneously contacting an object (a thin metal pole) (<a href=\"#pcbi.1002591.s007\" data-ref-type=\"supplementary-material\">Video S4<\/a>), and extremely fast motion (\\>10,000\u00b0\/second) (<a href=\"#pcbi.1002591.s008\" data-ref-type=\"supplementary-material\">Video S5<\/a>). Of the 4.5 million traced whiskers, 130 were incorrectly identified or not detected, less than one mistake per behavioral trial on average. Linking is robust to whiskers that occasionally leave the field of view (<a href=\"#pcbi.1002591.s009\" data-ref-type=\"supplementary-material\">Video S6<\/a>), and works well even at relatively low frame rates (100 fps; see <a href=\"#pcbi.1002591.s003\" data-ref-type=\"supplementary-material\">Text S1<\/a>). Lesser image quality will ultimately degrade results.\n\nThere are two principle sources of error in the linking. First, covariance between different features is ignored. For example, when the field of view is small, whiskers at high deflection angles might not be fully contained within the image. Any curve tracing one of these whiskers would appear shorter than the full whisker length. Under these conditions there is a strong correlation between angle and whisker length. The current model penalizes the shorter length because it is rarely observed; accounting for the covariance should eliminate this problem. Second, the estimated feature distributions are affected by systematic biases introduced from the heuristic used to initially guess whisker identity. A heuristic relying on a length threshold, like the one used here, may systematically reject these short curves at high deflection angle. This will result in a bias against strongly deflected whiskers.\n\nFundamentally, our linking method is applicable only to single rows of whiskers. The whiskers in videos imaging multiple rows of whiskers will be traced, but, because whisker images no longer maintain a strict anterior-posterior ordering, the linking will likely fail. Although this software was validated with images of one side of the mouse's whiskers, we have successfully tracked whiskers in images containing both whisker fields (<a href=\"#pcbi.1002591.s010\" data-ref-type=\"supplementary-material\">Video S7<\/a>). Also, the whiskers of freely moving rats and mice can be traced (<a href=\"#pcbi.1002591.s005\" data-ref-type=\"supplementary-material\">Videos S2<\/a>,<a href=\"#pcbi.1002591.s006\" data-ref-type=\"supplementary-material\">S3<\/a>). This suggests that it will be possible to use this software for two-dimensional tracking of single rows of whiskers in freely moving animals by incorporating head tracking \\[2\\], \\[11\\], \\[15\\], though we have not explicitly tested this.\n\nWe analyzed data acquired during learning of an object detection task (see <a href=\"#pcbi.1002591.s003\" data-ref-type=\"supplementary-material\">Text S1<\/a>) \\[17\\]. We tracked whiskers from four mice during the first 6 days of training (8148 trials, 3122 frames each). This allowed us to observe the changes in whisking behavior that emerge during learning of the task. We looked specifically at correct rejection trials (2485 trials), which were not perturbed by contact between object and whisker, to analyze the distribution of whisking patterns during learning. We characterized whisking by computing normalized histograms of whisker angle (1\u00b0 resolution) over time (100 ms bins) for a single representative whisker (C1). The angle and position of other whiskers were strongly correlated throughout (r^2^\\>0.95) in these trials. Two patterns emerged. After learning, two mice (JF25607 and JF25609) showed changes in whisking amplitude and mean angular position when the stimulus was present (<a href=\"#pcbi-1002591-g006\" data-ref-type=\"fig\">Figure 6A<\/a>). The other two mice (JF27332 and JF26706) did not show nearly as much stereotypy and appeared to rely on lower amplitude whisking (<a href=\"#pcbi-1002591-g006\" data-ref-type=\"fig\">Figure 6B<\/a>). All mice held their whiskers in a non-resting anterior position during most of the trial \\[1\\]. For JF25607 and JF25609, reduction in task error rate was correlated with an increase in mean deflection angle during stimulus presentation (r^2^\u200a=\u200a0.97,0.84 respectively) (<a href=\"#pcbi-1002591-g006\" data-ref-type=\"fig\">Figure 6C<\/a>). This correlation was smaller for the other two mice, which also learned the task but appeared to rely on a different whisking strategy.\n\nThe forces at the base of the whisker constitute the information available to an animal for somatosensory perception. These forces are proportional to changes in whisker curvature \\[16\\]. The instantaneous moment acting at a point on the whisker is proportional to the curvature change at that point. In contrast to whisker tracing software that represents whiskers as line segments\\[14\\], \\[15\\] or non-parametric cubic polynomials\\[13\\], the curves produced by our tracing algorithm provide a high-resolution representation of whisker shape, allowing measurement of varying curvature along the length of the whisker.\n\nTo illustrate this feature we measured curvature in the following manner: For each video frame (<a href=\"#pcbi-1002591-g007\" data-ref-type=\"fig\">Figure 7A<\/a>), the traced whisker midline (<a href=\"#pcbi-1002591-g007\" data-ref-type=\"fig\">Figure 7B<\/a>) was fit via least-squares to a parametric 5^th^ degree polynomial of the form:\n\n![](pcbi.1002591.e043.jpg)\n\nwhere \\[*x(t),y(t)*\\] are points that span the curve over the interval , and *a~i~,b~i~* are the fit parameters (<a href=\"#pcbi-1002591-g007\" data-ref-type=\"fig\">Figure 7C<\/a>). Curvature was measured at a specified distance along the curve from the follicle chosen for best signal to noise. The follicle was not imaged, and so the intersection between the curve and an arc circumscribing the face was used as a reference point instead. The curvature was then measured as the mean curvature inside a 1\u20132.5 mm long region about the point of interest where it was approximately constant. To ensure the measurement was not biased by shape outside the interval, another parametric polynomial fit (degree 2) was performed, but over the region of interest rather than the entire curve (<a href=\"#pcbi-1002591-g007\" data-ref-type=\"fig\">Figure 7D<\/a>). The follicle position was set by linearly extrapolating a set distance into the face. The point of whisker-pole contact was determined as the closest point to the center of the pole on the curve or on a line extrapolated from the nearest end of the curve (<a href=\"#pcbi-1002591-g007\" data-ref-type=\"fig\">Figure 7E<\/a>).\n\nTime series of curvature measurement (<a href=\"#pcbi-1002591-g007\" data-ref-type=\"fig\">Figure 7F<\/a>, measurements at 5 mm from follicle) can be used to identify most contact events. For whisker-pole contacts first made during a protraction, the first touch tends to be followed by a series of more forceful contacts. Contacts are correlated with relatively constant angular position of the whisker (<a href=\"#pcbi-1002591-g007\" data-ref-type=\"fig\">Figure 7G<\/a>). This indicates the mouse may press the whisker against the pole by translating the whisker pad rather than by rotating the whisker. The distribution of peak curvature-change during a whisker-pole contact indicates more the bulk of contacts were made during protraction and that these were more forceful (<a href=\"#pcbi-1002591-g007\" data-ref-type=\"fig\">Figure 7H<\/a>).\n\n## Availability\n\nThe software and documentation are freely available online (<http:\/\/whiskertracking.janelia.org>) as cross-platform (Windows, OS X, Linux) source code written in C with Python and Matlab interfaces included. Pre-built binaries are also available. A graphical user interface is provided to aid semi-automated tracing as well as the viewing and editing of results. The software is capable of analyzing 8-bit grayscale StreamPix (Norpix Sequence Format: SEQ), TIFF, MPEG, MOV, and AVI formatted video.\n\n# Supporting Information\n\nSample data and tutorial.\n\n(ZIP)\n\nClick here for additional data file.\n\nLine bias correction. (A) Raw data exhibits a fixed-pattern artifact where odd-numbered lines are systematically darker than bright lines. (B) This artifact is corrected by multiplying odd-lines by a factor estimated from the raw data itself. This removes the stripes without blurring or reducing the contrast of whiskers. Scale bar, 0.5 mm.\n\n(TIF)\n\nClick here for additional data file.\n\nMethodological details.\n\n(DOC)\n\nClick here for additional data file.\n\nVideo overlaid with high scoring seeds (eccentricity\\>0.99) for each frame. Pixels are colored according to the orientation indicated by the seed.\n\n(MP4)\n\nClick here for additional data file.\n\nResults of automatic tracing applied to video 500 Hz video (1.7 s duration) of a freely behaving rat exploring a corner with its full whisker field. The video was obtained with permission from the BIOTACT Whisker Tracking Benchmark \\[22\\] (Clip ID:behavingFullContact, courtesy of Igor Perkon and Goren Gordon). It has been cropped, and the frame rate has been changed for presentation.\n\n(MP4)\n\nClick here for additional data file.\n\nResults of automatic tracing applied to a video acquired at 100 Hz video (8 s duration) of a freely behaving mouse with its full whisker field. Background subtraction was performed before tracing to reduce the effects of non-uniform illumination. Because no image of the scene without the animal present was available, the background was estimated for each pixel as the maximum intensity observed at that point throughout the video. The video was obtained with permission from the BIOTACT Whisker Tracking Benchmark \\[22\\] (Clip ID: behavingMouse100, courtesy of Ehud Fonio and Ehud Ahissar). It has been cropped, and the frame rate has been changed for presentation.\n\n(MP4)\n\nClick here for additional data file.\n\nResults from a full trial. The results of automated tracing and linking applied to a video (500 Hz, 9.2 s duration) of a head fixed mouse trimmed to a single row of 4 whiskers interacting with a pole. Curves that were classified as whiskers are colored according to their identity, and otherwise they are not shown. Multiple whiskers simultaneously interact with the pole at 1.2\u20131.4 sec into the trial. Protracted bouts of whisking can be observed throughout the video.\n\n(MP4)\n\nClick here for additional data file.\n\nTracing and linking is robust to rapid changes in whisker angle. The results of automated tracing and linking applied to a video (500 Hz, 150 ms) of a head fixed mouse trimmed to a single row of 3 whiskers interacting with a pole. Curves that were classified as whiskers are colored according to their identity, and otherwise they are not shown. One whisker (red) has been trimmed so it cannot contact the pole. The green whisker presses against the pole, and quickly flicks past it as it is removed from the field. This is the fastest angular motion (16\u00b0\/ms) observed in the data set used to measure tracking accuracy.\n\n(MP4)\n\nClick here for additional data file.\n\nLinking is robust to whiskers that leave the field of view. The results of automated tracing and linking applied to a video (500 Hz, 190 ms) of a head fixed mouse trimmed to a single row of 3 whiskers interacting with a pole. Curves that were classified as whiskers are colored according to their identity, and otherwise they are not shown. Two whiskers (green and blue) are frequently occluded by the lick-port (black bar, lower right), but they are properly identified before and after such events.\n\n(MP4)\n\nClick here for additional data file.\n\nTracing and linking of whiskers bilaterally. The results of automated tracing and linking applied to a video (500 Hz, 1 s duration) of a bilateral view to a head fixed mouse trimmed to a single row of whiskers (2 on each side).\n\n(MP4)\n\nClick here for additional data file.\n\n# References\n\n[^1]: Conceived and designed the experiments: NGC DHO KS EWM. Performed the experiments: NGC DHO DH LP. Analyzed the data: NGC DHO DH LP AH SP KS. Contributed reagents\/materials\/analysis tools: NGC DHO DH LP. Wrote the paper: NGC KS EWM.","meta":{"dup_signals":{"dup_doc_count":110,"dup_dump_count":52,"dup_details":{"curated_sources":2,"2023-06":1,"2022-49":1,"2022-05":2,"2021-43":1,"2021-10":1,"2020-16":1,"2019-51":1,"2019-39":1,"2018-51":1,"2018-47":1,"2018-34":1,"2018-05":1,"2017-51":1,"2017-47":2,"2017-43":1,"2017-39":1,"2017-34":1,"2017-30":2,"2017-26":2,"2017-22":2,"2017-17":2,"2017-09":7,"2017-04":1,"2016-50":2,"2016-44":1,"2016-40":2,"2016-36":3,"2016-30":2,"2016-26":1,"2016-18":1,"2016-07":1,"2015-48":3,"2015-40":2,"2015-35":2,"2015-32":2,"2015-27":3,"2015-22":2,"2015-14":2,"2014-52":6,"2014-49":3,"2014-42":6,"2014-41":2,"2014-35":5,"2014-23":7,"2014-15":3,"2023-40":1,"2024-22":1,"2017-13":2,"2015-18":3,"2015-11":2,"2014-10":2,"2024-26":1}},"file":"PMC3390361"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  Medications necessary for disease management can simultaneously contribute to weight gain, especially in children. Patients with preexisting obesity are more susceptible to medication-related weight gain.\n  .\n  How equipped are primary care practitioners at identifying and potentially reducing medication-related weight gain? To inform this question germane to public health we sought to identify potential gaps in clinician knowledge related to metabolic adverse drug effects of weight gain.\n  .\n  # Methods\n  .\n  The study analyzed practitioner responses to the pre-activity questions of six continuing medical education (CME) activities from May 2009 through August 2010.\n  .\n  # Results\n  .\n  The 20,705 consecutive, self-selected respondents indicated varied levels of familiarity with adverse metabolic effects and psychiatric indications of atypical antipsychotics. Correct responses were lower than predicted for drug indications pertaining to autism (\u221217% predicted); drug effects on insulin resistance (\u221262% predicted); chronic disease risk in mental illness (\u221234% predicted); and drug safety research (\u221240% predicted). Pediatrician knowledge scores were similar to other primary care practitioners.\n  .\n  # Conclusions\n  .\n  Clinicians' knowledge of medication-related weight gain may lead them to overestimate the benefits of a drug in relation to its metabolic risks. The knowledge base of pediatricians appears comparable to their counterparts in adult medicine, even though metabolic drug effects in children have only become prevalent recently.\nauthor: Ingrid Kohlstadt; Gerold Wharton\ndate: 2013\ninstitute: 1Johns Hopkins Bloomberg School of Public Health, Center for Human Nutrition, 198 Prince George St, Annapolis MD 21401, Maryland, USA; 2The U.S. Food and Drug Administration, Office of Pediatric Therapeutics, Silver Spring, Maryland, USA\nreferences:\ntitle: Clinician uptake of obesity-related drug information: a qualitative assessment using continuing medical education activities\n\n# Background\n\nNo study to date assesses the knowledge base around medication-related weight gain in pediatric or adult primary care medicine. We therefore sought to characterize what practitioners know about metabolic drug effects in the context of clinical decision-making.\n\nInformed clinicians can often modify their patients' risk of adverse metabolic drug effects, even when medications are essential for disease management \\[1\\]. Practitioners can choose lowest effective dosing and therapies with fewer metabolic effects; treat underlying medical conditions which can contribute to weight gain, such as sleep apnea and hypothyroidism; correct nutritional deficiencies such as vitamins B~12~\\[2\\] and D \\[3\\] to facilitate lifestyle adherence; and counsel patients on drug-related increases in appetite, emphasizing adherence to medication and healthful lifestyle choices.\n\nAmong the patient groups most vulnerable to metabolic drug effects are children. Children are more susceptible to central nervous system effects of medications \\[4\\]. Some metabolic drug effects are unique to children at certain growth stages and demonstrate a prolonged effect \\[5,6\\]. Metabolic drug effects also tend to be delayed relative to the therapeutic benefit, especially in children. Concurrently, drug exposure is increasing in children, the age group with the fastest growing number of prescriptions \\[7\\], in part due to obesity-related chronic diseases. Preexisting overweight and obesity heighten vulnerability to metabolic drug effects.\n\nManaging adverse metabolic drug affects is relatively new to the practice of pediatrics. Historically pediatricians focused on medication-related weight loss and stunting, recorded as step-offs on patient growth charts. Today's pediatric practice may require as diligent a diagnosis and management of medication-related weight gain, especially since preexisting overweight and obesity, defined as a body mass index at or above the 85^th^ percentile, has reached approximately 32% of the U.S. population ages 2-19 \\[8,9\\].\n\nDisseminating drug safety updates to pediatricians holds other challenges as well. Safety information specific to children represents a recent advance. Practitioners may not realize they need to watch for such updates \\[10\\]. Metabolic drug effects specific to children and adolescents may be first identified years after a drug is on the market \\[11\\] because the metabolic effects in children tend to manifest beyond the timeframe of clinical trials. Disseminating drug safety information may be additionally complicated by practice patterns. For example, psychiatrists may diagnose and prescribe highly specialized treatment and look to primary care practitioners to monitor patients for adverse drug effects.\n\nClinicians draw on their knowledge base of adverse metabolic drug effects for clinical decision-making. Elevated and unique risks of metabolic drug effects and major shifts in disease prevalence and practice patterns in pediatrics together prompted our interest in confirming that primary care clinicians who care for children have a knowledge base comparable to their adult medicine counterparts.\n\n# Methods\n\n## CME partners\n\nContinuing medical education (CME) activities were developed in partnership with CME providers. Inclusion criteria for partners were: experience implementing pre-activity questions, having primary care practitioners as a target audience, willingness to co-develop programs relevant to medication-associated weight gain, providing free public access to associated media and print materials, and collaborating within time and budget constraints. Partners were selected across different media - audio, lectures, or web-based activities - Audio-Digest Foundation, Medscape CME, The Maryland Academy of Family Physicians, and The FDA Scientific Rounds Program.\n\n## Instrument development\n\nThe instrument in this study, pre-CME activity questions, measures practitioners' baseline knowledge relevant to the content of the CME activities. Pre-activity questions were 4-choice multiple choice questions or true-false questions. They were directed at clinical decision-making and were organized into four categories: 1) drug indications, 2) metabolic drug effects, 3) drug safety updates, and 4) patients most at risk. Each CME program partner selected among the pre-activity questions and adapted the wording to their standard format.\n\n## CME content\n\nThe 6 CME activities pertained to either atypical antipsychotic use in children or obesogenic medications in general. They were provided through the CME partners at varied intervals between June 2009 and August 2010. Each activity was an audio program, web-based program, or conference lecture; and awarded a maximum of 0.5 to 2 Category 1 CME credits. The program content, participant characteristics, and pre-activity questions are presented in Table\u00a0<a href=\"#T1\" data-ref-type=\"table\">1<\/a>.\n\nSummary of continuing medical education (CME) programs, June 2009 \u2013 August 2010\n\n| **CME partner and activity** | **Date, audience and promotions** | **Pre-activity questions** |\n|:---|:---|:---|\n| Activity 1: Maryland Academy of Family Physicians, 2009 Annual CME Assembly; Drug \u2013nutrient interactions | 6\/09 Conference attendees, primarily family physicians in the mid-Atlantic | 5 interspersed questions using an audience response system |\n| Activity 2: Audio-Digest Pediatrics on Atypical antipsychotics in children | 8\/09-8\/10 Subscribers, practitioners who care for children, approximately 75% physicians | 10 questions submitted by mail, fax, or website |\n| Activity 3: Audio-Digest Special Topics on Atypical antipsychotics in children \\* | 8\/09-8\/10 Responders to free CME post on the Audio-Digest homepage; American Academy of Pediatrics newsletter feature | 10 questions submitted via website |\n| Activity 4: Medscape CME on Atypical antipsychotics in children \\* | 11\/09-1\/10 Participants in Medscape's Psychiatry and Mental Health listserv with multiple cross-posts | 6 web-based interspersed questions |\n| Activity 5: Medscape CME on Medication-related weight gain \\* | 5\/10-8\/10 Participants in Medscape's Preventive Medicine and Public Health listserv with multiple cross-posts; American College of Preventive Medicine feature | 4 web-based interspersed questions |\n| Activity 6: Food and Drug Administration Scientific Rounds on Autism | 5\/10 FDA employees with regulatory and clinical interests | 3 questions using an audience response system, 1 which was developed to better understand responses from prior CME programs |\n\n\\*Three of the activities may be viewed online \\[12-14\\].\n\nIn order to compare the knowledge of practitioners specializing in pediatrics with adult medicine practitioners, we developed Activity 5 which is applicable to the care of children and adults.\n\nActivity 6 was the only program where the target audience was not primary care practitioners. The biweekly activity is attended by a diverse group of health care practitioners and scientists, all of whom work in regulation. The activity was included to better characterize practitioner knowledge of the autism indication for atypical antipsychotics.\n\n## Response analysis\n\nThe information used for the response analysis was obtained from the CME providers as anonymized source data with no way to match responses with individuals. No personal identifiers were used. The respondents were participating in CME activities, where responses to the related learning questions are routinely aggregated to inform future CME development and related research.\n\nWe analyzed the data with and without comparing it to predicted scores. Predicted scores facilitate comparison between multiple choice questions with four choices and binomial true-false questions, which differ in the likelihood of selecting a correct answer by chance alone. For this analysis, predicted scores were 70% for multiple choice and 85% for true-false questions. The basis for these numbers comes from Audio-Digest's overall average pretest scores, which are 70%, \\[personal communication August 2010\\] and the pedagogic intent of a CME to build on participants' existing practice-relevant knowledge.\n\nEach response holds an inherent error, since a participant with a constant knowledge base could score better or worse on the pre-activity depending on the circumstances at that moment. We estimated the two-tailed two standard deviations of this variability to be ten percent. We also analyzed the participants' responses to the choice identified as close to correct, also called the second best answer.\n\nSTATA\u00ae statistical software was used to run discrete-response regression analyses on pre-activity question responses. Probit regressions were used for binomial dependent variables, analyzing whether the respondent answered the CME question correctly. The probit models give a standard normal z-score rather than a t-statistic, with the total variability explained as a pseudo-R^2^ rather than a normal R^2^. McFadden's pseudo-R^2^ is reported. The probit analysis reports the overall significance of the model using an LR chi-square. The effect of a control variable in predicting correct responses (a certain percentage above\/below the average) is calculated as the difference in probability of getting a question correct versus a baseline probability. For this analysis, baseline probability is where all control variables are set to their population means.\n\nThe control variables used in the probit models were educational degree, medical specialty, and CME participation date. Geographic region was only provided by some respondents and was therefore not included in the analysis. The type of medical practice such as hospital-based or solo practice was not among the data obtained by the CME providers.\n\nPartial incomplete responses were included in the analysis. Having all pre-activity questions left blank was considered equivalent to nonparticipation, and these entries were excluded. Since the sample size of the distinct CME activities varied, both the unweighted and weighted averages of correct responses are reported.\n\n## Instrument validation\n\nTo assess the extent to which the pre-activity responses could be generalized among primary care practitioners, the responses were compared across the diverse CME programs detailed in Table\u00a0<a href=\"#T1\" data-ref-type=\"table\">1<\/a>.\n\nThe scores on pre-activity responses were compared to self-reported learning in Activities 2\u20133, where participants were asked, \"Please list one concept or strategy gained from this activity.\"\n\nParticipant evaluations of the CME programs were recorded, to confirm satisfactory evaluations. The rating of the CME activity on a 1\u20135 Likert scale is a composite score which reflects practice relevance and appropriate teaching level of target population.\n\nNot all participants completed all pre-activity questions. The data was analyzed both including and excluding question-specific non-responders, to detect a potential bias introduced by partial completion.\n\nActivities 2 and 3 were the longest-running programs, each offered for 13 months. They were analyzed for a temporal trend, since a news story or regulatory change during the interval could potentially change practitioner baseline knowledge or practice patterns.\n\n# Results\n\nThere were 20,705 participants in the combined six CME activities which spanned 15 months. Each participant answered one or more of the following questions.\n\n## Drug indications\n\nSee Table\u00a0<a href=\"#T2\" data-ref-type=\"table\">2<\/a>. For the first question, both the average correct response rate of 76% and the weighted correct response of 79% are within the predicted range. For the second question, the average correct response rate is 53% (17% below predicted) and the weighted average correct response rate is 52% (18% below predicted).\n\nResponses to multiple choice pre-activity questions on use of antipsychotic medications\n\n| **CME activity** | **Activity 2** | **Activity 3** | **Activity 4** |\n|:---|:---|:---|:---|\n| Sample size | *n*=1237 | *n*=611 | *n*=2400 |\n| Responses to: Which of the following is a labeled indication for one or more atypical antipsychotic drugs? |  |  |  |\n| A. Refractory epilepsy |  |  |  |\n| B. Refractory major depression in adolescents |  |  |  |\n| **C. Acute mania associated with bipolar-I disorder** |  |  |  |\n| D. Attention deficit\/hyperactivity disorder |  |  |  |\n| Incorrect (A) | 82 (7%) | 40 (6%) | 72 (3%) |\n| Incorrect (B) | 188 (15%) | 92 (15%) | 264 (11%) |\n| **Correct (C)** | **912 (74%)** | **439 (72%)** | **1992 (83%)** |\n| Incorrect (D) | 55 (4%) | 40 (6%) | 72 (3%) |\n| Correct vs. Predicted | +4% | +2% | +13% |\n| Responses to: Which of the following is a labeled indication for an atypical antipsychotic in a child, 7 years of age? |  |  |  |\n| **A. Irritability associated with autism** |  |  |  |\n| B. Acute bipolar mania |  |  |  |\n| C. Schizophrenia |  |  |  |\n| D. Generalized anxiety disorder |  |  |  |\n| **Correct (A)** | **777 (63%)** | **288 (47%)** | **1152 (48%)** |\n| Incorrect (B) | 197 (16%) | 114 (19%) | 456 (19%) |\n| Incorrect (C) | 222 (18%) | 189 (31%) | 720 (30%) |\n| Incorrect (D) | 38 (3%) | 19 (3%) | 72 (3%) |\n| Correct vs. Predicted | \u22127% | \u221223% | \u221222% |\n\nSee Table\u00a0<a href=\"#T3\" data-ref-type=\"table\">3<\/a>. The average correct response rate is 65% (20% below predicted) and the weighted average is 67% (18% below predicted).\n\nResponses to the true-false pre-activity question on use of antipsychotic medications\n\n| **CME activity** | **Activity 2** | **Activity 3** |\n|:---|:---|:---|\n| Sample size | *n*=1237 | *n*=611 |\n| Responses to: The intramuscular administration of at least one atypical antipsychotic agent has been approved by the FDA for use in children. True or false? |  |  |\n| Incorrect (True) | 341 (28%) | 265 (43%) |\n| **Correct (False)** | **896 (72%)** | **346 (57%)** |\n| Correct vs. Predicted | \u221213% | \u221228% |\n\nThe participants in Activity 6 were asked: Recommended treatment of autism includes all EXCEPT:\n\nA Correct nutritional deficiencies 6 (12%)\n\nB Treatment of concurrent attention-deficit hyperactivity disorder 5 (10%)\n\nC Prescribe atypical antipsychotics 29 (57%)\n\nD Use behavioral therapies following early diagnosis 11 (21%)\n\nThe rate of correct response, response C, is 57% (13% below predicted).\n\n## Adverse metabolic effects\n\nParticipants in Activity 5 were asked to respond to: After diagnosing Ed with metabolic syndrome, Ed's doctor advised him to reduce his weight by 10%, a total of 18 pounds, by diet and exercise. Which of the following medications potentially makes it more difficult for Ed to achieve his goal?\n\nA Angiotensin-converting enzyme inhibitors 6667 (41%)\n\nB Diuretic 1243 (8%)\n\nC Vitamin D 1106 (7%)\n\nD Biguanide 7021 (43%)\n\nThe rate of correct response, choice B, is 8% (62% below predicted). Specialty did not predict response to this question.\n\nParticipants in Activity 5 were also asked to respond to: Within months of being diagnosed with bipolar disorder at age 14, Sara gained 20 pounds. Which of the following is likely to contribute to her recent weight gain and body mass index of 28?\n\nA Vitamin D deficiency 952 (6%)\n\nB Atypical antipsychotic agent 10349 (63%)\n\nC An eating disorder 1072 (7%)\n\nD Psychostimulant agent 3948 (24%)\n\nThe average correct response rate of 63% falls within the predicted range. Predicted probability of answering the question correctly given the regression control variables is 65%. Analysis by specialty indicates that mental health specialists scored 28% better than average (z=27; p\\<0.01), family practitioners scored 14% higher (z=12; p\\<0.01), internal medicine specialists scored 9% higher (z=7; p\\<0.01), endocrinologists scored 8% higher (z=3; p\\<0.01). The regression explains 9% (pseudo-R^2^=0.09) of the total variability in responses and was very significant in predicting scores (LR chi-square=1833; p\\<0.01).\n\nTable\u00a0<a href=\"#T4\" data-ref-type=\"table\">4<\/a> indicates the responses to a pre-activity question on adverse drug effects. See Table\u00a0<a href=\"#T5\" data-ref-type=\"table\">5<\/a>. For the first question, the average correct response rate was 61% with a weighted correct response average of 67%. For the second question, the average correct response rate was 75% with a weighted average also of 75%. These are within the predicted range.\n\nResponses to the true-false pre-activity question on adverse drug effects\n\n| **CME activity** | **Activity 2** | **Activity 3** |\n|:---|:---|:---|\n| Sample size | *n*=1237 | *n*=611 |\n| Responses to: Hyperglycemia, hyperlipidemia, and elevated liver enzymes are labeled side effects for one or more atypical antipsychotics. True or false? |  |  |\n| **Correct (True)** | **1154 (93%)** | **582 (95%)** |\n| Incorrect (False) | 83 (7%) | 29 (5%) |\n| Correct vs. Predicted | +8% | +10% |\n\nResponses to multiple choice pre-activity questions on adverse drug effects\n\n| **CME activity** | **Activity 1** | **Activity 2** | **Activity 3** | **Activity 4** |\n|:---|:---|:---|:---|:---|\n| Sample size | *n*=39 | *n*=1237 | *n*=611 | *n*=2400 |\n| Responses to: Jeff is an athletic 14-year-old who has been diagnosed with schizophrenia and prescribed an atypical antipsychotic. You counsel the family about potential cardio-metabolic side effects. Which risk would you emphasize? |  |  |  |  |\n| A. Sudden death from myocardial infarction |  |  |  |  |\n| B. Cardiac arrhythmias |  |  |  |  |\n| **C. Increases in triglycerides, total cholesterol, and low-density lipoprotein** |  |  |  |  |\n| D. Because patient has normal weight, he is unlikely to experience significant weight gain |  |  |  |  |\n| Incorrect (A) | n\/a | 88 (7%) | 34 (6%) | 120 (5%) |\n| Incorrect (B) | n\/a | 173 (14%) | 132 (22%) | 408 (17%) |\n| **Correct (C)** | **n\/a** | **726 (59%)** | **288 (47%)** | **1848 (77%)** |\n| Incorrect (D) | n\/a | 245 (20%) | 156 (25%) | 48 (2%) |\n| Correct vs. Predicted | n\/a | \u221211% | \u221223% | +7% |\n| Responses to: Three months after initiating treatment with antipsychotic medication, Jeff has blood-work to monitor lipids, liver enzymes, and glucose. A work-up for what endocrine condition may additionally be indicated? |  |  |  |  |\n| A. Hypothyroidism |  |  |  |  |\n| B. **Hyperprolactinemia** |  |  |  |  |\n| C. Hyperparathyroidism |  |  |  |  |\n| D. Hyperthyroidism |  |  |  |  |\n| Incorrect (A) | 4 (10%) | 144 (12%) | 101 (17%) | 432 (18%) |\n| **Correct (B)** | **27 (75%)** | **974 (79%)** | **443 (73%)** | **1752 (73%)** |\n| Incorrect (C) | 3 (8%) | 57 (5%) | 22 (3%) | 72 (3%) |\n| Incorrect (D) | 5 (13%) | 55 (4%) | 44 (7%) | 144 (6%) |\n| Correct vs. Predicted | +5% | +9% | +3% | +3% |\n\n## Patients at increased risk\n\nResponses to a question about vulnerable populations are presented in Table\u00a0<a href=\"#T6\" data-ref-type=\"table\">6<\/a>. The average correct response rate is 36% (34% below predicted) with a weighted average of 33% (37% below predicted).\n\nResponses to the pre-activity question on vulnerable populations\n\n| **CME activity** | **Activity 1** | **Activity 2** | **Activity 3** | **Activity 4** | **Activity 5** |\n|:---|:---|:---|:---|:---|:---|\n| Sample size | *n*=45 | *n*=1237 | *N*=611 | *n*=2400 | *n*=16361 |\n| Responses to: Mental illness shortens lifespan as follows: |  |  |  |  |  |\n| **A. Patients with mental illness die 25 years earlier than the general population, mostly due to earlier onset of chronic medical conditions.** |  |  |  |  |  |\n| B. The average 15 years of potential life lost can be explained by premature death early in life. |  |  |  |  |  |\n| C. The causes of mortality are similar to the general population, but occur 10 years earlier on average. |  |  |  |  |  |\n| D. Suicide and accidents including motor vehicle accidents account for most of the 10 years of shorter life expectancy. |  |  |  |  |  |\n| **Correct (A)** | **13 (29%)** | **629 (51%)** | **215 (35%)** | **840 (35%)** | **5166 (32%)** |\n| Incorrect (B) | 9 (20%) | 285 (23%) | 184 (30%) | 144 (6%) | 1584 (10%) |\n| Next best answer (C) | 20 (44%) | 265 (21%) | 169 (28%) | 528 (22%) | 4557 (28%) |\n| Incorrect (D) | 3 (7%) | 55 (5%) | 42 (7%) | 888 (37%) | 4718 (29%) |\n| Correct vs. Predicted | \u221241% | \u221219% | \u221235% | \u221235% | \u221238% |\n\nFigure\u00a0<a href=\"#F1\" data-ref-type=\"fig\">1<\/a> illustrates the correct responses compared to the predicted responses for the pre-activity question on mental illness and chronic disease risk. The responses are presented across CME activities 1\u20135. Standard error bars are shown. Since one of the three incorrect responses (Choice C) in the question was close to the correct answer, it may reflect a stronger knowledge base than the other two incorrect choices. We therefore included this response in the figure.\n\nActivity 5's large sample size allowed for further analysis. Participants had a predicted probability of 31% in answering correctly. Participants specializing in mental health scored 14% higher than average (z=11; p\\<0.01) and family practitioners scored 4% higher (z=3; p\\<0.05). The probit regression explained 1% (pseudo-R^2^=0.01) of the total variability in the responses and was significant (LR chi-square=202, p\\<0.01).\n\n## Drug safety updates\n\nParticipants in Activity 5 were asked to respond to: Which of the following statements is correct?\n\nA. Comparative effectiveness trials are part of the drug approval process \\[5622 (34%)\\]\n\nB. Phase 3 clinical trials are powered to identify appetite-stimulating effects of medication \\[3346 (20%)\\]\n\nC. Incidence of weight gain can be calculated from a passive adverse events reporting system \\[4424 (27%)\\]\n\nD. Current legislation requires clinical trials in pediatric populations \\[2452 (15%)\\]\n\nOn average, 15% of participants answered correctly (55% below predicted) selecting choice D. Predicted probability of answering correctly given the regression variables is 14%. Analysis by specialty indicates that pediatricians scored 7% higher than average (z=6; p\\<0.01) and mental health specialists scored 2% higher (z=2; p\\<0.02). General practitioners scored 3% below average (z=\u22122; p\\<0.03) and emergency medicine specialists scored 4% lower (z=\u22122; p\\<0.04). The regression explained 2% of the total variability in answers (pseudo-R^2^=0.02) and was very significant (LR chi-square=242; p\\<0.01).\n\nNote that this question had the highest non-response rate, with 517 (3%) of participants leaving the question blank. Regression analysis excluding non-responders had the same significant outcome variables as the analysis which included non-responders.\n\nSee Table\u00a0<a href=\"#T7\" data-ref-type=\"table\">7<\/a>. The average correct response was 47% (23% below predicted) and the weighted average was 51% (19% below predicted).\n\nResponses to pre-activity questions on drug safety information\n\n| **CME activity** | **Activity 2** | **Activity 3** |\n|:---|:---|:---|\n| Sample size | n=1237 | n=611 |\n| Responses to: Practitioners are asked to report drug adverse events: |  |  |\n| **A. To MedWatch** |  |  |\n| B. Only when the adverse event is not specified on the drug label |  |  |\n| C. Within 30 days of occurrence, as required by law. |  |  |\n| D. Voluntarily, if observed within 30 days of the first dose |  |  |\n| **Correct (A)** | **698 (56%)** | **237 (39%)** |\n| Incorrect (B) | 85 (7%) | 53 (9%) |\n| Incorrect (C) | 203 (10%) | 174 (29%) |\n| Incorrect (D) | 243 (20%) | 146 (24%) |\n| Correct vs. Predicted | \u221214% | \u221231% |\n\nFor Activity 5 (*n*=16,361), the top three professions of participants were nurse practitioners (52%, *n*=8407), physicians (38%, *n*=6212), and physician assistants (3%, *n*=476). The top specialties were psychiatry\/mental health (12%, *n*=2022), family medicine (11%, *n*=1875), internal medicine (10%, *n*=1639), general practice (6%, *n*=946), and pediatrics (6%, *n*=906).\n\nWe controlled the regression analysis for predicting correct pre-activity responses for specialty, professional degree, and date of CME participation by quartile. The time of participation was included in the regression analysis because it explained a significant portion of the variability but yielded no clear pattern for interpretation. Results of the regression analysis follow each applicable question. The results of the analysis by specialty concur with the practice demands of each specialty. For example, family physicians, practitioners who follow patients across the lifespan, were more likely to correctly identify the profound extent to which mental illness shortens life expectancy due to chronic diseases.\n\n## Instrument analysis\n\nThe strength of the instrument is its ease of use in the context of CME programming, and its associated ability to identify trends in practitioner knowledge and some broad comparisons among practitioners. However since the instrument is comprised of multiple choice questions, responses to any one question are more appropriately viewed in the context of the full instrument.\n\nIn order to assess the variability of the instrument, responses were compared across CME programs which varied in content, timeframe, recruitment, and question administration. Figure\u00a0<a href=\"#F1\" data-ref-type=\"fig\">1<\/a> depicts the responses. Responses among CME programs varied within the pre-established +\/\u221210% test error, except for one program with a small sample size. The unweighted, correct response averages across CME programs are reported.\n\nTo assess the extent to which recruitment methods may influence the pre-activity responses, the overall scores of the two Audio-Digest programs were compared. The two programs differ only in how the participants were recruited. They were recruited as either, subscribers or one-time participants. The 10% difference in responses falls within the pre-established test error.\n\nPractice-relevance and the perception of the CME program's usefulness were considered in the instrument analysis. The participants in each of the 6 activities were asked to evaluate the program on a 1\u20135 Likert scale, 5 being the highest score. The ratings for each program ranged from 4.0 to 5.0, with an unweighted mean score of 4.5, suggesting that all were well-received and applicable to participants' clinical practice.\n\nThe pre-activity question responses were correlated with what participants said they learned from the activity. Participants in Activities 2 and 3 were asked to \"Please list one concept or strategy gained from this activity.\" The written responses fell into categories consistent with pre-activity responses: Pediatric indications (20), patient adherence (2), adverse effects (71), MedWatch reporting (9), drug interactions (5), and patient risk factor (8).\n\nA temporal trend in correct responses was not observed between the first three months and the total 13 months of the responses to Activities 2 and 3. Neither was any single news event or regulatory change identified which might be anticipated to influence practitioner knowledge on this topic during the study period.\n\n# Discussion\n\nThe childhood obesity epidemic is recent; however, practitioners who care for children appear as familiar with adverse metabolic drug effects as practitioners who care for adults. Those specializing in pediatrics performed better on a question about drug research, perhaps reflecting recent educational activities directed towards pediatricians.\n\nAcross medical specialties practitioner knowledge of medication-related weight gain was low in four areas of our study. Each of the knowledge gaps if practice relevant, would overestimate the medication's benefits or underestimate adverse metabolic effects. The net effect of each knowledge gap would therefore affect clinical decision-making in the same direction, potentially contributing to excess metabolic dysfunction. The four areas of low practitioner knowledge are as follows:\n\nResponses to questions about drug indications and the use of antipsychotics in autism suggest that some practitioners may mistake the management of aggressive symptoms for treatment of the underlying disease process. Additionally, new oral preparations are available for children who have difficulty swallowing pills. These preparations should be used before prescribing intramuscular preparations, which have greater metabolic effects and do not have pediatric use indications at the time this manuscript is written.\n\nAmong the questions pertaining to adverse metabolic effects, only 8% of practitioners selected the intended response that some diuretics have been associated with promoting insulin resistance. The 41% who incorrectly selected \"angiotensin-converting enzyme inhibitors\" are unlikely to have been aware that some diuretics promote insulin resistance and angiotensin converting enzymes, in contrast, may be insulin sensitizing \\[15\\], the distinction between these two antihypertensive therapies in a patient with metabolic syndrome would be practice relevant. Furthermore, these respondents may have erroneously equated the reduction in peripheral edema with meaningful, long-term weight loss among their patients. The 43% of practitioners who incorrectly selected \"biguanide\" may not have realized that metformin is in this medication class, so the response would have been more informative if the answer had read \"biguanide (metformin).\"\n\nResponses reflected low baseline knowledge of drug safety research and MedWatch, a passive surveillance program. It is possible that practitioners lack a framework for managing the escalating drug-related information. Our findings parallel those of a recent study of physician knowledge and adverse events reporting of dietary supplements \\[16\\].\n\nMental illness is associated with increased vulnerability to adverse metabolic effects. The profound chronic disease mortality among patients with mental illness was under-recognized across specialties as measured by our instrument. Awareness of the high mortality from chronic diseases among patients with mental illness might be unlikely to cause practitioners to alter the patient's psychiatric medications; however, it would guide overall care such as screening, referring, concurrent medication prescribing, and managing co-morbidities.\n\nThe knowledge gaps parallel the few peer-reviewed publications on medication-related weight gain, other than the atypical antipsychotics. Reviewing the proceeding of a large international conference on obesity \\[17\\] revealed a similar paucity of research and translational initiatives surrounding medication-related weight gain. Additionally, current drug product information and labeling lacks a consistent format or location for communicating the potential effects of a drug on the patient's appetite and underlying metabolism.\n\nPractitioners were familiar with the general indications for use of atypical antipsychotics in children and the adverse metabolic effects including prolactinemia, dyslipidemia, elevated liver enzymes, insulin resistance, and weight gain, findings which correlate with the lay and medical literature's recent attention to the topic \\[4\\]. Similarly, education initiatives about pediatric drug labeling have been directed to pediatricians and pediatricians were more knowledgeable than other practitioners about ongoing pharmacovigilance.\n\nThe instrument demonstrated internal consistency across diverse CME programs (Table\u00a0<a href=\"#T1\" data-ref-type=\"table\">1<\/a>), suggesting the findings may appropriately be generalized across U.S. primary care practitioners. The sampling frame captures participants across the United States, with diverse patient populations in diverse practice settings. The degrees of the participants, nurse practitioners, physician assistants, and medical doctors, correctly represent the educational diversity of primary care practitioners. Pediatricians scored as well as their adult medicine counterparts, suggesting that future initiatives could appropriately be directed to all primary care practitioners.\n\nAdditional merits of the instrument are that it can be implemented in a timely and cost-sensitive way. It can be applied to assess evidence-based practice knowledge \\[18\\]. Study findings can provide baseline data, by which to gauge the effectiveness of future interventions. The instrument also provides a continuing education curriculum developed free of industry interests. An internet curriculum on safe medication use measurably improved clinician practice choices \\[19,20\\].\n\nKnowledge is one of many clinical practice barriers to modifying medication-related weight gain, and merits incorporation into future initiatives. The findings, taken with the population prevalence of obesity, the emerging treatment options, and the central role of the primary care practitioner, suggest a significant prevention opportunity.\n\n# Conclusions\n\nPediatricians' knowledge base of adverse metabolic drug effects appears comparable to their counterparts in adult medicine. Regardless of medical specialty, practitioners participating in the CME programs reflected low knowledge on specific questions pertaining to drug indications, adverse metabolic effects, patient risk profiles and safety updates. Each of the four knowledge gaps would potentially influence clinical decision-making in the same manner, leading clinicians to overestimate the benefits of a drug in relation to its metabolic risks. Therefore future efforts to detail cross-specialty practitioner knowledge of metabolic drug effects and initiate education strategies to bolster knowledge could meaningfully contribute to obesity prevention.\n\n## Availability of supporting data\n\nThe full instrument (CME questions from all activities) is available at the journal's request.\n\n# Competing interests\n\nNeither author has financial or non-financial competing interests.\n\n# Authors' contributions\n\nIK developed the CME modules in collaboration with colleagues acknowledged elsewhere in the manuscript and published CME materials. She designed the study in collaboration with the Office of Pediatric Therapeutics and drafted the manuscript. GW participated in the design of the study and performed the statistical analysis. Both authors read and approved the final manuscript.\n\n# Author's information\n\nIK is a physician nutrition specialist board-certified in preventive medicine and public health. She is the editor of Advancing Medicine with Food and Nutrients, Second Edition (CRC Press, December 2012) and serves on the faculty of Johns Hopkins Bloomberg School of Public Health. As an inaugural FDA Commissioner's Fellow she worked within the Office of Pediatric Therapeutics on nutrition-related issues, which gave rise to this research collaboration.\n\n## Acknowledgements\n\nWe thank Anne Myers for her background work on pediatrician focus groups; Lon Osmond, Executive Director, Audio-Digest Foundation for his collaboration; Michelle Surrichio of the American College of Preventive Medicine for her technical assistance; and Rachel Barr of the Maryland Academy of Family Physicians for her conference preparations.\n\n## Disclaimer\n\nThis manuscript is a professional contribution developed by its authors. No endorsement by the FDA is intended or should be inferred.","meta":{"dup_signals":{"dup_doc_count":130,"dup_dump_count":41,"dup_details":{"curated_sources":2,"2021-49":1,"2019-26":1,"2019-13":1,"2019-04":1,"2018-47":1,"2018-39":1,"2018-30":2,"2018-13":1,"2017-47":1,"2017-34":1,"2017-26":1,"2017-17":1,"2017-09":25,"2017-04":1,"2016-50":1,"2016-44":1,"2016-36":1,"2016-30":1,"2016-22":1,"2016-18":1,"2015-48":4,"2015-40":3,"2015-35":4,"2015-32":4,"2015-27":4,"2015-22":4,"2015-14":2,"2014-52":4,"2014-49":6,"2014-42":9,"2014-41":3,"2014-35":6,"2014-23":7,"2014-15":4,"2023-50":1,"2015-18":4,"2015-11":4,"2015-06":4,"2014-10":3,"2013-48":2,"2024-30":1}},"file":"PMC3636058"},"subset":"pubmed_central"}
{"text":"abstract: Circulating interleukin (IL)-18 is elevated in obesity, but paradoxically causes hypophagia. We hypothesized that IL-18 may attenuate high-fat diet (HFD)-induced insulin resistance by activating AMP-activated protein kinase (AMPK). We studied mice with a global deletion of the \u03b1-isoform of the IL-18 receptor (IL-18R^\u2212\/\u2212^) fed a standard chow or HFD. We next performed gain-of-function experiments in skeletal muscle, in vitro, ex vivo, and in vivo. We show that IL-18 is implicated in metabolic homeostasis, inflammation, and insulin resistance via mechanisms involving the activation of AMPK in skeletal muscle. IL-18R^\u2212\/\u2212^ mice display increased weight gain, ectopic lipid deposition, inflammation, and reduced AMPK signaling in skeletal muscle. Treating myotubes or skeletal muscle strips with IL-18 activated AMPK and increased fat oxidation. Moreover, in vivo electroporation of IL-18 into skeletal muscle activated AMPK and concomitantly inhibited HFD-induced weight gain. In summary, IL-18 enhances AMPK signaling and lipid oxidation in skeletal muscle implicating IL-18 in metabolic homeostasis.\nauthor: Birgitte Lindegaard; Vance B. Matthews; Claus Brandt; Pernille Hojman; Tamara L. Allen; Emma Estevez; Matthew J. Watt; Clinton R. Bruce; Ole H. Mortensen; Susanne Syberg; Caroline Rudnicka; Julie Abildgaard; Henriette Pilegaard; Juan Hidalgo; Susanne Ditlevsen; Thomas J. Alsted; Andreas N. Madsen; Bente K. Pedersen; Mark A. FebbraioCorresponding author: Mark A. Febbraio, <firstname.lastname@example.com>, or Bente K. Pedersen, <email@example.com>.\ndate: 2013-09\nreferences:\ntitle: Interleukin-18 Activates Skeletal Muscle AMPK and Reduces Weight Gain and Insulin Resistance in Mice\n\nThe cytokine interleukin (IL)-18 was identified \u223c15 years ago as a cofactor that, together with IL-12, stimulates production of interferon-\u03b3 (1). This \u223c18-kDa cytokine, which has structural similarities to the IL-1 cytokine family, is widely expressed in many mammalian cells and tissues, including liver, adipose tissue, skeletal muscle, pancreas, brain, and endothelium (2). IL-18 is best known for its role in inflammation, whereby proinflammatory stimuli such as lipopolysaccharide, Fas ligand, and tumor necrosis factor-\u03b1 lead to caspase-1\u2013mediated cleavage of pro-IL-18 into mature IL-18. IL-18 then can signal via a heterodimer of the transmembrane IL-18 receptors (\u03b1 and \u03b2) and via a Toll-like receptor signaling cascade, ultimately leading to the activation of nuclear factor-\u03baB and subsequent regulation of gene transcription (3). Although this is the most characterized signaling pathway for this cytokine, it is worth noting that IL-18 also has been implicated in mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and signal transducer and activator of transcription (STAT) 3 signaling (4), which are all implicated in energy metabolism.\n\nIt is now well-accepted that obesity results in a state of low-grade chronic inflammation (5); therefore, it is not surprising that circulating IL-18 levels are elevated in human obesity (6) and in patients with type 2 diabetes (7). Somewhat paradoxically, the works of two separate groups have reported that mice with a global deletion of IL-18 become obese and insulin-resistant, whereas exogenous administration of recombinant IL-18 rescued this phenotype (8,9). These previous studies ascribed the mechanism of action of IL-18 in modulating nutrient homeostasis to be exclusively via neuronal control of food intake (8,9). However, in the earlier study (8), the authors demonstrated that the IL-18^\u2212\/\u2212^ mice displayed decreased peripheral insulin sensitivity and that IL-18 signals via STAT3 in the liver, increasing the possibility that IL-18 may play a role in peripheral energy metabolism because STAT3 plays a major role in maintaining metabolic homeostasis in the liver (10). In addition, we (11,12) and others (13,14) have shown that cytokines that result in activation of STAT3 via transmembrane receptor signaling can activate the AMP-activated kinase (AMPK) signaling pathway to enhance fat oxidation in skeletal muscle, thereby attenuating high-fat diet (HFD)-induced insulin resistance. Together, these previous studies increase the possibility that IL-18 may attenuate HFD-induced insulin resistance via affecting metabolic processes, such as activation of AMPK, in skeletal muscle. This is important from a therapeutic viewpoint because drugs that effectively modulate food intake via targeting the central nervous system have, to date, proven unsuccessful because of side effects associated with activation of the lateral hypothalamus (15). In the current study, we tested the hypothesis that IL-18 signaling can modulate nutrient homeostasis via mechanisms associated with peripheral energy metabolism. We show that IL-18 activates AMPK and increases lipid oxidation in skeletal muscle, implicating IL-18 in diet-induced obesity and insulin resistance.\n\n# RESEARCH DESIGN AND METHODS\n\n## Animal experimental protocols.\n\nFor the diet intervention study, 12-week-old *Il18R^\u2212\/\u2212^* (back-crossed 11 generations to C57BL\/6J) and wild-type C57BL\/6J mice were obtained from Charles River Laboratories (L'Arbresle, France). Subsequent to this initial cohort, we then performed experiments in *Il18R^\u2212\/\u2212^* and wild-type mice obtained from heterozygous mating. Eight-week-old C57BL\/6J mice (inbred; Herlev, Denmark) were used for a DNA electroporation study, and 13-week-old C57\/BL6 mice (inbred; Melbourne, Australia) were used for ex vivo experiments. All experiments were approved by The Animal Experiments Inspectorate in Denmark or the Baker IDI Alfred Medical Research and Education Precinct Animal Ethics Committee (or both) in accordance with the National Health and Medical Research Council of Australia Guidelines on Animal Experimentation. Mice were maintained on a 12-h light, 12-h dark cycle on a standard rodent chow diet (27%, 13%, and 60% kcal from protein, fat, and carbohydrates, respectively) or HFD composed of 60% calories from fat (Research Diets 12492; 20%, 60%, and 20% kcal from protein, fat, and carbohydrates, respectively) for 18 weeks (diet-induced obesity study) or 4 weeks (in vivo electroporation study).\n\nIn vivo electroporation experiments were performed as previously described (16). Briefly, the regulatory plasmid pTet-On was obtained from Clontech (Palo Alto, CA). Because IL-18 lacks a typical signal sequence, the V-J2-C region of murine immunoglobin k-chain was cloned upstream of the mature IL-18 sequence. The tibialis anterior muscle of each mouse was directly injected with 20 \u00b5L plasmid solution (0.5 \u00b5g\/\u00b5L) and electric pulsing was applied using 4-mm plate electrodes and an electric field of one high-voltage pulse (100 \u00b5s, 800 V\/cm) and one low-voltage pulse (400 ms, 100 V\/cm) (17). Induction of gene expression was obtained by administering drinking water consisting of distilled water containing 0.2 mg\/mL doxycycline (doxycycline hyclate; Sigma-Aldrich) (18).\n\nFor ex vivo experiments, mice were fed a standard chow diet and drinking water, available ad libitum. To examine palmitate metabolism, mice were first anesthetized and soleus muscles were carefully dissected into longitudinal strips from tendon to tendon. Strips were removed and \\[^14^C\\]palmitate oxidation was analyzed as previously described (19). The dose of 100 ng\/mL IL-18 was used and PBS served as placebo. The strips from the same animal were used for both IL-18 and PBS.\n\n## Skeletal muscle cell culture.\n\nTo examine whether IL-18 affected phosphorylation (Thr^172^) of AMPK and acetyl CoA carboxylase-\u03b2 (ACC\u03b2; Ser^79^), fully fused L6 myotubes were treated with recombinant rat IL-18 (MBL, Woburn, MA). Cells were treated with IL-18 for 10, 30, and 60 min at doses of 1 and 10 ng\/mL.\n\n## Analysis of body composition.\n\nWe measured body composition analysis in IL18R^\u2212\/\u2212^ by dual-energy X-ray absorptiometry using the Lunar PIXImus Mouse Densitometer (GE Medical Systems) and in IL-18 electroporated mice by using a Lunar Prodigy scanner with a small animal software application (GE Healthcare Systems). Animals were anesthetized by intraperitoneal injection of Hypnorm (0.4 mL\/kg; Janssen, Saunderton, U.K.) and Dormicum (2 mg\/kg; Roche, Basel, Switzerland), and laid flat on the scanning platform on their ventral side. To confirm dual-energy X-ray absorptiometry fat mass determination, mice were killed and two intra-abdominal fat pads (gonadal, retroperitoneal) and one subcutaneous fat pad (inguinal) were dissected and weighed.\n\n## Insulin tolerance tests.\n\nWe performed insulin tolerance tests in 7.5-month-old male mice 4 h after removal of food. Blood samples were obtained by tail cut and were analyzed for glucose content using a glucometer (Accu-chek Compact plus) immediately before and at 15, 30, 45, 60, 90, and 120 min after an intraperitoneal injection of insulin (0.75 units\/kg; Actrapid; Novo Nordisk).\n\n## Indirect calorimetry.\n\nMice were placed in a 16-chamber indirect calorimetry system (TSE Systems, Bad Homburg, Germany) cages for 10 days; the first 5 days were considered the acclimation phase and data were analyzed only for the last final days. After 3 days, the mice were fasted for 24 h. The V<span class=\"smallcaps\">o<\/span>~2~ (mL\/h\/kg), respiratory exchange ratio, and activity (beam breaks) were measured using the system. Mice had free access to food and water while in the chambers. Food intake was measured for the duration of data collection while mice underwent the indirect calorimetry measurements.\n\n## Plasma parameters analysis.\n\nWe collected blood samples from the tail vein of the mice into EDTA tubes and they were immediately spun at 6,000*g* for 10 min at 4\u00b0C and plasma was removed. Plasma was stored at \u221280\u00b0C until analysis. Plasma insulin was determined by ELISA (Crystal Chem). Blood glucose was measured by glucometer (Accu-chek Compact Plus). Adiponectin concentration was measured by RIA kit. Leptin, monocyte chemoattractant protein 1, plasminogen activator inhibitor 1, IL-6, and tumor necrosis factor-\u03b1 were measured by using a Lincoplex mouse serum adipokine panel (Linco).\n\n## Insulin signaling tissue collection.\n\nAnimals were anesthetized with an injection of Hypnorm (0.4 mL\/kg; Janssen, Saunderton, UK) and Dormicum (2 mg\/kg; Roche, Basel, Switzerland), and the gastrocnemius muscle as well as the right lobe of the liver (after a ligature around the blood vessel) were removed and stored in liquid nitrogen until further processing. This was followed by injection of insulin (1.5 units\/kg lean body mass) into the abdominal aorta and removal of the contralateral gastrocnemius and liver lobe occurred 2 min after injection. Samples were stored in liquid nitrogen until further processing.\n\n## RNA extraction and real-time quantitative PCR.\n\nMouse tissue was isolated, frozen in liquid nitrogen or in dry ice and absolute alcohol, and stored at \u221280\u00b0C until extraction. Total RNA was isolated from adipose tissue with TriZol (Life Technology), as described by the manufacturer; 500 ng RNA was reverse-transcribed to cDNA with the use of random hexamers (Taqman reverse-transcription reagents; Applied Biosystems). Real-time PCR was performed on an ABI PRISM 7900 sequence detector or 7500 fast sequence detector (Applied Biosystems). Each assay included (in triplicate) the following: a cDNA standard curve of five serial dilution points (range, 1\u20130.01); a no-template control; a no-reverse-transcriptase control; and 7.5 ng (0.375 ng for 18S rRNA) of each sample cDNA. For *18S rRNA, SREEBP1c*, fatty acid synthase (*FAS*), *HADB*, phosphoenolpyruvate carboxykinase (*PEPCK*), glucose-6 phosphate dehydrogenase, and carnitine palmitoyl transferase 1 (*CPT1*), the amplification mixtures were prepared with 2X Taqman Universal PCR master mix. All assay reagents were from Applied Biosystems. Primers and Taqman probes were designed for sterol regulatory\u2013element binding protein-1c and *FAS* using a mouse-specific database (ensemble.com) and Primer Express (Applied Biosystems) and primers for *CPT1* and *HADB* were designed using the free program primer3. The sequences to amplify a fragment of sterol regulatory\u2013element binding protein-1c was as follows: FP: 5\u2032 GACCACGGAGCCATGGAT; 3\u2032and RP: 5\u2032 GGCCCGGGAAGTCACTGT; 3\u2032 and TaqMan probe: 5\u2032 ACATTTGAAGACATGCTCCAGCTCATCAACA; 3\u2032, a fragment of *FAS* FP: 5\u2032 ATCCTGGAACGAGAACACGATCT 3\u2032; RP: 5\u2032 GGACTTGGGGGCTGTCGTGTCA; 3\u2032 and TaqMan probe: 5\u2032 CACGCTGCGGAAACTTCAGGAAATGT; 3\u2032, a fragment of *HAD* FP: GTGGAGAAGACCCTGAGCTA; RP: GCAAATCGGTCTTGTCTAGT; a fragment of *PEPCK* FP: GGCGGAGCATATGCT and RP: CCACAGGCACTAGGGAAGGC, a fragment of glucose-6 phosphate dehydrogenase FP: TCAACCTCGTCTTCAAGTGGATT; RP: GCTGTAGTAGTCGGTGTCCAGGA; CPTI FP: GTCGCTTCTTCAAGGTCTGG; and RP: AAGAAAGCAGCACGTTCCAT. Oligos for *SREBP1* and *FAS* were obtained from TaqCopenhagen (Copenhagen, Denmark) for *CPT1*, oligos for *HAD* were obtained from DNA technology (Aarhus, Denmark), and oligos for *PEPCK* and glucose-6 phosphate dehydrogenase were obtained from Geneworks (South Australia, Australia). The *18S rRNA* content was determined using a predeveloped assay reagent (Applied Biosystems). The relative concentrations of measured mRNAs were determined by plotting the threshold cycle versus the log of the serial dilution points, and the relative expression of the gene of interest was determined after normalization to *18S*, which was unaffected by genotype and diet.\n\n## AMPK activity and lipid measurements.\n\nAMPK activity was assayed in frozen tibialis anterior muscle homogenized in lysis buffer as described previously (12). Briefly, muscle lysate containing 200 \u03bcg protein was immunoprecipitated with antibody specific to the \u03b12 or \u03b11 catalytic subunit of AMPK and protein A\/G agarose beads. Beads were washed five times, and the activity of the immobilized enzyme was assayed based on the phosphorylation of substrate for AMP-activated protein kinase peptide (0.2 mmol\/L) by 0.2 mmol\/L ATP (containing 2 \u03bcCi \\[\u03b3-^32^P\\] ATP) in the presence and absence of 0.2 mmol\/L AMP. Label incorporation into the substrate for AMP-activated protein kinase peptide was measured on a Racbeta 1214 scintillation counter. For measurement of intramuscular triglycerides, freeze-dried muscle samples were dissected free of visible connective tissue and blood. Lipid was extracted by a Folch extraction, the triacylglycerol was saponified in an ethanol\/KOH solution at 60\u00b0C, and glycerol content was determined fluorometrically as described previously (12).\n\n## Protein analysis.\n\nTissue lysates (40 \u03bcg) were solubilized in Laemmli sample buffer and boiled for 5 min, resolved by SDS-PAGE on 10% polyacrylamide gels, transferred (semi-dry) to nitrocellulose membrane, blocked with 5% BSA, and immunoblotted with primary antibodies (2.5% BSA) overnight. After incubation in horseradish peroxidase-conjugated secondary antibody (2.5% BSA; Amersham Bioscience), the immunoreactive proteins were detected with enhanced chemiluminescence (Amersham Bioscience) and quantified by densitometry (ChemiDoc XRS). Membranes were stripped, washed, and reprobed for total protein content or housekeeping protein when appropriate. Total AKT was run on a separate gel and was not stripped. Samples from mice fed a chow or HFD were run on separate gels. The antibodies used for detection of total AKT and \u03b2-actin and phosphorylation of JNK1\/2, AKT, AMPK\u03b1, and ACC\u03b2 were purchased from Cell Signaling. The \u03b1-tubulin antibody was obtained from Sigma Aldrich.\n\n## Statistical analysis.\n\nAll analyses were performed using SAS 9.1. We performed comparison between two groups using unpaired Student *t* test and one-way ANOVA followed the Tukey post hoc test. Time-series data were analyzed with PROC MIXED. *P* \\< 0.05 was considered significant. When appropriate, values were logarithmically transformed to ensure normality and equal variance.\n\n# RESULTS\n\n## IL-18 receptor\u2013deficient mice are prone to weight gain that is not associated with hyperphagia.\n\nTo evaluate the role of IL-18 signaling in the etiology of body weight homeostasis, we first performed loss-of-function experiments by phenotyping mice with a global deletion of the \u03b1-isoform of the IL-18 receptor (IL-18R^\u2212\/\u2212^). Consistent with a previous study (8), IL-18R^\u2212\/\u2212^ mice become heavier than their wild-type counterparts (control \\[CON\\]) at \u223c6 months of age when fed a regular chow diet (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>A<\/em><\/a>).\n\nThe increase in body mass was attributable to an increase in adiposity because we observed differences in percent fat (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>B<\/em><\/a>) but not in percent fat-free mass (data not shown). This increase in adiposity was attributable to increases in visceral, but not subcutaneous, fat mass because both epididymal and retroperitoneal, but not inguinal, fat pad masses were higher in IL-18R^\u2212\/\u2212^ mice relative to CON (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>C<\/em><\/a>). Although both IL-18R^\u2212\/\u2212^ and CON mice had markedly increased body mass and fat pad mass when fed an HFD for 16 weeks, the differences observed when comparing genotypes on a regular chow diet were not evident when animals were fed an HFD (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>A\u2013D<\/em><\/a>). As discussed, previous studies have demonstrated that IL-18^\u2212\/\u2212^ mice are prone to weight gain because of hyperphagia (8,9). Although the earlier study reported weight gain and insulin resistance in IL-18R^\u2212\/\u2212^ mice, they did not make reference to altered feeding behavior in these animals (8). To determine whether the increase in adiposity observed in IL-18R^\u2212\/\u2212^ mice was attributable to an increase in food intake or a decrease in energy expenditure (or both), we next performed whole-body indirect calorimetry experiments. We observed no difference in food intake (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>E<\/em><\/a>), whole-body oxygen consumption (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>F<\/em><\/a>), or activity (data not shown) measured over 24 h when comparing IL-18R^\u2212\/\u2212^ mice with CON irrespective of diet. As expected, consumption of the HFD decreased whole-body RER, indicative of an increase in whole-body fat oxidation. When mice were provided with HFD ad libitum, average RER (over a 72-h period) was not different when comparing IL-18R^\u2212\/\u2212^ mice with CON irrespective of diet (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>H<\/em><\/a>). Of note, however, when mice were fasted and refed at the cessation of the 72-h period, IL-18R^\u2212\/\u2212^ mice displayed a significantly higher RER in both the fasted and refed conditions, relative to CON (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>H<\/em><\/a>).\n\n## IL-18R^\u2212\/\u2212^ mice are insulin-resistant.\n\nWe next examined whether IL-18R^\u2212\/\u2212^ mice were insulin-resistant. At 3 month of age, and before the IL-18R^\u2212\/\u2212^ mice became obese on a chow diet, there were no differences in insulin resistance as measured by an intraperitoneal tolerance test (ITT) (data not shown). However, with age and irrespective of diet, IL-18R^\u2212\/\u2212^ mice displayed whole-body insulin resistance as measured by both fasting hyperinsulinemia (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<em>A<\/em><\/a>) and impaired glucose clearance during an ITT (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<em>B<\/em><\/a> and [Supplementary Fig. 1](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1095\/-\/DC1)). We next examined insulin signaling in both muscle and liver by analyzing the phosphorylation of Akt (Ser^473^) before and 2 min after a bolus dose of insulin. Although insulin increased pAkt in the skeletal muscle (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<em>C<\/em><\/a>) and liver (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<em>E<\/em><\/a>) of CON animals fed a chow diet, this effect was markedly blunted in IL-18R^\u2212\/\u2212^ mice (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<em>C<\/em> and <em>E<\/em><\/a>). No differences in pAkt in either skeletal muscle (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<em>D<\/em><\/a>) or liver (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<em>F<\/em><\/a>) were observed when comparing IL-18R^\u2212\/\u2212^ with CON mice when fed an HFD.\n\n## IL-18R^\u2212\/\u2212^ mice store excess lipid in skeletal muscle and have inflamed livers and skeletal muscle.\n\nExcess adiposity often is associated with ectopic lipid storage in metabolic tissues such as liver and skeletal muscle, which can mediate insulin resistance either directly or via the upregulation of serine threonine kinases such as c-jun terminal kinase (JNK) and inhibitor of \u03baB kinase (5). Accordingly, we next measured intramyocellular and intrahepatic lipid content and the phosphorylation of JNK (Thr^183^\/Tyr^185^) and inhibitor of \u03baB kinase-\u03b1\u03b2 (Ser^180^\/ Ser^181^) in these tissues. Irrespective of diet, triacylglycerol content was higher in the skeletal muscles of IL-18R^\u2212\/\u2212^ relative to CON mice (<a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<em>A<\/em><\/a>). This was associated with elevated JNK (<a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<em>B<\/em><\/a>) but not with inhibitor of \u03baB-kinase ([Supplementary Fig. 2](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1095\/-\/DC1)) phosphorylation. Conversely, we did not observe any differences in intrahepatic triacylglycerol concentration when comparing IL-18R^\u2212\/\u2212^ with CON mice irrespective of diet (<a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<em>C<\/em><\/a>). Notwithstanding, phosphorylation of JNK (<a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<em>D<\/em><\/a>) and the mRNA expression of key fatty acid synthesis transcription factors\/enzyme sterol regulatory\u2013element binding protein-1c and *FAS* (<a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<em>E<\/em> and <em>F<\/em><\/a>) were elevated in the liver when comparing IL-18R^\u2212\/\u2212^ with CON mice when animals were fed a chow and an HFD. However, no differences were observed in the mRNA expression of key gluconeogenic enzymes *PEPCK* or glucose-6 phosphate dehydrogenase when comparing IL-18R^\u2212\/\u2212^ with CON mice irrespective of diet ([Supplementary Fig. 3](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1095\/-\/DC1)). Given that neither hepatosteatosis nor the expression of key enzymes involved in regulating hepatic glucose production was different when comparing IL-18R^\u2212\/\u2212^ with CON mice, it is unlikely that changes in liver insulin sensitivity were responsible for the reduced whole-body insulin sensitivity observed in IL-18R^\u2212\/\u2212^, although this possibility cannot be entirely ruled out.\n\n## IL-18R^\u2212\/\u2212^ mice have reduced AMPK signaling in metabolic tissues.\n\nBecause IL-18R^\u2212\/\u2212^ mice are prone to weight gain on a normal diet and ectopic lipid storage independent of feeding, we next examined whether pathways associated with lipid oxidation were impaired. One major pathway that regulates fatty acid oxidation is AMPK. AMPK phosphorylates ACC\u03b2, resulting in inhibition of ACC activity, which in turn leads to a decrease in malonyl CoA content, relieving inhibition of CPT1 and increasing fatty acid oxidation. No significant differences between phenotypes were observed when measuring the phosphorylation of AMPK (Thr^172^) (data not shown). However, the phosphorylation of ACC\u03b2 (Ser^218^), a downstream marker of AMPK activity, was reduced in the skeletal muscle, liver, and adipose tissue of IL-18R^\u2212\/\u2212^ when fed a chow diet (<a href=\"#F4\" data-ref-type=\"fig\">Fig. 4<em>A\u2013C<\/em><\/a>). In addition, this effect was maintained in skeletal muscle (<a href=\"#F4\" data-ref-type=\"fig\">Fig. 4<em>A<\/em><\/a>) and liver (<a href=\"#F4\" data-ref-type=\"fig\">Fig. 4<em>B<\/em><\/a>), but not adipose tissue (<a href=\"#F4\" data-ref-type=\"fig\">Fig. 4<em>C<\/em><\/a>) when mice were fed the HFD.\n\n## Exogenous IL-18 treatment increases AMPK signaling and fat oxidation in skeletal muscle in vitro and ex vivo.\n\nBecause we observed that IL-18R^\u2212\/\u2212^ mice become obese, store more lipid in skeletal muscle, and have defective ACC\u03b2 phosphorylation in this organ, we next performed in vitro and ex vivo experiments in muscle cells and whole muscle strips to confirm the role of IL-18 on AMPK signaling and fat oxidation in this important metabolic tissue. In initial experiments, we demonstrated that as little as 1.0 ng\/mL recombinant IL-18 protein was sufficient to phosphorylate both AMPK (Thr^172^) (<a href=\"#F5\" data-ref-type=\"fig\">Fig. 5<em>A<\/em><\/a>) and ACC\u03b2 (Ser^218^) (<a href=\"#F5\" data-ref-type=\"fig\">Fig. 5<em>B<\/em><\/a>) in L6 myotubes. We next performed experiments in isolated intact soleus muscle as previously reported (16,20,21). Treating these muscles with 100 ng\/mL recombinant IL-18 was sufficient to increase both palmitate oxidation (<a href=\"#F5\" data-ref-type=\"fig\">Fig. 5<em>C<\/em><\/a>) and AMPK phosphorylation (Thr^172^) (<a href=\"#F5\" data-ref-type=\"fig\">Fig. 5<em>D<\/em><\/a>).\n\n## Ectopic expression of IL-18 in a single tibialis anterior muscle is sufficient to protect against excess adipose tissue storage in mice fed an HFD.\n\nTo determine whether the results obtained in muscle cell and ex vivo muscle strips also were prevalent in vivo, we used the in vivo electroporation technique to overexpress *IL-18* cDNA in the tibialis anterior muscles of C57BL\/6J mice that were placed on an HFD diet for 4 weeks. Using this technique, we previously have observed a transfection efficiency of \u223c60%, as measured by the electroporation of a GFP construct as a control (21). IL-18 protein expression in the tibialis anterior was increased 30- to 40-fold above basal when compared with mice when the tibialis anterior was electroporated with an empty vector (sham) (<a href=\"#F6\" data-ref-type=\"fig\">Fig. 6<em>A<\/em><\/a>). No difference in body weight was observed when comparing IL-18 with sham electroporated mice (<a href=\"#F6\" data-ref-type=\"fig\">Fig. 6<em>B<\/em><\/a>). In accordance with this observation, adiposity was reduced when comparing IL-18 with sham electroporated mice at 4 weeks (<a href=\"#F6\" data-ref-type=\"fig\">Fig. 6<em>C<\/em> and <em>D<\/em><\/a> and [Supplementary Fig. 4](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1095\/-\/DC1)). However, this reduction in adiposity was insufficient to result in increased whole-body insulin sensitivity or glucose tolerance as measured by ITT and glucose tolerance test ([Supplementary Fig. 5](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1095\/-\/DC1)).\n\n## Ectopic expression of IL-18 in a single tibialis anterior muscle increases AMPK signaling and markers of lipid oxidation in this organ.\n\nWe next measured AMPK signaling and markers of lipid oxidation in IL-18 and sham electroporated muscles. Both AMPK activity (<a href=\"#F7\" data-ref-type=\"fig\">Fig. 7<em>A<\/em><\/a>) and ACC\u03b2 phosphorylation (Ser^218^) (<a href=\"#F7\" data-ref-type=\"fig\">Fig. 7<em>B<\/em><\/a>) were markedly elevated in IL-18 compared with sham electroporated muscles. In addition, we also observed increased mRNA abundance of \u03b2-hydroxyacyl-CoA-dehydrogenase, a key enzyme involved in mitochondrial function (<a href=\"#F7\" data-ref-type=\"fig\">Fig. 7<em>C<\/em><\/a>) and *CPT1* (<a href=\"#F7\" data-ref-type=\"fig\">Fig. 7<em>D<\/em><\/a>), the enzyme that controls the transfer of long-chain fatty acyl CoA into mitochondria and enhances rates of fatty acid oxidation in the IL-18 electroporated mice relative to sham mice. It is now well-known that skeletal muscle can act as an endocrine organ, producing \"myokines\" to result in tissue cross-talk (22). To examine whether IL-18 could act as a myokine when overexpressed in skeletal muscle, we examined circulating levels of IL-18 and markers of insulin sensitivity, fat oxidation, and inflammation in other tissues such as the liver and adipose. Despite the increase in intramuscular IL-18 expression with electroporation, plasma IL-18 was not elevated in the IL-18 electroporated mice relative to sham ([Supplementary Fig. 5](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1095\/-\/DC1)). Therefore, it was not surprising that pAkt, pAMPK, pACC, and pJNK were not altered in the liver or adipose tissue of IL-18 electroporated mice relative to sham mice ([Supplementary Figs. 5 and 6](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1095\/-\/DC1)). Together, these data provide evidence that IL-18 can activate AMPK in skeletal muscle in vivo.\n\n# DISCUSSION\n\nIL-18 signaling has been implicated in the etiology of metabolic homeostasis and insulin resistance; however, studies in IL-18\u2013deficient mice have suggested that IL-18 is required to prevent hyperphagia (8,9). In the current study, rather than use a model of genetic deletion of IL-18, we initially studied mice that harbor a global deletion of the functional IL-18R. We show that IL-18R^\u2212\/\u2212^ mice are prone to weight gain on a chow diet and develop insulin resistance, a phenotype that is associated with ectopic skeletal muscle lipid expression, inflammation, and reduced AMPK signaling.\n\nThe IL-18R^\u2212\/\u2212^ mice displayed significantly increased body weight and increased fat pad mass after \u223c26 weeks of age. Although others have shown that IL-18\u2013deficient mice display hyperphagia (8,9), we did not observe this in the IL-18R^\u2212\/\u2212^ mice. We were careful to monitor food intake in their normal habitat and during metabolic caging, so we are confident that the food intake between the IL-18R^\u2212\/\u2212^ and CON mice were comparable. Given the identical oxygen consumption between the IL-18R^\u2212\/\u2212^ and CON mice, why would the IL-18R^\u2212\/\u2212^ mice gain more weight if both energy input and expenditure were comparable? It should be noted that the difference in body weight when comparing the IL-18R^\u2212\/\u2212^ and CON mice was \u223c10% by the end of the study (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>A<\/em><\/a>), whereas the overall difference in percent body fat was \u223c4% (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>B<\/em><\/a>). This would equate to an approximate increase of 0.15 g fat or 0.70 kJ energy per day. The daily energy expenditure of a mouse has been estimated to be 42 kJ per day (23). Consequently, \\<2% difference in daily energy expenditure would be sufficient to result in the increased body weight but was unlikely to be detected with available techniques, such as metabolic caging. Similar problems have been encountered in other mouse models of obesity (23,24), and work from our group also has recently observed such an anomaly (25). Notwithstanding this apparent anomaly, it appears, based on the data we have reported, that the IL-18R^\u2212\/\u2212^ mice gain weight and store excess lipid in skeletal muscle, which results in whole-body and skeletal muscle insulin resistance.\n\nIt has been suggested that IL-18R also might be activated by ligands other than IL-18 (26,27), and this, potentially, could explain why hyperphagia previously was observed in IL-18\u2013deficient mice (8,9) but not in IL-18R^\u2212\/\u2212^ mice in the current study. Interestingly, when mice fed an HFD were fasted and refed, IL-18R^\u2212\/\u2212^ mice displayed a significantly higher RER in the refed condition (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>H<\/em><\/a>). This suggests that mice lacking the IL-18R cannot oxidize lipids and rely on utilization of carbohydrates as a preferred energy substrate during refeeding. This is supported by our gain-of-function data (IL-18 increased fat oxidation). Interestingly, Zorrilla et al. (9) observed that mice with IL-18 deficiency were not hyperphagic when fed an HFD and suggested that they differentially process carbohydrate-rich compared with lipid-rich diets or differentially use these macronutrients as fuel. Our study supports this hypothesis.\n\nBy performing whole-body insulin tolerance tests, we cannot ascertain whether the whole-body insulin resistance observed in the IL-18R^\u2212\/\u2212^ mice relative to control mice was attributable to insulin resistance in skeletal muscle, liver, or a combination of the two. However, several lines of evidence suggest that the defect in the IL-18R^\u2212\/\u2212^ mice was primarily in the skeletal muscle, and this is the rationale for choosing to study this organ in depth. First, it is well-acknowledged that ectopic lipid expression is a primary mechanism leading to insulin resistance (28). Whereas intramuscular triglycerides were elevated in the IL-18R^\u2212\/\u2212^ mice relative to CON mice, no such increase was observed in the liver (<a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<\/a>). Second, the mRNA expression of key gluconeogenic enzymes PEPCK and glucose-6 phosphate dehydrogenase were not different in the liver of the IL-18R^\u2212\/\u2212^ mice. Third, even though the IL-18R^\u2212\/\u2212^ mice were insulin-resistant on an HFD relative to CON mice, there was no evidence of decreased pAkt (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<em>F<\/em><\/a>) or of mRNA expression of *SREBP1* or *FAS* (<a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<em>E<\/em> and <em>F<\/em><\/a>) in the liver. Together, these data suggest that skeletal muscle was the origin of the primary defect, although we acknowledge that the effects of IL-18R^\u2212\/\u2212^ on the liver cannot be completely discounted because we did not directly measure insulin sensitivity in this organ.\n\nOf note was our observation that pJNK was markedly elevated in the livers of the IL-18R^\u2212\/\u2212^ mice relative to CON mice despite equivalent lipid content when animals were fed a chow diet (<a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<\/a>). Although this may seem counterintuitive, this observation is not novel. We previously have observed such a phenomenon in IL-6\u2013deficient mice (25). A potential mechanism recently has been proposed by Flavell et al., who demonstrated that NLRP6 and NLRP3 inflammasomes and, importantly, IL-18 negatively regulates nonalcoholic fatty liver disease\/nonalcoholic steatohepatitis progression as well as multiple aspects of metabolic syndrome via modulation of the gut microbiota not necessarily related to hepatosteatosis (29). Although speculative, the increased inflammation observed in the IL-18R^\u2212\/\u2212^ mice in the presence of relatively normal lipid levels may be related to such a mechanism.\n\nBased on our loss-of-function and gain-of-function models, IL-18 signaling is implicated in fatty acid oxidation rates in skeletal muscle as a result of activation of AMPK. As discussed, AMPK phosphorylates ACC\u03b2, resulting in inhibition of ACC activity, which in turn leads to a decrease in malonyl CoA content, relieving inhibition of CPT1 and increasing fatty acid oxidation. The phosphorylation of ACC\u03b2 was reduced in the skeletal muscle, liver, and adipose tissue of IL-18R^\u2212\/\u2212^ (<a href=\"#F4\" data-ref-type=\"fig\">Fig. 4<\/a>). Moreover, when cultured skeletal muscle cells or isolated skeletal muscle strips were treated with IL-18, phosphorylation of AMPK or ACC (or both) was increased and, in the case of intact ex vivo\u2013treated skeletal muscle, this increase was associated with enhanced fatty acid oxidation. Finally, when IL-18 was overexpressed in skeletal muscle in vivo, AMPK activity and ACC phosphorylation were increased, not only in the electoporated muscle (<a href=\"#F7\" data-ref-type=\"fig\">Fig. 7<em>A<\/em> and <em>B<\/em><\/a>). Taken together, the data provide evidence implicating IL-18 in the activation of AMPK.\n\nIt is now well-known that many cytokines, including leptin, adiponectin, ghrelin, IL-6, and ciliary neurotrophic factor can activate AMPK (30,31), but this is the first report indicating that IL-18 can act as an AMPK agonist. This observation, however, is consistent with these previous studies because IL-18 can act as an activator of STAT3 (4). Work from our group previously has shown that members of the IL-6 family of cytokines, which potently activate STAT3, also enhance fat oxidation via AMPK (11,12). Importantly, when mice that harbor a truncation of the COOH-terminal domain that eliminates these tyrosine residues on the gp130 receptor (gp130^\u2206STAT^ mice) are treated with CNTF, the phosphorylation of STAT3 is abolished, as is the activation of AMPK (12).\n\nAlthough feeding mice an HFD did not result in differences in body weight, fat mass, or insulin signaling in skeletal muscle and liver when comparing the genotypes, the IL-18R^\u2212\/\u2212^ mice nevertheless displayed elevated fasting insulin levels and impaired insulin tolerance as measured by an ITT (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<\/a>). Although speculative, this may be attributable to the fact that under HFD conditions, the activation of the AMPK pathway remained impaired in the IL-18R^\u2212\/\u2212^ mice, at least in skeletal muscle and liver (<a href=\"#F4\" data-ref-type=\"fig\">Fig. 4<\/a>), leading to elevated lipid levels in skeletal muscle of the IL-18R^\u2212\/\u2212^ mice under HFD conditions (<a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<\/a>).\n\nIn summary, we have identified that IL-18 can activate AMPK. Moreover, mice that harbor a genetic deletion of a functional IL-18R are prone to weight gain and development of insulin resistance and inflammation in important metabolic tissues such as skeletal muscle and liver. Therefore, our data add IL-18 to a growing list of catabolic proinflammatory cytokines that paradoxically are required to maintain pathways important for fatty acid oxidation and thus prevent insulin resistance.\n\n## ACKNOWLEDGMENTS\n\nThis study was supported, in part, by a grant from the National Health and Medical Research Council of Australia (NHMRC grant no. 526606). This study was further supported by the Danish Council for Independent Research\u2013Medical Sciences, the Commission of the European Communities (grant agreement no. 223576-MYOAGE), and by grants from the Novo Nordisk Foundation, H\u00f8rslevfonden, the Danish National Research Foundation (#10-083807), H\u00f8jmoseg\u00e5rdlegatet, Fonden for L\u00e6gevidenskabens Fremme, and Direkt\u00f8r Jacob Madsen og Hustru Olga Madsens Fond. The Centre of Inflammation and Metabolism (CIM) and The Rodent Metabolic Phenotyping Center is part of the UNIK Project: Food, Fitness & Pharma for Health and Disease (see [www.foodfitnesspharma.ku.dk](http:\/\/www.foodfitnesspharma.ku.dk)) supported by the Danish Ministry of Science, Technology, and Innovation. The CIM is a member of DD2-the Danish Center for Strategic Research in Type 2 Diabetes (the Danish Council for Strategic Research, grant no. 09-067009 and 09-075724). The Copenhagen Muscle Research Centre is supported by a grant from the Capital Region of Denmark. M.A.F. is a Senior Principal Research Fellow, M.J.W. is a Senior Research Fellow, and C.R.B. and V.B.M. are Career Development Fellows of the NHMRC. The CIM is supported by a grant from the Danish National Research Foundation (#02-512-55). B.L. was supported by a grant from the Danish National Research Foundation (#09-063656). B.L. received postdoctoral fellowship support from a grant from the Danish National Research Foundation.\n\nNo other potential conflicts of interest relevant to this article were reported.\n\nB.L. designed research, performed and\/or analyzed research, and wrote the manuscript. V.B.M., C.B., P.H., T.L.A., E.E., M.J.W., C.R.B., O.H.M., S.S., C.R., J.A., H.P., S.D., T.J.A., and A.N.M. performed and\/or analyzed research. J.H. contributed new reagents and analytical tools. B.K.P. designed research. M.A.F. designed research and wrote the manuscript. All authors contributed to the writing of the final submitted version of the manuscript. B.L. is the guarantor of this work and, as such, had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.\n\nThe authors acknowledge the assistance of Betina Mentz, Ruth Rousing, Hanne Villumsen, Lone Nielsen, Andreas Nygaard Madsen (University of Copenhagen), Steve Risis (Baker IDI Heart and Diabetes Institute), and the Rodent Metabolic Phenotyping Center (University of Copenhagen).\n\n# REFERENCES","meta":{"dup_signals":{"dup_doc_count":131,"dup_dump_count":50,"dup_details":{"curated_sources":3,"2022-40":1,"2021-49":1,"2021-43":1,"2019-43":1,"2019-09":1,"2018-51":1,"2018-43":1,"2018-34":1,"2018-26":3,"2018-17":3,"2018-09":1,"2018-05":3,"2017-47":3,"2017-43":5,"2017-39":3,"2017-34":1,"2017-30":9,"2017-26":2,"2017-22":2,"2017-17":7,"2017-09":4,"2017-04":10,"2016-50":3,"2016-44":4,"2016-40":3,"2016-36":3,"2016-30":3,"2016-26":3,"2016-22":3,"2016-18":2,"2016-07":2,"2015-48":3,"2015-40":2,"2015-35":3,"2015-32":1,"2015-27":1,"2015-22":2,"2015-14":2,"2014-52":3,"2014-49":1,"2014-42":3,"2014-41":3,"2014-35":1,"2014-23":2,"2023-50":1,"2017-13":4,"2015-18":2,"2015-11":1,"2015-06":2,"2024-18":1}},"file":"PMC3749341"},"subset":"pubmed_central"}
{"text":"author: Elaine R Mardis\ndate: 2010\ninstitute: 1The Genome Center at Washington University School of Medicine, 4444 Forest Park Blvd, St Louis, MO 63108, USA\nreferences:\ntitle: The \\$1,000 genome, the \\$100,000 analysis?\n\nHaving recently attended the Personal Genomes meeting at Cold Spring Harbor Laboratories (I was an organizer this year), I was struck by the number of talks that described the use of whole-genome sequencing and analysis to reveal the genetic basis of disease in patients. These patients included a child with irritable bowel disease, a child with severe combined immunodeficiency, two siblings affected with Miller syndrome, and several with cancers of different types. Although each presenter emphasized the rapidity with which these data can now be generated using next-generation sequencing instruments, they also listed the large number of people involved in the analysis of these datasets. The required expertise to 'solve' each case included molecular and computational biologists, geneticists, pathologists and physicians with exquisite knowledge of the disease and of treatment modalities, research nurses, genetic counselors, and IT and systems support specialists, among others. While much of the attendant effort was focused on the absolute importance of obtaining the correct diagnosis, the large number of specialists was critical for the completion of the data analysis, the annotation of variants, the interpretive 'filtering' necessary to deduce the causative or 'actionable' variants, the clinical verification of these variants, and the communication of results and their ramifications to the treating physician, and ultimately to the patient. At the end of the day, although the idea of clinical whole-genome sequencing for diagnosis is exciting and potentially life-changing for these patients, one does wonder how, in the clinical translation required for this practice to become commonplace, such a 'dream team' of specialists would be assembled for each case. In other words, even if the cost and speed of generating sequencing data continue their precipitous decreases, the cost of 'team' analysis seems unlikely to immediately follow suit. However, rather than predicting from this reasoning that widespread diagnosis by sequencing is unlikely to occur widely, it is perhaps more fruitful to predict, in my opinion, what is probably required for it to occur. I therefore offer the following as food for thought.\n\nOne source of difficulty in using resequencing approaches for diagnosis centers on the need to improve the quality and completeness of the human reference genome. In terms of quality, it is clear that the clone-based methods used to map, assign a minimal tiling path, and sequence the human reference genome did not yield a properly assembled or contiguous sequence equally across all loci. Lack of proper assembly is often due to collapsing of sequence within repetitive regions, such as segmental duplications, wherein genes can be found once the correct clones are identified and sequenced. At some loci, the current reference contains a single nucleotide polymorphism (SNP) that occurs at the minor allele frequency rather than being the major allele. In addition, some loci cannot be represented by a single tiling path and require multiple clone tiling paths to capture all of the sequence variations. All of these deficiencies and others not cited provide a less-than-optimal alignment target for next-generation sequencing data and can confound the analytical validity of variants necessary to properly interpret patient-derived data. Hence, although it is difficult work to perform, the ongoing efforts of the Genome Resource Consortium \\[1\\] to improve the overall completeness and correctness of the human reference genome should be enhanced.\n\nAlong these lines, although projects such as the early SNP Consortium \\[2\\], the subsequent HapMap projects \\[3-5\\], and more recently the 1,000 Genomes Project \\[6\\] have identified millions of SNPs in multiple ethnic groups, there is much more diversity to the human genome than single base differences. In some ways, the broader scope of 'beyond SNP' diversity of the genome across human populations remains mysterious, including common copy number polymorphisms, large insertions and deletions, and inversions. Mining the 1,000 Genomes data using methods to identify genome-wide structural variation should augment this considerably \\[7\\], with validation playing an important role, as many methods are still nascent. Lastly, devising clever ways to provide all such classes of variants as a 'searchable space' for sequence data alignment remains a significant challenge, as does the development of sequence alignment algorithms that facilitate the analysis of structurally complex loci.\n\nHow well do we understand the functions encoded by our genome? Certainly, comprehensive functional information about proteins, including the impact of mutations, is complete for relatively few genes. The development of high-throughput systems for biochemistry and enzymology could have a dramatic impact on this deficiency and would add vitality to these areas of scientific endeavor. Efforts that annotate regulatory protein binding sites, sites of RNA-mediated regulatory mechanisms, and other motifs that contribute to transcriptional regulation in the human genome must continue. Improved understanding of these regions, and thus their annotation, will require the power of model-organism-based systems to identify and characterize functional proteins or mechanisms that are shared with humans. We also must transfer these findings into human cell experimental systems that allow researchers to examine the impact of the mutations or other alterations of the genome on cellular pathways and the resulting disease biology. With functional consequences in hand, we will begin to understand and associate the clinical validity of genomic variants, effectively enabling the correlation of variant(s) with the resultant phenotype(s).\n\nIf our efforts to improve the human reference sequence quality, variation, and annotation are successful, how do we avoid the pitfall of having cheap human genome resequencing but complex and expensive manual analysis to make clinical sense out of the data? One approach would emphasize the development of 'clinical grade' interpretational analysis pipelines to perform much of the initial discovery from datasets derived from massively parallel sequencing \\[8\\]. Although such pipelines already exist in the research setting \\[9\\], manual checks and orthogonal validation of variants are required because of the ongoing development of the analytical approaches. Towards patient diagnoses, such validation could initially be performed in a clinical laboratory medicine setting, but ultimately we must develop sophisticated analytical approaches and quality filters that enable high-confidence variant detection solely from the primary data. All discovered variants would then be interpreted in the context of the ever-improving human genome annotation and evaluated in the contexts of medical genetics, of demonstrated clinical validity, and of the pharmaceutical databases (when appropriate), to identify causative or therapeutically actionable genes. Ultimately, as in medicine today, the results will require interpretation by a physician, which raises a separate but equally important issue: the significant need to develop and implement training programs in genomics for medical professionals. Pathologists and genetic counselors will be the first in line for training programs focused on genomic diagnostics, and improving the genomics education of medical students will also be a first priority. More challenging will be the genomics education of practicing physicians and other medical professionals, many of whom do not require genetics to perform their valuable role in health care daily, but who will be confronted in the near term by increasingly well informed patients who expect their doctors to be as well versed as they are about genome-guided diagnosis and treatment.\n\nA final word on the important topic of patient access to genome-guided medicine seems necessary and appropriate. The current high cost of whole-genome sequencing and analysis relative to most clinical diagnostic assays, coupled with the fact that these costs are not currently reimbursed by insurers, might mean that only those with the means to pay for the test will be allowed access. Perhaps worse, those with the fattest wallets might pay extra for a place higher in the queue, denying earlier access to patients who more desperately need the information. Although there are no easy answers here, one plausible solution might be the establishment of funds at major medical centers, where genome-guided medicine is likely to be practiced first, that pay for the genomic sequencing, diagnosis and associated costs and thus allow equitable access to this new assay.\n\n# Competing interests\n\nThe author declares that they have no competing interests.\n\n## Acknowledgements\n\nI thank Deanna Church, Timothy Ley and W Richard McCombie for their critical reading and suggestions.","meta":{"dup_signals":{"dup_doc_count":133,"dup_dump_count":47,"dup_details":{"curated_sources":2,"2019-18":2,"2019-09":1,"2019-04":2,"2018-47":2,"2018-43":1,"2018-39":1,"2018-34":1,"2018-30":1,"2018-26":1,"2018-17":2,"2018-09":2,"2017-39":2,"2017-34":1,"2017-30":1,"2017-22":1,"2017-17":1,"2017-09":5,"2017-04":1,"2016-50":1,"2016-44":2,"2016-40":2,"2016-36":8,"2016-30":6,"2016-07":4,"2015-48":3,"2015-40":1,"2015-35":3,"2015-32":3,"2015-27":3,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":4,"2014-42":9,"2014-41":6,"2014-35":4,"2014-23":6,"2014-15":5,"2019-30":1,"2017-13":1,"2015-18":4,"2015-11":3,"2015-06":3,"2014-10":3,"2013-48":4,"2013-20":4,"2024-22":1}},"file":"PMC3016626"},"subset":"pubmed_central"}
{"text":"date: 2018-05\ntitle: WHO and UNICEF issue new guidance to promote breastfeeding in health facilities globally\n\n**11 APRIL 2018 \\| GENEVA -** WHO and UNICEF today issued new ten-step guidance to increase support for breastfeeding in health facilities that provide maternity and newborn services. Breastfeeding all babies for the first 2 years would save the lives of more than 820 000 children under age 5 annually.\n\nThe Ten Steps to Successful Breastfeeding underpin the Baby-friendly Hospital Initiative, which both organizations launched in 1991. The practical guidance encourages new mothers to breastfeed and informs health workers how best to support breastfeeding.\n\nBreastfeeding is vital to a child's lifelong health, and reduces costs for health facilities, families, and governments. Breastfeeding within the first hour of birth protects newborn babies from infections and saves lives. Infants are at greater risk of death due to diarrhoea and other infections when they are only partially breastfed or not breastfed at all. Breastfeeding also improves IQ, school readiness and attendance, and is associated with higher income in adult life. It also reduces the risk of breast cancer in the mother.\n\n\"Breastfeeding saves lives. Its benefits help keep babies healthy in their first days and last will into adulthood,\" says UNICEF Executive Director Henrietta H. Fore. \"But breastfeeding requires support, encouragement and guidance. With these basic steps, implemented properly, we can significantly improve breastfeeding rates around the world and give children the best possible start in life.\"\n\nWHO Director-General Dr Tedros Adhanom Ghebreyesus says that in many hospitals and communities around the world, whether a child can be breastfed or not can make the difference between life and death, and whether a child will develop to reach his or her full potential.\n\n\"Hospitals are not there just to cure the ill. They are there to promote life and ensure people can thrive and live their lives to their full potential,\" says Dr Tedros. \"As part of every country's drive to achieve universal health coverage, there is no better or more crucial place to start than by ensuring the Ten Steps to Successful Breastfeeding are the standard for care of mothers and their babies.\"\n\nThe new guidance describes practical steps countries should take to protect, promote and support breastfeeding in facilities providing maternity and newborn services. They provide the immediate health system platform to help mothers initiate breastfeeding within the first hour and breastfeed exclusively for six months.\n\nIt describes how hospitals should have a written breastfeeding policy in place, staff competencies, and antenatal and post-birth care, including breastfeeding support for mothers. It also recommends limited use of breastmilk substitutes, rooming-in, responsive feeding, educating parents on the use of bottles and pacifiers, and support when mothers and babies are discharged from hospital.\n\n**Note to editors**\n\nThe Ten Steps are based on the WHO guidelines, issued in November 2017, titled Protecting, promoting and supporting breastfeeding in facilities providing maternity and newborn services.\n\nEarly initiation of breastfeeding, within one hour of birth, protects the newborn from acquiring infections and reduces newborn mortality. Starting breastfeeding early increases the chances of a successful continuation of breastfeeding. Exclusive breastfeeding for six months has many benefits for the infant and mother. Chief among these is protection against gastrointestinal infections and malnutrition, which are observed not only in developing but also industrialized countries.\n\nBreast-milk is also an important source of energy and nutrients in children aged 6\u201323 months. It can provide half or more of a child's energy needs between 6-12 months, and one-third of energy needs between 12-24 months. Breast-milk is also a critical source of energy and nutrients during illness, and reduces mortality among children who are malnourished.\n\nChildren and adolescents who were breastfed as babies are less likely to be overweight or obese.\n\nAvailable from: <http:\/\/www.who.int\/mediacentre\/news\/releases\/2018\/promote-breastfeeding-globally\/en\/>","meta":{"dup_signals":{"dup_doc_count":174,"dup_dump_count":48,"dup_details":{"curated_sources":2,"2023-40":4,"2023-23":1,"2023-14":3,"2023-06":1,"2022-49":2,"2022-40":3,"2022-33":1,"2022-27":2,"2022-21":1,"2022-05":1,"2021-43":2,"2021-39":4,"2021-25":2,"2021-21":2,"2021-17":2,"2021-10":2,"2021-04":2,"2020-50":3,"2020-45":1,"2020-40":5,"2020-34":2,"2020-29":3,"2020-16":1,"2020-10":3,"2020-05":2,"2019-51":1,"2019-43":1,"2019-39":5,"2019-35":3,"2019-30":2,"2019-26":5,"2019-22":5,"2019-18":12,"2019-13":7,"2019-09":8,"2019-04":10,"2018-51":9,"2018-47":14,"2018-43":5,"2018-39":3,"2018-34":4,"2018-30":1,"2018-26":5,"2018-17":10,"2024-30":2,"2024-26":2,"2024-18":1,"2024-10":2}},"file":"PMC6118173"},"subset":"pubmed_central"}
{"text":"abstract: The high levels of intelligence seen in humans, other primates, certain cetaceans and birds remain a major puzzle for evolutionary biologists, anthropologists and psychologists. It has long been held that social interactions provide the selection pressures necessary for the evolution of advanced cognitive abilities (the 'social intelligence hypothesis'), and in recent years decision-making in the context of cooperative social interactions has been conjectured to be of particular importance. Here we use an artificial neural network model to show that selection for efficient decision-making in cooperative dilemmas can give rise to selection pressures for greater cognitive abilities, and that intelligent strategies can themselves select for greater intelligence, leading to a Machiavellian arms race. Our results provide mechanistic support for the social intelligence hypothesis, highlight the potential importance of cooperative behaviour in the evolution of intelligence and may help us to explain the distribution of cooperation with intelligence across taxa.\nauthor: Luke McNally; Sam P. Brown; Andrew L. Jackson\\*Author for correspondence (<email@example.com>).\ndate: 2012-08-07\ninstitute: 1Department of Zoology, School of Natural Sciences, Trinity College Dublin, Dublin 2, Republic of Ireland; 2Centre for Biodiversity Research, Trinity College Dublin, Dublin 2, Republic of Ireland; 3Centre for Immunity, Infection and Evolution, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3JT, UK\nreferences:\ntitle: Cooperation and the evolution of intelligence\n\n# Introduction\n\nNatural selection never favours excess; if a lower-cost solution is present, it is selected for. Intelligence is a hugely costly trait. The human brain is responsible for 25 per cent of total glucose use, 20 per cent of oxygen use and 15 per cent of our total cardiac output, although making up only 2 per cent of our total body weight \\[1\\]. Explaining the evolution of such a costly trait has been a long-standing goal in evolutionary biology, leading to a rich array of explanatory hypotheses, ranging from evasion of predators to intelligence acting as an adaptation for the evolution of culture \\[2\u20134\\]. Among the proposed explanations, arguably the most influential has been the 'social intelligence hypothesis', which posits that it is the varied demands of social interactions that have led to advanced intelligence \\[4\u201312\\].\n\nIn recent years, the cognitive demands of reciprocity, one of the mechanisms posited as important in the maintenance of cooperation in humans and other intelligent taxa, have been suggested to be a causal factor in the evolution of advanced intelligence and human language. This has been particularly apparent in the evolutionary game theory literature, where conjecture regarding this relationship is frequent \\[13\u201316\\]. Indeed, there is a rich history of work relating intelligence and reciprocity in the game theory literature, though most of this work has focused on the cognitive abilities required for the evolution of cooperation, rather than the possible role that the negotiation of these interactions has in the evolution of intelligence \\[17\u201324\\]. As well as the cognitive abilities required for the coordination of partners during cooperative acts, both direct (decisions based on what you do to me) and indirect (decisions based on what you do to others) reciprocity have additional demands in terms of the ability to remember previous interactions and to integrate across these interactions to make decisions in cooperative dilemmas \\[25\u201331\\]. These cognitive demands, combined with the occurrence of cooperative behaviour between unrelated individuals in intelligent taxa, suggest that selection for these mechanisms of cooperation could, at least in part, be responsible for advanced cognitive abilities \\[26\\].\n\nThe many subfields within the social intelligence hypothesis have shown a rich elaboration of verbal arguments, and data from comparative studies support many of their predictions \\[32\u201334\\]. However, verbal reasoning and comparative analysis alone are not sufficient to assess the relative merit of competing hypotheses \\[35\\]; mechanistic models are needed to assess the plausibility of these different explanations for advanced cognition.\n\nHere, we use an artificial neural network model to focus on the potential for direct reciprocity, a behaviour that is widespread in humans, to select for advanced cognitive abilities. Rather than manufacturing some form of functional relationship between intelligence and fitness, we allow this relationship to emerge based on the demands of decision-making in two social dilemmas, and analyse the consequences for the evolution of intelligence.\n\n# Material and methods\n\n## The social dilemmas\n\nIn order to consider the dynamics of cooperative social interactions, we use the framework of two classic social dilemmas: the iterated prisoner's dilemma (IPD) and the iterated snowdrift game (ISD). In both games, two players must choose between cooperation and defection during repeated rounds. In the event of mutual cooperation or mutual defection, both players receive payoffs *R* or *P*, respectively, while a defector exploiting a cooperator gets *T* and the cooperator gets *S*. In the prisoner's dilemma, the benefit of an individual's cooperative behaviour goes to their opponent, while they pay all of the costs (e.g. food sharing, reciprocal coalitionary behaviour). This results in a payoff order of *T* \\> *R* \\> *P* \\> *S*. Here, the worst possible outcome for an individual is to cooperate while their opponent defects, while the best outcome is to defect while the opponent cooperates. In the snowdrift game, the benefits of cooperative behaviours are shared between opponents, and the costs are shared if both individuals cooperate (e.g. cooperative hunting, coalitionary behaviour with shared benefits). This results in a payoff order of *T* \\> *R* \\> *S* \\> *P*. Again, the best outcome for an individual is to defect while their opponent cooperates, though the worst possible outcome for an individual is for neither them nor their opponent to cooperate. In both games, the overall payoff (sum of both individual's payoffs) is greatest for mutual cooperation and lowest for mutual defection.\n\nAll of this means that the equilibrium frequency of cooperation for a single interaction (single-interaction Nash equilibrium) will be zero in the prisoner's dilemma but will be non-zero for a single-interaction snowdrift game \\[36\\]. These single-interaction Nash equilibria provide a useful benchmark against which to assess the effects of contingent behaviours (i.e. those that depend on the behaviour of others) in repeated interactions.\n\n## The neural network model\n\nAny attempt to define a metric of intelligence will always be a contentious matter. However, comparative studies across taxa have usually focused on two main classes of brain properties as proxies of intelligence: metrics based on relative or absolute size of the brain or certain brain regions, and metrics based on more specific properties such as numbers of cortical neurons \\[37\\]. It is with this tradition in mind that we develop our artificial neural network model, with evolving network structure, using the number of neurons, *i*, as our proxy for intelligence. Each individual can display varying levels of intelligence, from simply being characterized by a binary response of always cooperate or always defect to large neural networks that possess complex neuronal structure, allowing for computations to inform decisions based on payoffs and the integration of longer-term memory into their current decision-making processes.\n\nEach individual in our simulated populations possesses a neural network that determines their behaviour in social dilemmas (illustrated in <a href=\"#RSPB20120206F1\" data-ref-type=\"fig\">figure 1<\/a>). The networks each have two input nodes (which receive the payoffs of the individual and their opponent in the previous round as inputs) and one output node (giving the probability that they cooperate during their next interaction). The hidden layer of each individual's network has an evolving structure, possessing different numbers of cognitive and context nodes \\[38\\] (<a href=\"#RSPB20120206F1\" data-ref-type=\"fig\">figure 1<\/a>). Cognitive nodes allow for computation based on the values of network inputs and context nodes, which in turn allow for the build-up of memory based on previous states of their associated cognitive nodes.\n\nComputation in the network is implemented via synchronous updating of nodes. The value of each input node is passed to each of the network's cognitive nodes, multiplied by the weight linking the two nodes. Each cognitive node is also passed the current value of their associated context node (if they possess one) multiplied by the weight linking the two nodes. The cognitive nodes sum across all of the weighted values that they receive and pass this value through a sigmoidal squashing function, resulting in a value between 0 and 1, analogous to a probability of activation. All context nodes are then passed the value of their associated cognitive nodes. This allows the context nodes to build up memory of previous interactions without having to store the actual sequence of events that have occurred. The internal states of these context nodes could be considered analogous to emotional states. Finally, the values at all cognitive nodes are then passed to the output node (multiplied by their weights), summed and again passed through a sigmoidal squashing function. This output gives the probability that the individual will cooperate in the current round. As the sigmoidal function asymptotes to 0 at \u2013\u221e and 1 at \u221e, there will always be inherent noise in the network's probabilistic decision. This property of the function also means that it is easy to minimize noise in the network's behaviour if that behaviour shows a lack of contingency (as the node can always be near one of the asymptotes), while contingent behaviour will show greater noise (as switching is more difficult to achieve near the asymptotes). This formulation has intuitive appeal over simply adding extraneous noise to individual decisions, as in nature we would expect individuals to make few mistakes when their behaviour is non-contingent, while more complex decisions would be expected to be more error-prone. As the network cannot make decisions without an input, each individual has an additional trait encoding whether they cooperate or defect in the first round.\n\nWe allowed networks to evolve according to natural selection using a genetic algorithm where fitness is the mean payoff per round from the iterated games minus a penalty for the individual's intelligence, *i*. When individuals reproduce, mutations allow for the gain and loss of nodes from the hidden layer of their network with a fixed probability. Context nodes could only be gained if there was already a cognitive node present without an associated context node. The loss of a cognitive node with an associated node resulted also in the loss of the associated context node.\n\nThe addition of extra cognitive nodes gives networks the potential to perform complex computation based on payoffs by increasing the dimensions of internal representation of the network. The addition of context nodes gives the potential for the integration of longer-term memory of previous interactions in these computations. If an individual possessed no hidden layer nodes in its network, its behaviour in all rounds was decided by its first round move (i.e. they either always cooperated or always defected). The weights of each node in the network (arrowed lines in <a href=\"#RSPB20120206F1\" data-ref-type=\"fig\">figure 1<\/a>) and the threshold of each node (see the electronic supplementary material) were encoded as continuous genetic traits, again subject to mutation during reproduction. This means that, while the number of nodes in the network constrains the possible behavioural repertoire, it is the way that the constituent parts of the network interact that actually decides the individual's behaviour. In this way, our metric of intelligence assesses the potential for complex behaviour that the individual possesses, rather than the appropriateness or 'wisdom' of their behaviour, similarly to the measures of intelligence used in comparative studies.\n\n## Model implementation\n\nIn order to elucidate when selection favoured intelligence, we ran 10 replicates of our model for both the IPD and ISD, with each replicate lasting 50 000 generations. The payoff values used for all simulations were *R* = 6, *P* = 2, *T* = 7 and *S* = 1 for the IPD, and *R* = 5, *P* = 1, *T* = 8 and *S* = 2 for the ISD. The genetic algorithm was implemented as follows (see the electronic supplementary material for further details):\n\n1.  \u2014\u2002an initial population of random networks was generated;\n\n2.  \u2014\u2002each individual played every other individual in the population (50 individuals) in an IPD or ISD;\n\n3.  \u2014\u2002each individual network's fitness was calculated as their mean payoff per round minus a fitness penalty for their level of intelligence, *i*;\n\n4.  \u2014\u2002individuals were selected to reproduce asexually with probability proportional to their fitness;\n\n5.  \u2014\u2002newly produced offspring underwent mutation of their network weights, node thresholds and network structure with constant probabilities;\n\n6.  \u2014\u2002the previous generation died; and\n\n7.  \u2014\u2002the algorithm returned to step 2 until 50 000 generations was reached.\n\nDuring simulations we recorded the frequency of cooperation in the population, the intelligence of individuals (*i*) and assessments of the behaviour of individuals against a pre-determined test set of moves (see the electronic supplementary material). We then analysed the gradients of selection for intelligence across these simulations by taking selection for intelligence as the covariance between fitness and intelligence in any given generation \\[39\\]. As 50 million individual neural networks were simulated in our study, and individuals were not constrained to base their behaviour only on the previous move, our simulations generated a great diversity of strategies. In order to gain a coarse-grained overview of the strategic composition of the population, we clustered individuals based on their proximity to four canonical strategy types: always-defect-like, always-cooperate-like, tit-for-tat-like (do what your opponent did to you) and Pavlov-like (if your payoff is over a threshold, repeat your previous move). Assignment to each of these strategy types was based on which of these four strategies each individual network clustered closest to based on its behaviour against the test set. While this clustering is only a coarse-grained view, it allows assessment of the effects of shifts towards contingent cooperative strategies on selection for intelligence. Additionally, contingent human cooperation has previously clustered as either tit-for-tat-like or Pavlov-like \\[40\\], though longer-term memory is often included \\[41\\]. For full details of our data analysis, we direct readers to the electronic supplementary material.\n\n# Results\n\nOur model shows the spontaneous evolutionary emergence of behaviours similar to strategies known to perform well in the IPD and ISD, such as tit-for-tat and Pavlov, as well as simple always-cooperate or always-defect strategies (<a href=\"#RSPB20120206F2\" data-ref-type=\"fig\">figure 2<\/a>) \\[42\\]. Although our networks' behaviours are similar to these strategies, they often show integration over many previous rounds to decide on their next moves. For example, manual interrogation of networks revealed that, of the tit-for-tat type strategies that emerge, many are tit-for-2(or more)-tats, and many of the Pavlov-like strategies also show a threshold mechanism, switching to constant defection against opponents that show behaviour close to an always-defect strategy. Behaviour was observed that appeared to be close to many other strategies\u2014for example, grim variants (cooperate until the opponent defects, then defect forever), though often requiring more than one defection to trigger permanent defection; false cooperator (cooperate first then switch to defection), though often giving another cooperative move after many defections; and many other variants of tit-for-tat such as 2-tits-for-1-tat and 2-tits-for-2-tats. It is worth noting that strategies of these types that use longer-term memory are observed in behavioural experiments of repeated games with noise \\[41\\]. These responsive strategies require greater cognitive abilities in order to carry out computations based on payoffs, memorize past rounds and integrate across them to make decisions, in comparison with the lower requirements of simply always cooperating or defecting. We hasten to add, however, that the strategies emerging only resemble these strategies; the strategies vary in a continuous manner and often incorporate memory over more rounds. Our goal here is not to describe the strategies that can emerge in repeated games, as there is already extensive literature on this topic (see table 2.1 in \\[43\\]), but rather to elucidate the potential effects of their evolution on selection for cognitive capacities.\n\nIn order to elucidate the causal factors leading to the evolution of more complex strategies, we analyse the gradient of selection for intelligence in response to population features such as the prevalence of cooperative acts. We find that the selection for intelligence is maximized as the level of cooperation in the population moves above the single-interaction Nash equilibria towards more cooperative regimes (<a href=\"#RSPB20120206F3\" data-ref-type=\"fig\">figure 3<\/a>). In the IPD, this maximum occurs during increases in cooperation from the single-interaction Nash equilibrium, whereas in the ISD selection for intelligence is maximized at levels of cooperation just above the single-interaction Nash equilibrium. This discrepancy between the games is explained by the different natures of their single-interaction Nash equilibria. In the IPD, this equilibrium is zero, meaning that declining cooperation near this equilibrium is caused by increases in the frequency of individuals that always defect, requiring only little cognitive ability. In the ISD, the equilibrium is non-zero (0.23 in our simulations), meaning that decreases in cooperation back towards the equilibrium can be caused by 'meaner' contingent strategies (e.g. 2-tits-for-1-tat and false-cooperator variants), as well as individuals that always defect. As a result, transitions back to the single-interaction Nash equilibrium in the ISD can in principle select for intelligence, while this is very unlikely in the IPD.\n\nWe also find that increasing intelligence decreases the mean frequency of cooperative acts in the IPD (Spearman's rank correlation test; *\u03c1* = \u22120.2333, *p* \\< 0.001; <a href=\"#RSPB20120206F4\" data-ref-type=\"fig\">figure 4<\/a>*a*), while slightly increasing cooperation in the ISD (Spearmans' rank correlation test; *\u03c1* = 0.0089, *p* \\< 0.0001, <a href=\"#RSPB20120206F4\" data-ref-type=\"fig\">figure 4<\/a>*b*). Increasing intelligence increases the variance in the frequency of cooperative acts in the population in both the IPD (Breusch\u2013Pagan test; intercept = 0.0294, slope = 0.0582, *p* \\< 0.0001; <a href=\"#RSPB20120206F4\" data-ref-type=\"fig\">figure 4<\/a>*a*) and the ISD (Breusch\u2013Pagan test; intercept = 0.0309, slope = 0.0384, *p* \\< 0.001; <a href=\"#RSPB20120206F4\" data-ref-type=\"fig\">figure 4<\/a>*b*), showing that intelligence can facilitate greater extremes of cooperation. These results can be explained by assortment of individuals' cooperative acts \\[44\\]; the contingent strategies facilitated by increased intelligence allow an individual to increase the probability that they assort their cooperative acts with other cooperative acts. This leads to a synergistic process, where this increase in cooperation due to increased intelligence creates further opportunities for intelligent individuals to engage in mutual cooperation. However, as levels of cooperation increase further this feedback can break down, as there may be enough cooperation occurring for unconditional cooperators to increase in the population, allowing in turn for the invasion of unconditional defectors or 'meaner' intelligent strategies (e.g. grim variants, false-cooperator variants, etc.). This results in intelligence-facilitating cycles in the levels of cooperation seen in the population (<a href=\"#RSPB20120206F2\" data-ref-type=\"fig\">figure 2<\/a>), which increases both the variance in, and the maximum level of, cooperation.\n\nIn addition to dependency on the prevalence of, and change in, cooperative actions (<a href=\"#RSPB20120206F3\" data-ref-type=\"fig\">figure 3<\/a>), we find that intelligence can be subject to a Machiavellian runaway process \\[11,12\\]. In the ISD, as the frequency of contingent (intelligent) strategies increases, so too does selection for intelligence in the population (tit-for-tat-like strategies: Spearman's *\u03c1* = 0.2025, *p* \\< 0.0001; Pavlov-like strategies: *\u03c1* = 0.2352, *p* \\< 0.0001; see <a href=\"#RSPB20120206F5\" data-ref-type=\"fig\">figure 5<\/a>; electronic supplementary material, table S1 and figure S1). In the IPD, increasing frequencies of tit-for-tat and Pavlov reduces selection for intelligence at low levels of cooperation (frequency of cooperation \\<0.5; tit-for-tat: *\u03c1* = \u22120.0945, *p* \\< 0.0001; Pavlov: *\u03c1* = \u22120.0529, *p* \\< 0.0001), but does increase selection for intelligence when cooperation is more frequent (frequency of cooperation \u2265 0.5; tit-for-tat: *\u03c1* = 0.5491, *p* \\< 0.0001; Pavlov: *\u03c1* = 0.3187, *p* \\< 0.0001). The reason for this distinction between the IPD and the ISD is that there must already be some cooperation occurring for cooperative contingent strategies to be favoured via their ability to assort cooperative acts. In the ISD, the partially cooperative single-interaction equilibrium provides sufficient baseline cooperation for tit-for-tat and Pavlov to be favoured, whereas in the IPD the single-interaction equilibrium of zero cooperation means that unless contingent strategies (or random drift) have already increased cooperation, tit-for-tat-like and Pavlov-like strategies cannot be favoured, and hence cannot lead to an arms race. Note that it is still the case that intelligence is selected for in the IPD when cooperative acts are rare yet increasing (<a href=\"#RSPB20120206F3\" data-ref-type=\"fig\">figure 3<\/a>*a*). However, the cooperative acts that drive selection for intelligence in this case are generated by less cooperative contingent strategies, which cluster with always-defect as their closest pure strategy (*\u03c1* = 0.1095, *p* \\< 0.0001; see electronic supplementary material, table S1 and figure S1). This means that there is a succession in the arms race in the IPD, with 'mean' contingent strategies initially increasing selection for intelligence at low cooperation, and 'kind' contingent strategies increasing selection for intelligence as cooperation increases.\n\nIt is not any particular single strategy that drives these arms races; rather, as the complexity of the strategies in the population increases, there is selection for other complex strategies to outwit them. Unlike previous analyses where fixed strategies or strategies with constrained memory were used, our open-ended system allows for near infinite strategic variations to outwit opponents. In this way, selection for intelligence occurs owing to a constantly shifting strategic environment, where the 'best response' to the population of strategies can be shifting from generation to generation. Increases in memory allow for the potential of the recognition of opponents' strategies, allowing for the alteration of one's own strategy in response (e.g. Pavlov-like strategies that can recognize individuals that always defect). In turn this can allow for attempts to deceive opponents regarding one's own strategy (e.g. false cooperators).\n\n# Discussion\n\nIt is important here to note the closed nature of our model system; individuals can only choose within one social task (whether to cooperate or defect against another individual based on their behaviour in previous interactions with them). However, our results may apply in principle to other social scenarios where individuals use strategies to decide who to cooperate with or when to cooperate, such as indirect reciprocity \\[17,18\\], policing\/punishment \\[45,46\\] and partner choice \\[47\u201349\\]. Along with kin selection, these are the major mechanisms thought to lead to transitions to cooperative groups. As the intelligence of an individual increases, it is likely that more of these behavioural repertoires will become available to them, with increased intelligence acting as a pre-adaptation. For example, increased intelligence owing to selection for direct reciprocity could facilitate the evolution of indirect reciprocity or partner choice, highlighting the contingent nature of social evolution in multiple strategic dimensions \\[50\\]. The facilitation of new social behaviours due to emergent intelligence could allow for a perpetuated Machiavellian arms race leading to ever-greater levels of intelligence. Additionally, in our simulations populations evolved to play only a single game (either the IPD or ISD). The simultaneous play of multiple games could greatly increase strategic complexity, with the possibility of the integration of information from previous interactions in games with different payoff structures into the decision-making process.\n\nIt has previously been suggested that the pinnacles of cooperative behaviour in nature show a bimodal distribution with intelligence, with the most cooperative species displaying either limited cognition (e.g. microbes, social hymenoptera) or exceptional intelligence (e.g. humans and other primates, certain cetaceans and birds) \\[26\\]. It is clear in the former case that cooperation has evolved primarily owing to combinations of kin selection and ecological factors \\[51\\]. However, in the latter case, kin selection is not the only mechanism leading to cooperation, and may not even be the most important. A recent study has in fact suggested that relatedness was too low in human hunter\u2013gatherer groups for kin selection to drive the evolution of human cooperation \\[52\\]. In highly intelligent species, contingent behaviours (reciprocity, partner choice, etc.) appear to have been important in the evolution of cooperation \\[26\\]. Our results may help us to explain this pattern by showing that the selection for appropriate behavioural assortment of cooperative acts can lead to selection for greater cognitive abilities and Machiavellian arms races, and that intelligence facilitates greater extremes of cooperation. Additionally, although kin selection is still of importance in highly intelligent taxa, high relatedness may hinder the evolution of intelligence by driving unconditional cooperation to fixation in the population, without any need of contingent behaviours.\n\nA trait as complex as advanced intelligence is likely to have evolved owing to a combination of several factors rather than a single factor \\[4\\]. Along with the social intelligence hypothesis, many other theories attempting to explain the evolution of advanced intelligence have been suggested, among them that intelligence is an adaptation for tool use \\[53,54\\], that intelligence is an adaptation for social learning and the accumulation of culture \\[55\u201357\\], and that intelligence is the result of sexual selection \\[58\\]. All of these theories are supported by evidence from at least some of the most intelligent animals. However, the difficulty lies in disentangling the traits that are causal factors in the evolution of intelligence from those that are by-products of advanced intelligence. The combination of game theoretic frameworks and artificial neural network models presented here may provide a framework for the evaluation of the mechanistic strengths of these different hypotheses. While previous models have sought to relate cooperation and intelligence, the focus has most frequently been on the cognitive requirements of cooperation, rather than on the selection for intelligence. Many of these models have lacked an explicit brain structure \\[17\u201322\\], and among those studies that have used artificial neural networks, we know of no examples where the network structure was allowed to freely evolve or implications of selection for decision-making strategies for the evolution of intelligence were directly addressed \\[23,24\\]. While artificial neural networks are not real brains, relying on abstraction of the activity of millions of real neurons down to a manageable number of artificial neurons, they can provide insight into the dynamics of cognitive evolution and allow for the flexible evolution of behaviour \\[59\\]. Our results show that, in a freely evolving system, selection for efficient decision-making in social interactions can give rise to selection pressures for advanced cognition, supporting the view that the transition to the cooperative groups seen in the most intelligent taxa may be the key to their intellect.\n\n## Acknowledgments\n\nWe thank G. D. Ruxton, C. J. Tanner, I. Donohue, N. M. Marples, K. Irvine, R. Bshary and two anonymous reviewers for helpful comments on a previous version of this manuscript. We also thank F. J. Weissing, S. Gavrillets, J. A. R. Marshall and R. K\u00fcmmerli for helpful discussions. L.M. was supported by a scholarship from the Irish Research Council for Science, Engineering and Technology. S.P.B. was supported by the Wellcome Trust.\n\n# References","meta":{"dup_signals":{"dup_doc_count":173,"dup_dump_count":44,"dup_details":{"curated_sources":2,"2018-47":2,"2018-43":1,"2018-39":3,"2018-34":1,"2018-30":3,"2018-26":2,"2018-22":3,"2018-17":2,"2018-13":2,"2018-09":3,"2018-05":2,"2017-51":4,"2017-47":4,"2017-43":4,"2017-39":5,"2017-34":4,"2017-30":4,"2017-26":5,"2017-22":3,"2017-17":5,"2017-09":5,"2017-04":6,"2016-50":6,"2016-44":6,"2016-40":6,"2016-36":5,"2016-30":4,"2016-26":4,"2016-22":5,"2016-18":5,"2016-07":5,"2015-48":5,"2015-40":5,"2015-35":3,"2015-32":3,"2015-27":4,"2015-22":5,"2015-14":4,"2014-52":4,"2014-49":2,"2017-13":5,"2015-18":4,"2015-11":4,"2015-06":4}},"file":"PMC3385471"},"subset":"pubmed_central"}
{"text":"abstract: Two new approaches have identified physiological nonsense-mediated mRNA decay (NMD) substrates, and suggest that NMD functions as a multipurpose tool in the modulation of gene expression.\nauthor: Gabriele Neu-Yilik; Niels H Gehring; Matthias W Hentze; Andreas E Kulozik\ndate: 2004\ninstitute: 1Department of Pediatric Oncology, Hematology and Immunology, University of Heidelberg, Im Neuenheimer Feld 150, 69120 Heidelberg, Germany; 2Molecular Medicine Partnership Unit, EMBL\/University of Heidelberg, Im Neuenheimer Feld 156, 69120 Heidelberg, Germany; 3EMBL Heidelberg, Gene Expression Programme, Meyerhofstrasse 1, 69117 Heidelberg, Germany\nreferences:\ntitle: Nonsense-mediated mRNA decay: from vacuum cleaner to Swiss army knife\n\nNonsense-mediated mRNA decay (NMD) is a specific pathway for the degradation of mRNAs that have premature termination codons (PTCs) in their open reading frames (ORFs). Its importance is highlighted by its conservation in all eukaryotes. NMD counteracts the potentially harmful impact of mRNAs that have PTCs as a result of errors at various levels of gene expression, such as nonsense and frameshift mutations, transcriptional errors and faulty splicing. Thus, NMD serves as a 'cellular vacuum cleaner' that protects the cell from the potentially harmful effects of truncated proteins by eliminating mRNAs with PTCs in a sequence of events that is not yet fully understood. In recent years numerous biochemical and cell-biological investigations in *Saccharomyces cerevisiae* \\[1\\], *Drosophila melanogaster* \\[2\\], *Caenorhabditis elegans* \\[3\\] and human \\[4,5\\] cells have helped to elucidate some of the mechanistic details underlying the NMD pathway. A role for NMD in the regulation of mRNA metabolism beyond the mere vacuum cleaner function for faulty mRNAs has been suspected, and was foreshadowed by work on the splicing factor SC35 and some ribosomal proteins \\[6,7\\]. Now, 'genome-wide' approaches - one in yeast using microarrays and another *in silico*, analyzing information mined from mRNA and protein databases - have added powerful evidence to suggest that NMD may serve multiple purposes in gene expression \\[8-11\\].\n\n# Inspirations from yeast\n\nIn yeast, NMD depends on the expression of the Upf1, Upf2 (Nmd2) and Upf3 proteins. Single or simultaneous inactivation of the UPF genes stabilizes nonsense-containing mRNAs, indicating that their protein products interact functionally in the same pathway. He *et al*. \\[8\\] used high-density oligonucleotide arrays to analyze genome-wide expression profiles of yeast strains containing single deletions of the *UPF1*, *UPF2* or *UPF3* genes, as well as of the *DCP1* and *XRN1* genes which encode proteins with activities thought to be involved in the NMD pathway - an essential component of the mRNA decapping enzyme and the 5'-3' exonuclease, respectively. They also tested double deletions of the *XRN1* gene in combination with each of the UPF genes. Two-dimensional clustering analysis of the expressed genes for the *\u0394upf1*, *\u0394upf2* and *\u0394upf3* strains yielded several interesting results.\n\nThe deletion of *UPF1*, *UPF2* or *UPF3* generated nearly identical expression profiles. Thus, all three gene products act on the same targets, consistent with the function of Upf1, Upf2 and Upf3 in a single, linear pathway in yeast. The abundance of most mRNAs upregulated in Upf-deficient cells was also increased in *\u0394dcp1* and *\u0394xrn1* strains, suggesting that these mRNAs are largely degraded by decapping and subsequent 5'-3' exonucleolytic decay. This approach also identified a considerable number of NMD-regulated transcripts (765 out of the 7,839 genes represented on the microarrays) and showed that NMD substrates are generally expressed at below-average levels. In addition, most NMD substrates were found to be upregulated upon NMD inactivation, but some were downregulated, pointing to the existence of higher-order NMD targets (or additional functions of the Upf proteins in alternative gene-regulation pathways). Finally, only one third of the identified transcripts can be classified into structural or functional groups, some of which are surprising and hitherto unrecognized. Representatives of previously described NMD-substrate categories were identified, including mRNAs with nonsense mutations, transcripts resulting from faulty or regulated alternative splicing, mRNAs subject to leaky scanning during translation initiation, and mRNAs with an upstream ORF or with AATGA or ATGAA motifs immediately upstream of their translation initiation codons. More intriguing is the discovery of several new classes of NMD targets, including mRNAs that use translational +1 frameshifting, bicistronic mRNAs and, most interestingly, two classes of noncoding RNAs: pseudogene transcripts and transcripts encoded by transposable elements or their long terminal repeat (LTR) sequences.\n\nA significant fraction of the protein-encoding transcripts upregulated in the strains with mutations in the NMD-pathway genes \\[8\\] could be grouped into clusters of proteins that act in similar pathways. Among these are proteins coordinately involved in telomere maintenance, pre-mRNA splicing, peroxisomal function and DNA repair. This suggests the exciting possibility that NMD could orchestrate the expression of functional groups of genes. Several important results of the work by He *et al*. \\[8\\] confirm findings obtained by another lab using a similar approach several years ago \\[12\\]. Notably, the coordinate upregulation of genes involved in telomere maintenance by NMD inactivation has already sparked interesting follow-up investigations. The yeast NMD pathway has been shown to accelerate the rate of senescence promoted by the loss of the telomerase enzyme or by the erosion of telomeres that results from altering the stochiometry of telomere-cap components \\[13-15\\]. At least one likely primary target of NMD is the mRNA of Stn1p, an essential protein involved in chromosome-end protection. The observation by He *et al*. \\[8\\] that 35.9% of all ORFs encoded in the telomere region were upregulated in strains with mutations in components of the NMD pathway further illustrates that NMD controls many genes near telomere ends, although probably indirectly. These genes are usually silenced and may be derepressed when the protection of chromosome ends is disturbed by the loss of NMD. This enlightening example demonstrates how NMD can affect whole pathways by regulating the expression of one or a few primary target mRNAs, with consequences for groups of downstream secondary effectors. The discovery of additional examples of NMD-mediated control of functional pathways can be expected, promoting NMD to a gene-expression tool with many utilities. The cellular vacuum cleaner has therefore become a Swiss army knife (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>).\n\n# *In silico* veritas?\n\nIn a series of three recent publications \\[9-11\\] Steve Brenner and colleagues suggested, by data-mining, that one-third of reliably inferred human splice products form a major class of natural targets for NMD. Alternative splicing is thought to occur in 30-60% of human genes, expanding the coding repertoire of the limited number of genes in the genome and modulating tissue-specific and developmental gene functions. Brenner and colleagues \\[9-11\\] now suggest that alternative splicing provides a mechanism to generate PTC-containing splice products that are subsequently degraded by NMD and, as a consequence, that cooperation between alternative splicing and NMD pathways offers a major and currently underappreciated way to regulate gene expression.\n\nFor their *in silico* analysis, they mapped well-characterized human RefSeq \\[16\\] and LocusLink \\[17\\] mRNA sequences to genomic sequences, and then performed high-stringency alignments between these 'RefSeq-coding genes' and expressed sequence tags (ESTs). The reliably inferred splice variants were only accepted as likely NMD targets when they conformed to the '50-nucleotide rule': this hallmark of mammalian NMD predicates that stop codons located at least 50 nucleotides upstream of the last exon junction will be interpreted as 'premature' and trigger NMD. This approach leads to an underestimate of potential NMD targets, because some PTCs (for example, in T-cell receptor gene transcripts) will trigger NMD even when they are not followed by a sufficiently distant intron \\[18\\]. Moreover, Brenner and colleagues \\[9-11\\] also excluded mRNA variants that are indistinguishable from products of faulty splicing. These studies have unearthed several groups of functionally related proteins whose expression appears to be regulated by NMD, including translation factors and ribosomal proteins. This is in remarkable contrast to the yeast data of He *et al*. \\[8\\], where proteins with a function in translation were, if anything, underrepresented in the pool of NMD-regulated genes.\n\nNMD is not (yet) on everyone's mind. As a consequence, Brenner and colleagues \\[11\\] found several entries for truncated proteins in the Swiss-Prot database. In some of these cases the available experimental evidence confirms that the mRNAs that encode these truncated proteins are *bona fide* NMD substrates. We are left with a consolation and a surprise. The consolation is that traces of NMD can be uncovered even though they had been overlooked before. Having recently had to accept that the number of genes in the human genome is too limited to explain the far higher number of proteins (not to mention other gene products), we then had to learn that one plausible and elegant explanation lies in alternative splicing, which enables a gene to code for a whole family of related, or sometimes antagonistic, proteins. And now the surprise is that a large portion of this effort is supposedly expended only to direct many of the primary products to decay. Several conclusions can be drawn from these observations. First, databases need to be read and annotated with a full realization of the implications of NMD; and second, NMD seems to serve as a tool for rapidly switching off gene expression. This view extends the idea of NMD as a mechanism for ridding the cell of the potentially harmful products of faulty splicing. But there may be more to it. NMD rarely downregulates the expression of a transcript completely; more commonly, 10-30% of the PTC-containing transcripts survive and may allow the production of physiologically relevant levels of truncated protein products.\n\nFor NMD researchers it has always been hard to reconcile these observations with the presumed protective role of NMD, especially as very low levels of biological products can sometimes have enormous effects. Given the problems of detecting the low levels of proteins or peptides produced from downregulated transcripts, in addition to some lingering lack of awareness of NMD, examples to prove otherwise are hard to come by. A recent publication \\[19\\] describes a PTC-containing transcript of the high-affinity immunoglobulin E (IgE) receptor, Fc\u03b5RI\u03b2, arising from retention of an intron. This alternative transcript not only conforms to the 50-nucleotide rule but its expression levels are very low compared to those of the full-length transcript, as would be expected for an NMD target. Nonetheless, the truncated protein is not only detectable, it even competes effectively with the full-length protein to control Fc\u03b5RI\u03b2 expression on the cell surface. Thus, even low endogenous expression levels of NMD targets can suffice to generate a product with a biological function. A similar example of the utility of a *bona fide* NMD substrate is illustrated by the *unc-49* locus in *C. elegans*. This locus uses alternative splicing to produce three GABA-receptor subunits, two of which (A and B) undergo several splice events in their 3' UTRs, rendering them predicted NMD substrates \\[20\\]. While A-form transcripts are hardly detectable, B-form transcripts represent the most abundant form and code for a protein essential for the worm's locomotion. Either the B-form transcript escapes NMD, or the residual mRNA left after NMD suffices for the necessary protein production.\n\nAmong the examples presented by Brenner and colleagues \\[11\\] or in other studies \\[21\\] are genes with complex alternative splice patterns resulting in multiple transcript isoforms with or without PTCs. The lower abundance of these isoforms when compared to the full-length form supports the notion that they are targeted to the NMD pathway. But is the possibility that cells engage in complex alternative-splicing procedures generating multiple products, just to finally dispose of them, the only conceivable option? Four out of five PTC-containing isoforms of the mRNA for the LARD death receptor are readily detectable in non-activated lymphocytes, whereas only the full-length form is expressed in activated lymphocytes \\[11\\]. While it is entirely possible that the PTC-containing forms are generated to switch off receptor expression in resting lymphocytes, there are attractive alternatives: as in the case of the Fc\u03b5RI\u03b2 receptor \\[19\\], the PTC-containing mRNAs may produce proteins or peptides with relevant, although currently unknown, functions. Thus, quantitative control of the expression of low amounts of protein isoforms could represent yet another facet of the function of the NMD pathway.\n\nA role for NMD in controlling the levels of noncoding RNAs (including noncoding alternative splice products) must also be considered. RNA accounts for more than 95% of the human genome's output and there is increasing evidence that noncoding RNAs (including introns, and spliced and polyadenylated transcripts) can have a function, for example as a modulating network or an additional layer of information \\[22,23\\]. Importantly, noncoding RNAs have been discovered among natural NMD targets in yeast \\[8\\], where only a relatively small portion of the genome is transcribed into noncoding RNAs. What might be the role of NMD in mammals, which transcribe a far higher percentage of their genome into noncoding RNAs \\[22,24,25\\]? Clearly, the recent insights into the RNA-substrate spectrum of the NMD system should enhance the appreciation of NMD as a versatile, multipurpose mechanism that controls the transcriptome qualitatively and quantitatively.\n\n## Figures and Tables","meta":{"dup_signals":{"dup_doc_count":151,"dup_dump_count":47,"dup_details":{"curated_sources":4,"2023-14":1,"2022-27":1,"2022-05":1,"2020-34":1,"2019-51":1,"2019-43":1,"2019-26":1,"2019-18":1,"2019-09":2,"2018-51":2,"2018-43":2,"2018-34":1,"2018-22":1,"2018-05":2,"2017-39":1,"2017-30":2,"2017-22":1,"2017-09":10,"2016-50":1,"2016-44":2,"2016-40":1,"2016-36":11,"2016-30":8,"2016-22":1,"2016-18":1,"2015-48":4,"2015-40":2,"2015-35":6,"2015-32":4,"2015-27":4,"2015-22":4,"2015-14":4,"2014-52":4,"2014-49":4,"2014-42":9,"2014-41":7,"2014-35":6,"2014-23":6,"2014-15":4,"2023-40":1,"2015-18":3,"2015-11":4,"2015-06":3,"2014-10":3,"2013-48":4,"2013-20":3,"2024-10":1}},"file":"PMC395777"},"subset":"pubmed_central"}
{"text":"abstract: A report on the Human Genome Organisation (HUGO) 11th Human Genome Meeting, Helsinki, Finland, 31 May-3 June 2006.\nauthor: Kristine Kleivi\ndate: 2006\ninstitute: 1Medical Biotechnology, VTT Technical Research Center of Finland, 20521 Turku, Finland and Department of Genetics, Institute for Cancer Research, Rikshospitalet-Radiumhospitalet Medical Center, 0310 Oslo, Norway\ntitle: Advances in the genetics and epigenetics of gene regulation and human disease\n\nAt the recent annual meeting on the human genome in Helsinki, organized by the Human Genome Organisation (HUGO), close to 700 scientists gathered to present and discuss the latest advances in genome research. This report presents some selected highlights.\n\n# Genome variation, gene expression and disease susceptibility\n\nThrough their effects on gene expression, polymorphisms in the human genome can contribute to phenotypic variation and disease susceptibility. For many diseases, such as cancer, great effort is being made to study the sequence variants that contribute to disease susceptibility. The impact of genetic variation on common diseases was addressed by Kari Stefansson (deCODE Genetics, Reykjavik, Iceland), who gave an update on the identified sequence variants that may increase the risk of developing type 2 diabetes, prostate cancer, myocardial infarction, stroke and schizophrenia. In the past decades, type 2 diabetes has become a major health problem in the Western world, as both its incidence and its prevalence have increased rapidly. Stefansson reported his group's recent discovery of an inherited variant of the gene *TCF7L7*, encoding a protein called transcription factor 7-like 2 located on chromosome 10, which is estimated to account for about 20% of the diabetes cases. They have also showed an association between a common genetic variant in the microsatellite DG8S737 at chromosome band 8q24, which may contribute to the development of prostate cancers in European and African populations.\n\nSingle-nucleotide polymorphism (SNP) genotypes correlated with gene-expression data in breast tumors were presented by Vessela Kristensen (The Norwegian Radium Hospital, Oslo, Norway). For genotyping, she and her colleagues selected sets of genes involved in reactive oxygen species signaling (ROS) and the repair of DNA damage caused by ROS - that is, pathways that are generally affected by chemotherapy and radiation therapy. Using various statistical approaches, the genetic association between SNPs in genes involved in the ROS pathways and the expression levels of mRNA transcripts from a panel of breast cancer patients were assessed. Regulatory SNPs in the genes *EGF*, *IL1A*, *MAPK8*, *XPC*, *SOD2* and *ALOX12* were associated with alterations in the expression levels of several transcripts. Kristensen also showed that a set of SNPs were linked to a cluster of transcripts participating in the same functional pathway.\n\nThomas Hudson (McGill University, Montreal, Canada) described several resources and technologies that are available to study the impact of genome variation on gene expression. He and his colleagues systematically studied a subset of genes whose alleles show large differences in expression in lymphoblastoid cell lines. These data were integrated with HapMap data to search for haplotypes associated with mRNA expression at flanking genes. Hudson described the discovery of 16 loci harboring a common haplotype affecting the total expression of a gene, that is, all the alleles of the gene, and of 17 loci that affected relative allelic expression in heterozygous samples. To better understand the mechanisms controlling this gene expression, *cis*-acting polymorphisms need to be studied in the human genome in larger sample sets and tissue panels.\n\n# DNA methylation and epigenetic modification of the genome\n\nMethylation of CpG islands has an important role in controlling gene expression during mammalian development, and is frequently altered in diseases such as cancer. DNA methylation was extensively discussed in the meeting. For example, Carmen Sapienza (Temple University Medical School, Philadelphia, USA) reported that imprinted regions in humans are historical hotspots of recombination. Together with specific DNA sequences, epigenetic factors may have an important influence on the rate of meiotic recombination and the position of cross-overs. Using *in silico* and *in vitro* analyses, Sapienza's group have shown a relationship between increased rates of meiotic recombination and genomic imprinting. Imprinted regions showed more linkage disequilibrium, and had a significantly higher number of small haplotype blocks, than the non-imprinted regions. Their findings suggest that several factors, including both specific DNA sequences and epigenetics, are involved in controlling meiotic recombination in humans.\n\nNutritional influences during prenatal and early postnatal development may affect gene expression, and subsequently the phenotype, through epigenetic gene regulatory mechanisms. Nutrition is important in providing methyl donors for DNA, and some genes are especially sensitive to nutritional changes during embryogenesis. Rob Waterland (Baylor Collage of Medicine, Houston, USA) has used mouse models to show that some alleles are particularly susceptible to changes in methylation due to maternal nutrition. For example, supplementary nutrition can lead to increasing body weight across several generations of offspring. He and his colleagues postulate that maternal nutrition before and during pregnancy may affect the establishment of CpG methylation and the life-long expression of metastable epialleles (epigenetically modified alleles) in humans.\n\nWhereas the majority of CpG islands in the genome are normally unmethylated, a sizeable fraction is prone to methylation in various cell types and pathological conditions. Christoph Bock (Max Planck Institute for Informatics, Saarbr\u00fccken, Germany) described his group's work predicting CpG methylation on the basis of DNA sequence and genomic location. Using a bioinformatics approach, they were able to distinguish CpG islands that are prone to methylation from those that are not. For example, on chromosome 21, they were able to predict the CpG island methylation rate with 90% accuracy, which was later confirmed by *in vitro* analyses. This study revealed that the DNA composition of CpG islands, the sequence, the structure and the number of repeats play an important role in predisposing CpG islands to DNA methylation. Furthermore, these features can also be used to predict the CpG methylation pattern of the whole genome.\n\n# Regulatory genomics\n\nKnowledge about transcription factors, their binding specificities and the assembly of their binding sites to form tissue-specific enhancer elements is critical for understanding key regulatory mechanisms of human gene expression. Outi Hallikas (University of Helsinki, Finland) aimed to determine the binding specificities of transcription factors that are involved in growth control, and to find evolutionarily conserved enhancer elements that drive organ-specific expression of genes that regulate a cell's progression through the cell cycle. By using a high-throughput method for determining transcription factor binding sites, Hallikas reported the binding sites of the transcription factors GLIs, TCF4 and c-ETS1, which are involved in different signaling pathways such as those leading from the signal protein Wnt and the Ras\/MAPK intracellular signaling module. To identify the mammalian enhancer elements, Hallikas and colleagues have developed a new computational tool (Enhancer Element Locator; available online <http:\/\/www.cs.helsinki.fi\/u\/kpalin\/EEL\/>), and used it to predict active transcription factor binding sites. Validation of these in transgenic mice revealed the presence of enhancers in c-*Myc* and N-*Myc*, genes that play a role in growth control and tumorigenesis.\n\nFrom the same group, Mikael Bj\u00f6rklund presented a genome-wide RNA interference (RNAi) analysis of genes that are involved in cell-cycle control and cell-size regulation in *Drosophila*. Using flow cytometry, Bj\u00f6rklund and colleagues analyzed the RNAi-induced loss-of-function of 70% of the genes in *Drosophila*, including those conserved in humans, on cell-cycle progression by flow cytometry. Genes controlling several cellular processes were identified, including cell size, cytokinesis, apoptosis and phases of the cell cycle. In addition, a translational regulator (eIF-3p66) associated with the cyclin\/cyclin-dependent kinase pathway was identified.\n\nThe combination of RNAi and gene-expression profiling provides further insight into gene function and the regulatory networks controlling expression. Ilaria Piccini (Max Planck Institute for Molecular Genetics, Berlin, Germany) presented their genome-wide analysis of transcription factors in association with gene regulation. Using RNAi, Piccini and colleagues are knocking down the expression of 200 transcription factors involved in human developmental processes in a panel of cell lines, and investigating the downstream targets of these at the transcriptome level. They have initially focused on transcription factors encoded by chromosome 21, and as an example, Piccini described the identification of 72 potential target genes, containing both known and novel targets, that were dysregulated when expression of the transcription factor gene *BACH1* was silenced.\n\n# DNA amplifications in cancer genomes\n\nGene amplifications are seen in a variety of human cancers, and are often associated with poor clinical outcome for the patient. Target genes for amplified regions are often oncogenes, which may be used as therapeutic, prognostic and diagnostic targets. Therefore, increasing the knowledge of DNA copy number amplifications in human neoplasms is important. This issue was addressed by Samuel Myllykangas (University of Helsinki, Finland) who presented an extensive analysis of DNA copy number amplifications in human cancers. Using data from published comparative genomic hybridization studies, they performed an *in silico* analysis of DNA copy number changes in approximately 4,500 samples from over 70 different neoplasms. Computational analysis identified the different amplification hotspots, which were spread over large parts of the genome and frequently co-localized with known fragile sites, cancer genes and virus integration sites. Amplification of some chromosomal regions was observed in the majority of the cancers studied, whereas other amplifications were cancer-site specific. From the characteristic amplification profiles, Myllykangas showed that cancers with similar cellular origin and histology, such as breast and prostate adenocarcinomas, clustered together. Their discoveries show the relevance of global studies on DNA amplifications in human cancers, and suggest diagnostic and predictive possibilities.\n\nThe annual Human Genome Meeting of 2006 was an inspiring meeting, updating us on the latest knowledge in human genomics. The power of combining high-throughput experimental approaches with genome-wide bioinformatics, systems biology and data integration was emphasized. Thus, it was a successful demonstration of strategies that will be increasingly useful in human genetics in the future.","meta":{"dup_signals":{"dup_doc_count":103,"dup_dump_count":43,"dup_details":{"curated_sources":4,"2022-33":1,"2021-43":1,"2019-22":1,"2019-09":1,"2018-51":1,"2018-39":1,"2018-30":1,"2018-22":1,"2018-05":1,"2017-39":1,"2017-30":1,"2017-26":1,"2017-22":1,"2017-09":8,"2016-44":1,"2016-40":1,"2016-36":9,"2016-30":6,"2016-22":1,"2016-18":1,"2016-07":6,"2015-48":2,"2015-40":2,"2015-35":2,"2015-32":2,"2015-27":1,"2015-22":2,"2015-14":2,"2014-52":2,"2014-49":4,"2014-42":6,"2014-41":3,"2014-35":3,"2014-23":3,"2014-15":4,"2023-14":1,"2015-18":2,"2015-11":2,"2015-06":2,"2014-10":3,"2013-48":2,"2013-20":2,"2024-10":1}},"file":"PMC1779594"},"subset":"pubmed_central"}
{"text":"author: Raymond Hutubessy\ndate: 2013\ninstitute: 1Initiative for Vaccine Research, World Health Organization, 20 Avenue Appia, CH-1211, Geneva, Switzerland\ntitle: Q&A - Economic analyses for vaccine introduction decisions in low- and middle- income countries\n\n# Introduction\n\nRaymond Hutubessy is a senior health economist affiliated to the Immunization, Vaccines and Biologicals (IVB) Department of the World Health Organization (WHO), and is the executive secretary of the WHO Immunization and Vaccines related Implementation Research Advisory Committee (IVIR-AC). His main research interests focus on economic and financial analyses of vaccine introduction decisions in low- and middle-income countries (LMICs). In this Q&A, he will discuss the importance of this work in relation to the global context of vaccine introduction decisions (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>).\n\n## 1) What is the importance of conducting economic analyses for vaccine introduction decisions?\n\nVaccines for prevention of communicable diseases have been shown to be extremely effective in terms of health outcomes. Therefore, conducting economic analyses to get the most value for money from vaccine introduction decisions is of high importance; evidence and information resulting from these analyses are not the only input to the decision-making process for vaccine introduction decisions, but they are important ones.\n\nThe relationship between health and economic growth is one of the cornerstones of development economics: health status is determinant of productivity that can be shown to influence economic growth. Specifically, vaccines have a broader value in terms of their indirect effects (for example, herd immunity) and other externalities (for example, improvements in the cognitive development of children, higher school attendance and attainment, macroeconomic impact). Therefore, in addition to the traditional economic appraisals for vaccine introduction decisions it is useful to policy makers and other stakeholders involved with vaccine introduction decisions to demonstrate the broader added value of vaccines and investments in health in general.\n\nEconomic appraisals address different key issues with regard to decisions on vaccine introduction. These appraisals range from priority-setting issues across vaccines and other competing health interventions, to affordability and budget impact analysis, and costing and financing issues with regard to the introduction decisions of immunization programs. For these different policy questions, different analytical tools are available, such as cost-effectiveness analyses, costing studies, budget impact and optimization analysis.\n\n## 2) What are the main issues that should be considered?\n\nFirst, because many economic evaluations are based on analytical decision tools such as mathematical infectious disease models, costing tools, decision trees models and so on, transparency is needed on the choice of the modeling methodologies, parameters and country data used and assumptions made by the analyst. Standardization of methods of cost-effectiveness is therefore needed and analysts in the field should adhere to these guides. This allows users to make comparisons of different study results by different groups. The WHO, in addition to other organizations, has developed several guidelines on economic evaluations in health, and vaccines and immunization programs in particular.\n\nSecond, to be relevant, local decision makers have country-specific policy questions and therefore need contextualized study results driven by specific country data and information with regard to demographics, epidemiological and economic data, and local needs. However, this does not mean that for each country (in my field of work, this primarily involves LMICs), economists and analysts need to start from scratch - they can build on work from other groups who often put their models, including the computer program codes, in the public domain.\n\nIn light of this, efforts should be put into the collection of local data and building local technical capacity in LMICs so that they are able to perform their own analysis and interpret their own results with the aim of increasing local ownership of the evidence generated. The WHO, along with partners i.e. Pan American Health Organization (PAHO), Agence de M\u00e9dicine de Pr\u00e9ventive (AMP), Program for Appropriate Technology in Health (PATH), Sabin Vaccine Institute and USA Centers for Disease Control and Prevention, recently started the ProVac International Working Group to promote the use of economic analysis for vaccine introduction decisions in LMICs.\n\n## 3) Are there differences in conducting economic analyses for vaccine introduction decisions between higher income versus resource-limited settings?\n\nIn principle, the methods applied and tools used are similar in higher income versus resource-limited settings. However, because country demographics, disease burden, epidemiological and socioeconomic background, and health systems and infrastructure differ, the methods of measurement and valuation and the interpretation hence key drivers of results of economic evaluations will also differ.\n\nFor example, the price at which human papillomavirus (HPV) vaccination is considered to be cost-effective is heavily dependent on HPV prevalence and the existing local cervical cancer services and the ceiling cost-effectiveness or cost-effectiveness threshold (that is, societies' willingness to pay for an additional health gain). In high income countries, access to health care services is better and delivery systems of vaccines to reach adolescent girls are more advanced than in LMICs. As a result, the affordability question in such resource-rich scenarios focused around the relatively high public market prices of HPV vaccines, which may go up to 150 USD per dose.\n\nBy contrast, in countries eligible for the Global Alliance for Vaccines and Immunization's (GAVI Alliance) support, the manufacturers of one of the vaccines have offered an indicative price of 5 USD per dose. This is a 64% reduction on the lowest public prices. As a result, rather than the vaccine price *per se*, it is the securing of the delivery costs to get the vaccine from the port of entry to those girls in need that has become a main barrier in many of these countries. This barrier is in addition to other capacity issues, such as the existing delivery infrastructure being already over-stretched with traditional Expanded Programme on Immunization vaccines and other competing new vaccines, such as rotavirus and pneumococcal vaccines.\n\n## 4) Are there any specific ways in which these economic analyses have improved clinical outcomes?\n\nIn my opinion, economic analyses have helped to increased the level of health that health care spending can buy, and hence have aided promotion of the use of effective and cost-effective health interventions. For example, by identifying barriers and uptake issues of vaccine introduction decisions, economic analyses have contributed to bridge the link between the evidence on theoretical vaccine efficacy and real-life effectiveness. Another example is that economic analyses do not just appraise vertical disease programs but more often also have an integrated and combined disease program perspective. This has the potential to account for synergistic effects of disease programs and therefore will improve the overall public health impact.\n\n## 5) Where can I find more information?\n\nWHO and other partners have vaccine specific and generic websites on health economic information and initiativies. In addition, WHO published several key documents on vaccine economics.\n\n**WHO Initiative for Vaccine Research (IVR)** \\[<http:\/\/www.who.int\/vaccine_research\/implementation\/health_economics\/en\/index.html>\\]\n\n**CHOosing Interventions that are Cost Effective (WHO-CHOICE)** \\[<http:\/\/www.who.int\/choice\/en\/>\\]\n\n**WHO guide for standardization of economic evaluations of immunization programmes** \\[<http:\/\/whqlibdoc.who.int\/hq\/2008\/WHO_IVB_08.14_eng.pdf>\\]\n\n**Pan American Health Organization (PAHO ProVac)** \\[<http:\/\/new.paho.org\/provac\/>\\]\n\n**International Health Economics Association** \\[<https:\/\/www.healtheconomics.org\/>\\]\n\nJit M, Levin C, Brison M, Levin A, Resch S, Berkhof J, Kim J, Hutubessy R: **Economic analyses to support decisions about HPV vaccination in low- and middle-income countries: a consensus report and guide for analysts**. *BMC Med* 2013, **11**:23.\n\nHutubessy R, Levin A, Wang S, Morgan W, Ally M, John T, Broutet N: **A case study using the United Republic of Tanzania: costing nationwide HPV vaccine delivery using the WHO Cervical Cancer Prevention and Control Costing Tool**. *BMC Med* 2012, **10**:136.\n\nDeogaonkar R, Hutubessy R, van der Putten I, Evers S, Jit M: **Systematic review of studies evaluating the broader economic impact of vaccination in low and middle income countries**. *BMC Public Health* 2012, **12**:878.\n\nPostma MJ, Jit M, Rozenbaum MH, Standaert B, Tu HA, Hutubessy RC: **Comparative review of three cost-effectiveness models for rotavirus vaccines in national immunization programs; a generic approach applied to various regions in the world**. *BMC Med* 2011, **9**:84.\n\nHutubessy R, Henao AM, Namgyal P, Moorthy V, Hombach J: **Results from evaluations of models and cost-effectiveness tools to support introduction decisions for new vaccines need critical appraisal**. *BMC Med* 2011, **9**:55.\n\nJit M, Demarteau N, Elbasha E, Ginsberg G, Kim J, Praditsitthikorn N, Sinanovic E, Hutubessy R: **Human papillomavirus vaccine introduction in low-income and middle-income countries: guidance on the use of cost-effectiveness models**. *BMC Med* 2011, **9**:54.\n\nChaiyakunapruk N, Somkrua R, Hutubessy R, Henao AM, Hombach J, Melegaro A, Edmunds JW, Beutels P: **Cost effectiveness of pediatric pneumococcal conjugate vaccines: a comparative assessment of decision-making tools**. *BMC Med* 2011, **9**:53.\n\n# Author information\n\nRH is a staff member of the World Health Organization. The views expressed in this article is that of the author and do not necessarily represent the views of the World Health Organization.\n\n# Pre-publication history\n\nThe pre-publication history for this paper can be accessed here:\n\n<http:\/\/www.biomedcentral.com\/1741-7015\/11\/71\/prepub>","meta":{"dup_signals":{"dup_doc_count":118,"dup_dump_count":58,"dup_details":{"curated_sources":2,"2023-23":1,"2023-06":1,"2022-33":2,"2022-05":1,"2021-31":1,"2021-21":1,"2021-10":1,"2020-40":2,"2020-29":1,"2019-39":1,"2019-26":1,"2019-22":1,"2019-18":1,"2019-09":1,"2018-51":2,"2018-43":2,"2018-34":1,"2018-30":1,"2018-26":1,"2018-17":1,"2018-09":2,"2017-39":1,"2017-30":3,"2017-22":5,"2017-17":3,"2017-09":10,"2017-04":2,"2016-50":2,"2016-44":3,"2016-40":1,"2016-36":6,"2016-30":11,"2016-22":1,"2016-18":2,"2016-07":1,"2015-48":2,"2015-40":2,"2015-35":1,"2015-32":1,"2015-27":1,"2015-22":1,"2015-14":1,"2014-52":2,"2014-49":2,"2014-42":5,"2014-41":2,"2014-35":3,"2014-23":2,"2014-15":2,"2023-50":1,"2024-10":1,"2017-13":1,"2015-18":2,"2015-11":1,"2015-06":1,"2014-10":1,"2013-48":3,"2024-26":1}},"file":"PMC3599816"},"subset":"pubmed_central"}
{"text":"abstract: Nutrient timing is a popular nutritional strategy that involves the consumption of combinations of nutrients--primarily protein and carbohydrate--in and around an exercise session. Some have claimed that this approach can produce dramatic improvements in body composition. It has even been postulated that the timing of nutritional consumption may be more important than the absolute daily intake of nutrients. The post-exercise period is widely considered the most critical part of nutrient timing. Theoretically, consuming the proper ratio of nutrients during this time not only initiates the rebuilding of damaged muscle tissue and restoration of energy reserves, but it does so in a supercompensated fashion that enhances both body composition and exercise performance. Several researchers have made reference to an anabolic \"window of opportunity\" whereby a limited time exists after training to optimize training-related muscular adaptations. However, the importance - and even the existence - of a post-exercise 'window' can vary according to a number of factors. Not only is nutrient timing research open to question in terms of applicability, but recent evidence has directly challenged the classical view of the relevance of post-exercise nutritional intake with respect to anabolism. Therefore, the purpose of this paper will be twofold: 1) to review the existing literature on the effects of nutrient timing with respect to post-exercise muscular adaptations, and; 2) to draw relevant conclusions that allow practical, evidence-based nutritional recommendations to be made for maximizing the anabolic response to exercise.\nauthor: Alan Albert Aragon; Brad Jon Schoenfeld\ndate: 2013\ninstitute: 1California State University, Northridge, CA, USA; 2Department of Health Science, Lehman College, Bronx, NY, USA\nreferences:\ntitle: Nutrient timing revisited: is there a post-exercise anabolic window?\n\n# Introduction\n\nOver the past two decades, nutrient timing has been the subject of numerous research studies and reviews. The basis of nutrient timing involves the consumption of combinations of nutrients--primarily protein and carbohydrate--in and around an exercise session. The strategy is designed to maximize exercise-induced muscular adaptations and facilitate repair of damaged tissue \\[1\\]. Some have claimed that such timing strategies can produce dramatic improvements in body composition, particularly with respect to increases in fat-free mass \\[2\\]. It has even been postulated that the timing of nutritional consumption may be more important than the absolute daily intake of nutrients \\[3\\].\n\nThe post-exercise period is often considered the most critical part of nutrient timing. An intense resistance training workout results in the depletion of a significant proportion of stored fuels (including glycogen and amino acids) as well as causing damage to muscle fibers. Theoretically, consuming the proper ratio of nutrients during this time not only initiates the rebuilding of damaged tissue and restoration of energy reserves, but it does so in a supercompensated fashion that enhances both body composition and exercise performance. Several researchers have made reference to an \"anabolic window of opportunity\" whereby a limited time exists after training to optimize training-related muscular adaptations \\[3-5\\].\n\nHowever, the importance \u2013 and even the existence \u2013 of a post-exercise 'window' can vary according to a number of factors. Not only is nutrient timing research open to question in terms of applicability, but recent evidence has directly challenged the classical view of the relevance of post-exercise nutritional intake on anabolism. Therefore, the purpose of this paper will be twofold: 1) to review the existing literature on the effects of nutrient timing with respect to post-exercise muscular adaptations, and; 2) to draw relevant conclusions that allow evidence-based nutritional recommendations to be made for maximizing the anabolic response to exercise.\n\n## Glycogen repletion\n\nA primary goal of traditional post-workout nutrient timing recommendations is to replenish glycogen stores. Glycogen is considered essential to optimal resistance training performance, with as much as 80% of ATP production during such training derived from glycolysis \\[6\\]. MacDougall et al. \\[7\\] demonstrated that a single set of elbow flexion at 80% of 1 repetition maximum (RM) performed to muscular failure caused a 12% reduction in mixed-muscle glycogen concentration, while three sets at this intensity resulted in a 24% decrease. Similarly, Robergs et al. \\[8\\] reported that 3 sets of 12 RM performed to muscular failure resulted in a 26.1% reduction of glycogen stores in the vastus lateralis while six sets at this intensity led to a 38% decrease, primarily resulting from glycogen depletion in type II fibers compared to type I fibers. It therefore stands to reason that typical high volume bodybuilding-style workouts involving multiple exercises and sets for the same muscle group would deplete the majority of local glycogen stores.\n\nIn addition, there is evidence that glycogen serves to mediate intracellular signaling. This appears to be due, at least in part, to its negative regulatory effects on AMP-activated protein kinase (AMPK). Muscle anabolism and catabolism are regulated by a complex cascade of signaling pathways. Several pathways that have been identified as particularly important to muscle anabolism include mammalian target of rapamycin (mTOR), mitogen-activated protein kinase (MAPK), and various calcium- (Ca^2+^) dependent pathways. AMPK, on the other hand, is a cellular energy sensor that serves to enhance energy availability. As such, it blunts energy-consuming processes including the activation of mTORC1 mediated by insulin and mechanical tension, as well as heightening catabolic processes such as glycolysis, beta-oxidation, and protein degradation \\[9\\]. mTOR is considered a master network in the regulation of skeletal muscle growth \\[10,11\\], and its inhibition has a decidedly negative effect on anabolic processes \\[12\\]. Glycogen has been shown to inhibit purified AMPK in cell-free assays \\[13\\], and low glycogen levels are associated with an enhanced AMPK activity in humans *in vivo*\\[14\\].\n\nCreer et al. \\[15\\] demonstrated that changes in the phosphorylation of protein kinase B (Akt) are dependent on pre-exercise muscle glycogen content. After performing 3 sets of 10 repetitions of knee extensions with a load equating to 70% of 1 repetition maximum, early phase post-exercise Akt phosphorylation was increased only in the glycogen-loaded muscle, with no effect seen in the glycogen-depleted contralateral muscle. Glycogen inhibition also has been shown to blunt S6K activation, impair translation, and reduce the amount of mRNA of genes responsible for regulating muscle hypertrophy \\[16,17\\]. In contrast to these findings, a recent study by Camera et al. \\[18\\] found that high-intensity resistance training with low muscle glycogen levels did not impair anabolic signaling or muscle protein synthesis (MPS) during the early (4 h) postexercise recovery period. The discrepancy between studies is not clear at this time.\n\nGlycogen availability also has been shown to mediate muscle protein breakdown. Lemon and Mullin \\[19\\] found that nitrogen losses more than doubled following a bout of exercise in a glycogen-depleted versus glycogen-loaded state. Other researchers have displayed a similar inverse relationship between glycogen levels and proteolysis \\[20\\]. Considering the totality of evidence, maintaining a high intramuscular glycogen content at the onset of training appears beneficial to desired resistance training outcomes.\n\nStudies show a supercompensation of glycogen stores when carbohydrate is consumed immediately post-exercise, and delaying consumption by just 2 hours attenuates the rate of muscle glycogen re-synthesis by as much as 50% \\[21\\]. Exercise enhances insulin-stimulated glucose uptake following a workout with a strong correlation noted between the amount of uptake and the magnitude of glycogen utilization \\[22\\]. This is in part due to an increase in the translocation of GLUT4 during glycogen depletion \\[23,24\\] thereby facilitating entry of glucose into the cell. In addition, there is an exercise-induced increase in the activity of glycogen synthase\u2014the principle enzyme involved in promoting glycogen storage \\[25\\]. The combination of these factors facilitates the rapid uptake of glucose following an exercise bout, allowing glycogen to be replenished at an accelerated rate.\n\nThere is evidence that adding protein to a post-workout carbohydrate meal can enhance glycogen re-synthesis. Berardi et al. \\[26\\] demonstrated that consuming a protein-carbohydrate supplement in the 2-hour period following a 60-minute cycling bout resulted in significantly greater glycogen resynthesis compared to ingesting a calorie-equated carbohydrate solution alone. Similarly, Ivy et al. \\[27\\] found that consumption of a combination of protein and carbohydrate after a 2+ hour bout of cycling and sprinting increased muscle glycogen content significantly more than either a carbohydrate-only supplement of equal carbohydrate or caloric equivalency. The synergistic effects of protein-carbohydrate have been attributed to a more pronounced insulin response \\[28\\], although it should be noted that not all studies support these findings \\[29\\]. Jentjens et al. \\[30\\] found that given ample carbohydrate dosing (1.2 g\/kg\/hr), the addition of a protein and amino acid mixture (0.4 g\/kg\/hr) did not increase glycogen synthesis during a 3-hour post-depletion recovery period.\n\nDespite a sound theoretical basis, the practical significance of expeditiously repleting glycogen stores remains dubious. Without question, expediting glycogen resynthesis is important for a narrow subset of endurance sports where the duration between glycogen-depleting events is limited to less than approximately 8 hours \\[31\\]. Similar benefits could potentially be obtained by those who perform two-a-day split resistance training bouts (i.e. morning and evening) provided the same muscles will be worked during the respective sessions. However, for goals that are not specifically focused on the performance of multiple exercise bouts in the same day, the urgency of glycogen resynthesis is greatly diminished. High-intensity resistance training with moderate volume (6-9 sets per muscle group) has only been shown to reduce glycogen stores by 36-39% \\[8,32\\]. Certain athletes are prone to performing significantly more volume than this (i.e., competitive bodybuilders), but increased volume typically accompanies decreased frequency. For example, training a muscle group with 16-20 sets in a single session is done roughly once per week, whereas routines with 8-10 sets are done twice per week. In scenarios of higher volume and frequency of resistance training, incomplete resynthesis of pre-training glycogen levels would not be a concern aside from the far-fetched scenario where exhaustive training bouts of the same muscles occur after recovery intervals shorter than 24 hours. However, even in the event of complete glycogen depletion, replenishment to pre-training levels occurs well-within this timeframe, regardless of a significantly delayed post-exercise carbohydrate intake. For example, Parkin et al \\[33\\] compared the immediate post-exercise ingestion of 5 high-glycemic carbohydrate meals with a 2-hour wait before beginning the recovery feedings. No significant between-group differences were seen in glycogen levels at 8 hours and 24 hours post-exercise. In further support of this point, Fox et al. \\[34\\] saw no significant reduction in glycogen content 24 hours after depletion despite adding 165 g fat collectively to the post-exercise recovery meals and thus removing any potential advantage of high-glycemic conditions.\n\n## Protein breakdown\n\nAnother purported benefit of post-workout nutrient timing is an attenuation of muscle protein breakdown. This is primarily achieved by spiking insulin levels, as opposed to increasing amino acid availability \\[35,36\\]. Studies show that muscle protein breakdown is only slightly elevated immediately post-exercise and then rapidly rises thereafter \\[36\\]. In the fasted state, muscle protein breakdown is significantly heightened at 195 minutes following resistance exercise, resulting in a net negative protein balance \\[37\\]. These values are increased as much as 50% at the 3 hour mark, and elevated proteolysis can persist for up to 24 hours of the post-workout period \\[36\\].\n\nAlthough insulin has known anabolic properties \\[38,39\\], its primary impact post-exercise is believed to be anti-catabolic \\[40-43\\]. The mechanisms by which insulin reduces proteolysis are not well understood at this time. It has been theorized that insulin-mediated phosphorylation of PI3K\/Akt inhibits transcriptional activity of the proteolytic Forkhead family of transcription factors, resulting in their sequestration in the sarcoplasm away from their target genes \\[44\\]. Down-regulation of other aspects of the ubiquitin-proteasome pathway are also believed to play a role in the process \\[45\\]. Given that muscle hypertrophy represents the difference between myofibrillar protein synthesis and proteolysis, a decrease in protein breakdown would conceivably enhance accretion of contractile proteins and thus facilitate greater hypertrophy. Accordingly, it seems logical to conclude that consuming a protein-carbohydrate supplement following exercise would promote the greatest reduction in proteolysis since the combination of the two nutrients has been shown to elevate insulin levels to a greater extent than carbohydrate alone \\[28\\].\n\nHowever, while the theoretical basis behind spiking insulin post-workout is inherently sound, it remains questionable as to whether benefits extend into practice. First and foremost, research has consistently shown that, in the presence of elevated plasma amino acids, the effect of insulin elevation on net muscle protein balance plateaus within a range of 15\u201330 mU\/L \\[45,46\\]; roughly 3\u20134 times normal fasting levels. This insulinogenic effect is easily accomplished with typical mixed meals, considering that it takes approximately 1\u20132 hours for circulating substrate levels to peak, and 3\u20136 hours (or more) for a complete return to basal levels depending on the size of a meal. For example, Capaldo et al. \\[47\\] examined various metabolic effects during a 5-hour period after ingesting a solid meal comprised of 75 g carbohydrate 37 g protein, and 17 g fat. This meal was able to raise insulin 3 times above fasting levels within 30 minutes of consumption. At the 1-hour mark, insulin was 5 times greater than fasting. At the 5-hour mark, insulin was still double the fasting levels. In another example, Power et al. \\[48\\] showed that a 45g dose of whey protein isolate takes approximately 50 minutes to cause blood amino acid levels to peak. Insulin concentrations peaked 40 minutes after ingestion, and remained at elevations seen to maximize net muscle protein balance (15-30 mU\/L, or 104-208 pmol\/L) for approximately 2 hours. The inclusion of carbohydrate to this protein dose would cause insulin levels to peak higher and stay elevated even longer. Therefore, the recommendation for lifters to spike insulin post-exercise is somewhat trivial. The classical post-exercise objective to quickly reverse catabolic processes to promote recovery and growth may only be applicable in the absence of a properly constructed pre-exercise meal.\n\nMoreover, there is evidence that the effect of protein breakdown on muscle protein accretion may be overstated. Glynn et al. \\[49\\] found that the post-exercise anabolic response associated with combined protein and carbohydrate consumption was largely due to an elevation in muscle protein synthesis with only a minor influence from reduced muscle protein breakdown. These results were seen regardless of the extent of circulating insulin levels. Thus, it remains questionable as to what, if any, positive effects are realized with respect to muscle growth from spiking insulin after resistance training.\n\n## Protein synthesis\n\nPerhaps the most touted benefit of post-workout nutrient timing is that it potentiates increases in MPS. Resistance training alone has been shown to promote a twofold increase in protein synthesis following exercise, which is counterbalanced by the accelerated rate of proteolysis \\[36\\]. It appears that the stimulatory effects of hyperaminoacidemia on muscle protein synthesis, especially from essential amino acids, are potentiated by previous exercise \\[35,50\\]. There is some evidence that carbohydrate has an additive effect on enhancing post-exercise muscle protein synthesis when combined with amino acid ingestion \\[51\\], but others have failed to find such a benefit \\[52,53\\].\n\nSeveral studies have investigated whether an \"anabolic window\" exists in the immediate post-exercise period with respect to protein synthesis. For maximizing MPS, the evidence supports the superiority of post-exercise free amino acids and\/or protein (in various permutations with or without carbohydrate) compared to solely carbohydrate or non-caloric placebo \\[50,51,54-59\\]. However, despite the common recommendation to consume protein as soon as possible post-exercise \\[60,61\\], evidence-based support for this practice is currently lacking. Levenhagen et al. \\[62\\] demonstrated a clear benefit to consuming nutrients as soon as possible after exercise as opposed to delaying consumption. Employing a within-subject design,10 volunteers (5 men, 5 women) consumed an oral supplement containing 10 g protein, 8 g carbohydrate and 3 g fat either immediately following or three hours post-exercise. Protein synthesis of the legs and whole body was increased threefold when the supplement was ingested immediately after exercise, as compared to just 12% when consumption was delayed. A limitation of the study was that training involved moderate intensity, long duration aerobic exercise. Thus, the increased fractional synthetic rate was likely due to greater mitochondrial and\/or sarcoplasmic protein fractions, as opposed to synthesis of contractile elements \\[36\\]. In contrast to the timing effects shown by Levenhagen et al. \\[62\\], previous work by Rasmussen et al. \\[56\\] showed no significant difference in leg net amino acid balance between 6 g essential amino acids (EAA) co-ingested with 35 g carbohydrate taken 1 hour versus 3 hours post-exercise. Compounding the unreliability of the post-exercise 'window' is the finding by Tipton et al. \\[63\\] that immediate pre-exercise ingestion of the same EAA-carbohydrate solution resulted in a significantly greater and more sustained MPS response compared to the immediate post-exercise ingestion, although the validity of these findings have been disputed based on flawed methodology \\[36\\]. Notably, Fujita et al \\[64\\] saw opposite results using a similar design, except the EAA-carbohydrate was ingested 1 hour prior to exercise compared to ingestion immediately pre-exercise in Tipton et al. \\[63\\]. Adding yet more incongruity to the evidence, Tipton et al. \\[65\\] found no significant difference in net MPS between the ingestion of 20 g whey immediately pre- versus the same solution consumed 1 hour post-exercise. Collectively, the available data lack any consistent indication of an ideal post-exercise timing scheme for maximizing MPS.\n\nIt also should be noted that measures of MPS assessed following an acute bout of resistance exercise do not always occur in parallel with chronic upregulation of causative myogenic signals \\[66\\] and are not necessarily predictive of long-term hypertrophic responses to regimented resistance training \\[67\\]. Moreover, the post-exercise rise in MPS in untrained subjects is not recapitulated in the trained state \\[68\\], further confounding practical relevance. Thus, the utility of acute studies is limited to providing clues and generating hypotheses regarding hypertrophic adaptations; any attempt to extrapolate findings from such data to changes in lean body mass is speculative, at best.\n\n## Muscle hypertrophy\n\nA number of studies have directly investigated the long-term hypertrophic effects of post-exercise protein consumption. The results of these trials are curiously conflicting, seemingly because of varied study design and methodology. Moreover, a majority of studies employed both pre- and post-workout supplementation, making it impossible to tease out the impact of consuming nutrients after exercise. These confounding issues highlight the difficulty in attempting to draw relevant conclusions as to the validity of an \"anabolic window.\" What follows is an overview of the current research on the topic. Only those studies that specifically evaluated immediate (\u2264 1 hour) post-workout nutrient provision are discussed (see Table\u2009<a href=\"#T1\" data-ref-type=\"table\">1<\/a> for a summary of data).\n\nPost-exercise nutrition and muscle hypertrophy\n\n| **Study** | **Subjects** | **Supplementation** | **Protein matched with Control?** | **Measurement instrument** | **Training protocol** | **Results** |\n|:---|:---|:---|:---|:---|:---|:---|\n| Esmarck et al. \\[69\\] | 13 untrained elderly males | 10 g milk\/soy protein combo consumed either immediately or 2 hours after exercise | Yes | MRI and muscle biopsy | Progressive resistance training consisting of multiple sets of lat pulldown, leg press and knee extension performed 3 days\/wk for 12 wk | Significant increase in muscle CSA with immediate vs. delayed supplementation |\n| Cribb and Hayes \\[70\\] | 23 young recreational male bodybuilders | 1 g\/kg of a supplement containing 40 g whey isolate, 43 g glucose, and 7 g creatine monohydrate consumed either immediately before and after exercise or in the early morning and late evening | Yes | DXA and muscle biopsy | Progressive resistance training consisting of exercises for the major muscle groups performed 3 days\/wk for 10 wks | Significant increases in lean body mass and muscle CSA of type II fibers in immediate vs. delayed supplementation |\n| Willoughby et al. \\[71\\] | 19 untrained young males | 20 g protein or 20 g dextrose consumed 1 hour before and after exercise | No | Hydrostatic weighing, muscle biopsy, surface measurements | Progressive resistance training consisting of 3 sets of 6\u20138 repetitions for all the major muscles performed 4 days\/wk for 10 wks | Significant increase in total body mass, fat-free mass, and thigh mass with protein vs. carb supplementation |\n| Hulmi et al. \\[72\\] | 31 untrained young males | 15 g whey isolate or placebo consumed immediately before and after exercise | No | MRI, muscle biopsy | Progressive, periodized total body resistance training consisting of 2\u20135 sets of 5\u201320 repetitions performed 2 days\/wk for 21 wks. | Significant increase in CSA of the vastus lateralis but not of the other quadriceps muscles in supplemented group versus placebo. |\n| Verdijk et al. \\[73\\] | 28 untrained elderly males | 10 g casein hydrolysate or placebo consumed immediately before and after exercise | No | DXA, CT, and muscle biopsy | Progressive resistance training consisting of multiple sets of leg press and knee extension performed 3 days\/wk for 12 wks | No significant differences in muscle CSA between groups |\n| Hoffman et al. \\[74\\] | 33 well-trained young males | Supplement containing 42 g protein (milk\/collagen blend) and 2 g carbohydrate consumed either immediately before and after exercise or in the early morning and late evening | Yes | DXA | Progressive resistance training consisting of 3\u20134 sets of 6\u201310 repetitions of multiple exercises for the entire body peformed 4 days\/wk for 10 weeks. | No significant differences in total body mass or lean body mass between groups. |\n| Erskine et al. \\[75\\] | 33 untrained young males | 20 g high quality protein or placebo consumed immediately before and after exercise | No | MRI | 4-6 sets of elbow flexion performed 3 days\/wk for 12 weeks | No significant differences in muscle CSA between groups |\n\nEsmarck et al. \\[69\\] provided the first experimental evidence that consuming protein immediately after training enhanced muscular growth compared to delayed protein intake. Thirteen untrained elderly male volunteers were matched in pairs based on body composition and daily protein intake and divided into two groups: P0 or P2. Subjects performed a progressive resistance training program of multiple sets for the upper and lower body. P0 received an oral protein\/carbohydrate supplement immediately post-exercise while P2 received the same supplement 2 hours following the exercise bout. Training was carried out 3 days a week for 12 weeks. At the end of the study period, cross-sectional area (CSA) of the quadriceps femoris and mean fiber area were significantly increased in the P0 group while no significant increase was seen in P2. These results support the presence of a post-exercise window and suggest that delaying post-workout nutrient intake may impede muscular gains.\n\nIn contrast to these findings, Verdijk et al. \\[73\\] failed to detect any increases in skeletal muscle mass from consuming a post-exercise protein supplement in a similar population of elderly men. Twenty-eight untrained subjects were randomly assigned to receive either a protein or placebo supplement consumed immediately before and immediately following the exercise session. Subjects performed multiple sets of leg press and knee extension 3 days per week, with the intensity of exercise progressively increased over the course of the 12 week training period. No significant differences in muscle strength or hypertrophy were noted between groups at the end of the study period indicating that post exercise nutrient timing strategies do not enhance training-related adaptation. It should be noted that, as opposed to the study by Esmark et al. \\[69\\] this study only investigated adaptive responses of supplementation on the thigh musculature; it therefore is not clear based on these results whether the upper body might respond differently to post-exercise supplementation than the lower body.\n\nIn an elegant single-blinded design, Cribb and Hayes \\[70\\] found a significant benefit to post-exercise protein consumption in 23 recreational male bodybuilders. Subjects were randomly divided into either a PRE-POST group that consumed a supplement containing protein, carbohydrate and creatine immediately before and after training or a MOR-EVE group that consumed the same supplement in the morning and evening at least 5 hours outside the workout. Both groups performed regimented resistance training that progressively increased intensity from 70% 1RM to 95% 1RM over the course of 10 weeks. Results showed that the PRE-POST group achieved a significantly greater increase in lean body mass and increased type II fiber area compared to MOR-EVE. Findings support the benefits of nutrient timing on training-induced muscular adaptations. The study was limited by the addition of creatine monohydrate to the supplement, which may have facilitated increased uptake following training. Moreover, the fact that the supplement was taken both pre- and post-workout confounds whether an anabolic window mediated results.\n\nWilloughby et al. \\[71\\] also found that nutrient timing resulted in positive muscular adaptations. Nineteen untrained male subjects were randomly assigned to either receive 20 g of protein or 20 grams dextrose administered 1 hour before and after resistance exercise. Training consisted of 3 sets of 6\u20138 repetitions at 85%\u201390% intensity. Training was performed 4 times a week over the course of 10 weeks. At the end of the study period, total body mass, fat-free mass, and thigh mass was significantly greater in the protein-supplemented group compared to the group that received dextrose. Given that the group receiving the protein supplement consumed an additional 40 grams of protein on training days, it is difficult to discern whether results were due to the increased protein intake or the timing of the supplement.\n\nIn a comprehensive study of well-trained subjects, Hoffman et al. \\[74\\] randomly assigned 33 well-trained males to receive a protein supplement either in the morning and evening (n\u2009=\u200913) or immediately before and immediately after resistance exercise (n\u2009=\u200913). Seven participants served as unsupplemented controls. Workouts consisted of 3\u20134 sets of 6\u201310 repetitions of multiple exercises for the entire body. Training was carried out on 4 day-a-week split routine with intensity progressively increased over the course of the study period. After 10 weeks, no significant differences were noted between groups with respect to body mass and lean body mass. The study was limited by its use of DXA to assess body composition, which lacks the sensitivity to detect small changes in muscle mass compared to other imaging modalities such as MRI and CT \\[76\\].\n\nHulmi et al. \\[72\\] randomized 31 young untrained male subjects into 1 of 3 groups: protein supplement (n\u2009=\u200911), non-caloric placebo (n\u2009=\u200910) or control (n\u2009=\u200910). High-intensity resistance training was carried out over 21 weeks. Supplementation was provided before and after exercise. At the end of the study period, muscle CSA was significantly greater in the protein-supplemented group compared to placebo or control. A strength of the study was its long-term training period, providing support for the beneficial effects of nutrient timing on chronic hypertrophic gains. Again, however, it is unclear whether enhanced results associated with protein supplementation were due to timing or increased protein consumption.\n\nMost recently, Erskine et al. \\[75\\] failed to show a hypertrophic benefit from post-workout nutrient timing. Subjects were 33 untrained young males, pair-matched for habitual protein intake and strength response to a 3-week pre-study resistance training program. After a 6-week washout period where no training was performed, subjects were then randomly assigned to receive either a protein supplement or a placebo immediately before and after resistance exercise. Training consisted of 6\u2013 8 sets of elbow flexion carried out 3 days a week for 12 weeks. No significant differences were found in muscle volume or anatomical cross-sectional area between groups.\n\n# Discussion\n\nDespite claims that immediate post-exercise nutritional intake is essential to maximize hypertrophic gains, evidence-based support for such an \"anabolic window of opportunity\" is far from definitive. The hypothesis is based largely on the pre-supposition that training is carried out in a fasted state. During fasted exercise, a concomitant increase in muscle protein breakdown causes the pre-exercise net negative amino acid balance to persist in the post-exercise period despite training-induced increases in muscle protein synthesis \\[36\\]. Thus, in the case of resistance training after an overnight fast, it would make sense to provide immediate nutritional intervention--ideally in the form of a combination of protein and carbohydrate--for the purposes of promoting muscle protein synthesis and reducing proteolysis, thereby switching a net catabolic state into an anabolic one. Over a chronic period, this tactic could conceivably lead cumulatively to an increased rate of gains in muscle mass.\n\nThis inevitably begs the question of how pre-exercise nutrition might influence the urgency or effectiveness of post-exercise nutrition, since not everyone engages in fasted training. In practice, it is common for those with the primary goal of increasing muscular size and\/or strength to make a concerted effort to consume a pre-exercise meal within 1-2 hours prior to the bout in attempt to maximize training performance. Depending on its size and composition, this meal can conceivably function as both a pre- and an immediate post-exercise meal, since the time course of its digestion\/absorption can persist well into the recovery period. Tipton et al. \\[63\\] observed that a relatively small dose of EAA (6 g) taken immediately pre-exercise was able to elevate blood and muscle amino acid levels by roughly 130%, and these levels remained elevated for 2 hours after the exercise bout. Although this finding was subsequently challenged by Fujita et al. \\[64\\], other research by Tipton et al. \\[65\\] showed that the ingestion of 20 g whey taken immediately pre-exercise elevated muscular uptake of amino acids to 4.4 times pre-exercise resting levels during exercise, and did not return to baseline levels until 3 hours post-exercise. These data indicate that even minimal-to-moderate pre-exercise EAA or high-quality protein taken immediately before resistance training is capable of sustaining amino acid delivery into the post-exercise period. Given this scenario, immediate post-exercise protein dosing for the aim of mitigating catabolism seems redundant. The next scheduled protein-rich meal (whether it occurs immediately or 1\u20132 hours post-exercise) is likely sufficient for maximizing recovery and anabolism.\n\nOn the other hand, there are others who might train before lunch or after work, where the previous meal was finished 4\u20136 hours prior to commencing exercise. This lag in nutrient consumption can be considered significant enough to warrant post-exercise intervention if muscle retention or growth is the primary goal. Layman \\[77\\] estimated that the anabolic effect of a meal lasts 5-6 hours based on the rate of postprandial amino acid metabolism. However, infusion-based studies in rats \\[78,79\\] and humans \\[80,81\\] indicate that the postprandial rise in MPS from ingesting amino acids or a protein-rich meal is more transient, returning to baseline within 3 hours despite sustained elevations in amino acid availability. It thus has been hypothesized that a \"muscle full\" status can be reached where MPS becomes refractory, and circulating amino acids are shunted toward oxidation or fates other than MPS. In light of these findings, when training is initiated more than \\~3\u20134 hours after the preceding meal, the classical recommendation to consume protein (at least 25 g) as soon as possible seems warranted in order to reverse the catabolic state, which in turn could expedite muscular recovery and growth. However, as illustrated previously, minor pre-exercise nutritional interventions can be undertaken if a significant delay in the post-exercise meal is anticipated.\n\nAn interesting area of speculation is the generalizability of these recommendations across training statuses and age groups. Burd et al. \\[82\\] reported that an acute bout of resistance training in untrained subjects stimulates both mitochondrial and myofibrillar protein synthesis, whereas in trained subjects, protein synthesis becomes more preferential toward the myofibrillar component. This suggests a less global response in advanced trainees that potentially warrants closer attention to protein timing and type (e.g., high-leucine sources such as dairy proteins) in order to optimize rates of muscular adaptation. In addition to training status, age can influence training adaptations. Elderly subjects exhibit what has been termed \"anabolic resistance,\" characterized by a lower receptivity to amino acids and resistance training \\[83\\]. The mechanisms underlying this phenomenon are not clear, but there is evidence that in younger adults, the acute anabolic response to protein feeding appears to plateau at a lower dose than in elderly subjects. Illustrating this point, Moore et al. \\[84\\] found that 20 g whole egg protein maximally stimulated post-exercise MPS, while 40 g increased leucine oxidation without any further increase in MPS in young men. In contrast, Yang et al. \\[85\\] found that elderly subjects displayed greater increases in MPS when consuming a post-exercise dose of 40 g whey protein compared to 20 g. These findings suggest that older subjects require higher individual protein doses for the purpose of optimizing the anabolic response to training. Further research is needed to better assess post-workout nutrient timing response across various populations, particularly with respect to trained\/untrained and young\/elderly subjects.\n\nThe body of research in this area has several limitations. First, while there is an abundance of acute data, controlled, long-term trials that systematically compare the effects of various post-exercise timing schemes are lacking. The majority of chronic studies have examined pre- and post-exercise supplementation simultaneously, as opposed to comparing the two treatments against each other. This prevents the possibility of isolating the effects of either treatment. That is, we cannot know whether pre- or post-exercise supplementation was the critical contributor to the outcomes (or lack thereof). Another important limitation is that the majority of chronic studies neglect to match total protein intake between the conditions compared. As such, it's not possible to ascertain whether positive outcomes were influenced by timing relative to the training bout, or simply by a greater protein intake overall. Further, dosing strategies employed in the preponderance of chronic nutrient timing studies have been overly conservative, providing only 10\u201320 g protein near the exercise bout. More research is needed using protein doses known to maximize acute anabolic response, which has been shown to be approximately 20\u201340 g, depending on age \\[84,85\\]. There is also a lack of chronic studies examining the co-ingestion of protein and carbohydrate near training. Thus far, chronic studies have yielded equivocal results. On the whole, they have not corroborated the consistency of positive outcomes seen in acute studies examining post-exercise nutrition.\n\nAnother limitation is that the majority of studies on the topic have been carried out in untrained individuals. Muscular adaptations in those without resistance training experience tend to be robust, and do not necessarily reflect gains experienced in trained subjects. It therefore remains to be determined whether training status influences the hypertrophic response to post-exercise nutritional supplementation.\n\nA final limitation of the available research is that current methods used to assess muscle hypertrophy are widely disparate, and the accuracy of the measures obtained are inexact \\[68\\]. As such, it is questionable whether these tools are sensitive enough to detect small differences in muscular hypertrophy. Although minor variances in muscle mass would be of little relevance to the general population, they could be very meaningful for elite athletes and bodybuilders. Thus, despite conflicting evidence, the potential benefits of post-exercise supplementation cannot be readily dismissed for those seeking to optimize a hypertrophic response. By the same token, widely varying feeding patterns among individuals challenge the common assumption that the post-exercise \"anabolic window of opportunity\" is universally narrow and urgent.\n\n## Practical applications\n\nDistilling the data into firm, specific recommendations is difficult due to the inconsistency of findings and scarcity of systematic investigations seeking to optimize pre- and\/or post-exercise protein dosage and timing. Practical nutrient timing applications for the goal of muscle hypertrophy inevitably must be tempered with field observations and experience in order to bridge gaps in the scientific literature. With that said, high-quality protein dosed at 0.4\u20130.5 g\/kg of LBM at both pre- and post-exercise is a simple, relatively fail-safe general guideline that reflects the current evidence showing a maximal acute anabolic effect of 20\u201340 g \\[53,84,85\\]. For example, someone with 70 kg of LBM would consume roughly 28\u201335 g protein in both the pre- and post exercise meal. Exceeding this would be have minimal detriment if any, whereas significantly under-shooting or neglecting it altogether would not maximize the anabolic response.\n\nDue to the transient anabolic impact of a protein-rich meal and its potential synergy with the trained state, pre- and post-exercise meals should not be separated by more than approximately 3\u20134 hours, given a typical resistance training bout lasting 45\u201390 minutes. If protein is delivered within particularly large mixed-meals (which are inherently more anticatabolic), a case can be made for lengthening the interval to 5\u20136 hours. This strategy covers the hypothetical timing benefits while allowing significant flexibility in the length of the feeding windows before and after training. Specific timing within this general framework would vary depending on individual preference and tolerance, as well as exercise duration. One of many possible examples involving a 60-minute resistance training bout could have up to 90-minute feeding windows on both sides of the bout, given central placement between the meals. In contrast, bouts exceeding typical duration would default to shorter feeding windows if the 3\u20134 hour pre- to post-exercise meal interval is maintained. Shifting the training session closer to the pre- or post-exercise meal should be dictated by personal preference, tolerance, and lifestyle\/scheduling constraints.\n\nEven more so than with protein, carbohydrate dosage and timing relative to resistance training is a gray area lacking cohesive data to form concrete recommendations. It is tempting to recommend pre- and post-exercise carbohydrate doses that at least match or exceed the amounts of protein consumed in these meals. However, carbohydrate availability during and after exercise is of greater concern for endurance as opposed to strength or hypertrophy goals. Furthermore, the importance of co-ingesting post-exercise protein and carbohydrate has recently been challenged by studies examining the early recovery period, particularly when sufficient protein is provided. Koopman et al \\[52\\] found that after full-body resistance training, adding carbohydrate (0.15, or 0.6 g\/kg\/hr) to amply dosed casein hydrolysate (0.3 g\/kg\/hr) did not increase whole body protein balance during a 6-hour post-exercise recovery period compared to the protein-only treatment. Subsequently, Staples et al \\[53\\] reported that after lower-body resistance exercise (leg extensions), the increase in post-exercise muscle protein balance from ingesting 25 g whey isolate was not improved by an additional 50 g maltodextrin during a 3-hour recovery period. For the goal of maximizing rates of muscle gain, these findings support the broader objective of meeting total daily carbohydrate need instead of specifically timing its constituent doses. Collectively, these data indicate an increased potential for dietary flexibility while maintaining the pursuit of optimal timing.\n\n# Competing interests\n\nThe authors declare that they have no competing interests.\n\n# Authors' contribution\n\nAAA and BJS each contributed equally to the formulation and writing of the manuscript. Both authors read and approved the final manuscript.","meta":{"dup_signals":{"dup_doc_count":211,"dup_dump_count":85,"dup_details":{"curated_sources":4,"2023-40":1,"2023-23":3,"2023-14":2,"2022-49":1,"2022-40":2,"2022-33":3,"2022-27":1,"2022-21":3,"2021-49":1,"2021-43":2,"2021-39":1,"2021-31":3,"2021-25":1,"2021-21":2,"2021-17":1,"2021-10":2,"2020-50":1,"2020-40":1,"2020-34":1,"2020-29":1,"2020-24":1,"2020-16":3,"2020-10":1,"2020-05":1,"2019-47":1,"2019-43":2,"2019-35":1,"2019-30":2,"2019-22":1,"2019-13":2,"2019-09":1,"2019-04":1,"2018-51":2,"2018-47":1,"2018-39":2,"2018-30":4,"2018-26":1,"2018-22":2,"2018-17":2,"2018-13":4,"2018-09":2,"2017-51":5,"2017-47":1,"2017-43":4,"2017-39":2,"2017-34":4,"2017-30":1,"2017-26":5,"2017-22":4,"2017-17":1,"2017-09":5,"2017-04":3,"2016-50":3,"2016-44":3,"2016-40":3,"2016-36":3,"2016-30":4,"2016-26":3,"2016-22":3,"2016-18":3,"2016-07":2,"2015-48":3,"2015-40":3,"2015-35":3,"2015-32":3,"2015-27":3,"2015-22":3,"2015-14":2,"2014-52":3,"2014-49":4,"2014-42":8,"2014-41":4,"2014-35":6,"2014-23":6,"2014-15":3,"2023-50":1,"2024-22":2,"2024-18":4,"2024-10":3,"2017-13":3,"2015-18":2,"2015-11":2,"2015-06":2,"2014-10":1,"2013-48":1}},"file":"PMC3577439"},"subset":"pubmed_central"}
{"text":"abstract: Following extensive examination of published and unpublished materials, we provide a history of the use of dexamethasone in pregnant women at risk of carrying a female fetus affected by congenital adrenal hyperplasia (CAH). This intervention has been aimed at preventing development of ambiguous genitalia, the urogenital sinus, tomboyism, and lesbianism. We map out ethical problems in this history, including: misleading promotion to physicians and CAH-affected families; de facto experimentation without the necessary protections of approved research; troubling parallels to the history of prenatal use of diethylstilbestrol (DES); and the use of medicine and public monies to attempt prevention of benign behavioral sex variations. Critical attention is directed at recent investigations by the U.S. Food and Drug Administration (FDA) and Office of Human Research Protections (OHRP); we argue that the weak and unsupported conclusions of these investigations indicate major gaps in the systems meant to protect subjects of high-risk medical research.\nauthor: Alice Dreger; Ellen K. Feder; Anne Tamar-Mattis\ndate: 2012-07-31\nreferences:\nsubtitle: : An Ethics Canary in the Modern Medical Mine\ntitle: Prenatal Dexamethasone for Congenital Adrenal Hyperplasia\n\n# Introduction\/Our Backgrounds\n\nIn this article, we provide a condensed history of the use of prenatal dexamethasone for congenital adrenal hyperplasia, with an eye toward the ethically problematic aspects of this history. Congenital adrenal hyperplasia (CAH) is a disease of the endocrine system that can cause virilization (i.e., development of masculine traits) in female fetuses. In an attempt to prevent CAH-affected female fetuses from developing in a sexually atypical fashion, some physicians treat pregnant women \"at risk\" for having an affected daughter with the steroid dexamethasone. This intervention starts as soon as pregnancy is confirmed and continues throughout the pregnancy if the fetus is ultimately diagnosed as a CAH-affected female. If\u2014several weeks into the dosing\u2014the fetus is determined to be male or not CAH-affected, the intervention is immediately stopped, because the intention is only to alter the course of development in CAH-affected females.\n\nThis use of dexamethasone was first described in 1984 in *The Journal of Pediatrics* by Michel David and Maguelone Forest, French clinician-researchers. David and Forest reported apparent effectiveness of prenatal dexamethasone in eliminating genital virilization in a single girl affected by CAH (David and Forest 1984). In the nearly three decades since David and Forest's paper, many specialists have come to believe that prenatal dexamethasone for CAH constitutes the standard of care. A 2000\u20132001 survey of members of the European Society for Paediatric Endocrinology, representing 125 institutions, found that, \"\\[i\\]n 57\u00a0% of the centres prenatal diagnosis and treatment \\[of CAH with dexamethasone\\] are routine\" (Riepe et al. 2002, 199). A 2010 Continuing Medical Education review article in the *Obstetrical and Gynecological Survey* concluded: \"Given the data available at this moment, antenatal treatment with corticosteroids is recommended\" (Vos and Bruinse 2010, 203).\n\nBut what data are \"available at this moment\" such that prenatal dexamethasone for CAH can be recommended to obstetricians, and recommended by them to prospective patients?\n\nA systematic review and meta-analysis of this intervention, published in 2010 in *Clinical Endocrinology*, indicated that a search of the literature \"identified 1083 candidate studies for review; of which, only four studies were confirmed eligible\" for serious scientific consideration (Fern\u00e1ndez-Balsells et al. 2010, 438). That is to say, as late as 2010, less than one half of one percent of published \"studies\" of this intervention were regarded as being of high enough quality to provide meaningful data for a meta-analysis. Even these four studies were of low quality:\n\n> All the eligible studies were observational and were conducted by two groups of investigators (one from the US and one from Europe). \u2026 Studies lacked details regarding the use of methodological features that protect against bias. None of the studies reported blinding of the outcome assessors to the exposure (i.e., the researchers estimating each patient's degree of virilization). Loss to follow-up was, in most cases, substantial (Fern\u00e1ndez-Balsells et al. 2010, 438).\n\nIn spite of at least a thousand pregnant women likely having been exposed by the time of the review, the four studies judged worthy of inclusion in the meta-analysis covered only 325 pregnancies. Even more stunning, the meta-analysis revealed, \"*there were no data on long-term follow-up of physical and metabolic outcomes in children exposed to dexamethasone*\" prenatally for CAH (Fern\u00e1ndez-Balsells et al. 2010, 436, *emphasis added*). It was not that the data about long-term physical and metabolic outcomes were unclear; there simply were none available.\n\nToday, some clinicians promote prenatal dexamethasone for CAH as \"an excellent example of pharmacological therapy during pregnancy\" (Rosner et al. 2006, 803) and even as \"a paradigm of prenatal diagnosis and treatment\" (Nimkarn and New 2010a, 5). Yet the Endocrine Society Task Force that had commissioned the 2010 meta-analysis concluded: \"The evidence regarding fetal and maternal sequelae \u2026 is of low or very low quality due to methodological limitations and sample sizes\" (Speiser et al. 2010a, 4137). This would hardly seem to qualify prenatal dexamethasone for CAH as \"an excellent example\" or a \"paradigm\" of a prenatal pharmacological intervention. Indeed, for lack of quality clinical studies, the 2010 Task Force could not even say with any confidence whether prenatal dexamethasone works to reduce genital virilization. Notice the specific qualifications included in the Task Force's statement on efficacy: \"*\\[T\\]he groups advocating and performing prenatal treatment appear to agree* that it is effective in reducing and often eliminating virilization of female fetal genitalia and that the success rate is about 80\u201385\u00a0%\" (Speiser et al. 2010a, 4138, *emphasis added*).\n\nRegardless of some people's enthusiastic endorsements of the intervention, we show below that ethical debates within medicine about prenatal dexamethasone for CAH are actually not new. Those internal debates, however, have focused on potential risks and benefits to mothers and children exposed. There are a number of other ethical problems in the history of this intervention also deserving of attention, including: de facto experimentation on fetuses and pregnant women, largely outside of prospective long-term trials and without adequate informed consent; failure to appropriately collect and publish evidence when promoting and providing a high-risk intervention; use of medicine and public monies for research to prevent benign behavioral sex variations, including tomboyism and lesbianism (cf. Murphy 1997); and inadequacy in the United States of systems designed to protect subjects of medical experimentation, including especially pregnant women and their offspring.\n\nIn the United States, the use of prenatal dexamethasone remains \"off-label.\" This means that the indication has never received approval by the Food and Drug Administration (FDA).[^1] Nonetheless, we want to be clear: The problem we see with the use of prenatal dexamethasone for CAH is not per se that it is an off-label use; it is rather that\u2014as this paper documents\u2014prenatal dexamethasone for CAH has sometimes been promoted to prospective patients and clinicians in misleading ways, and sometimes promoted for uses that are not legitimately medical (e.g., for the prevention of tomboyism and lesbianism). Furthermore, this intervention\u2014*intended* to alter the course of fetal development\u2014has been \"studied\" in ways so slipshod as to breach professional standards of medical ethics.\n\nWe come to this work as history, philosophy, and legal scholars interested in the medical treatment of children with atypical sex, but also as women who have long advocated clinical reform in this general area. Late in 2009, clinicians working in the pediatric care of children born with sex anomalies made one of us (Dreger) aware of their growing alarm about prenatal dexamethasone for CAH. These clinicians were concerned that pregnant women at risk for having daughters with CAH were being given prenatal dexamethasone without being informed that (a) this use has consistently been labeled experimental by expert panels; (b) benefits and risks have not been established, due to inadequate scientific study; and (c) some children who had been exposed in utero were being studied retrospectively, in many cases years later, by the very clinicians who had been (and were still) actively promoting the use to pregnant women as \"safe for mother and child,\" to find out what the risks might really be.\n\nDreger (a historian) then reviewed the medical literature as well as Internet-based advertisements directed at affected families and became quite concerned. In December 2009 and January 2010, respectively, Dreger and UCLA pediatric geneticist Eric Vilain separately asked Mount Sinai School of Medicine pediatric endocrinologist Maria New, the most prominent promoter of this intervention, about the informed consent process she used. New is a highly distinguished pediatric endocrinologist and member of the National Academy of Sciences. By 2003, she had already publicly taken credit for having \"treated\" more than 600 pregnant women with dexamethasone in an attempt to prevent virilization in CAH-affected female fetuses (Kitzinger 2003), putting her efforts in this area well beyond any other clinical researcher's (see also New et al. 2001). Dreger's preliminary research indicated that, even while obtaining a federal grant promising to determine the actual safety and efficacy of prenatal dexamethasone for CAH through retrospective follow-up studies, New was functioning as a uniquely aggressive promoter of the intervention among parents and clinicians, repeatedly describing this intervention as \"safe for mother and child\" (New 2010a, \u00b64). But when Dreger and Vilain attempted to ask New about informed consent, both were rebuffed by New. Indeed, at the Miami medical conference session where Vilain pressed the issue (at New 2010b), New publicly admonished Vilain that his question to her was inappropriate.\n\nDreger then asked colleagues in bioethics and allied fields to join with her in raising concerns to the U.S. government regarding the potential failure to protect the rights of these women and their offspring. The second author of this paper (Feder) became the corresponding author on the resulting (February 2010) letters of concern from a total of 32 academicians to the Office of Human Research Protections (OHRP) and the FDA. The third author (Tamar-Mattis) handled most of our subsequent communications with federal agencies and conducted additional legal research.\n\nIn September 2010, the OHRP and FDA informed us they could find nothing worth pursuing further. The OHRP decided that abuse had not occurred because: (a) at least some of the pregnant women had been enrolled in IRB-approved studies when given the drug at Weill Cornell Medical College, where most of the interventions appear to have occurred while New worked there; (b) follow-up studies conducted at Mount Sinai School of Medicine under New had been IRB-approved; and (c) the fetal intervention itself does not require IRB oversight. The FDA explained that (to our surprise) regulations allow a clinician to promote an off-label use\u2014even an experimental use intended to alter fetal development\u2014as \"safe and effective\" so long as the clinician does not simultaneously work for the drug maker or count as an FDA-approved investigator of the drug (Borror 2010). (Dreger has made the agencies' full responses available at fetaldex.org.) Nevertheless, we show here that the material generated by the government's own investigations\u2014along with further scholarly inquiry on our part\u2014appear actually to *confirm* the concerns we expressed at the outset, suggesting a major failure of the layered systems designed to protect subjects of research, especially pregnant women and their fetuses.\n\nBecause the following history necessarily focuses attention on the actions of Maria New, we want to be sure that our readers appreciate that New's career has included critically important research and clinical care that has improved the lives, health, and fertility of a very large population. Her promotion of prenatal dexamethasone for CAH was no doubt motivated by a desire to improve the lives of her patients. The same beneficent attitude was likely present in all or most of the clinicians who used prenatal dexamethasone for CAH. But it is worth remembering that many cases in the history of medicine now rightly understood as ethically problematic were carried out by clinical researchers with good intentions (Eder 2011; Reverby 2009; Skloot 2010).\n\nIt has been impossible for us not to be aware that, as we have researched this fetal intervention, the medical world has been marking the 40^th^ anniversary of the 1971 publication of a study reporting a relatively high number of occurrences of a rare vaginal cancer in girls and young women who had been exposed in utero to diethylstilbestrol (DES). It was this small 1971 study\u2014eight subjects with adenocarcinoma of the vagina matched to 32 untreated controls\u2014that marked the beginning of the end of DES administration to pregnant women (Herbst, Ulfelder, and Poskanzer 1971). DES treatment during pregnancy had had admirable intentions, including primarily the prevention of miscarriage. Already by 1953, DES had actually been found ineffective for miscarriage prevention, but doctors continued to prescribe it to pregnant women anyway. Over the years, millions of fetuses were exposed, putting females and males at increased risk for rare cancers, genital anomalies, reproductive problems, etc. (Goodman, Schorge, and Greene 2011).\n\nAs we have spoken with others about how the use of prenatal dexamethasone for CAH has played out, it has been our experience that many have been skeptical that a DES-like scenario could occur with prenatal dexamethasone for CAH after what physicians learned from DES. We find ourselves wondering whether it is the assumption that \"we'll never do *that* again\" that paradoxically has blinded many clinicians to the striking parallels between DES and prenatal dexamethasone for CAH: Both DES and dexamethasone are powerful synthetic hormones; in both cases the practice involved administration of the intervention starting early in pregnancy; both were introduced into medical practice without much study as to efficacy and safety (Dreger et al. forthcoming).\n\nThree striking differences between these interventions are that: (1) this poorly studied yet widespread use of prenatal dexamethasone is happening *after* the lessons supposedly learned from DES; (2) while DES was never intended to alter fetal development, prenatal dexamethasone for CAH has explicitly aimed to do so; and (3) while DES was aimed at preventing fetal death, dexamethasone is directed at preventing something we would hope most people would understand to be substantially less dire, namely the development of atypical sex.\n\nYet rather than suggesting that the case of prenatal dexamethasone for CAH should be understood as one of the \"big\" stories of the history of medicine (like DES), we are suggesting something more disturbing: that this case appears to be representative of problems endemic in modern medicine, problems that threaten the health, lives, and rights of patients who continue to become unwitting subjects of (problematic) medical experimentation. Because so many systems of protection appear to have failed these women and children, we fear that prenatal dexamethasone for CAH is a canary in the modern medical mine.\n\n# CAH's Effects and the Goals of Using Prenatal Dexamethasone\n\nCongenital adrenal hyperplasia (CAH) is a genetic disease involving malfunction of the adrenal glands, endocrine organs that contribute to the production of sex steroids. CAH can occur in both males and females and can cause a number of metabolic problems, some of which may lead to postnatal adrenal crisis and death if left untreated. Because some forms of CAH are very dangerous, all U.S. states require newborn screening for CAH.\n\nThe prenatal administration of dexamethasone, a potent synthetic steroid of the glucocorticoid class, cannot prevent an affected child from being born with CAH. The intervention is aimed instead at causing CAH-affected female fetuses to develop in a more female-typical fashion than they otherwise might. Androgens contribute to sex differentiation, including in the brain and genitals; relatively low prenatal levels ordinarily result in a more female-typical development; relatively high levels usually result in male-typical development. In certain forms of CAH\u2014including 21-hydroxylase deficiency (21-OHD CAH), i.e., the type of CAH most at issue here\u2014the prenatal production of high levels of androgens may result in a genetic female (46,XX) fetus developing along a more masculine pathway neurologically and genitally. Prenatal dexamethasone is meant to engineer the CAH-affected female fetus's hormonal system to be typically female.\n\nThe intensity of CAH's effects on developing females varies. An affected genetic-female child might be born fairly female-typical, or she may be born with a large clitoris, labia that fuse and appear to form a scrotum, and an occluded or even absent lower vagina. Some affected genetic females have developed such masculine genitalia that they have been assumed at birth to be typical males and have been raised as boys (Eder 2011; Lee and Houk 2010). Newborn screening for CAH, aimed primarily at saving lives, has greatly reduced the likelihood of such children's conditions going undetected.\n\nAtypical clitorises and labia generally require no medical intervention for health. Despite evidence that elective genitoplasty may harm a girl (Crouch et al. 2004), pediatric urologists often perform surgery to \"feminize\" atypical clitorises and labia for what they call \"social reasons,\" including promotion of parent\u2013child bonding. They do so even while admitting we lack evidence that this approach is necessary or effective (Lee et al. 2006).\n\nSome CAH-affected girls are also born with a urogenital sinus, a condition in which the urethra and vagina are joined together. This creates potential for repetitive infections and also presents problems for sexual intercourse. Thus the urogenital sinus has traditionally been treated with pediatric surgery. It represents part of what prenatal dexamethasone is meant to prevent.\n\nWomen \"at risk\" of giving birth to a CAH-affected daughter are most often identified because they have already given birth to a CAH-affected child. Others are identified through genetic screening. Because \"the period during which the genitalia of a female fetus may become virilized begins only 6 \\[weeks\\] after conception, treatment must be instituted as soon as the woman knows she is pregnant\" (Speiser et al. 2010a, 4137; cf. Nimkarn and New 2007). Although this intervention is sometimes termed \"low-dose therapy\" (New 2010c), researchers estimate that \"the effective glucocorticoid doses reaching the fetus are 60\u2013100 times physiologic\" (Miller 2008, 17), meaning this intervention exposes the developing fetus to 60 to 100 times the normal level of glucocorticoids. The potential harms of prenatal dexamethasone represent a growing source of concern for clinical researchers because emerging research indicates glucocorticoids may alter \"fetal programming,\" potentially resulting in serious metabolic problems that will not become apparent until adulthood (Hirvikoski et al. 2007; Marciniak et al. 2011). Some animal studies, for example, suggest long-term risk to the cardiovascular system (Lajic, Nordenstr\u00f6m, and Hirvikoski 2011).\n\nCAH is an autosomal recessive disorder, so offspring of carrier parents have a 1 in 4 chance of having CAH and thus only a chance of 1 in 8 of being a CAH-affected female (since only half of the 1 in 4 will be females). While recent advances now may enable determination of fetal sex by the seventh week (Devaney et al. 2011), before 2011 the sex status of fetuses could not be determined reliably before 10 to 12\u00a0weeks. As a consequence, about 7 out of 8 (87.5\u00a0percent) of the individuals who have been exposed to many weeks of prenatal dexamethasone\u2014at 60 to 100 times normal levels\u2014never even had the condition that was being targeted with the prenatal intervention. These individuals have been \"necessarily\" exposed to risk in order to try to engineer the development of the 1 in 8 who would be CAH-affected females. Many of the clinicians who have raised ethical concerns about prenatal dexamethasone for CAH have specifically been concerned about the 87.5\u00a0percent (those 7 out of 8) exposed to the risk of first-trimester glucocorticoid \"therapy\" who stood no chance to benefit (e.g., Frias et al. 2001; Hirvikoski et al. 2007; Miller 2008; Speiser et al. 2010a).\n\nMost clinicians who have written about prenatal dexamethasone have spoken of its purpose as the prevention of ambiguous genitalia and the urogenital sinus. (Sometimes they speak of the urogenital sinus as part of \"ambiguous genitalia,\" and sometimes they speak of these as two sets of concerns.) But interestingly, some clinicians also speak of prenatal dexamethasone's purpose as the prevention of feminizing genital *surgeries*. So one elective risky intervention (prenatal dexamethasone) has been represented as necessary to prevent another (feminizing genital surgeries) (Nimkarn and New 2010a). Notably, in 2006, a major consensus of the American and European pediatric endocrine groups, known as \"the Chicago consensus,\" urged a more conservative approach to surgeries for genital anomalies, stating that, for girls, \"surgery should only be considered in cases of severe virilization\" (Lee et al. 2006, e491). But this recommendation does not appear to have had much (if any) effect on the promotion of prenatal dexamethasone as a preventive to \"feminizing\" surgery.\n\nStudies have shown that girls with 21-OHD CAH exhibit increased rates of what clinicians call \"behavioral masculinization,\" i.e., behaviors that are more male-typical. These girls are, on average, more interested in boy-typical play, hobbies, and subjects than non-affected females, less interested in becoming mothers, and more likely to grow up to be lesbian or bisexual (Meyer-Bahlburg 1999; Meyer-Bahlburg et al. 2006). The rate of adult identification as male is significantly higher in this group than in the general population of people with an XX karyotype; clinician-researchers report that about 5\u00a0percent of CAH-affected genetic-females may ultimately self-identify as male (Dessens, Slijper, and Drop 2005). For this reason, some clinicians have considered recommending raising highly virilized 46,XX CAH-affected babies as boys (Lee and Houk 2010; cf. Eder 2011).\n\nAlthough many researchers have hinted that prenatal dexamethasone might be good for preventing the \"behavioral masculinization\" associated with CAH (e.g., Lajic et al. 1998), this potential \"benefit\" has been spelled out most explicitly in the work of Maria New (Dreger, Feder, and Tamar-Mattis 2010). Writing with another pediatric endocrinologist in the 2010 *Annals of the New York Academy of Sciences* in an article that disregarded the 2006 Chicago consensus, New summed up the situation thus:\n\n> Without prenatal therapy, masculinization of external genitalia in females is potentially devastating. It carries the risk of wrong sex assignment at birth, difficult reconstructive surgery, and subsequent long-term effects on quality of life. Gender-related behaviors, namely childhood play, peer association, career and leisure time preferences in adolescence and adulthood, maternalism \\[interest in being a mother\\], aggression, and sexual orientation become masculinized in 46,XX girls and women with 21HOD deficiency. \u2026 Genital sensitivity impairment and difficulties in sexual function in women who underwent genitoplasty early in life have likewise been reported. We anticipate that prenatal dexamethasone therapy will reduce the well-documented behavioral masculinization and difficulties related to reconstructive surgeries (Nimkarn and New 2010a, 9).\n\nAmong those advocating prenatal dexamethasone, New seems to have been particularly concerned that CAH-affected girls may fail to grow up to be heterosexual wives and mothers. Speaking to a group of parents of children with CAH in 2001 at a meeting organized by the CARES Foundation, New showed a photo of a girl with ambiguous genitalia and said:\n\n> The challenge here is \u2026 to see what could be done to restore this baby to the normal female appearance which would be compatible with her parents presenting her as a girl, with her eventually becoming somebody's wife, and having normal sexual development, and becoming a mother. And she has all the machinery for motherhood, and therefore nothing should stop that, if we can repair her surgically and help her psychologically to continue to grow and develop as a girl (New 2001a).\n\nIn a 1999 paper entitled \"What Causes Low Rates of Child-Bearing in Congenital Adrenal Hyperplasia?\", New's chief collaborator in psychoneuroendocrine studies of CAH, Heino Meyer-Bahlburg of Columbia University, noted:\n\n> CAH women as a group have a lower interest than controls in getting married and performing the traditional child-care\/housewife role. As children, they show an unusually low interest in engaging in maternal play with baby dolls, and their interest in caring for infants, the frequency of daydreams or fantasies of pregnancy and motherhood, or the expressed wish of experiencing pregnancy and having children of their own appear to be relatively low in all age groups. (Meyer-Bahlburg 1999, 1845\u20131846).\n\nMeyer-Bahlburg posited that \"\\[l\\]ong term follow-up studies of the behavioral outcome will show whether \\[prenatal\\] dexamethasone treatment also prevents the effects of prenatal androgens on brain and behavior\" (1999, 1846).\n\nSurprisingly, results from our Freedom of Information Act (FOIA) requests\u2014made as part of our attempt to understand this history\u2014indicate that the U.S. National Institutes of Health (NIH) have funded New to see whether prenatal dexamethasone \"works\" to make more CAH-affected girls straight and interested in having babies. New's 1996 grant application states that\n\n> genital abnormalities and often multiple corrective surgeries needed affect social interaction, self image, romantic and sexual life, and fertility. As a consequence, many of these patients, and the majority of women with the salt-losing variant \\[of CAH\\], appear to remain childless and single. Preventive prenatal dexamethasone exposure is expected to improve this situation (New 1996a, 38).\n\nNew's NIH grant application specifically promised to try to determine \"the success of DEX in suppressing behavioral masculinization\" (New 1996b, 17).\n\n# A Long History of Ineffective Calls for Ethical, Scientifically Rigorous Studies\n\nThe 2010 systematic review and meta-analysis of prenatal dexamethasone for CAH (mentioned in our introduction) was commissioned by an Endocrine Society Task Force charged with developing new consensus guidelines for the treatment of CAH. That Task Force was in turn co-sponsored by the American Academy of Pediatrics, the Lawson Wilkins Pediatric Endocrine Society, the European Society for Paediatric Endocrinology, the Society of Pediatric Urology, the European Society of Endocrinology, the CARES Foundation, and the Androgen Excess and PCOS Society. Based on the systematic review and meta-analysis, in its 2010 practice guidelines the Task Force concluded\n\n> that prenatal therapy *continue* to be regarded as experimental. \u2026 We suggest that prenatal therapy be pursued through protocols approved by Institutional Review Boards \\[i.e., ethics committees\\] at centers capable of collecting outcomes data on a sufficiently large number of patients so that risks and benefits of this treatment can be defined more precisely (Speiser et al. 2010a, 4137, *emphasis added*).\n\nThe named authors of this 2010 consensus (including Meyer-Bahlburg) appeared to have recognized that their call for use only within scientifically-meaningful clinical trials pre-approved by ethics committees was not novel. In fact, expert panels have consistently judged prenatal dexamethasone for CAH to be risky and experimental. In 2002, a joint statement from the Lawson Wilkins Pediatric Endocrine Society and the European Society for Paediatric Endocrinology had already said something similar\u2014even stronger:\n\n> We believe that this specialized and demanding therapy should be undertaken by designated teams using nationally or multinationally approved protocols, subject to institutional review boards \\[IRBs\\] or ethics committees in recognized centers. Written informed consent must be obtained. \u2026 Families and clinicians should be obliged to undertake *prospective* follow-up of prenatally treated children whether they have CAH or not. The data should be entered into a central database *audited by an independent safety committee*. (Joint LWPES\/ESPE CAH Working Group 2002, 4049, *emphasis added*).\n\nAnd a year earlier, in a tense exchange of letters with New in the journal *Pediatrics*, representatives of the Section on Endocrinology and Committee on Genetics of the American Academy of Pediatrics had admonished New that\n\n> it remains the physician's ethical obligation to remain very cautious, even if using lower doses. The fact that only 1 in 8 treated pregnancies may benefit from the therapy confounds this equation even further. There is much information on the effect of glucocorticoids on the brain. Data continue to accumulate that indicate that high-dose glucocorticoid therapy is harmful for the developing (prenatal and postnatal) brain. Further, Dr. New herself coauthored a paper reporting increased frequency of white matter abnormalities and temporal lobe atrophy on magnetic resonance imaging in patients with CAH. Although cause and effect remain to be established, one must consider that glucocorticoid therapy may have played a role in causing these abnormalities (Frias et al. 2001, 805).\n\nThe AAP Committee concluded, in their response to New:\n\n> The maxim of \"first do no harm\" requires a cautious, long-term approach, which is why the Academy Committee unanimously agrees that prenatal glucocorticoid therapy for CAH should be confined to centers doing *controlled prospective, long-term studies*. The memory of the tragedies associated with prenatal use of dexamethasone and thalidomide demands no less (Frias et al. 2001, 805, *emphasis added*).\n\nA few months later, *Pediatrics* issued an erratum, correcting what may have been a Freudian slip; the AAP Committee had meant to say \"the memory of the tragedies associated with the prenatal use of *diethylstilbestrol (DES)* and thalidomide demands no less\" (*Pediatrics* 2001, 1450, *emphasis added*).\n\nOver the years, many individual researchers and clinicians expressed similar concerns about the use of prenatal dexamethasone for CAH outside of appropriate studies or, in some cases, about its use at all (Seckl and Miller 1997; Miller 1999; Ritzen 2001; Hughes 2003; Lajic et al. 2004; Hirvikoski et al. 2007). Even Forest, the French pioneer of the intervention, wrote in 2004, \"the prenatal treatment of CAH remains an experimental therapy and, hence, must only be done with fully informed consent in *controlled prospective* trials approved by human experimentation committees at centre's that see enough of these patients to collect meaningful data\" (2004, 479, *emphasis added*). By 2008, Walter Miller, distinguished professor of pediatrics and chief of endocrinology at the University of California\u2013San Francisco, declared, \"It is this author's opinion that this experimental treatment is not warranted and should not be pursued, even in prospective clinical trials\" (Miller 2008, 17). Miller later would add: \"It seems to me that the main point of prenatal therapy is to allay parental anxiety. In that construct, one must question the ethics of using the fetus as a reagent to treat the parent, especially when the risks are non-trivial\" (in Dreger 2010, \u00b620).\n\nIn spite of all these challenges and warnings, New and her collaborators in the United States appear never to have entered into a prospective, long-term, continuous study of the use of prenatal dexamethasone for CAH. New and her group do appear to have occasionally done some trials of prenatal dexamethasone during pregnancy, tracking outcomes up to the birth; among the 325 pregnancies included in studies seriously considered by the 2010 meta-analysis, 281 came from New's group (Fern\u00e1ndez-Balsells et al. 2010). Even those, however, were not studied in a matter adequate to establish efficacy or safety according to the standards of evidence-based medicine. The trials lacked adequate controls and methods to mitigate bias.\n\nMoreover, our recent FOIA findings on New's pregnancy trials of dexamethasone throw up multiple ethical red flags. For example, in her 1985 application to the Cornell IRB to study prenatal dexamethasone for CAH, New did not check the boxes indicating that her subject population included pregnant women and fetuses (New 1985, 2), even while, deeper in the application, she indicates that the study design calls for administration of dexamethasone starting at two to four\u00a0weeks of gestation (New 1985, 9e). The accompanying consent form describes this use as experimental, but then goes on to minimize the risks:\n\n> I understand that my participation in the project involves the following risks: transient and reversible suppression of the maternal and fetal adrenal gland. For this reason, extra doses of steroids will be administered to the mother at the time of delivery to cover for the additional stress. Although complications of glucocorticoid therapy (cleft palate, growth retardation, placental degeneration and fetal death) have been reported in laboratory animals, the doses used were extremely high. Congenital malformations associated with dexamethasone therapy are rare in humans, even when large doses are given. The pregnant women and fetuses treated to date with this regimen have not experienced complications (New 1985, 9e).\n\nA pregnant woman reading this might reasonably have read \"transient and reversible\" to mean that the risks included no long-term harms to her or her offspring.\n\nIn another New York Presbyterian Hospital\/Cornell Weill Medical College consent form for the prenatal administration of dexamethasone for CAH, marked \"IRB Approved\" and dated April 2004, New required pregnant women to sign a form saying they understood the use was \"experimental.\" But the form also advised the women: \"Over the last decade, treatment with dexamethasone in the prescribed doses has been shown to be effective in reducing the masculinization of the female fetus with CAH and has been shown to be safe for the fetus\" (New 2004).\n\nThus, it remains unclear\u2014but doubtful\u2014whether U.S. federal regulation 45 CFR 46 subpart B, which regulates experimentation on pregnant women, was consistently followed in this use of prenatal dexamethasone. Because the OHRP removed, from its FOIA response to us, sections of its correspondence with Weill Cornell that might have indicated how many women were included in pregnancy trials of prenatal dexamethasone conducted by New, we have been unable to determine how many of the 600-plus pregnant women and fetuses she says she had \"treated\" at Weill Cornell by 2003 had the benefit of IRB surveillance (such as it was). What we do know is that, given what we have seen, even those engaged in IRB-approved trials at Weill Cornell apparently cannot be said to have given informed consent.\n\nSo far as we can ascertain, there has been and remains an absence of IRB oversight for prenatal dexamethasone administration for CAH at Mount Sinai, where New began working in June 2004, in spite of the fact that New has claimed the exposure of fetuses at Mount Sinai as evidence of her research progress there. For example, in her 2006 grant \"progress report\" to the NIH, New indicated that she and her team had settled in at Mount Sinai and had been continuing the intervention there: \"The prenatal diagnosis and treatment program has galloped ahead so that we now have diagnosed and or treated 768 fetuses,\" including 27 fetuses exposed to dexamethasone from February 2005 to January 2006, i.e., when she was at Mount Sinai (New 2006, 3).\n\nOur FOIA digging also turned up a 2004 letter from Jeffrey Silverstein, chair of the Mount Sinai IRB, assuring the NIH that New had Mount Sinai IRB approval for a project called \"Prenatal Diagnosis and Treatment\" (Silverstein 2004); the accompanying documents indicate that the project specifically involved the use of prenatal dexamethasone for CAH. (New probably needed this IRB approval letter to continue her NIH funding.) Yet in his 2010 response to the OHRP on behalf of Mount Sinai in response to our letters of concern, Silverstein indicated that Mount Sinai did not believe IRB oversight of the decision to undertake this fetal intervention was needed, because New was not herself writing the prescriptions (Silverstein et al. 2010).\n\nMount Sinai's 2010 conclusions that New had not been doing research on those pregnancies appears to be based on the confusing premise that *other* doctors had been doing the prescribing to the pregnant women whose fetuses were then included in the growing numbers of New's subjects. Perhaps Mount Sinai's respondents to the OHRP in 2010 did not know that New had been reporting statistics on fetal exposure as part of the federally-funded research progress she described to the NIH as \"gallop\\[ing\\] ahead\" at Mount Sinai? But surely Silverstein, who lead-authored the Mount Sinai defense in 2010, should have known that he had himself assured the government in 2004 that New was intending prenatal exposure as part of a study supposedly under IRB supervision. If these fetuses were not regarded as research subjects locally in terms of having IRB protections, they appear to have at least been counted as research subjects federally.\n\nWith Meyer-Bahlburg, New has also conducted a few follow-up studies, mostly low-quality questionnaire studies in which parents have been asked, long-distance, to reply to a battery of questions. (The 2010 review found these of such low quality, they did not even bother to consider them for the meta-analysis, and Meyer-Bahlburg has admitted that \"fewer than 50\u00a0% of mothers and offspring have responded to questionnaires\" \\[Speiser et al. 2010b, under \"3. Prenatal Treatment of CAH\"\\].) For these studies, the researchers had IRB approval, but it does not appear that the families enrolled in these studies would have known that they might have been misled about the status of prenatal dexamethasone when it was administered to the pregnant women. Thus it is questionable whether their participation in the follow-up study can be called fully informed.\n\nSo far as we can ascertain, New was not alone in her less-than-rigorous approach to scientific study of prenatal dexamethasone for CAH. Indeed, although the intervention has been offered in many other nations, Sweden appears to be the only nation to have specifically restricted prenatal dexamethasone for CAH to women who agreed to participate in a continuous, prospective, long-term study of the intervention, a restriction instituted there in 1999. (Before that it could be obtained outside of trials.) The group has been led by Svetlana Lajic, associate professor of molecular medicine and surgery at the Karolinska University Hospital. In a recent exchange with one of us (Feder), Lajic revealed that the Swedish team has actually halted the intervention part of the study due to concerns about adverse effects.\n\nLajic wrote to Feder that \"due to our findings we have addressed the Regional Ethics Committee in Stockholm, in November 2010, and stated that due to possible adverse events we wish to put on hold further recruitment of patients to the on-going prospective study of prenatal DEX treatment of CAH\" (Lajic 2011). While our paper was in press, the Swedish team published news of this development, along with follow-up data on \"43 children treated in Sweden and Norway during 1985\u20131995\" (Hirvikoski et al. 2012, 1). When compared to controls,\n\n> \\[i\\]n general, treated children were born at term and were not small for gestational age. As a group, they did not exhibit teratogenous effects\/gross malformations, although eight severe adverse events were noted in the treated group, compared with one in the control group. Three children failed to thrive during the first year of life; in addition, one had developmental delay and hypospadias; one had hydrocephalus; two girls were born small for gestational age, and one of these girls was later diagnosed with mental retardation; and one child had severe mood fluctuations that caused hospital admission. In the control group, only one child was admitted because of Down's syndrome (Hirvikoski et al. 2012, 2).\n\nThe Swedish team has clearly been alarmed by the extraordinary number of serious medical problems among the small group of children treated prenatally.\n\nAs for the cognitive and behavioral outcomes, in their most recent report the Swedish team acknowledged that \"The small sample size, relatively high refusal rate, and the retrospective study design limited the conclusiveness of the results\" of their follow-up of the behavioral development in 40 Swedish children treated prenatally. Nevertheless, they could report that\n\n> \\[a\\]n adverse effect was observed in the form of impaired verbal working memory in CAH-unaffected short-term-treated cases \\[i.e., the children who were not the intended targets of the intervention\\]. The verbal working memory capacity correlated with the children's self-perception of difficulties in scholastic ability, another measure showing significantly lower results in CAH-unaffected, DEX-exposed children. These children also reported increased social anxiety. In the studies on gender role behavior, we found indications of more neutral behaviors in DEX-exposed boys (Hirvikoski et al. 2012, 2).\n\nIn other words, the boys appeared relatively less masculine\u2014another unintended effect.\n\nThe Swedish team argued that \"the\\[se\\] results cause concern because no side effects should be tolerated in CAH-unaffected children who do not benefit from the treatment per se\" (Hirvikoski et al. 2012, 2). Indeed, the team concluded its 2012 report with as strongly a worded ethics statement as has ever been issued by a team engaged in the use of prenatal dexamethasone for CAH: \"We find it unacceptable that, globally, fetuses at risk for CAH are still treated prenatally with DEX without follow-up\" (Hirvikoski et al. 2012, 2).\n\nAs we urged United States governmental agencies in 2010, the Swedish team now \"urge\\[s\\] the scientific community to perform additional retrospective studies, preferably on all treated children and young adults\" (Hirvikoski et al. 2012, 2). Yet we see no signs that rigorous, independently-audited, retrospective studies will be pursued in the United States, where the great majority of interventions appear to have occurred, largely under the guidance of Maria New. Although we find plenty of suggestions in New's grant materials and presentations that results from her retrospective follow-up studies are forthcoming, and although the NIH repeatedly renewed her prenatal dexamethasone research funding, little appears to have been produced in terms of longer-term data on prenatal dexamethasone for CAH, particularly with regard to long-term safety. We can find no publications resulting from New's 2007 Rare Diseases Clinical Research Network (RDCRN\/NIH) follow-up study protocol, except a paper she co-authored in a 2011 issue of *Advances in Experimental Medicine and Biology*. This paper purports to demonstrate the long-term safety of prenatal dexamethasone for CAH. But the article contains no methods section, no description of the study allegedly being reported, no description of the controls, nor even of the intervention. It consists simply of unexplained tables of data and unsupported narrative (New and Parsa 2011). Needless to say, it is not the kind of report that will ever make it into a meta-analysis.\n\nAlthough New had told the U.S. government in 1996 \"we propose to continue our studies of prenatal diagnosis and treatment\" (New 1996b, 61), we can find no evidence of there ever having been, in the United States, a reasonably-designed, IRB-approved, prospective, long-term study\u2014nothing like the rigorous approach taken in Sweden. In a recent debate with Dreger on this subject, Meyer-Bahlburg confirmed the total absence of prospective continuous studies in the United States and offered no indication that any has ever even been planned (Meyer-Bahlburg 2011). This is very concerning, and is even more worrisome when one considers the design of New's 2007 RDCRN\/NIH retrospective follow-up study (to be carried out with Meyer-Bahlburg). As we discovered from our FOIA requests, that protocol specifically states as an \"exclusion criteria for all groups\" this: \"mental impairment which prevents understanding of questionnaire\" (New 2007, 25).\n\nSo, while the researchers in Sweden are documenting cognitive impairment among those exposed to prenatal dexamethasone for CAH, researchers in America have specifically designed a follow-up study that *prevents detection* of certain negative effects on cognitive development.\n\n# Promotion of Prenatal Dexamethasone for CAH\n\nNew has a clear track record of promoting the use of prenatal dexamethasone for CAH as safe and effective to clinicians who might reasonably have expected her to represent accurately to them what was actually known and unknown. In a co-authored article for the 2000 edition of the *Cecil Textbook of Medicine*, New advised her colleagues: \"Prenatal treatment with dexamethasone has been shown to be safe and effective for both mother and child in the largest human studies\" (New and Josso 2000, 1302), implying that there were large human studies of sufficiently high quality to establish safety and efficacy. A few years later, New's discussion of prenatal dexamethasone (published with two co-authors) in the 2007 edition of the textbook *Pediatric Endocrinology* reads, \"we believe that proper prenatal treatment of fetuses at risk for CAH can be considered effective and safe. Long-term studies on the psychological development of patients treated prenatally are currently underway\"\u2014a tacit admission of more interest in outcomes related to gender identity and cognitive development than in metabolic outcomes like those involving cardiovascular function (New, Ghizzoni, and Lin-Su 2007, 237).\n\nNew's determined influence on prenatal treatment practices for CAH is also illustrated by the work she has published in *GeneReviews*, the NIH-sponsored free online textbook regarded as an authoritative source in prenatal counseling. Until recently (for reasons we explain below), New's article described prenatal dexamethasone for CAH as if it were standard practice, explaining precisely how clinicians in the field could implement the intervention. No mention was made of its experimental status, of the fact that the use is off-label, nor of the absence of evidence for its efficacy and safety (Nimkarn and New 2009; cf. Witchel and Miller 2012).\n\nIn her grants with the NIH, New also represented prenatal dexamethasone for CAH as having been shown safe and effective. For example, in her 2003 report, she indicated: \"Based on our experience and other large human studies, proper prenatal diagnosis and treatment of 21-OHD is safe for mother and child, and is effective\" (New 2003a, 98). In her NIH grant materials, New used the fact that \"\\[w\\]e are the only group in the U.S.A. routinely carrying out prenatal diagnosis and treatment of CAH\" as a major reason why the government should fund her studies on the \"large population of prenatally-treated infants\" she had \"accumulated\" (New 1996b, 2). Already by her 1996 report to the NIH, New was claiming her clinic \"receive\\[d\\] requests \\[for the intervention\\] from all over the U.S. and foreign countries at the rate of 3\u20134 per week\" (New 1996b, 61). A decade later, New's protocol for a follow-up study again indicated that her clinic had been drawing patients from all over the United States (New 2007, 25\u201326); recall that by 2006 she reported use of the intervention in 768 pregnancies (New 2006, 3).\n\nThe large draw of potential subjects for her grants perhaps occurred because New actively promoted prenatal dexamethasone for CAH as safe and effective not only to clinicians but also directly to parents, including through the CARES Foundation, a non-profit organization dedicated to supporting individuals and families with CAH and to advancing research and improved clinical practices (e.g., newborn screening). New's 2001 lecture to parents at CARES, quoted above, represents one example. Another appears in the CARES Foundation Winter 2003 newsletter, for which Elizabeth Kitzinger of Weill Cornell Medical College provided a short article effectively advertising New's clinic as *the* place to go for at-risk mothers:\n\n> In the United States, the only center routinely offering prenatal diagnosis and treatment is Dr. Maria New's clinic at New York Presbyterian Hospital-Weill Medical Center (Cornell) in New York City. Dr. New has treated over 600 pregnant women at risk for the birth of a CAH-affected child. \u2026 The results are remarkable. Dr. New maintains contact with all children treated prenatally, and has found no adverse developmental consequences. Thus, with nearly 20\u00a0years' experience, the treatment appears to be safe for mother and child, though there are endocrinologists who are wary of using dexamethasone prenatally even now (Kitzinger 2003, \u00b62\u2013\u00b63).\n\nSome parents reading this might hear in that last line a warning that not all clinicians were convinced this use was safe and effective. But others might reasonably read it as an indication that Dr. New would be the only clinician willing to help them. The text continues:\n\n> It is important to note that prenatal diagnosis and treatment should ONLY be done in a clinic like Dr. New's with long experience and commitment to follow-up. Only by tracking the growth of prenatally treated children can the long-term effects of treatment be exhaustively studied. Administering dexamethasone to achieve normal genitalia requires the judgment and experience of specialists. The benefits to families of classically affected girls cannot be underestimated. We hope that the availability of this treatment will be shared with all families at risk for the birth of CAH-affected children. (Kitzinger 2003, \u00b64, capitalization original).\n\nEven since the 2010 meta-analysis, the website of the Maria New Children's Hormone Foundation continues to claim \"the treatment has been found safe for mother and child\" (New 2010a, \u00b64). In 2010, our complaints to the FDA about advertisement of this off-label use as \"safe for mother and child\" were referred to Robert \"Skip\" Nelson, an FDA-based pediatrician and ethicist. As mentioned above, our discussions with Nelson clarified that regulations prohibit only two groups from advertising off-label uses as \"safe and effective\": employees of a drug's maker (which New is not) and FDA-approved investigators of drugs.\n\nAccording to Nelson, New did seek and obtain an IND (investigational new drug) exemption from the FDA in 1996 for prenatal dexamethasone for CAH; this would mean she sought and obtained the FDA's permission at that time for a specific study of this drug use. But as Nelson explained to us, since New does not *currently* have an approval from the FDA to study prenatal dexamethasone, she does not count as an FDA investigator, so she is not currently prohibited from calling this intervention \"safe and effective.\" In other words, because of a regulatory loophole in the United States, if you inappropriately investigate an off-label use of a drug, then you can also inappropriately advertise it\u2014even to pregnant women and even when the drug use is meant to alter the course of fetal development.\n\nNotably, we think Nelson may be incorrect in his claim that in 1996 the FDA granted New an IND exemption for the use of prenatal dexamethasone to prevent sex atypicality in CAH. Through the FOIA, we obtained the 1996 FDA letter cited by Nelson in 2010, and it actually refers only to a \"proposal to utilize dexamethasone to treat pregnant women with a \\[sic\\] congenital adrenal hyperplasia\" (Sobel 1996). It is entirely possible the 1996 letter referred to a study of the use of dexamethasone to treat women with CAH who became pregnant, i.e., an off-label use meant to treat a woman *herself* afflicted with CAH to keep *her* healthy during her pregnancy*,* an entirely different medical matter than an explicit attempt at fetal engineering wherein the mother is merely a carrier of CAH. There appears to be no evidence to support Nelson's representation of the 1996 IND exemption letter.\n\n# A Worrisome Track Record\n\nWe feel optimistic that our efforts to draw attention to this use of prenatal dexamethasone have increased the likelihood that physicians and researchers will respect the rights of the population exposed. Nevertheless, we feel pessimistic about ever knowing what really happened to most of those already exposed\u2014for weeks or months of pregnancy or fetal development. Recall that in 2002 a joint statement by the European and American pediatric endocrinology societies called for data on prenatal dexamethasone outcomes to be entered into \"a central database audited by an independent safety committee\" (Joint LWPES\/ESPE CAH Working Group 2002, 4049). But\u2014except for a still unpublished European study known as \"PREDEX,\" which enrolled only 24 subjects from 1999 to 2004, including subjects from the Swedish prospective cohort (Lajic et al. 2004)\u2014no such thing has happened. Instead, it appears from the 2007 RDCRN\/NIH study protocol that Maria New retains control of the database of contact information for what she reports is now more than 768 children and their mothers who have been exposed to this prenatal intervention through her clinics (New 2006, 3). It seems likely that that population represents at least a significant minority of those exposed. In fact, New's 2001 NIH \"application for continuation grant\" provides a table describing human subjects under the \"specific \\[study\\] aim\" called \"prenatal dx and treatment in families at risk,\" and there the total number of subjects is stated as 2,144 (New 2001b, 14).\n\nOur research has caused us substantial concern about what appears to be a pattern of misrepresentation by Maria New, even beyond what we take to be misrepresentations regarding the status of prenatal dexamethasone for CAH. As reported in *The Wall Street Journal* in 2005, one of New's NIH grants (which included work on CAH) formed the subject of a fraud suit brought against Weill Cornell Medical College\u2014a suit over \"phantom studies\" that resulted in a settlement whereby Cornell paid the government \\$4.4 million (Wysocki 2005). While the settlement required no admission of wrongdoing, it seems significant that, at the time of the fraud case, New's status at Cornell radically changed; substantial portions of her Cornell titles and salary appear to have been administratively withdrawn.[^2]\n\nAn oblique exchange in which we participated in 2010 raises additional concerns about New's track record for trustworthiness. In a project aimed at making known the experimental status of prenatal dexamethasone for CAH, Dreger wrote with Taylor Sale (Dreger's student and a genetic counselor) to the editors of *GeneReviews* to request that Dr. New's article in that publication be changed to reflect medical consensuses concerning prescription of prenatal dexamethasone for CAH (Dreger and Sale 2010). The editors reviewed the consensus statements Dreger and Sale provided them and then wrote to New with recommended changes. Some time later, one of the editors wrote to Dreger and Sale to report that: \"Dr. New's initial reply to our proposed edits to the 21-hydroxylase deficient \\[sic\\] CAH *GeneReview* is that 'Prenatal dexamethasone treatment has been FDA approved by Dr. Sobel.'\" The editor added, \"We are interested in your thoughts on \\[Dr. New's\\] comment\" (Dolan 2010). We informed the editors that Solomon Sobel was the FDA physician who had signed off in 1996 on a single IND exemption (Sobel 1996). As we surely did not need to explain to the *GeneReviews* editors, this did not qualify prenatal dexamethasone for CAH as \"FDA approved.\"\n\nToday, New's co-authored *GeneReviews* article on 21-OHD CAH says this: \"Prenatal treatment should continue to be considered experimental and should only be used within the context of a formal IRB-approved clinical trial\" (Nimkarn and New 2010b, \u00b61 under \"Therapies Under Investigation\"). Since the 2010 revision, the article no longer provides detailed instructions on how to conduct the intervention. And yet, the change reflected in the *GeneReviews* article does not appear to mark a major change in approach for New. Just after the revision of the *GeneReviews* piece, New published a short article in *The American Journal of Bioethics* entitled, \"Vindication of Prenatal Diagnosis and Treatment of Congenital Adrenal Hyperplasia with Low-Dose Dexamethasone.\" There she wrote: \"The recent reports by the Office of Human Research Protections and the FDA \u2026 make crystal clear that my research on prenatal treatment of CAH is and always has been both legally and ethically proper at every level\" (New 2010c, 68).\n\nIn spite of our petitions, the Office of Human Research Protections (OHRP) appears to be quite a bit less concerned than we are about the veracity of New's claims. For example, the agency appears to have accepted a claim that New had not been conducting fetal experimentation with dexamethasone at Mount Sinai, apparently without learning or considering that her 2006 NIH grant report described \"gallop\\[ing\\] ahead\" with the intervention and specified, as part of her \"research progress report,\" at least 27 fetuses exposed to prenatal dexamethasone for CAH since New had relocated her clinic to Mount Sinai (New 2006). As part of its investigation following our letters, the OHRP uncovered the deeply problematic IRB-approved consent forms from Weill Cornell mentioned above, yet the OHRP accepted New's claims that appropriate IRB oversight and informed consent had occurred. The OHRP staff did not indicate in their response to us how many of the pregnant women New claims to have \"treated\" were even in IRB-approved trials, nor did they address the problematic disjuncture between her own advertisement of prenatal dexamethasone as \"safe for mother and child\" and her simultaneous federally funded study to investigate whether in fact it is safe for mother and child.\n\nThis all seems particularly strange since Tamar-Mattis's research turned up two strongly-worded 2004 determination letters from the OHRP finding major faults with New's IRB-approved studies at Weill Cornell (Tamar-Mattis 2010). (OHRP determination letters are the final result of investigations that reveal problems in the conduct of research.) New was not the only researcher at Cornell named in the findings; in fact, the OHRP found the entire system for reviewing pediatric research at Cornell so problematic that they placed a restriction on Cornell's Federalwide Assurance and took the extraordinary step of requiring review by the IRB of all active research involving children (McNeilly 2004). The problems the OHRP found with protection for subjects in New's studies were particularly alarming: an informed consent form that did not include a description of the purpose of research, as required (McNeilly 2004, 5); consent forms that contained extremely complex language that a subject would be unlikely to understand (McNeilly 2004, 6); an informed consent form that did not state that \"the tests conducted for the protocol could be obtained outside the research\" (McNeilly 2004, 5); \"subjects enrolled outside the protocol age range prior to IRB review and approval\" (McNeilly 2004, 4); missing IRB records (McNeilly 2004, 3) and research initiated without first obtaining legally informed consent (McNeilly 2004, 3). The OHRP also noted that Weill Cornell had suspended New's research protocol in the fall of 2002 but did not report the suspension to the OHRP until June 2003 (McNeilly 2004, 4).\n\nWhy so much scrutiny of New and Weill Cornell by the OHRP in 2004 and so little by the OHRP in 2010? This seems to be part of a problematic trend forming at the OHRP\u2014again suggesting that prenatal dexamethasone for CAH is not a unique case but a bellwether. A March 2011 analysis from the *Report on Research Compliance* on the OHRP has found a substantial drop-off of OHRP determination letters in the last four\u00a0years: \"Agency observers and others expressed concern about the steep drops in letters and open cases, telling \\[the *Report on Research Compliance*\\] they raise questions about the agency's current commitment to serious oversight of human subjects research and investigations into possible wrongdoing\" (National Council of University Research Administrators 2011, 1). Notably, the \"possibility that compliance is increasing at institutions was not among the reasons OHRP cited for the drop, and funding is not a problem\" (National Council of University Research Administrators 2011, 1). Nor has the number of complaints made to the OHRP fallen.\n\n# Final Thoughts\n\nThe OHRP and FDA have not yet fully responded to our FOIA requests. (Dreger is presently suing to obtain the remainder of the documents.) But even what we already have found indicates that the government could well have made a case for inappropriate behaviors here and could have issued statements echoing at least some of our concerns about the way that prenatal dexamethasone for CAH has been administered and studied.\n\nDespite the disappointing responses from the OHRP and FDA, we do not regret raising the alarm as we did starting in January 2010. Doing so increased awareness among the affected population about the actual status of prenatal dexamethasone and has also shone light into corners we would not otherwise be able to view. For example, it made available to the public New's retrospective study protocol and IRB-approved consent forms, and it also led to a reporter for *Time* magazine finding women who indicated that they did not know, when given prenatal dexamethasone to attempt prevention of virilization in female fetuses, that it was an off-label and controversial drug use (Elton 2010). Without the investigation, this valuable information would have remained hidden from view.\n\nThe investigation also seems to have driven Mount Sinai to take action, judging from Silverstein's response to the OHRP:\n\n> The Committee \\[at Mount Sinai charged with responding\\] determined that there are widely differing opinions amongst the staff, with some staff members expressing significant concerns regarding the use of dexamethasone for the prenatal treatment of CAH. A particular concern is the current necessity to treat potentially unaffected fetuses until a diagnosis is determined. Therefore, the Committee concluded that the clinical use of dexamethasone in this situation should require a rigorous informed consent process with detailed documentation that the risks and benefits of this treatment have been clearly communicated to the parents making a decision to engage in prenatal treatment. The Committee also recommends that this issue be referred to the Medical Board of The Mount Sinai Hospital for further consideration of the consent issue. (Silverstein et al. 2010, 11).\n\nIt would appear, then, that Mount Sinai now shares our concerns about the practices associated with the administration of dexamethasone. Nevertheless, it is still the case that the Maria New Children's Hormone Foundation website declares that prenatal dexamethasone for CAH \"has been found safe for mother and child\" and provides a phone number to call to make an appointment (New 2010a, \u00b64). When the number is called, it rings to a clinic at Mount Sinai.\n\n# Epilogue\n\nAs our paper was in press, Maria New's research group, led by Meyer-Bahlburg, published a study including some cognitive outcome data for 67 children prenatally exposed to dexamethasone for CAH (Meyer-Bahlburg et al. 2012). The children studied came from New's database (Meyer-Bahlburg et al. 2012, line 92) and included eight CAH-affected girls who were \"long-term\" exposed in utero and 59 boys and CAH-unaffected girls who were \"short-term\" exposed (Meyer-Bahlburg et al. 2012, line 23). These children were compared to 73 unexposed controls (Meyer-Bahlburg et al. 2012, line 24).\n\nThe new paper concludes: \"Our studies do not replicate a previously reported adverse effect of short-term prenatal DEX exposure on working memory, while our findings on cognitive function in CAH girls with long-term DEX exposure contribute to concerns about potentially adverse cognitive aftereffects of such exposure\" (Meyer-Bahlburg et al. 2012, lines 34\u201336). The \"previously reported adverse effect of short-term prenatal DEX exposure\" was that reported by the Swedish team (Hirvikoski et al. 2007). But whereas the Swedish team employed a relatively rigorous design (a prospective, controlled, long-term study), in the new study from New's group, the 67 exposed children were selected via convenience sampling performed retrospectively.\n\nFurthermore, although the new Meyer-Bahlburg et al. publication purports to seek information on the effects of dexamethasone exposure (Meyer-Bahlburg et al. 2012, lines 26\u201327), among the eight girls long-term exposed the degree of exposure varies from a total of nine\u00a0weeks of fetal life to a total of 39\u00a0weeks (Meyer-Bahlburg et al. 2012, lines 117\u2013120), an exposure-length difference of more than four times, making it very difficult to establish meaningful dose-effect.\n\nIn marked contrast to the Swedish team, and as if to confirm the weakness of the study by New's group, Meyer-Bahlburg et al. found some \"positive\" cognitive outcomes in the short-term treated children. Understandably, they found this hard to explain. (No one suspects first-trimester glucocorticoid exposure at 60 to100 times normal levels to be good for children's brains.) In their discussion, the authors try to explain the lack of corroboration of their results by other studies or by logic (Meyer-Bahlburg et al. 2012, lines 278\u2013298), but they do not propose the most obvious explanation for this very strange finding: The paper's study population is a highly skewed sample.\n\nWe document here that New has claimed to have \"treated\" somewhere between 600 and 2,144 fetuses with dexamethasone for CAH, yet her group is now reporting cognitive outcomes on only 67 children in her database, a tiny fraction of those who ought to be available for study. Furthermore, based on New's claims to the public and to her granting agencies, her efforts should have produced between about 98 and 268 girls who were long-term dexamethasone-exposed, yet here her group reports on cognitive outcomes in only eight. Thus it would appear that the children included in the new study from Meyer-Bahlburg et al. represent somewhere between only 3 and 12 percent of the population prenatally exposed to dexamethasone under New's consultation.\n\nWhat the new paper from Meyer-Bahlburg et al. actually appears to replicate is our finding: that the approach taken by New and her collaborators to prenatal dexamethasone for CAH has been so scientifically weak as to be both clinically uninformative and profoundly unethical, especially in light of the history of DES.\n\nThe authors thank Heather Appelbaum, J.J. Burchman, James A. Bruce, Joel Frader, Susan Gilbert, Janet Green, Philip Gruppuso, Mary Ann Harrell, Shelley Harshe, Hilde Lindemann, Johanna Michael, Thad Morgan, Robert Nelson, Nigel Paneth, Taylor Sale, David Sandberg, Aron Sousa, Valerie Thonger, Kiira Triea, Katie Watson, Eric Vilain, two anonymous reviewers, and the journal's editors for assistance with this work. Funding for this project has been provided to Alice Dreger by the Office of the Provost of Northwestern University.\n\n### Open Access\n\nThis article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.\n\n# References\n\n[^1]: For an analysis of the problems with off-label usages, see Dresser and Frader (2009).\n\n[^2]: On February 12, 2003, New wrote to the NIH asking for money to support herself, under the NIH Merit Award system: \"As I am no longer Chairman of Pediatrics, Chief of Pediatric Endocrinology, and Program Director of the CCRC, I have much more time to devote to the research proposed in my Merit Award. \u2026 This is a hard time for me and I am deeply appreciative of your consideration\" (New 2003b). In a subsequent internal memo, the NIH staff \"enthusiastically support\\[ed\\]\" giving New more money under the Merit Award system, without mentioning *why* \"her circumstances changed abruptly this year when she had to relinquish the chairmanship of the Department of Pediatrics and the directorship of the Children's Clinical Research Center at Weill-Cornell Medical School and the salaries entailed in these positions\" (NIH Staff 2003). Shortly after, New was hired by Mount Sinai School of Medicine.","meta":{"dup_signals":{"dup_doc_count":122,"dup_dump_count":56,"dup_details":{"curated_sources":3,"2023-14":2,"2023-06":1,"2021-17":1,"2019-35":1,"2019-26":2,"2019-22":2,"2019-04":2,"2018-47":2,"2018-43":1,"2018-39":1,"2018-34":1,"2018-17":3,"2018-13":2,"2018-09":1,"2017-47":3,"2017-43":2,"2017-39":2,"2017-34":2,"2017-30":3,"2017-26":3,"2017-22":4,"2017-17":5,"2017-09":5,"2017-04":4,"2016-50":3,"2016-44":4,"2016-40":4,"2016-36":5,"2016-30":6,"2016-26":4,"2016-22":4,"2016-18":4,"2016-07":1,"2015-48":1,"2015-40":1,"2015-35":1,"2015-32":1,"2015-27":1,"2015-22":1,"2015-14":1,"2014-52":1,"2014-49":1,"2014-42":2,"2014-41":2,"2014-35":2,"2014-23":2,"2014-15":1,"2023-23":1,"2017-13":3,"2015-18":1,"2015-11":1,"2015-06":1,"2014-10":1,"2013-48":1,"2013-20":1,"2024-26":1}},"file":"PMC3416978"},"subset":"pubmed_central"}
{"text":"abstract: Flaws in the design, conduct, analysis, and reporting of randomised trials can cause the effect of an intervention to be underestimated or overestimated. The Cochrane Collaboration's tool for assessing risk of bias aims to make the process clearer and more accurate\nauthor: Julian P T Higgins; Douglas G Altman; Peter C G\u00f8tzsche; Peter J\u00fcni; David Moher; Andrew D Oxman; Jelena Savovi\u0107; Kenneth F Schulz; Laura Weeks; Jonathan A C SterneCorrespondence to: J P T Higgins <firstname.lastname@example.com>.\ndate: 2011\nreferences:\ntitle: The Cochrane Collaboration's tool for assessing risk of bias in randomised trials\n\n# Further details on the items included in risk assessment tool\n\nClick here for additional data file.\n\nRandomised trials, and systematic reviews of such trials, provide the most reliable evidence about the effects of healthcare interventions. Provided that there are enough participants, randomisation should ensure that participants in the intervention and comparison groups are similar with respect to both known and unknown prognostic factors. Differences in outcomes of interest between the different groups can then in principle be ascribed to the causal effect of the intervention.1\n\nCausal inferences from randomised trials can, however, be undermined by flaws in design, conduct, analyses, and reporting, leading to underestimation or overestimation of the true intervention effect (bias).2 However, it is usually impossible to know the extent to which biases have affected the results of a particular trial.\n\nSystematic reviews aim to collate and synthesise all studies that meet prespecified eligibility criteria3 using methods that attempt to minimise bias. To obtain reliable conclusions, review authors must carefully consider the potential limitations of the included studies. The notion of study \"quality\" is not well defined but relates to the extent to which its design, conduct, analysis, and presentation were appropriate to answer its research question. Many tools for assessing the quality of randomised trials are available, including scales (which score the trials) and checklists (which assess trials without producing a score).4 5 6 7 Until recently, Cochrane reviews used a variety of these tools, mainly checklists.8 In 2005 the Cochrane Collaboration's methods groups embarked on a new strategy for assessing the quality of randomised trials. In this paper we describe the collaboration's new risk of bias assessment tool, and the process by which it was developed and evaluated.\n\n# Development of risk assessment tool\n\nIn May 2005, 16 statisticians, epidemiologists, and review authors attended a three day meeting to develop the new tool. Before the meeting, JPTH and DGA compiled an extensive list of potential sources of bias in clinical trials. The items on the list were divided into seven areas: generation of the allocation sequence; concealment of the allocation sequence; blinding; attrition and exclusions; other generic sources of bias; biases specific to the trial design (such as crossover or cluster randomised trials); and biases that might be specific to a clinical specialty. For each of the seven areas, a nominated meeting participant prepared a review of the empirical evidence, a discussion of specific issues and uncertainties, and a proposed set of criteria for assessing protection from bias as adequate, inadequate, or unclear, supported by examples.\n\nDuring the meeting decisions were made by informal consensus regarding items that were truly potential biases rather than sources of heterogeneity or imprecision. Potential biases were then divided into domains, and strategies for their assessment were agreed, again by informal consensus, leading to the creation of a new tool for assessing potential for bias. Meeting participants also discussed how to summarise assessments across domains, how to illustrate assessments, and how to incorporate assessments into analyses and conclusions. Minutes of the meeting were transcribed from an audio recording in conjunction with written notes.\n\nAfter the meeting, pairs of authors developed detailed criteria for each included item in the tool and guidance for assessing the potential for bias. Documents were shared and feedback requested from the whole working group (including six who could not attend the meeting). Several email iterations took place, which also incorporated feedback from presentations of the proposed guidance at various meetings and workshops within the Cochrane Collaboration and from pilot work by selected review teams in collaboration with members of the working group. The materials were integrated by the co-leads into comprehensive guidance on the new risk of bias tool. This was published in February 2008 and adopted as the recommended method throughout the Cochrane Collaboration.9\n\n## Evaluation phase\n\nA three stage project to evaluate the tool was initiated in early 2009. A series of focus groups was held in which review authors who had used the tool were asked to reflect on their experiences. Findings from the focus groups were then fed into the design of questionnaires for use in three online surveys of review authors who had used the tool, review authors who had not used the tool (to explore why not), and editorial teams within the collaboration. We held a meeting to discuss the findings from the focus groups and surveys and to consider revisions to the first version of the risk of bias tool. This was attended by six participants from the 2005 meeting and 17 others, including statisticians, epidemiologists, coordinating editors and other staff of Cochrane review groups, and the editor in chief of the *Cochrane Library*.\n\n# The risk of bias tool\n\nAt the 2005 workshop the participants agreed the seven principles on which the new risk of bias assessment tool was based (box).\n\n## Principles for assessing risk of bias\n\n### 1. Do not use quality scales\n\nQuality scales and resulting scores are not an appropriate way to appraise clinical trials. They tend to combine assessments of aspects of the quality of reporting with aspects of trial conduct, and to assign weights to different items in ways that are difficult to justify. Both theoretical considerations10 and empirical evidence11 suggest that associations of different scales with intervention effect estimates are inconsistent and unpredictable\n\n### 2. Focus on internal validity\n\nThe internal validity of a study is the extent to which it is free from bias. It is important to separate assessment of internal validity from that of external validity (generalisability or applicability) and precision (the extent to which study results are free from random error). Applicability depends on the purpose for which the study is to be used and is less relevant without internal validity. Precision depends on the number of participants and events in a study. A small trial with low risk of bias may provide very imprecise results, with a wide confidence interval. Conversely, the results of a large trial may be precise (narrow confidence interval) but have a high risk of bias if internal validity is poor\n\n### 3. Assess the risk of bias in trial results, not the quality of reporting or methodological problems that are not directly related to risk of bias\n\nThe quality of reporting, such as whether details were described or not, affects the ability of systematic review authors and users of medical research to assess the risk of bias but is not directly related to the risk of bias. Similarly, some aspects of trial conduct, such as obtaining ethical approval or calculating sample size, are not directly related to the risk of bias. Conversely, results of a trial that used the best possible methods may still be at risk of bias. For example, blinding may not be feasible in many non-drug trials, and it would not be reasonable to consider the trial as low quality because of the absence of blinding. Nonetheless, many types of outcome may be influenced by participants' knowledge of the intervention received, and so the trial results for such outcomes may be considered to be at risk of bias because of the absence of blinding, despite this being impossible to achieve\n\n### 4. Assessments of risk of bias require judgment\n\nAssessment of whether a particular aspect of trial conduct renders its results at risk of bias requires both knowledge of the trial methods and a judgment about whether those methods are likely to have led to a risk of bias. We decided that the basis for bias assessments should be made explicit, by recording the aspects of the trial methods on which the judgment was based and then the judgment itself\n\n### 5. Choose domains to be assessed based on a combination of theoretical and empirical considerations\n\nEmpirical studies show that particular aspects of trial conduct are associated with bias.2 12 However, these studies did not include all potential sources of bias. For example, available evidence does not distinguish between different aspects of blinding (of participants, health professionals, and outcome assessment) and is very limited with regard to how authors dealt with incomplete outcome data. There may also be topic specific and design specific issues that are relevant only to some trials and reviews. For example, in a review containing crossover trials it might be appropriate to assess whether results were at risk of bias because there was an insufficient \"washout\" period between the two treatment periods\n\n### 6. Focus on risk of bias in the data as represented in the review rather than as originally reported\n\nSome papers may report trial results that are considered as at high risk of bias, for which it may be possible to derive a result at low risk of bias. For example, a paper that inappropriately excluded certain patients from analyses might report the intervention groups and outcomes for these patients, so that the omitted participants can be reinstated\n\n### 7. Report outcome specific evaluations of risk of bias\n\nSome aspects of trial conduct (for example, whether the randomised allocation was concealed at the time the participant was recruited) apply to the trial as a whole. For other aspects, however, the risk of bias is inherently specific to different outcomes within the trial. For example, all cause mortality might be ascertained through linkages to death registries (low risk of bias), while recurrence of cancer might have been assessed by a doctor with knowledge of the allocated intervention (high risk of bias)\n\nThe risk of bias tool covers six domains of bias: selection bias, performance bias, detection bias, attrition bias, reporting bias, and other bias. Within each domain, assessments are made for one or more items, which may cover different aspects of the domain, or different outcomes. Table 1<a href=\"#tbl1\" data-ref-type=\"table\"><\/a> shows the recommended list of items. These are discussed in more detail in the appendix on bmj.com.\n\n\u2002 Cochrane Collaboration's tool for assessing risk of bias (adapted from Higgins and Altman^13^)\n\n| Bias domain | Source of bias | Support for judgment | Review authors' judgment (assess as low, unclear or high risk of bias) |\n|----|----|----|----|\n| Selection bias | Random sequence generation | Describe the method used to generate the allocation sequence in sufficient detail to allow an assessment of whether it should produce comparable groups | Selection bias (biased allocation to interventions) due to inadequate generation of a randomised sequence |\n|  | Allocation concealment | Describe the method used to conceal the allocation sequence in sufficient detail to determine whether intervention allocations could have been foreseen before or during enrolment | Selection bias (biased allocation to interventions) due to inadequate concealment of allocations before assignment |\n| Performance bias | Blinding of participants and personnel\\* | Describe all measures used, if any, to blind trial participants and researchers from knowledge of which intervention a participant received. Provide any information relating to whether the intended blinding was effective | Performance bias due to knowledge of the allocated interventions by participants and personnel during the study |\n| Detection bias | Blinding of outcome assessment\\* | Describe all measures used, if any, to blind outcome assessment from knowledge of which intervention a participant received. Provide any information relating to whether the intended blinding was effective | Detection bias due to knowledge of the allocated interventions by outcome assessment |\n| Attrition bias | Incomplete outcome data\\* | Describe the completeness of outcome data for each main outcome, including attrition and exclusions from the analysis. State whether attrition and exclusions were reported, the numbers in each intervention group (compared with total randomised participants), reasons for attrition or exclusions where reported, and any reinclusions in analyses for the review | Attrition bias due to amount, nature, or handling of incomplete outcome data |\n| Reporting bias | Selective reporting | State how selective outcome reporting was examined and what was found | Reporting bias due to selective outcome reporting |\n| Other bias | Anything else, ideally prespecified | State any important concerns about bias not covered in the other domains in the tool | Bias due to problems not covered elsewhere |\n\n\\*Assessments should be made for each main outcome or class of outcomes.\n\nFor each item in the tool, the assessment of risk of bias is in two parts. The support for judgment provides a succinct free text description or summary of the relevant trial characteristic on which judgments of risk of bias are based and aims to ensure transparency in how judgments are reached. For example, the item about concealment of the randomised allocation sequence would provide details of what measures were in place, if any, to conceal the sequence. Information for these descriptions will often come from a single published trial report but may be obtained from a mixture of trial reports, protocols, published comments on the trial, and contacts with the investigators. The support for the judgment should provide a summary of known facts, including verbatim quotes where possible. The source of this information should be stated, and when there is no information on which to base a judgment, this should be stated.\n\nThe second part of the tool involves assigning a judgment of high, low, or unclear risk of material bias for each item. We define material bias as bias of sufficient magnitude to have a notable effect on the results or conclusions of the trial, recognising the subjectivity of any such judgment. Detailed criteria for making judgments about the risk of bias from each of the items in the tool are available in the *Cochrane Handbook.*13 If insufficient detail is reported of what happened in the trial, the judgment will usually be unclear risk of bias. A judgment of unclear risk should also be made if what happened in the trial is known but the associated risk of bias is unknown\u2014for example, if participants take additional drugs of unknown effectiveness as a result of them being aware of their intervention assignment. We recommend that judgments be made independently by at least two people, with any discrepancies resolved by discussion in the first instance.\n\nSome of the items in the tool, such as methods for randomisation, require only a single assessment for each trial included in the review. For other items, such as blinding and incomplete outcome data, two or more assessments may be used because they generally need to be made separately for different outcomes (or for the same outcome at different time points). However, we recommend that review authors limit the number of assessments used by grouping outcomes\u2014for example, as subjective or objective for the purposes of assessing blinding of outcome assessment or as \"patient reported at 6 months\" or \"patient reported at 12 months\" for assessing risk of bias due to incomplete outcome data.\n\n# Evaluation of initial implementation\n\nThe first (2008) version of the tool was slightly different from the one we present here. The 2008 version did not categorise biases by the six domains (selection bias, performance bias, etc); had a single assessment for blinding; and expressed risk of bias in the format '\"yes,\" \"no,\" or \"unclear\" (referring to lack of a risk) rather than as low, high, or unclear risk. The 2010 evaluation of the initial version found wide acceptance of the need for the risk of bias tool, with a consensus that it represents an improvement over methods previously recommended by the Collaboration or widely used in systematic reviews.\n\nParticipants in the focus groups noted that the tool took longer to complete than previous methods. Of 187 authors surveyed, 88% took longer than 10 minutes to complete the new tool, 44% longer than 20 minutes, and 7% longer than an hour, but 83% considered the time taken acceptable. There was consensus that classifying items in the tool according to categories of bias (selection bias, performance bias, etc) would help users, so we introduced these. There was also consensus that assessment of blinding should be separated into blinding of participants and health professionals (performance bias) and blinding of outcome assessment (detection bias) and that the phrasing of the judgments about risk should be changed to low, high, and unclear risk. The domains reported to be the most difficult to assess were risk of bias due to incomplete outcome data and selective reporting of outcomes. There was agreement that improved training materials and availability of worked examples would increase the quality and reliability of bias assessments.\n\n# Presentation of assessments\n\nResults of an assessment of risk of bias can be presented in a table, in which judgments for each item in each trial are presented alongside their descriptive justification. Table 2<a href=\"#tbl2\" data-ref-type=\"table\"><\/a> presents an example of a risk of bias table for one trial included in a Cochrane review of therapeutic monitoring of antiretrovirals for people with HIV.14 Risks of bias due to blinding and incomplete outcome data were assessed across all outcomes within each included study, rather than separately for different outcomes as will be more appropriate in some situations.\n\n\u2002Example of risk of bias table from a Cochrane review^14^\n\n| Bias | Authors' judgment | Support for judgment |\n|----|----|----|\n| Random sequence generation (selection bias) | Low risk | Quote: \"Randomization was one to one with a block of size 6. The list of randomization was obtained using the SAS procedure plan at the data statistical analysis centre\" |\n| Allocation concealment (selection bias) | Unclear risk | The randomisation list was created at the statistical data centre, but further description of allocation is not included |\n| Blinding of participants and researchers (performance bias) | High risk | Open label |\n| Blinding of outcome assessment (detection bias) | High risk | Open label |\n| Incomplete outcome data (attrition bias) | Low risk | Losses to follow-up were disclosed and the analyses were conducted using, firstly, a modified intention to treat analysis in which missing=failures and, secondly, on an observed basis. Although the authors describe an intention to treat analysis, the 139 participants initially randomised were not all included; five were excluded (four withdrew and one had lung cancer diagnosed). This is a reasonable attrition and not expected to affect results. Adequate sample size of 60 per group was achieved |\n| Selective reporting (reporting bias) | Low risk | All prespecified outcomes were reported |\n| Other bias | Unclear risk | No description of the uptake of the therapeutic drug monitoring recommendations by physicians, which could result in performance bias |\n\nPresenting risk of bias tables for every study in a review can be cumbersome, and we suggest that illustrations are used to summarise the judgments in the main systematic review document. The figure<a href=\"#fig1\" data-ref-type=\"fig\"><\/a> provides an example. Here the judgments apply to all meta-analyses in the review. An alternative would be to illustrate the risk of bias for a particular meta-analysis (or for a particular outcome if a statistical synthesis is not undertaken), showing the proportion of information that comes from studies at low, unclear, or high risk of bias for each item in the tool, among studies contributing information to that outcome.\n\n# Summary assessment of risk of bias\n\nTo draw conclusions about the overall risk of bias within or across trials it is necessary to summarise assessments across items in the tool for each outcome within each trial. In doing this, review authors must decide which domains are most important in the context of the review, ideally when writing their protocol. For example, for highly subjective outcomes such as pain, blinding of participants is critical. The way that summary judgments of risk of bias are reached should be explicit and should be informed by empirical evidence of bias when it exists, likely direction of bias, and likely magnitude of bias. Table 3<a href=\"#tbl3\" data-ref-type=\"table\"><\/a> provides a suggested framework for making summary assessments of the risk of bias for important outcomes within and across trials.\n\n\u2002Approach to formulating summary assessments of risk of bias for each important outcome (across domains) within and across trials (adapted from Higgins and Altman^13^)\n\n| Risk of bias | Interpretation | Within a trial | Across trials |\n|----|----|----|----|\n| Low risk of bias | Bias, if present, is unlikely to alter the results seriously | Low risk of bias for all key domains | Most information is from trials at low risk of bias |\n| Unclear risk of bias | A risk of bias that raises some doubt about the results | Low or unclear risk of bias for all key domains | Most information is from trials at low or unclear risk of bias |\n| High risk of bias | Bias may alter the results seriously | High risk of bias for one or more key domains | The proportion of information from trials at high risk of bias is sufficient to affect the interpretation of results |\n\n## Assessments of risk of bias and synthesis of results\n\nSummary assessments of the risk of bias for an outcome within each trial should inform the meta-analysis. The two preferable analytical strategies are to restrict the primary meta-analysis to studies at low risk of bias or to present meta-analyses stratified according to risk of bias. The choice between these strategies should be based on the context of the particular review and the balance between the potential for bias and the loss of precision when studies at high or unclear risk of bias are excluded. Meta-regression can be used to compare results from studies at high and low risk of bias, but such comparisons lack power, 15 and lack of a significant difference should not be interpreted as implying the absence of bias.\n\nA third strategy is to present a meta-analysis of all studies while providing a summary of the risk of bias across studies. However, this runs the risk that bias is downplayed in the discussion and conclusions of a review, so that decisions continue to be based, at least in part, on flawed evidence. This risk could be reduced by incorporating summary assessments into broader, but explicit, measures of the quality of evidence for each important outcome, for example using the GRADE system.16 This can help to ensure that judgments about the risk of bias, as well as other factors affecting the quality of evidence (such as imprecision, heterogeneity, and publication bias), are considered when interpreting the results of systematic reviews.17 18\n\n# Discussion\n\nDiscrepancies between the results of different systematic reviews examining the same question19 20 and between meta-analyses and subsequent large trials21 have shown that the results of meta-analyses can be biased, which may be partly caused by biased results in the trials they include. We believe our risk of bias tool is one of the most comprehensive approaches to assessing the potential for bias in randomised trials included in systematic reviews or meta-analyses. Inclusion of details of trial conduct, on which judgments of risk of bias are based, provides greater transparency than previous approaches, allowing readers to decide whether they agree with the judgments made. There is continuing uncertainty, and great variation in practice, over how to assess potential for bias in specific domains within trials, how to summarise bias assessments across such domains, and how to incorporate bias assessments into meta-analyses.\n\nA recent study has found that the tool takes longer to complete than other tools (the investigators took a mean of 8.8 minutes per person for a single predetermined outcome using our tool compared with 1.5 minutes for a previous rating scale for quality of reporting).22 The reliability of the tool has not been extensively studied, although the same authors observed that larger effect sizes were observed on average in studies rated as at high risk of bias compared with studies at low risk of bias.22\n\nBy explicitly incorporating judgments into the tool, we acknowledge that agreements between assessors may not be as high as for some other tools. However, we also explicitly target the risk of bias rather than reported characteristics of the trial. It would be easier to assess whether a drop-out rate exceeds 20% than whether a drop-out rate of 21% introduces an important risk of bias, but there is no guarantee that results from a study with a drop-out rate lower than 20% are at low risk of bias. Preliminary evidence suggests that incomplete outcome data and selective reporting are the most difficult items to assess; kappa measures of agreement of 0.32 (fair) and 0.13 (slight) respectively have been reported for these.22 It is important that guidance and training materials continue to be developed for all aspects of the tool, but particularly these two.\n\nWe hope that widespread adoption and implementation of the risk of bias tool, both within and outside the Cochrane Collaboration, will facilitate improved appraisal of evidence by healthcare decision makers and patients and ultimately lead to better healthcare. Improved understanding of the ways in which flaws in trial conduct may bias their results should also lead to better trials and more reliable evidence. Risk of bias assessments should continue to evolve, taking into account any new empirical evidence and the practical experience of authors of systematic reviews.\n\n## Summary points\n\n1.  Systematic reviews should carefully consider the potential limitations of the studies included\n\n2.  The Cochrane Collaboration has developed a new tool for assessing risk of bias in randomised trials\n\n3.  The tool separates a judgment about risk of bias from a description of the support for that judgment, for a series of items covering different domains of bias\n\nCite this as: *BMJ* 2011;343:d5928","meta":{"dup_signals":{"dup_doc_count":544,"dup_dump_count":89,"dup_details":{"curated_sources":2,"2023-50":10,"2023-40":13,"2023-23":11,"2023-14":8,"2023-06":10,"2022-49":9,"2022-40":8,"2022-33":16,"2022-27":10,"2022-21":15,"2022-05":11,"2021-49":13,"2021-43":12,"2021-39":16,"2021-31":6,"2021-25":8,"2021-21":9,"2021-17":16,"2021-10":7,"2021-04":5,"2020-50":8,"2020-45":5,"2020-40":9,"2020-34":11,"2020-29":7,"2020-24":8,"2020-16":10,"2020-10":15,"2020-05":10,"2019-51":7,"2019-47":10,"2019-43":12,"2019-39":8,"2019-35":5,"2019-30":9,"2019-26":8,"2019-22":4,"2019-18":7,"2019-13":4,"2019-09":4,"2018-51":7,"2018-47":2,"2018-43":4,"2018-39":3,"2018-34":3,"2018-30":2,"2018-26":3,"2018-17":3,"2018-13":1,"2018-05":2,"2017-51":1,"2017-47":6,"2017-43":4,"2017-39":2,"2017-34":6,"2017-30":1,"2017-26":7,"2017-22":3,"2017-17":2,"2017-09":2,"2016-50":1,"2016-36":4,"2016-30":3,"2016-26":2,"2016-22":3,"2016-18":2,"2016-07":3,"2015-48":3,"2015-40":3,"2015-35":2,"2015-32":3,"2015-27":2,"2015-22":3,"2015-14":2,"2014-52":3,"2014-49":5,"2014-42":7,"2014-41":4,"2014-35":5,"2014-23":5,"2024-30":8,"2024-26":4,"2024-22":4,"2024-18":4,"2024-10":4,"2017-13":7,"2015-18":3,"2015-11":3,"2015-06":2}},"file":"PMC3196245"},"subset":"pubmed_central"}
{"text":"date: 2020\ntitle: IN WHO GLOBAL PULSE SURVEY, 90% OF COUNTRIES REPORT DISRUPTIONS TO ESSENTIAL HEALTH SERVICES SINCE COVID-19 PANDEMIC\n\n**31 August 2020 News release** - WHO to roll out learning and monitoring tools to improve service provision during pandemic\n\nThe World Health Organization (WHO) today published a first indicative survey on the impact of COVID-19 on health systems based on 105 countries' reports. Data collected from five regions over the period from March to June 2020 illustrate that almost every country (90%) experienced disruption to its health services, with low- and middle-income countries reporting the greatest difficulties. Most countries reported that many routine and elective services have been suspended, while critical care - such as cancer screening and treatment and HIV therapy \u2013 has seen high-risk interruptions in low-income countries.\n\n\"The survey shines a light on the cracks in our health systems, but it also serves to inform new strategies to improve healthcare provision during the pandemic and beyond,\" said Dr Tedros Adhanom Ghebreyesus, WHO Director-General. \"COVID-19 should be a lesson to all countries that health is not an 'either-or' equation. We must better prepare for emergencies but also keep investing in health systems that fully respond to people's needs throughout the life course.\"\n\nServices hit across the board: Based on reports from key informants, countries on average experienced disruptions in 50% of a set of 25 tracer services. The most frequently disrupted areas reported included routine immunization \u2013 outreach services (70%) and facility-based services (61%), non-communicable diseases diagnosis and treatment (69%), family planning and contraception (68%), treatment for mental health disorders (61%), cancer diagnosis and treatment (55%).\n\nCountries also reported disruptions in malaria diagnosis and treatment (46%), tuberculosis case detection and treatment (42%) and antiretroviral treatment (32%). While some areas of health care, such as dental care and rehabilitation, may have been deliberately suspended in line with government protocols, the disruption of many of the other services is expected to have harmful effects on population health in the short- medium- and long-term.\n\nPotentially life-saving emergency services were disrupted in almost a quarter of responding countries. Disruptions to 24-hour emergency room services for example were affected in 22% of countries, urgent blood transfusions were disrupted in 23% of countries, emergency surgery was affected in 19% of the countries.\n\nDisruption due to a mix of supply and demand side factors. 76% of countries reported reductions in outpatient care attendance due to lower demand and other factors such as lockdowns and financial difficulties. The most commonly reported factor on the supply side was cancellation of elective services (66%). Other factors reported by countries included staff redeployment to provide COVID-19 relief, unavailability of services due to closings, and interruptions in the supply of medical equipment and health products.\n\nAdapting service delivery strategies. Many countries have started to implement some of the WHO recommended strategies to mitigate service disruptions, such as triaging to identify priorities, shifting to on-line patient consultations, changes to prescribing practices and supply chain and public health information strategies. However, only 14% of countries reported removal of user fees, which WHO recommends to offset potential financial difficulties for patients.\n\nThe pulse survey also provides an indication of countries' experiences in adapting strategies to mitigate the impact on service provision. Despite the limitations of such a survey, it highlights the need to improve real-time monitoring of changes in service delivery and utilization as the outbreak is likely to wax and wane over the next months, and to adapt solutions accordingly.\n\nTo that end, WHO will continue to work with countries and to provide supportive tools to address the fallout from COVID-19. Given countries' urgent demand for assistance during the pandemic response, WHO is developing the COVID19: Health Services Learning Hub, a web-based platform that will allow sharing of experiences and learning from innovative country practices that can inform the collective global response. WHO is also devising additional surveys at the sub-national level and in health facilities to gauge the longer-term impact of disruptions and help countries weigh the benefits and risks of pursuing different mitigation strategies.\n\nAvailable from: <https:\/\/www.who.int\/news-room\/detail\/31-08-2020-in-who-global-pulse-survey-90-of-countries-report-disruptions-to-essential-health-services-since-covid-19-pandemic>","meta":{"dup_signals":{"dup_doc_count":109,"dup_dump_count":23,"dup_details":{"curated_sources":1,"2023-50":4,"2023-40":6,"2023-23":1,"2023-14":3,"2023-06":3,"2022-49":2,"2022-40":4,"2022-33":1,"2022-27":3,"2022-21":3,"2022-05":3,"2021-49":1,"2021-43":1,"2021-39":5,"2021-31":5,"2021-25":5,"2021-21":6,"2021-17":7,"2021-10":5,"2021-04":9,"2020-50":6,"2020-45":13,"2020-40":12}},"file":"PMC7557558"},"subset":"pubmed_central"}
{"text":"author: Joshua P. Thaler; Stephan J. Guyenet; Mauricio D. Dorfman; Brent E. Wisse; Michael W. SchwartzCorresponding author: Michael W. Schwartz, <email@example.com>.\ndate: 2013-08\nreferences:\ntitle: Hypothalamic Inflammation: Marker or Mechanism of Obesity Pathogenesis?\n\nObesity and its associated metabolic and cardiovascular disorders are among the most challenging health problems confronting developed countries. Not only is obesity remarkably common, affecting more than one-third of U.S. adults, and very challenging to treat, but it is also tightly linked to type 2 diabetes and related metabolic disorders. A major obstacle to effective obesity treatment is that lost weight tends to be regained over time (1). Although the mechanisms underlying recovery of lost weight are incompletely understood, a large literature suggests that body fat stores are subject to homeostatic regulation, and that this process occurs in obese as well as normal-weight individuals. From this perspective, obesity can be viewed as a disorder in which the biologically defended level of body fat mass is increased. Recent findings implicate inflammation in key hypothalamic areas for body weight control in this process. In this review, we present an overview of energy homeostasis\u2014the biological process that underlies the control of body fat mass\u2014and describe evidence that defects in this regulatory system contribute to obesity pathogenesis. We then address molecular characteristics of hypothalamic inflammation and their implications for obesity pathogenesis (detailed more extensively in refs. 2,3), followed by evidence linking high-fat diet (HFD) feeding to neuropathological alteration of key hypothalamic areas controlling energy balance. We conclude by considering how cell-cell interactions may contribute to this deleterious hypothalamic response and the implications of these interactions for obesity pathogenesis.\n\n# The case for energy homeostasis\n\n## Homeostatic response to weight loss.\n\nFollowing a period of caloric restriction, lost weight is gradually but inexorably recovered in most individuals. This effect involves the capacity of the brain to sense the reduction of body energy stores and activate responses to compensate for this deficit. In simple terms, voluntary weight loss triggers increases of both appetite and energy efficiency, such that both sides of the energy balance equation shift in favor of weight gain (4,5). Perhaps the clearest example of this compensatory response to lost weight comes from the large clinical literature of diet studies in human subjects. Some 20 years ago, Safer (6) reported that the vast majority (\\>90%) of weight lost through hypocaloric dieting in humans is regained over a 5-year period. Even with the addition of exercise (which slows the rate of weight regain \\[7\\]), caloric restriction through lifestyle modification is a Sisyphean task over the long term. As an example, obese subjects enrolled in the lifestyle intervention arm of the landmark Diabetes Prevention Program (DPP) study averaged \u223c7% weight loss during the trial but regained virtually all of the weight lost over the full 10 years of postintervention monitoring (8). Lifestyle intervention in the follow-up Look AHEAD trial similarly failed to produce durable weight loss (9).\n\nRobust defense of body energy stores is similarly observed in conditions that promote weight loss by increasing energy expenditure rather than reducing intake, such as housing mice in a cold environment. To survive, a marked increase of energy expenditure is required to support thermogenesis and maintain core body temperature. Increased heat production occurs initially at the expense of modest body fat depletion, but as fat loss proceeds, food intake increases in a commensurate manner to offset further loss of body fat stores (10). Consequently, graded decreases of environmental temperature trigger proportionate increases of energy expenditure that are compensated by increased energy intake so as to maintain body fat stores.\n\n## Adaptive responses to weight gain.\n\nFrom the foregoing, it is evident that the energy homeostasis system responds robustly to states of negative energy balance, whether secondary to caloric restriction or increased energy expenditure. Does the energy homeostasis system also protect against weight gain? Available data from both animal models and humans support this possibility. A classic demonstration of this phenomenon was provided by the \"Experimental Obesity in Man\" studies performed at the University of Vermont almost 50 years ago (11). In these experiments, lean human volunteers were challenged to increase their body weight by 20% through overeating for a period of up to 6 months. Although overeating was initially accomplished without difficulty, the subjects' capacity to do so soon began to wane, presumably because counterregulatory mechanisms were engaged that reduce appetite. After reaching the prescribed 20% increase of body weight and being released from the experimental mandate to overeat, the subjects displayed several weeks of profound anorexia that caused the excess pounds to be shed. Only when body weight began to converge on preintervention values did food intake also return to normal levels. Similar observations were made in subsequent human overfeeding studies (12), and across a variety of animal models as well (13).\n\nIn the face of a challenge to body fat stores in either direction, therefore, robust homeostatic responses drive the return of body weight to its original, biologically defended level (sometimes referred to as a set-point). If normal-weight individuals defend against experimentally induced changes of body fat mass in either direction, it follows that obesity pathogenesis should involve impairment of the energy homeostasis system. Since overweight individuals appear, by and large, to defend their body fat mass as robustly as normal weight individuals (14), obesity does not appear to reflect a failure of energy homeostasis so much as the biological defense of an elevated level of body fat mass.\n\n# Obesity and energy homeostasis\n\nAn important strength of the homeostasis model for understanding obesity pathogenesis is that it offers a plausible explanation for why conservative approaches to weight loss fail over time. To understand how a slow, progressive increase in the defended level of body fat stores might occur, it is helpful to consider how the energy homeostasis system operates in normal-weight individuals.\n\n## How does energy homeostasis work?\n\nEnergy homeostasis involves an adiposity negative feedback system through which circulating signals such as the hormone leptin inform key brain centers about body energy stores (15). In response to a change of body fat mass, corresponding changes of plasma leptin levels and hence of leptin signaling in these brain areas elicit changes of both energy intake and energy expenditure that favor the return of body weight to preintervention levels. In response to weight loss due to caloric restriction, this model predicts that falling plasma levels of leptin and other adiposity negative feedback signals (e.g., insulin) elicit brain responses that minimize further weight loss and promote the eventual recovery of lost weight. Leptin also responds robustly to short-term changes of energy balance, prior to major changes in adiposity, and therefore integrates information about both long-term and short-term energy status (16,17).\n\nA growing number of leptin-responsive neuronal subsets have been identified, and their roles in adaptive changes of energy balance are increasingly well understood. Perhaps best studied of these are two leptin-sensitive neuronal subsets situated in the hypothalamic arcuate nucleus (ARC). Activation of neuropeptide Y and agouti-related peptide (NPY\/AgRP) neurons potently increases food intake and reduces energy expenditure through the paracrine action of the neuropeptides NPY and AgRP themselves and through inhibition of downstream neuronal activity via GABAergic signaling (15,18,19). Conversely, reduced intake and increased expenditure occurs with activation of proopiomelanocortin (POMC) neurons largely through the action of the anorexigenic neuropeptide \u03b1-melanocyte\u2013stimulating hormone (15) (although direct neurotransmitter action likely contributes as well \\[20\\]). Weight loss reduces leptin levels causing activation of NPY\/AgRP neurons and inhibition of POMC neurons (15). This combination potently favors positive energy balance and is sustained until lost weight has been regained.\n\nA large literature now indicates that proper functioning of these neurocircuits is required for normal energy homeostasis in rodent models, and many models of obesity caused by impairment of the leptin-melanocortin pathway now exist. A similar role was recently invoked for a distinct subset of leptin-sensitive ARC neurons containing neuronal nitric oxide synthetase. Deletion of leptin receptors from these neurons causes hyperphagia and obesity even more severe than that induced by defective melanocortin signaling (21). Neurons in other hypothalamic areas, including the ventromedial, lateral hypothalamic, and paraventricular nuclei, and in extrahypothalamic areas, including hindbrain (e.g., nucleus of the solitary tract), midbrain (ventral tegmental area), and forebrain (nucleus accumbens), are also critical for normal energy homeostasis.\n\nThat the aforementioned hypothalamic neurocircuits are essential for normal energy homeostasis in humans is supported by insights gained from rare monogenic forms of human obesity. Loss-of-function mutations affecting the leptin-melanocortin pathway, including genes encoding leptin, melanocortin-4 receptor (Mc4r), leptin receptor, and the prohormone convertase enzyme that processes POMC, together constitute the most prevalent, highly penetrant genetic causes of human obesity (22). However, \u223c95% of human obesity is due not to a single, highly penetrant mutation but rather to complex interactions between genetic and environmental factors that favor both weight gain and the defense of elevated body weight. Impaired leptin responsiveness is a feature of these more common forms of obesity as well and hence may contribute to obesity pathogenesis in susceptible individuals.\n\n## Hypothalamic inflammation and leptin resistance.\n\nLeptin resistance can be defined simply as an acquired or inherited defect in the response to leptin. This simplistic definition is problematic, however, since such leptin resistance can potentially arise from a long list of defects\u2014not only those affecting leptin receptor signal transduction, but at innumerable points downstream of or in parallel to leptin-mediated effects (23). For example, obesity due to Mc4r mutation, the most common monogenic form of human obesity, is characterized by leptin resistance, since leptin action on energy balance requires that it activate the melanocortin pathway. From this perspective, the term leptin resistance is easily misused, since a blunted feeding response to leptin is present in virtually all forms of obesity, save for that caused by leptin deficiency (23). This conundrum has to some extent confounded efforts to delineate underlying mechanisms.\n\nDespite this caveat, growing evidence suggests a causal role for leptin resistance in obesity pathogenesis and that obesity-associated hypothalamic inflammation can underlie this resistance. This hypothesis originated following clear evidence that inflammatory signaling contributes to obesity-associated insulin resistance in peripheral tissues such as liver, muscle, and adipose tissue (24). Among neuronal consequences of proinflammatory signaling is a disruption of intracellular signal transduction downstream of both insulin and leptin receptors via the insulin receptor substrate\u2013phosphatidylinositol 3-kinase pathway (2). Leptin signaling via the janus kinase\u2013signal transducer and activator of transcription pathway can also be impaired by cellular inflammation (25,26), suggesting that this mechanism may contribute not only to obesity-associated leptin and insulin resistance, but also to the associated increase in the defended level of body fat stores. However, dissociating cause and effect has been challenging, and evidence of leptin resistance caused by inflammation independently of obesity is lacking (though studies of aging, injury, and endotoxin-induced leptin resistance are suggestive of this possibility \\[27\u201329\\]). We favor the hypothesis that a vicious cycle exists involving obesity, leptin resistance, and inflammation. According to this model, diet-induced increases of inflammation beget a state of leptin resistance that promotes weight gain, which in turn triggers further inflammation and leptin resistance, eventually resulting in the biological defense of an elevated level of body fat. This perspective highlights the challenges inherent in determining the extent to which leptin resistance and inflammation are causes or consequences of weight gain, a challenge that cannot be met without methodologies that can distinguish cellular responses to diet from metabolic alterations induced by obesity itself.\n\n## Does hypothalamic inflammation cause obesity?\n\nThe first report of obesity-associated hypothalamic inflammation was in a rat model of diet-induced obesity (DIO) published in 2005 (30), and many investigators have since replicated this finding (2,3,31\u201336). The observation that genetic interventions that disrupt neuronal inflammation can block both obesity and hypothalamic leptin resistance during HFD feeding implicates this inflammation in obesity pathogenesis. For this to be the case, however, hypothalamic inflammation would have to occur prior to obesity onset, unlike the later onset of inflammation in peripheral tissues. This hypothesis is supported by our recent observation that in rats predisposed to DIO, expression of proinflammatory biomarkers increases in the mediobasal hypothalamus within 24 h of HFD onset (37). The mediobasal hypothalamus level of these markers increases more by day 3 and is followed by a transient normalization of inflammation, only to rise again in 2\u20133 weeks and remain elevated thereafter. Interestingly, this initial rise and fall of hypothalamic inflammatory markers follows a temporal pattern similar to that of caloric intake following the switch to an HFD.\n\nSeveral molecules and pathways have been identified as candidate mediators of hypothalamic inflammation during HFD feeding. Among these are the serine kinases c-Jun N-terminal kinase (Jnk) and\u00a0inhibitor of nuclear factor-\u03baB kinase (IKK\u03b2) as well as Toll-like receptor 4 (TLR4), the ceramide biosynthesis pathway, and the endoplasmic reticulum (ER) stress pathway (30\u201336). Each of these is upregulated in the rodent hypothalamus during DIO (30,36,38), and Jnk and IKK\u03b2 can inhibit insulin and leptin signaling by at least two mechanisms: induction of the signal termination molecule suppressor of cytokine signaling-3 and serine phosphorylation of the essential pathway intermediate insulin receptor substrate (3,24). Targeted disruption of each of these pathways in a hypothalamus-specific manner (by pharmacologic, viral gene transfer, or genetic means) limits the extent of HFD-induced obesity, hypothalamic leptin resistance, and systemic insulin resistance (30\u201336), implying a contributory role for these mechanisms of hypothalamic inflammation in the pathogenesis of DIO and its metabolic sequelae. Conversely, although acute or robust central nervous system inflammation is typically associated with anorexia and weight loss, interventions that generate more modest levels of hypothalamic inflammation (e.g., virally mediated neuronal expression of constitutively active IKK\u03b2, or central administration of either low-dose tumor necrosis factor-\u03b1 or the ER stress inducer thapsigargin) can produce mild obesity\/metabolic syndrome phenotypes (34,36,39,40). These data collectively suggest that in some contexts, hypothalamic inflammation is both necessary and sufficient for DIO. As such, an important research priority is to determine the relative importance of the many inflammatory signaling molecules and pathways implicated in promoting leptin resistance and obesity.\n\nAlthough these findings collectively offer evidence of a causal role for hypothalamic inflammation in the pathogenesis of DIO in rodent models, there are conflicting results that cast doubt on this hypothesis. First, several models of increased tissue inflammation (e.g., fasting in adipose tissue \\[41\\] and p50 knockout in liver \\[42\\]) show improved rather than worsened insulin sensitivity, suggesting that the relationship between hormone signaling and inflammation is context dependent. More importantly, a number of total-body proinflammatory cytokine and cytokine receptor knockout models (e.g., interleukin \\[IL\\]-1, IL-6, IL-1 receptor, and tumor necrosis factor-\u03b1 receptors \\[43\u201346\\]) display obesity phenotypes rather than being protected from DIO. Conversely, IL-1 receptor antagonist\u2013deficient mice show a lean, hypermetabolic phenotype with increased leptin sensitivity despite higher levels of IL-1 activity (47). Reconciling these results with the literature supporting a role for hypothalamic inflammation in DIO is inherently difficult because these are largely whole-body gene knockouts that affect multiple tissues and may cause developmental compensation. In many instances, the degree of change in inflammatory signaling in these models is much greater than that observed with HFD-induced obesity. Lastly, the hypothalamus was not specifically examined in these models. However, two recent studies have demonstrated dissociation between weight changes and alterations in hypothalamic inflammation. In the first, DIO-sensitive HFD-fed rats switched back to regular chow for 8 weeks were observed to lose all their excess weight despite sustained elevations in hypothalamic inflammatory gene expression (48). Conversely, neuronal peroxisome proliferator\u2013activated receptor-\u03b4 knockout mice are resistant to HFD-induced hypothalamic inflammation yet are more susceptible to DIO than wild-type controls (49). Thus, the evidence supporting a direct role of hypothalamic inflammation in promoting weight gain is clearly mixed. These considerations raise the possibility that HFD-induced hypothalamic inflammation is but one element of a more comprehensive injury process that itself contributes to obesity pathogenesis, a hypothesis that we explore further below.\n\n# Hypothalamic gliosis and neuron injury\n\nAt the cellular level, exposure of neurons to nutrient excess represents a significant stress that not only engages adaptive mechanisms such as autophagy and ER stress that limit neuronal damage, but also involves neighboring cell populations. More than 50% of the cellular composition of the brain is nonneuronal, including glial, vascular, and periventricular constituents (50). Astrocytes and microglia are the most abundant of these specialized cell types and, in addition to comprising much of the basic architecture of brain parenchyma, they maintain the blood-brain barrier, support neuronal metabolism, and both guard against and react to local tissue injury. To accomplish these various functions, both astrocytes and microglia are capable of remarkable plasticity, altering both their genetic programs and cellular morphologies (called reactive gliosis) to combat infection, support or consume damaged neurons, and direct the restorative process (51,52). Interestingly, rats receiving systemic leptin administration 3 h prior to cerebral ischemia had reduced infarct volume with signal transducer and activator of transcription-3 activation in astrocytes of the ischemic penumbra (53). Similarly, leptin administration and fasting reciprocally modulate glucose and glutamate transporter expression in hypothalamic astrocytes. These observations are suggestive of an interaction between nutritional status and glial cell function (54).\n\nWhereas the link between hypothalamic inflammation and obesity pathogenesis is the focus of a sizeable literature, the role of astrocytes and microglia in energy homeostasis is comparatively unexplored. Both cell types play crucial roles in synaptic remodeling (55), metabolic coupling at active synapses (56), and neurotransmitter reuptake (57), functions that ultimately influence neuronal activity and behavior. Astrocytes and microglia play central roles in the central nervous system response to injury but can provide either neuroprotection or perpetuate neurotoxicity depending on the nature of the underlying insult (51,58). In neurodegenerative conditions such as Alzheimer's disease, amyotrophic lateral sclerosis, and multiple sclerosis, microglia and astrocytes often accelerate the disease process by generating cytokines, reactive oxygen species, and other toxic mediators involved in clearing degenerating cells. By amplifying inflammatory signals in the hypothalamus analogously to the recruitment and activation of proinflammatory immune cells in adipose and other peripheral tissues (59,60), both cell types have the potential to impair neuronal function in ways that favor leptin resistance and associated weight gain. Lending support to this argument, TLR4, a putative mediator of saturated fatty acid\u2013induced inflammatory signaling, is abundantly expressed by microglia (33), and acute inactivation of TLR4 by central administration of an anti-TLR4 antibody can reduce weight gain during HFD feeding (33). In addition, reactive astrocytes and microglia accumulate in the hypothalamus during long-term HFD consumption at times when hypothalamic inflammation is clearly evident (33,37). In contrast, HFD-fed mice subjected to an involuntary exercise regimen exhibit modest improvements in glucose tolerance along with reduced hypothalamic microglial activation (61), suggesting a link between microglial phenotype and obesity-associated metabolic impairment. Finally, in a nonhuman primate model, microglial activation along with increased expression of inflammatory pathway genes was observed in fetuses from HFD-fed mothers, raising the possibility that hypothalamic microglia contribute to the effect of intrauterine programming to influence the adult phenotype (62).\n\nOther studies, however, suggest a more complex picture in which microglia and astrocytes play a protective role by limiting the deleterious hypothalamic consequences of consuming an HFD. First, mice with moderately increased production of IL-6 from astrocytes were protected from DIO, rather than being more susceptible (63). Moreover, adult rats overfed during the neonatal period manifest hypothalamic microglial activation (as evidenced by major histocompatibility complex class II expression) without local inflammation (64), and hypothalamic microglia from mice fed an HFD accumulate the generally anti-inflammatory molecule IgG (65). Finally, our recent work suggests that effects of short-term HFD feeding on hypothalamic inflammation and reactive gliosis are separable from one another (37). Specifically, whereas both processes were evident within the first week of HFD consumption, hypothalamic inflammation subsided over the next 2 weeks despite accumulation of enlarged microglia in the ARC that continued unabated.\n\nAlthough definitive answers are still awaited, these results are consistent with a model (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<\/a>) in which gliosis develops initially as a neuroprotective response to cope with neuronal stress induced by HFD feeding. In this scenario, glial responses initially constrain hypothalamic inflammatory signaling. With prolonged exposure to an HFD, however, astrocytes and\/or microglia may eventually convert to a more proinflammatory, neurotoxic phenotype. Detailed cellular phenotyping and carefully timed functional interventions should help clarify these possibilities.\n\n# Integrated model of neuronal injury and obesity pathogenesis\n\nLike cells in peripheral tissues of animals fed an HFD, hypothalamic neurons confronted by nutrient excess are proposed to respond by engaging mechanisms that limit excessive hormone- and nutrient-related signaling, manage organellar stress, and maintain structural integrity. At the same time, these neurons also integrate signaling inputs from neurons in other brain regions that may themselves be reacting to the change in diet. These various challenges to neuronal function are compatible with evidence that in rats predisposed to DIO, hypothalamic neurons initiate a stress response (as measured by up-regulation of the chaperone protein hsp72) within the first week of HFD exposure, close to the onset of hypothalamic inflammation (37). The accumulation of reactive astrocytes and microglia in the mediobasal hypothalamus occurs around the same time and precedes a subsequent reduction in the level of inflammation (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<\/a>).\n\nWith prolonged HFD exposure, however, POMC neurons exhibit increased autophagic activity and eventually (after 8 months) appear to decrease in number by 25% (37), consistent with prior reports of apoptosis of these neurons in mice with DIO (66). Based on these considerations, we speculate that hypothalamic inflammation during HFD feeding originates primarily within neurons rather than glial cells, and that the associated gliosis occurs in response to neuron injury and is initially neuroprotective. This hypothesis does not exclude the possibility that glial cells that respond directly to dietary components and\/or nutrient excess are also drivers of inflammation and\/or neuronal injury, but to date we are unaware of data that directly support this view. If chronic exposure to an HFD causes permanent damage to or death of neurons comprising energy balance neurocircuits, obesity susceptibility and other phenotypic outcomes may depend on the degree to which these neuronal subsets are susceptible to this type of stress. For example, if neuron injury and loss occur preferentially in POMC over AgRP neurons, the balance of functional anorexigenic to orexigenic inputs should shift so as to favor leptin resistance, excess weight gain, and an upward resetting of the defended level of body fat mass.\n\nAlthough the extent to which obesity and\/or HFD feeding impacts various hypothalamic cell populations in humans remains uncertain, early insights from brain imaging studies are beginning to support this type of neuropathological model. A retrospective analysis of magnetic resonance images from 34 subjects (BMI range 17.7\u201344.1 kg\/m^2^) revealed evidence of gliosis in the mediobasal hypothalamus that correlated with BMI (37), and a separate study reported an inverse correlation between systemic inflammation (measured as serum fibrinogen) and the integrity of brain structures involved in food reward and feeding behavior measured by magnetic resonance imaging in 44 overweight\/obese subjects (67). Brain imaging (including new approaches to the use of PET imaging to detect microglial activation) and other modalities have important potential to ascertain whether human obesity involves cumulative ARC neuronal damage and whether such an effect is reversible and\/or predictive of either future weight gain or weight regain following voluntary weight loss.\n\n# Summary\n\nOur understanding of obesity-associated hypothalamic inflammation\u2014its underlying causes, the contributions made by distinct cell types, the extent to which it is reflective of tissue injury versus repair, and its implications for obesity pathogenesis and treatment\u2014remains incomplete, but the field is evolving rapidly. Although hypothalamic inflammation shares several features with responses observed in peripheral tissues (e.g., comparable increases of some of the same inflammatory biomarkers, activation of local immune cells), its many unique features identify it as a fundamentally distinct process. For one, inflammation occurs much more rapidly in the hypothalamus than in peripheral tissues following the switch to an obesogenic HFD, such that it precedes weight gain. Available data are compatible with a model in which the initial cause of hypothalamic inflammation induced by HFD feeding involves injury to neurons that comprise energy balance neurocircuits. In turn, this injury may undermine homeostatic responses that protect against weight gain, thereby contributing to obesity pathogenesis. Additional studies are warranted to critically test this model and determine if therapeutic interventions targeting this process have a role in the future of obesity treatment.\n\n## ACKNOWLEDGMENTS\n\nThis work was supported by a National Institutes of Health (NIH) Fellowship Training Program Award (T32DK007247), NIH National Research Service Award (F32DK091989), the NIH-funded University of Washington Nutrition Obesity Research Center and the Diabetes Research Center, and NIH grants to M.W.S. (DK090320, DK083042, and DK052989) and J.P.T. (DK088872). J.P.T. was also supported by a Beginning Grant-in-Aid from the American Heart Association.\n\nM.W.S. has consulted for Santarus and Pfizer. No other potential conflicts of interest relevant to this article were reported.\n\nJ.P.T. and M.W.S. wrote the manuscript. S.J.G. and B.E.W. reviewed and edited the manuscript. M.D.D. contributed the figure and reviewed and edited the manuscript.\n\n# REFERENCES","meta":{"dup_signals":{"dup_doc_count":131,"dup_dump_count":58,"dup_details":{"curated_sources":4,"2021-49":1,"2021-31":1,"2021-21":1,"2021-17":1,"2020-50":1,"2020-24":1,"2020-16":1,"2019-43":3,"2019-35":1,"2019-30":1,"2019-04":1,"2018-43":1,"2018-34":1,"2018-26":3,"2018-22":1,"2018-17":2,"2018-13":1,"2018-09":1,"2018-05":3,"2017-47":4,"2017-43":1,"2017-39":3,"2017-34":1,"2017-30":4,"2017-26":2,"2017-22":2,"2017-17":5,"2017-09":4,"2017-04":5,"2016-50":3,"2016-44":4,"2016-40":3,"2016-36":3,"2016-30":2,"2016-26":3,"2016-22":3,"2016-18":3,"2016-07":3,"2015-48":2,"2015-40":1,"2015-35":1,"2015-32":3,"2015-27":2,"2015-22":2,"2015-14":2,"2014-52":3,"2014-49":2,"2014-42":2,"2014-41":4,"2014-35":3,"2014-23":2,"2022-05":1,"2024-22":1,"2017-13":4,"2015-18":3,"2015-11":3,"2015-06":1,"2024-26":1}},"file":"PMC3717869"},"subset":"pubmed_central"}
{"text":"author: PH Remans; JM van Laar; ME Sanders; P-P Tak; KA Reedquist\ndate: 2005\ninstitute: 1AMC, Amsterdam, The Netherlands; 2LUMC, Leiden, The Netherlands\ntitle: Synovial monocytes mediate Rap1-dependent oxidative stress in rheumatoid arthritis T lymphocytes\n\n# Background\n\nTransient production of reactive oxygen species (ROS) plays an important role in optimizing transcriptional and proliferative responses to T-cell receptor signaling. Conversely, chronic oxidative stress leads to mitogenic hyporesponsiveness and enhanced transcription of inflammatory gene products. It has recently been demonstrated that constitutive activation of the small GTPase Ras and simultaneous inhibition of Rap1 in synovial fluid (SF) T cells results in high intracellular ROS production, which is thought to underlie many of the functional abnormalities observed in these cells in rheumatoid arthritis.\n\n# Objectives\n\nTo identify the factor(s) responsible for modulation of intracellular ROS production in synovial T lymphocytes.\n\n# Methods\n\nPurified rheumatoid arthritis peripheral blood (PB) T cells were incubated in the presence of different cytokines, in 50% autologous SF, or with autologous SF monocytes for 72 hours. Activation status of Ras and Rap1 GTPases were determined using activation-specific probes for these GTPases. Oxidation of the dye DCF by FACS analysis was used to measure intracellular ROS production.\n\n# Results\n\nChronic stimulation of PB T cells for 72 hours with tumor necrosis factor alpha (TNF-\u03b1) or 50% autologous SF resulted in a slight increase in basal ROS production, but did not increase intracellular ROS production to levels found in SF T cells. Exposure of PB T cells to SF (but not PB) monocytes for 72 hours, however, led to a strong increase in ROS production in PB T cells, comparable with ROS levels in SF T cells. Moreover, similar Rap1 inhibition as found in SF T cells were observed in PB T cells after exposure to SF monocytes. To demonstrate that the inhibition of Rap1 is critical in the subsequent increase in ROS production, PB T cells were nucleofected with the constitutive active isoform of Rap1 (RapV12). In RapV12 nucleofected PB T cells the SF monocyte-induced ROS production was prevented. Cell\u2013cell contact is critical, since in PB T cells separated from SF monocytes by a transwell membrane, the inhibition of Rap1 was relieved, concomitant with an absence in excess ROS production. Additionally, we found that addition of 10 \u03bcg\/ml recombinant CTLA-4-Ig fusion protein also prevented oxidative stress in PB T cells exposed to SF monocytes, which suggested a central role for CD28. PB T cells were therefore stimulated with TNF-\u03b1, interferon gamma, IL-1\u03b2, or transforming growth factor beta, in the presence or absence of anti-CD28. Here we found that stimulation with anti CD28 by itself was sufficient to induce Rap1 inhibition and induce a moderate increase in ROS production. Co-incubation of PB T cells with TNF-\u03b1 strongly enhanced the intracellular ROS production.\n\n# Conclusion\n\n*In vitro* exposure of PB T cells from rheumatoid arthritis patients to synovial monocytes leads to a strong increase in intracellular ROS production. This is mediated by simultaneous Ras activation and inhibition of Rap1. Where Ras can be activated by a variety of stimuli, Rap1 inhibition is induced by SF monocytes through CD28 costimulatory signaling.","meta":{"dup_signals":{"dup_doc_count":103,"dup_dump_count":33,"dup_details":{"curated_sources":2,"2022-21":1,"2020-24":1,"2020-05":1,"2019-39":1,"2017-39":1,"2017-22":1,"2017-09":7,"2016-44":1,"2016-40":1,"2016-36":7,"2016-30":4,"2015-48":3,"2015-40":2,"2015-35":3,"2015-32":3,"2015-27":3,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":5,"2014-42":8,"2014-41":5,"2014-35":4,"2014-23":5,"2014-15":4,"2022-40":1,"2015-18":3,"2015-11":2,"2015-06":3,"2014-10":4,"2013-48":3,"2013-20":4,"2024-30":1}},"file":"PMC2834129"},"subset":"pubmed_central"}
{"text":"abstract: A report on the Cold Spring Harbor Laboratory meeting 'Systems Biology: Global Regulation of Gene Expression', Cold Spring Harbor, USA, 27-30 March 2008.\nauthor: Stein Aerts; Stefanie Butland\ndate: 2008\ninstitute: 1Laboratory of Neurogenetics, Department of Molecular and Developmental Genetics, VIB, Leuven B-3000, Belgium; 2Centre for Molecular Medicine and Therapeutics, CFRI, University of British Columbia, Vancouver, V5Z 4H4, Canada\ntitle: Sequencing the regulatory genome\n\nThe line between the biological and computational research communities has disappeared in the field of gene regulation. The group of regulatory biology researchers represented at the recent meeting at Cold Spring Harbor on systems biology shares the same goal: develop and apply experimental and computational technologies to decipher the genomic regulatory code and the gene regulatory networks that are the driving forces of development and evolution. As in previous years, important gaps in solving the complex problem of gene regulation were bridged. This year featured the emerging massively parallel sequencing technologies, which are now being applied to every conceivable step in the gene regulation process, from gene annotation and alternative splicing, to transcription factor binding, and chromatin structure. Topics covered at the meeting ranged widely and in this report, we give our impressions of some highlights in two dominant themes: gene regulation in a nuclear context and transcription factor binding specificity.\n\n# Genome geography in three dimensions\n\nWhen transcription factors are reading the genomic regulatory code to determine the complement of active genes in a cell at a given time, they can be aided, guided, or obstructed by the chromatin they operate on. To catch chromatin in the regulatory act, laboratories are sequencing the sites associated with histone modifications that mark repressive, activating, and bivalent chromatin states, high-resolution DNase I hypersensitive sites (DHSs) that mark accessible chromatin, possible insulator sites, sites bound by transcription factors and RNA polymerase II, and *in vivo* cross-linked sites that represent long-range regulatory interactions in a locus. In an increasing number of laboratories, the regulatory geography of the genome is now being assessed within the three-dimensional context of the nucleus.\n\nBas van Steensel (Netherlands Cancer Institute, Amsterdam, the Netherlands) provided an elegant picture of human gene regulation in three dimensions by identifying nuclear-lamina-associated domains (LADs) in interphase chromosomes. LADs range from 100 kb to 10 Mb and have sharp borders that define chromatin regions with distinctive characteristics: they tend to have fewer genes with lower expression levels compared with genes outside LADS, low RNA polymerase II occupancy, and enrichment of the repressive histone mark H3 trimethylated on lysine 27 (H3K27me3) at their borders. Thirty percent of LAD borders have at least one of three other marks: binding sites for the transcription factor CTCF (commonly held to act as insulators), a CpG island, or a promoter directing transcription away from the LAD.\n\nSites of chromatin accessibility across the human genome were precisely delineated by John Stamatoyannopoulos (University of Washington School of Medicine, St Louis, USA) in nine cell types by 'digital DNase I', an *in vivo* assay of DNase I hypersensitive sites identified by single-molecule sequencing. Stamatoyannopoulos has identified around 400,000 DHSs genome-wide, of which around 170,000 were highly regulated cell-type specific elements. A subset of these are organized into approximately 2,000 tissue 'regulons', each comprising a large cluster of lineage-specific elements spread out over tens or even hundreds of kilobases. Genes that were marked by these regulons in a given cell type showed striking over-representation of Gene Ontology terms for processes associated with the cell lineage in which they were observed. In addition to accessibility to DNase, active enhancers show specific histone modifications. Gary Hon (Ludwig Institute for Cancer Research, San Diego, USA) was able to distinguish cell-type specific enhancers from promoters by their enrichment for H3K4me1 over H3K4me3 modifications. He proposed that these enhancers are what drive cell-type specific patterns of gene expression.\n\nWith such data it is critical to determine how different regulon elements interact with each other to elicit a response. Job Dekker (University of Massachusetts Medical School, Worcester, USA) described his 'chromosome conformation capture carbon copy' (5C) method to detect many-by-many chromatin interactions for a picture of spatial conformation of genomic regions. His analysis of a 1 Mb region around the human beta-globin locus showed that an alternative promoter 250 kb upstream physically interacts with the globin locus control region. Dekker pointed out that \"simple models are insufficient\" for gene regulation, as CTCF sites, usually considered as insulators, at the beta-globin locus actually facilitate long-range interactions between promoters and enhancers.\n\n# Transcription factor binding specificity\n\nWe were reminded by Kevin Struhl (Harvard Medical School, Boston, USA) that the epigenetic states of chromatin cannot explain the specificity of gene expression, but are rather instructed by the sequence-specific transcription factors that translate the regulatory code and recruit chromatin-modifying activities. A cornerstone of our understanding of the regulatory language is the knowledge of a transcription factor's DNA-binding specificity. Significant progress has been achieved in deriving high-quality DNA-binding profiles through a variety of approaches with a large dose of collaboration, particularly with Martha Bulyk (Brigham & Women's Hospital and Harvard Medical School, Boston, USA) for protein-binding microarrays (PBMs). Scot Wolfe (University of Massachusetts Medical School, Worcester, USA) reported new binding profiles for 84 homeodomain transcription factors for *Drosophila melanogaster* through a bacterial one-hybrid system, while Gong-Hong Wei (University of Helsinki, Finland) described binding profiles for all 27 human and 26 mouse ETS family members using a microwell-based high-throughput assay and PBMs, and Christian Grove (University of Massa-chusetts Medical School, Worcester, USA) reported profiles for most of the basic helix-loop-helix (bHLH) dimers in *Caenorhabditis elegans* using a novel version of assay by PBMs. Timothy Hughes (University of Toronto, Canada) described profiles for 300 human and mouse transcription factors across 23 structural classes, including 168 profiles (of 175 total) for homeodomain transcription factors using PBMs.\n\nAll these profiles are highly conserved across species and can be ported between orthologous transcription factors when the DNA-contacting amino acids are conserved. This implies that a full compendium of transcripton factor binding specificities across all animals can be accomplished in the near future, with about one third being finished and released by these groups very soon. A question that remains is precisely how other contributors to specificity, such as transcription factor cooperativity, cell-type specific expression, variant or 'weak' recognition sites, and chromatin state together distinguish between correct target sites of related transcription factors that have virtually identical position weight matrices (PWMs).\n\nWhile the relationship between transcription factors and their binding profiles is well conserved, independent data from various speakers showed yet again that the locations of *bona fide* regulatory elements are not always conserved in an alignment between orthologous regions. This plasticity of transcription factor recognition sites between functionally conserved regulatory regions is still posing a challenge for their computational prediction. Pouya Kheradpour (Massa-chusetts Institute of Technology, Cambridge, USA) presented a pragmatic solution by allowing for movement of a predicted site in an alignment, which for many motifs resulted in increased recovery of conserved sites (sensitivity) at a given specificity. Furthermore, many nonconserved sites are located in transposable elements that are generally not under selection and are usually masked before sequence analysis. For example, Guillaume Bourque (Genome Institute of Singapore, Singapore) found that 43% of nonconserved p53-binding sites are repeat-associated. Ting Wang (University of California, Santa Cruz, USA) identified a similar proportion of p53-binding sites in human endogenous retrovirus long terminal repeats, and Stamatoyanno-poulos noted that around 10% of his DHSs map to transposable elements. Mobile elements thus provide an additional substrate for evolution of species-specific gene regulation.\n\n# What's next in transcriptional regulation?\n\nThe key challenge will be to combine the two topics highlighted in this report, namely determination of the specific binding sites for multiple transcription factors and the genome-scale characterization of chromatin states, and to link these with spatial and temporal differences in gene expression. Advances in measuring cell-type specific gene expression were shown by Bob Waterston (University of Washington, St Louis, USA), who is using automated image-processing tools to analyze three-dimensional movies of fluorescent-marker tagged transcription factors in *C. elegans* embryos. Comparing massive numbers of images, they can make direct quantitative comparisons of expression patterns of different transcription factors \"cell-by-cell, minute-by-minute\". On the same topic, Philip Benfey (Duke University, Durham, USA) has leveraged a compendium of gene-expression data at cell-type specific resolution for an entire organ. His group performed microarray experiments on diverse cell lineages across the radial and longitudinal axes of the *Arabidopsis* root. A complementary set of experiments on six different cell types showed that specific cell types respond uniquely to high-salt or low-iron stress conditions in terms of which genes are up- or down-regulated.\n\nRobert Kingston (Harvard Medical School, Boston, USA) is developing technologies for locus-specific chromatin isolation to get the complete list of players that bind *in vivo* to a regulatory locus. He presented a convincing proof of principle by isolating 95% of known telomere interactors and identifying new biologically relevant ones. A more classical way of determining the input of multiple transcription factors to a specific locus is by genetic screens. Results of a high-throughput assay were presented by Pinay Kainth (University of Toronto, Canada), who tested the input contributions of all nonessential yeast transcription factors and their potential regulators on 27 cell-cycle-specific promoters using quantitative fluorescence measurements. Although genetic perturbations that alter a promoter's output are not limited to the transcription factors that physically bind to the promoter, such data can approximate direct interactions, especially when combined with PWM-based motif predictions.\n\nOnce the transcription-factor-specific regulatory sites, chromatin accessibility, and long-range interactions are determined for a given cell state, one must still determine the *cis*-regulatory logic and the rate of transcription initiation that it produces. This is still a difficult problem addressed by only few groups, including that of Jason Gertz (Washington University School of Medicine, St Louis, USA), who reported the use of libraries of synthetic regulatory regions to examine putative roles of combinations of *cis*-elements even before they have been discovered in real enhancers. This approach provides a possible solution to the sparse sampling of sets of *in vivo* validated regulatory regions that produce a similar output.\n\nThis high-quality meeting of regulatory biology researchers indicates that we are taking important steps toward the construction of a powerful toolkit to identify and model *in vivo* regulatory interactions and networks. The strong proofs of principle demonstrated at this meeting, together with increased access to massively parallel sequencing platforms, anticipate an era in which systems geneticists will collaborate to perform gene-regulation experiments in unprecedented detail and scale to characterize their pet 'regulome', and niche biologists will apply these technologies to address specific hypotheses about development, health and disease.","meta":{"dup_signals":{"dup_doc_count":121,"dup_dump_count":47,"dup_details":{"curated_sources":3,"2020-50":1,"2020-24":1,"2019-47":1,"2019-35":1,"2019-26":1,"2019-13":1,"2019-04":1,"2018-51":1,"2018-47":1,"2018-43":1,"2018-39":1,"2018-30":1,"2018-26":1,"2018-09":1,"2017-39":1,"2017-22":1,"2017-09":8,"2016-44":1,"2016-40":1,"2016-36":8,"2016-30":8,"2016-22":1,"2016-18":1,"2016-07":5,"2015-48":3,"2015-40":1,"2015-35":2,"2015-32":3,"2015-27":3,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":4,"2014-42":8,"2014-41":4,"2014-35":5,"2014-23":5,"2014-15":3,"2022-27":1,"2024-10":1,"2015-18":2,"2015-11":3,"2015-06":3,"2014-10":3,"2013-48":2,"2013-20":3,"2024-30":1}},"file":"PMC2481419"},"subset":"pubmed_central"}
{"text":"author: Gregory A Petsko\ndate: 2010\ninstitute: 1Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02454-9110, USA\ntitle: A Faustian bargain\n\n# An open letter to George M Philip, President of the State University of New York At Albany\n\nDear President Philip,\n\nProbably the last thing you need at this moment is someone else from outside your university complaining about your decision. If you want to argue that I can't really understand all aspects of the situation, never having been associated with SUNY Albany, I wouldn't disagree. But I cannot let something like this go by without weighing in. I hope, when I'm through, you will at least understand why.\n\nJust 30 days ago, on October 1st, you announced that the departments of French, Italian, Classics, Russian and Theater Arts were being eliminated. You gave several reasons for your decision, including that 'there are comparatively fewer students enrolled in these degree programs.' Of course, your decision was also, perhaps chiefly, a cost-cutting measure - in fact, you stated that this decision might not have been necessary had the state legislature passed a bill that would have allowed your university to set its own tuition rates. Finally, you asserted that the humanities were a drain on the institution financially, as opposed to the sciences, which bring in money in the form of grants and contracts.\n\nLet's examine these and your other reasons in detail, because I think if one does, it becomes clear that the facts on which they are based have some important aspects that are not covered in your statement. First, the matter of enrollment. I'm sure that relatively few students take classes in these subjects nowadays, just as you say. There wouldn't have been many in my day, either, if universities hadn't required students to take a distribution of courses in many different parts of the academy: humanities, social sciences, the fine arts, the physical and natural sciences, and to attain minimal proficiency in at least one foreign language. You see, the reason that humanities classes have low enrollment is not because students these days are clamoring for more relevant courses; it's because administrators like you, and spineless faculty, have stopped setting distribution requirements and started allowing students to choose their own academic programs - something I feel is a complete abrogation of the duty of university faculty as teachers and mentors. You could fix the enrollment problem tomorrow by instituting a mandatory core curriculum that included a wide range of courses.\n\nYoung people haven't, for the most part, yet attained the wisdom to have that kind of freedom without making poor decisions. In fact, without wisdom, it's hard for most people. That idea is thrashed out better than anywhere else, I think, in Dostoyevsky's parable of the Grand Inquisitor, which is told in Chapter Five of his great novel, *The Brothers Karamazov*. In the parable, Christ comes back to earth in Seville at the time of the Spanish Inquisition. He performs several miracles but is arrested by Inquisition leaders and sentenced to be burned at the stake. The Grand Inquisitor visits Him in his cell to tell Him that the Church no longer needs Him. The main portion of the text is the Inquisitor explaining why. The Inquisitor says that Jesus rejected the three temptations of Satan in the desert in favor of freedom, but he believes that Jesus has misjudged human nature. The Inquisitor says that the vast majority of humanity cannot handle freedom. In giving humans the freedom to choose, Christ has doomed humanity to a life of suffering.\n\nThat single chapter in a much longer book is one of the great works of modern literature. You would find a lot in it to think about. I'm sure your Russian faculty would love to talk with you about it - if only you had a Russian department, which now, of course, you don't.\n\nThen there's the question of whether the state legislature's inaction gave you no other choice. I'm sure the budgetary problems you have to deal with are serious. They certainly are at Brandeis University, where I work. And we, too, faced critical strategic decisions because our income was no longer enough to meet our expenses. But we eschewed your draconian - and authoritarian - solution, and a team of faculty, with input from all parts of the university, came up with a plan to do more with fewer resources. I'm not saying that all the specifics of our solution would fit your institution, but the process sure would have. You did call a town meeting, but it was to discuss your plan, not let the university craft its own. And you called that meeting for Friday afternoon on October 1st, when few of your students or faculty would be around to attend. In your defense, you called the timing 'unfortunate', but pleaded that there was a 'limited availability of appropriate large venue options.' I find that rather surprising. If the President of Brandeis needed a lecture hall on short notice, he would get one. I guess you don't have much clout at your university.\n\nIt seems to me that the way you went about it couldn't have been more likely to alienate just about everybody on campus. In your position, I would have done everything possible to avoid that. I wouldn't want to end up in the 9th Bolgia (ditch of stone) of the 8th Circle of the Inferno, where the great 14th century Italian poet Dante Alighieri put the sowers of discord. There, as they struggle in that pit for all eternity, a demon continually hacks their limbs apart, just as in life they divided others.\n\nThe *Inferno* is the first book of Dante's *Divine Comedy*, one of the great works of the human imagination. There's so much to learn from it about human weakness and folly. The faculty in your Italian department would be delighted to introduce you to its many wonders - if only you had an Italian department, which now, of course, you don't.\n\nAnd do you really think even those faculty and administrators who may applaud your tough-minded stance (partly, I'm sure, in relief that they didn't get the axe themselves) are still going to be on your side in the future? I'm reminded of the fable by Aesop of the Travelers and the Bear: two men were walking together through the woods, when a bear rushed out at them. One of the travelers happened to be in front, and he grabbed the branch of a tree, climbed up, and hid himself in the leaves. The other, being too far behind, threw himself flat down on the ground, with his face in the dust. The bear came up to him, put his muzzle close to the man's ear, and sniffed and sniffed. But at last with a growl the bear slouched off, for bears will not touch dead meat. Then the fellow in the tree came down to his companion, and, laughing, said 'What was it that the bear whispered to you?' 'He told me,' said the other man, 'Never to trust a friend who deserts you in a pinch.'\n\nI first learned that fable, and its valuable lesson for life, in a freshman classics course. Aesop is credited with literally hundreds of fables, most of which are equally enjoyable - and enlightening. Your classics faculty would gladly tell you about them, if only you had a Classics department, which now, of course, you don't.\n\nAs for the argument that the humanities don't pay their own way, well, I guess that's true, but it seems to me that there's a fallacy in assuming that a university should be run like a business. I'm not saying it shouldn't be managed prudently, but the notion that every part of it needs to be self-supporting is simply at variance with what a university is all about. You seem to value entrepreneurial programs and practical subjects that might generate intellectual property more than you do 'old-fashioned' courses of study. But universities aren't just about discovering and capitalizing on new knowledge; they are also about preserving knowledge from being lost over time, and that requires a financial investment. There is good reason for it: what seems to be archaic today can become vital in the future. I'll give you two examples of that. The first is the science of virology, which in the 1970s was dying out because people felt that infectious diseases were no longer a serious health problem in the developed world and other subjects, such as molecular biology, were much sexier. Then, in the early 1990s, a little problem called AIDS became the world's number 1 health concern. The virus that causes AIDS was first isolated and characterized at the National Institutes of Health in the USA and the Institute Pasteur in France, because these were among the few institutions that still had thriving virology programs. My second example you will probably be more familiar with. Middle Eastern Studies, including the study of foreign languages such as Arabic and Persian, was hardly a hot subject on most campuses in the 1990s. Then came September 11, 2001. Suddenly we realized that we needed a lot more people who understood something about that part of the world, especially its Muslim culture. Those universities that had preserved their Middle Eastern Studies departments, even in the face of declining enrollment, suddenly became very important places. Those that hadn't - well, I'm sure you get the picture.\n\nI know one of your arguments is that not every place should try to do everything. Let other institutions have great programs in classics or theater arts, you say; we will focus on preparing students for jobs in the real world. Well, I hope I've just shown you that the real world is pretty fickle about what it wants. The best way for people to be prepared for the inevitable shock of change is to be as broadly educated as possible, because today's backwater is often tomorrow's hot field. And interdisciplinary research, which is all the rage these days, is only possible if people aren't too narrowly trained. If none of that convinces you, then I'm willing to let you turn your institution into a place that focuses on the practical, but only if you stop calling it a university and yourself the President of one. You see, the word 'university' derives from the Latin 'universitas', meaning 'the whole'. You can't be a university without having a thriving humanities program. You will need to call SUNY Albany a trade school, or perhaps a vocational college, but not a university. Not anymore.\n\nI utterly refuse to believe that you had no alternative. It's your job as President to find ways of solving problems that do not require the amputation of healthy limbs. Voltaire said that no problem can withstand the assault of sustained thinking. Voltaire, whose real name was Fran\u00e7ois-Marie Arouet, had a lot of pithy, witty and brilliant things to say (my favorite is 'God is a comedian playing to an audience that is afraid to laugh'). Much of what he wrote would be very useful to you. I'm sure the faculty in your French department would be happy to introduce you to his writings, if only you had a French department, which now, of course, you don't.\n\nI guess I shouldn't be surprised that you have trouble understanding the importance of maintaining programs in unglamorous or even seemingly 'dead' subjects. From your biography, you don't actually have a PhD or other high degree, and have never really taught or done research at a university. Perhaps my own background will interest you. I started out as a classics major. I'm now Professor of Biochemistry and Chemistry. Of all the courses I took in college and graduate school, the ones that have benefited me the most in my career as a scientist are the courses in classics, art history, sociology, and English literature. These courses didn't just give me a much better appreciation for my own culture; they taught me how to think, to analyze, and to write clearly. None of my sciences courses did any of that.\n\nOne of the things I do now is write a monthly column on science and society. I've done it for over 10 years, and I'm pleased to say some people seem to like it. If I've been fortunate enough to come up with a few insightful observations, I can assure you they are entirely due to my background in the humanities and my love of the arts.\n\nOne of the things I've written about is the way genomics is changing the world we live in. Our ability to manipulate the human genome is going to pose some very difficult questions for humanity in the next few decades, including the question of just what it means to be human. That isn't a question for science alone; it's a question that must be answered with input from every sphere of human thought, including - especially including - the humanities and arts. Science unleavened by the human heart and the human spirit is sterile, cold, and self-absorbed. It's also unimaginative: some of my best ideas as a scientist have come from thinking and reading about things that have, superficially, nothing to do with science. If I'm right that what it means to be human is going to be one of the central issues of our time, then universities that are best equipped to deal with it, in all its many facets, will be the most important institutions of higher learning in the future. You've just ensured that yours won't be one of them.\n\nSome of your defenders have asserted that this is all a brilliant ploy on your part - a master political move designed to shock the legislature and force them to give SUNY Albany enough resources to keep these departments open. That would be Machiavellian (another notable Italian writer, but then, you don't have any Italian faculty to tell you about him), certainly, but I doubt that you're that clever. If you were, you would have held that town meeting when the whole university could have been present, at a place where the press would be all over it. That's how you force the hand of a bunch of politicians. You proclaim your action on the steps of the state capitol. You don't try to sneak it through in the dead of night, when your institution has its back turned.\n\nNo, I think you were simply trying to balance your budget at the expense of what you believe to be weak, outdated and powerless departments. I think you will find, in time, that you made a Faustian bargain. Faust is the title character in a play by Johann Wolfgang von Goethe. It was written around 1800 but still attracts the largest audiences of any play in Germany whenever it's performed. Faust is the story of a scholar who makes a deal with the devil. The devil promises him anything he wants as long as he lives. In return, the devil will get - well, I'm sure you can guess how these sorts of deals usually go. If only you had a Theater department, which now, of course, you don't, you could ask them to perform the play so you could see what happens. It's awfully relevant to your situation. You see, Goethe believed that it profits a man nothing to give up his soul for the whole world. That's the whole world, President Philip, not just a balanced budget. Although, I guess, to be fair, you haven't given up your soul. Just the soul of your institution.\n\nDisrespectfully yours,\n\nGregory A Petsko","meta":{"dup_signals":{"dup_doc_count":420,"dup_dump_count":78,"dup_details":{"curated_sources":2,"2023-50":2,"2023-23":1,"2023-14":1,"2023-06":2,"2022-33":3,"2022-27":2,"2022-21":1,"2022-05":1,"2021-49":1,"2021-43":2,"2021-31":4,"2021-25":1,"2021-17":1,"2020-40":1,"2020-29":1,"2020-16":2,"2020-10":1,"2019-47":1,"2019-43":1,"2019-35":1,"2019-30":1,"2019-26":1,"2019-22":2,"2019-13":3,"2019-04":2,"2018-51":1,"2018-47":2,"2018-39":3,"2018-34":1,"2018-30":7,"2018-26":5,"2018-22":4,"2018-17":5,"2018-13":8,"2018-09":8,"2018-05":5,"2017-51":9,"2017-47":6,"2017-43":8,"2017-39":8,"2017-34":11,"2017-30":8,"2017-26":8,"2017-22":8,"2017-17":7,"2017-09":16,"2017-04":8,"2016-50":8,"2016-44":9,"2016-40":7,"2016-36":16,"2016-30":13,"2016-26":4,"2016-22":7,"2016-18":7,"2016-07":12,"2015-48":9,"2015-40":6,"2015-35":9,"2015-32":8,"2015-27":8,"2015-22":8,"2015-14":6,"2014-52":4,"2014-49":8,"2014-42":13,"2014-41":8,"2014-35":8,"2014-23":7,"2014-15":6,"2024-22":3,"2017-13":6,"2015-18":9,"2015-11":4,"2015-06":5,"2014-10":5,"2013-48":5,"2013-20":5}},"file":"PMC3218652"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  Of concern to health educators is the suggestion that college females practice diet and health behaviors that contradict the 2005 dietary guidelines for Americans. In this regard, there remain gaps in the research related to dieting among college females. Namely, do normal weight individuals diet differently from those who are overweight or obese, and are there dieting practices used by females that can be adapted to promote a healthy body weight? Since it is well recognized that females diet, this study seeks to determine the dieting practices used among normal, overweight, and obese college females (do they diet differently) and identify dieting practices that could be pursued to help these females more appropriately achieve and maintain a healthy body weight.\n  .\n  # Methods\n  .\n  A total of 185 female college students aged 18 to 24 years participated in this study. Height, weight, waist and hip circumferences, and skinfold thickness were measured to assess body composition. Surveys included a dieting practices questionnaire and a 30-day physical activity recall. Participants were classified according to body mass index (BMI) as normal weight (n = 113), overweight (n = 35), or obese (n = 21). Data were analyzed using JMP IN\u00ae software. Descriptive statistics included means, standard deviations, and frequency. Subsequent data analysis involved Pearson *X*^2^ and one-way analysis of variance with comparison for all pairs that were significantly different using Tukey-Kramer honestly significant difference test.\n  .\n  # Results\n  .\n  Outcomes of this study indicate the majority of participants (83%) used dieting for weight loss and believed they would be 2% to 6% greater than current weight if they did not diet; normal weight, overweight, and obese groups perceived attractive weight to be 94%, 85%, and 74%, respectively, of current weight; 80% of participants reported using physical activity to control weight, although only 19% exercised at a level that would promote weight loss; only two of 15 dieting behaviors assessed differed in terms of prevalence of use among groups, which were consciously eating less than you want (44% normal weight, 57% overweight, 81% obese) and using artificial sweeteners (31% normal weight and overweight, 5% obese); and the most prevalent explicit maladaptive weight loss behavior was smoking cigarettes (used by 9% of participants) and most unhealthy was skipping breakfast (32%).\n  .\n  # Conclusion\n  .\n  Collectively, results indicate female college students, regardless of weight status, would benefit from open discussions with health educators regarding healthy and effective dieting practices to achieve\/maintain a healthy body weight. The results are subject to replication among high school, middle-aged, and older females.\nauthor: Brenda M Malinauskas; Thomas D Raedeke; Victor G Aeby; Jean L Smith; Matthew B Dallas\ndate: 2006\ninstitute: 1Department of Nutrition and Hospitality Management, East Carolina University, Greenville, North Carolina, USA; 2Department of Exercise and Sport Science, East Carolina University, Greenville, North Carolina, USA; 3Department of Health Education, East Carolina University, Greenville, North Carolina, USA\nreferences:\ntitle: Dieting practices, weight perceptions, and body composition: A comparison of normal weight, overweight, and obese college females\n\n# Background\n\nTens of thousands of high school graduates will be heading out on their own this fall. Many will be confronting the rigors of higher education while adjusting to a new environment and new pressures. With all the changes, gaining weight is common. When you consider that more than 1.5 million students are entering U.S. colleges and universities each fall, by the following spring the weight gain entering collegians encounter is epidemic. Gaining weight during freshman year is so common that the phenomenon has its own nickname the \"Freshman 15\" and has been associated with the freshman experience for many years. Although unwanted weight gain can occur at any age, the transition from high school to college is a time of increased risk of weight gain among females.\n\nFemales, at a very young age, are concerned about body weight and place high importance on appearance, which is dramatically influenced by the media. A major messaging theme of teen magazine websites is the necessity to be beautiful \\[1\\]. Mooney et al \\[2\\] conducted interviews with home economic teachers and teen focus groups in Ireland and found that adolescent females are very conscious of their body image, are strongly influenced by high profile celebrities, and the primary motivations for wanting to be thin are to gain attention from males, approval from friends, and self confidence. Unfortunately, the desire to be beautiful may result in unwanted outcomes. For example, in a study by Monro et al \\[3\\] the authors reported an increase in appearance anxiety among female college students that resulted from viewing advertisements of idealized images. Wilson et al \\[4\\] reported that non-athlete college students reported more stress than athlete counterparts in areas of satisfaction with physical appearance, decisions about education, social conflicts regarding smoking, and financial burdens. Lastly, there are unique experiences of college females that may promote dieting, including fear of gaining weight, increased sense of independence that may promote experimenting with dieting (food restriction, use of supplements and fad diets), and changes to daily schedule that affect eating and exercise habits (\"pulling all-nighters\" to study for exams or complete course projects).\n\nEmergence of dieting among girls is most prevalent at 13 and 14 years of age and remains prevalent throughout adulthood \\[5\\]. Dieting has been reported among normal and underweight individuals, in addition to those who are overweight \\[6\\]. Despite the finding that dieting to control weight is often times ineffective; dieting remains popular among females \\[7\\]. When compared to college males, college females are more likely to actively diet, place high importance on appearance and the benefits of maintaining an ideal weight, and engage in unsafe dieting \\[8\\].\n\nGiven our culture, which suffers from a pathological emphasis on weight as a measure of a woman's worth, and given the continuing epidemic in our society of disordered eating, there are significant problems stemming from the idea of the \"Freshman 15\". The first problem is the assumption that excessive weight gain is inevitable during the college years. Second, for the many females already struggling with disordered eating, the construct of the \"Freshman 15\" becomes a terrifying probability that exacerbates an already difficult problem. For example, Patton \\[9\\] reported that girls with a history of severe dieting were 18 times more likely to develop eating disorders than girls with no history of dieting, and girls with a history of moderate dieting were five times more likely to develop eating disorders than girls who did not diet. Third, even for the college females who may not have significant issues with food or weight, the idea of the \"Freshman 15\" can play a significant role in solidifying the oppressive ideathat a female must be thin in order to qualify for success and happiness in our society. Such mentality can contribute significantly to the development of disordered eating throughout college.\n\nAt the core of what makes the \"Freshman 15\" concept so problematic is the reduction of female students to a single-dimension: body type. Given the myriad life issues that are relevant to women at the onset of the college years, to have a concept referring to the single superficial issue of body weight be so widespread and so influential is indicative of a larger cultural pathology. What is needed is a more aggressive message to the culture that contradicts this reductionistic message.\n\nIn this regard, there remain gaps in the research related to dieting among college females. Namely, do normal weight individuals diet differently from those who are overweight or obese, and are there dieting practices used by females that can be adapted to promote a healthy body weight? Because it is well recognized that females diet, this study seeks to determine the dieting practices used among normal, overweight, and obese college females (do they diet differently) and identify dieting practices that could be pursued to help these females more appropriately achieve and maintain a healthy body weight.\n\nOf concern to health educators is the suggestion that college females practice diet and health behaviors that contradict the 2005 dietary guidelines for Americans \\[10\\]. For example, physical exercise plays a role in weight management \\[10,11\\]. In an executive summary regarding treatment of overweight and obesity in adults, the National Heart, Lung, and Blood Institute in cooperation with the National Institutes of Diabetes and Digestive and Kidney Diseases report that an increase in physical activity is an important component of weight loss therapy and that sustained physical activity is helpful in prevention of weight regain \\[11\\]. Importantly, the Dietary Guidelines recommend adults engage in 60 to 90 minutes of daily moderate- to vigorous-intensity physical activity \\[10\\]. It is questionable if college females adhere to the public health message of higher intensity and longer duration physical activity to promote a healthy body weight \\[10,11\\]. The relationship between smoking and weight concerns has been explored. Potter et al \\[12\\] reported in a comprehensive review of smoking among adolescents that dieting behaviors, disordered eating symptoms, and general weight concerns were related to smoking among females.\n\nThe purpose of this study was to determine differences in dieting practices, weight perceptions, and body composition of normal weight, overweight, and obese female college students. The research questions under investigation seek to first, determine body composition, weight perceptions, and physical activity differences of normal weight, overweight, and obese college females. Second, identify perceived sources of pressure to be a certain weight among these groups. Third, identify weight loss behaviors ever used to consciously lose or control weight among groups. Fourth, identify the most prevalent weight control products and commercial diets used by female college students.\n\n# Methods\n\n## Design and sample\n\nThe study is a quasi-experimental design. The sample of convenience included students from five introductory nutrition courses taught by two instructors and invited to participate during the spring semester of 2005. The total sample recruited included 230 females, which represented 51% of all females who were enrolled in introductory nutrition courses spring 2005. Overall, 208 female participants completed the study, which was a 90% participation rate for females in the nutrition courses surveyed. The final sample size was 185, as 23 were excluded for the following reasons: age greater than 24 years (n = 14), BMI less than 18.5 kg\/m^2^ (n = 7), and reported pregnancy (n = 2).\n\nCourse enrollment ranged from 49 to 100 students. A scripted description of the study (read by the instructor at the beginning of a class period which highlighted the study protocol) and an electronic course announcement posted on Blackboard were the means used to inform students about the study. For the purpose of this study Blackboard is defined as a web-based education platform that aids course delivery; students are accustomed to checking Blackboard frequently for course information. Extra credit was available to students who completed the study. Males, females greater than 24 years of age, and females reporting current pregnancy were excluded from analysis.\n\n## Data collection and research instruments\n\nData collection occurred on two occasions. During the first meeting, students completed a survey regarding dieting practices, weight perceptions, and level of physical activity participation. During the second meeting, body composition assessments were completed. The university-affiliated institutional review board approved the project (University and Medical Center Institutional Review Board number 04\u20130487), and the project was carried out in compliance with the Helsinki Declaration. Signed informed consent was obtained from all participants.\n\nPrior to survey administration, one of two research assistants met with students during a class period, using a script to describe the study and provide instruction for completing the survey. The key points of the script were to provide uniform instruction for completing the study survey, encourage participants to answer all questions completely and truthfully, and to remind participants of the body composition assessment schedule. The survey took approximately 15 minutes to complete.\n\nThe survey included a dieting practices questionnaire that assessed demographic, dieting practices, weight perceptions, and perceived sources of pressure to be a certain weight, and a 30-day physical activity recall. The dieting practices questionnaire was developed by a Registered Dietitian and reviewed for content validity by two Registered Dietitians who practice in eating disorder treatment and nutrition counseling of college students. The questionnaire was adapted from one developed by Calderon et al \\[13\\] that investigated dieting practices among high school students. As a measure of physical activity, a 30 day self-report measure developed by Jackson et al \\[14\\] was used. To complete this measure, participants rate how active they have been over the past 30 days on a scale ranging from zero to seven. In general, scores of zero and one indicate that the person does not participate in regular activity. Participants who score two or three participate in some moderate activity whereas those who score four or above participate in some vigorous exercise. Baumgartner et al \\[15\\] reported that values four or above generally indicate that a person participates in physical activity on a regular basis. Regular activity participation, particularly vigorous exercise, often results in increased aerobic capacity. In support of the scale's validity, researchers have found that self-reported physical activity was a significant predictor of VO~2~ max \\[15\\].\n\nTo pilot test the survey, a small sample of 12 undergraduate female students completed the questionnaires. Minor syntax and formatting modifications were made to the dieting practices questionnaire based on their responses.\n\nOn the second occasion, participants reported to the lab for anthropometric measurements. Females with the first letter of their last name A to L reported after completing the second exam in their respective nutrition course, females with last name M to Z after the third. The lab was organized into two measurement stations that were separated by privacy screens. Weight and height were measured at the first station, circumference (waist and hip) and skinfold thickness (triceps, biceps, and thigh) measurements were obtained at the second station. Four research assistants (two for each station) were trained by a single anthropometrist. The same research assistant took all measurements for that \"station\" on each participant. Weight was measured to the nearest 0.1 kg (Tanita body composition analyzer, Arlington, IL), height (Seca portable height stadiometer, Leicester, England) and circumferences (non-flexible tape measure) to 0.1 cm, and skinfolds to 0.1 mm (Harpenden skinfold caliper, Vital Signs model 68875, Country Technology, Inc., Gays Mills, WI) using American College of Sports Medicine \\[16\\] procedures. Shoes, and as much clothing as was socially acceptable to the participant, were removed preceding body composition measurements. Participants were classified according to body mass index (BMI); BMI 18.5 to 24.9 kg\/m^2^ were classified as normal weight, 25 to 29.9 kg\/m^2^ as overweight, and 30 kg\/m^2^ or greater as obese \\[17\\].\n\n## Data analysis\n\nAnalyses were performed using JMP IN\u00ae software \\[18\\]. Descriptive statistics included means, standard deviations, and frequency. Subsequent data analysis involved Pearson *X*^2^ and one-way analysis of variance with comparison for all pairs that were significantly different using Tukey-Kramer honestly significant difference test. A significant level of .05 was used for statistical analysis.\n\n# Results\n\nOverall, 208 female participants completed the study, which was a 90% participation rate for females in the nutrition courses surveyed. However, 23 were excluded for the following reasons: age greater than 24 years (n = 14), BMI less than 18.5 kg\/m^2^ (n = 7), and reported pregnancy (n = 2). BMI less than 18.5 kg\/m^2^ is classified by the Center for Disease Control and Prevention \\[17\\] as an underweight condition. The final sample size was 185. Mean age of participants was 19.7 years (SD = 1.4). A majority (78%) of participants were White, 18% were Black, and 4% were American Indian, Asian, or Hispanic. Participants were predominantly freshmen and sophomores (70%), as expected because they were recruited from introductory nutrition courses.\n\nRegarding question one, body composition, weight perceptions, and physical activity of normal weight, overweight, and obese groups are reported in Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>. Despite significant differences among groups for weight, F(2, 166) = 171, p \\< .01, BMI, F(2, 166) = 272, p \\< .01, body fat, F(2, 166) = 96, p \\< .01, and waist-to-hip ratio, F(2, 166) = 53, p \\< .01, mean physical activity was similar among groups, F(2, 165) = 0.5, p = .61. Significant differences were found among groups for perceived healthy weight, F(2, 163) = 76, p \\< .01, and attractive weight, F(2, 160) = 74, p \\< .01, compared to current weight. Mean perceived healthy weight was 5% (normal weight), 13% (overweight), and 23% (obese) lower than current weight. Mean perceived attractive weight followed the same trend, and was 6%, 15%, and 26% lower than current weight for normal weight, overweight, and obese groups, respectively. All groups perceived natural weight to be greater than current weight, meaning if no attempt was made to control weight then weight would be 2% to 6% greater than current weight. The majority (80%) of participants reported using physical activity to control their weight. However, 32% did not participate in regularly programmed recreation, sport, or heavy physical activity, and only 19% of participants (21% normal weight, 21% overweight, and 5% obese groups) reported spending over 3 hours per week in vigorous aerobic activity.\n\nMean body composition, weight perceptions, and physical activity among normal weight, overweight, and obese female college students.\n\n|  | Weight classification |  |  |\n|----|----|----|----|\n|  |  |  |  |\n|  | Normal weight | Overweight | Obese |\n|  | n = 113 | n = 35 | n = 21 |\n| Variable | M \u00b1 SD | M \u00b1 SD | M \u00b1 SD |\n| Body composition |  |  |  |\n| Weight (pounds) | 130^a^ \u00b1 13 | 160^b^ \u00b1 18 | 213^c^ \u00b1 39 |\n| BMI (kg\/m^2^) | 21.9^a^ \u00b1 1.8 | 27.3^b^ \u00b1 1.3 | 35.3^c^ \u00b1 5.7 |\n| Body fat (%) | 28^a^ \u00b1 4 | 33^b^ \u00b1 4 | 38^c^ \u00b1 3 |\n| Waist-to-hip ratio | 0.72^a^ \u00b1 0.01 | 0.76^b^ \u00b1 0.05 | 0.83^c^ \u00b1 0.07 |\n| Weight perceptions |  |  |  |\n| Perceived healthy weight (pounds) | 123^a^ \u00b1 10 | 138^b^ \u00b1 17 | 164^c^ \u00b1 26 |\n| Perceived attractive weight (pounds) | 122^a^ \u00b1 11 | 136^b^ \u00b1 17 | 158^c^ \u00b1 29 |\n| Perceived natural weight (pounds) | 138^a^ \u00b1 18 | 169^b^ \u00b1 25 | 216^c^ \u00b1 42 |\n| Perceived healthy weight (% of current) | 95^a^ \u00b1 7 | 87^b^ \u00b1 6 | 77^c^ \u00b1 6 |\n| Perceived attractive weight (% of current) | 94^a^ \u00b1 7 | 85^b^ \u00b1 6 | 74^c^ \u00b1 8 |\n| Perceived natural weight (% of current) | 106 \u00b1 9 | 106 \u00b1 10 | 102 \u00b1 2 |\n| Physical activity | 4.1 \u00b1 2.5 | 3.8 \u00b1 2.5 | 3.5 \u00b1 2.5 |\n\nNote: Means in the same row having letters that do not share subscripts differ at *p* \\< .01 in the Tukey-Kramer honestly significant difference test. The higher the physical activity score, the greater intensity and amount of reported exercise over the past 30 days using a scoring system of 0 (do not participate regularly in planned exercise) to 7 (spend over 3 hours weekly in heavy exercise).\n\nConcerning question two, 83% of participants reported ever consciously trying to lose or control their weight, including 80% of normal weight, 91% of overweight, and 86% of obese participants. Mean age when dieting was initiated was 15.7 years (SD = 2.3), which was similar among groups, F(2, 137) = 1, p = .30. Fifty eight percent of participants reported pressure to be a certain weight; the primary sources were self (54%), media (37%), and friends (32%). Frequency distributions of \"yes\" response to these sources were similar among groups (see Table <a href=\"#T2\" data-ref-type=\"table\">2<\/a>). Other sources of pressure included significant other (reported as a source of pressure by 13% of participants), colleagues at work (5%), other athletes (3%), coach (2%), and family members (2%).\n\nPerceived sources of pressure to be a certain weight among normal weight, overweight, and obese female college students.\n\n| Variable         | Frequency \"yes\" response (%) | *X*^2^ | p (weight status) |\n|------------------|------------------------------|--------|-------------------|\n| Self             |                              | 2.5    | .29               |\n| Normal weight^a^ | 50                           |        |                   |\n| Overweight^b^    | 62                           |        |                   |\n| Obese^c^         | 43                           |        |                   |\n| Media            |                              | 0.9    | .63               |\n| Normal weight^a^ | 39                           |        |                   |\n| Overweight^b^    | 34                           |        |                   |\n| Obese^c^         | 29                           |        |                   |\n| Friends          |                              | 3.4    | .18               |\n| Normal weight^a^ | 31                           |        |                   |\n| Overweight^b^    | 37                           |        |                   |\n| Obese^c^         | 14                           |        |                   |\n\nNote: *X*^2^ (2, *N* = 166).\n\n^a^n = 113; ^b^n = 35; ^c^n = 21.\n\nReferring to question three, 15 dieting behaviors were assessed to determine if use differed among normal weight, overweight, and obese participants. Nine of the 15 behaviors had large enough sample size of \"yes\" response to analyze frequency distribution differences among groups; these are reported in Table <a href=\"#T3\" data-ref-type=\"table\">3<\/a>. The five most common behaviors used by all participants were exercising (80%), eating or drinking low fat or fat free versions of foods\/drinks (59%), consciously eat less than you want (51%), eating or drinking sugar free versions of foods\/drinks (43%), and count calories (40%). Eating less than you want and use of artificial sweeteners were the only behaviors for which frequency of use was significantly different among groups (see Table <a href=\"#T3\" data-ref-type=\"table\">3<\/a>). The other behaviors assessed, which had low frequency of use reported, included not eating foods with high glycemic index (used by 4% normal weight, 6% overweight, and 0% obese), smoke cigarettes (8%, 14%, and 5%), use laxatives after you eat (2%, 6%, and 5%), vomit after you eat (4%, 6%, and 5%), skip lunch (10%, 9%, 10%), and skip dinner (4%, 9%, 0%). Explicit maladaptive weight loss practices included smoking cigarettes (used by 9% of all participants), vomiting (5%) and use of laxatives (3%) after eating.\n\nWeight loss behaviors ever used to consciously lose or control weight of normal weight, overweight, and obese female college students.\n\n| Variable | Frequency \"yes\" response (%) | *X*^2^ | p (weight status) |\n|----|----|----|----|\n| Consciously eat less than you want |  | 10.1 | \\< .01 |\n| Normal weight^a^ | 44 |  |  |\n| Overweight^b^ | 57 |  |  |\n| Obese^c^ | 81 |  |  |\n| Count grams of fat |  | 1.0 | .60 |\n| Normal weight^a^ | 23 |  |  |\n| Overweight^b^ | 31 |  |  |\n| Obese^c^ | 24 |  |  |\n| Count net carbs |  | 2.3 | .32 |\n| Normal weight^a^ | 18 |  |  |\n| Overweight^b^ | 17 |  |  |\n| Obese^c^ | 5 |  |  |\n| Count calories |  | 0.2 | .89 |\n| Normal weight^a^ | 39 |  |  |\n| Overweight^b^ | 43 |  |  |\n| Obese^c^ | 43 |  |  |\n| Eat or drink low fat or fat free versions of foods\/drinks |  | 2.9 | .24 |\n| Normal weight^a^ | 57 |  |  |\n| Overweight^b^ | 71 |  |  |\n| Obese^c^ | 52 |  |  |\n| Eat or drink sugar free versions of foods\/drinks |  | 2.2 | .33 |\n| Normal weight^a^ | 43 |  |  |\n| Overweight^b^ | 49 |  |  |\n| Obese^c^ | 29 |  |  |\n| Exercise |  | 2.2 | .33 |\n| Normal weight^a^ | 77 |  |  |\n| Overweight^b^ | 89 |  |  |\n| Obese^c^ | 81 |  |  |\n| Use artificial sweeteners |  |  |  |\n| Normal weight^a^ | 31 | 6.3 | .04 |\n| Overweight^b^ | 31 |  |  |\n| Obese^c^ | 5 |  |  |\n| Skip breakfast |  |  |  |\n| Normal weight^a^ | 27 | 4.9 | .09 |\n| Overweight^b^ | 40 |  |  |\n| Obese^c^ | 48 |  |  |\n\nNote: *X*^2^ (2, N = 166).\n\n^a^n = 113; ^b^n = 35; ^c^n = 21.\n\nIn terms of question four, over-the-counter meal replacement drinks were used by 35% of participants, supplements (Hydroxycut, Stacker II, Xenadrine, CortiSlim, Trim Spa, Hot Rox) by 26%, meal replacement bars by 18%, dieter's tea, green tea, and green tea pills by 11%, and chromium picolinate by 3%. Physician prescribed weight loss pills were used by 3% of participants. In examining commercial diets, the most popular ones were Atkins or South Beach (20%) and Weight Watchers\u00ae (11%). Subway, Sugar Busters, and The Zone diet combined had been used by 7% of participants.\n\n# Discussion\n\nThe purpose of this study was to investigate dieting practices, weight perceptions, and body composition among normal weight, overweight, and obese college females. Findings from this study support the general belief that dieting by college females is a common weight management strategy, irrespective of weight status. Abraham and O'Dea \\[19\\] reported that females as young as 12 years of age had tried to lose weight, including 44% who dieted using food restriction and 78% who exercised to lose weight. Findings from the present study indicate that females continue to diet through college. Calderon et al \\[13\\] reported a greater prevalence of dieting among overweight (67% prevalence) versus at risk for overweight (25%) high school females, which is in contrast to the findings from the present study. We found 83% of college females reporting ever consciously trying to lose or control their weight, with similar prevalence between normal weight, overweight, and obese participants. The discrepancy between findings could be due to how dieting is defined by researchers and interpreted by study participants. Dieting strategies have become so \"main stream\" in our society, that one may not be aware, unless explicitly stated, behaviours that are being used to consciously lose or control ones' weight. For example, someone may have been drinking diet sodas to control\/lose their weight for so many years that they do not recognize this behaviour as a weight loss strategy unless it is pointed out to them. We classified dieting as any method of food restriction, use of supplements, or behaviours used to control one's weight. Although our questionnaire was adapted from the survey of Calderon and colleagues \\[13\\], we expanded upon behaviours and methods marketed for weight control that were popular at the time our study was conducted, including counting net carbs, eating\/drinking sugar free versions of foods\/drinks, avoiding foods with high glycemic index, and use of artificial sweeteners. Furthermore, a limitation of the study is that being a cross-sectional study design, we do not know if there exists a causal relationship between dieting and weight control. Longitudinal studies would be the best way to measure if dieting frequency among females and methods of dieting choices change throughout the lifespan and if there exists a causal relationship between dieting and weight control.\n\nWe found females perceive healthy and attractive weights to be lower than current weight, and that media influence contributes pressure to be a certain weight. Klesges et al \\[8\\] reported that college females are more likely than male counterparts to engage in healthy and unhealthy physical activity and food restriction behaviours and place higher importance on appearance benefits of maintaining an ideal body weight. Of great concern are the findings of Bacon et al \\[20\\] that indicated a significant negative correlation between bone mineral content and number of times on a weight loss diet among obese adult females. Additionally, Gingras et al \\[21\\] reported that adult female chronic dieters had low body satisfaction and suggested that it is important to address body image dissatisfaction with chronic dieting to improve health, regardless of body size of the dieter. Clearly, chronic dieting can have physical and emotional ramifications.\n\nMost important, many of the weight loss practices reported to be used by participants of the current study, if used appropriately, could promote healthy weight loss and be used long term to maintain a healthy weight. For example, the majority (80%) of participants reported using physical activity to control their weight. However, 32% did not participate in regularly programmed recreation, sport, or heavy physical activity, which is similar to the Behavioral Risk Factor Surveillance system finding that 15% to 34% of adults reported no leisure-time physical activity \\[22\\]. Holcomb et al \\[23\\] reported that body mass index, body fat, and waist-to-hip ratio were significantly lower as physical activity increases among premenopausal females. The researchers suggested that ability to increase daily physical activity to minimize fat accumulation could be a strong incentive to become more physically active. Strategies to improve nutrient intake and physical activity to promote health and weight control among females are warranted.\n\nThe current study makes an important contribution to the literature by narrowing the gap in research related to dieting among females. Namely, do normal weight individuals diet differently from those who are overweight or obese, and are there dieting practices used by females that can be adapted to promote a healthy body weight? Outcomes of interest to the health educator include: First, dieting practices are prevalent among female college students, regardless of body weight; Second, there were a number of dieting practices used that could promote effective weight loss and healthy weight maintenance if employed appropriately; Third, although many dieters stated they used physical activity for weight loss, the majority who used this method were not meeting physical activity goals for weight loss; and Fourth, the most prevalent explicit maladaptive weight loss behavior identified was smoking cigarettes and the most unhealthy dieting practice was skipping breakfast.\n\n# Conclusion\n\nThese findings suggest that health educators should promote education and intervention strategies for females that encourage appropriate weight control practices and dispel unhealthy and ineffective weight loss myths (using laxatives and skipping breakfast are effective weight control methods). Implications from this study are numerous. Female college students, regardless of weight status, would benefit from open discussions with health educators to identify healthy and unhealthy dieting practices they use. Throughout these discussions, females could identify healthy dieting practices that could be expanded upon to promote a healthy weight status and recognize the health consequences associated with using unhealthy dieting practices. Because this study involved only female college students at a state university the implications discussed here are subject to replication of the results. Beyond replication with other participants, researchers can consider how to effectively target specific dieting practices among females to promote healthy eating and exercise to promote and maintain a healthy weight. Future research should identify which dieting practices dieters report most content with and believe are most successful to promote short- and long-term weight management.\n\n# Competing interests\n\nThe author(s) declare that they have no competing interests.\n\n# Authors' contributions\n\nBMM conceived of the study, participated in its design, performed the statistical analysis, and drafted the manuscript. TAR participated in the design of the study and drafted the manuscript. VGA drafted the manuscript. JLS and MBD participated in coordination and data collection and helped to draft the manuscript. All authors read and approved the final manuscript.\n\n### Acknowledgements\n\nThis research was supported in part by undergraduate research assistantships, East Carolina University Honors Program. We thank Emily R. Bennett and Lauren B. Manning for their assistance with data acquisition.","meta":{"dup_signals":{"dup_doc_count":131,"dup_dump_count":36,"dup_details":{"curated_sources":2,"2023-40":2,"2023-14":1,"2022-40":1,"2022-27":2,"2022-05":1,"2021-49":1,"2021-31":2,"2021-17":1,"2021-04":1,"2020-40":1,"2020-29":1,"2020-10":1,"2019-51":1,"2019-43":1,"2017-17":1,"2015-48":5,"2015-40":2,"2015-35":5,"2015-32":3,"2015-27":4,"2015-22":6,"2015-14":4,"2014-52":6,"2014-49":8,"2014-42":11,"2014-41":7,"2014-35":7,"2014-23":5,"2014-15":8,"2015-18":5,"2015-11":4,"2015-06":5,"2014-10":5,"2013-48":5,"2013-20":5,"2024-18":1}},"file":"PMC1456978"},"subset":"pubmed_central"}
{"text":"author: Jacek R. Wi\u015bniewski\ndate: 2012-10-26\ninstitute: Department of Proteomics and Signal Transduction, Max-Planck-Institute for Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany; Email: <firstname.lastname@example.com>; Tel.: +49-89-85-78-2205; Fax: +49-89-85-78-2219\ntitle: Proteomes: A New Proteomic Journal\n\nIn the early years of proteomics, mass spectrometry served only as a technique in protein chemistry facilitating the characterization of purified proteins and mapping their posttranslational modifications (PTMs). A bit later this technique almost completely replaced Edman degradation and amino acid analysis. The continuous development of the mass spectrometry techniques created a huge analytical potential allowing the study of nearly complete proteomes in single experiments. This evolution distanced proteomics from protein chemistry and placed it in a novel position. Its capability to identify and quantify in parallel thousands of proteins and their modifications at minute sample amount requirements is one of the most fascinating technological advances in biology today.\n\nThere are several major areas of proteomics, such as mapping of proteomes, identification of posttranslational modifications and discovering interactions. Notably, proteomics has not only the capability to unveil protein composition but is also suitable for studying protein levels and their changes on a system wide scale. Currently, we can map more than 10,000 proteins in a single cell type and estimate the concentration of each protein. Even though some estimates are rough, they place proteomics closer to biochemistry and are helpful in better understanding a variety of phenomena in physiology, growth control and signaling.\n\nIdentification of posttranslational modification is one of the most attractive areas of proteomics. Expensive and laborious tasks that have been necessary for mapping PTMs using classical biochemistry, including labeling with radioactive isotopes, purification of peptides from isolated proteins purifications, and sequencing, were substituted by mass spectrometry based proteomics. Thanks to these developments, analysis of PTMs has become very popular, in particular because large numbers of modification sites can be identified in a relatively simple way by analyzing enriched fractions or just within complete cell lysates. In-depth proteomic studies allow mapping of thousands of phosphorylation, acetylation, or glycosylation sites occurring in a single population of cells or in a tissue, and position proteomics in a key role for studying modifications in a biological system's manner. Supplementing the identification with protein abundance and site-occupancy information will lead to validation of thermodynamic significance of individual modifications.\n\nAnalysis of clinical samples is another popular area of proteomics. Its landscape is shared by the identification of novel biomarkers and discovery of potential drug targets. The major challenges in this area are the limited availability of quality samples and the abundance of housekeeping proteins such as albumin in plasma, or titin in muscle tissue. The presence of such proteins impedes identification of lower abundant molecules that are the focus of analyses. Development of approaches to bypass these difficulties and to facilitate the discovery process is the aim of many researchers.\n\nAlthough proteomic technologies were frequently understood as something 'under construction' doubtless proteomic technologies are now ready to provide novel insights in biological processes. We launch *Proteomes* with the certainty that we can attract high quality manuscripts which will cover various fields of proteomics. We expect that *Proteomes* will publish articles in which proteomic technologies will play an essential role in solving questions and providing new insights in biology, biochemistry, and molecular biology.","meta":{"dup_signals":{"dup_doc_count":105,"dup_dump_count":41,"dup_details":{"curated_sources":2,"2018-17":1,"2018-05":1,"2017-47":2,"2017-39":1,"2017-30":2,"2017-26":1,"2017-22":1,"2017-17":2,"2017-09":1,"2017-04":2,"2016-50":2,"2016-44":3,"2016-40":3,"2016-36":3,"2016-30":3,"2016-26":3,"2016-22":3,"2016-18":1,"2016-07":1,"2015-48":3,"2015-40":3,"2015-35":3,"2015-32":3,"2015-27":2,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":3,"2014-42":7,"2014-41":4,"2014-35":4,"2014-23":5,"2014-15":4,"2018-26":1,"2015-18":3,"2015-11":3,"2015-06":3,"2014-10":2,"2013-48":2,"2013-20":2,"2017-13":1}},"file":"PMC5314490"},"subset":"pubmed_central"}
{"text":"abstract: John Westbrook is remembered.\nauthor: Christine Zardecki; Stephen K. BurleyCorrespondence e-mail: <email@example.com>\ndate: 2021-11-01\nreferences:\ntitle: John D. Westbrook Jr (1957\u20132021)\n\nJohn D. Westbrook Jr (1957\u20132021), Research Professor at Rutgers University and Data & Software Architect Lead for the RCSB PDB, passed away on 18 October 2021.<a href=\"#fig1\" data-ref-type=\"fig\"> \u25b8<\/a>\n\nHe was incredibly beloved and respected by his colleagues at Rutgers and worldwide, known for his dry wit and endless enthusiasm for thinking about all aspects of data and data management.\n\nJohn had a long and highly successful career developing ontologies, tools, and infrastructure in data acquisition, validation, standardization, and mining in the structural biology and life science domains. His work established the PDBx\/mmCIF data dictionary and format as the foundation of the modern Protein Data Bank (PDB) archive (wwPDB.org).\n\nMore than twenty-five years ago, while still a graduate student, John recognized the importance of a well defined data model to ensure high-quality and reliable structural information to data users. He was the principal architect of the mmCIF data representation for biological macromolecular data. Data are presented in either key-value or tabular form based on a simple, context-free grammar (without column width constraints). All relationships between common data items (*e.g.* atom and residue identifiers) are explicitly documented within the PDBx Exchange Dictionary (<https:\/\/mmcif.wwpdb.org>). The use of the PDBx\/mmCIF format enables software applications to evaluate and validate referential integrity within any PDB entry. A key strength of the mmCIF technology is the extensibility afforded by its rich collection of software-accessible metadata.\n\nThe current PDBx\/mmCIF dictionary contains more than 6200 definitions relating to experiments involved in macromolecular structure determination and descriptions of the structures themselves. The first implementation of this schema was used for the Nucleic Acid Database, a data resource of nucleic acid-containing X-ray crystallographic structures. Today, this dictionary underpins all data management of the PDB. Since 2014, it has served as the Master Format for the PDB archive. It also forms the basis of the Chemical Component Dictionary (<https:\/\/wwpdb.org\/data\/ccd>), which maintains and distributes small molecule chemical reference data in the PDB.\n\nIn 2011, the Worldwide Protein Data Bank (wwPDB) PDBx\/mmCIF Working Group was established to enable the direct use of PDBx\/mmCIF format files within major macromolecular crystallography software tools to provide recommendations on format extensions required for deposition of larger macromolecule structures to the PDB. This was a key step in the evolution of the PDB archive, which enabled studies of macromolecular machines, such as the ribosome, as single PDB structures (instead of split entries with atomic coordinates distributed among different entry files). In 2019, mandatory submission of PDBx\/mmCIF format files for deposition was announced (Adams *et al.*, 2019 \u25b8).\n\nTo ensure the success of the PDBx\/mmCIF dictionary and format, John worked with a wide range of community experts to extend the framework to encompass descriptions of macromolecular X-ray crystallographic experiments, 3D cryo-electron microscopy experiments, NMR spectroscopy experiments, protein and nucleic acid structural features, diffraction image data, and protein production and crystallization protocols. These efforts have recently focused on developing compatible data representations for X-ray free electron (XFEL) methods and integrative or hybrid methods (I\/HM). I\/HM structures, currently stored in the prototype PDB-Dev archive (<https:\/\/pdb-dev.wwpdb.org>), presented new challenges for data exchange among rapidly evolving and heterogeneous experimental repositories. Proper management of I\/HM structures in PDB-Dev also required extension of the PDBx\/mmCIF data dictionary to include coarse-grained or multiscale models, which will be essential for studying macromolecular structures *in situ* using cryo-electron tomography and other bioimaging methods.\n\nJohn contributed broadly to community data standards enabling interoperation and data integration within the biology and structural biology domains. His efforts have included (i) describing the increasing molecular complexity of macromolecular structure data, (ii) representing new experimental methodologies, including I\/HM techniques, and (iii) expanding the biological context required to facilitate broader integration with a spectrum of biomedical resources. John's work has been central to connecting crystallographic and related structural data for biological macromolecules to key resources across scientific disciplines. His efforts have been described in more than 120 peer-reviewed publications, one of which has been cited more than 21\u2005000 times according to the Web of Science (Berman *et al.*, 2000 \u25b8). Eight of his most influential published papers have appeared in the *Inter\u00adnational Tables for Crystallography*.\n\nJohn has also done yeoman service to the crystallographic community over many years and was recognized with the inaugural Biocuration Career Award from the International Society for Biocuration in 2016.\n\nFor the International Union of Crystallography, John served on the Commission for Maintenance of CIF Standard (COMCIFS), the Working Group on Data Diffraction Deposition (DDDWG), and the Committee on Data (CommDat). He also served as a Co-editor for *Acta Crystallo\u00adgraphica Section F*.\n\nJohn was a long-standing member of the American Crystallographic Association and served on the Data, Standards & Computing Committee. He also served on the Metadata Interest Group for the Research Data Alliance.\n\nJohn is survived by his wife, Bonnie J. Wagner-Westbrook, Ed.D. and his devoted Mother-in-Law, Joan N. Wagner of Clinton Twp., NJ; many cousins including Chandler Turner (of Portsmouth, VA), Ann (Turner) Heyes (of Tasmania, Australia), and Louise (Turner) Brown (of Oakland, CA).\n\n# References","meta":{"dup_signals":{"dup_doc_count":112,"dup_dump_count":15,"dup_details":{"curated_sources":2,"2023-50":11,"2023-40":10,"2023-23":13,"2023-14":6,"2023-06":12,"2022-49":6,"2022-40":4,"2022-33":2,"2022-27":1,"2022-21":1,"2024-30":6,"2024-26":9,"2024-22":4,"2024-18":10,"2024-10":15}},"file":"PMC8561733"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  Therapeutic targets have been defined for diseases like diabetes, hypertension or rheumatoid arthritis and adhering to them has improved outcomes. Such targets are just emerging for spondyloarthritis (SpA).\n  .\n  # Objective\n  .\n  To define the treatment target for SpA including ankylosing spondylitis and psoriatic arthritis (PsA) and develop recommendations for achieving the target, including a treat-to-target management strategy.\n  .\n  # Methods\n  .\n  Based on results of a systematic literature review and expert opinion, a task force of expert physicians and patients developed recommendations which were broadly discussed and voted upon in a Delphi-like process. Level of evidence, grade and strength of the recommendations were derived by respective means. The commonalities between axial SpA, peripheral SpA and PsA were discussed in detail.\n  .\n  # Results\n  .\n  Although the literature review did not reveal trials comparing a treat-to-target approach with another or no strategy, it provided indirect evidence regarding an optimised approach to therapy that facilitated the development of recommendations. The group agreed on 5 overarching principles and 11 recommendations; 9 of these recommendations related commonly to the whole spectrum of SpA and PsA, and only 2 were designed separately for axial SpA, peripheral SpA and PsA. The main treatment target, which should be based on a shared decision with the patient, was defined as remission, with the alternative target of low disease activity. Follow-up examinations at regular intervals that depend on the patient's status should safeguard the evolution of disease activity towards the targeted goal. Additional recommendations relate to extra-articular and extramusculoskeletal aspects and other important factors, such as comorbidity. While the level of evidence was generally quite low, the mean strength of recommendation was 9\u201310 (10: maximum agreement) for all recommendations. A research agenda was formulated.\n  .\n  # Conclusions\n  .\n  The task force defined the treatment target as remission or, alternatively, low disease activity, being aware that the evidence base is not strong and needs to be expanded by future research. These recommendations can inform the various stakeholders about expert opinion that aims for reaching optimal outcomes of SpA.\nauthor: Josef S Smolen; J\u00fcrgen Braun; Maxime Dougados; Paul Emery; Oliver FitzGerald; Philip Helliwell; Arthur Kavanaugh; Tore K Kvien; Robert Landew\u00e9; Thomas Luger; Philip Mease; Ignazio Olivieri; John Reveille; Christopher Ritchlin; Martin Rudwaleit; Monika Schoels; Joachim Sieper; Martinus de Wit; Xenofon Baraliakos; Neil Betteridge; Ruben Burgos-Vargas; Eduardo Collantes-Estevez; Atul Deodhar; Dirk Elewaut; Laure Gossec; Merryn Jongkees; Mara Maccarone; Kurt Redlich; Filip van den Bosch; James Cheng-Chung Wei; Kevin Winthrop; D\u00e9sir\u00e9e van der Heijde[^1]Correspondence to Professor Josef S Smolen, Division of Rheumatology, Department of Medicine 3, Medical University of Vienna, Waehringer Guertel 18-20, Vienna A-1090, Austria; <email@example.com>; <email@example.com>\ndate: 2014-01\ninstitute: 1Division of Rheumatology, Department of Medicine 3, Medical University of Vienna, Vienna, Austria; 22nd Department of Medicine, Hietzing Hospital Vienna, Vienna, Austria; 3Rheumazentrum Ruhrgebiet, Herne, Germany; 4Department of Rheumatology B, Cochin Hospital, Ren\u00e9 Descartes University, Paris, France; 5Division of Rheumatic and Musculoskeletal Disease, Leeds Institute of Molecular Medicine, University of Leeds, Chapel Allerton Hospital, Leeds, UK; 6Department of Rheumatology, St. Vincents University Hospital, Dublin, UK; 7Division of Rheumatology, Allergy, Immunology, University of California, San Diego, California, USA; 8Department of Rheumatology, Diakonhjemmet Hospital, Oslo, Norway; 9Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; 10Atrium Medical Center, Heerlen, The Netherlands; 11Clinic and Polyclinic of Dermatology, University of M\u00fcnster, M\u00fcnster, Germany; 12Swedish Medical Center and University of Washington, Seattle, Washington, USA; 13Rheumatology Department of Lucania, San Carlo Hospital of Potenza and Madonna delle Grazie Hospital of Matera, Potenza, Italy; 14Division of Rheumatology, University of Texas Health Science Center at Houston, Houston, Texas, USA; 15Allergy, Immunology and Rheumatology Division, The Center for Musculoskeletal Medicine, University of Rochester Medical Center, Rochester, New York, USA; 16Endokrinologikum Berlin, Berlin, Germany; 17Medical Department I, Rheumatology, Charit\u00e9 Campus Benjamin Franklin, Berlin, Germany; 18EULAR standing committee of People with Arthritis\/Rheumatism in Europe (PARE), Zurich, Switzerland; 19Rheumatology Department, Faculty of Medicine, Hospital General de M\u00e9xico Universidad Nacional Aut\u00f3noma de M\u00e9xico, Mexico City, Mexico; 20Department of Rheumatology, Reina Sofia University Hospital of C\u00f3rdoba\/IMBIC, C\u00f3rdoba, Spain; 21Division of Arthritis and Rheumatic Diseases, Oregon Health & Sciences University, Portland, USA; 22Laboratory for Molecular Immunology and Inflammation, Department of Rheumatology, Ghent University Hospital, Ghent, Belgium; 23Department of Rheumatology, Piti\u00e9 Salp\u00eatri\u00e8re Hospital, Pierre et Marie Curie University, Paris, France; 24Division of Allergy, Immunology and Rheumatology, Institute of Medicine, Chung Shan Medical University Hospital, Taichung, Taiwan; 25Department of Public Health and Preventive Medicine, Oregon Health & Science University, Portland, Oregon, USA; 26Department of Rheumatology, Leiden University Medical Center, Leiden, The Netherlands\nreferences:\ntitle: Treating spondyloarthritis, including ankylosing spondylitis and psoriatic arthritis, to target: recommendations of an international task force\n\nThe approaches to the diagnosis, therapy and follow-up of patients with ankylosing spondylitis (AS) and psoriatic arthritis (PsA) have undergone a number of paradigmatical changes over the last decade. Especially considerations of the disease spectrum of spondyloarthritis (SpA) have recently undergone remarkable changes. In addition to AS, defined by prevalent radiographic structural changes in the sacroiliac joints, non-radiographic axial SpA (axSpA) has been defined based on the absence of such changes but presence of sacroiliitis (as documented by MRI) and\/or human leukocyte antigen B27. The term axSpA, therefore, includes radiographic axSpA (AS) and non-radiographic axSpA. On this basis, new classification criteria have been established by Assessment of SpondyloArthritis international Society (ASAS),1 novel therapies have proven efficacious,2\u20136 MRI has been increasingly established as an imaging tool in SpA1 7 8 and new indices to assess disease activity have been developed.9\u201314 The novel approach to classification has also differentiated the two predominant manifestations of SpA, axial and\/or peripheral, and their potential parallel occurrence.15 The basis for the new classification lies in the sharing of characteristic features of SpA, such as sacroiliitis, spondylitis and enthesitis and common genetic markers and a positive family history. Furthermore, extramusculoskeletal manifestations such as psoriasis in PsA, a preceding gastrointestinal or urogenital infection as in the case of reactive arthritis (ReA), and chronic inflammatory bowel diseases (IBD) like Crohn's disease and ulcerative colitis, play a role in the definition of a clinical syndrome as belonging to the concept of SpA. For the classification of patients with PsA the Classification Criteria for *Psoriatic* Arthritis (CASPAR) criteria are well established.16 Since the presence of psoriasis plays a role in both criteria sets, the ASAS and the CASPAR criteria, there is some overlap between the two. There is no international agreement whether and how they can or should be differentiated. Finally, to account for therapeutic developments, management recommendations have recently been presented.17\u201320\n\nDespite all these advances, a variety of challenges exist when considering the management of patients with SpA,21\u201324 not least because the definition of a clear therapeutic target and strategies to reach such target are not yet optimally defined.\n\nIn many areas of medicine, such as diabetes care or cardiology, clear therapeutic targets are available.25\u201330 More recently, a treatment target has also been advocated for rheumatoid arthritis (RA), namely remission or low disease activity,31 32 a recommendation based on insights from various clinical trials as revealed by systematic literature reviews (SLRs).33 34 Much less information on the value of defining therapeutic targets is currently available for AS or PsA. Therefore, a task force was formed to discuss and develop a consensus on recommendations aimed at defining a treatment target for, and thus at improving the management of axial and peripheral SpA in clinical practice.\n\n# Methods\n\nThe consensus finding consisted of a three-step process. In a first step, the first and last author invited leading experts, defined on the basis of their citation frequency in the field and previous contributions to similar activities to form a steering committee. This steering committee, which included rheumatologists experienced in the care of patients with, and\/or clinical research in axial and\/or peripheral SpA (several of them Department chairs and thus in managerial functions), a dermatologist experienced in psoriasis, and patients being diagnosed with one of these diseases and\/or experienced in consensus finding processes, met in March 2011 in Vienna to discuss unmet needs in the therapeutic management of and the potential of using treatment targets in AS and PsA. To this end, the debate focused on axial and peripheral SpA separately in two breakout groups with a subsequent common assessment. In the course of these discussions there was unanimous agreement that defining therapeutic targets and an appropriate strategic treatment approach would be valuable, but that evidence for its validity may be lacking. Therefore it was decided to perform a SLR and respective PICO (Patient, Intervention, Control, Outcome) and search terms were formulated, in line with European League Against Rheumatism (EULAR) and Appraisal of Guidelines for Research and Evaluation recommendations.35 36 In the course of defining the scope of this activity, the target populations were also specified, namely health professionals involved in care of and patients affected by axial and\/or peripheral SpA. In addition, social security officials, hospital managers and policy makers at national and international levels were considered potential stakeholders in this activity.\n\nAt a subsequent meeting in November 2011 (Dusseldorf) comprising an expanded task force with increased international participation, the SLR was presented. These invitations were a consequence of the individuals' contributions to the field and deliberations among members of the steering committee. The literature search had revealed that currently no strategic trials addressing a target-oriented, steered therapy were published, although some indirect evidence on optimal therapeutic approaches was available to inform the next stages of the process.37 A major focus of discussion at this meeting, but also already at the steering committee meeting, was the question if diseases like AS, PsA, ReA and IBD arthritis should be seen as an entity or as different diseases. The respective decision would have a bearing to the consensus finding process, since it would mean to develop one, two or more documents. The initial deliberations tended toward separating the individual diseases for several reasons: (1) despite many commonalities, some important clinical manifestations are distinct between these conditions and certain health professionals (such as dermatologists and gastroenterologists) may not be sufficiently aware of the more unifying concept of SpA or its relevance when dealing with these conditions; (2) further, the existing distinction between PsA and AS is well known for and accepted by patients and changes in terminology may cause confusion regarding the understanding of their 'new' diagnosis; (3) to date, clinical trials have been performed almost entirely in individual subentities (AS, PsA) rather than SpA, and even most recently trials in a highly specific novel subset, non-radiographic axSpA, have been performed38; (4) the current drug approval process by regulatory agencies is also related to the individual diseases rather than SpA; and (5) there is some overlap between the different subgroups, but there are also major distinctions, for example, PsA with symmetric polyarthritis as the predominant feature would not fit well into either the axial or peripheral SpA group. Therefore, the provisional choice was to develop at least two documents, one for axSpA and one for PsA. The discussions took place in separate breakout sessions devoted to these topics and in a plenary session. At the plenary session, certain items were reformulated and reordered and two provisional sets of recommendations developed, with decisions made using a modified Delphi technique.32 The group then realised how similar the individual statements in each of the two documents were, but left further decisions to the next stage of the process.\n\nWith these two documents prepared and having in mind that peripheral SpA (such as ReA) had not yet been dealt with in detail, an even larger committee met in April 2012 (Amsterdam); its membership comprised the initial task force expanded by consideration of a more international scope to also include experts from Latin America and Asia, aside from previous participants from Europe and North America. Again, the scope and background of this activity was discussed and the provisional recommendations presented. The issue of disease definition and the need of developing one, two or three documents were addressed. The committee separated into three breakout groups discussing axSpA, peripheral SpA and PsA. In the course of the breakout discussions and the plenary session, the initial recommendations were amended and then votes cast. Importantly, when looking at the individual items, the participants felt that most of them were very similar and a broad decision was then taken to develop a single document comprising overarching principles and items common to SpA in general, but within that common document to develop a few individualised items for axSpA, PsA and peripheral SpA.\n\nEach statement, which had been formulated as a draft for voting in the course of the breakout sessions and by the whole task force, was subjected to voting as 'yes' (agreement with the wording) or 'no' (disagreement). Statements supported by \u226575% of votes were immediately accepted while those with \u226425% were rejected outright. Others were subjected to further discussion and subsequent voting, where \u226567% support or, in an eventual third round, a majority of \u226550% was needed.\n\nAfter the face-to-face meeting, the statements were distributed to the committee members by email for final comments. Only suggestions for improvements of clarity of wording or addressing redundancies were considered, while any changes to the meaning were not accepted.\n\nFinally, the group voted anonymously by email on the level of agreement, that is, strength of recommendation, with each of the derived bullet points (in the form it was ultimately agreed upon by the qualified majority of participants) using a 10-point numerical rating scale (1=do not agree at all, 10=agree completely).\n\n# Results\n\n## The evidence base\n\nThe SLR, which is published separately,37 revealed that in contrast to findings in RA33 no randomised controlled clinical trial has evaluated a targeted therapeutic approach in comparison with routine therapy. However, several publications had employed therapeutic targets and respective time requirements as endpoints or before escalating therapy, although this is often the placebo arm of a study that was allowed to escape or then escalated to active treatment. These comprised 14 studies in AS and 7 studies of PsA which were found suitable to inform the task force. Nevertheless, given the lack of studies evaluating target-steered versus non-steered treatment, the level of evidence for the developed recommendations is low and mainly based on expert consensus.\n\n## The consensus\n\nThe individual statements receiving a positive vote by the majority of the expert committee members comprise 5 overarching principles and 11 recommendations. The overarching principles and 9 of the statements are recommended for SpA in general, whereas the last 2 statements have been individualised for axSpA, peripheral SpA and PsA. The recommendations are shown in <a href=\"#ANNRHEUMDIS2013203419TB1\" data-ref-type=\"table\">table 1<\/a>. They are discussed in detail below and this detailed description should be regarded as part and parcel of the recommendations.\n\nRecommendations to treat all forms of Spondyloarthritis to target\n\n|  |  | LoE | GoR | SoR |\n|:---|:---|:---|:---|:---|\n| **Overarching principles** |  |  |  |  |\n| A. | The treatment target must be based on a shared decision between patient and rheumatologist | 5 | D | 9.7\u00b10.8 |\n| B. | SpA and PsA are often complex systemic diseases; as needed, the management of musculoskeletal and extra-articular manifestations should be coordinated between the rheumatologist and other specialists (such as dermatologist, gastroenterologist, ophthalmologist) | 5 | D | 9.5\u00b10.92 |\n| C. | The primary goal of treating the patient with SpA and\/or PsA is to maximise long-term health related quality of life and social participation through control of signs and symptoms, prevention of structural damage, normalisation or preservation of function, avoidance of toxicities and minimisation of comorbidities | 5\\* | D | 9.6\u00b10.67 |\n| D. | Abrogation of inflammation is presumably important to achieve these goals | 5\\* | D | 9.1\u00b11.04 |\n| E. | Treatment to target by measuring disease activity and adjusting therapy accordingly contributes to the optimisation of short term and\/or long term outcomes | 5\\* | D | 9.2\u00b11.11 |\n| **Recommendations** |  |  |  |  |\n| Common items for all forms of SpA |  |  |  |  |\n| 1\\. | A major treatment target should be clinical remission\/inactive disease of musculoskeletal involvement (arthritis, dactylitis, enthesitis, axial disease), taking extra-articular manifestations into consideration | 5\\* | D | 9.5\u00b10.77 |\n| 2\\. | The treatment target should be individualised according to the current clinical manifestations of the disease | 5 | D | 9.3\u00b11.03 |\n| 3\\. | Clinical remission\/inactive disease is defined as the absence of clinical and laboratory evidence of significant inflammatory disease activity | 5 | D | 9.0\u00b11.41 |\n| 4\\. | Low\/minimal disease activity may be an alternative treatment target | 5\\* | D | 9.4\u00b10.91 |\n| 5\\. | Disease activity should be measured on the basis of clinical signs and symptoms, and acute phase reactants | 5\\* | D | 9.4\u00b11.14 |\n| 6\\. | The choice of the measure of disease activity and the level of the target value may be influenced by considerations of comorbidities, patient factors and drug-related risks | 5 | D | 9.4\u00b11.02 |\n| 7\\. | Once the target is achieved, it should ideally be maintained throughout the course of the disease | 5\\* | D | 9.4\u00b10.76 |\n| 8\\. | The patient should be appropriately informed and involved in the discussions about the treatment target, and the risks and benefits of the strategy planned to reach this target | 5 | D | 9.8\u00b10.50 |\n| 9\\. | Structural changes, functional impairment, extra-articular manifestations, comorbidities and treatment risks should be considered when making clinical decisions, in addition to assessing measures of disease activity | 5 | D | 9.5\u00b10.81 |\n| Specific items for individual types of Spondyloarthritis |  |  |  |  |\n| \u2003\u2003\u2002*Axial Spondyloarthritis (including ankylosing spondylitis)* |  |  |  |  |\n| 10\\. | Validated composite measures of disease activity such as the BASDAI plus acute phase reactants or the Ankylosing Spondylitis Disease Activity Score, with or without measures of function such as BASFI, should be performed and documented regularly in routine clinical practice to guide treatment decisions; the frequency of the measurements depends on the level of disease activity | 5 | D | 9.3\u00b10.95 |\n| 11\\. | Other factors, such as axial inflammation on MRI, radiographic progression, peripheral musculoskeletal and extra-articular manifestations, as well as comorbidities may also be considered when setting clinical targets | 5\\* | D | 9.3\u00b10.80 |\n| *\u2003\u2003\u2002Peripheral Spondyloarthritis* |  |  |  |  |\n| 10\\. | Quantified measures of disease activity, which reflect the individual peripheral musculoskeletal manifestations (arthritis, dactylitis, enthesitis) should be performed and documented regularly in routine clinical practice to guide treatment decisions; the frequency of the measurements depends on the level of disease activity | 5 | D | 9.3\u00b10.85 |\n| 11\\. | Other factors. such as spinal and extra-articular manifestations, imaging results, changes in function\/quality of life, as well as comorbidities may also be considered for decision | 5 | D | 9.4\u00b10.78 |\n| \u2003\u2003\u2002*Psoriatic arthritis* |  |  |  |  |\n| 10\\. | Validated measures of musculoskeletal disease activity (arthritis, dactylitis, enthesitis, axial disease) should be performed and documented regularly in routine clinical practice to guide treatment decisions; the frequency of the measurements depends on the level of disease activity; cutaneous manifestations should also be considered | 5 | D | 9.4\u00b10.78 |\n| 11\\. | Other factors, such as spinal and extra-articular manifestations, imaging results, changes in function\/quality of life, as well as comorbidities may also be considered for decision | 5 | D | 9.3\u00b11.00 |\n\nAn asterisk in the LoE column denotes that for this item indirect evidence is available from the literature search which was nevertheless not sufficient for a higher grading of the evidence level.\n\nBASDAI, Bath Ankylosing Spondylitis Disease Activity Index; BASFI, Bath Ankylosing Spondylitis Functional Index; GoR, grade of recommendation; LoE, level of evidence; PsA, psoriatic arthritis; SoR, strength of recommendation (level of agreement); SpA, spondyloarthritis.\n\n## Overarching principles\n\nIn the Committee's view, a number of elements related to treating SpA are so representative of good clinical practice that they form a general framework for more specific recommendations. These were therefore termed overarching principles, and five such principles were developed and voted on.\n\n*A. The treatment target must be based on a shared decision between patient and rheumatologist*.\n\nPatient involvement in therapeutic decision-making has become a mandate in patient care, especially when dealing with chronic diseases. This is a general patient right, and has been shown to improve mutual understanding and outcome39\u201342 and is also increasingly recognised to be important in SpA.43\u201345 The committee was convinced that patients must be informed about the proposed treatment target, therapeutic options to reach the target and reasons for recommending the target also in light of the risk related to treatment and risk related to the disease; on the other hand, patients should actively participate in this discussion. This aspect is subsequently reinforced in recommendation number 8 and these two items received the highest level of agreement among all bullet points. The principle also specifically mentions the rheumatologist, since it is the rheumatologist who should coordinate treatment of patients with SpA. Evidence regarding RA suggests that patient outcome is better when care is provided by a rheumatologist,46 and this might also be so for the musculoskeletal manifestations of PsA and SpA.\n\n*B. SpA and PsA are often complex systemic diseases; as needed, the management of musculoskeletal and extra-articular manifestations should be coordinated between the rheumatologist and other specialists (such as dermatologist, gastroenterologist, ophthalmologist)*.\n\nThis item is supposed to inform patients, healthcare professionals with less experience in the care of SpA and non-medical stakeholders that patients with SpA are frequently suffering from extramusculoskeletal manifestations and are often in need of multidisciplinary care for optimal therapy. When multiorgan involvement is present, a harmonised approach among specialists is required which should ideally be coordinated by the rheumatologist, especially if the musculoskeletal involvement causes major complaints.\n\n*C. The primary goal of treating the patient with SpA and\/or PsA is to maximise long-term health related quality of life and social participation through control of signs and symptoms, prevention of structural damage, normalisation or preservation of function, avoidance of toxicities and minimisation of comorbidities*.\n\nThe significant burden of axSpA and PsA in terms of disability, loss of quality of life and work productivity has only recently been appreciated.47\u201352 This generally formulated item addresses the importance to control signs and symptoms like pain, structural changes such as ankylosis,53 54 comborbidities55\u201357 and the importance of focusing on the totality of disease manifestations and complications when determining the proposal for a treatment target.\n\n*D. Abrogation of inflammation is presumably important to achieve these goals*.\n\nPsA and SpA are inflammatory diseases and inflammation leads to their signs and symptoms, functional impairment as well as structural changes.7 11 58\u201361 Therefore, stopping inflammation appears to be of key importance to optimise outcome. Indeed, in many patients non-steroidal anti-inflammatory drugs (NSAIDs) can lead to cessation of signs and symptoms, normalisation of physical function and potentially inhibition of structural damage in the spine.62 63 Interference with the proinflammatory cytokine tumour necrosis factor (TNF) suppresses inflammation effectively and can lead to disappearance of signs and symptoms and maximal improvement of physical function. Thus, the task force was convinced that disappearance of inflammation conveys the best outcome. However, current evidence indicates that TNF inhibition does not prevent progression of structural changes in AS.64 65 Moreover, it has not been determined if a state of remission leads to better long-term outcome of SpA and\/or PsA than low disease activity. Thus, this item is somewhat more controversial than most other ones. Therefore the word 'presumably' was added. This point constitutes a backbone for some of the subsequent individual recommendations.\n\n*E. Treatment to target by measuring disease activity and adjusting therapy accordingly contributes to the optimisation of short and\/or long-term outcomes*.\n\nThe SLR has revealed that patients with AS who do not reach predefined, measurable treatment targets can achieve further improvement upon adaptation of their therapy. While for PsA this has not yet been established, the task force regarded the need to measure disease activity and amend therapy with persistently active disease as a general necessity and, therefore, as a principle.\n\nThe level of agreement with these five principles was very high, ranging between 9.1 (item D) and 9.7 (item A) on a scale of 10 (table), indicating that this large and quite heterogeneous task force had arrived at a quite unanimous view on the principal importance of certain approaches to treatment of SpA.\n\n## Recommendations\n\nAs mentioned previously, after intensive deliberations the committee had decided to create just one document covering axial and peripheral SpA, including PsA. To this end, nine unified recommendations and two additional items dealing separately with PsA, axial SpA and peripheral SpA were developed in the course of the discussions. These recommendations applied the results of the SLR, but given the low available evidence in the literature, they were mostly based on expert opinion, albeit consensual opinions of a large group of experts. The sequence of the recommendations follows a logical order, but also reflects the level of importance considered by the committee for each individual bullet point.\n\n### Common recommendations\n\n1\\. A major treatment target should be clinical remission\/inactive disease of musculoskeletal involvement (arthritis, dactylitis, enthesitis, axial disease), taking extra-articular manifestations into consideration (level of evidence (LoE): 5, grade of recommendation (GoR): D).\n\nHitherto, no clinical trial has compared outcomes of PsA or axSpA for progression of structural changes or improvement of physical function or quality of life when remission rather than another state was targeted. Definitions for remission (which is called inactive disease in AS) or at least minimal disease activity (MDA) exist for PsA and AS,12 66 67 but in contrast to RA the long-term benefits of remission have not yet been sufficiently established. Also, no clear definition of remission for extra-articular musculoskeletal features, such as enthesitis or dactylitis, is currently available. Moreover, it is not sufficiently known at present how remission of musculoskeletal symptoms relates to remission of skin disease in PsA or bowel disease in IBD. Therefore, the formulation of this first bullet point reflects the general lack of data, by saying '*a* major' rather than '*the* major' treatment goal, or by expanding the term 'clinical remission' to the somewhat less stringent term 'inactive disease'. However, the lack of thorough evidence and the unwillingness of the group to arrive at a more determined and clear verbalisation of this bullet point must not be mistaken as an uncertainty of the task force regarding the necessity of treating patients to become free of signs and symptoms of their peripheral joint or axial disease. On the contrary, the task force deemed this approach to be of utmost importance for short-term benefit and long-term outcome. Therefore it was placed as the first recommendation. Importantly, the group focussed the terms 'remission'\/'inactive disease' to the musculoskeletal manifestations of SpA and *not* the extramusculoskeletal abnormalities, although it clearly stated in the last part of this item that this must not be neglected in the therapeutic decision making. After several rounds of discussion, 83% of the participants agreed with the formulation of this bullet point and the strength of recommendation amounted to 9.5\u00b10.9.\n\n2\\. The treatment target should be individualised according to the current clinical manifestations of the disease (LoE: 5, GoR: D)\n\nThis item emphasises that every patient should be treated according to her or his current clinical manifestations and that in light of their heterogeneity each of these manifestations has to be accounted for when setting the therapeutic target. However, it also implies that at certain points in time the musculoskeletal symptoms may not be in the foreground, such as when extra-articular manifestations prevail and need appropriate attention. Again, no data exist in the literature to support or refute this recommendation which was voted for by 87% of the participants and attained a strength of recommendation (SoR) of 9.3\u00b11.0.\n\n*3. Clinical remission\/inactive disease is defined as the absence of clinical and laboratory evidence of significant inflammatory disease activity*.\n\nThis bullet point provides a definition for item 1 in order to clarify the definition of remission. Remission of an inflammatory rheumatic disease ideally comprises the absence of its signs and symptoms, the maximal improvement in physical function and halt of structural changes. While there is compelling evidence that these three characteristics go along with each other in RA, this is not yet sufficiently known in AS and PsA. However, in PsA, progression of joint damage is correlated with swollen joint counts and dactylitis59 68 and, therefore, it may be assumed that clinical remission will also lead to halt of structural progression. This is not quite clear in AS, since progression of syndesmophyte formation has been observed even when patients were in clinical remission on TNF inhibitors and formation of syndesmophytes occurred without presence of MRI inflammation.8 64 65 69 On the other hand, elevated levels of acute phase reactants (APRs) are associated with progression of structural changes in AS.70 Further, physical function and quality of life are related to symptoms of these diseases.71 For all these reasons the task force defined remission as stated above. Definitions of remission or partial remission are available for AS, when using mere patient reported outcomes67 or the more recently described Ankylosing Spondylitis Disease Activity Score (ASDAS).12 For PsA, remission criteria are not well established,66 especially given that in PsA most composite measures have been borrowed from RA while a composite measure specific for PsA has only recently been validated,10 but criteria for disease activity states have not yet been defined. Importantly, the term 'significant' was added deliberately, indicating that the presence of a minute extent of residual activity, such as the presence of a tender joint or residual swollen but painless joint, or residual axial pain that does not appear to relate to inflammation, would still be compatible with remission. On the other hand, the committee wished to have a stringent definition of remission which would not allow significant residual disease activity, such as several swollen joints or significant back pain, even if dramatically improved by therapy, to be called remission. Also, this bullet point speaks of 'clinical remission' indicating that clinical rather than imaging measures should be used to define remission, at least currently. In this respect it should be noted that MRI shows evidence of inflammation in AS and that a negative MRI can be regarded as imaging remission; however, the relationship between clinical and imaging remission still needs to be elaborated. Statement 3, which like the previous ones is based on expert opinion, received approval by 83% of the committee members and a SoR of 9.0\u00b11.4.\n\n4\\. Low disease activity\/MDA may be an alternative treatment target (LoE: 5; GoR: D)\n\nWhile remission (inactive disease) constitutes an ideal goal, clinical practice, stringently as it was defined in bullet point number 3 it may be difficult to achieve in many patients, especially those with established\/long-standing disease. Indeed, patients with axSpA with longer disease duration are less likely to attain partial remission than those with early disease.72 38 Thus, while remission is the ultimate and an ideal goal, low disease activity constitutes a useful alternative in the opinion of the task force, since it is assumed that physical function and quality of life may not be much worse than in remission and progression of structural damage, while possibly not halted, would be minimal. Indeed, as included in the bullet point, low disease activity can also be regarded as 'MDA'. Thus, minor residual signs and symptoms may still exist differentiating this state from inactive disease. Importantly, by stating that low disease activity is an alternative goal to remission, the committee also clearly implies that any other, higher state, even moderate disease activity, would not be acceptable and its presence should elicit therapeutic adaptations. More research will be needed to provide information on the optimal time point for achieving the treatment target. However, given that in clinical trials of AS maximal improvement was achieved between week 12 and week 24 regarding all outcome measures including ASAS partial remission4 73 74 and that similar observations have been made in PsA,2 75 76 a maximum of 6\u2005months for reaching the treatment target of low disease activity or remission seems appropriate, but it is advisable to adapt therapy earlier if no significant reduction in disease activity is observed within 3\u2005months. Recently, thresholds for disease activity states including inactive disease (equivalent to remission) have been defined for axSpA using the ASDAS,12 and ASAS definition of partial remission is also available67; a measure of MDA has been developed for PsA which is beginning to be used in clinical trials.77 78 Additional research in this respect will be required, especially for PsA and peripheral SpA. This item was accepted as defined by 79% of the participants and received a SoR of 9.4\u00b10.9.\n\n*5. Disease activity should be measured on the basis of clinical signs and symptoms, and APRs (LoE: 5, GoR: D)*.\n\nTraditionally, given that the spine is not as accessible to physical examination of signs of inflammation like a peripheral joint, disease activity in axSpA has been evaluated by employing the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) which comprises only patient reported variables related to symptoms of the disease. However, axSpA is an inflammatory disease with involvement of various inflammatory cells and cytokines.79 80 Indeed, inhibition of TNF is one of the mainstays of treatment today, leading to relief of symptoms, and inhibition of prostaglandin synthesis can reduce clinical symptoms, and retard progression of structural changes62 63; this appears to be particularly prominent in patients with elevated APRs, and the latter seem to be associated with progression of syndesmophyte formation.70 81 82 Consequently, disease activity assessment during follow-up and in the course of targeting a good outcome should comprise the assessment of clinical aspects of the disease as well as laboratory abnormalities, that is, APR measurement. This can be done separately by looking at for example, BASDAI and c-reactive protein (CRP), or by using a measure that comprises both aspects, such as ASDAS.11 Along the same line, PsA as an inflammatory and potentially destructive joint disease should be followed using measures that relate to the joints and the serological inflammatory response; these are contained in RA-related composite measures of disease activity, such as Disease Activity Score (DAS), DAS28 and Simplified Disease Activity Index (SDAI), but also in the Disease Activity in PsA and the psoriatic arthritis disease activity score.13 The current recommendation does not relate to systemic features of SpA, but solely to the musculoskeletal manifestations. For systemic features, other measures are needed and, according to the EULAR recommendations for treatment of PsA,18 this should be done in collaboration with respective specialists. Recommendation 5 received approval by 97% of participants and the SoR amounted to 9.4\u00b11.1.\n\n6\\. The choice of the measure of disease activity and the level of the target value may be influenced by considerations of comorbidities, patient factors and drug related risks (LoE: 5; GoR: D)\n\nPatients with chronic musculoskeletal diseases are frequently also suffering from comorbidities that (1) may be related to the overall spectrum of the disorder (such as IBD, uveitis or psoriasis), (2) may occur as a consequence of chronic inflammation (such as cardiovascular disease), (3) may be related to therapy (such as gastric ulcer or infection), or (4) just may occur concomitantly by chance. The presence of such comorbidities may alter the level of the treatment target, since the risk of aggravating the comorbid condition may outweigh the benefit conveyed by more intensive therapy to achieve the treatment target of the musculoskeletal manifestations. Further, the choice of follow-up measure may have to be changed under certain circumstances. For example, a concomitant disease which raises pain levels or APRs may influence the result of measuring disease activity. Likewise, when following patients on therapies that affect the APRs independently of clinical benefit one may have to reconsider the choice of a measure that contains an APR. Therefore, this point focuses on the application (and sometimes restricted applicability) of particular disease activity measures. This recommendation was approved by 97% of the task force members and received a SoR of 9.4\u00b11.0.\n\n7\\. Once the target is achieved, it should ideally be maintained throughout the course of the disease (LoE: 5; GoR: D)\n\nIt is clear that no patient's successfully targeted disease activity state should deteriorate during follow-up, since reactivation of disease may again lead to reduced quality of life and disability. There is evidence that on demand NSAID therapy, in contrast to regular NSAID treatment, is associated with progression of radiographic changes in AS63 and that stopping TNF-blocker therapy will lead to reactivation of AS83 and PsA.84 While the present consensus statement is not designed to provide recommendations on therapies with particular agents but rather on treatment strategies, the task force nevertheless points to the importance of maintaining the targeted therapeutic state once achieved and advises against stopping a successful therapy based on the available evidence. However, it has not yet been studied sufficiently if dose reduction or expansion of treatment intervals allows maintaining a good clinical state. Approval was given by 90% of the task force's members and the SoR amounted to 9.4\u00b10.8.\n\n8\\. The patient should be appropriately informed and involved in the discussions about the treatment target, and the risks and benefits of the strategy planned to reach this target (LoE: 5, GoR: D)\n\nWhile this statement has already been partly covered in the overarching principles, it was felt important to also raise this point in the context of the actual recommendations to bolster the importance of interaction between health professionals and their patients in all regards: setting and agreeing on the treatment target, discussing the strategies available to reach that target and the time it may take to attain it, and laying out the benefits and risks of the recommended treatment and consider the totality of clinical disease manifestations (including the extramusculoskeletal ones) and of comorbidities. This point also comprises the need to discuss steps to be taken if the treatment target is not achieved, such as adjustment of or switch to a new therapy. In this respect patient education programmes or booklets may provide additional helpful means. This item was approved by 81% of the participants and the SoR was 9.8\u00b10.5.\n\n*9. Structural changes, functional impairment, extra-articular manifestations, comorbidities and treatment risks should be considered when making clinical decisions, in addition to assessing measures of disease activity*.\n\nThis point is focused on considerations involved in therapeutic decision making. Although the last part of this recommendation emphasises the importance of regular assessment of disease activity with appropriate measures (see items 5 and 6), the first part suggests taking into account results of other investigations for treatment decisions, such as by imaging (especially structural changes in PsA), physical function and extra-articular manifestations. The latter comprise enthesitis or dactylitis as well as extramusculoskeletal disease. This is of importance, since treatment approaches to PsA will differ in the presence of enthesitis compared with patients who do not suffer from entheseal affection.18 Moreover, organ disease, such as lung involvement, aortitis, intestinal or skin manifestations as well as uveitis, may require involvement of other specialists (see overarching principle B). In particular uveitis can present across the spectrum of SpA and may reflect disease activity, and inflammatory bowel disease and psoriatic skin involvement must be considered in the respective disorders and do not strongly correlate with the degree or extent of musculoskeletal involvement. Risks and comorbidities are also reiterated here in the context of treatment decisions; before they were indicated with respect to the choice of measures of disease activity (see recommendation 6) and in relation to patient information (item 8). This recommendation achieved 100% agreement and a SoR of 9.5\u00b10.8.\n\n### Disease specific recommendations\n\nAs indicated above, items 10 and 11 have been formulated specifically for axSpA, PsA and peripheral SpA to account for the differences between certain characteristics of the different spondyloarthritides.\n\n#### Axial SpA (including AS)\n\n*10. Validated composite measures of disease activity such as BASDAI plus APRs or ASDAS, with or without measures of function such as Bath Ankylosing Spondylitis Functional Index (BASFI), should be performed and documented regularly in routine clinical practice to guide treatment decisions; the frequency of the measurements depends on the level of disease activity*.\n\nThis item is an expansion of recommendation numbers 5 and 9. It mentions those disease activity measures which have been repeatedly validated and are already in use in contemporary clinical trials. In line with recommendation 5, when BASDAI is employed, an APR, such as CRP or erythrocyte sedimentation rate, should also be determined. The ASDAS already comprises such a measure among its components.12 In addition, this recommendation also suggests the use of a particular functional measure, but other validated measures can also be applied (therefore the tem 'such as'). With highly active disease, follow-up examinations will have to be more frequent than with inactive disease\/remission. Moreover, the recommendation requests documentation of the measured results. Among task force members, 88% approved this item and SoR amounted to 9.3\u00b11.0.\n\n*11. Other factors such as axial inflammation on MRI, radiographic progression, peripheral musculoskeletal and extra-articular manifestations, may also be considered when setting clinical targets*.\n\nAgain, this recommendation expands item 9, but specifically mentions MRI as a highly valuable imaging method for the potential follow-up of axSpA. Likewise, non-axial disease manifestations will influence not only the therapeutic approach but have to be considered when setting treatment targets. Approval was voted for by 88% of the members and the SoR was 9.3\u00b10.8.\n\n#### Peripheral SpA\n\n*10. Quantified measures of disease activity, which reflect the individual peripheral musculoskeletal manifestations (arthritis, dactylitis, enthesitis) should be performed and documented regularly in routine clinical practice to guide treatment decisions; the frequency of the measurements depends on the level of disease activity*.\n\nThis recommendation, while expanding items 5, is here specifically tailored to peripheral SpA, such as ReA, IBD arthritis or former 'undifferentiated' peripheral SpA. Measures of disease activity are available and have been validated for the arthritis component of ReA9 and there exist measures for dactylitis and enthesitis which have not been primarily developed for peripheral SpA, but rather for PsA or AS.85\u201387 While they will have to be validated in peripheral SpA, they can be assumed useful for clinical practice until proven otherwise. Also this recommendation calls for documentation of the measured results. Of the participants, 100% approved this item and the SoR score achieved was 9.3\u00b10.9.\n\n*11. Other factors such as spinal and extra-articular manifestations, imaging results, changes in function\/quality of life, as well as comorbidities may also be considered for decision*.\n\nThis item reiterates and expands recommendation 9 and achieved 100% approval; the SoR was 9.4+0.8.\n\n#### Psoriatic arthritis\n\n*10. Validated measures of musculoskeletal disease activity (arthritis, dactylitis, enthesitis, axial disease) should be performed and documented regularly in routine clinical practice to guide treatment decisions; the frequency of the measurements depends on the level of disease activity; cutaneous manifestations should also be considered*.\n\nSpecific mention of skin disease tailors this recommendation to PsA, although skin may also be involved in axial and peripheral SpA. Further, in addition to arthritis, dactylitis and enthesitis, axial disease assessment is specifically brought forward here. Finally, a reminder is provided that the results of the various measures (and also the treatment target) should be documented. Clearly, with highly active disease patients should be seen frequently, such as monthly to every 3\u2005months, while with low disease activity or remission, follow-up examinations may be done only every 6\u201312\u2005months. However, skin involvement also has to be taken into account. The voting achieved 92% approval and the SoR amounted to 9.4\u00b10.8.\n\n*11. Other factors such as spinal and extra-articular manifestations, imaging results changes in function\/quality of life, as well as comorbidities may also be considered for decision*.\n\nAs for the other disease entities, this last recommendation expands item 9 and also the preceding one by reiterating the importance of comorbidities, and axial and soft tissue manifestations of PsA in the course of making treatment decisions. Approval was granted by 100% of the participants and the SoR amounted to 9.3\u00b11.0.\n\nA final anonymous vote on whether the task force members felt influenced by the fact that support for this activity was provided by a company, the result was 0.4\u00b11.3 (0 meaning no and 10 meaning heavy influence), indicating that the participants felt negligibly influenced.\n\n## Research agenda\n\nSince none of the recommendations is based on evidence, the research agenda has to comprise the search for evidence for all of them. However, beyond mere therapeutic aspects, insights into the relationships between individual musculoskeletal manifestations, damage and disability are still incomplete especially for the peripheral SpA, including PsA. <a href=\"#ANNRHEUMDIS2013203419TB2\" data-ref-type=\"table\">Table\u00a02<\/a> lists a research agenda as mentioned during the task force's meetings.\n\nResearch agenda\n\n| Topics | Specific questions |\n|:---|:---|\n| Composite activity measures (mainly PsA and peripheral SpA) | Validation where needed, definition of disease activity states and response categories |\n| Remission definition | Is it important that all clinical domains of axial SpA, peripheral SpA or PsA are in remission or is it sufficient to define some of them? |\n| Treatment target | Is there a difference in long-term outcome when comparing remission with low disease activity? |\n| Activity and damage | What is the progression of joint damage in different disease activity states in PsA? |\n| Disease duration | Are there differences in responsiveness and thus differences in attaining certain targets with different disease duration in PsA? |\n| Treatment to target | There is a need to design therapeutic trials that compare steered therapy aiming at remission or low disease activity with non-steered treatment (like TICORA)88 |\n| Axial involvement in PsA | Do spinal and peripheral involvements respond similarly or differently? |\n| Enthesitis, dactylitis | More data need to be attained on the response of dactylitis or enthesitis to different therapies |\n| Care by rheumatologist | Is care of axial SpA, peripheral SpA or PsA by a rheumatologist advantageous for outcomes when compared with care by non-rheumatologists? |\n| Maintenance of response | How can response be maintained? Can the dose of the therapy employed be reduced or the interval of applications be expanded and outcome maintained? |\n| Patient | Is outcome different when patients are informed in a structured way when compared with more general means of information? |\n\nPsA, psoriatic arthritis; SpA, Spondyloarthritis.\n\n# Discussion\n\nRecommendations to treat axSpA and PsA have been developed over the recent years.17\u201319 However, none of these addressed a clear therapeutic target and a strategy to reach this target. This has now been done in the present set of recommendations, and additional strategic aspects of treatment approaches are presented. Thus, the present consensus on treatment targets and general treatment approaches complements the published management recommendations,17\u201320 but a notable difference is the absence of suggestions or recommendations regarding a particular drug in any of the overarching principles or individual recommendations.\n\nTreatment recommendations should usually be based on evidence. However, where evidence is missing, expert opinion has to come into play. The recommendations presented here are not based on hard evidence, because strategic therapeutic trials, in which therapy was consistently adapted to reach a prespecified treatment target and compared with a non-steered approach, as performed in RA,88 89 are currently not available for axSpA, peripheral SpA or PsA, and other pertinent literature is scarce. While a SLR has provided indirect evidence from clinical trials which targeted specific endpoints37 and thus supplied some information towards the work of the task force, the individual recommendations can only be regarded as expert opinion (consensus) and therefore call for more research in the field.\n\nSo why then were the recommendations developed now rather than waiting for more evidence? Because the definition of a treatment target and strategy is timely at present in light of two major aspects: (1) a field like that of SpA should not stay behind other disease areas which have already defined their targets years to decades ago, when at least indirect evidence for the benefit of attaining certain treatment targets is available; and (2) the tremendous therapeutic advances of the past decade have greatly improved the chances of achieving good outcomes and, therefore, setting stringent treatment milestones has become a reality which should not be concealed. Moreover, in the course of its initial discussions on this issue, the task force felt that with these advances and the concomitant formulation of a research agenda, investigations towards providing respective evidence could be fostered and accelerated. Importantly, this view was shared among all task force members, which comprised patients and an international group of physicians with expertise in SpA.\n\nAt all three steps of this activity, which included initial discussions by the steering committee, formulation of recommendations by an expanded working group and development of treatment recommendations for all three entities, axSpA, peripheral SpA and PsA, unanimous agreement was attained. Moreover, all items achieved strong consensus in an anonymous voting process, with the lowest result being a mean of 9.0 on a scale of 0\u201310, indicating that the task force stood quite united behind the recommendations.\n\nThe complexity of the current endeavour resulted from the heterogeneity of the diseases covered. After long discussions and the intermediate development of more than one document, it was decided to produce a single set of recommendations for axSpA, peripheral SpA and PsA, in line with recent criteria for classification of SpA.15 Five overarching principles and nine recommendations were developed in common for all forms of SpA, including PsA. Only two recommendations were separately produced for axSpA, peripheral SpA and PsA, although their general scope was still very similar and differences only very subtle. The overall activity was partly influenced by the treat-to-target recommendations for RA.32\n\nSeveral of the recommendations stand out in their importance, while others can be seen as supportive or operational. A call for remission or inactive disease became item 1, because this was regarded the foremost treatment target. Indeed, we can anticipate that reducing inflammation and disease activity to the minimum is optimal for the patients, at least for their quality of life. However, the members were aware that remission may not be achievable in all patients and, therefore, formulated an alternative treatment target, especially for patients with long-standing disease, namely low disease activity (recommendation 4). Importantly, this acknowledgement indicates that disease activity states other than remission or low disease activity constitute unacceptable clinical states, unless justified because of other reasons, such as comorbidity (items 6, 9 and 11). Importantly, while validated measures of disease activity are available for PsA and ReA, disease activity states have not yet been sufficiently defined, contrasting the situation in AS. Another complexity relates to the necessity to use measures that reflect the individual manifestations of a patient which in some instances may involve assessment of peripheral joint disease, axial involvement, dactylitis and enthesitis. To identify an individual treatment goal can in itself be seen as an important part of a treatment strategy when an intervention is initiated and should be accompanied by a monitoring programme. It is also important that the agreed goal is documented in the records of the patient.\n\nPatient involvement in defining the treatment target and selection of therapies based on their risks and benefits was deemed so important, that it is stated in the first overarching principle and additionally in one of the recommendations.\n\nAs indicated above, given the small evidence base, the research agenda is of utmost importance. Research activities should focus on strategic therapeutic trials, and on addressing missing information, such as the definition of disease activity states in PsA.\n\nThe recommendations are summarised in a simplified form in an algorithm presented in <a href=\"#ANNRHEUMDIS2013203419F1\" data-ref-type=\"fig\">figure 1<\/a>. Like most types of recommendations, it will be necessary to revise the current document in due course, presumably in about 4 years or 5\u2005years or earlier, when significant evidence accumulates regarding the individual points of the recommendations. The task force hopes for an expansion of high quality research activities that either allow confirmation or modifications of its conclusions.\n\n# References\n\n[^1]: **Handling editor** Francis Berenbaum","meta":{"dup_signals":{"dup_doc_count":116,"dup_dump_count":55,"dup_details":{"curated_sources":2,"2023-50":3,"2023-40":3,"2023-23":2,"2022-40":2,"2022-33":3,"2022-27":4,"2022-21":4,"2022-05":2,"2021-49":1,"2021-43":2,"2021-39":5,"2021-31":4,"2021-25":1,"2021-21":3,"2021-17":6,"2021-10":3,"2021-04":2,"2020-50":1,"2020-45":2,"2020-40":1,"2020-34":2,"2020-29":1,"2020-16":2,"2020-10":1,"2020-05":3,"2019-47":2,"2019-39":1,"2019-26":2,"2019-22":1,"2019-18":2,"2019-13":1,"2019-09":1,"2019-04":2,"2018-51":3,"2018-47":2,"2018-39":3,"2018-30":1,"2018-26":1,"2018-22":1,"2018-13":2,"2018-09":1,"2017-51":2,"2017-47":1,"2017-43":2,"2017-39":1,"2017-34":3,"2017-26":2,"2017-22":1,"2017-17":1,"2017-09":1,"2024-30":3,"2024-26":1,"2024-22":4,"2024-18":2,"2017-13":1}},"file":"PMC3888616"},"subset":"pubmed_central"}
{"text":"abstract: Understanding basic neuronal mechanisms hold the hope for future treatment of brain disease. The 1st international conference on synapse, memory, drug addiction and pain was held in beautiful downtown Toronto, Canada on August 21\u201323, 2006. Unlike other traditional conferences, this new meeting focused on three major aims: (1) to promote new and cutting edge research in neuroscience; (2) to encourage international information exchange and scientific collaborations; and (3) to provide a platform for active scientists to discuss new findings. Up to 64 investigators presented their recent discoveries, from basic synaptic mechanisms to genes related to human brain disease. This meeting was in part sponsored by Molecular Pain, together with University of Toronto (Faculty of Medicine, Department of Physiology as well as Center for the Study of Pain). Our goal for this meeting is to promote future active scientific collaborations and improve human health through fundamental basic neuroscience researches. The second international meeting on Neurons and Brain Disease will be held in Toronto (August 29\u201331, 2007).\nauthor: Guo-Qiang Bi; Vadim Bolshakov; Guojun Bu; Catherine M Cahill; Zhou-Feng Chen; Graham L Collingridge; Robin L Cooper; Jens R Coorssen; Alaa El-Husseini; Vasco Galhardo; Wen-Biao Gan; Jianguo Gu; Kazuhide Inoue; John Isaac; Koichi Iwata; Zhengping Jia; Bong-Kiun Kaang; Mikito Kawamata; Satoshi Kida; Eric Klann; Tatsuro Kohno; Min Li; Xiao-Jiang Li; John F MacDonald; Karim Nader; Peter V Nguyen; Uhtaek Oh; Ke Ren; John C Roder; Michael W Salter; Weihong Song; Shuzo Sugita; Shao-Jun Tang; Yuanxiang Tao; Yu Tian Wang; Newton Woo; Melanie A Woodin; Zhen Yan; Megumu Yoshimura; Ming Xu; Zao C Xu; Xia Zhang; Mei Zhen; Min Zhuo\ndate: 2006\ninstitute: 1Department of Neurobiology, University of Pittsburgh, Pittsburgh, USA; 2Department of Psychiatry, Harvard University, Boston, USA; 3Department of Pediatrics, and Cell Biology and Physiology, Washington University in St. Louis, St. Louis, USA; 4Department of Pharmacology and Toxicology, Queen's University, Kingston, Canada; 5Department of Anesthesiology, Washington University in St. Louis, St. Louis, USA; 6Centre for Synaptic Plasticity, University of Bristol, Bristol, UK; 7Department of Biology, University of Kentucky, Lexington, USA; 8Department of Physiology and Biophysics, University of Calgary, Calgary, Canada; 9Department of Psychiatry, University of British Columbia, Vancouver, Canada; 10Institute for Molecular and Cell Biology, University of Porto, Porto, Portugal; 11Skirball Institute, New York University School of Medicine, New York, USA; 12Department of Oral and Maxillofacial Surgery, University of Florida, Gainesville, USA; 13Department of Pharmaceutical Health Care and Sciences, Kyushu University, Kyushu, Japan; 14NINDS, NIH, Bethesda, USA; 15Department of Physiology, Nihon University, Tokyo, Japan; 16Department of Physiology, University of Toronto, Toronto, Canada; 17Department of Biological Sciences, Seoul National University, Seoul, Korea; 18Department of Anesthesiology, Sapporo Medical University School of Medicine, Sapporo, Japan; 19Department of Agricultural Chemistry, Tokyo University of Agriculture, Tokyo, Japan; 20Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, USA; 21Division of Anesthesiology, Niigata University, Niigata, Japan; 22Department of Neuroscience and High Throughput Biology Center, Johns Hopkins University, Baltimore, USA; 23Department of Human Genetics, Emory University, Atlanta, USA; 24Department of Psychology, McGill University, Montreal, Canada; 25Department of Physiology, University of Alberta, Edmonton, Canada; 26Sensory Research Center, Seoul National University, Seoul, Korea; 27Department of Biomedical Sciences, University of Maryland, Baltimore, USA; 28Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Canada; 29Department of Neurobiology and Behavior, University of California, Irvine, USA; 30Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University, Baltimore, USA; 31Brain Research Center, University of British Columbia, Vancouver, Canada; 32NICHD, NIH, Bethesda, USA; 33Department of Cell and Systems Biology, University of Toronto, Toronto, Canada; 34Department of Physiology and Biophysics, State University of New York at Buffalo, Buffalo, USA; 35Department of Basic Medicine, Kyushu University, Kyushu, Japan; 36Department of Anesthesia and Critical Care, University of Chicago, Chicago, USA; 37Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, USA; 38Department of Psychiatry, University of Saskatchewan, Saskatoon, Canada\ntitle: Recent advances in basic neurosciences and brain disease: from synapses to behavior\n\n# Introduction\n\nOne key factor to promote the progress of science is to exchange scientific ideas and new discovery through meetings. Scientific meeting provides critical chance for investigators to communicate new ideas, discuss different\/conflicting results, and set up potential collaborations. Annual meeting of the American Society for Neuroscience (SfN) has served the community well in this aspect. However, with the increased membership and the scale of the meeting, SfN meetings only take place in a few cities in US in a rotated manner. Due to tight security control after 9\/11, many foreign investigators failed to obtain visiting visa to the meeting in a timely fashion. Considering these factors, a small scale of neuroscience meeting in a more relaxed city should provide better chance for investigators, in particular, principal investigators (PIs) to directly communicate with each other, face-to-face. The 1st international conference on synapse, memory, drug addiction and pain is designed to meet the need. The major aim of this meeting is to provide an opportunity for setting up global scientific exchanges, to provide an active stage for PIs to report novel or unpublished data, to bring neuroscientists working at different level of organism and systems together, and to promote research findings from junior and mid-career investigators.\n\nThe meeting is organized by Dr. Min Zhuo from the University of Toronto, with the help from Dr. Jianguo Gu from the University of Florida, and in part sponsored by Molecular Pain, University of Toronto (Faculty of Medicine, Department of Physiology as well as Center for the Study of Pain), and Olympus Inc.\n\nThere are four major themes for the meeting: synapse, Synaptic plasticity, memory, pain, and brain disease. Unlike other meetings, each speaker was given 15 min to talk, and 5 min for discussion. The time slot allowed for each speaker was tightly controlled by each chair (with a timer!), and few speakers went beyond the time permitted. The wine reception over the poster section provided a wonderful opportunity for further discussions. Taking advantage of the excellent location of the main campus in the downtown of Toronto, attendees also enjoyed the nice summer weather and wonderful food in Toronto. The Niagara Fall, a famous tour site, is above 90 min drive from the downtown of Toronto.\n\nStudents and post-doc fellows also presented over 50 posters on various topics. Among them, three posters were selected for best poster presentations and have been awarded with \\$250\u2013500.\n\n# Text\n\n## List of major themes, chairs, speakers and titles\n\n### Synapse\n\nChair: Min Li (USA)\n\n\u2022 Jens R Coorssen (University of Calgary, Calgary, Canada)\n\nThe role of cholesterol in synaptic release\n\n\u2022 Lin Mei (Medical College of Georgia, Augusta, USA)\n\nNeuregulin regulation of neuronal activity\n\n\u2022 Alaa El-Husseini (University of British Columbia, Vancouver, Canada)\n\nMechanisms that govern protein assembly at nascent neuronal contacts\n\n\u2022 Wen-Biao Gan (New York University School of Medicine, New York, USA)\n\nDendritic Spine Stability and Its Modification by Experience\n\n\u2022 Elise F Stanley (Toronto Western Research Institute, Toronto, Canada)\n\nThe presynaptic transmitter release site complex\n\nChair: Alaa El-Husseini (Canada)\n\n\u2022 Mei Zhen (University of Toronto, Toronto, Canada)\n\nSAD kinase regulates neuronal polarity and synapse formation\n\n\u2022 Robin L Cooper (University of Kentucky, Lexington, USA)\n\nEffects of the serotonergic system on physiology, development, learning and behavior of drosophila melanogaster\n\n\u2022 Min Li (Johns Hopkins University, Baltimore, USA)\n\nChemical regulation of membrane excitability\n\n\u2022 Shuzo Sugita (University of Toronto, Toronto, Canada)\n\nMolecular mechanism of GTP-dependent exocytosis\n\n\u2022 Lu-Yang Wang (University of Toronto, Toronto, Canada)\n\nConvergent pre- and post-synaptic adaptations for high-fidelity neurotransmission at the developing calyx of Held synapse\n\n\u2022 Wei-Yang Lu (University of Toronto, Toronto, Canada)\n\nPhysical Interaction between Acetylcholinesterase and Neurexins\n\n### Synaptic plasticity\n\nChair: Graham L Collingridge (UK) and Yu Tian Wang (Canada)\n\n\u2022 Eric Klann (Baylor College of Medicine, Houston, USA)\n\nTranslational Control during Hippocampal Synaptic Plasticity and Memory\n\n\u2022 Peter V Nguyen (University of Alberta, Edmonton, Canada)\n\nBeta-Adrenergic Receptors Recruit ERK and mTOR to Promote Translation-Dependent Synaptic Plasticity\n\n\u2022 Shao-Jun Tang (University of California, Irvine, USA)\n\nRegulation of Activity-Dependent Protein Synthesis in Dendrites\n\n\u2022 Yu Tian Wang (University of British Columbia, Vancouver, Canada)\n\nSynaptic plasticity in learning and memory\n\n\u2022 Graham L Collingridge (University of Bristol, Bristol, UK)\n\nGlutamate receptors and synaptic plasticity in the hippocampus\n\n\u2022 Michael W Salter (University of Toronto, Toronto, Canada)\n\nIns and outs of SRC regulation of NMDA receptors and synaptic plasticity\n\n\u2022 John F MacDonald (University of Toronto, Toronto, Canada)\n\nInhibitory Regulation of the Src Hub and LTP in CA1 Hippocampal Neurons\n\nChair: Michael W Salter (Canada)\n\n\u2022 Vadim Bolshakov (Harvard University, Boston, USA)\n\nSpatiotemporal asymmetry of associative synaptic plasticity in fear conditioning pathways\n\n\u2022 Guo-Qiang Bi (University of Pittsburgh, Pittsburgh, USA)\n\nDynamics and plasticity of reverberatory activity in small neuronal circuits\n\n\u2022 Melanie A Woodin (University of Toronto, Toronto, Canada)\n\nBidirectional spike-timing dependent plasticity of inhibitory transmission in the hippocampus\n\n\u2022 John Isaac (NIH, Bethesda, USA)\n\nKainate receptors in novel forms of long-term synaptic plasticity\n\n\u2022 Newton Woo (NIH, Bethesda, USA)\n\nRegulation of Bi-directional Plasticity by BDNF\n\n\u2022 Zhengping Jia (University of Toronto, Toronto, Canada)\n\nMolecular regulation of spine properties and synaptic plasticity\n\n### Pain\n\n**C**hair: Megumu Yoshimura (Japan)\n\n\u2022 Kazuhide Inoue (Kyushu University, Kyushu, Japan)\n\nP2X4: mechanisms of over expression in neuropathic pain state\n\n\u2022 Jianguo Gu (University of Florida, Gainesville, USA)\n\nTRPM8 and cold allodynia\n\n\u2022 Uhtaek Oh (Seoul National University, Seoul, Korea)\n\nTRPV1 and its Role for Inflammatory Pain\n\n\u2022 Vasco Galhardo (University of Porto, Porto, Portugal)\n\nImpairment in prefrontal-based emotional decision-making in rat models of chronic pain\n\n\u2022 Ke Ren (University of Maryland, Baltimore, USA)\n\nNeuronal\/glial cell interactions in CNS plasticity and persistent pain\n\nChair: Jianguo Gu (USA)\n\n\u2022 Yves De Koninck (Laval University, Quebec City, Canada)\n\nPlasticity of chloride homeostasis vs. plasticity of GABA\/glycine; who wins?\n\n\u2022 Megumu Yoshimura (Kyushu University, Kyushu, Japan)\n\nSynaptic mechanisms of acupuncture in the spinal dorsal horn revealed by in vivo patch-clamp recordings\n\n\u2022 Koichi Iwata (Nihon University, Tokyo, Japan)\n\nAnterior cingulate cortex and pain -its morphological feature and functional properties\n\n\u2022 Min Zhuo (University of Toronto, Toronto, Canada)\n\nCortical potentiation and its roles in persistent pain and fear\n\n\u2022 Vania A Apkarian (Northwestern University, Chicago, USA)\n\nChronic pain and emotional learning and memory\n\nChair: Uhtaek Oh (Korea)\n\n\u2022 Zhou-Feng Chen (Washington University in St. Louis, St. Louis, USA)\n\nLiving without serotonin: a genetic approach to study the roles of the serotonergic system in opioid analgesia and tolerance\n\n\u2022 Catherine M Cahill (Queen's University, Kingston, Canada)\n\nTrafficking of Delta Opioid Receptors in Chronic Pain\n\n\u2022 Hiroshi Ueda (Nagasaki University, Nagasaki, Japan)\n\nMolecular mechanisms of neuropathic pain \u2013 lysophosphatidic acid as the initiator\n\n\u2022 Yuanxiang Tao (Johns Hopkins University, Baltimore, USA)\n\nAre the PDZ domains at excitatory synapses potential molecular targets for prevention and treatment of chronic pain?\n\n\u2022 Tatsuro Kohno (Niigata University, Niigata, Japan)\n\nDifferent actions of opioid and cannabinoid receptor agonists in neuropathic pain\n\n\u2022 Ze'ev Seltzer (University of Toronto, Toronto, Canada)\n\nPower and limitations of the comparative approach that uses animal models to identify human chronic pain genes\n\n\u2022 Mikito Kawamata (Sapporo Medical University School of Medicine, Sapporo, Japan)\n\nGenetic variation in response properties of spinal dorsal horn neurons and rostral ventromedial medulla neurons in different mouse strains\n\n### Brain disease\n\nChair: Xiao-Ming Xu (USA)\n\n\u2022 Guojun Bu (Washington University in St. Louis, St. Louis, USA)\n\nLDL Receptor Family and Alzheimer's disease\n\n\u2022 Satoshi Kida (Tokyo University of Agriculture, Tokyo, Japan)\n\nMechanism of interaction between reconsolidation and extinction of contextual fear memory\n\n\u2022 Weihong Song (University of British Columbia, Vancouver, Canada)\n\nHypoxia facilitates Alzheimer's disease pathogenesis\n\n\u2022 Zhen Yan (State University of New York at Buffalo, Buffalo, USA)\n\nInteractions between Acetylcholine, Amyloid and Ion Channels in Alzheimer's Disease\n\n\u2022 Jian Feng (State University of New York at Buffalo, Buffalo, USA)\n\nAchilles' Heel of Midbrain Dopaminergic Neurons: Vulnerabilities and Defense Strategies\n\n\u2022 Xiao-Jiang Li (Emory University, Atlanta, USA)\n\nSynaptic toxicity of Huntington disease protein\n\nChair: Xiao-Jiang Li (USA)\n\n\u2022 Fang Liu (University of Toronto, Toronto, Canada)\n\nRegulation of dopamine reuptake by the direct protein-protein interaction between the dopamine D2 receptor and the dopamine transporter\n\n\u2022 Danny G Winder (Vanderbilt University School of Medicine, Nashville, USA)\n\nSynaptic plasticity in the bed nucleus of the stria terminalis: roles in addiction and anxiety\n\n\u2022 Ming Xu (University of Chicago, Chicago, USA)\n\nMolecular Mechanisms of neuronal plasticity induced by drugs of abuse\n\n\u2022 Xia Zhang (University of Saskatchewan, Saskatoon, Canada)\n\nTAT-3L4F, a novel peptide for the treatment of drug addiction\n\n\u2022 Evelyn K Lambe (University of Toronto, Toronto, Canada)\n\nHypocretin and nicotine excite the same thalamocortical synapses in prefrontal cortex: correlation with improved attention in rat\n\n\u2022 Wan Qi (University of Toronto, Toronto, Canada)\n\nRegulation of NMDA and GABA-A receptors by the tumor suppressor PTEN\n\n### Memory\n\nChair: Karim Nader (Canada)\n\n\u2022 Paul W Frankland (University of Toronto, Toronto, Canada)\n\nFunctional integration of adult-born granule cells into spatial memory networks in the dentate gyrus\n\n\u2022 Mara Dierssen (Centre for Genomic Regulation, Barcelona, Spain)\n\nDendritic pathology and altered structural plasticity in Down syndrome: In the search of candidate genes\n\n\u2022 Sheena A Josselyn (University of Toronto, Toronto, Canada)\n\nNeuronal memory competition: The role of CREB\n\n\u2022 Bong-Kiun Kaang (Seoul National University, Seoul, Korea)\n\nRole of a novel nucleolar protein ApLLP in synaptic plasticity and memory in Aplysia\n\n\u2022 Remi Quirion (Douglas Hospital Research Centre and INMHA, Montreal, Canada)\n\nNovel genes possibly involved in learning and memory\n\nChair: Bong-Kiun Kaang (Korea)\n\n\u2022 John C Roder (University of Toronto, Toronto, Canada)\n\nForward and reverse genetic screens in the mouse for mutants impaired in learning and memory\n\n\u2022 Karim Nader (McGill University, Montreal, Canada)\n\nIdentifying the neural mechanisms by which boundary conditions inhibit reconsolidation from occurring.\n\n\u2022 Yukio Komatsu (Nagoya University, Nagoya, Japan)\n\nRole of BDNF in the production of LTP at visual cortical inhibitory synapses\n\n\u2022 Xiao-Ming Xu (University of Louisville School of Medicine, Louisville, USA)\n\nSpinal cord injury repair: combinatorial strategies involving neuroprotection and axonal regeneration\n\n\u2022 Zao C Xu (Indiana University School of Medicine, Indianapolis, USA)\n\nSynaptic plasticity in pathological conditions\n\n# Abstracts\n\n\u2022 **Guo-Qiang Bi (Department of Neurobiology, University of Pittsburgh, Pittsburgh, USA) \u2013 Dynamics and plasticity of reverberatory activity in small neuronal circuits**\n\nThe concept of cell assembly was proposed by Hebb to provide an elementary structure for thought process and memory. The Hebbian cell assembly has two essential properties: 1. neuronal activity can reverberate in specific sequences within the assembly without sustained external drive; 2. synaptic modification resulted from the reverberatory activity further stabilizes the reverberation. Using whole-cell patch-clamp recording and simultaneous calcium imaging, we found that brief (e.g. 1-ms) stimulation of few neurons in a small network of about 100 cultured hippocampal neurons could trigger reverberatory activity in the network lasting for seconds. Such reverberatory activity consists of repeating motifs of specific patterns of population activation in the network. Paired-pulse stimuli with inter-pulse interval of \\~200\u2013400 ms are more effective in activating such oscillatory reverberation. Furthermore, repeated activation of reverberation with paired-pulse stimuli leads to long-term enhancement of subsequent activation by single stimuli. In addition, pairing a non-effective input (that does not activate network reverberation) into one neuron with an effective input (that activates reverberation) into another can convert the non-effective pathway into an effective one. Reverberatory circuits in vitro may serve as a prototype of Hebbian cell assembly for studies of the dynamics properties and underlying cellular mechanisms. (Supported by NIMH and Burroughs Wellcome Fund)\n\n\u2022 **Vadim Bolshakov (Department of Psychiatry, Harvard University, Boston, USA) \u2013 Spatiotemporal asymmetry of associative synaptic plasticity in fear conditioning pathways**\n\nLong-term potentiation (LTP) in afferent inputs to the amygdala serves an essential function in the acquisition of fear memory. The factors underlying input specificity of synaptic modifications implicated in the information transfer in fear conditioning pathways remain unknown. We now show that synapses in two auditory inputs converging on the same LA neuron utilize a form of the temporally asymmetric learning rule when the strength of na\u00efve synapses is only modified when a postsynaptic action potential closely follows the synaptic response. The stronger inhibitory drive in thalamic pathway, as compared to cortical input, hampers the induction of LTP at thalamo-amygdala synapses contributing to the spatial specificity of LTP in convergent inputs. These results indicate that spike timing-dependent synaptic plasticity in the LA is both temporarily and spatially asymmetric, which may contribute to the conditioned stimulus discrimination during fear behavior.\n\n\u2022 **Guojun Bu (Department of Pediatrics, and Cell Biology and Physiology, Washington University in St. Louis, St. Louis, USA) \u2013 LDL Receptor Family and Alzheimer's Disease**\n\nAmyloid-\u03b2 peptide (A\u03b2) production and accumulation in the brain is a central event in the pathogenesis of Alzheimer's disease (AD). Recent studies have shown that apolipoprotein E (apoE) receptors, members of the low-density lipoprotein receptor (LDLR) family, modulate A\u03b2 production as well as A\u03b2 cellular uptake. A\u03b2 is derived from proteolytic processing of amyloid precursor protein (APP), which interacts with several members of the LDLR family. Studies from our laboratory have focused on three members of the LDLR family, the LDLR-related protein (LRP), LRP1B, and the LDLR. Our *in vitro* cellular studies have shown that while LRP's rapid endocytosis facilitates APP endocytic trafficking and processing to A\u03b2, LRP1B's slow endocytosis inhibits these processes. In addition to modulating APP endocytic trafficking, LRP's rapid endocytosis also facilitates A\u03b2 cellular uptake by binding to A\u03b2 either directly or via LRP ligands such as apoE. Our *in vivo* studies using transgenic approach have shown that overexpression of LRP in CNS neurons increases cell-associated A\u03b2 and this increase correlates with an enhanced memory deficits in mice. We are currently investigating the cellular mechanisms by which LRP facilitates intraneuronal A\u03b2 accumulation, a pathological event that directly contributes to the early cognitive deficits seen in AD. Our preliminary results indicate that apoE plays an important role in intraneuronal A\u03b2 accumulation, likely by shuttling A\u03b2 into neurons via LRP-mediated pathways. We hypothesize that depending on the A\u03b2 species (A\u03b2 40 vs. A\u03b2 42), its aggregation states (monomers vs. oligomers), and the presence of apoE isoforms (apoE3 vs. apoE4), at least a portion of A\u03b2 that is internalized via an LRP-dependent pathway accumulates inside neurons. Molecular and cellular models underlying the mechanisms of LRP's involvements in AD will be presented and discussed.\n\n\u2022 **Catherine M Cahill (Department of Pharmacology and Toxicology, Queen's University, Kingston, Canada) \u2013 Trafficking of Delta Opioid Receptors in Chronic Pain**\n\nNeuropathic (NP) pain is defined as pain caused by a peripheral and\/or central nervous system lesion with sensory symptoms and signs and is estimated to affect more than 1.5% of Americans. Despite its prevalence and adverse impact on functionality and quality of life, it remains a significant challenge for physicians as it is typically refractory to traditional analgesics. However research increasingly suggests a therapeutic role of \u03b4OR agonists in treating chronic pain. Our research aims to understand the changes in \u03b4OR expression and function using both *in vivo* and *in vitro* techniques in an animal model of NP pain. NP, but not sham-operated, rats developed cold- and thermal-hyperalgesia as well as tactile allodynia. Intrathecal administration of a selective \u03b4OR agonist significantly alleviated these nociceptive behaviours and these effects were attenuated by a selective \u03b4OR antagonist. Real-time RT-PCR and western blotting experiments revealed no change in overall expression of \u03b4OR in the dorsal spinal cord however preliminary studies suggest that induction of NP pain may induce changes in subcellular localization of \u03b4ORs leading to enhanced analgesia.\n\n\u2022 **Zhou-Feng Chen (Department of Anesthesiology, Washington University in St. Louis, St. Louis, USA) \u2013 Living without serotonin: a genetic approach to study the roles of the serotonergic system in opioid analgesia and tolerance**\n\nNarcotics have long been used as an effective treatment for pain. The roles of the serotonergic (5-HT) system in opioid analgesia and tolerance, however, have been controversial. We have recently shown that the transcription factor Lm \u00d7 1b is essential for the development of 5-HT neurons. In the absence of Lm \u00d7 1b, all 5-HT neurons failed to develop in the raphe system. Because Lm \u00d7 1b-null mice die around birth, we have designed to strategy to delete Lm \u00d7 1b in 5-HT neurons only. Lm \u00d7 1b conditional knockout (CKO) mice lack all 5-HT neurons in the raphe system. Surprisingly, Lm \u00d7 1b CKO mice survive to the adulthood without motor deficiency. To assess the roles of the 5-HT system in opioid analgesia, we have examined the tail-flick responses of Lm \u00d7 1b CKO mice injected with mu-, kappa- and delta-opioid receptor agonists. In addition, we also examined the site of action of opioid receptor agonists by systemic, intrathecal and intracerebroventricular injections. These pharmacological studies revealed that the 5-HT system contributes differentially to opioid analgesia. Moreover, an examination of morphine analgesic tolerance in Lm \u00d7 1b CKO mice indicated that morphine tolerance is independent of the 5-HT system. These results should have important implications in our understanding of mechanisms of action for the 5-HT system in opioid analgesia and tolerance.\n\n\u2022 **Graham L Collingridge (Centre for Synaptic Plasticity, University of Bristol, Bristol, UK) \u2013 Kainate receptors: Functions and the discovery of novel antagonists.**\n\nLess is known about the role of kainate receptors compared with the other classes of ionotropic glutamate receptors in the CNS. However, recent studies, mainly employing the Lilly antagonist, LY382884, have identified several functions: For example, at mossy fibre synapses in the hippocampus these receptors function as facilitatory autoreceptors and are involved the induction of LTP. Kainate receptors also contribute to synaptic transmission at this synapse. Elsewhere kainate receptors can function as inhibitory autoreceptors and can regulate GABA transmission.\n\nWhilst LY382884 is a very useful antagonist it has a relatively narrow selectivity for GluR5-containing kainate receptors *versus* AMPA receptors. David Jane and his colleagues in Bristol have therefore developed a series of highly potent and specific GluR5 antagonists, the most potent of which is ACET. This compound should be extremely useful in investigating the role of kainate receptors in physiological and pathological functions in the CNS, for example, in neurodegeneration and neuropathic pain.\n\n\u2022 **Robin L Cooper (Department of Biology, University of Kentucky, Lexington, USA) \u2013 Effects of the serotonergic system on physiology, development, learning and behavior of drosophila melanogaster**\n\nThe serotonergic system in nervous tissue is known to play a vital role in development and behavior in simple to complex animal models. Using a simple model organism, Drosophila, the importance of serotonin (5-HT) circuitry in development and acute actions can be addressed. Also there are only four 5-HT receptors in the Drosophila genome, of which 5-HT2dro is known to be essential in the embryonic stages of development. Previously we have shown a physiological sensitivity of exogenous application of 5-HT on a sensory-CNS-motor circuit in semi-intact preparations of 3rd instar larvae. Now, using pharmacological manipulations and available receptor mutants for 5-HT2dro, we are studying the role of 5-HT in development, behavior and physiology of 3rd instar larvae. Para-chlorophenylalanine (p-CPA), is a blocker of 5-HT biosynthesis pathway and 3,4-Methylenedioxymethamphetamine (MDMA, Ecstasy), is a common drug of abuse in humans, which is known to compel mammalian serotonergic neurons to release 5-HT. When fed these compounds from 1st to 3rd instar a slowing of the growth occurred in a dose dependent manner. The rate of body wall and mouth hook movements were reduced in p-CPA and MDMA fed larvae. HPLC results showed lower amounts of 5-HT in larval brains for p-CPA but not MDMA fed larvae. An increase in sensitivity of sensory-CNS-motor circuit to 5-HT in drug fed larvae appears to be due an up regulation of 5-HT receptors. The antisense line for 5-HT2dro receptor also produces a delay in larval development. Preliminary data shows an impaired associative gustatory and olfactory learning behaviors in 3rd instar larvae with lower 5-HT or reduced expression of the 5-HT2dro receptor.\n\n\u2022 **Jens R Coorssen (Department of Physiology and Biophysics, University of Calgary, Calgary, Canada) -The role of cholesterol in synaptic release**\n\nFast, Ca^2+^ -triggered membrane merger defines regulated exocytosis. In native secretory vesicles, cholesterol (CHOL) functions in the fundamental fusion mechanism, and CHOL\/sphingomyelin \u2013 enriched microdomains define the efficiency (Ca^2+^ sensitivity and kinetics) of fusion. The role of CHOL in the fusion mechanism is mimicked by structurally dissimilar lipidic membrane components having spontaneous negative curvature (\/NC\/) = CHOL, and correlates quantitatively with the\/NC\/each contributes to the membrane (e.g. a-tocopherol and dioleoylphosphatidylethanolamine). Unable to substitute for CHOL in rafts, these lipids do not rescue fusion efficiency. Lipids of spontaneous\/NC\/\\< CHOL (e.g. dioleoylphosphatidic acid), do not support fusion. We have also identified com-parable molecular dependencies and relationships at the synapse, suggesting a conserved role for CHOL and the\/NC\/contributed. This quantitative relationship between\/NC\/and fusion appears most consistent with the stalk-pore model, demonstrating that\/NC\/itself is an essential component of the fundamental native fusion mechanism. The data also suggest that different fusion sites, vesicles, or secretory cells can use other lipidic components, in addition to sterols, to provide optimal local\/NC\/and even to modulate the fusion process.\n\n\u2022 **Alaa El-Husseini (Department of Psychiatry, University of British Columbia, Vancouver, Canada) \u2013 Elaboration of dendritic filopoidia is not a rate-limiting step for production of stable axonal-dendritic contacts.**\n\nDendritic filopodia are thought to play an active role in synaptogenesis and serve as precursors to spine synapses. However, this hypothesis is largely based on a temporal correlation between the onset of filopodia elaboration and synaptogenesis. We have previously demonstrated that the palmitoylated protein motifs of GAP-43 and paralemmin are sufficient to increase the number of filopodia and dendritic branches in neurons. Here we examined whether filopodia induced by these motifs, as well as those induced by cdc42 lead to the formation of stable synaptic contacts and the development of dendritic spines. Our analysis shows that expression of these filopodia inducing motifs (FIMs) or the constitutively active form of cdc-42 enhances filopodia motility, but reduces the probability of forming a stable axon-dendrite contact. Conversely, expression of neuroligin-1 a synapse inducing cell adhesion molecule, resulted in a decrease in filopodia motility, an increase in the number of stable axonal contacts, and the recruitment of synaptophysin positive transport packets. Postsynaptic scaffolding proteins such as Shank-1 that induce the maturation of spine synapses reduced filopodia number, but increased the rate at which filopodia transformed into spines. By following individual dendrites over a 2-day period we determined that relatively few sites with filopodia are replaced by spine synapses (\\~3%). These results suggest that high levels of filopodia elaboration and motility may not necessarily be a rate-limiting step for synapse formation, and that factors that control filopodia-process dynamics may participate in synapse formation by rapid stabilization of the initial contact between dendritic filopodia and axons.\n\n\u2022 **Vasco Galhardo (Institute for Molecular and Cell Biology, University of Porto, Porto, Portugal) \u2013 Impairment in prefrontal-based emotional decision-making in rat models of chronic pain**\n\nChronic pain is known to cause several cognitive deficits in human subjects. Among these deficits is the incapability of performing correctly in decision-making tasks that have a risk component, such as rewards of variable value. This cognitive impairment is known to occur after amygdalar or orbitofrontal lesions, where individuals are incapable of long-term planning and take high-risk decisions even if they lead to overall losses. It was recently shown that chronic pain patients also present this pattern of impaired decision-making (Apkarian et al, Pain, 108:129, 2004). However, no studies in chronic pain animal models have addressed poor performance in frontal-based cognitive tasks. For this reason we developed a novel behavioural task based on repetitive reward-based simple decisions, and studied its performance by both control, frontal-lesioned, and chronic pain animals (n = 6 per group). The task consisted on consecutive trials in which a rat entered an operant chamber and had to choose between two levers to recover a food reward. After each trial, the animal was removed to a contiguous chamber where he waited for a sound signal to begin a new trial. During the 15 days of the training phase both levers gave equal pseudo-random rewards: one food pellet in 8 of 10 presses, and no reward in the other two \u2013 low risk. In the trial probe one of the levers was modified to give 3 food pellets, but only in 3 of 10 visits \u2013 high risk. The pattern of 120 consecutive choices was used to calculate the lever-choice index (low risk minus high risk, divided by number of completed trials). In the first 60 trials all the animals (controls, lesioned and monoarthritic) reverently choose the large reward lever, but control animals reversed the pattern of choice in the second half of the session. When analyzing the last 30 entries, controls had a choice index of +0.42 \u00b1 0.17, while monoarthritic rats had -0.48 \u00b1 0.14, neuropathic rat had -0.53 \u00b1 0.21 and frontal-lesioned animals -0.58 \u00b1 0.31. We have shown for the first time that chronic pain induces complex changes in the cognitive neural processes that handle immediate decision-making in the rat (Support: FCT-POCI\/55811\/2004).\n\n\u2022 **Wen-Biao Gan (Skirball Institute, New York University School of Medicine, New York, USA) \u2013 Dendritic Spine Stability And Its Modification By Experience**\n\nThe nervous system requires not only synaptic plasticity for learning but also stability for long-term information storage. To study the degree of synaptic structural plasticity in intact animals, we developed a transcranial two-photon imaging technique to follow individual postsynaptic dendritic spines over time in transgenic mice over-expressing Yellow Fluorescent Protein. Using this technique, we found that in young adolescent mice (1-month-old), 13\u201320% of spines were eliminated and 5\u20138% were formed over 2 weeks in visual, barrel, motor and frontal cortices, indicating a cortical-wide loss of spines during this developmental period. In adult mice (4\u20136 months old), 3\u20135% of spines were eliminated and formed over 2\u20134 weeks in various cortical regions. When imaged over 19 months, only 26% of adult spines were eliminated and 19% were formed in barrel cortex. Thus, after a concurrent reduction in the number of spines in the diverse regions of young adolescent cortex, spines become remarkably stable and a majority of them can last throughout life.\n\nTo determine how spine dynamics are modified by experience, we examine the effect of long-term sensory deprivation via whisker trimming on dendritic spines in the barrel cortex. During young adolescence when a substantial net loss of spines occurs, we found that whisker trimming preferentially reduces the rate of on-going spine elimination than spine formation. This effect of deprivation diminishes as animals mature but still persists in adulthood. In addition, restoring sensory experience following adolescent deprivation accelerates spine elimination but has no significant effect on spine formation. The rate of spine elimination also decreases after chronic blockade of NMDA receptors with the antagonist MK801 and accelerates after drug withdrawal. These studies underscore the important role of sensory experience in spine elimination over the majority of an animal's life span, particularly during adolescence.\n\n\u2022 **Jianguo Gu (Department of Oral and Maxillofacial Surgery, University of Florida, Gainesville, USA) \u2013 TRPM8 and cold allodynia**\n\nPeripheral nerve injury often results in neuropathic pain manifested with both mechanical and thermal allodynia. Thermal allodynia of neuropathic pain conditions includes cold- and heat allodynia. While TRPV1 is found to be involved in heat allodynia, molecular mechanisms of cold allodynia remain unclear. Recently, transient receptor potential channel M8 (TRPM8 receptor) is found to be a cold- and menthol-sensing receptors expressed on a subpopulation of primary afferent fibers. Here we report the upregulation of TRPM8 expression on nociceptive-like afferent neurons following chronic constrictive nerve injury (CCI) rats that manifested with cold allodynia. We found not only the number of TRPM8-expressing neurons was increased, but also the responsiveness to cold and menthol became enhanced in these afferent neurons following CCI. These results suggest TRPM8 upregulation is associated with cold allodynia and may be an underlying mechanism of cold allodynia\n\n\u2022 **Kazuhide Inoue (Department of Pharmaceutical Health Care and Sciences, Kyushu University, Kyushu, Japan) \u2013 P2X4: mechanisms of over expression in neuropathic pain state**\n\nThere is abundant evidence that extracellular ATP and other nucleotides have an important role in pain signaling at both the periphery and in the CNS. Recent findings suggest that endogenous ATP and its receptor system might be involved in neuropathic pain. Neuropathic pain is often a consequence of nerve injury through surgery, bone compression, diabetes or infection. This type of pain can be so severe that even light touching can be intensely painful; unfortunately, this state is generally resistant to currently available treatments. We recently reported that the expression of P2X4 receptors in the spinal cord is enhanced in spinal microglia after peripheral nerve injury, and blocking pharmacologically and suppressing molecularly P2X4 receptors produce a reduction of the neuropathic pain behaviour (Nature 424,778\u2013783, 2003), and that brain-derived neurotrophic factor (BDNF) released from microglia by the stimulation of P2X4 causes the depolarizing shift in reversal potential of anion in LI neurons of rats with nerve injury (Nature, 438, 1017\u20131021, 2005), resulting in causing neuropathic pain. Understanding the key roles of these ATP receptors may lead to new strategies for controlling the pain.\n\n\u2022 **John Isaac (NINDS, NIH, Bethesda, USA) \u2013 Rapid, Activity-Dependent Plasticity in Timing Precision in Neonatal Barrel Cortex**\n\nDuring development neuronal networks acquire the ability to precisely time events. This is a critical developmental step since precise timing is required for information processing and plasticity in the adult brain. Despite this it is not known what process drives this maturation in timing. I will present recent work from my laboratory showing that long-term potentiation (LTP) induced at thalamocortical synapses in neonatal layer IV barrel cortex produces a rapid and dramatic improvement in input and output timing precision. LTP reduces the latency and variability of synaptically-evoked action potentials and reduces co-incidence detection for synaptic input. In contrast, LTP has only a small and variable effect on synaptic efficacy. This improvement in timing occurs during development, suggesting this process occurs in vivo in the developing barrel cortex. Thus, rather than increasing synaptic efficacy, the primary role of this form of neonatal LTP is to enable neurons to precisely time events.\n\n\u2022 **Koichi Iwata (Department of Physiology, Nihon University, Tokyo, Japan) \u2013 Anterior cingulate cortex and pain -its morphological feature and functional properties**\n\nIt is well known that the anterior cingulate cortex (ACC) has a variety of functions related to pain including pain perception. Many ACC neurons respond to noxious and non-noxious stimulation of the body. Most of these neurons have a large receptive field and increase their firing frequency as stimulus intensity increases. ACC nociceptive neurons have very specific morphological features, such as a small soma and a large number of spines on the dendritic trees, and axon collaterals spreading over a wide area of the ACC. In a retrograde trans-synaptic tracing study, we found that ACC neurons receive predominantly A-delta afferents inputs. We also analyzed the responses of ACC nociceptive neurons in awake behaving monkeys. A small number of ACC neurons modulated their activity during noxious heating of the facial skin. The neuronal activity was significantly higher when monkeys escaped from a noxious heat stimulus than when the monkeys detected a small change in temperature (T2) above a larger initial shift (T1). No relationship between firing frequency and detection latency of the T2 stimulation was observed. These findings suggest that ACC nociceptive neurons are involved in attention to pain and escape from pain, but not in the sensory-discriminative aspect of pain.\n\n\u2022 **Zhengping Jia (Department of Physiology, University of Toronto, Toronto, Canada) \u2013 Molecular regulation of spine properties and synaptic plasticity**\n\nThe dendritic spine is the major postsynaptic site of excitatory synapse and its changes are linked to synaptic plasticity, memory formation and various forms of mental and neurological disorders. However, the molecular mechanisms that govern spine development and regulation are poorly defined. We take genetic approaches in mice to identify and characterize the molecular signaling processes involved in the regulation of spine formation, spine morphology, and spine and synaptic plasticity. Specifically, we are interested in the signal transduction pathways stimulated by the Rho family small GTPases, key mediators of actin dynamics in response to various external stimuli. Our objective is to define the in vivo function and synaptic regulation of Rho signaling in the context of spine properties, hippocampal long-term potentiation and fear memory formation. The specific roles and the underlying mechanisms of various components required for normal Rho signaling will be discussed.\n\n\u2022 **Bong-Kiun Kaang (Department of Biological Sciences, Seoul National University, Seoul, Korea) \u2013 Role of a novel nucleolar protein ApLLP in synaptic plasticity and memory in Aplysia**\n\nIn Aplysia long-term synaptic plasticity is induced by serotonin (5-HT) or neural activity, and requires gene expression. Here, we demonstrate that ApLLP, a novel nucleolus protein is critically involved in both long-term facilitation (LTF) and behavioral sensitization. Membrane depolarization induced ApLLP expression, which activated ApC\/EBP expression through a direct binding to CRE. LTF was produced by a single pulse of 5-HT 30 min after the membrane depolarization. This LTF was blocked when either ApLLP or ApC\/EBP were blocked by specific antibodies. In contrast, ApLLP overexpression induced LTF in response to a single 5-HT treatment. Simultaneously, a siphon noxious stimulus (SNS) to intact Aplysia induced ApLLP and ApC\/EBP expression, and single tail shock 30 min after SNS transformed short-term sensitization to long-term sensitization of siphon withdrawal reflex. These results suggest that ApLLP is an activity-dependent transcriptional activator that switches short-term facilitation to long-term facilitation\n\n\u2022 **Mikito Kawamata (Department of Anesthesiology, Sapporo Medical University School of Medicine, Sapporo, Japan) \u2013 Genetic variation in response properties of spinal dorsal horn neurons and rostral ventromedial medulla neurons in different mouse strains**\n\nAlthough various methods of analgesia are currently used for persistent pain such as inflammatory pain and neuropathic pain, optimal pain therapy has still not been established. This may be at least in part related to variability of perceived pain among patients. Recent behavioral studies have shown that nociception in the mouse is heritable, which may reflect variable sensitivity to tissue injury-induced pain in humans (Mogil et al., 1996). Noxious information is transmitted through fine myelinated A\u03b4 and unmyelinated C afferents from the periphery to the superficial dorsal horn (SDH), especially to the substantia gelatinosa (SG, lamina II of Rexed) (Light and Perl, 1979). This sensory information is modified and integrated in the SG and consequently regulates the outputs of projection neurons located in lamina I and laminae V-VI (Cervero and Iggo, 1980; Eckert et al., 2003). In addition, the descending inhibitory influences from supraspinal structures, including the rostral ventromedial medulla (RVM), on SDH neurons are known to be modified under certain pathological conditions (Basbaum, 1973; Dubuisson and Wall, 1980; Laird and Cervero, 1990; Sandkuhler et al., 1995; Wall et al., 1999).\n\nThus, nociceptive network circuits in the central nervous system, including the SDH and RVM, may play an important role in different pain sensitivity in individuals. Our hypothesis is that SDH neurons and RVM neurons in different mouse strains may show different response properties following tissue injury and different sensitivity to analgesics depending on different genetic background. In order to prove this hypothesis, *in vivo* extracellular recordings and *in vivo* whole-cell patch-clamp recordings were made from SDH neurons located in deep laminae (laminae V-VI) and from superficial SDH neurons located in lamina II, respectively, in different strains of mice (A\/J, C57BL\/6J, and CBA\/J strains) before and after tissue injury induced by surgical incision and formalin injection according to previously described methods (Furue et al., 1999; Kawamata et al, 2005). In a separate study, single neuronal activity was isolated from different types of RVM neurons such as ON cells, OFF cells and NEURTRAL cells, and response properties of these neurons were determined before and after intraventricular injection of DAMGO or surgical injury.\n\nThe results have shown that different mouse strains have different sensitivities to postoperative pain and formalin-induced pain, reflecting different characteristics of SDH neurons in the strains following surgical incision and application of formalin. Responses of RVM neurons were also different in different mouse strains following surgical injury and that different mouse strains have different sensitivities to morphine application. The results suggest that pain intensity and pain mechanisms depend at least in part on genetic background of the individual. Furthermore, mechanisms of pain seen in a clinical setting may thus differ in individuals depending on the response properties of SDH neurons and RVM neurons.\n\n\u2022 **Satoshi Kida (Department of Agricultural Chemistry, Tokyo University of Agriculture, Tokyo, Japan) \u2013 Mechanism of interaction between reconsolidation and extinction of contextual fear memory**\n\nRetrieval of conditioned fear memory initiates two potentially dissociable but opposite processes; reconsolidation and extinction. Reconsolidation acts to stabilize, whereas extinction tends to weaken the expression of the original fear memory. To understand the mechanisms for the regulation of memory stability after the retrieval, we have investigated the relationship between reconsolidation and extinction using contextual fear conditioning, associative learning between context (conditioned stimulus; CS) and fear (unconditioned stimulus; US). We first examined effects of duration of re-exposure to CS on memory reconsolidation and extinction. Protein synthesis inhibition following short re-exposure (3 min) to CS disrupted the contextual fear memory, indicating short re-exposure induces memory reconsolidation. In contrast, protein synthesis inhibition following long-re-exposure (30 min) blocked memory extinction. Importantly, in extinction phases, contextual fear memory was intact even though protein synthesis was inhibited. Therefore, these observations suggested the interaction between memory reconsolidation and extinction phases. Indeed, memory extinction seems to be associated with regulation of fear memory stability after retrieval.\n\nTo further understand how extinction phase interacts with reconsolidation phase, we assume that molecules functioning on one phase should also function on another phase if interaction between reconsolidation and extinction phases is observed at the molecular level. Therefore, we compared molecular signatures of these processes using pharmacology and mouse genetics. Pharmacological experiments using antagonists for cannabinoid receptor 1 (CB1) and L-type voltage-gated calcium channels (LVGCCs), that play essential roles in memory extinction, indicated that both CB1 and LVGCCs are required for memory extinction but not consolidation and reconsolidation. More importantly, double injection of anisomycin and antagonists for either CB1 or LVGCCs prevents the disruption of the original memory by protein synthesis. These results suggest that CB1 and LVGCCs are required for not only memory extinction but also the destabilization of reactivated memory. We are now trying similar experiments using conditional CREB mutant mice.\n\nIn addition, to compare the brain regions associated with reconsolidation and extinction, we analyzed brain regions showing increase in CREB activity in reconsolidation and extinction phases by immunocytochemistry. We observed increase in phosphorylated CREB at serine 133 in amygdala and hippocampus following short re-exposure to CS inducing memory reconsolidation and in amygdala and prefrontal cortex following long re-exposure to CS inducing memory extinction. These observations suggest that acquisition of memory extinction prevent the activation of hippocampus, resulting in preservation of contextual fear memory.\n\nTaken together, these our findings indicate the interaction between memory extinction and regulation of memory stability at the molecular, anatomical and behavioral level. Further understanding the mechanisms of this interaction might make more clearly understand the significance of memory reconsolidation.\n\n\u2022 **Eric Klann (Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, USA) \u2013 Translational Control During Hippocampal Synaptic Plasticity and Memory**\n\nAltered gene expression is a hallmark of long-lasting synaptic plasticity and long-term memory. Regulation of local protein translation permits synapses to control synaptic efficacy independently of mRNA synthesis in the cell body. Recent studies, including several from this laboratory, have identified biochemical signaling cascades that couple neurotransmitter and neurotrophin receptors to the translation regulatory machinery in translation-dependent forms of synaptic plasticity and memory. In this presentation, these translation regulatory mechanisms and the signaling pathways that govern the expression of various forms of translation-dependent synaptic plasticity and memory will be discussed. In addition, synaptic plasticity and memory deficits in genetically engineered mice that lack specific translation factors and translation regulatory proteins will be discussed. These studies have revealed interesting links among the biochemical activities of translation factors, synaptic plasticity, and memory that are likely to be important for other forms of plasticity and behavior, such as those that underlie pain and drug addiction.\n\n\u2022 **Tatsuro Kohno (Division of Anesthesiology, Niigata University, Niigata, Japan) \u2013 Different actions of opioid and cannabinoid receptor agonists in neuropathic pain**\n\nPeripheral nerve injury causes neuropathic pain, which is characterized by hyperalgesia and allodynia to mechanical and thermal stimuli. Neuropathic pain has traditionally been considered opioid-resistant to intrathecal opioids; however, the efficacy of opioid in treating neuropathic pain is controversial. In contrast, increasing evidence indicates that cannabinoids are effective in alleviating neuropathic pain. We evaluated the effect of opioids and cannabinoids in two independent partial peripheral nerve injury models, the spared nerve injury (SNI) and the spinal nerve ligation (SNL) models. In both the SNI and SNL rat peripheral neuropathic pain models the presynaptic inhibitory effect of the \u03bc opioid receptor (MOR) agonist (DAMGO) on primary afferent-evoked excitatory postsynaptic currents (EPSCs) and miniature EPSCs in superficial dorsal horn neurons is substantially reduced, but only in those spinal cord segments innervated by injured primary afferents. The two nerve injury models also reduce the postsynaptic potassium channel opening action of DAMGO on lamina II spinal cord neurons, but again only in segments receiving injured afferent input. The inhibitory action of DAMGO on ERK (extracellular signal-regulated kinase) activation in dorsal horn neurons is also reduced in affected segments following nerve injury. MOR expression decreases substantially in injured dorsal root ganglion neurons (DRG), while intact neighboring DRGs are unaffected. In contrast to MOR agonist, the selective CB1 receptor agonist (ACEA) still suppressed C-fiber-induced ERK activation in dorsal horn neurons in injured spinal cord segments from SNL rats. These studies suggest that opioids may reduce sensitivity in those patients whose pain is generated mainly from injured nociceptor discharge. However, opioid may still be able to suppress neuropathic pain via acting on intact primary afferents or via supraspinal mechanisms. Because the efficacy of cannabinoid in suppressing C-fiber-induced ERK expression fully remains in the injured spinal segments after nerve ligation, our results support an undiminished potency of cannabinoid in attenuating neuropathic pain. Our data also suggest that there might be different regulatory mechanisms of opioids and cannabinoids for neuropathic pain.\n\n\u2022 **Min Li (Department of Neuroscience and High Throughput Biology Center, Johns Hopkins University, Baltimore, USA) \u2013 Chemical regulation of membrane excitability**\n\nBiological phenomena \u2013 ranging from neuronal action potential, to rhythmic cardiac contraction, to sensory transduction, to hormone secretion \u2013 are ultimately controlled by one class of proteins: the ion channels. Changes of ion channel activities by genetic mutations or by drugs are causes for human diseases and basis for therapeutics. Potassium channels are critical to a variety of biological processes and represent a very large class of ion channel proteins permeable to potassium ions. Of the more than 400 ion channel genes in the human genome, at least 167 are annotated potassium channels.\n\nThe regulation and biogenesis of potassium channels are important processes essential to the understanding of their physiological roles. Recent evidence indicates that cardiotoxicity of many human drugs for other intended targets is caused by inhibition of a subset of potassium channels through different mechanisms. These drugs are both chemically stable and economically available. Therefore, they represent useful chemical probes to investigate potassium channel regulation both at the molecular level and at the cell biological level. Using a combination of high throughput chemical biology approaches and detailed biochemical and electrophysiological analyses, we have screened and identified a number of regulatory compounds with unique mechanisms of action in regulating potassium channels.\n\n\u2022 **Xiao-Jiang Li (Department of Human Genetics, Emory University, Atlanta, USA) \u2013 Synaptic toxicity of Huntington disease protein**\n\nHuntington's disease (HD) is characterized by the selective loss of striatal projection neurons. In early stages of HD, neurodegeneration preferentially occurs in the lateral globus pallidus (LGP) and substantia nigra (SN), two regions where the axons of striatal neurons terminate. The unique neuronal structure, which is characterized by numerous neuronal processes that interact with each other at their terminals, may confer the preferential vulnerability to expanded polyQ proteins. In HD mice that precisely and genetically mimic the expression of full-length mutant huntingtin (htt) in HD patients, we found that degraded N-terminal fragments of htt preferentially forms aggregates in striatal neurons that are most affected in HD. More importantly, neuropil aggregates form preferentially in the processes of striatal neurons. In HD transgenic mice that express N-terminal mutant htt, the progressive formation of these neuropil aggregates correlates with disease progression. We also observed degenerated axons in which htt aggregates were associated with dark, swollen organelles that resemble degenerated mitochondria. These findings suggest that the early neuropathology of HD originates from axonal dysfunction and degeneration associated with htt neuropil aggregates.\n\n\u2022 **John F MacDonald (Department of Physiology, University of Toronto, Toronto, Canada) \u2013 Inhibitory Regulation of the Src Hub and LTP in CA1 Hippocampal Neurons**\n\nThe induction of long-term potentiation (LTP) at CA1 synapses of the hippocampus requires an influx of Ca^2+^ via N-methyl-d-aspartate receptors (NMDARs). High frequency stimulation depolarizes CA1 neurons, relieving the voltage-dependent block of NMDARs by Mg^2+^, permitting the entry of Ca^2+^ that is critical for this induction. Thus, NMDARs serve as co-incident detectors of the LTP-inducing afferent input to CA1 neurons. Enhanced activation of the non-receptor tyrosine kinase Src is also required for this co-incidence function; and, Src is the convergent target of a variety of G-protein coupled receptors (GPCRs) of the G\u03b1 q family (e.g. LPA, muscarinic, mGluR5 and PACAP receptors). These GPCRs stimulate a Src-dependent upregulation of NMDARs via a sequential activation of PKC and the non-receptor tyrosine kinase, Pyk2 which is also required for induction of LTP. Src therefore acts as a hub for the regulation of the induction of LTP at CA1 synapses.\n\nSignaling pathways which inhibit Src, and thereby inhibit the induction of LTP, have not been extensively studied. We have previously shown that platelet-derived growth factor receptors (PDGFR\u03b2) inhibit NMDARs in CA1 neurons by a PKA-dependent but Src-permissive mechanism. For example, in inside-out patches from cultured hippocampal neurons PKA fails to inhibit NMDAR channel activity unless it is first enhanced with a Src-activator peptide. Furthermore, we show that in hippocampal slices PDGFBB (receptor ligand) inhibits the induction of LTP. The initial step in this pathway requires tyrosine phosphorylation of tyrosine 1021 of the PDGFR which forms a SH2 docking site for PLC\u03b3. PLC\u03b3 interacts with another non-receptor kinase, Abelson kinase (Abl) which among other activities regulates PDGFR activity via a biochemical feedback. In recordings from single isolated CA1 pyramidal neurons we show that intracellular applications of Abl kinase strongly inhibit currents evoked by applications of NMDA. This inhibition is reversibly blocked by extracellular applications the PDGFR antagonist gleevec demonstrating the dependency of this response on PDGFR activity. How PDGFR, PLC\u03b3 and Abl kinase activity translates into inhibition of NMDARs is not fully understood and is currently under investigation.\n\n\u2022 **Karim Nader (Department of Psychology, McGill University, Montreal, Canada) \u2013 Identifying the neural mechanisms by which boundary conditions inhibit reconsolidation from occurring.**\n\nAlthough memory reconsolidation has been demonstrated in various learning tasks and animal models suggesting it is a fundamental process, reports of boundary conditions imply that reconsolidation is not ubiquitous. These boundary conditions, however, remain poorly defined at the behavioral, systems and molecular levels. Attempting to ameliorate this situation, we characterized reconsolidation of strong memories across all three levels of analysis. At the behavioral level we demonstrated that this boundary condition is transient, as infusions of anisomycin into lateral and basal amygdala in rats did not impair reconsolidation of overtrained auditory fear memories after 2 or 7 days, but did so after 30 or 60 days after training. At the systems level we showed that the hippocampus imposes the boundary condition on the amygdala, as the overtrained memory underwent reconsolidation 2 days after training in animals with pretraining dorsal hippocampus lesions. At the molecular level we demonstrated that the degree of expression of NR2B-containing NMDA receptors in the amygdala modulates reconsolidation of overtrained fear memories, as these receptors, which we previously have identified as being essential for the transformation of a consolidated memory back to a labile state, were down-regulated 2, but not 60 days after overtraining; furthermore, animals with pre-training hippocampus lesions, that did not exhibit the overtraining boundary conditions two days after training, had normal level of expression of NR2B subunits at that time-point. These findings make three conceptual advances in our understanding of reconsolidation: first, boundary conditions can be transient, second, boundary conditions can be imposed by other brain systems, and third, a mechanism mediating the manifestation of boundary conditions is down-regulation of the receptors that are critical for inducing reconsolidation.\n\n\u2022 **Peter V Nguyen (Department of Physiology, University of Alberta, Edmonton, Canada) \u2013 Beta-Adrenergic Receptors Recruit ERK and mTOR to Promote Translation-Dependent Synaptic Plasticity**\n\nA key question in neuroscience research is: How does activation of neuromodulatory receptors initiate protein synthesis during long-term synaptic plasticity? Activation of beta-adrenergic receptors can enhance long-term memory and modulate long-term synaptic plasticity in the mammalian hippocampus. Protein synthesis is required for the persistence of long-term potentiation (LTP) and for the consolidation of long-term memory. However, the intracellular signaling cascades that couple beta-adrenergic receptors to translation initiation and subsequent protein synthesis are unidentified. We used electrophysiological recordings in area CA1 of mouse hippocampal slices to investigate the recruitment of signaling cascades necessary for beta-adrenergic LTP. We found that maintenance of this LTP requires the extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin (mTOR) pathways, but not cAMP-dependent protein kinase (PKA). Consistent with these findings, treatment of hippocampal slices with isoproterenol, a beta-adrenergic agonist, increases phosphorylation of eukaryotic initiation factor 4E (eIF4E), the eIF4E kinase Mnk1, and the translation repressor, 4E-BP2. These translational regulators can be phosphorylated in an ERK- and mTOR-dependent manner. Moreover, activation of beta-adrenergic receptors eliminates deficits in late-LTP seen in transgenic mice that express reduced hippocampal PKA activity. Our results identify specific intracellular signaling pathways that link beta-adrenergic receptor activation at the membrane to translation initiation within the cytosol. More importantly, our data reveal a molecular mechanism for neuromodulatory control of protein synthesis during LTP, a process that is required for the formation of long-lasting memories. \\[Funded by Alberta Heritage Fdn. for Med. Res. and CIHR, NIH, NIMH, and the Fragile X Research Fdn\\].\n\n\u2022 **Uhtaek Oh (Sensory Research Center, Seoul National University, Seoul, Korea) \u2013 TRPV1 and its Role for Inflammatory Pain**\n\nCapsaicin (CAP) is a pungent ingredient in hot peppers. CAP has a unique action on the pain sensory system. CAP causes a pain when applied to the skin. The hyperalgesic action of CAP is mediated by the excitation of sensory neurons. CAP is known to activate ion channels that allow cation influxes, thus, depolarizing sensory neurons. CAP-activated ion channel along with its channel property was identified. The channel is a ligand-gated channel and permeable to various cations. The gene encoding for the CAP sensitive current was cloned and dubbed as VR1 (vanilloid receptor 1). Primary structure of VR1 shows that VR1 belongs to transient receptor potential (TRP) channel family, having 6 transmembrane domains with two long cytosolic amino acid sequences in each N- or C- terminus. According to the recently classified nomenclature, VR1 is now classified as TRPV1. Mice deficient of TRPV1 lacks thermal pain induced by inflammation. Thus, TRPV1 is most likely involved in the mediation of inflammatory pain. In the present symposium, I would like to introduce our research on TRV1, most notably, presenting evidence for the involvement of TRPV1 in mediating inflammatory pain signaling pathways.\n\nThe presence of TRPV1 receptor and its apparent role in pain suggests endogenous activator. Thus, endogenous activators of TRPV1 were searched. In our previous report, the hyperalgesic neural response such as c-fos expression in the dorsal horn of the spinal cord induced by inflammation is blocked by capsazepine, a CAP receptor blocker, suggesting that an endogenous capsaicin-like substance is produced and causes hyperalgesia by opening capsaicin-activated channels. Because ligands bind from the intracellular side of the channel, the endogenous ligands would likely be produced in the cell. We initially tested many intracellular messengers on the CAP channel to determine whether they activate the channel. We found that products of lipoxygenases (LO) are capable of activating the channel. Interestingly, products of LOs are implicated in mediating inflammatory nociception because various LO products are produced during inflammation and cause hyperalgesia when injected intradermally. In addition, products of LOs often function as intracellular messengers in neurons. Among their actions, products of LOs act directly on K+ channels in Aplysia sensory neurons (Piomellile et al., 1987) and mammalian cardiac muscle cells.\n\nIn the present seminar, we present evidence that products of LOs directly activate the CAP receptor in isolated membrane patches of sensory neurons. When applied to the bath of inside-out patches, 12-hydroperoxytetraenoic acid (12-HPETE) activates single-channel currents that were sensitive to capsazepine in isolated membrane patches. The IV curve of single-channel currents activated by 12-HPETE is outwardly-rectifying and identical to that obtained by CAP. The amplitude of single-channel currents activated by both 12-HPETE and CAP are not different. Theses results indicate that the channel currents activated by 12-HPETE are identical to those activated by CAP. The channels activated by 12-HPETE are permeable to various cations. LO products also activate TRPV1, the cloned CAP receptor, expressed HEK293 cells. Products of LOs other than 12-HPETE also activated the CAP channels. Among them, 12- and 15-HPETE, 5- and 15-(S)-hydroxyeicosatetraenoic acids, and leukotriene B4 possess the highest potency. Dose-response relationships reveal that the potencies of 12-HPETE, 15-HPETE, leukotrien B4, and 5-HETE are 8.0, 8.7, 9.2, and 11.7 \u03bcM, respectively, showing much lower potency than CAP. Anandamide, the endogenous ligand for cannabinoid receptors also activates the channel with half-maximal dose of 11.7 \u03bcM. Because prostaglandins (PGs) are known to be related to pain, various PGs are applied to the CAP receptors. PGs, however, fail to activate the channel. Other saturated or unsaturated fatty acids are also tested for its activation of CAP channels. They all fail to activate the channels.\n\nResults of our study indicate that CAP and various eicosanoids act on the capsaicin receptor, suggesting a structural similarity between CAP and eicosanoids. Thus, structures of eicosanoids and CAP in the energy-minimized state are superimposed to compare three-dimensional structures. Three-dimensional structures of 12-(S)-HPETE, 15-(S)-HPETE, 5-(S)-HETE, and LTB4 are compared with that of CAP. Interestingly, CAP in the energy-minimized state fits well to the S-shaped 12-HPETE. In particular, the phenolic hydroxide and amide moieties in CAP overlap precisely with the carboxylic acid and hydroperoxide moieties in 12-HPETE, respectively. The two key regions in CAP or 12-(S)-HPETE are known to have dipolar property that allows hydrogen bond interactions with the CAP receptor. In addition, the aliphatic chain region of the 12-(S)-HPETE fits well with the alkyl chain of CAP. In contrast, 15-HPETE, 5-HETE and LTB4, shared less structural similarity with CAP.\n\nBecause products of LO activate the channel, it seems obvious to ask what stimulates the LO\/TRPV1 pathway in order to cause pain. Although bradykinin (BK) is a powerful pain causing inflammatory mediator, but its activation mechanism of sensory neurons is not known. Because BK releases arachidonic acid, a key substrate for LO in sensory neurons, we hypothesized that BK activates TRPV1 via the PLA2\/LO pathway. In order to prove the hypothesis, we performed electrophysiological experiments, Ca2+-imaging, and chemical analysis of LO products. As a result, we observed that BK-evoked whole-cell currents recorded from sensory neurons were significantly reduced by capsazepine (CZP), a capsaicin receptor antagonist. In the skin nerve preparation, CZP and quinacrine, a PLA2 inhibitor, and NDGA, a LO inhibitor reduced BK-induced excitation of sensory nerves. In addition, quinacrine, NDGA and CZP blocked BK-induced Ca2+-influx. To examine if sensory neurons can, in fact, release the lipid products of LO by BK, we used HPLC-coupled with radioisotope to detect the lipid products. As results, we confirmed that 12-HETE, an immediate downstream metabolite of 12-HPETE was indeed released from sensory neurons after the BK application.\n\nIn addition, we also present unequivocal evidence that histamine, another inflammatory mediator, also uses the PLA2\/LO\/TRPV1 pathway for excitation of sensory neurons. Application of histamine caused influx of Ca2+, which was blocked by co-application of capsazepine or Ca2+-free condition. The Ca2+-influx induced by histamine was blocked by application of capsazepine. Likewise, the Ca2+-influx induced by histamine was also blocked by treatment of NDGA or quinacrine. Thus, these results now suggest that histamine activates TRPV1 by stimulation of PLA2 and LO. Because histamine is a major pruritogenic (itch causing) substance, identification of the histamine signaling pathway is much helpful to developing anti-pruritogenic substance to cure itch sensation in atopic dermatitis patients.\n\nThis study demonstrates that bradykinin and histamine excite sensory nerve endings by activating TRPV1 via production of 12-LO metabolites of arachidonic acid by activated PLA2. This finding identifies a mechanism that might be targeted in the development of new therapeutic strategies for the treatment of inflammatory pain or itch.\n\n\u2022 **Ke Ren (Department of Biomedical Sciences, University of Maryland, Baltimore, USA) \u2013 Neuronal\/glial cell interactions in CNS plasticity and persistent pain**\n\nNerve signals arising from sites of tissue injury lead to long-term changes in the central nervous system (CNS) referred to as central sensitization. Ample evidence indicates that central sensitization underlies mechanisms of persistent pain after injury. The emerging literature strongly implicates a role of neuronal\/glial cell interaction in central sensitization and hyperalgesia. Through still unknown mechanisms, glia can be activated after injury and release chemical mediators such as inflammatory cytokines that modulate neuronal activity and synaptic strength. Such glia-cytokine-neuron interactions may be critical in the chronic pain process. We tested this hypothesis in a rat model of synaptic plasticity and persistent pain. Tissue injury was produced by injecting complete Freund's adjuvant (CFA), an inflammatory agent, into the masseter muscle of the Sprague-Dawley rat. We first examined whether masseter inflammation induced glial activation in the spinal trigeminal complex (STC), the initial relay site for trigeminal nociceptive information. The results showed that masseter inflammation induced a selective and time-dependent increase in glial fibrillary acidic proteins (GFAP) levels, an indication of astroglial activation, in the STC. We next examined whether activation of glia by masseter inflammation is accompanied by an increase in inflammatory cytokine levels. Using Western blot and immunohistochemistry, an increase in IL-1beta in the STC was observed after masseter inflammation. The increase in IL-1beta was seen as early as 30 min after inflammation and lasted for about a week. Interestingly, the CFA-induced IL-1beta selectively colocalizes with GFAP, but not with NeuN, a neuronal marker, and CD11b, a marker of activated microglia. These results suggest that activated astrocytes are the source of IL-1beta release in the STC after masseter inflammation. To demonstrate the association of inflammation-induced cytokine release with glial activation, we tested the effect of propentofylline, a non-selective modulator of glia, on changes in GFAP and IL-1beta levels after masseter inflammation. Western blots showed that the propentofylline treatment blocked the increase in GFAP and IL-1beta after masseter CFA. We further showed that the increase in GFAP after masseter inflammation was blocked by local anesthesia of the injured site, suggesting its dependence on neuronal input. Interestingly, in a medullary slice preparation, substance P, a transmitter released from primary afferent terminals in the STC, induced an increase in GFAP and IL-1beta. These results are consistent with a role of neuronal signaling in triggering CNS glial activation. Finally, we tested the hypotheses that trigeminal glial activation and inflammatory cytokine release affect or facilitate neuronal plasticity through interactions with neuronal glutamate receptors. We administered IL-1receptor antagonist (ra) intrathecally via osmotic pumps at the level of the obex. The results showed that IL-1ra significantly attenuated behavioral hyperalgesia and blocked an increase in NMDA receptor phosphorylation after masseter inflammation. Our findings support a model of reciprocal neuron-glia interactions in the development of CNS plasticity and persistent pain. The model emphasizes activation of glia by injury-generated neuron input, concomitant cytokine release, and post-translational regulation of NMDA receptor sensitivity through IL-1receptor signaling. The outcome of these studies will help to identify novel targets and agents for clinical management of persistent pain. (Supported by NIH grants DE11964, DE15374, DA10275)\n\n\u2022 **John C Roder (Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Canada) \u2013 Forward and reverse genetic screens in the mouse for mutants impaired in learning and memory**\n\nLearning and memory in the mouse is a quantitative trait and genes account for 71% of the variance between strains. Our goal here is to identify new genes that contribute to learning and memory. We will employ a forward genetic screen using a chemical mutagen (ENU). A total of 2500 ENU mice were pre-screened for normal development. Of these, 10 showed deficits in context-dependent fear conditioning (\\< 2 sd from the mean), but normal cue-dependent freezing. A smaller screen was done on 100 mice. 2 showed deficits in performance on the hidden platform but normal performance on the visible platform (control). All these presumed mutants showed low heritability and penetrance and could not be mapped to chromosomal positions. At this point we revised our screen.\n\nA number of strains (n = 10) were compared in the water maze and the one showing optimal performance (129 S61SVE-v Tac) was chosen for ENU mutagenesis. In addition, we changed the screen to a much more difficult task and one that relied on a different sensory modality (sound) in trace conditioning.\n\nUpon screening 450 mice in trace only, one was obtained that showed no freezing. This mutant showed robust inheritance and penetrance and we are in the process of fine mapping, positional cloning and sequencing of the antifreeze locus. Verification of candidate genes will be carried out by BAC rescue of the mutant phenotype with the wildtype gene. Alternatively, the creation of the same mutant phenotype in wildtype mice by mutating the wild-type locus in ES cells.\n\nWe will carry out extensive neurobehavioural assays to determine if the learning and memory deficits are restricted to the hippocampus or are found in other brain regions as well (i.e. pre-frontal cortex, cerebellum, nucleus accumbens, brain stem, amygdala, striatum). Learning and memory mutants will be tested for their ability to form cognitive spatial maps in the hippocampus in vivo. Neuroanatomical studies will assess if development perturbation underlie these deficits. Gene expression and proteomic studies will identify where the gene is expressed and the biochemical pathway underlying its action. Modifier screens will be carried out to elucidate exciting new genetic pathways. The mutant genes we identify will be models for human genetic diseases that involve impairments in learning and memory. In these cases, the mutant mice will provide test beds for pre-clinical tests of cognitive enhancers in patients. In addition, they will suggest new targets for drug development.\n\n\u2022 **Michael W Salter (Department of Physiology, University of Toronto, Toronto, Canada) \u2013 Ins and outs of SRC regulation of NMDA receptors and synaptic plasticity**\n\nRegulation of postsynaptic glutamate receptors is one of the principal mechanisms for producing alterations of synaptic efficacy in the CNS. A growing body of evidence indicates that at glutamatergic synapses NMDA receptors are upregulated by Src family tyrosine kinases which are opposed by the action of tyrosine phosphatases, one of which has been identified as STEP. Src itself is expressed nearly ubiquitously in higher organisms with the highest levels of expression found in the CNS. Src represents a point through which multiple signaling cascades from, for example G-protein-coupled receptors, Eph receptors and integrins, converge to upregulate NMDA receptor activity. The upregulation of NMDARs by activation of Src participates in the induction of long-term potentiation of synaptic transmission in the hippocampus and in the spinal cord dorsal horn. We have determined that Src is anchored within the NMDA receptor complex by the protein ND2. Recently, we have found that interfering with the ND2-Src interaction *in vivo* prevents behavioural pain hypersensitivity. Thus, multiple mechanisms control Src in the NMDA receptor complex and disrupting Src-mediated enhancement of NMDA receptor function affects pathological plasticity in the CNS.\n\n\u2022 **Weihong Song (Department of Psychiatry, University of British Columbia, Vancouver, Canada) \u2013 Hypoxia facilitates Alzheimer's disease pathogenesis**\n\nThe molecular mechanism underlying the pathogenesis of majority of sporadic Alzheimer's disease (AD) cases is unknown. A history of stroke was found to be associated with development of some AD cases, especially in the presence of vascular risk factors. A reduced cerebral perfusion is a common vascular component among AD risk factors. Hypoxia is a direct consequence of hypoperfusion. We identified a functional hypoxia responsive element (HRE) in BACE1 promoter. Hypoxia increased APP CTF\u03b2 production by increasing BACE1 gene transcription and expression in *vitro* and in *vivo*. This paper showed that hypoxia facilitated AD pathogenesis. Under hypoxic condition APP23 mice, Swedish mutant APP transgenic mice, developed more neuritic plaques than normoxic mice. We found that hypoxia deteriorated the memory impairment in APP23 mice. Our results demonstrate that hypoxia facilitates AD pathogenesis and interventions that improve cerebral perfusion might benefit AD patients.\n\n\u2022 **Shuzo Sugita (Department of Physiology, University of Toronto, Toronto, Canada) \u2013 Molecular mechanism of GTP-dependent exocytosis**\n\nMany secretory cells utilize a GTP-dependent pathway to trigger exocytotic secretion. However, little is currently known about the mechanism by which this may occur. In the present study we attempted to identify the key signaling pathway that mediates GTP-dependent exocytosis. Incubation of permeabilized PC12 cells with soluble RalA GTPase strongly inhibited GTP-dependent exocytosis. A Ral-binding fragment from Sec5, a component of the exocyst complex, showed a similar inhibition. Point mutations in both RalA (RalA^E38R^) and the Sec5 (Sec5^T11A^) fragment which abolish the RalA-Sec5 interaction also abolished the inhibition of GTP-dependent exocytosis. In contrast the RalA and the Sec5 fragment showed no inhibition of Ca^2+^-dependent exocytosis, but cleavage of a SNARE (soluble-*N*-ethylmaleimide-sensitive factor attachment protein receptor) protein by Botulinum neurotoxin blocked both GTP- and Ca^2+^-dependent exocytosis. In stable RalA and RalB double knockdown cells, GTP-dependent exocytosis was severely reduced and was restored upon reintroducing expression of RalA or RalB by transfection. However, Ca^2+^-dependent exocytosis remained unchanged in the double-knockdown cells. Our results indicate that GTP- and Ca^2+^-dependent exocytosis use different sensors and effectors for triggering exocytosis while their final fusion steps are both SNARE-dependent. They also suggest that endogenous RalA and RalB function specifically as GTP sensors for the GTP-dependent exocytosis.\n\n\u2022 **Shao-Jun Tang (Department of Neurobiology and Behavior, University of California, Irvine, USA) \u2013 Regulation of Activity-Dependent Protein Synthesis in Dendrites**\n\nProtein synthesis in dendrites is essential for long-lasting synaptic plasticity, but little is known about how synaptic activity is coupled to mRNA translation. Using hippocampal neuron cultures and slices, we have investigated the role of glutamate receptors and mTOR signaling in control of dendritic protein synthesis. We find: 1) Specific antagonists of NMDA, AMPA and metabotropic glutamate receptors abolish glutamate-induced dendritic protein synthesis, whereas agonists of NMDA and metabotropic but not AMPA glutamate receptors activate protein synthesis in dendrites; 2) Inhibition of mTOR signaling, as well as its upstream activators, PI3K and AKT, block NMDA receptor-dependent dendritic protein synthesis. Conversely, activation of mTOR signaling induces dendritic protein synthesis; and 3) Dendritic protein synthesis activated by tetanus-mediated LTP induction in hippocampal slices requires NMDA receptors and mTOR signaling. These results suggest critical role of the NMDA receptor-mTOR signaling pathway in regulating protein synthesis in dendrites of hippocampal neurons.\n\n\u2022 **Yuanxiang Tao (Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University, Baltimore, USA) \u2013 Are the PDZ domains at excitatory synapses potential molecular targets for prevention and treatment of chronic pain?**\n\nThe PDZ (Postsynaptic density 95, Discs large, and Zonula occludens-1) domains are ubiquitous protein interaction modules often found among multi-protein signaling complexes at excitatory synapses. In the mammalian central nervous system, C-terminal motifs of N-methyl-d-aspartate (NMDA) receptor subunits NR2A and NR2B bind to the first and second PDZ domains of postsynaptic density (PSD)-95, PSD-93, and synaptic-associated protein (SAP)102, whereas C-terminal motifs of \u03b1-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluR2 interacts with the PDZ domain of protein interacting with C-kinase 1 (PICK1) and glutamate receptor interacting protein (GRIP). These PDZ-containing proteins not only are involved in synaptic trafficking of NMDA receptors and AMPA receptors but also couple the receptors to intracellular proteins and signaling enzymes, such as neuronal nitric oxide synthase (nNOS) and protein kinase C (PKC). Recent preclinical research shows that PSD-93, PSD-95, and PICK1 are highly expressed in the dorsal horn of the spinal cord. Immunocytochemical studies demonstrate that their immunoreactivities occur at a higher density in the superficial laminae and at a lower density in other laminae of the spinal dorsal horn. Spinal PSD-93 or PSD-95 deletion prevents NMDA receptor-dependent chronic pain from spinal nerve injury or injection of complete Freund's adjuvant (CFA) without affecting nociceptive responsiveness to acute pain. In addition, the disruption of the PDZ domain-mediated protein interaction between GluR2 and PICK1 in the spinal cord of rats or the knockout of PICK1 in mice has recently been shown to produce antinociceptive effects in AMPA receptor-dependent chronic pain caused by peripheral nerve injury and CFA, with preservation of acute pain transmission. Further studies have demonstrated that PSD-93 or PSD-95 deletion may alter the synaptic NMDA receptor expression and function in spinal cord neurons, which, in turn, may result in impaired NMDA receptor-dependent chronic pain. However, the underlying mechanism by which PICK1 lead to antinociception in chronic pain states is unclear. Our preliminary work indicates that CFA-induced chronic pain might increase time-dependent PKC phosphorylation of GluR2 Ser880, disrupt interaction of GluR2 with GRIP (but not with PICK1), and lead to PKC-dependent internalization of GluR2 (but not of GluR1) in the spinal cord neurons. GluR2 internalization might facilitate Ca2+ permeability and increase AMPAR function and neuronal activity, which may contribute to spinal central sensitization associated with chronic pain states. PICK1 deletion might reduce PKC phosphorylation of GluR2 Ser880 by blocking the recruitment of PKC to synaptic GluR2 and decrease PKC-dependent internalization of GluR2 in spinal cord neurons, which, in turn, might result in blunted AMPA receptor-dependent central sensitization in chronic pain. Therefore, it is very likely that PDZ domains at excitatory synapses may be new molecular targets for prevention and treatment of chronic pain.\n\n\u2022 **Yu Tian Wang (Brain Research Center, University of British Columbia, Vancouver, Canada) \u2013 Synaptic plasticity in learning and memory**\n\nSynaptic plasticity (i.e. a dynamic change in the strength of synaptic transmission between neurons), such as long-term potentiation (LTP) and depression (LTD) observed at the glutamatergic synapses of the CA1 region of the hippocampus, has long been proposed as a primary cellular mechanism for learning and memory. However, evidence for a definitive role of either LTP or LTD in learning and memory remains missing due to the lack of a specific inhibitor for LTP or LTD. Evidence accumulated in recent years strongly suggests that AMPA subtype glutamate receptors (AMPARs) are continuously cycling between the plasma membrane and intracellular compartments via vesicle-mediated plasma membrane insertion and endocytosis, and that facilitated AMPAR insertion and endocytosis at postsynaptic membranes contributes to the expression of LTP and LTD, respectively. Using a combination of recombinant receptor expression systems and hippocampal brain slice preparations, we were able to demonstrate that facilitated endocytosis of postsynaptic AMPARs during LTD is AMPAR GluR2 subunit-specific. These studies have lead us to develop a GluR2-derived interference peptide that, when delivered into neurons in the brain, can specifically block the expression of LTD without affecting normal basal synaptic transmission in many regions of the brain. Using the membrane-permeant form of the GluR2 peptide as a specific inhibitor of LTD, we were able to probe the role of LTD in freely moving rats with unprecedented specificity, and thereby provide evidence for the involvement of LTD in a number of learning and memory-related behaviours. Our work not only provides the first evidence for a definitive role of LTD in learning and memory, but also demonstrates the utility of peptides that disrupt AMPAR trafficking, the final step in the expression of synaptic plasticity, as tools to examine the critical role of LTD and\/or LTP in specific aspects of learning and memory in conscious animals.\n\n\u2022 **Newton Woo (NICHD, NIH, Bethesda, USA) \u2013 Regulation of Bi-directional Plasticity by BDNF**\n\nInitially characterized for its role in neuronal survival and differentiation, *B*rain *D*erived *N*eurotrophic *F*actor (BDNF) has emerged as a key regulator of synaptic plasticity, which is a persistent change in synaptic strength thought to underlie many cognitive functions. A salient characteristic of this ubiquitously expressed neurotrophin is that expression of BDNF is activity dependent, which has profound implications in development and neuronal plasticity. BDNF is synthesized as a precursor (proBDNF) that can undergo proteolytic cleavage to yield mature BDNF (mBDNF). Initially, the biological actions elicited by neurotrophins, including BDNF, were thought to only arise from the processed mature form. However, recent groundbreaking studies have demonstrated distinct biological roles for several proneurotrophins and their mature form via distinct receptor\/signaling cascades. This highlights the importance of the conversion of pro- to mature protein as a key regulatory step in neurotrophin actions. However, whether this proteolytic cleavage plays a role in synaptic plasticity has not been previously addressed. Here, I present evidence that such a conversion process from pro- to mature BDNF is important for one long-lasting form of synaptic plasticity, namely late-phase LTP (L-LTP). Application of strong theta-burst stimulation (TBS) induces L-LTP that is protein synthesis dependent. In BDNF +\/- mice, L-LTP is significantly impaired after TBS simulation. However, L-LTP can be rescued in BDNF +\/- mice when hippocampal slices were preincubated with mBDNF but not proBDNF. Subsequent experiments identified the tPA\/plasminogen system plays a critical role for both BDNF processing and L-LTP expression in the mouse hippocampus. To investigate the location of this conversion process, we performed several additional experiments using cleavage specific antibodies. It was discovered that in cultured hippocampal neurons, low frequency stimulation triggered proBDNF secretion, whereas high frequency stimulation induced the expression of mBDNF. Strikingly, tPA secretion only occurred after high frequency stimulation. Moreover surface staining of mBDNF was greatly enhanced upon depolarization. These results suggest that neuronal activity regulates the ratio of extracellular pro- to mature BDNF via tPA secretion. Finally, I present data that proBDNF facilitates hippocampal long-term depression (LTD). This facilitation of NMDAR LTD is dependent on the p75 neurotrophin receptor (p75^NTR^). Mice that lack p75^NTR^ exhibit a selective impairment in the NMDA-dependent form of long-term depression (LTD), but display normal expression of other hippocampal forms of synaptic plasticity. This selective deficit may be the result of a significant reduction in NR2B, a NMDA receptor subunit uniquely involved in LTD. Activation of p75^NTR^ by proBDNF enhanced hippocampal LTD. Our results challenge the classic view that the processed neurotrophins is the only functional form of neurotrophins to elicit biological actions and that an unexpected function of p75^NTR^ is to regulate the expression of hippocampal synaptic depression. Taken together, these results suggest a universal \"Yin-Yang\" model where pro- and mature- BDNF play diametrically opposite roles in synaptic plasticity.\n\n\u2022 **Melanie A Woodin (Department of Cell and Systems Biology, University of Toronto, Toronto, Canada) \u2013 Bidirectional spike-timing dependent plasticity of inhibitory transmission in the hippocampus**\n\nThe mammalian hippocampus, owing to its crucial role in memory, has been the primary focus of research into synaptic plasticity. Most studies have examined plasticity at excitatory (glutamatergic) synapses, despite the fact that neuronal output is not determined by the level of excitatory transmission alone, but by the levels of coincident excitatory and inhibitory transmission. In this study, we examined spike-timing dependent plasticity of GABAA receptor mediated inhibitory transmission in area CA1 of hippocampal slices from mature rats (6\u20138 weeks). Because the amplitude and reversal potential of GABAR currents are largely determined by intracellular chloride concentration, we first determined the GABAR reversal potential under conditions of intact intracellular chloride using the permeating agent gramicidin. Surprisingly, we found that GABAR reversal potential was \\~12 mV hyperpolarized compared to the reversal potential in a previous study of STDP of GABAR mediated transmission in P12-19 slices, as well as to our own recordings from P12-19 slices. We then performed a series of whole-cell recordings to determine the intacellular chloride concentration necessary to reproduce the GABAAR reversal potential measured with gramicidin. This allowed us to employ long-term, stable whole-cell recording to investigate whether a spike-timing protocol could induce changes in GABAAR reversal potential. Surprisingly, pairing of presynaptic stimulation with postsynaptic spiking led to bidirectional changes in the reversal potential, with the direction of change being dependent on the interval between pre- and post stimulation. When the postsynaptic neuron was made to fire bursts of action potentials 5 ms after presynaptic stimulation (correlated), at 5 Hz for 90 seconds, a depolarization of the reversal potential was seen. However, when the interval was lengthened to 100 ms (uncorrelated), a hyperpolarization of the reversal was seen. Due to the interplay between excitatory and inhibitory transmission, we suggest that this form of GABAergic plasticity may contribute to the enhancement of excitatory transmission under certain conditions.\n\n\u2022 **Zhen Yan (Department of Physiology and Biophysics, State University of New York at Buffalo, Buffalo, USA) \u2013 Interactions between Acetylcholine, Amyloid and Ion Channels in Alzheimer's Disease**\n\nIt has been well recognized that one prominent feature of Alzheimer's disease (AD) is the accumulation of b-amyloid (Ab), a major component of senile plaques. Another fundamental feature of AD is the severe degeneration of basal forebrain (BF) cholinergic neurons and deficient cholinergic functions in prefrontal cortex (PFC), a brain region implicated in high-level cognitive processes. We have found that cholinergic inputs from BF, by activating M1 muscarinic receptors in PFC pyramidal neurons, regulate the GABAA receptor channel, a key player in working memory, through a PKC\/Src-dependent mechanism. The M1 regulation of GABA transmission in PFC is impaired in the APP transgenic model of AD, due to the Ab interference with M1 activation of PKC. On the other hand, glutamate inputs from PFC, by activating Group III metabotropic glutamate receptors (mGluRIII) in BF neurons, suppresses NMDAR currents through an actin-dependent mechanism. Ab selectively disrupts mGluRIII regulation of NMDAR channels in BF cholinergic neurons, which may due to their sensitivity to Ab-induced cytoskeleton disintegration. Thus, our results have provided a potential mechanism for the synaptic failure of PFC pyramidal neurons and the selective degeneration of BF cholinergic neurons at the early stage of AD.\n\n\u2022 **Megumu Yoshimura (Department of Basic Medicine, Kyushu University, Kyushu, Japan) \u2013 Synaptic mechanisms of acupuncture in the spinal dorsal horn revealed by in vivo patch-clamp recordings**\n\nAccording to Chinese literatures, more than 300 acupoints have been described. Stimulation of each point elicits certain analgesia in the corresponding area. Physiological examinations have been made to unveil the mechanisms of the analgesic action of acupuncture, however, clear results have not been provided, because of difficulty in how to approach the changes of nociceptive transmission in CNS. One of promising approaches will be an in vivo patch-clamp recording from spinal dorsal horn neurons to see what is happening during acupuncture. Thus, we applied the in vivo patch-clamp recordings from substantia gelatinosa neurons that receive noxious inputs and observed a change in excitatory and inhibitory synaptic currents occuring spontaneously or evoked by stimulation of the skin in the receptive field. To enhance the nociceptive inputs from periphery, we used CFA induced inflammatory rats injected right hind paw. In this chronic pain model, spontaneous EPSCs with higher magnitude were observed in the majority of SG neurons. Application of acupuncture to the contralateral ST36 near the knee joint did not affect the spontaneous EPSCs. However, large amplitude of spontaneous IPSCs were elicited with the frequency of 2 to 10 Hz. Next, we tested the cell firing in the SG by stimulation of the skin with toothed forceps during the acupuncture. The skin-evoked spike firing was effectively inhibited reversibly by the acupuncture. In our previous slice experiments, noradrenaline and serotonin increased spontaneous IPSCs with large ampitude. Other possible candidates responsible for the depression of nociceptive inputs to the SG, such as dopamine, enkephalin, other opioids, substance P, CGRP did not increase the frequency and amplitude of spontaneous IPSCs.\n\n\u2022 **Ming Xu (Department of Anesthesia and Critical Care, University of Chicago, Chicago, USA) \u2013 Molecular Mechanisms of neuronal plasticity induced by drugs of abuse**\n\nDrug addiction is a brain disease that is characterized by the compulsive seeking and taking of a drug despite known adverse consequences. Drug addiction is also long-lasting with a high propensity to relapse. The brain dopaminergic system is a key neural substrate for mediating the actions of abused drugs. The development of drug addiction is thought to involve coordinated temporal and spatial actions of specific dopamine receptors, signaling molecules, and target molecules that change synaptic reorganizations in the brain. To dissect mechanisms underlying drug-induced neuroadaptations, we have made and analyzed D1 receptor mutant mice. We found that the D1 receptor mediates the locomotor-stimulating and rewarding effects of cocaine. The D1 receptor also mediates cocaine-induced changes in neuronal dendritic remodeling, ERK and CREB signaling, chromatin remodeling, and gene expression including c-*fos* and AP-1-regulated target genes. These results suggest that the D1 receptor is a major cell surface mediator for drug-induced behaviors and neuroadaptations, and that c-*fos-*regulated gene expression may contribute to the persistent nature of drug-induced behaviors. To investigate intracellular mechanisms of cocaine-induced persistent changes within D1 receptor-expressing neurons, we made D1 receptor neuron-specific c-*fos* mutant mice. We found that c-Fos contributes to the development and extinction of cocaine-induced conditioned place preference and behavioral sensitization, changes in dendritic reorganization, and regulation of immediate early genes and the expression of two classes of target genes that are involved in neurotransmission and neuronal connections. Noticeably, mutations of the D1 receptor gene and c-*fos* share several common consequences after repeated cocaine injections. Together, these findings suggest that the dopamine D1 receptor and c-Fos are a key receptor signaling system that contributes to persistent neuroadaptations to cocaine.\n\n\u2022 **Zao C Xu (Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, USA) \u2013 Synaptic plasticity in pathological conditions**\n\nSynaptic plasticity occurs during development and participates in physiological functions in adulthood during learning and memory. It has also been shown in pathological conditions such as epilepsy. To investigate the synaptic plasticity in neurodegenerative disorders and the underlying mechanisms, synaptic transmission and morphological changes were studied in neurotransplantations after excitotoxic lesion and in neurons after transient cerebral ischemia.\n\nFor transplantation studies, striatal primordium were collected from 16 d embryos and implanted into the striatum of adult Sprague-Dawley rats two days after kainic acid lesion. Intracellular recording in vivo and anterograde tracing experiments were performed 2\u20136 months after the transplantation. For ischemia studies, transient global ischemia was induced in adult Wistar rats. Electrophysiological recording and morphometry analysis of intracellularly stained neurons were performed at different intervals after ischemia.\n\nSpontaneous synaptic activities were greatly reduced in striatal grafts. Cortical or thalamic stimuli elicited monosynaptic excitatory postsynaptic potentials (EPSPs) from neurons in the graft. A late postsynaptic potential (L-PSP) was evoked from many graft neurons (17\/27) in addition to the initial EPSPs. Bursting action potentials were generated from the L-PSPs. Light and electron microscopic studies showed that the number of cortical and thalamic afferent fibers significantly reduced in the grafts. Some of these fibers formed dense clusters of terminals making multiple synapses on individual spines and dendrites. L-PSPs also could be evoked from neurons in the striatum and hippocampus following cerebral ischemia. Furthermore, the initial EPSPs were potentiated in ischemia-vulnerable neurons (spiny neurons in the striatum and CA1neurons in the hippocampus) but depressed or unchanged in ischemia-resistant neurons (large aspiny neurons in the striatum and CA3 neurons in the hippocampus) after ischemia. Quantitative analysis of 3-D reconstructed CA1 pyramidal neurons indicated that the total dendritic length in apical dendrites was significantly increased at 24 h after ischemia but remained about the same in basal dendrites. Such increase was due to the dendritic sprouting rather than dendritic extension, which occurred mainly in the middle segment of the apical dendrites.\n\nThese results demonstrate that the synaptic plasticity changes also occur in acute neurodegenerative disorders. The plasticity changes in striatal grafts might be the compensatory responses, whereas those in ischemic neurons might be associated with the selective neuronal damage after ischemic insults.\n\n\u2022 **Xia Zhang (Department of Psychiatry, University of Saskatchewan, Saskatoon, Canada) \u2013 Cannabinoid addiction and cannabinoid medicine**\n\nA. Suppression of cannabinoid rewarding effects and cannabinoid withdrawal syndrome respectively by the interfering peptide Tat-3L4F and lithium\n\nCannabinoids or marijuana is the most commonly used illicit drug in developed countries. The lifetime prevalence of marijuana dependence is the highest of all illicit drugs in the USA, but there is no effective medication available for treating marijuana addiction. Our recent two studies show potential strategies for treating marijuana addiction in humans. In the first study we found a physical interaction of the enzyme PTEN with a region in the third intracellular loop (3L4F) of 5-HT2C receptor (5-HT2cR) in cell cultures. PTEN limits agonist-induced phosphorylation of 5-HT2cR through its protein phosphatase activity. We then found the probable existence of PTEN:5-HT2cR complexes in putative dopaminergic neurons in the rat ventral tegmental area (VTA), a brain region in which virtually all abused drugs exert rewarding effects by activating its dopamine neurons. We next synthesized the interfering peptide Tat-3L4F, which is able to disrupt PTEN coupling with 5-HT2cR. Tat-3L4F or the 5-HT2cR agonist Ro600175 suppressed the increased firing rate of VTA dopaminergic neurons induced by delta9-tetrahydrocannabinol (THC), the psychoactive ingredient of marijuana. Using behavioral tests, we observed that Tat-3L4F or Ro600175 blocks conditioned place preference of THC, and that Ro600175, but not Tat-3L4F, produces anxiogenic effects, penile erection, hypophagia and motor functional suppression. These results suggest a potential strategy for treating cannabinoid addiction with the Tat-3L4F peptide. In the second study we demonstrate that lithium treatment prevented the cannabinoid withdrawal syndrome (CWS) in rats, which was accompanied by expression of the cellular activation marker Fos in oxytocin-immunoreactive neurons and a significant increase in oxytocin mRNA expression in the hypothalamic paraventricular and supraoptic nuclei. Lithium also significantly increased blood oxytocin levels. We suggest that the effects of lithium against the CWS are mediated by oxytocinergic neuronal activation and subsequent release and action of oxytocin within the CNS. This hypothesis is supported by further findings that the effects of lithium against the CWS were antagonized by an oxytocin antagonist and mimicked by oxytocin. These results led us to conduct a small-scale, pilot clinical study showing positive therapeutic effects of lithium against the CWS in patients with pure cannabinoid addiction.\n\nB. Promotion of hippocampal neurogenesis and suppression of anxiety and depression by cannabinoids. The adult hippocampus contains neural stem\/progenitor cells (NS\/PCs) capable of generating new neurons, i.e., neurogenesis. Most drugs of abuse examined to date decrease adult hippocampal neurogenesis, but the effects of cannabinoids remain unknown. We show that both embryonic and adult rat hippocampal NS\/PCs are immunoreactive for CB1 cannabinoid receptors. We then found that both the synthetic cannabinoid HU210 and an endogenous cannabinoid promote proliferation, but not differentiation, of cultured embryonic hippocampal NS\/PCs likely via a sequential activation of CB1 receptors, Gi\/o proteins, and ERK signaling. Chronic, but not acute, HU210 treatment promoted adult hippocampal neurogenesis and exerted anxiolytic- and antidepressant-like effects. X-irradiation of the hippocampus blocked both the neurogenic and behavioral effects of chronic HU210 treatment, suggesting that chronic HU210 treatment produces anxiolytic- and antidepressant-like effects likely via promotion of hippocampal neurogenesis.\n\n\u2022 **Mei Zhen (Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Canada) \u2013 SAD kinase regulates neuronal polarity and synapse formation**\n\nUsing *C. elegans* GABAergic neurons as a model system, we identified a Ser\/Thr kinase SAD-1 as a key player in establishing axon\/dendrite polarity and synaptic structures. In *C. elegans* the loss of the SAD-1 function leads to the accumulation of synaptic vesicles at dendritic regions of neurites, furthermore synaptic vesicles are loosely clustered at chemical synapses. Using genetic and biochemical approaches, we determined two separate genetic pathways through which SAD-1 kinase functions: During early differentiation of neurons, SAD-1 physically interacts with a scaffolding protein Neurabin to restrict the axonal fate of the developing neurites. After the establishment of axon and dendrites, SAD-1, restricted at presynaptic region by several presynaptic channels, negatively controls the incorporation of active zone proteins at chemical synapses. We are further delineating the activator and downstream effectors of the SAD kinase.\n\n\u2022 **Min Zhuo (Department of Physiology, University of Toronto, Toronto, Canada) \u2013 Cortical potentiation and its roles in persistent pain and fear**\n\nNeuronal synapses in the central nervous systems are plastic, and can undergo long-term changes throughout life. Studies of molecular and cellular mechanisms of such changes not only provide important insight into how we learn and store new knowledge in our brains, but also reveal the mechanisms of pathological changes occurring following a noxious stimulus such as pain and fear. Using integrative approaches including genetic, pharmacological, electrophysiological and behavioral studies, we explore the synaptic mechanisms for LTP and LTD in the cingulate and prefrontal cortex of adult mice. We found that activation of postsynaptic NMDA receptor is required for the induction of synaptic LTP. The expression of cingulate LTP is likely mediated by postsynaptic AMPA receptors, while presynaptic form of paired-pulse facilitation remained unchanged during synaptic potentiation. Activation of calcium-calmodulin stimulated adenylyl cyclase AC1 is required for the induction of LTP. Similar to the hippocampus, NMDA NR2A subtype receptor is required for the induction of LTP. NMDA NR2B receptors, however, also contribute to synaptic potentiation. Genetic reduction of NR2B expression or pharmacological inhibition of NR2B receptor by selective antagonists reduced behavioral contexual fear memory. The possible contribution of the ACC to the formation of fear memory is its role in pain perception. Supporting this hypothesis, inhibition of NMDA NR2NB receptors in the ACC inhibited behavioral sensitization to non-noxious stimuli. Our results provide strong evidence that synaptic potentiation within the cingulate\/prefrontal cortex play important roles physiological and pathological responses to noxious sensory stimuli and injury, including emotional fear and persistent pain.\n\n# Competing interests\n\nThe author(s) declare that they have no competing interests.\n\n# Authors' contributions\n\nEach author provided abstract for the 1st International Conference on Synapse, Memory, Drug Addiction and Pain, as indicated in the manuscript. MZ collected and organized abstracts for publishing. All authors read and approved the final manuscript.\n\n### Acknowledgements\n\nMZ is supported by grants from NINDS NS42722, the Canadian Institutes of Health Research, the EJLB-CIHR Michael Smith Chair in Neurosciences and Mental Health, and the Canada Research Chair.","meta":{"dup_signals":{"dup_doc_count":104,"dup_dump_count":47,"dup_details":{"curated_sources":4,"2023-14":1,"2022-33":1,"2022-21":1,"2021-43":1,"2020-29":1,"2019-51":1,"2019-35":1,"2019-22":1,"2019-13":1,"2019-04":1,"2018-47":1,"2018-39":1,"2018-30":1,"2018-22":1,"2018-13":1,"2017-47":2,"2017-34":2,"2017-26":1,"2017-22":1,"2017-17":1,"2017-09":1,"2016-22":1,"2016-18":1,"2015-48":3,"2015-40":2,"2015-35":3,"2015-32":4,"2015-27":2,"2015-22":4,"2015-14":3,"2014-52":3,"2014-49":4,"2014-42":9,"2014-41":4,"2014-35":4,"2014-23":6,"2014-15":3,"2023-40":1,"2024-10":1,"2017-13":1,"2015-18":3,"2015-11":3,"2015-06":3,"2014-10":2,"2013-48":3,"2013-20":3,"2024-26":1}},"file":"PMC1769477"},"subset":"pubmed_central"}
{"text":"abstract: A report on the Second EMBL\/EMBO Symposium on Functional Genomics: 'Exploring the Edges of Omics', European Molecular Biology Laboratory (EMBL), Heidelberg, Germany, 16-19 October 2004.\nauthor: Gino Poulin; Julie Ahringer\ndate: 2005\ninstitute: 1The Gurdon Institute and Department of Genetics, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK\ntitle: Living on the edge\n\nEMBL's recent symposium on functional genomics showed how this new field has matured. Work from a broad range of model organisms provided new biological insights, and a plethora of improved high-throughput technologies promised more of these in the future. We focus here on biological networks - a major theme of the meeting.\n\n# Network motifs\n\nThe identification of patterns in biological data can uncover mechanisms through which processes are regulated. Uri Alon (Weizmann Institute of Science, Rehovot, Israel) presented evidence that in gene-regulatory networks particular patterns of interconnections (network motifs) are enriched when compared to a randomized network. For example, the three-node feed-forward loop, in which transcription factor X regulates transcription factor Y and both jointly regulate gene Z, is a frequently used network motif; one example is in the L-arabinose utilization system in *Escherichia coli*. Studying this system *in vivo* Alon found that the feed-forward loop is protected against fluctuation of external signals and allows rapid shutdown of transcription. The identification of network motifs is important, as they are thought to perform specific information-processing tasks.\n\nDissecting networks involved in complex contexts such as animal development is a monumental task. Norbert Perrimon (Harvard Medical School\/Howard Hughes Medical Institute, Boston, USA) reported how his group is starting to tackle network complexity by carrying out genome-scale loss-of-function analysis in *Drosophila* cells using RNA interference (RNAi). The strategy is to perform multiple RNAi screens in different defined contexts (different cell lines or different stimuli) using sensitive and reliable reporter assays. Perrimon focused on canonical signaling pathways such as those involving Jaks and Stats, Wingless (Wg) and Hedgehog. From these systematic screens it appears that there are important overlaps between the pathways, and that the signaling components forming these pathways, are more numerous than expected. These findings were illustrated in a network topology map where, for example, 32 components are shared in the Wg and Hedgehog screens, but only two are shared between Wg, Hedgehog and Jak-Stat screens. To try and organize the data, a phenoprint matrix (a color-coded matrix that visually links phenotypes to genes) is being built, which at the moment encompasses about 20 genome-wide screens and more than 7,500 genes. This impressive work showing unexpected connections challenges our current view of how signal information is transduced to form an appropriate response.\n\nAn important role of biological networks is transcription regulation. Understanding how DNA-binding transcriptional regulators interpret the genome's regulatory code is essential. Richard Young (Whitehead and Broad Institutes, Massachusetts Institute of Technology, Cambridge, USA) reported the use of genome-wide location data (ChIP-chip) combined with phylogenetic conservation data to describe the promoter architecture and the global behavior of transcription factors in *Saccharomyces cerevisiae*. Four types of architectures were found: single regulators; repetitive binding motifs; multiple regulators; and co-occurring regulators. There are also four global behaviors: condition invariant (the transcription factor binds the same targets regardless of the environment tested); enabled (the transcription factor does not bind its target until enabled by the environment); expanded (the binding pattern is expanded by changes of environment); and altered (different targets depending on the environment). Of particular interest, it was estimated that 17% of DNA-binding factors are found on specific targets but wait for a signal before regulating transcription. This work will provide an excellent framework for modeling global gene expression in other eukaryotes.\n\n# Network hubs\n\nNetworks have particular nodes that are more highly connected than others; these nodes are called hubs. Marc Vidal (Dana-Farber Cancer Institute, Harvard Medical School, Boston, USA) described the use of large-scale yeast two-hybrid mapping to derive a protein-interaction network in which he found two types of hub, which behave differently. The first type is called the 'part' hub, and has numerous partners that interact with it simultaneously. The second type is the 'date' hub, which also has many potential partners, but where the interacting partners depend on location and time. The date hubs represent high-level connectors between structural or functional modules such as cellular organelles or particular pathways, whereas party hubs function inside these modules, at a lower level. In yeast, for example, calmodulin is a date hub that connects four different modules, while one of these modules, the endoplasmic reticulum, forms a party hub.\n\nStuart Kim (Stanford University, USA) has uncovered hubs through analyzing DNA microarray data for conserved gene co-regulation. These hubs, which he calls 'subunits' and 'integrators', also have different properties: subunit components are highly interconnected whereas integrator hubs have a central connection point with few connections between components. Also, subunit components are usually essential, whereas most integrator components are not, suggesting that these latter proteins may have partially redundant functions. He also presented evidence that newly evolved genes are not found in hubs. The uncovering of different properties for different types of hub is fundamental for further studies of biological networks.\n\n# The microRNA network\n\nMicroRNAs (miRNAs) regulate gene expression and are found in all metazoans studied so far. Three presentations addressed different aspects of miRNAs: identification of their targets; identification of novel miRNAs; and analysis of their biological functions. Steve Cohen (EMBL, Heidelberg, Germany) presented the results of systematic *in vivo* analysis of miRNA\/target pairing characteristics in *Drosophila*. It was determined that the 5' end of the miRNA is the most important in pairing and that a minimum of seven pairing nucleotides is required for silencing. Three types of target were also identified: canonical (perfect pairing), seed (fork-like) and compensatory (bubble-like). He estimated that half of the genes in the genome are regulated through miRNAs.\n\nHow miRNAs work is still not entirely defined. Ronald Plasterk (Hubrecht Laboratory, Utrecht, The Netherlands) has used RNAi in *Caenorhabditis elegans* to identify genes required for miRNA function. His laboratory used a reporter gene (*Hn-14::lacz*) that is regulated by the miRNA *let-7*. In this system, when *let-7* becomes expressed, the level of expression of the protein LACZ diminishes because of a translational inhibition of the reporter gene. Using a candidate-based approach that relies on previous genome-wide RNAi screens, 508 genes were tested by RNAi for causing an absence of silencing; 25 new genes were found with this property, one of which is the gene encoding the small ubiquitin-like modifier protein SUMO.\n\nVictor Ambros's laboratory (Dartmouth Medical School, Hanover, USA) is studying the biological function of miRNAs by generating deletions of the miRNA genes in *C. elegans*. He described how, by studying miRNA loss-of function phenotypes, miRNA activities have been grouped into four classes: coordinated (repression of multiple targets); collaborative (multiple miRNAs acting on common targets); redundant; and modulated. Redundancy within the *let-7* family was shown; double mutants between the two *let-7* family members *mir-48* and *mir-84* display a phenotype, but the single mutants do not.\n\nThe meeting was inspiring, presentations were of very high quality and participants were able to interact in a relaxed and comfortable atmosphere. The field of functional genomics has truly become 'functional' and we can look forward to hearing more at the next symposium.\n\n### Acknowledgements\n\nG.P. is supported by the Wellcome Trust and J.A. is a Wellcome Trust Senior Research Fellow (054523).","meta":{"dup_signals":{"dup_doc_count":106,"dup_dump_count":48,"dup_details":{"curated_sources":4,"2023-23":1,"2022-27":1,"2021-43":1,"2020-34":1,"2019-22":1,"2019-09":1,"2019-04":1,"2018-51":1,"2018-47":1,"2018-43":1,"2018-34":1,"2018-30":1,"2018-22":1,"2018-13":1,"2018-05":1,"2017-39":1,"2017-30":1,"2017-22":1,"2017-09":7,"2016-44":1,"2016-40":1,"2016-36":9,"2016-30":8,"2016-22":1,"2016-18":1,"2016-07":7,"2015-48":2,"2015-40":1,"2015-35":2,"2015-32":2,"2015-27":2,"2015-22":2,"2015-14":2,"2014-52":2,"2014-49":3,"2014-42":5,"2014-41":4,"2014-35":3,"2014-23":3,"2014-15":2,"2023-50":1,"2015-18":2,"2015-11":2,"2015-06":2,"2014-10":2,"2013-48":2,"2013-20":2,"2024-18":1}},"file":"PMC551530"},"subset":"pubmed_central"}
{"text":"date: 2017-05\ntitle: WHO LAUNCHES GLOBAL EFFORT TO HALVE MEDICATION-RELATED ERRORS IN 5 YEARS\n\n**29 MARCH 2017 \\| GENEVA\/BONN -** WHO today launched a global initiative to reduce severe, avoidable medication-associated harm in all countries by 50% over the next 5 years.\n\nThe Global Patient Safety Challenge on Medication Safety aims to address the weaknesses in health systems that lead to medication errors and the severe harm that results. It lays out ways to improve the way medicines are prescribed, distributed and consumed, and increase awareness among patients about the risks associated with the improper use of medication.\n\nMedication errors cause at least one death every day and injure approximately 1.3 million people annually in the United States of America alone. While low- and middle-income countries are estimated to have similar rates of medication-related adverse events to high-income countries, the impact is about twice as much in terms of the number of years of healthy life lost. Many countries lack good data, which will be gathered as part of the initiative.\n\nGlobally, the cost associated with medication errors has been estimated at US\\$ 42 billion annually or almost 1% of total global health expenditure.\n\n\"We all expect to be helped, not harmed, when we take medication,\" said Dr Margaret Chan, WHO Director-General. \"Apart from the human cost, medication errors place an enormous and unnecessary strain on health budgets. Preventing errors saves money and saves lives.\"\n\nEvery person around the world will at some point in their life take medicines to prevent or treat illness. However, medicines do sometimes cause serious harm if taken incorrectly, monitored insufficiently or as the result of an error, accident or communication problems.\n\nBoth health workers and patients can make mistakes that result in severe harm, such as ordering, prescribing, dispensing, preparing, administering or consuming the wrong medication or the wrong dose at the wrong time. But all medication errors are potentially avoidable. Preventing errors and the harm that results requires putting systems and procedures in place to ensure the right patient receives the right medication at the right dose via the right route at the right time.\n\nMedication errors can be caused by health worker fatigue, overcrowding, staff shortages, poor training and the wrong information being given to patients, among other reasons. Any one of these, or a combination, can affect the prescribing, dispensing, consumption, and monitoring of medications, which can result in severe harm, disability and even death.\n\nMost harm arises from systems failures in the way care is organized and coordinated, especially when multiple health providers are involved in a patient's care. An organizational culture that routinely implements best practices and that avoids blame when mistakes are made is the best environment for safe care.\n\nThe Challenge calls on countries to take early priority action to address these key factors: including medicines with a high risk of harm if used improperly; patients who take multiple medications for different diseases and conditions; and patients going through transitions of care, in order to reduce medication errors and harm to patients.\n\nThe actions planned in the Challenge will be focused on four areas: patients and the public; health care professionals; medicines as products; and systems and practices of medication. The Challenge aims to make improvements in each stage of the medication use process including prescribing, dispensing, administering, monitoring and use. WHO aims to provide guidance and develop strategies, plans and tools to ensure that the medication process has the safety of patients at its core, in all health care facilities.\n\n\"Over the years, I have spoken to many people who have lost loved ones to medication-related errors,\" said Sir Liam Donaldson, WHO Envoy for Patient Safety. \"Their stories, their quiet dignity and their acceptance of situations that should never have arisen have moved me deeply. It is to the memories of all those who have died due to incidents of unsafe care that this Challenge should be dedicated.\"\n\nThis challenge is WHO's third global patient safety challenge, following the Clean Care is Safe Care challenge on hand hygiene in 2005 and the Safe Surgery Saves Lives challenge in 2008.\n\nAvailable from: <http:\/\/www.who.int\/mediacentre\/news\/releases\/2017\/medication-related-errors\/en\/>","meta":{"dup_signals":{"dup_doc_count":140,"dup_dump_count":56,"dup_details":{"curated_sources":2,"2023-40":2,"2023-06":1,"2022-40":3,"2022-33":2,"2022-27":1,"2022-21":2,"2021-49":2,"2021-39":4,"2021-31":1,"2021-25":2,"2021-17":2,"2021-04":2,"2020-50":1,"2020-45":1,"2020-40":1,"2020-34":1,"2020-29":1,"2020-24":2,"2020-16":1,"2020-10":1,"2020-05":1,"2019-51":1,"2019-47":2,"2019-39":3,"2019-35":2,"2019-30":3,"2019-26":2,"2019-22":4,"2019-18":2,"2019-13":4,"2019-09":2,"2019-04":3,"2018-51":1,"2018-47":2,"2018-43":1,"2018-39":1,"2018-34":2,"2018-26":4,"2018-22":2,"2018-17":4,"2018-13":5,"2018-09":2,"2018-05":6,"2017-51":1,"2017-47":6,"2017-43":2,"2017-39":4,"2017-34":2,"2017-30":7,"2017-26":5,"2017-22":5,"2017-17":9,"2023-50":1,"2024-22":2,"2024-18":1,"2024-30":1}},"file":"PMC5447226"},"subset":"pubmed_central"}
{"text":"abstract: Primary malignant melanoma of the duodenum is an unusual oncologic entity. Patients usually present the similar clinical symptoms like other common tumors in this site. And there are no specific radiological features either. The cases with only little melanin pigment or without notable melanin pigment are very misleading, especially in small biopsies or frozen sections. Definite diagnosis depends on both careful histologic examination and the use of proper immunohistochemical stains. Moreover, detailed history and thorough investigation should be made to exclude the preexistence or coexistence of a primary lesion elsewhere. Herein we report the case of a 60-year-old male patient with primary malignant melanoma of the duodenum, which was misdiagnosed as lymphoma or undifferentiated carcinoma in frozen consultation. The patient had achieved disease-free survival for more than 46\u2009months without any evidence of recurrence after surgery.\n  .\n  # Virtual slides\n  .\n  The virtual slides for this article can be found here: <http:\/\/www.diagnosticpathology.diagnomx.eu\/vs\/1221457317710561>.\nauthor: Hongxia Li; Qinhe Fan; Zhen Wang; Hai Xu; Xiao Li; Weiming Zhang; Zhihong Zhang\ndate: 2012\ninstitute: 1Department of Pathology, the First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing 210029, P. R. China; 2Department of Radiology, the First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing 210029, P. R. China\nreferences:\ntitle: Primary malignant melanoma of the duodenum without visible melanin pigment: a mimicker of lymphoma or carcinoma\n\n# Background\n\nMalignant melanoma, originated from melanocyte, is not a common tumor and accounts for 1 to 3% of all malignancies \\[1\\]. However, it is the most common metastatic tumor of the gastrointestinal (GI) tract especially the small intestine and can present with fairly common constitutional symptoms \\[2\\]. Primary malignant melanoma originating in the small bowel, particularly in the duodenum, is extremely rare and very controversial. In addition, some cases with little melanin pigment or without visible melanin pigment are very misleading, especially in small biopsy specimens or frozen sections. Identifying areas showing possible melanin pigment and immunostains with specific markers for melanoma, such as HMB45, Melan-A and S-100 protein, are diagnostically important. We encountered a case of primary malignant melanoma of the duodenum (PMMD) without any visible melanin pigment and misdiagnosed as lymphoma or undifferentiated carcinoma in frozen consultation, to highlight its clinic-pathological features and the diagnostic pitfalls.\n\n# Case presentation\n\n## Clinical summary\n\nA 60-year-old Chinese male patient suffering from durative right abdominal pain and dark stools for one month, a recent episode of nausea and vomiting, was admitted to hospital in May 27, 2008. No history of fever, anorexia, hematemesis, radiating pain or weight loss was reported. Upper GI endoscopy revealed a malignant tumor at the descending part of the duodenum and the biopsy suspected poorly differentiated carcinoma. But there were no enough tumor cells for further immunohistochemical stain. In physical examination, there was no any obvious black nevus or nodule, nor swelled superficial lymph node. Routine hematological and biochemical studies showed no abnormalities. Serum detection showed tumor markers such as alpha fetoprotein (AFP), carcinoembryonic antigen (CEA), carbohydrate antigen 50 (CA-50), carbohydrate antigen 19\u20139 (CA-19-9) were all in the normal range. FOB (Fecal Occult Blood) Testing was positive. GI tract barium meal revealed a filling defect in the descendant duodenum, without obvious mucosal destruction and barium passed smoothly. Computed tomography scan of the abdomen disclosed the presence of a space-occupying lesion originating from the descending part of the duodenum with stenotic lumens (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>). An exploratory laparotomy was performed through a midline incision in Jun 2, 2008 and confirmed the presence of a solid tumor arising from the lateral part of the descendant duodenum, which invading the duodenal serosa. Enlarged mesentery lymph node and peripancreatic lymph node were identified. Because the surgeons suspected the tumor as lymphoma, so only a palliative operation (tumor resection) was done.\n\n## Pathologic findings\n\nIntraoperative consultation showed a tumor measuring 2.5\u2009cm\u2009\u00d7\u20091.5\u2009cm\u2009\u00d7\u20091\u2009cm, mesentery lymph node and peripancreatic lymph node measuring 3\u2009cm, 2\u2009cm in diameter respectively. The cut surfaces of the specimens were both pliable and gray red without pigment. Frozen sections were applied. Histologically, the lesion located under the enteric mucosa with diffusely infiltrative tumor cells, however, without obvious mucosal destruction (Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>A). The tumor cells were round, oval or polygonal epithelioid, showing markedly cytologic atypia with large eosinophilic nucleoli, abundant mitotic figures and moderate cytoplasm. But there was no obviously visible melanin pigment in cytoplasm (Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>B). Mesentery lymph node was positive for tumor cells (1\/1), whereas peripancreatic lymph node was negative (0\/1). According to above-mentioned features, pathologists consulted malignant tumor, prone to lymphoma or undifferentiated carcinoma. And the surgeons thought it might be lymphoma so only local tumor was resected.\n\nSubsequently, routine hematoxylin-eosin sections (formalin-fixed and paraffin-embedded) and immunostains were done. But the results of immunohistochemical stains didn't support the diagnoses of lymphoma and undifferentiated carcinoma. The tumor cells were strongly positive for melanoma marker (HMB45), Melan-A, S-100 protein (Figure <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>A-C) and vimentin, whereas CD20, CD45RO, CD3, CD79\u03b1, myeloperoxidase, terminal deoxynucleotidyl transferase, pan cytokeratin (AE1\/AE3), keratin 5\/6, keratin 7, synaptophysin, chromogranin A, and sarcoma markers such as smooth muscle actin, desmin, CD117, CD34 were all negative. A proliferative index of 60% was noted with Ki-67 immunostaining (Figure <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>D). So the diagnosis of malignant melanoma was confirmed.\n\nThorough postoperative systemic evaluations including a detailed history, clinical examination, endoscopic assessment, and radiologic imaging were done to rule out the presence of a primary cutaneous, anal, or ocular lesion, or a melanoma in any other site; however, no primary site was found. Therefore, the resected tumor was determined to represent a PMMD with mesentery lymph node metastasis. The patient refused chemotherapy and just used Chinese traditional medicine treatment intermittently to improve the immunity. Close follow-up showed that the patient is doing well with no evidence of loco regional recurrence or distant metastasis for more than 46\u2009months after surgery.\n\n# Discussion\n\nMalignant melanoma is a relatively rare tumor comprising 1-3% of all tumors and exhibits an unusual tendency to metastasize to the GI tract \\[3\\]. Although frequently seen in autopsy series in up to 50 to 60% of patients, only 2 to 5% of patients are diagnosed with metastatic malignant melanoma to the GI tract while they are still alive \\[4\\]. It is because that there are no specific symptoms of early development, which are general and constitutional; and even if metastases do occur, they are usually accompany with symptoms such as abdominal pain (62%), hemorrhage (50%), nausea and vomiting (26%), mass (22%), intestinal obstruction (18%), or intussusceptions (15%), and are frequently diagnosed in an emergency situation \\[5\\]. Metastasis to the GI tract is seen most frequently in the small intestine, followed by the colon, stomach, and rectum, but is rare in the esophagus. However, primary malignant melanoma originating in the small intestine and particularly in the duodenum is extremely rare, with only a few case reports \\[1,6,7\\].\n\nThere are different theories concerning the origin of primary malignant melanoma in the small bowel, though the controversy still exists over the presence of primary malignant melanoma in this area. Some proposes that malignant melanoma may originate from neural crest cells. These multipotential cells migrate through the body and can get to the bowel via the umbilical-mesenteric canal, where they later differentiate into specialized cells \\[8\\]. Although not consistently confirmed, another theory suggests that malignant melanoma might potentially develop from amine precursor uptake and decarboxylation (APUD) cells \\[9\\]. Besides, it is presumed that the small intestine normally contains melanoblasts, which may support the assumption of primary development of melanoma at this site.\n\nBefore making the diagnosis of primary malignant melanoma of the small bowel, thorough systemic examination must be done to rule out the possibility of metastasis from other sites, which may preferentially develop on the skin, retina, anal canal, or under the nail, and less frequently at other locations such as the esophagus, penis, or vagina. In addition, there should be no history of previous removal or spontaneous regression of any atypical melanocytic skin tumor \\[10\\]. The following diagnostic criteria have been occasionally proposed. According to Sachs *et al.*\\[8\\], primary malignant melanoma in the small bowel is diagnosed where there are: 1) biopsy-proven melanoma from the intestine at a single focus; 2) no evidence of disease in any other organs including the skin, eye and lymph nodes outside the region of drainage at the time of diagnosis; and 3) disease-free survival of at least 12\u2009months after diagnosis. According to Blecker \\[2\\], it is diagnosed when there is lack of concurrent or previous removal of a melanoma or melanocytic lesion from the skin and lack of any other organ involvement and in situ change in the overlying or adjacent GI epithelium. In this case, one single focus located in the lateral of the descendant duodenum, and thorough postoperative investigation including a detailed history, clinical examination, endoscopic assessment, and radiologic imaging failed to reveal any evidence of a pre-existing primary tumor or any other metastatic lesion. Moreover, the patient has survived for more than 46\u2009months with no evidence of loco regional recurrence or distant metastasis. Thus, we felt confident in supporting the diagnosis of PMMD.\n\nAlthough the possibility of primary malignant melanoma in the small intestine does exist, the incidence is extremely low. In addition, melanoma by itself is a great mimicker of other neoplastic conditions \\[11\\]. It is very easy to diagnose for the cases with obvious melanin pigment, but sometimes, the specimen only with little pigment or without any visible pigment, which may create a major diagnostic challenge, must be differentiated from other common intestinal tumors like lymphoma, carcinoma of poor differentiation, neuroendocrine tumor, leiomyosarcoma, GIST (Gastrointestinal Stromal Tumor), et al. More sections as far as possible and careful observation can help to find valuable diagnositic clues. Furthermore, immunohistochemical staining and electron microscope detection may play an important role for differential diagnosis. In our patient, the initial frozen sections revealed the tumor cells were relatively uniform with markedly cytologic atypia and abundant mitotic figures, so the pathologists favored lymphoma or carcinoma of poor differentiation, which were the most common malignancies of the duodenum. But further immunohistochemical stains provided no evidence for above diagnoses. By more careful observation, we found the discohesive tumor cells had one or more large eosinophilic nucleoli, which were one of the main features of melanoma apart from visible melanin pigment, so malignant melanoma - a very rare malignancy in this site should be taken into consideration. Expression of some specific markers such as HMB45, Melan-A and S-100 protein is required to confirm our conjecture.\n\nOptical treatment for malignant melanoma is an extensive and curative surgery, if possible, because other methods including adjuvant radiotherapy, chemotherapy and immunotherapy cannot offer definite treatment outcome. The time of diagnosis and the presence of metastases are supposed to represent major determinants of prognosis \\[10,12\\]. The median survival after curative resection of primary malignant melanoma of the small intestine is 49\u2009months \\[7\\]; the longest reported survival is 21\u2009years \\[12\\]. To the best of our knowledge, our patient refused chemotherapy and just used Chinese traditional medicine intermittently. Now he has achieved disease-free survival of more than 46\u2009months without any evidence of recurrence. The certain mechanism of Chinese traditional medicine is still unclear and always be regarded as complicated, sometimes even mysterious. Nowadays, one of generally accepted opinions is that it can enhance patients' immunity efficiently. But melanoma is destined to behave aggressively, and widespread metastases may present as a late complication. Furthermore, the Ki-67 labeling index in this case was greater than 60% and the patient had one mesentery lymph node involved. This highlights the need for close and long-term follow-up.\n\n# Conclusion\n\nIn conclusion, PMMD is an extremely rare neoplastic lesion of the alimentary tract. There are no specific symptoms and radiologic imaging. Especially, the cases with little melanin pigment or without visible melanin pigment are very misleading. Definite diagnosis depends on not only pathomorphology and immunohistochemical stains, but also detailed history and thorough investigation, the latter are more important to exclude the preexistence or coexistence of a primary lesion elsewhere.\n\n# Consent\n\nWritten informed consent was obtained from the patient for publication of this Case Report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.\n\n# Abbreviations\n\nPMMD: Primary malignant melanoma of the duodenum; GI: Gastrointestinal.\n\n# Competing interests\n\nThe authors declare that they have no competing interests.\n\n# Authors' contributions\n\nLH conceived and designed the case report and drafted the manuscript; ZZ designed the case report and contributed to the revisions and editing of the manuscript. FQ participated in the histopathological evaluation and gave the final diagnosis; WZ participated in the histopathological evaluation and supplied the literature review; XH provided the relevant radiological details; LX and ZW participated in immunohistochemical analysis. All authors have read and approved the final manuscript.","meta":{"dup_signals":{"dup_doc_count":125,"dup_dump_count":29,"dup_details":{"curated_sources":3,"2022-27":1,"2021-43":1,"2020-40":1,"2020-24":1,"2019-35":1,"2017-47":1,"2017-34":1,"2015-48":5,"2015-40":4,"2015-35":6,"2015-32":4,"2015-27":4,"2015-22":6,"2015-14":5,"2014-52":5,"2014-49":5,"2014-42":13,"2014-41":6,"2014-35":7,"2014-23":9,"2014-15":5,"2023-23":1,"2015-18":5,"2015-11":5,"2015-06":5,"2014-10":4,"2013-48":5,"2013-20":5,"2024-22":1}},"file":"PMC3472194"},"subset":"pubmed_central"}
{"text":"abstract: Repressingly, a robot scientist can generate functional genomic hypothesis and carry out experiments, increasing the working scientist's feelings of redundancy.\nauthor: Gregory A Petsko\ndate: 2004\ninstitute: 1Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02454-9110, USA\ntitle: Doctor Dunsel\n\n\"Dunsel ... is a term used by midshipmen at Starfleet Academy. It refers to a part which serves no useful purpose.\"\n\nMr Spock, in *Star Trek*, Episode 53: \"The Ultimate Computer\"\n\nParanoia began morphing into depression with the arrival of the 15 January 2004 issue of *Nature*. On page 247 was a paper by King *et al*. entitled 'Functional genomic hypothesis generation and experimentation by a robot scientist'. The paper describes an automated system that uses techniques from artificial intelligence to formulate hypotheses to explain observations. The system then devises experiments to test these hypotheses, and actually carries out the experiments using a simple laboratory robot. But that's not all. It then interprets the results so as to falsify any hypotheses not consistent with the data. Moreover, it can iterate this process, making it capable of developing and testing quite extensive models.\n\nIn the paper, the authors used this system to probe the genetic control of aromatic amino-acid biosynthesis in yeast, using various growth conditions and auxotrophic strains. The robot scientist took a series of systematic gene deletion strains and tried growing each in nutritional medium that lacked one of the intermediates in the pathway. If the deleted gene was required to make that intermediate, the strain would not grow and a component of the pathway would have been identified. The machine automatically examined the cultures to see how opaque they were, returned the results to the artificial intelligence package, and then received instructions for what experiments to perform to validate the hypotheses based on the results of the first round, and so on. The final result was the assembled pathway: the set of genes coding for the enzymes that control each step. The authors claim in the end that the automated system carried out the project just as efficiently - and more cost-effectively - than scientifically trained human volunteers.\n\n*Nature*, perhaps feeling guilty about the hordes of scientists who might be losing sleep over the prospect of having to go out and actually work for a living, tried to soften the blow with an editorial comment called 'Don't fear the Robot Scientist' (page 181 of the same issue) that completely missed the point. \"Contrary to first impressions,\" the commentator says cheerily, \"an automated system that designs its own experiments will benefit young molecular geneticists. At first glance, it seems to render obsolete the armies of postgrads and postdocs employed in the world's molecular-genetics laboratories.\"\n\nThat wasn't what was worrying me at all. Replacing my graduate students and postdocs with machines that would work around the clock and never pester me for more disk space on the computer or a new set of pipetmen; that would never complain about the temperature in the lab and never forget to clear up after themselves - that didn't sound so bad. It was the thought that it might eventually replace me that was frightening. After all, this thing didn't just carry out the experiments, it designed them and formulated hypotheses based on them. I thought I was supposed to do that.\n\n*Nature* continued, \"The team behind the Robot Scientist argues that such automation 'frees scientists to make the high-level creative leaps at which they excel'\". Well, the thing already plans, performs and interprets experiments. Just what leaps would those be, guys - designing the next generation of software for the robot? Still, I decided after an initial bad moment or ten, the robot was carrying out functional genomics. As we all know, genomics doesn't require real thought, just the semblance of it. Maybe I would have to surrender my genomics projects to some machine, but that only represented a part of my research effort. The rest of my work is structural biology, a branch of science of such technical sophistication and intellectual rigor that it could never be automated.\n\nThen the 10 February 2004 issue of *Proceedings of the National Academy of Sciences* arrived. On page 1537 - right after a paper of my own, to add insult to injury - was an article by James Holton and Tom Alber (who was once my graduate student, to add injury to insult) entitled 'Automated protein crystal structure determination using ELVES'. It describes an expert system that can fully automatically determine the crystal structure of a protein from the primary X-ray data. True, individual steps in this process had been automated for some time, and the ELVES system had already been used to carry out such steps or even groups of steps, but always under the user's direction. This was different: there was no human intervention at all. The system was able to solve the structure of a 12,000 molecular weight coiled-coil protein from crystallographic data sets in two different crystal forms following a single command that launched the program and directed it to the location of the data files. The entire process, including interpretation of the resulting electron density map and refinement of the atomic model to convergence, took 9.5 hours on a multi-processor computer for one of the crystal forms, and 165 hours - the thing must have stopped for coffee or something - for the other form. The authors concluded that \"high resolution structures with well-ordered metals can be determined automatically\". To be fair, the protein structure, being all helical, did not present any real challenges in the model-building stage, and the authors are commendably candid about the limitations of the method: \"ELVES is incapable of overcoming problems arising from poor data or inadequate phasing signal. Problems such as radiation damage, weak heavy atom signals, twinning, poor heavy atom models, low resolution, or crystal disorder that hinder crystallographic projects are not overcome by automation.\" Not yet, but just wait, I could hear them say *sotto voce*.\n\nSo, now I was about to be replaced as a crystallographer too. The year 2004 was sure turning out to be a terrific year. Well, strictly speaking I'm not paid just to do science anyway. Most of my salary comes from teaching undergraduates, and I consoled myself with the thought that I could always do more of that. Consoled myself, that is, until the arrival of last week's *Boston Globe* newspaper, with a story about a new effort at Massachusetts Institute of Technology (MIT) to revamp its undergraduate curriculum to take advantage of \"innovations in educational methods\". You know what that means - computer-based instruction. I could see it coming: once my lectures were all on the internet in interactive, self-test form, there would be no need for me to actually do any of the teaching myself anymore, or to be paid to do so - a fact I was sure would not be lost on any Brandeis administrator who might happen to read the article.\n\nFeeling now very much like a horse might have felt about the time Henry Ford began turning out Model Ts, I tried to find something - anything - that I could do that a machine couldn't. Suddenly, it came to me: writing papers and grants. I probably spend half my non-teaching time writing things, things with highly technical content that also have to be comprehensible to people in my field who aren't involved in the work I'm doing or am proposing to do. In fact, if I want to get a grant from a foundation or publish a paper in a high-profile, general journal like *Nature* or *Genome Biology*, I have to try to make this highly technical material comprehensible to people who aren't in my field at all. Automate that, if you can.\n\nWell, that may not be far off, actually. As Clive Thompson has pointed out (*The New York Times Magazine*, 14 December 2003), the music business is making strides towards doing something very like that. An artificial intelligence program called Hit Song Science from the Barcelona-based company Polyphonic HMI tries to determine whether a new song is going to be a hit. It uses a clustering algorithm to locate acoustic similarities between songs, similarities like common bits of rhythm, harmonies or keys. It compares these features of a new tune with all the Top 40 hits of the last 30 years; the closer the features of a new song are to a 'hit cluster', the more likely it is predicted, by the software, to be a hit. Thompson reports that the algorithm produces some strange groupings - the rock group U2 is similar to Beethoven, for example - yet it seems to work. A number of record companies are now using it to help pick which songs on a new album they will promote heavily. And, perhaps ominously, others are using it in the studio to tweak new songs as they are being recorded, changing various aspects of them to bring them closer to the hits in the nearest cluster. All well and good for the record companies, but it seems to me that this process is likely to take the spontaneity - and much of the novelty - right out of the music business. Hit songs tend to sound too much alike as it is, at least to this jaded listener; now they are going to be forced to sound even more alike. And clearly the same approach could be used, theoretically at least, to produce grants with a high probability of being funded, and scientific papers guaranteed to be accepted by top-rank journals. Hot Paper Science would cluster the titles, author names and affiliations, title words and key concepts that are shared by papers published in *Cell*, for example. One then only has to input one's own initial effort, 'The complete sequence of the gerbil genome' by Gregory A Petsko, *et al*., for example, and out would come 'Gerbil genome sequence: signal transduction pathways relevant to cancer, neurodegenerative diseases and apoptosis, with additional insights into systems biology and biodefense', plus a set of suggested coauthors that would help guarantee acceptance. The software would go on to write the paper, of course; submit it; and, if necessary, argue with the referees.\n\nWell, that was it, I thought. Before long, even my writing functions would be taken over by machines. I was rapidly being made redundant, as they say in the UK - a twentieth-century equivalent of Captain Kirk in the *Star Trek* episode \"The ultimate computer\", his command capabilities handled more efficiently by a machine programmed to replace human beings in space exploration, his plaintive (and sexist) cry, \"But there are some things men must do to remain men!\" drowned out by the bootsteps of the relentless march of automation.\n\nBut then something happened to lift my gloom and restore my self-esteem. It was the arrival of an e-mail reminding me about the curriculum committee meeting scheduled for that afternoon. Of course! I wasn't useless after all. In fact, real human scientists are indispensable, and always will be. Computers may be better at solving crystal structures, and robots may be better at doing genome-enabled, hypothesis-driven experiments - may even be better at interpreting them - and eventually there will probably be software that writes better papers and grants, but we humans can still waste enormous amounts of time at interminable committee meetings. No machine will ever be stupid enough to do that.","meta":{"dup_signals":{"dup_doc_count":124,"dup_dump_count":44,"dup_details":{"curated_sources":4,"2023-14":1,"2022-27":1,"2022-05":1,"2019-26":1,"2019-09":1,"2018-51":2,"2018-43":2,"2018-34":1,"2018-22":1,"2018-05":1,"2017-39":1,"2017-30":1,"2017-22":1,"2017-17":1,"2017-09":9,"2016-44":1,"2016-40":1,"2016-36":10,"2016-30":7,"2016-22":1,"2016-18":1,"2016-07":5,"2015-48":3,"2015-40":1,"2015-35":3,"2015-32":3,"2015-27":3,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":5,"2014-42":7,"2014-41":5,"2014-35":3,"2014-23":3,"2014-15":3,"2023-40":1,"2015-18":3,"2015-11":3,"2015-06":3,"2014-10":4,"2013-48":3,"2013-20":3,"2024-10":1}},"file":"PMC395772"},"subset":"pubmed_central"}
{"text":"author: Charles Darwin\ndate: 2010-10\ninstitute: First published in *Mind*, *2*, 285\u2013294. (reproduced from Christopher Green's Classics)\ntitle: <img src=\"ANS1017-0409-17-187-g001.jpg\" id=\"F1\" \/>A biographical sketch of an infant\n\nM. Taine's very interesting account of the mental development of an infant, translated in the last number of MIND (p. 252), has led me to look over a diary which I kept thirty-seven years ago with respect to one of my own infants. I had excellent opportunities for close observation, and wrote down at once whatever was observed. My chief object was expression, and my notes were used in my book on this subject; but as I attended to some other points, my observations may possibly possess some little interest in comparison with those by M. Taine, and with others which hereafter no doubt will be made. I feel sure, from what I have seen with my own infants, that the period of development of the several faculties will be found to differ considerably in different infants.\n\nDuring the first seven days various reflex actions, namely sneezing, hickuping, yawning, stretching, and of course sucking and screaming, were well performed by my infant. On the seventh day, I touched the naked sole of his foot with a bit of paper, and he jerked it away, curling at the same time his toes, like a much older child when tickled. The perfection of these reflex movements shows that the extreme imperfection of the voluntary ones is not due to the state of the muscles or of the coordinating centres, but to that of the seat of the will. At this time, though so early, it seemed clear to me that a warm soft hand \\[p. 286\\] applied to his face excited a wish to suck. This must be considered as a reflex or an instinctive action, for it is impossible to believe that experience and association with the touch of his mother's breast could so soon have come into play. During the first fortnight he often started on hearing any sudden sound, and blinked his eyes. The same fact was observed with some of my other infants within the first fortnight. Once, when he was 66 days old, I happened to sneeze, and he started violently, frowned, looked frightened, and cried rather badly: for an hour afterwards he was in a state which would be called nervous in an older person, for every slight noise made him start. A few days before this same date, he first started at an object suddenly seen; but for a long time afterwards sounds made him start and wink his eyes much more frequently than did sight; thus when 114 days old, I shook a paste-board box with comfits in it near his face and he started, whilst the same box when empty or any other object shaken as near or much nearer to his face produced no effect. We may infer from these several facts that the winking of the eyes, which manifestly serves to protect them, had not been acquired through experience. Although so sensitive to sound in a general way, he was not able even when 124 days old easily to recognise whence a sound proceeded, so as to direct his eyes to the source.\n\nWith respect to vision, \u2013 his eyes were fixed on a candle as early as the 9th day, and up to the 45th day nothing else seemed thus to fix them; but on the 49th day his attention was attracted by a bright-coloured tassel, as was shown by his eyes becoming fixed and the movements of his arms ceasing. It was surprising how slowly he acquired the power of following with his eyes an object if swinging at all rapidly; for he could not do this well when seven and a half months old. At the age of 32 days he perceived his mother's bosom when three or four inches from it, as was shown by the protrusion of his lips and his eyes becoming fixed; but I much doubt whether this had any connection with vision; he certainly had not touched the bosom. Whether he was guided through smell or the sensation of warmth or through association with the position in which he was held, I do not at all know.\n\nThe movements of his limbs and body were for a long time vague and purposeless, and usually performed in a jerking manner; but there was one exception to this rule, namely, that from a very early period, certainly long before he was 40 days old, he could move his hands to his own mouth. When 77 days old, he took the sucking bottle (with which he was partly fed) in his right hand, whether he was held on the left or right arm of his nurse, and he would not take it in his left hand \\[p. 287\\] until a week later although I tried to make him do so; so that the right hand was a week in advance of the left. Yet this infant afterwards proved to be left-handed, the tendency being no doubt inherited \u2013 his grandfather, mother, and a brother having been or being left-handed. When between 80 and 90 days old, he drew all sorts of objects into his mouth, and in two or three weeks' time could do this with some skill; but he often first touched his nose with the object and then dragged it down into his mouth. After grasping my finger and drawing it down into his mouth, his own hand prevented him from sucking it; but on the 114th day, after acting in this manner, he slipped his own hand down so that he could get the end of my finger into his mouth. This action was repeated several times, and evidently was not a chance but a rational one. The intentional movements of the hands and arms were thus much in advance of those of the body and legs; though the purposeless movements of the latter were from a very early period usually alternate as in the act of walking. When four months old, he often looked intently at his own hands and other objects close to him, and in doing so the eyes were turned much inwards, so that he often squinted frightfully. In a fortnight after this time (*i.e.* 132 days old) I observed that if an object was brought as near to his face as his own hands were, he tried to seize it, but often failed; and he did not try to do so in regard to more distant objects. I think there can be little doubt that the convergence of his eyes gave him the clue and excited him to move his arms. Although this infant thus began to use his hands at an early period, he showed no special aptitude in this respect, for when he was 2 years and 4 months old, he held pencils, pens, and other objects far less neatly and efficiently than did his sister who was then only 14 months old, and who showed great inherent aptitude in handling anything.\n\n*Anger*. \u2013 It was difficult to decide at how early an age anger was felt; on his eighth day he frowned and wrinkled the skin round his eyes before a crying fit, but this may have been due to pain or distress, and not to anger. When about ten weeks old, he was given some rather cold milk and he kept a slight frown on his forehead all the time that he was sucking, so that he looked like a grown-up person made cross from being compelled to do something which he did not like. When nearly four months old, and perhaps much earlier, there could be no doubt, from the manner in which the blood gushed into his whole face and scalp, that he easily got into a violent passion. A small cause sufficed; thus, when a little over seven months old, he screamed with rage because a lemon slipped away and he could not seize it with his hands. When eleven months old, if \\[p. 288\\] a wrong plaything was given to him, he would push it away and beat it; I presume that the beating was an instinctive sign of anger, like the snapping of the jaws by a young crocodile just out of the egg, and not that he imagined he could hurt the plaything. When two years and three months old, he became a great adept at throwing books or sticks, &c., at anyone who offended him; and so it was with some of my other sons. On the other hand, I could never see a trace of such aptitude in my infant daughters; and this makes me think that a tendency to throw objects is inherited by boys.\n\n*Fear*. \u2013 This feeling is probably one of the earliest which is experienced by infants, as shown by their starting at any sudden sound when only a few weeks old, followed by crying. Before the present one was 4 1\/2 months old I had been accustomed to make close to him many strange and loud noises, which were all taken as excellent jokes, but at this period I one day made a loud snoring noise which I had never done before; he instantly looked grave and then burst out crying. Two or three days afterwards, I made through forgetfullness the same noise with the same result. About the same time (*viz*. on the 137th day) I approached with my back towards him and then stood motionless; he looked very grave and much surprised, and would soon have cried, had I not turned round; then his face instantly relaxed into a smile. It is well known how intensely older children suffer from vague and undefined fears, as from the dark, or in passing an obscure corner in a large hall, &c. I may give as an instance that I took the child in question, when 2 1\/4 years old, to the Zoological Gardens, and he enjoyed looking at all the animals which were like those that he knew, such as deer, antelopes &c., and all the birds, even the ostriches, but was much alarmed at the various larger animals in cages. He often said afterwards that he wished to go again, but not to see \"beasts in houses\"; and we could in no manner account for this fear. May we not suspect that the vague but very real fears of children, which are quite independent of experience, are the inherited effects of real dangers and abject superstitions during ancient savage times? It is quite conformable with what we know of the transmission of formerly well-developed characters, that they should appear at an early period of life, and afterwards disappear.\n\n*Pleasurable Sensations*. \u2013 It may be presumed that infants feel pleasure whilst sucking and the expression of their swimming eyes seems to show that this is the case. This infant smiled when 45 days, a second infant when 46 days old; and these were true smiles, indicative of pleasure, for their eyes brightened and eyelids slightly closed. The smiles arose chiefly when looking at their mother, and were therefore probably of mental origin; \\[p. 289\\] but this infant often smiled then, and for some time afterwards, from some inward pleasurable feeling, for nothing was happening which could have in any way excited or amused him. When 110 days old he was exceedingly amused by a pinafore being thrown over his face and then suddenly withdrawn; and so he was when I suddenly uncovered my own face and approached his. He then uttered a little noise which was an incipient laugh. Here surprise was the chief cause of the amusement, as is the case to a large extent with the wit of grown-up persons. I believe that for three or four weeks before the time when he was amused by a face being suddenly uncovered, he received a little pinch on his nose and cheeks as a good joke. I was at first surprised at humour being appreciated by an infant only a little above three months old, but we should remember how very early puppies and kittens begin to play. When four months old, he showed in an unmistakable manner that he liked to hear the pianoforte played; so that here apparently was the earliest sign of an \u00e6sthetic feeling, unless the attraction of bright colours, which was exhibited much earlier, may be so considered.\n\n*Affection*. \u2013 This probably arose very early in life, if we may judge by his smiling at those who had charge of him when under two months old; though I had no distinct evidence of his distinguishing and recognising anyone, until he was nearly four months old. When nearly five months old, he plainly showed his wish to go to his nurse. But he did not spontaneously exhibit affection by overt acts until a little above a year old, namely, by kissing several times his nurse who had been absent for a short time. With respect to the allied feeling of sympathy, this was clearly shown at 6 months and 11 days by his melancholy face, with the corners of his mouth well depressed, when his nurse pretended to cry. Jealousy was plainly exhibited when I fondled a large doll, and when I weighed his infant sister, he being then 15 1\/2 months old. Seeing how strong a feeling jealousy is in dogs, it would probably be exhibited by infants at an earlier age than that just specified, if they were tried in a fitting manner.\n\n*Association of Ideas, Reason, &c.*\u2013 The first action which exhibited, as far as I observed, a kind of practical reasoning, has already been noticed, namely, the slipping his hand down my finger so as to get the end of it into his mouth; and this happened on the 114th day. When four and a half months old, he repeatedly smiled at my image and his own in a mirror, and no doubt mistook them for real objects; but he showed sense in being evidently surprised at my voice coming from behind him. Like all infants he much enjoyed thus looking at himself, and in less than two months perfectly understood that it was \\[p. 290\\] an image; for if I made quite silently any odd grimace, he would suddenly turn round to look at me. He was, however, puzzled at the age of seven months, when being out of doors he saw me on the inside of a large plate-glass window, and seemed in doubt whether or not it was an image. Another of my infants, a little girl, when exactly a year old, was not nearly so acute, and seemed quite perplexed at the image of a person in a mirror approaching her from behind. The higher apes which I tried with a small looking-glass behaved differently; they placed their hands behind the glass, and in doing so showed their sense, but far from taking pleasure in looking at themselves they got angry and would look no more.\n\nWhen five months old, associated ideas arising independently of any instruction became fixed in his mind; thus as soon as his hat and cloak were put on, he was very cross if he was not immediately taken out of doors. When exactly seven months old, he made the great step of associating his nurse with her name, so that if I called it out he would look round for her. Another infant used to amuse himself by shaking his head laterally: we praised and imitated him, saying \"Shake your head\"; and when he was seven months old, he would sometimes do so on being told without any other guide. During the next four months the former infant associated many things and actions with words; thus when asked for a kiss he would protrude his lips and keep still, \u2013 would shake his head and say in a scolding voice \"Ah\" to the coal-box or a little spilt water, &c., which he had been taught to consider as dirty. I may add that when a few days under nine months old he associated his own name with his image in the looking-glass, and when called by name would turn towards the glass even when at some distance from it. When a few days over nine months, he learnt spontaneously that a hand or other object causing a shadow to fall on the wall in front of him was to be looked for behind. Whilst under a year old, it was sufficient to repeat two or three times at intervals any short sentence to fix firmly in his mind some associated idea. In the infant described by M. Taine (pp. 254\u2013256) the age at which ideas readily became associated seems to have been considerably later, unless indeed the earlier cases were overlooked. The facility with which associated ideas due to instruction and others spontaneously arising were acquired, seemed to me by far the most strongly marked of all the distinctions between the mind of an infant and that of the cleverest full-grown dog that I have ever known. What a contrast does the mind of an infant present to that of the pike, described by Professor M\u00f6bius,\\[1\\] who during three whole months dashed and \\[p. 291\\] stunned himself against a glass partition which separated him from some minnows; and when, after at last learning that he could not attack them with impunity, he was placed in the aquarium with these same minnows, then in a persistent and senseless manner he would not attack them!\n\nCuriosity, as M. Taine remarks, is displayed at an early age by infants, and is highly important in the development of their minds; but I made no special observation on this head. Imitation likewise comes into play. When our infant was only four months old I thought that he tried to imitate sounds; but I may have deceived myself, for I was not thoroughly convinced that he did so until he was ten months old. At the age of 11 1\/2 months he could readily imitate all sorts of actions, such as shaking his head and saying \"Ah\" to any dirty object, or by carefully and slowly putting his forefinger in the middle of the palm of his other hand, to the childish rhyme of \"Pat it and pat it and mark it with T\". It was amusing to behold his pleased expression after successfully performing any such accomplishment.\n\nI do not know whether it is worth mentioning, as showing something about the strength of memory in a young child, that this one when 3 years and 23 days old on being shown an engraving of his grandfather, whom he had not seen for exactly six months, instantly recognised him and mentioned a whole string of events which had occurred whilst visiting him, and which certainly had never been mentioned in the interval.\n\n*Moral Sense*. \u2013 The first sign of moral sense was noticed at the age of nearly 13 months: I said \"Doddy (his nickname) won't give poor papa a kiss, \u2013 naughty Doddy\". These words, without doubt, made him feel slightly uncomfortable; and at last when I had returned to my chair, he protruded his lips as a sign that he was ready to kiss me; and he then shook his hand in an angry manner until I came and received his kiss. Nearly the same little scene recurred in a few days, and the reconciliation seemed to give him so much satisfaction, that several times afterwards he pretended to be angry and slapped me, and then insisted on giving me a kiss. So that here we have a touch of the dramatic art, which is so strongly pronounced in most young children. About this time it became easy to work on his feelings and make him do whatever was wanted. When 2 years and 3 months old, he gave his last bit of gingerbread to his little sister, and then cried out with high self-approbation \"Oh kind Doddy, kind Doddy\". Two months later, he became extremely sensitive to ridicule, and was so suspicious that he often thought people who were laughing and talking together were laughing at him. A little later (2 years and 7 1\/2 months old) I met him \\[p. 292\\] coming out of the dining room with his eyes unnaturally bright, and an odd unnatural or affected manner, so that I went into the room to see who was there, and found that he had been taking pounded sugar, which he had been told not to do. As he had never been in any way punished, his odd manner certainly was not due to fear, and I suppose it was pleasurable excitement struggling with conscience. A fortnight afterwards, I met him coming out of the same room, and he was eyeing his pinafore which he had carefully rolled up; and again his manner was so odd that I determined to see what was within his pinafore, notwithstanding that he said there was nothing and repeatedly commanded me to \"go away,\" and I found it stained with pickle-juice; so that here was carefully planned deceit. As this child was educated solely by working on his good feelings, he soon became as truthful, open, and tender, as anyone could desire.\n\n*Unconsciousness, Shyness*. \u2013 No one can have attended to very young children without being struck at the unabashed manner in which they fixedly stare without blinking their eyes at a new face; an old person can look in this manner only at an animal or inanimate object. This, I believe, is the result of young children not thinking in the least about themselves, and therefore not being in the least shy, though they are sometimes afraid of strangers. I saw the first symptom of shyness in my child when nearly two years and three months old: this was shown towards myself, after an absence of ten days from home, chiefly by his eyes being kept slightly averted from mine; but he soon came and sat on my knee and kissed me, and all trace of shyness disappeared.\n\n*Means of Communication*. \u2013 The noise of crying or rather of squalling, as no tears are shed for a long time, is of course uttered in an instinctive manner, but serves to show that there is suffering. After a time the sound differs according to the cause, such as hunger or pain. This was noticed when this infant was eleven weeks old, and I believe at an earlier age in another infant. Moreover, he appeared soon to learn to begin crying voluntarily, or to wrinkle his face in the manner proper to the occasion, so as to show that he wanted something. When 46 days old, he first made little noises without any meaning to please himself, and these soon became varied. An incipient laugh was observed on the 113th day, but much earlier in another infant. At this date I thought, as already remarked, that he began to try to imitate sounds, as he certainly did at a considerably later period. When five and a half months old, he uttered an articulate sound \"da\" but without any meaning attached to it. When a little over a year old, he used gestures \\[p. 293\\] to explain his wishes; to give a simple instance, he picked up a bit of paper and giving it to me pointed to the fire, as he had often seen and liked to see paper burnt. At exactly the age of a year, he made the great step of inventing a word for food, namely *mum*, but what led him to it I did not discover. And now instead of beginning to cry when he was hungry, he used this word in a demonstrative manner or as a verb, implying \"Give me food\". This word therefore corresponds with *ham* as used by M. Taine's infant at the later age of 14 months. But he also used *mum* as a substantive of wide signification; thus he called sugar *shu-mum*, and a little later after he had learned the word \"black,\" he called liquorice *black-shu-mum*, \u2013 black-sugar-food.\n\nI was particularly struck with the fact that when asking for food by the word *mum* he gave to it (I will copy the words written down at the time) \"a most strongly marked interrogatory sound at the end\". He also gave to \"Ah,\" which he chiefly used at first when recognising any person or his own image in a mirror, an exclamatory sound, such as we employ when surprised. I remark in my notes that the use of these intonations seemed to have arisen instinctively, and I regret that more observations were not made on this subject. I record, however, in my notes that at a rather later period, when between 18 and 21 months old, he modulated his voice in refusing peremptorily to do anything by a defiant whine, so as to express \"That I won't\"; and again his humph of assent expressed \"Yes, to be sure\". M. Taine also insists strongly on the highly expressive tones of the sounds made by his infant before she had learnt to speak. The interrogatory sound which my child gave to the word *mum* when asking for food is especially curious; for if anyone will use a single word or a short sentence in this manner, he will find that the musical pitch of his voice rises considerably at the close. I did not then see that this fact bears on the view which I have elsewhere maintained that before man used articulate language, he uttered notes in a true musical scale as does the anthropoid ape Hylobates.\n\nFinally, the wants of an infant are at first made intelligible by instinctive cries, which after a time are modified in part unconsciously, and in part, as I believe, voluntarily as a means of communication, \u2013 by the unconscious expression of the features, \u2013 by gestures and in a marked manner by different intonations, \u2013 lastly by words of a general nature invented by himself, then of a more precise nature imitated from those which he hears; and these latter are acquired at a wonderfully quick rate. An infant understands to a certain extent, and as \\[p. 294\\] I believe at a very early period, the meaning or feelings of those who tend him, by the expression of their features. There can hardly be a doubt about this with respect to smiling; and it seemed to me that the infant whose biography I have here given understood a compassionate expression at a little over five months old. When 6 months and 11 days old he certainly showed sympathy with his nurse on her pretending to cry. When pleased after performing some new accomplishment, being then almost a year old, he evidently studied the expression of those around him. It was probably due to differences of expression and not merely of the form of the features that certain faces clearly pleased him much more than others, even at so early an age as a little over six months. Before he was a year old, he understood intonations and gestures, as well as several words and short sentences. He understood one word, namely, his nurse's name, exactly five months before he invented his first word *mum*; and this is what might have been expected, as we know that the lower animals easily learn to understand spoken words.","meta":{"dup_signals":{"dup_doc_count":658,"dup_dump_count":73,"dup_details":{"curated_sources":4,"2023-23":1,"2022-49":1,"2022-33":1,"2022-27":1,"2021-49":1,"2021-39":2,"2021-31":2,"2021-25":1,"2021-10":2,"2020-40":2,"2020-24":2,"2020-05":1,"2019-47":1,"2019-43":1,"2019-30":1,"2019-26":1,"2019-18":2,"2019-13":1,"2019-09":2,"2019-04":2,"2018-47":1,"2018-43":3,"2018-34":3,"2018-30":2,"2018-26":2,"2018-22":5,"2018-17":8,"2018-13":3,"2018-09":7,"2018-05":6,"2017-51":2,"2017-47":10,"2017-43":6,"2017-39":10,"2017-34":4,"2017-30":7,"2017-26":8,"2017-22":8,"2017-17":11,"2017-09":12,"2017-04":13,"2016-50":13,"2016-44":14,"2016-40":14,"2016-36":14,"2016-30":14,"2016-26":12,"2016-22":14,"2016-18":14,"2016-07":16,"2015-48":15,"2015-40":12,"2015-35":14,"2015-32":15,"2015-27":14,"2015-22":15,"2015-14":11,"2014-52":15,"2014-49":22,"2014-42":39,"2014-41":24,"2014-35":22,"2014-23":25,"2014-15":24,"2023-50":1,"2024-10":8,"2017-13":10,"2015-18":13,"2015-11":13,"2015-06":14,"2014-10":17,"2013-48":15,"2013-20":12}},"file":"PMC4117005"},"subset":"pubmed_central"}
{"text":"abstract: The technological development of telemedicine has performed important progress, assuming a diagnostic relief role inside of the processes. Among the fields in fast evolution, telepathology is placed among those of greater interest. Up to some years ago, telepathology allowed us to observe at a distance and in real time, histological or cytological slides through the Internet, using a motorized microscope (dynamic telepathology). Currently, telepathology has completed an important step in ahead being possible to digitize completely a slide and to store it. This allows observation of the whole surface of histological or cytological slides remotely with a customary PC, without human intervention (virtual slide). The described systems have exclusive characteristics, so that a \"hybrid system\" supporting both technologies, turns out to be the best solution applicable in a wide range program. In order to realize the theoretical aspects previously described, we report an organizational model practicable and applicable to a territory in which three hospitals operate. An essential prerequisite in order to arrange an efficient telepathology system turns out to be one structured data transmission network, equipped with elevated guaranteed bandwidth, and one consolidated experience in the registration and management of digital images.\nauthor: Roberto Mencarelli; Adriano Marcolongo; Alessio Gasparetto\ndate: 2008\ninstitute: 1Department of Surgical Pathology, Azienda ULSS 18, Rovigo, Italy; 2General Manager, Azienda ULSS 18, Rovigo, Italy; 3Department of Information Technology \u2013 Azienda ULSS 18, Rovigo, Italy\nreferences:\ntitle: Organizational model for a telepathology system\n\n# Introduction\n\nThe acquisition of a telepathology system designed for a district territory that comprises three hospitals managed from two different health administrations (Adria and Rovigo) is inserted in a wide plan of local development of telemedicine. The challenge has been planned from the two administrations and fully financed by the Cariparo Foundation.\n\nIn order to achieve the implementation of this telepathology system there was a need for hospitals technology structures innovation. In particular: physical and logical structures for data transport, hardware modernization, software development, processes reorganization.\n\nThis technological innovation process is essential for developing and offering to citizens high quality and reliable health services. In particular, a telepathology system needs high hardware and network performance for processing, managing and archiving digital slide images.\n\nThe main aims, thanks to the introduction of telepathology, can be summarized as follows:\n\n\u2022 Increase of service quality offered to patients thanks to qualified second opinions;\n\n\u2022 Sharing and discussion of interesting pathological cases for increasing professional ability;\n\n\u2022 Creation of a permanent and available scientific repository.\n\n# Methods\n\nWe can distinguish three types of telepathology: static telepathology, dynamic telepathology, virtual slide telepathology. In this paper, we analyze the last two for describing how we can implement a \"hybrid telepathology system\" that takes advantage of the peculiarities of each one. The dynamic telepathology allows exploration, in real time, of the whole slide surface thanks to a robotized remote controlled microscope; this also permits us to change magnification, focus and digitize the interesting portion. The disadvantages regard the necessary presence of a technician for positioning the desired slide within the microscope and the discontinuous digitization in terms of area and magnification. In virtual slide telepathology, the slide is completely digitized and stored in a repository; this permits a single or multiple user consultation, in every time and without human intervention. The virtual slide allows the exploration of the whole slide surface with different magnification. There are many types of devices that can be used for slide digitization and the right option depends on workload and necessary scan rate \\[1\\]. Virtual slide telepathology has a a negligible disadvantage \\[2\\]: it is not possible to focus the areas that were not correctly acquired. During the scan process, the scanning device takes into account a finite point number for the focusing procedure; each point is characterized by its own focal plane. When the sample surface is irregular, the device uses proprietary algorithms to calculate an \"in focus surface\" above the slide; this allows an average good focus but locally a less accurate one. During this treatment, it can be noted that this problem is directly correlated with sample analysis type and grows with increasing histological or cytological sample surface irregularity. Another characteristic to take into account is the virtual slide file size; this depends principally on the following parameters: the resolution, the compression ratio, the compression algorithm and the color depth. Considering the resolution used for the histological and cytological slide scan (0,5 \u03bcm\/pixel \u2013 20\u00d7), the scan area maximum dimension (9 cm2), an adequate compression ratio (15:1) and an appropriate compression algorithm (JPEG2000) it can be possible to obtain averagely a 500 MB file even if the non compressed file is approximately 7.5 GB \\[3,4\\]. This file size allows, at present, a selected virtual slide storage for scientific archive creation but doesn't permit complete archiving of all slides; in fact, in case of complete archiving the storage device costs would be much too expensive. Certainly, in the future, the cost\/byte reduction for storage and an international agreement about the standard for this storage will permit all slides complete archiving, deleting the conservation necessity. This will lead to many advantages regarding clinical data availability and accessibility. In the end, the necessity of an adequate guaranteed data communication bandwidth for sharing virtual slide must be considered. This problem can be solved thanks to the use of an Image Server and the specific image format (image pyramid); this offers the possibility of selective visualization of images in terms of resolution and portion of interest, without the necessity of a complete transfer of virtual slide from Image Server to a local PC. The complete focusing of the whole slide surface is therefore the real problem to solve, while the archiving and the consultation of virtual slides are effectively faced thanks to scalable and high performance storage devices and dedicated Image Server.\n\nThere are mainly two types of second opinions: second opinions in real time on frozen section during surgery (intraoperative consultation) and second opinions on histological or cytological slides which require a complex interpretation. Each type of second opinion has specific characteristics that are directly correlated with previous discussion. In the first second opinion type the analyses are conducted on thick sample or with irregular surface (this arises from the methods through which the samples are obtained and the liquid amount contained into the tissue to analyze); for this reason the automatic scanner device (virtual slide telepathology) can't focus correctly on the whole slide surface but with a motorized microscope (dynamic telepathology) it is possible to focalize each sample surface point. Therefore, the dynamic telepathology represents an important tool for second opinion during intraoperative consultation, especially for the presence on the territory of two hospitals with surgery but without a resident pathologist. Regarding histological and cytological slides, the tissue samples are treated for obtaining thin sections; this deletes the trouble regarding focus due to irregular surface, as previously described. In this case the use of virtual slide telepathology is favorable because it allows the visualization of the morphological picture at any time, remotely with a customary PC, without human intervention. This allows one to obtain a second opinion on complex cases from an expert of personal choice and to perform an external quality control.\n\nBased on previous considerations and for obtaining an effective telepathology system, the best choice is a \"hybrid system\" composed of motorized microscopes, with remote control, and a scanner for slide digitization, in order to achieve the best characteristics from each system without respective disadvantages. This choice has been applied to Rovigo province health structures, in particular Rovigo, Adria and Trecenta hospitals. For storage purposes it has been used a NAS (Network Attached Storage) device (50 TB capacity \u2013 1 Gb\/s transfer rate). The previously described system is completely integrated with the CPOE (Computerized Physician Order Entry) based Hospital information System.\n\nThanks to complete slides digitization and the use of Image Server with high computational performances, it will be possible to apply filters to acquired images or to apply algorithms for calculating interesting quantities (e.g.: the cellular membrane distribution and continuity). These techniques adequately developed, tested and standardized will be the base for Computer Aided Diagnosis (CAD) introduction.\n\n# Conclusion\n\nThe implementation of a telepathology \"hybrid system\" adequately projected and integrated with the Hospital information System, leads to an increase in the service quality offered by the Pathological Anatomy department, principally thanks to: (a) qualified and real time second opinions on frozen section during surgery or histological-cytological consultation on slides which requires a complex interpretation; (b) continuous education process thanks to the sharing of interesting virtual slides and creation of a permanent scientific archive.\n\nThe design and implementation of a telepathology system must be based on real operative demands, supported by a technological structure that can guarantee a reliable and efficient service and projected using hospital experience on digital images acquisition and storage.\n\n### Acknowledgements\n\nThis article has been published as part of *Diagnostic Pathology* Volume 3 Supplement 1, 2008: New trends in digital pathology: Proceedings of the 9th European Congress on Telepathology and 3rd International Congress on Virtual Microscopy. The full contents of the supplement are available online at <http:\/\/www.diagnosticpathology.org\/supplements\/3\/S1>","meta":{"dup_signals":{"dup_doc_count":151,"dup_dump_count":50,"dup_details":{"curated_sources":2,"2022-49":1,"2022-21":1,"2021-39":1,"2020-40":1,"2020-29":1,"2020-16":1,"2019-30":1,"2019-22":1,"2019-13":1,"2019-04":1,"2018-47":1,"2018-39":1,"2018-30":1,"2018-17":1,"2018-09":1,"2017-39":1,"2017-30":1,"2017-22":1,"2017-09":13,"2017-04":1,"2016-50":1,"2016-44":1,"2016-40":1,"2016-36":1,"2016-22":1,"2016-18":1,"2015-48":5,"2015-40":3,"2015-35":5,"2015-32":5,"2015-27":5,"2015-22":5,"2015-14":5,"2014-52":5,"2014-49":3,"2014-42":14,"2014-41":8,"2014-35":5,"2014-23":8,"2014-15":6,"2023-14":1,"2024-10":1,"2017-13":1,"2015-18":5,"2015-11":5,"2015-06":3,"2014-10":4,"2013-48":5,"2013-20":3,"2024-26":1}},"file":"PMC2500113"},"subset":"pubmed_central"}
{"text":"abstract: This is the third in a series of articles, invited by the editors of *Diabetes*, that describes the research programs and aims of organizations committed to funding and fostering diabetes-related research. The first piece, contributed by the Juvenile Diabetes Research Foundation, appeared in the January 2012 issue of *Diabetes*. The second piece that describes the American Diabetes Association's research program appeared in the June 2012 issues of *Diabetes* and *Diabetes Care*.\nauthor: Judith E. Fradkin; Griffin P. RodgersCorresponding author: Judith Fradkin, <email@example.com>.\ndate: 2013-02\nreferences:\ntitle: Diabetes Research: A Perspective From the National Institute of Diabetes and Digestive and Kidney Diseases\n\nThe growing human and economic toll of diabetes has caused consternation worldwide. Not only is the number of people affected increasing at an alarming rate, but onset of the major forms of the disease occurs at ever younger ages. We now know that the reach of diabetes extends far beyond the classic acute metabolic and chronic vascular complications to increased risk of an ever-increasing array of conditions including Alzheimer disease, cancer, liver failure, bone fractures, depression, and hearing loss. In the U.S. one in three Medicare dollars is spent on care of people with diabetes, and the proportion of cardiovascular disease (CVD) risk attributable to diabetes is rising. While complications of diabetes may develop slowly over decades, antecedents of diabetes may lay in utero or early life. Thus the breadth of meaningful research extends across the life span, ranging from studies of how the in utero environment alters diabetes risk to improved understanding of the special needs of older patients with diabetes.\n\nSince the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) was established in 1950, we have seen huge progress in our ability to predict, classify, and treat diabetes and its complications as well as to prevent or delay type 2 diabetes. Landmark NIDDK-led clinical trials have demonstrated that glucose control can dramatically reduce diabetes complications, and lifestyle change producing modest weight loss, or the drug metformin, can substantially reduce development of type 2 diabetes. Were it not for this progress, the toll from rising rates of the major forms of diabetes would be much higher.\n\nDespite a challenging fiscal climate, the National Institutes of Health (NIH) expends over \\$1 billion on diabetes research annually. NIDDK accounts for about two-thirds of this total, and we are determined that these resources will be invested wisely and balanced among competing priorities. We must address the most compelling practical questions about clinical management and prevention of diabetes and its complications while also uncovering and exploiting novel pathways that will provide new targets and approaches to combat the disorder. Last year, a new strategic plan for diabetes research (1) was issued under NIDDK's leadership with input from over 100 scientists and multiple federal agencies and components of NIH. The plan highlights progress and opportunities in 10 key areas as well as resource and infrastructure needs. Conquering diabetes will require a wide range of expertise and talent from molecular and cell biology to behavioral and social sciences. Development and empowerment of this human capital is critical to this endeavor, including facilitating multidisciplinary collaborations and the application of new technologies to diabetes research. Here we will touch on highlights of our diabetes research priorities and initiatives, referring readers to the Diabetes Research Strategic Plan (1) for a more comprehensive analysis of advances and opportunities.\n\n# ENHANCING THE DIABETES RESEARCH WORKFORCE\n\nA well-trained and diverse scientific workforce is essential to our efforts to improve outcomes for people with or at risk for diabetes. To ensure a pipeline of new well-trained investigators in basic and clinical disciplines, NIDDK supports training grant, fellowship, and career award mechanisms to provide opportunities for investigators at all stages of the career trajectory. These are supplemented by programs targeted to specific needs, such as our medical student research program in diabetes, which allows medical students to conduct research under the direction of an established scientist at one of our seventeen NIDDK-funded Diabetes Research Centers; supplements to research and training grants to foster recruitment of underrepresented minority scientists to diabetes research; and institutional career development programs to attract pediatric endocrinologists to careers in childhood diabetes research. It is increasingly important to build multidisciplinary research teams and train multidisciplinary researchers. We are establishing interdisciplinary training grants to promote diabetes research training for bioengineers as well as career development programs in diabetes research for behavioral scientists. We will continue to foster the application of new expertise, for example in computational science and bioinformatics, to diabetes research problems. To help new investigators transition to independence, we also provide a less stringent pay line for early career investigators. We also invite new investigators with NIDDK research or career development grants to participate in NIDDK workshops designed to help them succeed as independent investigators.\n\n# FUNDAMENTAL RESEARCH\n\nTo uncover new approaches to prevention and therapy of diabetes and its devastating complications, NIDDK will continue to support a robust portfolio of investigator-initiated basic research. NIDDK has recently developed data on application and funding trends to help our research community understand the application and funding dynamics over recent years. This information is available at <http:\/\/www2.niddk.nih.gov\/Funding\/Grants\/FundingTrendsandValues.htm>. It shows that relative funding levels of most research categories have remained fairly stable since 2003, and demonstrates our continuing strong support of investigator-initiated research project grants or R01s and training and career development programs. Information on resources to empower researchers, such diabetes centers, human islets, mouse models, reagents, and databases, and on funding opportunities and staff to contact in specific program areas is available at <http:\/\/www2.niddk.nih.gov\/Research\/ScientificAreas\/Diabetes\/>.\n\n# DIABETES PREVENTION\n\nDiabetes prevention is a major public health challenge. For type 2 diabetes, the NIDDK-led Diabetes Prevention Program (DPP) demonstrated a dramatic effect of modest weight loss or the generic drug metformin in delaying or preventing type 2 diabetes (2). Ongoing studies are examining the durability of this risk reduction, the cost effectiveness of the interventions, and their impact in reducing diabetes complications. To facilitate translation of landmark clinical research into clinical practice and public health activities, NIDDK established a program to test practical, cost-effective approaches to deliver interventions proven efficacious in clinical trials for effectiveness in community and practice settings. One such NIDDK-supported study of a lifestyle change intervention delivered by YMCA fitness trainers (3) already is being rolled out nationwide by the YMCA with coverage from insurers such as United Health Group. In another promising approach, diabetes educators trained selected patients with well-controlled diabetes to serve as community health workers delivering a lifestyle intervention based on the DPP to community members with prediabetes (4). This NIDDK-funded research provides a basis for a new congressionally established National Diabetes Prevention Program at the Centers for Disease Control and Prevention (CDC) to foster delivery of evidence-based lifestyle change programs for people at high risk for type 2 diabetes. Given the sustained effort required to achieve and maintain lifestyle change, much additional research is needed to improve, disseminate, and evaluate type 2 diabetes prevention programs in the U.S. Approaches are also needed to reduce the development of risk factors for diabetes. Of particular importance are studies to reduce environmental exposures during pregnancy or childhood that may increase diabetes and obesity risk.\n\nThe incidence of type 1 diabetes is rising worldwide and the disease is occurring at younger ages suggesting an environmental trigger is responsible. Bold new programs aimed at preventing type 1 diabetes have been undertaken with support from the Special Statutory Funding Program for Type 1 Diabetes Research, which provides \\$150 million per year through 2013 for type 1 diabetes research. These special funds are in addition to the regular NIH appropriation. One program established under the program, The Environmental Determinants of Diabetes in the Young (TEDDY), has screened nearly half a million neonates to establish a cohort of over 8,000 at high genetic risk for type 1 diabetes. Participants will be followed from birth through 15 years of age to identify dietary, infectious, microbiome, or other environmental triggers of autoimmunity and type 1 diabetes and to study the interaction between environmental factors and specific genetic variations associated with disease risk. Identification of an infectious agent or dietary factor that triggers or protects against the disease would have immense implications for prevention through the development of a vaccine or dietary change. Also with special program support, the Type 1 Diabetes TrialNet is identifying individuals recently diagnosed with type 1 diabetes or at high risk of developing the disease and testing interventions to prevent diabetes or to slow its progression. Selective immune modulation has been shown to preserve insulin secretion in newly diagnosed patients, and TrialNet is exploring the use of one such agent, teplizumab, to prevent type 1 diabetes in individuals at very high short-term risk of type 1 diabetes. In the future, combination therapies aimed at modulating multiple steps of the toxic immune response and restoring immunoregulation may produce a clinically significant delay in onset and ultimately the prevention of type 1 diabetes.\n\n# CLINICAL TRIALS TO INFORM DIABETES MANAGEMENT\n\nWhile information on how to prevent and treat type 2 diabetes has grown rapidly, adequate data from rigorous clinical trials are not available to inform many routine decisions on care for patients with diabetes. Current guidelines are moving away from a \"one size fits all\" approach to incorporate factors such as diabetes duration and the presence of complications or other comorbidities. However, we lack information to individualize therapy based on demographic, physiologic, or genetic variation. Improved understanding of the genetic, physiologic, and environmental factors that underlie diabetes mellitus are necessary for more individualized diabetes treatment.\n\nNumerous drugs are approved for the treatment of type 2 diabetes, based largely on relatively short-term efficacy in glycemic reduction. However, it is not known whether particular drugs or drug combinations will have more durable effects in the maintenance of glucose control. NIDDK is supporting a large comparative effectiveness trial to inform the choice of second agent when metformin alone is inadequate for glycemic control. This multicenter randomized trial will provide information on health benefits as well as cost effectiveness of widely used treatments.\n\nSmall studies suggest it may be possible to preserve \u03b2-cell function during prediabetes and early in the course of type 2 diabetes. Major questions include the optimal timing of interventions, whether specific treatments have maximum benefit at different stages of the disease, and what patient characteristics influence the choice of initial therapy for individuals. A newly formed consortium will examine the approaches to the initial treatment of type 2 diabetes that may reverse or slow the decline in \u03b2-cell function over time.\n\nWhile major trials have established the importance of blood pressure and lipid control in reducing CVD in type 2 diabetes, much less is known about how cardiovascular risk factors should be managed in type 1 diabetes. When blood pressure and lipid lowering should begin and optimal therapeutic targets remain to be established. Although type 1 diabetes increases the risk of CVD as much as 10-fold compared to an age-matched population, testing practical approaches to mitigate this risk is challenging due to the low incidence of CVD in the younger type 1 diabetes population. Such trials may require the development of the validated biomarkers discussed below.\n\nDiabetes self-management training and promotion of effective self-care behaviors are vital to improving outcomes. The choices patients make daily about diet, physical activity, adherence to medications, self-monitoring, foot and dental care, and medical follow-up for early detection of complications are critical for improving diabetes outcomes. Research has established effective counseling and education strategies, including motivational interviewing, patient empowerment, and social and peer support. However, research is needed to expand the reach of such approaches to more patients and providers. NIDDK encourages research studying approaches such as group visits, telemedicine, and social media that may extend the impact of the limited workforce skilled in the provision of this care.\n\n# DIABETES IN SPECIAL POPULATIONS\n\nDiabetes spares no age, sex, racial or ethnic group, yet each such group faces special challenges. Intensive glycemic control in youth may afford lifelong protection from complications yet infancy and adolescence pose unique challenges in attaining such control. Treatment priorities and optimal glycemic, blood pressure, and cholesterol targets to prevent complications and maintain quality of life may differ for older adults or those with limited life expectancy. The *Eunice Kennedy Shriver* National Institute of Child Health and Human Development (NICHD)-led Hyperglycemia and Adverse Pregnancy Outcome (HAPO) study (5) showed that perinatal harm to mother and offspring occurs in pregnancy at lower levels of glycemia than previously appreciated. To identify gestational glycemic thresholds for longer-term effects on offspring and the risk of type 2 diabetes in mothers, NIDDK will support a follow-up study of this important cohort. NIDDK has also recently launched a study to explore approaches for the prevention of gestational diabetes mellitus. It also remains to be established what treatments during pregnancy mitigate perinatal and\/or long-term complications of gestational diabetes mellitus.\n\nThe biologic and environmental risk factors that contribute to underlying racial and ethnic disparities are poorly understood. The alarming emergence of type 2 diabetes in youth overwhelmingly occurs in minority populations. Ominous data on poor risk factor control portends very poor outcomes for this vulnerable population. To address this threat, NIDDK recently completed a major multicenter trial comparing three approaches to the treatment of type 2 diabetes in youth and a trial conducted in schools serving poor and minority children to prevent the development of risk factors for type 2 diabetes.\n\nAs better therapy for HIV infection, organ transplantation, cystic fibrosis, and other conditions improve survival, diabetes has emerged as an increasingly important complication. Critical questions must be addressed about optimal strategies to prevent and treat diabetes. For example, early diabetes diagnosis and treatment helped improve survival in people with cystic fibrosis\u2013related diabetes (CFRD). An NIDDK-funded trial showed that chronic weight loss can be reversed with the institution of insulin therapy but not with repaglinide early in the course of CFRD before fasting hyperglycemia develops (6). A current initiative will support research to understand the pathogenesis of CFRD, which affects half of adults with cystic fibrosis. NIDDK will also encourage research on treatment and prevention of diabetes in HIV patients.\n\nExceptional contributions to understanding diabetes in special populations have emerged from NIDDK's intramural research program. Spanning decades and generations, longitudinal studies of Pima Indians have uncovered physiologic, environmental, and genetic determinants of diabetes in the U.S. population with highest rates of type 2 diabetes. These studies have foreshadowed findings in the broader population, such as the contribution of intrauterine factors to childhood obesity and type 2 diabetes and the prognosis for these youth. Collaborations between intramural and extramural researchers studying individuals with severe insulin resistance and lipodystrophy have yielded important physiologic information and the emergence of leptin replacement therapy for lipodystrophy.\n\n# EPIDEMIOLOGY AND SURVEILLANCE IN DIABETES\n\nPopulation health data are essential to inform prevention strategies for diabetes and its complications, to identify trends in the development of diabetes and diabetes complications in the general population and in subpopulations, and to measure gaps in the translation of proven therapies into practice. NIDDK has partnered with CDC to support the SEARCH for Diabetes in Youth study. This multicenter study identifies cases of diabetes in people below 20 years of age in five geographically dispersed populations that encompass the ethnic diversity of the U.S.\n\nSEARCH has found that 1 out of every 523 persons under 20 years of age has diabetes, and 15,000 children are diagnosed with type 1 diabetes and 3,700 diagnosed with type 2 diabetes annually (7). SEARCH also provides data on trends in incidence and risk factor control in the pediatric diabetes population.\n\nSo that information on diabetes can be obtained at lower cost than if an independent study were initiated, NIDDK provides substantial support for the diabetes components of major CDC-led efforts including the National Health and Nutrition Evaluation Survey and the National Health Information Survey; partners with other components of NIH to enhance diabetes measures in ongoing studies; and offers support for investigator-initiated ancillary studies focused on diabetes. Of particular importance are collaborations with the National Heart, Lung, and Blood Institute (NHLBI) to address the increasing proportion of CVD attributable to diabetes and issues such as balancing the cardiometabolic risks and benefits associated with statin use.\n\nA third edition of the NIDDK publication *Diabetes in America* is currently in preparation. *Diabetes in America* provides a compilation and assessment of epidemiologic, public health, and clinical data on diabetes and its complications in the U.S.\n\n# RESEARCH TO PRACTICE TRANSLATION\n\nThere is a substantial gap between the results achieved in clinical trials and the outcomes in real-world settings. This is particularly true for the minority racial and ethnic groups and low socioeconomic status populations that suffer a disproportionate diabetes burden. Addressing this disparity is a major focus of our multipronged translation research program. Newly established Centers for Diabetes Translation Research will serve as a key component of our program to translate efficacious research findings into practice and the community to improve the health of Americans with\u2014or at risk for\u2014diabetes. In addition, R34 planning grants and R18 translational clinical trial grants offer a targeted mechanism to test strategies to improve the delivery of evidence-based therapies to improve diabetes management or prevention. These programs test innovative methods to improve clinical care and translate research findings into cost-effective and sustainable clinical treatment strategies, including community-based approaches to make preventive measures as widely accessible and practical as possible. NIDDK has also partnered with CDC to support the Natural Experiments for Translation in Diabetes (NEXT-D) Study, a research network designed to test the effectiveness and sustainability of population-targeted diabetes prevention and control policies emerging from health care systems, business and community organizations, and health care legislation. It includes large-scale natural experiments or effectiveness studies and rigorously designed prospective studies of diabetes prevention and control interventions. The National Diabetes Education Program, jointly sponsored by NIH and CDC, plays an important role in dissemination and translation of NIDDK-supported clinical research.\n\n# GENETICS OF TYPE 1 AND TYPE 2 DIABETES\n\nBecause knowledge of genetic risk factors has the potential for disease prediction, patient stratification, and insights into pathogenesis that can generate new approaches to prevention and treatment, NIDDK has expended considerable resources to apply state-of-the-art molecular and computational science to identify diabetes risk genes. Perhaps the most profound impact of these genetic discoveries has been in children with neonatal diabetes, who were often wrongly diagnosed with type 1 diabetes and treated with insulin. Now children with mutations in the genes encoding the SUR1 and Kir6.2 subunits of the potassium ion channel that regulates insulin secretion are treated more safely and effectively with sulfonylurea drugs rather than insulin (8). Genetic testing for type 1 diabetes risk has made possible the TEDDY study and TrialNet prevention studies described above. The NIDDK-led international Type 1 Diabetes Genetics Consortium helped increase the number of identified risk genes and gene regions from 3 only a decade ago to over 50 today. The challenge now is to understand how this variation contributes to disease pathogenesis opening up new therapeutic targets.\n\nFor type 2 diabetes, we are encouraged by the DPP finding that relative risk reduction with lifestyle change was as great in those carrying the high-risk TCF7L2 mutation as in other participants, despite higher rates of progression to diabetes. DPP also provided important pharmacogenetic data showing participants with a specific *KCNJ11* variant or alterations in metformin transport genes were less protected by metformin. These observations offer the possibility of individualizing therapy based on genotype (9). However, despite substantial progress in identifying genetic variation contributing to type 2 diabetes risk in populations of European origin, there is a critical lack of information about genetic variation contributing to type 2 diabetes in disproportionately affected minority populations. NIDDK has established a consortium of investigators to identify genetic variation contributing to type 2 diabetes in minorities. With known risk genes explaining only a small fraction of the genetic risk for type 2 diabetes, the identification of epigenetic changes that may play a role in the transmission of diabetes risk across generations is assuming increasing importance.\n\n# DIABETES COMPLICATIONS\n\nResearch has identified many pathways contributing to glucose-induced damage to endothelial cells including elevated flux through polyol and hexosamine pathways, accumulation of advanced glycation end products, activation of proinflammatory pathways, and inactivation of protein kinase C. Yet many questions remain about how these pathways may interact and converge to increase production of reactive oxygen species (ROS) in the mitochondria and how we can build on this new knowledge to develop therapies that can reduce ROS, reverse glycation, and lessen inflammation. Pathogenetic mechanisms, therapeutic targets, and biomarkers must be identified in specific tissues, including mechanisms of injury to specialized cells such as podocytes and pericytes. The relative importance of hyperglycemia and insulin resistance is of particular interest in understanding the link between diabetes and Alzheimer disease.\n\nA finite period of glycemic control has profound and long-lasting effects on the development of complications, a phenomenon termed metabolic memory (10). It has been proposed that the interaction of epigenetic changes with other persistent effects of hyperglycemia, such as glycation and oxidation of long-lived macromolecules, may explain this finding. Understanding pathways that contribute to metabolic memory may yield therapies targeted at the underlying molecular mechanisms. It may also help us learn about whether treatments that prevent the development of complications also prevent progression.\n\nThe course and development of long-term complications cannot be solely explained by the extent and duration of exposure to hyperglycemia. Genetic variation may explain why some people develop complications despite good control and others with poor control are protected. This information could yield undiscovered disease pathways and therapeutic targets, improve disease prediction over currently available clinical markers, and identify individuals for whom intensive therapy would be more or less beneficial. However, we know relatively little about genetic variation that may contribute to or protect from complications, and this is an important area for investigation.\n\nWe are also elucidating mechanisms that impair tissue repair and regeneration in diabetes, including dysfunction of endothelial and other stem cell populations. Identification of specific populations of stem or progenitor cells affected by diabetes and the extent to which damage is reversible is vital for understanding how complications of diabetes might be reversed by stimulating formation of normal new vessels and regrowth of nerves. Differentiation of induced pluripotent stem cells also holds promise for the repair of damaged tissues.\n\nWhile rates of blindness, kidney failure, amputation, and CVD have fallen substantially in those with diabetes, population-wide lowering of CVD has outpaced that in people with diabetes and the share of CVD in the U.S. attributable to diabetes is rising. To address this challenge, NIDDK and NHLBI have collaborated in a number of major multicenter trials to establish effective approaches to reducing CVD in those with type 2 diabetes, including Action for Health in Diabetes (Look AHEAD), Action to Control Cardiovascular Risk in Diabetes (ACCORD), and Bypass Angioplasty Revascularization Investigation in Type 2 Diabetes (BARI 2D). Results from ACCORD and other recent large clinical trials attempting to prevent CVD did not demonstrate a benefit of intensified near-normal glucose control on clinical CVD events in people with moderate to long-term diabetes duration and moderate to high CVD risk. More information is needed on the impact of diabetes duration and preexisting tissue damage on the ability to respond to therapies. Beyond the practical management questions addressed in clinical trials, new approaches to uncouple diabetes and CVD must be based on a mechanistic understanding of factors linking these conditions, including obesity, inflammation, insulin resistance, metabolic perturbations, altered coagulation, neuropathy, and nephropathy, and how the pathophysiology of atherosclerosis differs between people with type 1 and type 2 diabetes.\n\n# THE \u03b2-CELL\n\nImpaired insulin production is key to all forms of diabetes. The extent to which it is possible to preserve and\/or restore \u03b2-cell function early in the course of diabetes and whether \u03b2-cell recovery is possible later in the disease remains to be established. Both the nature and optimal timing of interventions to preserve \u03b2-cell function and the impact on clinical care and outcomes must be addressed. NIDDK has recently established the Restore Insulin SEcretion (RISE) consortium to explore approaches to remission of insulin secretory function early in type 2 diabetes. Investigators will study both pharmacologic interventions and bariatric surgery. The Type 1 Diabetes TrialNet studies approaches to slow \u03b2-cell loss early in the course of type 1 diabetes. Specific immunomodulatory therapies have been shown to preserve C-peptide, and additional strategies including intense metabolic control with continuous glucose monitoring and pump therapy at onset of disease are being investigated.\n\nMechanistic studies are necessary to understand why \u03b2-cells lose their ability to secrete insulin as well as the physiology underlying recovery and preservation of endogenous insulin secretion. Particularly encouraging is the finding of some residual C-peptide production in a substantial proportion of patients with long-established type 1 diabetes. Such individuals might be amenable to novel strategies under development to stimulate islet neogenesis with the potential to improve \u03b2-cell mass and function. The intriguing observation of diabetes remission after some forms of bariatric surgery must be investigated to establish durability of the effect and the characteristics of patients and procedures associated with remission. Elucidation of mechanisms underlying improved \u03b2-cell function after bariatric surgery is being pursued in both human studies and animal models potentially generating new approaches to restore \u03b2-cell function. Moreover, the recent identification of platelet-derived growth factor as a factor involved in the replication of \u03b2-cells in early life that are lost over time offers a potential pathway to \u03b2-cell regeneration (11).\n\nClinical studies of approaches to mitigate \u03b2-cell loss and\/or restore function could be accomplished much more efficiently if more reliable biomarkers or methods to image \u03b2-cell mass or function were available. The development of such tools has been and continues to be a high priority of NIDDK. It may be facilitated by the identification of proteins expressed in \u03b2-cells and \u03b2-cell surface markers and antibodies through the Beta Cell Biology Consortium (<http:\/\/www.betacell.org>). This consortium is pursuing a multifaceted approach to correct the loss of \u03b2-cell mass in diabetes, including cell reprogramming, regeneration, and replacement. It is also supporting research to develop mouse models in which development and function of human islets can be studied. Also, because of differences between murine and human islets, NIDDK has established a resource, the Integrated Islet Distribution Program (<http:\/\/iidp.coh.org>), to make human cadaveric islets available to the research community.\n\nThe Clinical Islet Transplantation Consortium, a joint effort of NIDDK and the National Institute of Allergy and Infectious Diseases (NIAID), is fostering development of islet transplantation as a cure for type 1 diabetic patients whose disease cannot be effectively managed with current methods of insulin administration or who have received a successful kidney transplant. Its ongoing trials aim to improve the methods of isolating and administering islets and minimizing the toxic effects of immunosuppressive drugs required for transplantation. NIDDK also supports collection, analysis, and communication of comprehensive and current data on all islet\/\u03b2-cell transplants performed in North America, as well as some European and Australian centers through the Collaborative Islet Transplant Registry. This clinical islet transplantation research is entirely supported through the special appropriation for type 1 diabetes research.\n\n# OBESITY\n\nA trans-NIH Task Force cochaired by NIDDK, NHLBI, and NICHD coordinates NIH efforts to identify genetic, behavioral, and environmental causes of obesity to understand how obesity leads to type 2 diabetes, CVD, and other serious health problems and to build on basic and clinical research findings to develop and study innovative prevention and treatment strategies. One key goal is to understand how biologic, cognitive, behavioral, social, and physical environmental factors interact to influence the development of obesity. For example, how do diet, exercise, and other factors influence reprogramming of neural circuits involved in regulating food intake and thermogenesis. Another goal is to identify distinct strategies that may be needed for achieving weight loss, maintaining weight loss, and preventing weight gain. Understanding responses to weight change that contribute to the very high recidivism to obesity may lead to effective strategies for maintenance of reduced body weight. These therapies might be quite different from those used to induce weight loss per se.\n\nChildhood obesity has fueled the rise of type 2 diabetes in teens and young adults. An NIDDK-led randomized trial testing an intervention to improve nutrition and physical activity in middle schools serving high-risk children demonstrated efficacy in secondary outcomes, reducing BMI *z*-score and other indices of adiposity. However, the primary outcome of the combined prevalence of overweight and obesity decreased in both the intervention and control schools perhaps due to information about the participating children's health, which was sent to all families (12). Family-based interventions have been successful for childhood weight control, but strategies for translation and widespread implementation of such interventions in high-risk populations remain to be developed. The roles of technologies such as smartphones and social networking and of community organizations must be evaluated for their potential to support individualized and tailored delivery of interventions outside of the clinical setting.\n\nAt the molecular level, there has been an explosion of knowledge about the mechanisms linking obesity to insulin resistance and excitement about potential therapeutic manipulation of adipose tissues based on the understanding of white adipose tissue heterogeneity, persistence of brown adipose tissue into adulthood, and metabolic flexibility of adipocytes. Tools and techniques have enabled researchers not only to define adipose anatomy and morphology but also to examine its dynamic function. Yet key questions remain about the mechanisms that determine adipocyte number and size, govern development and distribution, and link variation in body fat deposition to metabolic sequelae and macrophage recruitment and activation.\n\nParticularly challenging but of utmost importance are studies to understand mechanisms by which obesity, hyperglycemia, or other metabolic factors in pregnant women may predispose their offspring to obesity or diabetes. Research is needed on how placental biology and the intrauterine environment shape neural circuits, adipose tissue, and islet development in the fetus. Studies relating differences in energy homeostasis and body composition to genetic variation and defining the critical developmental periods for imprinting maladaptive metabolic changes will contribute to understanding how metabolic fate may be programmed early in development.\n\nThe finding that Roux-en-Y gastric bypass not only causes profound weight loss but can also restore euglycemia through mechanisms that appear independent of weight loss makes identification of these mechanisms a very high priority with the potential to uncover new therapeutic pathways to treat and possibly reverse type 2 diabetes. NIDDK is pursuing this through clinical research on the response to various bariatric surgical procedures and through murine studies in which examination of defined procedures in genetically altered mice enables examination of the roles of specific pathways in the metabolic outcomes of these surgeries. Understanding the hormonal and neural controllers of energy balance will be key to designing potential drug combinations targeting multiple components of the regulatory system with additive or synergistic effects.\n\n# DIABETES RESOURCES\n\nNIDDK supports numerous resources to improve the quality and multidisciplinary nature of research on diabetes by providing shared access to specialized resources. The NIDDK-supported Diabetes Research Centers, formerly known as Diabetes Endocrinology Research Centers and Diabetes Research and Training Centers, provide increased and cost-effective collaboration among multidisciplinary groups of investigators at institutions with an established, comprehensive research base in diabetes and related areas of endocrinology and metabolism. The National Mouse Metabolic Phenotyping Centers (<http:\/\/www.mmpc.org>) provide a range of complex exams used to characterize mouse metabolism, hormones, energy balance, eating and exercise, organ function and morphology, physiology, and histology. NIDDK supported research consortia, such as the Beta Cell Biology Consortium (<http:\/\/www.betacell.org\/>) and the Nuclear Receptor Signaling Atlas (<http:\/\/www.nursa.org\/>), provide data and reagents to the broader scientific community. Other important diabetes resources supported by NIDDK include a type 1 diabetes mouse repository at The Jackson Laboratory (<http:\/\/www.jax.org\/t1dr\/>) and Islet Cell Resource Centers (<http:\/\/icr.coh.org\/>) that provide human islets for research.\n\nSamples and data from the limited number of cohorts from large diabetes studies with well-characterized phenotypes at baseline and with longitudinal measurement of characteristics of interest are highly prized. To expand the usefulness of these major clinical studies by allowing the wider research community to access study materials beyond the end of the study or after a limited proprietary interval for ongoing studies, NIDDK has established biosample, genetics, and data repositories. Recently NIDDK has gone beyond the repository concept to create a living biobank, which provides investigators with the opportunity to obtain \"on-demand\" biological samples from selected individuals. This effort builds on the unique population of individuals at risk for the development of type 1 diabetes ascertained and monitored through TrialNet. The unprecedented availability of such samples and data may allow immunologists to understand early inciting events in type 1 pathogenesis.\n\n# APPLYING NEW TOOLS AND TECHNOLOGIES TO DIABETES RESEARCH AND PATIENT CARE\n\nLarge clinical trials have established the long-term benefits of intensive blood glucose control in lowing the risk of diabetes complications. However, insulin therapy is burdensome and limited by hypoglycemia. NIDDK has devoted considerable resources to develop technologies for accurate and rapid detection of glucose levels and appropriate adjustment of insulin delivery to create an artificial pancreas that simulates the functions of \u03b2-cells. Achieving this goal will require more accurate and robust glucose-sensing devices; more effective and rapidly acting insulin preparations; algorithms that align real-time glucose measurements with adjustment in insulin delivery; infusion devices that deliver insulin more effectively, conveniently, and physiologically; and fail-safe mechanisms to avoid hyper- or hypoglycemia. It will also be important to determine the benefit of combining insulin delivery with the delivery of the counterbalancing hormone glucagon to reduce hyperglycemia, and how timely transmission and remote interpretation of patient data may contribute to safety and efficacy. Research is ongoing to assess the capacity of current artificial pancreas technology to improve overall metabolism, increase patient well-being, restore hypoglycemia awareness, and preserve existing pancreatic \u03b2-cell function, as well as to understand the factors affecting its use and acceptance in different age-groups.\n\nAdvances in sensors, processors, memory storage, wireless communication, and Web-based data transport, processing, and sharing have applications not only to new therapies but also to many facets of diabetes research in free-living populations. These range from instruments that measure energy intake and physical activity to the application of continuous monitoring of blood glucose to enable exploration of questions about the impact on human health of glycemic excursions, which may not be captured by HbA~1c~ measurement. Such studies could address the question of whether and how hypoglycemia may contribute to CVD events. Studies of energy balance would benefit from tools to define the neural circuits and molecular mediators that regulate energy balance by sensing and responding to signals of energy status and tools to quantitate mitochondrial biogenesis and turnover and assess mitochondrial function. To assess the progression of diabetes, measures that directly measure \u03b2-cell mass are particularly important because current methods are all linked to \u03b2-cell function, which may have both reversible and irreversible components. Complications research could benefit from tools for the study of extracellular matrix proteins and their interactions with growth factors and circulating stem cells or for the study of epigenetic change or glycation and lipoxidation of proteins. Appropriate systems biology and computational tools are needed to facilitate the integration of sequencing, expression, proteome, and metabolome profiles to identify key biologic processes and their interactions. NIDDK seeks to foster development of paradigm-shifting technology and truly transformative tools through workshops, targeted funding opportunity announcements, and interdisciplinary research grants.\n\n# DIABETES AS A GLOBAL HEALTH ISSUE\n\nDiabetes is a universal problem. The impact of diabetes is rapidly growing among populations in developing and middle-income countries; without action, deaths and disability due to diabetes will continue to increase substantially. NIDDK promotes international collaboration between investigators in the U.S. and scientists in other countries to develop and test strategies to stem the epidemic of diabetes at home and globally. Many other countries have health care and medical records systems that are particularly useful for clinical research on diabetes. NIDDK has collaborated globally on type 1 diabetes research through networks such as TEDDY and TrialNet to expand access to research participants and gain insight from research collaborators. Genetic research on diabetes also knows no boundaries and our research efforts have benefited from combined analysis of international cohorts. Global collaboration on type 2 diabetes is particularly relevant to understanding diabetes in immigrant and minority populations in the U.S. International collaborations offer unique opportunities to compare effects of different environmental exposures and understand why specific populations may be particularly vulnerable to diabetes.\n\n# CONCLUSION\n\nDaunting as the challenge of diabetes appears, research has tremendously improved outcomes for people with the disorder. Were it not for declining rates of kidney disease, amputation, and CVD in people with diabetes, the burden associated with increased diabetes prevalence would be much greater. NIDDK recognizes the importance of collaboration with other components of NIH, other government agencies, and the diabetes voluntary organizations to realize the research progress. Our challenge is to stem the growing tide of diabetes through research ranging from understanding the fundamental processes underlying diabetes and its complications to studies of practical approaches to combat diabetes in medical and community settings.\n\n## ACKNOWLEDGMENTS\n\nNo potential conflicts of interest relevant to this article were reported.\n\n# REFERENCES","meta":{"dup_signals":{"dup_doc_count":134,"dup_dump_count":49,"dup_details":{"curated_sources":2,"2022-05":1,"2021-39":1,"2018-30":1,"2018-26":2,"2018-22":1,"2018-17":3,"2018-13":1,"2018-09":3,"2018-05":2,"2017-51":2,"2017-47":2,"2017-43":3,"2017-39":3,"2017-34":3,"2017-30":2,"2017-26":4,"2017-22":3,"2017-17":4,"2017-09":6,"2017-04":6,"2016-50":5,"2016-44":6,"2016-40":6,"2016-36":6,"2016-30":5,"2016-26":5,"2016-22":5,"2016-18":2,"2016-07":1,"2015-48":2,"2015-40":1,"2015-35":2,"2015-32":1,"2015-27":2,"2015-22":1,"2015-14":2,"2014-52":2,"2014-49":2,"2014-42":4,"2014-41":3,"2014-35":2,"2014-23":1,"2014-15":2,"2023-06":1,"2017-13":3,"2015-18":2,"2015-11":2,"2015-06":2,"2014-10":1}},"file":"PMC3554357"},"subset":"pubmed_central"}
{"text":"author: Eugene J. Barrett; Stephen RattiganCorresponding author: Eugene J. Barrett, <firstname.lastname@example.com>.\ndate: 2012-11\nreferences:\nsubtitle: : Its Measurement and Role in Metabolic Regulation\ntitle: Muscle Perfusion\n\nMethods for measuring muscle blood flow have been evolving over the past 120 years (1,2). Studies of hormonal regulation of muscle flow and metabolism began with the classical work by Andres et al. (3). Numerous diabetes investigators interested in muscle metabolism in vivo have estimated the net balance of glucose and other metabolites across a skeletal muscle bed or limb from the product of the arterial-venous concentration difference and the blood flow. In this review, drawing upon early studies, we will emphasize some of the principles and limitations of various techniques for measuring flow to estimate net exchange or rates of production or consumption of metabolites. <a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a> summarizes pertinent strengths and limitations of the most commonly used methods for estimating either muscle blood flow or perfusion. From later studies, we will deal more directly with the issue of how flow is hormonally regulated and the relationship between skeletal muscle flow regulation and metabolic regulation. That discussion will extend beyond flow alone as an important regulated variable, emphasizing instead perfusion, which encompasses both the rate and distribution of blood flow in a tissue. We will highlight some of the new methodologies that have helped clarify further the linkage between the regulation of skeletal muscle perfusion and metabolic function.\n\nMethods for measurement of limb and muscle blood flow\n\n![](2661tbl1)\n\n# Limb balance measurements identify sites of insulin action and resistance\n\nIt is appropriate to begin this discussion with the development of the forearm balance technique by investigators at Johns Hopkins in the early 1950s. These investigators put forward the hypothesis that through continuous infusion of a dye \"tracer\" (in this case Evans blue dye) that binds tightly and rapidly to serum proteins into the brachial artery and sampling from an ipsilateral antecubital vein, blood flow to the forearm could be quantified using simple spectrophotometric methods (3). They pointed out several advantages to the forearm for such studies, including *1*) that skeletal muscle makes up the preponderance (\\~80%) of the tissue mass of the forearm; *2*) the forearm's relatively small mass and slow blood flow allow infusion of very small amounts of dye, which minimizes the contribution of recirculating dye; and *3*) the vascular anatomy of the forearm is well understood and in \\>80% of individuals bifurcation of the brachial artery occurs below the antecubital crease, and therefore infusion of dye above the elbow should distribute to both the radial and ulnar vessels. Their measurements of flow corresponded well with the plethysmographic measurements that were available at that time. Plethysmography measures blood flow from the time-dependent increase in volume of a segment of a limb after venous outflow occlusion using either a strain gauge or other detection device. The development and application of plethysmographic limb flow measurements have recently been excellently reviewed (4). As there is no gold standard for measuring flow in clinical studies, cross-validation between methods provides needed assurance.\n\nIn these dye dilution studies, the issue of dye mixing in the brachial artery was extensively examined (3), as adequate mixing is clearly required for accurate blood flow measurements. Despite the finding that dye streaming occurred at the infusion rate used, it was observed by simultaneously sampling from multiple forearm veins that adequate mixing had occurred in most subjects. Interestingly, use of a jet injector to promote mixing of the infusate at the arterial injection site provoked downstream vasodilation (perhaps secondary to ATP or adenosine released by the endothelium traumatized by the jet shear) and was abandoned. Traction on the arterial catheter also altered downstream arterial resistance and flow, underscoring that care must be taken with this method.\n\nCombining this dye dilution method with arterial-venous (A-V) metabolite sampling allowed estimation of the substrate balance across the forearm (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<\/a>). These \"limb balance\" studies took advantage of the fact that the forearm receives only approximately one-fiftieth of the cardiac output (5). As a result, infusion of low doses of insulin (e.g., 0.05 mU\/min\/kg body wt) into the brachial artery provoked physiologically significant increases in plasma insulin concentrations bathing the forearm musculature, but when diluted in the whole-body plasma pool it had minimal or no effect on plasma glucose, potassium, or other metabolite concentrations. The same circumstance does not pertain for infusion of insulin into the femoral artery when leg balance measurements are made. The leg's greater mass and blood flow require higher rates of insulin infusion, and the insulin recirculates and affects plasma glucose and other metabolites. Using this forearm balance method a decade before they developed the insulin clamp (6), these investigators demonstrated that physiologic doses of insulin stimulated skeletal muscle glucose uptake under euglycemic conditions in humans (5,7) and that this action of insulin was impaired in obese adults (7).\n\nWithin a few years, other laboratories had begun to apply this limb balance method. In particular, the Cahill laboratory (8\u201310) at the Joslin Clinic and its diaspora (11\u201313) used limb balance measurements very effectively to study the actions of glucoregulatory hormones and exercise. Limb blood flow was measured by indocyanine green dye dilution or plethysmography. It is worthwhile noting that while these studies frequently ascribed the observed substrate use or production to muscle, other tissues within the limb can contribute to this measurement by either adding or removing metabolites and by contributing to the flow measurement. The former is perhaps less of an issue for the forearm where the deep antecubital vein cannulated in a retrograde fashion drains predominantly skeletal muscle, while the femoral vein is draining all muscle, adipose, and osseous tissues of the leg. However, to the extent that the A-V difference measurement selectively samples blood draining muscle (as it does in the forearm with retrograde deep forearm vein cannulation), multiplying the A-V difference for a metabolite by total forearm flow may not give an accurate balance if muscle and fat do not handle the metabolite in an identical fashion. It is important to recognize that when either forearm or leg metabolism is studied using the limb balance method, results are typically expressed as millimoles of substrate per minute per 100 mL of forearm or leg. As sex (14), age, and body habitus (15) influence the relative proportion of skeletal muscle versus adipose tissue present in the limb, caution needs to be used regarding ascribing metabolite exchange across the forearm or leg to a particular tissue type. In this regard, it bears keeping in mind that estimated rates of blood flow to subcutaneous adipose tissue and resting skeletal muscle are comparable (\u223c3\u20135 mL\/min\/100 g) (16) and glucose uptake to subcutaneous adipose tissue contributes significantly to body glucose disposal (17,18). Along this line, leg blood flow (milliliters per kilogram of leg) is not different between men and women, despite greater fat-free mass in males (19). These cautions and limitations pertain whether one is studying forearm or leg balances. With exercise, muscle blood flow can increase by \\>20-fold.\n\nUsing the leg balance method combined with the euglycemic insulin clamp, investigators demonstrated that leg glucose uptake could account for \u223c40% of body glucose disposal and suggested that muscle was principally responsible. Extrapolating from leg muscle mass to total body muscle, they concluded that during steady-state hyperinsulinemia, muscle accounted for 80\u201390% of glucose disposal (20). Leg glucose uptake was also found to be a major site of insulin resistance in both obesity and diabetes. The combined measurements of bulk blood flow together with the A-V substrate concentration differences proved to be a powerful tool for quantifying the metabolism of carbohydrates (21), fats (22), amino acids (23), and oxygen (24) by the limb tissues. Either dye dilution or plethysmographic methods were used. Whereas only modest data are available in the literature comparing these two methods for measurement of bulk blood flow, they do appear to yield comparable values for bulk limb blood flow (25).\n\nThe introduction of simultaneous A-V measurement of radiolabeled substrate with limb blood flow added yet another dimension to the limb balance technique. These methods were initially used to measure fatty acid uptake and oxidation by exercising muscle (26) and later to quantify both the transport and net uptake of glucose (27), free fatty acid (FFA) (28), and amino acids and subsequently to quantify rates of protein synthesis and degradation (29), of lipolysis and lipogenesis (22), and of oxidative and nonoxidative glucose disposal (30). Once again, the quantitative accuracy of these measurements hinges entirely on the measurement of blood flow. In aggregate, these studies demonstrated the great utility of the limb balance method to quantify metabolic events in peripheral tissue and to discover how they are regulated in health and disease.\n\nWith improvements in ultrasound methodology, Doppler flow measurements have become a third widely used method for measuring bulk limb blood flow, which has increasingly displaced the more invasive dye dilution technique. Doppler flow measurements were observed to correlate well with plethysmographic measurements (31).\n\nMuscle perfusion can be evaluated by various nuclear magnetic resonance (NMR) techniques. One of the most useful, because of its ability to determine both blood flow and flow distribution noninvasively, is arterial spin labeling (ASL). This technique can be used in humans and experimental animals (32). It has the potential to be directly compared with metabolism determined by other NMR techniques in the same region of tissue but not simultaneously (32). The ASL technique has been shown to correlate reasonably well (*r*^2^ = 0.85) with leg blood flow measured by venous plethysmography in humans (33) and well (*r*^2^ = 0.95) with limb blood flow in a rat-perfused hindlimb model (34). However, due to the expense of specialized NMR equipment and the substantial complexity of postacquisition data processing required, this technique has not been widely used by physiologists or clinical researchers.\n\nMuscle perfusion has also been measured by microdialysis where a semipermeable membrane is placed in the tissue and perfused with dialysate that contains markers such as ethanol or radiolabeled water. The exchange and recovery of these markers can be used to estimate total blood flow (35) and distribution of nutritive and nonnutritive blood flow (36). Combined with other tracers, the microdialysis technique can also provide information on the interstitial concentrations of metabolic and vasoactive moieties such as glucose, glycerol, lactate, amino acids, insulin, nitric oxide (NO), and adenosine (37). The technique, however, has several limitations that include its invasive nature; lack of spatial resolution, as it is difficult to determine the amount of tissue being sampled; and temporal resolution because of the long sampling times (minutes) required to collect sufficient dialysate for measurement.\n\n# Perfusion as A regulator of muscle metabolism\n\nA conceptual shift in the consideration of the role of blood flow in muscle metabolism occurred with studies from the laboratory of Alain Baron. He modified a method first used by Andersen and Saltin (38) for quantifying leg blood flow, which relied on a use of a thermodilution catheter introduced into the femoral vein (39). Thermodilution methods had previously been used extensively to measure blood flow in vascular beds other than skeletal muscle. While invasive, this methodology allowed very frequent measurements of blood flow during the course of prolonged metabolic balance studies. The design of the catheter allowed spraying of the chilled saline infusate to assure good mixing and nonlaminar flow prior to the temperature-sensing probe. With this technique, Baron observed that insulin not only increased glucose uptake by leg muscle in healthy individuals but also simultaneously increased blood flow. Further increasing flow with cholinergic stimulation during hyperinsulinemia significantly augmented leg glucose uptake (40). He and collaborators subsequently demonstrated that insulin increased flow by stimulation of NO production and that this flow increase was inhibited in type 2 and type 1 diabetes and obesity (39) and by experimentally induced insulin resistance (rev. in 41).\n\nThis work strongly suggested that insulin's action to promote the uptake of glucose, amino acids, and other substrates into muscle was aided by the concerted action of insulin on the vasculature to increase blood flow and presumably the delivery of insulin, glucose, and other metabolites to the muscle (41). The observation that this vascular action of insulin was blocked by inhibitors of NO synthase (NOS) (42\u201344) suggested that the endothelium was the target for insulin's vascular action. Subsequent studies in isolated endothelial cells demonstrated that insulin, acting via the phosphatidylinositol-3 kinase Akt pathway (45,46), specifically enhanced the activity of NOS (<a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<\/a>).\n\nWhile these observations of insulin's effect of increasing total limb blood flow were confirmed by a number of laboratories (47,48), concerns were raised and controversy ensued around both the reported need for prolonged, relatively high steady-state insulin concentrations to provoke insulin's vascular action relative to effects on glucose disposal (49,50) and the lack of effect of increasing flow pharmacologically on limb glucose uptake in insulin-resistant (51,52) subjects.\n\nStudies using positron emission tomography (PET) methods by investigators in Turku, Finland, were particularly informative, as PET allowed simultaneous measurements of perfusion, using labeled water (H~2~^15^O) and glucose (^18^F-deoxyglucose) within voxels of muscle, avoiding the issue of tissue heterogeneity and bulk flow distribution to multiple tissues in the limb (51,53).\n\nRadiolabeled or fluorescent microspheres have also been used in a number of animal studies to examine the effect of insulin on blood flow (54\u201356). As the technique requires tissue removal, it has not been applicable to clinical studies. However, results of published studies confirm that insulin can enhance skeletal muscle blood flow. Inasmuch as the microspheres used typically measure 15 \u00b1 3 \u00b5 in diameter, it is not clear that they have access to the capillary bed; they are more likely retained within the terminal arterioles. Multiple measurements can be made in a single animal provided different isotopes or fluorescent labels are available whose emissions can be distinguished by differences in energy spectrum. Of interest is the study by Liang et al. (55), which included measurements of the effects of insulin on multiple tissues. Insulin-induced increases in blood flow during euglycemia were most apparent in skeletal and cardiac muscle.\n\nSince the various laboratories examining the relationship between insulin's effect on limb blood flow and metabolism used different methods for measuring flow (e.g., strain gauge plethysmography, dye- or thermodilution, or PET), some of the discordant findings may have arisen from these measurements. This issue was never fully settled. However, additional opportunities became available with the development of techniques to measure the distribution of flow within a tissue rather than simply bulk flow to the tissue. The observation that the volume of muscle microvasculature perfused was regulated dates back to August Krogh (57). Changes in the volume of muscle microvasculature perfused could influence hormone and nutrient exchange within the muscle by altering the endothelial surface area exposed.\n\nAs shown in <a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<\/a>, the muscle microvasculature consists of third- and fourth-order arterioles, the capillary network, and small venules. Each terminal arteriole supplies multiple capillaries in an orderly arcade. At any given moment, only approximately one-third of the capillaries appear to be actively perfused in resting muscle (58). There is no conclusive evidence for the presence of smooth muscle \"sphincters\" at the capillary origin. Instead, it appears likely that many capillaries are functionally at least transiently not perfused because of *1*) the residual tone limiting flow to the terminal arteriole, *2*) the position of a particular capillary relative to the inflow into the arteriole, *3*) the interstitial pressure around the capillary, *4*) the intrinsic resistance of the capillary, and *5*) the residual pressure within the lumen of the draining venule. These factors in aggregate likely determine whether an individual capillary will be perfused at a particular time. Relaxation of the terminal arteriole, with consequent increases in precapillary pressure, will lead to perfusion of previously under- or unperfused capillaries. Time variable perfusion of terminal arterioles, a process termed \"vasomotion\" or \"flowmotion,\" appears to be due to the action of sympathetic nervous input, intrinsic myogenic responses in the vascular smooth muscle, and autoregulation by the endothelium within terminal aspects of the microvasculature conducting signals retrograde through gap junctions to signal to feed arterioles when some relaxation is needed to at least transiently restore perfusion (59). While intermittent perfusion of muscle microvasculature has been difficult to demonstrate in intravital microscopy studies of surgically exposed thin muscles (60), the use of noninvasive contrast ultrasound (see below) appears to have resolved this issue in larger muscle groups in both human and animal studies.\n\nJust over a decade ago, our laboratories began collaborating to develop and apply two methodologies for assessing the volume of vasculature perfused within muscle. The first relied on measurement of the A-V concentration difference of intravenously infused 1-methyl-xanthine (1-MX). Xanthine oxidase in the endothelial cell converts 1-MX to 1-methylurate, and the extent of metabolism provides an index of the endothelial surface perfused. Insulin was found in rats to increase 1-MX metabolism (61), and this was inhibited by factors that acutely provoked metabolic insulin resistance, including \u03b1-methyl serotonin (62), tumor necrosis factor-\u03b1 (63), elevated FFA concentrations (64), and NOS inhibition (65), and with chronic obesity (66). This method, however, was not found to be useful in humans because of the substantially lower extraction ratio for 1-MX across the skeletal muscle capillary bed.\n\nWe subsequently applied the technique of contrast-enhanced ultrasound (CEU) to the measurement of microvascular blood volume in both rodent and human studies. CEU was developed initially to image myocardial perfusion. The method relies on the insonation of intravenously infused, perfluorocarbon\u2013filled lipid microbubbles, which enhance the video intensity of ultrasound images. These microbubbles are typically 1\u20134 \u00b5 in diameter, remain within the vasculature, and have rheologic properties similar to erythrocytes. Most importantly, the microbubbles oscillate or rupture (depending on the energy of the ultrasound signal) when exposed to the ultrasound beam. This behavior is responsible for an intense reflected sound wave that enhances the image intensity.\n\nUnlike Doppler, plethysmography, PET, or tracer dilution methods, CEU does not measure volume flow, i.e., milliliters per minute per 100 mL tissue. Therefore, CEU cannot be used to provide an estimate of blood flow that could be coupled with A-V difference measurements to obtain a limb balance. Rather, it provides a sensitive index of the volume of microvasculature (and therefore the microvascular surface area available for nutrient exchange) that is perfused. It is most useful for comparing acute responses to interventions like increasing plasma insulin, exercise, feeding, etc. In this regard, it has proven quite useful for examining the action of insulin on the microvasculature. Similar to PET, CEU allows study of a region of interest within the muscle or adipose tissue. However, while PET signal intensity in muscle can be calibrated to the activity in blood measured simultaneously yielding a flow measurement, this is not available with CEU. However, CEU affords several advantages over PET scanning for clinical studies. It is less expensive, involves no radioisotopes, is portable, and the image analysis is substantially simpler. Perhaps most importantly, it allows separation of signals arising from larger conduit and feed arteries from signals arising from the microcirculation. This separation is based on capturing a \"replenishment curve,\" i.e., a time series of images that is initiated by instantaneously destroying all microbubbles in the volume being studied and following the microbubble replenishment and recovery of videointensity until it reaches a plateau. The plateau signal intensity is reached when all vessels in the tissue that are being perfused have again filled with blood containing microbubbles. Since flow in conduit and feed arteries is rapid, these fill quickly, while the capillaries and venules refill slowly. The image intensity created by vessels that fill rapidly can straightforwardly be subtracted from that seen at the plateau, with the difference being due to microvasculature containing microbubbles. The flow velocity for the replenishment curve is also provided by the image analysis, and flow is the product of flow velocity and volume perfused. However, both velocity and volume are measured in videointensity units, and their product is not readily converted to units of volume flow.\n\nIn a series of studies using either contrast ultrasound or the A-V concentration difference for 1-MX as an index of the endothelial surface perfused, we observed that insulin at physiologic concentrations enhanced the microvascular volume perfused within either human forearm (67) or rat hind leg muscle. This occurs within 15 min of onset of hyperinsulinemia (68,69) at modest physiologic insulin concentrations (70) and is blocked by inhibition of NOS (65). Importantly, loss of insulin's ability to enhance microvascular volume either by a blockade of NOS or as a result of endogenous insulin resistance secondary to obesity (71), diabetes (72), or experimental conditions (e.g., elevating FFA \\[73\\] or tumor necrosis factor-\u03b1 concentrations) impedes skeletal muscle glucose disposal. This recruitment process is illustrated in <a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<\/a>.\n\nThe simplest explanation for this apparent relationship between the microvascular volume perfused within skeletal muscle and metabolic insulin resistance arises from understanding that the delivery of glucose and insulin to skeletal muscle appears to be a limiting process for insulin action within skeletal muscle (6,74). Increasing the endothelial surface area and thereby enhancing insulin delivery could facilitate both the time of onset and the magnitude of the metabolic response to insulin. There may in addition be factors within the endothelium that relate to insulin transendothelial transport that are adversely affected by insulin resistance and contribute further to the metabolic disarray.\n\nThere are two other potential techniques that could be used to measure the volume of vasculature perfused within skeletal muscle. The first is an adaptation of PET in which ^15^O-labeled carbon monoxide is inhaled and binds rapidly to hemoglobin. In this manner, the PET tracer becomes a vascular tracer. With this technique, investigators have observed that insulin increases the intensity of the PET signal consistent with an increase in the volume occupied by vasculature within a given voxel. While statistically significant effects were seen, the magnitude of the effect is substantially less than is seen with CEU. This may relate to the fact that much of the PET signal appears to arise from larger blood vessels and there is no straightforward way to subtract out this component. Additionally, in order to have sufficient signal, the PET image is collected over a substantially longer period of time, and this exposure is longer than the mean transit time for blood traversing skeletal muscle. As a result, flowmotion within the tissue slice may lead to capturing signal from vessels that are only open a portion of the time. Perhaps because of these difficulties, very little has been done with this technique to examine microvascular perfusion within skeletal muscle using carbon monoxide\u2013labeled tracers.\n\nNear-infrared spectroscopy provides yet another potential methodology. This technique relies on the deeper penetration of near-infrared electromagnetic radiation into tissue compared with visible or ultraviolet light. It has been used extensively with pulse oximetry, and it can measure the relative amounts of oxyhemoglobin and desoxyhemoglobin. When coupled with venous occlusion, it can be used to determine the total rate of inflow of oxygenated blood to a muscle and potentially the microvascular volume filled. To our knowledge, it has not been applied to studies of insulin or low-intensity exercise\u2013mediated capillary recruitment within muscle. There is concern regarding how much contribution skin and subcutaneous adipose tissue make to the near-infrared spectroscopy signal, and this would be a particular issue in obese individuals and females.\n\n# Parallel effects of insulin and contraction on microvascular recruitment\n\nIt has long been known that exercise acts synergistically with hyperinsulinemia to enhance glucose disposal. Multiple mechanisms within muscle account for this including increased glycolytic activity, recruitment of GLUT4 transporters to the plasma membrane, and increased tissue perfusion. The impact of expanding microvascular volume by recruitment of previously unperfused or underperfused capillaries on delivery of insulin to muscle tissue can be appreciated from studies looking at the effect of microvascular recruitment induced by muscular contraction. We recently demonstrated that in the rat hind limb, even very modest contraction enhances the delivery of both insulin and albumin to the muscle interstitium (75). We were particularly intrigued by the parallel between low-dose insulin and very light exercise, which both stimulated microvascular recruitment within muscle without affecting total blood flow to the tissue. In contrast, more intense exercise (75,76) and higher insulin concentrations increase both microvascular recruitment and total blood flow. This suggests that there is an orderly, staged vascular response to either stimulus, with the microvasculature being more sensitive to either stimulus than larger resistance vessels, which regulate total flow.\n\nBoth microvascular recruitment and enhanced blood flow could increase the delivery of insulin, glucose, and other metabolites to skeletal muscle, but there may be some advantage to having a staged response with very modest increases in insulin or exercise. Capillary recruitment would not require simultaneous increases in cardiac output to prevent a fall in systemic arterial pressure. For insulin, this may be helpful particularly in the postprandial setting when there is already a demand for increased blood flow to splanchnic tissues. However, when the stimulus is particularly strong (as occurs with high concentrations of insulin or intense exercise), other systemic vascular regulatory responses must be called into play to maintain vascular homeostasis.\n\nIncreases in microvascular volume secondary to microvascular recruitment are not restricted to skeletal muscle. With use of contrast ultrasound, insulin has been observed to increase microvascular volume within cardiac muscle (77) and subcutaneous adipose tissue of the leg (78). Using an entirely different approach, investigators in the Netherlands have demonstrated increased numbers of perfused capillaries within the nail fold as captured by video microscopy in human skin (79). This method is unique in allowing direct quantitation of the number of perfused capillaries. Insulin can regulate this recruitment, but its impact is diminished in obesity and hypertension and by insulin resistance (80).\n\n## Closing remarks.\n\nAccurate measurements of blood flow are critical for in vivo quantitation of skeletal muscle fuel metabolism and how it is affected by insulin resistance, diabetes, or other metabolic disorders. Multiple methodologies are currently available for use by clinical and basic scientists performing studies of muscle metabolism (<a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>). Plethsymography, Doppler ultrasound, and dye dilution measurements are widely available, inexpensive, portable and not technically demanding and are consequently most commonly used. Other methods such as PET and magnetic resonance imaging, while more complex and expensive and not portable, give more detailed information about specific blood flow within a region of interest of tissue. This latter information is not available with the more readily performed methods. Doubtless, with improving technology these types of measurements will be made more often. Contrast ultrasound can provide unique information with regard to the perfusion of the microvasculature and a measurement of microvascular volume. However, CEU does not provide a measure of bulk flow. Using a variety of different techniques, investigators have clearly realized the intimate relationship between regulation of perfusion and metabolic regulation within muscle. In coming years, more will be learned with regard to the chemical signals responsible for the coordination of these two functions.\n\n## ACKNOWLEDGMENTS\n\nThis study was supported by National Institutes of Health grants DK-R01-057878 and R01 DK-073759 and American Diabetes Association Grant BS 06.\n\nNo potential conflicts of interest relevant to this article were reported.\n\nE.J.B. and S.R. reviewed literature and composed and edited the manuscript. E.J.B. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.\n\n# REFERENCES","meta":{"dup_signals":{"dup_doc_count":139,"dup_dump_count":41,"dup_details":{"curated_sources":2,"2018-26":2,"2018-22":3,"2018-17":3,"2018-13":2,"2018-09":1,"2018-05":2,"2017-51":3,"2017-47":2,"2017-43":5,"2017-39":2,"2017-34":4,"2017-30":3,"2017-26":5,"2017-22":2,"2017-17":7,"2017-09":6,"2017-04":7,"2016-50":4,"2016-44":7,"2016-40":7,"2016-36":6,"2016-30":6,"2016-26":6,"2016-22":5,"2016-18":2,"2016-07":2,"2015-48":1,"2015-40":1,"2015-35":1,"2015-32":1,"2015-27":2,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":2,"2014-42":3,"2014-41":3,"2014-35":1,"2017-13":5,"2015-18":3,"2015-11":1}},"file":"PMC3478558"},"subset":"pubmed_central"}
{"text":"abstract: Until the pathophysiology\/etiology of rheumatoid arthritis (RA) is better understood, treatment strategies must focus on disease management. Early diagnosis and treatment with disease-modifying antirheumatic drugs (DMARDs) are necessary to reduce early joint damage, functional loss, and mortality. Several clinical trials have now clearly shown that administering appropriate DMARDs early yields better therapeutic outcomes. However, RA is a heterogeneous disease in which responses to treatment vary considerably for any given patient. Thus, choosing which patients receive combination DMARDs, and which combinations, remains one of our major challenges in treating RA patients. In many well controlled clinical trials methotrexate and other DMARDs, including the tumor necrosis factor-\u03b1 inhibitors, have shown considerable efficacy in controlling the inflammatory process, but many patients continue to have active disease. Optimizing clinical response requires the use of a full spectrum of clinical agents with different therapeutic targets. Newer therapies, such as rituximab, that specifically target B cells have emerged as viable treatment options for patients with RA.\nauthor: Larry Moreland\ndate: 2005\ninstitute: 1University of Alabama at Birmingham School of Medicine, Birmingham, Alabama, USA\nreferences:\ntitle: Unmet needs in rheumatoid arthritis\n\n# Introduction\n\nCurrent treatment guidelines suggest that early diagnosis and initial treatment with disease-modifying antirheumatic drugs (DMARDs) are necessary to limit early joint damage and functional loss and to reduce mortality associated with rheumatoid arthritis (RA) \\[1\\]. The earlier use of methotrexate alone and in combination with other DMARDs is now the standard of care and has yielded better outcomes for patients with RA.\n\nHowever, RA is a heterogeneous disease, and patient responses to standard treatments are variable. Most recent clinical trials of newer DMARDs alone and in combination with methotrexate have shown that ACR50 response \u2013 which includes reducing the signs and symptoms of disease by 50%, according to criteria established by the American College of Rheumatology (ACR) \u2013 was achieved in less than two-thirds of the patients \\[2-5\\]. That leaves at least one-third of the most seriously affected patients with RA without an effective long-term treatment strategy. Until we are able to identify which patients will respond to which treatment, the availability of a variety of agents with different therapeutic targets offers the best opportunity to optimize clinical outcomes.\n\nRituximab, a chimeric anti-CD20 monoclonal antibody that has emerged as a potential treatment for RA via selective targeting of B lymphocytes, has been used extensively in the treatment of B cell malignancies. There is a growing body of evidence for the pathophysiologic role of B cells. Silverman and Carson \\[6\\] described that B lymphocytes can present immune-complexed antigens to autoreactive T cells; express adhesion and other co-stimulatory molecules that promote T cell activation; synthesize chemokines that induce leukocyte infiltration; produce factors that initiate and sustain angiogenesis and granulation tissue formation; and release autoantibodies that are directly or indirectly destructive to tissues and maintain a memory response to autoantigens. Apart from B cells and T cells, populations of monocytes, macrophages, endothelial cells, and fibroblasts have been implicated in the ongoing inflammatory process \\[7\\]. The availability of a broader spectrum of agents with different targeting mechanisms will provide more effective treatment options for diverse patient populations.\n\n# Overall picture of rheumatoid arthritis\n\nRA affects almost 1% of the adult population worldwide \\[1\\]. Clinicians have reason to be concerned when they manage a chronic and debilitating condition that requires aggressive, life-long management. When one looks at large cohort populations, patients with RA exhibit increased morbidity and mortality, compounded by a dramatic impact on quality of life. Approximately 80% of affected patients are disabled after 20 years \\[8\\], and life expectancy is reduced by an average of 3\u201318 years \\[9\\].\n\nThe management of RA has a marked impact in terms not only of the financial burden to the health care system but also of the financial burden to individual patients and their families. It has been estimated that the disorder costs the average individual up to US\\$8500 annually \\[10\\], with time lost from work ranging from 2.7 to 30 days \\[11\\].\n\n# Treatment advances over the past decade\n\nDuring the past 10 years or so, advances in the treatment of RA have underscored the role of methotrexate as a major cornerstone of therapy. However, many randomized controlled trials have demonstrated that methotrexate in combination with another DMARD is more effective than methotrexate monotherapy for many patient populations \\[3-5,12\\].\n\nIn a 2002 study, Kremer and colleagues \\[12\\] tested the hypothesis that adding leflumonide to the regimen of patients taking methotrexate alone would strengthen the clinical response. The team assigned 263 patients with RA to leflunomide plus methotrexate or methotrexate alone. At 24 weeks, 46.2% (60 of 130) of patients receiving the leflunomide\u2013methotrexate combination had achieved an ACR20 clinical response, as compared with 19.5% of the patients who had been maintained on a methotrexate\u2013placebo regimen (*P* \\< 0.001). In addition, they reported that 26.2% of the leflunomide patients achieved an ACR50 response, as compared with 6.0% of the patients in the methotrexate\u2013placebo arm (*P* \\< 0.001). This study was one of the first to show that the combination of the two biologic agents produced statistically significant and clinically meaningful improvement in patients with active RA.\n\nIn a 2-year, randomized, double-blind, placebo-controlled trial, O'Dell and colleagues \\[13\\] compared the effectiveness of methotrexate in combination with either hydroxychloroquine or sulfasalazine, as well as a combination of all three drugs. After 2 years, 55% of the 58 patients in the triple therapy arm achieved an ACR50 response; 40% of patients on hydroxychloroquine and methotrexate achieved an ACR50 response; and 29% of those on sulfasalazine and methotrexate achieved an ACR50 response. The differences between triple therapy and double therapy with sulfasalazine reached statistical significance (*P* = 0.005).\n\n# Biologic response modifiers\n\nEfforts to develop safer and more effective treatments for RA, based on an improved understanding of the role of inflammatory mediators, have been realized through the development of the biologic response modifiers. Some biologics have been approved for use in RA by the US Food and Drug Administration and the European Medicine Evaluation Agency, including etanercept (a soluble tumor necrosis factor \\[TNF\\]-\u03b1 type II receptor\u2013IgG~1~ fusion protein administered subcutaneously), infliximab (a chimeric \\[human and mouse\\] monoclonal antibody against TNF-\u03b1), and adalimumab (a human anti-TNF monoclonal antibody). These therapies have shown the ability to change dramatically the outcomes of the disease in some RA patients.\n\nResearchers have observed that interleukin (IL)-1, IL-6, and TNF-\u03b1 are important mediators that initiate and maintain inflammation in RA, resulting in cellular infiltration of synovium and damage, and the destruction of cartilage and bone \\[14\\]. TNF-\u03b1, a potent cytokine that exerts diverse stimulatory effects, is produced mainly by monocytes and macrophages, but also by B cells, T cells, and fibroblasts. Newly synthesized TNF-\u03b1 is expressed on the cell membrane and subsequently released through the cleavage of its membrane-anchoring domain by a serine metalloproteinase. Thus, inhibition of TNF-\u03b1 secretion may represent a therapeutic target.\n\nRA is believed to be initiated by CD4^+^ T cells, which amplify the immune response by stimulating other mononuclear cells, synovial fibroblasts, chondrocytes, and osteoclasts. Activated CD4^+^ T cells contribute to stimulation of osteoclastogenesis and activation of metalloproteinases responsible for the degradation of connective tissue, resulting in joint damage.\n\nClearly, there are many possible therapeutic targets, but inhibition of cytokines appears to offer an especially efficient approach to suppressing inflammation and preventing joint damage \\[14\\]. There are four biological therapies currently approved for RA: three TNF-\u03b1 inhibitors (infliximab, etanercept, and adalimumab) and one IL-1 inhibitor (anakinra).\n\nAbatacept, a cytotoxic T lymphocyte-associated antigen 4\u2013IgG~1~ (CTLA4\u2013Ig) fusion protein, known as a co-stimulation blocker, is administered in a 30 min infusion. Recent clinical trial data show that the combination of abatacept and methotrexate improves the signs and symptoms, physical functioning, and quality of life of patients with active RA \\[15\\].\n\n## Etanercept\n\nIn the Trial of Etanercept and Methotrexate with Radiographic Patient Outcomes (TEMPO), Klareskog and coworkers \\[16\\] enrolled 685 patients with active RA in a double-blind, randomized trial to determine the safety and efficacy of treatment with etanercept or methotrexate alone or in combination versus placebo for up to 52 weeks. Patients were randomly assigned to treatment with etanercept 25 mg subcutaneously twice a week, oral methotrexate monotherapy up to 20 mg every week, or a combination of the two agents.\n\nThe researchers found that the proportions of patients achieving ACR50 and ACR70 responses were consistently higher for the combination group than either the etanercept or methotrexate treatment arms throughout the study (Fig. <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>). At week 52, 69% of the patients in the combination protocol achieved an ACR50 response, as compared with 43% and 48% in the methotrexate and etanercept monotherapy groups, respectively (*P* \\< 0.0001). Moreover, after a 1-year period, 43% of the patients in the combination group had an ACR70 response, as compared with 19% and 24% in the methotrexate and etanercept monotherapy patients, respectively (*P* \\< 0.0001). Additionally, more than one-third of patients treated with the methotrexate\u2013etanercept combination protocol were in remission at 52 weeks.\n\nThe number of patients reporting infection or adverse events (AEs) was similar in all groups. Overall, Klareskog and colleagues reported that the combination of etanercept and methotrexate was significantly better in reducing disease activity, improving functional disability, and retarding radiographic progression compared with methotrexate or etanercept alone.\n\nA comparison of therapy with methotrexate and therapy with etanercept was also conducted by Bathon and colleagues \\[17\\]. They evaluated 632 patients with early RA who were given either twice weekly subcutaneous etanercept (10 mg or 25 mg) or weekly oral methotrexate (mean 19 mg\/week) for 12 months.\n\nCompared with patients who received methotrexate, patients taking the 25 mg dose of etanercept exhibited a more rapid rate of improvement, with significantly more patients having ACR20, ACR50, and ACR70 response improvement in disease activity during the first 6 months (*P* \\< 0.05). The mean increase in the erosion score during the first 6 months was 0.30 in the group assigned to receive 25 mg etanercept and 0.68 in the methotrexate group (*P* = 0.001). The respective increases during the first 12 months were 0.47 and 1.03 (*P* = 0.002).\n\nAmong patients who received the 25 mg dose of etanercept, 72% had no increase in the erosion score, as compared with 60% of patients in the methotrexate group (*P* = 0.007). This group of patients also had fewer AEs (*P* = 0.02) and fewer infections (*P* = 0.006) than did the group treated with methotrexate. Compared with oral methotrexate, subcutaneous etanercept acted more rapidly to decrease symptoms and slow joint damage in patients with early active RA.\n\nBathon and colleagues observed that the patients in the study were at risk for rapidly progressive joint damage. Their disease was predicted to progress without treatment at an estimated rate of four to five points per year on the Sharp erosion subscale, and four points per year on the Sharp joint-space narrowing subscale. The rates of progression for joint-space narrowing were low. Both etanercept and methotrexate prevented joint-space narrowing. The overall rates of erosion were also low, equivalent to the occurrence of one new erosion or the erosion of 20% of one joint every year in the methotrexate group and every 2 years in the group assigned to receive 25 mg etanercept. The effects of this dose of etanercept were evident sooner than were the effects of methotrexate, but the rates of change were similar in the two groups during the latter half of the study. Over a 1-year period, treatment with etanercept halted erosions in 72% of patients, whereas treatment with methotrexate halted erosions in 60% of patients \\[17\\].\n\n## Adalimumab\n\nIn a pivotal study \\[18\\], the combination of the biologic adalimumab and methotrexate, particularly at a higher dose of 40 mg every other week, yielded a statistically significant improvement compared with methotrexate plus placebo. In that multicenter, 52-week, double-blind, placebo-controlled study, 619 patients with active RA who had an inadequate response to methotrexate alone were randomly assigned to receive adalimumab 40 mg subcutaneously every other week (*n* = 207), adalimumab 20 mg subcutaneously every week (*n* = 212), or placebo (*n* = 200) plus concomitant treatment with methotrexate. The primary efficacy end-points were radiographic progression at week 52 (total Sharp score by a modified method); clinical response at week 24, defined as improvements that achieved at least an ACR20 response; and improvement in physical function at week 52, based on the disability index of the Health Assessment Questionnaire (HAQ). At week 52 there was statistically significantly less radiographic progression (Fig. <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>), as measured by change in total Sharp score, in the patients receiving adalimumab either 40 mg every other week (change \\[mean \u00b1 standard deviation\\] 0.1 \u00b1 4.8) or 20 mg weekly (0.8 \u00b1 4.9), as compared with that in the placebo group (2.7 \u00b1 6.8; *P* \\< 0.001).\n\nAt week 52, ACR50 responses were achieved by 42% and 38% of patients taking adalimumab 40 mg every other week and 20 mg weekly, respectively, but by 10% of patients taking placebo (*P* \\< 0.001). In terms of physical function at week 52, patients on combination therapy with adalimumab and methotrexate experienced statistically significant improvement (mean change in HAQ score -0.59 and -0.61, respectively, versus -0.25; *P* \\< 0.001).\n\nAdalimumab was generally well tolerated. Discontinuations occurred in 22% of adalimumab treated patients and in 30% of placebo treated patients. The rate of both serious AEs and nonserious AEs was similar between adalimumab and placebo groups. The proportion of patients reporting serious infections was higher in patients receiving adalimumab (3.8%) than in those receiving placebo (0.5%; *P* \\< 0.02) and was highest in the patients receiving 40 mg every other week.\n\nThe benefits of early treatment of RA with adalimumab, either alone or in combination with methotrexate, were supported more recently by the results of the PREMIER study \\[19\\]. In an analysis of almost 800 patients with a disease duration of less than 3 years, adalimumab 40 mg every other week plus methotrexate (escalated rapidly to 20 mg\/week) exhibited statistically significant improvement in ACR50 clinical response and amelioration of disease progression compared with either adalimumab or methotrexate alone. Of patients in the combination therapy group, 61% and 46% achieved ACR50 and ACR70 responses, respectively, as compared with 46% and 28% in the methotrexate group (*P* \\< 0.001) \u2013 a statistically significant difference that was sustained for up to 2 years. The difference in clinical response was also comparable between the combination arm of the trial and patients taking adalimumab alone. Moreover, at 2 years radiographic remission (as indicated by a Disease Activity Scale \\[DAS28\\] score \\< 2.6) was achieved by 50% of patients receiving combination therapy.\n\n## Infliximab\n\nMuch of the recent knowledge regarding the safety and efficacy of infliximab in combination with methotrexate emerged from analysis of data from the Anti-Tumor Necrosis Factor Trial in Rheumatoid Arthritis with Concomitant Therapy (ATTRACT) trial \\[20,21\\], the pivotal study that led to approval of the TNF-\u03b1 inhibitor infliximab in the USA and worldwide. ATTRACT demonstrated that the combination of methotrexate and infliximab, in particular, with highest doses given every 4\u20138 weeks, resulted in better clinical responses than methotrexate plus placebo.\n\nIn that study the investigators established that infliximab not only provided significant improvements in physical function and quality of life, but also succeeded in slowing or halting progressive joint damage and signs and symptoms of RA in patients who previously had an incomplete response to methotrexate alone.\n\nThe study included 428 patients who were randomly assigned to receive methotrexate plus placebo or infliximab at a dose of 3 mg\/kg or 10 mg\/kg plus methotrexate for 54 weeks, with an additional year of follow up. The protocol was later amended to allow for continued treatment during the second year.\n\nOf 259 patients who entered the second year of treatment, 216 continued to receive infliximab plus methotrexate for 102 weeks. Ninety-four of these 259 patients experienced a gap in therapy of greater than 8 weeks before continuing therapy. Infusions were administered at baseline, week 2, and week 6, followed by treatment every 4 weeks or every 8 weeks \u2013 alternating with placebo infusions in the interim 4-week visits \u2013 at a dose of 3 mg\/kg or 10 mg\/kg for a total of 102 weeks, which included the gap in therapy.\n\nThe results of the study showed that the infliximab plus methotrexate regimens led to significantly greater improvement in HAQ scores (*P* = 0.006) and in the Short Form 36-Item Health Survey (SF-36) physical component summary scores (*P* = 0.011), compared with the group of patients receiving monotherapy with methotrexate. There also was stability in the SF-36 mental component summary score among patients who received the infliximab plus methotrexate regimens. The median changes from baseline to week 102 in the total radiographic score were 4.25 for patients who received the methotrexate-only regimen and 0.50 for patients who received the infliximab plus methotrexate regimen. The proportion of patients achieving an ACR50 response at week 102 varied from 20% to 21% for the infliximab plus methotrexate groups, as compared with 6% for the methotrexate-only group. These data emphasize that the combination of infliximab plus methotrexate conferred significant, clinically relevant improvement in physical function and quality of life, accompanied by inhibition of progressive joint damage and sustained improvement in the signs and symptoms of RA among patients who previously had an incomplete response to methotrexate alone.\n\nThe ASPIRE (Active controlled Study of Patients receiving Infliximab for RA of Early onset) trial \\[5\\] randomly assigned patients with early RA to either methotrexate alone or methotrexate plus 3 mg\/kg or 6 mg\/kg infliximab at weeks 0, 2, and 6, and every 8 weeks thereafter through week 46. It revealed improvement in ACR scores in both combination treatment groups compared with the methotrexate arm (38.9% and 46.7%, versus 26.4%, respectively; *P* \\< 0.001 for both comparisons), significantly less radiographic progression at 6 months and 12 months, and improvement in physical function.\n\n## Anakinra\n\nTreatment with anakinra, the first IL-1 receptor antagonist, either alone or in combination with methotrexate, has emerged as an effective medication for patients with moderate-to-severe RA. In a study of 419 patients with active RA of duration greater than 6 months but less than 12 years \\[22\\], patients were randomly assigned to placebo or one of five doses of anakinra (0.04, 0.1, 0.4, 1.0 or 2.0 mg\/kg) plus methotrexate. Those assigned to the five anakinra regimens exhibited statistically significant (*P* = 0.001), dose-escalating efficacy in ACR20 responses as compared with the placebo plus methotrexate group after 12 weeks. The ACR20 response rates in the anakinra 1.0 mg\/kg (46%; *P* = 0.001) and 2.0 mg\/kg (38%; *P* = 0.007) dose groups were significantly better than those in the placebo group (19%). ACR20 responses at 24 weeks were consistent with those at 12 weeks. Other researchers have reported similar 6-month ACR20 (and ACR50) responses, as well as in individual components (i.e. HAQ, pain, C-reactive protein level, and erythrocyte sedimentation rate) \\[23\\].\n\n## Abatacept\n\nAbatacept (CTLA4\u2013Ig), a cytotoxic T-lymphocyte-associated antigen 4\u2013IgG~1~ fusion protein, is the first in a new class of drugs known as co-stimulation blockers that are being evaluated for the treatment of RA. Abatacept selectively modulates the co-stimulatory signal required for full T cell activation. The agent, which binds to CD80 and CD86 on antigen-presenting cells, blocking the engagement of CD28 on T cells and thus preventing T cell activation, acts earlier in the inflammatory cascade than do other biologic therapies by directly inhibiting the activation of T cells and the secondary activation of macrophages and B cells.\n\nThe efficacy of this novel therapy was tested by Kremer and colleagues \\[24\\], who randomly assigned patients with active RA despite methotrexate therapy to receive 2 mg\/kg CTLA4\u2013Ig (105 patients), 10 mg\/kg (115 patients), or placebo (119 patients) for 6 months. All patients also received methotrexate therapy during the study. Patients treated with 10 mg CTLA4\u2013Ig were more likely to have an ACR20 response than were patients who received placebo (60% versus 35%; *P* \\< 0.001). Significantly higher rates of ACR50 and ACR70 responses were seen in both CTLA4\u2013Ig groups than in the placebo group \\[24\\]. The group given 10 mg CTLA4\u2013Ig had clinically meaningful and statistically significant improvements in all eight subscales of the SF-36. CTLA4\u2013Ig was well tolerated, with an overall safety profile similar to that of methotrexate \\[15\\].\n\nRecently released phase III results from the Abatacept in Inadequate responders to Methotrexate (AIM) trial \\[24\\] found that 48.3% of patients achieved an ACR50 response after 1 year of therapy compared with 18.2% of patients who were given methotrexate injections (*P* \\< 0.001). At 6 months, the number of patients on abatacept who achieved an ACR70 response was 19.8%, compared with 6.5% of patients on methotrexate. After 1 year, 28.8% of patients had reached an ACR70 response, compared with 6.1% of patients on methotrexate. Both differences were statistically significant (*P* \\< 0.001).\n\n# Tumor necrosis factor-\u03b1: potential safety issues\n\nAlthough researchers, scientists, and clinicians are enthusiastic in their support of early intervention with TNF-\u03b1 inhibitors for patients with RA, safety issues remain an important consideration. Although infusion reactions and other AEs are infrequent, they may be very serious in some patients, in particular when complications associated with opportunistic infections occur. There is a need to follow patients very closely and to work with primary care physicians to see that these issues are addressed first and foremost.\n\nRare and serious AEs include infections (bacterial, fungal, or tubercular), demyelination, infusion related events, hematologic\/lymphoproliferative disorders, drug-induced systemic lupus erythematosus\/vasculitis, hepatotoxicity (infliximab), and potential congestive heart failure. The development of neutralizing antibodies also can be an issue in some patients and needs further exploration. Further studies are needed to determine whether some of these reported side effects are truly related to the TNF-\u03b1 inhibitor, or are a consequence of the disease itself and\/or comorbid conditions and concomitant medications.\n\n# Challenges\n\nTypically, clinicians have reserved biologics for those patients with severe disease who have failed other therapies. However, the emerging body of evidence suggests that practitioners should be moving toward treating earlier disease with these biologic agents in an effort to prevent structural damage. In addition, because of the costs associated with biologic therapy \u2013 often more than US\\$1000 per month \\[25\\] \u2013 and the potential risk for immune suppression, one of the key challenges that clinicians should address when considering the use of TNF-\u03b1 inhibitor therapy for active RA is how to determine which patients should be receiving which agents and in what combination. Importantly, the medical community must endeavor to identify those patients who will respond over the long term to these agents and weigh the risk\/benefit. Is it possible to pick out which patients are most likely to have that long-term response? There are numerous challenges and opportunities, as well as many unmet needs for patients with RA.\n\n# Conclusion\n\nRA is a hetereogeneous disease; there is substantial evidence that some patients respond adequately to a single DMARD, whereas others require a combination regimen. Reliable predictors of response are needed to guide therapeutic decision making, along with a firm definition of therapeutic goals. It is of equal importance to arrest the ongoing pre-existing damage or to intervene earlier to prevent damage. In addition, over the long term, as research and treatment become more aggressive, efficacy, toxicity, and costs must be balanced within the therapeutic equation to enhance the quality of life in patients with RA (Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>).\n\nTherapy for rheumatoid arthritis: unmet needs\n\n| Problem | Details |\n|----|----|\n| Heterogeneity of the disease | Some patients respond to DMARD monotherapy |\n|  | Not every patient responds to anti-TNF (mono or combination) therapy |\n|  | 'Remission' rates |\n|  | What is the pathology\/biologic process in each individual patient? |\n| Toxicities | Infections, immunogenecity, congestive heart failure, drug-induced vasculitis, etc |\n| Costs | Limits availability to may patients |\n\nDMARD, disease-modifying antirheumatic drug; TNF, tumor necrosis factor.\n\nThe next generation of therapies for RA will provide considerable opportunities. These include next generation TNF-\u03b1 inhibitors, anticytokines (anti-IL-6 receptor, anti-IL-15, and anti-IL-1), angiogenesis inhibitors, antiadhesion molecules, anti-T-cell co-stimulatory blockers (e.g. abatacept), anti-B-cell therapies (i.e. rituximab and belimumab), and many others.\n\n# Abbreviations\n\nACR = American College of Rheumatology; AE = adverse event; CTLA4\u2013Ig = cytotoxic T lymphocyte-associated antigen 4\u2013IgG~1~; DMARD = disease-modifying antirheumatic drug; HAQ = Health Assessment Questionnaire; IL = interleukin; RA = rheumatoid arthritis; SF-36 = Short Form 36-Item Health Survey; TNF = tumor necrosis factor.\n\n# Competing interests\n\nLM has received research grant support, and consulting and speaking fees from several companies in the past, including Abbott, Amgen, Centocor, Wyeth, Immunex, Bristol-Myers Squibb, Regeneron, Genentech, Merck, Pfizer, Boehringer Ingelheim, Roche and others.","meta":{"dup_signals":{"dup_doc_count":110,"dup_dump_count":30,"dup_details":{"curated_sources":1,"2023-23":1,"2022-33":1,"2021-39":1,"2021-17":1,"2021-04":1,"2020-29":1,"2019-51":1,"2017-09":13,"2016-44":1,"2016-40":1,"2016-36":12,"2016-30":8,"2016-22":1,"2016-18":1,"2016-07":13,"2015-48":3,"2015-40":2,"2015-35":3,"2015-32":3,"2015-27":3,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":5,"2014-42":5,"2014-41":4,"2014-35":4,"2014-23":6,"2014-15":4,"2023-40":1}},"file":"PMC2833970"},"subset":"pubmed_central"}
{"text":"abstract: A light-sensitive, externally powered microchip was surgically implanted subretinally near the macular region of volunteers blind from hereditary retinal dystrophy. The implant contains an array of 1500 active microphotodiodes ('chip'), each with its own amplifier and local stimulation electrode. At the implant's tip, another array of 16 wire-connected electrodes allows light-independent direct stimulation and testing of the neuron\u2013electrode interface. Visual scenes are projected naturally through the eye's lens onto the chip under the transparent retina. The chip generates a corresponding pattern of 38 \u00d7 40 pixels, each releasing light-intensity-dependent electric stimulation pulses. Subsequently, three previously blind persons could locate bright objects on a dark table, two of whom could discern grating patterns. One of these patients was able to correctly describe and name objects like a fork or knife on a table, geometric patterns, different kinds of fruit and discern shades of grey with only 15 per cent contrast. Without a training period, the regained visual functions enabled him to localize and approach persons in a room freely and to read large letters as complete words after several years of blindness. These results demonstrate for the first time that subretinal micro-electrode arrays with 1500 photodiodes can create detailed meaningful visual perception in previously blind individuals.\nauthor: Eberhart Zrenner; Karl Ulrich Bartz-Schmidt; Heval Benav; Dorothea Besch; Anna Bruckmann; Veit-Peter Gabel; Florian Gekeler; Udo Greppmaier; Alex Harscher; Steffen Kibbel; Johannes Koch; Akos Kusnyerik; Tobias Peters; Katarina Stingl; Helmut Sachs; Alfred Stett; Peter Szurman; Barbara Wilhelm; Robert Wilke\\*Author for correspondence (<email@example.com>).\ndate: 2011-05-22\ninstitute: 1Centre for Ophthalmology, University of T\u00fcbingen, Schleichstr. 12, 72076 T\u00fcbingen, Germany; 2Eye Clinic, University of Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany; 3Retina Implant AG, Gerhard-Kindler-Str. 8, 72770 Reutlingen, Germany; 4Department of Ophthalmology, Semmelweis University, Tomo u. 25-29, 1083 Budapest, Hungary; 5Steinbeis Transfer Centre Eyetrial at the Centre for Ophthalmology, Schleichstr. 12-16, 72076 T\u00fcbingen, Germany; 6Klinikum Friedrichstadt, Friedrichstr. 41, 01067 Dresden, Germany; 7NMI Natural and Medical Sciences Institute at the University of T\u00fcbingen, Markwiesenstr. 55, 72770 Reutlingen, Germany\nreferences:\ntitle: Subretinal electronic chips allow blind patients to read letters and combine them to words\n\n# Introduction\n\nRetinitis pigmentosa (RP) and age-related macular degeneration are diseases that predominantly affect photoreceptors of the retina and cause progressive vision loss\u2014leading eventually to blindness in over 15 million people worldwide \\[1\\]. Although blindness owing to photoreceptor degeneration presently remains incurable, inner retinal nerve cells may continue to function for many years despite neuronal remodelling \\[2\\]. While gene therapy and application of neuro-protective factors may help maintain vision in the early stages of degeneration, survival of the inner retina encouraged us \\[3\\] and others \\[4\u201311\\] to attempt a partial restoration of visual function using electric stimulation of the remaining retinal network.\n\nTwo fundamentally different approaches have been taken in this area: (i) implantation of electrode arrays which interface epiretinally with retinal ganglion cells that form the retinal output pathway \\[6\u20137,11\u201313\\], and (ii) implantation of microchips under the transparent retina to substitute the degenerated photoreceptors. The latter type of microchip senses light and generates stimulation signals simultaneously at many pixel locations, using microphotodiode arrays (MPDAs; \\[3,14\\]). While the first approach typically requires external image and data processing due to bypassing retinal image analysis, the second seeks to replace the function of degenerated photoreceptors directly by translating the light of the image falling onto the retina point by point into small currents that are proportional to the light stimulus. Ours is the only approach where the photodiode\u2013amplifier\u2013electrode set is contained within a single pixel of the MPDA such that each electrode provides an electrical stimulus to the remaining neurons nearby, thereby reflecting the visual signal that would normally be received via the corresponding, degenerated photoreceptor.\n\nOn the basis of *in vitro* measurements \\[15\\] and animal studies \\[16\\] our consortium developed a subretinal electronic implant that carefully accounts for biocompatibility \\[17\\], biostability, surgical feasibility by means of a transchoroidal surgical technique \\[18\\], safe threshold stimulation and dynamic range of stimulation and the limits of spatial resolution *in vitro* \\[19\\]. This report describes the results of a clinical pilot study, illustrating that subretinally implanted multi-electrode arrays restore sufficient visual function for object recognition and localization and for the performance of visual tasks essential in the daily lives of blind patients. The results of this pilot study provide strong evidence that the visual functions of patients blinded by a hereditary retinal dystrophy can, in principle, be restored to a degree sufficient for use in daily life.\n\n# The subretinal implant\n\nAs shown in <a href=\"#RSPB20101747F1\" data-ref-type=\"fig\">figure\u00a01<\/a>*a*, the tip of the implant consists of an MPDA with 1500 individual light-sensitive elements and a test field for direct stimulation (DS) with 4 \u00d7 4 electrodes for electrical, light-independent stimulation. Both are positioned on a thin polyimide foil (<a href=\"#RSPB20101747F1\" data-ref-type=\"fig\">figure\u00a01<\/a>*b*, far left). For details on the control unit that provides power and wireless control signals, see <a href=\"#RSPB20101747F2\" data-ref-type=\"fig\">figure\u00a02<\/a>*a*,*d* and electronic supplementary material, chapter 1*c*.\n\n## The microphotodiode array\n\nEach of the 1500 MPDA elements acts independently from its neighbours; four magnified elements (72 \u00d7 72 \u00b5m each) are shown in <a href=\"#RSPB20101747F1\" data-ref-type=\"fig\">figure\u00a01<\/a>*g*. Each element includes a light-sensitive photodiode (15 \u00d7 30 \u00b5m) that controls a differential amplifier (circuit shown as a sketch) whose output stage is coupled to a titanium nitride (TiN) electrode (50 \u00d7 50 \u00b5m), connected to the amplifier via the contact hole (details see electronic supplementary material, chapter 1*b*). Essentially, an image is captured several times per second simultaneously by all photodiodes. Each element ('pixel') generates monophasic anodic voltage pulses at its electrode. Thus, pixelized repetitive stimulation is delivered simultaneously by all electrodes to adjacent groups of bipolar cells \\[15,19\\], the amount of current provided by each electrode being dependent on the brightness at each photodiode. Light levels ranging across approximately 2 log units are converted to charge pulses by each pixel with a sigmoidal relationship and the sensitivity can be shifted manually by several log units (see electronic supplementary material, chapter 1, figure S1). The chip is estimated to cover a visual angle of approximately 11\u00b0 by 11\u00b0 (1\u00b0 approx. 288 \u00b5m on the retina). The distance between two MPDA electrodes corresponds to a visual angle of 15 min of arc.\n\n## The 4 by 4 test field for direct stimulation (DS test field)\n\nThe DS test field consists of 4 \u00d7 4 quadruple TiN electrodes (100 \u00d7 100 \u00b5m^2^, 280 \u00b5m apart laterally and 396 \u00b5m diagonally) for light-independent electrical stimulation (see <a href=\"#RSPB20101747F1\" data-ref-type=\"fig\">figure\u00a01<\/a>*c*). The DS test field was added for assessment of the electrode-interface characteristics and to study current injections and efficacy of pulses with different shapes and polarities other than those provided by the MPDA. In a limited spatial testing range simple patterns can be created with the DS test field as well (<a href=\"#RSPB20101747F1\" data-ref-type=\"fig\">figure\u00a01<\/a>*d*,*e* and *f*).\n\nThreshold voltage to elicit a percept was assessed in an up-and-down staircase procedure. Typical charge transfer of a single electrode at threshold was between 20 and 60 nC per pulse (for details, see electronic supplementary material, chapter 1*a*). The maximum charge density at the electrodes in the DS field was 600 \u00b5C cm^\u22122^. These values were well within commonly accepted safety limits and have been proven safe even for continuous retinal stimulation *ex vivo* \\[20\\].\n\nImpedance values of single electrodes were typically 300 k\u03a9 (at 1 kHz sinusoidal AC). Although regular impedance measurements in the patients were not conclusive, analysis of all available data showed that charge thresholds, but not voltage thresholds decreased significantly during the first days after implantation. Thereafter, both charge and voltage thresholds showed a slight tendency towards increasing values over the remaining implantation period.\n\n# Patients\n\nThe patients (two males and one female, age 40, 44 and 38, respectively) were blind owing to hereditary retinal degeneration (patients 1 and 2: RP, patient 3: choroideraemia) but had good central vision previously. Disease onset was reported by patient 2 at age 16, by patients 1 and 3 at age 6. They had lost their reading ability at least 5 years before implantation. Bright light stimulation mediated some limited light perception without any recognition of shapes in all three patients. They reported neither general diseases nor regular medication (for details see electronic supplementary material, chapter 2*c*).\n\n# Methods\n\n## Surgical procedure\n\nThe implant, protected by a long steel tube, was advanced through a retroauricular incision to the lateral orbital rim and guided inside the orbit to the surface of the eyeball (\\[21\\]; <a href=\"#RSPB20101747F2\" data-ref-type=\"fig\">figure\u00a02<\/a>*a*,*b*,*e*). The silicone cable (<a href=\"#RSPB20101747F2\" data-ref-type=\"fig\">figure\u00a02<\/a>*a*) was implanted subperiostally beneath the temporal muscle. The polyimide foil was then protected by a silicone tube and guided from the lateral orbital rim, where it was fixed, to the equator of the eye. Subsequently pars plana vitrectomy was performed. A localized retinal detachment was created by saline injection in the upper temporal quadrant above the planned scleral and choroidal incision area. After preparation of a scleral flap, the implant was advanced *ab externo* transchoroidally along a guiding foil into the subretinal space until it reached the preoperatively defined position (\\[22\\]; see electronic supplementary material, chapter 2*d*). Although putting a chip directly under the fovea has not turned out to be a surgical problem we had abstained in initial patients from placing the chip under the macula, but asked to place the chip closer and closer to the foveola as the surgical learning curve improved. Silicone oil was then injected into the vitreous cavity to support retinal reattachment. No serious adverse events were noted during the course of the study. For post-operative observations and consideration on surgical safety see electronic supplementary material, chapter 2*f*).\n\n## Psychophysical tests\n\nBeginning 7 to 9 days after surgery, tests with solely electrical stimuli were performed with the DS test field. Thereafter light evoked visual functions mediated by the MPDA-array were assessed using four psychophysical tests concerning light detection, basic temporal resolution, object localization and movement detection using the 'basic light and motion test' (BaLM \\[23\\]) described in electronic supplementary material, chapter 2*g*.\n\nIf passed successfully, three further steps followed: tests for recognition of stripe patterns (BAGA \\[24\\]), localization and recognition of objects common to daily life and visual acuity assessment (Landolt-C rings presented in an up-and-down staircase procedure to estimate the visual acuity in terms of maximum likelihood by means of FrACT test \\[25\\]). If these tasks were completed successfully, more challenging tasks were set (figures\u00a0<a href=\"#RSPB20101747F3\" data-ref-type=\"fig\">3<\/a>*a* and <a href=\"#RSPB20101747F4\" data-ref-type=\"fig\">4<\/a>*a*). Except for some optional tasks (indicated) well-established two- or four-alternative forced-choice methods (2AFC and 4AFC, respectively) were employed in order to test for statistical significance of a patient's performance. All tests were performed separately in two conditions: with 'Power ON' and 'Power OFF' ('baseline performance').\n\nMaximum screen luminance was approximately 3200 cd m^\u22122^ (for white light), neutral density filters (Schott NG filters 0.15\u20134 log U) served for attenuation (for details see electronic supplementary material, chapter 2).\n\n# Results\n\n## Electrical stimulation for pre-testing and learning via DS test field\n\nPulses of varying duration, polarity and shape were applied via the DS test field (16 electrodes, as shown in <a href=\"#RSPB20101747F1\" data-ref-type=\"fig\">figure\u00a01<\/a>*c*\u2013*f*) in a pre-testing routine. This procedure determined voltage thresholds for perception, accustomed the patients to electrically evoked visual impressions and tested retinal excitability and spatial resolution. An overview of the results including their statistical evaluation is given in a table presented in electronic supplementary material, chapter 3*a*.\n\nAll patients detected single-electrode single-pulse stimulation (0.5\u20136 ms pulses, typically 20\u201360 nC per electrode). Patients 1 and 2 consistently reported these stimuli as whitish round dot-like percepts, patient 3 reported percepts as elongated, short whitish\/yellowish lines. Upon activation of four electrodes with a single pulse, all three patients correctly distinguished vertical lines from horizontal lines within seconds and spontaneously reported them as straight. Patients 1 and 2 distinguished multiple single dots upon simultaneous activation of several electrodes in a diagonal row and reported dark areas separating the dots. Patient 3 saw diagonal lines formed by four electrodes, but not the dark areas between the dots.\n\nSimple patterns were also presented with the DS-array by pulsing electrodes sequentially (<a href=\"#RSPB20101747F1\" data-ref-type=\"fig\">figure\u00a01<\/a>*d*); each electrode was switched on for 3\u20136 ms at intervals of 208 ms. Patients 1 and 2 correctly reproduced these patterns after the first single presentation; patient 3 failed to do so. Upon presentation of a four-alternative, forced-choice (4AFC) paradigm, patients 1 and 2 reliably differentiated four different positions of the opening of the letter 'U' (73% and 88% correct responses, respectively, see electronic supplementary material, chapter 5, movie 1). Furthermore, patient 1 correctly distinguished 'U' from 'I' and even squares from triangles when only a single activated electrode differed in position (16\/16 correct, <a href=\"#RSPB20101747F1\" data-ref-type=\"fig\">figure\u00a01<\/a>*e*,*f*). Patient 2 correctly distinguished four letters individually presented randomly in 4AFC-mode (e.g. C,I,L,O (36\/36), I,L,V,T (10\/12)) in repetitive tests on different days (see electronic supplementary material, chapter 5, movie 2). He also distinguished sequential stimulation in clockwise versus anticlockwise direction (15 of 16 tests correct).\n\n## Light pattern perception with the microphotodiode array\n\nThe light-sensitive MPDA chip was operated at a sampling rate of 1 to 20 Hz with a pulse duration (PD) of 1\u20134 ms. The patient's head was comfortably positioned on a chin-rest (set up 1, <a href=\"#RSPB20101747F3\" data-ref-type=\"fig\">figure\u00a03<\/a>*a*), and refraction was corrected for the viewing distance of 60 cm. Chip settings were adjusted for a working range of 8\u2013800 cd m^\u22122^ white light or 1.2\u20134.3 cd m^\u22122^ red light (for details see electronic supplementary material, chapter 2*g*). All standardized testing was performed using a functional baseline control, i.e. performance was also tested with the chip switched off at random intervals unknown to patient and observer, as summarized in electronic supplementary material, table ST1.\n\n### Light perception and localization\n\nAll three patients were able to perceive light mediated by the chip. This was verified in task 1, using the BaLM-test in set up 1 (<a href=\"#RSPB20101747F3\" data-ref-type=\"fig\">figure\u00a03<\/a>*a*):\n\n1.  \u2014\u2002*BaLM flash test*: in task 1, the whole screen was illuminated briefly with one or two flashes (200 ms duration with 600 ms pause) after an auditory signal. All three patients passed this test for light detection (81.3%, 100% and 100% correct, respectively) and scored well-above chance rate; (*n* = 16; ON versus OFF: *p* = 0.00005, *t*-test).\n\n2.  \u2014\u2002*BaLM localization test*: when testing the ability to localize large bright areas in the visual field (small triangle in relation to a central fixation point in BaLM test) only patient 2 (87.5%; *n* = 16) passed the test successfully.\n\n3.  \u2014\u2002*BaLM movement test*: perception of movement was tested with a random dot pattern at an angular speed of 1.11\u00b0 s^\u22121^ moving in one of four directions (dot diameter 1.4 cm, average distance 1.5 cm (s.d. 0.26)), passed only by patient 2 (8 of 12, 4AFC, <a href=\"#RSPB20101747F3\" data-ref-type=\"fig\">figure\u00a03<\/a>*e*).\n\nIn task 2, spatial resolution was tested using grid patterns (<a href=\"#RSPB20101747F3\" data-ref-type=\"fig\">figure\u00a03<\/a>*b*). Bright lines of 0.6 cm width separated by 1.8 cm wide dark lines as well as bright lines of 0.8 cm width separated by 2.4 cm wide dark lines were presented at 63 cm distance. The orientation of these patterns was correctly recognized by patient 2. In terms of spatial frequency this corresponds to 0.46 cycles deg^\u22121^ (five of eight correct, 4AFC, *p* = 0.02) and 0.34 cycles deg^\u22121^ (four of four correct, 4AFC, *p* = 0.004), respectively (see electronic supplementary material, chapter 3, table ST1 and chapter 5, movie 3). Patient 3 succeeded at 0.22 cycles deg^\u22121^ (12 of 20, 4AFC, white light). Patient 1 had difficulty seeing the stripes, probably owing to her nystagmus, but distinguished horizontal from vertical lines projected onto her chip in a special set up using a fundus camera with comparable spatial arrangements and luminance.\n\nAs the spectral sensitivity of the chip is practically flat far into the infrared region, patients at several instances reported high sensitivity to infrared light.\n\n### Landolt C ring\n\nIn task 3, single letters and Landolt C rings were presented on the screen in various sizes (<a href=\"#RSPB20101747F3\" data-ref-type=\"fig\">figure\u00a03<\/a>*c*). Patients 1 and 3 discerned neither the Landolt C rings nor the letters and were accordingly not presented with tests of higher difficulty in set up 1. Patient 2, the only one with the chip placed under the macula, was quite successful and his visual performance is therefore described in greater detail below.\n\nOptimizing his implant settings resulted in an image recording time of 0.5 ms with a 7.5 Hz repetition frequency at a target luminance of 3.4 cd m^\u22122^ (red light), viewed with a correction of +7.0 dpt sph., \u22121.50 dpt cyl. at 121\u00b0. Landolt C rings (<a href=\"#RSPB20101747F3\" data-ref-type=\"fig\">figure\u00a03<\/a>*c*) were presented in an up-and-down staircase procedure (FrACT \\[25\\]; for details see electronic supplementary material, chapter 2). A maximum of 60 s was allowed for the patient to find each C-ring on the screen in his small visual field; failure to respond in time counted as mistake. Maximum visual acuity was up to 20\/1000 (log MAR =1.69)\u2014corresponding to a Landolt ring with 4.5 cm outer diameter and a gap of 9 mm, viewed at about 60 cm distance. In three other trials on different days he achieved log MAR values of 1.75, 1.94 and 1.86, respectively (see electronic supplementary material, chapter 5, movie 4).\n\nPatient 2 reliably differentiated also the letters L,I,T,Z on a screen (22 of 24, 4AFC, <a href=\"#RSPB20101747F3\" data-ref-type=\"fig\">figure\u00a03<\/a>*d*; 8.5 cm high, 1.7 cm line width, corresponding to a height of approximately 9\u00b0 of visual angle). He reported that having once found a letter, it appeared clearly in its natural form and was visible as a complete entity\u2014even during its first presentation.\n\n### Recognition of objects on a table\n\nIn the fourth task, the ability to perceive more naturalistic scenes was tested by a standardized set up at a dining table, assessed by an independent, professional mobility trainer (<a href=\"#RSPB20101747F4\" data-ref-type=\"fig\">figure\u00a04<\/a>*a*, for details see electronic supplementary material, chapter 2*g*). Patient 1 reliably localized a saucer, a square and a cup on the table; patient 3 correctly localized and differentiated a large plate from a saucer.\n\nPatient 2 localized, and moreover recognized and correctly differentiated square-, triangle-, circle-, rectangular-, and diamond-shapes, which differed only in shape but not in area from each other (<a href=\"#RSPB20101747F4\" data-ref-type=\"fig\">figure\u00a04<\/a>*b*, five of five correct, see electronic supplementary material, chapter 5, movie 5). Furthermore, he could localize and describe correctly a spoon, a knife, a cup (see electronic supplementary material, chapter 5, movie 6), as well as a banana and an apple (see electronic supplementary material, chapter 5, movie 7). Unlike the other dining table set ups this set up was entirely unknown to the patient and he was forced to make sense of an unfamiliar scene.\n\n### Optional tasks with letters, clock, grey papers of varying shades\n\nThe fifth group of tasks was performed only in patients who had successfully passed previous tasks. Patient 2 was able to distinguish between 16 different letters cut from white paper (5\u20138 cm high, font: Tahoma), placed on the black table (see <a href=\"#RSPB20101747F4\" data-ref-type=\"fig\">figure\u00a04<\/a>*c*, 22\/36 correct). The patient read letters (LOVE, MOUSE, SUOMI, etc.) correctly (five of five), also repeatedly on several days. He noted spelling mistakes in his name MIIKKA (mentioning that one 'I' and one 'K' were missing) when he first saw this word (see electronic supplementary material, chapter 5, movie 8), i.e. he perceived both individual letters and continuous, meaningful words\u2014a prerequisite for reading.\n\nAs an additional task, a clock face was presented with two hands (6 \u00d7 1.5 cm for the hours, 12 \u00d7 1.5 cm for the minutes, <a href=\"#RSPB20101747F4\" data-ref-type=\"fig\">figure\u00a04<\/a>*d*). Patient 2 was asked to indicate clock times set to full quarter hours. The patient correctly recognized 11 of 12 possible settings. Patient 2 also distinguished seven out of nine contrast differences of 15 per cent among nine neighbouring cards (10 \u00d7 10 cm, presented in 2AFC mode, *p* = 0.07) with linearly scaled shades of grey varying from 3 to 35 cd m^\u22122^ (<a href=\"#RSPB20101747F4\" data-ref-type=\"fig\">figure\u00a04<\/a>*e*).\n\nAll patients showed distinct learning effects which, while they could not be quantified in this first pilot study, are reported as 'spontaneous observations' in electronic supplementary material, chapter 3*d*.\n\n### Pupillary reflexes\n\nPupillary constriction in response to light as an objective measure of MPDA efficacy was assessed by infrared pupillography (for methods and recordings, see electronic supplementary material, chapter 2*i*). The amplitude of pupillary constriction was clearly more pronounced when the chip was activated (see electronic supplementary material, chapter 2, figure S2). In all three patients the chip-on condition improved pupil reaction and was always accompanied by subjective light perception. An analysis of variance was calculated for the constriction amplitudes of all three patients (with chip-on or chip-off) and patient as factors (sum of squares 0.184, *F* = 6.48, *p* = 0.022).\n\n# Discussion\n\n## The general approaches to retinal prostheses\n\nA number of research groups have taken up the challenge of developing a retinal prosthesis. Rizzo *et al.* \\[4\\] and Weiland *et al.* \\[26\\] have reported on first trial stimulations of the retina with single epiretinal electrodes. Chow *et al*. \\[27\\] were the first to subretinally implant well-tolerated multiphotodiode arrays, intending to use the energy created by incident light for neuronal stimulation directly without amplification. However, owing to insufficient energy from the small light sensors these failed to restore vision. Second Sight (Medical Products Inc., Sylmar, CA) has a multicentre study running with the epiretinal ARGUS II device with 60 electrodes; some patients were reported to recognize large single letters by scanning them with rapid head movements \\[28\\]. Clinical studies with epiretinal electrode arrays were also performed by Koch *et al.* \\[29\\] and Richard *et al.* \\[30\\]. Other groups developed approaches with electrodes placed between sclera and choroid \\[8,10\\]. These groups argue that this 'suprachoroidal' approach may have the benefit of being less invasive, therefore bearing fewer risks in terms of surgical procedures. At this time, as only limited peer-reviewed information is available from ongoing clinical trials using subretinal, epiretinal and suprachoroidal approaches, it is too early to compare the final long-term outcome of the various designs. All have inherent theoretical advantages and disadvantages; basic differences and their consequences are pointed out in the following.\n\n*Epiretinal implants* seek to interact directly with the retinal output neurons; the image processing of the complex inner retinal network must be performed externally. The processing of camera-captured images can be more easily adjusted to account for individual electrode thresholds. However, the number of simultaneously addressed electrodes is limited by present technology. Several groups have developed externally powered, fully implantable epiretinal systems with arrays of up to 60 microelectrodes \\[7,28\u201332\\]. Although they have reported promising results, even for long term use, the low number of electrodes limit visual performance to object localization and shape perception \\[33\\]. Yanai *et al*. \\[6\\] reported no difference in patient performance when a single pixel or multiple pixels were activated using a prototype of the ARGUS I implant. In *epiretinal* implants that use head mounted cameras, eye movements are not correlated to the visually perceived scene. Such a mismatch of visual and proprioceptive information must render object localization difficult \\[34\\].\n\n*Subretinal approaches*, in contrast, replace in principle only the lost function of diseased photoreceptors; thus, the remaining network of the inner retina can be used for more natural processing of the image as it is forwarded, point\u2013by-point, several times per second to inner retinal neurons. Although the surgical procedure may be more demanding, the number of pixels can be much higher, presently limited only by the size of an implant and the spatial spread of electrical stimulation. Fixation of the chip in the subretinal space is easier and, once positioned, the chip remains in place, tightly connected to the inner retina without the need for scleral tacks as used in epiretinal approaches. Moreover, our subretinal implant (Retina Implant AG, Reutlingen, Germany) is the only one so far, where the image receiver array moves exactly with the eye. This has practical implications, outlined below, as natural eye movements can be used to find and fixate a target. On the other hand, the duration of our study was limited owing to time constraints of a transdermal cable; other studies have reported longer implantation times \\[33\\]. Moreover, the range of variations in online image processing is small in devices that work quasi-autonomously under the retina.\n\n*Suprachoroidal implants*, although bearing lower surgical risks, are located further away from target cells. This may result in high stimulation thresholds, increased power consumption, and certainly loss of spatial resolution. While the surgery is easier and less invasive, the location between highly light absorbing sclera and choroid does not allow the implantation of a light sensitive array that moves with the eye.\n\nIn the following sections, the results obtained in our subretinal study are discussed in more detail.\n\n## The spatial domain\n\nUsing simulated prosthetic vision Perez *et al*. \\[35\\] have shown that the precision in recognition tasks with normal sighted subjects increased with a density of pixels up to 1000 in a 10\u00b0 \u00d7 7\u00b0 visual field on the retina. Thus, at least several hundred electrodes should be employed to provide significant vision\u2014a daunting technical barrier \\[35\\]. The present study\u2014the first to successfully employ electronic arrays with such a large number of electrodes\u2014presents proof-of-concept that such devices can restore useful vision in blind human subjects, even though the ultimate goal of broad clinical application will take time to develop.\n\nThe size of the visual field (11\u00b0 \u00d7 11\u00b0) in our patients, although small, is sufficient for orientation and object localization, as is well established in patients with peripheral retinal dystrophies. Reading requires a field of 3 by 5 degrees according to Aulhorn \\[36\\].\n\nInter-individual variations in visual performance among the patients of this study can be assumed to result from their respective stages of degeneration \\[2\\], the duration of their blindness, and the retinal localization of the implant, although presently no convincing correlation can be established. Clearly spatial reorganization of the retina takes place; however, it is very slow, taking decades. As the inner retina is not dependent on choroidal perfusion, it also survives the complete loss of the choroid\u2014as seen in our patient with choroideraemia. This also explains why blockage of choroido-retinal transport by our implant does not affect survival of the inner retina.\n\nIn our study, precise localization of the microelectrode array under the fovea appeared important for the restoration of useful percepts via spatially ordered electrical stimulation. High spatial resolution and the ability to read are restricted in normal observers to the central retina (5\u00b0 \u00d7 3\u00b0), which is significantly over-represented in the visual cortex relative to more peripheral areas of the retina.\n\n## The temporal domain and the problem of image fading\n\nTemporal resolution was investigated over a range from 1 to 20 Hz. When applying continuous electrical stimuli via the *DS-array* at a fixed retinal location with PD of 1\u20134 ms, patient percepts faded after approximately 15 s when presented at a 0.3 Hz repetition rate; after approx. 2 s at 2 Hz; and after approx. 0.5 s at 10 Hz. This is in close accordance with the observations of Perez *et al*. \\[37\\] with *epiretinal* ARGUS II devices that an image stabilized on the retina quickly disappears; to restore the image required a movement of the image across the retina, by means of rapid head shaking. Similarly, Jensen & Rizzo \\[38\\] observed in rabbit retina that the retinal response to a second or third electrical pulse rapidly decreases as compared to the first pulse with increasing repetition rates; apparently inner retina neurons suffer from a prolonged inhibition if stimulated electrically under conditions where the surrounding network under the electrode is being activated as a whole. By contrast, objects like grating patterns or letters can be perceived continuously with our light sensitive *subretinal MPDA*. Patients see the image constantly as a complete entity without head movements\u2014even on the first day of stimulation. The source of this difference can be found in involuntary eye movements controlled by the superior colliculus. Even during fixation, our eyes continuously make slight movements (slow drifts and microsaccades up to 50 min of arc and 1 to 3 Hz) that refresh the image by constantly changing the activated photoreceptor population\u2014even during strict fixation \\[39\\]. Objects viewed by our patients\u2014with the chip moving in synchronization with natural eye movement\u2014dynamically activate a range of adjacent pixels on the chip, as eye movements and microsaccades continuously shift the 'electrical image' on the retina for about 1\u20133 pixels, thus preventing mechanisms of local adaptation and image fading. Details on the role and magnitude of microsaccades in relation to pixel size are outlined in electronic supplementary material, chapter 3*e* (figure S3).\n\n## The cellular 'interface'\n\n*In vitro* experiments have shown that subretinal stimulation, at least at threshold, preferentially stimulates bipolar cells \\[15,19\\]. This may be one reason for the correct retinotopic perceptions reported in this study, since local excitation of small groups of bipolar cells is recognized in the brain at the correct position in the visual field. By contrast, epiretinal stimulation of ganglion cell fibres may result in disparities between stimulation location and perceived visual field location because the axons of RGCs course across the retina on their way into the brain via the optic nerve. On the other hand, none of the different approaches has principal problems with addressing simultaneously ON and OFF neurons (see electronic supplementary material, chapter 3*c*).\n\n## Learning and cognition\n\nWith the subretinal approach and its retinotopically correct spatial transmission, no long-term learning procedure was necessary to enable the patients to recognize shapes correctly. Even at the first trial with the DS test field or with the MPDA, patients were able to correctly perceive the complete entity of an object in the presented physical geometric form, the bright parts appearing whitish or yellowish, the dark parts as grey or black; there were no reports on colour sensations although in very rare and brief instances coloured tinges were noticed by patients.\n\nThe observation that patient 2 could readily name an object upon its first presentation to his visual field is of particular importance, and is in line with our observation of retinotopically correct perception from DS experiments and from the other patients who recognized a line and its direction clearly. This does not mean that the patients had undisturbed percepts. Patients reported some wobbling of the image, probably owing to a relatively low image capture frequency (5\u20137 Hz) to which they adapted quickly.\n\nAs expected, patient performance improved over time. Practising with the MPDA between 4 to 6 h daily, they had to learn to control their eye position because each object was presented within a relatively small field of vision (11\u00b0 \u00d7 11\u00b0). Patient 2 reported that the two lines of the letter L were initially moving slightly independently of each other, but that they appeared connected at the corner after approximately one week. Apparently the binding of correlated motion cues can be regained quickly (see electronic supplementary material, chapter 3*d* and chapter 5, movie 9). If patients were asked to point to an object they had discovered there was clearly improvement of visuomotor abilities within a week.\n\n## Future concepts\n\n*Methodological and technical aspects*: our first approach was designed as a short duration study of up to several weeks in only a few patients in order to achieve a proof-of-concept for a cable bound version of a subretinal active implant. Our ongoing follow-up study is employing the next-generation system (Alpha IMS; \\[40\\], produced by Retina Implant AG, Reutlingen, Germany), where an encapsulated secondary coil for power and signal transmission is positioned subdermally behind the ear, with a primary coil clipped magnetically on top. We also anticipate that lateral processing in terms of mutual inhibition of pixels, as performed in centre-surround receptive field processing will improve contrast vision and spatial resolution. Penetrating three-dimensional electrodes as developed by various groups may improve the contact to the bipolar cell layer but may be more damaging to the retina.\n\n# Conclusion\n\nThis study demonstrated that subretinal micro-electrode arrays can restore visual percepts in patients blind from hereditary retinal degenerations to such an extent that localization and recognition of objects can provide useful vision, up to reading letters. Despite all remaining biological and technical challenges, our results offer hope that restoration of vision in the blind with electronic retinal prostheses is a feasible way to help those who cannot profit from emerging gene therapy and\/or the application of neuroprotective agents. The advantage of our approach is that all parts of the device can be implanted invisibly in the body, that inner retina processing can be used and that a continuous, stable image with unmatched spatial resolution is perceived. Still further development is necessary to provide long term stability, improved contrast, spatial resolution and increased field size through multiple chip implantation. Nevertheless, the present study provides proof-of-concept that electronic subretinal devices have the potential to improve visual function from a state of complete blindness to one of low vision that allows localization and recognition of objects up to reading capability.\n\n## Acknowledgements\n\nWe are very grateful to all who contributed to the 'SUBRET' project; for names of contributors, funding organizations and disclosure of interest we refer to electronic supplementary material.\n\n# References","meta":{"dup_signals":{"dup_doc_count":189,"dup_dump_count":45,"dup_details":{"curated_sources":2,"2018-47":3,"2018-43":1,"2018-39":2,"2018-34":2,"2018-30":3,"2018-26":2,"2018-22":3,"2018-17":4,"2018-13":4,"2018-09":3,"2018-05":4,"2017-51":5,"2017-47":4,"2017-43":6,"2017-39":4,"2017-34":4,"2017-30":5,"2017-26":5,"2017-22":4,"2017-17":7,"2017-09":6,"2017-04":6,"2016-50":3,"2016-44":9,"2016-40":6,"2016-36":6,"2016-30":5,"2016-26":5,"2016-22":5,"2016-18":5,"2016-07":5,"2015-48":5,"2015-40":5,"2015-35":5,"2015-32":3,"2015-27":3,"2015-22":4,"2015-14":4,"2014-52":4,"2014-49":5,"2018-51":1,"2017-13":3,"2015-18":4,"2015-11":2,"2015-06":3}},"file":"PMC3081743"},"subset":"pubmed_central"}
{"text":"date: 2018-06\ntitle: 9 out of 10 people worldwide breathe polluted air, but more countries are taking action\n\n2 May 2018 - WAir pollution levels remain dangerously high in many parts of the world. New data from WHO shows that 9 out of 10 people breathe air containing high levels of pollutants. Updated estimations reveal an alarming death toll of 7 million people every year caused by ambient (outdoor) and household air pollution.\n\n\"Air pollution threatens us all, but the poorest and most marginalized people bear the brunt of the burden,\" says Dr Tedros Adhanom Ghebreyesus, Director-General of WHO. \"It is unacceptable that over 3 billion people \u2013 most of them women and children \u2013 are still breathing deadly smoke every day from using polluting stoves and fuels in their homes. If we don't take urgent action on air pollution, we will never come close to achieving sustainable development.\"\n\n7 million deaths every year\n\nWHO estimates that around 7 million people die every year from exposure to fine particles in polluted air that penetrate deep into the lungs and cardiovascular system, causing diseases including stroke, heart disease, lung cancer, chronic obstructive pulmonary diseases and respiratory infections, including pneumonia.\n\nAmbient air pollution alone caused some 4.2 million deaths in 2016, while household air pollution from cooking with polluting fuels and technologies caused an estimated 3.8 million deaths in the same period.\n\nMore than 90% of air pollution-related deaths occur in low- and middle-income countries, mainly in Asia and Africa, followed by low- and middle-income countries of the Eastern Mediterranean region, Europe and the Americas.\n\nAround 3 billion people \u2013 more than 40% of the world's population \u2013 still do not have access to clean cooking fuels and technologies in their homes, the main source of household air pollution. WHO has been monitoring household air pollution for more than a decade and, while the rate of access to clean fuels and technologies is increasing everywhere, improvements are not even keeping pace with population growth in many parts of the world, particularly in sub-Saharan Africa.\n\nWHO recognizes that air pollution is a critical risk factor for noncommunicable diseases (NCDs), causing an estimated one-quarter (24%) of all adult deaths from heart disease, 25% from stroke, 43% from chronic obstructive pulmonary disease and 29% from lung cancer.\n\nMore countries taking action\n\nMore than 4300 cities in 108 countries are now included in WHO's ambient air quality database, making this the world's most comprehensive database on ambient air pollution. Since 2016, more than 1000 additional cities have been added to WHO's database which shows that more countries are measuring and taking action to reduce air pollution than ever before. The database collects annual mean concentrations of fine particulate matter (PM10 and PM2.5). PM2.5 includes pollutants, such as sulfate, nitrates and black carbon, which pose the greatest risks to human health. WHO air quality recommendations call for countries to reduce their air pollution to annual mean values of 20 \u00b5g\/m3 (for PM10) and 10 \u00b5g\/m3 (for PM25).\n\n\"Many of the world's megacities exceed WHO's guideline levels for air quality by more than 5 times, representing a major risk to people's health,\" says Dr Maria Neira, Director of the Department of Public Health, Social and Environmental Determinants of Health, at WHO. \"We are seeing an acceleration of political interest in this global public health challenge. The increase in cities recording air pollution data reflects a commitment to air quality assessment and monitoring. Most of this increase has occurred in high-income countries, but we hope to see a similar scale-up of monitoring efforts worldwide.\"\n\nWhile the latest data show ambient air pollution levels are still dangerously high in most parts of the world, they also show some positive progress. Countries are taking measures to tackle and reduce air pollution from particulate matter. For example, in just two years, India's Pradhan Mantri Ujjwala Yojana Scheme has provided some 37 million women living below the poverty line with free LPG connections to support them to switch to clean household energy use. Mexico City has committed to cleaner vehicle standards, including a move to soot-free buses and a ban on private diesel cars by 2025.\n\nMajor sources of air pollution from particulate matter include the inefficient use of energy by households, industry, the agriculture and transport sectors, and coal-fired power plants. In some regions, sand and desert dust, waste burning and deforestation are additional sources of air pollution. Air quality can also be influenced by natural elements such as geographic, meteorological and seasonal factors.\n\nAir pollution does not recognize borders. Improving air quality demands sustained and coordinated government action at all levels. Countries need to work together on solutions for sustainable transport, more efficient and renewable energy production and use and waste management. WHO works with many sectors including transport and energy, urban planning and rural development to support countries to tackle this problem.\n\nKey findings:\n\nWHO estimates that around 90% of people worldwide breathe polluted air. Over the past 6 years, ambient air pollution levels have remained high and approximatively stable, with declining concentrations in some part of Europe and in the Americas.\n\nThe highest ambient air pollution levels are in the Eastern Mediterranean Region and in South-East Asia, with annual mean levels often exceeding more than 5 times WHO limits, followed by low and middle-income cities in Africa and the Western Pacific.\n\nAfrica and some of the Western Pacific have a serious lack of air pollution data. For Africa, the database now contains PM measurements for more than twice as many cities as previous versions, however data was identified for only 8 of 47 countries in the region.\n\nEurope has the highest number of places reporting data.\n\nIn general, ambient air pollution levels are lowest in high-income countries, particularly in Europe, the Americas and the Western Pacific.In cities of high-income countries in Europe, air pollution has been shown to lower average life expectancy by anywhere between 2 and 24 months, depending on pollution levels.\n\n\"Political leaders at all levels of government, including city mayors, are now starting to pay attention and take action,\" adds Dr Tedros. \"The good news is that we are seeing more and more governments increasing commitments to monitor and reduce air pollution as well as more global action from the health sector and other sectors like transport, housing and energy.\"\n\nThis year WHO will convene the first Global Conference on Air Pollution and Health (30 October \u2013 1 November 2018) to bring governments and partners together in a global effort to improve air quality and combat climate change. <http:\/\/www.who.int\/airpollution\/events\/conference\/en\/>\n\nAvailable from: <http:\/\/www.who.int\/news-room\/detail\/02-05-2018-9-out-of-10-people-worldwide-breathe-polluted-air-but-more-countries-are-taking-action>","meta":{"dup_signals":{"dup_doc_count":184,"dup_dump_count":52,"dup_details":{"curated_sources":2,"2023-50":3,"2023-40":3,"2023-23":2,"2023-14":7,"2023-06":1,"2022-49":3,"2022-40":4,"2022-33":1,"2022-27":3,"2022-21":3,"2022-05":3,"2021-49":2,"2021-43":2,"2021-39":7,"2021-31":4,"2021-25":2,"2021-21":3,"2021-17":3,"2021-10":3,"2021-04":4,"2020-50":4,"2020-45":2,"2020-40":5,"2020-34":1,"2020-29":4,"2020-24":1,"2020-16":2,"2020-10":2,"2020-05":1,"2019-51":2,"2019-47":3,"2019-43":4,"2019-39":3,"2019-35":7,"2019-30":1,"2019-26":6,"2019-22":5,"2019-18":4,"2019-13":2,"2019-09":8,"2019-04":7,"2018-51":2,"2018-47":9,"2018-43":3,"2018-39":4,"2018-34":8,"2018-30":4,"2018-26":1,"2018-22":10,"2024-22":1,"2024-10":2,"2024-26":1}},"file":"PMC6146220"},"subset":"pubmed_central"}
{"text":"abstract: # Objective\n  .\n  To characterise the clinical features of patients admitted to hospital with coronavirus disease 2019 (covid-19) in the United Kingdom during the growth phase of the first wave of this outbreak who were enrolled in the International Severe Acute Respiratory and emerging Infections Consortium (ISARIC) World Health Organization (WHO) Clinical Characterisation Protocol UK (CCP-UK) study, and to explore risk factors associated with mortality in hospital.\n  .\n  # Design\n  .\n  Prospective observational cohort study with rapid data gathering and near real time analysis.\n  .\n  # Setting\n  .\n  208 acute care hospitals in England, Wales, and Scotland between 6 February and 19 April 2020. A case report form developed by ISARIC and WHO was used to collect clinical data. A minimal follow-up time of two weeks (to 3 May 2020) allowed most patients to complete their hospital admission.\n  .\n  # Participants\n  .\n  20\u2009133 hospital inpatients with covid-19.\n  .\n  # Main outcome measures\n  .\n  Admission to critical care (high dependency unit or intensive care unit) and mortality in hospital.\n  .\n  # Results\n  .\n  The median age of patients admitted to hospital with covid-19, or with a diagnosis of covid-19 made in hospital, was 73 years (interquartile range 58-82, range 0-104). More men were admitted than women (men 60%, n=12\u2009068; women 40%, n=8065). The median duration of symptoms before admission was 4 days (interquartile range 1-8). The commonest comorbidities were chronic cardiac disease (31%, 5469\/17\u2009702), uncomplicated diabetes (21%, 3650\/17\u2009599), non-asthmatic chronic pulmonary disease (18%, 3128\/17\u2009634), and chronic kidney disease (16%, 2830\/17\u2009506); 23% (4161\/18\u2009525) had no reported major comorbidity. Overall, 41% (8199\/20\u2009133) of patients were discharged alive, 26% (5165\/20\u2009133) died, and 34% (6769\/20\u2009133) continued to receive care at the reporting date. 17% (3001\/18\u2009183) required admission to high dependency or intensive care units; of these, 28% (826\/3001) were discharged alive, 32% (958\/3001) died, and 41% (1217\/3001) continued to receive care at the reporting date. Of those receiving mechanical ventilation, 17% (276\/1658) were discharged alive, 37% (618\/1658) died, and 46% (764\/1658) remained in hospital. Increasing age, male sex, and comorbidities including chronic cardiac disease, non-asthmatic chronic pulmonary disease, chronic kidney disease, liver disease and obesity were associated with higher mortality in hospital.\n  .\n  # Conclusions\n  .\n  ISARIC WHO CCP-UK is a large prospective cohort study of patients in hospital with covid-19. The study continues to enrol at the time of this report. In study participants, mortality was high, independent risk factors were increasing age, male sex, and chronic comorbidity, including obesity. This study has shown the importance of pandemic preparedness and the need to maintain readiness to launch research studies in response to outbreaks.\n  .\n  # Study registration\n  .\n  ISRCTN66726260.\nauthor: Annemarie B Docherty; Ewen M Harrison; Christopher A Green; Hayley E Hardwick; Riinu Pius; Lisa Norman; Karl A Holden; Jonathan M Read; Frank Dondelinger; Gail Carson; Laura Merson; James Lee; Daniel Plotkin; Louise Sigfrid; Sophie Halpin; Clare Jackson; Carrol Gamble; Peter W Horby; Jonathan S Nguyen-Van-Tam; Antonia Ho; Clark D Russell; Jake Dunning; Peter JM Openshaw; J Kenneth Baillie; Malcolm G SempleCorrespondence to: M G Semple <firstname.lastname@example.com> (or [@TweedieChap](https:\/\/twitter.com\/tweediechap) on Twitter)\ndate: 2020\nreferences:\ntitle: Features of 20\u2009133 UK patients in hospital with covid-19 using the ISARIC WHO Clinical Characterisation Protocol: prospective observational cohort study\n\n# Introduction\n\nThe outbreak of disease caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was declared a pandemic by the World Health Organization on 11 March 2020.1 The WHO situation report dated 30 April 2020 stated 3\u2009090\u2009445 people had confirmed coronavirus disease 2019 (covid-19) and 217\u2009769 people had died across the world.2\n\nIn the wake of the influenza A H1N1 pandemic (2009) and the emergence of Middle East respiratory syndrome coronavirus (2012), it was recognised that the effectiveness of a response to a future pandemic threat would critically depend on the speed and focus of that response. The United Kingdom set up and maintained a \"sleeping\" prepandemic suite of protocols, documents, and agreements in preparation for future outbreaks. The International Severe Acute Respiratory and emerging Infections Consortium (ISARIC) WHO Clinical Characterisation Protocol UK (CCP-UK) study was a core component of this portfolio.3 Further details about ISARIC WHO CCP-UK can be found at <https:\/\/isaric4c.net> and in the online supplement.\n\nIn response to the emergence of SARS-CoV-2 and its pandemic potential, the ISARIC WHO CCP-UK study was activated on 17 January 2020, in time to enrol the first wave of patients with covid-19 admitted to hospitals in England and Wales. The first confirmed patient with covid-19 in the UK was reported on 31 January 2020.\n\nHospital admission rates for patients with covid-19 have been difficult to estimate because rates depend on the prevalence of community testing and admission criteria, which vary between countries. However, an estimated one in 10 to one in five adults have illnesses of sufficient severity to warrant hospital admission.4 Patients have mostly been admitted with severe acute respiratory infection or severe acute respiratory syndrome according to the previous WHO case definitions.5 6 The provision of intensive care also varies between countries. Studies first from China, and more recently from Europe and the United States, have found rates of admission to intensive care range from 5% to 32%.7 8 Old age, chronic major comorbidity, and male sex have consistently been associated with increased mortality.9 10 11 12\n\nIn this first report of the ISARIC WHO CCP-UK study, we characterise the clinical features of patients admitted to hospital with covid-19 in England, Scotland, and Wales during the growth phase of the first wave of this outbreak, up to 19 April 2020. Future reports will include Northern Ireland. We describe all patient outcomes as known on 3 May 2020 and explore risk factors associated with mortality in hospital.\n\n# Methods\n\n## Study design and setting\n\nThe ISARIC WHO CCP-UK (National Institute for Health Research Clinical Research Network Central Portfolio Management System ID: 14152) study is an ongoing prospective cohort study in 208 acute care hospitals in England, Scotland, and Wales. The protocol (supplementary material 2), revision history, case report form (version 9.2; supplementary material 3), information leaflets, consent forms and details of the Independent Data and Material Access Committee are available online.21\n\n## Participants\n\nInclusion criteria were people of all ages who were admitted to one of 208 acute care hospitals in England, Scotland, and Wales with proven or high likelihood of infection with a pathogen of public health interest, defined as SARS-CoV-2 for this event by Public Health England. Reverse transcriptase polymerase chain reaction was the only mode of testing available during the period of study. The decision to test was at the discretion of the clinician attending the patient, and not defined by protocol. The enrolment criterion \"high likelihood of infection\" reflects that a preparedness protocol cannot assume that a diagnostic test will be available for an emergent pathogen. Site training emphasises that only patients who tested positive for covid-19 were eligible for enrolment.\n\nNational guidance was provided by Public Health England and other UK public health agencies that advised who to test based on clinical case definitions for possible covid-19 (online supplement). We also included patients who had been admitted for a separate condition but had tested positive for covid-19 during their hospital stay. We collected additional biological samples for research purposes when consent was given (please see online supplement for details of consent procedures and biological samples). These samples are currently undergoing analysis and we will present the results when they become available. Patients were only enrolled during their index admission. We used three tiers in the ISARIC WHO CCP-UK protocol. Patients in tier 0 had clinical information from their routine health records uploaded into the case report form. Consent was not required for collection of depersonalised routine healthcare data for research in England and Wales. A waiver for consent was given by the Public Benefit and Privacy Panel in Scotland. Tier 1 and 2 of the protocol involve additional biological sampling for research purposes for which consent by, or assent for, participants was obtained.\n\n## Data collection\n\nWe collected baseline demographic data on a paper case report form (version 9.2; supplementary material 2) that was developed by ISARIC and WHO for use in outbreak investigations. Data were uploaded from admission, and usually before hospital episodes were complete, to a REDCap database (Research Electronic Data Capture, Vanderbilt University, US, hosted by University of Oxford, UK). We aimed to record measures of illness severity and routine blood test results at a minimum of four time points: day of hospital admission (day 1), day 3, day 6, day 9, and day of any admission to critical care. We recorded relevant treatments that patients received in hospital, level of care (ward based, high dependency unit, or intensive care unit), complications, and details of discharge or death while in hospital. Further information about these variables can be found in the online supplement.\n\n## Outcomes\n\nThe main outcomes were critical care admission (high dependency unit or intensive care unit) and mortality in hospital or palliative discharge. We chose a priori to restrict analysis of outcomes to patients who were admitted more than two weeks before data extraction (3 May 2020) to enable most patients to finish their hospital admission.\n\n## Bias\n\nResearch nurses relied on local covid-19 test reports to enrol patients. Capacity to enrol was limited by staff resources at times of high covid-19 activity. Otherwise we are unable to comment on the potential selection bias of our cohort. We are in the process of linking to routine administrative healthcare data and will be able to make comparisons at that point.\n\n## Missing data\n\nThe nature of the study means that a large amount of data were missing, particularly during the later parts of the growth curve of the UK outbreak. Because this paper is mainly descriptive, we have not performed any imputation for missing data, and describe the data as they stand. To reduce the impact of missing data on outcome analyses, we restricted these analyses to patients who had been admitted for at least two weeks before data extraction.\n\n## Statistical analyses\n\nContinuous data are summarised as median (interquartile range) and categorical data as frequency (percentage). For univariate comparisons, the Mann-Whitney U test or Kruskal-Wallis test were used. We compared categorical data by using the \u03c7^2^ test.\n\nWe used several approaches to model survival. Discharge from hospital was considered an absorbing state, meaning that once discharged, patients were considered no longer at risk of death. Patients who were discharged were not censored and held within the risk set, therefore accounting for the competing risk of discharge on death. We checked this approach by using a formal Fine and Gray competing risks approach. Hierarchical Cox proportional hazards approaches included geographical region (clinical commissioning group or health board) as a random intercept. We used a parsimonious criterion based model building approach based on several principles: clinically relevant explanatory variables were identified a priori for exploration; population stratification was incorporated; interactions were checked at first order level; final model selection was informed by log likelihood tests and the concordance statistic, with appropriate assumptions checked including the distribution of residuals and requirement for proportional hazards. We set statistical significance at 5%. All tests were two sided. We analysed data by using R (R Core Team version 3.6.3, Vienna, Austria), with packages including tidyverse, finalfit, survival, cmprsk, and coxme.\n\n## Patient and public involvement\n\nThis was an urgent public health research study in response to a Public Health Emergency of International Concern. Patients or the public were not involved in the design, conduct, or reporting of this rapid response research.\n\n# Results\n\nOn behalf of ISARIC WHO CCP-UK, 2468 research nurses, administrators, and medical students enrolled 20\u2009133 patients who were admitted with covid-19 to 208 hospitals in England, Scotland, and Wales between 6 February and 14:00 on 19 April 2020 (<a href=\"#tbl1\" data-ref-type=\"table\">table 1<\/a> and fig E1). This figure represents 34% of the 59\u2009215 covid-19 admissions in these countries. The median time from onset of symptoms of covid-19 in the community to presentation at hospital was 4 days (interquartile range 1-8; n=16\u2009221).\n\nBaseline characteristics of 20\u2009133 patients with coronavirus disease 2019 stratified by sex. Data are numbers (percentages) unless stated otherwise\n\n| Characteristics | Male | Female | All |\n|----|----|----|----|\n| Total No (%) | 12\u2009068 (59.9) | 8065 (40.1) | 20\u2009133 |\n| Age at admission (n=20\u2009133) |  |  |  |\n| \u2003Median (interquartile range) | 72.0 (58.0-81.0) | 74.0 (58.0-84.0) | 72.9 (58.0-82.0) |\n| Age (n=20\u2009133) |  |  |  |\n| \u2003\\<18 | 180 (1.5) | 130 (1.6) | 310 (1.5) |\n| \u200318-39 | 534 (4.4) | 533 (6.6) | 1067 (5.3) |\n| \u200340-50 | 888 (7.4) | 530 (6.6) | 1418 (7.0) |\n| \u200350-59 | 1728 (14.3) | 980 (12.2) | 2708 (13.5) |\n| \u200360-69 | 2115 (17.5) | 1181 (14.6) | 3296 (16.4) |\n| \u200370-79 | 2972 (24.6) | 1720 (21.3) | 4692 (23.3) |\n| \u2003\u226580 | 3651 (30.3) | 2991 (37.1) | 6642 (33.0) |\n| Any comorbidity (n=18\u2009525) |  |  |  |\n| \u2003No | 2591 (23.4) | 1570 (21.1) | 4161 (22.5) |\n| \u2003Yes | 8492 (76.6) | 5872 (78.9) | 14\u2009364 (77.5) |\n| Chronic cardiac disease (n=17\u2009702) |  |  |  |\n| \u2003No | 7086 (66.8) | 5147 (72.6) | 12\u2009233 (69.1) |\n| \u2003Yes | 3527 (33.2) | 1942 (27.4) | 5469 (30.9) |\n| Chronic pulmonary disease, not asthma (n=17\u2009634) |  |  |  |\n| \u2003No | 8616 (81.7) | 5890 (83.1) | 14\u2009506 (82.3) |\n| \u2003Yes | 1931 (18.3) | 1197 (16.9) | 3128 (17.7) |\n| Asthma (n=17\u2009535) |  |  |  |\n| \u2003No | 9274 (88.6) | 5721 (80.9) | 14\u2009995 (85.5) |\n| \u2003Yes | 1192 (11.4) | 1348 (19.1) | 2540 (14.5) |\n| Smoker (n=14\u2009184) |  |  |  |\n| \u2003Never smoked | 5030 (58.8) | 3938 (69.9) | 8968 (63.2) |\n| \u2003Former smoker | 2972 (34.8) | 1392 (24.7) | 4364 (30.8) |\n| \u2003Yes | 549 (6.4) | 303 (5.4) | 852 (6.0) |\n| Chronic kidney disease (n=17\u2009506) |  |  |  |\n| \u2003No | 8792 (84.0) | 5884 (83.5) | 14\u2009676 (83.8) |\n| \u2003Yes | 1671 (16.0) | 1159 (16.5) | 2830 (16.2) |\n| Diabetes without complications (n=17\u2009599) |  |  |  |\n| \u2003No | 8254 (78.3) | 5695 (80.7) | 13\u2009949 (79.3) |\n| \u2003Yes | 2290 (21.7) | 1360 (19.3) | 3650 (20.7) |\n| Diabetes with complications (n=17\u2009516) |  |  |  |\n| \u2003No | 9628 (91.8) | 6589 (93.8) | 16\u2009217 (92.6) |\n| \u2003Yes | 860 (8.2) | 439 (6.2) | 1299 (7.4) |\n| Obesity (n=16\u2009081) |  |  |  |\n| \u2003No | 8725 (90.6) | 5671 (87.8) | 14\u2009396 (89.5) |\n| \u2003Yes | 900 (9.4) | 785 (12.2) | 1685 (10.5) |\n| Chronic neurological disorder (n=17\u2009382) |  |  |  |\n| \u2003No | 9222 (88.6) | 6189 (88.7) | 15\u2009411 (88.7) |\n| \u2003Yes | 1181 (11.4) | 790 (11.3) | 1971 (11.3) |\n| Dementia (n=17\u2009459) |  |  |  |\n| \u2003No | 9211 (88.2) | 5888 (83.9) | 15\u2009099 (86.5) |\n| \u2003Yes | 1232 (11.8) | 1128 (16.1) | 2360 (13.5) |\n| Malignancy (n=17\u2009354) |  |  |  |\n| \u2003No | 9251 (89.2) | 6360 (91.0) | 15\u2009611 (90.0) |\n| \u2003Yes | 1117 (10.8) | 626 (9.0) | 1743 (10.0) |\n| Moderate or severe liver disease (n=17\u2009360) |  |  |  |\n| \u2003No | 10\u2009181 (98.0) | 6869 (98.5) | 17\u2009050 (98.2) |\n| \u2003Yes | 204 (2.0) | 106 (1.5) | 310 (1.8) |\n| Mild liver disease (n=17\u2009331) |  |  |  |\n| \u2003No | 10\u2009195 (98.3) | 6855 (98.5) | 17\u2009050 (98.4) |\n| \u2003Yes | 174 (1.7) | 107 (1.5) | 281 (1.6) |\n| Chronic haematological disease (n=17\u2009328) |  |  |  |\n| \u2003No | 9951 (96.0) | 6684 (96.0) | 16\u2009635 (96.0) |\n| \u2003Yes | 415 (4.0) | 278 (4.0) | 693 (4.0) |\n| Rheumatological disorder (n=17\u2009289) |  |  |  |\n| \u2003No | 9562 (92.4) | 6031 (86.9) | 15\u2009593 (90.2) |\n| \u2003Yes | 787 (7.6) | 909 (13.1) | 1696 (9.8) |\n| Malnutrition (n=16\u2009695) |  |  |  |\n| \u2003No | 9768 (97.8) | 6531 (97.4) | 16\u2009299 (97.6) |\n| \u2003Yes | 222 (2.2) | 174 (2.6) | 396 (2.4) |\n| Previous immunosuppressant drug treatment (n=18\u2009009) |  |  |  |\n| \u2003Yes | 876 (8.1) | 791 (11.0) | 1667 (9.3) |\n| \u2003No | 9339 (86.6) | 6032 (83.5) | 15\u2009371 (85.4) |\n| \u2003Not applicable | 573 (5.3) | 398 (5.5) | 971 (5.4) |\n| Previous anti-infective treatment (n=18\u2009017) |  |  |  |\n| \u2003No | 1940 (18.0) | 1311 (18.2) | 3251 (18.0) |\n| \u2003Yes | 8285 (76.8) | 5520 (76.4) | 13\u2009805 (76.6) |\n| \u2003Not applicable | 569 (5.3) | 392 (5.4) | 961 (5.3) |\n| AIDS\/HIV (n=17\u2009251) |  |  |  |\n| \u2003No | 10\u2009259 (99.5) | 6909 (99.6) | 17\u2009168 (99.5) |\n| \u2003Yes | 55 (0.5) | 28 (0.4) | 83 (0.5) |\n\n## Age and sex\n\nThe median age of patients was 73 years (interquartile range 58-82, range 0-104; <a href=\"#f1\" data-ref-type=\"fig\">fig 1<\/a>); 310 patients (1.5%) were less than 18 years old and 194 (1.0%) were less than 5 years old. More men (59.9%, n=12\u2009068) than women (40.1%, n=8065) were admitted to hospital with covid-19. One hundred women (10%) of reproductive age (n=1033) were recorded as being pregnant.\n\n## Symptoms\n\nThe most common symptoms were cough (68.9%, 12\u2009896\/18\u2009730), fever (71.6%, 12\u2009499\/17\u2009452), and shortness of breath (71.2%, 12\u2009107\/16\u2009999; <a href=\"#f2\" data-ref-type=\"fig\">fig 2<\/a>, top left panel), though these data reflect the case definition. Only 4.5% (855\/19\u2009178) of patients reported no symptoms on admission. We found a high degree of overlap between the three most common symptoms (<a href=\"#f2\" data-ref-type=\"fig\">fig 2<\/a>, lower left panel).\n\nClusters of symptoms on admission were apparent (fig E2). The most common symptom cluster encompassed the respiratory system: cough, sputum, shortness of breath, and fever. We also observed three other clusters: one encompassing musculoskeletal symptoms (myalgia, joint pain, headache, and fatigue); a cluster of enteric symptoms (abdominal pain, vomiting, and diarrhoea); and less commonly, a mucocutaneous cluster. Twenty nine per cent (5384\/18\u2009605) of all patients complained of enteric symptoms on admission, mostly in association with respiratory symptoms; however, 4% of all patients described enteric symptoms alone.\n\n## Comorbidities\n\n<a href=\"#f2\" data-ref-type=\"fig\">Figure 2<\/a> (top right panel) and <a href=\"#tbl1\" data-ref-type=\"table\">table 1<\/a> show major comorbidities recorded on admission. The most common major comorbidities were chronic cardiac disease (30.9%, 5469\/17\u2009702), diabetes without complications (20.7%, 3650\/17\u2009599), chronic pulmonary disease excluding asthma (17.7%, 3128\/17\u2009634), chronic kidney disease (16.2%, 2830\/17\u2009506), and asthma (14.5%, 2540\/17\u2009535). Of 18\u2009525 patients, 22.5% (4161) had no documented major comorbidity. There was little overlap between the three most common comorbidities (<a href=\"#f2\" data-ref-type=\"fig\">fig 2<\/a>, lower right panel).\n\nSix per cent (852\/14\u2009184) of patients were current smokers, 30.8% (4364) were previous smokers, and 63.2% (8968) had never smoked. Figure E3 shows the pattern of major comorbidity stratified by age.\n\n## Level of care\n\nA high proportion of patients required admission to high dependency or intensive care units (17%, 3001\/18\u2009183; <a href=\"#f3\" data-ref-type=\"fig\">fig 3<\/a>), and 55% (9244\/16\u2009849) received high flow oxygen at some point during their admission. Sixteen per cent of patients (2670\/16\u2009805) were treated with non-invasive ventilation, while 10% (1658\/16\u2009866) received invasive ventilation.\n\n## Patient outcomes\n\nOverall, 41% (8199\/20\u2009133) of patients were discharged alive, 26% (5165\/20\u2009133) died, and 34% (6769\/20\u2009133) continued to receive care at the date of reporting (<a href=\"#f4\" data-ref-type=\"fig\">fig 4<\/a>). The median age of patients who died in hospital from covid-19 in the study was 80 years, and only 11% (559\/4880) of these patients had no documented major comorbidity.\n\nFor patients who received only ward care, 47% (7203\/15\u2009297) were discharged alive, 26% (3954\/15\u2009297) died, and 27% (4140\/15\u2009297) remained in hospital at the date of reporting. As expected, outcomes were worse for those who needed higher levels of care.\n\nOf patients admitted to critical care (high dependency unit or intensive care unit), 28% (826\/3001) were discharged alive, 32% (958\/3001) died, and 41% (1217\/3001) continued to receive care at the date of reporting. Although the patients who received mechanical ventilation were younger than the overall cohort (61 years, interquartile range 52-69), only 17% (276\/1658) had been discharged alive by 19 April 2020, 37% (618\/1658) had died, and 46% (764\/1658) continued to receive care.\n\nLength of stay increased with age for patients discharged alive (fig E4). For patients who died, we found no association between age and time to death, with around 80% dying before day 14 of hospital admission.\n\n## Association of pre-existing patient characteristics and survival\n\nThe online supplement (table E4) describes univariable and multivariable associations with mortality. <a href=\"#f5\" data-ref-type=\"fig\">Figure 5<\/a> shows variables that remained significant in the multivariable model. Increasing age was a strong predictor of mortality in hospital after adjusting for major comorbidity (reference age \\<50 years): 50-59 years, hazard ratio 2.63 (95% confidence interval 2.06 to 3.35, P\\<0.001); 60-69 years, 4.99 (3.99 to 6.25, P\\<0.001); 70-79 years, 8.51 (6.85 to 10.57, P\\<0.001); \u226580 years, 11.09 (8.93 to 13.77, P\\<0.001). Female sex was associated with lower mortality (0.81, 0.75 to 0.86, P\\<0.001). Chronic cardiac disease, chronic non-asthmatic pulmonary disease, chronic kidney disease, obesity, chronic neurological disorder (such as stroke), dementia, malignancy, and liver disease were also associated with increased hospital mortality. An interactive infographic is available at <https:\/\/isaric4c.net\/info>. This information must not be used as a predictive tool in practice or to inform individual treatment decisions.\n\n# Discussion\n\n## Principal findings\n\nPatients with covid-19 usually presented with fever, cough, and shortness of breath, and met the WHO case definitions for severe acute respiratory infection or severe acute respiratory syndrome. The most common previous major comorbidities were chronic cardiac disease, diabetes, and chronic non-asthmatic pulmonary disease. Seventeen per cent of patients were admitted to critical care (high dependency unit or intensive care unit). Mortality in hospital was at least 26%, with 34% of the cohort still in hospital at the time of analysis; these proportions increased with escalating level of care. Factors associated with mortality in hospital were increasing age, male sex, and major comorbidities (cardiac disease, non-asthmatic pulmonary disease, kidney disease, liver disease, malignancy, obesity, and dementia).\n\nThe data presented in this study describe patients admitted to hospital during the growth phase of the SARS-CoV-2 pandemic in the UK. The first 101 patients were enrolled in the early phase of the outbreak as part of a high consequence infectious disease containment strategy that ended on 10 March 2020. These patients and others who were identified through screening in hospital, or who contracted covid-19 after admission (hospital acquired infection), are included in the 855 patients who were admitted without covid-19 symptoms. The impact these patients have had on the overall cohort characteristics has diminished as numbers have increased, and we believe it is important to keep these patients in the study. Other patients in our cohort without covid-19 symptoms are those who were diagnosed with the disease at the discretion of the clinician looking after them while staying in hospital for other reasons.\n\nThe pattern of disease we describe broadly reflects the pattern reported globally.7 Patients in our study had a higher median age and higher rates of chronic obstructive pulmonary disease and asthma than patients in China8 and the US.11 12 The prevalence of obesity in our study (11%) was considerably lower than the overall UK prevalence (29%).13 This proportion could reflect the relatively elderly male population admitted to hospital and misclassification or under reporting by admitting physicians. Our patients presented with a relatively short time interval between onset of symptoms and admission to hospital, which might also be a function of the older and vulnerable patient population.\n\nThe current case definition of cough and fever, if strictly applied, would miss 7% of our inpatients. A smaller proportion, 4% of patients, presented with enteric symptoms only. This figure could be an underestimate because these patients fall outside standard criteria for testing. This enteric presentation risks misclassification of patients, and assignment to non-covid-19 care areas, which could pose a nosocomial transmission risk. Severe SARS-CoV-2 infections are rare in people younger than 18 years, comprising only 1.5% of those admitted to hospital. Only 1.0% of those in our study were younger than 5 years. The J shaped age distribution is starkly different to the U shaped age distribution seen in seasonal influenza and the W shaped distribution observed in the 2009 influenza pandemic.14 The reason why SARS-CoV-19 has mostly spared children is not clear, but we speculate this could be because angiotensin converting enzyme 2 receptors are expressed differently in younger lungs.\n\nOther studies have not widely reported that obesity as recognised by clinical staff is associated with mortality in hospital after adjustment for other comorbidities, age, and sex. Obesity was recognised as a risk factor in the 2009 influenza A H1N1 pandemic, but not for the 2012 Middle East respiratory syndrome coronavirus.15 16\n\nThe proportion of pregnant women in our cohort was small (10%), similar to the estimated proportion of pregnant women in the community.15 Pregnancy was not associated with mortality, in apparent contrast to influenza.17\n\n## Comparison with other studies\n\nThe proportion of patients admitted to critical care in our study was similar to that reported in Italy (17%),18 19 and New York (14.2%),11 12 but higher than China.8 At the time of enrolment, the Intensive Care Society had issued guidance to its members that there would be no rationing of critical care admission until all capacity in the country had been exhausted. As far as we are aware, critical care capacity was not exceeded in the UK during the period of the study. We do not believe that any equipment shortages existed during this period that might have prompted more aggressive futility discussions.\n\nMortality in our cohort was high in patients admitted to general wards who were not admitted to critical care, which suggests that advanced care planning occurred. We were unable to capture treatment limiting decisions about level of care. The high median age of patients who died in the cohort (80 years) could partly explain the high mortality rate. Mortality rates were extremely high for patients who received invasive mechanical ventilation in the intensive care unit compared with the 2009 influenza A H1N1 pandemic, for which mortality in intensive care was 31%.15 Our data were in line with the initial ICNARC (intensive care national audit and research centre) audit reports, which represent intensive care units in England, Wales, Scotland, and Northern Ireland.20\n\nOutcome analyses only included patients who were admitted before 19 April to allow most patients to complete their hospital admission. However, an inherent reporting bias exists because the sickest of patients, particularly those admitted to intensive care, have the longest hospital stays; mortality rates in hospital could therefore increase. These mortality rates were considerably higher than the 24% mortality rate in hospital seen in patients in intensive care units in Italy19 and the US.11 12 The lower rate in the US could in part be explained by differences in healthcare systems and the proportion of intensive care unit beds to hospital beds between the two countries. In Italy, a lower proportion of patients received mechanical ventilation, and most of their patients (72%) remained in hospital at the time of the analysis.19\n\nThe finding of independent associations of advancing age, male sex, chronic respiratory (non-asthmatic) disease, chronic cardiac disease, and chronic neurological disease with mortality in hospital is in line with early international reports.9 10 However, although age adjusted mortality rates are high in elderly patients, most of these patients were admitted to hospital with symptoms of covid-19 and would not have been in hospital otherwise. Enhanced severity in male patients was seen across all ages.\n\n## Strengths and limitations of study\n\nISARIC WHO CCP-UK stood ready to conduct large scale studies of pandemic outbreaks for eight years, enabling us to enrol 34% of all patients with covid-19 admitted to 208 acute care hospitals across England, Wales and Scotland in the early phase of the pandemic.\n\nOur study has some limitations. We do not currently have data on the inpatients that were not enrolled, or people managed in community settings, such as usual domestic residences and older people's care homes. We are unable to comment on community risk factors that drive hospital admission except by inference from expected representation at admission. We will be linking to routine administrative healthcare datasets which will enable us to assess the presence of any selection bias.\n\nA large amount of data were missing and we suggest there are two main reasons for this. Firstly, enrolment occurred in the nonlinear growth phase of the outbreak, and outcomes for recent admissions have not been reported yet; these admissions account for 18% of the total number of patients enrolled. Secondly, the research network was dealing with unprecedented numbers of patients at a time when many were seconded to clinical practice or themselves off sick. This study is ongoing, and further data are being added to case report forms.\n\nWe suggest it is possible that the sickest patients were enrolled in our study, and this could partly explain our high mortality rates in hospital. Some of the sickest patients in the study had the longest lengths of hospital stay and we do not have outcome data for all of these patients yet.\n\n## Conclusions and policy implications\n\nThis large and rapidly conducted study of patients admitted to hospital in England, Wales, and Scotland with covid-19 shows the importance of putting plans in place for the study of epidemic and pandemic threats, and the need to maintain these plans. Our study identifies sectors of the population that are at greatest risk of a poor outcome, and reports the use of healthcare resources. Most patients with covid-19 experience mild disease. However, in our cohort, of those who were admitted to hospital two weeks before data extraction, less than half have been discharged alive and a quarter have died. The remainder continued to receive care at the date of reporting. Seventeen percent of patients admitted to hospital required critical care. Factors associated with mortality in hospital were increasing age, male sex, obesity, and major comorbidities.\n\nISARIC Coronavirus Clinical Characterisation Consortium21 investigators have submitted regular reports to the UK Government's New and Emerging Respiratory Virus Threats Advisory Group (NERVTAG)22 and the Scientific Advisory Group for Emergencies (SAGE).23 Patient level data have been shared and independently analysed by the Scientific Pandemic Influenza Group on Modelling (SPI-M)24 and other investigators. Aggregated data have been shared with WHO in the ISARIC covid-19 report.\n\nStudies such as this cannot be developed, approved, and opened from the start of a pandemic in time to inform case management and public health policy. Our study has shown the importance of forward planning and investment in preparedness studies. Over the next few months we will issue reports in *The BMJ* on specific topics and analyses that are key to understanding the impact of covid-19 and focus on improving patient outcomes.\n\n### What is already known on this topic\n\n1.  Observational studies in China have reported risk factors associated with severe covid-19 that requires hospital admission\n\n2.  Studies describing the features and outcomes of patients with severe covid-19 who have been admitted to hospital in Europe are lacking\n\n3.  Older male adults, people with diabetes, hypertension, cardiovascular disease, or chronic respiratory disease are at greater risk of severe covid-19 that requires hospital admission and higher levels of care, and are at higher risk of death\n\n### What this study adds\n\n1.  This rapid prospective investigation of patients with covid-19 admitted to hospital in England, Wales, and Scotland showed that obesity, chronic kidney disease, and liver disease were also associated with increased hospital mortality\n\n2.  Obesity is a major additional risk factor that was not highlighted in data from China\n\n3.  Severe covid-19 leads to a prolonged hospital stay and a high mortality rate; over a quarter of inpatients in this study had died at the time of reporting, and nearly a third remained in hospital\n\nThe study protocol is available at <http:\/\/isaric4c.net\/protocols>; study registry <https:\/\/www.isrctn.com\/ISRCTN66726260>. This work uses data provided by patients and collected by the NHS as part of their care and support \\#DataSavesLives. We are extremely grateful to the 2648 frontline NHS clinical and research staff and volunteer medical students who collected these data in challenging circumstances; and the generosity of the patients and their families for their individual contributions in these difficult times. We also acknowledge the support of Jeremy J Farrar, Nahoko Shindo, Devika Dixit, Nipunie Rajapakse, Piero Olliaro, Lyndsey Castle, Martha Buckley, Debbie Malden, Katherine Newell, Kwame O'Neill, Emmanuelle Denis, Claire Petersen, Scott Mullaney, Sue MacFarlane, Chris Jones, Nicole Maziere, Katie Bullock, Emily Cass, William Reynolds, Milton Ashworth, Ben Catterall, Louise Cooper, Terry Foster, Paul Matthew Ridley, Anthony Evans, Catherine Hartley, Chris Dunn, D Sales, Diane Latawiec, Erwan Trochu, Eve Wilcock, Innocent Gerald Asiimwe, Isabel Garcia-Dorival, J Eunice Zhang, Jack Pilgrim, Jane A Armstrong, Jordan J Clark, Jordan Thomas, Katharine King, Katie Neville, Alexandra Ahmed, Krishanthi S Subramaniam, Lauren Lett, Laurence McEvoy, Libby van Tonder, Lucia Alicia Livoti, Nahida S Miah, Rebecca K Shears, Rebecca Louise Jensen, Rebekah Penrice-Randal, Robyn Kiy, Samantha Leanne Barlow, Shadia Khandaker, Soeren Metelmann, Tessa Prince, Trevor R Jones, Benjamin Brennan, Agnieska Szemiel, Siddharth Bakshi, Daniella Lefteri, Maria Mancini, Julien Martinez, Angela Elliott, Joyce Mitchell, John McLauchlan, Aislynn Taggart, Oslem Dincarslan, Annette Lake, Claire Petersen, Scott Mullaney, and Graham Cooke.\n\nISARIC Coronavirus Clinical Characterisation Consortium (ISARIC4C): Consortium lead investigator: J Kenneth Baillie; chief investigator: Malcolm G Semple; co-lead investigator: Peter JM Openshaw; ISARIC clinical coordinator: Gail Carson; co-investigators: Beatrice Alex, Benjamin Bach, Wendy S Barclay, Debby Bogaert, Meera Chand, Graham S Cooke, Annemarie B Docherty, Jake Dunning, Ana da Silva Filipe, Tom Fletcher, Christopher A Green, Julian A Hiscox, Antonia Ying Wai Ho, Peter W Horby, Samreen Ijaz, Saye Khoo, Paul Klenerman, Andrew Law, Wei Shen Lim, Alexander J Mentzer, Laura Merson, Alison M Meynert, Mahdad Noursadeghi, Shona C Moore, Massimo Palmarini, William A Paxton, Georgios Pollakis, Nicholas Price, Andrew Rambaut, David L Robertson, Clark D Russell, Vanessa Sancho-Shimizu, Janet T Scott, Tom Solomon, Shiranee Sriskandan, David Stuart, Charlotte Summers, Richard S Tedder, Emma C Thomson, Ryan S Thwaites, Lance CW Turtle, Maria Zambon; project managers: Hayley E Hardwick, Chloe Donohue, Jane Ewins, Wilna Oosthuyzen, Fiona Griffiths; data analysts: Lisa Norman, Riinu Pius, Tom M Drake, Cameron J Fairfield, Stephen Knight, Kenneth A Mclean, Derek Murphy, Catherine A Shaw; data and information system managers: Jo Dalton, Michelle Girvan, Egle Saviciute, Stephanie Roberts, Janet Harrison, Laura Marsh, Marie Connor; data integration and presentation: Gary Leeming, Andrew Law, Ross Hendry; material management: William Greenhalf, Victoria Shaw, Sarah McDonald; local principal investigators: Kayode Adeniji, Daniel Agranoff, Ken Agwuh, Dhiraj Ail, Ana Alegria, Brian Angus, Abdul Ashish, Dougal Atkinson, Shahedal Bari, Gavin Barlow, Stella Barnass, Nicholas Barrett, Christopher Bassford, David Baxter, Michael Beadsworth, Jolanta Bernatoniene, John Berridge, Nicola Best, Pieter Bothma, David Brealey, Robin Brittain-Long, Naomi Bulteel, Tom Burden, Andrew Burtenshaw, Vikki Caruth, David Chadwick, Duncan Chambler, Nigel Chee, Jenny Child, Srikanth Chukkambotla, Tom Clark, Paul Collini, Graham Cooke, Catherine Cosgrove, Jason Cupitt, Maria-Teresa Cutino-Moguel, Paul Dark, Chris Dawson, Samir Dervisevic, Phil Donnison, Sam Douthwaite, Ingrid DuRand, Ahilanadan Dushianthan, Tristan Dyer, Cariad Evans, Chi Eziefula, Chrisopher Fegan, Adam Finn, Duncan Fullerton, Sanjeev Garg, Sanjeev Garg, Atul Garg, Effrossyni Gkrania-Klotsas, Jo Godden, Arthur Goldsmith, Clive Graham, Elaine Hardy, Stuart Hartshorn, Daniel Harvey, Peter Havalda, Daniel B Hawcutt, Antonia Ho, Maria Hobrok, Luke Hodgson, Anita Holme, Anil Hormis, Michael Jacobs, Susan Jain, Paul Jennings, Agilan Kaliappan, Vidya Kasipandian, Stephen Kegg, Michael Kelsey, Jason Kendall, Caroline Kerrison, Ian Kerslake, Oliver Koch, Gouri Koduri, George Koshy, Shondipon Laha, Susan Larkin, Tamas Leiner, Patrick Lillie, James Limb, Vanessa Linnett, Jeff Little, Michael MacMahon, Emily MacNaughton, Ravish Mankregod, Huw Masson, Elijah Matovu, Katherine McCullough, Ruth McEwen, Manjula Meda, Gary Mills, Jane Minton, Mariyam Mirfenderesky, Kavya Mohandas, James Moon, Elinoor Moore, Patrick Morgan, Craig Morris, Katherine Mortimore, Samuel Moses, Mbiye Mpenge, Rohinton Mulla, Michael Murphy, Megan Nagel, Thapas Nagarajan, Mark Nelson, Igor Otahal, Mark Pais, Selva Panchatsharam, Hassan Paraiso, Brij Patel, Justin Pepperell, Mark Peters, Mandeep Phull, Stefania Pintus, Jagtur Singh Pooni, Frank Post, David Price, Rachel Prout, Nikolas Rae, Henrik Reschreiter, Tim Reynolds, Neil Richardson, Mark Roberts, Devender Roberts, Alistair Rose, Guy Rousseau, Brendan Ryan, Taranprit Saluja, Aarti Shah, Prad Shanmuga, Anil Sharma, Anna Shawcross, Jeremy Sizer, Richard Smith, Catherine Snelson, Nick Spittle, Nikki Staines, Tom Stambach, Richard Stewart, Pradeep Subudhi, Tamas Szakmany, Kate Tatham, Jo Thomas, Chris Thompson, Robert Thompson, Ascanio Tridente, Darell Tupper-Carey, Mary Twagira, Andrew Ustianowski, Nick Vallotton, Lisa Vincent-Smith, Shico Visuvanathan, Alan Vuylsteke, Sam Waddy, Rachel Wake, Andrew Walden, Tony Whitehouse, Paul Whittaker, Ashley Whittington, Meme Wijesinghe, Martin Williams, Lawrence Wilson, Sarah Wilson, Stephen Winchester, Martin Wiselka, Adam Wolverson, Daniel G Wooton, Andrew Workman, Bryan Yates, Peter Young.\n\nExtra material supplied by authors\n\nWeb appendix: Supplementary material 1\u2014online supplement\n\nWeb appendix: Supplementary material 2\u2014protocol\n\nWeb appendix: Supplementary material 3\u2014case report form","meta":{"dup_signals":{"dup_doc_count":166,"dup_dump_count":31,"dup_details":{"curated_sources":2,"2023-50":9,"2023-40":3,"2023-23":4,"2023-14":4,"2023-06":9,"2022-49":7,"2022-40":4,"2022-33":11,"2022-27":11,"2022-21":10,"2022-05":8,"2021-49":10,"2021-43":5,"2021-39":7,"2021-31":5,"2021-25":4,"2021-21":9,"2021-17":7,"2021-10":4,"2021-04":1,"2020-50":2,"2020-45":4,"2020-40":1,"2020-34":3,"2020-29":3,"2020-24":2,"2024-30":3,"2024-26":3,"2024-22":4,"2024-18":4,"2024-10":3}},"file":"PMC7243036"},"subset":"pubmed_central"}
{"text":"abstract: 1.  IVIG, combined with moderate-dose of corticosteroids, might improve patient outcomes.\n  .\n  2.  The use of corticosteroids might accelerate recovery from COVID-19.\n  .\n  3.  No controlled clinical trials exist on the use of corticosteroids for COVID-19.\n  .\n  4.  IL6 correlates with severity, criticality, viral load, and prognosis of COVID-19.\n  .\n  5.  Tocilizumab, an anti-IL6, can confer benefit in patients with COVID-19 and high IL6.\nauthor: Amene Saghazadeh; Nima Rezaei\u204eCorresponding author at: Children's Medical Center Hospital, Dr. Qarib St, Keshavarz Blvd, Tehran 14194, Iran.\ndate: 2020-05-08\nreferences:\ntitle: Towards treatment planning of COVID-19: Rationale and hypothesis for the use of multiple immunosuppressive agents: Anti-antibodies, immunoglobulins, and corticosteroids\n\n# Introduction\n\nSeveral generations have been exposed to COVID-19 under different conditions of life at varying locations around the world. As long as the COVID-19 continues to spread, its power of genome modification would probably be increased. What concerns us more is that the 2019-nCoV, by the process of modification of genome structure, might become more and more fitted to humans to profoundly affect those who have already escaped \u2013 children and young people without a pre-existing condition. No one can tell how much time it takes to reach a new level of perfection, and that the development of vaccines for active immunization is a long process.\n\nMoreover, most of the best available anti-viral agents are not helpful in the treatment of COVID-19. A randomized controlled trial of 199 hospitalized adults with confirmed COVID-19 demonstrated no actual difference between patients who received lopinavir-ritonavir and patients who received standard care alone in clinical improvement, death rate, and positive virus test rate at day 28 \\[1\\]. Here we review intermediate indicators of the pathogenesis of COVID-19 that have the potential of being considered for the treatment of COVID-19.\n\n# SARS-CoV2 cell entry is not complex\n\n<a href=\"#f0005\" data-ref-type=\"fig\">Fig. 1<\/a> is a schematic illustration of the minimum proteins required to mediate the SARS-CoV2 cell entry. The trimeric, transmembrane spike (S) glycoprotein of the virus, SARS-2-S, includes two main functional subunits: S1 and S2. The former, in turn, consists of the four core domains, S1~A~, S1~B~, S1~C~, and S1~D~, that contribute to the attachment to the surface receptor of target cells. Then, the latter coordinates the fusion of viral and cellular membranes. Receptor binding activates proteases that can carry out proteolytic cleavage of the S protein. In coronaviruses, cleavage occurs at two sites: the S1\/S2 junction and at the S2\u2032, a region close to the viral fusion peptide \\[2\\]. Proteolytic cleavage of the S protein causes conformational changes so that they cannot revert to the original structure and profound enough to prime the S2 subunit for the fusion of viral and cellular membranes.\n\n# Certain characteristics of SARS-CoV2\n\n## SARS-CoV2 has acquired an S glycoprotein that highly underwent genetic variation and glycosylation\n\n### Polybasic cleavage site\n\nAs evidenced by sequence analysis, there is a residue insertion formed of four amino acids (12 nucleotides) at the boundary between S1 and S2 subunits of the SARS-CoV 2 S. It defines a polybasic furin cleavage site of RRAR for the human SARS-CoV 2 that was absent in human SARS-CoV, bat SARS-like CoVs, and pangolin SARS-like CoV while might be present in other species \\[3\\]. After the introduction of mutation to the residue insertion and furin cleavage site, the S1\/S2 cleavage of the SARS-CoV 2 S did not longer take place. However, the SARS-CoV 2 S entry raised for VeroE6 cells and remained high in BHK cells that express human ACE. Therefore, it seems that SARS-CoV2 transmissibility does not depend on the S1\/S2 cleavage.\n\nA polybasic cleavage site explains a virus that is highly-pathogenic for humans while it is low-pathogenic for other species. For example, using reverse genetic tools, an avian paramyxovirus type 7 (APMV-7) was developed by mutating the fusion (F) protein cleavage site \\[4\\]. The constructed APMV-7 showed furin cleavage and increased replication and syncytium formation in cell cultures. However, chicken exposed to the virus did not exhibit infection.\n\n### Glycosylation\n\nGlycosylation and its related products, i.e., glycans, introduce changes to the viral envelope that make the virus fitted for interaction with the host cell membrane \\[5\\]. Generally, glycans are oligosaccharides linked to the dense decoration of the spike glycoprotein. In particular, these oligosaccharides have shown to influence the folding of the S protein and proteolytic process so that they facilitate the virus cell entry. Moreover, a virus with the glycosylated glycoprotein gains an extra feature for an escape from the immune responses. Therefore, glycans are a good target for vaccine design.\n\nTwo main types of glycans are N-linked and O-linked glycans. Both are released from glycoproteins. Whereas enzymes fulfill the construction of N-glycans, chemical methods perform the release of O-glycans. N-glycans are linked to the amino acid asparagine (Asn) residues (Asn-any amino acids except for proline- Ser or Thr) utilizing an N-glycosidic bond, mostly N-acetylglucosamine. O-glycans are attached to the amino acid serine (Ser) and threonine (Thr) residues by the addition of an N-acetyl galactosamine (GalNAc). For example, N-glycans exist in Hendra virus, SARS-CoV, influenza virus, hepatitis virus, HIV-1, and West Nile virus \\[6\\], and O-glycans have occurred in the Ebola virus.\n\nThe 2019-nCoV S protein includes 13 and 9N-linked glycosylation sequons in the S1 and S2 subunit, respectively \\[3\\]. All of these have previously occurred in the SARS-CoV S glycoprotein, except for four-linked glycosylation sequons in the S1. Also, due to the existence of an amino acid proline in the polybasic cleavage site, which makes the inserted sequence PRRA, there are three O-linked glycans introduced to the 2019-nCoV RBD residues S673, T678, and S686 \\[7\\].\n\n### SARS-CoV2 receptor binding domain contains six amino acids providing favorable positions for binding to human ACE2\n\nWhen compared to SARS-CoV S~B~, the motif binding the human ACE2 to the SARS-CoV 2 S~B~ showed a relatively higher binding affinity for human ACE2 as indicated in smaller equilibrium dissociation constant (2.9\u00a0nM vs. 7.7\u00a0nM) \\[3\\]. Structural analysis has demonstrated that fourteen positions critically take part in the receptor-binding domain of the SARS-CoV S~B~ that contain eight conserved (T402, Y436, Y440, N473, Y475, T486, G488, and Y491) positions and six semi-conserved (R426 to N448, Y442 to L464, L472 to F495, N479 to Q502, Y484 to Q507, and T487 to N510) substitutions with respect to the SARS-CoV 2 S~B~.\n\n### SARS-CoV2 receptor binding domain contains cyclic regions that can make interaction with cell-surface GRP78\n\nPep42 is a cyclic oligopeptide that, with its hydrophobic character, can selectively interact with cell surface glucose\u2010regulated protein 78 (GRP78), a member of 70\u00a0kDa heat shock proteins. GRP78, also known as BiP or HSPA5, under endoplasmic reticulum stress, can be translocated from the endoplasmic reticulum to the membrane and helps to maintain cellular integrity under physiologic and pathological stress. It critically contributes to various functions ranging from protein folding, transportation, and degradation to cell-signaling, proliferation, survival, apoptosis, inflammation, and immunity. The expression of GRP78 decreases with age.\n\nOn the 2019-nCoV spike protein, 13 disulfide bonds are corresponding to 13 different cyclic regions thought to be similar to the cyclic form of Pep42 \\[8\\]. Among these, four regions I-IV take place in the outer surface of a putative receptor-binding domain (RBD) on the viral spike., including C361, C379, C391, and C480. These regions share sequence similarity with Pep42, ranging from 15.38% to 46.15%. However, only one of them, i.e., region IV (GRAVY\u00a0=\u00a00.08), is a hydrophobic region, like Pep42 (GRAVY\u00a0=\u00a01.1). Structural models evaluate the energy contribution for region IV as a part of region III to the GRP78 to be about (\u22129.8 out of \u221214.0\u00a0kcal\/mol), and the docking platform proposes region IV as the best region binding to GRP78. The region IV can be linked to the substrate-binding domain \u03b2 (SBDB) of GRP78 using five H-bonds (through P479, N481, E484, and N487) and four hydrophobic interactions (through T478, E484, and F486).\n\n# Passive immunization\n\nThere are two main ways to induce protection against infections: active and passive immunization. Active immunization comes to exist when the body's own immune system can produce antibodies actively following exposure to a viral antigen. On the contrary, there is a passive working mode of the immune system that would appear following the transfer of antibodies that act directly to neutralize viral infectivity.\n\n## Serum therapy\n\n### Convalescent plasma from patients recovered from COVID-19\n\n#### Hypothesis: Plasma from patients recovered from COVID-19 contain antibodies against 2019-nCoV\n\nA look at the history of viral outbreaks offers convalescent plasma as the only remedy to avoid further fatalities. The most recent examples include the pandemic of influenza A H1N1 (H1N1pdm09) in virus infection 2009, the Western African Ebola virus epidemic in 2014, and the outbreak of MERS-CoV in 2015 \\[9\\]. Meta-analysis studies have shown a reduced risk of mortality in patients with SARS-CoV and influenza receiving convalescent plasma \\[10\\].\n\n#### Rationale: Convalescent plasma from patients recovered from COVID-19 can treat patients with severe COVID-19\n\nA pilot study \\[11\\] recently has investigated the safety and effect of convalescent plasma that contain antibody levels higher than 1:640 combined with regular anti-viral agents and standard supportive care on clinical outcomes of ten patients with severe COVID-19. The study showed clinical improvement of all the ten patients accompanied by an increase in lymphocyte count and a decrease in CRP. Following transfusion of convalescent plasma, all the seven patients who were SARS-CoV2 RNA positive before transfusion of convalescent plasma turned SARS-CoV2 negative. There were no control groups receiving convalescent plasma alone or standard therapy without convalescent therapy to evaluate the main effect of convalescent plasma.\n\n### Serum from bats with SARS-like CoV\n\n#### Hypothesis: whole-genome sequencing and phylogenetic analysis shed light on the origin of the 2019-nCoV virus \u2013 It has been probably introduced from bats to men\n\nGenome sequencing of a fecal bat sample, Rp3, could detect an isolate of coronaviruses, which was almost identical to the causative agent of the SARS-CoV outbreak of 2002\u20132003. Hence, it attained the name SARS-like coronavirus isolate Rp3 (SL-CoV Rp3) \\[12\\]. The bronchoalveolar lavage fluid (BALF) of a patient with SARS contained the 2019-nCoV. RNA sequencing could reveal about 90% similarity in nucleotides of the novel coronavirus and of SARS-like coronavirus that had previously related to bats \\[13\\], \\[14\\]. In particular, the S protein of the 2019-nCoV has a high sequence identity of 80\u201398% with the S protein of bat SARS-like CoVs, such as SARSr-CoV ZXC21 S, ZC45 S, and RaTG13 \\[3\\]. Moreover, in phylogenomic trees, branches for the 2019-nCoV are of greater length than those for the 2003 SARS-CoV, and therefore more favorable to bats.\n\n#### Rationale: Bat serum is not able to efficiently neutralize SARS-CoV\n\nThe range of bats, belonging to the genus *Rhinolophus* (horseshoe bats) and the family *Rhinolophidae*, produce the SARS-CoV antibody \\[12\\]. Polymerase chain reaction (PCR) will confirm the presence of SARS-CoV nucleocapsid (N) and polymerase (P) proteins in fecal samples if an individual bat being seropositive for SARS-CoV \\[12\\]. There is a significant degree of resemblance of greater than 90% in the nucleotide sequence of the viral genomes between SL-CoV Rp3 \\[12\\] and the Tor2 strain of SARS-CoV \u2013 which was isolated in Toronto \\[15\\]. The differences in the genome sequences of SARS-CoV in the two species occur merely in the S gene \u2013 which encodes the S1 domain of the coronavirus spike protein and contains regions with high mutation rates \\[12\\]. The coronaviruses commonly possess five open reading frames (ORF) that correlate with the production of the replicase polyprotein (P), the spike (S), envelope (E), and membrane (M) glycoproteins and the nucleocapsid (N) protein. The human SARS-CoV Tor2 and bat SL-CoV Rp3 strains remain more than 90% identical at the proteins P, E, M, and N. the protein S consists of two main domains: 1) the S1 domain conveys the role of receptor binding and 2) the S2 domain assumes the role of the fusion of viral and host-cell membranes. In particular, the human SARS-CoV Tor2 strain shows a noticeable degree of difference in the S1 domain from the bat SL-CoV Rp3 strain. This diversity would suffice to produce functional differences between the species, and is an apparent reason why bat sera having high levels of cross-reactive antibodies not acted efficiently to neutralize SARS-CoV.\n\n### Serum from convalescent SARS patients\n\n#### Hypothesis: SARS-CoV and SARS-CoV2 are ideally similar in the structure and the cell entry receptor and protease\n\nSARS-CoV and SARS-CoV2 share absolutely the same cleavage junctions, almost the same sequence (96%) of their main protease, a high degree (76%) of similarity in the amino acid sequence of their S protein, a similar S2\u2032 cleavage site, a similar spectrum of cells they can enter, and the similarity of the most residues essential for binding ACE2 \\[16\\], \\[17\\], \\[18\\]. Also, both of them utilize the same domain of S1~B~ to interact with the ACE2 receptor. However, they differ in proteolytic processing to some degree. Study \\[16\\] of the human embryonic kidney (HEK) cell line, 293\u00a0T, has shown that a signal for the S2 subunit is present in cells inoculated with SARS-2-S, but not in cells inoculated with SARS-S.\n\nTwo main proteases for both SARS-S and SARS-2-S are endosomal cysteine proteases cathepsin B and L (CatB\/L) and the transmembrane protease, serine 2TMPRSS2 \\[16\\]. In 293\u00a0T cells lacking 2TMPRSS2, blocking CatB\/L activity through increasing the endosomal pH by ammonium chloride could significantly limit the entry of both SARS-S and SARS-2-S. In TMPRSS2\u00a0+\u00a0Caco-2 cells, the effect of ammonium chloride existed to a lesser extent. A combination of camostat mesylate, a blocker of TMPRSS2, and E-64d, an inhibitor of CatB\/L, yielded the complete inhibition of SARS-2-S entry in TMPRSS2\u00a0+\u00a0Caco-2 cells. In both the human lung cancer cell line Calu-3 and the primary human lung cells, there was a reduction of the entry of both SARS-S and SARS-2-S by camostat mesylate, indicating that SARS-S and SARS-2-S partially require TMPRSS2 for a lung infection.\n\n#### Rational: Serum from convalescent SARS patients is able to neutralize SARS-CoV2 efficiently\n\nAntiserum that contains antibodies against human ACE2 could hinder the entry of both SARS-S and SARS-2-S pseudotypes while not affected the entry of VSV-G and MERS-S pseudotypes. It supports the notion that SARS-S and SARS-2-S utilize the same primary entry receptor, i.e., ACE2, which is different from the primary receptors VSV-G and MERS-S engage for cell entry that is LDLR and DPP4, respectively.\n\nSera from three convalescent SARS patients reduced the SARS-S entry and, to e lesser degree, the SARS-2-S entry. The patient serum effect was in a dose-dependent manner \\[16\\].\n\n### Serum from rabbits immunized with SARS\n\n#### Hypotheses: Serum from rabbits immunized with SARS is more effective than serum from convalescent SARS patients\n\nParaoxonases (PON) are mammalian enzymes associated with anti-oxidant and anti-inflammatory effects \\[19\\]. Rabbit PON differs from human PON in terms of more activity and more stability under the circumstances \\[20\\].\n\n#### Rational: Serum from rabbits immunized with the S1 subunit of SARS-S is able to neutralize SARS-CoV2 very efficiently\n\nSera from rabbits immunized with the S1 subunit of SARS-S could effectively reduce the entry of both SARS-S and SARS-2-S \\[16\\]. When compared with patient serum, rabbit serum revealed to us higher efficiency in inhibition of SARS-2-S entry at the same concentration.\n\n## Intravenous immunoglobulins (IVIG)\n\n### Hypothesis: IVIG contains a large pool of human antibodies\n\nIVIG is an immunomodulatory treatment currently useful for a variety of human diseases that share an idiopathic origin, ranging from autoimmune disorders to primary antibody deficiencies. Also, IVIG has shown promising results in case of severe (such as sepsis, Parvovirus B19 infection, West Nile virus encephalitis, HIV, Clostridium difficile infections, Mycobacterium avium, Mycobacterium tuberculosis, and Nocardia infections) and recurrent infections in primary antibodies deficiencies \\[21\\].\n\nMost patients develop antibodies against the NP and RBD of 2019-nCoV during the second week after infection onset \\[22\\]. Analysis of serum samples collected 14 or more days after symptom onset revealed detection of IgG and IgM antibodies against NP in 94% and 88% and RBD in 100% and 94% among patients with COVID19. Studies consistently show that increased immunoglobulin levels accompany the transition from early to late course of COVID19. It poses the possibility that IVIG therapy might help to accelerate recovery from COVID-19.\n\n### Rationale: IVIG might help to improve the outcome of patients with COVID-19\n\nThe study \\[23\\] included ten patients with COVID-19 who demonstrated worsening symptoms, e.g., decreased lymphocyte count and decreased PaO2\/FIO2 ratio and oxygen saturation, following treatment with a short-term moderate-dose corticosteroid (methylprednisolone 80\u00a0mg\/d) plus immunoglobulin (10\u00a0g\/d). After switching to the double dose of 1600\u00a0mg\/d methylprednisolone plus 20\u00a0g\/d immunoglobulin, all of the patients improved in the clinical, laboratory, and paraclinical outcomes.\n\nPassive immunization protects against disease, and so it should be administered as early as possible when the patient is diagnosed. Studies show that the viral RNA of 2019-nCoV reaches its peak during the first week and then gradually decreases and that IgG and IgM begin to rise from the 10th day so that most patients have anti-viral antibodies by the 14th day.\n\n# Recombinant type I IFN\n\n## Hypothesis: IFNs play a role in anti-viral immunity\n\nType I IFNs play a primary role in the inhibition of viral replication through the coordination of anti-viral immune responses and modulation of inflammatory responses \\[24\\].\n\n## Rational: Recombinant type I IFN can effectively inhibit SARS-CoV2 replication\n\nIn 24\u201348\u00a0h after infection, SARS-CoV2 replication reaches titer that causes the cytopathic effect in Vero E6 cells, which express ACE2 and thus are susceptible to SARS-CoV2 infection \\[25\\]. It is similar to the viral replication kinetics for SARS-CoV \\[25\\]. Pretreatment with recombinant type I IFN could effectively inhibit SARS-CoV2 replication at both 24 and 48\u00a0h after infection \\[25\\]. For SARS-CoV, such an effect was present at 24\u00a0h but absent at 48\u00a0h after infection \\[25\\]. The difference between SARS-CoV2 and SARS-CoV in response to type I IFN treatment may be due to differences in structural proteins of these viruses, including NSP3, ORF3b, and ORF6 \\[25\\].\n\n# Development of a human monoclonal antibody (mAb)\n\n## Targeting the S protein\n\n### Hypothesis: A mAb against the binding domain of the virus can inhibit SARS-CoV2 infection\n\nRecently, hybridoma supernatants containing antibody repertoires from immunized transgenic mice that express the human immunoglobulin heavy and light chains and rat origin immunoglobulin constant regions were used to detect antibodies that can cross neutralize SARS-S and SARS-2-S \\[26\\]. One chimeric 47D11 H2L2 antibody displayed such a cross-neutralizing activity, decreased syncytia formation induced by SARS-S and SARS2-S, and could protect VeroE6 cells against SARS-S and SARS2-S pseudotyped virus \\[26\\]. It may lie in its similar affinities for interacting with the same domain of the S1 subunit, i.e., S1~B~, of each SARS-S and SARS-2-S.\n\n### Rational: The chimeric 47D11 H2L2 might not be effective in human lung cells as it is *in vitro*\n\n47D11 carried a higher affinity for interacting with the S2 subunit of SARS-S than that of SARS-2-S. It is important that for both SARS-S and SARS-2-S, the binding of the 47D11 antibody to the target \u2013 the S1~B~ domain \u2013 does not block the binding of S1~B~ and S2 to ACE2 receptor \\[26\\]. By contrast, neutralizing antibodies that specifically target SARS-S could compete with S1~B~ and S2 for binding to ACE2.\n\n## Targeting pro-inflammatory cytokines\n\n### Hypothesis: A mAb against IL6 can attenuate hyper inflammation\n\nTocilizumab, also known as atlizumab, is a humanized anti-human IL6 receptor antibody approved by FDA for several inflammatory and autoimmune diseases severe, such as cytokine release syndrome, rheumatoid arthritis, giant cell arteritis, polyarticular juvenile idiopathic arthritis, and systematic juvenile idiopathic arthritis. It is safe and effective for both adults and children two years of age and older.\n\n### Rationale: Tocilizumab can treat lung injury in patients with critical and severe COVID-19\n\nIn the study \\[27\\], 21 patients with COVID-19 whose condition was severe or critical received one or two doses of Tocilizumab plus standard therapy. Patients who had a mean IL6 level of more than 100\u00a0pg\/ml before tocilizumab treatment showed improvement in clinical symptoms and peripheral oxygen saturation and normalization for lymphocyte proportion and CRP levels. Also, lung lesion opacity was absorbed in 90% of patients. Neither serious adverse effects nor deaths occurred with tocilizumab treatment.\n\nThere are ongoing clinical trials for tocilizumab treatment in patients with moderate and severe COVID-19. Currently, the use of Tocilizumab is recommended for patients with COVID-19 who have warning signs of hyper inflammation, as can be measured by IL6, ferritin, platelet counts, inflammatory markers, and H score \\[28\\].\n\n# Corticosteroids\n\n## Hypothesis: Corticosteroids can modulate inflammation\n\nCorticosteroids are commonly used for modulation of a variety of inflammatory conditions. In addition to a daily regimen, they can be used in the form of pulse therapy to treat flares of autoimmune diseases. However, caution in the use of corticosteroids is needed due to the potential serious side effects associated with corticosteroid drugs and that corticosteroids generally suppress the immune system. The latter means that corticosteroids modulate hyper inflammation and, on the other hand, inhibit immune responses that are vital for the host defense against the virus \\[29\\].\n\n## Rationale: Corticosteroids might help accelerate recovery from COVID-19\n\nThe study \\[30\\] investigated the effect of inhaled corticosteroids ciclesonide, cortisone, prednisolone, dexamethasone, and fluticasone on the replication of the MERS-CoV. Among the four compounds, the only ciclesonide was capable of inhibiting viral replication. Also, ciclesonide induced a significant inhibition of viral replication of other human coronaviruses, such as HCoV-229E and SARS-CoV, and another positive-strand RNA virus, rubella virus, while not affect the viral replication of negative-strand RNA viruses, e.g., influenza and respiratory syncytial virus. For the MERS-CoV, a nonstructural protein 15 (NSP15) appeared to act as the target of ciclesonide. An amino acid substitution in the NSP15 conferred resistance of the mutated MERS-CoV to ciclesonide. Mometasone could help deal effectively with the mutated MERS-CoV. For the SARS-CoV2, all three ciclesonide, mometasone, and lopinavir were able to inhibit viral replication to a similar degree. Interestingly, their effect was more noticeable than serine protease inhibitors, e.g., nafamostat and camostat in cells that Vero cells that express TMPRSS2. It indicates the tendency of the SARS-CoV2 to enter the cell through the cathepsin\/endosomal pathway rather than through the TMPRSS2\/cell surface pathway.\n\nThe study \\[31\\] included 46 patients with severe COVID-19, of these 26 patients received methylprednisolone (1\u20132\u00a0mg\/kg\/d for 5\u20137\u00a0days), and 20 patients received standard therapy without methylprednisolone. The first group achieved faster improvement in clinical symptoms (fever and peripheral oxygen saturation) and lung lesions detected by CT imaging. However, two deaths occurred in the first group and one death in the second group. Moreover, the two groups did not differ in laboratory parameters, including WBC, lymphocyte count, monocyte count, and cytokines (IL-2, IL-4, IL-6, and IL-10) six days after treatment.\n\nThere is a report of the patient with COVID-19 treated with methylprednisolone since day 8 of the disease course. However, his situation worsened and developed respiratory failure and died on day 14 \\[32\\].\n\n# Eggs for increasing ACE2 and copper\n\nEgg ovotransferrin contains an angiotensin-converting enzyme (ACE) inhibitory peptide, known as IRW, that has shown to decrease blood pressure in hypertensive rats \\[33\\]. Through the up-regulation of ACE2, E-cadherin, ABCB-1, and IRF-8, IRW can decrease RAS activity, hyperplasia, and vascular inflammation and aid differential regulation of leukocytes. On the other hand, by the down-regulation of pro-inflammatory molecules, e.g., ICAM-1 and VCAM-1, IRW can reduce the recruitment of leukocytes and vascular inflammation.\n\nCopper might help the immune system to combat viral infections. A High-content of copper exists in eggs and eggshells of domestic birds. In particular, the eggshell of pigeon (4 $\\pm$ 0.29\u00a0\u03bcg\/g) and the egg of quail (4.67 $\\pm$ 1.08\u00a0\u03bcg\/g) contains high concentrations of copper \\[34\\].\n\n# Conclusion\n\nThe novel coronavirus, SARS-CoV2, can cause a potentially fatal disease, COVID-19, in humans. The infection of human cells by SARS-CoV2 includes two sequential steps: attachment of the virus to the surface receptor of target cells and the fusion of viral and host membranes. The former requires at least a receptor-binding domain on the SARS-CoV2 Spike protein that can interact with a cell surface receptor, for example, ACE2, expressed on human cells. The latter requires at least the host protease(s) to mediate proteolytic cleavage of the SARS-CoV2 Spike protein into S1 and S2 subunits and consequently promote the fusion of viral and host membranes. Also, SARS-CoV2 possesses a polybasic cleavage site that can explain the high pathogenicity of SARS-CoV2, N-glycans and O-glycans that make the dense decoration of SARS-CoV2 S protein, and cyclic regions that can interact with cell-surface GRP78. Essential elements that process SARS-CoV2 cell entry and specific characteristics that allow SARS-CoV2 to escape the immune system have the potential as targets for COVID-19 therapy.\n\nLack of specific treatments for COVID-19 and the very time-consuming process of vaccine development lead us to trust traditional notions of immunization using passive transfer of humoral immunity. Passive immunization can be done using plasma therapy and IVIG therapy. Plasma from patients recovered from COVID-19 that contains antibodies against SARS-CoV2 has shown promising results in patients with severe COVID-19. Also, SARS-CoV and SARS-CoV2 are ideally similar in the structure and the cell entry receptor and protease. Studies show that serum from convalescent SARS patients and serum from rabbits immunized with SARS are both able to neutralize SARS-CoV2 efficiently. However, serum from bats immunized with SARS-lice coronavirus SL-CoV Rp3 could not exert such an effect. It is due to a noticeable degree of difference in the S1 domain in the S1 domain between the bat SL-CoV Rp3 strain and SARS-CoV2. A short-term moderate dose of IVIG combined with moderate-dose of corticosteroids might improve patient outcomes. Studies show that the viral RNA of SARS-CoV2 reaches its peak during the first week and then gradually decreases and that IgG and IgM begin to rise from the 10th day so that most patients have anti-viral antibodies by the 14th day. Passive immunization protects against disease, and so it should be administered as early as possible when the patient is diagnosed.\n\nEvidence links COVID-19 to variable degrees of inflammation. Corticosteroids offer a potent anti-inflammatory option. However, they do not dictate precise actions and might cause suppression of anti-viral immune responses as well. Studies show that the use of corticosteroids might accelerate recovery from COVID-19. There are no controlled clinical trials that show whether the use of corticosteroids can reduce COVID-19-related death. Moreover, the pro-inflammatory cytokine IL6 is the best-documented cytokine in COVID-19 correlated with severity, criticality, viral load, and prognosis of patients with COVID-19. Tocilizumab, a monoclonal antibody against IL6, could confer clinical benefit in patients with high IL6 levels.\n\n# References","meta":{"dup_signals":{"dup_doc_count":1000,"dup_dump_count":22,"dup_details":{"curated_sources":2,"2023-50":9,"2023-40":6,"2023-23":35,"2023-14":64,"2023-06":3,"2022-49":6,"2022-40":16,"2022-33":50,"2022-27":117,"2022-21":116,"2022-05":93,"2021-49":114,"2021-43":35,"2021-39":57,"2021-31":164,"2021-25":11,"2021-21":1,"2024-30":2,"2024-26":39,"2024-22":16,"2024-18":39,"2024-10":5}},"file":"PMC7205724"},"subset":"pubmed_central"}
{"text":"author: Gunther Eysenbach\ndate: 1999\nreferences:\ntitle: Welcome to the Journal of Medical Internet Research\n\nWelcome to the \"Journal of Medical Internet Research\" - JMIR, the first international scientific peer-reviewed journal on all aspects of research, information and communication in healthcare using Internet and Intranet-related technologies.\n\nWhy does the world need the JMIR? The Internet - and more specifically, the World-Wide-Web - has an impact on many areas of medicine - broadly we can divide them into \"clinical information and telemedicine\", \"medical education and information exchange\" and \"consumer health informatics\":\n\n-   First, Internet protocols are used for clinical information and communication. In the future, Internet technology will be the platform for many telemedical applications.\n\n-   Second, the Internet revolutionizes the gathering, access and dissemination of non-clinical information in medicine: Bibliographic and factual databases are now world-wide accessible via graphical user interfaces, epidemiological and public health information can be gathered using the Internet, and increasingly the Internet is used for interactive medical education applications.\n\n-   Third, the Internet plays an important role for consumer health education, health promotion and teleprevention. (As an aside, it should be emphasized that \"health education\" on the Internet goes beyond the traditional model of health education, where a medical professional teaches the patient: On the Internet, much \"health education\" is done \"consumer-to-consumer\" by means of patient self support groups organizing in cyberspace. These patient-to-patient interchanges are becoming an important part of healthcare and are redefining the traditional model of preventive medicine and health promotion).\n\nAll these aspects of \"cybermedicine\" have implications for consumer empowerment and evidence-based medicine: The Internet (or Intranets) enables health professionals to access clinical data just in time, it allows health professionals to access the evidence on the efficacy of available interventions, and finally, it empowers consumers to actively take part in the decision making process.\n\nClearly, the medical use of the Internet presents enormous opportunities and challenges. The need for research and rapid publication of the findings is obvious. Research in this area should go beyond mere development and provision of technical solutions; it should also address social and human factors, and evaluate the impact of the Internet on society and health care, and public health.\n\nJMIR wishes to publish papers that help physicians and consumers to maximize the use of the Internet. We invite researchers to evaluate the effectiveness and efficiency of Internet communications in health care. We encourage publishers of Internet health information to apply rigorous research methods (such as randomization of users) to evaluate different methods and determinants of communication effectiveness. We invite researchers to compare the effectiveness of communication and information on the Internet with other (traditional) methods of communication. We call for papers that describe and evaluate the effects of the Internet on the patient-physician relationship and the impact on public health. We invite papers that describe the use of the Internet for evidence-based medicine, for example work that demonstrates the development and dissemination of clinical guidelines using the Internet. We wish to receive papers on ethical and legal problems, as well as cross-border and cross-cultural issues, affecting medicine on the Internet, and papers describing possible solutions to the problem of equity of information access. We would like to receive systematic studies examining the quality of medical information available in various online venues. We encourage thought regarding methods of evaluation, quality assessment and improvement of Internet information. We would like to receive proposals for standards in the field of medical publishing on the Internet, including self-regulation issues, policies and guidelines to provide reliable healthcare information. We encourage researchers to experiment with online questionnaires and other data collection experiments such as medical surveys and psychological tests. We would like to publish innovative approaches to use the Internet for healthcare research, examples might include clinical studies, drug reaction reporting and surveillance systems. We would like to publish comments and papers on electronic medical publishing and the use of the Internet for traditional scholarly publishing. We welcome descriptions of websites with innovative content or form, but authors should always make attempts to evaluate the impact of their work, for example trying to determine basic information about user demographics and traffic, where appropriate.\n\nAs publishers of a journal *about* the Internet, we are also dedicated to using and experimenting with the Internet as a medium itself. Obviously, we are utilizing the Internet for communication with authors (which is done exclusively by email), and communication with external reviewers, but we also intend to experiment with some novel methods of peer-review. We further invite authors to experiment with innovative methods to communicate their findings, for example submitting HypER-papers (Hypertext Enriched Research Papers) \\[1\\] or by the inclusion of animated figures (animated gifs), audio and video into their documents, or by attaching original data which could be downloaded and possibly dynamically analyzed using JAVA applets.\n\nNew Internet standards and tools are developing at a breathtaking pace, and many Internet trends have a half-life of less than 6 months. We think that traditional paper journals are simply too slow for the fast-moving field of Internet-technologies. One of our editors reported that an article submitted to a leading medical informatics journal was only published 2 years later - obviously an unacceptable (and unnecessary) delay. Thus, we are trying to publish fast - usually our peer-review time is 1-2 weeks, and we publish all e-papers as soon as they have been accepted (though the printed version may be published later). We will follow a dual publishing strategy - full articles will be published on the Internet, all abstracts and short important articles will be published as a printed version, mainly for archiving and indexing purposes. Our peer-review process will be rigorous and constructive, helping authors to improve their manuscripts and guaranteeing a high-quality journal. We eagerly look forward to your contributions.\n\nGunther Eysenbach MD\n\n*Editor,*\n\n*Journal of Medical Internet Research*","meta":{"dup_signals":{"dup_doc_count":123,"dup_dump_count":57,"dup_details":{"curated_sources":2,"2023-06":3,"2022-40":2,"2022-21":1,"2022-05":2,"2020-40":1,"2020-34":3,"2020-05":4,"2019-47":1,"2019-35":1,"2019-30":1,"2019-22":2,"2018-51":2,"2018-47":1,"2018-34":1,"2018-22":1,"2018-09":1,"2018-05":1,"2017-47":1,"2017-39":1,"2017-30":2,"2017-22":1,"2017-17":1,"2017-09":2,"2017-04":2,"2016-50":2,"2016-44":2,"2016-40":2,"2016-36":2,"2016-30":3,"2016-26":2,"2016-22":2,"2016-18":2,"2016-07":3,"2015-48":3,"2015-40":2,"2015-35":3,"2015-32":3,"2015-27":3,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":5,"2014-42":9,"2014-41":1,"2014-35":2,"2014-23":2,"2014-15":1,"2023-23":1,"2024-22":2,"2017-13":1,"2015-18":2,"2015-11":3,"2015-06":3,"2014-10":3,"2013-48":2,"2013-20":2,"2024-26":1}},"file":"PMC1761703"},"subset":"pubmed_central"}
{"text":"author: Arnold von Eckardstein; Jerzy-Roch NoferCorresponding author: Arnold von Eckardstein, <email@example.com>.\ndate: 2013-08\nreferences:\ntitle: Frail HDLs and Stiff Arteries in Type 2 Diabetes in Juveniles\n\nLowering of LDL cholesterol plasma levels with statins reduces coronary heart disease (CHD) event rates by up to 50% (1), implying a residual cardiovascular risk of the same magnitude despite treatment. Moreover, statins increase the risk of type 2 diabetes mellitus (T2DM), especially in patients showing components of the metabolic syndrome (2). Hence, there is considerable need for novel therapeutic regimens improving CHD prevention without increasing the risk of T2DM.\n\nHDLs are an interesting target for this objective. Most observational studies and meta-analyses thereof demonstrated the inverse relationship of HDL cholesterol (HDL-C) levels with the CHD risk (3) as well as T2DM and its vascular complications (4,5). HDL particles exert various potentially antiatherogenic (6\u20138) and antidiabetogenic activities (4). Atherosclerotic lesions were decreased or even reversed in animals by transgenic overexpression or application of exogenous apolipoprotein (apo) A-I, which constitutes the most abundant protein of HDL (6). Animal experiments also provided evidence that HDL improves the function and survival of pancreatic \u03b2-cells and glucose uptake into muscle, liver, and adipose tissue (4). In humans, artificially reconstituted HDL particles reduced coronary plaque volume (9,10) and improved glycemia (11). In contrast to these promising results, addition of fenofibrate, niacin, torcetrapib, or dalcetrapib to statins failed to reduce cardiovascular risk beyond that provided by statin treatment alone despite increasing HDL-C (12\u201315). Moreover, alterations in HDL-C, either associated with mutations in the human genome or provoked in genetic mouse models, did not consistently translate into opposite changes of cardiovascular risk and atherosclerotic plaque load, respectively (16,17).\n\nBecause of these controversial data, the suitability of HDL as a therapeutic target has been increasingly questioned. However, it is important to emphasize that interventional trials and Mendelian randomization studies targeted HDL-C, which neither exerts nor reflects any of the potentially antiatherogenic activities of HDL (6). In a prototypic HDL particle, two to five molecules of apoA-I and \u223c100 molecules of phosphatidylcholine form an amphipathic shell in which several molecules of unesterified cholesterol are imbedded and it surrounds a core of cholesterol esters (18). Molar differences in the content of these major protein and lipid constituents produce considerable heterogeneity of HDL in shape, size, density, and charge (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<\/a>). The macroheterogeneity of HDL is further compounded by quantitatively minor proteins, lipids, or microRNAs (19\u201321), many of which contribute to the potentially antiatherogenic and antidiabetogenic properties of HDL. Additional HDL microheterogeneity is a consequence of various inflammatory diseases, including T2DM or CHD, which lead to the loss of or structural modification of typical HDL constituents or the acquisition of atypical HDL constituents (22). Several alterations of HDL structure and composition have been associated with the loss of potentially vasoprotective functions, such as stimulation of cholesterol efflux (7) and endothelium-dependent vasodilation (8), independently of plasma HDL-C levels. Importantly, the plasma concentrations of many microcomponents of HDL amount to only \u22641 \u03bcmol\/L. Hence, they are three to four orders of magnitude lower than those of HDL-C (usually \\>1 mmol\/L) and one to two orders of magnitude lower than those of apoA-I (50 \u03bcmol\/L) or HDL particles (10\u201320 \u03bcmol\/L). Accordingly, these microcomponents are nonrandomly distributed among HDL subclasses and are not recovered by measurements of HDL-C, apoA-I, or HDL subclasses.\n\nMany laboratories worldwide currently are searching for functional biomarkers of HDL, which are more closely related to atherosclerosis and cardiovascular outcomes than HDL-C. One promising approach in this direction has been undertaken by Gordon et al. (23), who investigated the association of HDL subclasses and their proteomes with the presence of T2DM and obesity in adolescents and with pulse wave velocity (PWV), a noninvasive measure of vascular stiffness and hence a surrogate of atherosclerosis. Among 12 HDL subfractions identified by the authors, large HDL particles showed greatest differences between T2DM patients and nondiabetic controls. Compared with those in healthy controls, these subfractions were deprived of several proteins, including apoA-I, apoA-II, apoE, apoM, and paraoxonase 1 (PON1) in T2DM patients. These results in humans closely correspond to recent findings by Kothapalli et al. (24) showing increased arterial stiffness in apoE-deficient mice and favorable effects on arterial elasticity exerted by apoE-containing HDL particles. In addition to changes in protein composition, the phospholipid content of large HDL subfractions showed a significant inverse correlation with PWV. In agreement with protective functionality, large HDL particles were enriched in the sphingosine-1-phosphate\u2013binding lipocalin apoM (25) and the antioxidative enzyme PON1 (26) in nondiabetic subjects. In contrast, cholesterol concentrations in smaller HDL particles showed positive correlations with PWV, suggesting adverse effects on vascular health. Of note, HDL-C did not show any significant correlation with PWV.\n\nThe study has several strengths. By investigating young patients with diabetes and controls, Gordon et al. (23) minimized the effect of confounders complicating data interpretation in diabetic adults. By using gel filtration rather than ultracentrifugation, the authors eliminated artifacts arising from protein and lipid displacement by shear forces or high ion concentrations. It is noteworthy that the authors retrieved several proteins, including apoM and PON1, in large HDL particles that were previously assigned to small HDL particles by ultracentrifugation (27). The risk of recording false-positive results after using the comprehensive proteomic approach was limited by the stringent selection of those proteins for statistical analyses, which were identified by previous proteomic examination of HDL.\n\nGeneral limitations of these explorative studies are the cross-sectional design and the small number of patients. As acknowledged by the authors, statistical association does not imply causality. In this respect, it will be interesting to test the effect of large HDL fractions on endothelial functions, which were previously found to be modulated by PON1 or apoM (8,25,26). The expansion to prospective studies will require methodological advancements permitting analyses of hundreds or even thousands of samples. To this end, a refined proteomic and lipidomic examination of large HDL particles might help to identify proteins or lipids that can be specifically targeted by high-throughput technologies such as immunoassays or single-reaction monitoring mass spectrometry. It also should be emphasized that the enzymatic assay used by the authors, which quantifies choline rather than phospholipids, neither discriminates between phosphatidylcholines, lysophoshatidylcholines, plasmalogens, and sphingomyelins nor records noncholine phospholipids such as phosphatidylethanolamines, phosphatidylserines, and sphingosine-1-phosphate. Another limitation of the study is that the mass spectrometry approach measured only relative concentrations. Interestingly, however, this semiquantitative approach unraveled reduced peptide signals in HDL from T2DM patients. It is possible that posttranslational protein modifications altered the mass of peptides and thereby prevented their recording by the assigned molecular mass. This explanation is congruent with previous findings showing enhanced glycation, nitration, chlorination, sulfoxidation, or carbamylation in HDL from diabetic subjects (22). Such modifications may offer new opportunities for use as biomarkers (7,8,22).\n\nIn conclusion, the study by Gordon et al. (23) provides new insights into the molecular heterogeneity of HDL and its association with T2DM and atherosclerosis. Apart from reproducing and extending these findings in larger observational studies, it will be important to resolve the structure, function, and metabolism of large HDL fractions. Further structure\u2013function studies may help to select molecules or modifications within HDL, which can be used as biomarkers for identification, treatment stratification, and monitoring of patients at increased risk for cardiovascular diseases or diabetes mellitus.\n\n## ACKNOWLEDGMENTS\n\nA.v.E. is supported by grants from the Swiss National Science Foundation (3100A0-116404\/1, 3100A0-130836\/1), the FP7 Programme of the European Commission (RESOLVE, 305707), and the Zurich Center of Integrative Human Physiology, and has received honoraria for lectures and advisory activities from Hoffmann-La Roche, Merck Sharpe & Dohme, and AstraZeneca. J.-R.N. is supported by grants from the German Foundation for Pathobiochemistry and Molecular Diagnostics, the Italian Ministry for Education, University, and Research (IDEAS RBID08777T), Novartis Germany, Actelion Pharmaceuticals Germany, and Siemens Healthcare Diagnostics. No other potential conflicts of interest relevant to this article were reported.\n\n# REFERENCES","meta":{"dup_signals":{"dup_doc_count":138,"dup_dump_count":46,"dup_details":{"curated_sources":2,"2022-05":1,"2020-05":1,"2018-26":1,"2018-22":3,"2018-17":1,"2018-13":2,"2018-05":2,"2017-51":2,"2017-47":1,"2017-43":7,"2017-39":1,"2017-34":3,"2017-30":5,"2017-26":4,"2017-22":2,"2017-17":8,"2017-09":4,"2017-04":10,"2016-50":4,"2016-44":4,"2016-40":4,"2016-36":4,"2016-30":3,"2016-26":3,"2016-22":3,"2016-18":3,"2016-07":3,"2015-48":3,"2015-40":3,"2015-35":3,"2015-32":2,"2015-27":3,"2015-22":3,"2015-14":2,"2014-52":4,"2014-49":5,"2014-42":3,"2014-41":2,"2014-35":1,"2023-06":1,"2024-26":2,"2024-22":1,"2017-13":2,"2015-18":3,"2015-11":1,"2015-06":3}},"file":"PMC3717845"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  Filarial nematodes, including *Brugia malayi*, the causative agent of lymphatic filariasis, undergo molting in both arthropod and mammalian hosts to complete their life cycles. An understanding of how these parasites cross developmental checkpoints may reveal potential targets for intervention. Pharmacological evidence suggests that ecdysteroids play a role in parasitic nematode molting and fertility although their specific function remains unknown. In insects, ecdysone triggers molting through the activation of the ecdysone receptor: a heterodimer of EcR (ecdysone receptor) and USP (Ultraspiracle).\n  .\n  # Methods and Findings\n  .\n  We report the cloning and characterization of a *B. malayi* EcR homologue (*Bma-EcR*). Bma-EcR dimerizes with insect and nematode USP\/RXRs and binds to DNA encoding a canonical ecdysone response element (EcRE). In support of the existence of an active ecdysone receptor in *Brugia* we also cloned a *Brugia rxr* (retinoid X receptor) homolog (*Bma-RXR*) and demonstrate that Bma-EcR and Bma-RXR interact to form an active heterodimer using a mammalian two-hybrid activation assay. The Bma-EcR ligand-binding domain (LBD) exhibits ligand-dependent transactivation via a GAL4 fusion protein combined with a chimeric RXR in mammalian cells treated with Ponasterone-A or a synthetic ecdysone agonist. Furthermore, we demonstrate specific up-regulation of reporter gene activity in transgenic *B. malayi* embryos transfected with a luciferase construct controlled by an EcRE engineered in a *B. malayi* promoter, in the presence of 20-hydroxy-ecdysone.\n  .\n  # Conclusions\n  .\n  Our study identifies and characterizes the two components (*Bma-EcR* and *Bma-RXR*) necessary for constituting a functional ecdysteroid receptor in *B. malayi*. Importantly, the ligand binding domain of BmaEcR is shown to be capable of responding to ecdysteroid ligands, and conversely, ecdysteroids can activate transcription of genes downstream of an EcRE in live *B. malayi* embryos. These results together confirm that an ecdysone signaling system operates in *B. malayi* and strongly suggest that Bma-EcR plays a central role in it. Furthermore, our study proposes that existing compounds targeting the insect ecdysone signaling pathway should be considered as potential pharmacological agents against filarial parasites.\nauthor: George Tzertzinis; Ana L. Ega\u00f1a; Subba Reddy Palli; Marc Robinson-Rechavi; Chris R. Gissendanner; Canhui Liu; Thomas R. Unnasch; Claude V. Maina\\* E-mail: <firstname.lastname@example.com>[^1][^2]\ndate: 2010-03\ninstitute: 1 New England Biolabs, Ipswich, Massachusetts, United States of America; 2 Department of Entomology, University of Kentucky, Lexington, Kentucky, United States of America; 3 Department of Ecology and Evolution, University of Lausanne, Lausanne, Switzerland; 4 Global Health Infectious Disease Research Program, College of Public Health, University of South Florida, Tampa, Florida, United States of America; University of Pittsburgh, United States of America\nreferences:\ntitle: Molecular Evidence for a Functional Ecdysone Signaling System in *Brugia malayi*\n\n# Introduction\n\nHuman filarial parasitic nematodes are responsible for two chronic severely debilitating tropical diseases: lymphatic filariasis and onchocerciasis. The global efforts in the treatment and control of the spread of infection for both parasites so far have resulted in limited success. Also, the widespread use of the few available specific drugs for fighting these diseases raises the possibility of the development of drug resistance \\[1\\]. With 140 million cases of infection worldwide, and over a billion people at risk of contracting these debilitating diseases \\[2\\], the development of a wide range of therapeutic interventions and treatment options is urgent.\n\nFilarial parasites spend portions of their life cycle in obligate mammalian and insect hosts. The completion of a successful life cycle requires the passage of the developing nematode through four molts, two in the mammalian host and two in the arthropod host. The transmission of the parasite from one host to the other initiates a rapid molt, indicating that the developmental cues that trigger molting are closely tied to the integration of the parasitic larva into a new host environment. Inhibition of molting would result in the arrest of the life cycle in either the mammalian or insect host and the prevention of both pathology and\/or the infective cycle. Thus, the study of the molting process in filarial nematodes could point to specific targets for drug development.\n\nMolting in ecdysozoans \\[3\\] has been best characterized in insects. 20-hydroxyecdysone (20E) acts as the temporal signal to initiate molting, regulates embryogenesis, and coordinates tissue-specific morphogenetic changes in insects \\[4\\]\u2013\\[6\\]. Ecdysone signaling is regulated by the activity of a heterodimeric receptor composed of two nuclear receptor proteins EcR and USP, although the hormone binding function resides only within EcR \\[7\\]\u2013\\[10\\]. After ligand binding, EcR\/USP activates a cascade of gene expression whose end result is the execution of molting \\[11\\].\n\nThree alternatively spliced mRNA isoforms of *EcR* have been identified in *Drosophila* \\[12\\]. Mutations in these different *EcR mRNA* isoforms result in a range of phenotypes that includes lethality at the embryonic, larval and pupal stages, disruption of salivary gland degeneration \\[13\\], aberrant neuronal remodeling during metamorphosis \\[14\\], and changes in female fecundity and vitellogenesis \\[15\\].\n\nEcR and USP, as well as a number of the proteins involved in the ecdysone-signaling cascade, are members of the nuclear receptor (NR) superfamily \\[10\\],\\[16\\]\u2013\\[18\\]. NRs are characterized by significant amino acid sequence similarities in two key functional domains: the DNA binding domain (DBD), which directs the sequence-specific DNA-binding of the receptor, and the ligand binding domain (LBD), which mediates dimerization, ligand binding and transcriptional activation \\[19\\]\u2013\\[20\\]. Some nuclear receptors have been shown to interact with a number of small molecule ligands such as metabolites and hormones, and these interactions are important for regulation of their activity. Other NRs are considered orphan receptors and are either not ligand-regulated or their cognate ligands have yet to be identified \\[19\\]. Homologs of the insect NRs that function downstream of EcR and USP have also been identified in filarial parasites (21, 22; Ega\u00f1a, Gissendanner and Maina, unpublished results) as well as in the free-living nematode *C. elegans* \\[23\\]\u2013\\[26\\]. Surprisingly, however, homologs of EcR or RXR\/USP are apparently absent in the exceptionally large *C. elegans* NR family \\[23\\].\n\nIn filarial nematodes the molecular triggers of molting remain largely unknown. As in insects, a possible candidate for a signal that controls molting in *B. malayi*, the causative agent of lymphatic filariasis, is the steroid hormone 20E. Both free and conjugated ecdysteroids have been identified in the larvae of several parasitic nematodes including *Dirofilaria immitis* and *Onchocerca volvulus* \\[27\\]\u2013\\[29\\]. In addition, ecdysteroids have been shown to exert biological effects on several nematodes. For example, in *Nematospiroides dubius* \\[30\\] and *Ascaris suum* \\[31\\] molting can be stimulated *in vitro* by low concentrations of ecdysteroids. Also, molting of third stage larvae of *D. immitis* can be stimulated with 20E and RH5849, an ecdysone agonist \\[32\\],\\[33\\]. The arrest at the pachytene stage of meiosis is abrogated when *D. immitis* ovaries are cultured *in vitro* with ecdysone and *B. pahangi* adult females can be stimulated to release microfilaria when cultured *in vitro* with ecdysone \\[34\\].\n\nThere appears to be a physiological connection between the filarial parasite and its arthropod host that may involve ecdysteroid signaling. Uptake of microfilaria (L1) by a feeding female mosquito at the time of a bloodmeal coincides with an increase in the production of mosquito ecdysteroids that results in the initiation of mosquito oocyte maturation \\[35\\]. Concurrent with this increase in ecdysteroid concentration in the mosquito host, larvae initiate a molt transition from L1 to L2 and later from L2 to L3, the infectious stage of the parasite. These observations suggest a potential role for ecdysone in the regulation of molting and other developmental processes in filarial nematodes.\n\nWe previously identified an *rxr* homolog in the dog filarial parasitic nematode *D. immitis* and demonstrated its ability to dimerize with an insect EcR and function in Schneider S2 cells \\[36\\]. We extend this work here with the identification and characterization of *EcR* and *rxr* homologs from *B. malayi*. Bma-EcR and Bma-RXR share some of the biochemical properties of insect EcR and RXR and show differences that appear to be nematode specific.\n\n# Methods\n\n## Parasites, RNA isolation and reverse transcription\n\n*Brugia malayi* adult males, females or L1 larvae (TRS Labs, Athens, GA) were frozen in liquid nitrogen and ground with a pestle and mortar. Total RNA was purified from the pulverized tissue using RNAwiz (Ambion). RNA was quantified with a spectrophotometer and its quality assessed by gel electrophoresis. One \u00b5g of total RNA per isolation was reverse-transcribed using the ProtoScript first strand cDNA synthesis kit (New England Biolabs) following the manufacturer's protocol.\n\n## Cloning of *Bma-EcR*, *Ov-RXR* and *Bma-RXR*\n\nThe genomic library from *B. malayi* in pBeloBAC vector gridded on Nylon filters (Filarial Genome Network, FGN, (<http:\/\/www.nematodes.org\/fgn\/index.shtml>) was screened using a cDNA fragment from a *D. immitis* EcR homolog (*Di-EcR*) (C. Shea, J. Richer and C. V. Maina, unpublished results) as a probe. Three positive BACs were identified and the individual corresponding bacterial clones were cultured. The inserts were confirmed to contain identical or overlapping sequences by restriction digestion analysis. One 11kb *Xba*I fragment identified by southern blot hybridization with the *Di-EcR* probe was subcloned into Litmus 28i and the insert was sequenced using GPS\u00ae-1 Genome Priming System (NEB) as directed by the manufacturer. PCR primers were designed to amplify *Bma-EcR* using sequence from the identified exons \\[37\\]. The primers used to amplify the full ORF were: 5\u2032 \u2013 GGC GCT AGC ATG ACT ACA GCA ACA GTA ACA TAT CAT GAG TT \u2013 3\u2032 (Nco-MMT-5); 5\u2032 \u2013 GGC CTC GAG CGA TTC TAT GGA TAG CCG GTT GAG GTT \u2013 3\u2032 (Xho-GYP-3). To determine the expression pattern and identify alternate isoforms of *Bma-EcR*, adult female, male, L1, L2, L3 cDNA libraries (FGN) were screened using the following primers: 5\u2032 \u2013 GGG TAA TTC CTA CCA ACA GCT - 3\u2032(GNS); 5\u2032 \u2013 CAA GGG TCC AAT GAA TTC ACG AT \u2013 3\u2032 (GPL) corresponding to a fragment of the LBD from amino acids GNSYQQ to REFIGPL'. Additional sequence to extend the *Bma-EcR* isoforms identified was obtained by PCR, combining the latter two primers with the T3 and T7 promoter primers as their sequence is present in the library vector.\n\nAn *O. volvulus* L3 cDNA library (FGN) was screened by PCR using the following primers: 5\u2032 \u2013 GAT CTT ATC TAT CTA TGC CGA GAA ,\u2013 3\u2032; 5\u2032 \u2013 TAC TTT GAC ATT TGC GGT AAC GAC \u2013 3\u2032 corresponding to the amino acid sequence DLIYLCRE and RYRKCQSM of the conserved DNA binding domain of DiRXR-1 respectively. Additional *Ov-rxr* sequence was obtained by PCR using the same primers in combination with the T7 and T3 promoter primers. Candidate clones were identified by hybridization with a fragment of DiRXR-1 sequence. An amplified fragment from this library contained sequence corresponding to the *Ov-rxr* A\/B and C domains.\n\nUsing BLAST, the *Di-rxr-1* sequence \\[36\\] was used to screen the *B. malayi* genome sequence available from The Institute for Genomic Research (TIGR) parasites database (<http:\/\/blast.jcvi.org\/er-blast\/index.cgi?project=bma1>). This analysis resulted in the identification of several exons encoding an 1189 bp fragment of open reading frame (ORF) that corresponded to a putative homolog of *rxr* in *Brugia* (*Bma-RXR*). Based on the genomic sequence we designed PCR primers and used them to amplify the expected *Bma-RXR* mRNA using a nested PCR approach. One \u00b5l of a reverse transcription reaction from female total RNA was used as the template for the first round of PCR carried out with primers 5\u2032 \u2013 CGA TCT ATG CCC ATC AGA TTG-3\u2032 (LCP) and 5\u2032 \u2013 CAC AAT GCA AGC TAA GAG ATC G \u2013 3\u2032(RSL) at 46\u00b0C annealing temperature. Six percent of the first round PCR reaction was used as a template in a second round of PCR with primers 5\u2032 \u2013 CGA TTT AAC TCC AAA TGG AAG TCG \u2013 3\u2032 (DLT) and 5 \u2032\u2013 AGC AAA GCG TTG AGT TTG TGT TGG \u2013 3\u2032 (PTQ) at 47\u00b0C annealing temperature. Using the sequence obtained, primers were designed to extend the 5\u2032 of the coding sequence using a semi-nested PCR approach in combination with the 5\u2032 splice leader SL1 primer. As above, two rounds of PCR were employed, in the first round using the SL1 primer (5\u2032 \u2013 GGT TTA ATT ACC CAA GTT TGA G - 3\u2032) and primer 5\u2032 \u2013 GAT GCT CGA TCA CCG CAT ATT GCA CAA ATG - 3\u2032 (CAI) at 68\u00b0C annealing temperature and for the second round the SL1 primer and primer 5\u2032- TGG CAT ACA GTG TCA TAT TTG GTG TTG TGC - 3\u2032 (STT) at 66\u00b0C annealing temperature. The 3\u2032 coding sequence was obtained by 3\u2032 RACE using the First Choice RLM-RACE kit (Ambion) with *Bma-RXR* primers 5\u2032 \u2013 GGC TCT AAT GCT ACC ATC ATT TAA TGA A - 3\u2032 (ALM), and 5\u2032 \u2013 GAA GAT CAA GCT CGA TTA ATA AGA TTT GGA - 3\u2032 (EDQ) following the manufacturer's protocol. For each amplified fragment several clones were sequenced. The positions of the primers used are indicated by short arrows over the corresponding amino acid sequence in the alignment Figures.\n\n## Northern blot analyses\n\nTen \u00b5g of total RNA from adult male, adult female and microfilaria were used to carry out northern blot analyses using the NorthernMax-Gly kit (Ambion). The *Bma-EcR* and *Bma-RXR* probes were 1kb DNA fragments from the respective coding regions labeled using random priming with the NEBlot kit (NEB) and ^32^P-dATP (NEN-Dupont). The sizes of the hybridizing RNA species were estimated using an RNA ladder that was run adjacent to the samples as a reference.\n\n## Phylogenetic analyses\n\nPredicted amino acid sequences of cloned cDNAs were aligned with all nuclear receptor sequences from Swissprot and GenBank, using Muscle \\[38\\] with default options. A complete phylogeny of all the nuclear receptor super-family was built with PhyML \\[39\\]. Subsequently, phylogenies of the relevant sub-families were constructed with 1000 bootstrap replicates. Well aligned sites were selected with GBLOCKS \\[40\\], with relaxed options to allow a few gaps per column of the alignment. In each case PhyML was run with rate heterogeneity with 4 classes, parameter alpha estimated from the data, BIONJ starting tree. Support for nodes was estimated by Approximate Likelihood-Ratio Test (aLRT) \\[41\\].\n\n## Protein-protein interaction by GST-pull-down assays\n\nA cDNA fragment of *Bma-EcR* encoding aa 152\u2013465 (upstream of the C-domain to the end of the predicted ORF) was amplified by PCR using primers 5\u2032 \u2013 AGC TTC CAT GGC AGC TGA AGA AGG TCA ATC TAA TGG CGA CAG TGA GT \u2013 3\u2032 (536 to 557 of EF362469) and Xho-GYP-3 (See cloning of *Bma-EcR*). The fragment was cloned in frame with GST in the vector pGEX-KG \\[42\\]. The fusion protein was produced in *E. coli* BL21 by induction at 30\u00b0C with 0.1 mM IPTG and purified on Glutathione Sepharose beads (Pharmacia) as directed by the manufacturer. Recombinant *Di-rxr-1* and *Aausp* (gift from A. Raikhel, University of California Riverside) in pcDNA-3 (Invitrogen) were transcribed and translated *in vitro* in rabbit reticulocyte lysates using the TNT T7 coupled transcription-translation system (Promega) in the presence of ^35^S-Methionine (Amersham Biosciences) as recommended by the manufacturer. Glutathione resin beads loaded with 1 \u00b5g of GST:Bma-EcR fusion protein were incubated for 1 h at 4\u00b0C with 5 \u00b5l of rabbit reticulocyte lysate containing labeled proteins (Di-RXR-1 or AaUSP) in a total volume of 10 \u00b5l of binding buffer (20 mM Tris, 1 mM EDTA, 1 mM DTT, 10% glycerol, 150 mM sodium chloride, 0.5 mg\/ml of BSA, complete protease inhibitor cocktail (Sigma). The beads were washed twice with binding buffer and three times with buffer without BSA, then incubated with 10 mM reduced glutathione to elute the proteins, and centrifuged. Supernatants were mixed with loading buffer and analyzed by SDS-PAGE. Signals were detected by autoradiography of the dried gels.\n\n## DNA binding by electrophoretic mobility shift assays (EMSA)\n\nThe ecdysone response element (PAL-1) described by Hu et al. \\[43\\] was produced by annealing two synthetic oligonucleotides: 5\u2032 \u2013 TTG GAC AAG GTC AGT GAC CTC CTT GTT CT \u2013 3\u2032 and its complement (with two overhanging Ts at each 3\u2032 end). PAL-1 was labeled with ^32^P-dATP (NEN-Dupont) using Klenow polymerase (New England Biolabs) and purified by spin column G50 chromatography (Amersham Biosciences). A cDNA fragment containing the complete coding region of *Bma-EcRA* was cloned in pcDNA-3 (Invitrogen) using the *Nhe*I and *Xho*I restriction sites. Two additional constructs containing *Bma-EcRB* and *C* respectively were also cloned using the same strategy. The three *Bma-EcR* isoforms were transcribed and translated *in vitro* in rabbit reticulocyte lysates using the TNT T7 coupled transcription-translation system (Promega) following the manufacturer's protocol. The translation yield of each construct was assessed by labeling a portion of the reaction with ^35^S-Methionine and analyzing the products after gel electrophoresis and autoradiography. Binding reactions were performed at room temperature in 10mM Tris-HCl pH 7.5, 50 mM NaCl, 10 mM MgCl~2~, 0.5 mM DTT, 0.025 mM EDTA, 4% glycerol, 0.2 \u00b5g\/\u00b5L poly dI-poly-dC, 0.13 \u00b5g\/\u00b5L BSA, 0.05% NP40, with 13 fmol\/\u00b5l labeled PAL-1 and 3.5 \u00b5L TNT reaction mixture containing the corresponding proteins (0.5 \u00b5LAaUSP and 1.5 or 2.5 \u00b5L Bma-EcR), in 15 \u00b5L total volume for 20 min before loading in a 6% native TBE gel (Invitrogen). Signals were detected by autoradiography of the dried gels.\n\n## Constructs and transactivation assays in mammalian cells\n\nTo construct GAL4:Bma-EcR and VP16:Bma-RXR, the DEF domains of *Bma-EcR* and *Bma-RXR* were PCR amplified and cloned into pM and pVP16 vectors (EcR residues 259\u2013565; RXR residues 191 to 464) respectively (Clontech). The construct VP16:Lm-HsRXREF (Chimera 9) has been previously described \\[44\\]. pFRLUC, encoding firefly luciferase under the control of the GAL4 response element (Stratagene Cloning Systems) was used as a reporter.\n\nFifty thousand NIH 3T3 cells per well in 12-well plates were transfected with 0.25 \u00b5g of receptor(s) and 1.0 \u00b5g of reporter constructs using 4 \u00b5l of SuperFect (Qiagen). After transfection, the cells were grown in medium containing ligands for 24\u201348 hours. A second reporter, Renilla luciferase (0.1 \u00b5g), expressed under a thymidine kinase constitutive promoter was cotransfected into cells and was used for normalization. The cells were harvested, lysed and the reporter activity was measured in an aliquot of lysate. Luciferase activity was measured using Dual-luciferaseTM reporter assay system from Promega Corporation (Madison, WI, USA). The results are reported as averages of normalized luciferase activity and the error bars correspond to the standard deviation from multiple assays. The ligands used were: RG-102240, a synthetic stable diacylhydrazine ecdysone agonist \\[N-(1,1-dimethylethyl)-N\u2032-(2-ethyl-3-methoxybenzoyl)-3,5-dimethylbenzohydrazide\\] also known as GS-E or RSL1, (RheoGene, New England Biolabs) and Ponasterone-A (Invitrogen). The ligands were applied in DMSO at the indicated final concentrations and the final concentration of DMSO was maintained at \\<0.1%.\n\n## Constructs and transactivation assays in *Brugia malayi* embryos\n\nIn order to construct an ecdysteroid response reporter for *Brugia malayi* the repeat domain of the *B. malayi* 12 kDa small ribosomal subunit gene promoter \\[45\\] (construct BmRPS12 (\u2212641 to \u22121)\/luc) was replaced (in both orientations) with the PAL-1 EcRE shown to be recognized by Bma-EcR *in vitro*. Previous studies have shown that the repeat acts as a transcriptional enhancer. Outward facing primers flanking the repeat domain containing synthetic *Spe*I sites at their 5\u2032 ends were used in an inverse PCR reaction employing BmRPS12 (\u2212641 to \u22121) as a template \\[46\\]. The resulting amplicons were purified using the QiaQuick PCR cleanup kit (Qiagen). The purified amplicons were digested with *Spe*I, gel purified, self-ligated and transformed into *E. coli*. The resulting construct was designated BmRPS12 -rep. A double stranded oligonucleotide consisting of five tandem repeats of the EcRE: ctag(GGACA<u>AGGTCA<\/u>G<u>TGACCT<\/u>CCTTGTTC) 5\u00d7 with *Spe*I overhangs was then ligated into the *Spe*I site of BmRPS12 -rep. The insertions in the forward and reverse orientations were designated BmRPS12-EcRE and BmRPS12-EcRE-rev respectively.\n\nConstructs were tested for promoter activity in transiently transfected *B. malayi* embryos essentially as previously described \\[47\\]. In brief, embryos were isolated from gravid female parasites and transfected with BmRPS12-EcRE (or BmRPS12-EcRE-rev) mixed with a constant amount of a transfection control, consisting of the BmHSP70 promoter fragment driving the expression of renilla luciferase (construct BmHSP70 (\u2212659 to \u22121)\/ren). Following a rest of five minutes, the transfected embryos were transferred to embryo culture media (RPMI tissue culture medium containing 25 mM HEPES, 20% fetal calf serum, 20 mM glucose, 24 mM sodium bicarbonate, 2.5 mg ml-1 amphotericin B, 10 units ml-1 penicillin, 10 units ml-1 streptomycin and 40 mg ml\/L gentamycin), supplemented with 1 \u00b5M 20-OH ecdysone dissolved in 50% ethanol or solvent control. Transfected embryos were maintained in culture for 48 hours before being assayed for transgene activity. Firefly luciferase activity was normalized to renilla luciferase activity in each sample to control for variations in transfection efficiency. Firefly\/renilla activity ratios for each sample were further normalized to the activity ratio from embryos transfected in parallel in each experiment with the parental construct BmRPS12 -rep. This permitted comparisons of data collected in experiments carried out on different days.\n\n## Statistical analysis\n\nEach construct was tested in two independent experiments, with each experiment containing triplicate transfections of each construct to be analyzed. The statistical significance of differences noted between the activity in the control and experimental transfections was determined using Dunnett's test, as previously described \\[47\\].\n\n## Sequence accession numbers\n\nThe nucleotide sequences for Bma-EcR isoform A, Bma-EcR isoform C, Bma-RXR and Ovnhr-4 have been deposited in the GenBank database under GenBankAccession Numbers: EF362469, EF362470, EF362471, and EF362472.\n\n# Results\n\n## Bma-EcR cloning and genomic structure\n\nA candidate *EcR* homolog was first identified from *D. immitis* using degenerate PCR primers based on insect EcRs (C. Shea, J. Richer and C.V. Maina, unpublished results). Using sequences from the *D. immitis EcR* homolog, genomic libraries from *B. malayi* available from the Filarial Genome Network (FGN) were screened. A strongly hybridizing BAC was identified and sequenced. This BAC contained a gene that encodes a protein with strong similarities to the *EcR* branch of nuclear receptors (see below). We designated this gene as *Bma*-EcR (to distinguish it from *Bombyx mori* EcR \\[37\\]). Using sequences corresponding to the predicted *Bma-EcR* exons, PCR primers were designed and used to screen larval and adult cDNA libraries. This library survey revealed *Bma-EcR* expression in L1, L3, and L4 larval stages, as well as in adult males and females (data not shown). In the microfilaria (L1) library, using primers from the putative ligand-binding domain (LBD) encoding region, two alternatively spliced mRNA isoforms of *Bma-EcR* (isoforms A and C) were identified (<a href=\"#pntd-0000625-g001\" data-ref-type=\"fig\">Fig. 1A<\/a>). *Bma-EcRA* is the isoform containing the longest ORF (597 a.a.) with an intact LBD. *Bma-EcRC* contains exon 6 with a 29-nucleotide deletion that results in a reading frame shift that generates a premature stop codon and truncation of the LBD at helix 5 (<a href=\"#pntd-0000625-g001\" data-ref-type=\"fig\">Fig. 1<\/a>). This is the result of an alternative splice site within exon 6. In addition to these confirmed isoforms, a splice site consensus sequence was identified within exon 5 at the end of the DNA-binding domain (DBD) that, if used, would result in the omission of ten amino acids from the C-terminal extension of the DBD (indicated by an arrow in <a href=\"#pntd-0000625-g001\" data-ref-type=\"fig\">Figure 1A<\/a>). This type of spliced mRNA isoform has been identified in *D. immitis* (C. Shea, J. Richer and C. V. Maina, unpublished results). We were unable to clone such an isoform (EcRB) from *B. malayi* by RT-PCR. However, we cannot exclude the possibility that *Bma-EcRB* is expressed in specific tissues or developmental stages not represented in the libraries or RNA used.\n\n## Sequence and phylogenetic analysis of Bma-EcR\n\nBma-EcR shows strongest similarity to the NR1H group of nuclear receptors typified by the insect EcRs and mammalian FXR and LXR receptors (<a href=\"#pntd-0000625-g001\" data-ref-type=\"fig\">Figs. 1B<\/a>, <a href=\"#pntd-0000625-g002\" data-ref-type=\"fig\">2<\/a>). The strongest similarity is in the DBD which contains the canonical C~4~ zinc finger structure of nuclear receptors. This domain is 10 amino acids longer in Bma-EcR than in the homologous region of the other EcRs. However, as indicated above, exclusion of these 10 amino acids by alternative splicing of this site (isoform B) would result in a better alignment of Bma-EcR with the other EcRs (<a href=\"#pntd-0000625-g001\" data-ref-type=\"fig\">Fig. 1B<\/a>). The LBD shows significant similarity in the regions encoding helices 3\u201310 \\[48\\]. An exception is found in the region of helix 11\u201312 (<a href=\"#pntd-0000625-g001\" data-ref-type=\"fig\">Fig. 1B<\/a>). Helix 12 of insect EcRs contains the AF2 motif, responsible for ligand-dependent transcriptional activation. Although predictions of secondary structure of the *Bma-EcR* LBD protein sequence indicate helical folding of putative helices 3\u201310, no helical propensity is predicted in the region of helix 12 (data not shown). Immediately following the helix 12 region a glutamine-rich helical segment is present. Glutamine-rich sequences are often associated with transcription activation domains \\[49\\]. These differences make Bma-EcR an unusual member of the receptor family that perhaps uses a different mechanism for ligand-dependent activation.\n\nGlobal phylogenetic analysis (see Supporting Information <a href=\"#pntd.0000625.s001\" data-ref-type=\"supplementary-material\">Fig. S1<\/a>) places *Bma-EcR* with arthropod EcRs. The position of *Bma-EcR* is strongly supported (99% aLRT support) in a phylogenetic tree of the sub-family (<a href=\"#pntd-0000625-g002\" data-ref-type=\"fig\">Figures 2<\/a> and <a href=\"#pntd.0000625.s002\" data-ref-type=\"supplementary-material\">S2<\/a>). The branch leading to *Bma-EcR* is long, indicating a relatively derived sequence, but not more derived than that of dipteran EcRs, for example. Separate analysis of the DBD and LBD produced similar tree topologies, especially concerning the position of *Bma-EcR*.\n\n## BmaEcR expression analysis\n\nNorthern blot analysis was used to establish the expression pattern of *Bma-EcR* in *Brugia* adult females, males, and L1 microfilaria. The fragment used as the probe encompassed the coding sequence common to both mRNA isoforms identified. A predominant species of approximately 3.75\u20134 Kb was present in all RNA samples tested (<a href=\"#pntd-0000625-g003\" data-ref-type=\"fig\">Fig. 3<\/a>), which was consistent with our detection by RT-PCR of the *Bma-EcR* isoforms in libraries from those same stages, and implies the existence of longer 5\u2032 and\/or 3\u2032 untranslated regions than are present in our cloned cDNA species. Shorter minor RNA species are detectable which may indicate the existence of additional isoforms (<a href=\"#pntd-0000625-g003\" data-ref-type=\"fig\">Fig. 3<\/a>).\n\n## Bma-EcR dimerization with RXR and USP\n\nEcRs heterodimerize with Ultraspiracle (USP) proteins to form functional ecdysone receptors that bind to ecdysteroid ligands and ecdysone response elements (EcREs) \\[35\\]. In order to test whether Bma-EcR heterodimerizes with a canonical insect USP or its filarial homologue Di-RXR-1 \\[36\\], an *in vitro* binding assay was carried out. *In vitro* translated ^35^S-labeled Di-RXR-1 or *Aedes aegypti* USP (AaUSP) were incubated with GST or GST:Bma-EcR fusion proteins immobilized on glutathione-beads. Specific bands corresponding to the full length AaUSP and Di-RXR-1 were detected bound to GST:Bma-EcR (<a href=\"#pntd-0000625-g004\" data-ref-type=\"fig\">Fig. 4A<\/a>). No binding to GST alone was detected with either protein bait. While the *in vitro* translation of AaUSP resulted in the production of a major protein species of the predicted full length AaUSP (<a href=\"#pntd-0000625-g004\" data-ref-type=\"fig\">Fig. 4A<\/a>, lane 1), *in vitro* translation of Di-RXR-1 produced multiple protein species (<a href=\"#pntd-0000625-g004\" data-ref-type=\"fig\">Fig. 4<\/a> lane 4) including one corresponding to full-length Di-RXR-1 (ca 55 kD), which specifically bound to GST:Bma-EcR (<a href=\"#pntd-0000625-g004\" data-ref-type=\"fig\">Fig. 4A<\/a> lane 6). These results indicate that Bma-EcR protein, like EcR, is capable of heterodimerization with USP protein *in vitro*.\n\n## BmaEcR DNA binding properties\n\nHaving established that Bma-EcR can dimerize with USP\/RXRs, we investigated the DNA binding properties of the two isolated protein isoforms of *Bma-EcR* (Forms A and C) to a palindromic ecdysone response element (PAL-1 EcRE) based on the *Drosophila hsp27* ecdysone response gene \\[43\\], using EMSA. In addition to the cloned *Bma-EcRA* and *Bma-EcRC* mRNA isoforms, a construct lacking the 10 amino acids downstream of the zinc finger domain was engineered (putative Bma-EcRB). The three Bma-EcR isoforms and AaUSP (as the heterodimerization partner) were produced in rabbit reticulocyte lysates and their relative amounts were estimated using ^35^S-met labeling and autoradiography (<a href=\"#pntd.0000625.s003\" data-ref-type=\"supplementary-material\">Fig S3<\/a>). An equal amount of AaUSP-containing reticulocyte lysate was incubated with increasing amounts of each Bma-EcR isoform preparation and ^32^P-labeled EcRE prior to analysis by native polyacrylamide gel electrophoresis. AaUSP produces a specific band with the EcRE as has been shown before \\[50\\] (<a href=\"#pntd-0000625-g004\" data-ref-type=\"fig\">Fig. 4B<\/a>, dot), which migrates faster than a nonspecific band produced by the reticulocyte lysate (<a href=\"#pntd-0000625-g004\" data-ref-type=\"fig\">Fig. 4B<\/a>, asterisk). Both Bma-EcRA and -B produced an additional slower migrating band consistent with a heterodimer bound to the probe (<a href=\"#pntd-0000625-g004\" data-ref-type=\"fig\">Fig. 4B<\/a>, lanes 2\u20135, arrow). In contrast, no additional band is detected with Bma-EcRC (<a href=\"#pntd-0000625-g004\" data-ref-type=\"fig\">Fig. 4B<\/a>, lanes 6\u20137). This result is not unexpected given that Bma-EcRC, which contains a premature stop codon and encodes a protein with a truncated LBD, lacks essential structural features for heterodimerization. Neither Bma-EcRA nor Bma-EcRB bound substantially to the EcRE in the absence of AaUSP (<a href=\"#pntd-0000625-g004\" data-ref-type=\"fig\">Fig. 4B<\/a>, lanes 8\u20139). This *in vitro* analysis of Bma-EcR heterodimerization with AaUSP and binding to an EcRE suggests that Bma-EcR has DNA-binding properties similar to those of ecdysone receptors.\n\n## Cloning of a *B. malayi* RXR homolog\n\nThe dimerization properties of Bma-EcR and the identification of *rxr* \\[36\\] and *EcR* homologs in the dog filarial parasite *D. immitis* pointed to the likelihood that an *rxr* homolog also exists in other filarial nematodes. Using degenerate PCR primers we were able to clone a fragment with high sequence similarity to Di-RXR-1 from *O. volvulus* cDNA (see Experimental Procedures; sequence deposited in GenBank ). In *B. malayi*, however, although we searched for an RXR type receptor in the genomic libraries available using *Di-rxr-1* as a probe, no strongly hybridizing sequences were detected. While this work was in progress genomic data from the *B. malayi* genome project became available, which provided us with an alternative route to clone the *B. malayi* RXR\/USP \\[51\\]. Using the sequence information from the other filarial species as well as the *Brugia malayi* genome project we designed a combined RT-PCR and RACE approach (described in detail in Experimental Procedures) that allowed us to obtain clones for the *B. malayi* homolog of RXR which we named *Bma-RXR*. The longest cDNA sequence identified for *Bma-RXR* is 1398 bp and encodes a 465 amino acid protein that has strong similarity to *D. immitis* Di-RXR-1 (100% amino acid identity in the DBD and 83% in the LBD). The amino acid sequence similarity between the *B. malayi* and *D. immitis* RXRs substantially deteriorates in the last exon. Interestingly, the last exon corresponds to the helix 12 region of the LBD where the activation function AF-2 usually resides (<a href=\"#pntd-0000625-g005\" data-ref-type=\"fig\">Fig. 5<\/a>). This LBD region is also highly dissimilar between the filarial nematode RXRs and their homologs in other non-nematode species. Notably the motif LIRVL consistent with the RXR AF2 is found in Bma-RXR but not Di-RXR-1.\n\n## Phylogenetic analysis of Bma-RXR\n\nSimilarly to Bma-EcR, global phylogenetic analysis places Bma-RXR together with USPs and RXRs (Supplementary material <a href=\"#pntd.0000625.s001\" data-ref-type=\"supplementary-material\">Figs. S1<\/a> and <a href=\"#pntd.0000625.s002\" data-ref-type=\"supplementary-material\">S2<\/a>.). Using HNF4s as the outgroup, there is 100% aALRT (approximate Likelihood Ratio Test ) support to place Bma-RXR in the USP\/RXR sub-family (<a href=\"#pntd-0000625-g006\" data-ref-type=\"fig\">Fig. 6<\/a>). Relationships among arthropod USP\/RXRs and Bma-RXR are not well resolved (aALRTs under 50%), but Bma-RXR groups strongly with Di-RXR-1. The grouping of Bma-RXR among USP\/RXRs remains the same whether the *Schistosoma mansoni* sequences are included or not in the tree (data not shown). The *Schistosoma* sequences are extremely divergent, to the extent of not being phylogenetically informative \\[52\\],\\[53\\], and branch at the base of the tree. While it is known that dipteran and lepidopteran USPs evolve especially fast \\[53\\], Di-RXR-1 and Bma-RXR appear to have evolved even faster. Separate phylogenies of the DBD and LBD (not shown) indicate that this is entirely due to a very derived LBD. This observation is consistent with the alignment. The DBD on the other hand has evolved slowly, like the DBDs of its homologs in other species.\n\n## Bma-RXR expression\n\nExpression of *Bma-RXR* was analyzed in adult females, males, and L1 larvae by Northen blot analysis (<a href=\"#pntd-0000625-g007\" data-ref-type=\"fig\">Fig. 7<\/a>). A \u223c5kb RNA species was clearly detected in female and male RNA samples. Low levels of the \u223c5 kb *Bma-RXR* RNA species were also observed in the L1 RNA sample. Two additional *Bma-RXR* bands of approximately 3.75 kb and 3 kb were also detected in adult females. The presence of *Bma-RXR* mRNA in males and L1 larvae was in agreement with RT-PCR results (data not shown).\n\n## Bma-EcR heterodimerization and ligand-dependence *in vivo*\n\nTo further characterize the properties of Bma-EcR we tested whether Bma-EcR, by virtue of its LBD, is capable of forming a dimer with Bma-RXR, its putative native partner, to constitute a functional receptor and transduce the hormonal signal of ecdysteroids in a cellular context. The assay we employed takes advantage of the fact that the LBD of nuclear receptors can function in a modular fashion fused to heterologous DNA binding domains such as the GAL4 DBD \\[43\\],\\[44\\]. In order to test the ability of Bma-EcR LBD to activate transcription of a reporter gene in response to a particular hormone ligand, NIH 3T3 cells were co-transfected with GAL4:Bma-EcR(LBD) in combination with RXR LBDs fused to VP16. In addition to the Bma-RXR(LBD) we tested human HsRXR and Hs-LmRXR(LBD) (a chimeric human-locust LBD). The latter was selected because it shows no constitutive dimerization and high ligand-dependent activity when partnered with other ecdysone receptors \\[44\\] (and ). The transfected cells were tested for trans-activation in the absence or presence of either the ecdysteroid Ponasterone-A or the synthetic ecdysone agonist RSL1 by assaying luciferase activity.\n\nSignificant transactivation was detected when GAL4:Bma-EcR(LBD) was partnered with Bma-RXR(LBD) (<a href=\"#pntd-0000625-g008\" data-ref-type=\"fig\">Fig. 8A<\/a>). The addition of RSL1 (<a href=\"#pntd-0000625-g008\" data-ref-type=\"fig\">Fig. 8B<\/a>) or Ponasterone A (data not shown) had no further stimulatory effect on the detected activity. These data demonstrate that Bma-EcR and Bma-RXR are *bona fide* nuclear receptor partners and that, like their insect counterparts, they avidly dimerize in the absence of ligand. The ligands apparently cannot appreciably increase the heterodimer's ability to activate transcription above that of the VP16 activation domain in this assay.\n\nSignificant ligand-dependent transcriptional activation of luciferase was detected, however, when GAL4:Bma-EcR(LBD) was partnered with the chimeric VP16:Hs-LmRXR and treated either with Ponasterone-A or RSL1 (<a href=\"#pntd-0000625-g008\" data-ref-type=\"fig\">Fig. 8C<\/a>). This is likely the result of ligand-dependent dimerization of the two receptor LBD fusions and subsequent trans-activation *via* the VP16 activation domain. This result clearly demonstrates the ability of Bma-EcR LBD to transduce the action of the ecdysteroid Ponasterone-A and the ecdysteroid agonist, RSL1 in the transfected cells.\n\nThe dimerization and transactivation studies presented here show that Bma-EcR is able to heterodimerize with Bma-RXR in a cellular context and capable of triggering a transcriptional response in an ecdysteroid-specific manner. These observations taken together along with their expression profile suggest that Bma-EcR and Bma-RXR have the prerequisite functional properties to constitute a functional *Brugia malayi* ecdysone receptor.\n\n## Ecdysone-dependent transcription in *B. malayi*: A reporter assay\n\nThe existence of homologs for both protein components of Ecdysone Receptor in *B. malayi* which possess functional dimerization and DNA binding properties, and the earlier pharmacological observations by H. Rees \\[33\\],\\[34\\] suggest that ecdysone could function as a transcriptional regulatory ligand in *B. malayi*. To directly test this hypothesis, we employed a recently established transient transformation technique to explore whether ecdysteroids can activate transcription in *B. malayi* using a reporter assay. Recent studies have demonstrated that the 5\u2032 UTR of the gene encoding the 12 kDa small subunit ribosomal protein of *B. malayi* (BmRPS12) was capable of acting as a promoter when used to drive the expression of a luciferase reporter gene in transiently transfected *B. malayi* embryos \\[45\\]. The BmRPS12 promoter contains 5 \u00be copies of an almost exact 44 nt repeat that acts as an enhancer element \\[45\\]. This promoter construct driving the expression of firefly luciferase (construct BmRPS12 (\u2212641 to \u22121)\/luc) was used to develop a reporter for *B. malayi* in which the enhancer repeat element was replaced with canonical ecdysone response elements (EcREs). We constructed the EcRE-BmRPS12-luciferase reporter (as described in <a href=\"#s2\" data-ref-type=\"sec\">Methods<\/a>) using the PAL-1 element that Bma-ECR is capable of binding *in vitro* (<a href=\"#pntd-0000625-g004\" data-ref-type=\"fig\">Fig. 4B<\/a>). This construct was tested for transcriptional activity in transfected *B. malayi* embryos, which were exposed to 20-OH ecdysone (20-E), or solvent alone, before being assayed for luciferase reporter activity. As shown in <a href=\"#pntd-0000625-g009\" data-ref-type=\"fig\">Fig. 9A<\/a> ecdysteroid treatment resulted in a significant increase of reporter gene activity in cultures exposed to 20-E relative to control cultures (transfected in parallel with the same construct but exposed to solvent alone). This response to 20-E requires the presence of the EcRE sequence, since a construct lacking the EcRE did not exhibit any increase in luciferase activity in response to 20-E. Similarly, the response was strictly dependent on hormone, as constructs containing the EcRE produced levels of activity that were not significantly different from those obtained with the construct lacking the EcRE, in the absence of 20-E. Constructs containing the EcRE in both orientations were equally responsive to 20-E treatment, in keeping with previous studies demonstrating the symmetric nature of the binding of nuclear hormone receptors to their cognate response elements \\[43\\]. The response to the 20-OH ecdysone was dose-dependent, reaching a plateau at 5 \u00b5M (<a href=\"#pntd-0000625-g009\" data-ref-type=\"fig\">Fig. 9B<\/a>). These results provide molecular evidence for the function of ecdysone in transcriptional responses of *B. malayi* and reveal the functional operation of a corresponding signaling system.\n\n# Discussion\n\nMolting in ecdyzosoans has been studied most extensively in insects. In insects EcR and USP initiate the transduction of the molt-triggering signal \\[4\\],\\[9\\]. Molting progression is mediated by the expression and activation of a number of well-characterized genes, including additional nuclear receptors \\[16\\],\\[17\\],\\[54,\\]. In contrast, in nematodes molting initiation and the molecular signaling responsible for its progression are only now starting to be understood. An RNAi screen in *C. elegans* for genes that are involved in molting has revealed a large number of \"molting\" genes, which encode proteins ranging from transcription factors and intercellular signaling molecules to proteases and protease inhibitors. However, no signal has been specifically identified as being the putative molting trigger \\[55\\]. Expression profiles of *C. elegans* \"ecdysone cascade\" nuclear receptors during molting cycles parallel the expression of their homologs in insects \\[56\\], and *nhr-23*, *nhr-25*, *nhr-41*, and *nhr-85*, the *C. elegans* orthologs of *DHR3*, *Ftz-F1*, *DHR78*, and *E75*, respectively, have been shown to be important for proper molting and\/or dauer larva formation \\[26\\], \\[56\\]\u2013\\[58\\]. The fact that the *C. elegans* genome contains no identifiable homologs of *EcR* or *rxr* \\[23\\] and that no ecdysteroids have been identified in this nematode, has led to the suggestion that ecdysone itself is unlikely to be the molting hormone in this free living nematode \\[55\\].\n\nOur previous studies demonstrated the existence of an *rxr* homolog in the canine filarial nematode *D. immitis* \\[36\\]. The isolation of *Di-rxr-1* indicated that, in contrast to *C. elegans*, filarial nematodes might contain different sets of NRs. The isolation of homologs of *EcR* and *rxr* in *Brugia malayi* presented here demonstrates that filarial nematodes express both components of the ecdysone receptor and these nuclear receptors show dimerization, DNA binding, and hormone-binding characteristics similar to those of the canonical insect ecdysone receptors. Our phylogenetic analyses place the two receptors in the corresponding branches of the superfamily tree. They also indicate a rapid evolution of the LBDs. The LBDs of nematode RXRs are extremely divergent, on a similar scale to that of *Schistosoma* RXR LBD. Subsequent to our identification of *EcR* and *rxr* homologs in *Brugia*, the sequencing of the genome was completed, identifying additional putative nuclear receptors in the ecdysone signaling cascade \\[51\\].\n\nWe cloned two *Bma-EcR* and one *Bma-rxr* mRNA isoforms. Northern blot analyses revealed *Bma-EcR* and *Bma-rxr* expression in adult males, females and L1s. In addition, RT-PCR analyses indicate that *Bma-EcR* is also present in L1, L2 and L3 larval stages. Since females contain developing embryos, it is not possible to differentiate between embryonic and female-specific expression of these two nuclear receptors in *B. malayi*. In insects *EcR* has been shown to be critical for both embryonic development and oogenesis \\[15\\],\\[59\\],\\[60\\] and in filarial nematodes ecdysone treatment releases meiotic arrest and stimulates microfilaria release \\[34\\]. Expression of *EcR* and *rxr* homologs in *B. malayi* females points to possible functions of the ecdysone receptor also in nematode oogenesis and\/or embryogenesis.\n\nThe expression pattern of *Bma-RXR* differs somewhat from the expression pattern of the other filarial *rxr* identified to date, *Di- rxr-1*, which is expressed in males but not females \\[36\\]. In insects the rxr homologue \"*Ultraspiracle*\" (USP) is considered the main functional partner of EcR and as such its expression overlaps with that of *EcR* \\[5\\]. This also seems to be the case in *B. malayi*, where we observed that at least one isoform of *Bma-rxr* has an overlapping expression pattern with *Bma-EcR*. However, two other *Bma-rxr* isoforms appear to be specifically expressed only in females.\n\nThe sequence differences of *B. malayi* and *D. immitis* RXR may mirror differences in expression patterns of the two RXR homologues. Whether these differences in sequence and expression pattern correlate with differences in ligand interaction and\/or function remains an open question.\n\nBoth Bma-EcRA and a putative isoform B are able to bind a canonical ecdysone response element (EcRE) when partnered with USP. The question of whether isoform B exists in *B. malayi* (as in *D. immitis*) remains unanswered. We have shown that such an isoform is biochemically active, being able to dimerize with an insect USP and bind EcRE *in vitro*. Furthermore, isoform B is the most similar to the insect EcRs. Bma-EcRB contains a shorter (i.e. canonical) \"T-box\" region than Bma-EcRA (<a href=\"#pntd-0000625-g001\" data-ref-type=\"fig\">Fig. 1<\/a>). The \"T-box\" region has been described as being able to modulate DNA binding to extended hormone response elements \\[61\\]. The presence of possible sequence variation in the \"T-box\" region in these two *Bma-EcR* variants could point to the possibility of differences in isoform-specific interactions with DNA target sequences.\n\nBma-EcRC contains a truncated LBD, and it is similar in organization to the estrogen-alpha variant Delta-5, which displays dominant-negative activity \\[62\\]. As we have shown, isoform C is unable to dimerize with a *bona fide* USP to bind the palindromic EcRE. These data suggest that Bma-EcRC may carry out a novel function that is independent of any interactions with an RXR partner. Establishing the role of *Bma-EcRC* is the aim of future investigations.\n\nThe sequence in the region of helices 11\u201312 in the LBD of *B. malayi* and *D. immitis* EcR and RXR homologues is strikingly divergent when compared to each other and to other EcRs and RXRs respectively. The most prominent feature in Bma-EcR is the absence of conserved helix 12 residues. This difference raises the question of what constitutes a functional activation function corresponding to AF2 in these nematode members of the nuclear receptor family. Our transcriptional activation assay results clearly show that the two receptors can dimerize and that the LBD of Bma-EcR is capable of transducing an ecdysteroid signal in a cellular context. Even though our analysis was carried out in a heterologous system, this type of assay has been shown to be highly informative for LBD-ligand interactions \\[44\\]. In this system, however, strong constitutive dimerization of receptor partners can obscure possible transcriptional effects of the ligand. Our results obtained with the chimeric RXR-LBD (which confers low constitutive dimerization) as a partner, indicate that the Bma-EcR LBD does show an ecdysteroid response. Evidence of hormone binding from these transactivation assays and the absence of a recognizable AF2 motif in Bma-EcR suggest that this receptor utilizes different features to achieve equivalent transcriptional functions than its insect counterparts.\n\nThe identification of the putative ecdysone receptor components presented here provides strong support to the long standing hypothesis that ecdysteroids play a role in filarial nematode embryogenesis and molting similar to their role in insects.\\[4\\],\\[32\\]. Ecdysteroids have been detected in a number of nematodes (reviewed by Barker and Rees, \\[32\\]). When *in vitro* cultivation of *Onchocerca volvulus* microfilaria was attained, it was observed that the addition of 20E to the culture media resulted in L1 larva progressing to the infective L3 stage \\[63\\]. This observation is consistent with the fact that after the bloodmeal, mosquitoes raise their ecdysteroid level, which correlates with the subsequent rapid molting of the ingested L1 larvae to the L2 stage. We attempted to directly demonstrate that ecdysone can act as a transcriptional trigger *in vivo* using a transient transformation reporter assay. Indeed, significant activity was observed in response to ecdysone . Our transgenic *Brugia* experiments confirm the *in vivo* functionality of both a consensus EcRE and 20-hydroxyecdysone in measurable transcriptional activity. Although we present no data to establish that the observed activation is mediated by the receptor(s) we have cloned, our results in conjunction with previous studies on this subject confirm that filarial nematodes in particular, contain and express the gene components of a functional ecdysone signaling system that is quite similar to that of other ecdysozoa. The role of this signaling system in filarial development will be the subject of further studies. Furthermore, the existence of a functional ecdysone signaling pathway in filarial nematodes does point to the possibility of using a novel approach for the development of drugs to fight filariasis based on testing of pre-existing compounds that specifically target the ecdysone pathway \\[64\\].\n\n# Supporting Information\n\nPhylogenetic tree of all Nuclear receptors. Maximum likelihood phylogenetic tree generated with all nuclear receptor sequences obtained from SwissProt and GenBank constructed as described in the <a href=\"#s2\" data-ref-type=\"sec\">Methods<\/a>. The positions of Bma-EcR and Bma-RXR reported here are indicated by arrows. The accession numbers and the statistical aLRT support for the branches are indicated.\n\n(0.23 MB TIF)\n\nClick here for additional data file.\n\nSub-trees containing EcRs and RXRs. Sub-trees from the phylogeny of <a href=\"#pntd.0000625.s001\" data-ref-type=\"supplementary-material\">Figure S1<\/a> containing all EcRs (left) or all RXRs (right). The accession numbers and the statistical aLRT support for the branches are indicated.\n\n(0.27 MB TIF)\n\nClick here for additional data file.\n\nIn vitro translated proteins used in <a href=\"#pntd-0000625-g004\" data-ref-type=\"fig\">Figure 4<\/a>. SDS-PAGE of ^35^S-labeled in vitro translated proteins used in <a href=\"#pntd-0000625-g004\" data-ref-type=\"fig\">Figure 4<\/a> showing size and relative amounts of the three Bma-EcR isoforms and AaUSP. One \u00b5L of each in vitro translated protein was analyzed by autoradiography of the dried gel.\n\n(0.13 MB TIF)\n\nClick here for additional data file.\n\nHsLmRXR-VP16 (LBD) chimera activates the reporter upon RSL1 treatment only in the presence of a responsive EcR heterodimer partner. Transactivation assay with the chimeric VP16:Hs-LmRXR(LBD) used in <a href=\"#pntd-0000625-g008\" data-ref-type=\"fig\">Figure 8<\/a>. The same construct was transfected along with a Gal4: CfEcR(LBD) fusion or alone in NIH-3T3 cells using the same experimental protocols as for <a href=\"#pntd-0000625-g008\" data-ref-type=\"fig\">Figure 8<\/a>. Activation of the reporter is observed only upon induction with the ecdysone agonist RSL-1. CfEcR(LBD) encodes the LBD of the ecdysteroid receptor from *Choristoneura fumiferana* \\[44\\].\n\n(0.07 MB DOC)\n\nClick here for additional data file.\n\nList of accession numbers for all EcR and RXR sequences used in the phylogenetic analyses shown in <a href=\"#pntd-0000625-g002\" data-ref-type=\"fig\">Figures 2<\/a> and <a href=\"#pntd-0000625-g006\" data-ref-type=\"fig\">6<\/a>.\n\n(0.07 MB DOC)\n\nClick here for additional data file.\n\nWe thank the filarial genome network for resources, Dr. A. Raikhel, UC Riverside, for generously providing AaUSP constructs, Dr. C. Shea for reagents and helpful comments on the manuscript. We are also grateful to Dr. Donald Comb for his continuous support of this project.\n\n# References\n\n[^1]: **\u00a4:** Current address: Department of Biology, University of Louisiana at Monroe, Monroe, Louisiana 71209, United States of America\n\n[^2]: Conceived and designed the experiments: GT TRU CVM. Performed the experiments: GT ALE SRP CRG CL. Analyzed the data: GT ALE MRR CRG TRU CVM. Contributed reagents\/materials\/analysis tools: SRP TRU. Wrote the paper: GT ALE. Cloning of BmaEcR, OvRXR, construction of plasmids, and protein-DNA binding: GT. Cloning of BmaRXR, Northern blots: ALE. Mammalian cell 2-hybrid assays: SRP. Phylogenetic analysis of sequence data: MRR. Cloning of BmaRXR: CRG. Promoter for *Brugia* reporter assay and *Brugia* biolistic transformation assays: TRU. Cloning of nematode receptors, sequence analysis: CVM.","meta":{"dup_signals":{"dup_doc_count":106,"dup_dump_count":27,"dup_details":{"curated_sources":3,"2017-51":1,"2017-43":1,"2017-22":1,"2017-09":11,"2016-44":1,"2016-36":2,"2016-30":6,"2016-07":2,"2015-48":5,"2015-40":3,"2015-35":5,"2015-32":4,"2015-27":3,"2015-22":3,"2015-14":2,"2014-52":2,"2014-49":3,"2014-42":6,"2014-41":6,"2014-35":4,"2014-23":6,"2014-15":7,"2019-26":1,"2015-18":6,"2015-11":4,"2015-06":2,"2014-10":6}},"file":"PMC2834746"},"subset":"pubmed_central"}
{"text":"abstract: The Hypertext atlas of Dermatopathology, the Atlas of Fetal and Neonatal Pathology and Hypertext atlas of Pathology (this one in Czech only) are available at <http:\/\/www.muni.cz\/atlases>. These atlases offer many clinical, macroscopic and microscopic images, together with short introductory texts. Most of the images are annotated and arrows pointing to the important parts of the image can be activated.\n  .\n  The Virtual Microscope interface is used for the access to the histological images obtained in high resolution using automated microscope and image stitching, possibly in more focusing planes. Parts of the image prepared in advance are downloaded on demand to save the memory of the user's computer. The virtual microscope is programmed in JavaScript only, works in Firefox\/Mozilla and MSIE browsers without need to install any additional software.\nauthor: Josef Feit; Lud\u011bk Matyska; Vladim\u00edr Ulman; Luk\u00e1\u0161 Hejtm\u00e1nek; Hana Jedli\u010dkov\u00e1; Marta Je\u017eov\u00e1; Mojm\u00edr Moulis; V\u011bra Feitov\u00e1\ndate: 2008\ninstitute: 1Institute of Pathology, Masaryk University, Brno, Czech Republic; 2Faculty of Informatics, Masaryk University, Brno, Czech Republic; 3Dept. of Dermatovenerology,, St. Anna Hospital, Masaryk University, Brno, Czech Republic; 4Dept. of Radiology, St. Anna Hospital, Masaryk University, Brno, Czech Republic\nreferences:\ntitle: Virtual microscope interface to high resolution histological images\n\n# Introduction\n\nThe Hypertext Atlas of Dermatopathology \\[1\\] is available on the Internet since 2008. It contains 4840 clinical, macroscopic and histologic images. Recently the Atlas of Fetal and Neonatal Pathology is available as well. The Atlas of Pathology for pre-graduate students of medicine is available as well (in Czech only) and new atlases are under preparation (today with about 2300 images).\n\nThe atlases contain annotated images (arrows pointing to important parts of the images can be activated) and short introductory texts. Histological images are taken using motorized microscope to take image parts, which are later stitched together. The image stitching is based on analysis of overlapping parts of individual image tiles and joined together using the gradient running on randomly generated curve to obtain the best cosmetic results.\n\n# Methods\n\n## Hardware, image acquisition\n\nLeica DMLA microscope with a set of PlanApo lenses (HC Fluotar 5\/0.15, HC PlanApo 10\/0.30, 20\/0.50, 40\/0.70, 100\/1.35 and a Plan 2\/0.07 lenses) equipped with the Nikon DMX-1200 digital camera is used to obtain image parts at the resolution at 1200 \u00d7 1020 pixels, 3 \u00d7 8 bit colour. Motorized stage (Merzh\u00e4user) is automatically moved from one image to another. The system is controlled by Lucia DI (LIM, Prague). Home made software is used to create composed, very large pictures and to prepare the virtual microscope image stacks.\n\n## Source texts of the atlas\n\nThe atlas source texts are in XML data format. Programs (written mostly in Perl 5.8 programming language) are used to parsing and checking the document structure and to generate the final HTML files, which are uploaded on the server. The overall size of the atlases is about 110 GB of data.\n\n## Image post-processing, virtual microscope\n\nEach image part can be taken in one or more focusing levels. This image stack can be processed by pan-focusing function, which selects sharp areas of each image from the stack to create one image tile and overcoming the problem of image artefacts caused by uneven slides. This feature is especially useful if only slides of suboptimal quality are available (slides of rare cases, from old slide collections etc.). We use usually 3 levels, their distance of focusing planes varies according to the objective used.\n\nAlternatively the whole image stacks are taken and saved. The resulting images are created from stitching tiles from each plane separately. This approach allows creation of multilevel images, which can be focused. This approach we use especially in images taken in high resolution using 100\u00d7 immersion lens, as in bone marrow biopsies. We use usually 5 or 7 focusing planes, sometimes more (up to 13).\n\nAfter stitching each image is digitally manipulated (colour correction, sharpening), archived and cut into pieces, side of which are of multiples 256. Larger pieces 512 and more) are converted into 256 parts. All these image parts are saved into structured directories.\n\nThe browser loads proper parts of image according to current viewport, magnification and focusing level. It reacts to user's actions (image dragging, magnification changing, change in the size of the virtual microscope window, focusing) through catching events, calculates the names of corresponding new image tiles, which are loaded and added into the DOM of the image being displayed. The image parts, which got out of he viewport, are released from memory.\n\nThe individual parts can be stored on the server or locally on the disc. No special server application is needed. The magnification can be changed, images can be dragged and the images saved in stacks can be focused. This approach is used especially in images t Users can change the size of the virtual microscope window up to the full screen.\n\n# Results\n\nOur atlases are available at <http:\/\/www.muni.cz\/atlases>. The access requires registration, which is free. In January 2008 about 1450 users were registered. Combined size of all the atlases is over 110 GB of data.\n\nThe virtual microscope interface works reasonably smooth. This approach does not require installation of any additional software (but the JavaScript in the browser must be enabled). MSIE since the version 4 and Mozilla family of Internet browsers are supported.\n\nThe interface consists of several windows: the text of the atlas, the window with an image in basic (900 px) size with possible activation of arrows, list of signs and window with the virtual microscope with magnification and focus changer (see the Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>). Users can open more images at the same time for easy comparing.\n\n# Discussion\n\nPublishing teaching materials in the Internet has many advantages. In properly designed publication systems the complexity does not grow with extent of the source texts and images. It is easy to publish new version, to add new materials or reflect comments and desires of the students. The quality of the images is very high, usually much better than in printed textbooks and their number is virtually unlimited. Moreover, virtual microscope offers new experience to the students (dragging, focusing, magnification changing), leading to more active approach to learning \\[2\\]. Virtual slides can capture whole tissue specimens, not only selected areas (\"negative\" areas are important as well) and can be used in quizzes as well. In our teaching labs we do not use microscopes any more. Digital slides do not worn out, can be easily replaced if new, more instructive case is available and can be accessed any time from anywhere. Virtual microscope is suitable for preparing diagnostic seminars and reference collections. One slide is enough to prepare the case, so even small specimens can be used for seminar without the danger of cutting through the tissue without having representative specimen for each student or participant.\n\n# Conclusion\n\nOur atlases are continuously upgraded and expanded. In addition to the above-mentioned Atlas of Dermatopathology and Atlas of Fetopathology and Neonatal Pathology we are preparing new atlases (of muscle pathology and bone marrow biopsy). In future image sharing of our images will be possible as well, so that other teachers will be able to include links to images in our atlases, comment them according to their taste and still have access to all the features of the virtual microscope.\n\n### Acknowledgements\n\nThis work was supported by Project MediGRID (1ET202090537) of the Information Society program.\n\nThis article has been published as part of *Diagnostic Pathology* Volume 3 Supplement 1, 2008: New trends in digital pathology: Proceedings of the 9th European Congress on Telepathology and 3rd International Congress on Virtual Microscopy. The full contents of the supplement are available online at <http:\/\/www.diagnosticpathology.org\/supplements\/3\/S1>","meta":{"dup_signals":{"dup_doc_count":176,"dup_dump_count":54,"dup_details":{"curated_sources":3,"2023-14":1,"2022-49":1,"2022-21":1,"2021-39":1,"2021-10":1,"2020-40":1,"2020-16":1,"2019-47":1,"2019-39":1,"2019-26":1,"2019-18":1,"2019-09":1,"2018-51":1,"2018-43":1,"2018-34":1,"2018-26":1,"2018-13":1,"2017-47":1,"2017-34":1,"2017-26":1,"2017-22":1,"2017-17":1,"2017-09":14,"2017-04":1,"2016-50":1,"2016-44":1,"2016-40":1,"2016-36":1,"2016-30":1,"2016-22":1,"2016-18":1,"2016-07":14,"2015-48":5,"2015-40":5,"2015-35":5,"2015-32":5,"2015-27":4,"2015-22":5,"2015-14":5,"2014-52":5,"2014-49":7,"2014-42":14,"2014-41":5,"2014-35":6,"2014-23":9,"2014-15":6,"2023-50":1,"2015-18":4,"2015-11":5,"2015-06":5,"2014-10":4,"2013-48":5,"2013-20":4,"2024-22":1}},"file":"PMC2500118"},"subset":"pubmed_central"}
{"text":"abstract: With increasing dietary cholesterol intake the liver switches from a mainly resilient to a predominantly inflammatory state, which is associated with early lesion formation.\nauthor: Robert Kleemann; Lars Verschuren; Marjan J van Erk; Yuri Nikolsky; Nicole HP Cnubben; Elwin R Verheij; Age K Smilde; Henk FJ Hendriks; Susanne Zadelaar; Graham J Smith; Valery Kaznacheev; Tatiana Nikolskaya; Anton Melnikov; Eva Hurt-Camejo; Jan van der Greef; Ben van Ommen; Teake Kooistra\ndate: 2007\ninstitute: 1Department of Vascular and Metabolic Diseases, TNO-Quality of Life, BioSciences, Gaubius Laboratory, Zernikedreef 9, 2333 CK Leiden, The Netherlands; 2Department of Vascular Surgery, Leiden University Medical Center, Albinusdreef 2, 2300 RC Leiden, The Netherlands; 3Department of Physiological Genomics, TNO-Quality of Life, BioSciences, Utrechtseweg 48, 3704 HE Zeist, The Netherlands; 4GeneGo Inc., Renaissance Drive, St Joseph, MI 49085, USA; 5Department of Analytical Research, TNO-Quality of Life, Quality and Safety, Utrechtseweg 48, 3704 HE Zeist, The Netherlands; 6AstraZeneca, CV&GI Research, Silk Road Business Park, Macclesfield, Cheshire SK10 2NA, UK; 7Vavilov Institute for General Genetics, Russian Academy of Science, Gubkin Street 3, 117809 Moscow, Russia; 8AstraZeneca CV&GI Research, 43183 M\u00f6lndal, Sweden\nreferences:\ntitle: Atherosclerosis and liver inflammation induced by increased dietary cholesterol intake: a combined transcriptomics and metabolomics analysis\n\n# Background\n\nAtherosclerosis is a multifactorial disease of the large arteries and the leading cause of morbidity and mortality in industrialized countries \\[1\\]. There is ample evidence that hypercholesterolemia (that is, elevated plasma levels of low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL)) induced by genetic modification or enhanced intake of dietary lipids is a major causative factor in atherogenesis \\[2,3\\]. It is equally clear that from the very beginning of lesion formation, atherogenesis requires an inflammatory component, which is thought to drive the progression of the disease \\[4,5\\]. Indeed, some of the variation in the rate of lesion progression in different individuals may relate to variations in their basal inflammatory state \\[6,7\\]. However, while the inflammatory processes in the complex evolution of the lesion from the early fatty streak to a fibrous plaque are considered self-perpetuating phenomena, the initial trigger and origin of the inflammatory component in hypercholesterolemia remains enigmatic \\[6,8\\].\n\nRecent observations by us and others suggest that the liver plays a key role in the inflammatory response evoked by dietary constituents (reviewed in \\[8,9\\]). For example, liver-derived inflammation markers such as C-reactive protein (CRP) and serum amyloid A (SAA) increase rapidly (within days) after consumption of an excess amount of dietary lipids \\[8,10\\], and thus by far precede the onset of early aortic lesion formation \\[8\\]. These findings suggest that nutritional cholesterol itself may contribute to the evolution of the inflammatory component of atherogenesis. We postulate that pro-atherogenic inflammatory factors originate at least partly from the liver. We also hypothesize that these factors come into play at high dietary cholesterol doses because of the exponential rather than linear nature of the relationship between cholesterol intake (measured as cholesterol plasma levels) and atherosclerotic lesion size \\[11,12\\] as specified in more detail in Additional data file 1.\n\nIn this study we sought evidence for the hypothesis that inflammation and hypercholesterolemia are not separate factors, but closely related features of the same trigger, dietary cholesterol. In particular, using a variety of newly developed functional bioinformatics tools, we addressed the question of how the liver responds to increasing dietary cholesterol loads at the gene transcription level and analyzed how hepatic cholesterol metabolism is linked to the hepatic inflammatory response, including underlying regulatory mechanisms. Notably, all analyses were performed at a very early stage of the atherogenic process (that is, after 10 weeks of cholesterol feeding) to limit potential feedback reactions from the vessel wall.\n\nAn established model for cholesterol-induced atherosclerosis, ApoE\\*3Leiden transgenic (E3L) mice, allowed the application of experimental conditions that mimic the human situation: E3L mice display a lipoprotein profile similar to that of humans suffering from dysbetalipoproteinemia and develop atherosclerotic lesions that resemble human lesions with regard to morphology and cellular composition \\[13,14\\]. E3L mice were exposed to increasing doses of dietary cholesterol (as the only dietary variable modulated), and liver genome and metabolome datasets were analyzed in a unique context, that is, at the time point of first lesion development. Advanced (functional) bioinformatical analysis allowed us to merge metabolome and transcriptome datasets and to analyze pathways and biochemical processes comprehensively. Recent advances in systems biology (for example, new biological process software for network building and data mining) have enabled us to discover significant relationships and to identify transcriptional master regulators that control gene alteration and are ultimately responsible for effects at the process level.\n\n# Results\n\n## Effect of dietary cholesterol load on plasma lipids and early atherosclerosis\n\nTreatment of female E3L mice with a cholesterol-free (Con), a low-cholesterol (LC) or a high-cholesterol (HC) diet resulted in total plasma cholesterol concentrations that stabilized at 5.9 \u00b1 0.3 mM, 13.3 \u00b1 1.9 mM and 17.9 \u00b1 2.4 mM, respectively. The increase in plasma cholesterol in the LC and HC groups was confined to the pro-atherogenic lipoprotein particles VLDL and LDL (Figure <a href=\"#F1\" data-ref-type=\"fig\">1a<\/a>). High-density lipoprotein (HDL) levels and plasma triglyceride levels were comparable between the groups (Figure <a href=\"#F1\" data-ref-type=\"fig\">1a<\/a> and Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>). The plasma levels of alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT), two markers of liver function, were comparable in the Con and LC groups and slightly elevated in the HC group.\n\nEffects of dietary cholesterol on plasma lipids and inflammation markers\n\n|                           | Con        | LC           | HC              |\n|---------------------------|------------|--------------|-----------------|\n| Body weight (start) (g)   | 20.3 \u00b1 1.5 | 20.8 \u00b1 1.5   | 20.6 \u00b1 0.9      |\n| Body weight gain (g)      | 0.4 \u00b1 0.7  | 0.7 \u00b1 0.8    | 0.6 \u00b1 0.5       |\n| Food intake (g\/day)       | 2.6 \u00b1 0.2  | 2.9 \u00b1 0.3\\*  | 2.5 \u00b1 0.2^\u2020^    |\n| Plasma cholesterol (mM)   | 5.9 \u00b1 0.3  | 13.3 \u00b1 1.9\\* | 17.9 \u00b1 2.4\\*^\u2020^ |\n| Plasma triglyceride (mM)  | 1.7 \u00b1 0.4  | 2.3 \u00b1 0.3    | 2.1 \u00b1 0.7       |\n| Plasma E-selectin (\u03bcg\/ml) | 44.3 \u00b1 2.3 | 44.3 \u00b1 6.3   | 55.1 \u00b1 8.5\\*^\u2020^ |\n| Plasma SAA (\u03bcg\/ml)        | 2.8 \u00b1 0.6  | 4.7 \u00b1 1.7    | 8.3 \u00b1 2.7\\*^\u2020^  |\n| Plasma ALAT (U\/mL)        | 48 \u00b1 44    | 45 \u00b1 22      | 75 \u00b1 23         |\n| Plasma ASAT (U\/mL)        | 260 \u00b1 123  | 237 \u00b1 57     | 569 \u00b1 221\\*^\u2020^  |\n\nThree groups of female E3L mice were fed either a cholesterol-free (Con) diet or the same diet supplemented with 0.25% (LC) or 1.0% (HC) w\/w cholesterol. Listed are the average body weight at the start (t = 0) of the experimental period together with the body weight gain, the average daily food intake and the average plasma levels of cholesterol, triglycerides, E-selectin, serum amyloid A (SAA), alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT). All data are mean \u00b1 standard deviation. \\**P* \\< 0.05 versus Con; ^\u2020^*P* \\< 0.05 versus LC (ANOVA, least significant difference *post hoc* test).\n\nAfter 10 weeks of dietary treatment, the animals were euthanized to score early atherosclerosis. Longitudinally opened aortas of the Con and LC groups were essentially lesion-free (*en face* oil red O-staining), while aortas of the HC group already contained lesions (not shown). Consistent with the presence of atherosclerosis in the HC group, the vascular inflammation marker E-selectin was elevated only in this group (Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>). Early onset of atherosclerosis was analyzed in more detail in the valve area of the aortic root (Figure <a href=\"#F1\" data-ref-type=\"fig\">1b<\/a>), a region in which lesions develop first \\[15\\]. The total cross-sectional lesion area under basal conditions was 1,900 \u00b1 900 \u03bcm^2^ (Con group; Figure <a href=\"#F1\" data-ref-type=\"fig\">1c<\/a>). Compared to the Con group, the lesion area was relatively moderately increased in the LC group (4.2-fold; *P* \\< 0.05) and strongly increased in HC (19.5-fold; *P* \\< 0.05), confirming the exponential rather than linear relationship between total plasma cholesterol levels and the lesion area.\n\nNext, lesions in the aortic root were graded according to the classification of the American Heart Association. Under control conditions (Con); only about 10% of the aortic segments contained lesions, all of which were very mild type I lesions not identified by *en face* staining (Figure <a href=\"#F1\" data-ref-type=\"fig\">1d<\/a>). In the LC group, more (40%) aortic segments showed lesions, of which 38% were mild type I-III lesions and 2% were severe type IV lesions. In the HC group, 81% of the aortic segments displayed lesions, most of which were mild lesions (76% type I-III lesions; 5% type IV). The predominance of mild-type lesions confirms an early stage of atherosclerotic disease in all groups. Notably, a positive association was observed between the cross-sectional lesion area and the plasma levels of SAA, an inflammation marker formed in liver. SAA was significantly elevated in the HC group, pointing to a hepatic inflammatory response to cholesterol feeding (Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>) that is associated with early atherosclerotic lesion formation.\n\n## Analysis of the hepatic gene response to increasing doses of dietary cholesterol\n\nTo get insight into the complex traits underlying the (patho)physiological response of the liver to dietary cholesterol, whole-genome and metabolome measurements were made. Compared to the Con group, a relatively small number of genes (551) significantly changed with LC treatment (Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>). HC treatment modulated most (440 out of 551) of these genes and, additionally, affected 1,896 other genes. The individual gene expression profiles within a treatment group were very similar and formed clusters as confirmed by hierarchical clustering analysis (not shown). Differences in gene expression between the treatment groups were validated and confirmed for a selected group of genes by RT-PCR (Additional data file 2).\n\nStandard Gene Ontology (GO) biological process annotation allowed categorization of 52% of the differentially expressed genes based on their biological function (Table <a href=\"#T2\" data-ref-type=\"table\">2<\/a>). LC treatment predominantly affected genes belonging to lipid and lipoprotein metabolism, protein metabolism, carbohydrate metabolism, energy metabolism and transport. HC treatment affected the same GO groups but, additionally, also genes relevant to immune and inflammatory responses, cell proliferation, apoptosis, cell adhesion and cytoskeleton integrity (Table <a href=\"#T2\" data-ref-type=\"table\">2<\/a> and Additional data file 3).\n\nOverview of genes that are differentially expressed in response to cholesterol\n\n|  | LC |  |  | HC |  |  |\n|----|----|----|----|----|----|----|\n|  |  |  |  |  |  |  |\n| GO category | Up | Down | Total | Up | Down | Total |\n| Lipid and lipoprotein metabolism (includes cholesterol and steroid metabolism) | 8 | 50 | 58 | 37 | 114 | 151 |\n| Protein metabolism (includes protein folding and breakdown) | 34 | 14 | 48 | 143 | 98 | 241 |\n| Other metabolism (includes carbohydrate metabolism) | 32 | 19 | 51 | 122 | 130 | 252 |\n| Generation of precursor metabolites and energy | 10 | 15 | 25 | 24 | 47 | 71 |\n| Transport | 31 | 15 | 46 | 125 | 77 | 202 |\n| Immune and stress response\/inflammation | 19 | 7 | 26 | 99 | 49 | 148 |\n| Cell proliferation\/apoptosis | 9 | 3 | 12 | 37 | 18 | 55 |\n| Cell adhesion\/cytoskeleton | 10 | 1 | 11 | 76 | 8 | 84 |\n\nDifferentially expressed genes of LC and HC groups (ANOVA FDR \\< 0.05 and *t*-test compared to Con group *P* \\< 0.01) were analyzed according to standard GO biological process annotation and grouped into functional categories.\n\nTo refine the liver transcriptome data analysis and to define which biological processes are switched on\/off with increasing dietary cholesterol loads, we performed gene enrichment analysis in four different functional ontologies: biological processes, canonical pathway maps, cellular processes and disease categories using MetaCore\u2122. This allowed us to analyze functionally related genes (for example, genes belonging to a specific biochemical process) as a whole. Table <a href=\"#T3\" data-ref-type=\"table\">3<\/a> summarizes the significantly changed biological processes for the LC and HC groups. Four key ('master') process categories were affected by cholesterol feeding: lipid metabolism; carbohydrate and amino acid metabolism; transport; immune and inflammatory responses.\n\nAnalysis of processes that are changed significantly upon treatment with dietary cholesterol\n\n|  |  |  | Differentially expressed (%) |  |\n|----|----|----|----|----|\n|  |  |  |  |  |\n| Master process | Subprocess (child terms) | Number of genes measured | LC | HC |\n| Lipid metabolism |  | 264 | 8.7\\* | 24.2\\* |\n|  | Fatty acid metabolism, fatty acid beta-oxidation | 8 | 0.0 | 50.0\\* |\n|  | Triacylglycerol metabolism | 7 | 0.0 | 57.1\\* |\n|  | Cholesterol metabolism | 27 | 33.3\\* | 33.3\\* |\n|  | Cholesterol biosynthesis | 7 | 71.4\\* | 57.1\\* |\n|  | Lipoprotein metabolism | 18 | 16.7\\* | 44.4\\* |\n|  | Lipid biosynthesis | 105 | 11.4\\* | 23.8\\* |\n|  |  |  |  |  |\n| Immune response |  | 297 | 3.0 | 12.1\\* |\n|  | Antigen presentation, exogenous antigen | 10 | 10.0 | 70.0\\* |\n|  | Antigen processing | 17 | 5.9 | 35.3\\* |\n|  | Acute-phase response | 11 | 9.1 | 36.4\\* |\n|  |  |  |  |  |\n| General metabolism |  | 3,600 | 3.3 | 13.1\\* |\n|  | Cellular polysaccharide metabolism | 19 | 5.3 | 26.3\\* |\n|  | Polysaccharide biosynthesis | 9 | 0.0 | 33.3\\* |\n|  | Cofactor metabolism | 116 | 5.2 | 21.6\\* |\n|  | Regulation of translational initiation | 9 | 0.0 | 44.4\\* |\n|  | Amino acid metabolism | 103 | 2.9 | 20.4\\* |\n|  |  |  |  |  |\n| Transport |  | 1,119 | 2.9 | 14.3\\* |\n|  | Intracellular protein transport | 161 | 3.7 | 19.9\\* |\n|  | Golgi vesicle transport | 16 | 6.3 | 37.5\\* |\n|  | Mitochondrial transport | 11 | 18.2\\* | 54.5\\* |\n\nMaster processes and their subprocesses (child terms) are listed together with the number of genes measured (third column). Percentages reflect the fraction of genes differentially expressed (within a specific process or pathway) in the LC and HC groups compared to the Con group. Relevant biological processes were identified in GenMAPP by comparison of the set of differentially expressed genes (ANOVA; *P* \\< 0.01 and FDR \\< 0.05) with all genes present on the array. \\*Biological processes with a Z-score \\>2 and a PermuteP \\< 0.05.\n\nIn the LC group, most significant effects occurred within the master process of lipid metabolism. Important subprocesses (that is, processes in which more than 10% of process-related genes changed significantly) were lipid biosynthesis, lipoprotein metabolism, cholesterol metabolism and cholesterol biosynthesis (Table <a href=\"#T3\" data-ref-type=\"table\">3<\/a>). The overall functional effect of LC treatment can be summarized as a substantial down-regulation of cholesterol and lipid metabolism. This adaptive response of the liver indicates metabolic liver resilience up to doses of 0.25% (w\/w) cholesterol.\n\nA further increase of dietary cholesterol (1% w\/w; HC group) intensified the changes in gene expression seen with LC treatment, indicating further metabolic adaptation. For example, all individual genes of the cholesterol biosynthesis pathway were down-regulated to a greater extent by HC than by LC treatment (see pathway map in Additional data file 4a): the gene of the rate-limiting enzyme of this pathway, *HMG CoA reductase* (*HDMH*), was down-regulated 2.8-fold and 10.6-fold by LC and HC treatments, respectively. Similarly, genes relevant for lipid and lipoprotein metabolism, *LDL receptor* (LC group, 1.3-fold down-regulated; HC group, 1.9-fold down-regulated) and *lipoprotein lipase* (LC group, 1.8-fold up-regulated; HC group, 5.5-fold up-regulated), were dose-dependently modulated.\n\nBesides marked effects on 'lipid metabolism', HC treatment induced significant changes in the master processes: 'general metabolism', 'transport' and 'immune and inflammatory response' (Table <a href=\"#T3\" data-ref-type=\"table\">3<\/a>). In particular, HC treatment enhanced the subprocesses involved in translational initiation, Golgi vesicle transport, mitochondrial transport, antigen presentation, antigen processing and acute phase response by affecting the expression of more than 35% of the genes in these subprocesses.\n\nSignificantly, HC but not LC dietary stress activated specific inflammatory pathways (that is, the platelet-derived growth factor (PDGF), interferon-\u03b3 (IFN\u03b3), interleukin-1 (IL-1) and tumor necrosis factor-\u03b1 (TNF\u03b1) signaling pathways; Figure <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>). Activation of these inflammatory pathways with HC treatment leads to a significant up-regulation of MAP kinases, complement factors and acute phase proteins such as SAA. HC treatment significantly up-regulates, for example, all four *SAA* isotype genes (Figure <a href=\"#F4\" data-ref-type=\"fig\">4a<\/a>), which is consistent with the observed changes in SAA protein concentrations in plasma (compare Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>).\n\nMore generally, HC treatment induced many genes, the gene products of which reportedly or putatively initiate or mediate inflammatory events (Additional data file 3), including genes encoding proteases, complement components, chemokines and their receptors, heat shock proteins, adhesion molecules and integrins, acute phase proteins, and inflammatory transcription factors, altogether indicating a profound reprogramming of the liver towards an inflammatory state not observed with LC treatment.\n\nIn separate experiments using female E3L mice, the hepatic inflammatory response to cholesterol-feeding was analyzed in more detail, including dose-dependency and time course. Plasma SAA served as a marker and readout of liver inflammation. Feeding of cholesterol at doses up to 0.50% w\/w did not alter plasma SAA levels (Figure <a href=\"#F4\" data-ref-type=\"fig\">4b<\/a>). At higher cholesterol doses (\u22650.75% w\/w), plasma SAA levels increased markedly. A time course study with 1% w\/w cholesterol (HC diet) showed that plasma SAA levels started to increase after 2 weeks and that plasma SAA levels continued to increase over time (Figure <a href=\"#F4\" data-ref-type=\"fig\">4c<\/a>). Together, these refined analyses indicate that the liver is resilient up to a cholesterol dose of about 0.50% w\/w (adaptive response) and that the inflammatory response evoked with higher cholesterol concentrations starts within two weeks of starting the HC diet.\n\nEnrichment analysis with disease categories confirmed the activation of many signaling and effector pathways relevant for inflammation and immunity by HC, but not by LC, treatment. The most affected (that is, activated at the gene expression level) disease categories with HC treatment were interrelated cardiovascular disorders and (auto) immune diseases, including cerebral and intracranial arterial diseases, cerebral amyloid angiopathy, hepatocellular carcinoma, and hepatitis (Additional data file 4b).\n\nAltogether, this global analysis shows that the liver responds to a low load of dietary cholesterol mainly by adapting its metabolic program, whereas at a high cholesterol load the liver is much more extensively reprogrammed, and, in addition to metabolic adaptations, expresses genes involved in inflammatory stress.\n\n## Analysis of diet-dependent metabolic changes in liver and plasma\n\nTo verify whether the switch from metabolic adaptation (with LC treatment) to hepatic inflammatory stress (with HC treatment) is also reflected at the metabolite level, we performed a comprehensive HPLC\/MS-based lipidome analysis (measurement in total of about 300 identified di- and triglycerides, phosphatidylcholines, lysophosphatidylcholines, cholesterol esters) on liver homogenates of Con, LC and HC groups, and corresponding plasma samples.\n\nThe individual metabolite fingerprints within a treatment group were similar and formed clusters as assessed by principal component analysis (PCA; Figure <a href=\"#F5\" data-ref-type=\"fig\">5<\/a>). The clusters of the Con and LC groups overlapped partly, demonstrating that the Con and LC groups have a similar intrahepatic lipid pattern. This indicates that the metabolic adjustments on the gene level in the LC group were efficacious and enabled the liver to cope with moderate dietary stress. The HC cluster did not overlap with the clusters of the Con group, demonstrating that the switch to a proinflammatory liver gene expression profile is accompanied by development of a new metabolic hepatic state, which differs significantly from the metabolic state at baseline (Con group).\n\n## Identification of transcriptional regulators that control the hepatic response to cholesterol\n\nTo identify the transcription factors and underlying regulatory mechanisms that govern the hepatic response to LC and HC stress, we performed a combined analysis of the liver transcriptome and metabolome dataset. Functional networks allowed the identification of transcriptional key ('master') regulators relevant for liver resilience and liver inflammation.\n\nThe adaptation of hepatic lipid metabolism to LC stress was mainly controlled by retinoid X receptor (RXR), SP-1, peroxisome proliferator activated receptor-\u03b1 (PPAR\u03b1), sterol regulatory element binding protein (SREBP)1 and SREBP2 (networks not shown), which are established positive regulators of genes involved in cholesterol biosynthesis \\[16\\]. Combined analysis of genome and metabolite datasets revealed that the intrahepatic level of eicosapentaenoic acid, a suppressor of SREBP1 \\[17\\], was increased, providing a molecular explanation for the observed down-regulation of genes involved in cholesterol biosynthesis (Additional data file 5).\n\nA subsequent network analysis of HC-modulated genes allowed the identification of transcription factors that mediate the evolution of hepatic inflammation and are ultimately responsible for the effects on the process level. HC-evoked changes require specific transcriptional master regulators, some of which are established in this context (nuclear factor kappa B (NF-\u03baB), activator protein-1 (AP-1), CAAT\/enhancer-binding protein (C\/EBP)\u03b2, p53), and others that are new (CREB-binding protein (CBP), hepatocyte nuclear factor-4\u03b1(HNF4\u03b1), SP-1, signal transducer and activator of transcription-3\/-5 (STAT-3\/-5), Yin Yang-1 (YY1); Figure <a href=\"#F6\" data-ref-type=\"fig\">6<\/a> and Additional data file 6).\n\nConsistent with this, the identified transcription factors control the expression of genes encoding acute phase response proteins, complement factors, growth factors, proteases, chemokine receptors and factors stimulating cell adhesion, as confirmed by data mining. Most importantly, HC treatment induced genes whose products can act extracellularly (Additional data file 7) and possess reportedly pro-atherogenic properties. Examples include complement components (C1qb, C1qR, C3aR1, C9), chemoattractant factors (ccl6, ccl12, ccl19), chemoattractant receptors (CCR2, CCR5), cytokines inducing impaired endothelial barrier function (IFN-\u03b3), adhesion regulators (integrin \u03b22, integrin \u03b25, CD164 antigen\/sialomucin, junction adhesion molecule-2), growth factors (PDGF, vascular endothelial growth factor (VEGF)-C, transforming growth factor (TGF)-\u03b2), proteases involved in matrix remodeling during atherogenesis (cathepsins B, L, S and Z; matrix metalloprotease-12), and cardiovascular risk factors\/inflammation markers (haptoglobin, orosomucoid 2, fibrinogen-like protein 2, \u03b11-microglobulin). This upregulation of pro-atherogenic candidate genes in the HC group is consistent with the observed enhanced early atherosclerosis found in this group.\n\nExpansion of the lipid and inflammatory networks revealed that hepatic lipid metabolism is linked to the hepatic inflammatory response via specific transcriptional regulators that control both processes. Among these dual regulators were CBP, C\/EBPs, PPAR\u03b1 and SP-1 (Table <a href=\"#T4\" data-ref-type=\"table\">4<\/a>). The presence of molecular links between lipid metabolism and inflammation raises the possibility that specific intervention with an anti-inflammatory compound may, in turn, affect plasma cholesterol levels. In a first attempt to test this possibility, female E3L mice were fed a HC diet to increase plasma cholesterol levels (from 5.3 mM to 19.3 mM) and systemic inflammation (SAA from 1.7 \u03bcg\/ml to 9.2 \u03bcg\/ml). Then, animals were treated with the same HC diet supplemented with salicylate, an inhibitor of NF-\u03baB signaling, or vehicle. While plasma cholesterol and SAA levels remained elevated in the vehicle-treated group, the salicylate-treated group displayed significantly lower plasma SAA levels (7.7 \u03bcg\/ml; *P* \\< 0.05) and significantly reduced cholesterol levels (9.9 mM; *P* \\< 0.05) demonstrating that specific intervention into the inflammatory component does indeed affect plasma cholesterol.\n\nIdentified master regulators that control inflammatory reprogramming of the liver\n\n| Transcription factor | Regulator of\/node point for | Example of downstream effects |\n|----|----|----|\n| AP-1 (c-jun\/c-fos) | Inflammation | Mmp-12, col1a1, hsp27 |\n| CREP binding protein (CBP) | Lipid, inflammation, immune response, cell proliferation | Very broadly acting coactivator (can bind to SREBPs) |\n| C\/EBPs | Lipid, inflammation, energy metabolism | Acute phase genes (for SAA, CRP, fibrinogen), hepatic gluconeogenesis and lipid homeostasis, energy metabolism (PEPCK, FAS), TGF-\u03b2 signaling |\n| Forkhead transcription factor FOXO1 | Lipid, inflammation\/proliferation | Glycolysis, pentose phosphate shunt, and lipogenic and sterol synthetic pathways, LPL (via SHP) |\n| NF-\u03baB | Inflammation | SAA, CD83, CD86, CCR5, VEGF-C |\n| PPAR\u03b1\/RXR\u03b1 | Lipid, inflammation | LPL, ABCA1, macrophage activation, glucose homeostasis |\n| p53 | Inflammation | HSP27, HSPA4, IFI16, IBP3, RBBP4 |\n| SMAD3 | Inflammation | Proteases and growth factors (via TGF-\u03b2 signaling) |\n| SP-1 | Lipid, inflammation | ABCA1, ICAM-1, cellular matrix genes COL1A1, COL1A2 |\n| SREBP-1\/-2 | Lipid, inflammation | Sterol biosynthesis genes, LDLR, link to C\/EBP\u03b1 |\n| STAT1\/3\/5 | Inflammation | Acute phase genes |\n| YY1 | Inflammation\/proliferation | Inflammatory response genes (SAA, vWF, CCR5), cellular matrix genes |\n\nBiological networks were generated using MetaCore\u2122 software and transcriptional master regulators were identified in significant gene networks (*P* \\< 0.05).\n\n# Discussion\n\nDevelopment of atherosclerotic lesions requires a lipid component (hypercholesterolemia) and an inflammatory component. In the present study, we demonstrate that high dose dietary cholesterol (HC diet) strongly induces early atherosclerotic lesion formation in a humanized model for atherosclerosis, E3L mice. This is not, or is only slightly, the case with a cholesterol-free (Con) or low dose cholesterol (LC) diet. Importantly, Con, LC, and HC diets dose-dependently increase plasma cholesterol levels, but only HC treatment induces a marked systemic inflammatory response, which precedes lesion formation and is related to liver inflammation. We employed newly developed (functional) systems biology technologies to unravel how increasing the dose of dietary cholesterol affects liver homeostasis and evokes hepatic inflammation. The following important findings were made. The liver absorbs escalating doses of dietary cholesterol primarily by adjusting the expression level of genes involved in lipid metabolism, as revealed by advanced gene expression analysis. This metabolic resilience is confirmed by analysis of metabolites in liver. At high doses of dietary cholesterol, the liver also develops an inflammatory stress response, which is characterized by up-regulation of pro-atherogenic candidate genes and activation of (at least four distinct) inflammatory pathways. The evolution of hepatic inflammation involves specific transcriptional regulators, several of which have been newly identified in this study. Interestingly, some of these transcription factors have a dual role and control both hepatic lipid metabolism and hepatic inflammation, indicating that the same regulatory mechanisms underlie these processes and thereby link the two processes.\n\nThe present study delineates, to our knowledge for the first time, the genome-wide response of the liver to increasing doses of dietary cholesterol, with specific attention to inflammatory processes, and in relation to early atherosclerotic lesion formation. The liver responds to moderate elevations in dietary cholesterol (LC diet) by adjusting major metabolic processes related to lipid metabolism. For example, the expression of genes involved in endogenous cholesterol synthesis (for example, *HMG-CoA reductase*) and cholesterol uptake from plasma (for example, *LDLR*) is diminished. At high loads of dietary cholesterol (HC diet), the liver strives for homeostasis by intensifying the changes in gene expression observed with the LC diet. Similar dose-dependent effects of dietary cholesterol have been reported by others \\[18\\] but the number of studies that assess dose-dependent effects of dietary cholesterol is relatively small and analyses are restricted to a limited number of genes. Our genome-wide approach is comprehensive and demonstrates that metabolic processes as a whole are adjusted at the level of gene expression.\n\nImportantly, the adjustment at the gene level is efficacious only up to a certain degree of cholesterol stress: while low loads of cholesterol are fully absorbed (consider the comparable intrahepatic lipidome fingerprints for the LC and Con groups), exposure to high loads of dietary cholesterol in the HC group significantly altered the liver lipidome, despite further intensified adjustment of gene expression. Our combined analysis of genes and functional readouts (lipid metabolites) clearly demonstrates that a dose of 1% w\/w of cholesterol, which is typically used to induce experimental dyslipidemia and atherosclerosis in mice \\[13,19,20\\], is an extreme condition because the metabolic resilience of the liver is already overstretched.\n\nConcomitant with the adjustment of metabolic genes to HC dietary stress, HC treatment also evokes a hepatic inflammatory response. The development of an inflammatory gene expression profile upon feeding of a diet containing cholesterol has also been reported by others. For example, Tous *et al*. \\[21\\] showed that atherosclerosis-prone apoE-\/- mice receiving a high fat\/high cholesterol diet develop an impairment of liver histology consisting of fat accumulation, macrophage proliferation, and inflammation, and that there is a chronological and quantitative relationship between liver impairment and the formation of atheromatous lesions. Vergnes *et al*. \\[22\\] showed that cholesterol and cholate components of the atherogenic diet have distinct pro-atherogenic effects on gene expression and particularly that cholesterol is required for induction of genes involved in acute inflammation in C57BL\/6J mice. Recinos *et al*. \\[23\\] reported that liver gene expression in LDLR-\/- mice is associated with diet and lesion development and demonstrated the induction of components of the alternative component pathway. Zabalawi *et al*. \\[24\\] showed the induction of fatal inflammation in LDLR-\/- and ApoAI-\/-LDLR-\/- mice fed dietary fat and cholesterol. However, the exact molecular inflammatory pathways switched on\/off by dietary cholesterol have remained unknown. While some of the above studies employing microarray analysis have examined some of the individual components of the inflammatory response to cholesterol, we have set out to generate a holistic profile of the complex, interrelated nature of the response of the liver to cholesterol. Advanced pathway analysis combined with functional network building enabled us to unravel four key inflammatory pathways (IFN\u03b3, TNF\u03b1, IL-1, and PDGF pathways) that play central roles in the evolution of cholesterol-induced inflammation in the liver. Further research is necessary to resolve the sequence of events over time (for example, which pathway is switched on first). Remarkably, these pathways are also critical for lesion development in the vessel wall, suggesting that the inflammatory response to cholesterol stress described herein for the liver may involve similar routes in other tissues and, as such, has more general significance.\n\nOur results suggest that hepatic inflammatory response may be causatively related to lesion initiation in the aorta, because pro-atherogenic candidate genes (that is, genes encoding candidate inflammatory components reportedly or putatively involved in early lesion formation) were found to be upregulated specifically in the HC group but not, or only slightly, in the LC group. The presence of a 'hepatic source' for inflammatory factors in HC stress may also explain the observed exponential (rather than linear) increase in lesion formation seen with increasing dietary cholesterol loads \\[11,12\\]. Consistent with this notion is the view that the inflammatory arm of atherogenesis is a principle driving force of lesion development.\n\nAn inflammatory reprogramming of the liver has also been observed in C57BL\/6J mice treated with a 1.25% w\/w cholesterol diet resulting in total plasma cholesterol concentrations of 3.6 mM \\[22\\], that is, a level comparable to the Con group in our study. Unlike that in E3L mice, the total plasma cholesterol in C57BL\/6J mice is mainly confined to HDL, an anti-atherogenic, anti-inflammatory lipoprotein facilitating transport of cholesterol from the periphery back to the liver. The fact that mice with a strongly different lipoprotein profile (E3L, LDLR-\/- and C57BL\/6) show a similar hepatic inflammatory response to cholesterol feeding indicates that the observed inflammatory effect of dietary cholesterol is a general phenomenon and not restricted to the model of dysbetalipoproteinemia used herein. Also, it suggests that the influx of dietary cholesterol into the liver (via chylomicrons) rather than plasma cholesterol is key to the inflammatory response of the liver. This supposition would also be in accord with the rapidity of the effect: in a time-resolved analysis of plasma SAA during atherogenesis, we report here a strong elevation of plasma SAA within two weeks of cholesterol-feeding in female E3L mice. This is also in line with the inflammatory reprogramming of C57BL6\/J mice within three weeks \\[22\\] and clearly demonstrates that the hepatic inflammatory response precedes the formation of atherosclerotic lesions, suggesting that dietary cholesterol can be an important trigger and a possible source of the inflammatory component of atherosclerotic disease. In the present study, the liver function markers ALAT and ASAT remained within the normal levels stipulated for the function criteria for donor livers \\[25\\], indicating normal liver function under the experimental conditions applied in this study. Our results do not exclude the possibility, however, that sterols may oxidize and become toxic and that the oxidized sterols contribute, at least partly, to the inflammatory effects observed by us and others.\n\nInflammation may also arise from established risk factors other than high plasma cholesterol (for example, hypertension, diabetes\/hyperglycemia). Dietary glucose can modulate the mRNA expression and serum concentrations of immune parameters but these alterations rapidly normalize in normoglycemic subjects \\[26\\]. In the case of an impaired metabolic state, however, postprandial hyperglycemia increases the magnitude and duration of systemic inflammatory responses, which probably promotes the development of cardiovascular disease.\n\nOur results show that the evolution of hepatic inflammation is controlled by specific transcriptional regulators, some of which are well known in the context of cholesterol-inducible inflammation (SREBPs, NF-\u03baB, AP-1, C\/EBPs), while others have been newly identified in the present study (CBP, HNF4\u03b1, SP-1, STAT-3\/-5, YY1). Interestingly, some of these factors may also represent molecular links between lipid\/cholesterol metabolism and inflammation. Supportive evidence for an interrelationship between liver metabolism and inflammation also comes from pharmacological intervention studies. On the one hand, cholesterol-lowering drugs reduce the general inflammatory status and the expression of liver-derived inflammation markers in E3L mice (compare to the pleiotropic effects of statins) \\[7,14,27\\] The anti-inflammatory IKK\u03b2-inhibiting compound salicylate \\[28,29\\] reduces plasma cholesterol in the same mouse model (this paper) indicating that modulation of cholesterol levels via inflammation may be possible as well. A hypocholesterolemic effect of salicylate has also been reported in catfish \\[30\\] and salicylate was found to inhibit hepatic lipogenesis in isolated rat hepatocytes *in vitro* \\[31\\]. Prigge and Gebhard \\[32\\] showed that acetylsalicylate (aspirin), a classical inhibitor of COX1 and COX2 \\[29\\], induces biliary cholesterol secretion in the rat, an effect that may contribute to the cholesterol-lowering effect seen with compounds of the salicylate category: in diabetic human subjects, very high doses of aspirin (around 7 g\/d) were associated with a 15% reduction of total plasma cholesterol and CRP \\[29\\].\n\n# Conclusion\n\nWe demonstrate that dietary cholesterol is not only a lipid risk factor but also a trigger of hepatic inflammation and, as such, also involved in the evolution of the inflammatory arm of atherosclerotic disease. A certain degree of genetic resilience and elasticity allows the liver to cope with moderate cholesterol stress, but high loads of cholesterol result in an inflammatory pro-atherogenic response (involving specific pathways and transcriptional regulators), which enhances early lesion formation. Our findings that cholesterol and inflammation are closely linked via specific transcriptional master regulators might lead to new strategies for future therapeutic intervention.\n\n# Materials and methods\n\n## Animals and diets\n\nFemale E3L mice were used at the age of 12 weeks for all experiments. Animal experiments were approved by the Institutional Animal Care and Use Committee of The Netherlands Organization for Applied Scientific Research (TNO) and were in compliance with European Community specifications regarding the use of laboratory animals.\n\nA group of E3L mice (*n* = 17) was treated with a cholesterol-free diet (diet T; Hope Farms, Woerden, The Netherlands) for 10 weeks (Con group). The major ingredients of diet T (all w\/w) were cacao butter (15%), corn oil (1%), sucrose (40.5%), casein (20%), corn starch (10%) and cellulose (6%). Two other groups (*n* = 17 each) received the same diet but supplemented with either 0.25% w\/w cholesterol (LC group) or 1.0% w\/w cholesterol (HC group). After ten weeks of diet feeding, animals were euthanized under anesthesia to collect livers, hearts and aortas. Tissues were snap-frozen in liquid nitrogen and stored at -80\u00b0C until use.\n\nTo assess the effect of salicylate on plasma levels of inflammation markers and cholesterol, two groups of female E3L mice (*n* = 10; 12 weeks old) were treated with the HC diet for 3 weeks. Then, HC dietary treatment was either continued (vehicle control group) or animals were fed HC supplemented with 0.12% w\/w salicylate (equaling a dose of 145 mg\/kg\/day) for 8 weeks. Plasma samples were obtained by tail bleeding without fixation of the test animals to minimize stress.\n\n## Analyses of plasma lipids and proteins\n\nTotal plasma cholesterol and triglyceride levels were measured after 4 h of fasting, using kits No.1489437 (Roche Diagnostics, Almere, The Netherlands) and No.337-B (Sigma, Aldrich Chemie BV, Zwijndrecht, The Netherlands) \\[33\\]. For lipoprotein profiles, pooled plasma was fractionated using an \u1ea2KTA FPLC system (Pharmacia, Roosendaal, The Netherlands) \\[9\\]. The plasma levels of SAA were determined by ELISA as reported \\[14\\]. Plasma ALAT and ASAT levels were determined spectrophotometrically using a Reflotron system (Roche Diagnostics) \\[9\\].\n\nFor lipiodomics analysis of liver homogenates and plasma samples, electrospray liquid chromatography mass spectroscopy (LC-MS) analysis was applied \\[34\\]. Briefly, samples (5 \u03bcl) were incubated with 200 \u03bcl isopropanol and a mixture of internal standards (heptadecanoyl-lysophosphatidylcholine, di-lauroyl-phosphatidylcholine, heptadecanoyl-cholesterol and tri-heptadecanoyl-glycerol; Sigma, St Louis, MO, USA)). After vortexing, the lipids were extracted and isolated by centrifugation (lipids in isopropanol phase). Electrospray LC-MS lipid analysis was performed on a Thermo LTQ apparatus equipped with a Surveyor HPLC system (Thermo Electron, San Jose, CA, USA). The samples were measured in fully randomized sequences. Quality control samples, prepared from a single pool of E3L mouse reference tissue, were analyzed at regular intervals (bracketing 10 samples). The LC-MS raw data files were processed using a software developed by TNO (IMPRESS) to generate comprehensive peak tables (m\/z value, retention time and peak area). Data were then subjected to retention time alignment of peaks, internal standard correction of peak areas and quality control resulting in a final lipidomics dataset.\n\nThe obtained lipidomics dataset was analyzed and visualized by PCA essentially as described \\[35\\]. Prior to analysis, the data were mean-centered and auto-scaled to ensure an equal contribution of all lipid measurements to the PCA-model.\n\n## Analyses of atherosclerosis\n\nHearts were fixed and embedded in paraffin to prepare serial cross sections (5 \u03bcm thick) throughout the entire aortic valve area for (immuno) histological analysis. Cross sections were stained with hematoxylin-phloxine-saffron, and atherosclerosis was analyzed blindly in four cross-sections of each specimen (at intervals of 30 \u03bcm) as reported \\[14,36\\]. QWin-software (Leica) was used for morphometric computer-assisted analysis of lesion number, lesion area, and lesion severity as described in detail elsewhere \\[7\\]. Significance of difference was calculated by one-way analysis of variance (ANOVA) test followed by a least significant difference *post hoc* analysis using SPSS 11.5 for Windows (SPSS, Chicago, IL, USA). The level of statistical significance was set at \u03b1 \\< 0.05.\n\n## Nucleic acid extraction and gene expression analysis\n\nTotal RNA was extracted from individual livers (*n* = 5 per group) using RNAzol (Campro Scientific, Veenendaal, The Netherlands) and glass beads according to the manufacturer's instructions. The integrity of each RNA sample obtained was examined by Agilent Lab-on-a-chip technology using the RNA 6000 Nano LabChip kit and a bioanalyzer 2100 (both Agilent Technologies, Amstelveen, The Netherlands). The quality control procedure is described in Additional data file 8. The One-Cycle Target Labeling and Control Reagent kit (Affymetrix \\#900493) and the protocols optimized by Affymetrix were used to prepare biotinylated cRNA (from 5 \u03bcg of total RNA) for microarray hybridization (*n* = 5 per group). The quality of intermediate products (that is, biotin-labeled cRNA and fragmented cRNA) was again controlled using the RNA 6000 Nano Lab-on-a-chip and bioanalyzer 2100. Microarray analysis was carried out using an Affymetrix technology platform and Affymetrix GeneChip^\u00ae^ mouse genome 430 2.0 arrays (45,037 probe sets; 34,000 well-characterized mouse genes). Briefly, fragmented cRNA was mixed with spiked controls, applied to Affymetrix Test chips, and good quality samples were then used to hybridize with murine GeneChip^\u00ae^ 430 2.0 arrays. The hybridization, probe array washing and staining, and washing procedures were executed as described in the Affymetrix protocols, and probe arrays were scanned with a Hewlett-Packard Gene Array Scanner (Leiden Genome Technology Center, Leiden, The Netherlands).\n\n## Gene expression data analysis\n\nRaw signal intensities were normalized using the GCRMA algorithm (Affylm package in R). Datasets are freely accessible online through ArrayExpress \\[37\\]. Normalized signal intensities below 10 were replaced by 10. Probe sets with an absent call in all arrays were removed before further analysis of the data. RT-PCR was performed essentially as described \\[27,38\\] to validate and confirm differences in gene expression between the treatment groups.\n\nStatistical analysis was performed in BRB ArrayTools (Dr Richard Simon and Amy Peng Lam \\[39\\]). Con, LC and HC groups were tested for differentially expressed genes using class comparisons with multiple testing corrections by estimation of false discovery rate (FDR). Differentially expressed genes were identified at a threshold for significance of \u03b1 \\< 0.01 and a FDR \\< 5%. Within the set of differentially expressed genes, a Student's *t*-test was carried out to analyze differential expression of individual genes between the cholesterol-fed groups and the Con group. Differences of *P* \\< 0.01 versus Con were considered significant.\n\nFor biological interpretation of the differentially expressed genes, software tools GenMAPP and MetaCore\u2122 (GeneGo Inc., St Joseph, MI, USA) were used. Enrichment of biological processes (GO annotation) was analyzed in GenMAPP, biological processes in GenMAPP with a Z-score \\>2 and PermuteP \\< 0.05 were considered as significantly changed. In MetaCore\u2122, enrichment analysis \\[40\\] of four independent ontologies was performed. In addition to biological process gene ontology, data were also analyzed in canonical pathway maps, GeneGo-cellular processes and disease categories.\n\nDistribution by canonical pathway maps reveals the most significant signaling and\/or metabolic pathways. Experimental data are visualized as red\/blue thermometers pointing up\/down, and signifying up\/down-regulation of the map objects. Distribution by GeneGo processes provides the most significant functional process categories enriched with experimental data. GeneGo processes represent comprehensive pre-built process-specific networks, in which all objects are interconnected by experimentally validated interactions. The up- and down-regulated genes are visualized as red or blue circles, respectively. The disease categories represent sets of genes associated with certain diseases. Gene enrichment analysis shows the relative enrichment of the up- and down-regulated genes with the genes from different disease categories. As in the case of process enrichment, this procedure is carried out by p value distribution.\n\nThe biological networks were assembled from manually curated protein-protein, protein-DNA and protein-ligand (metabolite) interactions, which are accumulated in the MetaCore\u2122 database. Each edge or link on the network is based on small-experiment data referenced in the corresponding literature. The legend for MetaCore\u2122 Networks from the MetaCore\u2122 guideline is provided in Additional data file 6d. For the generation of functional networks, transcriptome and metabolome datasets were merged, allowing combined analysis. Networks were generated using the Shortest path (SP) algorithm, which links the nodes from experimental datasets by the shortest directed graphs, allowing up to two additional steps using interactions and nodes from the MetaCore\u2122 database. To present most of the relevant network data on the same figure, we used the add\/expand function and the Merge Networks feature. The resulting networks provide links based on the known interaction data not only between the nodes from the query data set(s), but also between the nodes that regulate the given genes or metabolites. Network nodes with available experimental data are distinguished with red or blue circles, representing up- or down-regulation, respectively.\n\n# Abbreviations\n\nALAT, alanine aminotransferase; AP, activator protein; ASAT, aspartate aminotransferase; CBP, CREB-binding protein; C\/EBP, CAAT\/enhancer-binding protein; Con, cholesterol-free; CRP, C-reactive protein; E3L, ApoE\\*3Leiden transgenic; FDR, false discovery rate; GO, Gene Ontology; HC, high-cholesterol; HDL, high-density lipoprotein; HNF, hepatocyte nuclear factor; IFN, interferon; IL, interleukin; LC, low-cholesterol; LC-MS, liquid chromatography mass spectroscopy; LDL, low-density lipoprotein; NF-\u03baB, nuclear factor kappa B; PCA, principal component analysis; PDGF, platelet-derived growth factor; PPAR, peroxisome proliferator activated receptor; RXR, retinoid X receptor; SAA, serum amyloid A; SREBP, sterol regulatory element binding protein; STAT, signal transducer and activator of transcription; TGF, transforming growth factor; TNF, tumor necrosis factor; VLDL, very low-density lipoprotein; YY, Yin Yang.\n\n# Authors' contributions\n\nRK provided the conceptual background to the analysis, interpreted the results and wrote the manuscript. LV did the *in vivo* atherosclerosis studies, performed the assays and interpreted the data. MvE performed the computational analysis including biological processes and assisted with manuscript writing. YN coordinated the software development and the multidimensional analysis of biological processes using (pathway) networks. NC supervised the work and coordinated the lipidomics and genomics analyses. EV developed the lipidomics methodology and performed the lipidomics measurements. AS coordinated the multivariate statistical analysis and drafted the manuscript. HH helped with data interpretation and evaluated the manuscript. SZ performed animal experiments and quantified plasma inflammation markers. GS participated in designing the experiment and manuscript writing. VK developed the tools for multidimensional data analyses and performed computations. TN assisted with the preparation of the figures of networks and pathways. AM assisted in data interpretation and bioinformatical techniques for gene ontology analyses. EH participated in designing the study and manuscript preparation. JG coordinated the development of the metabolomics technologies and critically evaluated the manuscript. BO led the bioinformatical analyses, developed the concepts for integrated data analysis and drafted the manuscript. TK initiated the study, interpreted the data and helped with manuscript writing. All authors read and approved the final manuscript.\n\n# Additional data files\n\nThe following additional data are available with the online version of this paper. Additional data file <a href=\"#S1\" data-ref-type=\"supplementary-material\">1<\/a> shows the exponential positive correlation between atherosclerotic lesion area and total plasma cholesterol in female E3L mice. Additional data file <a href=\"#S2\" data-ref-type=\"supplementary-material\">2<\/a> shows the validation and confirmation of Affymetrix microarray gene expression data by RT-PCR analysis. Additional data file <a href=\"#S3\" data-ref-type=\"supplementary-material\">3<\/a> is a table of the genes (including GenBank identification number and the gene symbol) that are differentially expressed with increasing doses of dietary cholesterol. Additional data file <a href=\"#S4\" data-ref-type=\"supplementary-material\">4<\/a> shows the canonical pathway analysis for cholesterol metabolism and analysis of the gene expression data based on GO annotation with disease categories (MetaCore\u2122 software, GeneGO). Additional data file <a href=\"#S5\" data-ref-type=\"supplementary-material\">5<\/a> shows the comprehensive network analysis (functional OMICs analysis) by merging gene expression datasets with the metabolite datasets using MetaCore\u2122 network software. Additional data file <a href=\"#S6\" data-ref-type=\"supplementary-material\">6<\/a> shows the biological networks of differentially expressed genes in the HC group allowing the identification of transcriptional master regulators. Additional data file <a href=\"#S7\" data-ref-type=\"supplementary-material\">7<\/a> is a table lisitng cholesterol-induced factors with reported extracellular function. Additional data file <a href=\"#S8\" data-ref-type=\"supplementary-material\">8<\/a> describes the quality control analysis steps for RNA samples prior to hybridization on Affymetrix microarrays usig Agilent Lab-on-a-chip technology.\n\n# Supplementary Material\n\n###### Additional data file 1\n\nExponential positive correlation between atherosclerotic lesion area and total plasma cholesterol in female E3L mice.\n\nClick here for file\n\n###### Additional data file 2\n\nValidation and confirmation of Affymetrix microarray gene expression data by RT-PCR analysis.\n\nClick here for file\n\n###### Additional data file 3\n\nGenes (including GenBank identification number and the gene symbol) that are differentially expressed with increasing doses of dietary cholesterol.\n\nClick here for file\n\n###### Additional data file 4\n\nCanonical pathway analysis for cholesterol metabolism and analysis of the gene expression data based on GO annotation with disease categories (MetaCore\u2122 software, GeneGO).\n\nClick here for file\n\n###### Additional data file 5\n\nComprehensive network analysis (functional OMICs analysis) by merging gene expression datasets with the metabolite datasets using MetaCore\u2122 network software.\n\nClick here for file\n\n###### Additional data file 6\n\nBiological networks of differentially expressed genes in the HC group allowing the identification of transcriptional master regulators.\n\nClick here for file\n\n###### Additional data file 7\n\nCholesterol-induced factors with reported extracellular function.\n\nClick here for file\n\n###### Additional data file 8\n\nQuality control analysis steps for RNA samples prior to hybridization on Affymetrix microarrays usig Agilent Lab-on-a-chip technology.\n\nClick here for file\n\n### Acknowledgements\n\nThis study was supported by the Dutch Organization for Scientific Research (NWO; grant VENI 016. 036.061 to RK), the Dutch Heart Foundation (NHS; grant 2002B102 to LV), and the TNO research program NISB (to RK, ME, NC, EV, AS, JG and TK). We are grateful to Maren White for helpful discussions and critical reading of the manuscript. We thank AstraZeneca for supporting this study. We thank Ally Perlina, Wilbert Heijne, Robert-Jan Lamers, Annie Jie and Karin Toet for excellent bioinformatical and analytical help. The authors gratefully acknowledge grant support from The European Nutrigenomics Organisation (NuGO, CT-2004-505944; Focus Team 'Metabolic Stress and Disease to RK, TK and SZ).","meta":{"dup_signals":{"dup_doc_count":141,"dup_dump_count":32,"dup_details":{"curated_sources":4,"2022-21":1,"2020-45":1,"2020-24":1,"2019-47":1,"2019-39":1,"2017-09":18,"2016-44":1,"2016-40":1,"2016-36":25,"2016-30":18,"2015-48":3,"2015-40":2,"2015-35":3,"2015-32":3,"2015-27":2,"2015-22":3,"2015-14":2,"2014-52":3,"2014-49":5,"2014-42":8,"2014-41":5,"2014-35":3,"2014-23":4,"2014-15":5,"2023-23":1,"2015-18":3,"2015-11":3,"2015-06":3,"2014-10":2,"2013-48":3,"2013-20":2,"2024-22":1}},"file":"PMC2375038"},"subset":"pubmed_central"}
{"text":"author: Etzel Carde\u00f1a\\*Correspondence: <firstname.lastname@example.com>[^1][^2][^3]\ndate: 2014-01-27\ninstitute: Department of Psychology, Lund UniversityLund, Sweden\nreferences:\ntitle: A call for an open, informed study of all aspects of consciousness\n\nScience thrives when there is an open, informed discussion of all evidence, and recognition that scientific knowledge is provisional and subject to revision. This attitude is in stark contrast with reaching conclusions based solely on a previous set of beliefs or on the assertions of authority figures. Indeed, the search for knowledge wherever it may lead inspired a group of notable scientists and philosophers to found in 1882 the Society for Psychical Research in London. Its purpose was \"to investigate that large body of debatable phenomena\u2026 without prejudice or prepossession of any kind, and in the same spirit of exact and unimpassioned inquiry which has enabled Science to solve so many problems.\" Some of the areas in consciousness they investigated such as psychological dissociation, hypnosis, and preconscious cognition are now well integrated into mainstream science. That has not been the case with research on phenomena such as purported telepathy or precognition, which some scientists (a clear minority according to the surveys conducted <http:\/\/en.wikademia.org\/Surveys_of_academic_opinion_regarding_parapsychology>) dis-miss *a priori* as pseudoscience or illegitimate. Contrary to the negative impression given by some critics, we would like to stress the following:\n\n1.  Research on parapsychological phenomena (psi) is being carried out in various accredited universities and research centers throughout the world by academics in different disciplines trained in the scientific method (e.g., circa 80 Ph.D.s have been awarded in psi-related topics in the UK in recent years). This research has continued for over a century despite the taboo against investigating the topic, almost complete lack of funding, and professional and personal attacks (Carde\u00f1a, 201). The Parapsychological Association has been an affiliate of the AAAS since 1969, and more than 20 Nobel prizewinners and many other eminent scientists have supported the study of psi or even conducted research themselves (Carde\u00f1a, 2013).\n\n2.  Despite a negative attitude by some editors and reviewers, results supporting the validity of psi phenomena continue to be published in peer-reviewed, academic journals in relevant fields, from psychology to neuroscience to physics e.g., (Storm et al., 2010; Bem, 2011; Hameroff, 2012; Radin et al., 2012).\n\n3.  Increased experimental controls have not eliminated or even decreased significant support for the existence of psi phenomena, as suggested by various recent meta-analyses (Sherwood and Roe, 2003; Schmidt et al., 2004; B\u00f6sch et al., 2006; Radin et al., 2006; Storm et al., 2010, 2012, 2013; Tressoldi, 2011; Mossbridge et al., 2012; Schmidt, 2012).\n\n4.  These meta-analyses and other studies (Blackmore, 1980)suggest that data supportive of psi phenomena cannot reasonably be accounted for by chance or by a \"file drawer\" effect. Indeed, contrary to most disciplines, parapsychology journals have for decades encouraged publication of null results and of papers critical of a psi explanation (Wiseman et al., 1996; Sch\u00f6nwetter et al., 2011). A psi trial registry has been established to improve research practice <http:\/\/www.koestler-parapsychology.psy.ed.ac.uk\/TrialRegistryDetails.html>.\n\n5.  The effect sizes reported in most meta-analyses are relatively small and the phenomena cannot be produced on demand, but this also characterizes various phenomena found in other disciplines that focus on complex human behavior and performance such as psychology and medicine (Utts, 1991; Richard and Bond, 2003).\n\n6.  Although more conclusive explanations for psi phenomena await further theoretical and research developments, they do not *prima facie* violate known laws of nature given modern theories in physics that transcend classical restrictions of time and space, combined with growing evidence for quantum effects in biological systems (Sheehan, 2011; Lambert et al., 2013).\n\nWith respect to the proposal that \"exceptional claims require exceptional evidence,\" the original intention of the phrase is typically misunderstood (Truzzi, 1978). Even in its inaccurate interpretation what counts as an \"exceptional claim\" is far from clear. For instance, many phenomena now accepted in science such as the existence of meteorites, the germ theory of disease, or, more recently, adult neurogenesis, were originally considered so exceptional that evidence for their existence was ignored or dismissed by contemporaneous scientists. It is also far from clear what would count as \"exceptional evidence\" or who would set that threshold. Dismissing empirical observations *a priori*, based solely on biases or theoretical assumptions, underlies a distrust of the ability of the scientific process to discuss and evaluate evidence on its own merits. The undersigned differ in the extent to which we are convinced that the case for psi phenomena has already been made, but not in our view of science as a non-dogmatic, open, critical but respectful process that requires thorough consideration of all evidence as well as skepticism toward both the assumptions we already hold and those that challenge them.\n\nDaryl Bem, Professor Emeritus of Psychology, Cornell University, USA\n\nEtzel Carde\u00f1a, Thorsen Professor of Psychology, Lund University, Sweden\n\nBernard Carr, Professor in Mathematics and Astronomy, University of London, UK\n\nC. Robert Cloninger, Renard Professor of Psychiatry, Genetics, and Psychology, Washington University in St. Louis, USA\n\nRobert G. Jahn, Past Dean of Engineering, Princeton University, USA\n\nBrian Josephson, Emeritus Professor of Physics, University of Cambridge, UK (Nobel prizewinner in physics, 1973)\n\nMenas C. Kafatos, Fletcher Jones Endowed Professor of Computational Physics, Chapman University, USA\n\nIrving Kirsch, Professor of Psychology, University of Plymouth, Lecturer in Medicine, Harvard Medical School, USA, UK\n\nMark Leary, Professor of Psychology and Neuroscience, Duke University, USA\n\nDean Radin, Chief Scientist, Institute of Noetic Sciences, Adjunct Faculty in Psychology, Sonoma State University, USA\n\nRobert Rosenthal, Distinguished Professor, University of California, Riverside, Edgar Pierce Professor Emeritus, Harvard University, USA\n\nLothar Sch\u00e4fer, Distinguished Professor Emeritus of Physical Chemistry, University of Arkansas, USA\n\nRaymond Tallis, Emeritus Professor of Geriatric Medicine, University of Manchester, UK\n\nCharles T. Tart, Professor in Psychology Emeritus, University of California, Davis, USA\n\nSimon Thorpe, Director of Research CNRS (Brain and Cognition), University of Toulouse, France\n\nPatrizio Tressoldi, Researcher in Psychology, Universit\u00e0 degli Studi di Padova, Italy\n\nJessica Utts, Professor and Chair of Statistics, University of California, Irvine, USA\n\nMax Velmans, Professor Emeritus in Psychology, Goldsmiths, University of London, UK\n\nCaroline Watt, Senior Lecturer in Psychology, Edinburgh University, UK\n\nPhil Zimbardo, Professor in Psychology Emeritus, Stanford University, USA\n\nAnd\u2026\n\nP. Baseilhac, Researcher in Theoretical Physics, University of Tours, France\n\nEberhard Bauer, Dept. Head, Institute of Border Areas of Psychology and Mental Hygiene, Freiburg, Germany\n\nJulie Beischel, Adjunct Faculty in Psychology and Integrated Inquity, Saybrook University, USA\n\nHans Bengtsson, Professor of Psychology, Lund University, Sweden\n\nMichael Bloch, Associate Professor of Psychology, University of San Francisco, USA\n\nStephen Braude, Professor of Philosophy Emeritus, University of Maryland Baltimore County, USA\n\nRichard Broughton, Senior Lecturer, School of Social Sciences, University of Northampton, UK\n\nAntonio Capafons, Professor of Psychology, University of Valencia, Spain\n\nJames C. Carpenter, Adjunct Professor of Psychiatry, University of North Carolina, Chapel Hill, USA\n\nAllan Leslie Combs, Doshi Professor of Consciousness Studies, California Institute of Integral Studies, USA\n\nDeborah Delanoy, Emeritus Professor of Psychology, University of Northampton, UK\n\nArnaud Delorme, Professor of Neuroscience, Paul Sabatier University, France\n\nVilfredo De Pascalis, Professor of General Psychology, \"La Sapienza\" University of Rome, Italy\n\nKurt Dressler, Professor in Molecular Spectroscopy Emeritus, Eidg. Techn. Hochschule Z\u00fcrich, Switzerland\n\nHoyt Edge, Hugh H. and Jeannette G. McKean Professor of Philosophy, Rollins College, USA\n\nSuitbert Ertel, Emeritus Professor of Psychology, University of G\u00f6ttingen, Germany\n\nFranco Fabbro, Professor in Child Neuropsychiatry, University of Udine, Italy\n\nEnrico Facco, Professor of Anesthesia and Intensive Care, University of Padua, Italy\n\nWolfgang Fach, Researcher, Institute of Border Areas of Psychology and Mental Hygiene, Freiburg, Germany\n\nHarris L. Friedman, Former Research Professor of Psychology, University of Florida, USA\n\nAlan Gauld, Former Reader in Psychology, University of Nottingham, UK\n\nAntoon Geels, Professor in the Psychology of Religion Emeritus, Lund University, Sweden\n\nBruce Greyson, Carlson Professor of Psychiatry and Neurobehavioral Sciences, University of Virginia, Charlottesville, USA\n\nErlendur Haraldsson, Professor Emeritus of Psychology, University of Iceland, Iceland\n\nRichard Conn Henry, Academy Professor (Physics and Astronomy), The Johns Hopkins University, USA\n\nDavid J. Hufford, University Professor Emeritus, Penn State College of Medicine, USA\n\nOscar Iborra, Researcher, Department of Experimental Psychology, Granada University, Spain\n\nHarvey Irwin, former Associate Professor, University of New England, Australia\n\nGraham Jamieson, Lecturer in Human Neuropsychology, University of New England, Australia\n\nErick Janssen, Adjunct Professor, Department of Psychology, Indiana University, USA\n\nPer Johnsson, Head, Department of Psychology, Lund University, Sweden\n\nEdward F. Kelly, Research Professor in the Department of Psychiatry and Neurobehavioral Sciences, University of Virginia, Charlottesville, USA\n\nEmily Williams Kelly, Research Assistant Professor in the Department of Psychiatry and Neurobehavioral Sciences, University of Virginia, Charlottesville, USA\n\nHideyuki Kokubo, Researcher, Institute for Informatics of Consciousness, Meiji University, Japan\n\nJeffrey J. Kripal, J. Newton Rayzor Professor of Religious Studies, Rice University, USA\n\nStanley Krippner, Professor of Psychology and Integrated Inquiry, Saybrook University, USA\n\nDavid Luke, Senior Lecturer, Department of Psychology and Counselling, University of Greenwich, UK\n\nFatima Regina Machado, Researcher, Universidade de S\u00e3o Paulo, Brasil\n\nMarkus Maier, Professor in Psychology, University of Munich, Germany\n\nGerhard Mayer, Researcher, Institute of Border Areas of Psychology and Mental Hygiene, Freiburg, Germany\n\nAntonia Mills, Professor First Nations Studies, University of Northern British Columbia, Canada\n\nGarret Moddel, Professor in Electrical, Computer, & Energy Engineering, University of Colorado, Boulder, USA\n\nAlexander Moreira-Almeida, Professor of Psychiatry, Universidade Federal de Juiz de Fora, Brasil\n\nAndrew Moskowitz, Professor in Psychology and Behavioral Sciences, Aarhus University, Denmark\n\nJulia Mossbridge, Fellow in Psychology, Northwestern University, USA\n\nJudi Neal, Professor Emeritus of Management, University of New Haven, USA\n\nRoger Nelson, Retired Research Staff, Princeton University, USA\n\nFotini Pallikari, Professor of Physics, University of Athens, Greece\n\nAlejandro Parra, Researcher in Psychology, Universidad Abierta Interamericana, Argentina\n\nJos\u00e9 Miguel P\u00e9rez Navarro, Lecturer in Education, International University of La Rioja, Spain\n\nGerald H. Pollack, Professor in Bioengineering. University of Washington, Seattle, USA\n\nJohn Poynton, Professor Emeritus in Biology, University of KwaZulu-Natal, South Africa\n\nDavid Presti, Senior Lecturer, Neurobiology and Cognitive Science, University of California, Berkeley, USA\n\nThomas Rabeyron, Lecturer in Clinical Psychology, Nantes University, France\n\nInmaculada Ramos Lerate, Researcher in Physics, Alba Synchrotron Light Source, Barcelona, Spain.\n\nChris Roe, Professor of Psychology, University of Northampton, UK\n\nStefan Schmidt, Professor, Europa Universit\u00e4t Viadrina, Germany\n\nGary E. Schwartz, Professor of Psychology, Medicine, Neurology, Psychiatry, and Surgery, University of Arizona, USA\n\nDaniel P. Sheehan, Professor of Physics, University of San Diego, USA\n\nSimon Sherwood, Senior Lecturer in Psychology, University of Greenwich, UK\n\nChristine Simmonds-Moore, Assistant Professor of Psychology, University of West Georgia, USA\n\nM\u00e1rio Sim\u00f5es, Professor in Psychiatry. University of Lisbon, Portugal\n\nHuston Smith, Prof. of Philosophy Emeritus, Syracuse University, USA\n\nJerry Solfvin, Associate Professor in Indic Studies, University of Massachusetts, Dartmouth, USA\n\nLance Storm, Visiting Research Fellow, University of Adelaide, Australia\n\nJeffrey Allan Sugar, Assistant Professor of Clinical Psychiatry, University of Southern California, Los Angeles, USA\n\nNeil Theise, Professor of Pathology and Medicine, The Icahn School of Medicine at Mount Sinai, USA\n\nJim Tucker, Bonner-Lowry Associate Professor of Psychiatry and Neurobehavioral Sciences, University of Virginia, USA\n\nYulia Ustinova, Associate Professor in History, Ben-Gurion University of the Negev, Israel\n\nWalter von Lucadou, Senior Lecturer at the Furtwangen Technical University, Germany\n\nMaurits van den Noort, Senior Researcher, Free University of Brussels, Belgium\n\nDavid Vernon, Senior Lecturer in Psychology, Canterbury Christ Church University, UK\n\nHarald Walach, Professor, Europa Universit\u00e4t Viadrina, Germany\n\nHelmut Wautischer, Senior Lecturer in Philosophy, Sonoma State University, USA\n\nDonald West, Emeritus Professor of Clinical Criminology, University of Cambridge, UK\n\nN.C. Wickramasinghe, Professor in Astrobiology, Cardiff University, UK\n\nFred Alan Wolf, formerly Professor in physics at San Diego State University, the Universities of Paris, London, and the Hebrew University of Jerusalem\n\nRobin Wooffitt, Professor of Sociology, University of York, UK\n\nWellington Zangari, Professor in Psychology, University of Sao Paulo, Brazil\n\nAldo Zucco, Professor, Dipartimento di Psicologia Generale, Universit\u00e0 di Padova, Italy\n\nAlthough for practical reasons only one author is listed, the call is a collective creation of the co-signatories.\n\n# References\n\n[^1]: This article was submitted to the journal Frontiers in Human Neuroscience.\n\n[^2]: Edited by: Christian Agrillo, University of Padova, Italy\n\n[^3]: Reviewed by: Imants Baruss, King's University College at The University of Western Ontario, Canada","meta":{"dup_signals":{"dup_doc_count":126,"dup_dump_count":67,"dup_details":{"curated_sources":2,"2023-50":2,"2023-40":2,"2023-23":1,"2023-06":3,"2022-49":2,"2022-40":3,"2022-33":1,"2022-27":1,"2022-21":1,"2022-05":2,"2021-49":1,"2021-43":2,"2021-39":1,"2021-31":1,"2021-21":1,"2021-17":2,"2021-10":1,"2021-04":1,"2020-45":1,"2020-40":2,"2020-29":1,"2020-24":1,"2020-10":2,"2020-05":1,"2019-47":2,"2019-43":2,"2019-39":1,"2019-35":2,"2019-30":2,"2019-26":2,"2019-22":1,"2019-18":2,"2019-09":2,"2018-51":3,"2018-47":1,"2018-43":2,"2018-39":1,"2018-34":2,"2018-30":1,"2018-26":4,"2018-22":1,"2018-13":5,"2018-05":3,"2017-51":2,"2017-47":1,"2017-43":5,"2017-39":2,"2017-34":2,"2017-30":2,"2017-26":2,"2017-22":4,"2017-17":2,"2017-09":3,"2017-04":1,"2016-50":1,"2016-44":1,"2016-40":1,"2016-36":2,"2016-30":2,"2016-22":1,"2014-15":3,"2024-26":2,"2024-22":2,"2024-18":1,"2024-10":1,"2017-13":5,"2014-10":1}},"file":"PMC3902298"},"subset":"pubmed_central"}
{"text":"abstract: This is a report on a 37-patient continuation study of the open ended, Omega-3 Fatty Acid (O-3FA) add-on study. Subjects consisted of the original 19 patients, along with 18 new patients recruited and followed in the same fashion as the first nineteen. Subjects carried a DSM-IV-TR diagnosis of Bipolar Disorder and were visiting a Mood Disorder Clinic regularly through the length of the study. At each visit, patients' clinical status was monitored using the Clinical Monitoring Form. Subjects reported on the frequency and severity of irritability experienced during the preceding ten days; frequency was measured by way of percentage of days in which subjects experienced irritability, while severity of that irritability was rated on a Likert scale of 1 \u2013 4 (if present). The irritability component of Young Mania Rating Scale (YMRS) was also recorded quarterly on 13 of the 39 patients consistently. Patients had persistent irritability despite their ongoing pharmacologic and psychotherapy.\n  .\n  Omega-3 Fatty Acid intake helped with the irritability component of patients suffering from bipolar disorder with a significant presenting sign of irritability. Low dose (1 to 2 grams per day), add-on O-3FA may also help with the irritability component of different clinical conditions, such as schizophrenia, borderline personality disorder and other psychiatric conditions with a common presenting sign of irritability.\nauthor: Kemal Sagduyu; Mehmet E Dokucu; Bruce A Eddy; Gerald Craigen; Claudia F Baldassano; Ay\u015feg\u00fcl Y\u0131ld\u0131z\ndate: 2005\ninstitute: 1University of Missouri \u2013 Kansas City, Missouri, 8801 West 148^th^ Terrace, Overland Park, KS 66221, USA; 2Washington University, School of Medicine, Department of Psychiatry, Campus Box: 8134, 660 South Euclid Avenue, St. Louis, Missouri, 63110, USA; 3Department of Psychiatry, School of Medicine, University of Missouri-Kansas City, Resource Development Institute, 601 Walnut Street, Kansas City, MO 64106, USA; 4Mood Disorders Psychopharmacology Unit, University Health Network, Toronto Western Hospital, 399 Bathurst Street, ECW-3D-010, Toronto, Ontario M5T 2S8, Canada; 5Mood and Anxiety Disorders Clinic, Department of Psychiatry, University of Pennsylvania, 3535 Market Street, 2nd floor, Philadelphia, PA 19104, USA; 6Dokuz Eyl\u00fcl Medical School, Department of Psychiatry, \u0130zmir, Turkey\nreferences:\ntitle: Omega-3 fatty acids decreased irritability of patients with bipolar disorder in an add-on, open label study\n\n# Introduction\n\nAccording to the United States National Institute of Mental Health (NIMH), Bipolar Disorder (BPD), also known as manic-depressive illness, is a serious medical illness that causes shifts in a person's mood, energy, and ability to function. Different from the normal ups and downs that everyone goes through, the symptoms of bipolar disorder are severe. Bipolar disorder is a complex, chronic condition associated with considerable morbidity and mortality, including a high rate of suicide. Bipolar disorder causes dramatic mood swings from overly \"high\" and\/or irritable to sad and hopeless, and then back again, often with periods of normal mood in between. Severe changes in energy and behavior go along with these changes in mood. The periods of highs and lows are called episodes of mania and depression. Most people with bipolar disorder can achieve substantial stabilization of their mood swings and related symptoms over time with proper treatment. A strategy that combines medication and psychosocial treatment is optimal for managing the disorder over time.\n\n# Background\n\nOmega-3 fatty acids (0-3FA) may have a beneficial effect on irritable mood. Low O-3FA levels in red blood cell membranes of depressed patients hint that O-3FA may be helpful in treating mood disorders \\[1\\]. A recent article has given an excellent review of O-3FA and studies showing their effectiveness in depression, bipolar disorder and aggression \\[2\\]. In this article, two published studies are discussed that have reported on similar therapeutic effects of O-3FA \\[2\\]. One placebo-controlled study of 20 patients revealed that ethyl ester of eicosapentaenoic acid (E-EPA) was effective in stabilizing the moods of depressed patients \\[3\\]. Another report, a double-blind, placebo controlled study (N = 22\/19), measured the effect of O-3FA docosahexaenoic acid (DHA) on the aggressive tendencies of college students. The O-3FA DHA group (1.5\u20131.8 g O-FA DHA\/day) did not display any increase in aggressive tendencies when external stressors peaked, while the placebo group displayed a significant increase in their aggressive tendencies under similar circumstances \\[4\\]. In a recent study, 25% of 111 patients with bipolar-I disorder who met criteria for a DSM-IV major depressive episode also experienced substantial irritability in the absence of associated symptoms of mania. These findings suggest that abnormal irritability is not limited to mania or mixed states \\[5\\]. However, recent studies give caution that at a 6 gram per day average daily dose, as a single agent, omega 3 fatty acids may not be as effective as an antidepressant \\[6-9\\].\n\nO-3FA may also help with the irritability component of different clinical conditions, such as depression, mania, schizophrenia, borderline personality disorder and other psychiatric conditions with a common presenting sign of irritability. Numerous other conditions have an irritability component, including Borderline Personality Disorder, Alzheimer's disease, Premenstrual Dysphoric Disorder, to name a few \\[10-12\\]. There is one report suggesting beneficial effect of Omega-3 Fatty acid treatment for Borderline Personality Disorder. This double-blind, placebo-controlled pilot study specifically showed that EPA may influence both aggression and depression \\[12\\]. Although attention-deficit\/hyperactivity disorder (ADHD) also has an irritability component, recent publications bring doubt to the O3FA connection in ADHD \\[13,14\\].\n\nA recent, open ended, O-3FA add-on study has shown beneficial effect of O-3FA on irritability in 19 patients with mood disorders \\[15\\]. These patients had already been receiving different combinations of pharmacotherapy and talk therapy. Despite their treatment, the irritability component of their illness was still causing social, occupational and other life disturbances. Hence, they were chosen for the O-3FA add-on component of the study. In the nineteen-patient phase of the study, bipolar patients of every subtype, ages 18 to 65 years, with significant irritability were studied. All patients received a systematic assessment battery at entry and were treated by a psychiatrist, trained to deliver care and measure outcomes in patients with bipolar disorder, consistent with expert recommendations. At every follow-up visit, the treating psychiatrist completed a standardized assessment and assigns a clinical status based on DSM-IV criteria. Patients had independent evaluations at regular intervals throughout the study and remain under the care of the same treating psychiatrist while receiving variable medications and talk therapy, depending on their need \\[15\\]. In the 19-patient study, a paired sample t-test revealed a large decrease in the percent of days irritable after O-3FA was administered. Before treatment, the mean irritability percentage was 81.05 (SD = 23.31) and after treatment the mean irritability percentage dropped to 30.00 (SD = 36.67). Despite the small number of patients in the study (n = 19), the difference between means was statistically significant (t (18) 4.512, p \\< .001). Using a paired sample t-test, a significant difference was also found between the highest irritability score (mean = 2.79; SD = 0.92) and the last recorded irritability (mean = 0.79; SD = 0.85) while taking O-3FA (t(18) = 8.270; p \\< .001) \\[15\\].\n\n# Methods\n\nThis is a report on a 37-patient continuation phase of the open ended, O-3FA add-on study. Subjects consisted of the original 19 patients, in addition to the 18 new patients recruited and followed in the same fashion as the first nineteen \\[15\\]. Subjects carried a DSM-IV-TR \\[16\\] diagnosis of Bipolar Disorder and were visiting a Mood Disorder Clinic regularly throughout the length of the study. At each visit, patients' clinical status was monitored using the Clinical Monitoring Form \\[17\\]. Subjects reported on the frequency and severity of irritability experienced during the preceding ten days; frequency was measured by way of percentage of days in which subjects experienced irritability, while severity of that irritability was rated on a Likert scale of 1 \u2013 4 (if present). The irritability component of Young Mania Rating Scale \\[18\\] (YMRS) was also recorded quarterly on 13 of the 39 patients consistently. The patients were asked about general dietary omega-3 intake before the fish oil was added on, and basic nutritional guidance was given to subjects at the clinic. Patients in general were not heavy fish\/product consumers.\n\n## Dosage\n\nStarting dose and last maintenance dose were available for 37 subjects (Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>). Subjects self-medicated, and therefore, the last maintenance dose of O-3FA was chosen by each subject. The mean starting dose was 1824.32 mg (SD 1075.07), and the mean for the last maintenance dose was considerably higher at 2878.38 mg (SD 2011.79). The increase was statistically significant using a paired sample t-test (t = -3.44, 36df, p = .001).\n\n# Statistical Results\n\n## Percentage of Irritable (Days)\n\nThe initial mean was 63.51 (SD 34.17), indicating that on average, subjects were irritable for about six of the previous ten days. The mean for the last recorded percentage was less than half of the initial score: 30.27 (SD 34.03). The decrease was found to be statistically significant using a paired sample t-test (t = 4.36, 36 df, p \\< .001). The difference between the distributions was examined using the non-parametric sign test. The number of negative differences (25) significantly exceeded positive differences (7); there were five ties, and the pre\/post distributions were significantly different (p \\< .003).\n\n## YMRS Irritability Sub-score\n\nThirty four subjects had initial and last recorded YMRS irritability sub-scores. As with the above means there was a sizable decrease. The initial mean score was 3.18 (SD 1.09). The mean for the last recorded percentage was 1.68 (SD 1.89). The decrease was found to be statistically significant using a paired sample t-test (t = 4.21, 33 df, p \\< .001).\n\n## YMRS Total Score\n\nStarting and last recorded YMRS scores were available for 34 subjects. The mean starting score 10.71 (SD 6.77), and the mean for the last recorded score was 4.85 (SD 5.63). The decrease found to be statistically significant using a paired sample t-test (t = 4.14, 33 df, p \\< .001).\n\n## Severity\n\nThirty six subjects had initial and last recorded severity scores on the ADE. Again, a decrease was found. The initial mean score was 2.14 (SD 1.22). The mean for the last recorded score was 0.94 (SD 0.92). This decrease was found to be statistically significant using a paired sample t-test (t = 5.23, 35 df, p \\< .001).\n\n## Composite: Severity and Irritability\n\nAs an exploratory measure, a composite score was created by multiplying the ADE severity score, which has a maximum of 4 points, by the percentage of the ten days prior to measurement which the patient was rated as irritable. The initial mean on this composite was 159.72. As with other measures, there was wide variation: SD = 122.92. The mean for this measure on the last recorded scores was percentage was about one-fourth of the initial score: 43.89 (SD 64.38). The decrease was found to be statistically significant using a paired sample t-test (t = 5.00, 35 df, p \\< .001).\n\n## Last Recorded Maintenance Dose and Percentage of Irritability After\n\nBecause of apparent wide variation on these two measures and a concern that outliers may have affected some results, the last recorded irritability scores were plotted against the maintenance dose. This revealed a rather bimodal pattern, in which relatively lower irritability measures (\u2264 50%) clustered in the quadrant with lower dosage levels (\u2264 4,000 mg).\n\n## Duration and YMRS Total\n\nIn response to a similar observation regarding wide variation in the last recorded values (84 days to 5.5 years) the values were also plotted. A clearly bimodal pattern appeared in which 11 subjects (about one-third of study participants) clustered in the quadrant representing short duration (\\<500 days) and higher YMRS totals (\\>7). The remaining two-thirds of subjects clustered in the quadrant representing short duration and lower YMRS totals (\\<6).\n\n## Subject Weight\n\nThe mean start weight was 176.97 lbs (SD 43.13), and the mean for the last weight recorded was slightly higher at 178.59 lbs (SD 43.24). The increase was not statistically significant.\n\n# Follow-up Subjects\n\nFollow-up information, recorded after the collection of the \"last\" scores for most of the above variables, was available for 13 of the 37 subjects. Final YMRS total or scale scores were not available for this sub-group.\n\n## Omega 3 Duration\n\nThe final date recorded for the duration of O3 was derived based from an O3 start date and a \"final\" date recorded for O3. The time period ranged 84 days to 1995 days (5.46 years). The mean duration of O3 for this group was 439.62 days (SD = 487.46).\n\n## Dosage\n\nFor these subjects, the mean starting dose was 1807.69 mg (SD 990.34), and the mean for the last maintenance dose was higher at 2615.38 mg (SD 1894.66). The increase was not significant.\n\n## Percentage Irritable (Days)\n\nThe initial mean was 82.31 (SD 20.88). The mean for the last recorded percentage was dramatically lower: 25.38 (SD 32.04). The decrease was found to be statistically significant using a paired sample t-test (t = 6.52 12 df, p \\< .001). The difference between the distributions was examined using a sign test. The number of negative differences (12) significantly exceeded positive differences (0); there was one tie, and the pre\/post distributions were significantly different (p \\< .001).\n\n## Severity\n\nThe initial mean score for the 13 subjects with final scores was 2.69 (SD 0.95). The mean for the final score was 0.77 (SD 0.83). This decrease was found to be statistically significant using a paired sample t-test (t = 6.22, 12 df, p \\< .001).\n\n## Composite: Severity and Irritability\n\nAn exploratory composite score, described above, was also created for the subjects with final scores. For these subjects, the initial mean was higher than that of the total group, 223.08. Again, there was wide variation: SD = 104.19. The mean for this measure on the last recorded scores was percentage was much lower that the initial score: 33.08 (SD 39.87). The decrease was found to be statistically significant using a paired sample t-test (t = 6.70, 12 df, p \\< .001).\n\n## Weight\n\nFor these 13 subjects, the mean start weight was 166.23 lbs (SD 35.68), and the mean for the final weight recorded was also slightly higher at 168.23 lbs (33.62). As with the previous finding regarding weight, the increase was not statistically significant.\n\n# Results\n\nOmega-3 Fatty Acids added onto the existing treatment helped with the irritability component of a significant percentage of patients suffering from bipolar disorder with a persistent sign of irritability.\n\n# Discussion\n\nAs seen from the standard deviations of several of the variables discussed here, measures ranged widely. This creates difficulty in using descriptive data, such as means, to adequately portray subject attributes and performance. Using data reduction techniques or grouping subjects according to high and low scores on various attributes may be one way to increase the descriptiveness, which would be possible and more reasonable with a larger pool of subjects.\n\nA potential limitation or interpretive consideration merits discussion. For many of the variables discussed above, noticeable differences in measures were observed between the \"starting\" versus \"last recorded\" group (n = 37) and the \"starting\" versus \"final\" measures group (n = 13). Given these differences and the smaller number of subjects in the second set of comparisons, \"starting\" versus \"final\" comparisons should be interpreted with caution until differences inherent in this \"final\" subgroup (n = 13) are more clearly understood. This is clearly seen in the results of sign tests, in which the apparent magnitude of the \"final\" effects is pronounced.\n\nStatistically significant within-subjects differences were found in several independent variables. This is especially notable given the small number of subjects. The preliminary findings suggest that a rigorously designed study tailored especially to the examination of the effects of O-3FA is warranted.\n\nThe majority of data were collected within an ongoing \"best-practice, outpatient bipolar disorder study\" that involved medications and talk therapy which we have not reported or discussed herein. Results must, therefore, be interpreted with caution.\n\nThere are several mechanisms through which O-3FA are theorized to help with mood, irritability, aggression etc. Suggested theories of mechanism converge on the theory of nerve cell membrane stabilization. A recent study has come closest to showing physical proof of effectiveness of O-3FA through indirect demonstration of greater membrane fluidity, as detected by reductions in Tesla-2 (T2) values in MRI scans \\[19\\]. The overlapping beneficial effects of antipsychotics, antidepressants, anticonvulsants, O-3FA, and nonpsychoactive cannabinoids, as they relate to pain, stroke, schizophrenia, psychoneuroimmunology, Alzheimer's disease, and stress, may be because of their common effects at protein kinases, thus affecting the structure and function of the cell membrane and the cell \\[20\\]. These changes should help the cell operate within an optimal level of excitation, which may be related to emerging evidence that these therapeutic agents have neuroprotective value \\[20\\]. A recent randomized placebo controlled double blind intervention study suggests an adaptogenic role for O-3FA in stress \\[21\\].\n\nWe would like to discuss briefly the issue of daily dosing of O-3FA for nutrition and medicinal purpose: Recent studies give caution that at a 6 gram per day average daily dose, as a single agent, omega 3 fatty acids may not be as effective as an antidepressant \\[6-9\\]. However, these studies may have given too high of a dose of O-3FA, above 6 grams daily, with possibly beyond a therapeutic window of effectiveness for O-3FA. Our scatter plots indicate that the optimum effective dose for irritability is at 1\u20132 gram of EPA plus DHA per day, which would be the dosing we suggest. A recent exploratory dose study of O-3FA for schizophrenic patients showed that 2 g\/day EPA-treated patients had lower symptom scores, and needed less medication greatest. In this study, there was a positive relationship between improvement on rating scales and rise in red blood cell arachidonic acid concentration as well \\[22\\].\n\nThe United States (US) accounts for more than 51% of the 430.3 billion dollar expended on pharmaceutical products worldwide each year \\[23\\]. World healthcare society first needs access to low-cost, nontoxic, non-expert-dependent interventions to ensure basic health outcomes. Food may represent the most cost-effective means of promoting public health \\[23\\]. The American Heart Association recommends consumption of two servings of fish per week for persons with no history of coronary heart disease and at least one serving of fish daily for those with known coronary heart disease \\[24\\]. Approximately 1 g per day of EPA acid plus DHA acid is recommended for cardioprotection \\[24\\]. Higher dosages of omega-3 fatty acids are required to reduce elevated triglyceride levels (2 to 4 g per day) and to reduce morning stiffness and the number of tender joints in patients with rheumatoid arthritis (at least 3 g per day) \\[24\\].\n\nWe conclude that it is beneficial in many ways to establish a regular intake of 1\u20132 g per day EPA acid plus DHA, similar to daily intake of vitamins with minerals. Dietary interventions to remedy omega-3 deficiency is necessary \\[23\\]. It is time for more aggressive funding for research into medicinal foods, such as omega-three fatty acids \\[23\\].\n\n## Figures and Tables\n\nInitial, Last and Final Omega 3 Dosages (mg). summarizes dosage under three conditions. Figures for the Initial Dose include two subjects (n = 39) for whom no corresponding follow-up data were available.\n\n| Initial, Last Recorded and Final Omega 3 Dosages (mg) |  |  |  |\n|----|----|----|----|\n|  | Initial | Last Recorded | Final^T1^ |\n|  |  |  |  |\n| n | 39 | 37 | 13 |\n| Mean | 1833.33 | 2878.38 | 2615.38 |\n| Mode | 1000^T2,\\ T3^ | 1000 | 2000 |\n| Median | 2000 | 2000 | 2000 |\n| SD | 1071.91 | 2011.79 | 1894.66 |\n\nT1 = Final group results (n = 13) are discussed below.\n\nT2 = Multiple modes exist. The smallest value is shown.\n\nT3 = One gram (1,000 miligram) of fish oil; of which about 180 milligrams is (eicosapentaenoic acid) EPA and 120 milligrams is DHA (docosahexaenoic acid), (for a total of 300 milligrams of omega 3's) in each clear capsule.","meta":{"dup_signals":{"dup_doc_count":160,"dup_dump_count":55,"dup_details":{"curated_sources":5,"2022-27":1,"2021-49":1,"2021-10":1,"2021-04":1,"2020-45":1,"2020-34":1,"2020-24":1,"2019-47":1,"2019-43":1,"2019-30":1,"2019-22":1,"2019-13":1,"2019-04":1,"2018-51":1,"2018-43":1,"2018-34":1,"2018-30":1,"2018-22":1,"2018-13":1,"2017-47":2,"2017-34":1,"2017-26":1,"2017-22":1,"2017-09":13,"2017-04":1,"2016-50":1,"2016-44":1,"2016-40":1,"2016-36":1,"2016-30":1,"2016-22":1,"2016-18":1,"2015-48":5,"2015-40":3,"2015-35":5,"2015-32":5,"2015-27":3,"2015-22":5,"2015-14":4,"2014-52":5,"2014-49":6,"2014-42":9,"2014-41":8,"2014-35":9,"2014-23":7,"2014-15":7,"2023-50":1,"2017-13":1,"2015-18":4,"2015-11":5,"2015-06":3,"2014-10":5,"2013-48":5,"2013-20":4,"2024-26":1}},"file":"PMC549076"},"subset":"pubmed_central"}
{"text":"abstract: Vocal learning is relatively common in birds but less so in mammals. Sexual selection and individual or group recognition have been identified as major forces in its evolution. While important in the development of vocal displays, vocal learning also allows signal copying in social interactions. Such copying can function in addressing or labelling selected conspecifics. Most examples of addressing in non-humans come from bird song, where matching occurs in an aggressive context. However, in other animals, addressing with learned signals is very much an affiliative signal. We studied the function of vocal copying in a mammal that shows vocal learning as well as complex cognitive and social behaviour, the bottlenose dolphin (*Tursiops truncatus*). Copying occurred almost exclusively between close associates such as mother\u2013calf pairs and male alliances during separation and was not followed by aggression. All copies were clearly recognizable as such because copiers consistently modified some acoustic parameters of a signal when copying it. We found no evidence for the use of copying in aggression or deception. This use of vocal copying is similar to its use in human language, where the maintenance of social bonds appears to be more important than the immediate defence of resources.\nauthor: Stephanie L. King; Laela S. Sayigh; Randall S. Wells; Wendi Fellner; Vincent M. Janike-mail: <firstname.lastname@example.com>\ndate: 2013-04-22\ninstitute: 1Sea Mammal Research Unit, School of Biology, University of St Andrews, St Andrews, Fife KY16 8LB, UK; 2Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA; 3Chicago Zoological Society, c\/o Mote Marine Laboratory, 1600 Ken Thompson Parkway, Sarasota, FL 34236, USA; 4The Seas, Epcot, Walt Disney World Resort, 2016 Avenue of the Stars, EC Trl. W-251, Lake Buena Vista, FL 32830, USA\nreferences:\ntitle: Vocal copying of individually distinctive signature whistles in bottlenose dolphins\n\n# Introduction\n\nVocal production learning enables animals to copy novel sounds in their environment or to develop their own distinctive calls, avoiding overlap with those heard before \\[1\\]. Most commonly, vocal learning leads to convergence in sound parameters between individuals. A good example of this can be found in bird song dialects \\[2\\] or in the development of group-specific contact calls \\[3\u20137\\]. The exchange of such shared calls between individuals can be aggressive or affiliative in nature. While contact calls are known to be affiliative \\[7\\], song type matching in song birds tends to have an aggressive connotation \\[8\\]. Song sparrows, for example, use song type matching when defending their territory against an unknown male, but avoid it when interacting with known neighbours with whom they use more subtle repertoire matching \\[9,10\\]. Repertoire matching, i.e. the use of a shared song type while avoiding a reply with the same song type, may allow the addressing of a neighbour in a more affiliative or neutral way.\n\nIn most instances, these interactions occur with calls that are shared by more than one individual. In the case of contact calls, the common call belongs either to a group or a pair of animals. In bird song, animals have individual repertoires where each song type is shared with other individuals, but the overall composition of the repertoire may be unique. Production rates for each shared call or song type are usually similar across the individuals that share it. Individual call or song types survive in populations as cultural traditions that can outlive the animals that produce them at any one time \\[11\\].\n\nThe signature whistle of the bottlenose dolphin stands out from these examples in that it seems to be more individually specific. Bottlenose dolphins produce a large variety of narrow-band frequency-modulated whistles and pulsed sounds for communication \\[12\\]. As part of their repertoire, each individual also develops an individually distinctive signature whistle \\[13,14\\] that develops under the influence of vocal learning \\[15\u201317\\]. Individuals listen to their acoustic environment early in life and then develop their own novel frequency modulation pattern or contour for their signature whistle \\[15\\]. The result is a novel and unique modulation pattern that identifies the individual even in the absence of general voice cues \\[18\\]. Interindividual variation in signature whistles is much larger than that found in recognition signals of other species \\[19\\].\n\nBottlenose dolphins live in fluid fission\u2013fusion societies with animals forming a variety of different social relationships \\[20\\]. This social organization, coupled with restrictions in underwater vision and olfaction, has led to natural selection favouring designed individual signature whistles \\[12,14\\] instead of relying on the by-product distinctiveness of voice features \\[19\\]. The signature whistle tends to be the most commonly used whistle in each individual's repertoire accounting for around 50 per cent of all whistles produced by animals in the wild \\[21\\]. Bottlenose dolphins are, however, able to learn new sounds throughout their lives \\[22\\], and conspecifics occasionally imitate the signature whistles of others \\[23\\]. Thus, one animal's signature whistle can form a minor part of another animal's vocal repertoire as a result of copying \\[17,23,24\\]. Signature whistle copying is, however, rare \\[23,25\u201327\\], albeit significantly more common than expected by chance \\[25\\]. As such, each signature whistle forms only a major part of one animal's repertoire, allowing it to be a label for that particular individual when copied.\n\nNevertheless, the function of copying events remains unclear. It has been argued that copying of signature whistle types is equivalent to addressing other individuals. Such addressing can be affiliative or aggressive. Unlike songbirds, delphinids are not territorial and do not sing. Instead, they use their acoustic signals in the context of social interactions and group cohesion \\[12\\]. Bottlenose dolphins have low rates of aggression towards close associates and higher ones towards social competitors, for example among male alliances \\[20\\]. Investigating who is copying who can therefore give us information on the signal value of copying. In addition to affiliative and aggressive functions, a third hypothesis for whistle copying is that it is used as a deceptive form of signalling \\[28\\]. For example, deceptive signature whistle copying by male dolphins could allow them to gain access to females guarded by other males or to avoid directed aggression from a male alliance \\[29\\]. It appears that copies are sufficiently rare to allow for such a use without jeopardizing the reliability of signature whistles as identity signals.\n\nTo investigate these three hypotheses, the occurrence of signature whistle copying was studied in captive and briefly captured and subsequently released wild bottlenose dolphins. We hypothesized that if signature whistle copying is affiliative it should only occur between close associates. Alternatively, copying in an aggressive context should be more common between animals that are less closely associated. Furthermore, copies used in a deceptive way should ideally not be recognizable as copies, whereas in affiliative or aggressive contexts, they could be recognizable as such. We also investigated the temporal aspects of whistle copying given the importance of signal type matching in other species.\n\n# Material and methods\n\n## Social and acoustic data from the wild\n\nData were collected from wild bottlenose dolphins around Sarasota Bay, FL, USA between 1984 and 2009. The amount of time animals are sighted together can be used to give a measure of their association. The half-weight ratio coefficients of association (CoA) \\[30\\] is defined as CoA = 2*N~ab~*\/*N~a~* + *N~b~*~,~ in which *N~ab~* is the number of times individuals A and B have been seen together, *N~a~* is the number of times individual A has been seen without B, and *N~b~* is the number of times individual B has been seen without A. CoAs were calculated for all study animals from data gained during regular, systematic photographic identification surveys of dolphins. CoAs given for each pair of animals caught together are from the year the recordings were taken. Wild bottlenose dolphin acoustic recordings were collected during capture\u2013release events for health assessments and life-history studies in Sarasota Bay \\[31\\]. One such event takes on average 108 min from the time the net is set to the time the individual is released. During these events, animals were physically restrained and frequently out of visual sight, but not acoustic range, of one another. The signature whistle of an individual is the most common whistle type emitted in such isolation conditions \\[14\\]. The Sarasota Dolphin Research Programme has now accumulated a catalogue of whistles from over 250 individual dolphins from the resident community in Sarasota Bay since 1975 \\[14\\], many of which were recorded in multiple capture\u2013release sessions. We compared all whistles produced by an individual with the signature whistles of all others in the same capture set in order to identify copying events. Ages of animals were known from long-term observations \\[32\\] or from analysing growth rings in teeth \\[33\\].\n\nThe vocalizations of each individual were recorded via a suction cup hydrophone, allowing the identification of the caller for each recorded call. Either custom-built or SSQ94 hydrophones were used (High Tech Inc.). Between 1984 and 2004, the acoustic recordings were taken with either Marantz PMD-430 or Sony TC-D5M stereo-cassette recorders (frequency response of recording system: 0.02\u201318 kHz \u00b1 5 dB) or Panasonic AG-6400 or AG-7400 video-cassette recorders (frequency response of recording system: 0.02\u201325 kHz \u00b1 3 dB). For recordings taken from 2005 onwards, a Sound Devices 744T digital recorder was used (sampled at 96 kHz, 24-bit, frequency response of recording system: 0.02\u201348 kHz \u00b1 1 dB).\n\nThe first step of analysis consisted of visual comparisons of spectrograms of 205 h and 23 min of acoustic recordings of temporarily caught and released, wild bottlenose dolphins by one observer in order to identify copying events within each capture set. The total recording time inspected in this way was 110 h and 55 min for pairs of animals caught together with low association levels (CoA \\< 0.5) and 94 h and 28 min for pairs of animals caught together with high association levels (CoA \\> 0.5). The second step involved a detailed analysis of 32 h and 12 min (<a href=\"#RSPB20130053TB1\" data-ref-type=\"table\">table 1<\/a>) of recordings where vocal copying had been found. These contained a total of 10 219 whistles, which is the dataset on which this in-depth analysis is based.\n\nPairs of animals involved in signature whistle copying events, with the animal producing copies in bold. The mean similarity values are given for each animal's signature whistle when compared with the vocal copy. The copier's own signature whistles had low similarity scores with the copy while the signature whistles of the copied animals had high similarity scores with the copies (see the electronic supplementary material, figure S1).\n\n| pair | sex | relationship | CoA | age | recording time (min) | no. of vocal copies | average similarity values |\n|:---|:---|:---|----|----|----|----|----|\n| **1. Calvin Ranier** | MM | associates | 1^a^ | 1528 | 7070 | 13\u2014 | 1.54.5 |\n| **2a. FB26** **2b. FB48** | MM | alliance partners | 0.8 | 3129 | 93101 | 385 | 1.0\/3.2^b^1.0\/3.5^b^ |\n| **3. FB114** FB20 | MM | associates | 0.07 | 1615 | 5195 | 4\u2014 | 2.43.3 |\n| **4. FB90**FB122 | FM | mothercalf | 0.98 | 254 | 9292 | 17\u2014 | 1.33.3 |\n| **5. FB65**FB67 | FF | calfmother | 0.67 | 621 | 7070 | 1\u2014 | 1.23.6 |\n| **6. FB228**FB65 | MF | calfmother | 0.95 | 521 | 106106 | 8\u2014 | 1.13.5 |\n| **7. FB5**FB55 | FF | mothercalf | 1.0 | 293 | 8585 | 17\u2014 | 1.33.3 |\n| **8a. FB358b. FB93** | FF | mothercalf | 0.9 | 323 | 9292 | 24 | 1.7\/3.7^b^2.5\/3.2^b^ |\n| **9. FB71**FB95 | FF | mothercalf | 1.0 | 281 | 9797 | 13\u2014 | 1.03.3 |\n| **10. FB5**FB155 | FF | mothercalf | 0.56 | 292 | 7979 | 40\u2014 | 1.03.5 |\n| **11. FB9**FB177 | FF | mothercalf | 0.9 | 20 | 105105 | 9\u2014 | 1.23.4 |\n\n^a^These animals were permanent residents in a captive facility.\n\n^b^Where both animals copied one another the average similarity value for that animal's own signature with the copy it produced of the other animal's signature whistle is given first (low number) followed by the average similarity value for that animal's own signature whistle with the copy produced by the other animal in the pair (larger number).\n\n## Social and acoustic data from captivity\n\nTo investigate the social context of copying, four captive adult males were recorded at The Seas Aquarium, Lake Buena Vista, FL, USA, during May\u2013June 2009. One male, Ranier, was estimated to be 28 years old and was collected at approximately 3 years of age in the northern Gulf of Mexico. The other males were Calvin (15 years old), Khyber (18 years old) and Malabar (8 years old), who were all captive born. All four animals had been together for 3.5 years at the start of the study; Ranier and Calvin had been together for 6 years. Vocalizations of these dolphins were recorded with two HTI-96 MIN hydrophones (frequency response: 0.002\u201330 kHz\u00b11 dB) and two CRT hydrophones (C54 series; frequency response: 0.016\u201344 kHz\u00b13 dB) onto a Toshiba Satellite Pro laptop using a four channel Avisoft v. 416 U<span class=\"smallcaps\">ltrasound<\/span>G<span class=\"smallcaps\">ate<\/span> recording device (sampled at 50 kHz, 8 bit).\n\nA total recording time of 16 h for the four males was analysed. The length of recording time when copying between pairs could be identified (as determined by their positions in the pool system) was as follows: 16 h (100%) for Ranier and Calvin, Ranier and Malabar, Khyber and Calvin and Khyber and Malabar; 14 h (87.3%) for Ranier and Khyber and 2 h (12.7%) for Calvin and Malabar. The caller was identified, using passive acoustic localization \\[23\\]. The social association of male pairs at The Seas was evaluated by measuring synchrony in their swimming patterns \\[34\\]. A focal animal instantaneous sampling method was used with an observation period of 7.5 min and a 15 s interval. At each 15 s interval, the focal animal's synchrony status was assessed relative to each other animal in the group. Observations took place 5 days per week between 08.00 and 18.00, and each animal served as the focal animal once each day in an order determined by a balanced, randomly ordered schedule. Observations were made between January 2009 and June 2009 when all four dolphins were together in the same pool.\n\n## Identifying copying events\n\nInitially, one observer (S.L.K.) compared all whistles in a given captured or captive group with each other, and identified all occurrences where the same whistle type was being produced by more than one animal by inspecting spectrograms (fast Fourier transform (FFT) length 512, overlap 100%, Blackmann\u2013Harris window) in Adobe A<span class=\"smallcaps\">udition<\/span> v. 2.0 (Adobe Systems). Five naive human observers, blind to context and animal identity, were then used to rate the similarity of each copy of a signature whistle to the original signature whistle (the whistle as produced by its owner) and to the copier's own signature whistle. Visual classification was used as it is more reliable than computer-based methods in dolphin whistle classification \\[14,35\\] and is frequently used in animal communication studies \\[2,36\\]. The five observers were given the extracted contours (frequency modulation pattern) of the whistles as plots of frequency versus time and were asked to rate whistle similarity using a five-point similarity index ranging from 1 (dissimilar) to 5 (similar). Only copied whistles that reached a mean similarity score of more than 3 with the original signature whistle and less than 3 with the copier's own signature whistle were deemed copies and included in the analysis. A value of 3 indicates a relatively high similarity as indicated in previous studies \\[25,29,37\\].\n\n## Acoustic analysis\n\nThe whistle contours of every copy as well as of randomly chosen exemplars of signature whistles of both interacting individuals were extracted using a supervised contour extraction programme \\[38\\], with a time resolution of 5 ms. From the contours, the following parameters were measured: start frequency, end frequency, minimum frequency, maximum frequency, frequency range, duration and mean frequency. One further parameter, number of loops, was read directly from the spectrogram where applicable. A loop was defined as a repeated modulation pattern within a signature whistle that could be separated by periods of stereotyped, discrete segments of silence. These periods of silence were taken to be 250 ms or less, which is the maximum inter-loop interval found in this population \\[39\\].\n\n## Statistical analysis\n\nAll statistical procedures were conducted in R (R project for statistical computing; GNU project). Acoustic parameters were analysed by first testing for normality using the Lilliefors (Kolmogorov\u2013Smirnov) test. Depending upon the outcome, either the Mann\u2013Whitney test or a Welch's *t*-test was used to compare differences between parameters of the copies with the original signature whistles and the copier's own signature whistle. A sampling statistic was then created by multiplying these test statistics together, which created a combined test statistic for all parameters. This allowed comparisons of overall difference between two whistle types. A permutation test was used to shuffle the acoustic parameter measurements of the copies with those of the original signature whistles within each pair of animals. This was carried out to test whether the combined acoustic parameter statistic was significantly different from a random distribution. Ten thousand permutations were performed to calculate the distribution of the test statistic under the null hypothesis (random distribution), and the observed test statistic was then compared with this random distribution. A two-tailed test was used with a Bonferroni-adjusted significance level of *p* \\< 0.002. In addition, all parameters were used in a non-metric multi-dimensional scaling analysis with a good STRESS fit of 0.04.\n\nA permutation test was also used to test whether signal copying only occurred between affiliated pairs of animals. This involved shuffling the CoAs of the pairs of animals who produced vocal copies (*n* = 11) with those that did not (*n* = 191). Many of the individuals who copied were also in pairs with other animals where copying was not present. The sampling statistic of interest was the mean CoA for the pairs involved in signal copying. Ten thousand permutations were performed to calculate the distribution of the test statistic under the null hypothesis that the CoAs of copiers were randomly distributed. The observed test statistic was then compared with the random distribution.\n\nPermutation tests were also performed on the timing of copies after the original signature whistle. The times of copies (*n* = 108) were shuffled with the times of the copier's own signature whistles given in response to the copied signature whistles (*n* = 1651). The random distribution was calculated from 10 000 permutations under the null hypothesis that there was no difference between the timing of copies of signature whistles after the occurrence of the template whistle and the timing of the copier's own signature whistle after the occurrence of the template whistle. The observed test statistic (mean time between original signature whistle and copy) was compared with the random distribution.\n\n# Results\n\n## Who copies whom?\n\nIn total, 85 different capture\u2013release events of wild dolphins were analysed, comprising 121 individuals in different group compositions. Of these individuals, 48 were sampled on more than one occasion (range: 2\u20137). Of the 85 capture\u2013release events analysed, 11 consisted of single male\u2013male pairs, 31 consisted of single mother\u2013calf pairs and the remaining 43 consisted of groups of different compositions. These compositions included two or more adults of the same or both sexes, mother\u2013calf pairs with other adults and groups of mother\u2013calf pairs.\n\nAs in previous studies \\[14,40\\], each bottlenose dolphin almost exclusively used its own, individually distinctive signature whistle during capture\u2013release events. Whistle rates were generally high at these events, with a mean of 5.3 whistles per minute per individual. In 10 of 85 different capture\u2013release sets, however, individuals were found occasionally copying the signature whistle of another animal in the set (mean rate in sets with copying: 0.18 copies per minute per individual). This occurred in 10 of 179 pairs of animals recorded from 1988 through 2004, consisting of two of the 11 male\u2013male pairings and eight of the 31 mother\u2013calf pairs. In some instances, both members of a pair copied one another (<a href=\"#RSPB20130053F1\" data-ref-type=\"fig\">figure 1<\/a> and <a href=\"#RSPB20130053TB1\" data-ref-type=\"table\">table 1<\/a>; electronic supplementary material, figure S1). The total number of individuals who produced vocal copies was therefore 12. The five human judges who viewed frequency contour plots to quantify similarity of the copies with both the originals and the copier's own signature whistles showed statistically significant agreement (*\u03ba* = 0.42, *z* = 29.9, *p* \\< 0.0001) \\[41\\]. Similarity values for all copies are given in <a href=\"#RSPB20130053TB1\" data-ref-type=\"table\">table 1<\/a>.\n\nThe results of a permutation test clearly showed that signature whistle copying occurred between closely affiliated pairs of animals (*p* = 0.0006). The mean half-weight coefficient of association (CoA; which can range from 0 to 1) for the 10 pairs of animals that copied was 0.8, whereas the mean CoA for non-copiers was 0.4 (<a href=\"#RSPB20130053F2\" data-ref-type=\"fig\">figure 2<\/a>). Interestingly, there were also three instances of copying of whistles that were not signatures between two adult, wild females of low association (see the electronic supplementary material, figure S2). These animals also produced their own signature whistles but no signature whistle copies.\n\nIn recordings of four aquarium housed males (forming six possible pairs) at The Seas, one pair also engaged in signature whistle copying. These two individuals showed high levels of synchronous behaviour (23% of 285 min of observation time) in the pool. Synchrony is a sign of social bonding in male bottlenose dolphins \\[34\\]. One exchange of signature whistle copying between these males was 30 s in duration: both males emitted the signature whistle of one of them in an interactive sequence consisting of 13 and 11 renditions respectively (see the electronic supplementary material, figure S3). Copying in these individuals was not accompanied by aggressive behaviour (total observation time 16 h with 13 copies produced). The synchrony of the other male pairs was generally lower (7\u201313% of the observation time). One other pair, however, had a high level of synchrony (26%) but did not engage in whistle copying. Thus, copying does not necessarily occur in bonded males.\n\n## How accurate are vocal copies?\n\nFrequency parameter measurements of copies produced by 11 animals (one captive and 10 wild animals; two wild copiers were excluded owing to small sample sizes) revealed consistent differences between signature whistle copies and the original, copied signature whistle (<a href=\"#RSPB20130053TB2\" data-ref-type=\"table\">table 2<\/a> and <a href=\"#RSPB20130053F3\" data-ref-type=\"fig\">figure 3<\/a>). While the overall frequency modulation pattern of the copied whistle showed high similarity to the original (<a href=\"#RSPB20130053F1\" data-ref-type=\"fig\">figure 1<\/a>), copiers introduced consistent variation in single acoustic parameters such as the start or end frequency (see the electronic supplementary material, table S1). In these parameters, copies were often closer to other whistle contours than to the copied signature whistle (<a href=\"#RSPB20130053F3\" data-ref-type=\"fig\">figure 3<\/a>). Individuals varied in the parameters modified; on average 4.4 parameters (range: 1\u20136) differed significantly between the copy and the original signature whistle. Copies most frequently differed from the original (for 10 of 11 copiers) in mean frequency and maximum frequency (see the electronic supplementary material, table S1). Over half of the copiers also produced copies that differed significantly from the original signature whistle in end frequency (six of 11 copiers) and frequency range (seven of 11 copiers). The copies were equally likely to be higher or lower in frequency than the original. In addition to frequency parameters, one adult male, FB26, altered the number of loops in a multi-looped whistle in his copies of the signature whistle of his alliance partner, adult male FB48. Although FB48 varied his number of loops (range: 3\u20136), FB26 almost always produced a three-looped copy. The number of loops in FB26's copies and FB48's originals differed significantly (Mann\u2013Whitney: *W* = 152.5, *N*~1~ = 38, *N*~2~ = 35, *p* \\< 0.0001). All of the signature whistle copies also differed significantly from those of the copiers' own signature whistles in some parameters (mean number of parameters different = 3.54; range: 1\u20137), whereas other parameters of a copy resembled those of the copier's own signature whistle (mean = 2; range: 0\u20135).\n\nTest statistics for all acoustic parameter measurements combined for each copy and original signature whistle comparison. Shown are the sampling statistic of actual combined parameter measurements (observed), and the mean test statistic of combined parameter measurements under the null hypothesis based on 10 000 permutations (expected). Differences between acoustic parameter measurements of vocal copies and original signature whistles are significant at a level of *p* \\< 0.002.\n\n|  | observed test statistic | expected test statistic | *p* |\n|:---|:---|----|----|\n| Ranier versus copy of Ranier | \u22127.52 | \u22120.002 | 0.002 |\n| FB48 versus copy of FB48 | 0.19 | \u22120.007 | 0.12 |\n| FB26 versus copy of FB26 | 559 | 0.025 | \\<0.0001 |\n| FB20 versus copy of FB20 | 166 | 0.43 | 0.0031 |\n| FB122 versus copy of FB122 | 0.27 | 0.003 | 0.1 |\n| FB65 versus copy of FB65 | 1004 | 0.03 | \\<0.0001 |\n| FB55 versus copy of FB55 | 24 000 | 0.016 | \\<0.0001 |\n| FB35 versus copy of FB35 | 125 | \u22120.01 | \\<0.0001 |\n| FB95 versus copy of FB95 | \u22121439 | \u22120.01 | \\<0.0001 |\n| FB155 versus copy of FB155 | 3 071 589 | 1.85a | \\<0.0001 |\n| FB177 versus copy of FB177 | \u22122646 | \u22120.0003 | \\<0.0001 |\n\n## Vocal matching\n\nTo further investigate whether copies were emitted in response to the identified model (referred to as the original signature whistle), we investigated whether they were temporally correlated and thus occurred in vocal matching interactions. Vocal matching can be described as a receiver responding to a signal by changing some features of its own vocal behaviour in order to imitate the preceding signal. Bottlenose dolphins had very high vocalization rates during these capture\u2013release events, so it was difficult to judge whether whistles were produced in response to those of other animals. An investigation into the timing of signature whistle copies, however, revealed that the mean time between an original signature whistle and its copy was significantly less than the mean time between an original signature whistle and a copier's own signature whistle (0.94 versus 2.55 s; permutation, *p* \\< 0.0001). In the long-term captive males, vocal rates were lower, and the matching pattern was clearer: almost all copying events occurred within 1 s after the emission of the original signature whistle by its owner, indicating copies were directed towards the owner of the original signature whistle.\n\n# Discussion\n\nWe conducted a large-scale analysis on the occurrence of vocal copying in wild bottlenose dolphins that were briefly caught, sampled and released. This dataset offered a unique opportunity to study the vocal interactions between individuals whose vocal repertoires \\[14,40\\] and association patterns had been well documented over decades in the wild \\[32,42\\]. In line with previous studies \\[23,25,26\\], we found whistle copying to be rare. This is consistent with the idea that signature whistles are used to indicate identity, because such a system would not be sustainable with high copying rates. While a copy could be recognizable as such if it occurred only in specific contexts, aquatic organisms usually have only limited contextual information with the acoustic signals they receive. Frequent copying of signature whistles would therefore render the identity information of the whistle unreliable. The rare copying of signature whistles may, however, be particularly suited to addressing close associates \\[23\u201325\\].\n\nWe found that copying occurred primarily in matching interactions between animals with high CoAs outside aggressive contexts, demonstrating that it is an affiliative signal. All pairs of animals that produced signature whistle copies were close associates, with only one pair having a low CoA for the year prior to recording. However, these two males were each other's closest male associate in the 4 year period prior to the recording. Many of the copiers were mother\u2013calf pairs, with both mothers and calves likely to copy one another. While most female calves' signature whistles are distinct from their mothers', males sometimes do sound like their mothers \\[37\\]. The signature whistles of the male calves in this study, however, did not resemble those of their mothers (see <a href=\"#RSPB20130053F1\" data-ref-type=\"fig\">figure 1<\/a> and electronic supplementary material, figure S1). Signature whistles of male alliance partners also tend to become more alike over time \\[43\\]. In this study, however, males continued producing their own, non-identical, signature whistles as well as copying the finer details of each other's preferred whistle type. Thus, age, sex and relatedness were not significant factors for the results presented here.\n\nWe found no evidence for a deceptive function of signature whistle copies. In animals that are capable of vocal learning, variations can be introduced into a copied signal, allowing encoding of additional information. Bottlenose dolphins produced accurate copies of the frequency modulation pattern of a whistle (<a href=\"#RSPB20130053F1\" data-ref-type=\"fig\">figure 1<\/a>), but introduced fine-scale differences in some acoustic parameters (<a href=\"#RSPB20130053TB2\" data-ref-type=\"table\">table 2<\/a> and <a href=\"#RSPB20130053F3\" data-ref-type=\"fig\">figure 3<\/a>). As a result, signature whistle copies were clearly recognizable as such. Copies may even carry identity information of the copier, as some individuals maintained some frequency parameters of their own signature whistles in their copies (see the electronic supplementary material, table S1). While these variations may appear subtle, they were generally outside the acoustic variations used by the signature whistle owner itself. Dolphins are clearly capable of detecting such differences in the fundamental frequency as well as the upper harmonics \\[44,45\\]. Hence, these copies cannot function in a deceptive manner. Only animals that are familiar with the whistle of the owner would, however, be able to recognize copies. In encounters with unknown animals, a high rate of copying would still lead to confusion, arguing for low rates of copying overall. In fact, wild bottlenose dolphins do not copy signature whistles when encountering other groups of dolphins at sea \\[46\\].\n\nThree lines of evidence suggest that active selection may have resulted in the variation found in signature whistle copies. First, bottlenose dolphins are capable of producing almost perfect copies of model sounds \\[22\\], suggesting that the variation is not due to limits on copying performance. Second, in experimental copying studies, bottlenose dolphins sometimes alter parameters of copies from one session to the next, and subsequently only produce copies with these novel parameter values \\[47\\]. Third, it has been shown that some dolphins introduce novel components such as sidebands to whistle copies, while they are perfectly capable of producing whistles without sidebands at these frequencies \\[24\\]. Thus, it is unlikely that variations introduced to copies are merely errors or reflect limitations in copying performance.\n\nA role of vocal learning in the development of signals used in group cohesion and the maintenance of social bonds can be found in a number of social species \\[3\u20137,48,49\\]. The bottlenose dolphin signature whistle stands out in that it is invented by its main producer and can only be shared by animals who had experience with the inventor. Besides humans, bottlenose dolphins appear to be the other main example of affiliative copying with such individually specific learned signals, although some parrot species do use vocal learning to develop labels for social companions \\[50\u201352\\] and therefore deserve further investigation in this context. Further studies are also needed to elucidate whether copying such signals is different from sharing learned contact calls or adjusting acoustic parameters in communal displays as found in other birds and primates. Bottlenose dolphins can be trained to use vocal copies of novel, arbitrary sounds to refer to objects \\[22\\]. It is not yet known whether they use learned signals in this way in their own communication system. However, bottlenose dolphins have been found to copy signature whistles of animals that are not present in their group \\[27\\]. It is possible that signature whistle copying represents a rare case of referential communication with learned signals in a communication system other than human language \\[12\\]. Future studies should look closely at the exact context, flexibility and role of copying in a wider selection of species to assess its significance as a potential stepping stone towards referential communication.\n\n## Acknowledgements\n\nWe thank Ana Catarina Alves, Aasta Eik-Nes, Thomas G\u00f6tz, Teresa Gridley, Mike Lonergan, Silvana Neves, Cornelia Oedekoven, Peter McGregor and Peter Tyack for help and advice during this study. Mike Beecher and three anonymous reviewers have provided helpful comments on the manuscript. We thank Walt Disney World Animals, Science and Environment and The Seas, Epcot, Walt Disney World Resorts, Lake Buena Vista, FL, USA for letting us work with their animals, especially Heidi Harley, Andy Stamper, Patrick Berry and Jane Davis. Fieldwork in Sarasota Bay would not have been possible without the efforts of a large team of biologists, veterinarians and dolphin handlers, including the staff of the Sarasota Dolphin Research Programme, Blair Irvine, Michael Scott, Jay Sweeney and a team of experienced volunteers. Ages of some of the Sarasota Bay dolphins were provided by Aleta Hohn. This work was supported by a BBSRC Doctoral Training Grant, Dolphin Quest, the Chicago Zoological Society, the National Oceanic and Atmospheric Administration (NOAA) Fisheries Service, Disney's Animals, Science and Environment, Dolphin Biology Research Institute, Mote Marine Laboratory, Harbor Branch Oceanographic Institute and a Royal Society University Research Fellowship and a Fellowship of the Wissenschaftskolleg zu Berlin to V.M.J. Work was conducted under NOAA Fisheries Service Scientific Research permit nos 417, 655, 945, 522\u20131569 and 522-1785 (to R.S.W.), IACUC approval through Mote Marine Laboratory, and approval of Disney's Animal Care and Welfare Committee. All data are archived and accessible at the Sea Mammal Research Unit, University of St Andrews.\n\n# References","meta":{"dup_signals":{"dup_doc_count":162,"dup_dump_count":39,"dup_details":{"curated_sources":2,"2018-43":2,"2018-34":2,"2018-26":4,"2018-17":2,"2018-13":1,"2018-09":3,"2017-51":1,"2017-47":3,"2017-43":1,"2017-39":3,"2017-34":1,"2017-30":4,"2017-26":2,"2017-22":2,"2017-17":5,"2017-09":6,"2017-04":6,"2016-50":5,"2016-44":5,"2016-40":5,"2016-36":6,"2016-30":7,"2016-26":5,"2016-22":5,"2016-18":5,"2016-07":6,"2015-48":5,"2015-40":7,"2015-35":5,"2015-32":5,"2015-27":5,"2015-22":5,"2015-14":4,"2014-52":5,"2014-49":8,"2017-13":2,"2015-18":5,"2015-11":4,"2015-06":3}},"file":"PMC3619487"},"subset":"pubmed_central"}
{"text":"author: Elizabeth R. Seaquist; Dianne Figlewicz Lattemann; Roger A. DixonCorresponding author: Elizabeth R. Seaquist, <firstname.lastname@example.com>.\ndate: 2012-12\ntitle: American Diabetes Association Research Symposium: Diabetes and the Brain\n\nFrom 28\u201330 October 2011, more than 100 people assembled in Alexandria, Virginia, to participate in an American Diabetes Association\u2013sponsored research symposium entitled \"Diabetes and the Brain.\" The objective of the symposium was to discuss the role of the brain in normal and abnormal metabolism and to consider the impact of diabetes on cerebral structure and function. Symposium participants were particularly interested in understanding how abnormalities in brain metabolism could affect the development of diabetes and obesity and how these diseases could, in turn, affect learning and memory. The growing epidemic of diabetes brought urgency to the meeting because of the ever-increasing number of people placed at risk for the cerebral complications of the disease. In this report, we present meeting highlights in five related areas as follows: *1)* metabolism, blood flow, and epigenetics; *2*) glucose sensing and hypoglycemia counterregulation; *3)* insulin resistance and action in the brain; *4)* neurocognition and imaging; and *5*) energy homeostasis.\n\n# METABOLISM, BLOOD FLOW, AND EPIGENETICS\n\nThe symposium opened with an outstanding review of cerebral metabolism by Gerald Dienel, PhD, from the University of Arkansas. He emphasized that glucose is the major fuel for the adult brain and that it has multifunctional roles in brain function. Metabolism of glucose via the glycolytic, pentose phosphate shunt, and tricarboxylic acid cycle pathways provides ATP for energy, NADPH for defense against oxidative stress, and carbon for biosynthesis of amino acids and sugars used for synthesis of glycoproteins and glycolipids and for de novo synthesis of tricarboxylic acid cycle-derived neurotransmitters glutamate and \u03b3-aminobutyric acid (GABA) (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<\/a>). Glycogen is stored predominately in astrocytes, and the CO~2~ fixation reaction required for glutamate biosynthesis occurs in astrocytes. Local rates of blood flow and glucose utilization are closely linked to local activities of brain cells, and metabolic rates increase with increased functional activity and decline when cellular signaling is reduced. Energy metabolism and neurotransmission are closely interrelated because glutamate, GABA, acetylcholine, and glycine are synthesized via glucose metabolic pathways, and catecholamine synthesis and degradation rely on oxygen. Metabolic brain imaging and magnetic resonance spectroscopic studies take advantage of the obligatory use of glucose as the predominant brain fuel, and labeled glucose or labeled glucose analogs are used to assay rates of specific reactions or pathways in living brain. Different labeled precursors are used to measure major pathway fluxes in neurons (glucose) and astrocytes (acetate), and they are used for trafficking of metabolites between these two major cell types. In experimental diabetes, changes in the metabolic rate of glucose in the brain are small, but increased brain glucose levels increase production of sorbitol, elevate oxidative stress, impair gap junctional communication among astrocytes, and cause other abnormalities. Although cognitive capability and other brain functions are affected by diabetes, its impact on the brain does not appear to be as severe as it is on peripheral organs.\n\nEric Newman, PhD, from the University of Minnesota, discussed the regulation of cerebral blood flow in the retina and used it as a model for regulation of brain blood flow. He emphasized that neuronal activity in the brain leads to localized increases in blood flow, a process termed functional hyperemia. Recent work suggests that activity-dependent increases in blood flow are mediated largely by a feedforward mechanism, whereby transmitter release from active neurons results in release of vasodilatory agents, such as arachidonic acid and its metabolites, either from activated glial cells or from other neurons. He went on to say that the regulation of central nervous system (CNS) blood flow is disrupted in diabetic patients. In diabetes, these changes have been best-characterized in retinas in which decreases in light-evoked vasodilation are seen, even before overt retinopathy is observed. Loss of the functional hyperemia response may result in hypoxia and may contribute to development of diabetic retinopathy.\n\nDr. Newman presented results from a recently completed study in his laboratory, where he found that light-evoked vasodilation was substantially reduced in rats with diabetes for a duration of 7 months. Loss of vasodilation reproduced the disruption of functional hyperemia observed in patients with diabetes. Inducible nitric oxide synthase (iNOS) was upregulated in these animals. He suggested that this response could be responsible for the reduction of functional hyperemia in diabetes, particularly because inhibiting iNOS with aminoguanidine reversed its loss. Dr. Newman concluded that targeting iNOS or its downstream signaling pathway with selective inhibitors to restore functional hyperemia ultimately might be an effective therapy for treating diabetic retinopathy.\n\nGiulio Maria Pasinetti, MD, PhD, from Mount Sinai School of Medicine, focused on epigenetics (DNA methylation) in mouse models of diet-induced obesity. In the Tg2576 mouse, a high-fat diet for up to 5 months resulted in increased soluble \u03b2-amyloid in hippocampus and cortex (key CNS sites for learning and cognition) and impaired spatial memory behavioral performance. Using a model of the metabolic syndrome (increased body weight; glucose intolerance; and elevated blood pressure, plasma insulin, and cholesterol) developed in the wild-type C57BL\/6J mouse, his group observed impairment in hippocampal electrophysiological measures that may be linked to impaired memory consolidation. Dr. Pasinetti summarized progress in identification of genes that are targets for DNA epigenetic modification and altered expression in the CNS of the mouse model of metabolic syndrome. These include genes for mitochondrial energy metabolism that are downregulated by diet-induced obesity secondary to hypermethylation of specific promoter sequences. A caloric restriction mimetic, Combi-phenol, protects against or normalizes several sequelae of the metabolic syndrome in this model, including brain mitochondrial dysfunction, cognitive behavioral performance, and CNS network connectivity evaluated with diffusion tensor imaging. The Combi-phenol is being studied in phase I and II clinical trials for safety, measurement of biomarkers, and memory assessments as a potential therapeutic or therapeutic adjunct for Alzheimer disease (AD).\n\n# GLUCOSE SENSING AND HYPOGLYCEMIA COUNTERREGULATION\n\nBrain glucose-sensing mechanisms in the brain were the topic of the presentation by Vanessa Routh, PhD, from the New Jersey Medical School. She explained that the ventromedial hypothalamus (VMH) contains both glucose-excited and glucose-inhibited neurons that respond to physiologically relevant changes in glucose. Glucose-excited neurons increase firing as glucose is increased through a mechanism that involves closure of the K~ATP~ channel. The phenotype of these neurons remains uncertain, but some believe they may be producing pro-opiomelanocortin (POMC). Glucose-inhibited neurons reduce firing in response to an increase in glucose. This action appears to be mediated by AMP kinase and neuronal NOS and couples to a chloride channel. Glucose-inhibited neurons are distributed throughout the VMH, including approximately half of the neuropeptide Y (NPY)\u2013producing neuronal population. The ability of the glucose-inhibited neurons to sense and respond to glucose parallels the ability of the brain to sense and respond to fasting and hypoglycemia in normal rodents. Moreover, the response of glucose-inhibited and glucose-excited neurons to decreased glucose can be blunted by satiety signals such as leptin or insulin and can be increased by the orexigenic peptide ghrelin. On the basis of these results, Dr. Routh proposed the hypothesis that glucose-sensing neurons are important for sensing decreased glucose and initiating compensatory responses under conditions of energy deficit, such as fasting or hypoglycemia. However, during normal energy balance, it is important that glucose-sensing neurons do not respond to small glucose decreases associated with meal-to-meal fluctuations in glucose because such a response could lead to inappropriate signals of overall energy deficit. As a result, satiety hormones mask and orexigenic agents enhance the responsiveness of glucose-sensing neurons to reductions in glucose.\n\nStephanie Amiel, MD, from King's College London School of Medicine, presented a lecture entitled \"Neural Circuits Involved in Hypoglycemia Counterregulation.\" She reviewed the physiology underlying the counterregulatory response to hypoglycemia and then presented neuroimaging studies in humans that have helped visualize the global brain response to hypoglycemia. Many cerebral regions have been shown to participate in the detection of hypoglycemia and coordination of the counterregulatory response, including the hypothalamus, the thalamus, the hippocampus, and higher brain centers. With a new understanding of the parts of the brain involved in the counterregulatory response to hypoglycemia, she proposed that new therapeutic avenues to prevent and treat hypoglycemia may be possible.\n\nHow these different brain regions may detect and respond to hypoglycemia was discussed by E.R.S. MD, from the University of Minnesota, in her lecture entitled \"Mechanisms Underlying the Development of Hypoglycemia-Associated Autonomic Failure.\" She began her presentation by emphasizing that hypoglycemia-associated autonomic failure (HAAF) is a clinically significant problem that occurs in patients with type 1 and advanced type 2 diabetes. Recurrent hypoglycemia leads to HAAF because it shifts the glucose threshold that elicits adrenomedullary response (i.e., lower and lower blood sugars required to elicit response) and reduces the magnitude of hormonal counterregulatory response while having no effect on glucose level associated with neuroglycopenia symptoms. HAAF is reversible by avoiding hypoglycemia.\n\nThe mechanisms responsible for the development of HAAF remain uncertain, but at least three have been proposed. One potential mechanism is that hypoglycemia induces a change in brain glucose metabolism in such a way to prevent regulatory centers from sensing a change in blood glucose concentration during systemic hypoglycemia. A second potential mechanism is that the brain may increase the utilization of alternative fuels such as lactate and glycogen during hypoglycemia and therefore may not experience energy deficit in the setting of systemic hypoglycemia. A third mechanism suggests that changes in downstream signaling of GABA and opioids that link detection of hypoglycemia to counterregulatory response are altered in the setting of recurrent hypoglycemia.\n\n# INSULIN RESISTANCE AND INSULIN ACTION IN THE BRAIN\n\nC. Ronald Kahn, MD, from the Joslin Clinic and the Harvard Medical School, discussed diabetes, insulin action, and the brain. One approach to identifying pathways through which insulin alters brain metabolism has been creation of mice in which the insulin receptor is knocked out only in neural tissue. These mice show increased food intake, increased adipose mass, hyperleptinemia, and insulin resistance; however, behaviorally, they are the same as wild-type mice at a young age. However, as they age, they begin to behave abnormally in open field tests (interpreted as a sign of anxiety), in tail suspension tests (interpreted as a sign of depression), and in forced swimming tests (interpreted as a loss of motivation). How the loss of insulin action causes these behavior changes remains unknown, but preliminary studies show that these mice have decreased phosphorylation of glycogen synthase kinase 3 and increased phosphorylation of \u03c4, two proteins known to be important in the pathogenesis of AD. Dr. Kahn's group recently performed a gene expression analysis on the hypothalamus from streptozotocin-treated C57BL\/6 mice (a model for insulin-deficient diabetes) and found that genes involved in cholesterol biosynthesis are suppressed relative to controls. In addition, rates of cholesterol synthesis and the synaptosomal cholesterol content are decreased in the brains of these diabetic mice, as are levels of sterol regulatory element\u2013binding protein 2 and srebp cleavage-activating protein (SCAP) mRNA and protein, two important regulators of cholesterol synthesis.\n\nMark W.J. Strachan, MD, from the Western General Hospital, Edinburgh, Scotland, also discussed insulin action in the brain in his lecture entitled \"Emerging Data on the Relationship Between Insulin Resistance and Neurovascular Disorders.\" Insulin receptors are found in high concentrations within the limbic system of the brain. In vitro, insulin affects neuronal excitability and synaptic plasticity. Direct relationships have been shown between insulin action and the pathogenesis of AD. The \u03b2-amyloid oligomers, which accumulate in the brain in AD, are intimately linked with neuronal dysfunction and cause a rapid and substantial loss of cell surface insulin receptors in hippocampal neuronal cultures. The development of insulin resistance in the brain then can set-up a vicious cycle because resultant activation of glucose synthase kinase 3 promotes hyperphosphorylation of \u03c4 protein and altered processing of amyloid precursor protein, which increases \u03b2-amyloid oligomer production. Moreover, insulin resistance (through attendant hyperinsulinemia) may reduce the ability of insulin-degrading enzyme in the brain to break-down \u03b2-amyloid oligomers. Proving the existence of these mechanisms and pathways in humans has been challenging, but insulin concentrations in the cerebrospinal fluid do appear lower in people with severe AD. In addition, administration of intravenous insulin (with concurrent glucose to maintain euglycemia) has been shown to enhance cognitive function. Direct administration of insulin via the intranasal route has a favorable impact on cognition and may provide new opportunities for treatment. Therapies designed to improve cerebral insulin action have produced mixed results. Administration of rosiglitazone was not of cognitive benefit to people with AD, perhaps because it does not cross the blood--brain barrier, whereas an exercise intervention did lead to improvement in insulin sensitivity and cognitive function in older adults.\n\nSuzanne Craft, PhD, from the University of Washington, presented a programmatic analysis of a specific mechanism that may link type 2 diabetes with pathological brain changes, cognitive impairment, and even AD-related dementia. A key target (if not culprit) is insulin secretion and action. Dysregulation or other abnormalities in brain insulin play a role in normal aging-related cognitive decline, exacerbated cognitive decline with type 2 diabetes, and (when severe) mild cognitive impairment and AD. The two forms of insulin dysregulation that increase the risk of neurocognitive dysfunction, insulin resistance and peripheral hyperinsulinemia, also were discussed by other speakers in this symposium. Dr. Craft detailed several mechanisms through which insulin resistance and diabetes may increase the risk of cognitive impairment and AD. In addition to reduced brain insulin uptake, these specific mechanisms include: *1*) impaired glucose and lipid metabolism; *2)* disrupted regulation of \u03b2-amyloid clearance (i.e., a hallmark of AD neuropathology); *3)* increased inflammation; and *4)* compromised vascular function (e.g., microvascular lesions). Notably, insulin receptors are distributed in several critical brain regions (e.g., hippocampus, frontal lobe), and insulin performs several crucial brain cognition functions (e.g., increasing neurotransmitter levels, glucose utilization). The fact that numerous specific mechanisms can independently and perhaps interactively interrupt signaling in any of these regions creates a formidable challenge for clinical research.\n\nIf insulin resistance is a mechanism through which diabetes may compromise neurocognitive functioning, then there may be preventable or modifiable factors that lead to insulin dysregulation as well as therapeutics that may improve insulin resistance. Regarding the former, Dr. Craft reported that epidemiological studies suggest that the expanding prevalence of conditions related to insulin resistance in western societies may be attributable to decreased levels of physical activity and increased dietary intake of calories and saturated fats. Arguably, healthier lifestyles may be protective factors operating in several specific mechanisms through which insulin resistance can lead to neurocognitive dysfunction. Regarding the latter, Dr. Craft described her own research on the therapeutic strategy of administering intranasal insulin in an effort to improve neural insulin signaling. In her project known as the Study of Nasal Insulin to Fight Forgetfulness (SNIFF), she reported promising outcomes, including improved memory performance and improved performance in activities of daily living.\n\n# NEUROCOGNITION AND IMAGING\n\nChristopher M. Ryan, PhD, from the University of Pittsburgh, presented a historically informative tour of the major issues and strategies for assessing cognitive function in diabetic patients. Although the field has benefited from a surge of interest in recent years, it has come to recognize that improvements can be implemented across research laboratories in crucial aspects such as: *1*) establishing diagnostic standards and procedures for evaluating equivalences and systematic variations in clinical groups; *2*) reducing inconsistencies in the cognitive testing batteries deployed (e.g., standardization, theoretical breadth, measurement purity, manifest vs. latent variable representations, and validity issues); *3*) improving research sensitivity to special population characteristics that affect diabetes--cognition relationships (e.g., children, older adults, multiple impairments); and *4*) addressing the importance of longitudinal follow-up for understanding the trajectories of cognitive change. Dr. Ryan, who was an early contributor and still is a continuing contributor to the field, focused on identifying a framework for representing some of the major approaches to selecting cognitive measures for research in diabetes and reviewing a large number of frequently used cognitive batteries.\n\nRegarding the framework, Dr. Ryan described several related approaches. One approach emphasizes identifying relatively nonspecific cognitive markers of diabetes-related dysfunction. Neurocognitive speed has proven to be the most sensitive marker across multiple studies, even while using tasks that range widely in terms of their process purity. In a complementary approach, researchers use cognitive and neuropsychological test batteries to identify neurocognitive or clinical phenotypes (e.g., mild cognitive impairment) associated with diabetes. Results using this approach have improved as the batteries become more contemporary, comprehensive, and multidimensional. However, Dr. Ryan cautioned that it is crucial to map the expected cognitive effects to the brain changes (e.g., vascular) on the basis of theoretical and clinical data and to use batteries that are age-appropriate and change-sensitive to evaluate these relationships and performances at different phases of the life span.\n\nBrian M. Frier, MD, from the Royal Infirmary of Edinburgh, Scotland, shifted the focus from extant approaches and available test batteries to the correlative topic of observed cognitive effects of diabetes and their potential mechanisms. He began by noting that the \"unsystematic\" nature of early research in the area was likely attributable to cross-study variations and within-study limitations in sample sizes, cognitive batteries, and measurement of relevant covariates. Accordingly, Dr. Frier supplemented the typical organizing scheme of diabetes type. Rather than summarizing similar and dissimilar cognitive effects of type 1 and type 2 diabetes, he began by presenting the main observed (or to-be-tested) risk factors for cognitive decrements as associated with each. Both sets of risk factors include a genetic predisposition (although this remains an understudied aspect for both). For type 1 diabetes, the other risk factors include recurrent hypoglycemia, chronic hyperglycemia, long duration, and early age of onset, as well as a group of diabetes complications. Not surprisingly, for type 2 diabetes the list is much longer and more diverse (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<\/a>). It includes vascular issues (microvascular and macrovascular disease, hypertension), acute hyperglycemia, recurrent hypoglycemia, hyperinsulinemia, dyslipidemia, amyloid deposition in the brain, and inflammatory mediators, as well as drug therapy issues and psychosocial factors such as depression. The implicit message is that diabetes per se may affect neurocognitive performance in a variety of ways but does so not just through the separate prisms of diabetes type. It is also influenced within type by a diverse set of barely overlapping but likely interacting influences.\n\nFor both disease types, there is consistent evidence that neurocognitive speed is affected, particularly straightforward indicators of mean performance rates that usually show slower average speeds for diabetic patients relative to controls. In addition, some downstream indicators of basic cognitive resources (e.g., executive function) and products (e.g., episodic memory) that may require speed for efficient performance are often implicated in research with both types of diabetes. According to Dr. Frier's review, several of the promising risk factors for cognitive decrements in type 1 diabetes have not yet been confirmed empirically. One confirmed factor among children and adolescents is early disease onset (up to age 7), a factor that is also linked to progress and participation in schooling and social development. Regarding research on cognitive effects of type 2 diabetes, a key challenge is to differentiate neurocognitive changes that characterize normal aging from those exacerbated by diabetes or associated with progressive neurodegenerative decline. Researchers are increasingly considering the roles of modulating factors. In addition to age, sex, and education, these include the specific risk factors in <a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<\/a>, numerous comorbidities (e.g., obesity, vascular dysfunction), treatment and adherence (e.g., diet, oral agents), and exposure to risk-enhancing substances (alcohol, smoking, cholesterol). In summary, Dr. Frier's recommendations for future research included examining multiple influences on longer-term progressive or accelerated declines in cognitive functioning. We await further results from prospective studies that feature larger sample sizes, broader coverage of key cognitive functions, and markers of important risk and mediator factors, all measured across extended bands of adulthood.\n\nThe presentation by Geert Jan Biessels, MD, PhD, from the University Medical Centre Utrecht, addressed etiology, risk factors, and biomarkers of diabetes-related cognitive dysfunction with an emphasis on novel opportunities provided by longitudinal research methods. A first important point is that cognitive dysfunction in diabetes should be characterized not just in terms of implicated aspects of cognition (e.g., episodic memory or neurocognitive speed) or even magnitude of the concurrent deficit Rather because most observed decrements are modest in magnitude, dysfunction also should be defined by characteristics of change, including direction, slope, trajectory, and outcome. Whereas for some diabetic patients neurocognitive deficits may decline only slowly over long periods of adulthood, others may experience trajectories that lead beyond normal decline to frank cognitive impairment and even dementia. Dr. Biessels reported that although diabetic patients who exhibit sustained modest decrement profiles of both types of diabetes (1 and 2) and of any age, those who experience more rapid neurocognitive decline are more likely to be patients with type 2 diabetes who eventually have development of overt dementia.\n\nDr. Biessels' second major point was that the two types of cognitive dysfunction in diabetes may differ not only in terms of temporal and outcome characteristics but also in terms of associated biomarkers, risk factors, and etiological influences. In addition, Dr. Biessels noted that these dynamic and interactive processes can be directly examined with multiple assessments and appropriate statistical analyses. He described one promising longitudinal study that may yield data to answer many of the questions raised in his presentation. It features abundant risk factor data on large samples of type 2 diabetic patients from the Kaiser Permanente Northern California Diabetes Registry. Finally, a summary point from his biomarker and etiology discussion merits special attention. Dr. Biessels noted an emerging theory that may imply that the two types of diabetes--cognition trajectories may differ in part in the extent to which an additional (diabetes and immediate complications) \"hit\" from substantial brain pathology is absorbed by some patients. For example, a mild stroke or increased rates of amyloid deposition may accelerate the transition of some type 2 diabetic patients from normal cognition to impairment or dementia.\n\nGail Musen, PhD, from the Joslin Diabetes Center, highlighted the value of using neuroimaging techniques to shed light on neural correlates of cognitive dysfunction in diabetes. A principal rationale is that diabetes-related changes in the brain may be detectable with recent technology early in diabetes-related degenerative processes and that they may also precede and be differentially associated with specific diabetes-related cognitive manifestations and decremental patterns. Dr. Musen commented on the recent progress and future promise in this area, attributable in part to new and more widely available neuroimaging techniques. For example, early diabetes--brain research on white matter hyperintensities (often observed with magnetic resonance imaging) revealed mixed patterns across studies. Using the more sensitive, flexible, and objective diffusion tensor imaging, researchers can examine and compare a number of white matter abnormalities and connectivity patterns. These lines of investigation were not previously available because of limitations in imaging technology. In addition, voxel-based morphometry studies of cortical thickness (including both gray and white matter density) are being used to compare diabetic patients and healthy controls. Dr. Musen reported new research from her team on one promising avenue that may eventually link diabetes more closely with cognitive impairment and dementia risk. In normal adults, the default mode network is typically deactivated during goal-directed cognitive activity, with variations on activation and metabolism patterns for AD patients. She described one study in which similar disruptions occurred for type 1 diabetic patients and an effort to investigate similar issues with type 2 diabetes (a well-known risk factor for AD). Dr. Musen concluded her presentation with several forward-looking recommendations, including identification of neural mechanisms associated with potential cognitive dysfunctions in diabetes, pursuing this goal with larger-scale studies (in terms of both sample sizes and assessment waves), and collection of repeated assessments of target (neuroimaging, cognitive performance) and covariate (diabetes-related biomarkers) measures.\n\n# ENERGY HOMEOSTASIS\n\nD.F.L., PhD, from the University of Washington, presented translational research demonstrating the effects of insulin and leptin on reward behavior and dopamine function in the rat. Her work has focused predominately on the rewarding value of sucrose, based on findings that sucrose drives motivation for intake of mixtures of sugar, fat, and flavor. She reviewed the CNS anatomy that mediates reward behavior and energy homeostasis and made three key points. First, these sets of circuitries are substantially interconnected. Second, specific sites within both sets of circuitries play a role in mediating different aspects of reward behavior. Third, insulin and leptin can decrease reward behavior through actions on both reward and energy homeostatic circuitries. The neurotransmitter dopamine plays a central role in reward and motivation. D.F.L. reviewed the effect of insulin to increase dopamine reuptake, which curtails dopamine signaling. There is evidence that this effect is attributable to increased cell-surface cycling of the dopamine reuptake transporter, and that this cycling is dependent on activation of phosphatidylinositol 3 kinase mechanisms. Additionally, it has been shown that leptin can decrease dopamine neuronal activity. Together, both hormones act to dampen dopamine signaling in normal animals. D.F.L. finished by reporting that exposure to a moderate-fat diet results in increased motivation for sucrose and resistance to insulin and leptin effects in rats that are metabolically normal and preobese. She concluded with examples from current human imaging studies suggesting that the effects of insulin and insulin resistance on reward circuitry in rodents are relevant for humans.\n\nTony Lam, PhD, from the University of Michigan, reviewed work from his laboratory in a lecture entitled \"Gut--Brain Signaling.\" He has shown that the influx of fatty acids into the duodenum leads to the release of cholecystokinin, subsequent activation of vagal afferents, and transmission of the signal to the nucleus of the solitary tract. As a result, the gut-brain-liver axis is activated to reduce hepatic glucose production. In addition, metabolism of lipids and glucose in the hypothalamus activates protein kinase C and K~ATP~ channels that regulate hepatic glucose production. Diabetes and obesity alter these nutrient-sensing pathways in both the gut and the brain, which leads to abnormalities in glucose homeostasis.\n\nJoel Elmquist, DVM, PhD, from the University of Texas Southwestern Medical Center, reviewed the anatomical, synaptic, and cellular functions of the mediobasal hypothalamic circuitry in the coordinated regulation of multiple components of energy homeostasis. This circuitry has now been characterized for reciprocal regulatory interactions in locus, as well as for receiving and transmitting multiple signals from other CNS sites and from the periphery. Research has focused on the roles of (orexigenic) NPY\/agouti-related protein (AGRP) and (anorexigenic) POMC neurons. Dr. Elmquist provided a historical perspective on the field that laid the groundwork for the utility of genetically modified mouse models to determine specific neuronal and functional aspects of leptin activity. Specific ablation of leptin receptors in POMC neurons, which produce the neuropeptide melanocortin, results in modest obesity with decreased energy expenditure and locomotor activity but no effect on food intake, along with mild glucose intolerance and insulin resistance. Specific reinstatement of functional leptin receptors in these POMC neurons normalizes peripheral glucose homeostasis and glucagon release. Interestingly, a model in which both insulin and leptin receptors were ablated in POMC neurons did not result in strong effects on body weight but showed profound glucose dyshomeostasis and reduced fertility. This suggested coordinated action of insulin and leptin signaling in POMC neurons that regulate reproductive and hepatic function. However, leptin receptor effects are distributed throughout a network; for example, the leptin receptors expressed on steroidogenic factor-1 neurons of the VMH appear to regulate energy expenditure. The melanocortin receptor subtype 4 (MCR4) mediates many of the energy homeostatic effects of melanocortin, and new evidence suggests that MCR4 activity in multiple central and peripheral nervous system sites is important for regulation of energy homeostasis. Thus, MCR4 receptors expressed in SIM1 neurons of the paraventricular nucleus of the hypothalamus and the amygdala correct hyperphagia in MCR4-null mice. MCR4 receptors expressed in cholinergic neurons of the sympathetic nervous system increase energy expenditure and regulate hepatic glucose production, and MCR4 receptors expressed in the cholinergic neurons of the parasympathetic nervous system may regulate endocrine pancreatic function.\n\nTamas Horvath, DVM, PhD, from Yale University, presented his work examining synaptic interactions directly between POMC and other local neurons. The AGRP\/NPY neuron releases the inhibitory neurotransmitter GABA. In addition to a projection to the paraventricular nucleus of the hypothalamus, the AGRP\/NPY neuron sends a GABAergic projection to local POMC neurons in the arcuate nucleus. This local circuit is a direct target for leptin action as well as for the orexigenic hormone\/neuropeptide, ghrelin. Recent work from Horvath's group has demonstrated ghrelin stimulation of UCP2 expression. Studies in a UCP2 knockout model have shown an impairment of ghrelin-stimulated electrical activity and mitochondriogenesis in NPY\/AGRP neurons. Ghrelin effects have been observed in other CNS sites, i.e., the hippocampus and the dopaminergic neuron in the substantia nigra pars compacta. Taken together, the group's recent findings clearly delineate a key role of the UCP2 protein in many aspects of CNS function and its modulation by nutritional and metabolic status.\n\nThe theme of hypothalamic\/brain\/periphery networking was further emphasized in the presentation by Joseph Bass, MD, PhD, from Northwestern University, that focused on circadian regulation of energy homeostasis. Dr. Bass reviewed the discovery of clock proteins in the 1990s, which set the stage for research on circadian function and its regulation in the subsequent years. In mammals, including humans, the CNS \"master clock\" has been identified as the suprachiasmatic nucleus (SCN), and circadian rhythms are encoded by the sequential and reciprocal expression of transcription activators, CLOCK\/BMAL1, that induce expression of the transcription repressors, PER1\u20133\/CRY1\u20133, that inhibit CLOCK\/BMAL1 expression. The cycle of expression takes approximately 24 h and is entrained by light. The SCN has connections with key homeostatic nuclei in the hypothalamus, including the arcuate, paraventricular nucleus, ventromedial hypothalamus (VMH), lateral hypothalamus, and dorsal medial hypothalamus (DMH). Thus, there is direct input on behavior, activity, metabolism, and the hypothalamic-pituitary-adrenal axis, which in turn is a key diurnal regulator of activity and metabolism. Importantly, it is now appreciated that \"clock proteins\" are expressed in nearly all cells. Peripheral tissue clocks are entrained to food availability, adrenal glucocorticoids, and temperature, all of which can reset peripheral clocks. Although research is only beginning to uncover the complex crosstalk between stimuli and peripheral clocks, molecules that reflect metabolic status such as NAD and the nutrient sensor AMP kinase, relay information to the clocks and connectivity is bidirectional. A current line of investigation is examining the coupling of metabolic processes and CNS clock function. A high-fat diet in an animal model has been shown to increase period length of the daily activity rhythm and to phase-shift metabolic gene expression in the liver and adipose as well as in the hypothalamus. The CNS locus of this effect (SCN per se, or SCN projections) remains to be determined but clearly demonstrates metabolite\/CNS clock interaction. Finally, sleep and activity abnormalities such as narcolepsy and genetic evidence in humans with altered sleep patterns suggest that pursuit of this research and links between clock activities and metabolic disease will reveal the importance of clock\/environmental coordination to human metabolic health.\n\n# CONCLUSIONS\n\nThe workshop brought together scientists and clinicians working in disparate areas such as metabolism, psychology, imaging, and neuroscience who were all united by a desire to understand how diabetes affects cerebral structure and function. In the opening lecture, Dr. Gerald Dienel reported anecdotally that early in his career it was unusual for basic neuroscientists to consider diabetes as an influence on cognitive functioning, much less a risk factor for neurodegenerative disease. By the end of the workshop, new collaborations were formed that will support future multidisciplinary research in this area. Ideally, this work should be based on the following principles: both \"neuro\" and \"cognitive\" phenomena must be clinically relevant, theoretically selected, domain-specific, and carefully measured; research should emphasize, whenever possible, continuous (rather than categorical) assessments of phenomena related to diagnosis, performance, structure, modulating factors, and clinical outcomes; investigation should attend to a broad range of potential modulators, including proximal (e.g., insulin resistance), somatic or distal (e.g., functional biomarkers), comorbidities (e.g., vascular disruptions, inflammation), therapy--social (e.g., treatment availability and adherence, depression), and genetic susceptibilities (e.g., genetic polymorphisms related to diabetes, AD, obesity); work should continue to examine the complex systems through which endocrine and neural systems regulate energy metabolism; and, whenever possible, scientists should study these relationships dynamically using longitudinal follow-up assessments and appropriate change-sensitive statistical analyses.\n\n## ACKNOWLEDGMENTS\n\nNo potential conflicts of interest relevant to this article were reported.\n\nE.R.S., D.F.L., and R.A.D. contributed to the drafting and revising of the manuscript.\n\nThe authors gratefully acknowledge the organizational support of Shirley Ash (American Diabetes Association).","meta":{"dup_signals":{"dup_doc_count":145,"dup_dump_count":42,"dup_details":{"curated_sources":4,"2021-21":1,"2018-26":1,"2018-22":3,"2018-17":2,"2018-13":3,"2018-05":3,"2017-51":3,"2017-47":1,"2017-43":6,"2017-39":1,"2017-34":5,"2017-30":4,"2017-26":6,"2017-22":1,"2017-17":6,"2017-09":6,"2017-04":10,"2016-50":5,"2016-44":6,"2016-40":6,"2016-36":5,"2016-30":5,"2016-26":6,"2016-22":5,"2016-18":2,"2016-07":2,"2015-48":3,"2015-40":3,"2015-32":2,"2015-27":2,"2015-22":3,"2015-14":2,"2014-52":3,"2014-49":2,"2014-42":2,"2014-41":4,"2014-35":2,"2023-23":1,"2017-13":1,"2015-18":3,"2015-11":3,"2024-18":1}},"file":"PMC3501868"},"subset":"pubmed_central"}
{"text":"abstract: # Objective\n  .\n  To examine whether business improvement districts (BID) contributed to greater than expected declines in the incidence of violent crimes in affected neighbourhoods.\n  .\n  # Method\n  .\n  A Bayesian hierarchical model was used to assess the changes in the incidence of violent crimes between 1994 and 2005 and the implementation of 30 BID in Los Angeles neighbourhoods.\n  .\n  # Results\n  .\n  The implementation of BID was associated with a 12% reduction in the incidence of robbery (95% posterior probability interval \u22122 to 24) and an 8% reduction in the total incidence of violent crimes (95% posterior probability interval \u22125 to 21). The strength of the effect of BID on robbery crimes varied by location.\n  .\n  # Conclusion\n  .\n  These findings indicate that the implementation of BID can reduce the incidence of violent crimes likely to result in injury to individuals. The findings also indicate that the establishment of a BID by itself is not a panacea, and highlight the importance of targeting BID efforts to crime prevention interventions that reduce violence exposure associated with criminal behaviours.\nauthor: John MacDonald; Daniela Golinelli; Robert J Stokes; Ricky Bluthenthal**Correspondence to** Dr John M MacDonald, Department of Criminology, University of Pennsylvania, McNeil Building, Suite 483 3718 Locust Walk, Philadelphia, PA 19104-6286, USA; <firstname.lastname@example.com>\ndate: 2010-06-29\ninstitute: 1University of Pennsylvania, Philadelphia, Pennsylvania, USA; 2RAND Corporation, Santa Monica, California, USA; 3Drexel University, Philadelphia, Pennsylvania, USA\nreferences:\ntitle: The effect of business improvement districts on the incidence of violent crimes\n\nResearch indicates the importance of community-level attributes for explaining the incidence of interpersonal violence and crime in neighbourhoods, but there are few examples of effective community-level violence prevention interventions.1\u20133 Several studies suggest that implementation of the community economic development model of the business improvement district (BID) reduces crime in affected neighbourhoods.4 5 The BID model relies on special assessments levied on commercial properties located within designated business areas to augment services typically provided by public agencies, including sanitation, public safety, place marketing and planning efforts.6 Although managed and operated by private sector non-profit organisations, the majority of BID are public entities, chartered and regulated by local governments.7 The services delivered through BID assessment schemes, however, do not typically replace current public services. BID services typically are directed towards sanitation and security of common public space areas such as sidewalks (and not interior spaces), analogous to the common area service arrangements seen in home owners' associations.8 BID often focus their budgets on providing private security to their business locales and surrounding neighbourhoods, as a basic level of enhancement to publicly funded police services.9\n\nOne of the more rigorous evaluations of BID by Brooks5 indicated that their adoption in areas of Los Angeles was associated with a significant drop in the number of serious crimes reported to the police between 1990 and 2002, controlling for time stable differences between areas and in comparison with neighbourhoods that proposed BID but did not end up adopting them. Given that the adoption of a BID in Los Angeles requires extensive support from business and property owners (eg, at least 15% of the business owners or 50% of the property owners must sign supporting petitions) and a laborious process of legal and legislative oversight, the simple proposed adoption of a BID may not provide a strong comparison group.10 11 The actual process of BID adoption is, by itself, a signal of commitment from business merchants and landowners to promote economic development through various community change activities. Even after taking into account time stable area differences in the average volume of crime, poverty rates and other neighbourhood features, it is difficult to reconcile whether establishing a BID is independent from other facets of community change that may presage drops in crime. A detailed analysis of budget data and observations of neighbourhoods in Los Angeles where BID are situated, for example, showed that their priorities of spending were correlated with observable indicators of neighbourhood physical decay and surrounding economic conditions.12 Therefore, using the BID area before BID implementation may be a more appropriate comparison group.\n\nWe rely on a pre\u2013post intervention design to assess the effects of BID on the incidence of violent crimes. We use the year of implementation to reflect the exposure to the BID intervention and examine the pre\u2013post changes in the incidence of violent crimes in affected neighbourhoods, controlling for overall time trends.\n\n# Methods\n\n## Design\n\nWe examined BID effects by modelling the pre\u2013post changes in the incidence or rate of violent crimes from 1994 to 2005 for all neighbourhood areas exposed to BID. Between 1996 and 2003, a total of 30 separate BID were implemented in Los Angeles. The unit of analysis is any of the 30 neighbourhood areas that eventually adopted a BID in Los Angeles during the period of observation. <a href=\"#tbl1\" data-ref-type=\"table\">Table 1<\/a> reports the number of BID areas that became fully operational at any given year. We consider a BID fully operational if its implementation occurred for the entire calendar year. The formulation of BID in Los Angeles requires a formalised and uniform planning and adoption stage that is structured by law. Details are provided elsewhere.12 For all the areas that eventually adopted a BID in Los Angeles, there are at least 2\u2005years' worth of data during which no BID (pre) was operational and, similarly, at least 2\u2005years of data during which all the BID were fully operational (post). We make use of this type of interrupted time series to estimate the average BID effect on the rate of violent crimes.\n\nBID by year of observation in Los Angeles\n\n<table>\n<thead>\n<tr>\n<th>Year<\/th>\n<th>No of BID started<\/th>\n<th>BID area<\/th>\n<\/tr>\n<\/thead>\n<tbody>\n<tr>\n<td>1994<\/td>\n<td>\u2013<\/td>\n<td><\/td>\n<\/tr>\n<tr>\n<td>1995<\/td>\n<td>\u2013<\/td>\n<td><\/td>\n<\/tr>\n<tr>\n<td>1996<\/td>\n<td>2<\/td>\n<td>Wilshire Centre<br \/>\nFashion District<\/td>\n<\/tr>\n<tr>\n<td>1997<\/td>\n<td>2<\/td>\n<td>Hollywood Entertainment I<br \/>\nSan Pedro<\/td>\n<\/tr>\n<tr>\n<td>1998<\/td>\n<td>6<\/td>\n<td>Los Feliz Village<br \/>\nLarchmont Village<br \/>\nDowntown Centre<br \/>\nFigueroa Corridor<br \/>\nCentury Corridor<br \/>\nGreater Lincoln Heights<\/td>\n<\/tr>\n<tr>\n<td>1999<\/td>\n<td>11<\/td>\n<td>Granada Hills<br \/>\nCanoga Park<br \/>\nVan Nuys Boulevard Auto Row<br \/>\nTarzana<br \/>\nStudio City<br \/>\nHollywood Media<br \/>\nWestwood Village<br \/>\nHistoric Core (Downtown)<br \/>\nToy District<br \/>\nDowntown Industrial<br \/>\nJefferson Park<\/td>\n<\/tr>\n<tr>\n<td>2000<\/td>\n<td>2<\/td>\n<td>Chatsworth<br \/>\nSherman Oaks<\/td>\n<\/tr>\n<tr>\n<td>2001<\/td>\n<td>4<\/td>\n<td>Encino<br \/>\nLos Angeles Chinatown<br \/>\nWilmington<br \/>\nLincoln Heights Industrial<\/td>\n<\/tr>\n<tr>\n<td>2002<\/td>\n<td>2<\/td>\n<td>Northridge<br \/>\nHighland Park<\/td>\n<\/tr>\n<tr>\n<td>2003<\/td>\n<td>1<\/td>\n<td>Reseda<\/td>\n<\/tr>\n<tr>\n<td>2004<\/td>\n<td>0<\/td>\n<td>\u2013<\/td>\n<\/tr>\n<tr>\n<td>2005<\/td>\n<td>0<\/td>\n<td>\u2013<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\nSource: Los Angeles City Clerk's Office.\n\nBID, business improvement district.\n\n## Data\n\nThe data consist of the yearly counts of officially recorded violent felony crimes (homicide, rape, robbery and aggravated assault) by the Los Angeles Police Department that were aggregated from police reporting districts to corresponding BID areas. If a reporting district is present in a BID this district is counted as receiving the BID intervention. A total of 179 reporting districts are present in the 30 BID neighbourhood areas. We focus primarily on the counts of robbery, because this crime is less susceptible to underreporting by the police, is more likely than other crimes to occur in public settings between strangers and just over 30% of people who are robbed experience an injury.13\u201315 Nearly one out of five robberies nationally result in a serious injury that requires medical treatment, such as a gunshot wound, knife\/stab\/slash wound, broken bones or teeth, internal injury and\/or loss of consciousness.16 17 The estimated social costs of an average robbery are high, with an average cost of US\\$39\u2008287 (in 2005 dollars) if one includes monetary costs associated with medical and emergency services, lost productivity, mental health and general quality of life.18 By focussing our analysis primarily on violent crimes, and in particular robbery, we are offering a closer look at the effective role that BID expend on crime prevention efforts that attempt to improve the social control of public space areas through environmental design modifications and spending on private security. We do not examine property crime offences because they are less likely to be reported to the police, many occur in private settings (eg, larceny\/theft) and are not 'street crimes' directly subject to BID efforts to enhance social control in public spaces.\n\n## Time trends\n\n<a href=\"#tbl2\" data-ref-type=\"table\">Table 2<\/a> presents a summary of the average frequency of robbery and all violent crimes for areas exposed to BID compared with non-BID areas that incorporate the rest of Los Angeles. The simple linear trend of these data indicates that BID areas experienced greater, on average, yearly reductions in the incidence of robbery and violence than non-BID areas. For example, the average yearly reduction in robbery was 1.9 in BID areas, compared with 1.2 in non-BID areas. The log of the average robbery counts indicates a 7% reduction in BID areas compared with a 5.7% reduction in non-BID areas. During the 12-year period, however, the average yearly count of reported violent offences dropped by 58% for Los Angeles as a whole, suggesting that it is important to take into account this secular trend in assessing the effects of BID on violent crimes.\n\nAverage crimes, by year in BID and non-BID reporting districts\n\n| Year         | Non-BID areas (n=893) |            | BID areas (n=179) |            |\n|--------------|-----------------------|------------|-------------------|------------|\n|              | Robbery               | Violent    | Robbery           | Violent    |\n|              | M (median)            | M (median) | M (median)        | M (median) |\n| 1994         | 25.88 (17)            | 64.27 (42) | 41.6 (35)         | 86.53 (66) |\n| 1995         | 24.01 (16)            | 60.89 (41) | 41.56 (35)        | 89.75 (74) |\n| 1996         | 21.06 (13)            | 54.70 (35) | 34.3 (29)         | 77.35 (61) |\n| 1997         | 16.85 (11)            | 49.14 (33) | 29.37 (24)        | 70.52 (57) |\n| 1998         | 13.06 (8)             | 42.99 (28) | 22.34 (17)        | 59.77 (51) |\n| 1999         | 11.82 (7)             | 40.58 (26) | 19.98 (16)        | 56.46 (46) |\n| 2000         | 12.90 (8)             | 43.63 (26) | 21.66 (18)        | 60.26 (51) |\n| 2001         | 15.43 (10)            | 49.38 (31) | 26.20 (21)        | 68.74 (55) |\n| 2002         | 14.05 (8)             | 44.56 (26) | 24.83 (20)        | 62.94 (54) |\n| 2003         | 13.60 (8)             | 42.24 (26) | 23.92 (19)        | 60.21 (51) |\n| 2004         | 11.81 (7)             | 36.65 (22) | 19.43 (15)        | 49.12 (44) |\n| 2005         | 11.49 (7)             | 26.49 (15) | 18.06 (14)        | 37.91 (34) |\n| Average      | 16.00 (9)             | 46.29 (28) | 26.94 (10)        | 64.96 (52) |\n| Linear trend | \u22121.2                  | \u22122.5       | \u22121.9              | \u22123.6       |\n\nEach column presents the average (M) and median (50th percentile) values for the number of incidents in business improvement district (BID) and non-BID areas.\n\n## Statistical model\n\nWe used a Bayesian hierarchical model to assess the pre\u2013post effects of BID adoption in areas that were exposed to BID. We estimate the BID effect in each individual area and the average BID effect across all areas. We model the number of reported robberies and violent crimes with a Poisson distribution with mean (*\u03bb~it~*) for each of the 30 BID areas (*i*) over a 12-year (*t*) time period. We include a random effect parameter for areas to scale the time trend for the volume of crimes in each BID area, and to account for time invariant differences across the 30 BID areas. We account for the overall 12-year crime time trend in Los Angeles with a natural cubic spline.19 As the yearly incidence of violent crimes for Los Angeles as a whole was declining over the 12-year study period, it is important to account for the secular trend in the model so as not to overestimate the BID effect. The population at risk of violent crimes in each BID area is unknown, representing a mix of residents and non-residents (eg, shoppers). We bypass the problem of having to estimate the population at risk of violent crimes by comparing every BID area with itself. Under the assumption that the population at risk does not change over time, we propose a model that has as the main parameter of interest for area *i* the ratio (*K~i~*) of the crime rate if a BID was adopted divided by the crime rate that the trends predict would have happened if the BID had not been adopted. If *K~i~* is less than 1, this indicates that the presence of a BID in area (*i*) is associated with a reduction in the incidence of violent crimes for that area. We use a Bayesian hierarchical model to assess individual effects across each BID area (*i*) and to approximate the average overall BID effect (*\u03bc~K~*) across all 30 areas.20 Full technical details on the model and specification of priors are available elsewhere.12\n\nThe yearly violent crime trends for a sample of 12 of the 30 BID areas are displayed in <a href=\"#fig1\" data-ref-type=\"fig\">figure 1<\/a> and show the timing of each of the BID interventions (denoted by a vertical line). <a href=\"#fig1\" data-ref-type=\"fig\">Figure 1<\/a> provides a visual sense for the fact that the Bayesian hierarchical model is estimating an interrupted time series for each unique BID area and then pooling the effect across all areas. The Poisson model implies the counterfactual that the rate of violent crimes in an area after the BID becomes fully operational is proportional to what the rate would have been in that area had the BID not been implemented.\n\n# Results\n\n<a href=\"#tbl3\" data-ref-type=\"table\">Table 3<\/a> reports the overall BID effect for the incidence of robbery and violent crimes in terms of the percentage reductions (posterior mean) associated with the adoption of BID and the 95% posterior probability intervals. <a href=\"#tbl3\" data-ref-type=\"table\">Table 3<\/a> also reports the posterior probability of observing an overall BID effect on reducing the incidence of robbery and violent crimes (P(*\u03bc~K~*\\<1), where *\u03bc~K~* represents the overall effect across the 30 BID areas. We find that the posterior probability of a BID effect is 0.96. In other words, there is strong evidence that BID reduced the robbery rate. The estimated percentage reduction: obtained as one minus the posterior mean (*\u03bc~K~*) indicates a 12% average reduction (95% posterior probability interval \u22122 to 24) in the incidence of robbery associated with the implementation of BID. For the total incidence of violence BID effects are in the same direction, but the statistical evidence is not as strong (P(*\u03bc~K~* \\<1)=0.91), and indicates an 8% average reduction (95% posterior probability interval \u22125 to 21) in the total incidence of violence associated with the adoption of BID.\n\nEstimated percentage reduction in reported violent crimes from BID\n\n| Outcomes | Posterior mean<a href=\"#table-fn1\" data-ref-type=\"table-fn\">*<\/a> | 95% Posterior probability interval | Posterior probability BID effect (P(\u03bc~*K*~ \\<1)) |\n|----|----|----|----|\n| Robbery | 12 | (\u22122,24) | 0.96 |\n| Violent | 8 | (\u22125,21) | 0.91 |\n\nPosterior mean reflects the percentage reduction as calculated by (1\u2212*\u03bc~K~*)\u00d7100.\n\nBID, business improvement district.\n\nGiven that the observed probability of an overall BID effect was strongest for robbery, <a href=\"#tbl4\" data-ref-type=\"table\">table 4<\/a> reports the BID area-specific effects for official reports of robbery in terms of percentage reductions in robberies (*1\u2013K~i~)*. The individual BID area results for robbery show that for 14 of the 30 areas the posterior probability of observing a BID effect is 0.90 or higher. In terms of effect sizes for these 14 areas, the robbery rate is reduced by a high of 27% in the Century Corridor BID to a low of 14% in the Downtown Industrial BID. For another two BID areas, the observed posterior probability is more than 0.80, which still provides evidence for the presence of a BID effect in the expected direction. The BID effects appear to be most pronounced for the incidence of robbery, which one would expect is likely to be affected by environmental features of the neighbourhoods, such as hiring private security officers, which are the focus of BID efforts to control public space areas. Overall, there seems to be evidence in the data that the BID in Los Angeles had an effect in reducing the incidence of reported robberies.\n\nArea-specific estimates of BID effects on robbery\n\n| BID name | Posterior mean (1\u2013*K~i~*) | BID effect (p=*K~i~*\\<1) | 95% Posterior CI |\n|----|----|----|----|\n| Granada Hills | 18 | 0.93 | \u22126, 37 |\n| Chatsworth | 5 | 0.65 | \u221220, 25 |\n| Northridge | 18 | 0.94 | \u22125, 36 |\n| Reseda | 15 | 0.90 | \u22129, 33 |\n| Canoga Park | 3 | 0.60 | \u221224, 25 |\n| Van Nuys | 26 | 0.99 | 7, 41 |\n| Tarzana | \u221210 | 0.25 | \u221244, 16 |\n| Encino | 11 | 0.76 | \u221222, 35 |\n| Sherman Oaks | 10 | 0.76 | \u221218, 31 |\n| Studio City | 9 | 0.76 | \u221220, 31 |\n| Los Feliz Village | 21 | 0.98 | 1, 39 |\n| Highland Park | 11 | 0.83 | \u221214, 30 |\n| Hollywood Entertainment | 9 | 0.80 | \u221216, 28 |\n| Hollywood Media | 15 | 0.95 | \u22125, 32 |\n| Larchmont Village | 34 | 0.99 | 5, 53 |\n| Wilshire Centre | 4 | 0.63 | \u221225, 26 |\n| Los Angeles Chinatown | 21 | 0.98 | 0, 38 |\n| Westwood Village | 21 | 0.97 | \u22121, 39 |\n| Downtown Centre | 7 | 0.74 | \u221217, 25 |\n| Historic Core | 1 | 0.55 | \u221221, 21 |\n| Toy District | 8 | 0.77 | \u221216, 27 |\n| Fashion District | \u221224 | 0.05 | \u221263, 5 |\n| Downtown Industrial | 14 | 0.90 | \u20138, 31 |\n| Figueroa Corridor | 20 | 0.96 | \u22122, 36 |\n| Jefferson Park | 17 | 0.95 | \u22124, 33 |\n| Century Corridor | 27 | 1.00 | 8, 43 |\n| Wilmington | \u20137 | 0.28 | \u221234, 14 |\n| San Pedro | 8 | 0.75 | \u221218, 29 |\n| Lincoln Heights | 11 | 0.77 | \u221220, 34 |\n| Greater Lincoln Heights | 25 | 1.00 | 6, 41 |\n\n*K~i~*=ratio of robbery crimes (post-business improvement district (BID)\/pre-BID). Bold indicates a BID with a probability of a BID effect of \u22650.90.\n\nGiven the size of the BID effects appear to be most pronounced for robbery, this raises the question of how much BID spending occurs in relation to social costs saved by reducing robberies. Multiplying our estimated 12% (annual) reduction to the average incidence of robberies (*M*=160.7) associated with BID implementation to the estimated social costs (US\\$39\u2008287 in 2005 dollars)18 of an average robbery shows a marginal cost saving of approximately US\\$757\u2008611 (in 2005 dollars) (annually). Given that the average annual budget of the 30 BID in Los Angeles was approximately US\\$736\u2008670 (in 2005 dollars),12 this suggests that a sizeable social cost\u2013benefit of BID implementation can be attributed to the reductions in robbery alone.\n\n## Additional tests\n\nWe also conducted several additional tests on our measures of violent crime and the methods for approximating BID effects. Homicides, for example, are the most accurately reported violent crimes, but we did not discuss the yearly trends in homicide because the counts are so low. The average number of homicides per year in neighbourhoods associated with BID is less than one, and the median (50th percentile) is zero. The point estimates from replicating our model for homicide counts varies widely, and the probability of detecting a BID effect is low (P(\u03bc~K~\\<1)=0.43)), suggesting that BID have no appreciable effect on homicide. In particular, the homicide model indicates a 5% increase associated with BID adoption but with a large CI (95% posterior probability interval \u221250 to 29), resulting from low counts and imprecision in our estimate. A combined estimate of the count of robbery and homicide together was statistically identical to that of robbery alone, suggesting that the BID adoption effect observed is driven by the rate of robberies.\n\nWe also constructed alternative model specifications in line with previous work that Brooks5 used on an analysis of the effects of BID on reported serious crimes in Los Angeles during earlier years. We compared the estimated effect of BID implementation on robberies and all reported violent crimes using all police reporting districts in Los Angeles as the unit of analysis, including those that do not intersect BID areas. We then included a dummy variable denoting the timing of BID, adjacent neighbourhoods to BID as control variables, and fixed-effect terms (dummy variables) for each individual reporting district, year, and their interactions (reporting district\\*year). Our results from these specifications were sensitive to the parameterisation of the outcomes. If we relied on ordinary least squares regression we found a statistically significant BID effect in reducing the mean incidence of robberies (b=\u22124.63; p\\<0.001) and all violent crimes (b=\u22127.33; p\\<0.001) by approximately 16% and 11%, respectively. However, if we relied on a Poisson regression model the results were substantially lower and were only marginally significant for robberies (b=\u22120.02; p=0.07) and total violence (b=\u22120.01; p=0.09), reducing the respective incidence by 3% and 2%. We think this sensitivity test provides further justification for our use of a simpler model that estimates only the BID effects for those areas that eventually adopted BID, rather than assigning BID effects to the entire city of Los Angeles.\n\n## Limitations\n\nThe Bayesian hierarchical model provides an estimate of the effect of BID on the incidence of robbery and violent crimes in areas that were exposed to BID. Like all models this approach has several limitations. First, the model assumes that the population at risk of violence does not change once a BID starts. It is, however, possible that the establishment of a BID could change the population at risk of violent crimes in a number of ways. If, for example, there is a substantial increase in the number of shoppers or new residents because a BID was implemented then even a substantial decline in the incidence of violence would be offset by an increase in the denominator for the population at risk. Such an increase in the population at risk of violence would lead one to conclude that the adoption of the BID did not have an effect in reducing violence. Assuming that the population at risk coincides with the residential population in an area would be incorrect, as it is almost certain that successfully implemented BID attract a larger number of people for commerce to areas.\n\nSecond, the model assumes that the level to which violent crimes are reported to the police does not change systematically with the adoption of a BID. If, however, the adoption of a BID implies an increase in local merchants' and residents' willingness to report crimes to the police and an increased response from the police to combat crime, then the violent crime reports may actually increase as a function of the implementation of a BID. If this were the case, the adopted model would suggest that adopting a BID has the effect of increasing the incidence of violent crimes. Given that the findings suggest an overall effect of BID on reducing the robbery rate and marginal effects for all violent crimes, we have some confidence in these results.\n\nIn addition, if one assumes that BID areas have unique features in terms of the businesses that operate and the communities that encourage their establishment, constructing a group of comparison areas that are matched to the BID areas with respect to certain demographic features of area residents would represent a less conservative test of the effects of BID. We think that the areas that will eventually adopt a BID are the best comparison group for those areas that have already adopted a BID, as there are clearly features of BID areas that are unique in their ability to get a majority of landowners and merchants interested in their adoption. At the same time, our analysis offers no prescription for the various mechanisms by which BID impact robbery rates. BID adopt a variety of tactics, such as mobilising the police, hiring private security officers, street cleaning and environmental redesign to increase a sense of cleanliness and safety of BID areas. Unfortunately, the tactics adopted by each BID area are complex and not easy to approximate in a statistical model.\n\n# Discussion and conclusions\n\nThe results from this study suggest that BID reduce the rate of robbery crimes in affected Los Angeles neighbourhoods. The overall effect of BID on robberies is consistent with the efforts that many of these Los Angeles BID expend on improving the physical appearance of their areas to make them more attractive to commercial business and less attractive to potential offenders (eg, painting over graffiti, increased street lighting, closed-circuit television, or CCTV, cameras). The size of the BID effect on robberies varies across the 30 BID areas and appears to indicate a greater than expected reduction in robberies in those located in neighbourhoods that have undergone significant patterns of economic development or invested heavily in crime prevention. For example, BID area-specific effects were apparent in Jefferson Park and Figueroa Corridor, which are situated close to the University of Southern California, in areas of notable gentrification and economic development. Hollywood Media and Larchmont also exhibited BID-area-specific effects on the incidence of robberies and are situated in neighbourhoods undergoing gentrification. BID effects are present in Century Corridor, Figueroa Corridor and Hollywood Media, all of which invest heavily in crime prevention through hiring private security officers and other activities. 10\u201312 Los Angeles BID spend a considerable share of their resources hiring private security or public ambassadors who focus on keeping streetscapes clean and safe, thereby increasing the level of social control in public spaces. Approximately 13 of the 30 BID in Los Angeles spend more than US\\$200\u2008000 a year (2005 dollars) on such 'clean' and 'safe' efforts. These strategies are closely linked to research and theory on crime prevention through environmental design to reduce opportunities for crime and violence and, in particular, robbery.21\u201323\n\nGiven the limited budgets and staff of many BID, it is of no surprise that the mere presence of a BID is not uniformly associated with a reduced incidence of violence. Some BID spend as much as half their annual budgets on crime prevention and environmental redesign or beautification efforts. BID crime prevention activities may also garner additional resources from the police, as the police now have an active 'partner' in a community. Other established Los Angeles BID have relatively small budgets and focus their efforts primarily on place promotion in an effort to foster improved commercial activity for their constituent businesses.12 While the protocol for establishing a BID in Los Angeles is uniform and codified into law,12 the dosage of tactics to improve neighbourhood environments varies between BID areas. Relying on a conservative estimate of pre\u2013post changes in reported violent crimes for only those areas that adopted BID we found significant overall effects on robbery, with some BID areas exhibiting greater effects than others. We cannot say whether spending on private security or economic development efforts caused these reductions or are merely correlated with them. This study relied on observational data, which limits our ability to infer as to whether the correlations observed are causally related. We attempted to remove the potential selection effects of establishing a BID by estimating pre\u2013post effects on reported violent crimes for only those areas that eventually adopted a BID. In the absence of an experimental design in which BID are randomly assigned to neighbourhoods we do not know whether BID activities actually caused the declines in robbery rates.\n\nThe efforts spent by BID in Los Angeles on economic development activities and social control efforts that focus on crime prevention, beautification and advocating for more public safety and sanitation services to many blighted sections of Los Angeles12 are associated with a reduction in the incidence of robberies. This information can assist in designing and testing the feasibility of BID as a community-level violence prevention intervention.\n\n###### What is already known on this subject\n\n-   The incidence of violence is associated with neighbourhood environments.\n\n-   BID focus on public safety.\n\n-   Community economic development models may help reduce crime.\n\n###### What this study adds\n\n-   The first systematic look at the effects of BID on violent crimes.\n\n-   Rigorous methodology for assessing the effects of BID on violent crimes.\n\n# References","meta":{"dup_signals":{"dup_doc_count":153,"dup_dump_count":53,"dup_details":{"curated_sources":2,"2023-06":1,"2022-33":2,"2022-27":1,"2020-45":2,"2020-40":1,"2019-26":1,"2018-51":1,"2018-43":1,"2018-26":1,"2018-22":2,"2018-17":1,"2018-13":1,"2018-05":2,"2017-51":1,"2017-43":4,"2017-39":2,"2017-34":3,"2017-30":1,"2017-22":4,"2017-17":1,"2017-09":5,"2017-04":2,"2016-50":1,"2016-44":4,"2016-40":4,"2016-36":3,"2016-30":3,"2016-26":2,"2016-22":3,"2016-18":3,"2016-07":4,"2015-48":4,"2015-40":2,"2015-35":2,"2015-32":3,"2015-27":4,"2015-22":3,"2015-14":4,"2014-52":3,"2014-49":5,"2014-42":9,"2014-41":6,"2014-35":7,"2014-23":7,"2014-15":6,"2023-50":1,"2015-18":4,"2015-11":3,"2015-06":4,"2014-10":2,"2013-48":3,"2013-20":1,"2024-10":1}},"file":"PMC2976613"},"subset":"pubmed_central"}
{"text":"abstract: Pollination improves the yield of most crop species and contributes to one-third of global crop production, but comprehensive benefits including crop quality are still unknown. Hence, pollination is underestimated by international policies, which is particularly alarming in times of agricultural intensification and diminishing pollination services. In this study, exclusion experiments with strawberries showed bee pollination to improve fruit quality, quantity and market value compared with wind and self-pollination. Bee-pollinated fruits were heavier, had less malformations and reached higher commercial grades. They had increased redness and reduced sugar\u2013acid\u2013ratios and were firmer, thus improving the commercially important shelf life. Longer shelf life reduced fruit loss by at least 11%. This is accounting for 0.32 billion US\\$ of the 1.44 billion US\\$ provided by bee pollination to the total value of 2.90 billion US\\$ made with strawberry selling in the European Union 2009. The fruit quality and yield effects are driven by the pollination-mediated production of hormonal growth regulators, which occur in several pollination-dependent crops. Thus, our comprehensive findings should be transferable to a wide range of crops and demonstrate bee pollination to be a hitherto underestimated but vital and economically important determinant of fruit quality.\nauthor: Bj\u00f6rn K. Klatt; Andrea Holzschuh; Catrin Westphal; Yann Clough; Inga Smit; Elke Pawelzik; Teja Tscharntkee-mail: <firstname.lastname@example.com>\ndate: 2014-01-22\ninstitute: 1Agroecology, Department of Crop Sciences, University of G\u00f6ttingen, Grisebachstrasse 6, 37077 G\u00f6ttingen, Germany; 2Centre for Environmental and Climate Research, University of Lund, S\u00f6lvegatan 37, 22362 Lund, Sweden; 3Department of Animal Ecology and Tropical Biology (Zoology III), Biocenter, Am Hubland, University of W\u00fcrzburg, 97074 W\u00fcrzburg, Germany; 4Quality of Plant Products, Department of Crop Sciences, Carl-Sprengel-Weg 1, 37075 G\u00f6ttingen, Germany\nreferences:\ntitle: Bee pollination improves crop quality, shelf life and commercial value\n\n# Introduction\n\nAgricultural production forms one of the most important economic sectors \\[1\\]. The quantity of most crop species is increased by pollination \\[2,3\\], which is a highly important, but also seriously endangered \\[4\\] ecosystem service. More than 75% of the 115 leading crop species worldwide are dependent on or at least benefit from animal pollination, whereas wind and self-pollination are sufficient for only 28 crop species \\[2\\]. Thereby, animal pollination contributes to an estimated 35% of global crop production \\[2\\]. It is mostly pollination-dependent crops such as fruits that contribute to a healthy human diet by providing particularly high amounts of essential nutrients such as vitamins, antioxidants and fibre \\[5,6\\]. Berries especially have been found to benefit human health and are increasingly used for therapies against chronic diseases and even cancer \\[5\\]. First attempts for sustaining pollination and other ecosystem services have been aligned in a strategic plan of the Convention on Biological Diversity in Nagoya in 2010. However, recent decisions, such as the new Common Agricultural Policy of the European Union (EU), still endanger ecosystem services by promoting high-intensity agricultural management. Thus, the value of pollination and other ecosystem services is still underestimated or even disregarded in national and international policies. In this study, we expand our knowledge of the underestimated benefits of bee pollination by experimentally quantifying its impacts on crop quantity, quality, shelf life and market value. This should contribute to a better understanding of its monetary and social importance, thereby enhancing a sustainable implementation in future policies. We used strawberries (*Fragaria x ananassa* DUCH.), a crop whose worldwide cultivation is on the increase \\[1\\], as a model system.\n\nStrawberry plants flower in several successive flowering periods within a season, with flowers becoming smaller over time \\[7\\]. Varieties are self-compatible in most cases, and stigmas become receptive before the anthers of the same flower release pollen, so that allogamy is favoured. Bee pollination increases strawberry weight and shape. Effects depend on varieties \\[8,9\\], presumably owing to differences in pollinator attraction \\[10\\] and their dependence on cross-pollination \\[7\\]. Recent findings about metabolic processes in strawberries support the idea that pollination may also impact shelf life \\[11\u201314\\]. Owing to high fruit sensitivity to fungal infections and mechanical injuries, strawberry fruits have a short shelf life \\[12\\]. More than 90% of fruits can become non-marketable after only 4 days in storage \\[15\\]. Several studies addressed the potential elongation of the shelf life of strawberries with modified storage procedures \\[15\u201319\\], which highlights how economically important this problem is. Crop features allowing longer storage and thereby, reducing post-harvest losses in supermarkets and households are of major interest worldwide \\[20\\], but have so far not been analysed in terms of pollination. Shelf life and pathogenic susceptibility of strawberries are mostly related to fruit firmness \\[15\\], but surface colour and sugar\u2013acid\u2013ratios are also involved \\[15\u201319\\]. Fruit colour further determines consumers perception and influences their purchasing behaviour \\[19\\], but has never been related to animal pollination. In addition, only few studies report a relation of pollination to firmness \\[21\u201323\\] and sugar contents \\[22,24\u201327\\] of fruits. Hence, comprehensive economic gains of bee pollination are largely unknown and in particular, the potential effect on commercially important parameters of the overall fruit quality has not yet been explored.\n\nWe set up a field experiment with nine commercially important strawberry varieties and assessed the influence of self, wind and bee pollination on strawberry fruits using exclusion treatments. We expected that: (i) bee-pollinated fruits would have higher numbers of fertilized achenes, the true 'nut' fruits of the strawberry, owing to higher pollination success compared with wind- and self-pollination; (ii) bee pollination would therefore lead to fruits with higher commercial value compared with wind- and self-pollinated fruits, owing to less malformations improving commercial grades and higher fruit weight; as well as (iii) higher firmness and longer shelf life; and (iv) bee-pollinated fruits should have a more intense red colour and lower sugar\u2013acid\u2013ratios, thus improving the post-harvest quality of strawberries.\n\n# Methods\n\n## Experimental set-up\n\nIn 2008, we planted nine commercially important strawberry varieties of *Fragaria x ananassa* DUCH. (Darselect, Elsanta, Florence, Honeoye, Korona, Lambada, Salsa, Symphony, Yamaska) on an experimental field. The field was subdivided in 12 plots, and nine rows per plot planted with 18 plants of a single variety per row. All varieties were present in all plots. The sequence of the rows within the plots was randomized. The field was surrounded by two further rows of strawberries to weaken edge effects. Five honeybee hives (*Apis mellifera* L.) and approximately 300 trap nests dominated by *Osmia bicornis* L., have been established for several years close to the field to ensure stable pollination services. Experiments were conducted in 2009 in the first yield year using exclusion treatments on two plants per variety and plot. Following the consecutive flowering periods of strawberries \\[7\\], all buds of a plant were covered with Osmolux bags (Pantek, Montesson, France) to allow only self-pollination (self-pollination treatment), gauze bags (mesh width 0.25 mm) to allow self- and wind pollination (wind pollination treatment) or remained uncovered to allow additional insect pollination (bee pollination treatment), respectively. Osmolux bags are semipermeable for water and steam, so that microclimate differences between bagged and unbagged flowers were kept at a minimum. Gauze bags do not create an atmosphere closed from outside the bags and thus have no influence on microclimate. Bags were removed directly after fruit set, when petals began to wither and fall off the flower, and the first approach of a fruit was visible, about 7 days after flower opening. At least 50 fruits per variety and treatment were harvested at maturity. All analyses except the titratable acid content were conducted on the same day of harvesting to avoid influence on post-harvest quality owing to water loss and metabolic procedures.\n\nWe collected insect pollinators under favourable weather conditions (*T* \\> 17\u00b0C; low cloud cover; wind speed less than 4 m s^\u22121^) in 2010, using standardized transect walks. Four strawberry varieties (Honeoye, Elsanta, Korona, Lambada) were selected based on their flowering time, so that all other varieties were flowering at the same time as at least one of these four varieties. Thus, pollinators were collected across the entire flowering season of the commercial strawberry field. Each transect consisted of one row of strawberries per plot of each of the selected varieties. On each of 4 days, four different plots of the experimental field were randomly selected and insects pollinating strawberry flowers were collected using sweep nets on each of the four selected varieties. Each transect was visited for 10 min. Pollinators were identified to species level, and data were pooled across all varieties.\n\n## Commercial value\n\n### Weight and commercial grades\n\nWe calculated the commercial value of each fruit based on fruit weight (BA2001 S, Sartorius, G\u00f6ttingen, Germany) and the market value of strawberry fruits, which is based on the availability of fruits on the market and commercial grades. Fruits were sorted into commercial grades, owing to aberrations in shape (deformations), colour (areas with yellow or green colour) and size (fruit diameter), following the official trade guidelines \\[28\\]. B.K.K. was trained by experienced strawberry growers and colleagues on how to apply the EU trade guidelines. Fruits with no or only slight deformations, with minimal areas of yellow or green colour which did not affect their general appearance, and with a minimum diameter of 18 mm, were sorted into grade extra\/one. Fruits showing distinct deformations and larger areas with yellow or green colour, but that had a minimum diameter of 18 mm were classified as grade two. Non-marketable fruits had strong deformations, large areas of yellow or green colour or were of a diameter smaller than 18 mm. Aberrations in colour usually occurred in combination with fruit deformations and were thus not treated separately. Following the above-mentioned Commission's regulation, grades extra and one can be treated separately, but are used combined in practice. We calculated proportions of fruits for each commercial grade and pollination treatment across all varieties (<a href=\"#RSPB20132440F2\" data-ref-type=\"fig\">figure 2<\/a>*b*) and also separately for each variety (see the electronic supplementary material, S2).\n\nWe obtained the commercial value of each fruit by multiplying its weight with its market value per gram \\[29\\]. The latter was assessed based on harvest time and commercial grades. Harvest time influences the market value of strawberry fruits owing to the availability of fruits on the market: the more fruits that are available, the lower the market value. Thus, fruits that are sorted into lower commercial grades have lower market values. Finally, we extrapolated commercial value to 1000 fruits for a better relationship to market situations.\n\n### Firmness and shelf life\n\nWe bisected fruits and measured firmness at the centre of each half according to Sanz *et al.* \\[17\\] with the following modifications: we fitted the texture analyser (TxT2, Stable Micro System, Surrey, UK) with a 5 mm diameter probe and a 25 kg compression cell, and used a maximum penetration of 4 mm.\n\n## Post-harvest quality\n\nColourimetric analysis was applied according to Caner *et al.* \\[19\\] at two opposite sides of the centre of each fruit in the Lab-colour space using a portable colourimeter (CR-310 Chromameter, Konica Minolta, Badhoevedorp, The Netherlands). The total soluble solids are strongly correlated to the total sugar content of a solution and were measured using a handheld refractometer (HRH30, Kr\u00fcss, Hamburg, Germany). Measurements for each fruit were conducted twice and repeated when the values differed by more than 0.2 Brix. Fruits were freeze-dried (Epsilon 2-40, Christ, Osterode, Germany), and all samples from the same plant were pooled and milled. To account for an average water content of 82%, which was analysed on a sample of 250 fruits, 0.18 g of each freeze-dried sample was diluted in 20 ml distilled water and titrated according to Caner *et al.* \\[19\\].\n\n## Pollination success\n\nWe used at least eight fruits from each variety and treatment to analyse the number of fertilized achenes per fruit, which represent pollination success. Each fruit was blended in 100 ml distilled water for two minutes (Speedy Pro GVA 1, Krups, Offenbach, Germany). Fertilized achenes are heavier than water and sink to the bottom, whereas aborted achenes are lighter and accumulate at the water surface. Fertilized achenes were counted (Contador, Pfeuffer, Kitzingen, Germany) after drying for 48 h at 85\u00b0C.\n\n## Statistical analysis\n\nIn the case of repeated measurements per fruit, we calculated mean values for fruit characteristics. We fitted linear mixed-effects models with random effects allowing treatment slopes and intercepts to vary among varieties \\[30\\], using R \\[31\\]. To account for space and time errors and unbalance in the data, the random part was completed by two further terms: the interaction of plot, variety and plant, whereas flowering period was included as a crossed random effect. Response variables were commercial value per fruit, fruit weight, firmness, surface colour values (red colour, brightness, yellow colour) and number of fertilized achenes. In the models with sugar\u2013acid\u2013ratio as response variable, only plot and variety were used to complete the random part, because sugar\u2013acid\u2013ratios were calculated based on arithmetic means per plant.\n\nBee, wind and self-pollination treatments were used as fixed effect levels. To test whether pollination treatments differed and whether there was a main effect of all pollination treatments across all varieties, a model with unpooled treatment levels (full model), models with successively pooled treatment levels and a model without treatment as fixed effect were compared \\[30\\] using second order Akaike's information criterion (AICc) and likelihood \\[32\\]. This allowed us to test whether all treatments, specific treatment levels only, or none of the treatments had an effect on the response variables. The latter case was taken to indicate that treatment effects were specific to the variety and without a shared common effect between varieties. Residuals were inspected for constant variance, and transformations were used to account for non-normality and heterogeneity, where necessary. Main effect values and parameter estimates were extracted from the model and used for plotting after back transformation.\n\nPearson's chi-squared analysis was used to calculate differences between pollination treatments in the number of fruits for each commercial grade. Differences were shown in proportions for better illustration (<a href=\"#RSPB20132440F1\" data-ref-type=\"fig\">figure 1<\/a>*b*).\n\n# Results\n\n## Commercial value\n\n### Weight and commercial grades\n\nStrawberry flowers were mainly visited by bees (98.5%). Wild bees were most abundant (64.6%), whereas *A. mellifera* L. (33.9%) and non-bee pollinators (flies: 1.6%) were found less often (<a href=\"#RSPB20132440TB1\" data-ref-type=\"table\">table 1<\/a>). The solitary wild bee *O. bicornis* L. (52.0%) was the most abundant pollinator, whereas other wild bee species accounted for less than 5% of the bee community.\n\nPollinators visiting strawberry flowers on the experimental field. (To identify the main pollinators of strawberry flowers on the experimental field, four varieties were randomly selected and insects visiting strawberry flowers were collected. Sweep netting was conducted for 10 minutes on four transects that were randomly selected on each of four different days in 2010. Strawberries were mainly pollinated by solitary wild bees with *O. bicornis* L. being the most frequent species, while honeybees (*Apis mellifera* L.) and non-bee pollinators were less abundant.)\n\n| species                      | abundance | proportion | functional group |\n|:-----------------------------|-----------|------------|:-----------------|\n| *Osmia bicornis* L.          | 66        | 52.0       | wild bee         |\n| *Apis mellifera* L.          | 43        | 33.9       | honeybee         |\n| *Bombus terrestris* L.       | 5         | 3.9        | wild bee         |\n| *Andrena flavipes* Panz.     | 3         | 2.4        | wild bee         |\n| *Merodon equestris* F.       | 2         | 1.6        | fly              |\n| *Andrena gravida* Imhoff     | 2         | 1.6        | wild bee         |\n| *Bombus hypnorum* L.         | 1         | 0.8        | wild bee         |\n| *Bombus lapidarius* L.       | 1         | 0.8        | wild bee         |\n| *Bombus lucorum* L.          | 1         | 0.8        | wild bee         |\n| *Bombus pascuorum* Scop.     | 1         | 0.8        | wild bee         |\n| *Bombus pratorum* L.         | 1         | 0.8        | wild bee         |\n| *Andrena chrysosceles* Kirb. | 1         | 0.8        | wild bee         |\n| total wild bees              | 82        | 64.6       | \u2014                |\n| total honeybees              | 43        | 33.9       | \u2014                |\n| total non-bees (flies)       | 2         | 1.6        | \u2014                |\n\nBee pollination resulted in strawberry fruits with the highest commercial value (<a href=\"#RSPB20132440F1\" data-ref-type=\"fig\">figure 1<\/a>*a*). On average, bee pollination increased the commercial value per fruit by 38.6% compared with wind pollination and by 54.3% compared with self-pollination. Fruits resulting from wind pollination had a 25.5% higher market value than self-pollinated fruits. Pollination treatments were stronger than differences between varieties and thus had a main effect across all varieties (see <a href=\"#RSPB20132440TB2\" data-ref-type=\"table\">table 2<\/a> for AICc and likelihood values). Our results suggest that altogether, bee pollination contributed 1.12 billion US\\$ to a total of 2.90 billion US\\$ made with commercial selling of 1.5 million tonnes of strawberries in the EU in 2009 \\[1\\]\u2014but so far without consideration of the monetary value provided by enhanced shelf life (see below). Price and marketability of strawberries depend on commercial grades of fruit quality (shape, size and colour) \\[28\\]. Malformations, in particular, are a common problem affecting strawberry price and marketability \\[33\\]. Our experiment showed that pollination treatments significantly differed in the number of fruits for each commercial grade (**\u03c7**~4~^2^ = 60.504; *p* \\< 0.001, *n* = 1895). Bee pollination reduced malformations and thus enhanced marketability in all varieties except the variety Symphony (<a href=\"#RSPB20132440F1\" data-ref-type=\"fig\">figure 1<\/a>*b*; see the electronic supplementary material, S2 for variety values). The highest proportion of bee pollinated fruits was assigned to the best grade extra\/one, whereas non-marketable fruits formed the smallest fraction. By contrast, wind and self-pollination led to high proportions of non-marketable fruits. When compared with wind and self-pollination, bee pollination not only improved fruit shape, but also fruit weight (<a href=\"#RSPB20132440F1\" data-ref-type=\"fig\">figure 1<\/a>*c*). Bee-pollinated fruits were on average 11.0% heavier than wind-pollinated and 30.3% heavier than self-pollinated fruits. Pollination treatments were stronger than differences between varieties and thus had a main effect across all varieties (see <a href=\"#RSPB20132440TB2\" data-ref-type=\"table\">table 2<\/a> for AICc and likelihood values).\n\nDelta AICc values and likelihood resulting from model comparisons. (AICc = 0 indicates the model with the highest explanatory power. Lower delta AICc and higher likelihood indicate better explanatory power of a model. Likelihood was calculated for models with delta AICc of less than seven \\[32\\]. Best explaining models are highlighted in italics. Sample sizes are given in brackets behind fruit parameters. None, no treatment level pooled; sans, model without fixed effect.)\n\n| fruit parameter | pooled levels |  |  |  |  |\n|:---|:---|:---|:---|:---|:---|\n|  | none | bee = wind | wind = self | bee = self | sans |\n| commercial value (*n* = 1892) |  |  |  |  |  |\n| *\u2003*AICc | *0* | 4.512 | 0.173 | 3.527 | 2.501 |\n| *\u2003*likelihood | *0.403* | 0.042 | 0.370 | 0.069 | 0.115 |\n| fruit weight (*n* = 1895) |  |  |  |  |  |\n| *\u2003*AICc | *0* | 4.162 | 3.507 | 4.872 | 3.137 |\n| *\u2003*likelihood | *0.627* | 0.078 | 0.109 | 0.055 | 0.131 |\n| shelf life (*n* = 1268) |  |  |  |  |  |\n| *\u2003*AICc | *0* | 0.347 | 1.791 | 7.218 | 5.273 |\n| *\u2003*likelihood | *0.431* | 0.362 | 0.174 | \u2014 | 0.031 |\n| red colour (*n* = 1279) |  |  |  |  |  |\n| *\u2003*AICc | 1.428 | 1.608 | *0* | 2.021 | 0.323 |\n| *\u2003*likelihood | 0.155 | 0.142 | *0.317* | 0.115 | 0.270 |\n| sugar\u2013acid\u2013ratio (*n* = 345) |  |  |  |  |  |\n| *\u2003*AICc | 2.128 | 3.244 | *0* | 1.247 | 1.147 |\n| *\u2003*likelihood | 0.131 | 0.075 | *0.378* | 0.203 | 0.213 |\n| pollination success (*n* = 356) |  |  |  |  |  |\n| *\u2003*AICc | *0* | 4.267 | 9.192 | 8.704 | 7.290 |\n| *\u2003*likelihood | *0.894* | 0.106 | \u2014 | \u2014 | \u2014 |\n\n### Shelf life\n\nBee pollination strongly impacted the shelf life of strawberries by improving their firmness (<a href=\"#RSPB20132440F2\" data-ref-type=\"fig\">figure 2<\/a>*a*). The firmness values of each treatment and variety were related to shelf life, measured as the number of days until 50% of fruits had been lost owing to surface and fungal decay (see the electronic supplementary material, S3). Higher firmness resulting from bee pollination potentially elongated the shelf life of strawberry fruits by about 12 h compared with wind pollination, and by more than 26 h compared with self-pollination. After 4 days in storage, only 29.4% of the wind-pollinated fruits and none self-pollinated fruit were still marketable, whereas, at the same time, 40.4% of the bee-pollinated fruits remained in a marketable condition. Thus, bee pollination accounted for a decrease of at least 11.0% in fruit losses during storage. These findings suggest that the value for bee pollination calculated in \u00a73a(i) has to be increased to accommodate this impact on the shelf life of strawberries. Hence, pollination benefits on the shelf life of strawberries potentially added another 0.32 billion US\\$ to the commercial value of strawberry pollination (without shelf-life effects: 1.12 billion US\\$). In total, bee pollination contributed 1.44 billion US\\$ to a total of 2.90 billion US\\$ made with the commercialization of 1.5 million tonnes of strawberries in the EU in 2009 \\[1\\]. Pollination treatments had a main influence on shelf life across all varieties (see <a href=\"#RSPB20132440TB2\" data-ref-type=\"table\">table 2<\/a> for AICc and likelihood values). Varieties producing fruits with high firmness benefitted most from bee pollination.\n\n## Post-harvest quality\n\nIn most varieties, bee-pollinated fruits had a more intense red colour compared with fruits resulting from wind and self-pollination (<a href=\"#RSPB20132440F2\" data-ref-type=\"fig\">figure 2<\/a>*b*). Self-pollinated fruits of the varieties Lambada and Symphony showed the most intense red colour in the self-pollination treatment. The bee pollination treatment differed from the two other pollination treatments across all varieties, whereas strong variety differences imped a difference between wind and self-pollination treatments (see <a href=\"#RSPB20132440TB2\" data-ref-type=\"table\">table 2<\/a> for AICc and likelihood values). The brightness of bee- and wind-pollinated fruits was similar and highly correlated to yellow colour intensity (see the electronic supplementary material, S4 and S5). Both colour properties did not differ between bee and wind pollination, but self-pollinated fruits were darker and had less intense red colour. Thus, bee pollination resulted in bright fruits with a more intense red colour than wind pollination fruits, whereas self-pollinated fruits were darker and less red (<a href=\"#RSPB20132440F2\" data-ref-type=\"fig\">figure 2<\/a>*b* and the electronic supplementary material, S4).\n\nSenescence of strawberries is not only related to losses in firmness and colour changes, but also to increasing sugar\u2013acid\u2013ratios. Bee-pollinated fruits generally had a lower sugar\u2013acid\u2013ratio compared with wind- and self-pollinated fruits across all varieties (<a href=\"#RSPB20132440F2\" data-ref-type=\"fig\">figure 2<\/a>*c*), but fruits of the varieties Elsanta and Symphony had a higher sugar\u2013acid\u2013ratio with bee pollination. The difference between wind and self-pollination remained variety-dependent (see <a href=\"#RSPB20132440TB2\" data-ref-type=\"table\">table 2<\/a> for AICc and likelihood values), whereas the sugar\u2013acid\u2013ratio of fruits resulting from bee pollination differed from both other treatments across all varieties.\n\n## Pollination success\n\nPollination success was related to the number of fertilized achenes dependent on pollination treatments. Bee pollination was much more efficient than wind and self-pollination, resulting in a higher number of fertilized achenes per fruit across all varieties (<a href=\"#RSPB20132440F3\" data-ref-type=\"fig\">figure 3<\/a>; see <a href=\"#RSPB20132440TB2\" data-ref-type=\"table\">table 2<\/a> for AICc and likelihood values). Bee pollination on average increased the number of fertilized achenes by about 26.8% compared with wind pollination and about 61.7% compared with self-pollination. Wind-pollinated fruits had a 47.7% higher number of fertilized achenes than fruits resulting from self-pollination. This confirms our findings to be true effects of bee pollination.\n\n# Discussion\n\nWe found bee pollination, which was mainly conducted by solitary wild bees, to play a key role for several features of the quantity and quality of strawberry fruits. Bee pollinated fruits showed less malformations, greater fruit weight and longer shelf life, resulting in higher commercial value as well as improved post-harvest quality by more intensive red colour and lower sugar\u2013acid\u2013ratios than fruits resulting from wind and self-pollination.\n\nThe mechanism behind the benefits of strawberry pollination by bees is based on the fertilization of the true 'nut' fruits of the strawberry, the achenes \\[11\u201314\\]. During their visits, bees allocate pollen homogeneously on the receptacles, increasing the number of fertilized achenes per fruit \\[34\\]. While unfertilized achenes resulting from insufficient pollination have no physiological functionality, fertilized achenes produce the plant hormone auxin \\[35\\], which mediates the accumulation of gibberellic acid \\[14\\]. Together, these plant hormones induce fruit growth by improving cell progeny and size, thereby enhancing the weight of strawberry fruits \\[12\\]. This further improves fruit quality and thereby commercial grades \\[12\\] by preventing malformations, which are caused by areas of unfertilized and thus physiologically inactive achenes \\[33\\].\n\nHow can pollination induce a longer shelf life in strawberries? The shelf life of strawberries and other fruits is mostly dependent on their firmness \\[15,33\\], which is also functionally based on fertilized achenes \\[33\\] and thus dependent on successful pollination. Auxin and gibberellic acid delay fruit-softening and thereby enhance firmness and shelf life, by limiting the expression of several fruit-softening proteins, the so-called expansins \\[11\\]. Higher levels of both plant hormones also increase the post-harvest quality of strawberries. Although auxin alone reduces the accumulation of anthocyanins \\[11\\], high levels of both auxin and gibberellic acid can, in conjunction, increase anthocyanin accumulation \\[12\\]. In contrast to firmness and colour changes, sugar\u2013acid\u2013ratios of strawberries are not directly affected by auxin and gibberellic acid \\[12\\]. But higher firmness of fruits is associated with more stable cell walls which might reduce respiration, which is known to limit metabolic processes affecting sugar and acid contents during storage \\[19\\]. Indirect positive effects of pollination are therefore probable.\n\nPlant hormones that can influence the quality of fruits and vegetables are known to occur not only in strawberries, but also in several other crops \\[36\\] that require animal pollination \\[7\\]. Crops such as coffee \\[37\\] and blueberry \\[38\\] benefit from animal pollination in terms of fruit set and fruit size; and it has been shown elsewhere that fruit shape can benefit from increased animal pollination \\[7\\]. This indicates that our findings may be transferable to a high variety of crops and that animal pollination may largely contribute to crop quality. However, only few studies have focused to date on effects of pollination other than the effects on crop yield and fruit set. It has been shown that the sugar content of loquats \\[24,26\\], vine cactus \\[25\\] and oriental melon \\[22\\] as well as the firmness of oriental melon \\[22\\] and cucumber \\[23\\] can be increased by animal pollination. Contrasting results are available for the tomato, whereas Al-Attal *et al.* \\[21\\] showed that pollination increased the firmness of tomatoes in greenhouses, but pollination had no effect on the firmness of cherry tomatoes under field conditions \\[27\\]. Oilseed rape is another important crop whose quality benefits from insect pollination by higher oil content and lower chlorophyll content \\[39\\]. These results support the assumption of a general impact of pollination on multiple aspects of crop quality. However, such comprehensive findings about the benefits of pollination on crop quality, yield and commercial value as in our study, which can be mechanistically well linked to formerly reported physiological processes, have never, to our knowledge, been reported before.\n\nOur results showed strawberries to be almost exclusively visited by bees, with solitary wild bees being most abundant. This contrasts with earlier findings, where honeybees were the most common pollinators of strawberries and other crops \\[7\\] and further shows that the wild bee pollination can be important for crop production, if wild bees are abundant close to crop fields. Wild bee pollinators have already been shown to be effective crop pollinators \\[40\\], including strawberries \\[41\\]. Additional experiments are required to assess the current abundance of wild bee pollinators and thus their importance for strawberry production on commercial strawberry fields under conventional management conditions.\n\nIn our study, we used an innovative approach to the calculation of the commercial value of pollination by considering not only overall yield \\[2,3\\] but also crop quality in terms of trade classes, shelf life and changing market values. Shelf life is a major factor determining the commercial value of pollination. Globally, between one-third and a half of all fruits and vegetables are lost due to mechanical damage and deterioration during handling, transport and storage directly after harvest, or wasted at retailer and consumer levels \\[42\\]. This illustrates the commercial and social importance of crop shelf life and the far-reaching impact of pollination deficits.\n\nOf course, our calculations may still underestimate the commercial value of bee pollination as they are not considering commercial pollination benefits related to colour, sugar\u2013acid\u2013ratio and other taste components.\n\n# Conclusion\n\nIn conclusion, our results showed that crop pollination is of higher economic importance than hitherto thought. Plant hormones, the production of which is mediated by pollination, occur in several other pollination-dependent fruits and vegetables \\[36\\]. This highlights the major importance of animal pollination for crop quality in other crops in addition to strawberries. Quality improvements of crops can greatly affect marketability and contribute to reducing food loss and waste. In the industrialized countries, 30\u201350% of all crops are thrown away at retail and consumer levels \\[20,42\\]. Under the current scenario of rapid human population increase and global food demand \\[43\\], achieving high quality and quantity of crops is a pressing issue. Our study suggests that comprehensive analyses of the benefits of pollination for animal-dependent crops, which comprise 70% of all major crop species \\[2\\], may clearly increase estimates of the economic value of this ecosystem service. Pollination appears to be economically much more important than previously recognized and needs better support through adequate agricultural management and policy.\n\n## Acknowledgements\n\nWe thank, K. M. Krewenka, J. Fr\u00fcnd, C. Scherber, M. von Fragstein, G. Everwand, B. Scheid, K. Klatt and H. P.-G. Klatt for comments on the manuscript and N. Bl\u00fcthgen, D. Kleijn and L. Garibaldi for reviewing the manuscript prior to submission. We thank the members, students and technicians of the agroecology group for their field assistance.\n\n## Funding statement\n\nThis work has been supported by the German Research Foundation (DFG) and the SAPES research environment. Data can be accessed on request from the Agroecology at G\u00f6ttingen University (see <http:\/\/www.uni-goettingen.de\/en\/74726.html> for contact details) and 3TU.Datacentrum (see <http:\/\/datacentrum.3tu.nl\/en\/home\/>).\n\n# References","meta":{"dup_signals":{"dup_doc_count":118,"dup_dump_count":43,"dup_details":{"curated_sources":2,"2018-43":1,"2018-39":2,"2018-30":2,"2018-26":1,"2018-22":1,"2018-17":1,"2018-13":2,"2018-09":1,"2018-05":2,"2017-51":2,"2017-47":2,"2017-43":2,"2017-39":3,"2017-34":2,"2017-30":1,"2017-26":2,"2017-22":2,"2017-17":1,"2017-09":5,"2017-04":3,"2016-50":4,"2016-44":5,"2016-40":4,"2016-36":4,"2016-30":7,"2016-26":4,"2016-22":4,"2016-18":3,"2016-07":3,"2015-48":4,"2015-40":2,"2015-35":3,"2015-32":4,"2015-27":2,"2015-22":3,"2015-14":3,"2014-52":4,"2014-49":5,"2018-47":1,"2017-13":3,"2015-18":3,"2015-11":1,"2015-06":2}},"file":"PMC3866401"},"subset":"pubmed_central"}
{"text":"abstract: # Introduction\n  .\n  National Health Service hospitals and government agencies are increasingly using mortality rates to monitor the quality of inpatient care. Mortality and Morbidity (M&M) meetings, established to review deaths as part of professional learning, have the potential to provide hospital boards with the assurance that patients are not dying as a consequence of unsafe clinical practices. This paper examines whether and how these meetings can contribute to the governance of patient safety.\n  .\n  # Methods\n  .\n  To understand the arrangement and role of M&M meetings in an English hospital, non-participant observations of meetings (n=9) and semistructured interviews with meeting chairs (n=19) were carried out. Following this, a structured mortality review process was codesigned and introduced into three clinical specialties over 12\u2005months. A qualitative approach of observations (n=30) and interviews (n=40) was used to examine the impact on meetings and on frontline clinicians, managers and board members.\n  .\n  # Findings\n  .\n  The initial study of M&M meetings showed a considerable variation in the way deaths were reviewed and a lack of integration of these meetings into the hospital's governance framework. The introduction of the standardised mortality review process strengthened these processes. Clinicians supported its inclusion into M&M meetings and managers and board members saw that a standardised trust-wide process offered greater levels of assurance.\n  .\n  # Conclusion\n  .\n  M&M meetings already exist in many healthcare organisations and provide a governance resource that is underutilised. They can improve accountability of mortality data and support quality improvement without compromising professional learning, especially when facilitated by a standardised mortality review process.\nauthor: Juliet Higginson; Rhiannon Walters; Naomi Fulop**Correspondence to** Professor Naomi Fulop, Department of Applied Health Research, University College London, 1-19 Torrington Place, London WC1E 7HB, UK; <firstname.lastname@example.com>\ndate: 2012-05-03\ninstitute: NIHR King's Patient Safety and Service Quality Research Centre, King's College London, London, UK\nreferences:\ntitle: Mortality and morbidity meetings: an untapped resource for improving the governance of patient safety?\n\n# Introduction\n\nIn recent years, there has been an increasing international interest in using mortality rates to monitor the quality of hospital care.1 2 Concern about patient safety and scrutiny of mortality rates intensified in the UK with the extensive coverage of investigations into National Health Service (NHS) hospital failures and the Dr Foster report with its patient safety rating for NHS trusts.3 4 As a consequence, boards of healthcare organisations now require assurance that the care they provide is safe and that patients are not dying through failure of their services. Many trusts include mortality rates in their performance scorecards or dashboards and actively engage with national patient safety improvement initiatives, such as Patient Safety First and Safer Patient Initiative, to reduce mortality rates.5 6\n\nA forum that has traditionally reviewed in-hospital deaths is the long-standing Mortality and Morbidity (M&M) meeting established by surgeons to further professional education. In regularly reviewing deaths and complications, these meetings have the potential to provide accountability and the necessary improvement measures required for patient safety as well as professional learning. How effective they are in fulfilling these additional roles remains unexplored.\n\nIn many countries, M&M meetings are embedded within the medical curriculum for doctors in training.7 Junior doctors present cases to other doctors for reflection on diagnostic or treatment decision-making, and in return they receive clinicopathological wisdom and learn presentation skills. In the past, the brief discussions between the clinicians about the causes of death were thought to be effective peer review and an adequate means of changing practice.8 9 Little attention was paid to analysing the causes of deaths for quality improvement.10 11 Studies have shown that for M&M meetings to facilitate improvement and be more than a forum for peer review, they need to be structured and systematic in reviewing and discussing deaths, directing discussions towards improving system and process variations.12\u201314 Studies recommended that to support this, junior doctors' training should include more focus on systematic process change and less on medical error in M&M meetings.15\u201317\n\nHistorically, M&M meetings have been led and attended only by the medical profession and have remained autonomous, with knowledge not being available or shared with other professions or across the wider hospital governance framework. This 'silo' working has led to a lack of organisational learning and accountability.18 Increasingly hospitals are beginning to integrate M&M meetings into their governance processes, by making them mandatory and more accountable for reviewing deaths and taking corrective action should adverse events arise.19\u201321 To support this, the US Agency for Healthcare Research and Quality has produced web-based guidance for case analysis.22\n\nTraditionally, adverse outcomes discussed at M&M meetings have been attributed to individual competence in treating patients rather than the system or process failures involved with the care.11 23 Although both contribute to errors, the focus on individuals has led clinicians to fear embarrassment and loss of reputation, making them reluctant to speak openly about errors at meetings.24 25 This defensive behaviour is thought to be counterproductive to eliminating adverse events and assuring safe care.26 27\n\nIn light of this evidence we wanted to see whether and how M&M meetings in an English teaching hospital could facilitate quality improvement, be accountable and provide assurance within the organisation's governance processes. Using a structured mortality review process as a facilitating mechanism, we wanted to assess what impact this would have on the original focus of the meetings and on professional learning; to explore how hospital staff viewed the changes; and evaluate the potential that a different format of M&M meeting could offer the organisation.\n\n# Methods\n\n## Setting\n\nThe participating organisation was an English NHS teaching hospital offering specialist tertiary services in addition to general and surgical care. The study was carried out in close collaboration with the hospital to inform and support other strategies for reducing mortality rates. The single case study allowed us to study both horizontally (ie, across divisions) and vertically (up and down the managerial and professional hierarchies) within the organisation and this approach allowed us to make analytic generalisations rather than statistical ones.28\n\n## Initial assessment\n\nDuring 2009, we studied the characteristics and processes of all the hospital M&M meetings and their position within the governance framework using non-participant observations at meetings (n=9) and semistructured interviews with meeting chairs and governance managers (n=19). The results showed considerable variation in meeting structure, format and case presentations, confirming the findings from previous studies. We found that the responsibility for managing these meetings was devolved to clinical groups and as a consequence they had developed independently and in individual ways. There was no formal reporting structure from these meetings into the wider hospital governance to inform or assure the board that deaths were not occurring as a result of unsafe care.\n\nFollowing this we codeveloped a standardised mortality review form and database with hospital staff, based on recommendations in the literature and drawing on the method of examining death used by the National Confidential Enquiry into Patient Outcome and Death and the Scottish Audit of Surgical Mortality.29\u201332 The review form focused on whether the death was avoidable; on issues arising from the care of the patient; and on whether these could have contributed to the patient's death. It recorded where actions were necessary to address any problems; whether an adverse incident report was needed; and who was going to take any actions forward (Mortality review form: Appendix 1, web only). We developed and piloted the review form and database which were then introduced into three clinical specialties selected purposively for their patient cohort. This process was carried out from January to December 2010 during which time we studied whether and how this standardised mortality review process supported M&M meetings to contribute to wider governance processes.\n\n## Participants\n\nFive care groups across two divisions agreed to participate in the study. Three care groups agreed to adopt a standardised mortality review process, two from a general medical division and one from a specialist division. One group in each division agreed to be a study control and not implement the review process but participate in the evaluation process. The two clinical divisions were selected to participate because they regularly experienced high numbers of deaths albeit for contrasting reasons. Patients admitted to the general medical division had a range of complex medical problems and multiple comorbidities, were often frail and elderly and had numerous possible causes of death. In contrast, patients admitted into the second division, which offered specialist tertiary services, had a narrower range of specialty-specific causes of death, such as acute or end-stage organ failure.\n\n## Data collection\n\nWe used qualitative methods, interviews and observations to understand how and whether the standard mortality review process was or was not used, and how and whether it supported M&M meetings in contributing to governance.\n\nForty semistructured interviews were carried out by the researcher (JH), including frontline staff (n=32) and at senior executive level (n=8). (Interview schedule: Appendix 2, web only). Participants were recruited with the aim of acquiring a broad sample across professional and occupational groups (Sampling matrix: Appendix 3, web only). Interviews were carried out with Chairs of M&M meetings (n=5) as well as a range of consultants (n=6), junior doctors (n=6), nursing staff (n=7) who attended the meetings, and some who were not invited, to capture a wide range of experiences and views. Managers and senior clinicians (n=8), senior executives and board members (n=8) were selected because of their governance role.\n\nInterviewees were provided with study details when they were invited to participate and were assured of anonymity and confidentiality. Consent to participating and being recorded was obtained at the start of the interview. Interviews lasted approximately 45\u2005min. Frontline staff interviewees were asked to identify the role and format of M&M meetings; the importance of quality improvement in making care safer; views about the introduction of the review form and process; and the governance of M&M meeting outcomes and accountability. Senior executives were asked what patient safety data they currently received; the role that M&M meetings played in providing that data; and how they perceived the potential for a standardised process for reviewing deaths in the hospital governance of patient safety.\n\nIn addition to the interviews, we used non-participant observations at M&M (n=26) and governance (n=4) meetings to provide background and context to the interviewees' comments. Consent for these was obtained prior to commencing the data collection.\n\n## Data analysis\n\nObservations were organised in Microsoft Excel. Data from interviews were dual coded (JH and RW), generating inductive and deductive themes, which were agreed with a third researcher (NF) who read a sample of transcripts. Data were organised and analysed using NVivo 9 software.\n\n# Findings\n\nTwo of the three specialties implemented the review process throughout the study. The remaining specialty helped with the development of the process but refrained from testing the final version. We present our findings under the following three main headings: how M&M meetings contributed to the governance of patient safety within the hospital; how M&M meetings provided a resource for learning and accountability; and what impact the standardised review process had on both activities.\n\n# The contribution of M&M meetings to the governance of mortality data\n\nDuring the study, changes occurred in the governance of mortality data at both trust and divisional levels of the organisation as shown in <a href=\"#fig1\" data-ref-type=\"fig\">figure 1<\/a>. The left hand side of the figure outlines the prestudy governance processes for mortality data within the general medical division and hospital and shows no upward reporting from divisions to the hospital board and only informal cascading of information down to frontline staff. The right hand side shows the changes to governance introduced during the course of the study.\n\nA monthly high-level trust safety committee was established to monitor externally published risk adjusted mortality rates, investigate outliers identified by the national regulator (the Care Quality Commission) and receive 6-monthly divisional reports from M&M meetings. It provided quarterly reports and assurance to a quality subcommittee of the board. M&M meetings within each division began reporting quarterly to the divisional risk and governance committee, using outcome measures produced from the standardised review process. Improvement measures determined by the M&M meetings were communicated to the frontline staff through newly established ward-based specialty governance and clinical management meetings. This strengthening of divisional and hospital governance arrangements may have been shaped in part by participation in, and feedback from, the study and a greater awareness of the potential of M&M meetings to contribute to governance.\n\nAll interviewees agreed that senior executives and board members should be accountable for the safety of patient care, especially when 'things went wrong', and that reporting outcomes from M&M meetings to a trust committee necessary. Illustrative interview quotes are shown in <a href=\"#box1\" data-ref-type=\"boxed-text\">box 1<\/a>.\n\n###### The contribution of Mortality and Morbidity meetings to the governance of mortality data\n\n'Obviously, it's very important for the trust to know what's going on mortality-wise across the entire trust. And actually I don't think you can have meetings that are about such a significant area of care without that going to a higher group of people. It's all very well to say yes well we've got mortality meetings in all of the divisions and they're all going fine, but how does anybody know? \u2026 Because at the end of the day they're going to be the ones who are answering the phone when the press sort of ring up and say ah we hear that your standardised mortality ratio is through the roof!' (Consultant).\n\n'I think that information from these meetings can provide assurance that we don't have excess deaths. It helps us to pre-empt problems perhaps before we are alerted. By looking at morbidity cases we can also try to avoid problems before they lead to a fatality.' (Chair, Safety Monitoring Committee).\n\n'I think that it's really important to \u2026 know that we have learned from the experience, and so how you capture that and reassure management that we won't make the same mistake again is really important\u2026.' (Board member).\n\nM&M participants viewed reporting to this Safety Monitoring Committee as acceptable provided that the environment was non-judgemental and understanding of the case mix of their patients. Some clinicians and managers appreciated the committee's focus on data while others wished for an opportunity to share learning from other specialties, escalate unresolved issues and receive support for change. This was contrary to the views of two senior executives who thought the role of the committee was to monitor mortality rates, and that it was the divisions' responsibility to address quality and safety issues.\n\n# M&M meetings as a resource for learning, improvement and accountability\n\nObservations of M&M meetings during January to December 2010 showed considerable variation in the structure, organisation and the process of reviewing deaths as shown in <a href=\"#tbl1\" data-ref-type=\"table\">tables 1<\/a> and <a href=\"#tbl2\" data-ref-type=\"table\">2<\/a>. Responsibility for the meetings was devolved to the specialties which had led them to be non-standard and autonomous.\n\nSummary of the structure and format of Mortality and Morbidity meetings\n\n| Meeting characteristics | General medical division |  |  | Specialist division |  |\n|----|----|----|----|----|----|\n|  | Test group 1 | Test group 2 | Control 1 | Test group 3 | Control 2 |\n| Frequency | Weekly | Monthly | Monthly | Monthly and then weekly | Not formalised |\n| Timing | Working hours | Lunchtime | Lunchtime | Lunchtime | Working hours |\n| Length | 20\u201330\u2005min | 1\u2005h | 1\u2005h | 3\u2005h then 1\u2005h | 1\u2005h |\n| Venue | Departmental seminar room | Lecture theatre in medical school | Hospital committee room | Departmental seminar room | Departmental seminar room |\n| Reason for organisation | Part of a management meeting | Part of a grand round programme | Capacity and semiformal setting | Local and easily accessible | Part of the professional development programme |\n| Attendees | Consultants, a junior doctor representative, clinical director, senior nurses, managers | Consultants, junior doctors, senior nurse, governance managers | Consultants, junior doctors | Consultants, junior doctors, clinical director, nurses | Consultants, junior doctors, clinical director nurses, pharmacists and dietician |\n| Number of attendees | 12 | 15\u201335 | 18\u201320 | 24\u201341 | 25 |\n\nSummary of the review processes carried out by Mortality and Morbidity (M&M) meetings\n\n|  | General medical division |  |  | Specialist division |  |\n|----|----|----|----|----|----|\n|  | Test group 1 | Test group 2 | Control 1 | Test group 3 | Control 2 |\n| Source of mortality data for meetings | Departmental database | Inpatient episode data | Inpatient discharge data | Inpatient episode data | Departmental database |\n| How meetings informed | M&M coordinators extracted cases from database | Hospital intelligence unit notified M&M coordinators | Software programme designed by M&M co-chair | Hospital intelligence unit notified M&M coordinators | M&M coordinators extracted cases from database |\n| Numbers of cases reviewed | 1\u20136 | 10\u201342 | 8\u201315 | 2\u20137 | 5\u20136 |\n| Criteria for selection | All deaths | All deaths | All deaths | All deaths | 'Interesting' cases |\n| Review process | Standardised mortality review | Standardised mortality review | Departmental structured process | No formalised structure | No formalised structure |\n| Meeting records | Standardised mortality review and minutes | Standardised mortality process | Excel spreadsheet | Minutes | Minutes |\n| Issues identified and recorded | Yes | Yes | Yes | Yes | Yes |\n| Actions specified | Yes | Yes | Yes | Yes | Yes |\n| Actions assigned | Yes | Yes | No | No | No |\n| Actions followed up at next meeting | No | No | Yes | No | No |\n| Record circulated | Minutes yes | No | Yes | Available for viewing but not circulated | Available at next meeting |\n\n## Meeting frequency\n\nClinical staff found weekly meetings helpful as the cases were still fresh in their minds and the small number allowed time for indepth discussion. However, the chair of a meeting that met monthly suggested that weekly meetings were less 'special' and would lose their impact.\n\n## Venue\n\nTo encourage attendance, meetings were held at lunchtime or during working hours as part of the department's education programme. One specialty held their M&M review as part of a larger management meeting that considered adverse incidents, complaints and other 'bad bits' of the service. Incorporating mortality reviews had the benefit of integrating and addressing all risk issues together, especially as it had 'all the right people round the table' to achieve changes. It reduced the number of meetings that clinicians with managerial responsibilities had to attend but had the disadvantage of not being open to all clinical staff.\n\nThe various ways in which M&M meetings reviewed deaths is summarised in <a href=\"#tbl2\" data-ref-type=\"table\">table 2<\/a>. One control group M&M meeting had already identified a need for a structured approach to their meetings and developed its own system for categorising deaths. However, this system did not identify the specific failures in care or action needed to address them.\n\n## Case presentations\n\nGenerally, junior doctors were tasked with reviewing deaths prior to meetings and presenting a summary of each case at the meetings. In one instance, 40 cases were presented in an hour and was 'mind-numbingly boring' according to one clinician. There was no time for indepth discussions making the review a tick box ceremony with errors liable to be 'swept under the table'. As peer review was also important to clinicians, many felt that all deaths should be presented to a wide audience. Achieving a balance between scrutinising all deaths by more than one person, and selecting and discussing avoidable deaths in depth, appeared to be challenging.\n\n## Participation and culture\n\nBoth doctors and nurses reported that it was important to know which cases were going to be discussed at meetings. They wanted to familiarise themselves with the relevant case details to 'defend' any course of action that they had, or had not taken and for nurses especially, to give them the confidence to participate. All interviewees stressed that M&M meetings should be blame-free to facilitate improvement and accountability, although some were not sure that this was true of their meetings. One control group had made a deliberate decision to develop a safe and non-critical environment before considering any other aspect of the meeting.\n\n## Accountability\n\nAll the participants saw learning and improvement in care as the purpose of M&M meetings and an essential part of the clinical activity. Many saw them as having an additional governance role as shown in <a href=\"#box2\" data-ref-type=\"boxed-text\">box 2<\/a>. There were no reports that the educational and learning role of the M&M meeting was compromised by a greater focus on accountability.\n\n###### Mortality and Morbidity (M&M) meetings as a resource for learning and accountability\n\n'I think the potential for them (M&M meetings) is absolutely massive. I think if we have a culture of openness and discussing these cases that are very difficult in a very frank robust way, and junior doctors and hopefully other staff come to that and see that discussion, take (learning) away with them, then I think that's very powerful. And I think that if we're able to look at trends and patterns, then you can also use that to influence policy or practice within the division and within our wards and within our daily work.' (Consultant).\n\n'In terms of reporting, I think \u2026 the Trust should know exactly what is happening just as we should as clinicians' (Chair, M&M meeting).\n\n'Everything should be transparent; what you are doing well and what can be improved. If there are failings in the system it should be readily available for board members.' (Junior doctor).\n\n'I think the big driver (for M&M meetings) would be about improving patient outcomes\u2026and linking it to the governance agenda, patient safety and patient experience and that would be how I, and ward managers, should be engaged in the process.' (Nurse).\n\n'I guess if I look back I kind of wonder how on earth we thought we had assurance of any description before we started having robust M&Ms. I guess the reality is we didn't question too hard and had we questioned we'd have said no we didn't have any assurance. So they have a very important role.' (Senior manager).\n\n# The impact of the standardised mortality review process\n\n## Improved structure to meetings\n\nCase reviewers liked how the standardised process directed the line of questioning and made it easier to extract the relevant information from case notes. Some thought it was helpful as a teaching aid for junior doctors. It highlighted and captured areas of concern and helped to focus and structure the meeting: 'helps stop the rambling'. Many thought this was what was needed in their meetings, as described in <a href=\"#box3\" data-ref-type=\"boxed-text\">box 3<\/a>.\n\n###### The impact of the standardised process on Mortality and Morbidity meetings\n\n'I think they've become much more structured, and again that's been partly helped by the fact that we have a form that actually provides a framework. I think without that things become very woolly; people can kind of get bogged down in looking at the micro aspects of morbidity and mortality, and not necessarily the bigger picture.' (Manager).\n\n## Improved case review\n\nThe review process brought standardisation to the examination of deaths and provided a framework that could be applied to patient deaths in different circumstances. It also provided an assurance that all deaths were reviewed in the same way, a fact that board members viewed positively. It gave the meetings more significance and made them 'official', reported one person, 'not just a paper exercise'. To maintain transparency in meetings clinicians suggested that the review form should be completed openly in the meeting.\n\n## Improved records\n\nThe electronic record of the standardised mortality review facilitated easier reporting and the identification of themes of issues over time. Clinicians spoke of its capacity to formalise 'organisational memory', those issues that arose infrequently and were only remembered by long-standing members of staff. Actions needed and assigned were more formally recorded, and in doing so improved the likelihood of corrective measures being undertaken. Equally important was the paper trail it provided of how problems were addressed and practice had changed.\n\n## Improved reporting and governance\n\nChairs and managers of the participating groups were especially positive about the support the process offered in producing performance reports for the trust Safety Monitoring Committee. M&M meeting outcomes\u2014the number of avoidable deaths, contributory factors and actions taken to address any quality issues\u2014were included in their twice-yearly report and were later developed into a standard reporting template by the committee.\n\nOne of the intervention groups in the general medical division identified a continuing problem with care which was identified through a more structured mortality review and addressed through an improved governance structure. This is outlined in <a href=\"#box4\" data-ref-type=\"boxed-text\">box 4<\/a>.\n\n###### An example of a change in practice in the division with improved governance\n\nAt a monthly departmental Mortality and Morbidity (M&M) meeting, all 22 deaths from the previous month were presented. These cases had been reviewed by two junior doctors in consultation with the M&M meeting chair. The junior doctors presented a summary of each case on a single PowerPoint slide, describing clinical details and causes of death. In four of the 22 cases, where the patients were not expected to die, the meeting chair led a greater examination and discussion. In one of these cases, an acutely ill patient admitted to a medical ward had not received the appropriate timely specialist input despite a referral having been made. The M&M meeting agreed that this omission constituted poor clinical care which potentially contributed to the death. 'Process issues and communication' were attributed to the lack of response to the referral. The case was also reviewed in the corresponding specialist M&M meeting.\n\n### Action\n\n1.  Specialty consultant invited to the next M&M meeting for an open discussion.\n\n2.  Referral guidelines to be produced jointly by the two departments.\n\n### Problem repeated\n\n1.  Failure to get prompt specialist input on a further occasion.\n\n### Action\n\n1.  Chair raised the issue at a quarterly divisional M&M meeting: clinical director advised specialist divisions of the need for compliance with new referral guidance.\n\n2.  Chair reported problem in his 6-monthly feedback to the trust Safety Monitoring Committee for hospital-wide compliance with the referral guidance.\n\n## Standardised process\n\nMany clinicians, and especially board members, saw the benefit of having a standardised process across the hospital to provide reassurance that all deaths were being reviewed in the same way. The majority of those interviewed wanted to see the review process extended to all hospital M&M meetings. Some participants of the control group M&M meetings, who had no experience of using the process, felt that if it was rigorously completed, the data would provide 'clear performance metrics which could be hugely valuable to the organisation'.\n\n## Concerns\n\nThere were a few less-positive comments from staff who had not experienced the review process first-hand. The specialty that decided not to continue testing the study review felt it would not advance their own process and might limit the openness of the discussion, thus jeopardising the 'fragile culture' of their meeting.\n\nSome meeting participants expressed concern that completing the review might become the focus of the meeting rather than it being an aid to direct discussion. Extra administrative support might also be needed. Others suggested that specialties might wish to add specialty-specific questions to the core template. Overall, the concerns were outweighed by support for the process.\n\n# Discussion\n\nM&M meetings are an established part of medical education and have existed for decades as forums for doctors to review and present cases for biomedical exploration. Their potential for quality improvement depends on them being more structured and reviewing deaths in a more systematic way. Changes to the curriculum of junior doctors' professional education have done much to promote this.24 33 34 Studies have also shown how specialty or departmental M&M meetings have improved quality by adopting structured reviews.14 23 35 Our findings build on these and show how M&M meetings can be integrated across the whole hospital and contribute to the accountability for deaths and governance of patient safety. Meetings that adopt a standardised and systematic review process can focus on systems and process failures; provide a record of meeting outcomes and follow-up actions taken to address failures; and facilitate the reporting of meeting outcomes and assurance to the board.\n\nWe had anticipated that there might be greater resistance from clinicians, who would want to maintain their autonomy and not want to codify and share sensitive data.36 37 Our findings show considerable support from them for both the standardised review process and the wider governance role of M&M meetings. There may be several reasons for a greater acceptance than expected. Health professionals are expected to take responsibility for governance of their own clinical practice and are increasingly aware that hospitals are being monitored for the quality of their care.38 Many are being co-opted into managerial roles such as clinical directors, medical directors and chairs of governance committees and are accountable for the governance of quality and safety data.36 39 This additional managerial responsibility encourages clinicians to value systems that make the provision of governance data easier.\n\nThe standard review process was generally accepted because it met with clinicians' requirements to capture the tacit nature of case context and complexity.18 26 Unlike many checklists it was adapted to their particular contextual needs which gave them ownership.40 Close collaboration during its development also helped to increase engagement and was seen less as a top-down imposition.41 42 Health professionals welcomed the focus on systems and process failures and away from individual competence, while senior executives and board members appreciated the standardisation and safety monitoring that the process supported.43 Suggestions for an M&M meeting review process that facilitates both learning and assurance, based on our findings, are listed in <a href=\"#box5\" data-ref-type=\"boxed-text\">box 5<\/a>.\n\n###### Suggestions for using Mortality and Morbidity (M&M) meetings for governance of patient safety\n\n-   Invite all types of health professionals to M&M meetings and notify them in advance which cases are to be reviewed and discussed.\n\n-   Employ a short, standardised review process that highlights avoidable deaths and contributory factors, allowing for staff involvement in the design and flexibility for specialty-specific questions as necessary.\n\n-   Allow adequate time for case discussions using the review questions as an aid.\n\n-   Encourage a focus on systems and process variations and not individual competence.\n\n-   Carry out the review openly, summarising actions at the end of the meeting and tracking them at the following one.\n\n-   Record meeting outcomes electronically to facilitate audit and performance data.\n\n-   Integrate M&M meetings into the wider governance structure and monitor meeting outcomes for shared learning and assurance.\n\nAlthough this is a single case study, personal communications with medical directors of four other NHS trusts championing patient safety helped place our findings into context. As with our case study, responsibility for M&M meetings in these hospitals had previously been devolved to clinicians and provided no assurance that deaths were being reviewed in a systematic and rigorous way. Outcomes from meetings were neither standardised nor integrated into the hospital governance. In the smaller hospitals, there was an increased focus on reviewing and following up deaths but this was carried out by individual senior clinicians. However, the larger hospitals were beginning to see the need for a more effective way of reviewing larger numbers of deaths and the important role that M&M meetings could play, and were introducing standard trust-wide review processes.\n\n# Conclusion\n\nM&M meetings have the potential to contribute to the governance of patient safety. They exist in many healthcare organisations and are a governance resource that is generally underutilised. They can improve the accountability of mortality data and support quality improvement without compromising professional learning, especially when facilitated by a standardised mortality review process.44\n\nThe research team would like to thank all the hospital staff who contributed to the research, and members of the PSSQ team for their helpful comments on earlier drafts of the paper.\n\n# References","meta":{"dup_signals":{"dup_doc_count":131,"dup_dump_count":62,"dup_details":{"curated_sources":2,"2023-50":2,"2022-40":1,"2022-21":1,"2022-05":1,"2021-49":1,"2021-43":1,"2021-17":1,"2020-29":2,"2020-16":1,"2020-05":1,"2019-47":1,"2019-43":2,"2019-39":2,"2019-18":1,"2018-30":2,"2018-26":1,"2018-17":1,"2018-13":1,"2018-09":1,"2018-05":1,"2017-51":2,"2017-47":1,"2017-43":3,"2017-39":4,"2017-30":4,"2017-22":4,"2017-17":3,"2017-09":5,"2017-04":1,"2016-50":2,"2016-44":3,"2016-40":3,"2016-36":3,"2016-30":2,"2016-26":1,"2016-22":2,"2016-18":2,"2016-07":2,"2015-48":2,"2015-40":1,"2015-35":2,"2015-32":2,"2015-27":1,"2015-22":3,"2015-14":2,"2014-52":2,"2014-49":2,"2014-42":7,"2014-41":4,"2014-35":2,"2014-23":5,"2014-15":7,"2024-22":1,"2024-18":1,"2017-13":1,"2015-18":2,"2015-11":1,"2015-06":2,"2014-10":3,"2013-48":2,"2013-20":1,"2024-30":1}},"file":"PMC3382446"},"subset":"pubmed_central"}
{"text":"abstract: **Trisha Greenhalgh and colleagues** argue that, although evidence based medicine has had many benefits, it has also had some negative unintended consequences. They offer a preliminary agenda for the movement's renaissance, refocusing on providing useable evidence that can be combined with context and professional expertise so that individual patients get optimal treatment\nauthor: Trisha Greenhalgh; Jeremy Howick; Neal MaskreyCorrespondence to: T Greenhalgh <email@example.com>\ndate: 2014\nreferences:\ntitle: Evidence based medicine: a movement in crisis?\n\nIt is more than 20 years since the evidence based medicine working group announced a \"new paradigm\" for teaching and practising clinical medicine.1 Tradition, anecdote, and theoretical reasoning from basic sciences would be replaced by evidence from high quality randomised controlled trials and observational studies, in combination with clinical expertise and the needs and wishes of patients.\n\nEvidence based medicine quickly became an energetic intellectual community committed to making clinical practice more scientific and empirically grounded and thereby achieving safer, more consistent, and more cost effective care.2 Achievements included establishing the Cochrane Collaboration to collate and summarise evidence from clinical trials;3 setting methodological and publication standards for primary and secondary research;4 building national and international infrastructures for developing and updating clinical practice guidelines;5 developing resources and courses for teaching critical appraisal;6 and building the knowledge base for implementation and knowledge translation.7\n\nFrom the outset, critics were concerned that the emphasis on experimental evidence could devalue basic sciences and the tacit knowledge that accumulates with clinical experience; they also questioned whether findings from average results in clinical studies could inform decisions about real patients, who seldom fit the textbook description of disease and differ from those included in research trials.8 But others argued that evidence based medicine, if practised knowledgably and compassionately, could accommodate basic scientific principles, the subtleties of clinical judgment, and the patient's clinical and personal idiosyncrasies.1\n\nTwo decades of enthusiasm and funding have produced numerous successes for evidence based medicine. An early example was the British Thoracic Society's 1990 asthma guidelines, developed through consensus but based on a combination of randomised trials and observational studies.9 Subsequently, the use of personal care plans and step wise prescription of inhaled steroids for asthma increased,10 and morbidity and mortality fell.11 More recently, uptake of the UK National Institute for Health and Care Excellence guidelines for prevention of venous thromboembolism after surgery has produced significant reductions in thromboembolic complications.12\n\nDespite these and many other successes, wide variation in implementing evidence based practice remains a problem. For example, the incidence of arthroscopic washout of the knee joint, whose benefits are unproved except when there is a known loose body, varies from 3 to 48 per 100\u2009000 in England.13 More fundamentally, many who support evidence based medicine in principle have argued that the movement is now facing a serious crisis (box 1).14 15 Below we set out the problems and suggest some solutions.\n\n# Box 1: Crisis in evidence based medicine?\n\n-   The evidence based \"quality mark\" has been misappropriated by vested interests\n\n-   The volume of evidence, especially clinical guidelines, has become unmanageable\n\n-   Statistically significant benefits may be marginal in clinical practice\n\n-   Inflexible rules and technology driven prompts may produce care that is management driven rather than patient centred\n\n-   Evidence based guidelines often map poorly to complex multimorbidity\n\n# Distortion of the evidence based brand\n\nThe first problem is that the evidence based \"quality mark\" has been misappropriated and distorted by vested interests. In particular, the drug and medical devices industries increasingly set the research agenda. They define what counts as disease (for example, female sexual arousal disorder, treatable with sildenafil16 and male baldness, treatable with finasteride17) and predisease \"risk states\" (such as low bone density, treatable with alendronate).18 They also decide which tests and treatments will be compared in empirical studies and choose (often surrogate) outcome measures for establishing \"efficacy.\"19\n\nFurthermore, by overpowering trials to ensure that small differences will be statistically significant, setting inclusion criteria to select those most likely to respond to treatment, manipulating the dose of both intervention and control drugs, using surrogate endpoints, and selectively publishing positive studies, industry may manage to publish its outputs as \"unbiased\" studies in leading peer reviewed journals.20 Use of these kinds of tactic in studies of psychiatric drugs sponsored by their respective manufacturers enabled them to show that drug A outperformed drug B, which outperformed drug C, which in turn outperformed drug A.21 One review of industry sponsored trials of antidepressants showed that 37 of 38 with positive findings, but only 14 of 36 with negative findings, were published.22\n\nEvidence based medicine's quality checklists and risk of bias tools may be unable to detect the increasingly subtle biases in industry sponsored studies.23 Some so called evidence based policies (such as dementia case finding for the over 75s and universal health checks for the over 40s in the UK) seem to be based largely on political conviction.24 25 Critics have condemned the role of the drug industry in influencing the policy makers who introduced them.26\n\n# Too much evidence\n\nThe second aspect of evidence based medicine's crisis (and yet, ironically, also a measure of its success) is the sheer volume of evidence available. In particular, the number of clinical guidelines is now both unmanageable and unfathomable. One 2005 audit of a 24 hour medical take in an acute hospital, for example, included 18 patients with 44 diagnoses and identified 3679 pages of national guidelines (an estimated 122 hours of reading) relevant to their immediate care.27\n\n# Marginal gains and a shift from disease to risk\n\nEvidence based medicine is, increasingly, a science of marginal gains\u2014since the low hanging fruit (interventions that promise big improvements) for many conditions were picked long ago. After the early big gains of highly active antiretroviral therapy for HIV28 and triple therapy for *Helicobacter pylori* positive peptic ulcer,29 contemporary research questions focus on the marginal gains of whether these drug combinations should be given in series or in parallel and how to increase the proportion of patients who take their complex medication regimen as directed.30 31\n\nLarge trials designed to achieve marginal gains in a near saturated therapeutic field typically overestimate potential benefits (because trial samples are unrepresentative and, if the trial is overpowered, effects may be statistically but not clinically significant) and underestimate harms (because adverse events tend to be underdetected or underreported). The 74 year old who is put on a high dose statin because the clinician applies a fragment of a guideline uncritically and who, as a result, develops muscle pains that interfere with her hobbies and ability to exercise, is a good example of the evidence based tail wagging the clinical dog. In such scenarios, the focus of clinical care shifts insidiously from the patient (this 74 year old woman) to the population subgroup (women aged 70 to 75) and from ends (what is the goal of investigation or treatment in this patient?) to means (how can we ensure that everyone in a defined denominator population is taking statins?).\n\nAs the examples above show, evidence based medicine has drifted in recent years from investigating and managing established disease to detecting and intervening in non-diseases. Risk assessment using \"evidence based\" scores and algorithms (for heart disease, diabetes, cancer, and osteoporosis, for example) now occurs on an industrial scale, with scant attention to the opportunity costs or unintended human and financial consequences.26\n\n# Overemphasis on following algorithmic rules\n\nWell intentioned efforts to automate use of evidence through computerised decision support systems, structured templates, and point of care prompts can crowd out the local, individualised, and patient initiated elements of the clinical consultation.8 For example, when a clinician is following a template driven diabetes check-up, serious non-diabetes related symptoms that the patient mentions in passing may not by documented or acted on.32 Inexperienced clinicians may (partly through fear of litigation) engage mechanically and defensively with decision support technologies, stifling the development of a more nuanced clinical expertise that embraces accumulated practical experience, tolerance of uncertainty, and the ability to apply practical and ethical judgment in a unique case.33\n\nTemplates and point of care prompts also contribute to the creeping managerialism and politicisation of clinical practice.8 As Harrison and Checkland observe: \"As the language of EBM becomes ever more embedded in medical practice, and as bureaucratic rules become the accepted way to implement 'the best' evidence, its requirements for evidence are quietly attenuated in favour of an emphasis on rules.\"34\n\nFor example, the Quality and Outcomes Framework (QOF) in UK general practice is incentivised by financial \"quality points\" and administered largely by non-clinical staff who generate these points by recalling patients for structured reviews and checks. QOF has been associated with significant improvements in blood pressure control, especially in deprived populations.35 But its downside is an audit driven, technocratic exercise in which few patients are offered personalised shared decision making with a senior clinician before having the recommended tests and treatments, and in which clinical consultations are continually interrupted by pop-up point of care prompts.32 36\n\n# Poor fit for multimorbidity\n\nFinally, as the population ages and the prevalence of chronic degenerative diseases increases, the patient with a single condition that maps unproblematically to a single evidence based guideline is becoming a rarity. Even when primary studies were designed to include participants with multiple conditions, applying their findings to patients with particular comorbidities remains problematic. Multimorbidity (a single condition only in name) affects every person differently and seems to defy efforts to produce or apply objective scores, metrics, interventions, or guidelines.37 Increasingly, the evidence based management of one disease or risk state may cause or exacerbate another\u2014most commonly through the perils of polypharmacy in the older patient.38\n\n# Return to real evidence based medicine\n\nTo address the above concerns, we believe it is time to launch a campaign for real evidence based medicine (box 2).\n\n## Box 2: What is real evidence based medicine and how do we achieve it?\n\n### Real evidence based medicine:\n\n-   Makes the ethical care of the patient its top priority\n\n-   Demands individualised evidence in a format that clinicians and patients can understand\n\n-   Is characterised by expert judgment rather than mechanical rule following\n\n-   Shares decisions with patients through meaningful conversations\n\n-   Builds on a strong clinician-patient relationship and the human aspects of care\n\n-   Applies these principles at community level for evidence based public health\n\n### Actions to deliver real evidence based medicine\n\n-   Patients must demand better evidence, better presented, better explained, and applied in a more personalised way\n\n-   Clinical training must go beyond searching and critical appraisal to hone expert judgment and shared decision making skills\n\n-   Producers of evidence summaries, clinical guidelines, and decision support tools must take account of who will use them, for what purposes, and under what constraints\n\n-   Publishers must demand that studies meet usability standards as well as methodological ones\n\n-   Policy makers must resist the instrumental generation and use of \"evidence\" by vested interests\n\n-   Independent funders must increasingly shape the production, synthesis, and dissemination of high quality clinical and public health evidence\n\n-   The research agenda must become broader and more interdisciplinary, embracing the experience of illness, the psychology of evidence interpretation, the negotiation and sharing of evidence by clinicians and patients, and how to prevent harm from overdiagnosis\n\n## Individualised for the patient\n\nReal evidence based medicine has the care of individual patients as its top priority, asking, \"what is the best course of action for this patient, in these circumstances, at this point in their illness or condition?\"39 It consciously and reflexively refuses to let process (doing tests, prescribing medicines) dominate outcomes (the agreed goal of management in an individual case). It engages with an ethical and existential agenda (how should we live? when should we accept death?) and with that goal in mind, carefully distinguishes between whether to investigate, treat, or screen and how to do so.40\n\nTo support such an approach, evidence must be individualised for the patient. This requires that research findings be expressed in ways that most people will understand (such as the number needed to treat, number needed to harm, and number needed to screen41) and that practitioners, together with their patients, are free to make appropriate care decisions that may not match what \"best (average) evidence\" seems to suggest.\n\nImportantly, real shared decision making is not the same as taking the patient through a series of if-then decision options. Rather, it involves finding out what matters to the patient\u2014what is at stake for them\u2014and making judicious use of professional knowledge and status (to what extent, and in what ways, does this person want to be \"empowered\"?) and introducing research evidence in a way that informs a dialogue about what best to do, how, and why. This is a simple concept but by no means easy to deliver. Tools that contain quantitative estimates of risk and benefit are needed, but they must be designed to support conversations not climb probability trees.\n\n## Judgment not rules\n\nReal evidence based medicine is not bound by rules. The Dreyfus brothers have described five levels of learning, beginning with the novice who learns the basic rules and applies them mechanically with no attention to context.42 The next two stages involve increasing depth of knowledge and sensitivity to context when applying rules. In the fourth and fifth stages, rule following gives way to expert judgments*,* characterised by rapid, intuitive reasoning informed by imagination, common sense, and judiciously selected research evidence and other rules.\n\nIn clinical diagnosis, for example, the novice clinician works methodically and slowly through a long and standardised history, exhaustive physical examination, and (often numerous) diagnostic tests.43 The expert, in contrast, makes a rapid initial differential diagnosis through intuition, then uses a more selective history, examination, and set of tests to rule in or rule out particular possibilities. To equate \"quality\" in clinical care with strict adherence to guidelines or protocols, however robust these rules may be, is to overlook the evidence on the more sophisticated process of advanced expertise.\n\n## Aligned with professional, relationship based care\n\nReal evidence based medicine builds (ideally) on a strong interpersonal relationship between patient and clinician. It values continuity of care and empathetic listening, especially for people who are seriously and incurably sick.44 Research evidence may still be key to making the right decision\u2014but it does not determine that decision. Clinicians may provide information, but they are also trained to make ethical and technical judgments, and they hold a socially recognised role to care, comfort, and bear witness to suffering.45 The challenges of self management in severe chronic illness, for example, are not merely about making treatment choices but about the practical and emotional work of implementing those choices.46 As serious illness is lived, evidence based guidelines may become irrelevant, absurd, or even harmful (most obviously, in terminal illness).\n\n## Public health dimension\n\nAlthough we have focused on individual clinical care, there is also an important evidence base relating to population level interventions aimed at improving public health (such as pricing and labelling of consumables, fluoridation of water, and sex education). These are often complex, multifaceted programmes with important ethical and practical dimensions, but the same principles apply as in clinical care. Success of interventions depends on local feasibility, acceptability, and fit with context\u2014and hence on informed, shared decision making with and by local communities, using summaries and visualisations of population level metrics.47\n\n# Delivering real evidence based medicine\n\nTo deliver real evidence based medicine, the movement's stakeholders must be proactive and persistent. Patients (for whose care the movement exists) must demand better evidence, better presented, better explained, and applied in a more personalised way with sensitivity to context and individual goals.48 There are already some models of good practice here. In arthritis, for example, patient advocacy groups that emphasise the importance of experiential evidence and patient centred strategies have existed for over 30 years and have influenced the choice of outcome measures used in comparative effectiveness studies.49 Patient input has refocused several NICE guidelines (for example, on psoriasis).50\n\nThird sector advisory and advocacy groups such as the UK's Consumer Association ([www.which.co.uk](http:\/\/www.which.co.uk\/)), Picker Institute ([www.pickereurope.org](http:\/\/www.pickereurope.org\/)), and Sense About Science ([www.senseaboutscience.org](http:\/\/www.senseaboutscience.org\/)) have a crucial role in educating citizens and contributing to public debate about the use and abuse of evidence. The James Lind Alliance ([www.lindalliance.org](http:\/\/www.lindalliance.org\/)) brings patients, carers, and clinicians together to prioritise research questions. Such groups must remain, as far as possible, independent of vested interests and aware of the distorting influence of tied funding.\n\n## Training must be reoriented from rule following\n\nCritical appraisal skills\u2014including basic numeracy, electronic database searching, and the ability systematically to ask questions of a research study\u2014are prerequisites for competence in evidence based medicine.6 But clinicians need to be able to apply them to real case examples.51\n\nToo often, teaching resources use schematic, fictionalised vignettes in which the sick patient is reduced to narrative \"factoids\" that can populate a decision tree or a score sheet in an objective structured clinical examination. Rather than focus on these tidy textbook cases, once they have learnt some basic rules and gained some experience, students should be encouraged to try intuitive reasoning in the clinic and at the bedside, and then use formal evidence based methods to check, explain, and communicate diagnoses and decisions.43 They must also be taught how to share both evidence and uncertainty with patients using appropriate decision aids52 and adapt their approach to individual needs, circumstances, and preferences.39\n\nLikewise, there is a strong argument for extending the continuing medical education curriculum beyond \"evidence updates.\" Peer observation and review, reflective case discussion in small groups (with input from patients who want to articulate their experiences, choices, and priorities) and ongoing conversations with fellow professionals can help hone and maintain the ability to manage the challenges of applying evidence based medicine in the real world.53 The linking together of educational theory, cognitive psychology, information mastery, and implementation science into a coherent approach that supports front line decision making with patients54 is rarely taught in practice.\n\n## Evidence must be usable as well as robust\n\nAnother precondition for real evidence based medicine is that those who produce and summarise research evidence must attend more closely to the needs of those who might use it. Lengthy and expensive reviews that are \"methodologically robust\" but unusable in practice often fail to inform, inspire, or influence.55 A recent systematic review of diabetes risk scores revealed that the authors of most studies were primarily concerned with the intellectual concept of improving the predictive value of the score but had given little or no thought to how their score might be used, by whom, or for what\u2014nor what the implications would be for real people who would be designated \"at risk\" by the score.56\n\nEvidence users include clinicians and patients of varying statistical literacy, many of whom have limited time or inclination for the small print.41 Different approaches such as brief, plain language summaries for the non-expert (as offered by NICE), visualisations,57 infographics,52 option grids,58 and other decision aids59 should be routinely offered and widely used. Yet currently, only a fraction of the available evidence is presented in usable form, and few clinicians are aware that such usable shared decision aids exist.\n\n## Publishers must raise the bar\n\nThis raises an imperative for publishing standards. Just as journal editors shifted the expression of probability from potentially misleading P values to more meaningful confidence intervals by requiring them in publication standards,60 so they should now raise the bar for authors to improve the usability of evidence, and especially to require that research findings are presented in a way that informs individualised conversations.\n\nGiven that real evidence based medicine is as much about when to ignore or over-ride guidelines as how to follow them, those who write guidelines should flag up the need for judgment and informed, shared decision making. The American College of Cardiology recently published new cholesterol guidelines;61 *JAMA* followed with a pragmatic, patient focused article on how to apply this guideline and when to consider ignoring it, including an online visualisation tool to support conversations with patients.62 As the authors commented, \"the target for performance measures is not the percentage of patients who . . . are prescribed statins, but the proportion of eligible patients who participate in shared decision making about statin use.\"62 Their approach deserves to be emulated widely.\n\n## Research must transcend conflicts of interest\n\nTo support real evidence based medicine, and in particular to reassure policy makers, clinicians, and the public that research and the guidance derived from it can be trusted,63 the infrastructure for research and guideline development must show the highest standards of probity. Independent funding of national bodies for medical research is crucial.\n\n## Broader, more imaginative research is needed\n\nThe research agenda for real evidence based medicine is much broader than critical appraisal and draws on a wider range of underpinning disciplines. For example, it should include the study of the patient's experience of illness and the real life clinical encounter for different conditions and in different circumstances. The field would be enriched, for example, by qualitative research to elucidate the logic of care\u2013that is, the numerous elements of good illness management that are complementary to the application of research evidence.64\n\nWe need to gain a better understanding (perhaps beginning with a synthesis of the cognitive psychology literature) of how clinicians and patients find, interpret, and evaluate evidence from research studies, and how (and if) these processes feed into clinical communication, exploration of diagnostic options, and shared decision making.54 Deeper study is also needed into the less algorithmic components of clinical method such as intuition and heuristic reasoning, and how evidence may be incorporated into such reasoning.43\n\nIn relation to producing usable evidence, we need to identify how to balance gold standard systematic reviews with pragmatic, rapid reviews that gain in timeliness and accessibility what they lose in depth and detail.65 In the same vein, we need research on how and in what circumstances to trade detail for brevity in developing guidelines. We need to develop decision aids that support clinicians and patients to clarify the goals of care, raise and answer questions about the quality and completeness of evidence, and understand and contextualise estimates of benefit and harm. We also need to improve both the usefulness and ease of use of these and other evidence based tools (models, scores, algorithms, and so on) including the intellectual, social, and temporal demands they make on users and the resource implications for the healthcare organisation and system.\n\nIn the educational field, it is time we extended the evidence base for integrated curriculums that promote reflection and case discussion alongside the application of evidence.66 Discussions on how to interpret and apply evidence to real cases, and the sharing of collective knowledge and expertise in the form of \"mindlines\" among clinicians53 or within illness communities67 may provide useful data sources for such studies. It is by studying these more sophisticated forms of knowing that we are likely to determine how best to produce expert clinicians and expert patients, and to prevent the harms that arise from overdiagnosis, overtreatment, and overscreening.33\n\nIn relation to effectiveness, we need greater attention to postmarketing research in day to day hospital and primary care settings to confirm that subsequent experience replicates the results of licensing trials. This will allow gold standard tests and their cut-off points for ruling out diagnoses and treatments to be revised to minimise overdiagnosis or underdiagnosis.43\n\nFinally, in relation to the collective effort to prevent the misappropriation of the evidence based quality mark, a key research priority remains the study of hidden biases in sponsored research\u2014for example, by refining the statistical techniques for challenging findings that appear too good to be true.\n\n# Conclusion\n\nMuch progress has been made and lives have been saved through the systematic collation, synthesis, and application of high quality empirical evidence. However, evidence based medicine has not resolved the problems it set out to address (especially evidence biases and the hidden hand of vested interests), which have become subtler and harder to detect. Furthermore, contemporary healthcare's complex economic, political, technological and commercial context has tended to steer the evidence based agenda towards populations, statistics, risk, and spurious certainty. Despite lip service to shared decision making, patients can be left confused and even tyrannised when their clinical management is inappropriately driven by algorithmic protocols, top-down directives and population targets.\n\nSuch problems have led some to argue for the rejection of evidence based medicine as a failed model. Instead we argue for a return to the movement's founding principles\u2014to individualise evidence and share decisions through meaningful conversations in the context of a humanistic and professional clinician-patient relationship (box 2). To deliver this agenda, evidence based medicine's many stakeholders\u2014patients, clinicians, educators, producers and publishers of evidence, policy makers, research funders, and researchers from a range of academic disciplines\u2014must work together. Many of the ideas in this paper are not new, and a number of cross sector campaigns with similar goals have already begun (box 3). We hope that our call for a campaign for real evidence based medicine will open up debate and invite readers to contribute (for example, by posting rapid responses on bmj.com).\n\n## Box 3: Campaigns aligned with real evidence based medicine\n\n1.  *Too much medicine*\u2014A rapidly growing movement, led jointly by clinicians, academics and patients, aims to reduce harm from overdiagnosis, overscreening, and overtreatment.26 33 The second of what will hopefully be an annual \"preventing overdiagnosis\" conference will be held in Oxford in September 2014 ([www.preventingoverdiagnosis.net](http:\/\/www.preventingoverdiagnosis.net\/))\n\n2.  *All trials* ([www.alltrials.net](http:\/\/www.alltrials.net\/))\u2014an international initiative to ensure that all clinical trials are registered at inception and no findings are withheld from publication\n\n3.  *Reducing waste and increasing value in medical research* ([www.thelancet.com\/series\/research](http:\/\/www.thelancet.com\/series\/research))*\u2014*A recent *Lancet* series highlighting the waste and loss of value caused by research that addresses the wrong questions, uses inappropriate study designs; is weighed down by bureaucracy, or is so badly or inaccessibly reported that practitioners and policymakers simply cannot apply it\n\n4.  *Improving publishing standards* ([www.icmje.org\/urm_main.html](http:\/\/www.icmje.org\/urm_main.html) )*\u2014*A campaign by the International Committee of Medical Journal Editors to improve the quality and transparency of medical publishing by discouraging ghost-writing and raising the standards for declarations of conflicts of interest\n\n5.  *Integrated medical education\u2014*Campaign to strengthen the integration of the different components of the curriculum by developing bedside clinical skills, understanding and applying research evidence, and reflecting and deliberating about complex cases68 69\n\nCite this as: *BMJ* 2014;348:g3725","meta":{"dup_signals":{"dup_doc_count":178,"dup_dump_count":82,"dup_details":{"curated_sources":2,"2023-50":3,"2023-40":4,"2023-23":4,"2023-14":2,"2023-06":1,"2022-49":1,"2022-40":3,"2022-33":3,"2022-27":4,"2022-21":4,"2022-05":3,"2021-49":3,"2021-43":3,"2021-39":4,"2021-31":1,"2021-25":4,"2021-21":2,"2021-17":3,"2021-10":4,"2021-04":1,"2020-50":2,"2020-45":2,"2020-40":1,"2020-34":3,"2020-29":2,"2020-24":1,"2020-10":2,"2020-05":3,"2019-51":2,"2019-47":2,"2019-43":1,"2019-39":2,"2019-30":1,"2019-26":1,"2019-18":2,"2019-13":1,"2019-09":2,"2019-04":1,"2018-51":2,"2018-47":1,"2018-43":3,"2018-34":2,"2018-26":3,"2018-22":1,"2018-17":2,"2018-09":4,"2018-05":1,"2017-51":2,"2017-47":2,"2017-43":3,"2017-39":3,"2017-34":1,"2017-30":4,"2017-26":4,"2017-22":4,"2017-17":4,"2016-36":1,"2016-30":1,"2016-26":1,"2016-22":1,"2016-18":1,"2016-07":1,"2015-48":1,"2015-40":1,"2015-35":1,"2015-32":1,"2015-27":1,"2015-22":1,"2015-14":1,"2014-52":1,"2014-42":1,"2014-41":2,"2014-35":2,"2014-23":1,"2024-30":2,"2024-26":4,"2024-18":1,"2024-10":1,"2017-13":7,"2015-18":3,"2015-11":3,"2015-06":1}},"file":"PMC4056639"},"subset":"pubmed_central"}
{"text":"abstract: On April 25th 1953, three publications in *Nature* forever changed the face of the life sciences in reporting the structure of DNA. Sixty years later, Raymond Gosling shares his memories of the race to the double helix.\nauthor: Naomi Attar\ndate: 2013\ninstitute: 1Genome Biology, BioMed Central, 236 Gray's Inn Road, London WC1X 8HB, UK\nreferences:\ntitle: Raymond Gosling: the man who crystallized genes\n\n*It has not escaped our attention that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material*.\n\nJames D Watson and Francis HC Crick \\[1\\]\n\nBy including this statement in their April 25th 1953 *Nature* article describing a model for the structure of DNA, Watson and Crick made one of the great understatements in history. In that moment, the seeds for the double helix's infamy - alongside the names 'Watson' and Crick' - were sown. Lesser known outside scientific circles is that this article did not include one iota of experimental data: Watson and Crick, who were based at the University of Cambridge's Cavendish laboratory, contributed deductive reasoning alone to the double helix model, albeit reasoning of an undoubtedly Nobel Prize-worthy standard. Instead, as has now been described many times, the model relied on X-ray diffraction data obtained by others, at King's College, and these data did not reach Watson and Crick by entirely wholesome means \\[2\\]. To add to the insult, Watson and Crick's report of the double helix did not fully credit the work of King's as being essential to the construction of their model, although the King's team did enjoy co-publication of their data alongside the double helix article, in the form of two articles in the same issue of *Nature* \\[3,4\\]. One of these articles described the X-ray diffraction work performed by senior researcher Rosalind E Franklin, together with PhD student Raymond G Gosling, and contained the highest quality diffraction patterns yet achieved for DNA \\[3\\]. It was these data that had proved invaluable in Watson and Crick's quest for the double helix.\n\nEarlier still, before Franklin arrived at King's, Gosling had achieved a major breakthrough in the search for DNA's structure when he became the first person to crystallize genes, under the guidance of Maurice Wilkins, who was the lead author of the other King's article to accompany Watson and Crick's model \\[4\\].\n\nWatson published his controversial memoir of the discovery, aptly named 'The Double Helix', in the 1960s \\[5\\], and in doing so propelled the story to worldwide fame, establishing DNA's structure as an icon of science in the popular imagination. However, events were relayed in Watson's book very much from his own point of view and at times, it has been argued, even verged on the fictitious.\n\nAside from Watson, Ray Gosling is the only surviving member of the select group of seven scientists to feature as an author on one of the three *Nature* articles. Gosling and his wife, Mary, were kind enough to welcome *Genome Biology* into their home, where he shared with us his perspective of the events of 60 years ago.\n\nElsewhere, *Genome Biology* has marked the anniversary by canvassing our Editorial Board for their opinions on the most important advances in the field since 1953 \\[6\\].\n\n# The accidental biophysicist\n\nSomething curious happened in scientific research in the mid-20th Century. Biology had been the neglected sibling of chemistry and, especially, physics, which had until then monopolized the glitz and glamor of scientific inquiry. Perhaps it was the sense that many of the big questions in physics had now been tackled, or a philosophical shift brought about by the experience of the dark forces of war and fascism in the 1930s and 40s, or perhaps it was just a simple matter of improved methodology; whatever the cause, history shows that many physicists and chemists began to become excited by biology at this time, and to turn their attention to addressing biological questions. Nobel Prize-winning physicist Erwin Schr\u00f6dinger is often credited with kick-starting this trend in his book 'What is life?' \\[7\\], which is said to have inspired even Crick and Watson themselves in their quest for the double helix.\n\nSo was Prof (Emeritus) Raymond Gosling DSc FKC (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>) similarly inspired when he opted to pursue a PhD in biophysics, in work that would culminate in establishing the molecular structure of DNA? Not a bit of it! Not only was Gosling unaware of Schr\u00f6dinger's work until \"much later\", he had not in fact originally wanted to become a scientist at all: \"I wanted to do Medicine, but Father said we couldn't afford Medicine because it would take *x* years to qualify and so on. And so the next best thing to doing medicine I thought was to do a fundamental subject like physics. I was very attracted by the thought that the Scots always refer to physics as 'natural philosophy'.\"\n\nAnd so a career in physics beckoned. But, having graduated from University College London in 1949, Gosling found himself limited in his options. Edward Andrade was the head of the Physics Department, and if Gosling had stayed on at University College, he would have been bound by Andrade's interests in viscosity and this \"didn't appeal.\" Instead, he was tempted by developments across town at King's College, rivals-in-chief to his own *alma mater* (Box 1), where John Randall (Box 2) had recently taken the Wheatstone Chair of Physics. Instead of being deterred by the rivalry, Gosling impishly thought to himself that it would be \"rather fun\" to join Randall's laboratory.\n\n# John Randall: the unsung hero of the double helix\n\nIf there's one message that Raymond Gosling would like you to take away from this article, it is that the role of John Randall in the pursuit of the double helix cannot be overstated, and that Randall has lamentably not been adequately credited in most tellings of the story. Gosling feels so strongly on this subject that he recently wrote to *The Times* to emphasize the point, prompted by an article that had recognized Rosalind Franklin's contribution but that had omitted Randall's.\n\nRandall's motto at the Medical Research Council (MRC) Biophysics Unit that he headed at King's College was 'to bring the logi of physics to the graphi of biology'. This interdisciplinary philosophy was at the time decidedly modern, and his switch from the questions of physics to those of biology was also, according to Gosling, \"ahead of the curve\". Randall's modernity extended to his respect for female scientists, many of whom he recruited at King's, as exemplified by his long-standing working relationship with Honor Bell. This facet of Randall's modern outlook was to prove very important with his later recruitment of Rosalind Franklin (Box 2).\n\n\"The people in the University and a few other establishments tended to make fun of Randall's approach, his claim that you would need to have different disciplines working together, and they called it 'Randall's Circus'. Now, that's what attracted me in the first place! I heard about this strange bald-headed little man with a Napoleonic complex who was running the circus in biophysics, and it sounded wonderful to me!\"\n\nSo was it the rebellion that Gosling was attracted to? \"Yeah, absolutely!\"\n\nRandall's most important legacy was his firm belief that DNA must be the agent of genetic inheritance, a concept not widely recognized at the time (despite good evidence to support it \\[8\\]), and his consequent determination to discover the structure of what he considered to be the genetic material. This was Randall's circus, and he was the ringmaster - and the ringmaster masterfully guided his troupe in its quest.\n\n# Maurice Wilkins stays awake\n\nAfter spending a year simultaneously studying zoology at Birkbeck and working as a medical physicist at Middlesex Hospital, prompted by Randall's insistence that he first learn some biology, Gosling joined the MRC Unit as a PhD student. At the outset, he worked alongside Maurice Wilkins (Box 2), another physicist-cum-biologist, and a veteran of the Manhattan Project racked with feelings of guilt. Wilkins and Gosling used ram's sperm as a source of DNA, following Randall's idea that \"ram's sperm have very flat heads - unlike human sperm, which is like a rugby football\", the benefit of which was that \"the long chains must lie in the plane of a ram's sperm head.\" But Gosling's attempts to obtain X-ray diffraction patterns from these sperm did not meet with much success.\n\nIt would take a series of what Gosling describes as \"serendipitous\" events to bring about a change in their fortunes (\"most of my life has been beset or encouraged by serendipitous acts,\" he says). There happened to be one man who could produce a sample of DNA of far superior quality to that produced by any other laboratory, and that man was Swiss biochemist Rudolf Signer. By one or two strokes of luck, Gosling managed to obtain a fair quantity of this DNA: \"Signer gave a lecture at the Royal Society on this method that he'd developed to separate out the DNA from the nuclear protein and so produce high molecular weight pure DNA. Signer asked at the end of the lecture if anybody would like some of this material, and he had a specimen tube full of this freeze-dried material. Only two people put their hand up. I'm glad to say that Maurice was awake enough to put his hand up!\"\n\nWas that often not the case? \"It was often not the case! He rushed down to the front and got half of all there was, which turned out to be very necessary.\"\n\nInitially, Gosling only wanted to use Signer's DNA as a control to determine which of six methods he had devised for making ram's sperm lie flat was most successful. Wilkins had \"a steady hand enough to pull fibers of 5 to 10 \u03bcm. Maurice pulled by wrapping them round a paperclip and then sort of - very scientific! - pushing it open to make them taut. I managed to get him to produce at least 35, so this is a 35 fiber specimen. And little blobs of LePage's cement, priced 6d in Woolworths down The Strand, pulling the fibers together. Very scientific!\"\n\nThe act of pulling the fiber orientated the molecules along the fiber axis, akin to the orientation that flattening ram's sperm was aiming for. Gosling took the fibers from the Signer sample to the basement of the Chemistry Department, which housed a Raymax X-ray tube. \"The first thing I produced was even fuzzier than my ram's sperm! Randall was most amused, and he was delighted to be able to point out that I'd missed a trick because the material that was in my DNA was largely carbon, nitrogen and oxygen, which was just the same as the atoms in the air inside the camera.\" The result was a diffuse back-scattering of X-rays, which fogged the film, and so Gosling was instructed to displace all the air with hydrogen. And this is where the next piece of serendipity steps in.\n\n# Serendipity, my dear Watson!\n\n\"This Raymax tube was already in a frightening place, it was three floors below the ground level, which was the level of the Thames, about 50 yards away, because it was the basement of the Chemistry Department. And it was lead-lined so that the X-rays should be shut in because there were various lecture theatres nearby. So having realized that I needed to keep a watchful eye on the amount of hydrogen I was filling the room up with - so that I wasn't going to blow myself up and repeat the Hindenberg Disaster - I bubbled the hydrogen through water to help judge when the camera had been swept clean of air. It just so happened that this produced enough water vapor in the camera to be taken up by the fibers and produce crystallites. It turns out that freeze-dried DNA from Signer's preparation would form micro-crystallites in a humidity of 92%, and that was by serendipity alone that I just hit that value.\"\n\nGosling is in no doubt that this was \"the most exciting thing that's happened to me before or since!\" He can still remember the moment clearly: \"standing in the dark room outside this lead-lined room, and looking at the developer, and up through the developer tank swam this beautiful spotted photograph, you are familiar with them now I'm sure. It took 90-something hours to take the photograph, again, pot luck. But it really was the most wonderful thing. And I knew at the time that what I'd just done was to produce a crystalline state in these fibers, and if then the DNA was the gene material, I must be the first person ever to make genes crystallize.\"\n\nDid Gosling realize at that moment that it would from then onwards just be a matter of time, that the structure of DNA was now in his grasp? \"Yes. Yes, that was why I could truly say it was my 'Eureka!' moment. I went back down the tunnels over to the Physics Department, where Wilkins used to spend his life, so he was still there. Wilkins realized even more certainly than I did that we had just crystallized genes. As with Randall, he was convinced that the DNA was the genetic material, and now he was convinced that the DNA could be made to crystallize. I can still remember vividly the excitement of showing this thing to Wilkins and drinking his sherry by the glass... by the gulpful.\"\n\nInterestingly, Gosling's account diverges somewhat from that given by Wilkins in his Nobel lecture (Box 3). Another - understandable - omission from Wilkins' lecture is a somewhat unorthodox, and nevertheless essential, contribution he made to Gosling's first X-ray images of crystalline DNA (Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>). Gosling had \"sealed the conventional camera onto its base and the lid and so forth with vacuum wax and stuff that you used in order to keep air out. This was to keep the hydrogen in, of course. The collimator was made of heavy brass and although I could seal it to the outside of the camera, there was no way I could think of to really prevent the gas coming out of the collimator tube. To my great surprise, when I was showing him how far I had got, this rather shy Assistant Director of the MRC Unit said: 'Try this.' And he pulled out from his pocket a packet of Durex.\" By a quirky twist of irony, the introduction of condoms to the story occurs after Gosling had ceased working on sperm, having switched to Signer's DNA, which was derived from calf thymus.\n\nDoes the 'Assistant Director' refer to Maurice Wilkins? \"Yes. But he was painfully shy.\"\n\nBut he was such a dedicated scientist that he was willing to risk the embarrassment... \"To risk everything, yes, that's right! It was extraordinary, really.\"\n\nThe condom did the trick, and Gosling was able to produce some high quality X-ray images. This is where Jim Watson first enters the picture: \"there was a conference in Naples that was on the structure of biologically active molecules. Randall was invited to talk and said that he couldn't, so he sent Wilkins, and Wilkins showed our beautiful picture and said: 'Look boys!' Or rather more... I mean his lectures were as dry as dust, so in a dusty sort of way, he made it clear that they had crystallized the genetic material. Now, Watson was in the audience, and I was told by somebody else who was there that Watson up until then had been doing his usual trick of pretending to read the newspaper while everybody gave of their best results and so forth. And he actually... when the picture came up on the screen in Wilkins' lecture, he actually put his paper down. And so he was convinced then and there, that if the material could be crystallized, then the structure could be found and it was just a short step from one to the other.\"\n\nWatson, having seen the fruits of Gosling's serendipity, asked Wilkins if he could join the MRC Unit, but was refused \"because Wilkins was afraid of him. He's quite scary, old Jim, on full flight.\" Instead, he approached Bragg (Box 2) at Cambridge's Cavendish laboratory, where - by another stroke of serendipity (Box 4) - he ended up allocated a desk next to Francis Crick. And the rest, as they say, is history...\n\n# Rosalind Franklin: a friction engineered by Randall?\n\nMeanwhile, Randall was not convinced that Wilkins and Gosling would ever learn enough crystallography \"to be able to solve this spotty diagram.\" For this reason, Rosalind Franklin was recruited to the project; Gosling believes that her appointment is another key pillar of Randall's contribution to the double helix story, as her experimental talent proved invaluable.\n\nFamously, Franklin and Wilkins enjoyed - or, rather, did not enjoy - a very fractious relationship. Gosling sees it as a \"pure personality clash\", as well as a very unfortunate misunderstanding, which only came to light many years later. \"The whole trouble was that there was a meeting in Randall's office, where Rosalind turned up and Alex Stokes and myself were invited along to meet her, and Wilkins was off somewhere else. In his autobiography, I think he says that he was away in Wales with a new girlfriend. But that was the key to what followed, he wasn't there.\" At this meeting, Gosling was assigned to work under Franklin, having previously been working with Wilkins.\n\n\"It was a very curious thing. Randall actually wrote to Rosalind saying that she would be asked to direct the X-ray crystallographic work on the Signer DNA material, and I didn't know that he'd done that.\" As with Gosling, Wilkins himself was not made aware of this letter until many years later.\n\nGosling believes that the misunderstanding was \"deliberate\" on Randall's part, rather than an unfortunate oversight. When he eventually discovered the truth of the matter, Gosling was \"really shocked\" because \"it was against all Randall's principles as I understood them. Up until then, everybody would freely discuss their work and interact with Wilkins probably more often than interacting with Randall directly.\"\n\nPerhaps most peculiar in the whole episode is that Randall and Wilkins had known each other for many years, and had been close colleagues at several institutions. So what was Randall's motivation? \"He definitely subscribed to the divide and rule principle, as lots of people did. He thought it would make them competitive and improve their work.\"\n\nAnother school of thought might be that Randall did not think Wilkins up to the job of the matter at hand, but did not want to confront him directly about these concerns. However, when this theory is put to him, Gosling is unsure. But what is for certain is that Franklin had very much been told by Randall - \"this very dynamic Head of Department\" - that she was a post-graduate fellow and it was her research, not Wilkins'.\n\n\"Wilkins came back to the lab after a few days and, you know, said, 'How are you?' and 'What are you doing' sort of thing, and got her back up. It was very unfortunate.\" Gosling, in the dark as much as everyone else, joined others in attributing this response to a Bolshie streak in Franklin's character, and also had sympathy for Wilkins, having previously worked closely with him. Now that he was Franklin's student, Gosling was very much caught in the middle: \"It was terrible, terrible. I spent my life going from one to the other, giving messages, trying to play the peacemaker.\"\n\nEventually, the dispute between Wilkins and Franklin resulted in Randall suggesting to Franklin that she had better leave, even though the DNA work was not yet complete - although he was good enough to \"fix her up with his old pal \\[JD\\] Bernal\" at Birkbeck.\n\nLater on, Wilkins was \"beset by worry that he had been responsible for not integrating Rosalind into the group\" and it very much lingered with him, to the extent that, for the rest of his life, he would frequently ask Gosling whether he had been unkind to her.\n\n# The wrong model\n\nAll Hollywood script writers worth their salt know that a successful plot is built around a boy meeting a girl, losing the girl and then winning the girl back. Change a few nouns, and you pretty much have the story of the double helix: Crick and Watson 'discover' the structure, lose the 'discovery' when it turns out to be wrong, and then win back the discovery by coming up with a better model. Where they erred was in rushing to triumphalism with the first, incorrect, model: \"we suddenly received a call, in '51 I think it was, from Crick - from Maurice. Crick had got in touch with Maurice to say that he hoped he didn't mind, but they had built - him and Jim had built - a model of DNA as a double helix, following the results that we had deduced in structure 'B'. And would we like to go to Cambridge to see it?\"\n\nAccording to Gosling, it was clear to him then that the King's data, such as it was at the time, had fed into Watson and Crick's model. Nevertheless, Gosling, Franklin and Wilkins, together with their King's colleagues Bill Seeds and Geoffrey Brown, took the Liverpool Street train \"with a heavy heart\" to Cambridge. But, upon arrival, it was immediately apparent that Watson and Crick had made some elementary mistakes, in both senses of the word.\n\n\"We arrived in the lab to be shown the model and to the absolute relief of Rosalind and myself - I don't know about Wilkins, what he thought at the time, because I was dealing with my own thoughts and not observing other people - the boys had built a model with the phosphate linkages going up the middle of the thing, which gave it, of course, rigidity, and so you could hang all the nucleotides and things off the ends of the ionic chain. That must be wrong, because we knew that the water went into that phosphate-oxygen group, and there was an ionic linkage there between the sodium - it was the sodium salt of DNA - and the phosphate group, and you got eight molecules of water going in, quite a lot of water that would go in and come out very easily, as we had shown. So it meant that whatever the structure was, those phosphate groups had to be on the outside. And so we were delighted, and Bragg was embarrassed because it wasn't done to actually work on another man's problem.\"\n\nWatson and Crick's recklessness, in playing their cards so early, was pounced upon by Franklin, who \"tore the model apart point by point.\" As Gosling notes with some amusement, Crick was later to comment that Franklin's demolition of the model was the only time he ever saw Jim Watson at a loss for words. \"And I can believe it!\"\n\nSo did Franklin deliver her criticism with obvious relish, or did she play it straight? \"Oh, no, with obvious relish! She reminded me very much of a particular lady in the University of St Andrews Physics Department that I worked in when I left King's, in which she'd turn up at seminars by new PhD students or the like and she would tear their suggestions apart. 'You're wrong, and you're wrong for the following reasons, one, two, three, four...'\"\n\nGosling had, like Franklin, realized instantly that the model was wrong but did not join her in skewering its inadequacies. Why not? \"I left it to her. I didn't need to discuss it at all, I mean she was...she was on top of her form! My word, no.\"\n\nPerhaps the famously negative portrayal of Franklin in Jim Watson's book 'The Double Helix' was payback for this moment? \"Yes. Oh, I'd never thought of it, but yes, that's true. The humiliation. He must have felt - that's the word - he must have felt humiliated. Who the hell is this woman telling me... Yes, you can see it more clearly looking back, can't you?\"\n\nRather than focus on Jim Watson's humiliation, however, Gosling was at the time \"just happy that it meant that Rosalind and I could go back to the Strand and just get on with doing the mathematics. And it was ours to take as long as we liked.\"\n\nWatson and Crick's misplaced haste really did seem to have handed the game to King's. When Wilkins reported what had happened to Randall, he was \"furious and stormed off to Cambridge to see Sir Lawrence, and Lawrence was apologetic and actually forbade the lads from doing any more work on DNA, and that it was a King's problem and that Crick had plenty to do on hemoglobin and that he should concentrate on getting his PhD.\"\n\n# The American competition\n\nWith the ban imposed upon Watson and Crick, DNA would have remained a King's problem, were it not for Caltech's Linus Pauling (Box 2) and, moreover, the inexplicable indiscretion of Pauling's son Peter. Not content with having discovered pretty much everything else going in chemistry and molecular biology, Pauling had turned his attention to the structure of DNA. A physicist by training, Pauling had got under the skin of atoms and molecules with great success to describe the nature of the chemical bond and the secondary structure of proteins, and much else besides. So he seemed like a good bet in the race to discover the structure of DNA.\n\nPauling's son Peter was at the time based in Cambridge, and somehow managed to leak the news of his father's interest in the question to Jim Watson. The prospect of losing to Pauling, who had already beaten him to the alpha-helix and beta-sheet, was too much for Bragg, and he allowed Watson and Crick to start work on DNA again.\n\nIt so happens that the first papers Pauling wrote on the subject actually contained the same mistake made by Watson and Crick, in that the phosphates were on the inside. Further, his proposed structure was a triple, rather than a double, helix \\[7,8\\]. It was very wide of the mark, but the threat of Pauling's intellectual prowess was not to be underestimated.\n\nDoes Gosling believe that Bragg's reaction was justified? After all, a draft manuscript written by Franklin shows that she had already drawn many correct conclusions about DNA's structure, including its double helical nature. Would Pauling have arrived at the model before King's with Cambridge out of the race? \"That's the problem. That's the \\$64,000 question I've been asked so many times. 'How long would it have taken you?' And I don't know.\"\n\n# King's loses the race\n\nFranklin's methodological approach was not that of someone in a race to the prize, but instead favored slow, steady progress. Franklin's skills as a chemist had borne fruit in determining that there were two crystalline forms of DNA, dependent on the humidity; the respective forms at lower and higher humidity were christened by Gosling and Franklin 'A' and 'B'. It was accepted that 'B' would be the *in vivo* form, due to its formation in humid conditions, and Franklin had taken an exceptionally high quality X-ray diffraction pattern of this form ('photo 51', see Figure <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>), which proved invaluable to Watson and Crick. From the King's data, it was clear that 'B' was helical, but this could not be said with certainty for 'A' (Figure <a href=\"#F4\" data-ref-type=\"fig\">4<\/a>).\n\nFranklin had set herself the task of deducing the structure of 'A' from first principles, using Patterson functions, and she was having quite some success in doing so. \"We were the first people ever to do a cylindrical Patterson. But now nobody does it, because the computer would do it for you in a twink. And so you would probably go straight to an atomic density map, rather than vectors.\"\n\nBut why the focus on 'A', given that this was known not to be the biologically relevant form? \"Well, that's hindsight, isn't it. Her answer would have been, 'we've got over 100 diffraction spots, so we can do all the mathematics of the Patterson function.'\" Such data were not available for 'B', and Franklin's preference for sticking to first principles, and aversion to playing about with models and \"guesswork\", was the deciding factor.\n\nAnother obstacle for Gosling was his ignorance of space groups. \"Alex Stokes (Box 2) had taught me enough crystallography that I had been able, before Rosalind was even appointed, to index the spotty picture that I had produced, and find the unit cell and therefore the symmetry that the molecule must exhibit. I got the space group right, I got all the major indices right. But what I didn't realize was that the C2 space group meant that there must be a dyad axis perpendicular to the fiber axis. The density values, which determine how many strings of the molecule are per unit cell, could in our case be two or four, and we were getting an answer for the density, which was a bit difficult to measure, between two and three. Now Crick realized immediately from my unit cell data that there must be a dyad axis and therefore, if this double diamond was showing a helical pattern, it meant there was a double helix.\"\n\nThe reason Crick knew all about this space group was that he was studying, under Bragg, the structure of hemoglobin, which just so happens to share the same space group. So another bit of serendipity? \"Absolutely.\"\n\nThe slow pace at King's was no match for the fury of Watson and Crick's efforts, and so it came to pass that Gosling, Franklin and company were called up to Cambridge a second time...\n\n# That 'eureka!' moment\n\n\"We went up, saw the structure, we came back to King's and looked at our Pattersons, and every section of our Pattersons we looked at screamed at you, 'double helix!' And it was just there! - once you knew what to look for. It was amazing.\"\n\nWhen Gosling saw this second structure - the double helix we are now so familiar with - for the first time, was it as obvious to him that this model was right as it had been that the first model was wrong? \"Absolutely. Absolutely, because it was so elegant. And it also explained the diffraction pattern as being such a clear double helix because the phosphate groups were on the outside, the sodium then was ionically bonded to the phosphorous, and the eight molecules of water went into the same group. So you got this enormous scattering power, due to the electronic number of oxygen, nitrogen and carbon.\"\n\nIt wasn't just the phosphates on the outside that made the model so convincing, but also the \"stuff in the middle.\" For one thing, it made a lot of sense that the identities of the nucleotides could be changed without impacting on the overall structure. For another, the nucleotides' \"stair rods looked identical to the X-rays because of the way they were bonded.\" More importantly, Watson and Crick took Chargaff's (Box 2) finding that \"no matter which DNA he studied, there was always a 1:1 ratio of adenine to cytosine, and guanine to thymine, and that meant that these were identical\" and incorporated it into the model in the form of complementary base pairing. With this seemingly simple rule, suddenly \"the solution to DNA's reproductivity was so simple.\"\n\nChargaff's ratio was already in the public domain, before Watson and Crick's model was unveiled, but \"no one had made the connection\" that this meant the DNA structure would rely on complementary base pairing, nor that this \"was necessary to explain how the DNA replicated.\"\n\nSo, given that the double helix and all its features were already present in Franklin and Gosling's data, but had somehow been opaque to them, perhaps it was a good thing that Watson and Crick worked on their model, even if by surreptitious means, and the injustice really was more the lack of attribution? Gosling agrees. \"I think so. We would have, I think, got there eventually. But Crick himself has said, and Wilkins has agreed with him, that if they hadn't built the model, we would have got there at King's, but not in one fell swoop, that it would have come out in dribs and drabs about the various aspects.\"\n\nAnd, as a consequence, history would have been deprived of a singularly exciting moment? \"That's right. That 'eureka!' moment, as Jim himself admits to, when you know that that specific pairing is there, and it's in the literature! And then, I can quite sympathize with him in a way, such a very strange feeling. Because I think it was Bernard Shaw who said that very few people are lucky enough to have an original thought in their whole lives. But there I was presented with mine, the crystallization of DNA, as he was, on a plate. Dong! Light bulb!\"\n\nGosling readily offers his admiration for Crick and Watson's achievement: \"they'd not only put together a model showing that the DNA was in the form of a double helix, but that it had this dyad axis and that this meant that these stair rods of the nucleotides had to be specifically paired - now that was worth a Nobel Prize.\"\n\nSimilarly, Franklin reacted to this second model with a level of grace to equal the schadenfreude with which she had destroyed the first. \"If you look at the BBC 'Secret of Life' documentary, which is absolutely brilliant - it really is a wonderful thing - they have a shot there, which is of course made up, but it has Rosalind Franklin by herself looking at the model in Crick's lab. And Bragg comes in and says something like, 'Do you have any regrets...' - or something like that - '...Miss Franklin.' And Miss Franklin apparently said, 'No, we all stand on each other's shoulders.' And that stuck in my mind. Whether she said it or not, I don't know. But that was her attitude that she took when she and I were discussing it.\"\n\nTo her credit, Franklin \"absolutely\" put science before ego. However, at the time, she was not aware of the extent to which Watson and Crick had based their model upon her data. Does Gosling believe she came to know about this before her death in 1958? \"Yes. Oh, she did know about that.\"\n\n# Obscurity and infamy\n\nPerhaps surprisingly, given the iconic nature of the double helix today, the 1953 discovery did not initially have much impact, as Gosling saw it. An illustration of this might be the difficulty he had finding work following his PhD - there were no opportunities for him to stay at King's, which he would have gladly done.\n\n\"I talked about DNA for about a year after I wrote my PhD because I was convinced that it would only take a year... that the structure of DNA now being known, the ability to control carcinogenic activity within any tissue you like would be available in two or three years' time. And when two or three years came and went, and nobody had shown that you could... 'Alright, so a double helix, so what?'\"\n\nDue to Franklin's enforced move to Birkbeck, Gosling ended up writing his thesis away from King's, under her guidance. This annoyed Randall somewhat, as he was \"of the old school, who... in those days, nobody but the Professor had PhD research students.\" Randall's response to the situation was to appoint himself as Gosling's internal *viva* (thesis defense) examiner, and he roped in Bernal as the external examiner. Oddly, when you consider that the content of his thesis amounted to a not insignificant contribution to the discovery of DNA's structure, the prospect of his *viva* with Randall \"scared the pants off\" Gosling. In part, he explains, this was because his studentship had begun by \"trying to establish what the problem was\", due to the \"amorphous\" state of biophysics, rather than starting off with a \"ready-made problem\", as his friends in Ingold's Chemistry Department at University College had done.\n\nGiven that his studentship had been under Randall's direction, the idea that Randall would then find fault with Gosling's rationale in the *viva* is hard to resolve, outside of a Kafka novel perhaps. \"Yes! Very Kafkaesque, yes.\"\n\nBut, still, he surely cannot have been too afraid of defending a thesis describing such successful work? \"Well, I can tell you, and I'll tell anybody, that the big difference between writing a paper - especially if you have co-authors - is you can always, when the discussion gets too heated, say you didn't write that bit, it was him! But with a thesis, you're aware that as you are writing each word, it can be attributed to you. And that's an odd feeling, especially when you know who your examiners are.\"\n\nGosling's fear did not end with the anticipation of his *viva*, but progressed into the examination itself. \"Randall and Bernal had obviously had a very good lunch. I went in there at 2 o'clock, and at half past five, they were still going on talking about the origin of life. I was hardly asked a question. The two of them were at it hammer-and-tongs. I was terrified. I was just terrified, because I was convinced that Bernal was a genius - one of only three I have met in my life, together with Crick and Haldane (Box 5).\"\n\nHaving become disillusioned about the failure of the double helix to make much of an impression, Gosling lost interest and drifted away from the field. He had in any case wanted to move closer to his original dream, medicine, and did indeed go on to lead a successful career as a medical physicist (Box 6).\n\nBut an unexpected development suddenly cast the double helix in the spotlight, and spawned a legend that solidified its place in popular culture. Jim Watson wrote a book. In Gosling's eyes, \"that's what did it\" for the double helix. And that's where Gosling's praise for Watson's book - 'The Double Helix' \\[5\\] - ends.\n\n\"That book is a novel. A very successful novel, but it is a novel. Wilkins and Crick wrote to Harvard saying they should not publish this book, and they didn't.\" But someone else did.\n\nAnother unhappy reader of 'The Double Helix' was Max Perutz. While Watson and Crick obtained some of the King's data through Crick's friendship with Maurice Wilkins (\"Innocently. Because he'd always discussed his work, as Crick had with him, from their undergraduate days.\"), other data came from a progress report submitted to the MRC, which Watson had obtained from Perutz.\n\n\"I actually had a letter that I can no longer find from Perutz saying that he wanted me to be assured that he did not rush down the corridor waving the MRC report containing our contribution about the size and shape of the molecule. It's true they had actually got it from him. But the way Watson tells it, it's sort of Archimedes getting out of the bathtub again, you know.\"\n\nKey for Crick and Wilkins was their dismay at the portrayal of Rosalind Franklin, which amounted to what you might describe as an *ad hominem* character assassination, and a wildly inaccurate one at that. Worse still, Franklin had died of ovarian cancer several years earlier, and so was not able to defend herself. Was Gosling also angry about how she had been portrayed? \"Yes. Very much so.\"\n\nBut he is gratified that many people rose to Rosalind's defense (\"her part in it now is if anything overplayed!\"), and in particular that Brenda Maddox's \"first class\" biography of Franklin was able to set the record straight \\[2\\]. Unwittingly, Watson is responsible for the widespread admiration with which Franklin is viewed today, which is to a large degree a reaction to his book.\n\nHow did Gosling feel about his own portrayal in 'The Double Helix'? Was he concerned that his role had been underplayed? \"No, not really. But I felt that Watson was so busy criticizing poor Rosalind that he didn't mention and give credit to the work of Alex Stokes and Wilkins and myself. And Randall.\"\n\nDespite his strong reservations about 'The Double Helix', Gosling acknowledges that Watson has much to be complimented for. \"He is very precocious, he is one year younger than me, which annoys me - ha! - and he is without doubt a very lively mind and his ability to spot that specific pairing was worth the Nobel Prize.\"\n\n# Reconnecting with DNA\n\nAs mentioned previously, Gosling's subsequent career deviated from biology (Box 6), and he didn't follow the progress of molecular biology during this time (\"not at all!\"). This scientific realignment proved problematic on one occasion during his tenure at the University of the West Indies, when he was asked to deliver a lecture to the Trinidad campus on what his discovery was leading to. \"I hadn't the foggiest! So I had to bone up, and that was incredible.\"\n\nAsked jocularly whether he contacted Francis Crick for assistance, Gosling replies that, actually, he did! \"Well, yeah, I did have a conversation with Francis. And it was... the whole thing was fascinating, I mean to look into this new world.\"\n\nWhen Gosling hears of developments that have come from his work at King's, does he feel connected to it? \"No, I feel detached, I really do - it's gone way ahead of where I was. But I realize that I don't go to the literature enough to say that I have kept up.\"\n\nThe 50th anniversary of the double helix reignited the world's interest in Ray Gosling, and suddenly he was invited to events alongside Nobel laureates and other giants of the molecular biology world - most of whom he had never met before, nor even been aware of their work. Gosling was particularly taken with Alec Jeffreys (\"a very nice chap\") and Paul Nurse (\"a very witty fellow indeed\"), and was \"very flattered\" when Nurse invited him to contribute a brief memoir of the double helix to a time capsule for The Francis Crick Institute.\n\nHave any DNA-based developments caused him concern, in the same vein as the conflicted feelings felt by Wilkins and some other alumni of the Manhattan Project? \"No. But I do very much feel that I can't stress enough the critical stage we're at. Up until now evolution has occurred spontaneously, set in motion in all sorts of ways. And what is happening now is that our species are the first ever to have their hands on the levers controlling evolution. If you are a pessimist, you would say the glass is half empty, and you would think it's rather a sinister situation. So you have to take an optimistic point of view, at least I think you do.\"\n\nWork on transgenic animals has made a strong impression on Gosling (he highlights the example of so-called spidergoats), as has the notion of an embryo with three genetic parents, in which maternal chromosomes and mitochondrial DNA originate from different individuals.\n\nReturning to his favorite theme of Randall, Gosling recalls how in later years the ringmaster of the double helix discovery focused his work on cilia. \"Randall again was ahead of his time, wasn't he? In concentrating on the cilia. Isn't that fascinating? How long's the old boy been dead? '84, gosh. He would have been so pleased, wouldn't he...\"\n\n# Box 1. 'The best rag I ever came across'\n\nTo illustrate the level of treachery that his move from University College to King's College might have been perceived as, Gosling relates a practical joke that took place in the 1930s: \"The engineers at University College got into the front reception hall in King's and cut a hole in the belly of Reggie the Lion, who's their mascot, and filled it with rotten vegetables that they'd got from Covent Garden, and sealed it up and painted it and put it back where it was, which was on a special plinth above the entrance doors. And it was weeks while the beadles looked for the source of the smell, and that was I think the best rag that I ever came across.\"\n\n# Box 2. Watson and Crick's key accomplices - willing and unwitting - in the quest for the double helix\n\n## John Randall\n\nA physicist by background, Randall was \"ahead of the curve\" in setting his sights on biological questions. He established the MRC Biophysics Unit at King's, where his modern approach translated to interdisciplinary research with an unusually large, for the time, quotient of female scientists. Randall was \"autocratic\" and had a \"Napoleonic complex\", and the Unit was very much his \"circus\". But Gosling has immense admiration for Randall, and believes that his drive and vision were the magic ingredient that led to the double helix discovery, rendering him the great \"unsung hero\" of the story.\n\n## Maurice Wilkins\n\nA long-standing colleague of Randall at a number of institutions, Wilkins became his right-hand man once recruited to the Unit, where he served as Assistant Director. A dedicated scientist, the \"painfully shy\" Wilkins guided Gosling's early work at King's. Wilkins had first attempted to study DNA using ultraviolet light techniques, but without much success. One of the two King's articles co-published with Watson and Crick's work in *Nature*'s April 25th 1953 edition described Wilkins' work, together with Alex Stokes and Herbert Wilson, on the structure of DNA. This publication focused on how to interpret X-ray diffraction patterns and on showing that DNA from various species adopts the same structure.\n\nFor his contributions to the discovery of the double helix, Wilkins shared the Nobel Prize in Physiology or Medicine for 1962 with Watson and Crick (Box 3).\n\n## Rosalind Franklin\n\nRosalind Franklin was recruited to King's, where Gosling worked under her, by Randall and was key to obtaining the quality of X-ray diffraction pattern necessary to determine the structure of DNA. She had come to Randall's attention for her work on the properties of coal as part of the war effort. At the time, it was \"very unusual\" for a woman to have a senior research role at King's, although not unusual in Randall's laboratory (see text), and many outside Randall's circle were taken aback by it.\n\nFranklin was \"a very good experimental scientist, as you had to be if you were a woman in those days.\" She fell out with Maurice Wilkins from the beginning of her time at King's, largely due to a misunderstanding engineered by Randall (see text), and then earned Jim Watson's enmity when she proved a caustic foe in the race for the double helix. Franklin viewed her study of the tobacco mosaic virus, performed at Birkbeck under Bernal after her DNA research, as her life's greatest work.\n\nFranklin, like Gosling, was a Londoner, although Franklin hailed from the affluent neighborhood of Notting Hill, as the daughter of a merchant banker, whereas Gosling had more modest origins in the suburbs.\n\nFranklin died of ovarian cancer in 1958 at the age of 37, nearly five years to the day after the publication of the double helix. It is thought that Franklin's work with X-rays might have contributed to her death. Several years later, Jim Watson included a very negative account of Franklin in his book, 'The Double Helix' \\[5\\], but was met with much opprobrium for doing so. A more recent biography by Brenda Maddox has done much to raise the profile of Franklin and to emphasize the importance of her contribution to the double helix discovery \\[2\\].\n\nAs Nobel Prizes are not awarded posthumously, Franklin was not eligible to be included in the 1962 Nobel Prize shared by Wilkins, Watson and Crick.\n\n## Alex Stokes\n\nAlex Stokes developed mathematical methods for interpreting X-ray diffraction patterns while at King's MRC Unit; without this know-how, Gosling would not have been able to make sense of his DNA data. Stokes was a co-author, with Wilkins and Herbert Wilson, of one of the three articles on DNA structure co-published in *Nature* on April 25th 1953.\n\n## Rudolf Signer\n\nAs with Randall, Gosling considers Signer to be an \"unsung hero\" of the double helix story. Signer was a Swiss biochemist, based at the University of Bern, who could produce a DNA sample of far superior quality to any other available at the time. Maurice Wilkins obtained such a sample, derived from calf thymus, after Signer generously offered it to all takers at a London lecture.\n\n## Lawrence Bragg\n\nLawrence Bragg was awarded the Nobel Prize in Physics, together with his father William Bragg, at the tender age of 25, making him the youngest ever recipient of this prize to date. Bragg's Nobel was in recognition for the methodology of studying crystal structures by X-ray diffraction, which the Braggs had developed at the University of Leeds. It was also at Leeds that William Astbury obtained early X-ray diffraction patterns of (non-crystalline) DNA.\n\nBy the 1950s, Bragg was serving as the Head of the Cavendish Laboratory at Cambridge, where both Crick and Watson worked under him. Also based at Bragg's Cavendish for a time was Peter Pauling, son to Linus, who leaked information of his father's interest in the question of DNA's structure. A fourth key player to work under Bragg at the Cavendish was the chemist Jerry Donohoe, who set Watson and Crick right about errors they were making in the likely chemical form of DNA's bases.\n\n## Linus Pauling\n\nPauling is most notable for his work on the nature of the chemical bond and on the secondary structure of proteins, and was one of the most prolific scientists of the 20th Century, in terms of major advances credited to his name.\n\nPauling began work on the structure of DNA while King's were working on the problem, and Crick and Watson used the specter of defeat at the hands of Pauling - who had beaten Bragg to the alpha-helix and beta-sheet - to goad Bragg into allowing them to restart work on DNA's structure. As an American based in California, Pauling also added an element of transatlantic competition to the race, although Cambridge-based Jim Watson was of course himself also American.\n\nPauling published a model for DNA's structure in February 1953. However, the model did not come close to the true structure, with the most obvious mistakes being the number of strands - he had proposed a triple, rather than a double, helix - and the location of the phosphates on the interior of the helix.\n\nPauling is the only person in history to have won two individual Nobel Prizes - these were for Chemistry (1954) and Peace (1962), the latter as a result of his political activism, some aspects of which had famously brought him unwelcome attention from the United States government, in the form of travel restrictions. In part for this reason, Gosling was never able to meet Pauling in person.\n\n## Erwin Chargaff\n\nChargaff had discovered the 1:1 ratio that existed between the complementary base pairs in DNA, but had not made the leap from that discovery to the rules of base pairing. Chargaff personally communicated his discovery - dubbed 'Chargaff's rules' - to Watson and Crick, and was \"as cross as two sticks\" not to be included in Watson's book.\n\nOriginally from the present-day Ukraine, and having spent time at various European research institutions, Chargaff relocated to New York to escape the rise of Nazism. It was here, at Columbia University, that he determined 'Chargaff's rules'.\n\n# Box 3. Serendipity or sagacity?\n\nGosling claims here and elsewhere \\[11\\] that he was the first person to crystallize DNA purely by a stroke of luck: he had used water to monitor hydrogen and, serendipitously, the humidity absorbed by the DNA fibers from this water resulted in crystallization (see text).\n\nIn his Nobel lecture \\[12\\], however, Gosling's colleague Maurice Wilkins gives a different version of events: 'One reason for this success was that we kept the fibres moist. We remembered that, to obtain detailed X-ray patterns from proteins, \\[JD\\] Bernal had kept protein crystals in their mother liquor. It seemed likely that the configuration of all kinds of water-soluble biological macromolecules would depend on their aqueous environment.'\n\n# Box 4. Watson and Crick: the perfect pairing\n\nGosling relates how, having been refused entry to King's, Jim Watson \"was nonetheless convinced that he had to learn some basic crystallography, and then he'd be able to find the structure of DNA.\" He approached Sir Lawrence Bragg and asked to join his Cavendish Laboratory at the University of Cambridge, and was accepted (\"you didn't turn down a pair of willing hands which came self-funded.\")\n\nBecause of the way the space requirements of his workers happened to be at that time, Bragg gave Watson a spare desk next to Francis Crick (\"a thoroughly nice man\"), who was \"already getting Sir Lawrence rather mad because - as Bragg was heard to say several times - the man never stopped talking. And this is true, the man was incredibly wired for looking at exciting new developments.\" Crick had just come across the argument about whether DNA or protein was the genetic material, and had come to the conclusion it was the DNA. \"And here is this pop-eyed chap from America turning up, who is saying that is exactly what it is and the people in King's have got it, and we should build models - that's the way to go.\"\n\nAs with the base pairs they discovered, Gosling considers Watson and Crick to be the perfect complement to one another: Crick's \"genius\" and Watson's \"persistence\".\n\nGosling notes the contrast between the experimentalists at King's and Watson and Crick, who \"never did an experiment in their lives, it was all deductive powers of reasoning.\" Nevertheless, Gosling believes that those powers were very much worthy of the Nobel Prize in Physiology or Medicine awarded to them, alongside Maurice Wilkins, in 1962.\n\n# Box 5. An encounter with JB Haldane\n\nGosling believes he has met three geniuses in his lifetime: Francis Crick, JD Bernal and JB Haldane. \"Those three, I was lucky to meet. I mean, I don't think there are very many scientists who have the privilege.\"\n\nHe first met Haldane when an undergraduate at University College, but a later encounter was much more memorable.\n\n\"JB Haldane came to a conversazione at the Royal Institution, and it was time for the eaties to be dished out, and so I was the one who got the short straw and had to stay by the model in case anybody came to look at it. And there it was, this lovely double helix. And this Haldane shuffled up and started to roll a cigarette, and apparently he used the cheapest possible tobacco and Rizla paper, and made his own, like he was a student. Nowadays, you would have accused him of making a reefer, a joint. But he rolled it, took a few puffs and said, 'Well, now you will have to find an untw... tw... ...twiddlase.' Because he had an awful stammer, and that I remember vividly. I stood there awestruck, thinking about an untwiddlase! I mean, this was before the concept of telomeres. But he was on it. Incredible man.\"\n\n# Box 6. Gosling after DNA\n\nAfter the initial double helix data had been published, Gosling and Franklin finished up their research into the structure of DNA with another article in *Nature* \\[13\\]. Gosling continued to work in crystallography for a few years, focusing on the structure of nucleotides, but spent most of his career as a medical physicist, developing devices for the study and diagnosis of atherosclerosis.\n\nThe idea to perform this work originated from a discussion he had while based at the University of the West Indies. \"Again, a somewhat serendipitous situation developed, in that I got to know very well indeed the senior lecturer in morbid anatomy. He wanted an explanation as to why the fatty plaques should develop in arteries where the blood was moving fastest, that this was an anomalous situation, and all of the work that was being done on atherosclerosis - on atherogenesis, if you like - at that time was being done by biochemists. And he said, surely, isn't there a big hole here for someone who is a biophysicist to look at the characteristics of the pulsatile flow, and that must play a part in the formation of these plaques.\"\n\nGosling began the project while on a sabbatical back in Randall's laboratory at King's and, while there, Randall persuaded him to change tack. \"One day in my little lab, Randall appeared. And he had this terrifying habit of doing just that. I mean, he walked around with hush puppies and you couldn't hear him coming, and suddenly you were in an empty lab and then the next thing you know, he's at your elbow. Terribly dangerous! But what he said was illuminating. He'd come down to find me because he wanted me to stop building glass tubes with lumps inside, because if I did, and if I was successful in replicating the conditions, all that would happen is that some bloody physiologist would come along and pooh pooh the whole thing because it was not like the *in vivo* state, and that what I ought to do is to build an animal model and observe it directly in a living situation. And then he disappeared.\"\n\nGosling took Randall's advice and, when he returned to Jamaica, switched to working on cockerels, which turned out to be the best animal model available. Later, he continued his work on atherosclerosis at Guy's Hospital, London, where he developed ultrasound devices for the analysis and diagnosis of atheromatous plaques. This phase of his career included an important discovery.\n\n\"As the lumps developed, so it changed the elasticity of the artery wall. As you know, you get hardening of the arteries as they build up with atheromatous plaques, but we were able to show that, before that happens, they get three times more distensible, which was an unlooked for, unknown thing - counterintuitive.\"\n\nGosling found the direct impact he could see his work making in medical physics more satisfying than fundamental biology, and he also preferred the more steady rate of progress to the manic ups and downs of his years as a crystallographer. His time at King's, therefore, was a preamble to a rewarding and successful career that led Gosling in a very different direction to his fellow actors in the double helix story.\n\nNow retired, Gosling's contribution to science has been recognized in the form of election as a Fellow of King's College and the award of a DSc from the University of the West Indies. Inexplicably, Gosling has never received recognition in the Queen's honors system from the British government, although he was invited to meet Prime Minister Tony Blair at Downing Street in honor of the double helix's 50th anniversary.","meta":{"dup_signals":{"dup_doc_count":177,"dup_dump_count":51,"dup_details":{"curated_sources":4,"2023-23":1,"2022-33":1,"2021-43":1,"2020-34":1,"2020-24":1,"2019-51":1,"2019-47":2,"2019-39":1,"2019-35":1,"2019-30":1,"2019-13":1,"2019-04":2,"2018-47":2,"2018-43":2,"2018-34":2,"2018-26":1,"2018-17":1,"2018-09":1,"2018-05":1,"2017-47":1,"2017-39":1,"2017-34":1,"2017-22":2,"2017-09":18,"2016-40":1,"2016-36":17,"2016-30":16,"2016-22":1,"2016-18":1,"2016-07":12,"2015-48":4,"2015-40":1,"2015-35":4,"2015-32":5,"2015-27":4,"2015-22":4,"2015-14":4,"2014-52":3,"2014-49":5,"2014-42":9,"2014-41":7,"2014-35":4,"2014-23":5,"2014-15":2,"2023-40":1,"2015-18":4,"2015-11":4,"2015-06":4,"2014-10":2,"2013-48":1,"2024-10":1}},"file":"PMC3663117"},"subset":"pubmed_central"}
{"text":"abstract: A report of the Biochemical Society\/Wellcome Trust meeting 'Protein Evolution - Sequences, Structures and Systems', Hinxton, UK, 26-27 January 2009.\nauthor: John W Pinney; Michael PH Stumpf\ndate: 2009\ninstitute: 1Centre for Bioinformatics, Division of Molecular Biosciences, Imperial College London, Wolfson Building, London SW7 2AZ, UK\ntitle: Evolving proteins at Darwin's bicentenary\n\nThe effects of natural selection are ultimately mediated through protein function. The traditional view that selection on proteins is primarily due to the effects of mutations on protein structure has, however, in recent years been replaced by a much richer picture. This modern perspective was in evidence at a recent meeting on protein evolution in Hinxton, UK. Here we report some of the highlights.\n\nUnsurprisingly, Charles Darwin featured at lot at the meeting. Evolutionary arguments are all-pervasive in the biomedical and life sciences and this is particularly true for the analysis of proteins and their role in cell and molecular biology. From initial investigations of individual proteins in the 1940s and 1950s, which were motivated by even earlier work on blood groups, we can now routinely collect information from a large number of sequenced genomes to help us understand the evolution of proteins in terms of their sequences, structures and functions, and their roles as parts of biological systems.\n\n# Comparative evolution\n\nThe primacy of comparative, and thus evolutionary, arguments in the analysis of proteins and their structure was emphasized by Tom Blundell (University of Cambridge, UK), who reviewed almost 40 years of structural bioinformatics. He noted that in the early studies of insulin structure, the common ancestry of all life on Earth meant that lessons learned in the context of one species were transferable to other species. This in turn meant that sequence data could be linked to structure more directly through comparative arguments than would have been possible using biophysical or biochemical arguments. Despite vast increases in computational power and experimental resolution, this continues to be the case to the present day.\n\nThe explosion in available whole-genome data has provided us with a much richer understanding of genomic aspects of protein evolution. This was highlighted by Chris Ponting (University of Oxford, UK), who contrasted the distributions of proteins and protein family members in the human and mouse genomes. Such a comparison reveals high levels of sequence duplication - probably in line with what might be expected, given recent findings of copy-number variation - and suggests a scenario where ancient single-copy genes are only rarely gained or lost. Members of larger gene families, however, have experienced much more frequent gene duplication and loss; this may reflect the role of such gene families in adaptive evolution, as seen in the rapid evolution of the androgen-binding proteins in mouse.\n\nThe theme of adaptation was elaborated on by Bengt Mannervik (Uppsala University, Sweden), who focused on the evolution of enzymes, a class of proteins with perhaps uniquely well-characterized functionality. Here, he argued, the relative trade-off between substrate specificity and enzymatic activity has given rise to a quasi-species-like evolutionary scenario: abundant protein polymorphisms underlie a complex population of functional enzymatic variants. Such diversity in the metabolic functions available within the population may presumably help to buffer changes in the environment encountered during evolution.\n\nAraxi Urrutia (University of Bath, UK) addressed predominantly the link between gene and protein expression and evolutionary conservation and adaptation. As she pointed out, there is clear emerging evidence that highly expressed genes in humans share certain characteristics such as short intron lengths and higher codon-usage bias and favor less metabolically expensive amino acids. This affects the rate at which protein-coding genes evolve in a manner independent of protein structure. Moreover, this level of selection also appears to depend on the genomic context, as patterns of expression of neighboring genes are statistically correlated.\n\n# Insights from structure\n\nAlso fundamental to protein activity is post-translational modification, notably phosphorylation. This is a field of enormous biomedical importance, as kinase and phosphatase activities crucially regulate signaling and metabolic processes. The structural work of Louise Johnson (University of Oxford, UK) and colleagues bridges 'classical' structural biology and systems biology, and she discussed the structural factors underlying the regulation of kinases and phosphorylation. These comprehensive analyses are now also beginning to reveal how biochemical compounds can affect kinase regulation in a manner that may become clinically exploitable.\n\nKeeping to the structural theme, Christine Orengo (University College London, UK) discussed the phenomenal insights that have been gained recently into the evolution of protein domain superfamilies and the ensuing effects that this can have on protein structure, active sites, and ultimately, function. For example, the analysis clearly reveals common structural cores that are shared across the members of the same superfamily but may be modified in individual members. Orengo documented how such differences in the HUP superdomain family lead to differences in the participation of paralogs in protein complexes and biological processes following duplication.\n\nAlex Bateman (Wellcome Trust Sanger Institute, UK) further elaborated on the evolution of families of protein domains. Such a domain-centric point of view adds a valuable and useful perspective. Yet even at the level of shuffling these protein building blocks, the picture becomes more detailed as the available evolutionary resolution increases: for example, the frequency of changes in domain architecture is seen to approximately double following a gene duplication event as compared with a speciation event.\n\n# Protein evolution *in vitro* and *in vivo*\n\nUsing extensive and genome-wide data from yeast and humans, Laurence Hurst (University of Bath, UK) demonstrated the substantial role of non-structural selection pressures, such as those imposed by transcription and translation, on the evolutionary dynamics of proteins.\n\nTaking these into account results in a much richer picture of protein evolution, with the contribution of splicing-related constraints being particularly pronounced in mammals. Surprisingly, perhaps, these constraints show the same relative importance for protein evolution as aspects of gene expression do, as discussed by Urrutia. This is in stark contrast to the traditional amino-acid-centered view of protein evolution.\n\nUsing analogies with mountaineering, Dan Tawfik (Weizmann Institute, Rehovot, Israel) covered the exciting opportunities afforded by experimental studies of protein evolution. Evolution has sometimes been viewed previously as an observational and mathematical discipline rather than one characterized by experimental work. Tawfik showed how it is possible to explore evolutionary trajectories through the space of possible protein folds or functions in far more detail than had previously been thought possible. One of the exciting possibilities emerging from this work is that we will be able to study the interplay between neutral evolution and the various factors influencing selection. There is already good direct experimental detail from these laboratory studies that demonstrate the link between the rate of protein evolution and 'functional promiscuity' and conformational variability.\n\nOne of us (MPHS) described the phage-shock stress response in *Escherichia coli* as an example in which the loss and gain of proteins across bacterial species can only be understood in the context of mechanistic models of the system itself. Loss of individual genes can compromise the functionality of the stress response, which can only be tolerated under certain ecological conditions. As a result, it appears that either the complete set of proteins contributing to the stress response is maintained in bacterial genomes, or all are lost together. This all-or-nothing scenario is probably inextricably linked to the ecological niches inhabited by the bacteria.\n\nDavid Robertson (University of Manchester, UK) discussed how patterns of gene duplication and diversification have shaped the global structure of protein-protein interaction networks, as well as many of their detailed features. In contrast to previous work, this detailed analysis of the protein-interaction network in *Saccharomyces cerevisiae* clearly shows that the coevolution of interacting proteins cannot simply be explained by observed protein-protein interactions. What emerges from this and related studies is that many of the high-level models of network evolution proposed only a few years ago are too simplistic for dealing with such highly contingent and complex processes. Robertson concluded with a discussion of the evolutionary history of human disease genes, which also highlights the importance of historical levels of gene duplication, and reinforces the need for nuanced assessment of the different factors affecting protein evolution.\n\nDiscussing the physical interations of kinases, Mike Tyers (University of Edinburgh, UK) described an exciting new experimental mapping study of physical protein-protein interactions of kinases. The experimental determination of these, frequently weak, protein interactions poses many challenges, requiring considerable reworking of existing platforms for proteomics, but the information produced is expected to be of great value to systems biologists. Preliminary results already suggest that the wealth of material expected from this survey will aid our understanding of the molecular mechanisms involved in these processes.\n\nTwo hundred years after the birth of Charles Darwin, we understand a great deal about the processes of evolution and how they have shaped the diversity of life on Earth. The application of the simple idea of \"descent with modification\" to proteins, their structures, expression patterns, interactions and ultimately their emergent functions continues to produce fundamental insights into how biological systems evolve. But the picture emerging from this unprecedented access to molecular data at all levels of cellular organization is much more nuanced than we would have thought possible only a few years ago.","meta":{"dup_signals":{"dup_doc_count":101,"dup_dump_count":46,"dup_details":{"curated_sources":2,"2021-43":1,"2021-31":1,"2020-24":1,"2019-30":1,"2019-22":1,"2019-09":1,"2018-51":1,"2018-43":1,"2018-30":1,"2018-17":1,"2018-09":1,"2017-47":1,"2017-34":1,"2017-22":1,"2017-17":1,"2017-09":7,"2016-50":1,"2016-44":1,"2016-40":1,"2016-36":9,"2016-30":7,"2016-22":1,"2016-18":1,"2016-07":4,"2015-48":2,"2015-40":1,"2015-35":2,"2015-32":2,"2015-27":2,"2015-22":2,"2015-14":2,"2014-52":2,"2014-49":4,"2014-42":7,"2014-41":4,"2014-35":1,"2014-23":3,"2014-15":4,"2023-50":1,"2015-18":2,"2015-11":2,"2015-06":2,"2014-10":2,"2013-48":2,"2013-20":2,"2024-26":1}},"file":"PMC2688924"},"subset":"pubmed_central"}
{"text":"abstract: # OBJECTIVE\n  .\n  To review the evidence about the impact of hypoglycemia on patients with diabetes that has become available since the past reviews of this subject by the American Diabetes Association and The Endocrine Society and to provide guidance about how this new information should be incorporated into clinical practice.\n  .\n  # PARTICIPANTS\n  .\n  Five members of the American Diabetes Association and five members of The Endocrine Society with expertise in different aspects of hypoglycemia were invited by the Chair, who is a member of both, to participate in a planning conference call and a 2-day meeting that was also attended by staff from both organizations. Subsequent communications took place via e-mail and phone calls. The writing group consisted of those invitees who participated in the writing of the manuscript. The workgroup meeting was supported by educational grants to the American Diabetes Association from Lilly USA, LLC and Novo Nordisk and sponsorship to the American Diabetes Association from Sanofi. The sponsors had no input into the development of or content of the report.\n  .\n  # EVIDENCE\n  .\n  The writing group considered data from recent clinical trials and other studies to update the prior workgroup report. Unpublished data were not used. Expert opinion was used to develop some conclusions.\n  .\n  # CONSENSUS PROCESS\n  .\n  Consensus was achieved by group discussion during conference calls and face-to-face meetings, as well as by iterative revisions of the written document. The document was reviewed and approved by the American Diabetes Association's Professional Practice Committee in October 2012 and approved by the Executive Committee of the Board of Directors in November 2012 and was reviewed and approved by The Endocrine Society's Clinical Affairs Core Committee in October 2012 and by Council in November 2012.\n  .\n  # CONCLUSIONS\n  .\n  The workgroup reconfirmed the previous definitions of hypoglycemia in diabetes, reviewed the implications of hypoglycemia on both short- and long-term outcomes, considered the implications of hypoglycemia on treatment outcomes, presented strategies to prevent hypoglycemia, and identified knowledge gaps that should be addressed by future research. In addition, tools for patients to report hypoglycemia at each visit and for clinicians to document counseling are provided.\nauthor: Elizabeth R. Seaquist; John Anderson; Belinda Childs; Philip Cryer; Samuel Dagogo-Jack; Lisa Fish; Simon R. Heller; Henry Rodriguez; James Rosenzweig; Robert VigerskyCorresponding author: Elizabeth R. Seaquist, <firstname.lastname@example.com>.\ndate: 2013-05\nreferences:\ntitle: Hypoglycemia and Diabetes: A Report of a Workgroup of the American Diabetes Association and The Endocrine Society\n\nIn 2005, the American Diabetes Association Workgroup on Hypoglycemia released a report entitled \"Defining and Reporting Hypoglycemia in Diabetes\" (1). In that report, recommendations were primarily made to advise the U.S. Food and Drug Administration (FDA) on how hypoglycemia should be used as an end point in studies of new treatments for diabetes. In 2009, The Endocrine Society released a clinical practice guideline entitled \"Evaluation and Management of Adult Hypoglycemic Disorders,\" which summarized how clinicians should manage hypoglycemia in patients with diabetes (2). Since then, new evidence has become available that links hypoglycemia with adverse outcomes in older patients with type 2 diabetes (3\u20136) and in children with type 1 diabetes (7,8). To provide guidance about how this new information should be incorporated into clinical practice, the American Diabetes Association and The Endocrine Society assembled a new Workgroup on Hypoglycemia in April 2012 to address the following questions:\n\n1.  How should hypoglycemia in diabetes be defined and reported?\n\n2.  What are the implications of hypoglycemia on both short- and long-term outcomes in people with diabetes?\n\n3.  What are the implications of hypoglycemia on treatment targets for patients with diabetes?\n\n4.  What strategies are known to prevent hypoglycemia, and what are the clinical recommendations for those at risk for hypoglycemia?\n\n5.  What are the current knowledge gaps in our understanding of hypoglycemia, and what research is necessary to fill these gaps?\n\n# How should hypoglycemia in diabetes be defined and reported?\n\nHypoglycemia puts patients at risk for injury and death. Consequently the workgroup defines iatrogenic hypoglycemia in patients with diabetes as all episodes of an abnormally low plasma glucose concentration that expose the individual to potential harm. A single threshold value for plasma glucose concentration that defines hypoglycemia in diabetes cannot be assigned because glycemic thresholds for symptoms of hypoglycemia (among other responses) shift to lower plasma glucose concentrations after recent antecedent hypoglycemia (9\u201312) and to higher plasma glucose concentrations in patients with poorly controlled diabetes and infrequent hypoglycemia (13).\n\nNonetheless, an alert value can be defined that draws the attention of both patients and caregivers to the potential harm associated with hypoglycemia. The workgroup (1) suggests that patients at risk for hypoglycemia (i.e., those treated with a sulfonylurea, glinide, or insulin) should be alert to the possibility of developing hypoglycemia at a self-monitored plasma glucose\u2014or continuous glucose monitoring subcutaneous glucose\u2014concentration of \u226470 mg\/dL (\u22643.9 mmol\/L). This alert value is data driven and pragmatic (14). Given the limited accuracy of the monitoring devices, it approximates the lower limit of the normal postabsorptive plasma glucose concentration (15), the glycemic thresholds for activation of glucose counterregulatory systems in nondiabetic individuals (15), and the upper limit of plasma glucose level reported to reduce counterregulatory responses to subsequent hypoglycemia (11). Because it is higher than the glycemic threshold for symptoms in both nondiabetic individuals and those with well-controlled diabetes (9,13,14), it generally allows time to prevent a clinical hypoglycemic episode and provides some margin for the limited accuracy of monitoring devices at low-glucose levels. People with diabetes need not always self-treat at an estimated glucose concentration of \u226470 mg\/dL (\u22643.9 mmol\/L). Options other than carbohydrate ingestion include repeating the test in the short term, changing behavior (e.g., avoiding driving or elective exercise until the glucose level is higher), and adjusting the treatment regimen. Although this alert value has been debated (9,13,14), a plasma concentration of \u226470 mg\/dL (\u22643.9 mmol\/L) can be used as a cut-off value in the classification of hypoglycemia in diabetes.\n\nConsistent with past recommendations (1), the workgroup suggests the following classification of hypoglycemia in diabetes:\n\n### 1) Severe hypoglycemia.\n\nSevere hypoglycemia is an event requiring assistance of another person to actively administer carbohydrates, glucagon, or take other corrective actions. Plasma glucose concentrations may not be available during an event, but neurological recovery following the return of plasma glucose to normal is considered sufficient evidence that the event was induced by a low plasma glucose concentration.\n\n### 2) Documented symptomatic hypoglycemia.\n\nDocumented symptomatic hypoglycemia is an event during which typical symptoms of hypoglycemia are accompanied by a measured plasma glucose concentration \u226470 mg\/dL (\u22643.9 mmol\/L).\n\n### 3) Asymptomatic hypoglycemia.\n\nAsymptomatic hypoglycemia is an event not accompanied by typical symptoms of hypoglycemia but with a measured plasma glucose concentration \u226470 mg\/dL (\u22643.9 mmol\/L).\n\n### 4) Probable symptomatic hypoglycemia.\n\nProbable symptomatic hypoglycemia is an event during which symptoms typical of hypoglycemia are not accompanied by a plasma glucose determination but that was presumably caused by a plasma glucose concentration \u226470 mg\/dL (\u22643.9 mmol\/L).\n\n### 5) Pseudo-hypoglycemia.\n\nPseudo-hypoglycemia is an event during which the person with diabetes reports any of the typical symptoms of hypoglycemia with a measured plasma glucose concentration \\>70 mg\/dL (\\>3.9 mmol\/L) but approaching that level.\n\n## The challenge of measuring glucose accurately\n\nCurrently, two technologies are available to measure glucose in outpatients: capillary measurement with point-of-care (POC) glucose meters (self-monitored blood glucose \\[SMBG\\]) and interstitial measurement with continuous glucose monitors (CGMs), both retrospective and real time. The International Organization for Standardization (ISO) and FDA standards require that POC meters' analytical accuracy be within 20% of the actual value in 95% of samples with glucose levels \u226575 mg\/dL and \u00b115 mg\/dL for samples with glucose \\<75 mg\/dL. Despite this relatively large permissible variation, Freckmann et al. (16) found that only 15 of 27 meters on the market in Europe several years ago met the current analytical standards of \u00b115 mg\/dL in the hypoglycemia range, 2 of 27 met \u00b110 mg\/dL, and none were capable of measuring \u00b15 mg\/dL.\n\nThe need for accurate meters in the \\<75 mg\/dL range is essential in insulin-treated patients, whether they are outpatients or inpatients, but it is less important in those outpatients who are on medications that rarely cause hypoglycemia. In critical care units, where the accuracy of POC meters is particularly crucial, their performance may be compromised by medications (vasopressors, acetaminophen), treatments (oxygen), and clinical states (hypotension, anemia) (17). Karon et al. (18) translated these measurement errors into potential insulin-dosing errors using simulation modeling and found that if there were a total measurement error of 20%, 1- and 2-step errors in insulin dose would occur 45% and 6% of the time, respectively, in a tight glycemic control protocol. Such imprecision may affect the safe implementation of insulin infusion protocols in critical care units and may account in part for the high hypoglycemia rates in most trials of inpatient intensive glycemic control.\n\nRetrospective and real-time CGMs represent an evolving technology that has made considerable progress in overall (point + rate) accuracy. However, the accuracy of CGMs in the hypoglycemic range is poor as demonstrated by error grid analysis (19,20). With existing real-time CGMs, accuracy can be achieved in only 60\u201373% of samples in the range of 40\u201380 mg\/dL (21,22). Because the accuracy of CGMs, like POC meters, is negatively affected by multiple factors in hospitalized patients and they are calibrated with POC meters affected by those same factors, CGMs are not recommended for glycemic management in hospitalized patients at this time (17).\n\n# What are the implications of hypoglycemia on both short- and long-term outcomes in people with diabetes?\n\nIatrogenic hypoglycemia is more frequent in patients with profound endogenous insulin deficiency\u2014type 1 diabetes and advanced type 2 diabetes\u2014and its incidence increases with the duration of diabetes (23). It is caused by treatment with a sulfonylurea, glinide, or insulin and occurs about two to three times more frequently in type 1 diabetes than in type 2 diabetes (23,24). Event rates for severe hypoglycemia for patients with type 1 diabetes range from 115 (24) to 320 (23) per 100 patient-years. Severe hypoglycemia in patients with type 2 diabetes has been shown to occur at rates of 35 (24) to 70 (23) per 100 patient-years. However, because type 2 diabetes is much more prevalent than type 1 diabetes, most episodes of hypoglycemia, including severe hypoglycemia, occur in people with type 2 diabetes (25).\n\nThere is no doubt that hypoglycemia can be fatal (26). In addition to case reports of hypoglycemic deaths in patients with type 1 and type 2 diabetes, four recent reports of mortality rates in series of patients indicate that 4% (27), 6% (28), 7% (29), and 10% (30) of deaths of patients with type 1 diabetes were caused by hypoglycemia. A temporal relationship between extremely low subcutaneous glucose concentrations and death in a patient with type 1 diabetes who was wearing a CGM device and was found dead in bed has been reported (31). Although profound and prolonged hypoglycemia can cause brain death, most episodes of fatal hypoglycemia are probably the result of other mechanisms, such as ventricular arrhythmias (26). In this section, we will consider the effects of hypoglycemia on the development of hypoglycemia unawareness and how iatrogenic hypoglycemia may affect outcomes in specific patient groups.\n\n## Hypoglycemia unawareness and hypoglycemia-associated autonomic failure\n\nAcute hypoglycemia in patients with diabetes can lead to confusion, loss of consciousness, seizures, and even death, but how a particular patient responds to a drop in glucose appears to depend on how frequently that patient experiences hypoglycemia. Recurrent hypoglycemia has been shown to reduce the glucose level that precipitates the counterregulatory response necessary to restore euglycemia during a subsequent episode of hypoglycemia (10\u201312). As a result, patients with frequent hypoglycemia do not experience the symptoms from the adrenergic response to a fall in glucose until the blood glucose reaches lower and lower levels. For some individuals, the level that triggers the response is below the glucose level associated with neuroglycopenia. The first sign of hypoglycemia in these patients is confusion, and they often must rely on the assistance of others to recognize and treat low blood glucose. Such individuals are said to have developed hypoglycemia unawareness. Defective glucose counterregulation (the result of loss of a decrease in insulin production and an increase in glucagon release along with an attenuated increase in epinephrine) and hypoglycemia unawareness (the result of an attenuated increase in sympathoadrenal activity) are the components of hypoglycemia-associated autonomic failure (HAAF) in patients with diabetes. HAAF is a form of functional sympathoadrenal failure that is most often caused by recent antecedent iatrogenic hypoglycemia (25) and is at least partly reversible by scrupulous avoidance of hypoglycemia (32\u201334). Indeed, HAAF has been shown to be maintained by recurrent iatrogenic hypoglycemia (33,34). The development of HAAF is associated with a 25-fold (35) or greater (36) increased risk of severe hypoglycemia during intensive glycemic therapy. It is important to distinguish HAAF from classical autonomic neuropathy, which may occur as one form of diabetic neuropathy. Impaired sympathoadrenal activation is generally confined to the response to hypoglycemia, and autonomic activities in organs such as the heart, gastrointestinal tract, and bladder appear to be unaffected.\n\nClinically, HAAF can be viewed as both adaptive and maladaptive. On the one hand, patients with hypoglycemia unawareness and type 1 diabetes appear to perform better on tests of cognitive function during hypoglycemia than do patients who are able to detect hypoglycemia normally (37). In addition, the time necessary for full cognitive recovery after restoration of euglycemia appears to be faster in patients who have hypoglycemia unawareness than in patients with normal detection of hypoglycemia (37). The HAAF habituation of the sympathoadrenal response to recurrent hypoglycemic stress in humans (38) may be analogous to the phenomenon of habituation of the hypothalamic-pituitary-adrenocortical response to recurrent restraint stress in rats (39). Rats subjected to recurrent moderate hypoglycemia had less brain cell death (40) and less mortality (41) during or following marked hypoglycemia than those not subjected to recurrent hypoglycemia.\n\nOn the other hand, HAAF is clearly maladaptive since defective glucose counterregulation and hypoglycemia unawareness substantially increase the risk of severe hypoglycemia with its morbidity and potential mortality (26). A particularly low plasma glucose concentration might trigger a robust, potentially fatal sympathoadrenal discharge. Life-threatening episodes of hypoglycemia need not be frequent to be devastating.\n\n## Impact of hypoglycemia on children with diabetes\n\nHypoglycemia is a common problem in children with type 1 diabetes because of the challenges presented by insulin dosing, variable eating patterns, erratic activity, and the limited ability of small children to detect hypoglycemia. The infant, young child, and even the adolescent typically exhibit unpredictable feeding\u2014not eating all the anticipated food at a meal and snacking unpredictably between meals\u2014and have prolonged periods of fasting overnight that increase the risk of hypoglycemia. Selecting the correct prandial dose of insulin is therefore often difficult. Very low insulin requirements for basal and mealtime dosing in the infant and young child frequently require use of miniscule basal rates in pump therapy and one-half unit dosing increments with injections. Management rarely requires the use of diluted insulin, e.g., 10 units per mL. Infants and toddlers may not recognize the symptoms of hypoglycemia and lack the ability to effectively communicate their distress. Caregivers must be particularly aware that changes in behavior such as a loss of temper may be a sign of hypoglycemia.\n\nPuberty is associated with insulin resistance, while at the same time the normal developmental stages of adolescence may lead to inattention to diabetes and increased risk for hypoglycemia. As children grow, they often have widely fluctuating levels of activity during the day, which puts them at risk for hypoglycemia. Minimizing the impact of hypoglycemia on children with diabetes requires the education and engagement of parents, patients, and other caregivers in the management of the disease (42,43).\n\nThe youngest patients are most vulnerable to the adverse consequences of hypoglycemia. Ongoing maturation of the central nervous system puts these children at greater risk for cognitive deficits as a consequence of hypoglycemia (44). Recent studies have examined the impact of hypoglycemia on cognitive function and cerebral structure in children and found that those who experience this complication before the age of 5 years seem to be more affected than those who do not have hypoglycemia until later (7). The long-term impact of hypoglycemia on cognition before the age of 5 years is unknown.\n\n## Impact of hypoglycemia on adults with type 1 diabetes\n\nLandmark data on the impact of hypoglycemia on adults with type 1 diabetes come from the Diabetes Control and Complications Trial (DCCT) and its follow-up study, where cognition has been systematically measured over time. In this cohort, performance on a comprehensive battery of neurocognitive tests at 18 years of follow-up was the same in participants with and without a history of severe hypoglycemia (28). Despite such reassuring findings, recent investigation with advanced imaging techniques has demonstrated that adults with type 1 diabetes appear to call upon a greater volume of the brain to perform a working memory task during hypoglycemia (45). These findings suggest that adults with type 1 diabetes must recruit more regions to preserve cognitive function during hypoglycemia than adults without the disease. More work will be necessary to understand the significance of these observations on the long-term cognitive ability of adults with type 1 diabetes.\n\n## Impact of hypoglycemia on patients with type 2 diabetes\n\nThere is growing evidence that patients with type 2 diabetes might be particularly vulnerable to adverse events associated with hypoglycemia. Over the last decade, three large trials examined the effect of glucose lowering on cardiovascular events in patients with type 2 diabetes: ACCORD (Action to Control Cardiovascular Risk in Diabetes), ADVANCE (Action in Diabetes and Vascular Disease: Preterax and Diamicron MR Controlled Evaluation), and VADT (Veterans Affairs Diabetes Trial). Between them, a total of 24,000 patients with high cardiovascular risk were randomly assigned to either intensive glycemic control or standard therapy (3\u20135). In each, subjects who were randomly assigned to the intensive arm experienced more episodes of hypoglycemia than did those who were randomly assigned to the standard treatment arm. In the ACCORD trial, subjects who were randomly assigned to the intensive arm also experienced a 20% increase in mortality, and the glycemic control study was stopped early due to this finding. A relationship between mortality and randomization to intensive glucose control was not observed in ADVANCE or VADT, although VADT was underpowered to explore this relationship. A number of explanations have been offered to explain the findings of ACCORD, including chance, greater weight gain, and specific medication effects, but perhaps the most convincing candidate was hypoglycemia, which was threefold higher in the intensive arm of ACCORD (4).\n\nIn the opinion of the blinded adjudication committee assigned to investigate mortality in ACCORD, hypoglycemia was judged to have a definite role in only one death, a probable role in three deaths, and a possible role in 38 deaths (46), which represents a role in less than 10% of the deaths recorded in the study population while the glycemic intervention was active. The investigators thus suggest that hypoglycemia at the time of death was probably not responsible for the increased mortality rate in the intensive arm of ACCORD. Since glycemia was not measured at the time of death in any of the ACCORD subjects, we may never know. However, the potential lethal mechanisms that might be provoked by hypoglycemia could cause mortality downstream of the hypoglycemic event, increasing the difficulty in establishing cause and effect.\n\nAll three trials clearly demonstrated that an episode of severe hypoglycemia was associated with an increased risk of subsequent mortality. In ACCORD, those who had one or more severe hypoglycemic episodes had higher rates of death than those without such episodes across both study arms (hazard ratio 1.41 \\[95% CI 1.03\u20131.93\\]) (46). One-third of all deaths were due to cardiovascular disease, and hypoglycemia was associated with higher cardiovascular mortality. In VADT, a recent severe hypoglycemic event was the strongest independent predictor of death at 90 days (3). In ADVANCE, where rates of hypoglycemia were low, a similar pattern was found (47). Of course, in post hoc analyses a causal relationship cannot be established with certainty. It is possible that the association between hypoglycemia and death may be merely an indicator for vulnerability for death from any cause.\n\nThe relationship between hypoglycemia and subsequent cognitive function in patients with type 2 diabetes has also been investigated. In a large population study, hypoglycemic episodes that required hospitalization or a visit to the emergency department between 1980 and 2002 were associated with approximately double the risk of incident dementia after 2003 (6). However, since the study population did not undergo detailed tests of cognitive function prior to 2003, it is possible that those with incident dementia actually had mild cognitive dysfunction prior to experiencing the episode(s) of severe hypoglycemia. The possibility that mild cognitive dysfunction might increase the risk of experiencing severe hypoglycemia has been supported by analyses from the ACCORD study (48). In the ACCORD MIND (Memory IN Diabetes) study, in which cognitive function was assessed longitudinally, no difference was noted in the rate at which cognitive performance declined over time in subjects randomly assigned to the intensive versus the standard glucose arms despite the fact that they experienced three times as much hypoglycemia (49). Future investigation will need to address this question because the existing data are somewhat contradictory.\n\n## Impact of hypoglycemia on the elderly\n\nPatients in the older age-groups are especially vulnerable to hypoglycemia. Epidemiological studies show that hypoglycemia is the most frequent metabolic complication experienced by older adults in the U.S. (50). Although severe hypoglycemia is common in older individuals with both type 1 and type 2 diabetes, patients with type 2 diabetes tend to have longer hospital stays and greater medical costs. The most significant predictors of this condition are advanced age, recent hospitalization, and polypharmacy, as shown in a study of Tennessee Medicare patients (51). Age-related declines in renal function and hepatic enzyme activity may interfere with the metabolism of sulfonylureas and insulin, thereby potentiating their hypoglycemic effects. The vulnerability of the elderly to severe hypoglycemia may be partially related to a progressive age-related decrease in \u03b2-adrenergic receptor function (52). Age-related impairment in counterregulatory hormone responses has been described in elderly patients with diabetes, especially with respect to glucagon and growth hormone (53). Symptoms of neuroglycopenia are more prevalent (54). With the prolonged duration of type 2 diabetes as is often seen in the elderly patient, the glucagon response to hypoglycemia is virtually absent (55). The intensification of glycemic control in the elderly patient is associated with an increased reduction in the plasma glucose thresholds for epinephrine release and for the appearance of hypoglycemia (56). As a result, changes in the level of glycemic control have a marked impact on the risk of developing hypoglycemia in the elderly.\n\nOlder adults with diabetes have a disproportionately high number of clinical complications and comorbidities, all of which can be exacerbated by and sometimes contribute to episodes of hypoglycemia. Older adults with diabetes are at much higher risk for the geriatric syndrome, which includes falls, incontinence, frailty, cognitive impairment, and depressive symptoms (57). The cognitive and executive dysfunction associated with the geriatric syndrome interferes with the patient's ability to perform self-care activities appropriately and follow the treatment regimen (58).\n\nTo minimize the risk of hypoglycemia in the elderly, careful education regarding the symptoms and treatment of hypoglycemia, with regular reinforcement, is extremely important because of the recognized gaps in the knowledge base of these individuals (59). In addition, it is important to assess the elderly for functional status as part of the overall clinical assessment in order to properly apply individualized glycemic control goals. Arbitrary short-acting insulin sliding scales, which are used much too often in long-term care facilities (60), should be avoided, and glyburide should be discontinued in favor of shorter-acting insulin secretagogues or medications that do not cause hypoglycemia. The recently published 2012 Beers list of prohibited medications in long-term care facilities specifically lists insulin sliding scales and glyburide as treatment modalities that should be avoided (61). Complex regimens requiring multiple decision points should be simplified, especially for patients with decreased functional status. In addition, caregivers and staff in long-term care facilities need to be educated on the causes and risks of hypoglycemia and the proper surveillance and treatment of this condition.\n\n## Impact of hypoglycemia on hospitalized patients\n\nPersons with diabetes are three times more likely to be hospitalized than those without diabetes, and approximately 25% of hospitalized patients (including people without a history of diabetes) have hyperglycemia (62\u201365). Inpatient hyperglycemia has been associated with prolonged hospital length of stay and with numerous adverse outcomes including mortality (64,66\u201368). The understandable zeal to minimize the adverse consequences of inpatient hyperglycemia, together with the demonstration that intensive glycemic control improved outcomes in surgical intensive care unit (ICU) patients (69), led to widespread adoption of aggressive glucose management among ICU patients. However, subsequent studies showed that such aggressive lowering of glycemia in the ICU is not uniformly beneficial, markedly increases the risk of severe hypoglycemia, and may be associated with increased mortality (70\u201374).\n\nThe true incidence and prevalence of hypoglycemia among hospitalized patients with diabetes are not known precisely. In a retrospective study of 31,970 patients admitted to the general wards of an academic medical center in 2007, a total of 3,349 patients (10.5%) had at least one episode of hypoglycemia (\u226470 mg\/dL) (75). In another review of 5,365 inpatients admitted to ICUs, 102 (1.9%) had at least one episode of severe hypoglycemia (\\<40 mg\/dL) (76). The risk factors for inpatient hypoglycemia include older age, presence of comorbidities, diabetes, increasing number of antidiabetic agents, tight glycemic control, septic shock, renal insufficiency, mechanical ventilation, and severity of illness (75,76). With regard to impact, a retrospective analysis of 4,368 admissions involving 2,582 diabetic patients admitted to the general ward indicated that severe hypoglycemia (\u226450 mg\/dL) was associated with increased length of stay and greater odds of inpatient death and death within 1 year of hospital discharge (77).\n\n## Impact of hypoglycemia during pregnancy\n\nMaintaining blood glucose control in pregnancy as close to that of healthy pregnant women is important in minimizing the negative effects on the mother and the fetus (78). This is true for women with pregestational type 1 or type 2 diabetes, as well for those with gestational diabetes mellitus. Normal blood glucose levels during pregnancy are 20% lower than in nonpregnant women (79), making the definition and detection of hypoglycemia more challenging. For women with type 1 diabetes, severe hypoglycemia occurs 3\u20135 times more frequently in the first trimester and at a lower rate in the third trimester when compared with the incidence in the year preceding the pregnancy (80). Risk factors for severe hypoglycemia in pregnancy include a history of severe hypoglycemia in the preceding year, impaired hypoglycemia awareness, long duration of diabetes, low HbA~1c~ in early pregnancy, fluctuating plasma glucose levels, and excessive use of supplementary insulin between meals. Surprisingly, nausea and vomiting during pregnancy did not appear to add significant risk. When pregnant and nonpregnant women are compared with CGM, mild hypoglycemia (defined by the authors as blood glucose \\<60 mg\/dL) is more common in all pregnant women, but equally so regardless of whether or not they have diabetes, either pregestational or gestational (81). Hypoglycemia is generally without risk for the fetus as long as the mother avoids injury during the episode. For women with preexisting diabetes, insulin requirements rise throughout the pregnancy and then drop precipitously at the time of delivery of the placenta, requiring an abrupt reduction in insulin dosing to avoid postdelivery hypoglycemia. Breastfeeding may also be a risk factor for hypoglycemia in women with insulin-treated diabetes (82).\n\n## Impact of hypoglycemia on quality of life and activities of daily living\n\nHypoglycemia and the fear of hypoglycemia have a significant impact on quality-of-life measures in patients with both type 1 and type 2 diabetes (83). Nocturnal hypoglycemia in particular may impact one's sense of well-being on the following day because of its impact on sleep quantity and quality (84). Patients with recurrent hypoglycemia have been found to have chronic mood disorders including depression and anxiety (85,86), although it is hard to establish cause and effect between hypoglycemia and mood changes. Interpersonal relationships may suffer as a result of hypoglycemia in patients with diabetes. In-depth interviews of a small group of otherwise healthy young adults with type 1 diabetes revealed the presence of interpersonal conflict including fears of dependency and loss of control. These adults also reported difficulty talking about issues related to hypoglycemia with significant others (87). This difficulty may carry over to their work life, where hypoglycemia has been linked to reduced productivity (88). Hypoglycemia also impairs one's ability to drive a car (89\u201391), and many jurisdictions require documentation that severe hypoglycemia is not occurring before persons with diabetes are permitted to have a license to operate a motor vehicle (92). However, impaired awareness of hypoglycemia has not consistently been associated with an increased risk of car collisions (92\u201395).\n\n# What are the implications of hypoglycemia on treatment targets for patients with diabetes?\n\nThe glycemic target established for any given patient should depend on the patient's age, life expectancy, comorbidities, preferences, and an assessment of how hypoglycemia might impact his or her life. This patient-centered approach requires that clinicians spend time developing an individualized treatment plan with each patient. For very young children, the risks of severe hypoglycemia on brain development may require a strategy that attempts to avoid hypoglycemia at all costs. For healthy adults with diabetes, a reasonable glycemic goal might be the lowest HbA~1c~ that does not cause severe hypoglycemia, preserves awareness of hypoglycemia, and results in an acceptable number of documented episodes of symptomatic hypoglycemia. With current therapies, a strategy that completely avoids hypoglycemia may not be possible in patients with type 1 diabetes who strive to minimize their risks of developing the microvascular complications of the disease. However, glycemic goals might reasonably be relaxed in patients with long-standing type 1 diabetes and advanced complications or in those who are free of complications but have a limited life expectancy because of another disease process. In such patients, the glycemic goal could be to achieve glucose levels sufficiently low to prevent symptoms of hyperglycemia.\n\nFor patients with type 2 diabetes, the risk of hypoglycemia depends on the medications used (96). Early in the course of the disease, most patients are treated with lifestyle changes and metformin, neither of which causes hypoglycemia. Therefore, an HbA~1c~ of \\<7% is appropriate for many patients with recent-onset type 2 diabetes. As the disease progresses, it is likely that medications that increase the risk of hypoglycemia will be added. This, plus the presence of complications or comorbidities that limit life expectancy, means that glycemic goals may need to be less aggressive. While the benefits of achieving an HbA~1c~ of \\<7% may continue to be advocated for patients with type 2 diabetes at risk for microvascular complications and with sufficient life expectancy, less aggressive targets may be appropriate in those with known cardiovascular disease, extensive comorbidities, or limited life expectancy.\n\nOlder individuals with gait imbalance and frailty may experience a life-changing injury if they fall during a hypoglycemia episode, so avoiding hypoglycemia is paramount in such patients. Patients with cognitive dysfunction may have difficulty adhering to a complicated treatment strategy designed to achieve a low HbA~1c~ (48). Such patients will benefit from a simplification of the treatment strategy with a goal to prevent hypoglycemia as much as possible. Furthermore, the benefits of aggressive glycemic therapy in those affected are unclear.\n\n# What strategies are known to prevent hypoglycemia, and what are the clinical recommendations for those at risk for hypoglycemia?\n\nRecurrent hypoglycemia increases the risk of severe hypoglycemia and the development of hypoglycemia unawareness and HAAF. Effective approaches known to decrease the risk of iatrogenic hypoglycemia include patient education, dietary and exercise modifications, medication adjustment, careful glucose monitoring by the patient, and conscientious surveillance by the clinician.\n\n## Patient education\n\nThere is limited research related to the influence of self-management education on the incidence or prevention of hypoglycemia. However, there is clear evidence that diabetes education improves patient outcomes (97\u201399). As part of the educational plan, the individual with diabetes and his or her domestic companions need to recognize the symptoms of hypoglycemia and be able to treat a hypoglycemic episode properly with oral carbohydrates or glucagon. Hypoglycemia, including its risk factors and remediation, should be discussed routinely with patients receiving treatment with insulin or sulfonylurea\/glinide drugs, especially those with a history of recurrent hypoglycemia or impaired awareness of hypoglycemia. In addition, patients must understand how their medications work so they can minimize the risk of hypoglycemia. Care should be taken to educate patients on the typical pharmacokinetics of these medications. When evaluating a patient's report of hypoglycemia, it is important to adopt interviewing approaches that guide the patient to a correct identification of the precipitating factors of the episodes of hypoglycemia. Such a heuristic review of likely factors (skipped or inadequate meal, unusual exertion, alcohol ingestion, insulin dosage mishaps, etc.) in the period prior to the event can deepen the patient's appreciation of the behavioral factors that predispose to hypoglycemia.\n\nThere is convincing evidence that formal training programs that teach patients to replace insulin \"physiologically\" by giving background and mealtime\/correction doses of insulin can reduce the risk of severe hypoglycemia. The Insulin Treatment and Training programs developed by M\u00fchlhauser and Berger (100) have reported improved glycemic control comparable with DCCT while reducing the rates of severe hypoglycemia (101,102). These programs have been successfully delivered in other settings (103,104) with comparable reductions in hypoglycemic risk (105). Patients with frequent hypoglycemia may also benefit from enrollment in a blood glucose awareness training program. In such a program, patients and their relatives are trained to recognize subtle cues and early neuroglycopenic indicators of evolving hypoglycemia and respond to them before the occurrence of disabling hypoglycemia (106,107).\n\n## Dietary intervention\n\nPatients with diabetes need to recognize which foods contain carbohydrates and understand how the carbohydrates in their diet affect blood glucose. To avoid hypoglycemia, patients on long-acting secretagogues and fixed insulin regimens must be encouraged to follow a predictable meal plan. Patients on more flexible insulin regimens must know that prandial insulin injections should be coupled to meal times. Dissociated meal and insulin injection patterns lead to wide fluctuations in plasma glucose levels. Patients on any hypoglycemia-inducing medication should also be instructed to carry carbohydrates with them at all times to treat hypoglycemia.\n\nThe best bedtime snack to prevent overnight hypoglycemia in patients with type 1 diabetes has been investigated without clear consensus (108\u2013112). These conflicting reports suggest that the administration of bedtime snacks may need to be individualized and be part of a comprehensive strategy (balanced diet, patient education, optimized drug regimens, and physical activity counseling) for the prevention of nocturnal hypoglycemia.\n\n## Exercise management\n\nPhysical activity increases glucose utilization, which increases the risk of hypoglycemia. The risk factors for exertional hypoglycemia include prolonged exercise duration, unaccustomed exercise intensity, and inadequate energy supply in relation to ambient insulinemia (113,114). Postexertional hypoglycemia can be prevented or minimized by careful glucose monitoring before and after exercise and taking appropriate preemptive actions. Preexercise snacks should be ingested if blood glucose values indicate falling glucose levels. Patients with diabetes should carry readily absorbable carbohydrates when embarking on exercise, including sporadic house or yard work. Because of the kinetics of rapid-acting and intermediate-acting insulin, it may be prudent to empirically adjust insulin doses on the days of planned exercise, especially in patients with well-controlled diabetes with a history of exercise-related hypoglycemia.\n\n## Medication adjustment\n\nHypoglycemic episodes that are not readily explained by conventional factors (skipped or irregular meals, unaccustomed exercise, alcohol ingestion, etc.) may be due to excessive doses of drugs used to treat diabetes. A thorough review of blood glucose patterns may suggest vulnerable periods of the day that mandate adjustments to the current antidiabetes regimen. Such adjustments may include substitution of rapid-acting insulin (lispro, aspart, glulisine) for regular insulin, or basal insulin glargine or detemir for NPH, to decrease the risk of hypoglycemia. Continuous subcutaneous insulin infusion offers great flexibility for adjusting the doses and administration pattern of insulin to counteract iatrogenic hypoglycemia (115). For patients with type 2 diabetes, sulfonylureas are the oral agents that pose the greatest risk for iatrogenic hypoglycemia and substitution with other classes of oral agents or even glucagon-like peptide 1 analogs should be considered in the event of troublesome hypoglycemia (96). Interestingly, successful transplantation of whole pancreata or isolated pancreatic islet cells in patients with type 1 diabetes (116\u2013118) results in marked improvements in glycemic control and near abolition of iatrogenic hypoglycemia.\n\nPatients who develop hypoglycemia unawareness do so because of frequent and recurrent hypoglycemia. To avoid such frequent hypoglycemia, adjustments in the treatment regimen that scrupulously avoid hypoglycemia are necessary (<a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>). In published studies, this has required frequent (almost daily) contact between clinician and patient, and adjustments to caloric intake and insulin regimen based on blood glucose values (10,119,120). With this approach, restoration of autonomic symptoms of hypoglycemia occurred within 2 weeks, and complete reversal of hypoglycemia unawareness was achieved by 3 months. In some but not all reports, the recovery of symptoms is accompanied by the improvement in epinephrine secretion (32,33,120,121). The return of hypoglycemic symptom awareness was associated with a modest increase (\u223c0.5%) in HbA~lc~ values (33), but others have reported no loss of glycemic control (32,34).\n\nApproach to restore recognition of hypoglycemia in patients with HAAF\n\n![](1384tbl1)\n\n## Glucose monitoring\n\nGlucose monitoring is essential in managing patients at risk for hypoglycemia. Patients treated with insulin, sulfonylureas, or glinides should check their blood glucose whenever they develop the symptoms of hypoglycemia in order to confirm that they must ingest carbohydrates to treat the symptoms and collect information that can be used by the clinician to adjust the therapeutic regimen to avoid future hypoglycemia. Patients on basal-bolus insulin therapy should check their blood glucose before each meal and figure this value into the calculation of the dose of rapid-acting insulin to take at that time. Such care in dosing will likely reduce the risk of hypoglycemia.\n\nRecent technological developments have provided patients with new tools for glucose monitoring. Real-time CGM, by virtue of its ability to display the direction and rate of change, provides helpful information to the wearer leading to proactive measures to avoid hypoglycemia, e.g., when to think about having a snack or suspending insulin delivery on a pump. The CGM's audible and\/or vibratory alarms may be particularly helpful in avoiding severe hypoglycemia at night and restoring hypoglycemic awareness. With the low-glucose alarms set at 108 mg\/dL, 4 weeks of real-time CGM use restored the epinephrine response and improved adrenergic symptoms during a hyperinsulinemic hypoglycemic clamp in a small group of adolescents with type 1 diabetes and hypoglycemic unawareness (122).\n\nThe artificial pancreas, which couples a CGM to an insulin pump through sophisticated predictive algorithms, holds out the promise of completely eliminating hypoglycemia. Several internationally collaborative groups are working on various approaches to the artificial pancreas. The first step in this direction is the low-glucose suspend pump that is available in Europe and currently in clinical trials in the U.S. This device shuts off insulin delivery for up to 2 h once the interstitial glucose concentration reaches a preset threshold and reduces the duration of nocturnal hypoglycemia (123).\n\n## Clinical surveillance\n\nClinicians and educators must assess the risk of hypoglycemia at every visit with patients treated with insulin and insulin secretagogues. An efficient way to begin this assessment might be to have the patient complete the questionnaire shown in <a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a> while in the waiting room. Review of the completed questionnaire will help the clinician learn how often the patient is experiencing symptomatic and asymptomatic hypoglycemia, ensure the patient is aware of how to appropriately treat hypoglycemia, and remind both parties of the risks associated with driving while hypoglycemic. To ensure that hypoglycemia has been adequately addressed during a visit, providers may want to use the Hypoglycemia Provider Checklist (<a href=\"#T3\" data-ref-type=\"table\">Table 3<\/a>).\n\nHypoglycemia Patient Questionnaire\n\n![](1384tbl2)\n\nHypoglycemia Provider Checklist\n\n![](1384tbl3)\n\nA careful review of the glucose log collected by the patient should also be done at each visit. The date, approximate time, and circumstances surrounding recent episodes of hypoglycemia should be noted, together with information regarding the awareness of the warning symptoms of hypoglycemia. A reliable history of impaired autonomic responses (tremulousness, sweating, palpitations, and hunger) during hypoglycemia may be the most practical approach to making the diagnosis of hypoglycemia unawareness. If symptoms are absent or if frequent episodes of recurrent hypoglycemia occur within hours to days of each other, it is likely that the patient has HAAF. Other historical clues such as experiencing more than one episode of severe hypoglycemia that required the assistance of another over the preceding year or a family report that they are recognizing more frequent episodes of hypoglycemia may also provide clues that the patient has developed hypoglycemia unawareness. A self-reported history of impaired or absent perception of autonomic symptoms during hypoglycemia correlates strongly with laboratory confirmation of hypoglycemia unawareness (33,121,124,125).\n\n# What are the current knowledge gaps in our understanding of hypoglycemia, and what research is necessary to fill these gaps?\n\nSince the publication of the previous report from the Workgroup on Hypoglycemia in 2005 (1), much has been learned about the impact of hypoglycemia on patient outcomes. However, hypoglycemia continues to cause considerable morbidity and even mortality in patients with diabetes. If patients are to benefit from the reduction in microvascular complications that follows from achieving near-normal levels of glycemia, additional research will be necessary to prevent them from experiencing hypoglycemia and HAAF. First, new surveillance methods that provide consistent ways of reporting hypoglycemia must be developed so that the impact of any intervention to prevent and treat hypoglycemia can be fully assessed. Greater attention must be focused on understanding which patients are most at risk for hypoglycemia and on developing new educational strategies that effectively reduce the number of episodes experienced by at-risk patients. New therapies that do not cause hypoglycemia, including an artificial pancreas, need to be developed for both type 1 and type 2 diabetes. The technologies used to monitor blood glucose must become more accurate, more reliable, easier to use, and less expensive. The mechanisms that render patients unable to increase glucagon secretion in response to hypoglycemia and that are responsible for the development of HAAF must be identified so strategies can be developed to ensure that patients always experience early warning signs of impending neuroglycopenia. The impact of hypoglycemia on short-term outcomes such as mortality and long-term outcomes such as cognitive dysfunction need to be better defined, and the mechanisms for these associations need to be understood. Focused research in these priority areas will address our knowledge gaps about hypoglycemia and ultimately reduce the impact of iatrogenic hypoglycemia on patients with diabetes.\n\nThe workgroup meeting was supported by educational grants to the American Diabetes Association from Lilly USA, LLC and Novo Nordisk and sponsorship to the American Diabetes Association from Sanofi. The sponsors had no input into the development of or content of the report. No other potential conflicts of interest relevant to this article were reported.\n\nThe workgroup members thank Stephanie Kutler and Meredith Dyer of The Endocrine Society and Sue Kirkman, MD, of the American Diabetes Association for staff support.\n\n# References","meta":{"dup_signals":{"dup_doc_count":161,"dup_dump_count":79,"dup_details":{"curated_sources":2,"2023-40":3,"2023-14":1,"2023-06":1,"2022-49":1,"2022-40":1,"2022-21":2,"2022-05":1,"2021-49":3,"2021-43":3,"2021-39":3,"2021-31":2,"2021-25":2,"2021-21":1,"2021-17":3,"2021-10":4,"2021-04":2,"2020-50":2,"2020-45":6,"2020-40":4,"2020-34":3,"2020-29":2,"2020-24":5,"2020-16":3,"2020-10":2,"2020-05":1,"2019-51":1,"2019-47":3,"2019-43":5,"2019-39":4,"2019-35":1,"2019-26":1,"2019-13":2,"2019-09":1,"2019-04":3,"2018-51":2,"2018-47":1,"2018-43":1,"2018-39":3,"2018-34":2,"2018-26":3,"2018-17":2,"2018-09":1,"2018-05":2,"2017-51":1,"2017-47":2,"2017-43":1,"2017-39":2,"2017-34":1,"2017-30":3,"2017-22":4,"2017-17":1,"2017-09":3,"2017-04":3,"2016-50":2,"2016-44":3,"2016-40":2,"2016-36":2,"2016-30":1,"2016-26":1,"2016-22":1,"2016-18":1,"2016-07":1,"2015-48":1,"2015-35":1,"2015-32":1,"2015-22":1,"2014-52":1,"2014-49":2,"2014-42":3,"2014-41":2,"2014-35":2,"2014-23":1,"2014-15":1,"2024-26":2,"2017-13":3,"2015-18":1,"2015-11":1,"2015-06":1,"2014-10":1}},"file":"PMC3631867"},"subset":"pubmed_central"}
{"text":"author: David S Pisetsky\ndate: 2013\ninstitute: 1Duke University Medical Center, 151G, Durham VA Medical Center, Durham, NC 27705, USA\nreferences:\ntitle: The Choosing Wisely initiative: Does it have your back?\n\nAs a subspecialty, rheumatology demands knowledge, intuition, and judgment to go along with a broad appreciation of the nuances and mysteries of internal medicine. Creativity is also part of the practice since many conditions in the purview of rheumatology lack established therapies that have received US Food and Drug Administration approval or testing in a controlled trial. Therefore, rheumatologists must create treatment plans, often on the fly (or by the seat of their pants), producing a succession of n-of-1 trials in their practices. As if their current attributes were not enough, rheumatologists, like other providers, must now have wisdom and practice not only smartly but wisely. Such is the direction of Choosing Wisely.\n\nFor those of you who are not familiar with Choosing Wisely, this initiative of the American Board of Internal Medicine Foundation is a very timely and important effort to help reduce the cost of health care by encouraging providers to adopt more judicious and evidence-based ways to diagnose and treat illness. To meet this goal, professional medical organizations and societies are identifying situations in which cost reduction can be achieved by avoiding the overuse of low-yield (but expensive) diagnostic tests or prescription of therapies of marginal or questionable efficacy.\n\nAlthough the goal of Choosing Wisely is highly laudable, many of the items targeted for reduction or elimination at this time are not that controversial or radical, nor will they require a major change in practice patterns of most providers. In common parlance, these items are low-hanging fruit. As a first step in cost containment, the Choosing Wisely initiative has stayed away (probably wisely) from big drivers of costs in the US, like the price of drugs, fragmentation of care, and lack of adequate preventative services. Certainly, in the US, the problem of uninsured patients continues to be vexing and could require the wisdom of Solomon as state governments go their various ways in response to Obamacare.\n\nBy its nature, the Choosing Wisely initiative is public and political as well as medical, designed to show the citizenry, including the government and payers, that physicians are willing to consider cost and partner with patients to constrain spending. This spending seems forever to increase to levels that everyone calls unsustain able, although, so far, the body politic has not screamed 'Enough is enough' and meant it. For its part, the American College of Rheumatology followed a rigorous and thoughtful approach to designate practices for the Choosing Wisely campaign (Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>). Because the recommendations came from a Delphi process of organization members, the choices are informed and reasonable. Having never ordered Lyme serologies or used magnetic resonance imaging to stage rheumatoid arthritis, I may have already demonstrated my wisdom.\n\nThe American College of Rheumatology's top 5 list for choosing wisely\n\n|  |\n|----|\n| 1\\. Do not test anti-nuclear antibody (ANA) subserologies without a positive ANA and clinical suspicion of immune-mediated disease. |\n| 2\\. Do not test for Lyme disease as a cause of musculoskeletal symptoms without an exposure history and appropriate examination findings. |\n| 3\\. Do not perform magnetic resonance imaging of the peripheral joints to routinely monitor inflammatory arthritis. |\n| 4\\. Do not prescribe biologic agents for rheumatoid arthritis before a trial of methotrexate (or other conventional non-biologic disease-modifying anti-rheumatic drug). |\n| 5\\. Do not routinely repeat dual x-ray absorptiometry scans more often than once every 2 years. |\n\nReproduced with permission from John Wiley & Sons \\[8\\].\n\nIn the case of musculoskeletal disease, however, other groups have weighed in. The American College of Physicians says, 'Don't obtain imaging studies in patients with non-specific low back pain', whereas the American Academy of Family Physicians says, 'Don't do imaging for low back pain within the first six weeks, unless red flags are present'. The recommendations on back pain may not be easy to follow, because in the real world, patients with back pain can present with confusing signs and symptoms and often cannot date the onset of their problems. Furthermore, red flags are fortunately rare.\n\nAs I have learned many times, I do not know what nonspecific back pain is or how to differentiate it from the pain of a spondyloarthropathy, which, after all, can be pretty 'non-specific'. I am not alone in my uncertainty. I know a very experienced and savvy rheumatologist who missed the diagnosis of ankylosing spondylitis in himself, thinking that the nagging ache in the lower spine resulted from the pounding of too much tennis and jogging. He is now on a tumor necrosis factor blocker and doing smashingly, wondering how he had missed the diagnosis for over five years.\n\nOur unit had its discussion of Choosing Wisely in a dreary conference room with three large bookcases filled with outdated editions of major texts. Our unit also includes clinical immunology and allergy, and one of our faculty members questioned the American Academy of Allergy, Asthma and Immunology recommendation about the use of spirometry in diagnosing and managing asthma. Similarly, I raised concerns about logistical issues in waiting until a fluorescent anti-nuclear antibody test is positive before ordering specific anti-nuclear antibodies.(Does that mean another clinic visit, which can be associated with a hefty facility fee?) Nevertheless, we all got on board and pledged to Choose Wisely in the future.\n\nAlthough I support the Choosing Wisely initiative, I wish that the recommendations did not have such an Old Testament quality and embody, like the Ten Commandments, so many *don'ts*. I did find the words 'refrain' and 'avoid' while searching the lists on the Choosing Wisely website, but a few *do's* would have been nice. Also, I think that more recommendations on specific treatments to use and not eschew would have been very helpful given the enormous range of drugs now on the market.\n\nAs many rheumatologists know, back pain presents a conundrum not only because diagnosis is difficult but because treatment is difficult. For many patients, acute and chronic back pain can be severe, debilitating, and disabling. Often, such pain will not respond adequately to analgesics such as acetaminophen or a non-steroidal anti-inflammatory agent or measures such as heat or massage. Opioids then become an option, but a decision to prescribe opioids may be become problematic in the framework of Choosing Wisely, which suggests limiting diagnostic evaluations for back pain at least initially.\n\nBecause of the many societal as well as medical issues associated with controlled substances, physicians like to pursue at least some diagnostic testing prior to prescribing opioids, especially in individuals at high risk for drug side effects (for example, older individuals who could fall) or for complications of dependency and addiction. Recognizing the huge problem of opioid abuse in the population and the pressures on providers to prescribe these agents, the Federation of State Medical Boards has issued recommendations on prescribing controlled substances for pain; the recommendations emphasize the importance of a thorough patient evaluation in determining the need for a controlled substance. I wonder whether a history and physical exam are enough to make this judgment in the absence of some kind of imaging of the back.\n\nFor Choosing Wisely to be successful, the participation of patients is critical since they must partner with providers to accept a different kind of medical care at a time of cost containment and evolving expectations. Patients with pain, however, represent a special group because of the burden of their symptoms and the pressing need for relief. Unless a patient feels satisfied, it is not unusual for them to try a variety of interventions in quick succession, going from an orthopedist to a rheumatologist to a chiropractor to an acupuncturist. Indeed, studies suggest that 1 in 4 patients with pain will seek a new provider at least three times because they feel that their care is not optimal, meaning that their pain has not been adequately relieved.\n\nIn such a potentially conflicted setting, the patient's perception of the provider is critical. In my experience, patients appreciate and accept treatment recommendations when time is spent on the visit, complaints are taken seriously, and, yes, diagnostic testing is performed. Sometimes imaging is performed for no other reason than to allay patient worry and reduce fear and anxiety about the meaning of a surge of excruciating pain. While the yield on x-rays may be low, an imaging study may be important to signal a commitment to find the diagnosis and develop a treatment plan that is based on the best evidence. Not infrequently, this plan will consider the risks and benefits of opioids if prescribed and, importantly, address psychological and life-style issues that may lead to abuse.\n\nAt present, many of the items in Choosing Wisely relate to diagnosis, and imaging is at the top of the list because of its expense. Clearly, there are many ways to reduce cost, whether by limiting the number of tests or reducing the cost of each. To me, both options should be on the table as this initiative goes forward. Certainly, choices should be the subject of intensive research that can fall under the rubric of comparative effectiveness. Given the number of skilled investigators, registries, and outcome assessment tools available for innovative real-world studies, recommendations on choices can be based not only on wisdom but on science.\n\nChoosing Wisely is an excellent idea and the first attempts are in the right direction. My hope is that, on the next round, we confront some real choices - hard ones - and figure out what wisdom really is when it comes to the practice of medicine.\n\n# Competing interests\n\nThe author declares that they have no competing interests.","meta":{"dup_signals":{"dup_doc_count":106,"dup_dump_count":39,"dup_details":{"curated_sources":2,"2022-33":1,"2019-22":1,"2019-09":1,"2018-51":2,"2018-43":1,"2018-34":1,"2018-22":1,"2018-13":1,"2017-47":1,"2017-34":1,"2017-26":1,"2017-22":1,"2017-09":10,"2016-40":1,"2016-36":9,"2016-30":8,"2016-22":1,"2016-07":11,"2015-48":2,"2015-40":1,"2015-35":2,"2015-32":2,"2015-27":2,"2015-22":2,"2015-14":2,"2014-52":2,"2014-49":4,"2014-42":5,"2014-41":4,"2014-35":3,"2014-23":3,"2014-15":4,"2023-40":1,"2024-18":2,"2015-18":2,"2015-11":2,"2015-06":2,"2014-10":2,"2013-48":2}},"file":"PMC3978556"},"subset":"pubmed_central"}
{"text":"abstract: U87MG is a commonly studied grade IV glioma cell line that has been analyzed in at least 1,700 publications over four decades. In order to comprehensively characterize the genome of this cell line and to serve as a model of broad cancer genome sequencing, we have generated greater than 30\u00d7 genomic sequence coverage using a novel 50-base mate paired strategy with a 1.4kb mean insert library. A total of 1,014,984,286 mate-end and 120,691,623 single-end two-base encoded reads were generated from five slides. All data were aligned using a custom designed tool called BFAST, allowing optimal color space read alignment and accurate identification of DNA variants. The aligned sequence reads and mate-pair information identified 35 interchromosomal translocation events, 1,315 structural variations (\\>100 bp), 191,743 small (\\<21 bp) insertions and deletions (indels), and 2,384,470 single nucleotide variations (SNVs). Among these observations, the known homozygous mutation in *PTEN* was robustly identified, and genes involved in cell adhesion were overrepresented in the mutated gene list. Data were compared to 219,187 heterozygous single nucleotide polymorphisms assayed by Illumina 1M Duo genotyping array to assess accuracy: 93.83% of all SNPs were reliably detected at filtering thresholds that yield greater than 99.99% sequence accuracy. Protein coding sequences were disrupted predominantly in this cancer cell line due to small indels, large deletions, and translocations. In total, 512 genes were homozygously mutated, including 154 by SNVs, 178 by small indels, 145 by large microdeletions, and 35 by interchromosomal translocations to reveal a highly mutated cell line genome. Of the small homozygously mutated variants, 8 SNVs and 99 indels were novel events not present in dbSNP. These data demonstrate that routine generation of broad cancer genome sequence is possible outside of genome centers. The sequence analysis of U87MG provides an unparalleled level of mutational resolution compared to any cell line to date.\nauthor: Michael James Clark; Nils Homer; Brian D. O'Connor; Zugen Chen; Ascia Eskin; Hane Lee; Barry Merriman; Stanley F. Nelson\\* E-mail: <firstname.lastname@example.com>[^1]\ndate: 2010-01\ninstitute: 1Department of Human Genetics, University of California Los Angeles, Los Angeles, California, United States of America; 2Department of Computer Science, University of California Los Angeles, Los Angeles, California, United States of America; University of Washington, United States of America\nreferences:\ntitle: U87MG Decoded: The Genomic Sequence of a Cytogenetically Aberrant Human Cancer Cell Line\n\n# Introduction\n\nGrade IV glioma, also called glioblastoma multiforme (GBM), is the most common primary malignant brain tumor with about 16,000 new diagnoses each year in the United States. While the number of cases is relatively small, comprising only 1.35% of primary malignant cancers in the US \\[1\\], GBMs have a one-year survival rate of only 29.6%, making it one of the most deadly types of cancer \\[2\\]. Recent clinical studies demonstrate improved survival with a combination of radiation and Temozolomide chemotherapy, but median survival time for GBM patients who receive therapy is only 15 months \\[3\\]. Due to its highly aggressive nature and poor therapeutic options, understanding the genetic etiology of GBM is of great interest and therefore, GBM has been selected as one of the three initial cancer types to be thoroughly studied in the TCGA program \\[4\\].\n\nTo that end, numerous cell line models of GBM have been established and used in vast numbers of studies over the years. It is well recognized that cell line models of human disorders, especially cancers, are an important resource. While these cell lines are the basis of substantial biological insight, experiments are currently performed in the absence of genome-wide mutational status as no cell line that models a human disease has yet had its genome fully sequenced. Here, we have sequenced the genome of U87MG, a long established cell line derived from a human grade IV glioma used in over 1,700 publications \\[5\\]. A wide range of biological information is known about this cell line. The U87MG cell line is known to have a highly aberrant genomic structure based on karyotyping, SKY \\[6\\], and FISH \\[7\\]. However, these methods neither provide the resolution required to visualize the precise breakpoint of a translocation event, nor are they generally capable of identifying genomic microdeletions (deletions on the order of a megabase or less in size) in a whole genome survey of structural variation. SNP genotyping microarrays can be used to detect regions of structural variation in the forms of loss of heterozygosity (LOH) and copy number (CN) based on probe intensity, but do not reveal chromosomal joins. To assess the genomic stability of U87MG, the genome was genotyped by Illumina Human 1M-Duo BeadChip microarray. In spite of being cultured independently for several years, the regions of LOH and the CN state of our U87MG genome matched exactly with data retrieved from the Sanger COSMIC database for U87MG \\[8\\], which had been assayed on an Affymetrix Genome-Wide Human SNP Array 6.0. This suggests that although U87MG bears a large number of large-scale chromosomal aberrations, it has been relatively stable for years and is not rapidly changing. This suggests that prior work on U87MG may be reinterpreted based on the whole genome sequence data presented here.\n\nThe first draft of the consensus sequence of the human genome was reported in 2001 \\[9\\],\\[10\\]. The first individual human diploid sequence was sequenced using capillary-based Sanger sequencing \\[11\\]. Since then, a few additional diploid human genomes have been published utilizing a variety of massively parallel sequencing techniques to sequence human genomes to varying degrees of coverage, variant discovery, and quality typically costing well over \\$200,000 and several machine months of operation \\[12\\]\u2013\\[16\\]. For the sequencing of U87MG, we utilized ABI SOLiD technology, which uses a ligation-based assay with two-base color-encoded oligonucleotides that has been demonstrated to allow highly accurate single nucleotide variant (SNV) and insertion\/deletion (indel) detection \\[17\\]. Additionally, long mate-paired genomic libraries with a mean insert size of 1\u20132kb allowed higher clone coverage of the genome, which improved our ability to identify genomic structural variations such as interchromosomal translocations and large deletions. While longer insert sizes would improve resolution of some structural variants, during genomic shearing the highest density of large fragments occurs at 1.5kb, allowing a sufficiently complex library to be generated from only 10 micrograms of genomic DNA while still being well powered to identify structural variations. Here, we demonstrate that aligning the two-base color-encoding data with BFAST software and decoding during alignment allows for highly sensitive detection of indels, which have in the past been difficult to detect by short read massively parallel sequencing.\n\nFor cancer sequencing, it is important to assess not only SNVs, but indels, structural variations and translocations, and it is preferable to extract this information from a common assay platform. A major characteristic of the U87MG cell line that differentiates it from the samples used in other whole genome sequencing projects published thus far is its highly aberrant genomic structure. Due to its heavily rearranged state, we thoroughly and accurately assessed each of these major classes of mutations and demonstrated that small indels, large microdeletions and interchromosomal translocations are actually the major categories of mutations that affect known genes in this cancer cell line. These analyses provide a model for other genome sequencing projects outside major genome centers of how to both thoroughly sequence and assess the mutational state of whole genomes.\n\n# Results\n\n## Data Production\n\nFrom ten micrograms of input genomic DNA, we performed two and a half full sequencing runs on the ABI SOLiD Sequencing System, for a total of five full slides of data \\[17\\]. Utilizing the ABI long mate-pair protocol, we produced 1,014,984,286 raw 50bp mate-paired reads (101.5Gb). In some cases the bead was recognized by the imaging software for only one read, thereby producing an additional 120,691,623 single end reads (6.0Gb). In aggregate, we generated a total of 107.5Gb of raw data (<a href=\"#pgen-1000832-t001\" data-ref-type=\"table\">Table 1<\/a>).\n\n10.1371\/journal.pgen.1000832.t001\n\n###### Genome sequencing summary.\n\n<img src=\"pgen.1000832.t001\" id=\"pgen-1000832-t001-1\" \/>\n\n|  |  |\n|:---|:--:|\n| **Sequencing Libraries** | 1 |\n| **SOLiD Runs (Slides)** | 2.5 (5) |\n| **Strategy** | 2\u00d750 |\n| **Mate-paired reads passing quality filter (total bases)** | 1,014,984,286 (101.5Gb) |\n| **Single-end reads passing quality filter (total bases)** | 120,691,623 (6.0Gb) |\n| **Mate-paired reads uniquely aligned by BFAST (bases)** | 390,064,184 (39.06Gb) |\n| **Unpaired reads uniquely aligned by BFAST (bases)** | 266,635,829 (13.33Gb) |\n| **Single-end reads uniquely aligned by BFAST (bases)** | 62,336,824 (3.12Gb) |\n| **Total bases uniquely aligned by BFAST** | 55.51Gb |\n\nWe also performed an exon capture approach designed to sequence the exons of 5,253 genes (10.7Mb) annotated in the Wellcome Trust Sanger Institute Catalogue of Somatic Mutations in Cancer (COSMIC) V38 \\[8\\], Cancer Gene Census, Cancer Genome Project Planned Studies and The Cancer Genome Atlas (TCGA) \\[4\\] GBM gene list using a custom-created Agilent array. This approach used the Illumina GAII sequencing system \\[18\\] to sequence captured DNA fragments using a paired end sequencing protocol. This resulted in 9,948,782 raw 76bp paired end reads (1.51Gb), and a mean base coverage of 29.5\u00d7. These reads were used to calculate concordance rates with the larger whole genome sequence dataset.\n\nThe Blat-like Fast Accurate Search Tool (BFAST) \\[19\\] version 0.5.3 was used to align 107.5Gb of raw color space reads to the color space conversion of the human genome assembly hg18 from University of California, Santa Cruz (<http:\/\/hgdownload.cse.ucsc.edu\/goldenPath\/hg18\/bigZips\/>, based on the March 2006 NCBI build 36.1). Duplicate reads, typically from the same initial PCR fragment during genomic library construction, were inevitable and accounted for 16.4% of the total aligned data. These were removed using the alignment filtering utility in the DNAA package (<http:\/\/dnaa.sourceforge.net>). A total of 390,604,184 paired end reads (39.06Gb), 266,635,829 (13.33Gb) unpaired reads, and 62,336,824 (3.12Gb) single end reads were successfully mapped to a unique location in the reference genome with high confidence for a total of 55.51Gb of aligned sequence (<a href=\"#pgen-1000832-t001\" data-ref-type=\"table\">Table 1<\/a>). For the exon capture dataset, we uniquely aligned 8,142,874 paired end reads (1.2Gb) and 1,097,000 (83Mb) unpaired reads for a total of 1.32Gb of raw aligned sequence (<a href=\"#pgen-1000832-t002\" data-ref-type=\"table\">Table 2<\/a>). Using the ABI SOLiD reads, we identified small insertions and deletions (indels), single nucleotide variants (SNVs), and structural variants such as large-scale microdeletions and translocation events. The exon capture Solexa reads were used to validate SNVs identified in the SOLiD sequencing.\n\n10.1371\/journal.pgen.1000832.t002\n\n###### Exon capture sequencing summary.\n\n<img src=\"pgen.1000832.t002\" id=\"pgen-1000832-t002-2\" \/>\n\n|  |  |\n|:---|:--:|\n| **Sequencing Libraries** | 1 |\n| **Illumina Runs (Lanes)** | 1\/8 (1) |\n| **Strategy** | 2\u00d776 |\n| **Number of bases targeted** | 10752923 |\n| **Mate-paired reads passing quality filter (total bases)** | 9,948,782 (1.51Gb) |\n| **Mate-paired reads uniquely aligned by BFAST (bases)** | 8,142,874 (1.2Gb) |\n| **Unpaired reads uniquely aligned by BFAST (bases)** | 1,097,000 (83Mb) |\n| **Total bases uniquely aligned by BFAST** | 1.32Gb |\n| **Total targeted bases sequenced** | 317,017,503 |\n| **Mean coverage within targeted bases** | 29.5\u00d7 |\n\nThe overall pattern of base sequence coverage from the shotgun reads changes across the genome, and as expected is highly concordant with the copy number state as determined by Illumina 1M Duo and Affymetrix 6.0 SNP analysis (<a href=\"#pgen-1000832-g001\" data-ref-type=\"fig\">Figure 1<\/a>). Regions of two normal copies, such as chromosome 3, showed even base sequence coverage across their entire length (12.4 reads\/base, excluding centromeric and telomeric regions which are not represented accurately in hg18). Meanwhile, regions with one-copy state according to the SNP chip, such as the distal q-arm of chr11 and the distal p-arm of chr6, show about half the base sequence coverage (7.2 reads\/base) as a predicted two-copy region. Likewise, predicted three-copy state regions, such as the distal q-arm of chr13, show about 1.5 times the base sequence coverage of a predicted two-copy region. A complete deletion spanning the region on chromosome 9 that includes the CDKN2A gene is also seen in both the SNP chip and ABI SOLiD base sequence coverage. These data show at a very large scale that sequence placement is generally correct and supports the copy number state calls from the array based data.\n\n## Variant Discovery\n\nSingle nucleotide variants (SNVs) and small insertions and deletions ranging from 1 to 20 bases (indels) were identified from the alignment data using the MAQ consensus model \\[20\\] as implemented in the SAMtools software suite \\[21\\]. SAMtools produced variant calls, zygosity predictions, and a Phred-scaled probability that the consensus is identical to the reference. To improve the reliability of our variant calls, variants were required to have a Phred score of at least 10 and further needed to be observed greater than or equal to 4 but less than 60 times and at least once on each strand.\n\nIn total, we identified 2,384,470 SNVs meeting our filtering criteria. Of these, 2,140,848 (89.8%) were identified as exact matches to entries in dbSNP129 \\[22\\]. Exact matches had both the variant and observed alleles in the dbSNP entry, allowing for the discovery of novel alleles at known SNP locations. In total, 243,622 SNVs (10.2%) were identified as novel events not previously recorded in dbSNP 129. This rate of novel variant discovery is consistent with other whole human normal genome sequences of European ancestry relative to dbSNP \\[12\\]. These SNVs were further characterized based on zygosity predictions from the MAQ consensus model, separating SNVs into homozygous or heterozygous categories (<a href=\"#pgen-1000832-t003\" data-ref-type=\"table\">Table 3<\/a>). The observed diversity value for SNVs (\u03b8~SNV~, number of heterozygous SNVs\/number of base pairs) across autosomal chromosomes was 4.4\u00d710^\u22124^, which is generally consistent with the normal human genome variation rate.\n\n10.1371\/journal.pgen.1000832.t003\n\n###### SNV filtering and quantification.\n\n<img src=\"pgen.1000832.t003\" id=\"pgen-1000832-t003-3\" \/>\n\n| SNV Classification | Total | In dbSNP 129 | Not in dbSNP 129 |\n|:---|:--:|:--:|:--:|\n| **Variants meeting filter criteria** | 2,384,470 | 2,140,848 | 243,622 |\n| **Synonymous or not coding region** | 2,375,812 | 2,133,226 | 242,586 |\n| **Coding region & non-synonymous** | 8,658 | 7,622 | 1,036 |\n| **Splice site mutations** | 151 | 132 | 19 |\n| **Heterozygous splice site mutations** | 62 | 47 | 15 |\n| **Homozygous splice site mutations** | 89 | 85 | 4 |\n| **Premature stop** | 134 | 93 | 41 |\n| **Heterozygous premature stop** | 82 | 48 | 34 |\n| **Homozygous premature stop** | 52 | 45 | 7 |\n| **Non-synonymous** | 8,538 | 7,518 | 1,020 |\n| **Heterozygous non-synonymous** | 4,005 | 3,134 | 871 |\n| **Homozygous non-synonymous** | 4,533 | 4,384 | 149 |\n\nFor small (\\<21bp) insertions and deletions, 191,743 events were detected with 116,964 not previously documented in dbSNP 129. The same criteria as used for SNVs was used for determining if an indel was novel and they were further classified as homozygous or heterozygous using the SAMtools variant caller (<a href=\"#pgen-1000832-t004\" data-ref-type=\"table\">Table 4<\/a>). The observed diversity value (\u03b8~indel~, number of heterozygous indels\/number of base pairs) across autosomal chromosomes was 0.38\u00d710^\u22124^.\n\n10.1371\/journal.pgen.1000832.t004\n\n###### Indel filtering and quantification.\n\n<img src=\"pgen.1000832.t004\" id=\"pgen-1000832-t004-4\" \/>\n\n| Indel Classification | Total | In dbSNP 129 | Not in dbSNP 129 |\n|:---|:--:|:--:|:--:|\n| **Variants meeting filter criteria** | 191,743 | 74,779 | 116,964 |\n| **Synonymous or not coding region** | 191,359 | 74,643 | 116,716 |\n| **Coding region & non-synonymous** | 384 | 136 | 248 |\n| **Splice site mutations** | 84 | 34 | 50 |\n| **Heterozygous splice site mutations** | 20 | 7 | 13 |\n| **Homozygous splice site mutations** | 64 | 27 | 37 |\n| **Heterozygous premature stop** | 91 | 15 | 76 |\n| **Homozygous premature stop** | 94 | 40 | 54 |\n| **Heterozygous non-synonymous** | 168 | 45 | 123 |\n| **Homozygous non-synonymous** | 193 | 86 | 107 |\n| **Heterozygous frameshift** | 141 | 33 | 108 |\n| **Homozygous frameshift** | 179 | 80 | 99 |\n| **Heterozygous in-frame indels** | 26 | 11 | 15 |\n| **Homozygous in-frame indels** | 14 | 5 | 9 |\n\nA subset of 38 variants meeting genome-wide filtering criteria, including a 20-base deletion, was tested by PCR and Sanger sequencing with 34 being validated. In summary, 85.2% of SNVs (23\/27), and 100% of small insertions (3\/3), deletions (4\/4), translocations (3\/3) and microdeletions (1\/1) were validated in this manner (<a href=\"#pgen.1000832.s005\" data-ref-type=\"supplementary-material\">Table S1<\/a>). While this is a small sample, it demonstrates an overall low false positive rate.\n\n## Indel Size Distribution\n\nThe size distribution of indels identified in U87MG is generally consistent with previous studies on coding and non-coding indel sizes in non-cancer samples \\[23\\]\u2013\\[25\\]. Small deletion sizes ranged from 1 to 20 bases in size and their distribution approximates a power law distribution in concordance with previous findings \\[23\\] (<a href=\"#pgen-1000832-g002\" data-ref-type=\"fig\">Figure 2A<\/a>). There is a small deviation from the power law distribution with an excess of 4-base indels in U87MG's non-coding regions (<a href=\"#pgen-1000832-g002\" data-ref-type=\"fig\">Figure 2A<\/a>, red bars) \\[11\\],\\[26\\].\n\nA similar trend is seen with insertions in non-coding sequence with the maximum observed insertion size of 17 bases (<a href=\"#pgen-1000832-g002\" data-ref-type=\"fig\">Figure 2B<\/a>, red bars). The maximum insertion size observed is less than the maximum deletion size because it is easier to align longer deletions than it is to align insertions. Some small insertions and deletions are likely to be larger than the upper limit of 17 and 20 bases actually observed, but the 50-base read length limits the power to align such reads directly.\n\nIn coding regions, there is a bias towards events that are multiples of 3-bases in length that maintain the reading frame despite variant alleles, suggesting that many of these are polymorphisms (<a href=\"#pgen-1000832-g002\" data-ref-type=\"fig\">Figure 2A<\/a>-deletions, <a href=\"#pgen-1000832-g002\" data-ref-type=\"fig\">Figure 2B<\/a>-insertions, blue bars). In non-coding regions, only 10.8% of indels are a multiple of 3 bases in size, while in coding regions, 27.0% are 3, 6, 9, 12 or 15 bases in size. This trend is expected based on past observations of non-cancer samples \\[11\\],\\[26\\].\n\n## Nucleotide Substitution Frequencies\n\nObserved SNV base substitution patterns were consistent with common mutational phenomena in both coding sequences and genome wide. As expected, the predominant nucleotide substitution seen in SNVs is a transition, changing purine for purine (A\\<-\\>G) or pyrimidine for pyrimidine (C\\<-\\>T). Previous studies have observed that two out of every three SNPs are transitions as opposed to transversions \\[27\\], and we observed that 67.4% of our SNVs were transitions, while 32.6% were transversions, a 2.07\u22361 ratio. (<a href=\"#pgen-1000832-g003\" data-ref-type=\"fig\">Figure 3<\/a>) However, in coding regions, there appears to be an increase in C-\\>T\/G-\\>A transitions and a decrease in T-\\>C\/A-\\>G transitions, whereas genome-wide these transitions were approximately equivalent.\n\n## Estimation of Genomic Coverage\n\nTo assess the coverage depth of the U87MG genome sequence, we followed Ley at al. \\[13\\] and required detection of both alleles at most positions in the genome. We utilized the Illumina 1M-Duo BeadChip to find reliably sequenced positions in the genome with an understanding that this may lead to bias towards more unique regions of the genome. In order to best use the SNP genotyping array data, we included only those regions that are diploid based on normal frequency of heterozygous calls and copy number assessment. This effectively permitted us to use the heterozygous calls for assessing accuracy of the short read data for variant calling (<a href=\"#pgen-1000832-g001\" data-ref-type=\"fig\">Figure 1<\/a>). Only SNPs both observed to be heterozygous and that the Illumina genotyping chip called 'high quality' were used, which provided a total of 219,187 high quality heterozygous SNPs for comparison. 99.71% of these were sequenced at least once. After applying variant detection filtering criteria (see <a href=\"#s4\" data-ref-type=\"sec\">Materials and Methods<\/a>) and assessing concordance between the sequence calls and genotyping array calls, 93.71% of the genome was sequenced at sufficient depth to call both alleles of the diploid genome. This is roughly equivalent to the likelihood of sufficient sampling of the whole genome when repeats and segmental duplications are excluded.\n\nNotably, a variant allele was observed at every position called heterozygous by SNP chip, while a reference allele was observed at 201,414 (97.94%) positions. In other words, the SNV detection algorithm uniformly miscalled the homozygous variant allele. Filtering for quality causes a bias toward identifying SNVs at sites that have higher coverage. That said, after SNV quality filtering, diploid coverage of the cytogenetically normal portions of the genome was 10.85\u00d7 for each allele, which is clearly adequate for calling over 90% of the base variant positions on each allele at high accuracy.\n\nBecause the positions of the genome included on SNP arrays is not a random sampling of the genome, we also assessed mapping coverage genome-wide. Of all bases in the haploid genome, 78.9% of the whole reference genome was covered by at least one reliably placed read. Of that portion of the genome, 91.9% of all bases were effectively sequenced based on passing variant calling filters (Phred\\>10, \\>4\u00d7 coverage, \\<60\u00d7 coverage). Thus, a total of 72.5% of the whole genome was sequenced, including repeats and duplicated regions, which is typical of short sequence shotgun approaches.\n\n## Exon Capture Cross-Validation of Sequence Variants\n\n10.9Mb of genomic sequence was targeted consisting of the amino acid encoding exons of 5,235 genes and were sequenced to a mean coverage of 30\u00d7 using the Illumina GAII sequencer. Given the larger variability of coverage from the capture data, only a subset of these bases (8.5Mb) was evaluable to determine the false positive variant detection rate from the complete genomic sequence data. This region contained 1,621 SNPs present in dbSNP129. Within the 8.5Mb of common and well-covered sequence in the genomic sequence data and the capture sequence, there were 1,780 SNVs called from the genomic sequence. The same non-reference allele was concordantly observed at 1,631 positions within the capture data. At 149 positions, the non-reference allele was not observed in the capture data, but the reference allele was detected. However, the mean coverage at these 149 positions was significantly lower than that of the other 1,631 positions (p\u200a=\u200a0.0003), suggesting that the non-reference allele was not adequately covered and is under called in the capture data. Moreover, of the 1,621 dbSNPs in the region, the capture adequately covered only 1,515. In these data there was a bias for the pull down data to under observe the non-reference allele (<a href=\"#pgen.1000832.s001\" data-ref-type=\"supplementary-material\">Figure S1<\/a>). The 106 dbSNP positions detected in the ABI whole genome sequence dataset were observed to all call the reported alternate allele from dbSNP. In theory, if these were errors, then non-reference base calls should be randomly distributed to the three alternate base calls. Thus, no discrepancies are reliably identified within the dbSNP overlap when a variant was called in the ABI genomic sequence data.\n\nThere were a total of 100 novel SNVs detected in the ABI genomic sequence dataset that were also very well evaluated in the Illumina pull down data with at least 20 high quality Illumina reads, such that the ABI sequence could be well validated. Of these, 2 of the 100 discovered variants in the genomic sequence dataset were not observed in the Illumina pull down sequencing dataset. Thus, of the entire 8.5mb interval there are 2 unconfirmed variants for an estimated false positive error rate of about 3\u00d710^\u22127^ for the whole interval. Alternatively viewed, there were 100 novel SNVs, with a 2% error rate in those novel positions. Thus, the de novo false discovery rate may be as high as 2%. Extrapolating to the whole set of 243,622 novel SNVs, we expect up to 4,872 false positives SNVs. These observations are roughly concordant with a sampling of 37 novel SNVs (not in dbSNP) in the whole genome set selected for testing by Sanger sequencing. Of these, 34 out of 37 (92%) were validated.\n\n## Individual Genome Comparison\n\nThere are now several publicly available complete genomes sequenced on next generation platforms. We compared the SNVs discovered in U87MG to two of these published genomes: the James D. Watson genome \\[12\\] and the first Asian genome (YanHuang) \\[14\\]. Further, we simultaneously compared each of these to dbSNP version 129 \\[22\\]. Compared with dbSNP, 10.2% of U87MG SNVs, 9.5% of Watson SNVs, and 12.0% of YanHuang SNVs were not present within dbSNP (<a href=\"#pgen-1000832-g004\" data-ref-type=\"fig\">Figure 4<\/a>). As U87MG was derived from a patient of Caucasian ancestry, which is confirmed by genotyping, it is unsurprising to see a higher overlap with dbSNP for U87MG than for YanHuang. Between the three genomes themselves, 44.7% of U87MG SNVs overlapped with Watson SNVs while 60.0% of SNVs were in common with YanHuang SNVs. Only 8.5% of dbSNP SNVs were shared between Watson and U87MG, while 11.3% of them were shared between YanHuang and U87MG. Thus, there is not a substantially higher amount of SNVs in the U87MG cancer genome relative to normal genomes.\n\n## Structural Variation Identification\n\nWe utilized the predictable insert distance of mate-paired sequence fragments to directly observe structural variations in U87MG. Our target insert size of 1.5kb gave us a normal distribution of paired end insert lengths ranging from 1kb to 2kb with median around 1.25kb and mean around 1.45kb in the actual sequence data (<a href=\"#pgen.1000832.s002\" data-ref-type=\"supplementary-material\">Figure S2<\/a>). We identified 1,314 large structural variations, including 35 interchromosomal events, 599 complete homozygous deletions (including a large region on chromosome 9 containing CDKN2A\/B, which commonly experience homozygous deletions in brain cancer), 361 heterozygous deletion events, and 319 other intrachromosomal events (<a href=\"#pgen-1000832-t005\" data-ref-type=\"table\">Table 5<\/a>). The 599 complete microdeletions summed up to approximately 5.76Mb of total sequence, while the 361 heterozygous microdeletions summed to 5.36Mb of total sequence. Most of the microdeletions were under 2kb in total size. Because of the high sequence coverage and mate pair strategy each event was supported by an average of 138 mate pair reads. Mispairing of the mate pairs did occur occasionally due to molecular chimerism in the library fabrication process, but such reads occur at a low frequency (\\<1\/40 of the reads). Thus, the true rearrangement\/deletion events were highly distinct from noise in well-mapped sequences. Interchromosomal events included translocations and large insertion\/deletion events where one part of a chromosome was inserted into a different chromosome, sometimes replacing a segment of DNA. All together, these structural variations show a highly complex rearrangement of genomic material in this cancer cell line (<a href=\"#pgen-1000832-g005\" data-ref-type=\"fig\">Figure 5<\/a>). All identified structural variants are summarized in <a href=\"#pgen.1000832.s006\" data-ref-type=\"supplementary-material\">Table S2<\/a>. We note as well that even when breakpoints are within genome-wide common repeats there can be sufficient mapping information to reliably identify the translocation breakpoint (<a href=\"#pgen.1000832.s003\" data-ref-type=\"supplementary-material\">Figure S3<\/a>).\n\n10.1371\/journal.pgen.1000832.t005\n\n###### Structural variations detected.\n\n<img src=\"pgen.1000832.t005\" id=\"pgen-1000832-t005-5\" \/>\n\n| Type | \\# of events | \\# that span genes (%) | \\# of affected genes (%) |\n|:---|:--:|:--:|:--:|\n| **Complete deletion** | 599 | 95 (15.9%) | 145 |\n| **Heterozygous deletion** | 361 | 58 (16.0%) | 91 |\n| **Interchromosomal translocation** | 35 | 32 (91.4%) | 35 |\n| **Other intrachromosomal events** | 319 | 146 (45.8%) | 166 |\n\nThe thirty-five interchromosomal events often coincided with positions of copy number change based on the average base coverage (<a href=\"#pgen-1000832-g005\" data-ref-type=\"fig\">Figure 5<\/a>). <a href=\"#pgen-1000832-g006\" data-ref-type=\"fig\">Figure 6<\/a> shows two interchromosomal events between chromosomes 2 and 16. The events on chromosome 16 are less than 1kb apart while those on chromosome 2 are about 160kb apart. Based on the average base coverage, there appears to be a loss of genomic material between the event boundaries on their respective chromosomes, shifting from two to one copy. Although we are unable to determine the origin of such an event, it appears that there was an interchromosomal translocation between chromosomes 2 and 16 with a loss of the DNA between the identified regions on each chromosome.\n\nA subset of 3 translocations were confirmed by amplifying DNA from the breakpoint-spanning region by polymerase chain reaction and sequencing by dideoxy Sanger sequencing (<a href=\"#pgen.1000832.s005\" data-ref-type=\"supplementary-material\">Table S1<\/a>). Each confirmed the predicted breakpoint to within 100 nucleotides of the correct position. In a subset of cases, unmapped short read fragments could be identified from the shotgun short read data that span the breakpoint and are concordant at base resolution with Sanger sequencing of PCR amplified product spanning the breakpoint\n\n## Genes Affected by Mutations in Coding Sequence\n\nThe SNVs and indels identified in U87MG were assessed for their potential to affect protein-coding sequence. We considered variants predicted to be homozygous and to affect the coding sequence of a gene through a frameshift, early termination, intron splice site, or start\/stop codon loss mutation as causing a complete loss of that protein. We chose to focus on homozygous null mutations for two major reasons. First, this is an interesting set of genes that we can predict from the whole genome data are non-functional within this commonly used cell line. Although heterozygous mutations can certainly affect gene products in multiple ways, it is difficult to assess their effect from genomic data alone. Second, by cross-referencing such null mutations with known regions of common mutation in gliomas we can pick out specific candidates that are of interest to the glioma community.\n\nOf the 2,384,470 SNVs and 191,743 small indels in U87MG, a total of 332 genes are predicted to have loss-of-function, homozygous mutations as a consequence of small variants (<a href=\"#pgen.1000832.s007\" data-ref-type=\"supplementary-material\">Table S3<\/a>). Of these, 225 genes contained variants matching alleles annotated in dbSNP (version 129), while 107 contained novel variants not observed in dbSNP.\n\nWe further divided these homozygous mutant genes by variant type. Of genes mutated by SNVs, 146 contained variants present in dbSNP while only 8 were knocked out by variants not in dbSNP. The ratio of known SNPs causing loss-of-function mutations to total known SNPs (146\/2,140,848\u200a=\u200a6.82\u00d710^\u22125^) was not significantly different from the ratio of novel SNVs causing loss-of-function mutations over total novel SNVs (8\/243,622\u200a=\u200a3.28\u00d710^\u22125^; p\u200a=\u200a0.04). This indicates that many of the possible de novo point mutations may indeed be rare inherited variants made homozygous by chromosomal loss of the normal allele.\n\nIn contrast to the trend in SNVs, small indels that homozygously mutated genes were more often novel. There were 79 genes predicted homozygously mutated by indel variants reported in dbSNP while 99 were predicted mutated by novel indels. Despite this trend, however, there was not a significant enrichment of deleterious indels among the novel indels (99\/191,743\u200a=\u200a5.16\u00d710^\u22124^) compared to the known indels (79\/116,964\u200a=\u200a6.75\u00d710^\u22124^; p\u200a=\u200a0.08) This suggests that the difference in ratios of novel versus documented SNVs (8 vs. 146) and indels (99 vs. 79) is the result of compositional bias in dbSNP129, which contains a far greater number of SNPs compared to indels.\n\nWe also assessed the structural variants in U87MG for whether or not they were likely to affect a gene. Two different criteria were used to determine if translocations and microdeletions impacted a coding region, both predicted to produce an aberrant or nonfunctional protein. Using the UCSC known gene database, we identified 35 genes affected by interchromosomal translocations, 145 affected by complete deletions, 91 affected by heterozygous deletions and 166 affected by other intrachromosomal translocations (<a href=\"#pgen-1000832-t004\" data-ref-type=\"table\">Table 4<\/a>).\n\nInterchromosomal translocation events were significantly enriched for occurring at positions where they would affect genes with 32 out of 35 events (91.4%) occurring within 1kb of a gene (p\\<0.0001), while only 44.1% of the reference genome is within 1kb of a known gene. In total, intrachromosomal events did not display this enrichment with 145\/319 (45.5%) falling within 1kb of a gene (p\u200a=\u200a0.67). However, we ran a set of simulations to assess whether microdeletions were enriched to overlap exons because we noted that 585 of our 599 complete microdeletions were less than 10kb in length with a mean size of 1.8kb. We ran 100,000 simulations randomly placing 600 microdeletions of 2kb lengths and determined how many times a microdeletion spanned an exon. In this way, we demonstrated that complete (homozygous) microdeletions under 10kb in size spanned exons slightly more often than by chance with a simulated p-value of .046. Similar assessment of microdeletions greater than 10kb in size did not find evidence of enrichment. These findings suggest that small microdeletions may preferentially occur within genes as opposed to being randomly distributed across the genome, but the signal is not strong from the available data. Genes affected by structural variations are summarized in <a href=\"#pgen.1000832.s008\" data-ref-type=\"supplementary-material\">Table S4<\/a>.\n\n## Annotation of Relevant Mutated Genes\n\nThe annotation tool DAVID was used to further examine the biological significance of the list of likely knockout mutations (including genes affected by SNVs, indels, microdeletions and translocation events) using the EASE analysis module. After gene ontology (GO) analysis, 18 GO terms were nominally enriched and associated with the mutated gene with a p-value \\<\u200a=\u200a0.01 (<a href=\"#pgen.1000832.s009\" data-ref-type=\"supplementary-material\">Table S5<\/a>). These GO enrichments include cell adhesion (GO:0007155 and GO:0022610), membrane (GO:0044425), and protein kinase regulator activity (GO:0019887).\n\nThe list of genes was also compared to the list of cancer-associated genes maintained by the Cancer Gene Census project (<http:\/\/www.sanger.ac.uk\/genetics\/CGP\/Census\/>). For SNVs and small indels, eight were observed in the census list, but this is not unexpected given the large number of mutations found in this cell line (p\u200a=\u200a0.21). Two CGC genes were affected by complete microdeletions (CDKN2A and MLLT3), and one gene each was affected by heterozygous microdeletions (IL21R) and interchromosomal translocations (SET). These included genes previously annotated as mutated in instances of T cell prolymphocytic leukemia (TCRA and MLLT3), glioma (PTEN), endometrial cancer (PTEN), anaplastic large-cell lymphoma (CLTCL1), prostate cancer (ETV1 & PTEN), Ewing sarcoma (FLI1 and ETV1), desmoplastic small round cell tumor (FLI1), acute lymphocytic leukemia (FLI1 and MLLT3), clear cell sarcoma (FLI1), sarcoma (FLI1), myoepithelioma (FLI1), follicular thyroid cancer (PAX8), non-Hodgkin lymphoma (IL21R), acute myelogenous leukemia (SET), fibromyxoid sarcoma (CREB3L2), melanoma (XPC), and multiple other tumor types (PTEN and CDKN2A).\n\nWe also explored the overlap of genes with mutations in GBMs according to the Cancer Genome Atlas (TCGA) with those we predicted are homozygously loss-of-function mutated in U87MG (<a href=\"#pgen.1000832.s009\" data-ref-type=\"supplementary-material\">Table S5<\/a>). Seven genes mutated in U87MG by SNVs or indels were also found mutated within the TCGA sample (PTEN, LTF, KCNJ16, ABCA13, FLI1, MLL4, DSP). This overlap is not statistically significant (p\u200a=\u200a0.16). Ten additional genes overlapped, including two genes mutated by interchromosomal translocations (CNTFR, ELAVL2), three genes mutated by intrachromosomal translocations (ANXA8, LRRC4C, ALDH1A3), and five by homozygous microdeletions (CDKN2A, CDKN2C, MTAP, IFNA21, TMBIM4).\n\nFinally, in order to place the homozygous mutations of U87MG in context relative to GBM mutational patterns as a whole, the Genomic Identification of Significant Targets in Cancer (GISTIC) method \\[28\\] was applied to 293 glioblastoma samples with genome wide copy number information available from the TCGA. This yielded a list of significant, commonly deleted regions present across glioblastomas as a group and highlights genes commonly mutated in GBMs. These data indicate that all or parts of chromosomes 1, 6, 9, 10, 13, 14, 15, and 22 are commonly deleted within GBMs as a group. In total, these regions comprise 915,306,764 bases, covering roughly 30 percent of the genome. In order to highlight genes homozygously mutated in U87MG that are within the regions of common loss, we cross-referenced these lists and found that 62\/332 (19%) are within the GISTIC defined regions. This does not suggest a significant overlap of homozygously mutated genes in U87MG with commonly deleted regions, but those mutated genes that do overlap may be of increased relevance to cancer. Two of the 62 genes are also in the Cancer Gene Census: PTEN and TCRA. We propose that a subset of the genes mutated in U87 within these commonly deleted regions may be the specific targets of mutation and should be assessed on larger sample sets. (<a href=\"#pgen.1000832.s009\" data-ref-type=\"supplementary-material\">Table S5<\/a> and <a href=\"#pgen.1000832.s004\" data-ref-type=\"supplementary-material\">Figure S4<\/a>).\n\n# Discussion\n\nReported individual human genome sequencing projects using massively parallel shotgun sequencing with alignment to the human reference genome clearly indicate the practicality of individual whole genome sequencing. However, the monetary cost of data generation, data analysis issues, and the time it takes to perform the experiments have remained substantial limitations to general application in many laboratories. Here we demonstrate enormous improvements in the throughput of data generation. Using a mate-pair strategy and only ten micrograms of input genomic DNA, we generated sufficient numbers of short sequence reads in approximately 5 weeks of machine operation with a total reagent cost of under \\$30,000. We believe this makes U87MG the least expensive published genome sequenced to date signaling that routine generation of whole genomes is feasible in individual laboratories. Further, the two-base encoding strategy employed within the ABI SOLiD system is a powerful approach for comprehensive analysis of genome sequences and, in concert with BFAST alignment software, is able to identify SNVs, indels, structural variants, and translocations.\n\nOf particular interest in whole-genome resequencing studies such as this one is how much raw data must be produced to sequence both alleles using a shotgun strategy. Here, 107.5Gb of raw data was generated. Of this, 55.51Gb was mapped to unique positions in the reference genome. In effect, this results in a mean base coverage of 10.85\u00d7 per allele within non-repetitive regions of the genome. Repetitive regions are of course undermapped, as their unique locations are more difficult to determine. This level of oversampling is adequate for high stringency variant calling (error rate less than 5\u00d710^\u22126^) at 93.71% of heterozygous SNP positions. There may be some biases in library generation resulting in bases that are not successfully covered even if they are relatively unique, but solutions to this may be found in performing multiple sequencing runs with varied library designs, as suggested in other studies \\[17\\].\n\nWith rapid advances in the generation of massively parallel shotgun short reads, one of the major computational problems faced is the rapid and sensitive alignment of greater than 1 billion paired end reads needed to resequence an individual genome. We demonstrate a practical solution using BFAST, which was able to perform fully gapped local alignment on the two-base encoded data to maximize variant calling in less than 4 days on a 20-node 8-core computer cluster.\n\nComparing U87MG SNVs with the James Watson \\[12\\] and YanHuang \\[14\\] genome projects' SNVs displays differences in SNV detection between the three projects. Being derived from a Caucasian individual, U87MG and James Watson are expected to share more SNVs than U87MG and YanHuang. However, when we compared SNVs between U87MG and these two genomes, more SNVs were actually shared between U87MG and YanHuang. Meanwhile, the YanHuang project called significantly more SNVs in total than both our U87MG sequencing project and the James Watson project. These results stress that utilizing different sequencing platforms (U87MG-ABI SOLiD, James Watson-Roche 454, YanHuang-Illumina Solexa), alignment tools (U87MG-BFAST, James Watson-BLAT, YanHuang-SOAP) and analytical approaches results in finding different quantities of SNVs. The higher genomic coverage in our U87MG sequence relative to James Watson and the increased sensitivity of BFAST relative to BLAT and SOAP were counted on to find highly robust variants. This is particularly important when sequencing a cancer genome because of the interest in finding novel cancer mutations as opposed to common polymorphisms.\n\nThe genomic sequence demonstrates global differences in variant type across the coding and non-coding portions of the human genome. By increasing the sensitivity of indel detection, we revealed that small indels have mutated genes at a higher rate than SNVs. A larger proportion of the indels identified are predicted to cause a protein coding change compared to SNVs (178\/191,743 indels vs. 154\/2,384,470 SNVs).\n\nIn U87MG, there is a relative increase in 4-base indels genome-wide, which has been observed in other normal genomes \\[23\\]\u2013\\[25\\] (<a href=\"#pgen-1000832-g002\" data-ref-type=\"fig\">Figure 2<\/a>, red bars). However, indels found in coding regions exhibit a bias toward events that are multiples of 3-bases in length (<a href=\"#pgen-1000832-g002\" data-ref-type=\"fig\">Figure 2<\/a>, blue bars) presumably selected to maintain reading frame. Thus, many of these events are likely to be polymorphisms and not disease related genomic mutations \\[25\\]. Similarly, the nucleotide substitution frequencies demonstrate a bias in coding regions compared to non-coding. Two-thirds of the substitutions were transitions genome-wide, as expected \\[27\\], but there was an enrichment of CG-\\>TA transitions in coding regions (<a href=\"#pgen-1000832-g003\" data-ref-type=\"fig\">Figure 3<\/a>). It is well established that the most common source of point mutations and SNPs in primates is deamination of methyl-cytosine (meC), causing transition to a thymine (T) \\[16\\],\\[29\\], and there is circumstantial evidence of that in U87MG's genome as well.\n\nThe resolution of genome-wide chromosomal rearrangements is substantially improved by the mate-pair strategy, coupled with sensitive and independent alignment of the short 50-base reads (<a href=\"#pgen-1000832-g005\" data-ref-type=\"fig\">Figure 5<\/a>). Based on published SKY data, we anticipated 7 interchromosomal breakpoints \\[6\\]. However, whole-genome mate-paired sequence data revealed the precise chromosomal joins of 35 interchromosomal events, which account for previously observed chromosomal abnormalities in U87MG but at additional finer scale resolution (<a href=\"#pgen-1000832-g005\" data-ref-type=\"fig\">Figure 5<\/a>, <a href=\"#pgen-1000832-g006\" data-ref-type=\"fig\">Figure 6<\/a>, <a href=\"#pgen-1000832-g007\" data-ref-type=\"fig\">Figure 7<\/a>). The translocation events were enriched in genic regions with 32\/35 (91.4%) occurring within 1kb of genes. A weaker, but still noticeable enrichment over genes occurs with microdeletions as well, which are generally missed by other experimental techniques like DNA microarrays. Thus, within the overall mutational landscape of this cancer cell line, translocations and structural variants preferentially occurred over genes, supporting a model where cancer mutations occur via structural instability rather than novel point mutations.\n\nDelving into the functional effects of the mutations in U87MG through gene ontology and cross-referencing the literature, we found a large number of known and predicted cancer mutations present in the cell line. There is always a concern when dealing with a cancer cell line that mutations will be more related to its status as a cell line than to the cancer it was derived from. While this remains a concern, the large number of predicted and known cancer genes present in U87MG suggests other genes mutated in it have relevance to cancer as well. Using GISTIC to find regions with common deletions in glioma samples, we highlight 60 genes that are mutated in U87MG and are located in regions that are commonly deleted in GBMs that are not included within the Cancer Gene Census list as potential candidate mutational targets in GBMs (<a href=\"#pgen.1000832.s009\" data-ref-type=\"supplementary-material\">Table S5<\/a>).\n\nCancer cell lines are commonly used as laboratory resources to study basic molecular and cellular biology. It is clearly preferable to have complete genomic sequence for these valuable resources. U87MG is the most commonly studied brain cancer cell line and is highly cytogenetically aberrant. While this made the sequencing and mutational analysis more challenging, it serves as a model for future cultured cell line genomic sequencing. Through custom analyses, we found that the mutational landscape of the U87MG genome is vastly more complicated than we would have expected based on the variants discovered in previously published genomes. It is our hope that the increased genomic resolution presented here will direct researchers and clinicians in their work with this brain cancer cell line to create more effective experiments and lead to a greater ability to draw meaningful conclusions in the future.\n\n# Materials and Methods\n\n## Data Sources\n\nThe NCBI reference genome (build 36.1, hg18, March 2006), genome annotations, and dbSNP version 129 were downloaded from the UCSC genome database located at <http:\/\/genome.ucsc.edu>. A local mirror of the UCSC genome database (hg18) was used for the subsequent analysis of variants using included gene models and annotations. The Watson genome variants were downloaded from Cold Spring Harbor Laboratory (<http:\/\/jimwatsonsequence.cshl.edu>) with bulk data files available from <ftp:\/\/jimwatsonsequence.cshl.edu\/jimwatsonsequence\/>. The YanHuang variants were downloaded from the Beijing Genomics Institute at Shenzhen (<http:\/\/yh.genomics.org.cn\/>) with bulk data files available from <http:\/\/yh.genomics.org.cn\/download.jsp>.\n\n## Sample Preparation\n\nU87MG cells were ordered from ATCC (HTB-14) and cultured in a standard way. Genomic DNA was isolated from cultured U87MG cells using Qiagen Gentra Puregene reagents. DNA was stored at \u221220C until library generation.\n\n## ABI Sequencing\n\nLong-Mate-Paired Library Construction: The U87MG genomic DNA 2\u00d7 50bp long mate-paired library construction was carried out using the reagents and protocol provided by Applied Biosystems (SOLiD 3 System Library Preparation Guide). A similar protocol was reported previously \\[17\\]. Briefly, 45ug of genomic DNA was fragmented by HydroShear (Digilab Genomic Solutions Inc) to 1.0\u20132.5kb. The fragmented DNA was repaired by the End-It DNA End-Repair Kit (Epicentre). Subsequently, the LMP CAP adaptor was ligated to the ends. DNA Fragments between 1.2\u20131.7kb were selected by 1.0% agarose gel to avoid concatamers and circularized with a biotinylated internal adaptor. Non-circularized DNA fragments were eliminated by Plasmid-Safe ATP-Dependent DNase (Epicentre) and 3ug of circularized DNA was recovered after purification. Original DNA nicks at the LMP CAP oligo\/genomic insert border were translated into the target genomic DNA about 100bp by nick translation using E. coli DNA polymerase I. Fragments containing the target genomic DNA and adaptors were cleaved from the circularized DNA by single-strand specific S1 nuclease. P1 and P2 adaptors were ligated to the fragments and the ligated mixture was used to create two separate libraries with 10 cycles of PCR amplification. Finally, 250\u2013300bp fragments were selected to generate mate paired sequencing libraries with average target genomic DNA on each end around 90bp by excision from PAGE gel and use as emulsion PCR template. Templated Beads Preparation: The templated beads preparation was performed using the reagents and protocol from the manufacturer (Applied Biosystems SOLiD 3 Templated Beads Preparation Guide). SOLiD 3 Sequencing: The 2\u00d750b mate-paired sequencing was performed exactly according to the Applied Biosystems SOLiD 3 System Instrument Operation Guide and using the reagents from Applied Biosystems.\n\n## Exon Pull-Down Capture Sequencing with Illumina GAII\n\nWe used an array pull-down capture strategy established in our lab \\[30\\]. An Agilent custom array for capturing 5,253 \"cancer-related\" genes was designed through Agilent e-array system ([www.agilent.com](http:\/\/www.agilent.com)). Only the amino acid encoding regions were targeted with 60mer oligos spaced center-to-center 20\u201330bp. The probes were randomly distributed across two separate 244K arrays. The library for cancer gene capture sequencing was generated following the standard Illumina paired-end library preparation protocol. 5ug of genomic DNA was used for the starting material and 250\u2013300bp fragments were size-selected during the gel-extraction step. In the last step, 18 cycles of PCR were performed in multiple tubes to yield 4ug of product and mixed with 50ug of Human Cot-1 DNA (Invitrogen), 52ul of Agilent 10\u00d7 Blocking Agent, 260ul of Agilent 2\u00d7 Hybridization Buffer and 10\u00d7 molar concentration of unpurified Illumina paired-end primer pairs custom made according to the sequences provided by Illumina (Oligonucleotide sequences, 2008, Illumina, Inc: available on request from Illumina). The mix was then diluted with elution buffer for the final volume of 520ul and then incubated at 95\u00b0C for 3 min and 37\u00b0C for 30min. 490ul of the hybridization mix was added to the array and hybridized in the Agilent hybridization oven (Robins Scientific) for 65 hrs at 65\u00b0C, 20rpm. After hybridization, the array was washed according to the Agilent wash procedure A protocol. The second wash was extended to 5 minutes to increase the wash stringency. After washing, the array was stripped by incubating it in the Agilent hybridization oven at 95\u00b0C for 10min, 20rpm with 1.09\u00d7 Titanium Taq PCR Buffer (Clonetech). After the incubation and collection of the solution, 4 tubes of PCR were performed with each tube containing 96ul of the collected solution, 1ul of dNTPs (10mM each), 1ul of Titanium Taq (Clonetech) and Solexa primers, 1ul each. 15 cycles of PCR was performed at the following condition: 30sec at 95\u00b0C, (10 sec at 95\u00b0C, 30 sec at 65\u00b0C, 30 sec at 72\u00b0C)\u00d718 cycles, 5 min at 72\u00b0C and hold at 4\u00b0C. The amplified product was purified using QIAquick PCR Purification Kit and eluted in 30ul of EB. After confirming the size of the amplicon on 2% agarose gel and measuring the concentration, the amplicon was diluted to 10nM, the working concentration for cluster generation. The Illumina flowcell was prepared according to the manufacturer's protocol and the Genome Analyzer was run using standard manufacturer's recommended protocols. The image data produced were converted to intensity files and were processed through the Firecrest and Bustard algorithms (1.3.2) provided by Illumina to call the individual sequence reads.\n\n## ABI SOLiD Sequence Alignment and Consensus Base Calling\n\nWe used Blat-like Fast Accurate Search Tool version 0.5.3 (BFAST <http:\/\/bfast.sourceforge.net>) \\[19\\] to perform sequence alignment of the two-base encoded reads off the ABI SOLiD to the NCBI human reference genome (build 36.1). Utilizing the local alignment algorithm included in BFAST \\[31\\], we were able to simultaneously decode the short reads, while searching for color errors (encoding errors), base changes, insertions, and deletions.\n\nWe found candidate alignment locations (CALs) for each end independently. We utilized ten indexes to be robust to up to six color errors, equating to a 12% per-read error rate:\n\n1111111111111111111111\n\n111110100111110011111111111\n\n10111111011001100011111000111111\n\n1111111100101111000001100011111011\n\n111111110001111110011111111\n\n11111011010011000011000110011111111\n\n1111111111110011101111111\n\n111011000011111111001111011111\n\n1110110001011010011100101111101111\n\n111111001000110001011100110001100011111\n\nWe also set parameters to use only informative keys when looking up reads in each index (BFAST parameter -K 8), and to ignore reads with too many CALs aggregated across all indexes (BFAST parameter -M 384). If reads mapped to greater than 384 locations, then they were categorized as 'unmapped'. We then performed local alignment for each of the returned CALs, simultaneously decoding the read from color space searching for color errors (encoding errors), base changes, insertions, and deletions \\[31\\]. We choose the \"best scoring\" alignment, accepting an alignment only if it was at least the equivalent edit distance of two color errors away from the next best alignment. This is approximately similar to a 'mapping quality' of 20 or better from the MAQ program output, for reference. We removed duplicate reads using the alignment filtering utility found in DNAA (<http:\/\/dnaa.sourceforge.net>). For single-end and mate-paired reads where only one end mapped, we removed duplicates based on reads having identical stat positions. For mate-paired reads, we removed duplicates where both ends had the same start position.\n\n## Illumina Genome Analyzer Sequence Alignment\n\nIllumina generated sequence was aligned to the NCBI human reference genome (build 36.1) using BFAST with the following parameters applied. Each end of the fragment library was mapped independently to identify CALs, utilizing ten indexes to be robust to errors and variants in the short (typically 36bp) reads:\n\n1111111111111111111111\n\n1111101110111010100101011011111\n\n1011110101101001011000011010001111111\n\n10111001101001100100111101010001011111\n\n11111011011101111011111111\n\n111111100101001000101111101110111\n\n11110101110010100010101101010111111\n\n111101101011011001100000101101001011101\n\n1111011010001000110101100101100110100111\n\n1111010010110110101110010110111011\n\nWe also set parameters to use only informative keys when looking up reads in each index (BFAST parameter -K 8), and to ignore reads with too many CALs aggregated across all indexes (BFAST parameter -M 1280). We then performed a standard local alignment for each CAL. Reads were declared mapped if a single unique best scoring alignment was identified within the genome. Duplicate reads were filtered out in the same manner as for the ABI SOLiD data.\n\n## Single Nucleotide Variant and Small Insertion and Deletion Detection\n\nTo find SNVs including SNPs and small indels, we assumed the MAQ consensus-calling model \\[20\\] utilizing the implementation in SAMtools \\[21\\]. We used a value of 0.0000007 for the prior of a difference between two haplotypes (-r parameter). This was chosen based on ROC analysis of a test dataset (data not shown).\n\n## Structural Variation Detection\n\nStructural variations were detected using custom algorithms designed to comprehensively search for groups of mate-pair reads with aberrant paired-end insert size distributions that are consistently identifying a unique structural variant in the genome. We utilized the \"dtranslocations\" utility in the DNAA package (<http:\/\/dnaa.sourceforge.net>) for the primary structural variation candidate search. The utility first selected all pairs for which each end is uniquely mapped to a single location in the human genome and for which the mate-pair reads are not positioned in the expected size range relative to the consensus genome. Then we filter out false positives that are not consistent with a chromosomal difference on an allele. Briefly, the genome was divided into 500-base bins sequentially stepped 100-bases apart from their start positions. Each bin was then paired with other bins on the basis of containing similar 'mismapped' mate-pair reads. The aberrant mate-paired reads were defined as reads that were mapping less than 1000 or greater than 2000 bases apart within the reference genome sequence, which is selected based on the insert size distribution calculated from the aggregate dataset (<a href=\"#pgen.1000832.s002\" data-ref-type=\"supplementary-material\">Figure S2<\/a>). These were then rank-ordered based on the number of mate-pairs meeting criteria, and the destination bin with the most reads within it was paired with a given source bin to create a 'binset'. Binsets containing less than 4 reads were filtered out, removing 98.3% of the candidates based on having too little evidence supporting them. The resulting list of filtered binsets was then scanned for clusters of binsets. Binset clusters are groups of binsets where the source bins occur within 2000 bases of each other and the destination bins occur within 2000 bases of each other. Redundant binsets were combined and those binset clusters that contain too few (less than 9 binsets spanning at least 1000 bases) or too many binsets (greater than 29 binsets spanning at most 3000 bases\u2014higher is impossible given our insert size distribution) were removed as artifacts. The resulting binset clusters represent the reads immediately flanking structural breakpoint events. This detection process is currently being automated as Breakway (<http:\/\/breakway.sourceforge.net>), but was done using custom scripts at the time of analysis.\n\nThe structural variations were then separated into interchromosomal and intrachromosomal events. Intrachromosomal events of less than 1Mb are assessed for deletion status by averaging base coverage within the bounds of the event and comparing it to base coverage 200kb outside the event on both sides. Those that have average interior base coverage less than 25% of the average exterior base coverage are classified as \"complete\" deletions. Those with average interior base coverage between 25% and 75% that of average exterior base coverage are classified as \"heterozygous deletions\" (deletions of at least one copy of the region, but with at least one copy remaining).\n\n## Genes Affected by Mutations in Coding Sequence\n\nVariant calls from the SAMtools pileup tool were first loaded into a SeqWare QueryEngine database and subsequently filtered to produce BED files. This filtering criteria required that a variant be seen at least 4 times and at most 60 times with an observation occurring on each strand at least once. For SNVs we further enforced the criteria that SNVs should only be called in reads lacking indels and the last 5 bases of the reads were also ignored. This reduced the likelihood that spurious mismappings were used to predict SNVs and eliminated the lowest quality bases from consideration. For small indels (\\<21bp) we enforced a slightly different filter by requiring that any reads supporting an indel were only allowed to contain one contiguous indel and these reads were not considered if the indel occurred on either the beginning or end of the read. These criteria, like the SNV criteria, were used to reduce the likelihood of using mismapped reads or locally misaligned reads in the variant calling algorithm. The elimination of reads with indels at the beginning or end of the read was intended to remove potential alignment artifacts caused by ambiguous gap introduction due to lack of information at the ends to guide proper alignment. Together, these filtering criteria reduced the likelihood that sequencing errors were identified as SNV or indel variants. We used scripts available in the BFAST toolset and SeqWare Pipeline to filter and annotate the variant calls. Variants passing these filters were further annotated by their overlap with dbSNP version 129. Variants were required to share the same genomic position as a dbSNP entry along with matching the allele present in the database to be considered overlapping. Mapping to dbSNP allowed us to filter out known SNPs from de novo variants.\n\nFiltered SNV and indel variants were then analyzed for their affect within the genome that is annotated with gene models. This analysis used scripts from the SeqWare Pipeline project and gene models downloaded from the UCSC hg18 human genome annotation database. Six different gene model sets from hg18 were considered: UCSC genes (knownGene), RefSeq genes (refGene, <http:\/\/www.ncbi.nlm.nih.gov\/RefSeq>), Consensus Coding Sequence genes (ccdsGene, <http:\/\/www.ncbi.nlm.nih.gov\/CCDS>), Mammalian Gene Collection genes (mgcGenes, <http:\/\/mgc.nci.nih.gov>), Vertebrate Genome Annotation genes (vegaGene, <http:\/\/vega.sanger.ac.uk>), and Ensembl genes (ensGene, <http:\/\/www.ensembl.org>). Each variant was evaluated for overlap with genes from each of the 6 gene models. If overlap was detected the variant was examined and tagged with one or more of the following terms depending on the nature of the event: \"utr-mutation\", \"coding-nonsynonymous\", \"coding-synonymous\", \"abnormal-ref-gene-model-lacking-stop-codon\", \"abnormal-ref-gene-model-lacking-start-codon\", \"frameshift\", \"early-termination\", \"inframe-indel\", \"intron-splice-site-mutation\", \"stop-codon-loss\", and\/or \"start-codon-loss\". The variant was also tagged with the gene symbol and other accessions to facilitate lookups. This information was loaded into a SeqWare QueryEngine database to allow for querying and filtering of the variants as needed.\n\nGenes affected by structural variations were assessed in two ways depending on the structural variation type. For interchromosomal translocation events, a gene was considered \"affected\" when either end of an interchromosomal translocation event fell in a genic region (including the entire coding region plus 1kb up- or down-stream of the gene's coding region). The same criteria were used for all intrachromosomal translocation events. For events that were classified as complete or heterozygous deletions, a gene was considered affected if all or part of a coding exon was deleted.\n\n## Annotation of Relevant Mutated Genes\n\nHomozygous SNVs, small indels, large deletions, and translocation events for variants that included predicted coding sequence changes were tallied. This became a reference list of variants with serious homozygous mutations that likely completely disrupted, or \"knocked out\", the normal function or synthesis of the target protein.\n\nFor the SNVs and small indels, a \"knockout\" variant was defined as a homozygous call by the SAMtools variant caller where the variant was predicted by the SeqWare Pipeline scripts to change coding sequence with one or more of the following annotations: \"early-termination\", \"frameshift\", \"intron-splice-site-mutation\", \"start-codon-loss\", and\/or \"stop-codon-loss\". The \"early-termination\" event represented a stop codon introduced upstream of the annotated stop codon. The \"frameshift\" represented an indel that resulted in a shifting of the reading frame of the gene resulting in, typically, early termination and non-sense coding sequence. The \"intron-splice-site-mutation\" referred to a mutation in the two consensus splice site intronic bases flanking exons (GT at the 5\u2032 splice site and AG at the 3\u2032 splice site). Finally, \"stop-codon-loss\" and \"start-codon-loss\" simply refer to variants that interrupt the stop or start codons. We chose to not include \"coding-nonsynonymous\" and \"inframe-indel\" annotations in this list of knocked out variants because, while potentially serious as these mutations are, they are not guaranteed to result in an unexpressed or non-functional protein. However, homozygous frameshift, early termination, splice site, and stop\/start codon loss mutations are very likely to interrupt a gene's expression and translation to functional protein.\n\nAs described above, large microdeletions that removed all or part of an exon and interchromosomal translocation events that fell within 1kb of a gene's coding region were also classified as mutated genes.\n\nOnce suspect knockout variants were identified, a mapping process was used to translate one or more variants to the gene symbol. This mapping allowed us to condense multiple variants affecting multiple gene models to a more abbreviated list of gene symbols likely to be affected by these knockout mutations. The mapping from variants to gene symbols used variants identified with gene models from the refGene and the knownGene tables in the UCSC hg18 database and mapped these variants to gene symbols using queries against the name field of the knownGene table and the alias field of the kgAlias table. The UCSC table browser was used to accomplish these queries and map the knownGene identifiers to gene symbols via the kgXref table. A similar approach was used for homozygous large-scale microdeletions and translocation events.\n\n## DAVID\/EASE Analysis\n\nThe list of knockout genes was uploaded to the Database for Annotation, Visualization, and Integrated Discovery (DAVID, version 2008) to identify enriched Gene Ontology (GO) terms \\[32\\]\u2013\\[33\\]. Overlap with GO terms from the biological process, cellular component, and molecular function ontologies were considered. The default parameters were used and a p-value cutoff of \\<\u200a=\u200a0.01 was considered significant.\n\n## Cancer Gene Census\n\nThe overlap between the Cancer Gene Census genes and those identified as knockouts in U87MG were compared. The Cancer Gene Census project is an ongoing effort to catalog genes with mutations that have been implicated in cancer \\[34\\]. It is a highly curated list that includes annotations for each gene including tumor types, class of mutations, and other genetic properties. We used the gene symbol list from the September 30^th^, 2009 complete working list, which includes 412 gene symbols.\n\n## TCGA\n\nThe overlap between mutations in the Cancer Genome Atlas (TCGA) and those identified as knockouts in U87MG was analyzed. TCGA is an ongoing effort to understand the molecular basis of cancer through large-scale copy number analysis, expression profiling, genome sequencing, and methylation studies among other techniques \\[4\\]. It provides information on mutations found by Sanger sequencing on many patient samples. For glioblastoma this includes sequence data aberrations detected in 158 patient samples and 1,177 genes.\n\n## Genomic Identification of Significant Targets in Cancer\n\nThe Genomic Identification of Significant Targets in Cancer (GISTIC) method was used to find significant areas of deletion in 293 samples from the TCGA \\[24\\]. The GISTIC technique was designed to identify and analyze chromosomal aberrations across a set of cancer samples, based on the amplitude of the aberrations as well as the frequency across samples. This approach produced a series of commonly deleted regions across the set of TCGA GBMs. To calculate the areas of deletion, we used 293 Affymetrix SNP 6.0 samples segmented using the GLAD SNP analysis module \\[35\\]. Default parameters of GISTIC were used. GISTIC produces peak limits, wide peak limits, and in addition broader region limits. These commonly deleted broader regions were then scanned for predicted knockout genes in U87MG.\n\n## Indel Size Distribution and Nucleotide Substitution Frequencies\n\nThe distribution of small indel sizes was examined for both deletions and insertions. Indels classified as affecting coding-sequence by the SeqWare Pipeline (see above) were compared to those outside coding regions. Raw counts were collected, recalculated as percents of total, and compared directly.\n\nSimilarly, nucleotide substitution frequency was examined for SNVs from U87MG both genome-wide and only in coding regions. Once binned appropriately, the SNV nucleotide substitutions were counted, tallied in a table, and graphed as percents of total.\n\n## Individual Genome Comparison\n\nVariants from the Watson and Yan Huang genome were downloaded from each respective project from the following URLs: <ftp:\/\/jimwatsonsequence.cshl.edu\/jimwatsonsequence\/watson-454-snp-v01.txt.gz> and <http:\/\/yh.genomics.org.cn\/do.downServlet?file=data\/snps\/yhsnp_add.gff>. These files contained variant calls for each genome along with annotations describing the variant as novel or occurring in dbSNP. The Watson genome only contained SNV calls so our comparison was limited to just SNVs. The Yan Huang genome also contained calls indicating heterozygous or homozygous. However, a variant was considered to match between genomes regardless of zygosity state. We compared the overlap of the U87MG genome, dbSNP and each of these genomes in turn. SNVs from U87MG that were considered for comparison had to meet our criteria; variants had to be observed at least 4 times, at most 60 times, at least once per strand, and with a minimum phred score of 10. SNVs in the three-way comparison were said to match if the position and allele matched between the genomes. If both variants matched between U87MG and the other genome and one was annotated in dbSNP, then the other was considered in dbSNP as well. If neither contained annotations from dbSNP the variant was considered novel. A similar process was carried out for variants distinct to each genome. The results were recorded as Venn diagrams showing the overlap between dbSNP, U87MG, and the Watson or Yan Huang genome.\n\n## Illumina SNP Chip\n\nGenomic DNA from U87MG was submitted to the Southern California Genotyping Consortium to be run on the Illumina Human 1M-Duo BeadChip, which consists of 1,199,187 probes scattered across the human genome. The Illumina Beadstudio program was used to analyze the resulting intensity data. Loss of heterozygosity was determined by analyzing B-allele frequency as determined by the Beadstudio program. Normal two-copy regions of the genome are represented by long stretches of probes with B-allele frequencies of 0, 0.5 or 1. Regions of LOH, on the other hand, deviate from this pattern significantly. Copy number was determined by looking at probe intensity.\n\n## Sanger Sequencing Validation\n\nPrimers for validation were designed by targeting regions immediately flanking the event predicted by our whole genome sequence analysis using the Primer3 tool (<http:\/\/frodo.wi.mit.ed\/primer3\/>). Polymerase chain reaction was performed following standard protocols using Finnzymes Phusion Hot-Start High Fidelity polymerase. Products were run on 2% agarose gel electrophoresis and product purity and size was assessed by staining with ethidium bromide. Sanger sequencing was performed at the UCLA Genotyping and Sequencing core facility using an ABI 3730 Capillary DNA Analyzer. Sequence trace files were analyzed using Geospiza FinchTV. Validation status and PCR primers are listed in <a href=\"#pgen.1000832.s005\" data-ref-type=\"supplementary-material\">Table S1<\/a>.\n\n## Data Deposition and Availability\n\nIntensities, quality scores, and color space sequence for the genomic sequence of U87 SOLiD were uploaded to the Sequence Read Archive under the accession SRA009912.1\/Sequence of U87 Glioblastoma Cell-line. Intensities, quality scores, and nucleotide space sequence for the exon capture U87 Illumina sequence were also uploaded to the Short Read Archive under the same accession. For both datasets, alignment files have been uploaded to the Short Read Archive as additional analysis results.\n\nVariant calls for both datasets are available via a SeqWare QueryEngine web service at <http:\/\/genome.ucla.edu\/U87>. This tool allows for querying the variants using a variety of search criteria including coverage, mutational consequence, gene symbol, and others. SeqWare QueryEngine produces results in both BED and WIG format making it compatible with the majority of genome browsers such as the UCSC genome and table browsers. Variant data will be uploaded to SRA as metadata along with the raw sequences. For the whole genome SOLiD alignment, small indels (\\<21bp), SNVs, large deletions, and translocation events can be queried. For the exon capture Illumina alignment, small indels and SNVs can be queried.\n\n## Software Availability\n\nMost software used for this project is open-source and freely available. We created two software projects that were instrumental in the analysis of the U87MG data: BFAST and SeqWare. The color- and nucleotide-space alignment tool BFAST can be downloaded from <http:\/\/bfast.sourceforge.net> and many of our alignment filtering as well as the primary step in structural variation detection can be found in the DNAA package at <http:\/\/dnaa.sourceforge.net>. The SeqWare software project was used throughout the analysis of variant calls. We used the SeqWare LIMS tool for sample tracking, the SeqWare Pipeline analysis programs for annotating variants with dbSNP status and mutational consequence predictions, and SeqWare QueryEngine was used to database and query variant calls and annotations. This software and documentation can be downloaded from <http:\/\/seqware.sourceforge.net>.\n\n# Supporting Information\n\nConcordance between Solexa capture data and SOLiD whole genome data. The left plot displays the SNP call concordance between each experiment (Solexa capture data in blue, SOLiD whole genome data in red) with the Illumina 1M Beadchip microarray for the 8.5Mb of sequence pulled down in the capture experiment. The right plot displays concordance of the non-reference (mutant) allele calls with the array data for those regions.\n\n(0.43 MB TIF)\n\nClick here for additional data file.\n\nPaired end insert size distribution. Empirical paired end insert size distribution for reads where both ends aligned with duplicates removed.\n\n(0.41 MB TIF)\n\nClick here for additional data file.\n\nAlignment is robust against genome-wide repeat elements. Circos plot \\[35\\] of reads spanning a complete microdeletion on chromosome 2, bases 201855000\u2013201858000, are shown in dark blue, with the normal reads in the surrounding region in light blue. The green plot shows base-coverage at each position. The outermost track shows the structure of a gene, CASP8, overlapping this region (large boxes-exons, lines-introns). The track containing black and red boxes shows genome-wide repeat elements (black-LINE, red-SINE). Note the high density of reads even over conserved LINE elements. Some SINE elements do demonstrate a drop in alignments, but these do not prevent the identification of structural variation-spanning reads.\n\n(0.21 MB TIF)\n\nClick here for additional data file.\n\nCommonly deleted regions in GBM according to GISTIC. This deletion plot shows significant regions of deletion in 293 GBM samples from the TCGA. The top of the plot shows the G-score and the bottom shows the q-values. G-score reflects the frequency and amplitude of the deletion. Q-values greater than 0.25 were considered significant. Overlap of genes mutated in U87 via SNVs or Indels and broad regions of deletion are considered to be likely cancer targets. This includes all or part of chromosomes 1, 6, 9, 10, 13, 14, 15, and 22.\n\n(0.43 MB TIF)\n\nClick here for additional data file.\n\nPCR and dideoxy sequencing validation. A list of the variants that were validated by PCR and dideoxy sequencing including primers used, varient location, and validation status.\n\n(0.03 MB XLS)\n\nClick here for additional data file.\n\nStructural variants in U87MG. All structural variants listed as regions immediately flanking the genomic breakpoint.\n\n(0.18 MB XLS)\n\nClick here for additional data file.\n\nGenes knocked out by SNVs\/Indels. List of all genes predicted to be knocked out by SNVs and Indels in U87MG.\n\n(0.20 MB XLS)\n\nClick here for additional data file.\n\nGenes affected by structural variants. List of all genes predicted to be affected by structural variants in U87MG.\n\n(0.48 MB XLS)\n\nClick here for additional data file.\n\nAnnotation of mutated genes. Lists of genes predicted to be mutated in U87MG annotated by various cancer-related gene databases.\n\n(0.17 MB XLS)\n\nClick here for additional data file.\n\nWe would like to acknowledge Bret Harry and Jordan Mendler for computational support and for maintaining our computer cluster and pipeline.\n\n# References\n\n[^1]: Conceived and designed the experiments: MJC NH BDO ZC HL BM SFN. Performed the experiments: MJC BDO ZC HL. Analyzed the data: MJC NH BDO AE HL BM SFN. Contributed reagents\/materials\/analysis tools: MJC NH BDO ZC HL BM SFN. Wrote the paper: MJC NH BDO ZC AE HL BM SFN.","meta":{"dup_signals":{"dup_doc_count":104,"dup_dump_count":34,"dup_details":{"curated_sources":3,"2021-21":1,"2021-17":1,"2020-45":1,"2019-51":1,"2019-35":1,"2018-47":1,"2018-13":2,"2018-05":1,"2017-39":1,"2017-26":3,"2017-22":4,"2017-17":3,"2017-09":2,"2016-40":1,"2016-36":2,"2016-30":2,"2016-18":1,"2016-07":1,"2015-48":1,"2015-22":3,"2014-52":7,"2014-49":3,"2014-42":7,"2014-41":8,"2014-35":7,"2014-23":8,"2014-15":5,"2023-14":1,"2017-13":6,"2015-18":6,"2015-11":4,"2015-06":1,"2014-10":4,"2013-48":1}},"file":"PMC2813426"},"subset":"pubmed_central"}
{"text":"author: Liza Gross\ndate: 2006-09\ntitle: Evolution of Neonatal Imitation\n\nHumans do it. Chimps do it. Why shouldn't monkeys do it, too? Mimicry exists throughout the animal kingdom, but imitation with a purpose\u2014matching one's behavior to others' as a form of social learning\u2014has been seen only in great apes. (Mockingbirds can imitate an impressive number of other birds' songs, but they can't mimic you sticking out your tongue like a chimp can.) This matching behavior likely helps individuals conform to social norms and perform actions in the proper context. It's generally believed that monkeys do not imitate in this way. However, the discovery that rhesus monkeys have \"mirror neurons\"\u2014neurons that fire both when monkeys watch another animal perform an action and when they perform the same action\u2014suggests they possess the common neural framework for perception and action that is associated with imitation.\n\nMost studies exploring the early signs of matching behavior have focused on humans. A landmark 1977 study by Andrew Meltzoff and Keith Moore showed that 12- to 21-day-old infants could imitate adults who pursed their lips, stuck out their tongue, opened their mouth, and extended their fingers. They later found similar results in newborns, demonstrating that imitation is innate, not learned. A handful of studies on newborn chimps found a similar capacity for imitating human facial gestures. In a new study, Pier Ferrari, Stephen Suomi, and colleagues explored the possibility that imitation evolved earlier in the primate tree by studying neonatal imitation in rhesus monkeys, which split from the human lineage about 25 million years ago. They found that rhesus infants can indeed imitate a subset of human facial gestures\u2014gestures the monkeys use to communicate. The first investigation of neonatal imitation outside the great ape lineage, their study suggests that the trait is not unique to great apes after all.\n\nFerrari et al. tested 21 baby rhesus monkeys' response to various experimental conditions at different ages (one, three, seven, and 14 days old). Infants were held in front of a researcher who began with a passive expression (the baseline condition) and then made one of several gestures, including tongue protrusion, mouth opening, lip smacking, and hand opening.\n\nDay-old infants rarely displayed mouth opening behavior, but smacked their lips frequently. When experimenters performed the mouth opening gesture, infants responded with increased lip smacking but did not increase any other behavior. None of the other stimuli produced significant responses. But by day 3, matched behaviors emerged: infants stuck out their tongues far more often in response to researchers' tongue protrusions compared with control conditions, and smacked their lips far more often while watching researchers smacking theirs. (Watch an infant imitating mouth opening at DOI: [10.1371\/journal.pbio.0040302.sv001](10.1371\/journal.pbio.0040302.sv001).) By day 7, the monkeys tended to decrease lip smacking when humans performed the gesture, and by two weeks, all imitative behavior stopped.\n\nInfant rhesus monkeys, these results suggest, have a narrow imitation window that opens three days after birth, when they can reproduce human tongue protrusion and lip smacking. This imitation period is much longer in humans (two to three months) and chimps (about two months). It's possible that rhesus babies show more varied and prolonged imitative behavior in response to mom or other monkeys than to human experimenters, who may not provide the most relevant biological cues. But this narrow window does comport with the development schedule of rhesus monkeys, which is much shorter than that of humans and chimps.\n\nMany questions remain about the neural mechanisms of neonatal imitation. The researchers argue that their results support a resonance mechanism linked to mirror neurons, which have recently been identified while monkeys observe others' lip smacking and tongue protrusion. In this model, observing human mouth gestures directly activates mirror neurons in the monkeys' brain, ultimately leading to a replication of the gesture.\n\nHuman babies can imitate an adult's facial gesture a day after seeing it, which may help them identify individuals. For rhesus monkeys, lip smacking (which often alternates with tongue protrusion) accompanies grooming sessions and signals affiliation\u2014an important social cue for a species that is often described as \"despotic and nepotistic.\" Picking up these social gestures early in life may well facilitate the animal's early social relations (primarily with the mother) and assimilation into the social fabric of the group, providing a mechanism for distinguishing friend from foe. It will be interesting to test the extent of imitation in monkeys with more complex social dynamics. While the social life of rhesus monkeys may not demand the more sophisticated repertoire of behaviors seen in great apes, they seem to be hard-wired for imitation just like apes.","meta":{"dup_signals":{"dup_doc_count":106,"dup_dump_count":43,"dup_details":{"curated_sources":4,"2022-33":2,"2021-31":2,"2021-25":1,"2021-10":1,"2020-45":1,"2020-24":1,"2019-18":1,"2018-51":2,"2018-34":1,"2017-30":2,"2017-17":4,"2017-09":5,"2017-04":2,"2016-50":1,"2016-44":2,"2016-40":2,"2016-36":2,"2016-30":5,"2016-26":2,"2016-22":2,"2016-18":1,"2016-07":2,"2015-48":3,"2015-40":2,"2015-35":2,"2015-32":3,"2015-27":1,"2015-22":4,"2015-14":2,"2014-52":4,"2014-49":4,"2014-42":4,"2014-41":2,"2014-35":3,"2014-23":8,"2014-15":2,"2023-40":1,"2015-18":3,"2015-11":1,"2015-06":2,"2014-10":3,"2013-48":2,"2013-20":2}},"file":"PMC1560175"},"subset":"pubmed_central"}
{"text":"abstract: We investigated the association of glycemia and 43 genetic risk variants for hyperglycemia\/type 2 diabetes with amino acid levels in the population-based Metabolic Syndrome in Men (METSIM) Study, including 9,369 nondiabetic or newly diagnosed type 2 diabetic Finnish men. Plasma levels of eight amino acids were measured with proton nuclear magnetic resonance spectroscopy. Increasing fasting and 2-h plasma glucose levels were associated with increasing levels of several amino acids and decreasing levels of histidine and glutamine. Alanine, leucine, isoleucine, tyrosine, and glutamine predicted incident type 2 diabetes in a 4.7-year follow-up of the METSIM Study, and their effects were largely mediated by insulin resistance (except for glutamine). We also found significant correlations between insulin sensitivity (Matsuda insulin sensitivity index) and mRNA expression of genes regulating amino acid degradation in 200 subcutaneous adipose tissue samples. Only 1 of 43 risk single nucleotide polymorphisms for type 2 diabetes or hyperglycemia, the glucose-increasing major C allele of rs780094 of *GCKR,* was significantly associated with decreased levels of alanine and isoleucine and elevated levels of glutamine. In conclusion, the levels of branched-chain, aromatic amino acids and alanine increased and the levels of glutamine and histidine decreased with increasing glycemia, reflecting, at least in part, insulin resistance. Only one single nucleotide polymorphism regulating hyperglycemia was significantly associated with amino acid levels.\nauthor: Alena Stan\u010d\u00e1kov\u00e1; Mete Civelek; Niyas K. Saleem; Pasi Soininen; Antti J. Kangas; Henna Cederberg; Jussi Paananen; Jussi Pihlajam\u00e4ki; Lori L. Bonnycastle; Mario A. Morken; Michael Boehnke; P\u00e4ivi Pajukanta; Aldons J. Lusis; Francis S. Collins; Johanna Kuusisto; Mika Ala-Korpela; Markku LaaksoCorresponding author: Markku Laakso, <email@example.com>.\ndate: 2012-07\nreferences:\ntitle: Hyperglycemia and a Common Variant of *GCKR* Are Associated With the Levels of Eight Amino Acids in 9,369 Finnish Men\n\nInsulin regulates carbohydrate, lipid, protein, and amino acid metabolism (1). Insulin inhibits proteolysis and associated release of amino acids and stimulates amino acid uptake and protein synthesis in skeletal muscle (2,3). Selected amino acids, however, enhance insulin secretion (4,5) or modulate insulin sensitivity (6\u201310), the two main mechanisms in the regulation of glucose homeostasis.\n\nA recent study reported that three branched-chain amino acids (BCAAs), valine, leucine and isoleucine, and two aromatic amino acids, phenylalanine and tyrosine, predicted type 2 diabetes (11). The risk of diabetes was fivefold higher in individuals in the top quartile of a combination of three amino acids (isoleucine, phenylalanine, and tyrosine) compared with individuals in the lowest quartile. Although some small studies have reported that the levels of amino acids differ between individuals with normal and abnormal glucose tolerance (12,13), previous studies have not investigated the levels of amino acids across the entire range of glucose tolerance.\n\nAmino acids modulate insulin action on glucose transport (9,14\u201316) and gluconeogenesis (17). High levels of BCAAs, especially leucine, have been shown to associate with insulin resistance (16,18,19) or insulin-resistant states, including diabetes (6,12,13). BCAAs have been shown to downregulate insulin action on glucose uptake by inhibiting critical steps in the postreceptor insulin signaling cascade (10), although other studies have concluded that leucine and isoleucine stimulate glucose uptake (7,8,20). Gluconeogenic amino acids (mainly alanine and glutamine) can enhance hepatic glucose production and thus lead to hyperglycemia (21,22). Finally, amino acids such as arginine, glutamine, leucine, and phenylalanine directly stimulate insulin secretion (4).\n\nType 2 diabetes is a complex metabolic disease with a significant genetic component, and \\>40 gene loci associated with the risk of type 2 diabetes or hyperglycemia have been identified (23\u201326). The mechanisms by which these loci contribute to the risk of diabetes are only partially known. There are no previous studies on the association of these gene variants with the levels of amino acids.\n\nThe aims of our study were *1*) to investigate the relationship between the levels of amino acids and fasting and 2-h glucose across the entire range of glucose tolerance, *2*) to investigate the role of insulin sensitivity and insulin secretion in this relationship, *3*) to investigate the relationship between insulin sensitivity and adipose tissue mRNA expression of genes implicated in the catabolism of amino acids, and *4*) to investigate whether any of 43 risk single nucleotide polymorphisms (SNPs) for type 2 diabetes or hyperglycemia affect serum amino acid levels.\n\n# RESEARCH DESIGN AND METHODS\n\n## Subjects and clinical measurements.\n\nThe study included 9,369 nondiabetic or newly diagnosed type 2 diabetic men from the population-based Metabolic Syndrome in Men (METSIM) Study (mean \u00b1 SD age, 57 \u00b1 7 years; BMI, 27.0 \u00b1 4.0 kg\/m^2^). The study design has been described in detail elsewhere (27).\n\nGlucose tolerance was evaluated according to the American Diabetes Association criteria (28). A total of 3,026 subjects (32.3%) had normal glucose tolerance (NGT), 4,327 (46.2%) had isolated impaired fasting glucose (IFG), 312 (3.3%) had isolated impaired glucose tolerance (IGT), 1,058 (11.3%) had IFG and IGT, and 646 (6.9%) had newly diagnosed type 2 diabetes. Additional analyses were performed in 1,775 nondiabetic subjects re-examined during an ongoing follow-up METSIM study, of which 375 maintained NGT, 1,249 remained nondiabetic, and 151 developed new type 2 diabetes during the mean follow-up of 4.7 \u00b1 1.0 years. None of 9,369 subjects was receiving antidiabetic treatment. BMI was calculated as weight (kg) divided by height (m) squared. The study was approved by the ethics committee of the University of Kuopio and Kuopio University Hospital and conducted in accordance with the Helsinki Declaration.\n\n## Amino acid measurements.\n\nA high-throughput serum nuclear magnetic resonance (NMR) platform operating at 500 MHz was used for amino acid quantification (29). Fasting serum samples collected at the baseline study were stored at \u221280\u00b0C and thawed overnight in a refrigerator before sample preparation. Aliquots of each sample (300 \u03bcL) were mixed with sodium phosphate buffer (300 \u03bcL). A proton NMR spectrum was acquired where most spectral signals from the macromolecules and lipoprotein lipids were suppressed to enhance detection of the amino acid signals. The eight amino acids (alanine, phenylalanine, valine, leucine, isoleucine, tyrosine, histidine, glutamine) were quantified in standardized concentration units. Details of the NMR experimentation and amino acid quantification have been described previously (29,30).\n\n## Insulin sensitivity and insulin secretion indices.\n\nResults of oral glucose tolerance testing (OGTT) were used to calculate the Matsuda index of insulin sensitivity (ISI) as 10,000\/\u221a (fasting insulin \u00d7 fasting glucose \u00d7 mean insulin during OGTT \u00d7 mean glucose during OGTT) (31). An index of early-phase insulin secretion during an OGTT, insulin area under the curve (InsAUC)~0\u201330~\/glucose (Glu)AUC~0\u201330~, was calculated as (fasting insulin + 30-min insulin)\/(fasting glucose + 30-min glucose) (pmol\/mmol) (27).\n\n## Genotyping.\n\nGenotyping of 43 SNPs (29 SNPs associated with risk for type 2 diabetes and 14 associated with increased fasting or 2-h glucose in an OGTT) (23\u201326) was performed using the Applied Biosystems TaqMan Allelic Discrimination Assay at the University of Eastern Finland or the Sequenom iPlex Gold SBE assay at the National Human Genome Research Institute at the National Institutes of Health. The TaqMan genotyping call rate was 100%, and the discordance rate was 0% among 4.5% DNA samples genotyped in duplicate. The Sequenom iPlex call rate was 90.2\u201396.9%, and the discordance rate was 0% among 4.2% DNA samples genotyped in duplicate. All SNPs were in Hardy-Weinberg equilibrium at the significance level corrected for multiple testing by Bonferroni method (*P \\<* 0.0012). Descriptive data for individual SNPs are shown in [Supplementary Table 1](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db11-1378\/-\/DC1).\n\n## Gene expression analysis.\n\nTotal RNA was isolated from 200 subcutaneous fat biopsy samples of METSIM participants using Qiagen miRNeasy kit according to manufacturer's instructions. RNA integrity number values were assessed with the Agilent Bioanalyzer 2100. High-quality samples (RNA integrity number \\>7.0) were used for transcriptional profiling with the Illumina Human HT-12 v3 Expression BeadChip. Genome Studio software (2010.v3) was used for obtaining fluorescent intensities. The HT-12 BeadChip contains 48,804 expression and 786 control probes. Expression data from 19,306 probes were removed because of *1*) failure of the probe to align to a genomic or transcriptomic location; *2*) alignment of the probe to multiple genomic or transcriptomic locations; or *3*) presence of SNPs in the probe sequence that may affect hybridization efficiency using the methodology developed by Barbosa-Morais et al. (32). The remaining 29,497 probes were processed using nonparametric background correction, followed by quantile normalization with control and expression probes using the *neqc* function in the *limma* package (R v2.13.0) (33). The 16,223 probes with detection *P* values \\<0.01 in any of the 200 samples were used for further analysis. Gene expression data have been deposited to Gene Expression Omnibus (GEO) with the accession number GSE32512.\n\n## Statistical analysis.\n\nStatistical analyses were conducted using SPSS 17 software (SPSS, Chicago, IL). All amino acids, BMI, Matsuda ISI, and early-phase insulin secretion index (InsAUC~0\u201330~\/GluAUC~0\u201330~) were log-transformed to correct for their skewed distribution. Amino acids were compared across the fasting and 2-h glucose categories using the general linear model adjusted for age and BMI, or additionally for Matsuda ISI or InsAUC~0\u201330~\/GluAUC~0\u201330~ indices. *P* \\< 0.003 (corrected for 16 tests by Bonferroni method) was considered statistically significant. Associations between amino acid levels and indices of insulin sensitivity and insulin secretion were evaluated with Pearson correlation coefficients. The association of amino acid levels with newly developed type 2 diabetes was tested with logistic regression adjusted for confounding factors. Correlations between gene expression levels and phenotypes were calculated using the Pearson correlation coefficient. We used the Benjamini\u2013Hochberg false discovery rate (FDR) method (34) to correct for multiple comparisons, and considered an FDR-adjusted *P*~FDR~ \\< 0.05 statistically significant.\n\nFor genetic association analysis, unstandardized effect sizes (B \\[SE\\]) per copy of the minor allele were estimated by linear regression analysis adjusted for age and BMI, using untransformed dependent variables, and percentages of B from the mean were calculated. *P* values were calculated using logarithmically transformed variables when appropriate. A *P* \\< 1.45 \u00d7 10^\u22124^ adjusted for multiple comparisons by Bonferroni method was considered to be statistically significant given a total of 344 tests performed (8 traits \u00d7 43 SNPs). We had \u226580% power to detect changes in the mean trait value from 1.4 to 5.9% per copy of the minor allele at the significance level of 0.05, depending on the minor allele frequency ([Supplementary Fig. 1](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db11-1378\/-\/DC1)). Hardy-Weinberg equilibrium was evaluated by \u03c7^2^ test.\n\n# RESULTS\n\n## Hyperglycemia and the levels of eight amino acids.\n\nWe generated categories of fasting (FPG) and 2-h (2hPG) plasma glucose (by 0.5 and 1.0 mmol\/L steps, respectively) to investigate the relationship between amino acid levels and glycemia in participants with normoglycemia, IFG, IGT, and type 2 diabetes. Categories with FPG \\<5.0 mmol\/L and 2hPG \\<5.0 mmol\/L were set as the reference categories. Across the FPG categories, we observed a significant (*P* \\< 0.003) increase in isoleucine level of 38% in the highest glucose category versus the reference category (*P* = 6.7 \u00d7 10^\u221210^ adjusted for age and BMI), tyrosine (+21%, *P* = 1.8 \u00d7 10^\u221215^), alanine (+17%, *P* = 4.3 \u00d7 10^\u221242^), phenylalanine (+15%, *P* = 1.1 \u00d7 10^\u22129^), and leucine (+19%, *P* = 4.2 \u00d7 10^\u22124^) levels, and a significant decrease in histidine (\u22129%, *P* = 3.3 \u00d7 10^\u22124^) and glutamine (\u221222%, *P* = 1.9 \u00d7 10^\u221248^; <a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>A<\/em><\/a>). Similar trends were seen across the 2hPG categories, with a significant increase in isoleucine (+38%, *P* = 2.7 \u00d7 10^\u221258^), tyrosine (+30%, *P* = 1.1 \u00d7 10^\u221210^), alanine (+14%, *P* = 2.3 \u00d7 10^\u221246^), phenylalanine (+14%, *P* = 1.1 \u00d7 10^\u221213^), and leucine (+15%, *P* = 1.0 \u00d7 10^\u221218^) levels, and a significant decrease in histidine (\u22125%, *P* = 9.1 \u00d7 10^\u22126^) and glutamine (\u221212%, *P* = 1.4 \u00d7 10^\u221231^) levels with higher 2hPG (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>B<\/em><\/a>). These effects were more significant in obese (BMI \u226527 kg\/m^2^) than in nonobese (BMI\\<27 kg\/m^2^) participants, although the trends were similar in both groups ([Supplementary Fig. 2](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db11-1378\/-\/DC1)). Overall, most amino acids increased, whereas glutamine and histidine decreased with higher FPG and 2hPG.\n\n<a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a> shows that all amino acids that were increased in hyperglycemia negatively correlated with Matsuda ISI (*r* \u2264 \u22120.3, *P* \u2264 1 \u00d7 10^\u2212154^) and positively (*r* \u2265 0.2, *P* \u2264 3 \u00d7 10^\u221285^) with the InsAUC~0\u201330~\/GluAUC~0\u201330~ index of early-phase insulin secretion. For glutamine, which was decreased in hyperglycemia, the correlations were weaker and in the opposite direction (*r* = 0.17 for Matsuda ISI and \u22120.12 for InsAUC~0\u201330~\/GluAUC~0\u201330~). Correlations of amino acid levels (except for histidine) with InsAUC~0\u201330~\/GluAUC~0\u201330~ were weaker than correlations with Matsuda ISI and were largely attenuated after the adjustment of InsAUC~0\u201330~\/GluAUC~0\u201330~ for Matsuda ISI. Furthermore, additional adjustment for Matsuda ISI attenuated or abolished most of the associations between glucose categories and amino acid levels (with the exception of valine and histidine), whereas adjustment for InsAUC~0\u201330~\/GluAUC~0\u201330~ attenuated only the associations for histidine ([Supplementary Table 2](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db11-1378\/-\/DC1)). Thus, insulin sensitivity seemed to at least partly explain the relationship between glucose and amino acid levels.\n\nPearson correlations between the levels of eight amino acids and indices of insulin sensitivity (Matsuda ISI) and early-phase insulin secretion (InsAUC~0\u201330~\/GluAUC~0\u201330~) in nondiabetic METSIM participants\n\n![](1895tbl1)\n\n## Association of eight amino acid levels with risk of type 2 diabetes.\n\nWe investigated the relationship between amino acid levels and the development of type 2 diabetes during a 4.7-year follow-up, including 526 re-examined METSIM participants (375 with NGT at baseline and follow-up examinations, and 151 with type 2 diabetes). Of eight amino acids measured at baseline, high levels of alanine (*P* = 6.7 \u00d7 10^\u22125^), leucine (*P* = 0.005), isoleucine (*P* = 3.3 \u00d7 10^\u22125^), tyrosine (*P* = 0.001), and phenylalanine (*P* = 0.048) were significantly associated with incident diabetes (adjusted for age and BMI; <a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a>). Additional adjustment for Matsuda ISI alone, or Matsuda ISI and InsAUC~0\u201330~\/GluAUC~0\u201330~, but not for InsAUC~0\u201330~\/GluAUC~0\u201330~ alone, abolished statistical significances, suggesting that the association of these amino acids with incident diabetes was mostly explained by insulin resistance. Further adjustment for fasting hyperglycemia did not essentially change the association, but adjustment for 2-h glucose made the association for alanine statistically significant (*P* = 0.027). The elevated baseline glutamine level was significantly associated with a decreased risk of newly developed type 2 diabetes (*P* = 4.1 \u00d7 10^\u22126^), and this association persisted after the adjustment for Matsuda ISI (*P* = 1.5 \u00d7 10^\u22124^) or InsAUC~0\u201330~\/GluAUC~0\u201330~ (*P* = 6.7 \u00d7 10^\u22126^) or both (*P* = 0.048). Additional adjustment for fasting glucose abolished this association (*P* = 0.051). The levels of glutamine were significantly lower at baseline in 440 participants who developed abnormal glucose tolerance (IGT and\/or IFG, diabetes) at follow-up, compared with 375 participants who remained normoglycemic (155.6 vs. 159.6; *P* = 0.019, adjusted for age and BMI; [Supplementary Table 3](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db11-1378\/-\/DC1)). We also performed statistical analyses in a larger sample, including all 1,624 reexamined participants who did not develop diabetes during the follow-up and 151 participants who developed type 2 diabetes. The results remained essentially similar, although *P* values were less significant.\n\nAssociation between the baseline levels of eight amino acids and newly developed type 2 diabetes during the follow-up of the METSIM study participants (logistic regression adjusted for age, BMI, and additional covariates)\n\n![](1895tbl2)\n\n## Gene expression of genes involved in amino acid metabolism in relation to insulin sensitivity.\n\nAnalysis of microarray data from subcutaneous adipose tissue samples of 200 METSIM participants showed that Matsuda ISI correlated significantly with mRNA levels of several genes involved in the metabolism of alanine, including a key enzyme, alanine aminotransferase (*r* = 0.46, *P*~FDR~ = 9.7 \u00d7 10^\u22129^); glutamine, including a key enzyme, glutamine synthetase (*r* = 0.44, *P*~FDR~ = 4.0 \u00d7 10^\u22128^); BCAA, including key enzymes of BCAA degradation, branched chain amino-acid transaminase 2 (*r* = 0.36, *P*~FDR~ = 2.5 \u00d7 10^\u22125^) and branched-chain \u03b1-keto acid dehydrogenase (BCKDH) A and B (*r* = 0.45 and 0.35, *P*~FDR~ = 1.3 \u00d7 10^\u22128^ and 3.0 \u00d7 10^\u22125^); and phenylalanine, tyrosine, and histidine ([Supplementary Table 4](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db11-1378\/-\/DC1)). The most consistent results were observed for the BCAA degradation, as enzyme mRNAs correlated positively with Matsuda ISI at almost all steps of the metabolic pathway ([Supplementary Fig. 3](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db11-1378\/-\/DC1)). <a href=\"#F2\" data-ref-type=\"fig\">Figure 2<\/a> shows that the mRNA levels of three key enzymes in BCAA degradation also correlated negatively with ISI, FPG, and 2hFP levels. However, these associations disappeared when controlled for Matsuda ISI. These results indicate that the association between insulin sensitivity and amino acid catabolism (especially BCAA catabolism) also exists at the mRNA level and add further evidence that insulin sensitivity is likely contributing to higher BCAA levels in hyperglycemia.\n\n## Risk variants for type 2 diabetes and\/or hyperglycemia and the levels of eight amino acids.\n\nOf the 43 SNPs investigated, only *GCKR* rs780094 was significantly associated with the levels of several amino acids after Bonferroni correction for multiple testing (*P* \\< 1.45 \u00d7 10^\u22124^) (<a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<\/a> and [Supplementary Table 1](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db11-1378\/-\/DC1)). The glucose-increasing C (major) allele was associated with lower levels of alanine (effect size \u22121.9% per C allele, *P* = 1.6 \u00d7 10^\u221211^) and isoleucine (\u22122.3%, *P* = 3.1 \u00d7 10^\u22126^), and higher levels of glutamine (+1.2%, *P* = 1.0 \u00d7 10^\u22126^ adjusted for age and BMI). The C allele also had nominally significant effects on leucine (\u22121.2%, *P* = 0.001), tyrosine (+1.1%, *P* = 0.001), and histidine (+0.8%, *P* = 0.003). These associations did not change significantly after additional adjustment for FPG, 2hPG, or Matsuda ISI (data not shown), traits known to be modulated by SNPs of *GCKR*. Furthermore, the association of amino acids with newly developed type 2 diabetes was not affected by further adjustment for rs780094.\n\nA number of nominally significant associations of other SNPs with amino acids were found; the top ranking was rs8042680 (*PRC1*). The type 2 diabetes risk allele (A) was nominally associated with higher levels of leucine (+1.2%, *P* = 0.002), isoleucine (+1.7%, *P* = 0.001), and valine (+1.2%, *P* = 0.001; [Supplementary Table 1](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db11-1378\/-\/DC1)). The most consistent nominally significant associations were seen for rs75789326 (*IRS1*). The nonrisk allele (G) was associated with lower leucine (\u22120.8%, *P* = 0.019), isoleucine (\u22121.5%, *P* = 0.003), tyrosine (\u22120.7%, *P* = 0.037), valine (\u22120.7%, *P* = 0.047), and histidine (\u22120.5%, *P* = 0.039) levels.\n\n# DISCUSSION\n\nThis is the first large, population-based study aiming to investigate the relationship between hyperglycemia and 43 risk SNPs for type 2 diabetes\/hyperglycemia and amino acid levels. We observed that with increasing FPG and\/or 2hPG, the levels of alanine, valine, leucine, isoleucine, phenylalanine, and tyrosine increased, whereas the levels of histidine and glutamine decreased. Significant correlations between insulin sensitivity and mRNA expression of genes regulating amino acid metabolism were found. Only 1 SNP (rs780094 in *GCKR*) of 43 risk SNPs for type 2 diabetes or hyperglycemia was significantly associated with the levels of several amino acids.\n\n## Hyperglycemia and levels of eight amino acids.\n\nWe demonstrated that the levels of alanine, phenylalanine, valine, leucine, isoleucine, and tyrosine increased and the levels of histidine and glutamine decreased in hyperglycemia, indicating that the changes in amino acid levels parallel closely the changes in FPG and 2hPG levels. Similar trends were also observed in normoglycemia, suggesting that even mild elevations of glucose levels result in changes in amino acid levels. The largest and most significant increase across the FPG and 2hPG categories was observed for isoleucine (increased up to 38 and 30%, respectively, compared with the reference category), and alanine (increased up to 17 and 14%), and the largest decrease was seen for glutamine (by 22 and 12%, respectively). We also showed that obesity did not have a major effect on these associations, although the effects of hyperglycemia on the levels of amino acids tended to be more pronounced in obese individuals.\n\nThe levels of all amino acids correlated inversely (except for glutamine correlating positively) with insulin sensitivity (Matsuda ISI). The strongest correlation was found for isoleucine (*r* = \u22120.49), a BCAA previously shown to be a potent activator of glucose uptake in skeletal muscle (7,8). Insulin resistance is likely to be an important mechanism explaining these associations because the adjustment for Matsuda ISI attenuated or abolished most of the associations between glucose (especially 2hPG) and amino acid levels. Further evidence for the role of insulin resistance mediating the associations of amino acids with hyperglycemia comes from our 4.7-year prospective follow-up of the METSIM cohort. We demonstrated that alanine, leucine, isoleucine, tyrosine, and glutamine were associated with incident type 2 diabetes. Adjustment for insulin sensitivity abolished significant associations, with the exception of glutamine, indicating that insulin resistance is likely to play an important role in the risk of type 2 diabetes induced by amino acids. Insulin secretion or hyperglycemia did not significantly modify these associations. Our study not only supports the recent observations of a relationship between amino acid levels with the risk of diabetes (11) but also offers a possible underlying mechanism to explain this relationship.\n\nOur study found that the levels of BCAAs (particularly isoleucine) were strongly correlated with mRNA levels of genes involved in BCAA catabolism. BCAAs are thought to be early indicators of insulin resistance (6,10,18,35). High levels of BCAAs promote insulin resistance by interfering with the insulin-signaling pathway (10) or by directly inhibiting muscle glucose transport and\/or phosphorylation in skeletal muscle (9), whereas insulin resistance in turn may contribute to high levels of amino acids by increased release of BCAAs (and other amino acids) due to impaired ability of insulin to suppress proteolysis in skeletal muscle (36). BCAAs are oxidized in skeletal muscle and adipose tissue (37) to form alanine and glutamine (38). Oxidation of BCAAs was increased in individuals with type 2 diabetes (21). In animal models of type 2 diabetes, BCAA catabolism is downregulated due to low activity of BCKDH complex (39), the key enzyme of BCAA oxidation. In our study, adipose tissue mRNA expression of *BCKDHA* and *BCKDHB* (encoding \u03b1 and \u03b2 polypeptides of BCKDH E1) as well as of *BCAT2* (branched chain amino-acid transaminase 2 mitochondrial, the first enzyme in BCAA catabolism) and other genes involved in BCAA catabolism correlated positively with Matsuda ISI and negatively with glucose levels. This suggests that the insulin resistance-related decrease in degradation of BCAAs could be one of the mechanisms leading to the elevation of BCAA levels in hyperglycemia in humans. Other possible mechanisms for the elevation of BCAAs in hyperglycemia could be a decreased uptake and increased release of BCAAs from skeletal muscle due to increased protein catabolism in insulin resistance.\n\nAlanine is a nonessential amino acid synthesized from pyruvate and amino acids (mainly BCAAs) primarily in skeletal muscle and gut and used for gluconeogenesis in the liver. Alanine is the main precursor for gluconeogenesis in the liver (38,40) and a stimulator of glucagon secretion (41). Therefore, high plasma levels of alanine may contribute especially to fasting hyperglycemia by enhancing gluconeogenesis. Our study shows that elevated levels of alanine in hyperglycemia, both in obese and nonobese individuals, could be mediated by insulin resistance, which is supported by a significant positive correlation between insulin sensitivity and mRNA level of alanine aminotransferase, the key enzyme in alanine metabolism.\n\nGlutamine is a nonessential gluconeogenic amino acid synthesized from glucose or amino acids or released by proteolysis from skeletal muscle (42). Unlike alanine, glutamine is primarily taken up by the gut and kidneys, where it is processed to glucose via the gluconeogenesis pathway. Glucagon, but not insulin sensitivity, seems to play a role in the uptake of glutamine by the gut (43). Differential regulation of glutamine metabolism, compared with other amino acids, could possibly explain the opposite associations between glutamine and glucose metabolism. We found that plasma levels of glutamine decreased with elevated levels of FPG and 2hPG and that a low level of glutamine was significantly associated with future diabetes, independently of obesity and insulin resistance. Baseline glutamine levels were already decreased in normoglycemic participants who developed abnormal glucose tolerance at follow-up compared with those who remained normoglycemic. Our results are in agreement with previous studies showing that patients with type 2 diabetes (12) and lean insulin-resistant normoglycemic offspring of patients with type 2 diabetes have lower levels of glutamine than normoglycemic individuals (44).\n\n## Risk variants for type 2 diabetes and\/or hyperglycemia and the levels of eight amino acids.\n\nA common variant in *GCKR* was the only SNP among 43 loci associated with the levels of several amino acids. *GCKR* encodes the glucokinase regulatory protein, which inhibits the effects of glucokinase (GCK) on glycogen synthesis and glycolysis in the liver (45). SNPs at the *GCKR* locus have been associated with fasting glycemia (24,46), risk of type 2 diabetes (46,47), insulin resistance (46\u201348), and elevated hepatic glucose uptake (49). In our study, the glucose-increasing major C allele of the intronic SNP rs780094 was significantly and unexpectedly associated with decreased levels of alanine and isoleucine and elevated levels of glutamine. Mechanisms explaining the relationship of rs780094 of *GCKR* with amino acids are unknown, but the association with low levels of alanine could be a consequence of reduced glycolysis induced by *GCKR* resulting in decreased production of pyruvate and its conversion to alanine. On the basis of our study, it remains unclear whether the effects of rs780094 of *GCKR* on amino acid metabolism are primary or secondary. Although in our study the C allele of rs780094 was associated with both FPG and 2hPG, as well as with Matsuda ISI, the association of *GCKR* with the levels of amino acids was independent of these variables (data not shown). The *GCKR* variant did not affect the association of amino acids with newly developed type 2 diabetes, which agrees with its modest effect on amino acid levels.\n\nAlthough several type 2 diabetes\/hyperglycemia risk SNPs showed nominally significant associations with the levels of selected amino acids, the most consistent results were found for rs7578326 near *IRS1*. The major A allele previously linked to type 2 diabetes risk in a genome-wide association study (26) was nominally associated with elevated levels of several amino acids (valine, leucine, isoleucine, tyrosine, histidine). *IRS1* encodes the insulin receptor substrate 1, an important component of the insulin-signaling pathway, and its gene variant affects insulin resistance and adiposity (50). The association of *IRS1* with amino acid levels, if confirmed, could contribute to evidence for a causal relationship between insulin resistance and amino acid metabolism. The type 2 diabetes risk allele of the rs8042680 in *PRC1*, encoding protein regulator of cytokinesis 1, was associated specifically but nominally with higher levels of all three BCAAs. *PRC1* is not known to have a function in amino acid metabolism. These results suggest that the relationship between the levels of glucose and amino acids could be determined, at least in part, by genes regulating hyperglycemia or the risk of diabetes.\n\nThis study has limitations. Only Finnish men were included, and therefore, we do not know whether our results are applicable to women and to different ethnic or racial groups. We had only a modest statistical power to demonstrate statistically significant associations of gene variants with amino acids.\n\nIn conclusion, our large, population-based study shows that levels of branched-chain, aromatic amino acids and alanine increase whereas glutamine and histidine decrease with increasing FPG and\/or 2hPG levels. These associations seemed to be mediated by insulin resistance, at least in part, supported by correlations of expression of genes involved in amino acid metabolism with the Matsuda ISI. Among the 43 loci associated with risk for hyperglycemia or type 2 diabetes, only *GCKR* rs780094 was significantly associated with several amino acids, especially with the levels of alanine.\n\n## ACKNOWLEDGMENTS\n\nThis work was supported by the Academy of Finland (A.S., M.L., SALVE program to M.A.-K.), the Finnish Diabetes Research Foundation (M.L.), the Finnish Cardiovascular Research Foundation (M.L., M.A.-K.), the Jenny and Antti Wihuri Foundation (A.J.K.), an EVO grant from the Kuopio University Hospital (5263), DK062370 to M.B., 1Z01 HG000024 to F.S.C., and National Institutes of Health Grant HL-095056 to P.P. and Grant HL-28481 to P.P. and A.J.L.\n\nNo potential conflicts of interest relevant to this article were reported.\n\nA.S. wrote the manuscript and researched the data. M.C., N.K.S., P.P., and A.J.L. performed the mRNA experiments, analyzed the data, and reviewed and edited the manuscript. P.S. conceived, designed, and performed the NMR experiments, analyzed the data, and reviewed and edited the manuscript. A.J.K. analyzed the NMR data, contributed analysis tools, and reviewed and edited the manuscript. H.C., J.Pa., and J.Pi. researched the data and reviewed and edited the manuscript. L.L.B., M.A.M., and F.S.C. designed and performed genotyping and reviewed and edited the manuscript. M.B. contributed analysis tools and reviewed and edited the manuscript. J.K. designed the study and reviewed the manuscript. M.A.-K. conceived and designed the NMR experiments, analyzed the data, and reviewed and edited the manuscript. M.L. designed the study, contributed to discussion, and reviewed and edited the manuscript. M.L. is the guarantor of this work and, as such, had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.\n\n# REFERENCES","meta":{"dup_signals":{"dup_doc_count":150,"dup_dump_count":63,"dup_details":{"curated_sources":2,"2022-21":1,"2021-31":3,"2021-21":2,"2021-17":1,"2020-29":1,"2020-24":1,"2020-16":1,"2020-10":1,"2019-51":2,"2019-47":1,"2019-43":3,"2019-35":2,"2019-30":2,"2019-26":2,"2019-22":1,"2019-13":2,"2019-04":1,"2018-47":1,"2018-39":1,"2018-30":1,"2018-26":1,"2018-22":3,"2018-17":2,"2018-13":2,"2018-09":2,"2018-05":2,"2017-51":3,"2017-47":2,"2017-43":4,"2017-39":2,"2017-34":2,"2017-30":4,"2017-26":3,"2017-22":2,"2017-17":4,"2017-09":3,"2017-04":7,"2016-50":4,"2016-44":4,"2016-40":3,"2016-36":3,"2016-30":3,"2016-26":3,"2016-22":3,"2016-18":3,"2015-48":3,"2015-40":3,"2015-35":1,"2015-32":1,"2015-27":1,"2015-22":3,"2015-14":2,"2014-52":3,"2014-49":2,"2014-42":5,"2014-41":3,"2014-35":3,"2014-23":3,"2022-40":1,"2017-13":4,"2015-18":3,"2015-11":2,"2015-06":1}},"file":"PMC3379649"},"subset":"pubmed_central"}
{"text":"abstract: A report on the 13th International Congress on Yeasts (ICY), held at Madison, Wisconsin, USA, 26-30 August 2012.\nauthor: Daniela Delneri\ndate: 2012\ninstitute: 1Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PL, UK\ntitle: The yeast factory\n\nAlmost 400 scientists attended the 13th International Congress on Yeasts (ICY), where advances in yeast physiology, genomics, biotechnology, ecology and taxonomy were reported. In addition, the meeting celebrated the achievements accomplished with yeast in the past two decades, with the keynote speaker, Steve Oliver (University of Cambridge, UK), giving an overview of the scientific findings in yeast genomics, starting from the pioneering sequencing of *Saccharomyces cerevisiae* chromosome III in 1992 to the recent translational studies using yeast as a model to predict copy number variation phenotypes in human cancer cell lines. The plenary speaker Bernard Dujon (Institut Pasteur, France) also superbly summarized the history of comparative genomics with yeasts, the achievements of the G\u00e9nolevures consortium (<http:\/\/www.genolevures.org\/>) and the evolutionary insights gained in the hemiascomycetes lineage.\n\nHere, I highlight some contributions from three key themes of the meeting, including genomic diversity, comparative studies and evolution; biogeography; and taxonomy.\n\n# Genomic diversity, comparative analysis and evolution\n\n*S. cerevisiae* yeasts form diversified groups, usually defined by the source from which they were isolated. Justin Fay (Washington University, USA) analyzed a highly differentiated group of *S. cerevisiae* isolated from grapes to assess whether the level of phenotypic variation between natural and domesticated strains could be a consequence of limited gene flow between sympatric wild and vineyard strains. Chardonnay must (pressed grapes) was fermented with both oak tree isolates and wine strains of yeast, and differences in the aroma and flavor of the resulting wine were assessed by 51 human volunteers. Not only was the oak tree wine the least palatable, but genome comparative analysis also showed that the wild isolates were exchanging genes with vineyard strains, potentially contaminating wine production with off-flavors. Fay concluded there is no clear link between the genetic diversification of vineyard yeasts and domestication.\n\nUnderstanding the molecular basis of phenotypic differences among yeast strains may lead to the engineering of new strains of biotechnological relevance. Amparo Querol (IATA-CSIC, Valencia, Spain) presented her studies on differential expression profiles during wine fermentation of 29 yeast strains (14 fermentative yeasts and 15 wild isolates). The detection of differences in transcript levels between homologous genes from natural and wine strains can help the selection of properties that are desirable in enology (fermentation at low temperature, high production of glycerol and lower ethanol yield).\n\nComparative genomics studies have also explored the evolution and reconstruction of yeast carbon metabolism. Jure Piskur (Lund University, Sweden) investigated the origin of the Crabtree effect, which enables some species of yeast to produce ethanol, rather than biomass, at high sugar concentrations, by analyzing 40 different yeasts in the Saccharomycetes class, covering 200 million years of history. Interestingly, he found that species that originated before the whole genome duplication, such as *Lachancea* species, were 'Crabtree positive', producing as much ethanol as biomass. He concluded that the ability to produce ethanol from sugar is independent from the whole genome duplication, and originated about 120 million years ago, when the first modern flowering plants and fruits appeared on our planet.\n\nDawn Thompson (Broad Institute, USA) compared transcriptome data for 15 fungal species grown in a variety of conditions in order to study how regulation evolves and how network rewiring can lead to phenotypic adaptation. Growth rate, expression profiles, metabolites and nucleosome organization profiles were analyzed and compared to reconstruct molecular networks of ancestral and extant species.\n\nInterspecies hybridization is a source of phenotypic diversity for the colonization of new environments. Eladio Barrio (University of Valencia, Spain) analyzed the genomes of hybrids between *S. cerevisiae* and *Saccharomyces kudriavzevii*, focusing on mitochondrial inheritance. There was a tendency for the hybrids to lose the portion of the genome derived from *S. kudriavzevii*, but also to retain the mitochondria from this species. Phylogenetic reconstruction of mitochondria based on the cytochrome-c oxidase subunit II (*COX2*) sequence showed that mitochondrial DNA often recombines in a region between *COX2* and the *ORF1* sequence (a large ORF related to the group I introns), and that introgression from *S. paradoxus* is present.\n\nSerge Casaregola (CIRM-Levures, INRA, France) carried out a population genomics study of two species of the CTG clade (yeasts that translate CTG as serine instead of leucine), *Millerozyma farinosa* and *Debaryomyces hansenii*, and showed that several cryptic species, which could not be distinguished using ribosomal DNA analysis, were present. New hybrids, such as those between *M. farinosa* and *Millerozyma miso*, were also discovered. These hybrids seemed to undergo loss of heterozygosis readily during sporulation. Casaregola concluded that the diversity among yeasts of the CTG clade is wider than previously thought, with the coexistence of many cryptic species, haploids, heterozygote diploids and hybrids.\n\nYeast has long been used as model to study the phenotypic effects of copy number variation and mitochondrial diseases. Maitreya Dunham (University of Washington, USA) analyzed the origins and consequences of copy number variation in yeast strains grown in a chemostat under different nutritional limitations. By introducing a collection of barcoded plasmids containing a copy of every gene into the yeast cells, she was able to study the effect of duplications. She identified a set of genes whose copy number increase was beneficial to the cell in all the conditions tested, and others that were environment-dependent. Dunham also reported a new inverted tandem repeat structure that is inconsistent with the currently accepted mechanisms of DNA repair. She proposed a new model for origin-dependent inverted repeat amplification, suggesting that such tandem architecture can be caused by an error in the DNA replication fork.\n\nMonique Bolotin-Fukuhara (Universit\u00e9 Paris Sud, France) described the generation of a collection of yeast strains with mutations in mitochondrial transfer RNAs that mimicked human pathological diseases. She showed that the severity of the human phenotype was recapitulated in yeast, and that the nature of the mitochondrial sequence neighboring the engineered mutations affected the severity of the phenotype.\n\n# Biogeography\n\nSignificant advances on the ecology and natural distribution of *Saccharomyces* *sensu stricto* species were made recently. Jos\u00e9 Paulo Sampaio (Universidade Nova de Lisboa, Portugal), explained how the belief that *Saccharomyces* was a purely domesticated organism, living mainly in human-made environments such as fermenting grapes, has hampered the detection of truly ecological species for several years. Improvements in the isolation methodologies from natural substrates are gradually changing this situation, allowing the identification of several new yeast isolates. In fact, yeasts are widely distributed on oaks and on species of the closely related *Nothofagus* genus, found in Patagonia, the Philippines, South Australia and New Zealand. The cryotolerant species *Saccharomyces arboriculus*, *Saccharomyces eubayanus* and *Saccharomyces uvarum* have all been isolated from *Nothofagus* species. Sampaio's study of *S. uvarum* biogeography has shown the existence of three different populations, found in Europe, South America and Australasia, respectively. Interestingly, the Australasian population is highly divergent, and hybrid crosses of Australasian strains with those from the South American populations have as little as 36% spore viability. Although *Saccharomyces paradoxus* and *S. cerevisiae* seem to have a global distribution, other lineages such as *S. kudriavzevii* and *Saccharomyces mikatae* were isolated from restricted geographical areas.\n\nChristopher Hittinger (University of Wisconsin, USA) reported on the discovery and the genome analysis of *S. eubayanus* (isolated from *Nothofagus* sp. in Patagonia), the missing parental contributor to the lager-brewing yeast *Saccharomyces pastorianus*. A total of 200 *S. eubayanus* and *S. uvarum* strains were isolated in Argentina and were sequenced to determine the genetic diversity and population structure of this species. Interestingly, Hittinger pointed out that both *S. eubayanus* and *S. uvarum* have retained some parts of the RNA interference machinery in their genomes, such as the *Dicer 1* (*DCR1*) gene, which are lost in the other *Saccharomyces sensu stricto* species. Hittinger also mentioned the *Saccharomyces sensu stricto* consortium (SSS), which includes improved and assembled genome sequences for these yeasts (<http:\/\/www.saccharomycessensustricto.org>).\n\n# Taxonomy\n\nThe theme of taxonomy was an important part of the meeting given the rapid and increasing number of newly discovered yeast species. In his plenary lecture, Teun Boekhout (CBS-KNAW, Utrecht, The Netherlands) called for a revision of the phylogenetic classification not only of the basidiomycetous yeasts, but also of the highly polyphyletic *Candida* genus, in which the majority of species are harmless and have no relation to the human pathogen *Candida albicans*. The systematics of yeast species taxonomy should be carried out using refined multigene phylogenies or whole genome analysis, because the morphological and physiological criteria are often misleading, conflicting with the gene tree. It follows that there is a need for whole genome sequencing projects at the species level, building on from the existing genomic characterization of several strains belonging to *S. cerevisiae* and *S. paradoxus* species (<http:\/\/www.sanger.ac.uk\/research\/projects\/genomeinformatics\/sgrp.html>). Boekhout also discussed his large-scale effort to determine unusual phenotypes in less explored non-conventional yeasts that may be of industrial relevance (the EU Cornucopia project).\n\nVincent Robert (CBS-KNAW, Utrecht, the Netherlands) gave an update on the DNA barcoding of all the 9,000 strains in the CBS collection by sequencing the D1\/D2 and ITS region of the ribosomal DNA. This barcode method is useful to revise identifications and select strains for further exploration. Moreover, MALDI-TOF (matrix-assisted laser desorption\/ionization time-of-flight mass spectrometry) techniques, based on differential peptide fingerprinting patterns, are being developed and applied for strain classification and quality control analysis of the CBS collection.\n\nThe recent discovery of the new species *S. eubayanus*, one of the original parents of *S. pastorianus*, necessitated a revision of the nomenclature and phylogeny of the *sensu strictu* species, with the old *Saccharomyces bayanus* var. *uvarum* becoming a recognized species called *S. uvarum*, and the old *S. bayanus* var. *bayanus* becoming *S. bayanus*, presenting a hybrid genome from different strains and species belonging to *S. uvarum*, *S. eubayanus* and *S. cerevisiae*. Huu-Vang Nguyen (INRA, AgroParisTech, Thiverval-Grignon, France) showed that a small number of specific markers (*NTS2*, *MAL31*, *MEL1*, *MTY1*) are sufficient to discriminate between pure lines of *S. uvarum* and *S. eubayanus* and the hybrids *S. bayanus* and *S. cerevisiae*. By PCR and restriction fragment length polymorphism analysis, he was able to reclassify the *S. bayanus* strains CBS 424, CBS 425 and CBS 3008, showing that they were inbred lines of *S. uvarum* and *S. eubayanus*, and that they did not contain the *S. cerevisiae* portion of the genome usually present in *S. bayanus* strains. This method is a quick and reliable approach to differentiating between different hybrids and species belonging to the *Saccharomyces* *sensu stricto* group.\n\nIn conclusion, the ICY meeting showed that there is an exciting future for yeast research, with an ever expanding repertoire of species and strains to study and set of genomics tools with which to do so.","meta":{"dup_signals":{"dup_doc_count":127,"dup_dump_count":52,"dup_details":{"curated_sources":2,"2023-23":1,"2022-27":1,"2021-49":1,"2020-24":1,"2019-30":1,"2019-22":1,"2019-09":1,"2018-51":2,"2018-39":2,"2018-30":1,"2018-22":1,"2018-09":1,"2018-05":1,"2017-51":1,"2017-47":1,"2017-43":1,"2017-39":1,"2017-34":1,"2017-30":1,"2017-26":1,"2017-22":1,"2017-17":1,"2017-09":11,"2017-04":1,"2016-50":1,"2016-44":1,"2016-36":9,"2016-30":8,"2016-22":1,"2016-18":1,"2016-07":5,"2015-48":3,"2015-40":2,"2015-35":3,"2015-32":3,"2015-27":3,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":3,"2014-42":5,"2014-41":5,"2014-35":5,"2014-23":3,"2014-15":3,"2023-40":1,"2015-18":3,"2015-11":3,"2015-06":3,"2014-10":3,"2013-48":2,"2017-13":1}},"file":"PMC3491391"},"subset":"pubmed_central"}
{"text":"abstract: Recent advances in shotgun sequencing and computational methods for genome assembly have advanced the field of metagenomics, the culture-independent cloning and analysis of microbial DNA extracted directly from an environmental sample, to provide glimpses into the life of uncultured microorganisms.\nauthor: Patrick D Schloss; Jo Handelsman\ndate: 2005\ninstitute: 1Department of Plant Pathology, University of Wisconsin, Madison, WI 53706, USA\nreferences:\ntitle: Metagenomics for studying unculturable microorganisms: cutting the Gordian knot\n\nThe estimate that fewer than 1% of the prokaryotes in most environments can be cultivated in isolation \\[1\\] has produced a quandary: what is the significance of the field of modern microbial genomics if it is limited to culturable organisms? Until recently, this limitation meant that the genomes of most microbial life could not be dissected because more than half of the known bacterial phyla contain no cultured representatives, and the archaeal kingdoms are likewise dominated by uncultured members. The problem can be likened to the Gordian knot of Greek legend, which was impossible to unravel. The knot, which was constructed with interwoven strands with no ends exposed, served as a source of great pride of the citizens of Gordium where it was displayed. It was Alexander the Great who finally cut the massive knot and called the act his greatest victory. One strategy to expose the rest of the microbial world to the eye of the microbiologist - analogous to attempting to untie the knot - is to coax more bacteria into pure culture. The alternative approach - which could cut through it as Alexander the Great did - is metagenomics.\n\nMetagenomics is the culture-independent analysis of a mixture of microbial genomes (termed the metagenome) using an approach based either on expression or on sequencing \\[2,3\\]. Recent studies in the Sargasso Sea \\[4\\], acid mine drainage \\[5\\], soil \\[6\\], and sunken whale skeletons \\[6\\] have used the shotgun-sequencing approach to sample the genomic content of these varied environments. In each study, environmental samples were obtained and the microbial DNA was extracted directly from the sample, sheared, cloned into *Escherichia coli*, and random clones were sequenced. In some of the studies sequence overlaps were then used to assemble contigs or scaffolds of genomic sequence. The Sargasso Sea study \\[4\\] resulted in nearly 2,000,000 random sequence reads, a massive total \\[7\\]; the acid-mine-drainage community sequence, more modest in size but impressive in the analytical insights gained, was based on 100,000 sequence reads (Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>). Assembling so many sequence reads, while simultaneously accounting for heterogeneities between genomes, introduced unique challenges for each study. In the hyper-diverse soil metagenomic sequencing project, fewer than 1% of the 150,000 sequence reads could be assigned to a contig \\[6\\], whereas the acid-mine-drainage sequencing project successfully assigned 85% of the sequence reads to one of 1,183 scaffolds \\[5\\]. The genome sequences of uncultured microorganisms residing in mixed communities can now realistically be determined.\n\n# A simple oceanic community\n\nThe most extensive metagenomic sequencing effort has been the attempt by Venter *et al.* \\[4\\] to sequence the prokaryotic genomes in the water of the Sargasso Sea, a well characterized region of the Atlantic near Bermuda that has unusually low nutrient levels; this study has already spawned numerous other meta-analyses (for example, \\[8-11\\]). Among one billion nucleotides of sequenced DNA, Venter *et al.* \\[4\\] identified more than 1.2 million open reading frames (ORFs), including 782 that had significant similarity to rhodopsin-like proteins. This was a surprise because the rhodopsins were previously thought to be present in only a small group of organisms, and the Sargasso Sea study broadened the spectrum of species known to have them. One intriguing problem in metagenomics is that most ORFs cannot be assigned to gene families of known function \\[2\\]. In the Sargasso Sea sequences, for instance, 69% of the ORFs had no known function \\[4\\]. This analysis points to a major limitation in annotating sequences from uncultured microorganisms: if no relative of the organism being sequenced has ever been sequenced, then the likelihood of matching each of the newly identified genes to genes of known function is low. The choice of database used for comparison determines the answer, as demonstrated by the identification by Venter *et al.* \\[4\\] of 16S rRNA sequences from the Sargasso Sea by querying a database containing only 16S rRNA gene sequences from genome sequences of Bacteria and Archaea. As they limited the comparative database to cultured microorganisms, it was not surprising that they did not identify any 16S rRNA gene fragments from any phyla with no cultured representatives. A further limitation of this study was presented by Delong \\[12\\], who pointed out that the two genomes that Venter *et al.* \\[4\\] were able to complete were probably contaminants in the sea-water sample. Obvious examples of assembly error (for example, contigs containing bacterial 5S and 23S rRNA genes adjacent to an archaeal 16S rRNA gene) suggest an insidious assembly problem throughout the sequence collection \\[12\\]. Perhaps the next stage of the project will profit from the mistakes of this 'pilot' sequencing attempt \\[4\\].\n\n# An even simpler biofilm community\n\nAlthough the nutrient-limited Sargasso Sea was selected for metagenomics because it was thought to contain a simple community \\[4\\], the community was not simple enough to allow assembly of most of the sequence reads into contigs. Tyson *et al.* \\[5\\] selected a far simpler community, that of a biofilm found in the very acidic waste water from an iron mine (termed acid mine drainage), which contains three bacterial and three archaeal lineages. By grouping the assembled contigs into 'bins' according to their GC content and the number of reads per contig, they were able to assign each bin to an organism. The near-complete genome sequences (ten-fold coverage) of *Ferroplasma* type II and *Leptospirillum* group II members enabled Tyson *et al.* \\[5\\] conceptually to model the metabolic processes that each genome contributes to the broader community.\n\nThis thorough sequencing and metabolic analysis provided the starting point for a 'proteogenomic' analysis. Protein was extracted from biofilms found in the acid mine drainage and digested with trypsin \\[13\\]. Applying shotgun mass spectrometry to the fragmented proteins, Ram *et al.* \\[13\\] obtained a sequence of part of the proteome. By combining the proteome and metagenome sequences \\[5\\], they linked one or more peptide sequences to approximately 49% of the ORFs from the five dominant genomes \\[13\\]. The most powerful outcome of this analysis was the identification from the *Leptospirillum* group II sequences of a novel acid-stable iron-oxidizing c-type cytochrome with an adsorption maximum wavelength at 579 nm (Cyt~579~). Cyt~579~ is the primary iron-oxidizing enzyme in the microbial community and mediates the rate-limiting step in acid production. In this relatively simple community, the proteogenomic approach enabled Ram *et al.* \\[13\\] to quantify protein production from each ORF, validate the DNA-derived metabolic model, and identify a process that potentially acts as a keystone for the whole ecosystem.\n\n# First metagenomic analyses of complex microbial communities\n\nA fundamental challenge in understanding microbial communities is to chronicle genetic conservation across time and location and to delineate the smallest complement of genes conserved in genomes across different communities \\[4\\]. Tringe *et al.* \\[6\\] tackled this problem by sequencing microbial communities sampled both from soil from a Minnesota farm and from three deep-sea communities living on sunken whale skeletons ('whale-fall') and comparing them with the Sargasso Sea sequence collection. ORFs from each metagenomic sequence were assigned to clusters of orthologous genes (COGs \\[14\\]), operons, pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) \\[15\\], and COG functional categories \\[14\\].\n\nThe relative enrichment found using each of the four annotation methods between the Sargasso Sea, deep-sea whale fall, and Minnesota farm soil was then determined, resulting, in essence, in an *in silico* subtractive hybridization. The over-representation of rhodopsin ORFs in the Sargasso Sea and ORFs encoding cellobiose phosphorylase in the Minnesota farm soil make biological sense, because marine microorganisms are more likely to use light-driven energy transduction systems and soil microorganisms are more likely to encounter plant-derived oligosaccharides such as cellobiose. The large number of ORFs of no known function that were over-represented in each community may indicate as-yet unknown functional systems. Generating copious sequence information from a community is intrinsically valuable, but this comparative analysis \\[6\\] is a worthy example of how metagenomics may move beyond descriptive, annotation-based analyses toward meaningful inference about ecological phenomena.\n\n# Dealing with complexity and contamination\n\nApplication of molecular biology methods to cultured organisms has led to striking insights into the life of microbes in mono-species culture. But genomics has failed to elucidate the functions of microbial communities, where most microorganisms on Earth spend most of their time and that provide the platform from microorganisms shape plant, animal, environmental and human health. Metagenomics, coupled with gene arrays, proteomics, expression-based analyses, and microscopy, will give insights into problems such as genome evolution and the membership of particular niches that are currently hindered by our inability to culture most microorganisms in pure culture \\[16\\]. To realize the full potential of metagenomics, however, a number of obstacles need to be overcome. Perhaps the most significant of these is the microbial complexity in most communities. The successful analysis of the acid mine drainage community was predicated on its simplicity. In contrast, the Minnesota farm soil probably contains more than 5,000 species and 10^4^-10^5^ strains, making it inevitable that the over 150,000 sequence reads could not be assembled into contigs \\[6\\] (Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>). It is likely that 2-5 gigabase-pairs of sequence are necessary to obtain eight-fold coverage of the dominant species in the community, suggesting that inventive approaches are needed to enrich DNA sequences from less abundant organisms or from members that are unique to a community \\[3\\].\n\nAnother focus for improvement in metagenomics is the use of robust sampling and DNA-extraction procedures. Methodology that guards against contamination such as that revealed in the Sargasso Sea samples is essential. Making the metagenomic studies ecologically meaningful will require sampling strategies that account for spatial and temporal variability, thereby enabling comparisons between communities \\[17\\]. These comparisons will also require standardized and aggressive methods for extracting DNA. It is unfortunate that all of the large metagenomic sequencing projects used chemical extraction methods to obtain DNA, whereas the technique of 'bead beating', which applies high shear forces to cells, is more effective than chemical lysis methods at breaking tough cells (for example, \\[18\\]). The studies that used chemical lysis methods therefore include DNA from only a subset of the organisms that can be accessed by modern methods.\n\nThis is an exciting time for metagenomics, as many projects are underway to sequence the metagenomes of biologically interesting environments. The US Joint Genome Institute (JGI) has essentially sequenced the metagenomes of the microbial communities associated with two extinct ancient cave bears, which contained less than 2 and 6% cave bear DNA, respectively \\[19\\]. The JGI is also currently sequencing metagenomic DNA for more than ten studies through their scientific Community Sequencing Program \\[20\\], and the J. Craig Venter Foundation is sequencing the metagenomes of samples taken along a path intended to simulate the voyage of Darwin's ship *The Beagle*, as well as samples of New York City's air \\[21\\]. A future prospect is completing the human genome by sequencing the metagenome of the 10^12^ microbial cells that are associated with the human body \\[22\\]. Each of these studies will unearth secrets unique to the environment being examined, and comparison of results of these studies will provide a meta-understanding of the recurrent and unique themes in community structure and function.\n\n## Figures and Tables\n\nSummary of metagenomic sequencing projects\n\n| Community | Estimated species richness | Thousands of sequence reads | Total DNA sequenced (Mbp) | Sequence reads in contigs (%) |\n|----|----|----|----|----|\n| Acid mine drainage | 6 | 100 | 76 | 85 |\n|  |  |  |  |  |\n| Deep sea whale fall |  |  |  |  |\n| Sample 1 | 150 | 38 | 25 | 43 |\n| Sample 2 | 50 | 38 | 25 | 32 |\n| Sample 3 | 20 | 40 | 25 | 47 |\n|  |  |  |  |  |\n| Sargasso Sea |  |  |  |  |\n| Samples 1-4 | 300 per sample | 1,662 | 1,361 | 61 |\n| Sample 5-7 | 300 per sample | 325 | 265 | \\<1 |\n|  |  |  |  |  |\n| Minnesota farm soil | \\>3,000 | 150 | 100 | \\<1 |","meta":{"dup_signals":{"dup_doc_count":133,"dup_dump_count":54,"dup_details":{"curated_sources":2,"2023-23":1,"2022-27":1,"2021-43":1,"2021-17":1,"2021-10":2,"2021-04":2,"2020-45":1,"2020-29":1,"2020-10":1,"2019-47":1,"2019-35":1,"2019-26":1,"2019-22":1,"2019-09":1,"2018-51":1,"2018-43":1,"2018-34":1,"2018-22":1,"2018-05":1,"2017-39":1,"2017-34":1,"2017-26":1,"2017-17":1,"2017-09":9,"2016-44":1,"2016-40":1,"2016-36":9,"2016-30":9,"2016-22":1,"2016-18":1,"2016-07":2,"2015-48":3,"2015-40":1,"2015-35":3,"2015-32":3,"2015-27":3,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":6,"2014-42":9,"2014-41":5,"2014-35":5,"2014-23":3,"2014-15":3,"2023-40":1,"2017-13":1,"2015-18":3,"2015-11":3,"2015-06":3,"2014-10":3,"2013-48":3,"2013-20":2,"2024-18":1}},"file":"PMC1273625"},"subset":"pubmed_central"}
{"text":"abstract: We need to familiarize ourselves with the facts of evolution, so that we can mount a spirited defense against creationism and the forces of ignorance.\nauthor: Gregory A Petsko\ndate: 2008\ninstitute: 1Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02454-9110, USA.\ntitle: It is alive\n\nThey're at it again. Armed with another new idea from the Discovery Institute, that bastion of ignorance, right-wing political ideology, and pseudo-scientific claptrap, the creationist movement has mounted yet another assault on science. This time it comes in two flavors: propaganda and legislative.\n\nThe propaganda is in the form of a poorly written, badly acted movie produced by Ben Stein, an attorney and entertainment figure who once served as a speechwriter for US Presidents Gerald Ford and Richard Nixon. As if working for Nixon didn't do enough to demonstrate his faulty judgment, he has become an ardent critic of evolution and an advocate for 'intelligent design', which is creationism poorly disguised as 'science'. He co-wrote and stars in the film *Expelled: No Intelligence Allowed*, which attempts to link evolution to the eugenics movement in Nazi Germany and to the Holocaust, and portrays advocates of intelligent design as champions of academic freedom and victims of discrimination by the scientific community. The famous evolutionary biologist and atheist Richard Dawkins has a spirited attack on the film on his website <http:\/\/richarddawkins.net>, and there's also a lively critique from the National Center for Science Education <http:\/\/www.expelledexposed.com>.\n\nFortunately, the film is sinking faster than the *Lusitania*. As far as I can discover, it has grossed less than US\\$8 million in ticket sales to date, far less than its cost, and is playing to virtually empty houses in the few theaters that are still showing it. Whether this is because people recognize its ideas as rubbish or because it is simply a bad movie, I don't know. So we can probably ignore it, as it so richly deserves. But the legislative attack is much more serious.\n\nOn 11 June 2008, the Louisiana House of Representatives voted 94:3 in favor of a bill that would promote 'critical thinking' by students on topics such as evolution, the origins of life, global warming, and human cloning. The Louisiana Senate already passed a similar bill, Senate Bill 733, by a vote of 35:0, but an amendment adopted by the House, which would allow the state Board of Elementary and Secondary Education to prohibit supplemental materials it deems inappropriate, means that the Senate must pass the bill again. If they do, and this seems a certainty, then the bill will be sent to Louisiana governor Bobby Jindal, at 36 the youngest governor in the United States and the first Indian-American to serve as the head of a state government. A former Hindu who converted to Catholicism in high school, Jindal attended Oxford University on a Rhodes Scholarship. Jindal was a biology major at Brown University, so he should understand the science at stake here, but he opposes stem-cell research and has publicly supported the teaching of 'intelligent design' in public schools. He has not stated whether or not he will sign Bill 733. A fascinating subtext to this story is that Jindal is reportedly under consideration by Republican presidential nominee John McCain as a possible vice-presidential nominee.\n\nThe bill is cleverly worded: it states in section 1C that it \"shall not be construed to promote any religious doctrine, promote discrimination for or against a particular set of religious beliefs, or promote discrimination for or against religion or nonreligion.\" In an interview with the conservative newspaper *The Washington Times* (12 June 2008), Jason Stern, vice-president of the Louisiana Family Forum, a Christian right-wing lobby group, insisted \"It's not about a certain viewpoint. It's allowing \\[teachers\\] to teach the controversy.\"\n\nLet me say this as clearly as possible, so there can be no mistake about what I mean: there is no controversy. Just because a few misguided so-called scientists question the validity of the concept of evolution doesn't mean there is a controversy. There are still some people who believe the Earth is flat (there's even a 'Flat Earth Society'), but that doesn't mean that a grade-school science teacher should teach his or her students that the Earth might be flat. The fact that some people believe nonsense does not give that nonsense scientific validity. A challenge to existing scientific principles must be based on evidence, not on belief, and there isn't a shred of evidence to support either creationism or intelligent design. Those ideas belong in a religion or philosophy class, not in a science class.\n\nBy the way, speaking of religion class, if we accept the creationists' own rationale for this bill, then shouldn't right-wing fundamentalist Christian schools be forced to 'teach the controversy' about religion? It's a much more controversial subject than science. Shouldn't their students be forced to consider the possibility that there is no God, or that the Muslim faith, or the Hindu faith, or the Jewish faith might be the true one? Or that there are so many different translations and versions of the Bible that there is no way of knowing which one is the 'word of God'? You can see how quickly their argument breaks down.\n\nWhat about the academic freedom argument? If someone wants to teach creationism in a science class, shouldn't they have the right to do so? Certainly - if they want to get fired. Because if they do that they deserve to get fired. It has nothing to do with academic freedom; it's about basic competence. Consider, for example, a science teacher who taught that the Sun revolves around the Earth. Even the intelligent-design advocates would probably have to admit that such a science teacher was incompetent and ought to be dismissed. That teacher might counter with a claim that his or her academic freedom was being infringed, but no court would uphold it, any more than a court would uphold a similar claim from a history teacher who taught that the Allies lost World War 2 or that Napoleon Bonaparte was emperor of Japan. Science, and history, may welcome speculation, but the speculation must be based on facts, and when it isn't, then it doesn't belong in that subject. Any 'science' teacher who teaches that the Earth might have been created about 6,000 years ago and that all the material evidence that it's billions of years old is controversial is simply incompetent. If the state of Louisiana wants its children taught by such people then they deserve the kind of workforce and citizenry they are going to get.\n\nIt's worth pointing out that in 1987, in the case of Edwards *versus* Aguillard, the US Supreme Court ruled as unconstitutional the idea of equal time for \"creation science\" and evolution in biology classes. That precedent will almost certainly be used as the basis for a constitutional challenge to the Louisiana law if it passes. Also, in the state of Pennsylvania, the 'Kitzmiller *versus* Dover' case in 2005 put to rest the idea of intelligent design as an alternative to evolution being taught in biology classes - the judge there, in a brilliantly reasoned opinion, demonstrated that intelligent design was just creationism by another name. Although not a Supreme Court case, this decision was strong enough to cause creation science advocates to switch tactics to arguments about academic freedom, the focus of the current legislation at issue in Louisiana.\n\nLest you think this is merely some Bible Belt aberration, let me assure you that the creationists are marshalling this argument in other states as well. In Michigan, Senate Bill 1361, introduced in the Michigan Senate on 3 June 2008, and referred to the Senate Committee on Education, is yet another 'academic freedom' bill aimed squarely at the teaching of evolution. Identical to Michigan House Bill 6027, which is still in the House Committee on Education, Senate Bill 1361 would, if enacted, require state and local administrators \"to create an environment within public elementary and secondary schools that encourages pupils to explore scientific questions, learn about scientific evidence, develop critical thinking skills, and respond appropriately and respectfully to differences of opinion about controversial issues\" and \"to assist teachers to find more effective ways to present the science curriculum in instances where that curriculum addresses scientific controversies\" by allowing them \"to help pupils understand, analyze, critique, and review in an objective manner the scientific strengths and scientific weaknesses of existing scientific theories pertinent to the course being taught.\" And in Texas (why is it not a shock that the state that gave us George W Bush would show up here), the Texas State Board of Education is again considering mandating a science curriculum that teaches the \"strengths and weaknesses\" of evolution. On 7 June 2008, the Houston Chronicle wrote that \"strengths and weaknesses\" language is \"a 'teach the controversy' approach, whereby religion is propounded under the guise of scientific inquiry\". The editorial went on to say: \"What students really need is to be able to study science from materials that have not been hijacked by creationists whose personal agenda includes muddying the science curriculum. Creationism is not a 'system of science'.\"\n\nAs scientists, we need to protest with our feet and our wallets. I am about to become the president of the American Society for Biochemistry and Molecular Biology, a scientific society with about 12,000 members. Our 2009 annual meeting is scheduled to take place in New Orleans. If Bill 733 becomes law in Louisiana, it will be too late to move the meeting to another state. But we need to see to it that no future meeting of our society will take place in Louisiana as long as that law stands, nor should we hold it in any other state (are you listening, Michigan and Texas?) that passes a similar law. I call upon the presidents of the American Chemical Society, the American Association of Immunologists, the Society for Neuroscience, and all the other scientific societies in the US and around the world to join me in this action and make clear to the state legislators in Louisiana, the governor of the state, and the mayor and business bureau of New Orleans that this will be the consequence. You can do the same. Governor Jindal can be reached through his website <http:\/\/www.bobbyjindal.com> and Ray Nagin, mayor of New Orleans, can be reached through the Mayor's office <http:\/\/www.cityofno.com\/Portals\/Portal35\/portal.aspx>.\n\nIn its ability to rise again just when we think we've got it licked, creationism is like Frankenstein's monster. \"Come see, villagers! It is alive!\" We'll never be rid of it by being silent and doing nothing, so one important thing is to force governments that ally themselves with this monster to pay for their folly by denying them our business. In addition, we must all arm ourselves with the one weapon we have that, in the end, the monster cannot overcome: the truth. All of us need to familiarize ourselves with the facts of evolution so that we can mount a spirited defense against the forces of ignorance and the charlatans who would exploit human insecurity and need for certainty.\n\nCarl Sagan memorably called science \"a candle in the dark\". Well, the darkness is always around us, closer than you think sometimes. Yes, it is alive. Creationism's alive because some of our fellow men and women keep it alive. In the dark.","meta":{"dup_signals":{"dup_doc_count":119,"dup_dump_count":47,"dup_details":{"curated_sources":2,"2020-29":1,"2020-16":1,"2019-47":1,"2019-43":1,"2019-35":1,"2019-26":1,"2019-13":1,"2019-04":1,"2018-51":1,"2018-47":1,"2018-43":1,"2018-39":1,"2018-30":1,"2018-26":1,"2018-09":1,"2017-43":1,"2017-22":1,"2017-09":7,"2016-44":1,"2016-36":8,"2016-30":7,"2016-22":1,"2016-18":1,"2016-07":4,"2015-48":3,"2015-40":3,"2015-35":3,"2015-32":3,"2015-27":3,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":3,"2014-42":8,"2014-41":3,"2014-35":5,"2014-23":4,"2014-15":5,"2022-27":1,"2024-10":1,"2015-18":3,"2015-11":2,"2015-06":3,"2014-10":2,"2013-48":3,"2013-20":3,"2024-26":1}},"file":"PMC2481410"},"subset":"pubmed_central"}
{"text":"abstract: In face of the multiple controversies surrounding the DSM process in general and the development of DSM-5 in particular, we have organized a discussion around what we consider six essential questions in further work on the DSM. The six questions involve: 1) the nature of a mental disorder; 2) the definition of mental disorder; 3) the issue of whether, in the current state of psychiatric science, DSM-5 should assume a cautious, conservative posture or an assertive, transformative posture; 4) the role of pragmatic considerations in the construction of DSM-5; 5) the issue of utility of the DSM - whether DSM-III and IV have been designed more for clinicians or researchers, and how this conflict should be dealt with in the new manual; and 6) the possibility and advisability, given all the problems with DSM-III and IV, of designing a different diagnostic system. Part I of this article will take up the first two questions. With the first question, invited commentators express a range of opinion regarding the nature of psychiatric disorders, loosely divided into a realist position that the diagnostic categories represent real diseases that we can accurately name and know with our perceptual abilities, a middle, nominalist position that psychiatric disorders do exist in the real world but that our diagnostic categories are constructs that may or may not accurately represent the disorders out there, and finally a purely constructivist position that the diagnostic categories are simply constructs with no evidence of psychiatric disorders in the real world. The second question again offers a range of opinion as to how we should define a mental or psychiatric disorder, including the possibility that we should not try to formulate a definition. The general introduction, as well as the introductions and conclusions for the specific questions, are written by James Phillips, and the responses to commentaries are written by Allen Frances.\nauthor: James Phillips; Allen Frances; Michael A Cerullo; John Chardavoyne; Hannah S Decker; Michael B First; Nassir Ghaemi; Gary Greenberg; Andrew C Hinderliter; Warren A Kinghorn; Steven G LoBello; Elliott B Martin; Aaron L Mishara; Joel Paris; Joseph M Pierre; Ronald W Pies; Harold A Pincus; Douglas Porter; Claire Pouncey; Michael A Schwartz; Thomas Szasz; Jerome C Wakefield; G Scott Waterman; Owen Whooley; Peter Zachar\ndate: 2012\ninstitute: 1Department of Psychiatry, Yale School of Medicine, 300 George St., Suite 901, New Haven, CT 06511, USA; 2Department of Psychiatry and Behavioral Sciences, Duke University Medical Center, 508 Fulton St., Durham, NC 27710, USA; 3Department of Psychiatry and Behavioral Neuroscience, University of Cincinnati College of Medicine, 260 Stetson Street, Suite 3200, Cincinnati, OH 45219, USA; 4Department of History, University of Houston, 524 Agnes Arnold, Houston, 77204, USA; 5Department of Psychiatry, Columbia University College of Physicians and Surgeons, Division of Clinical Phenomenology, New York State Psychiatric Institute, 1051 Riverside Drive, New York, NY 10032, USA; 6Department of Psychiatry, Tufts Medical Center, 800 Washington Street, Boston, MA 02111, USA; 7Human Relations Counseling Service, 400 Bayonet Street Suite \\#202, New London, CT 06320, USA; 8Department of Linguistics, University of Illinois, Urbana-Champaign 4080 Foreign Languages Building, 707 S Mathews Ave, Urbana, IL 61801, USA; 9Duke Divinity School, Box 90968, Durham, NC 27708, USA; 10Department of Psychology, Auburn University Montgomery, 7061 Senators Drive, Montgomery, AL 36117, USA; 11Department of Clinical Psychology, The Chicago School of Professional Psychology, 325 North Wells Street, Chicago IL, 60654, USA; 12Institute of Community and Family Psychiatry, SMBD-Jewish General Hospital, Department of Psychiatry, McGill University, 4333 cote Ste. Catherine, Montreal H3T1E4 Quebec, Canada; 13Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine at UCLA, 760 Westwood Plaza, Los Angeles, CA 90095, USA; 14VA West Los Angeles Healthcare Center, 11301 Wilshire Blvd, Los Angeles, CA 90073, USA; 15Department of Psychiatry, SUNY Upstate Medical University, 750 East Adams St., \\#343CWB, Syracuse, NY 13210, USA; 16Irving Institute for Clinical and Translational Research, Columbia University Medical Center, 630 West 168th Street, New York, NY 10032, USA; 17New York Presbyterian Hospital, 1051 Riverside Drive, Unit 09, New York, NY 10032, USA; 18Rand Corporation, 1776 Main St Santa Monica, California 90401, USA; 19Central City Behavioral Health Center, 2221 Philip Street, New Orleans, LA 70113, USA; 20Center for Bioethics, University of Pennsylvania, 3401 Market Street, Suite 320 Philadelphia, PA 19104, USA; 21Department of Psychiatry, Texas AMHSC College of Medicine, 4110 Guadalupe Street, Austin, Texas 78751, USA; 22Silver School of Social Work, New York University, 1 Washington Square North, New York, NY 10003, USA; 23Department of Psychiatry, NYU Langone Medical Center, 550 First Ave, New York, NY 10016, USA; 24Department of Psychiatry, University of Vermont College of Medicine, 89 Beaumont Avenue, Given Courtyard N104, Burlington, Vermont 05405, USA; 25Institute for Health, Health Care Policy, and Aging Research, Rutgers, the State University of New Jersey, 112 Paterson St., New Brunswick, NJ 08901, USA\nreferences:\ntitle: The six most essential questions in psychiatric diagnosis: a pluralogue part 1: conceptual and definitional issues in psychiatric diagnosis\n\n# General Introduction\n\nThis article has its own history, which is worth recounting to provide the context of its composition.\n\nAs reviewed by Regier and colleagues \\[1\\], DSM-5 was in the planning stage since 1999, with a publication date initially planned for 2010 (now rescheduled to 2013). The early work was published as a volume of six white papers, *A Research Agenda for DSM-V* \\[2\\] in 2002. In 2006 David Kupfer was appointed Chairman, and Darrel Regier Vice-Chairman, of the DSM-5 Task Force. Other members of the Task Force were appointed in 2007, and members of the various Work Groups in 2008.\n\nFrom the beginning of the planning process the architects of DSM-5 recognized a number of problems with DSM-III and DSM-IV that warranted attention in the new manual. These problems are now well known and have received much discussion, but I will quote the summary provided by Regier and colleagues:\n\nOver the past 30 years, there has been a continuous testing of multiple hypotheses that are inherent in the *Diagnostic and Statistical Manual of Mental Disorders*, from the third edition (DSM-III) to the fourth (DSM-IV)... The expectation of Robins and Guze was that each clinical syndrome described in the Feighner criteria, RDC, and DSM-III would ultimately be validated by its separation from other disorders, common clinical course, genetic aggregation in families, and further differentiation by future laboratory tests--which would now include anatomical and functional imaging, molecular genetics, pathophysiological variations, and neuropsychological testing. To the original validators Kendler added differential response to treatment, which could include both pharmacological and psychotherapeutic interventions... However, as these criteria have been tested in multiple epidemiological, clinical, and genetic studies through slightly revised DSM-III-R and DSM-IV editions, the lack of clear separation of these syndromes became apparent from the high levels of comorbidity that were reported... In addition, treatment response became less specific as selective serotonin reuptake inhibitors were found to be effective for a wide range of anxiety, mood, and eating disorders and atypical antipsychotics received indications for schizophrenia, bipolar disorder, and treatment-resistant major depression. More recently, it was found that a majority of patients with entry diagnoses of major depression in the Sequenced Treatment Alternatives to Relieve Depression (STAR\\*D)study had significant anxiety symptoms, and this subgroup had a more severe clinical course and was less responsive to available treatments... Likewise, we have come to understand that we are unlikely to find single gene underpinnings for most mental disorders, which are more likely to have polygenetic vulnerabilities interacting with epigenetic factors (that switch genes on and off) and environmental exposures to produce disorders. \\[\\[2\\], pp. 645-646\\]\n\nAs the work of the DSM-5 Task Force and Work Groups moved forward, a controversy developed that involved Robert Spitzer and Allen Frances, Chairmen respectively of the DSM-III and DSM-IV Task Forces. The controversy began with Spitzer's Letter to the Editor, \"DSM-V: Open and Transparent,\" on July 18, 2008 in *Psychiatric Times* \\[3\\], detailing his unsuccessful effort to obtain minutes of the DSM-5 Task Force meetings. In ensuing months Allen Frances joined him in an exchange with members of the Task Force. In a series of articles and blog postings in *Psychiatric Times*, Frances (at times with Spitzer) carried out a sustained critique of the DSM-5 work in which he focused both on issues of transparency and issues of process and content \\[4-16\\]. The latter involved the Task Force and Work Group efforts to address the problems of DSM-IV with changes that, in Frances' opinion, were premature and not backed by current scientific evidence. These changes included new diagnoses such as mixed anxiety-depression, an expanded list of addictive disorders, the addition of subthreshold conditions such as Psychosis Risk Syndrome, and overly inclusive criteria sets - all destined, in Frances' judgment, to expand the population of the mentally ill, with the inevitable consequence of increasing the number of false positive diagnoses and the attendant consequence of exposing individuals unnecessarily to potent psychotropic medications. The changes also included extensive dimensional measures to be used with minimal scientific foundation.\n\nFrances pointed out that the NIMH was embarked on a major effort to upgrade the scientific foundation of psychiatric disorders (described below by Michael First), and that pending the results of that research effort in the coming years, we should for now mostly stick with the existing descriptive, categorical system, in full awareness of all its limitations. In brief, he has argued, we are not ready for the \"paradigm shift\" hoped for in the 2002 *A Research Agenda*.\n\nWe should note that as the DSM-5 Work Groups were being developed, the Task Force rejected a proposal in 2008 to add a Conceptual Issues Work Group \\[17\\] - well before Spitzer and Frances began their online critiques.\n\nIn the course of this debate over DSM-5 I proposed to Allen in early 2010 that we use the pages of the Bulletin of the Association for the Advancement of Philosophy and Psychiatry (of which I am Editor) to expand and bring more voices into the discussion. This led to two issues of the Bulletin in 2010 devoted to conceptual issues in DSM-5 \\[18,19\\]. (Vol 17, No 1 of the AAPP Bulletin will be referred to as Bulletin 1, and Vol 17, No 2 will be referred to as Bulletin 2. Both are available at <http:\/\/alien.dowling.edu\/~cperring\/aapp\/bulletin.htm.>) Interest in this topic is reflected in the fact that the second Bulletin issue, with commentaries on Frances' extended response in the first issue, and his responses to the commentaries, reached over 70,000 words.\n\nAlso in 2010, as Frances continued his critique through blog postings in *Psychiatric Times*, John Sadler and I began a series of regular, DSM-5 conceptual issues blogs in the same journal \\[20-33\\].\n\nWith the success of the Bulletin symposium, we approached the editor of PEHM, James Giordano, about using the pages of PEHM to continue the DSM-5 discussion under a different format, and with the goal of reaching a broader audience. The new format would be a series of \"essential questions\" for DSM-5, commentaries by a series of individuals (some of them commentators from the Bulletin issues, others making a first appearance in this article), and responses to the commentaries by Frances. Such is the origin of this article. (The general introduction, individual introductions, and conclusion are written by this author (JP), the responses by Allen Frances.\n\nFor this exercise we have distilled the wide-ranging discussions from the Bulletin issues into six questions, listed below with the format in which they were presented to commentators. (As explained below, the umpire metaphor in Question 1 is taken from Frances' discussion in Bulletin 1.)\n\n1\\) How to Choose Among the Five Umpires of Epistemology?\n\nAre DSM diagnoses more like constructs or more like diseases? We would like to have the positions of each of the five epistemological umpires stated as clearly as possible.\n\nUmpire 1) There are balls and there are strikes and I call them as they are.\n\nUmpire 2) There are balls and there are strikes and I call them as I see them.\n\nUmpire 3) There are no balls and there are no strikes until I call them.\n\nUmpire 4) There are balls and there are strikes and I call them as I use them.\n\nUmpire 5) Don't call them at all because the game is not fair.\n\nCould you please state the position of the umpire which you endorse?\n\n2\\) What is a Mental Disorder?\n\nIt has been difficult to reach agreement on a definition of mental disorder. Could you comment on this problem, or offer what you think is an adequate definition of the concept, mental disorder?\n\n3\\) What are the Benefits and Risks of Conservatism?\n\nGiven the state of the science of psychiatric disorders, should we design DSM-5 in a conservative manner, with minimal change, or do the state of psychiatric science and the problems in DSM-IV dictate major change?\n\n4\\) Is Pragmatism Practical?\n\nWhat roles do science and pragmatism play in the construction of DSM-5? Does our science allow us to make major decisions on a scientific basis? What role do pragmatic considerations play, both when the science is strong and when the science is weak?\n\n5\\) How Compatible are All the Purposes of DSM?\n\nIs there a conflict over utility in the DSMs? The authors of DSM-III, DSM-IV, and DSM-5 intend the manuals to be useful for both clinicians and researchers. Is there a conflict between what is useful for clinicians and what is useful for researchers? Which group is served better by DSM-III and DSM-IV, and by the prospective changes in DSM-5?\n\n6\\) Is DSM the Only Way to do Diagnosis?\n\nGiven the problems in DSM-III, DSM-IV, and (likely) in DSM-5, would you argue for an alternative, more rational diagnostic system than the DSM? Could you describe it? Would your alternative system simply replace the DSM or restructure it in a major way?\n\nAs will become apparent in what follows, these six questions are in multiple ways interrelated, and for that reason a response to one of the questions is often relevant to another of the questions. This is, for instance, quite obvious with Questions 1 and 2. What you think a mental disorder is will affect how you define the notion of mental disorder. Question 4 quickly enters this discussion. Should pragmatic, in addition to purely scientific, considerations enter into your effort to describe and define mental illness? Under Question 1, for instance, Harold Pincus offers a \"pragmatic\" response that could easily be placed under Question 4.\n\nAnd now let's bring in Question 3 - whether to take a conservative or activist attitude toward changes in DSM-5. Don't forget that threading its way through all of these questions is the dissatisfaction and disappointment with the scientific status of DSM-III and IV. That troubled status clearly played a role in the epistemological (and ontological) discussion in Question 1, the definitional issue of Question 2, and the pragmatic aspect of Question 4. It is emblematic of the complexity of these discussions that the same troubled state of the current nosology will lead Scott Waterman in an activist direction in Question 3 and Michael Cerullo in a conservative direction.\n\nThe final two questions take us in somewhat other directions, but both are related to the discussions that precede them. Question 5, about utility, raises major issues concerning how the manual is actually used, and for whom it is really designed - again, questions related to those of scientific status, definition, pragmatic considerations, and finally attitudes toward change. With this question it's hard to find anyone wanting to defend the premise of DSM-III and IV (and apparently DSM-5) that the manuals are equally useful for clinicians and researchers.\n\nFinally with Question 6 we have an ultimate question - whether the current state of the DSMs warrants a total overhaul. With Ronald Pies we have an individually imagined overhaul; with Joel Paris we have a commentary on DSM-5's effort at revision, and with Michael First's presentation of the NIMH Research Domain Criteria project (RDoC), we have NIMH's response - that the diagnostic manuals of the future may not resemble the DSMs as we know them.\n\nWe should not expect from this or any other publication final answers to the questions of psychiatric classification. The questions are too large, and our expectations have to be more modest. What we know is that the goals of DSM-III & IV have not been achieved and that we are left with more immediate questions as to how to proceed with the current revision, DSM-5. Responses to these questions are understandably mixed. What we hope from this article is to keep the discussion going, and perhaps to move it forward a bit.\n\nFinally, because of the total size of this exercise, \"The Six Most Essential Questions In Psychiatric Diagnosis: A Pluralogue\" will be published in four parts: each of the first three covering two questions and the final part a general conclusion. Thus this article, Part 1, covers the first two questions.\n\n# Question \\#1: How do we Choose Among the Five Umpires of Epistemology?\n\n*Are DSM diagnoses more like constructs or more like diseases? We would like to have the positions of each of the five epistemological umpires stated as clearly as possible*.\n\n*Umpire 1) There are balls and there are strikes and I call them as they are*.\n\n*Umpire 2) There are balls and there are strikes and I call them as I see them*.\n\n*Umpire 3) There are no balls and there are no strikes until I call them*.\n\n*Umpire 4) There are balls and there are strikes and I call them as I use them*.\n\n*Umpire 5) Don't call them at all because the game is not fair*.\n\n*Could you please the position of the umpire which you endorse?*\n\n## Introduction\n\nQuestion \\#1 involves both ontological and epistemological issues: what are psychiatric disorders, and how do we know them? Framing these questions with the metaphor of umpires and balls and strikes comes from Allen Frances's response to commentaries in Bulletin 1, \"DSM in Philosophyland: Curiouser and Curiouser.\" That response offered the positions of three umpires: the realist first umpire, the nominalist second umpire, and the constructionist third umpire. The author sided with Umpire 2, espousing a nominalist stance to the effect that he knows that there is real psychopathology out there but has no guarantee that his diagnostic constructs sort it out correctly. He wrote: \"This brings us to me a (call'um as I see'um) second umpire. In preparing DSM-IV, I had no grand illusions of seeing reality straight on or of reconstructing it whole cloth from my own pet theories. I just wanted to get the job done - produce a useful document that would make the fewest possible mistakes, and create the fewest problems for patients\" (Bulletin 1, p. 22).\n\nFor this article we have added two more umpires: a pragmatist fourth umpire and a fifth umpire who rejects the entire exercise. We were motivated to add these umpires by the fact that some of the responses required them.\n\nFurther, we recognize that in asking respondents to choose one position and defend it, we have made an unreasonable demand. Why should an individual not say, I'm a combination of these two umpires, or, I'm a lot of this umpire and a little of that, or finally, I'm a first umpire if we're talking about Huntington's disease, but a second umpire if we're talking about schizoaffective disorder. So, quite understandably, in some our responses we witness the same problem we have with our diagnoses: comorbidity - in this case epistemological (or ontologic) comorbidity rather than diagnostic comorbidity.\n\nIn this debate over the nature of psychiatric disorders we experience a tension among the umpires that reflects the status of nosologic science. On the one hand our patients suffer greatly from psychiatric symptoms, and it seems wildly foolish to theorize away their suffering. On the other hand our efforts to organize and classify their suffering can seem arbitrary and confusing. We organize or categorize a symptom cluster and give it a diagnostic name, and it overlaps with another cluster. Or a patient simply has symptoms of both. We start off with the expectation that there will be a match-up between therapeutic agent and diagnostic cluster, and we discover that, at the extreme, most of our pharmacologic agents seem to treat most of our disorders. Finally, we somehow want to resolve this confusion by getting at the underpinnings of the identified disorders, and we discover that the genetics and neuroscience don't support our groupings.\n\nIn view of this confusion it's not surprising that opinion divides itself in various ways. Focus on the real suffering out there, along with a conviction that the diagnostic clusters reflect distinct, real conditions, and you end up as a first umpire. Focus on that suffering with uncertainty about the isomorphism between label and disorder, and you become a second umpire. Switch your focus onto the arbitrariness of the labeling, and you end up questioning whether there is anything but the labeling and become a third umpire. Or switch away from the issues of these umpires onto the effects of one label versus another, and you are now a fourth umpire. Finally, decide that it's all nonsense, and you are our fifth umpire.\n\n## Commentary: A Game for Every Kind of Umpire (Almost)\n\nPeter Zachar, Ph.D. and Steven G. Lobello, Ph.D.\n\nAuburn University Montgomery Department of Psychology.\n\nOne might think that a philosophical pragmatist should identify with either the pragmatist or the nominalist position in Allen Frances's clever analogy, but that isn't the case. From a pragmatist perspective, philosophical -isms such as realism, pragmatism, nominalism, and constructionism are conceptual distinctions that we make for certain purposes. The question is what information or response options are gained from making these distinctions that would not be gained were other distinctions made.\n\nFor example, let's take the pragmatist's view that *I call balls and strikes as I use them*. If taken too literally this is a recipe for a shallow utilitarianism. One of the ethical principles of umpires is to try to make the game as fair as possible - so every batter and pitcher should face the same strike zone (for that umpire). An umpire should attempt to call the pitches as they are (to the best of his ability), and not widen the zone for batters he favors and narrow it for those he does not. Also, in most games, a degree of unreliability in deciding what counts as a ball or strike may not matter, but it can matter a lot in big games. Presumably every psychiatric patient should be treated like a big game, but with 15 minute medication management sessions that is not likely the case. So a kind of realist attitude is important for keeping the game fair. This is true of psychiatric nosology as well. We should always attempt to classify the world as it is not how we want it to be. A pragmatist would not deny the spirit of this ethic.\n\nMost pragmatists would point out that the purpose of the strike zone is to assure that the batter has a chance to hit the ball well enough to get on base. He cannot do so if the pitch is too high, in the dirt, or wide of the plate. This makes the strike zone a practical kind. There are also practical constraints on the strike zone's location that create a kind of objectivity - but beyond that there is no gold standard. Furthermore, it is not true that every pitch that goes through the zone on the way to the catcher is a strike. For example, spit balls have such unpredictable trajectories that batters have very little chance of hitting them, and they are therefore illegal whether or not they are in the zone. Psychiatry lacks fixed gold standards as well, and the social implications of giving a diagnosis that is contrary to the purpose of diagnosing can also affect whether something is considered to be an official disorder (e.g., pedophilia).\n\nWhat of the nominalists who say *I call balls and strikes as I see them*? Perhaps a better way to think about nominalists is that they deny both that the criteria for balls and strikes were created by the Platonic baseball gods and that competent umpires can recognize what is naturally a ball and naturally a strike. Cousins to the pragmatist, the nominalists say that what exist are particular pitches, and we tend to group them into the ball category or the strike category for various and sundry reasons. Very different pitches like fast balls, curve balls and sliders can all be strikes. These groupings can also be altered. For example up until the 1920s the spit ball was a legitimate pitch (as homosexuality was once considered a legitimate psychiatric disorder).\n\nSo nominalists and pragmatists are uncomfortable when realists start talking about fixed world structures and natural kinds. There are, however, *kinds* - fastballs, curve balls, etc. With the realists, the pragmatists and nominalists recognize the value of understanding the causal mechanisms that produce these kinds (e.g., Vaseline helps you throw good spit balls), but individual pitches can be grouped in a plurality of ways.\n\nThe constructionist position is the easiest to defend in this example because baseball is a social construction, and like other social constructions such as the U. S. Government and currency, baseball is a real thing. So what information do we gain from the constructionist analysis? Rather than saying *There are no balls and strikes until \"I\" call them*, it is more accurate to say that social construction is a historical and community activity. Baseball proper did not exist in 1800 and a pretty good story can be told about the social and economic factors that helped shape the game we have today. A similar narrative could be developed for psychiatry, for example, there is a good story to be told about how degeneration theory in the 19^th^ century and pharmaceutical marketing practices in the 20^th^ century both shaped the classification system. Social constructionists would also point out that something like the introduction of the designated hitter was not a deductive consequence of the rules of the game. Its legitimacy has to be understood with respect to the baseball community and its chosen authorities. Something similar is true of the scientific community and its designated authorities, including the process by which the DSM and the ICD is developed. The pragmatists consider this useful information.\n\nFinally we come to the Szasian. It is a category mistake to lump a political and ethical position such as *I refuse to play because the game is not fair* with realism, pragmatism, nominalism and constructionism. Anti-psychiatry is better considered a behavioral option available to a disillusioned realist. In terms of baseball, the claim would be that in the rest of sports, things like field goals and holes-in-one are objectively fixed, but there is so much variation between umpires in terms of the strike zone, that any rational person would see that the so-called objectivity of the game is a myth. Other like-minded critics would point out that there seems to be statistical evidence that the strike zone gets wider when the count is full - which keeps the game exciting. It is also economically convenient for the sport as a whole if pitchers are allowed some leeway when being close to throwing perfect games and batters allowed leeway when being close to breaking hitting records. Field goals and holes-in-one do not work like that, say the critics, yet baseball wants its consumers to think it is like those other sports. Perhaps the best argument against the Szaszian view is to point out that if they studied football and golf more closely, they might see that things are not as always as objective over there as they assume. Baseball should not be evaluated with respect to an idealized image of other sports just as psychiatry should not be evaluated with respect to an idealized image of other medical specialties.\n\n## Commentary: Mental Disorders, Like Diseases, Are Constructs. So What?\n\nClaire Pouncey, M.D., Ph.D.\n\nPhiladelphia, PA.\n\nThe literature on the philosophy of psychiatric nosology often conflates questions of ontology - i.e., whether mental disorders exist as abstract entities- with questions of epistemology - i.e., how we can know anything about them if they do. To ask whether mental disorders are (actual) diseases or (mere) constructs confuses these two types of questions about mental disorders, as I will use the first three umpire positions to illustrate. This error is prevalent in academic discussions about psychiatric nosology.\n\nOntologic commitments are basic metaphysical commitments about what exists in the world. Most of us, by virtue of the fact that we operate in our physical and social worlds as we do, are committed to the existence of intersubjectively appreciable mid-level objects, such as plants, buildings, bodies of water, and other persons, to name just a few. That is, we are realists *about* (and realism is always local to a particular question) mid-level objects, as evidenced by our behaviors.\n\nIt is easier to be skeptical (a.k.a. antirealist) about invisible, microscopic, macroscopic, and abstract objects. Most of us are ontologically committed to the existence of oxygen, given what we know about basic physiology and the chemistry of our natural environment, although it is microscopic in its elemental form and undetectable by the senses in its macroscopic form. Our commitments to microscopic entities such as muons, macroscopic entities such as red giants, intangible phenomena such as global warming, or second-order (categorical) entities such as phyla may be much weaker, and more prone to debate.\n\nMental disorders generate ontological skepticism on several levels. First, they are abstract entities that cannot be directly appreciated with the human senses, even indirectly, as we might with macro- or microscopic objects. Second, they are not clearly natural processes whose detection is untarnished by human interpretation, or the imposition of values. Third, it is unclear whether mental disorders should be conceived as abstractions that exist in the world apart from the individual persons who experience them, and thus instantiate them. Together, these reasons to doubt the ontic status of mental disorders become quite persuasive.\n\nSetting ontological antirealism aside, we can ask epistemological questions separately: if we assume that mental disorders do exist as abstract entities, how do we go about studying them, and on what basis can we possibly gain genuine knowledge about them? Even if we collectively agree that, for example, a particular person at a given time were experiencing a major depressive episode, on what grounds can we know that 'major depressive disorder' exists as an abstract entity? On what grounds can we infer that the broader class 'mood disorders', or 'mental disorders' as the most general class, exist as further abstractions? Epistemic realists may be realists about *Hector's* depression, about the existence of an abstract entity that is major depressive disorder, or about the existence of mental disorders in the world generally. They may not be realists about all three. Similarly, epistemic antirealists may doubt one or more of these commitments.\n\nUmpire \\#1 is both an ontological realist and an epistemological realist about balls and strikes in baseball. Balls and strikes are real things (events) that exist (happen) in the world, and Umpire 1 has the means and ability to detect them in accurate and unbiased ways: \"There are balls and there are strikes and I call them as they are.\" This tends to be the position attributed to psychiatry. Psychiatry's rhetoric, if not the actual commitments of all practitioners, says both that mental disorders are abstract entities that exist in the world and manifest in individual persons, and that these processes can be intersubjectively appreciated and elucidated as they truly are. Let's call this the Strong Realist position.\n\nSuch confidence is not exhibited by Umpire \\#2, who shares the ontological realism of Umpire \\#1, but not the epistemological realism. In tempering his epistemological position to \"I call them as I see them,\" Umpire \\#2 maintains that balls and strikes exist apart from his perception of them, but softens his position to recognize that he may not always perceive them as they exist in the world. That is, Umpire \\#2 is *ontologically* committed to the existence of balls and strikes, but does not assume that he always has epistemic access to that reality. Let's call this the Strong Realist\/Weak Constructivist position.\n\nUmpire \\#3 is an ontological and an epistemological antirealist about balls and strikes: no balls or strikes exist in the world regardless of who thinks they might. In calling them, the umpire constructs the truth. This is not necessarily to say that all his calls are unfounded fictions, but rather it is to say that although the umpire describes his perceptions as accurately he can, there is no ultimate, underlying reality to which those perceptions could be compared, even in the absence of epistemic limitations. Let's call this the Strong Constructivist position.\n\nPsychiatry's strongest critics tend to make strong constructivist arguments: mental disorders do not exist, so any diagnosis, treatment intervention, or research finding is exempt from ultimate confirmation or refutation. In their strongest form, calling mental disorders 'constructs' *is* meant to communicate that they are mere fictions, completely unfounded medical lore. However, note that on the Strong Realist\/Weak Constructivist view this is not the case. Calling a mental disorder a 'construct' does not imply invention so much as it serves as a reminder that our epistemic access to the reality of things is always limited. On this view, every abstract entity is a construct, and constructs can be legitimate objects of scientific investigation. Often, there is broad agreement about the nature of scientific constructs, such as phyla, subatomic particles, or diseases, even if the construct is construed as a working hypothesis, or a category of disparate entities that does not lend itself to simple definition or characterization. On this view, mental disorders are like diseases: they are a heterogeneous class of abstract entities that have uncertain ontic status apart from the persons who instantiate them. In formalizing its nosology, psychiatry is trying to call them as we see them.\n\n## Commentary: Why Umpires Don't Matter\n\nNassir Ghaemi, M.D.\n\nTufts University Department of Psychiatry.\n\nNietzsche said truth is a mobile army of metaphors. If you get your metaphor wrong, you'll miss the truth. I think this is the case with the umpire metaphor that seems to be the central concept underlying the thinking of my interlocutor. I think it is simply wrong-headed. It sets up psychiatry and science and knowledge as a game, where the rules can be changed, and where there may be no truth. If you are a postmodernist extremist, this may make sense. But if you accept that there are truths in the world (such as that if you take very high doses of lithium, you will get toxicity), then it makes no sense.\n\nA mistaken metaphor has no response except to say that it is mistaken.\n\nBefore offering a better metaphor, let me say that I accept the realist position, that is, that diseases exist independent of me and you that are expressed as psychiatric symptoms like the chronic delusions of schizophrenia, or the mood states of manic-depression. To prove this fact, I suggest three approaches. One, suggested by Paul McHugh, is to actually see people who have these symptoms, the old kick the table test of realism. The second is to debate the merits of the positions pro and con; I won't do so here, but I think others have done so in reasonably persuasive ways, such as Roth and Kroll's *Reality of Mental Illness*. The third is to apply the pragmatic test, and see the consequences of one position or the other. I accept the realist view in at least some psychiatric diseases, but I would add that if one does not, he or she should think of the consequences. I don't see how one can reject the reality of psychiatric disease, and still practice psychiatry, especially with the use of harmful drugs.\n\nThis metaphor brings out those stark choices, as well as provides further rationale for the reality of at least some psychiatric diseases based on how matters have gone in other examples of similar problems in the history of science and medicine.\n\nHere then is a better metaphor for understanding psychiatric nosology, one that I heard from Kenneth Kendler and which I am expanding here. In a presentation on \"epistemic iteration,\" building on work in history of science, Kendler described how we can understand any scientific process as involving an approximation of reality through successive stages of knowledge. The main alternative to this process is \"random walk\" where there is no trend toward any goal in the process of scientific research. Kendler points out that epistemic iteration won't work if there are no real psychiatric illnesses. If these are all, completely and purely, nothing but social constructions, figments of our cultural imaginations, then there is no point to scientific research at all. (I would add: to be honest doctors, we should stop thereby killing patients with our toxic drugs - since all drugs are toxic - stop taking their money to buy our large houses, and retire.) The random walk model is a dead end for any ethical practice of medicine, because if there is no truth to the matter, then we should not claim to have any special knowledge about the truth.\n\nIf there is a reality to any psychiatric illness, then epistemic iteration makes sense, and indeed it has been the process by which much scientific knowledge has been obtained in the past. Take temperature. A long process evolved before we arrived at the expansion of mercury as a good way to measure temperature. There was a reality: there is such a thing as hot and cold temperatures. How we measured that reality varied over time, and we gradually have evolved at a very good way of measuring it. Temperature is not the same thing as mercury expansion: our truth here is not some kind of mystical absolute knowledge. But it is a true knowledge.\n\nA similar rationale may apply to psychiatric diseases. We may, over time, approximate what they are, with our tools of knowledge, if we try to do so in a successive and honest manner, seeking to really know the truth, rather than presuming it does not exist.\n\nThe better metaphor, then, which captures epistemic iteration versus random walk alternatives would be to think of a surface, and a spot on that surface, which we can label X, representing the true place we want our disease definition (see figure). If we were God, we would know that X is the right way to describe the disease. Let A be our current knowledge. How do we get from A to X. One way is to go from A to B, from B to C, from C to D, in a zig zag pattern, as our research takes us in different directions, but gradually and successively closer to X. This is epistemic iteration.\n\nThe random walk pattern would involve the same starting point A, and multiple movements to B, C, and D, but with no endpoint, because no X would exist (see figure). In this process, movement is random, there is no reality pulling scientific research towards it, like gravity pulling objects closer, and there is no end, and no truth. If this is the nature of things, then our profession has to admit to everyone everywhere that this is what we are doing. We should then give up any claims to specific knowledge and stop treating - and harming - people.\n\nThe history of medicine and the history of science gives many examples of both approaches. So the question really is an ontological one: do mental illnesses exist as realities in the external world, as biological diseases independent of our social constructs and personal beliefs? The umpire metaphor assumes, but does not answer, that question. The epistemic iteration metaphor shows how the answer to that question faces us with two opposed choices about how we understand science and psychiatry. If psychiatry is like the rest of medicine, if there are some psychiatric diseases that are independent biological realities just as there are some medical diseases, then the epistemic iteration metaphor would seem valid in some cases, and the umpire metaphor, useless as it is, should be discarded. Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>.\n\n## Commentary: The Three Umpires of Metaphysics\n\nMichael Cerullo, M.D.\n\nUniversity of Cincinatti Department of Psychiatry.\n\nThe debate about the nature of the external world has been a central question of metaphysics since the first pre-Socratic philosophers. Most working scientists and philosophers today would be classified as modern realists who believe there is an independent objective external reality. Within the realist camp there is further debate about just how much we can know about absolute reality. Immanuel Kant termed the underlying reality of the world \"the thing in itself\" (*das Ding an sich*) and believed we could never truly know this ultimate reality \\[34\\]. Opposed to the realists are the anti-realists who hold that there is no independent objective reality separate from our own subjective experience. Allen Frances' umpire analogy is a good way to frame the major positions in this debate \\[2\\](Francis 2, 21-25). Frances' first umpire who believes there are balls and strikes and calls them as they are is a modern realist. Umpire two is a Kantian realist who believes there are balls and strikes but can only call them as she sees them. Umpire three is an anti-realist who believes there are no balls and strikes until he calls them.\n\nThese days it is hard to seriously defend an anti-realist position in science. Neuroscientists contend that all behavior, from depression to extroversion to a dislike of tomatoes, is dependent and explainable by the workings of the brain. On the other hand there is still a real debate as to whether subatomic particles are the final bedrock of reality or a mere appearance of a deeper reality (strings? more particles all the way down?). However this latter Kantian uncertainty doesn't seem to have much relevance to the debate about the brain. After all, it doesn't seem to make any difference in our understanding of neurons if their atoms are ultimately made of strings or point particles.\n\nOutside of metaphysics there is another parallel to the umpire analogy in epistemology. Within epistemology there is a subfield interested in the taxonomy of illness. The two major groups in this debate are the naturalists and normativists \\[35,36\\]. Naturalists believe disease can be defined objectively as a breakdown in normal biology. The naturalist position corresponds to the first umpire. Normativists believe our definitions of disease are subjective and culturally driven and thus identify with the third umpire. The second umpire seems to mix elements of both epistemological positions.\n\nMy own sympathies lie with modern realism when it comes to behavior and a combination of normativist and naturalist positions when defining disease. Although there is physical explanation for all behavior (hence my realist position), not everything in the universe is physical. Definitions of disease require value judgments, and while each value judgment surely has a physical explanation in the brain, nothing physical can decide which judgment is correct. Even in areas of medicine outside psychiatry there is often a strong normativist element in how diseases are defined. Many diseases such as hypertension or hypercholesterolemia require making arbitrary cut off points in laboratory values. Deciding these cut off points requires making hard decisions about public health and considering the risk\/benefit ratio of any decision. There is clearly a strong normativist element in theses definitions, yet clearly that does not make them bad or incorrect descriptions. Many psychiatric diseases also have a similar logic. While everyone has some sad mood or anxiety there are obvious extremes which are justifiably labeled as mood or anxiety disorders. Once again there may be certain arbitrary cut off points when deciding how much sadness or anxiety is too much but that does not invalidate these definitions anymore so that it would the \"physical\" illness listed above. This being said, there are also many diseases that are much better defined from a more naturalist perspective. For example, in psychiatry schizophrenia seems to be better defined from the naturalist perspective along with other physical diseases like Parkinson's disease or dementia. It seems easier to define these diseases using the naturalist ideal of disease as a breakdown in the \"typical\" human biology.\n\nThe lesson in these debates is that psychiatrists (and the public) should recognize that all definitions of disease have normativist and naturalist elements even in a world described by a scientific realism. None of Frances' umpires fits with my combined metaphysical and epistemological positions. Therefore I suggest a different umpire, one who believes in an objective physical world that we can access to determine exactly what are balls and strikes. Yet it is the umpire and players who first must choose the rules of the game, some of which may always seem arbitrary but the majority of which are constrained by the physics of balls and bats and the semantic and historical notions of games and baseball.\n\n## Commentary\n\nJerome C. Wakefield, Ph.D., D.S.W.\n\nSilver School of Social Work and Department of Psychiatry, New York University.\n\nRegarding the Umpires: First, to avoid confusion, one has to distinguish the role of Umpire calls within the rules of baseball from the call as an attempt to state what happened. The Umpire calls them as he\/she sees them, with the goal of getting it right - and understands that the way it looks can be misleading. But, whether correct or incorrect, the Umpire's call \"stands\" despite any later evidence that emerges to the contrary, and to that extent the call constitutes\/constructs the game's reality. Diagnosis, too, has dual aspects - a game in which one plays by the rules to justify reimbursement, and a hypothesis about what is going on in the patient. I focus on the hypothesis-testing aspects of both Umpire calls and the DSM.\n\nIn attempting to make a call that reflects the truth, Umpires 1 and 3 embrace intellectual doctrines designed to deal with their epistemic anxieties - Umpire 1 can't stand uncertainty, and Umpire 3 can't stand the arrogance that comes from Umpire 1's certainty. Ironically, Umpire 1 and Umpire 3 fall into the same fallacy, that of collapsing ontology and epistemology into one. Umpire 1 naively sees his\/her judgment as being a direct impression of reality without epistemic mediation, thus epistemological uncertainty is avoided. Umpire 3 sees his\/her judgment as creating or constituting \"reality\" from his\/her perspective, so again epistemological uncertainty is avoided. On the other hand, Umpire 2, while closest to the correct approach, describes his\/her reality and his\/her perception in a rather disconnected way.\n\nSo, I vote for Umpire 1.5 (humble realism): There are balls and there are strikes (plus some ontologically fuzzy cases), and based on how I see them and any other available evidence, I call them as I believe they are, and because the evidence in these cases is usually a pretty good indicator of reality, calling them as I see them usually equals calling them as they are. But, I can be wrong! The truth does not necessarily correspond to my call, and fresh evidence can always be brought to bear to help get closer to the truth.\n\nCommon sense offers the best guide here. Recently, Tigers' pitcher Armando Galarraga was one pitch away from achieving baseball immortality with a perfect game, an extremely rare event. In a close call at first base, Umpire Jim Joyce called the runner safe, destroying Galarraga's chance. But, as everyone saw from the instant reply, in fact the runner, Jason Donald, was out. Jim Joyce said to the press; \"I just cost that kid a perfect game... I thought (Donald) beat the throw. I was convinced he beat the throw, until I saw the replay... It was the biggest call of my career and I kicked the (expletive) out of it.\" He then went to Galarraga and explained what he saw, and made it clear that he was wrong (\"Imperfect\" Umpire Apologizes by Steve Adubato, Ph.D., Star-Ledger). Fortunately for the lessons we and our kids take away from baseball, Joyce was not Umpire 1 or 2 or 3, but humble realist Umpire 1.5 who understood the possibility of error inherent in the attempt for mind to represent reality.\n\nAs to the other part of the question, the dichotomy between constructivism and realism is a false one. Our diagnostic categories are constructs (as are all concepts) intended in the long run to refer to underlying diseases\/disorders. Current DSM diagnoses are constructs that are starting points for a recursive process aimed at getting at disorders. We somewhat misleadingly refer to them now as \"disorders,\" although frequently we acknowledge that one of these categories likely encompasses many disorders. Close attention to the way revise our views and the grounds on which our judgments are made suggests that the individuation of disorders ultimately depends on the individuation of dysfunctions (see the answer to question 6).\n\n## Commentary\n\nJoseph Pierre, M.D.\n\nUCLA Department of Psychiatry.\n\nConsider the brief history of Pluto as a planet, as told in the recently published book, *How I Killed Pluto and Why It Had It Coming* \\[37\\]. A few thousand years ago, during the era of Greek geocentrism, the Earth was considered to be the center of the universe, while the sun and moon were regarded as two of the seven planets that orbited around it. Later in the 16^th^ century, as Copernicus' mathematical models of heliocentrism were embraced, the Earth and the sun traded categories at the expense of the moon. The subsequent discoveries of Uranus in 1781, Neptune in 1846, and Pluto in 1930 resulted in the total of nine planets that most of us learned about in elementary school. However, in 2006, Pluto was officially downgraded from classification as a planet, in part because of the discovery in 2005 of a larger mass of rock and ice called \"Xena\" orbiting not that far away. Now our children will be taught that there are only eight planets, and will perhaps eventually learn that there are also heavenly bodies called \"dwarf planets,\" among them Pluto and Eris (the new, official name for \"Xena\").\n\nTo anyone that really relies on taxonomy in their daily work, it inevitably becomes apparent that such efforts at classification never seem to do a perfect job of \"carving nature at its joints.\" This is especially true with scientifically-based taxonomies - they change based on the evolution of underlying definitions; new categories and sub-categories emerge while previous entities are re-categorized in order to accommodate new data; and challenges to classification at border-zones linger on. Although this kind of change sometimes causes the general public to regard science with skepticism, it is this very adaptability in the face of new data that is the strength of science and the feature that most distinguishes it from dogma.\n\nThe belief that this dynamic process is both acceptable and necessary for the Diagnostic and Statistical Manual of Mental Disorders (DSM) would seem to place myself in the category of Allen Frances' \"Umpire \\#2,\" where I suspect the vast majority of clinicians reside. Still, since I have just suggested that reality often defies simple classification, allow me to state my position more clearly. I believe that psychiatric disorders do exist and that they are brain-mediated diseases (leaving aside for the moment the challenge of defining \"disease\") with genetic, biologic, and environmental etiologies and influences. The disorders (not diseases) cataloged in the DSM represent our best attempts at achieving consensus definitions of these conditions, seriously limited as we are by diagnosis that is based almost exclusively on describing manifest symptoms. Because of this limitation, it is unavoidable that psychiatric diagnosis is overly simplistic, just as many medical diagnoses would still be if not for technology-driven discoveries about pathophysiology. As such, DSM diagnoses are constructs, and DSM-IV's chief utility is as a \"good enough rough guide for clinical work \\[38\\].\"\n\nAs an imperfect work in progress, the DSM-IV contains diagnostic constructs of variable validity. In the tradition of Umpire \\#1, I believe that many of the disorders in DSM do a good job of describing the essential symptomatic features of what are probably \"real diseases\" (e.g. obsessive-compulsive disorder). However, I can also acknowledge the concerns of Umpire \\#3, including that some DSM disorders may tread dangerously close to pathological labeling of socially unacceptable behaviors (e.g. paraphilias) \\[39\\], while others might be better understood as \"culture-bound syndromes\" (e.g. anorexia) \\[40\\].\n\n## Commentary\n\nGary Greenberg, Ph.D.\n\nNew London, CT.\n\n\"There are no balls or strikes until I call them\" is not the postmodern fantasia that it sounds, nor is it a throwback to the idealism that Samuel Johnson refuted so thoroughly by kicking Bishop Berkeley in the knee. Or, to put it another way, it is neither the death knell of psychiatry nor a straw man for psychiatrists to use to refute their critics.\n\nWhat it is, really, is just plain common sense. To question diagnosis is not to question the existence of suffering, or of the mind that gives us the experience of suffering, or of the value of sorting it into category. It is merely to point out that before we can do that sorting, we have to posit those categories. Where do they come from? Are there really diseases in nature?\n\nConsider this question. What is the difference, from nature's point of view, between the snapping of a branch of an old oak tree and the snapping of a femur of an old man? We rightly recoil from the suggestion that there is no difference, and yet to assume that there is *in nature* a difference is to assume that nature cares about us enough to provide us with categories of broken hips. There is ample evidence, most stunningly Darwinian theory, that this is not true. Nature is indifferent. Unlike Major League Baseball, nature doesn't provide the rules by which the world can be divided into balls and strikes.\n\nIf there is a difference between the hip and the branch, it is surely to be found in the difference between the man and the tree, which is that the man is capable of caring about his femur, as are the people that love him. The only reason to distinguish one break from the other is to create a category--*intracapsular transcervical fracture, Stage II*, let's say. Naming the suffering, we bring it into the human realm. (It is not a coincidence that the authors of Genesis tell us that the first task given to Adam and Eve in Eden was to name the creatures of the earth; naming is how we put our stamp on the world.) By inventing categories like this one, we give ourselves a way to get hold of it, which in medicine means among other things a way to talk to other professionals about it, a way to determine treatment options, and a way to provide a prognosis to the patient and family. What we don't do is to discover that nature intends hips to break in certain ways, that there exist in nature intracapsular transcervical fractures and trochanteric fractures, any more than nature provides a branch with different ways to snap off a tree.\n\nThis much is uncontroversial, largely because whether you buy the argument or not, you are still going to treat the problem more or less the same way. The difference between fracture as a man made and a natural category is trivial, unless you're in a philosophical argument. But when it comes to psychiatry, something changes. To call a snapped femur an illness is to make only the broadest assumptions about human nature--that it is in our nature to walk and to be out of pain. To call fear *generalized anxiety disorder* or sadness accompanied by anhedonia, disturbances in sleep and appetite, and fatigue *depression* requires us to make much tighter, and more decisive, assumptions about who we are, about how we are supposed to feel, about what life is for. How much anxiety is a creature cognizant of its inevitable death supposed to feel? How sad should we be about the human condition? How do you know that?\n\nTo create these categories is to take a position on the most basic, and unanswerable, questions we face: what is the good life, and what makes it good? It's the epitome of hubris to claim that you have determined scientifically how to answer those questions, and yet to insist that you have found mental illnesses in nature is to do exactly that. But that's not to say that you can't determine scientifically patterns of psychic suffering as they are discerned by people who spend a lot of time observing and interacting with sufferers. The people who detect and name those patterns cannot help but organize what they observe according to their lived experience. The categories they invent then allow them to call those diseases into being. They don't make the categories up out of thin air, but neither do they find them under microscopes, or under rocks for that matter. That's what it means to say that the diseases don't exist until the doctors say they do. Which doesn't mean the diseases don't exist at all, just that they are human creations, and, at their best, fashioned out of love.\n\nIf psychiatry were to officially recognize this fundamental uncertainty, then it would become a much more honest profession--and, to my way of thinking, a more noble one. For it would not be able to lose sight of the basic mystery of who we are and how we are supposed to live.\n\n## Commentary\n\nHarold A Pincus, M.D.\n\nColumbia University Department of Psychiatry.\n\nThe fourth umpire has a very pragmatic perspective and understands that a classification of diagnostic categories is used for many different purposes by many different groups and individuals. Umpire 4 also understands that these various \"user groups\" approach their tasks with varying empirical, philosophical and historical backgrounds and, and with this proliferation of users and backgrounds, there needs to be a balance between (to mix metaphors) letting \"a thousand flowers bloom\" - creating a Tower of Babel with little ability to effectively communicate among these groups - and a single approach that cannot be tailored to particular needs. From this perspective, there is a recognition that the world has changed and the management of information has become the pre-eminent task of a classification system, overshadowing (but also enhancing), the clinical, research and educational goals of a classification. As such, the ICD\/DSM should serve a critical translation function to anchor communications among multiple user groups that apply psychiatric classification in their day to day functions.\n\nThis information management goal intersects with multiple user groups in terms of:\n\n-health policy\n\n-clinical decision making\n\n-quality measurement\n\n-epidemiology\n\n-educational certification\/accreditation\n\n-multiple areas of research from genetics to psychopharmacology to cognitive science, etc.\n\nThe way this would work is that the ICD\/DSM classification would remain relatively stable, serving as a kind of \"Rosetta Stone\" to facilitate communication among the various user groups. Each individual user \"tribe\" (or individual scientist) would be free to identify various alternative classifications. However, all journals or other public reporting mechanisms would require that any clinical population also be described in the ICD\/DSM classification in addition to whatever tribal criteria for the \"Syndrome XYZ\", 70% met ICD\/DSM criteria for GAD, 40% OCD, and 30% Anxiety Disorder, NOS). Changes in future (descriptive) classifications should be infrequent and guided by a highly conservative process that would only incorporate changes with strong evidence that they:\n\n1\\. Enhance overall communication among the \"tribes\"\n\n2\\. Enhance clinical decision-making\n\n3\\. Enhance patient outcomes\n\nHowever, ICD\/DSM would have a section describing the relationships among the various tribal concepts that could be updated on a more frequent basis.\n\nNote that this approach gives up the ideal (or even a focus) on validity, per se. Maintaining effective communication (most notably, effective use, reliability and understandability) and clinical utility \\[41\\] (either the more limited improvement of clinical and organizational decision-making processes or the ideal of outcomes improvement) become the principal goals of the classification. In other words, while a psychiatric classification must be useful for a variety of purposes, it cannot be expected to be simultaneously at the forefront of, for example, neurobiology and genetics, psychoanalysis, and the education of mental health counselors, primary care providers and psychologists.\n\nHowever, multiple groups can continue their work on epistemic iteration using genetic approaches and others can develop ways to better measure quality or costs of care and yet others can study dimensional ratings of personality. However, each tribal group would need to be able to communicate across the commons using the \"Rosetta Stone\". Thus, we would not be wobbling toward the asymptote of true validity, but, instead, be very slowly, but continually, rising toward the goal of better outcomes for patients.\n\n## Commentary\n\nThomas Szasz, M.D.\n\nSUNY Upstate Medical University.\n\nI thank Dr. James Phillips for inviting me to comment on this debate. I am pleased but hesitant to accept, lest by engaging in a discussion of the DSM (the American Psychiatric Association's *Diagnostic and Statistical Manual of Mental Disorders*) I legitimize the conceptual validity of \"mental disorders\" as medical diseases, and of psychiatry as a medical specialty.\n\nPsychiatrists and others who engage in this and similar discussions accept psychiatry as a science and medical discipline, the American Psychiatric Association (APA) as a medical-scientific organization, and the DSM as a list of \"disorders,\" a weasel word for \"diagnoses\" and \"diseases,\" which are different phenomena, not merely different words for the same phenomenon.\n\nIn law, the APA is a legitimating organization and the DSM a legitimating document. In practice, it is the APA and the DSM that provide medical, legal and ethical justification for physicians to diagnose and treat, judges to incarcerate and excuse, insurance companies to pay, and a myriad other social exchanges to be transacted. Implicitly, if not explicitly, the debaters's task is to improve the \"accuracy\" of the DSM as a \"diagnostic instrument\" and increase its power as a document of legitimation.\n\nLong ago, having become convinced of the fictitious character of mental disorders, the immorality of psychiatric coercions and excuses, and the frequent injuriousness of psychiatric treatments, I set myself a very different task: namely, to delegitimize the legitimating authorities and agencies and their vast powers, enforced by psychiatrists and other mental health professionals, mental health laws, mental health courts, and mental health sentences.\n\nIn *Psychiatry: The Science of Lies*, I cite the warning of John Selden, the celebrated seventeenth-century English jurist and scholar: \"The reason of a thing is not to be inquired after, till you are sure the thing itself be so. We commonly are at, *what's the reason for it?* before we are sure of the thing.\" In psychiatry it is usually impossible to be sure of \"'what a thing itself really is,\" because \"the thing itself\" is prejudged by social convention couched in ordinary language and then translated into pseudo-medical jargon.\n\nSeventy-five years ago, in my teens, I suspected that mental illness was a bogus entity and kept my mouth shut. Twenty-five years later, more secure in my identity, I said so in print. Fifty years later, in the tenth decade of my life, I am pleased to read Dr. Allen Frances candidly acknowledging: \"Alas, I have read dozens of definitions of mental disorder (and helped to write one) and I can't say that any have the slightest value whatever. Historically, conditions have become mental disorders by accretion and practical necessity, not because they met some independent set of operationalized definitional criteria. Indeed, the concept of mental disorder is so amorphous, protean, and heterogeneous that it inherently defies definition. This is a hole at the center of psychiatric classification.\" This is as good as saying, \"Mental illness, there ain't no such thing,\" and still remain loyal to one's profession.\n\nThe fallacy intrinsic to the concept of mental illness - call it mistake, mendacity, metaphor, myth, oxymoron, or what you will - constitutes a vastly larger \"problem\" than the phrase \"a hole at the center of psychiatric classification\" suggests. The \"hole\" - \"mental illness\" as medical problem - affects medicine, law, education, economics, politics, psychiatry, the mental health professions, everyday language - indeed the very fabric of contemporary Western, especially American, society. The concept of \"psychiatric diagnosis,\" enshrined in the DSM and treated by the discussants as a \"problem,\" is challenging because it is also a solution, albeit a false one.\n\nMedicalization, epitomized by psychiatry, is the foundation stone of our modern, secular-statist ideology, manifested by the Therapeutic State. The DSM, though patently absurd, has become an utterly indispensable legal-social tool.\n\nIdeologies - supported by common consent, church, state, and tradition - are social facts\/\"truths.\" As such, they are virtually impervious to criticism and possess very long lives. The DSM is here to stay and so is the intellectual and moral morass in which psychiatry has entwined itself and the modern mind.\n\n## Commentary: On Inviting the Gorilla to the Epistemological Party\n\nElliott Martin, M.D.\n\nYale University Department of Psychiatry.\n\nWhat makes the epistemological umpire analogy so enticing is its capacity for adaptation, the fact that the strike zone must be different for every batter. If I call 'em as I see 'em, then of course what is a ball thrown to one batter may be a strike thrown to another. As applied to the broadly descriptive nosology of DSM IV there is hardly an argument to be made against this. But let's add a missing piece to the scenario. Let's cast the eight hundred pound gorilla in the analogy, the insurers, as 'the owner'. More specifically, let's call the beast 'the hometown owner'. And then let's say the umpire's salary is paid by the owner.\n\nWith the game yet played on rural fields, before the advent of electronic pitch-tracking devices, before the price of every pitch was calculated, before the global media contracts, the strike zone was a sacred space, the tiny, arbitrary, marked off piece of ether from which intimacy the entire game was decided. Before the 'owners' blew the entire field up to stadium-size the game was about conceptualization and process; before psychiatry was snatched up by the insurers the pathologies were sought in subjectivity over objectivity. Artfulness existed alongside science. What, after all, did psychiatrists care for nosology before the rise of private insurance over the past several decades? Disordered thinking, as opposed to ordered thinking, was just that. Slapping a name on it did little to change the fact. One man's depression is another man's 'blues', and what does the patient care for the label?\n\n'Carving nature' does require a measure of reliability, true, but the only conversations I have had in which I have coughed up the full DSM criteria have been those over the telephone, most often in the emergency room, with insurance reviewers 'objectively' determining, from up to thousands of miles away, whether a particular patient warrants two days or three days in which to be cured. And at that, for the benefit and safety of my patients, my strike zone widens tremendously after five minutes, and my diagnoses tend to reduce to the very non-DSM, if at times heavily punctuated, 'imminently suicidal!' or 'imminently homicidal!'. The arguments tend to end there, and it is apparent that what is missing in the epistemological umpire analogy is the hard baseball rule against arguing balls and strikes.\n\nAs a former academic, however, I simply have to believe that there is an inherent value in the pursuit of knowledge for knowledge's sake, that all sciences, veiled or not, are interwoven, regardless of the current paradigms, and the loss of even one is somehow crippling to the others. But 'the owners', despite the fact that they stand oblivious, willfully or not, to the devastation they create, can no longer be ignored in these arguments. Whatever the historical mechanisms, the pursuit of knowledge has come up hard against the pursuit of profit in these last few decades. I contend that the process of classification is the process that, if not created by, than at least has been manipulated ever since by the owners. As students of the human mind, arbitrary classification of disorders of the mind does not inform us; it informs the gorilla. Describing 'normalcy' and 'variants thereof' only serve to destroy further an already hobbled subjectivity. Nosology destroys narrative, and where formerly our patients were more appropriately likened to novels, they are now become, for the ease of illiterate overlords, more like newspapers.\n\nAs the noted Assyriologist, Jean Bottero, put it in defense of his own limited field, \"Yes, the university of sciences is useless; for profit, yes, philosophy is useless, anthropology is useless, archaeology, philology, and history are useless, oriental studies and Assyriology are useless, entirely useless. That is why we hold them in such high esteem!\" \\[\\[42\\], p. 25\\] Psychiatry finds itself in a unique position among the 'useless' sciences. Like the umpire offered a bribe by the owner, if the field chooses utterly to subserve profit it likely stands to gain tremendously. If the field chooses to uphold an ideal of humanism in the face of gorilla-ism then we will likely be faced with the same fate as philosophy, anthropology, archaeology, philology, and history. In which case let us all call 'em as we see 'em, keep the paperwork tidy, and at the very least be ever mindful of the watchful gaze of the gorilla.\n\n## Allen Frances responds: There Is A Time And Place For Every Umpire\n\nNone of the five umpires is completely right all of the time. And none is totally wrong all of the time. Each has a season and appropriate time at the plate.\n\nForty years ago, Umpires 1, 3, and 5 were in competitive ascendance. The nascent school of biological psychiatry was a confident Umpire 1- convinced that mental disorders would soon yield their secrets and be as fully understood as physical illnesses. In fact, there was a heated controversy whether the new diagnostic manual (DSM III) then being prepared was a catalog of 'disorders' or of their much preferred term 'diseases'.\n\nIn sharp contrast, the competing models that dominated psychiatry forty years ago were very much like the skeptical Umpires 3 and 5- in their different ways, all were nihilistic about the value or reality of psychiatric diagnosis. Psychoanalysis dealt with highly inferential concepts impossible to reduce to reliable diagnosis. Family, group, and community psychiatry went so far as to deny that the individual patient was a proper or very relevant unit for diagnostic assessment, preferring models that diagnosed the system at larger aggregates of interpersonal affiliation. When Szasz, then as now, decried the 'myth of mental illness', there was little coherent opposition outside the group of the smugly confident pioneers of biological psychiatry (who soon would be hoisted by their own petard).\n\nThe years have not been kind to umpires 1, 3, and 5. Each still stakes some small claim to attention, but umpire 2 now clearly rules and welcomes the collaboration of his close cousin, the ever practical umpire 4.\n\nWhy the revolution in epistemological sentiment? Biological psychiatry helped spark a wondrous neuroscience revolution that is perhaps the most thrilling focus of twenty first century biological science. But the findings have revealed a remarkably complex brain unwilling to yield any simple answers. There is thus far almost no translation from the glory of basic science discovery to the hard slog of understanding the etiology and pathogenesis of the 'mental disorders'. These no longer seem at all reducible to simple diseases, but rather are better understood as no more than currently convenient constructs or heuristics that allow us to communicate with one another as we conduct our clinical, research, educational, forensic, and administrative work.\n\nMost hard core biological psychiatrists have lost heart in the na\u03c2ve faith of umpire 1 that he can define simple models of illness. Those who were hunting (and reporting) the gene or genes for schizophrenia, bipolar, and other disorders have been forced repeatedly to retract and eat humble pie. Initial findings never achieved replication for what became the obvious reason that there is no 'disease' of schizophrenia- that instead schizophrenia is better understood as just a construct (albeit it a very useful one) with hundreds of different 'causes'.\n\nMeanwhile the diagnostic nihilism of Umpires 3 and 5 also became less relevant when DSM III proved that psychiatric diagnosis could be a reliable and useful tool of communication.\n\nUmpire 2 now rules. Mental disorders are no more and no less than constructs. And Umpire 4 is quick to point out that they are very useful constructs. The current dominance of Umpires 2 and 4 is temporary, and certainly not complete. In some very gradual and piecemeal way, the future holds hope for an increased role for Umpire 1. As we slowly discover the biology of mental disorders, small subunits will cohere around a common pathogenesis and declare themselves as a disease. This is beginning to happen for the dementias of the Alzheimer's type. But it will always be necessary to retain the corrective voices of the skeptical Umpires 3 and 5- to remind us just how little we know and how feeble are our tools for knowing.\n\n### Reply to Drs Zachar and Lobello\n\nThank you for your contribution which I received after writing my own. You have stated my position with much greater clarity and erudition than I could muster.\n\n### Reply to Dr Pouncey\n\nThank you for your clarification of the Umpire metaphor. Your analysis nicely demonstrates the similarities and the differences in the positions of Umpires 1 and 2- both accept the possibility of an independent reality, but differ sharply in there estimation of our current ability to apprehend it.\n\n### Reply to Dr Ghaemi\n\nDr Ghaemi sets up a false and totally unnecessary dichotomy between his true believer version of realism and what he calls \"taking a random walk\". It is possible, indeed necessary, to take a very modest position regarding the current state of certitude of psychiatric knowledge on the causes of psychopathology without assuming that we know nothing or are walking totally blind or that our constructs have no current heuristic value. Umpire 2's honest admission that he can do no better than call them as he sees them does not deny the possibility of real strikes and real balls- it just states the very constrained limits of our apprehension. I have no problem at all with the metaphor of epistemic iteration- it is obviously the route of all science. But let's realize how early in the path we are and how uncertain is its best direction.\n\n### Reply to Dr Cerullo\n\nHow comforting to be a first umpire. I admire the magisterial confidence of Dr Cerullo's statement, \"Most working scientists and philosophers would be classified as modern realists who believe there is an independent objective external reality\". I wish I could feel so firmly planted in a \"real\" world and possess such na\u03c2ve faith in mankind's capacity to apprehend its contours. Alas, as I read it, the enormous expansion of human knowledge during the last hundred years is enough to make umpire 1's head spin with confusion. The more we learn, the more we discover just how much we don't (and perhaps can't) know. Einstein gave us a four dimensional world that even physicists have trouble visualizing. Then the string theorists made it exponentially more complicated by expanding the dimensions into double figures and introducing conceptions of reality that may or may not ever be testable. The quantum theorists describe a \"spooky\" (Einstein's term) and inherently uncertain world that lends itself to extremely accurate large n prediction, but totally defies our intuitive understanding of the specific mechanics. It also turns out that we are pathetically limited in our sensory capacities, even when they are extended with our most powerful sensing instruments. Evolution allows us to detect only 4% of our universe, the rest of energy and matter being \"dark\" to us. Indeed, there may be a vast multiplicity of multiverses out there and we may never know them. So I don't see human beings as having great status as judges of reality- we are like mice describing the proverbial elephant- having available only fallible and very temporary constructs.\n\nTo get back to our umpires, the connections between brain functioning and psychiatric problems are definitely real, but they are so complex and heterogeneous as to defy any simple \"realist\" faith that we are close to seeing them straight on, much less solving them.\n\n### Response to Dr Wakefield\n\nDrs Wakefield and Pouncey have made many of the same important points. Dr Wakefield's \"humble realism\" (associated with an honest and flexible willingness to admit fallibility and the possibility of error) works for a great baseball umpire and is not a bad model for a psychiatric diagnostician. The difference between umpire 2 and umpire 1.5 depends on how close you think our field is to understanding the reality of psychopathology. I am even more humble than Dr Wakefield and will stick with umpire 2.\n\n### Reply to Dr Pierre\n\nI agree.\n\n### Reply to Dr Greenberg\n\nIn defending Umpire 3, Dr Greenberg assumes a grandly, neutral view of man's place in the world and makes clear how limited are our abilities in naming and classifying its manifestations. Greenberg rightly suggests that the distinction between a broken branch and a broken femur may be extremely meaningful to the patient and his doctor, but is really trivial in the grand scheme of an indifferent nature. He might equally have pointed out that from a bacteria's perspective, pneumonia is not a disease- it is just an opportunity for a good feed. Diseases, according to Greenberg's argument, are no more than human constructs made up de novo by us as inherently self interested third umpires.\n\nFrom Greenberg's lofty perch, mankind's attempts to label do seem pathetically self referential and solipsistic, extremely limited in their apprehension of reality (even assuming that there is a graspable reality ready to be apprehended). But it seems to me that his level of philosophic detachment works only in the exalted theoretical realms, and contrary (to his statement) fails badly to do justice to the needs and opportunities of our everyday, \"common sense\" world.\n\nGreenberg and I do agree completely on several points: 1) if mother nature had the gift of speaking our language and the motivation to do so, she would probably indicate she couldn't care less about our names and that she doesn't feel particularly well described by them; 2) our categories are no more than tentative approximations and are subject to distortion by personal whims, cultural values local to time and place, ignorance, and the profit motive; and, 3) psychiatry's names should be used with special caution because they lack strong external validators, carry great social valence, and describe very fuzzy territorial boundaries.\n\nWhere my umpire 2 position differs from Greenberg's umpire 3 is in our relative estimations of how closely our names and constructs can ever come to approximating an underlying reality. My umpire 2 position is skeptical about umpire 1's current ability \"to call them as they are\" and advises modesty in the face of the brain's seemingly inexhaustible complexity. But I remain hopeful that there is a reality and that, at least at the human level, it will eventually become more or less knowable. We may never fully figure out the origin and fate of the universe or the loopy weirdness of the quantum world. But the odds are that decades (or centuries) of scientific advance will gradually elucidate the hundreds (or thousands) of different pathways responsible for what we now crudely call \"schizophrenia\".\n\nGreenberg is more skeptical than I about the progress of science and is, at heart, a platonic idealist who finds life cheapened by excessive brain materialism. He sees psychiatric disorders as no more than human constructs - metaphors, some of which are useful, some harmful. His umpire 3 does not does not believe the glory and pain of human existence can or should be completely reduced to the level of chemical reactions or neuronal misconnections. This is a fair view for poets and philosophers (and Greenberg is both), but I see a ghost in his machine and dispute that allowing it in makes \"common sense\".\n\n### Reply to Dr Pincus\n\nThank you for inventing the fourth umpire. Dr Pincus is the most practical of men and he has created a handy metaphor for describing the ultimate goal of any DSM- to be useful to its users. There is only one problem with the fourth umpire's position- but it is a big one. There is no external check on his discretion, no scientific or value system that guides what is useful. Everything depends on the skill and goodwill of the umpire. In the wrong hands pragmatism can have dreadful consequences- commissars who treat political dissent as mental illness or judges who psychiatrically commit run of the mill rapists to keep them off the streets. But to ignore the practical consequences of psychiatric decisions leads to its own set of abuses- most recently diagnostic inflation and excessive treatment.\n\n### Reply to Dr Szasz\n\nI have enormous respect for intellectual reach and depth of Dr Szasz' critique of psychiatric diagnosis and for the moral power of his lifelong efforts to prevent its misuse. He skillfully undercut the pretentions of the Umpire I position at a time when its biological proponents were at their triumphalist peak, loudly trumpeting that they were close to finding the gene for schizophrenia and to elucidating its brain lesions. He anticipated and exposed the naivete of these overly ambitious and misleading claims. He has fought the good fight to protect the rights, dignity, and personal responsibility of those deemed to be \"mentally ill\". My argument with Dr Szasz is that he goes too far and draws bright lines where there are shades of gray. Surely, he is right that schizophrenia is no \"disease\", but that does not mean it is a \"myth\". Surely, he is right that psychiatric diagnosis can be misused and misunderstood, but that doesn't mean it is useless or can be dispensed with. Dr Szasz is correct in defining the many of problems with psychiatric diagnosis, but doesn't have alternative solutions. There is a baby in there with the bath water he is so eager to discard.\n\n### Reply to Dr Martin\n\nI agree that we can't always assume the Umpires are acting only from the purist and most disinterested of motives. Games can be fixed for financial gain and psychiatry operates in a real world of large drug company, insurance, and publishing profits. My experience has been that the actual framers of DSM IV and of DSM 5 have not been shills for industry- but that heavy drug marketing has led to much over-diagnosis using DSM IV and that the risks are greatly heightened because of the new diagnoses being suggested for DSM 5. Dr Martin's comment makes clear that we must be aware the diagnosis of a given patient can be distorted by real world economic factors and must be ever vigilant to protect the integrity of the process.\n\n# Question \\#2: What is a Mental Disorder?\n\n*It has been difficult to reach agreement on a definition of mental disorder. Could you comment on this problem, or offer what you think is an adequate definition of the concept, mental disorder?*\n\n## Introduction\n\nOn the face of it this is a strange question. As treating clinicians we surely should be able to offer a definition of what it is we treat. As researchers surely we should be able to define the object of our research. And finally as philosophers writing about mental illness, surely we should be able to provide a definition of the object of our investigation. So why is it so difficult to accomplish these tasks? Allen Frances has puzzled over this question, and as he indicates below, it leads him into Humpty Dumpty's world of \"shifting, ambiguous, and idiosyncratic word usages.\"\n\nFailures to accomplish a consensually accepted definition lead in two directions: give up or keep trying. The first approach is represented by Warren Kinghorn, who argues in his commentary that we won't achieve the desired definition and don't need it anyway - any more than other specialists do for their work - and thus should abandon the effort to try yet again to get it right in DSM-5.\n\nThe opposite approach is represented in different ways: by Jerome Wakefield, on the one hand, and Stein and colleagues, on the other. In his contribution to this article Wakefield presents the evolution-based harmful dysfunction definition of mental illness for which he is justly well-known. In this contribution he argues that the varied positions of figures like Allen Frances and Kenneth Kendler depend implicitly on the HD understanding and definition of mental disorder.\n\nStein and colleagues (not represented in this article) \\[43\\] take another approach in trying to improve the DSM-IV definition by operationalizing it, and then going to work on the operationalized definition. They tweak some of the DSM-IV (definitional) criteria as well as adding further criteria, e.g., acknowledging the normative, value-laden aspect of many diagnoses. In their effort to improve the DSM-IV definition, they address many of the complaints lodged against DSM-IV (co-morbidity, poor separation between diagnoses, poor separation from normality, etc.) In trying to be comprehensive, they include issues of clinical utility, scientific accuracy through validators, and pragmatic concern for patient outcome. They do not, however, deal with the issue discussed in Question 5, namely, that these can be conflicting agendas, and that at times we effectively prioritize one over the other.\n\nLife never being as neat as one would like it, Allen Frances and Joseph Pierre defeat my effort of a simple dichotomy and occupy a middle ground between the alternatives of giving up on a definition or trying to improve it. Each argues for attempting a definition but assures us that it will be a messy undertaking.\n\nAnd that conclusion reminds me that in this search for an adequate definition of mental disorder it would be useful to invoke Wittgenstein's notion of family resemblances. In discussing the essence of language or language games, Wittgenstein writes:\n\nInstead of producing something common to all that we call language, I am saying that these phenomena have no one thing in common which makes us use the same word for all,-but that they are *related* to one another in many ways. And it is because of this relationship, or these relationships, that we call them all \"language.\"...\n\nI can think of no better expression to characterize these similarities than \"family resemblances\"; for the various resemblances between members of a family: build, features, colour of eyes, gait, temperament, etc. etc. overlap and criss-cross in the same way.-And I shall say: 'games' form a family. \\[\\[44\\], pp. 31-32\\].\n\nThe Wittgensteinian approach would represent the middle ground of Frances and Pierre. In technical DSM terminology, diagnoses don't all share the same properties or validators; they resemble one another because they share some. A diagnosis might have a place in the nosology because of \"historical accretion,\" but might lose it because it doesn't meet standards of other current validators. Examples are the paraphilias and conduct disturbances, both of which may lose their admission tickets because of excessive normative valence and inadequate internal distress. Borderline Personality Disorder (now Borderline Type) is being retained presumably because descriptively it covers a lot of the symptomatic morass otherwise not covered; but with its known problems - heterogeneous presentation, excessive comorbidity, lack of genetic or pathophysiologic foundation - it will probably collapse and be carved up eventually for lack of real validators, i.e., not enough validators in common with other members of the DSM family. And finally, to underline Pierre's point, distinguishing a validator from a value is an ambiguous enterprise.\n\nI readily acknowledge that the family resemblance model will not satisfy those in search of a tighter definition of what constitutes a psychiatric disorder. Its main virtue is that it reflects how we actually \"define\" - and in the absence of a tighter definition, will continue to \"define\" - psychiatric disorders.\n\n## Commentary\n\nJerome C. Wakefield, Ph.D., D.S.W.\n\nSilver School of Social Work and Department of Psychiatry, New York University.\n\nAl Frances's powerful writings on psychiatric diagnosis offer vigorous arguments about what should and should not be diagnosed as psychiatrically disordered (not only regarding DSM-5 overreaching; consider his adding the clinical significance criterion to most diagnostic criteria sets in DSM-IV). Yet, Frances vehemently denies that there is any coherent concept underlying our judgments of what is and is not a disorder. This may save him from a troublesome additional debate, but, as observed by some commentators, it undercuts the coherence let alone force of his critique of false-positive implications of DSM-5 proposals.\n\nDespite his disavowals, Frances's arguments derive their enormous power from an implicit reliance on common intuitions about the concept of disorder as failure of biologically designed human nature. Sometimes this implicit appeal emerges explicitly, as in Frances's \\[45\\] explanation of why he rejects DSM-5's proposed approach to behavioral addiction: \"The fundamental problem is that repetitive (even if costly) pleasure seeking is a ubiquitous part of human nature.... The evolution of our brains was strongly influenced by the fact that, until recently, most people did not get to live very long. Our hard brain wiring was built for short term survival and propagating DNA- not for the longer term planning that would be desirable now that we have much lengthened lifespans.... This type of hard wiring was clearly a winner in the evolutionary struggle when life was \"nasty, brutish, and short\". But it gets us into constant trouble in a world where pleasure temptations are everywhere and their long term negative consequences should count for more than our brains are wired to appreciate.\"\n\nNotice that Ken Kendler et al. \\[46\\], on the extreme opposite side of the DSM-5 debate, implicitly appeal to the same biological-design criterion when explaining why fearful distress in reaction to real danger is not a disorder: \"An individual experiencing a panic attack after just barely escaping a fatal climbing accident would not be considered psychiatrically disordered because the mechanism for panic attacks probably evolved to prepare us for such situations of real danger\" (p. 771).\n\nSo, what is the concept of disorder to which Frances and Kendler implicitly appeal? The DSM's definition of disorder says that a disorder exists only when symptoms are caused by a dysfunction in the individual and lead to certain forms of harm, such as distress or impairment. Observing that the concept of \"dysfunction\" was left unelaborated and that distress or disability are not the only harms that would warrant diagnosis, I proposed what I labeled the \"harmful dysfunction\" (HD) analysis of the concept of disorder \\[47-52\\].\n\nThe harmful dysfunction analysis maintains that the concept of disorder has two components, a factual component and a value component. To be a disorder, a condition must satisfy both components. The value or \"harm\" component refers to negative or undesirable or harmful conditions, which applies to most symptomatic conditions. Obviously, who gets to make the judgment that a condition is harmful and on what grounds (especially in a pluralistic society) is a complex issue. But the basic point is that no condition, even if a clear biological malfunction, is a disorder if it is not considered in some sense harmful to the individual or society. This is the basis for the \"clinical significance\" requirement.\n\nThe factual component requires that the condition must involve a failure of some mental mechanism to perform one of its natural, biologically designed functions. This is highly inferential and speculative and fuzzy at this stage of knowledge of mental processes, but it is the conceptual target at which we aim nonetheless. Indeed, although both the notions of dysfunction and harm are fuzzy concepts, as long as they determine a range of clear cases on either side of the disorder\/non-disorder boundary, they can provide a cogent and useful conceptual structure. Other useful categorical distinctions - such as between night and day, or child and adult - also have fuzzy boundaries, and pragmatic considerations determine specifically where the dividing line is drawn (see Question 1).\n\nToday, we understand that human nature -specifically, species-typical biological design -- is due to evolution through natural selection. So, dysfunction in the sense relevant to judgments of medical disorder consists of failure of internal mechanisms to perform evolved functions. The \"dysfunction\" component of the analysis means that, as far as the legitimate application of the concept of disorder goes, disorder cannot be manufactured from personal or social values and used as a cover for \"treatment\" in service of social control. The \"dysfunction\" requirement places a limit on what can be legitimately said to be a disorder, and explains why many negative conditions are false positives.\n\nHow can we test this account of the generally shared meaning of our distinction between disorder and non-disorder? One way is to see if it is consistent with some of our shared intuitions. Consider some simple examples of conditions NOT considered disorders. Neither illiteracy nor an immigrant's inability to speak the local language are considered disorders, yet are terribly impairing, potentially distressing, and disadvantageous mental conditions (whereas dyslexia and aphasia are disorders). Being a \"night person\" rather than a \"morning person\" in a 9-to-5-structured culture is potentially disadvantageous, but considered a normal variation. Fertility when pregnancy is unwanted, pregnancy when children are unwanted, and pain during childbirth are all undesirable and potentially harmful conditions commonly treated by physicians but not considered disorders. Neither debilitating fatigue after exertion nor sleep - probably the single most massively impairing human condition of all, rendering virtually everyone semi-paralyzed and periodically hallucinating for one-third of their lifespans -- are seen as disorders. Nor is delinquent behavior by rambunctious teenagers or grief after a loved one's death.\n\nThe source of these classificatory judgments cannot be reduced to personal or social values. Many of these non-disordered conditions are personally and\/or socially undesirable. There is some additional element operating here to explain these judgments. The common element is that we consider all these conditions to be part of the way human beings are designed to function, however problematic they may be in our current environment.\n\nMoreover, our intuitions are that entire cultures can be incorrect about their disorder judgments. Victorians' deeply held values and beliefs led them to classify masturbation and female clitoral orgasm as disorders, some ante-bellum Southern physicians considered slaves who ran away from their masters to be psychiatrically disordered (\"drapetomania\"), and Soviet psychiatrists treated political dissidents as disordered. We believe that these diagnostic judgments, although consistent with the respective cultures' values, were incorrect -- not correct \"for them\" and incorrect for us, but just plain wrong. The factual component of \"disorder\" explains how this can be so.\n\nNow, consider the types of conditions to be found in the DSM. At this point in the history of psychiatry, we are about where Hippocrates was in formulating diagnostic categories. We know virtually nothing about the underlying natures of mental mechanisms, but from circumstantial evidence and indirect inferences, we infer what conditions are likely disorders. The superordinate categories in the DSM - e.g., psychotic (thought) disorders, anxiety disorders, mood (sadness\/elation emotion) disorders, sexual disorders, sleep disorders, and so on - correspond to human systems about which we feel we can pretty reliably infer that they are biologically designed to operate in certain ways, and we can recognize in a range of cases when something has gone wrong - albeit with large fuzzy boundaries and large ranges of uncertain cases given our ignorance.\n\nAnother way of assessing the HD analysis is by whether it accomplishes certain important goals motivating such an analysis. At a minimum, an analysis of the concept of mental disorder should do four things:\n\n\\(1\\) Explain widely shared classificatory judgments about whether or not problematic conditions are disorders.\n\n\\(2\\) Explain why mental disorders are disorders in the same sense as physical disorders, thus why psychiatry is part of medicine.\n\n\\(3\\) Explain the distinction between control of socially undesirable mental conditions versus treatment of mental disorder.\n\n\\(4\\) Offer a fruitful way of thinking about research.\n\nThe first three goals are addressed above. As to research, the HD analysis explains the primary goal as seeking to understand mental mechanisms and their functions, and ultimately to identify and distinguish specific mental dysfunctions, yielding an \"etiological\" classification.\n\nFrances's end run around the concept of disorder is accomplished partly by his insisting on a scientific-sounding \"cost-benefit analysis\" of each DSM-5 proposal. This is a useful rubric for raising concerns and is of course an improvement over wanton unreflective pathologization. In drawing boundaries in some fuzzy domains, the concept of disorder offers little guidance, and perhaps cost-benefit analysis is all we can do. However, generally reframing the process of deciding whether to pathologize a condition as a cost-benefit analysis, if taken seriously, is not only intellectually unsupportable but dangerous, for the same reasons why one would not want cost-benefit analysis to determine whether an accused was judged guilty in a trial - namely, one loses any protection against social control aspirations. After all, for all we know a careful cost-benefit analysis might show that from a social perspective \"treating\" the Soviet dissidents for psychosis, slaves who ran away for \"drapetomania,\" and women for \"pathological\" clitoral orgasms was justifiable in the respective social circumstances--but it was incorrect use of diagnosis nonetheless, because the individuals so labeled (in many cases) did not actually suffer from disorders. Just as the health professions spring from the concept of disorder, restraint of efforts to use the health professions for social control also spring from this crucial concept. To deny the existence and importance of the concept of mental disorder in a discussion of the validity of diagnostic criteria in psychiatry is sort of like a teacher denying there is knowledge or ignorance before entering the classroom or a judge denying there is criminal guilt or innocence before rendering a verdict.\n\nI believe that, despite his misstep on the concept of disorder, Frances is entirely correct in his relentless focus on false positives as the Achilles heel of the DSM-5 effort. The reason why this is so is not simply a matter of preventing repetitions of the \"false epidemics\" of the past due to criteria revisions. The deeper point is that the very aspirations of the editors of DSM-5 for a paradigm shift towards etiologically based diagnosis - a shift for which we are not yet ready - depends in the long run on distinguishing etiologies. That means distinguishing different dysfunctions as well as distinguishing normal non-dysfunctions from dysfunctions that underlie similar symptom presentations.\n\nBecause we are not yet ready to distinguish dysfunctions, DSM-5 must remain theory neutral. However, a serious conceptual-validity review could have allowed us to do considerably better in distinguishing dysfunction from likely non-dysfunction for many categories. Instead, the DSM-5 work groups are expanding diagnoses so that more individuals coming into consultation can be labeled with a disorder, without careful conceptual analysis. Given this focus, the DSM-5 is likely to take psychiatry further away from the editors' stated goal. The DSM-5 could have made progress towards etiologically grounded categories by eliminating some of the false positives that afflict the manual's operational criteria sets, placing them under separate V Code categories. With false positives swamping many categories, psychiatric science will continue to flounder, unable to distinguish dysfunction etiologies because its criteria cannot in many cases even distinguish internal dysfunction where something has gone wrong from intense but normal biologically designed reactions to events.\n\n## Commentary: Definitions of \"Mental Disorder:\" Elusion and Illusion\n\nWarren Kinghorn, M.D.\n\nDuke University Department of Psychiatry.\n\nThe definitions of \"mental disorder\" which have appeared in each edition of the *DSM* since *DSM-III* (hereafter referred to as the \"*DSM* definitions\") are both carefully crafted and widely ignored. Pioneered by Spitzer and Endicott as a means for demarcating (true) mental disorders from non-pathological conditions in the wake of controversy over the status of homosexuality in *DSM-II* \\[53\\], the *DSM* definitions have from the beginning, and despite copious qualification and caveat, proved controversial and largely irrelevant to the practical use of the *DSM*. Specific criticisms of the *DSM* definitions, such as their circular use of \"clinically significant,\" are by now commonplace \\[54,55\\]. The *DSM-5* Task Force has proposed an updated definition which seeks to improve on prior definitions and which emphasizes the pragmatic and method-driven nature of psychiatric nosology \\[43\\]. The *DSM-5* proposal is worthy of sustained critical consideration which I do not offer here. In this brief account, rather, I wish to offer three reasons why future editions of the *DSM* should not include a definition of \"mental disorder.\" In each case, I argue, the *DSM* definitions *seem* to contribute to a certain good, but this is illusion.\n\nFirst, the *DSM* definitions *seem* only to describe the terrain of what is already considered \"mental disorder,\" innocuously supplying rough logical boundaries to psychiatric praxis without limiting or shaping it. But this is an illusion. Although, as Sadler \\[54\\] points out, the *DSM* definitions have not in practice influenced the way that new diagnoses are incorporated into the *DSM*, they do provide a regulative language for how one speaks about the mental disorders that are already there. Questions, for example, about whether major depressive disorder most properly inheres in an individual or in a group (or even in a society; \\[56\\]) run counter to the methodological individualism of the *DSM*, enshrined in its definitions of mental disorder, and are therefore difficult to ask without bringing the entire *DSM* project into question. This should not be the case; it is precisely by excluding useful questions that the *DSM* renders itself an obstacle to nosological advance rather than a catalyst to it.\n\nSecond, the *DSM* definitions of mental disorder *seem* to demarcate a safe conceptual territory within human life and experience within which the medical model can properly rule. The unspoken but nonetheless persuasive model seems to run something like: *Why should a psychiatric technology (medication, ECT, manual-driven psychotherapy, etc.) be deployed within this situation?* A: Because it's a mental disorder, and one uses psychiatric treatments for mental disorders. *Q: But how do we know that this is a mental disorder?* A: Well, it's in the *DSM*, and besides, it fits into the conceptual space which the *DSM* defines as \"mental disorder.\" But this, too, is illusory and deceptive. The deployment of psychiatric technology is not justified because a particular condition is demarcated as \"mental disorder\" but because, after all goods are weighed and all options considered, the use of technology is prudentially indicated. Whether the condition is classified as \"mental disorder\" has little to do with this particular question. Although the *DSM* definitions importantly exclude certain situations (e.g., primary social deviance) from the medical model, it would be better for the *DSM* simply to stipulate these ethical commitments rather than to embed them within the definition of \"mental disorder.\" Moreover, the fact that a certain condition satisfies the *DSM* definitions does not serve as *prima facie* justification of the deployment of technology for that condition, and the *DSM* should not collude in any contrary assumption.\n\nFinally, the *DSM* definitions *seem* to focus diverse mental health professionals on a common moral project. Clinicians may disagree about etiology and treatment, that is, but can at least join together in stamping out \"mental disorder\" as described in the generously broad *DSM* definitions. But this also is illusion, proved empirically to be so by the failure of every *DSM* definition to achieve widespread consensus and destined to be proved again in the inevitable failure of the *DSM-5* definition. The reason for this is not that the definitions are poorly crafted (quite the opposite) but that such consensus, within the contemporary mental health landscape, is not a conceptual possibility. For example, with regard to the *DSM-5* proposal - the best definition to date - particular clinicians are certain to reject not only nosological individualism but also the foundational assumptions behind \"underlying psychobiological dysfunction,\" the exclusion of expectable responses to common stressors and losses, and the distinction between \"behavioral\" and \"psychological.\" Furthermore, even if consensus on a formal definition were attainable, it would accomplish little since agreement about \"dysfunction\" and \"impairment\" and \"deviance\" can be no stronger than correlative agreement about proper human functioning in a particular situation (such that, e.g., what one clinician judges as impairment, another judges as unreasonable expectation). There is, unfortunately, no more agreement about proper human function than about the (closely related) nature of \"mental health.\"\n\nCrafting a definition of mental disorder is a useful thought-experiment for psychiatric nosologists and debating such definitions is great fun for the philosophically-minded. But in a document as influential and generally accessible as the *DSM*, such definitions elude and mislead more than they illumine. They should therefore be honorably retired.\n\n## Commentary\n\nJoseph Pierre, M.D.\n\nUCLA Department of Psychiatry.\n\nDeveloping an ironclad definition of mental illness (or for that matter the more general notion of disease) is indeed a daunting task \\[43,57-59\\]. Most attempts at a medical model definition are based on some variation on the concept of \"something that has gone wrong biologically with an individual that results in distress or functional impairment,\" but problems with this approach quickly emerge. First, we have no firm explanations for what's biologically wrong in mental disorders \\[57\\] - no \"underlying psychobiological dysfunctions\" \\[43\\] have yet been elucidated (and as the joke goes, in the few cases where such \"lesions\" have been identified, they transform from psychiatric disorders into neurologic diseases). Second, the concepts of \"wrongness\" and \"dysfunction\" are unavoidably value-laden \\[57,60\\], while \"distress\" and \"suffering\" are subjective and relativistic \\[43,61\\]. Third, distinguishing between mental illness and \"problems of living,\" \"expectable responses to common stressors\" (or extraordinary stressors), and \"social deviance or conflicts with society\" is at best challenging \\[43\\], given the inextricable, reciprocal relationship between individuals and their environments\/cultures \\[58\\]. Therefore, in the absence of lab tests to detect biological lesions, psychiatric diagnosis inevitably rests upon some kind of \"judgment call\" on the part of a clinician and the inescapable conclusion that \"no definition perfectly specifies precise boundaries for the concept of 'mental disorder \\[43\\].'\"\n\nAs suggested earlier, clinicians do not in general fret over what does or does not constitute a disease. If, for example, a patient's arm is broken in a car accident, a doctor doesn't lose sleep pondering whether this represents \"broken bone disorder\" or simply an expected response to an environmental stressor - the bone is set and the arm is casted. For psychiatrists, ever since Freud's development of \"The Talking Cure,\" the business of psychiatry has increasingly shifted from asylum care of patients with psychosis to outpatient treatment of the \"worried well\" \\[62\\]. Likewise, we now live in a society that regards the attainment of happiness as a worthwhile goal in life, if not an entitlement \\[63\\]. Therefore, mental disorder or not, clinicians working in \"mental health\" see it as their calling to try to improve the lives of whomever walks through their office door seeking help.\n\nBut clinical decisions are not the only decisions that hinge upon diagnosis. The underappreciated challenge to defining a mental disorder in the real world stems from the many different questions that are ultimately being asked of diagnosis:\n\nShould \"disorder X\" be treated?\n\nWhat is the best way to treat \"disorder X\"?\n\nShould screening for \"disorder X\" be implemented in the community in the interest of treatment and prevention?\n\nShould special school services be offered for children with \"disorder X?\"\n\nShould insurance companies reimburse for care based on \"disorder X?\"\n\nShould research funding be granted to study \"disorder X?\"\n\nShould a patient population based on \"disorder X\" be selected for etiologic research?\n\nShould someone with \"disorder X\" who has committed a crime be sentenced to prison or to involuntary psychiatric treatment?\n\nThe far-reaching implications of these questions render the distinction between mental illness and normality far more significant than when considered in a clinical vacuum, especially in the era of rationed healthcare services, insurance reimbursement, and competitive research funding. For the clinician and patient, erring on the side of assigning, or receiving, a diagnosis of a mental disorder has become incentivized, but future economic and policy decisions may necessitate a more conservative threshold for defining mental illness \\[61\\]. Therefore, the many questions asked of diagnosis cannot be answered by any single definition of mental illness, or by simply referring to the DSM. Instead, and in particular as DSM-5 embraces the concept of diagnostic spectra in which the borders between pathology and normality are stretched, wide-ranging consideration and context-specific analyses by clinicians, patients and their families, researchers, DSM architects, and policy-makers will be a vital, ongoing process that shapes the fate of modern psychiatry.\n\n## Commentary\n\nJohn Chardavoyne, M.D.\n\nYale University Department of Psychiatry.\n\nHow should American psychiatry define \"mental disorder?\" There is a varied array of dimensions that are emphasized in the various definitions, including the biological basis of psychiatric illness; the behavioral manifestations; the severity of impairment; the level of distress; and the differentiation between pathology versus normalcy. In this commentary I will provide examples of these different aspects, suggest reasons for the confusion, and propose the beginning of a more integrated approach.\n\nFirst, let's review elements of a few of the definitions that have been proposed. The DSM-IV definition emphasizes explicitly that it characterizes disorders, not people; that there are biological, psychological, or behavioral dysfunctions that result in a psychological or behavioral syndrome; this creates impairment or distress; it is not a result from discord between the individual and society; and that these problems are not culturally-sanctioned. <http:\/\/www.DSM5.org\/ProposedRevisions\/Pages\/proposedrevision.aspx?rid=465#>. The proposed changes for DSM-V include that there is psychobiological dysfunction that results in a psychological or behavioral syndrome; that there is evidence of distress or impairment in functioning; that the response is not expectable and not culturally-sanctioned; it is not a result from discord between the individual and society; and that there is diagnostic validity and utility. <http:\/\/www.DSM5.org\/ProposedRevisions\/Pages\/proposedrevision.aspx?rid=465>. According to the National Alliance on Mental Illness, mental illness is defined with an emphasis on the medical model. <http:\/\/www.nami.org\/Content\/NavigationMenu\/Inform_Yourself\/About_Mental_Illness\/About_Mental_Illness.htm>. The International Classification of Diseases-10 definition highlights the symptoms. <http:\/\/www.who.int\/classifications\/icd\/en\/bluebook.pdf>.\n\nThese definitions reflect the difficulties in adequately describing the nature of a \"mental disorder.\" They reflect the uncertainty about how biology results in psychiatric manifestations and vice versa and difficulties in the following: the way to differentiate pathology from normalcy, how to categorize the syndromes, and the location of the source of dysfunction. Along these lines, a chief concern is the emphasis on overt behaviors to classify mental disorders. Despite how patients report subjective experiences and distress, the overt behaviors have been used as the major indicators of disorder. The difficulties related to quantifying the first-person subjective experience compared to the third-person objective observations (overt behaviors) manifest here. The Psychodynamic Diagnostic Manual reintroduces the subjective experience into assessment \\[64\\].\n\nThis begs the question of the direction that psychiatry wants to go. Do psychiatrists want to be able to treat an individual with manic symptoms and also someone with intimacy problems? Presumably the former would have more behavioral markers than the latter. However, how can one quantify the suffering of one versus the other? Simply because there are not as many behavioral manifestations, does that mean that the person is not suffering sufficiently to warrant treatment by a psychiatrist? Equally important, does that suggest that insurance companies continue to determine reimbursement for treatment based on behaviors without considering level of suffering and other factors that contribute towards psychological dysfunction like thoughts, feelings, and relationships?\n\nAnother element to consider when defining \"mental disorder\" is the fact that there is a person foremost who has the signs and symptoms of a mental disorder. As referenced earlier, the DSM-IV states that the definition focuses on the disorder and not the person. <http:\/\/www.DSM5.org\/ProposedRevisions\/Pages\/proposedrevision.aspx?rid=465#>. This portends various problems because psychiatrists treat people. This fact can be lost in this age of fifteen-minute medication checks and insurance pressures. The essential aspect of the help is the relationship so acknowledgement of the patient as a person with an illness should be added to the definition of mental disorder. \"... When therapists apply manualized treatments to selected symptom clusters without addressing the complex person who experiences the symptoms and without attending to the therapeutic relationship that supports the treatment, therapeutic results are short-lived and rates of remission are high.\" \\[64\\]Admittedly, what complicates this issue is our lack of understanding of how brain processes (ion fluxes, neurotransmission, etc) result in consciousness, intentionality, thoughts, and the subjective experience of emotions and how these psychological states affect brain processes. Along this vain, perhaps the use of \"mental\" should be reconsidered because it implies, intentionally or not, the separation of mind and brain. Perhaps \"neuro-mental\" could be considered? The definition possibly could begin like this: \"An individual (with hopes, dreams, disappointments, and feelings like his treaters) is considered to have a neuro-mental disorder when....\"\n\nThe definition of a mental disorder will evolve over time as our knowledge advances and cultures evolve. It may be worthwhile to recognize that a definition will be merely a synthesis of the various dialectical poles and will require repeated adjustments over time. If we're willing to change our understanding of the definition, then perhaps we'll be willing to better understand our patients.\n\n## Commentary: The Difficulties of Defining a Mental Disorder in DSM-III\n\nHannah S. Decker, Ph.D.\n\nUniversity of Houston.\n\nI am commenting on this question as a historian of psychiatry who is writing a book on the making of DSM-III.\n\nThe first two DSMs in 1952 and 1968 eschewed a definition of mental disorder befitting their modest origins from nomenclatures that served primarily \"the \\[statistical\\] needs and case loads of public mental hospitals\" \\[\\[65\\], p. vi\\]. But the editor of the third edition (1980), Robert L. Spitzer, had more ambitious goals for the manual resulting in a volume that was over three times the size of DSM-II and had dozens of new diagnoses. It was Spitzer's plan to include in DSM-III a definition of \"mental illness\" as a subset of \"medical illness.\" Circumstances forced him to abandon this type of definition, but there is a definition of \"mental disorder\" in DSM-III, prefaced by the familiar caveat, \"there is no satisfactory definition that specifies precise boundaries for the concept 'mental disorder'\" \\[\\[66\\], p. 5\\].\n\nSpitzer wanted to establish that, without any doubt, psychiatry was a part of medicine. He had initially thought seriously about mental disorders even before he was appointed the head of the Task Force. In 1973, he had brokered the removal of the diagnosis of homosexuality as a mental disorder from DSM-II, and the controversy surrounding the event sensitized him to the subject of what constituted a mental disorder. He soon found impediments to his goal of establishing definitions of medical and mental disorder in DSM-III. Still, at every turn he persevered because he envisioned the issuance of the new diagnostic manual as having intellectual goals far larger than its being a diagnostic classification. Spitzer wanted DSM-III to play a role in combating the anti-psychiatry movement of the 1960s and early '70s and to refute critics such as Thomas Szasz who said mental illness was a myth.\n\nI would like to spell out briefly the obstacles that lay in the path of an agreed-upon definition of a mental disorder. At the annual meeting of the American Psychiatric Association in May 1976, Spitzer and Jean Endicott, a close colleague on the DSM-III Task Force, put forth their definitions of medical and mental disorders. The reaction was quite negative. As Spitzer later reported: \"Some questioned the need and wisdom of having any definition. Many argued that the definition proposed was too restrictive, and if officially adopted, would have the potential for limiting the appropriate activities of our profession... they also felt that it was out of keeping with trends in medicine that emphasize the continuity of health and illness\" \\[\\[67\\], p. 16\\]. (This continues to be an important question in current debates over what diagnoses should be in DSM-5. Allen Frances, in particular, has argued against pathologizing what he sees as aspects of normality, \"everyday incapacity,\" in his words.)\n\nThen, Spitzer encountered strenuous opposition from psychologists to the notion that mental disorders were medical disorders. This was a turf issue, with the psychologists fearing that they would lose the right to treat mental disorders if they were defined as medical. In June 1976, a conference was held in St. Louis on \"Critically Examining DSM-III in Midstream.\" Dr. Maurice Lorr, representing the American Psychological Association, \"expressed the view that mental disorders (as medical disorders) should be limited to those conditions for which a biological etiology or pathophysiology could be demonstrated.\" In addition, just two months earlier, a former president of the American Psychological Association had been quite blunt in expressing his view that DSM-III was \"turning every human problem into a disease, in anticipation of the shower of health plan gold that is over the horizon\" \\[\\[67\\], p. 36\\].\n\nIn spite of these disagreements, Spitzer, as was his wont, did not surrender easily. He returned the next year to bolster his arguments. This was at the yearly meeting of the American Psychopathological Association, an organization of preeminent American psychiatrists dedicated to research on human behavior. In 1977 it devoted its annual conference to \"Critical Issues in Psychiatric Diagnosis.\" Spitzer and Endicott not only presented retooled definitions of both medical and mental disorders, but Spitzer, as an editor of the 1978 published proceedings of the conference, now took the opportunity to remind his readers of the blows psychiatry had endured in the 1960s and early '70s: \"The very concept of psychiatric illness has been under considerable attack in recent years. This attack has largely depended upon studies derived from the social sciences. Some have taken the stand that what are called mental illnesses are simply those particular groups of behaviors that certain societies have considered deviant and reprehensible.\" Spitzer believed that this rejection of the legitimacy of psychiatry was partly owed to the fact that \"no generally agreed upon definition of mental illness has been propounded that is not open to the criticisms of cultural relativism\" \\[\\[68\\], p. 5\\].\n\nIn addition to his conviction that DSM-III, with its new diagnostic criteria, would bring diagnostic reliability to psychiatry, Spitzer conceived of the new DSM as a weapon that could repel psychiatry's cultural challengers. The new manual would thus have a potential of historical proportions. Nevertheless, although Spitzer labored mightily to develop \"mental illness\" as a subset of \"medical illness,\" he was ultimately forced to bow both to the opinions of his psychiatric colleagues, who had philosophical and practical objections to his definition, and to the demands of the psychologists that mental illnesses be labeled \"mental disorders.\" The upshot was that mental disorders did not get to be defined as medical disorders.\n\nThe attempts of Robert Spitzer--a psychiatrist of considerable accomplishment in many areas of the profession--to establish a definition of a mental disorder, illustrate the complexities of arriving at one that is intellectually satisfying, clinically useful, and practically acceptable. Nevertheless, he did include a definition of mental disorder in DSM-III under the category of \"Basic Concepts\" \\[\\[66\\], pp. 5-6\\]. DSM-IV \\[\\[69\\], pp. xxi-xxii\\], with some changes, essentially preserved Spitzer's definition, which also forms the basis of the definition planned for DSM-5 \\[70\\] (See also Stein et al \\[43\\]. The individuals addressing this latest revision include the habitual warning: \"No definition perfectly specifies precise boundaries for the concept of either 'medical disorder' or 'mental\/psychiatric disorder'\" \\[70\\].\n\n## Allen Frances responds: Mental Disorder Defies Definition\n\nHumpty Dumpty: \"When I choose a word it means just what I choose it to mean\"\n\nWhen it comes to defining the term \"mental disorder\" or figuring out which conditions qualify, we enter Humpty's world of shifting, ambiguous, and idiosyncratic word usages. This is a fundamental weakness of the whole field of mental health.\n\nMany crucial problems would be much less problematic if only it were possible to frame an operational definition of mental disorder that really worked. Nosologists could use it to guide decisions on which aspects of human distress and malfunction should be considered psychiatric- and which should not. Clinicians could use it when deciding whether to diagnose and treat a patient on the border with normality. A meaningful definition would clear up the great confusion in the legal system where matters of great consequence often rest on whether a mental disorder is present or absent.\n\nAlas, I have read dozens of definitions of mental disorder (and helped to write one) and I can't say that any have the slightest value whatever. Historically, conditions have become mental disorders by accretion and practical necessity, not because they met some independent set of operationalized definitional criteria. Indeed, the concept of mental disorder is so amorphous, protean, and heterogeneous that it inherently defies definition. This is a hole at the center of psychiatric classification\n\nAnd the specific mental disorders certainly constitute a hodge podge. Some describe short term states, others lifelong personality. Some reflect inner misery, others bad behavior. Some represent problems rarely or never seen in normals, others are just slight accentuations of the everyday. Some reflect too little control, others too much. Some are quite intrinsic to the individual, others are defined against varying and changing cultural mores and stressors. Some begin in infancy, others in old age. Some affect primarily thought, others emotions, yet others behaviors, others interpersonal relations, and there are complex combinations of all of these. Some seem more biological, others more psychological or social.\n\nIf there is a common theme it is distress and disability, but these are very imprecise and nonspecific markers on which to hang a definition. Ironically, the one definition of mental disorder that does have great and abiding practical meaning is never given formal status because it is tautological and potentially highly self serving. It would go something like \"Mental disorder is what clinicians treat and researchers research and educators teach and insurance companies pay for.\" In effect, this is historically how the individual mental disorders made their way into the system.\n\nThe definition of mental disorder has been elastic and follows practice rather than guides it. The greater the number of mental health clinicians, the greater the number of life conditions that work their way into becoming disorders. There were only six disorders listed in the initial census of mental patients in the mid nineteenth century, now there are close to three hundred. Society also has a seemingly insatiable capacity (even hunger) to accept and endorse newly defined mental disorders that help to define and explain away its emerging concerns.\n\nAs a result, psychiatry is subject to recurring diagnostic fads. Were DSM5 to have its way we would have a wholesale medicalization of everyday incapacity (mild memory loss with aging); distress (grief, mixed anxiety depression); defects in self control (binge eating); eccentricity (psychotic risk); irresponsibility(hypersexuality); and even criminality (rape, statutory rape). Remarkably, none of these newly proposed diagnoses even remotely pass the standard loose definition of \"what clinician's treat\". None of these \"mental disorders\" has an established treatment with proven efficacy. Each is so early in development as to be no more than \"what researchers research\" - a concoction of highly specialized research interests.\n\nWe must accept that our diagnostic classification is the result of historical accretion and accident without any real underlying system or scientific necessity. The rules for entry have varied over time and have rarely been very rigorous. Our mental disorders are no more than fallible social constructs.\n\nDespite all these limitations, the definitions of mental disorders contained in the DSM's are necessary and do achieve great practical utility. The DSM provides a common language for clinicians, a tool for researchers, and a bridge across the clinical\/research interface. It offers a textbook of information for educators and students. It contains the coding system for statistical, insurance, and administrative purposes. DSM diagnoses also often play an important role in both civil and criminal legal proceedings. The DSM system is imperfect, but indispensable.\n\nIt is undoubtedly a failing on my part, but I find myself unable to take much interest in efforts to define mental disorder. My too practical temperament prefers to spend my too limited time on earth attending to concrete and soluble problems and studiously avoids the abstract and the insoluble. Defining mental disorder in a useful way clearly lies above my intellectual pay grade.\n\nThis is not to say that the question is uninteresting or unimportant. Would that there were a workable definition of mental disorder. We could then comfortably decide which of the proposed mental disorders need be included in the DSM, which aspects of human suffering and deviance are best left out. We could also come to a ready judgment about each individual potential 'patient'- who best qualifies for diagnosis and treatment, who is best left to his own devices.\n\nAlas, however, the sheep and the goats refuse to declare themselves in any convenient and discernible way. The definitions of mental disorder offered here make perfect sense in the abstract, but provide no guidance on how to make concrete decisions. They do not tell us, for example, whether mixed anxiety depression or binge eating or the early forgetting of advanced years are disorders or facts of life. They do not guide us in diagnosing the many people who populate the fuzzy boundary between mental disorder and normality.\n\nSeeing no practical consequence, I have no opinion on the fine points of definition- since these seem to be of only academic interest. Mental disorder is (like 'disease' and 'obscenity' or 'love') something you hope you can spot when you see it, but by implicit rules that inherently are poorly defined and ever shifting.\n\n### Reply to Dr Wakefield\n\nIf anyone in the world could usefully define mental disorder, it would be Jerry Wakefield. He has tried long, hard, skillfully, even brilliantly and has come up with a definition that works extremely well on paper. His \"harmful dysfunction\" and evolutionary perspective provide the best possible abstract definition of mental disorder. The problem is that Dr Wakefield's definition is not operational in a way that provides guidance on the two questions that most count: 1) Is this proposed new diagnosis a mental disorder that should be included in the official nomenclature? 2) Does this person have sufficient psychiatric problems to warrant a diagnosis of mental disorder? Unfortunately, neither question lends itself to his definitional solution. As Dr Wakefield himself points out, \"both the notions of dysfunction and harm are fuzzy concepts\" that are only useful to \"determine a range of clear cases on either side of the disorder\/nondisorder boundary.\" We are left to settle the crucial and frequent tough fuzzy boundary questions in what remains a necessarily unsatisfactory, ad hoc, and often idiosyncratic manner.\n\nDr Wakefield seems to accept the necessity of my less abstract \"cost\/benefit analysis\" approach for reducing the reckless DSM 5 diagnostic exuberance at the fuzzy boundary with normality. But he goes on rightly to criticize its susceptibility to misuse in the service of social control or economic manipulation. These issues are taken up in more detail in question \\#4 on the limitations of pragmatics in framing the diagnostic system.\n\nDr Wakefield and I agree completely on the most important question facing psychiatry today- the risk of false positives and excessive treatment. Diagnostic inflation has been a huge problem in the way DSM IV has been used. It will be greatly amplified by the many new high prevalence diagnoses being suggested for DSM 5. Unfortunately, no available definition of mental disorder has been able to withstand the pressure of lowered diagnostic standards and drug company advertising.\n\n### Response to Dr Kinghorn\n\nI agree that the DSM's gain little from attempting to provide an abstract definition of mental disorder and that having a useless definition may be worse than offering no definition at all. Also, let's remember that we are not alone in being definitionally challenged- there is really no good operational definition in medicine for \"disease\" or \"illness.\"\n\n### Response to Dr Pierre\n\nI agree completely with Dr Pierre's eloquent critique of the definitions of mental disorder and fully endorse his concern that such consequential decisions rest on such a fragile tissue. It will be important to get the widest input on all the crucial questions raised by Dr Pierre. We can't rely on any definition of mental disorder, nor can we trust the wisdom of experts on that disorder or of any single professional association. The decisions about what constitutes a mental disorder require the same safety care as the FDA devotes before allowing the introduction of a new drug.\n\n### Reply to Dr Chardavoyne\n\nI understand Dr Chardavoyne's regret that DSM may seem to lose the person in its effort to define the disorder. I just don't see a solution within the diagnostic system- which necessarily has to focus on symptom similarities, rather than the particularities and idiosyncrasies that make each of us who we are. Holding fast to the person is the crucial task of every clinician, but it is not something the DSM can help with.\n\n### Reply to Dr Decker\n\nHannah Decker does us a great service by recalling and recording attempts to define mental disorder. I am afraid, however, that this is a situation in which knowing a problematic past is insufficient to avoid repeating it. There will always be a strong desire to define 'mental disorder' because it is so important in setting the boundary with normality. But all efforts at universal definition will fail because the concept is so inherently fuzzy and situation bound. The only consolation is that 'medical illness' is equally vague and hard to define.\n\n# Conclusion\n\nThe two questions covered in this article form a natural pair. How you define mental disorder (Question 2) will certainly depend on what you think mental disorders are and how we are able to know about them (Question 1). I will briefly summarize the discussion developed in these questions and save a larger review for the general conclusion.\n\nAs indicated above in the General Introduction, the startling failure of research to validate the DSM categories of DSM-III and DSM-IV has led to a conceptual crisis in our nosology: what exactly is the status of DSM diagnoses? Do they identify real diseases, or are they merely convenient (and at time arbitrary) ways of grouping psychiatric symptoms? These are the issues dealt with in Question 1, framed in the umpire metaphor introduced by Allen Frances in his \"DSM in Philosophyland\" piece published in Bulletin 1 and commented on at length in Bulletin 2. The commentaries in this article roughly follow the positions of the five imagined umpires, although, as explained above, most of us will not restrict ourselves to a purist version of one of the umpires. Indeed, Frances himself, while stating a clear point of view, acknowledges that each umpire position captures a bit of the truth.\n\nThe first two commentaries address Question 1 in a broad way, commenting on the process of deciding about the merits of the various positions. Peter Zachar and Stephen Lobello are scholars who take their baseball seriously and weave the metaphor into a complex analysis in which their pragmatic (practical kinds) perspective subsumes all the umpires, including the pragmatic fourth umpire. In her commentary Claire Pouncey doesn't quite assume a position but provides a clear presentation of the differences among the first three umpires. She begins with the clarification that the umpire question involves both ontology and epistemology: what is there, and can I know it. Umpire 1 is a Strong Realist - both an ontological realist and an epistemological realist. Umpire 2 is a Strong Realist\/Weak Constructivist - an ontological realist and an epistemological less-than-realist. Umpire 3 is a Strong Constructivist - an ontological anti-realist and an epistemological anti-realist.\n\nIn his commentary Nassir Ghaemi offers a spirited defense of a realist, first-umpire position, challenging those who don't accept the reality of mental illnesses as to what they're doing treating patients. I am calling him a first umpire, but he rejects the umpire metaphor, offering in its stead Kenneth Kendler's notion of \"epistemic iteration.\"\n\nThe next three commentators assume some variation on the \"nominalist\" second umpire position. Michael Cerullo invokes the naturalist\/normativist debate, a distinction that echoes Jerome Wakefield's harmful dysfunction notion of psychiatric disorders. Cerullo argues that all diseases, including psychiatric disorders, have natural and normativist aspects, although some lean more toward the naturalist dimension and others toward the normativist dimension.\n\nIn his contribution Jerome Wakefield follows with a thorough presentation of his well-known harmful dysfunction understanding of mental disorders. For purposes of the umpire discussion he locates his HD umpire in a humble realist 1.5 position - nominalism with a tilt toward realism.\n\nFinally, Joseph Pierre invokes the fate of the planet Pluto to point to the reality of things studied by science and reminds us of the biological reality of mental disorders; but, acknowledging the uncertainties of our knowledge, he assumes the second umpire position. Like the others in the second umpire group, he notes that some psychiatric disorders make more claim on a first umpire stance than others.\n\nGary Greenberg boldly assumes the third umpire position, even invoking Samuel Johnson's kick in a face-off with Ghaemi's use of the kick to defend the first umpire. Greenberg argues that the human interest is so powerful in determining what counts as disease and what does not that honesty drives us to the constructivist stance.\n\nIn his commentary Harold Pincus elaborates the very diverse ways in which concepts of mental disorders are used by an assortment of user groups, leading him to emphasize the fourth umpire, pragmatist, position toward psychiatric conditions. He argues cogently that validity as we now know it will not be a meaningful concept in the future.\n\nFinally, with his usual energy and without any indication of retreat, Thomas Szasz comfortably assumes the position of fifth umpire and reviews the stance toward psychiatric disorders he familiarized us with fifty years ago.\n\nAnd still finally, in a reflection that probably belongs best with the fifth umpire, Elliott Martin argues that the insurance industry has so co-opted the nosology that we might consider it the only umpire in the game.\n\nIn his response to the commentaries on Question 1, Allen Frances begins by noting that \"\\[n\\]one of the five umpires is completely right all of the time. And none is totally wrong all of the time. Each has a season and appropriate time at the plate.\" He then proceeds to a historical perspective, noting that in the heyday of biological psychiatry forty years ago, Umpire 1, 3, and 5 were ascendant. On the one hand, the biological psychiatrists were confident that the realist position of Umpire 1 would prevail. And on the other hand, they were challenged by a broad range of skeptics occupying the positions of the Umpires 3 and 5. In Frances' account that has all changed. Chastened by the failures of biological psychiatric to produce, but convinced of the reality of psychiatric illness, we as a majority have gravitated toward the position of Umpire 2 - there is certainly psychiatric illness, but the categories of DSM-III and IV may not have carved those infamous joints correctly. Frances offers a guarded defense of the categories, nonetheless, arguing that, until further science has settled the issue of what are valid categories, the current ones serve a useful function of organizing the clinical phenomena which we confront in our work. The pragmatic Umpire 4 thus has a say in our current efforts to diagnose. \"Mental disorders are no more and no less than constructs. And Umpire 4 is quick to point out that they are very useful constructs.\" Frances ends on an optimistic note that with more scientific clarity in the future, we can anticipate that Umpire 1 will gradually assume prominence over Umpire 2.\n\nWith the second question, definition, Frances can certainly claim more experience that most of us because of the time he put in grappling with this question in DSM-IV. His dissatisfaction with his own work product, and his skepticism about ever getting it right are certainly revealing - and consistent with his response to the first question. He points to the heterogeneity of what gets called a mental disorder, as well as to the unavoidable fact that many have been admitted to the club through the distinctly unscientific process of historical accretion. All that said, he argues that the DSM categories serve a very important role in facilitating communication among mental health professional and are thus necessary, however imperfect and imprecise. He concludes on a note of flagging interest in settling this question.\n\nThe commentators have stretched themselves to the imaginable extremes in tackling this question. The majority, along with Frances, view the DSM as a very motley assortment of behaviors and states of mind, and they see the DSM definition as trying to accommodate what we in fact treat, as opposed to leading us to decide what we *should* treat. The exception is Jerome Wakefield, who has argued persuasively for some time - and rehearses the main features of his argument here - that we can provide a clear of definition of mental disorder with the notion of harmful dysfunction. This is a definition that covers both the scientific and normative aspects of mental disorder, and that purports to guide us rather than follow us in our practice.\n\nThe commentaries at the other end of the spectrum start with Warren Kinghorn, who argues that since the DSM definition accomplishes nothing, even what it minimally claims to accomplish - organizing the terrain and establishing common goals of practice - we should acknowledge that we don't use it, don't need it, and should just retire it.\n\nJoseph Pierre offers another argument for the impossibility of developing an adequate definition - poor science, value intrusion, ever broadening parameters of practice - and also reminds us that general medicine does quite well without an official definition. In spite of his cogent argument for the failed project of definition, he appears to stop short of Kinghorn's recommendation, and without mustering Frances' defense, appears to be in favor of having the definition, however inadequate. That is presumably because, unlike, Kinghorn, he feels that an official definition can have significant consequences for the field of psychiatry.\n\nJohn Chardavoyne, makes a plea for escaping the inadequate science by reorienting the definition away from the disease and back to the person. In doing this he retains a definition but stands somewhat outside the debate that has engaged Frances and the other commentators.\n\nIn a final commentary Hannah Decker takes a look back and reviews Robert Spitzer's struggles to develop a definition of mental illness for DSM-III - a commentary that prompts Allen Frances to remark that this is a situation in which even a thorough examination of the past may not improve our performance in the present.\n\n# Competing interests\n\nMF is an external consultant to the NIMH Research Domain Criteria (RDoC) Project. NG has research grants from Pfizer and Sunovion, and is a research consultant for Sunovion. MS is a consultant for AstraZeneca, Merck, Novartis and Sunovion. Other authors report no competing interests.\n\n# Authors' contributions\n\nJP(Phillips) wrote the general General Introduction and Conclusion, as well as the introductions to the individual conclusions. AF wrote the Responses to Commentaries. MC, JC, HD, MF, NG, GG, AH, WK, SL, EM, AM, JP(Paris), RP, HP, DP, CP, MS, TS, JW, SW, OW, PZ wrote the commentaries. All authors read and approved the final manuscript.","meta":{"dup_signals":{"dup_doc_count":142,"dup_dump_count":74,"dup_details":{"curated_sources":4,"2023-14":2,"2022-49":1,"2022-21":1,"2021-17":1,"2021-10":1,"2020-50":1,"2020-40":1,"2020-29":2,"2020-24":1,"2020-16":1,"2020-10":1,"2020-05":1,"2019-47":4,"2019-43":1,"2019-39":2,"2019-30":1,"2019-22":1,"2019-18":1,"2019-13":1,"2019-09":2,"2019-04":1,"2018-51":2,"2018-47":1,"2018-43":1,"2018-34":1,"2018-30":3,"2018-26":1,"2018-17":1,"2018-13":3,"2018-09":3,"2018-05":1,"2017-51":1,"2017-47":3,"2017-43":2,"2017-39":2,"2017-34":3,"2017-30":2,"2017-26":3,"2017-22":5,"2017-17":2,"2017-09":3,"2017-04":3,"2016-50":4,"2016-44":3,"2016-40":3,"2016-36":3,"2016-30":5,"2016-26":2,"2016-22":2,"2016-18":2,"2015-48":3,"2015-40":1,"2015-35":1,"2015-32":1,"2015-27":2,"2015-22":1,"2015-14":1,"2014-52":1,"2014-49":1,"2014-42":4,"2014-41":2,"2014-35":1,"2014-23":2,"2014-15":2,"2024-18":1,"2024-10":1,"2017-13":5,"2015-18":2,"2015-11":1,"2015-06":2,"2014-10":1,"2013-48":1,"2013-20":2,"2024-22":1}},"file":"PMC3305603"},"subset":"pubmed_central"}
{"text":"date: 2009-04-10\ntitle: Findings of scientific misconduct.\n\n# Findings of Scientific Misconduct\n\nNotice Number: NOT-OD-09-082\n\nKey Dates**  \n**Release Date: April 9, 2009\n\nIssued by**  \n**Department of Health and Human Services\n\nNotice is hereby given that the Office of Research Integrity (ORI) and the Assistant Secretary for Health have taken final action in the following case:\n\nRobert B. Fogel, M.D., Harvard Medical School and Brigham and Women's Hospital: Based on information that the Respondent volunteered to his former mentor on November 7, 2006, and detailed in a written admission on September 19, 2007, and ORI's review of Joint Inquiry and Investigation reports by Harvard Medical School (HMS) and the Brigham and Women's Hospital (BWH), the U.S. Public Health Service (PHS) found that Dr. Robert B. Fogel, former Assistant Professor of Medicine and Associate Physician at HMS, and former Co-Director of the Fellowship in Sleep Medicine at BWH, engaged in scientific misconduct in research supported by National Heart, Lung, and Blood Institute (NHLBI), National Institutes of Health (NIH), awards P50 HL60292, R01 HL48531, K23 HL04400, and F32 HL10246, and National Center for Research Resources (NCRR), NIH, award M01 RR02635.\n\nPHS found that Respondent engaged in scientific misconduct by falsifying and fabricating baseline data from a study of sleep apnea in severely obese patients published in the following paper: Fogel, R.B., Malhotra, A., Dalagiorgou, G., Robinson, M.K., Jakab, M., Kikinis, R., Pittman, S.D., and White, D.P. \\`\\`Anatomic and physiologic predictors of apnea severity in morbidly obese subjects.' Sleep 2:150-155, 2003 (hereafter referred to as the \\`\\`Sleep paper'); and in a preliminary abstract reporting on this work.\n\nSpecifically, PHS found that for the data reported in the Sleep paper, the Respondent:\n\nChanged\/falsified roughly half of the physiologic data\n\nFabricated roughly 20% of the anatomic data that were supposedly obtained from Computed Tomography (CT) images\n\nChanged\/falsified 50 to 80 percent of the other anatomic data\n\nChanged\/falsified roughly 40 to 50 percent of the sleep data so that those data would better conform to his hypothesis.\n\nRespondent also published some of the falsified and fabricated data in an abstract in Sleep 24, Abstract Supplement A7, 2001.\n\nDr. Fogel has entered into a Voluntary Settlement Agreement in which he has voluntarily agreed, for a period of three (3) years, beginning on March 16, 2009:\n\n\\(1\\) To exclude himself from serving in any advisory capacity to PHS, including but not limited to service on any PHS advisory committee, board, and\/or peer review committee, or as a consultant;\n\n\\(2\\) That any institution that submits an application for PHS support for a research project on which the Respondent's participation is proposed or that uses the Respondent in any capacity on PHS supported research, or that submits a report of PHS-funded research in which the Respondent is involved, must concurrently submit a plan for supervision of the Respondent's duties to the funding agency for approval; the supervisory plan must be designed to ensure the scientific integrity of the Respondent's research contribution; a copy of the supervisory plan must also be submitted to ORI by the institution; the Respondent agrees that he will not participate in any PHS-supported research until such a supervisory plan is submitted to ORI; and\n\n\\(3\\) To ensure that any institution employing him submits, in conjunction with each application for PHS funds or report, manuscript, or abstract of PHS-funded research in which the Respondent is involved, a certification that the data provided by the Respondent are based on actual experiments or are otherwise legitimately derived and that the data, procedures, and methodology are accurately reported in the application or report. The Respondent must ensure that the institution sends the certification to ORI.\n\nFOR FURTHER INFORMATION CONTACT:**  \n** Director**  \n** Division of Investigative Oversight**  \n** Office of Research Integrity**  \n** 1101 Wootton Parkway, Suite 750**  \n** Rockville, MD 20852**  \n** (240) 453-8800.","meta":{"dup_signals":{"dup_doc_count":101,"dup_dump_count":41,"dup_details":{"curated_sources":2,"2022-33":1,"2018-30":1,"2018-13":1,"2018-05":1,"2017-47":1,"2017-34":1,"2017-30":1,"2017-22":1,"2017-17":1,"2017-09":2,"2017-04":1,"2016-50":1,"2016-44":1,"2016-40":1,"2016-36":1,"2016-30":2,"2016-26":1,"2016-22":1,"2016-18":1,"2016-07":3,"2015-48":3,"2015-40":3,"2015-35":4,"2015-32":4,"2015-27":3,"2015-22":3,"2015-14":4,"2014-52":3,"2014-49":2,"2014-42":2,"2014-41":6,"2014-35":5,"2014-23":6,"2014-15":4,"2022-49":1,"2015-18":3,"2015-11":3,"2015-06":3,"2014-10":5,"2013-48":4,"2013-20":4}},"file":"PMC4259702"},"subset":"pubmed_central"}
{"text":"author: Govindarajan Rajagopalan; Yogish C. Kudva; Chella S. DavidCorresponding author: Chella S. David, <firstname.lastname@example.com>\ndate: 2012-07\nreferences:\ntitle: Is HOT a Cool Treatment for Type 1 Diabetes?\n\nType 1 diabetes (T1D) is an organ-specific autoimmune disease characterized by immune-mediated destruction of the insulin-producing \u03b2-cells of the islets of Langerhans that results in life-long insulin dependence (1). Given the immunological nature of the disease, numerous antigen-specific as well as nonantigen-specific tolerance induction or immune deviation strategies have been developed as treatments for T1D. Although successful in experimental models, results of studies to translate these strategies to humans have been discouraging (2\u20135). This has prompted researchers to explore safer, broader, and more effective immunotherapeutic approaches to prevent, treat, and\/or revert T1D. In this issue of *Diabetes*, Faleo et al. (6) show compelling data regarding use of hyperbaric oxygen therapy in autoimmune diabetes using nonobese diabetic (NOD) mice. This model spontaneously develops T1D, a feature that closely mimics human disease. Faleo et al. (6) show that hyperbaric oxygen therapy (HOT) significantly protects from T1D when initiated early in the disease course, but not after its onset, suggesting that this approach could be useful in high-risk individuals.\n\nAccording to the Undersea and Hyperbaric Medical Society, HOT is a process wherein patients breathe 100% oxygen (atmospheric air contains \\~21% oxygen) while inside a treatment chamber at a pressure higher than at sea level (usually 2.5 times greater pressure). In 1662, Henshaw, a British clergyman, first used pressurized atmospheric air to treat certain ailments. Since that time, HOT has been used to treat various conditions such as decompression sickness, arterial gas embolism, and carbon monoxide poisoning (with or without cyanide poisoning), and to facilitate wound healing (7). It should be noted that inhalation of 100% oxygen at 1 normal atmosphere of pressure or exposing isolated parts of the body to 100% oxygen does not constitute HOT.\n\nFaleo et al. (6) exposed prediabetic NOD mice to pressurized 100% oxygen (HOT-100%), 21% oxygen (HOT-21%), or oxygen-depleted air supplemented with pure oxygen (HOT-12%) for 60 min every day for varying periods of time. T1D risk was assessed using different models. In the cyclophosphamide-accelerated model, more than 75% of control, HOT-12%, and HOT-21% mice developed hyperglycemia, compared with less than 50% of HOT-100% mice. The severity of insulitis was also reduced in the HOT-100% group. Even though HOT was not administered on the day of cyclophosphamide therapy, the possibility that HOT could have influenced the metabolism of cyclophosphamide (8), and thereby indirectly influenced the incidence of accelerated hyperglycemia, was not addressed by the authors.\n\nIn the spontaneous model, when HOT was initiated at 4 weeks of age, 85% of untreated control NOD mice developed hyperglycemia by 35 weeks compared with 65% of mice in the HOT-100% group. Although this finding was statistically significant, a considerable percentage\u201465%\u2014of this inbred, homogenous mouse strain still developed hyperglycemia with HOT-100%, an observation that raises questions about the likely effectiveness of HOT among more heterogeneous human T1D populations. Data from a key experiment\u2014the effect of HOT-100% on the incidence of hyperglycemia when administered close to onset of diabetes in NOD mice (13 weeks of age)\u2014were not reported. In this group, HOT-100% attenuated insulitis and significantly delayed the onset of hyperglycemia when administered along with glucagon-like peptide (GLP)-1 analog and exenatide (EXN) delivered by mini-osmotic pumps. Inclusion of a group receiving HOT-100% and vehicle or a scrambled peptide, instead of EXN, would have strengthened the inference that HOT-100% is more effective when combined with EXN. Overall, the findings in this report suggest that only prolonged exposure to HOT-100% that is initiated at a very young age reduced T1D risk in this mouse model.\n\nThe mechanisms by which HOT provided its beneficial effects in T1D or even in other models of inflammation are not fully understood. HOT can suppress inflammation by modulating the expression of integrins (9) and probably other adhesion molecules. This would interfere with homing of inflammatory cells to islets, thereby reducing insulitis. In support of this hypothesis, HOT-100%\u2013treated mice had reduced insulitis. Furthermore, expression of CD62L, a lymphocyte homing marker, was altered in the spontaneous, but not cyclophosphamide-accelerated, T1D model.\n\nHOT could modulate inflammation through hypoxia-inducible factor (HIF)-1 (10). Hypoxia increases HIF expression, whereas hyperoxia represses HIF. HIF-1 can induce interleukin (IL)-17 and inhibit Foxp3^+^ regulatory T-cell (Treg) development (11). Therefore, by reducing the expression of HIF-1 (12,13), HOT could inhibit the Th17 pathway and promote Treg development, thereby protecting from T1D. Although HIF was not investigated in this report, increased Treg in HOT-100% mice supports this theory. However, it remains to be elucidated whether HOT-100% mediates its effects directly through increasing Treg numbers and functions and\/or through suppressing IL-17. HIF is also induced rapidly in transplanted islets and promotes islet apoptosis (14,15). Therefore, repression of HIF by HOT may promote \u03b2-cell survival and play a beneficial role in diabetes. Reduced apoptosis and increased proliferation of \u03b2-cells in HOT-100% mice support this theory. Paradoxically, HOT can also increase HIF expression (16), and HIF can have anti-inflammatory activities in T1D (17). Therefore, the interplay between HOT and HIF in the T1D settings remains to be determined and is a topic ripe for further investigation.\n\nHOT is also known to increase levels of reactive oxygen species (ROS) as well as reactive nitrogen species (RNS) (18). Both ROS and RNS participate in innumerable physiological and pathological processes (19). Of relevance, ROS are known to damage \u03b2-cells and participate in inflammatory processes (20), which could be counterproductive in T1D patients receiving HOT. Therefore, the roles of ROS and RNS in HOT need to be thoroughly investigated. Furthermore, this study showed that HOT-100% suppressed the responses of T cells to a potent mitogenic stimulation (anti-CD3) and elevated the levels of IL-10. Induction of such generalized immune suppression by HOT could be a potential drawback in humans, particularly in children, because this might predispose to infections, diminish the ability to clear infections, and mount adequate protective immunity after immunizations.\n\nIn conclusion, this descriptive work by Faleo et al. (6) addresses the potential benefits of HOT in T1D. Molecular oxygen is a fundamental component of various biochemical processes, and its use as a therapeutic agent could have diverse effects (21), both positive and negative. A detailed investigation is warranted to understand its mechanisms of actions as well as possible side effects (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<\/a>). In T1D, HOT is ineffective once the autoimmunity has progressed to a prediabetic stage, making its utility in patients with existing T1D questionable. When started early in the disease course, HOT reduced the incidence of T1D only by \\~20% in NOD mice. However, in humans, this could be a significant number. Therefore, given the established safety and limited side effects of HOT in humans, its translation to a pilot human clinical trial, either alone or with other immunomodulatory agents, at least in high-risk individuals (those with a first-degree relative with T1D or who express susceptible HLA alleles, etc.), is a possibility.\n\n## ACKNOWLEDGMENTS\n\nNo potential conflicts of interest relevant to this article were reported.\n\n# REFERENCES","meta":{"dup_signals":{"dup_doc_count":118,"dup_dump_count":45,"dup_details":{"curated_sources":2,"2019-35":1,"2019-26":1,"2019-13":1,"2018-26":2,"2018-22":1,"2018-17":3,"2018-09":2,"2018-05":2,"2017-51":2,"2017-47":1,"2017-43":5,"2017-39":1,"2017-34":2,"2017-30":4,"2017-26":4,"2017-22":1,"2017-17":6,"2017-09":4,"2017-04":6,"2016-50":3,"2016-44":4,"2016-40":3,"2016-36":3,"2016-30":3,"2016-26":3,"2016-22":3,"2016-18":2,"2016-07":3,"2015-48":3,"2015-40":2,"2015-35":2,"2015-32":1,"2015-27":3,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":1,"2014-42":1,"2014-41":5,"2014-35":2,"2019-43":1,"2017-13":3,"2015-18":3,"2015-11":1,"2015-06":3}},"file":"PMC3379652"},"subset":"pubmed_central"}
{"text":"date: 2004\ntitle: Editorial\n\nFollowing an overwhelming vote by the US House of Representatives urging the National Institutes of Health (NIH) to develop an Open Access strategy, the NIH has recently invited comment on its plans to enhance access to the research that it funds. Under the proposed scheme, NIH-funded researchers would have to provide electronic copies of the final accepted versions of each of their manuscripts, for archiving along with any supplementary information in PubMed Central \\[<http:\/\/www.pubmedcentral.nih.gov>\\]. Six months after publication of the research in question - or sooner if the publisher agrees - the provisional copies would be made publicly available at no charge to readers.\n\n*Journal of Biology* heartily supports the NIH proposal, which brings us one step closer to the immediate availability of all peer-reviewed research free of charge. Indeed, as Open Access pioneers, BioMed Central and *Journal of Biology* already provide PubMed Central with final full text and PDF versions of all published research articles immediately, and we encourage all publishers to follow suit. In an ideal future, the electronic version of each research article would be the final and definitive form - easily archived, centrally searchable and available at the click of a mouse to all who would read it, be they scientists or members of the public. Of course, print would still play an important role, but printed articles will no longer constitute the historic record of the work. And moving away from the printed article in favor of its online incarnation makes sense for other reasons too: electronically, researchers can display all relevant data instead of an edited subset, and moving images and other web-only formats can be easily integrated. It will no longer be possible to represent a complete research article accurately on the printed page.\n\nIn keeping with this shift in emphasis, *Journal of Biology* is now presenting its printed articles in a new light. Since its inception, the journal has eschewed the rigidity of producing regular issues, instead producing collections of articles grouped by their focus on a key piece of research, not necessarily by publication date. Each printed collection serves to draw readers' attention to the definitive online content, which is freely available on the journal's website or in central archives such as PubMed Central. In recognition of the non-traditional way that *Journal of Biology* has always conceptually clustered its articles, the cover of the bound printed articles now sports a bold new design - do please look out for it in print and on the journal's website.\n\nSince its launch in June 2002, nearly a million copies of *Journal of Biology* have been distributed in print to life scientists worldwide, entirely free of charge. But the journal and its publisher, BioMed Central, are committed to building a sustainable Open Access business model. This should primarily ensure that research articles are immediately and permanently available online without charge, as well as being deposited into permanent repositories - both of which provide far more efficient ways of disseminating, retrieving, and searching for scientific information. For this reason, the journal has now changed its approach to print distribution. Some readers will continue receiving complimentary copies as before, either with *The Scientist* or individually, but for others who wish to receive articles in print, a modest annual subscription charge of \\$50 (\u00a330\/\u20ac40) will be levied - see \\[<http:\/\/jbiol.com\/subscribe>\\]. These funds will help to maintain the quality of the reprints and to cover mailing costs. The journal also offers special print rates to librarians and institutions, so please encourage your library or head of institute to sign up. Of course, all of the content of *Journal of Biology* will continue to be available free of charge online.\n\n*Journal of Biology* urges other funding bodies and policy makers to follow the lead of the NIH, the UK Parliament, the Howard Hughes Medical Institute, The Wellcome Trust and the signatories of the Berlin Declaration \\[<http:\/\/www.zim.mpg.de\/openaccess-berlin>\\], and to encourage the researchers they fund to publish their results in a way that promotes public availability of scientific information. At the same time, it should be recognized that we still have a long way to go before every research article is free for anyone to read online on the day it is published. 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{"text":"abstract: Decreased collateral vessel formation in diabetic peripheral limbs is characterized by abnormalities of the angiogenic response to ischemia. Hyperglycemia is known to activate protein kinase C (PKC), affecting the expression and activity of growth factors such as vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF). The current study investigates the role of PKC\u03b4 in diabetes-induced poor collateral vessel formation and inhibition of angiogenic factors expression and actions. Ischemic adductor muscles of diabetic *Prkcd*^+\/+^ mice exhibited reduced blood reperfusion, vascular density, and number of small vessels compared with nondiabetic *Prkcd*^+\/+^ mice. By contrast, diabetic *Prkcd*^\u2212\/\u2212^ mice showed significant increased blood flow, capillary density, and number of capillaries. Although expression of various PKC isoforms was unchanged, activation of PKC\u03b4 was increased in diabetic *Prkcd*^+\/+^ mice. VEGF and PDGF mRNA and protein expression were decreased in the muscles of diabetic *Prkcd*^+\/+^ mice and were normalized in diabetic *Prkcd*^\u2212\/\u2212^ mice. Furthermore, phosphorylation of VEGF receptor 2 (VEGFR2) and PDGF receptor-\u03b2 (PDGFR-\u03b2) were blunted in diabetic *Prkcd*^+\/+^ mice but elevated in diabetic *Prkcd*^\u2212\/\u2212^ mice. The inhibition of VEGFR2 and PDGFR-\u03b2 activity was associated with increased SHP-1 expression. In conclusion, our data have uncovered the mechanisms by which PKC\u03b4 activation induced poor collateral vessel formation, offering potential novel targets to regulate angiogenesis therapeutically in diabetic patients.\nauthor: Farah Lizotte; Martin Par\u00e9; Benoit Denhez; Michael Leitges; Andr\u00e9anne Guay; Pedro GeraldesCorresponding author: Pedro Geraldes, <firstname.lastname@example.com>.\ndate: 2013-08\nreferences:\ntitle: PKC\u03b4 Impaired Vessel Formation and Angiogenic Factor Expression in Diabetic Ischemic Limbs\n\nThe main long-term complications from diabetes are vascular diseases, which are in turn the main causes of morbidity and mortality in diabetic patients (1). Diabetic vascular complications affect several important organs, including the retina, kidney, and arteries (2,3). Peripheral vascular diseases are the major risk factor for nontraumatic lower limb amputation in patients with diabetes (4), characterized by collateral vessel development insufficient to support the loss of blood flow through occluded arteries in the ischemic limbs (5). Multiple abnormalities in the angiogenic response to ischemia have been documented in the diabetic state and depend on complex interactions of multiple growth factors and vascular cells.\n\nExperiments to improve angiogenesis and vascular cell survival by local infusion of vascular endothelial growth factor (VEGF) or angiopoietin by increasing its expression have also been reported in nondiabetic animal models (6,7). Moreover, animal studies have used platelet-derived growth factor (PDGF) to improve collateral vessel formation and vascular healing in the diabetic state (8). Clinical trials using recombinant growth factors have noted transient improvement of myocardial and distal leg circulation (9\u201311). However, these favorable vascular effects appeared to produce limited clinical benefits (12). Local administration of growth factors, such as VEGF by gene therapy in the setting of diabetes, does not appear to have the beneficial long-term effects seen in the absence of diabetes or to improve quality of life (13,14). One potential problem with normalizing VEGF or PDGF action alone is that a variety of growth factors may be needed to establish and maintain the capillary bed.\n\nVarious studies have clearly identified that the expression of growth factors, such as VEGF, PDGF, and stromal-derived factor-1 (SDF-1), are critically important in the formation of collateral vessels in response to ischemia (15\u201317). Previous studies suggested that hyperglycemia attenuates VEGF production and levels in myocardial tissue and in animal models of wound repair (5,18). Furthermore, decreased VEGF and PDGF expression in the peripheral limbs and nerves of diabetic animals and rodents has been reported (19\u201321). Although the underlying mechanism of reduction of VEGF and PDGF expression in diabetes is not clear, it is well-known that the major inducers of VEGF and PDGF (i.e., hypoxia and oxidants) can both play a role in diabetes. We and other researchers have reported that variation in PDGF signaling, rather than expression, is linked to morphological abnormalities in the retina and in collateral capillary formation in an ischemic limb model of diabetic animals (22,23). Clearly, poor collateral vessel formation during diabetes-induced ischemia is attributable to the lack of production and\/or action of critical growth factors such as VEGF and PDGF. Therefore, further studies of the basic mechanisms of hyperglycemia-induced activation of toxic metabolites, such as activation of protein kinase C (PKC), are needed to identify how these proteins contribute to growth factor deregulation.\n\nPKC, a member of a large family of serine\/threonine kinases, is involved in the pathophysiology of vascular complications. When activated, PKC phosphorylates specific serine or threonine residues on target proteins that vary, depending on cell type. PKC has multiple isoforms that function in a wide variety of biological systems (24). PKC activation increases endothelial permeability and decreases blood flow and the production and response of angiogenic growth factors that contribute to the loss of capillary pericytes, retinal permeability, ischemia, and neovascularization (25\u201329).\n\nPrevious data have demonstrated that high glucose levels in smooth muscle cells activate PKC\u03b1, -\u03b2, -\u03b4, and -\u03b5 but not the atypical PKC\u03b6 (30,31). In general, high levels of glucose-induced PKC activation cause vascular dysfunction by altering the expressions of growth factors such as VEGF, PDGF, transforming growth factor-\u03b2, and others (32\u201334). PKC\u03b4 has been proposed to participate in smooth muscle cell apoptosis, and deletion of this PKC isoform led to increased arteriosclerosis (35). Moreover, we previously demonstrated that diabetes-induced PKC\u03b4 activation generates PDGF unresponsiveness, causing pericyte apoptosis, acellular capillaries, and diabetic retinopathy (23). We therefore hypothesized that PKC\u03b4 activation could be involved in proangiogenic factor inhibition that triggers poor collateral vessel formation in diabetes.\n\n# RESEARCH DESIGN AND METHODS\n\n## Reagents and antibodies.\n\nPrimary antibodies for immunoblotting were obtained from commercial sources, including actin (horseradish peroxidase \\[HRP\\]; I-19), SHP-1 (C19), VEGF (147), PKC\u03b1 (C-20), PKC\u03b2 (C-18), PKC\u03b5 (C-15), and nitric oxide synthase (NOS) 3 (C-20) antibodies from Santa Cruz Biotechnology Inc.; phospho (p)-tyrosine, p-PKC\u03b4 (Thr 505), PKC\u03b4, p-VEGF receptor 2 (VEGFR2; Y1175), VEGFR2, p-PDGF receptor-\u03b2 (PDGFR-\u03b2; Tyr 1009), and PDGFR-\u03b2 antibodies from Cell Signaling; anti-\u03b1 smooth muscle actin from Abcam; polyclonal antibody against protein-tyrosine phosphatase 1B (PTP1B) and CD31 from BD Bioscience; SHP-2 and SHP-1 antibodies from Millipore; and rabbit and mouse peroxidase-conjugated secondary antibody from GE Healthcare Bio-Sciences. All other reagents used, including EDTA, leupeptin, phenylmethylsulfonyl fluoride, aprotinin, and Na~3~VO~4~, were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise stated.\n\n## Animal and experimental design.\n\nC57BL\/6J mice (6 weeks old) were purchased from The Jackson Laboratory (Bar Harbor, ME) and bred in our animal facility. *Prkcd*^\u2212\/\u2212^ mice, described previously and provided by Dr. Michael Leitges (35), were generated by the insertion of a LacZ\/neo cassette into the first transcribed exon of the PKC\u03b4 gene. This insertion abolished the transcription of PKC\u03b4, leading to a null allele. *Prkcd*^\u2212\/\u2212^ mice with mixed background of 129SV and C57BL\/6J strains were crossbred for 10 generations (F12) with wild-type C57BL\/6J background from The Jackson Laboratory. Animals were rendered diabetic for a 2-month period by intraperitoneal streptozotocin injection (50 mg\/kg in 0.05 mol\/L citrate buffer, pH 4.5; Sigma) on 5 consecutive days after an overnight fast; control mice were injected with citrate buffer. Blood glucose was measured by Glucometer (Contour, Bayer Inc.). Throughout the study period, animals were provided with free access to water and standard rodent chow (Harlan Teklad, Madison, WI). All experiments were conducted in accordance with the Canadian Council of Animal Care and University of Sherbrooke guidelines.\n\n## Hind limb ischemia model.\n\nWe assessed blood flow in nondiabetic and *Prkcd*^+\/+^ and *Prkcd*^\u2212\/\u2212^ mice diabetic for 2 months. Animals were anesthetized, and the entire lower extremity of each mouse was shaved. A small incision was made along the thigh all the way to inguinal ligament and extending superiorly toward the mouse abdomen. The femoral artery was isolated from the femoral nerve and vein and ligated distally to the origin of the arteria profunda femoris. The incision was closed by interrupted 5-0 sutures (Syneture).\n\n## Laser Doppler perfusion imaging and physical examination.\n\nHind limb blood flow was measured using PIM3 laser Doppler perfusion imaging (Perimed Inc.). Consecutive perfusion measurements were obtained by scanning the region of interest (hind limb and foot) of anesthetized animals. Measurements were performed before and after artery ligation and on postoperative days 7, 14, 21, and 28. To account for variables that affect blood flow temporally, the results at any given time were expressed as a ratio against simultaneously obtained perfusion measurements of the right (ligated) and left (nonligated) limb. Tissue necrosis was scored to assess mice that had to be killed during the course of the experiment due to necrosis\/loss of toes.\n\n## Histopathology and TUNEL assay.\n\nRight and left abductor muscles from *Prkcd*^+\/+^ and *Prkcd*^\u2212\/\u2212^ mice were harvested for pathological examination. Sections were fixed in 4% paraformaldehyde (Sigma-Aldrich) for 18 h and then transferred to 90% ethanol for light microscopy and immunohistochemistry. Paraformaldehyde-fixed tissue was embedded in paraffin, and 6-\u00b5m sections were stained with hematoxylin and eosin (Sigma). Apoptotic cells were detected using the TACS 2 Tdt-Fluor in situ apoptosis detection kit (Trevigen, Gaithersburg, MD) according to the manufacturer's instructions.\n\n## Immunofluorescence.\n\nAdductor muscles were blocked with 10% goat serum for 1 h and exposed in sequence to primary antibodies (CD31 and \u03b1-smooth muscle actin, 1:100) overnight, followed by incubation with secondary antibodies Alexa-647 conjugated anti-rabbit IgG and Alexa-594 conjugated anti-mouse (1:500; Jackson ImmunoResearch Laboratories). Confocal images were captured on a Zeiss LSM 410 microscope; images of one experiment were taken at the same time under identical settings and handled in Adobe Photoshop similarly across all images.\n\n## Immunoblot analysis.\n\nAdductor muscles were lysed in Laemmli buffer (50 mmol\/L Tris \\[pH 6.8\\], 2% SDS, and 10% glycerol) containing protease inhibitors (1 mmol\/L phenylmethylsulfonyl fluoride, 2 \u03bcg\/mL aprotinin, 10 \u03bcg\/mL leupeptin, 1 mmol\/L Na~3~VO~4~; Sigma). Protein amount was measured with a BCA kit (Bio-Rad). The lysates (10\u201320 \u00b5g protein) were separated by SDS-PAGE, transferred to polyvinylidene fluoride membrane, and blocked with 5% skim milk. Antigens were detected using anti-rabbit HRP-conjugated antibody for other Western blotting and detected with the ECL system (Pierce Thermo Fisher, Piscataway, NJ). Protein content quantification was performed using computer-assisted densitometry with Image J software (National Institutes of Health).\n\n## Real-time PCR analysis.\n\nReal-time PCR was performed to evaluate mRNA expressions of PKC\u03b1, PKC\u03b2, PKC\u03b4, PKC\u03b5, VEGF, PDGF, KDR\/Flk-1, PDGFR-\u03b2, endothelial NOS (eNOS), SDF-1, FGF2, SHP-1, SHP-2, and PTP1B of nonischemic and ischemic limbs. Total RNA was extracted from adductor muscles with TRI-REAGENT, as described by the manufacturer and RNeasy mini kit (Qiagen, Valencia, CA). The RNA was treated with DNase I (Invitrogen) to remove any genomic DNA contamination. Approximately 1 \u03bcg RNA was used to generate cDNA using SuperScript III reverse transcriptase and random hexamers (Invitrogen) at 50\u00b0C for 60 min. PCR primers and probes are listed in [Supplementary Table 1](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1432\/-\/DC1). Glyceraldehyde-3-phosphate dehydrogenase and 18S ribosomal RNA expression were used for normalization. PCR products were gel purified, subcloned using a QIA quick PCR Purification kit (Qiagen), and sequenced in both directions to confirm identity.\n\n## Nuclear extract and nonradioactive transcription factor assay.\n\nAdductor muscles were lysed and nuclear-specific proteins isolated using the NucBuster Protein Extraction Kit (Novagen, Madison, WI) according to the manufacturer's instructions. Detection of hypoxia-inducible factor-1\u03b1 (HIF-1\u03b1) in the nucleus was quantified using the nonradioactive transcription factor assay kit (Cayman, Ann Arbor, MI). Briefly, nuclear protein (20 \u00b5g) was incubated for 24 h in a 96-well plate containing immobilized specific double-stranded DNA consensus sequence of the HIF-1\u03b1 response element. HIF-1\u03b1 contained in the nuclear extract was linked specifically to the HIF-1\u03b1 response element. Wells were washed five times, and the HIF transcription factor complex was detected by addition of a specific primary antibody directed against HIF-1\u03b1 and incubated for 1 h. Wells were washed five times and exposed with secondary antibody conjugated to HRP for 1 h. Wells were then washed five times, and developing agent was added to provide a sensitive colorimetric readout at 450 nm (Infinite M200; Tecan Group Ltd., M\u00e4nnedorf, Switzerland) to quantify nuclear HIF-1\u03b1 levels.\n\n## Statistical analyses.\n\nThe data are shown as mean \u00b1 SD for each group. Statistical analysis was performed by unpaired *t* test or by one-way ANOVA, followed by the Tukey test correction for multiple comparisons. All results were considered statistically significant at *P* \\< 0.05.\n\n# RESULTS\n\n## Deletion of PKC\u03b4 improved reperfusion and vascular response ischemia on diabetic limbs.\n\nNondiabetic and diabetic male *Prkcd*^\u2212\/\u2212^ mice and control littermates (*Prkcd*^+\/+^) underwent unilateral right femoral artery ligation. Body weight and fasting glucose levels were measured when mice were killed ([Supplementary Table 2](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1432\/-\/DC1)). Blood flow reperfusion was assessed using the PIM 3 laser Doppler imaging system (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>A<\/em><\/a>). Diabetic *Prkcd*^+\/+^ mice exhibited reduced blood reperfusion of the ischemic limb compared with nondiabetic *Prkcd*^+\/+^ mice (*P* = 0.0046). In contrast, reperfusion of blood flow of diabetic *Prkcd*^\u2212\/\u2212^ mice was significantly improved (*P* = 0.0003) compared with diabetic *Prkcd*^+\/+^ mice and similar to nondiabetic *Prkcd*^+\/+^ and *Prkcd*^\u2212\/\u2212^ mice 28 days after the ligation (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>B<\/em><\/a>). Because diabetic patients are at high risk of lower limb amputation, we assessed limb necrosis and apoptosis. Impaired reperfusion in ischemic limbs of diabetic *Prkcd*^+\/+^ mice was associated with elevated tissue necrosis, amputation (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>C<\/em><\/a>), and apoptosis (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<\/a>) compared with nondiabetic counterparts.\n\nOne main effect of hypoxia is to induce angiogenesis and to promote new capillary formation. To test whether activation of PKC\u03b4 is responsible for poor collateral vessel formation in diabetes, we measured capillary density and capillary diameter in the ischemic adductor muscles. <a href=\"#F3\" data-ref-type=\"fig\">Figure 3<\/a> demonstrated that the adductor muscles of diabetic *Prkcd*^+\/+^ mice displayed a 31% vascular density reduction compared with nondiabetic *Prkcd*^+\/+^ mice. The decline of capillary density was accompanied with a 50% reduction in number of vessels with a diameter of 50 \u00b5m or less. Interestingly, diabetic *Prkcd*^\u2212\/\u2212^ mice showed a significant increase in capillary density and number of vessels with a diameter of less than 50 \u00b5m compared with diabetic *Prkcd*^+\/+^ mice (<a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<em>D<\/em><\/a>).\n\n## PKC\u03b4 is activated in diabetic ischemic limb.\n\nHyperglycemia is known to activate multiple PKC isoforms, preferably the \u03b2 and \u03b4 isoforms, in vascular cells. Expression of various isoforms of PKC was assessed by quantitative PCR in muscle tissues (<a href=\"#F4\" data-ref-type=\"fig\">Fig. 4<\/a>). Compared with nondiabetic *Prkcd*^+\/+^ mice, mRNA expression of PKC\u03b2 and \u03b4 was modestly increased in adductor muscles of diabetic *Prkcd*^+\/+^ mice and unchanged in *Prkcd*^\u2212\/\u2212^ mice (<a href=\"#F4\" data-ref-type=\"fig\">Fig. 4<em>B<\/em> and <em>D<\/em><\/a>). There was no significant difference in the mRNA expression of PKC\u03b1 and -\u03b5 (<a href=\"#F4\" data-ref-type=\"fig\">Fig. 4<em>A<\/em> and <em>C<\/em><\/a>). Although diabetic *Prkcd*^+\/+^ mice did not exhibit higher levels of protein expression of PKC\u03b1, -\u03b22, -\u03b5, or -\u03b4 isoforms, adductor muscles of *Prkcd*^+\/+^ mice showed a significant and impressive increase of PKC\u03b4 phosphorylation (Thr 505; *P* = 0.0040), as a marker of PKC\u03b4 activation, 28 days after unilateral femoral artery ligation compared with nondiabetic *Prkcd*^+\/+^ mice (<a href=\"#F5\" data-ref-type=\"fig\">Fig. 5<\/a>).\n\n## Inhibition of PKC\u03b4 promotes proangiogenic growth factor expression and activation.\n\nTo explain how the absence of PKC\u03b4 improved reperfusion in diabetic ischemic limbs, we performed a wide analysis of the gene and protein expression of angiogenic-related factors and their receptors. Quantitative gene expression analyses by real-time PCR indicated that VEGF-A, PDGF-B, and PDGFR-\u03b2 mRNA expression was significantly decreased in the adductor muscles of diabetic mice by 46, 30, and 63%, respectively, compared with nondiabetic *Prkcd*^+\/+^ mice (<a href=\"#F6\" data-ref-type=\"fig\">Fig. 6<em>A<\/em>, <em>C<\/em>, and <em>D<\/em><\/a>). The reduction of these genes in diabetic *Prkcd*^+\/+^ mice was not observed in diabetic *Prkcd*^\u2212\/\u2212^ mice. Moreover, mRNA expression of VEGFR2 (KDR\/Flk-1), PDGF-B, and PDGFR-\u03b2 was significantly upregulated in diabetic *Prkcd*^\u2212\/\u2212^ compared with diabetic *Prkcd*^+\/+^ mice (<a href=\"#F6\" data-ref-type=\"fig\">Fig. 6<em>B\u2013D<\/em><\/a>). These results suggest that impaired PDGF and VEGF expression by PKC\u03b4 activation might be the contributing factor for poor collateral vessel formation in diabetes. Expression of other angiogenic factors, such as SDF-1, FGF-2, and eNOS, as well as transcriptional factor activity of HIF-1\u03b1, was unchanged within all groups of mice (<a href=\"#F6\" data-ref-type=\"fig\">Fig. 6<em>E\u2013H<\/em><\/a> and [Supplementary Fig. 1](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1432\/-\/DC1)). In contrast to 4 weeks after femoral artery ligation, transcriptional factor activity and mRNA levels of HIF-1\u03b1 were significantly decreased in diabetic *Prkcd*^+\/+^ mice compared with nondiabetic *Prkcd*^+\/+^ and diabetic *Prkcd*^\u2212\/\u2212^ mice ([Supplementary Figs. 2 and 3](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1432\/-\/DC1)).\n\n## VEGFR2 and PDGFR-\u03b2 activation is decreased in diabetic ischemic muscles.\n\nTo further investigate the mechanisms of impaired angiogenic response to restore blood flow in diabetes, the expression, activation, and signaling pathway of VEGF-A and PDGF-B and their respective receptors (VEGFR2 and PDGFR-\u03b2) were examined. Protein expression of PDGF-B was significantly decreased in diabetic versus nondiabetic adductor muscles of wild-type animals. In contrast, VEGF-A and PDGF-B protein expression was elevated in the ischemic limb of the diabetic PKC\u03b4 null mice (<a href=\"#F7\" data-ref-type=\"fig\">Fig. 7<em>A<\/em> and <em>B<\/em><\/a>). Phosphorylation of VEGFR2 and PDGFR-\u03b2 was inhibited in ischemic adductor muscles of diabetic mice compared with nondiabetic *Prkcd*^+\/+^ mice. However, activation of Src was elevated in adductor muscles of diabetic *Prkcd*^+\/+^ mice compared with nondiabetc *Prkcd*^+\/+^ and *Prkcd*^\u2212\/\u2212^ mice (<a href=\"#F7\" data-ref-type=\"fig\">Fig. 7<em>B<\/em><\/a>). Interestingly, tyrosine phosphorylation of VEGFR2 and PDGFR\u03b2, as well as PLC\u03b31, Akt, and ERK phosphorylation, was greatly enhanced in *Prkcd*^\u2212\/\u2212^ mice compared with diabetic *Prkcd*^+\/+^ mice (<a href=\"#F7\" data-ref-type=\"fig\">Fig. 7<em>A<\/em> and <em>B<\/em><\/a>). We did not observe any changes in the eNOS protein expression among experimental groups (<a href=\"#F7\" data-ref-type=\"fig\">Fig. 7<em>A<\/em><\/a>).\n\n## Expression of SHP-1 caused VEGFR2 and PDGFR-\u03b2 inactivation.\n\nWe have previously shown that activation of PKC\u03b4 leads to increased expression of SHP-1, which inhibits the PDGF-signaling pathway and promotes retinal pericyte apoptosis in diabetic animals. To determine whether SHP-1 is implicated in PKC\u03b4-induced VEGFR2 and PDGFR-\u03b2 dephosphorylation in diabetic ischemic adductor muscles, we measured SHP-1 expression by quantitative PCR and immunoblot analysis. <a href=\"#F8\" data-ref-type=\"fig\">Figure 8<em>A<\/em> and <em>B<\/em><\/a> indicates that mRNA expression of SHP-1, but not SHP-2 or PTP1B, is elevated in diabetic *Prkcd*^+\/+^ mice, whereas SHP-1 is clearly downregulated in *Prkcd*^\u2212\/\u2212^ mice. We confirmed through immunoblot analysis that SHP-1 protein expression was elevated by 2.3-fold in ischemic adductor muscles of diabetic *Prkcd*^+\/+^ mice compared with nondiabetic *Prkcd*^+\/+^ mice. The increase expression of SHP-1 was not observed in diabetic *Prkcd*^\u2212\/\u2212^ mice (<a href=\"#F8\" data-ref-type=\"fig\">Fig. 8<em>C<\/em><\/a>). No change was detected in the protein expression of SHP-2 and PTP1B within all groups of mice (<a href=\"#F8\" data-ref-type=\"fig\">Fig. 8<em>D<\/em><\/a>).\n\n# DISCUSSION\n\nDiabetes is associated with the progression of vascular complications, such as peripheral arterial disease, and is a major risk factor for lower limb amputations (4). In the current study, we have demonstrated that activation of PKC\u03b4 diminishes the expression of VEGF and PDGF, two critical proangiogenic factors contributing to poor capillary formation and blood flow reperfusion of the ischemic limbs. In addition to reducing expression of VEGF and PDGF, phosphorylation of VEGF and PDGF receptors was abrogated in diabetic ischemic muscles compared with nondiabetic ischemic muscles. The inhibition of growth factor receptor phosphorylation was associated with the upregulation of SHP-1 expression, which has been reported to deactivate tyrosine kinase receptors such as VEGF and PDGF receptors. Overall, deletion of PKC\u03b4 prevents the reduction of VEGF and PDGF expression and re-establishes KDR\/Flk-1 and PDGFR-\u03b2 phosphorylation, favoring new capillary formation and blood flow reperfusion.\n\nWound healing is a complex, well-orchestrated, and dynamic process that involves a coordinated and precise interaction of various cell types and mediators. Given the fundamental contribution of VEGF and PDGF to the angiogenic process, the mechanism by which activation of PKC\u03b4 isoform prevents growth factors expression and signaling actions may provide a better understanding of how diabetes reduces collateral vessel formation in the ischemic limb. In this study, we demonstrated that PKC\u03b4 is activated in diabetic ischemic muscles and reduced blood flow reperfusion, contributing to tissue necrosis, amputation, and apoptosis. Previous studies have reported that PKC\u03b4 is involved in vascular cell apoptosis. PKC\u03b4 activates p-38, mitogen-activated protein kinase, p53, and caspase-3 cleavage to favor endothelial (36) and smooth muscle cell apoptosis (37,38). Therefore, deletion of PKC\u03b4 may enhance vascular cell migration and proliferation, two significant steps in the formation of new blood vessels.\n\nTotal expression of PKC isoform in ischemic muscles was slightly affected by diabetes, probably because mRNA and protein analyses were performed 28 days after femoral artery ligation. However, phosphorylation of PKC\u03b4 on threonine 505, a phosphorylation site within the activation loop, clearly suggests that PKC\u03b4 is activated in the muscles of diabetic ischemic limbs compared with nondiabetic muscles. Previous data showed that the inhibition of PKC\u03b4, using an isozyme-specific peptide, improved the number of microvessels and cerebral blood flow after acute focal ischemia in normotensive rats (39). Our data demonstrate that deletion of PKC\u03b4 restores blood flow perfusion in diabetic ischemic muscles by promoting the number of capillaries and reducing tissue apoptosis.\n\nThe reduction of VEGF and PDGF receptor expression and the downstream signaling pathway is associated with impaired angiogenesis process in diabetic foot ulcer and ischemic diseases. Our results indicate that diabetes-induced PKC\u03b4 activation decreases VEGF, PDGF, KDR\/Flk-1, and PDGFR-\u03b2 mRNA expression in the ischemic limb, which is completely restored in PKC\u03b4-null mice. Interestingly, impaired angiogenic response in ischemic arterial diseases of type 1 and type 2 diabetes is associated with VEGF inhibition in endothelial cells and monocytes (13,40). It is possible that the ablation of PKC\u03b4 may also affect VEGF signaling in monocytes, which may contribute to vessel formation abnormalities. However, this assumption will need further investigation.\n\nHIF-1\u03b1 is a master regulator of angiogenic factors in response to tissue hypoxia. Previous study showed that HIF-1\u03b1 gene transfer increased recovery of limb perfusion, increasing eNOS activation and vessel density (41). In our study, however, the increase in the expression of VEGF in muscles of PKC\u03b4-deficient mice may not have been entirely due to upregulation of HIF-1\u03b1. Because protein extraction was performed 4 weeks after the femoral artery ligation, it is possible that the expression of HIF-1\u03b1 could have returned to basal levels. This hypothesis is supported by results obtained 2 weeks after the surgery. Our data demonstrated that HIF-1\u03b1 transcriptional factor activity and mRNA expression were increased in nondiabetic and diabetic PKC\u03b4-null mice 2 weeks only after surgery ([Supplementary Figs. 2 and 3](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1432\/-\/DC1)). Besides VEGF and PDGF expression, our data suggest that PKC\u03b4 activation disrupts VEGF and PDGF signaling, whereas in PKC\u03b4-deficient mice, the activity of VEGFR2, PDGFR-\u03b2, PLC\u03b31, Akt, and ERK is enhanced. Surprisingly, Src phosphorylation was increased in the ischemic muscles of diabetic wild-type mice even if PDGFR-\u03b2 activity was reduced. However, a previous study reported that reactive oxygen species (ROS) production induced Src phosphorylation (42). Because ROS are massively produced in ischemic and hyperglycemic conditions, it is probable that ROS production is responsible for the Src phosphorylation seen in diabetic wild-type mice.\n\nThere is strong evidence that progenitor cell recruitment and homing participate in angiogenesis and wound repair, which are guided by SDF-1 (43). Although the number of progenitor cells is reduced in diabetic mice, inadequate progenitor cell mobilization has been proposed as one potential mechanism of impaired angiogenesis (44). However, our results did not observe any change in SDF-1 expression in PKC\u03b4-null mice, suggesting that mobilization and local trafficking of progenitor cells to the ischemic site was not affected by the PKC\u03b4 isoform.\n\nDespite advances in revascularization techniques, limb salvage and pain relief cannot be achieved in many diabetic patients with diffuse peripheral vascular disease. VEGF-mediated gene therapy has shown promising results as an innovative method in the treatment of severe cardiovascular diseases. However, a randomized study of gene therapy failed to meet the primary objective of significant amputation reduction (45). During the 10-year follow-up period, no significant differences were detected in the number of amputations or causes of death with the use of transient VEGF-A\u2013mediated gene therapy. One reason for this lack of improvement is perhaps because neovascularization requires the interaction of multiple growth factors that can promote, in a synergic manner, new and mature blood vessels. Enhancing the responsiveness of diabetic vascular cells to proangiogenic factors may offer a potential new approach to treat peripheral arterial diseases. Protein tyrosine phosphatase is a group of proteins that is critical in abating cell response to growth factors by inhibiting tyrosine kinase phosphorylation. Our results demonstrated that SHP-1 expression was increased in diabetic ischemic muscles and was responsible for VEGF and PDGR receptor dephosphorylation.\n\nAlthough not significant, a slight rise in SHP-2 (18%) and PTP1B (37%) expression was observed in diabetic PKC\u03b4-null mice. Previous studies have shown that PDGF activation enhanced SHP-2 and PTP1B activity (46,47), which may explain our results. We have reported that activation of PKC\u03b4 induces the expression of SHP-1 in cultured pericytes exposed to high glucose concentrations and inhibits the PDGF signaling pathway contributing to pericyte apoptosis (23). Others studies have also shown that SHP-1 is a negative regulator of VEGF signal transduction and inhibits endothelial cell proliferation (48,49). Interestingly, silencing SHP-1 increased phosphorylation of KDR\/Flk-1 and markedly enhanced capillary density in a nondiabetic hind limb ischemia model (50). However, our current study does not provide a direct link between SHP-1 expression and reduced angiogenesis, which will require further investigations. Nevertheless, our findings have identified PKC\u03b4, and potentially SHP-1, as potential therapeutic targets for the treatment of diabetic peripheral arterial diseases and cardiovascular complications.\n\nIn summary, we have provided evidence that PKC\u03b4 is activated by diabetes in ischemic muscles and induced SHP-1 expression, contributing to VEGF and PDGF unresponsiveness and poor angiogenesis response. Although various therapies are partly successful in restoring blood flow to the affected tissues, there is no effective strategy to specifically produce new functional vessels to dismiss diabetic ischemic stress. Our data enhance our understanding of the mechanisms underlying poor collateral vessel formation induced by PKC activation and may offer potential novel targets to regulate angiogenesis therapeutically in patients with diabetes.\n\n## ACKNOWLEDGMENTS\n\nThis study was supported by grants from the Canadian Diabetes Association, Fonds de Recherche du Qu\u00e9bec\u2013Sant\u00e9, and Diab\u00e8te Qu\u00e9bec to P.G. and was performed at the Centre de Recherche Clinique \u00c9tienne-Le Bel, a research center funded by the Fonds de Recherche du Qu\u00e9bec\u2013Sant\u00e9. P.G. is currently the recipient of a Scholarship Award from the Canadian Diabetes Association and the Canadian Research Chair in Vascular Complications of Diabetes.\n\nNo potential conflicts of interest relevant to this article were reported.\n\nF.L., M.P., B.D., and P.G. performed experiments and analyzed the data. M.L. provided the *Prkcd*-deficient mice. A.G. performed animal care and researched data. F.L. and P.G. wrote the manuscript. P.G. is the guarantor of this work, and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.\n\nThe authors gratefully acknowledge Marie-\u00c9laine Clavet (Montreal Heart Institute) for her assistance with histochemistry technics.\n\n# REFERENCES","meta":{"dup_signals":{"dup_doc_count":147,"dup_dump_count":49,"dup_details":{"curated_sources":2,"2020-16":2,"2018-47":1,"2018-43":1,"2018-34":1,"2018-30":1,"2018-22":3,"2018-17":1,"2018-13":3,"2018-09":1,"2018-05":1,"2017-51":2,"2017-47":3,"2017-43":7,"2017-39":3,"2017-34":1,"2017-30":12,"2017-26":2,"2017-22":3,"2017-17":13,"2017-09":4,"2017-04":14,"2016-50":3,"2016-44":4,"2016-40":4,"2016-36":4,"2016-30":3,"2016-26":4,"2016-22":4,"2016-18":2,"2016-07":1,"2015-48":2,"2015-40":1,"2015-35":1,"2015-32":3,"2015-27":2,"2015-22":3,"2015-14":1,"2014-52":3,"2014-49":4,"2014-42":1,"2014-41":2,"2014-35":1,"2021-21":1,"2024-22":1,"2017-13":2,"2015-18":3,"2015-11":2,"2015-06":3,"2024-26":1}},"file":"PMC3717846"},"subset":"pubmed_central"}
{"text":"abstract: We present the first ever global account of the production, use, and end-of-life fate of all plastics ever made by humankind.\nauthor: Roland Geyer; Jenna R. Jambeck; Kara Lavender Law\\*Corresponding author. Email: <firstname.lastname@example.com>\ndate: 2017-07\nreferences:\ntitle: Production, use, and fate of all plastics ever made\n\n# INTRODUCTION\n\nA world without plastics, or synthetic organic polymers, seems unimaginable today, yet their large-scale production and use only dates back to \\~1950. Although the first synthetic plastics, such as Bakelite, appeared in the early 20th century, widespread use of plastics outside of the military did not occur until after World War II. The ensuing rapid growth in plastics production is extraordinary, surpassing most other man-made materials. Notable exceptions are materials that are used extensively in the construction sector, such as steel and cement (*1*, *2*).\n\nInstead, plastics' largest market is packaging, an application whose growth was accelerated by a global shift from reusable to single-use containers. As a result, the share of plastics in municipal solid waste (by mass) increased from less than 1% in 1960 to more than 10% by 2005 in middle- and high-income countries (*3*). At the same time, global solid waste generation, which is strongly correlated with gross national income per capita, has grown steadily over the past five decades (*4*, *5*).\n\nThe vast majority of monomers used to make plastics, such as ethylene and propylene, are derived from fossil hydrocarbons. None of the commonly used plastics are biodegradable. As a result, they accumulate, rather than decompose, in landfills or the natural environment (*6*). The only way to permanently eliminate plastic waste is by destructive thermal treatment, such as combustion or pyrolysis. Thus, near-permanent contamination of the natural environment with plastic waste is a growing concern. Plastic debris has been found in all major ocean basins (*6*), with an estimated 4 to 12 million metric tons (Mt) of plastic waste generated on land entering the marine environment in 2010 alone (*3*). Contamination of freshwater systems and terrestrial habitats is also increasingly reported (*7*\u2013*9*), as is environmental contamination with synthetic fibers (*9*, *10*). Plastic waste is now so ubiquitous in the environment that it has been suggested as a geological indicator of the proposed Anthropocene era (*11*).\n\nWe present the first global analysis of all mass-produced plastics ever made by developing and combining global data on production, use, and end-of-life fate of polymer resins, synthetic fibers, and additives into a comprehensive material flow model. The analysis includes thermoplastics, thermosets, polyurethanes (PURs), elastomers, coatings, and sealants but focuses on the most prevalent resins and fibers: high-density polyethylene (PE), low-density and linear low-density PE, polypropylene (PP), polystyrene (PS), polyvinylchloride (PVC), polyethylene terephthalate (PET), and PUR resins; and polyester, polyamide, and acrylic (PP&A) fibers. The pure polymer is mixed with additives to enhance the properties of the material.\n\n# RESULTS AND DISCUSSION\n\nGlobal production of resins and fibers increased from 2 Mt in 1950 to 380 Mt in 2015, a compound annual growth rate (CAGR) of 8.4% (table S1), roughly 2.5 times the CAGR of the global gross domestic product during that period (*12*, *13*). The total amount of resins and fibers manufactured from 1950 through 2015 is 7800 Mt. Half of this\u20143900 Mt\u2014was produced in just the past 13 years. Today, China alone accounts for 28% of global resin and 68% of global PP&A fiber production (*13*\u2013*15*). Bio-based or biodegradable plastics currently have a global production capacity of only 4 Mt and are excluded from this analysis (*16*).\n\nWe compiled production statistics for resins, fibers, and additives from a variety of industry sources and synthesized them according to type and consuming sector (table S2 and figs. S1 and S2) (*12*\u2013*24*). Data on fiber and additives production are not readily available and have typically been omitted until now. On average, we find that nonfiber plastics contain 93% polymer resin and 7% additives by mass. When including additives in the calculation, the amount of nonfiber plastics (henceforth defined as resins plus additives) manufactured since 1950 increases to 7300 Mt. PP&A fibers add another 1000 Mt. Plasticizers, fillers, and flame retardants account for about three quarters of all additives (table S3). The largest groups in total nonfiber plastics production are PE (36%), PP (21%), and PVC (12%), followed by PET, PUR, and PS (\\<10% each). Polyester, most of which is PET, accounts for 70% of all PP&A fiber production. Together, these seven groups account for 92% of all plastics ever made. Approximately 42% of all nonfiber plastics have been used for packaging, which is predominantly composed of PE, PP, and PET. The building and construction sector, which has used 69% of all PVC, is the next largest consuming sector, using 19% of all nonfiber plastics (table S2).\n\nWe combined plastic production data with product lifetime distributions for eight different industrial use sectors, or product categories, to model how long plastics are in use before they reach the end of their useful lifetimes and are discarded (*22*, *25*\u2013*29*). We assumed log-normal distributions with means ranging from less than 1 year, for packaging, to decades, for building and construction (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<\/a>). This is a commonly used modeling approach to estimating waste generation for specific materials (*22*, *25*, *26*). A more direct way to measure plastic waste generation is to combine solid waste generation data with waste characterization information, as in the study of Jambeck *et al.* (*3*). However, for many countries, these data are not available in the detail and quality required for the present analysis.\n\nWe estimate that in 2015, 407 Mt of primary plastics (plastics manufactured from virgin materials) entered the use phase, whereas 302 Mt left it. Thus, in 2015, 105 Mt were added to the in-use stock. For comparison, we estimate that plastic waste generation in 2010 was 274 Mt, which is equal to the independently derived estimate of 275 Mt by Jambeck *et al.* (*3*). The different product lifetimes lead to a substantial shift in industrial use sector and polymer type between plastics entering and leaving use in any given year (tables S4 and S5 and figs. S1 to S4). Most of the packaging plastics leave use the same year they are produced, whereas construction plastics leaving use were produced decades earlier, when production quantities were much lower. For example, in 2015, 42% of primary nonfiber plastics produced (146 Mt) entered use as packaging and 19% (65 Mt) as construction, whereas nonfiber plastic waste leaving use was 54% packaging (141 Mt) and only 5% construction (12 Mt). Similarly, in 2015, PVC accounted for 11% of nonfiber plastics production (38 Mt) and only 6% of nonfiber plastic waste generation (16 Mt).\n\nBy the end of 2015, all plastic waste ever generated from primary plastics had reached 5800 Mt, 700 Mt of which were PP&A fibers. There are essentially three different fates for plastic waste. First, it can be recycled or reprocessed into a secondary material (*22*, *26*). Recycling delays, rather than avoids, final disposal. It reduces future plastic waste generation only if it displaces primary plastic production (*30*); however, because of its counterfactual nature, this displacement is extremely difficult to establish (*31*). Furthermore, contamination and the mixing of polymer types generate secondary plastics of limited or low technical and economic value. Second, plastics can be destroyed thermally. Although there are emerging technologies, such as pyrolysis, which extracts fuel from plastic waste, to date, virtually all thermal destruction has been by incineration, with or without energy recovery. The environmental and health impacts of waste incinerators strongly depend on emission control technology, as well as incinerator design and operation. Finally, plastics can be discarded and either contained in a managed system, such as sanitary landfills, or left uncontained in open dumps or in the natural environment.\n\nWe estimate that 2500 Mt of plastics\u2014or 30% of all plastics ever produced\u2014are currently in use. Between 1950 and 2015, cumulative waste generation of primary and secondary (recycled) plastic waste amounted to 6300 Mt. Of this, approximately 800 Mt (12%) of plastics have been incinerated and 600 Mt (9%) have been recycled, only 10% of which have been recycled more than once. Around 4900 Mt\u201460% of all plastics ever produced\u2014were discarded and are accumulating in landfills or in the natural environment (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<\/a>). Of this, 600 Mt were PP&A fibers. None of the mass-produced plastics biodegrade in any meaningful way; however, sunlight weakens the materials, causing fragmentation into particles known to reach millimeters or micrometers in size (*32*). Research into the environmental impacts of these \"microplastics\" in marine and freshwater environments has accelerated in recent years (*33*), but little is known about the impacts of plastic waste in land-based ecosystems.\n\nBefore 1980, plastic recycling and incineration were negligible. Since then, only nonfiber plastics have been subject to significant recycling efforts. The following results apply to nonfiber plastic only: Global recycling and incineration rates have slowly increased to account for 18 and 24%, respectively, of nonfiber plastic waste generated in 2014 (figs. S5 and S6). On the basis of limited available data, the highest recycling rates in 2014 were in Europe (30%) and China (25%), whereas in the United States, plastic recycling has remained steady at 9% since 2012 (*12*, *13*, *34*\u2013*36*). In Europe and China, incineration rates have increased over time to reach 40 and 30%, respectively, in 2014 (*13*, *35*). However, in the United States, nonfiber plastics incineration peaked at 21% in 1995 before decreasing to 16% in 2014 as recycling rates increased, with discard rates remaining constant at 75% during that time period (*34*). Waste management information for 52 other countries suggests that in 2014, the rest of the world had recycling and incineration rates similar to those of the United States (*37*). To date, end-of-life textiles (fiber products) do not experience significant recycling rates and are thus incinerated or discarded together with other solid waste.\n\nPrimary plastics production data describe a robust time trend throughout its entire history. If production were to continue on this curve, humankind will have produced 26,000 Mt of resins, 6000 Mt of PP&A fibers, and 2000 Mt of additives by the end of 2050. Assuming consistent use patterns and projecting current global waste management trends to 2050 (fig. S7), 9000 Mt of plastic waste will have been recycled, 12,000 Mt incinerated, and 12,000 Mt discarded in landfills or the natural environment (<a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<\/a>).\n\nAny material flow analysis of this kind requires multiple assumptions or simplifications, which are listed in Materials and Methods, and is subject to considerable uncertainty; as such, all cumulative results are rounded to the nearest 100 Mt. The largest sources of uncertainty are the lifetime distributions of the product categories and the plastic incineration and recycling rates outside of Europe and the United States. Increasing\/decreasing the mean lifetimes of all product categories by 1 SD changes the cumulative primary plastic waste generation (for 1950 to 2015) from 5900 to 4600\/6200 Mt or by \u22124\/+5%. Increasing\/decreasing current global incineration and recycling rates by 5%, and adjusting the time trends accordingly, changes the cumulative discarded plastic waste from 4900 (for 1950 to 2015) to 4500\/5200 Mt or by \u22128\/+6%.\n\nThe growth of plastics production in the past 65 years has substantially outpaced any other manufactured material. The same properties that make plastics so versatile in innumerable applications\u2014durability and resistance to degradation\u2014make these materials difficult or impossible for nature to assimilate. Thus, without a well-designed and tailor-made management strategy for end-of-life plastics, humans are conducting a singular uncontrolled experiment on a global scale, in which billions of metric tons of material will accumulate across all major terrestrial and aquatic ecosystems on the planet. The relative advantages and disadvantages of dematerialization, substitution, reuse, material recycling, waste-to-energy, and conversion technologies must be carefully considered to design the best solutions to the environmental challenges posed by the enormous and sustained global growth in plastics production and use.\n\n# MATERIALS AND METHODS\n\n## Plastic production\n\nThe starting point of the plastic production model is global annual pure polymer (resin) production data from 1950 to 2015, published by the Plastics Europe Market Research Group, and global annual fiber production data from 1970 to 2015 published by The Fiber Year and Tecnon OrbiChem (table S1). The resin data closely follow a second-order polynomial time trend, which generated a fit of *R*^2^ = 0.9968. The fiber data closely follow a third-order polynomial time trend, which generated a fit of *R*^2^ = 0.9934. Global breakdowns of total production by polymer type and industrial use sector were derived from annual market and polymer data for North America, Europe, China, and India (table S2) (*12*, *13*, *19*\u2013*24*). U.S. and European data are available for 2002 to 2014. Polymer type and industrial use sector breakdowns of polymer production are similar across countries and regions.\n\nGlobal additives production data, which are not publicly available, were acquired from market research companies and cross-checked for consistency (table S3) (*17*, *18*). Additives data are available for 2000 to 2014. Polymer type and industrial use sector breakdowns of polymer production and the additives to polymer fraction were both stable over the time period for which data are available and thus assumed constant throughout the modeling period of 1950\u20132015. Any errors in the early decades were mitigated by the lower production rates in those years. Additives data were organized by additive type and industrial use sector and integrated with the polymer data. *P*~*i*~ (*t*) denotes the amount of primary plastics (that is, polymers plus additives) produced in year *t* and used in sector *i* (fig. S1).\n\n## Plastic waste generation and fate\n\nPlastics use was characterized by discretized log-normal distributions, LTD~i~ (*j*), which denotes the fraction of plastics in industrial use sector *i* used for *j* years (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<\/a>). Mean values and SDs were gathered from published literature (table S4) (*22*, *25*\u2013*29*). Product lifetimes may vary significantly across economies and also across demographic groups, which is why distributions were used and sensitivity analysis was conducted with regard to mean product lifetimes. The total amount of primary plastic waste generated in year *t* was calculated as PW (*t*) = $\\sum_{\\mathit{i} = 1}^{8}\\sum_{\\mathit{j} = 1}^{65}\\mathit{P}_{\\mathit{i}}(\\mathit{t} - \\mathit{j}) \\bullet \\text{LTD}_{\\mathit{i}}(\\mathit{j})$ (figs. S3 and S4). Secondary plastic waste generated in year *t* was calculated as the fraction of total plastic waste that was recycled *k* years ago, SW (*t*) = \\[PW (*t* \u2212 *k*) + SW (*t* \u2212 *k*)\\]\\[RR (*t* \u2212 *k*)\\], where *k* is the average use time of secondary plastics and RR (*t* \u2212 *k*) is the global recycling rate in year *t* \u2212 *k*. Amounts of plastic waste discarded and incinerated are calculated as DW(*t*) = \\[PW(*t*) + SW(*t*) \u2219 DR(*t*) and IW(*t*) = \\[PW(*t*) + SW(*t*)\\] \u2219 IR(*t*), with DR(*t*) and IR(*t*) being the global discard and incineration rates in year *t* (fig. S5). Cumulative values at time *T* were calculated as the sum over all *T* \u2212 1950 years of plastics mass production. Examples are cumulative primary production $\\text{CP}_{\\mathit{i}}(\\mathit{T}) = \\sum_{\\mathit{t} = 1950}^{\\mathit{T}}\\mathit{P}_{\\mathit{i}}(\\mathit{t})$ and cumulative primary plastic waste generation, $\\text{CPW}(\\mathit{T}) = \\sum_{\\mathit{t} = 1950}^{\\mathit{T}}PW(\\mathit{t})$ (<a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<\/a>).\n\n## Recycling, incineration, and discard rates\n\nTime series for resin, that is, nonfiber recycling, incineration, and discard rates were collected separately for four world regions: the United States, the EU-28 plus Norway and Switzerland, China, and the rest of the world. Detailed and comprehensive solid waste management data for the United States were published by the U.S. Environmental Protection Agency dating back to 1960 (table S7) (*34*). European data were from several reports by PlasticsEurope, which collectively cover 1996 to 2014 (*12*, *13*, *38*). Chinese data were synthesized and reconciled from the English version of the China Statistical Yearbook, translations of Chinese publications and government reports, and additional waste management literature (*35*, *36*, *39*\u2013*41*). Waste management for the rest of the world was based on World Bank data (*37*). Time series for global recycling, incineration, and discard rates (fig. S5) were derived by adding the rates of the four regions weighted by their relative contribution to global plastic waste generation. In many world regions, waste management data were sparse and of poor quality. For this reason, sensitivity analysis with regard to waste management rates was conducted.\n\nThe resulting global nonfiber recycling rate increased at a constant 0.7% per annum (p.a.) between 1990 and 2014. If this linear trend is assumed to continue, the global recycling rate would reach 44% in 2050. The global nonfiber incineration rate has grown more unevenly but, on average, increased 0.7% p.a. between 1980 and 2014. Assuming an annual increase of 0.7% between 2014 and 2050 yielded a global incineration rate of 50% by 2050. With those two assumptions, global discard rate would decrease from 58% in 2014 to 6% in 2050 (fig. S7). The dashed lines in <a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<\/a> are based on those assumptions and therefore simply forward projections of historical global trends and should not be mistaken for a prediction or forecast. There is currently no significant recycling of synthetic fibers. It was thus assumed that end-of-life textiles are incinerated and discarded together with all other municipal solid waste.\n\n# Supplementary Material\n\n###### http:\/\/advances.sciencemag.org\/cgi\/content\/full\/3\/7\/e1700782\/DC1\n\n## Acknowledgments\n\nWe thank C.-J. Simon and R. Nayaran for providing polymer production data and G.L. Mahoney for designing <a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<\/a>. We especially thank I. Creelman and R. Song for data collection and model implementation, E. Smith for assistance in compiling the U.S. Environmental Protection Agency and China solid waste management data, S. Wang for researching China plastic waste management and translation of articles, J. Chamberlain for researching additives, and I. MacAdam-Somer for researching synthetic fibers. This work benefitted from helpful discussions with T. Siegler and C. Rochman and comments from two anonymous reviewers. **Funding:** This work was conducted within the Marine Debris Working Group at the National Center for Ecological Analysis and Synthesis, University of California, Santa Barbara, with support from Ocean Conservancy. R.G. was supported by the NSF Chemical, Bioengineering, Environmental and Transport Systems grant \\#1335478. **Author contributions:** R.G. led the research design, data collection, model development, calculations, interpretation of results, and writing of the manuscript; J.R.J. contributed to research design, data collection, model development, and interpretation of results; K.L.L. contributed to research design, analysis and interpretation of results, and writing of the manuscript. **Competing interests:** The authors declare that they have no competing interests. **Data and materials availability:** All data needed to evaluate the conclusions in the paper are present in the paper and\/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.\n\n### SUPPLEMENTARY MATERIALS\n\nSupplementary material for this article is available at <http:\/\/advances.sciencemag.org\/cgi\/content\/full\/3\/7\/e1700782\/DC1>\n\nfig. S1. Global primary plastics production (in million metric tons) according to industrial use sector from 1950 to 2015.\n\nfig. S2. Global primary plastics production (in million metric tons) according to polymer type from 1950 to 2015.\n\nfig. S3. Global primary plastics waste generation (in million metric tons) according to industrial use sector from 1950 to 2015.\n\nfig. S4. Global primary plastics waste generation (in million metric tons) according to polymer type from 1950 to 2015.\n\nfig. S5. Estimated percentage of global (nonfiber) plastic waste recycled, incinerated, and discarded from 1950 to 2014 \\[(*12*, *13*, *34*\u2013*42*) and table S7\\].\n\nfig. S6. Annual global primary and secondary plastic waste generation TW (*t*), recycling RW (*t*), incineration IW (*t*), and discard DW (*t*) (in million metric tons) from 1950 to 2014.\n\nfig. S7. Projection of global trends in recycling, incineration, and discard of plastic waste from 1980 to 2014 (to the left of vertical black line) to 2050 (to the right of vertical black line).\n\ntable S1. Annual global polymer resin and fiber production in million metric tons (*12*\u2013*15*).\n\ntable S2. Share of total polymer resin production according to polymer type and industrial use sector calculated from data for Europe, the United States, China, and India covering the period 2002\u20132014 (*12*, *13*, *19*\u2013*24*).\n\ntable S3. Share of additive type in global plastics production from data covering the period 2000\u20132014 (*17*, *18*).\n\ntable S4. Baseline mean values and SDs used to generate log-normal product lifetime distributions for the eight industrial use sectors used in this study (*22*, *25*\u2013*29*).\n\ntable S5. Global primary plastics production and primary waste generation (in million metric tons) in 2015 according to industrial use sector.\n\ntable S6. Global primary plastics production and primary waste generation (in million metric tons) in 2015 according to polymer type\/additive.\n\ntable S7. Additional data sources for U.S. plastics recycling and incineration.\n\ntable S8. Complete list of data sources.\n\n# REFERENCES AND NOTES","meta":{"dup_signals":{"dup_doc_count":103,"dup_dump_count":36,"dup_details":{"curated_sources":1,"2021-31":3,"2021-25":7,"2021-21":1,"2021-17":5,"2021-04":6,"2020-50":2,"2020-45":6,"2020-40":2,"2020-34":7,"2020-29":2,"2020-24":1,"2020-16":4,"2020-10":5,"2020-05":1,"2019-51":5,"2019-47":3,"2019-43":4,"2019-39":3,"2019-35":5,"2019-30":2,"2019-26":7,"2019-22":1,"2019-18":1,"2019-13":1,"2019-09":3,"2018-51":1,"2018-47":1,"2018-43":1,"2018-39":1,"2018-34":5,"2018-26":1,"2018-22":1,"2018-13":1,"2017-51":1,"2017-43":1,"2017-34":1}},"file":"PMC5517107"},"subset":"pubmed_central"}
{"text":"date: 2018-04\ntitle: Individualized, supportive care key to positive childbirth experience, says WHO\n\n**15 FEBRUARY 2018 \\| GENEVA -** AWHO has issued new recommendations to establish global care standards for healthy pregnant women and reduce unnecessary medical interventions.\n\nWorldwide, an estimated 140 million births take place every year. Most of these occur without complications for women and their babies. Yet, over the past 20 years, practitioners have increased the use of interventions that were previously only used to avoid risks or treat complications, such as oxytocin infusion to speed up labour or caesarean sections.\n\n\"We want women to give birth in a safe environment with skilled birth attendants in well-equipped facilities. However, the increasing medicalization of normal childbirth processes are undermining a woman's own capability to give birth and negatively impacting her birth experience,\" says Dr Princess Nothemba Simelela, WHO Assistant Director-General for Family, Women, Children and Adolescents.\n\n\"If labour is progressing normally, and the woman and her baby are in good condition, they do not need to receive additional interventions to accelerate labour,\" she says.\n\nChildbirth is a normal physiological process that can be accomplished without complications for the majority of women and babies. However, studies show a substantial proportion of healthy pregnant women undergo at least one clinical intervention during labour and birth. They are also often subjected to needless and potentially harmful routine interventions.\n\nThe new WHO guideline includes 56 evidence-based recommendations on what care is needed throughout labour and immediately after for the woman and her baby. These include having a companion of choice during labour and childbirth; ensuring respectful care and good communication between women and health providers; maintaining privacy and confidentiality; and allowing women to make decisions about their pain management, labour and birth positions and natural urge to push, among others.\n\n**Every labour is unique and progresses at different rates**\n\nThe new WHO guideline recognizes that every labour and childbirth is unique and that the duration of the active first stage of labour varies from one woman to another. In a first labour, it usually does not extend beyond 12 hours. In subsequent labours it usually does not extend beyond 10 hours.\n\nTo reduce unnecessary medical interventions, the WHO guideline states that the previous benchmark for cervical dilation rate at 1 cm\/hr during the active first stage of labour (as assessed by a partograph or chart used to document the course of a normal labour) may be unrealistic for some women and is inaccurate in identifying women at risk of adverse birth outcomes. The guideline emphasizes that a slower cervical dilation rate alone should not be a routine indication for intervention to accelerate labour or expedite birth.\n\n\"Many women want a natural birth and prefer to rely on their bodies to give birth to their baby without the aid of medical intervention,\" says Ian Askew, WHO Director, Department of Reproductive Health and Research. \"Even when a medical intervention is wanted or needed, the inclusion of women in making decisions about the care they receive is important to ensure that they meet their goal of a positive childbirth experience.\"\n\n**High quality care for all women**\n\nUnnecessary labour interventions are widespread in low-, middle- and high-income settings, often putting a strain on already scarce resources in some countries, and further widening of the equity gap.\n\nAs more women give birth in health facilities with skilled health professionals and timely referrals, they deserve better quality of care. About 830 women die from pregnancy- or childbirth-related complications around the world every day \u2013 the majority could be prevented with high-quality care in pregnancy and during childbirth.\n\nDisrespectful and non-dignified care is prevalent in many health facilities, violating human rights and preventing women from accessing care services during childbirth. In many parts of the world, the health provider controls the birthing process, which further exposes healthy pregnant women to unnecessary medical interventions that interfere with the natural childbirth process.\n\nAchieving the best possible physical, emotional, and psychological outcomes for the woman and her baby requires a model of care in which health systems empower all women to access care that focuses on the mother and child.\n\nHealth professionals should advise healthy pregnant women that the duration of labour varies greatly from one woman to another. While most women want a natural labour and birth, they also acknowledge that birth can be an unpredictable and risky event and that close monitoring and sometimes medical interventions may be necessary. Even when interventions are needed or wanted, women usually wish to retain a sense of personal achievement and control by being involved in decision making, and by rooming in with their baby after childbirth.\n\nThe data also highlight the need for rapid scale-up of research. There have been some encouraging signs in funding available for investment in research for a cure for dementia in recent years, but much more needs to be done. The number of articles in peer-reviewed journals on dementia in 2016 was close to 7000. This compares with more than 15 000 for diabetes, and more than 99 000 for cancer during the same year. Research is needed not only to find a cure for dementia, but also in the areas of prevention, risk reduction, diagnosis, treatment and care.\n\nThe Observatory will provide a knowledge bank where health and social care authorities, medical professionals, researchers and civil society organizations will be able to find country and regional dementia profiles, global reports, policy guidance, guidelines and toolkits on dementia prevention and care.\n\n**Dementia**\n\nDementia is an umbrella term for several diseases that are mostly progressive, affecting memory, other cognitive abilities and behaviour and interfering significantly with a person's ability to maintain the activities of daily living. Women are more often affected than men. Alzheimer's disease is the most common type of dementia and accounts for 60\u201370% of cases. The other common types are vascular dementia and mixed forms.\n\nAvailable from: <http:\/\/www.who.int\/mediacentre\/news\/releases\/2018\/positive-childbirth-experience\/en\/>","meta":{"dup_signals":{"dup_doc_count":118,"dup_dump_count":47,"dup_details":{"curated_sources":2,"2023-50":3,"2023-40":1,"2023-23":1,"2023-14":1,"2023-06":2,"2022-49":1,"2022-33":1,"2022-05":1,"2021-43":1,"2021-39":1,"2021-31":3,"2021-25":1,"2021-21":2,"2021-17":2,"2021-04":2,"2020-50":1,"2020-45":2,"2020-40":3,"2020-29":2,"2020-24":1,"2020-16":2,"2020-10":2,"2020-05":2,"2019-51":5,"2019-43":2,"2019-39":1,"2019-35":7,"2019-26":3,"2019-22":3,"2019-18":5,"2019-13":2,"2019-09":3,"2019-04":7,"2018-51":3,"2018-47":2,"2018-43":5,"2018-39":2,"2018-34":5,"2018-30":1,"2018-26":3,"2018-22":4,"2018-17":2,"2018-13":7,"2024-26":1,"2024-18":2,"2024-10":2,"2024-30":1}},"file":"PMC5938665"},"subset":"pubmed_central"}
{"text":"abstract: Using a genome-scale approach to study transcription levels in a human CD8^+^ T-cell clone, a recent study has suggested that the repertoire of molecules on the surface of T cells is close to being completely characterized.\nauthor: Joerg Ermann; Chan D Chung; C Garrison Fathman\ndate: 2004\ninstitute: 1Division of Immunology and Rheumatology, Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA\nreferences:\ntitle: Scratching the (T cell) surface\n\nOver the last few years technological advances have made it possible to study, in parallel, the expression of thousands of genes in cells, tissues or organisms. While this genome-scale approach to gene-expression analysis has been touted by some as the new 'golden era' of hypothesis-unlimited discovery-driven research \\[1\\], it is apparent that our ability to make sense of the vast accumulations of data does not always keep up with our ability to generate them. Many groups have used technologies such as microarrays or serial analysis of gene expression (SAGE) to analyze gene expression in cells of the immune system. A recent article by Evans *et al*. in *Immunity* \\[2\\] attempting to expand beyond the simple description of differential gene-expression patterns arrives at a bold conclusion. The article's title is intriguing - *The T-cell surface - how well do we know it?* - and the conclusion is that we know it quite well.\n\nNot surprisingly, molecules on the surface of T cells are of great interest to immunologists. Much information has been generated about T-cell-specific surface molecules and their function since the first monoclonal antibodies against leukocyte surface markers were made in the late 1970s. Attempts to compare the specificities of different monoclonal antibodies and to identify their targets led to the development of the cluster of differentiation (CD) nomenclature \\[3\\]. Most of the CD antigens turned out to be proteins (a few CD antibodies detect carbohydrate modifications) and the respective genes have been cloned in both humans and mice. Over time the CD system thus transformed into a classification for leukocyte surface molecules, rather than antibodies. Currently, there are 247 assigned CDs \\[4\\]. Almost 200 additional molecules are being considered for CD status during the current Eighth International Workshop on Human Leukocyte Differentiation Antigens that will culminate in the HLDA8 Conference in Adelaide, Australia in December 2004 \\[5\\].\n\nThe molecules on the surface of T cells belong to very diverse structural and functional classes, and include components of the immunoreceptors on T, B and NK cells, adhesion molecules, and cytokine and chemokine receptors. Some CDs have proven to be useful markers of subpopulations of cells with strikingly different functions. Most T cells express CD3 and an \u03b1\u03b2 T-cell receptor (TCR) paired with either the CD4 or the CD8 molecule as co-receptor. CD4^+^ \u03b1\u03b2 T cells recognize antigens presented by antigen-presenting cells in the context of major histocompatibility complex (MHC) class II molecules. Their main effector function is the production of cytokines and the facilitation of immune responses. A subclass of CD4^+^ T cells that has recently gained attention comprises regulatory\/suppressor T cells, which are characterized by the constitutive expression of CD25 - the \u03b1-chain of the interleukin 2 (IL-2) receptor. These cells are thought to negatively regulate immune responses and to prevent uncontrolled autoimmunity. CD8^+^ \u03b1\u03b2 T cells recognize antigens in the context of MHC class I molecules, which are expressed on most somatic cells. Upon activation, CD8^+^ T cells develop into cytolytic T lymphocytes (CTLs) ready to kill cells infected by intracellular pathogens, such as viruses, or eradicate tumors cells.\n\nIn their article on the T-cell surface, Evans *et al*. \\[2\\] used SAGE to study gene expression in a human CD8^+^ CTL clone. The SAGE library that was generated (referred to as the CTL library) contained 71,174 SAGE tags representing 20,204 distinct sequences. This number was estimated to cover all of the transcripts whose expression level was at or above 0.008% of the transcriptome (that is having at least 22 copies per cell). The library included 111 genes with, or being considered for, CD status. Several pairwise comparisons with unrelated SAGE libraries were then performed.\n\nThe central analysis of the paper starts with a comparison of the CTL library with a library derived from cerebellum; 1,098 transcripts were significantly more abundant in the CTL library. Among these, about a quarter of the transcripts with known function coded for proteins involved in protein\/mRNA synthesis, a result that was thought to reflect the proliferating versus non-proliferating character of the two cell\/tissue types (CTL versus cerebellum). Interestingly, the set of genes that was highly differentially expressed in the CTLs was enriched for surface markers, signaling molecules and soluble mediators. In an attempt to find a core set of CTL-specific genes, additional comparisons were performed between the CTL library and SAGE libraries from ovary epithelium (as a type of proliferating cell) and a panel of tumor libraries. This resulted in a shortened list of 387 CTL-specific transcripts.\n\nNotably, at all stages of comparison 42-45% of the transcripts lacked an assigned function. Of the known genes in the final list of 387 specific transcripts in the CTL library, 27% were cell-surface molecules, including TCR components, CD2, CD5, and CD8. Evans *et al*. \\[2\\] then asked how many of the unknown CTL-specific transcripts encoded surface molecules. Sequences representing UniGene clusters \\[6\\] were analyzed for signatures of surface molecules by domain analysis, looking for transmembrane regions or other domains characteristic of leukocyte surface molecules, and by BLAST searches for related genes with known function. Surprisingly, only 2 of the 97 (2%) UniGene clusters analyzed showed some potential for encoding novel surface molecules. The authors therefore concluded that \"the cell-type-specific composition of the resting CD8^+^ T-cell surface is now largely defined.\"\n\nHow complete and cell-type-specific is this list of 387 genes? The CTL SAGE library of 20,204 transcripts represents the transcriptome of the CTL clone. A detailed discussion of the limitations of the SAGE method is beyond the scope of this article; suffice it to say that it is possible that functionally important genes may be missing from the library due to low mRNA expression levels, chance or because they lack the target sequence for the tagging enzyme used in the SAGE protocol \\[7\\]. In order to get to the final set of 378 CTL-specific genes Evans *et al*. \\[2\\] eliminated all transcripts that were present at comparable levels in unrelated libraries. This powerful approach seems to have validated itself by the fact that TCR components and other principal T-cell markers were present in the shortlist of 387 genes. The method used is not unbiased, however. The choice of libraries (in this case cerebellum, ovary epithelium, and a panel of tumor cell lines that were not specified further), data quality, and the algorithms used for comparison can be expected to have an enormous impact on the results obtained. It is easy to see how functionally important genes might get lost because they are expressed at sufficiently high levels in one of the cell or tissue types used for comparison. A list of cell-type-specific genes derived by this method of successive *in silico* subtraction defines a cell-type-specific gene-expression pattern against the transcriptional background of the cell or tissue types used for comparison. It is not a list of all genes relevant for cell-type-specific function.\n\nThe major finding of the Evans *et al*. study \\[2\\] is that among the 387 CTL-specific transcripts 27% of the known genes encoded cell-surface molecules, whereas only 2% of the unknown genes showed some potential in that regard. The implication is that the catalog of CTL surface molecules is close to being complete. While it is not unreasonable to assume that the concerted efforts over the last two decades to characterize surface molecules on leukocytes have led to a situation where most CTL-specific surface molecules are known \\[8\\], some questions remain. Is this finding unique for CTLs (or for leukocytes in general)? What would be the result of a similar analysis in, for instance, ovary epithelium? Were there unknown cell-surface molecules in the CTL SAGE library? If so, at what point of the stepwise subtraction process did these transcripts get eliminated? It has been noted that leukocytes share many surface molecules with neuronal cells and epithelial cells \\[8\\], the very cells used for subtraction by Evans *et al*. \\[2\\]. An alternative experimental approach to analyzing the incidence of unknown cell-surface molecules might be to generate SAGE libraries from microsomal and free-ribosomal mRNA pools generated through equilibrium density centrifugation. This approach has been demonstrated to discriminate secretory and cell-surface molecules from nonsecretory proteins quite efficiently \\[9,10\\]. One would expect a significantly lower percentage of unknown transcripts in the secretory\/surface molecule fraction.\n\nOnly the CTL SAGE library was actually generated by Evans *et al*. \\[2\\]. The other libraries used for comparison were derived from publicly available databases. Open access to primary gene-expression data is essential, not only for enabling researchers to reproduce published analyses, but also to allow for novel experimental approaches that incorporate relevant data generated by others. Important information can be gained by comparing genome-wide expression data across large numbers of samples. In a recent, extreme example, 3,283 DNA microarrays from human, *Drosophila*, *Caenorhabditis elegans* and yeast were used to define evolutionarily conserved genetic modules of co-expressed genes \\[11\\]. SAGE data have been publicly available on SAGEMap \\[12,13\\] for a number of years. Microarray data are far more complex, but a standard for the annotation of microarray data (Minimal Information About a Microarray Experiment; MIAME) \\[14\\] and a platform-independent data exchange format (Microarray Gene Expression Markup Language; MAGE-ML) \\[15\\] have been developed. Furthermore, public repositories for microarray data such as ArrayExpress \\[16,17\\] and GeoBus \\[18,19\\] are now available.\n\nSAGE has the advantage over current microarray technology of measuring absolute transcript abundance. Nevertheless, there are some limitations as to what can be said about the T-cell surface by studying mRNA levels. First, for a number of surface molecules, such as CD45, a variety of functionally important splice variants have been described \\[20\\] that cannot be distinguished by the 3' SAGE tag. Second, mRNA levels correlate poorly with protein abundance \\[21\\]. Third, posttranslational protein modifications can be functionally relevant; for example, glycosylation of CD8 has been demonstrated to affect thymocyte selection by influencing activation thresholds \\[22\\]. Fourth, T-cell activation involves re-localization of surface molecules leading to the formation of the immunological synapse, a supramolecular cluster at the contact zone between antigen-presenting cell and the T cell \\[23\\]. These early events precede changes in gene expression.\n\nFinally, it seems important to note that the T-cell surface is an abstraction. T cells comprise quite different subsets of cells at variable activation states. As pointed out by Evans *et al*. \\[2\\], the finding that most of the molecules on the T-cell surface appear to be known applies strictly only to a resting CD8^+^ T-cell clone *in vitro*. 'The T-cell surface - how well do we know it?' is an important question on our way into the post-genomic era of immunology. But even with complete lists of the genes expressed in certain T-cell subpopulations, much more needs to be learned about the regulation and complex interactions of the proteins they encode. We are just scratching the (T cell) surface.","meta":{"dup_signals":{"dup_doc_count":140,"dup_dump_count":48,"dup_details":{"curated_sources":2,"2020-34":1,"2019-22":1,"2018-47":1,"2018-39":1,"2018-22":1,"2018-09":1,"2018-05":2,"2017-51":1,"2017-43":1,"2017-39":2,"2017-34":1,"2017-30":2,"2017-26":1,"2017-22":1,"2017-17":2,"2017-09":3,"2017-04":2,"2016-50":3,"2016-44":2,"2016-40":2,"2016-36":6,"2016-30":4,"2016-26":2,"2016-22":3,"2016-18":3,"2016-07":5,"2015-48":4,"2015-40":3,"2015-35":4,"2015-32":3,"2015-27":5,"2015-22":4,"2015-14":3,"2014-52":4,"2014-49":3,"2014-42":9,"2014-41":4,"2014-35":3,"2014-23":6,"2014-15":3,"2021-49":1,"2017-13":2,"2015-18":4,"2015-11":4,"2015-06":4,"2014-10":4,"2013-48":4,"2013-20":3}},"file":"PMC395726"},"subset":"pubmed_central"}
{"text":"abstract: Chronic consumption of diets high in resistant starch (RS) leads to reduced fat cell size compared to diets high in digestible starch (DS) in rats and increases total and meal fat oxidation in humans. The aim of the present study was to examine the rate of lipogenesis in key lipogenic organs following a high RS or DS meal. Following an overnight fast, male Wistar rats ingested a meal with an RS content of 2% or 30% of total carbohydrate and were then administered an i.p bolus of 50 \u03bcCi ^3^H~2~O either immediately or 1 hour post-meal. One hour following tracer administration, rats were sacrificed, a blood sample collected, and the liver, white adipose tissue (WAT), and gastrocnemius muscle excised and frozen until assayed for total ^3^H-lipid and ^3^H-glycogen content. Plasma triglyceride and NEFA concentrations and ^3^H-glycogen content did not differ between groups. In all tissues, except the liver, there was a trend for the rate of lipogenesis to be higher in the DS group than the RS group which reached significance only in WAT at 1 h (p \\< 0.01). On a whole body level, this attenuation of fat deposition in WAT in response to a RS diet could be significant for the prevention of weight gain in the long-term.\nauthor: Janine A Higgins; Marc A Brown; Leonard H Storlien\ndate: 2006\ninstitute: 1University of Colorado Health Sciences Center, Denver, Colorado 80262, USA; 2Metabolic Research Centre, Faculty of Health & Behavioural Sciences, University of Wollongong, NSW 2522, Australia; 3AstraZeneca International, M\u00f6lndal, Sweden\nreferences:\ntitle: Consumption of resistant starch decreases postprandial lipogenesis in white adipose tissue of the rat\n\n# Findings\n\nIt has been reported that chronic resistant starch (RS) feeding in rats causes a decrease in adipocyte cell size, a decrease in fatty acid synthase expression, and reduced whole-body weight gain relative to digestible starch (DS) feeding \\[1,2\\]. Additionally, in healthy adults, a single RS meal caused a substantial elevation in total and meal fat oxidation compared to a DS meal \\[3\\]. These data suggest that RS intake may influence postprandial lipid metabolism. The aim of the present study was to examine the rate of lipogenesis in key lipogenic organs acutely following a RS or DS meal.\n\nMale Wistar rats (*Rattus norvegicus*) were obtained from the Animal Resource Center (Murdoch, Western Australia) and were housed in groups of three at the University of Wollongong, Animal House. The rats were maintained at 22\u00b0C on a 12-h light\/dark cycle (light cycle from 0700\u20131900 h), with free access to a standard laboratory chow (Young Stock Feed, Young, Australia) and water. The study was conducted according to the National Health and Medical Research Council (NH&MRC; Australia) code of practice for the care and use of animals for scientific purposes. Test Diets were prepared as previously described \\[5\\]. As a percentage of total energy, all diets contained 67% carbohydrate (57% starch; 10% sucrose), 22% protein, and 11% fat. All diets were identical in composition except for the percentage of RS and DS included in the starch component. For the RS diet, the starch used was a natural high amylose starch, Hi-Maize 957\u2122 (National Starch and Chemical Co), which is 60% amylose\/40% amylopectin, versus waxy cornstarch, which is 0% amylose\/100% amylopectin, for the DS diet. Diets were presented to the animals in an unprocessed, unpelleted form so the starches were not subjected to cooking or extrusion. The dietary fiber level of the starches was determined using the Association of Analytical Chemistry (AOAC) enzymatic-gravimetric method. Total dietary fiber (dry solids) was lower (approximately 1%) for the DS starch than the RS starch which contained 19.3% fiber.\n\nTwo weeks prior to a meal test, rats were presented with test diet ad libatum overnight to familiarize them with this diet and prevent neophobia on the test day. Twice over the next week, rats were starved for 5 h then placed in testing cages and presented with a meal of similar size to that which they would receive during the meal test (approximately 0.4 g of diet). Rats had no exposure to test diet or disruption of normal eating habits for seven days prior to the meal test. For all tests, rats were starved overnight (12\u201315 hours). The following morning, rats were placed in 25 \u00d7 25-cm wire testing cages and allowed 30 min to acclimate before testing commenced. The rat was then presented with a test meal of 1.0 g carbohydrate\/kg body weight (1.49 g diet\/kg body weight). Rats were allowed 15 minutes to consume all food presented to them. Rats were injected with 50 \u03bcCi ^3^H~2~O either immediately following food consumption (1 h groups) or 1 h post-ingestion (2 h groups). One hour after ^3^H~2~O administration, rats were sacrificed by i.p. injection of 250 mg\/kg pentobarbitone sodium. 400 \u03bcl blood was collected and the gastrocnemius muscle, liver, epidydimal white adipose tissue (WAT), and interscapular brown adipose tissue (BAT) were excised and immediately freeze-clamped. All tissues were stored at -80\u00b0C until assayed for lipid and glycogen content. Plasma was pipetted into fresh tubes and stored at -20\u00b0C for subsequent analysis of triglyceride and non-esterified fatty acid (NEFA) concentrations.\n\nAll plasma samples were assayed in duplicate. Plasma triglycerides were measured using a colorimetric assay kit supplied by Boehringer Mannheim (Germany), according to the manufacturer's instructions. Plasma NEFAs were estimated using a NEFA C kit (Wako Pure Chemicals Inc., Japan). All colorimetric assays were analyzed using a BioRad (model 550) microplate reader (Hercules, CA).\n\nEstimation of total lipogenesis and glycogenesis was conducted as previously described \\[4\\]. Briefly, 100\u2013200 mg of tissue was weighed out and completely digested in 1 M KOH by heating at 70\u00b0C for 40 min. Half of this tissue digest was spotted onto filter paper, air dried, and glycogen precipitated using ethanol. The remainder of the tissue digest was added to a chloroform\/methanol mixture, agitated and the lipid extracted using three hexane washes. Pooled hexane layers from each tissue were dried under nitrogen, suspended in scintillant and counted. Note that glycogen measurements were not performed on adipose tissue samples due to their very low total glycogen content.\n\nResults are expressed as means \u00b1 SEM using Statistics for Windows (Analytical Software, Tallahassee, FL), unless otherwise indicated. Statistical differences between diet groups were determined using an ANOVA with repeated measures and protected least significant difference (PLSD) test for comparison of means.\n\nThere was no difference in body weight between the groups used (330 \u00b1 18 g and 328 \u00b1 20 g for the DS and RS groups, reapectively). All animals ate at least 87% of the diet presented to them. There was no difference in total test meal consumption between groups. Animals consumed 93% (0.376 g), 95% (0.384 g), 92% (0.372 g), and 96% (0.388 g) of food presented for the RS 1 h, DS 1 h, RS 2 h, DS 2 h groups, respectively.\n\nPlasma triglyceride and NEFA concentrations did not differ between groups (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>). Triglyceride concentrations at baseline, 1 h, and 2 h were 0.39 \u00b1 0.04, 0.63 \u00b1 0.11, and 0.54 \u00b1 0.11 mmol\/L for the DS group vs 0.38 \u00b1 0.05, 0.65 \u00b1 0.07, and 0.67 \u00b1 0.09 mmol\/L for the RS group. NEFA data showed similar trends.\n\nIn all tissues, except the liver, there was a trend for the rate of lipogenesis to be higher in the DS group than the RS group. This trend reached significance only in WAT at 1 h (p \\< 0.01; Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>). There was no significant difference in the rate of glycogensis in the liver or gastrocnemius in response to a DS or RS meal at any time point investigated (data not shown).\n\nThe amount of carbohydrate fed per meal in this study is comparable to that used in oral glucose tolerance tests by other investigators (eg. \\[4\\]). The average meal size received during this study was 0.38 g. The small size of this meal facilitated relatively uniform and complete consumption of the meal by all animals. In addition, a small meal size reflects the nibbling nature of a rat's natural eating pattern and is therefore physiologically appropriate for use in a rodent model.\n\nOver the 2 h postprandial period, there was approximately a 30% reduction in the amount of lipogenesis, exclusively in white adipose tissue, in response to a RS diet relative to a DS diet. This number is reflective of data in humans which shows a 20\u201330% increase in postprandial fat oxidation following a RS meal \\[3\\]. As rats are 'nibblers', they will conceivably consume a meal of the size fed in this study many times over the course of a day. There fore, the observed 30% reduction in lipid synthesis in response to a RS diet could reduce adipose tissue mass in the long-term.\n\nFurther studies need to be conducted to elucidate the mechanism\/s behind the observed decrease in lipogenesis in WAT in response to RS consumption. As no differences in circulating plasma triglyceride or NEFA concentrations was observed, it is possible that differences in circulating insulin concentration play a role in this effect. We have previously shown that RS consumption, at the level fed in the present study, decreases circulating plasma insulin without changing plasma glucose concentration in rats \\[6\\]. It could be argued that this attenuation in insulin concentration due to RS consumption is effecting WAT lipogenic enzyme activity as it has previously been shown that these enzymes are more insulin responsive in WAT than liver which could explain the tissue specificity of the observed response.\n\nKabir et al \\[2\\] have previously reported that long-term feeding of a high RS diet reduces fat cell size relative to a high DS diet. Data presented here indicate that a high RS meal reduces total fat synthesis in white adipose tissue following a meal relative to a high DS meal. This decrease in lipid synthesis could indeed contribute to control of fat cell size in RS fed rats in the long-term. Decreased lipogenesis selectively in WAT and a reduction in adipocyte cell size in response to RS feeding have broad implications for the treatment and prevention of obesity. Furthermore, increasing the RS intake of a typical Western diet would be easy to achieve through the consumption of natural foods with a high RS content and\/or the consumption of commercially produced foods which are fortified with high RS starches, such as breads, pasta, and cereals. RS can even be easily incorporated into foods such as candy and pizza dough, making increased consumption an achievable possibility.\n\n# Abbreviations\n\nRS, resistant starch\n\nDS, digestible starch\n\nWAT, white adipose tissue\n\nBAT, brown adipose tissue\n\nNEFA, non-esterified fatty acid\n\n# Competing interests\n\nThe author(s) declare that they have no competing interests.\n\n# Authors' contributions\n\nJH conceived of the study design and was responsible for overall study coordination, conducting rat meal tests\/sacrifice, data analysis, and manuscript preparation. MB was responsible for conducting rat meal tests\/sacrifice, statistical analyses, and contributed to manuscript preparation. LS conducted rat tissue extractions and contributed to the study design and manuscript preparation.\n\n### Acknowledgements\n\nStarches were donated by National Starch and Chemical Company, Australia. Vitamin and mineral mixes were a gift from Millmaster Feeds, Enfield, Australia.","meta":{"dup_signals":{"dup_doc_count":109,"dup_dump_count":40,"dup_details":{"curated_sources":2,"2021-49":1,"2020-34":1,"2020-10":1,"2019-47":1,"2019-43":1,"2019-35":1,"2018-05":1,"2017-30":1,"2017-17":1,"2017-09":11,"2017-04":1,"2016-50":1,"2016-44":1,"2016-40":1,"2016-36":1,"2016-30":1,"2016-22":1,"2016-18":1,"2015-48":3,"2015-40":2,"2015-35":4,"2015-32":4,"2015-27":3,"2015-22":4,"2015-14":2,"2014-52":4,"2014-49":5,"2014-42":9,"2014-41":4,"2014-35":5,"2014-23":6,"2014-15":5,"2022-27":1,"2015-18":3,"2015-11":3,"2015-06":2,"2014-10":3,"2013-48":3,"2013-20":3,"2024-18":1}},"file":"PMC1618391"},"subset":"pubmed_central"}
{"text":"abstract: # OBJECTIVE\n  .\n  To forecast the number of U.S. individuals aged \\<20 years with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM) through 2050, accounting for changing demography and diabetes incidence.\n  .\n  # RESEARCH DESIGN AND METHODS\n  .\n  We used Markov modeling framework to generate yearly forecasts of the number of individuals in each of three states (diabetes, no diabetes, and death). We used 2001 prevalence and 2002 incidence of T1DM and T2DM from the SEARCH for Diabetes in Youth study and U.S. Census Bureau population demographic projections. Two scenarios were considered for T1DM and T2DM incidence: *1*) constant incidence over time; *2*) for T1DM yearly percentage increases of 3.5, 2.2, 1.8, and 2.1% by age-groups 0\u20134 years, 5\u20139 years, 10\u201314 years, and 15\u201319 years, respectively, and for T2DM a yearly 2.3% increase across all ages.\n  .\n  # RESULTS\n  .\n  Under scenario 1, the projected number of youth with T1DM rises from 166,018 to 203,382 and with T2DM from 20,203 to 30,111, respectively, in 2010 and 2050. Under scenario 2, the number of youth with T1DM nearly triples from 179,388 in 2010 to 587,488 in 2050 (prevalence 2.13\/1,000 and 5.20\/1,000 \\[+144% increase\\]), with the greatest increase in youth of minority racial\/ethnic groups. The number of youth with T2DM almost quadruples from 22,820 in 2010 to 84,131 in 2050; prevalence increases from 0.27\/1,000 to 0.75\/1,000 (+178% increase).\n  .\n  # CONCLUSIONS\n  .\n  A linear increase in diabetes incidence could result in a substantial increase in the number of youth with T1DM and T2DM over the next 40 years, especially those of minority race\/ethnicity.\nauthor: Giuseppina Imperatore; James P. Boyle; Theodore J. Thompson; Doug Case; Dana Dabelea; Richard F. Hamman; Jean M. Lawrence; Angela D. Liese; Lenna L. Liu; Elizabeth J. Mayer-Davis; Beatriz L. Rodriguez; Debra Standiford; Corresponding author: Giuseppina Imperatore, <firstname.lastname@example.com>.Received 6 April 2012 and accepted 4 September 2012.\ndate: 2012-12\nreferences:\nsubtitle: : Dynamic modeling of incidence, mortality, and population growth\ntitle: Projections of Type 1 and Type 2 Diabetes Burden in the U.S. Population Aged \\<20 Years Through 2050\n\nDiabetes is one of the most common and costly chronic pediatric diseases (1). The SEARCH for Diabetes in Youth study (SEARCH) estimated that in 2001 about 154,000 individuals in the U.S. aged \\<20 years were living with diabetes and that each year approximately 15,000 youth aged \\<20 years are being diagnosed with type 1 diabetes mellitus (T1DM) and 3,700 with type 2 diabetes mellitus (T2DM) (2,3). Assessing the future burden of diabetes in youth by diabetes type is crucial for implementing public health primary and secondary prevention programs and planning health care delivery services.\n\nA number of studies have estimated the burden of diagnosed diabetes through 2050 in the U.S (4,5). A limitation is that they were not able to separate the contribution of T1DM from T2DM to the projected diabetes burden. Although the majority of adults with diabetes have T2DM, the majority of youth with diabetes currently have T1DM. On the other hand, T2DM may be becoming more common in adolescents, especially among minority youth (2,3).\n\nThere is substantial variation in the incidence of T1DM and T2DM across the major racial\/ethnic groups in the U.S. The incidence of T1DM is highest among non-Hispanic whites (NHWs) and lowest in American Indians (3). In contrast, T2DM disproportionally affects individuals from all racial\/ethnic minority groups (3). Therefore, changes in the race\/ethnicity distribution of the U.S. population will substantially impact the absolute number of individuals living with T1DM or T2DM. This makes even more compelling the need for diabetes type\u2013specific projections.\n\nTo overcome the limitations of previous studies and provide contemporary estimates of the national type-specific burden of diabetes in youth, we constructed a system of dynamic equations that incorporate diabetes prevalence and incidence, as well as birth, migration, and mortality estimates. These equations model the future burden of diabetes in U.S. youth aged \\<20 years through 2050. In addition, we perform sensitivity analyses to assess the impact of increases in the incidence and\/or changes in the risk of mortality separately for T1DM and T2DM.\n\n# RESEARCH DESIGN AND METHODS\n\n## Data sources\n\nThe data sources for this study include the U.S. Census Bureau (6) and the SEARCH study (2,3,7\u201311). Census data include estimates of the 2001 U.S. population by ages (0, 1, 2,\u2026,19 years), race\/ethnicity (NHW, non-Hispanic black \\[NHB\\], Hispanic, Asian and Pacific Islander \\[API\\], American Indian\/Alaska Native \\[AIAN\\]), and sex, as well as projection estimates of births, deaths, and net migration by the same dimensions for the years 2002\u20132050. For each of these components of population change\u2014fertility, mortality and net migration\u2014the census applied three different assumptions to forecast the future population size. We used the series using the middle assumption for each of these components, also designated as the middle series. SEARCH data include diabetes prevalence in 2001 and incidence from 2002 to 2007 collected from geographically defined populations in Ohio, Colorado, South Carolina, and Washington, as well as Indian Health Service beneficiaries from four American Indian populations, and enrollees in managed health care plans in California and Hawaii. SEARCH is a multicenter study that began in 2001 and is conducting population-based ascertainment of youth aged \\<20 years with clinically diagnosed, nongestational diabetes. Institutional review board(s) for each site approved the study protocol. A detailed description of the SEARCH study has been published elsewhere (2,3).\n\n## Statistical analysis\n\n### 2001 prevalence estimation.\n\nWe used Poisson regression to estimate T1DM and T2DM prevalence as a function of age (0,1,\u2026,19 years), race\/ethnicity (NHW, NHB, Hispanic, API, AIAN), and sex. The Bayesian information criterion was used to select the best fitting models (12). The Bayesian information criterion selects a model from a collection of possible nonnested models by maximizing the likelihood but with a penalty for higher dimensional models. Posterior predictive checking was used to assess model consistency with the data (13). We used the deviance function as a test measure and calculated the \"Bayesian *P* value.\" Bayesian *P* values do not work like the more common frequentist *P* values; values between 0.1 and 0.9 indicate good model fit (14). Models were fit using Bayesian methods in WinBUGS (15). The final models included a cubic spline for age (knots at 0, 9, and 19 years), race\/ethnicity, and sex. Posterior predictive checking yielded Bayesian *P* values equal to 0.39 and 0.22, indicating good model fit for T1DM and T2DM, respectively. Our estimates of the 2001 T1DM and T2DM prevalence were the means of the posterior distributions obtained from fitting the final models.\n\n### 2002 incidence rate estimation.\n\nFrom a population size of 30,549,412 person-years in 2002 through 2007, SEARCH identified 6,164 incident cases of T1DM and 1,534 incident cases of T2DM. We used Poisson regression to estimate T1DM and T2DM incidence as a function of age (0,1,\u2026,19 years), race\/ethnicity (NHW, NHB, Hispanic, API, AIAN), sex, and calendar year. We were primarily interested in incidence estimates for the year 2002. Including 6 years of data with random effects by year improved the year 2002 estimates by \"borrowing strength\" from the other years. The final models included a cubic spline for age (knots at 0, 9, and 19 years), race, sex, age by sex interactions, and random effects of calendar year. Posterior predictive checking yielded Bayesian *P* values equal to 0.29 and 0.12, indicating good model fit for T1DM and T2DM, respectively. Our estimates of the 2002 T1DM and T2DM incidence rates were the means of the posterior distributions obtained from fitting the final models.\n\n### Projection model.\n\nWe constructed dynamic models consisting of systems of difference equations similar to models described previously (16,17). In these models, the U.S. population aged 0 to 19 years is modeled at 1-year intervals starting at year 2001 and ending at year 2050. Specifically, we defined numbers of individuals in various disease states (diabetes, no diabetes, and death). The mathematical details are presented in [Supplementary Appendix 1](http:\/\/care.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/dc12-0669\/-\/DC1).\n\n### Sensitivity analyses.\n\nWe conducted sensitivity analyses by varying both relative risks of death in youth with diabetes versus nondiabetic youth and incidence rate projections for T1DM and T2DM. Two scenarios were considered for relative risks of death: *1*) the relative risks of death for youth in the age-group \\<20 years with T1DM or T2DM are equal to one, i.e., death rates by age, race\/ethnicity, and sex for persons with and without diabetes are equal to the census projected death rates; *2*) death rates of youth with diabetes are higher than those without diabetes with a relative risk equal to 1.5. Two scenarios for T1DM incidence rates were considered: *1*) constant incidence over time at 2002 levels (baseline scenario); *2*) yearly percentage increases of 3.5, 2.2, 1.8, and 2.1% by age-groups 0\u20134 years, 5\u20139 years, 10\u201314 years, and 15\u201319 years (as seen in a previous study from Colorado \\[18\\]), respectively, were applied to all 10 race-sex combinations. Correspondingly, two scenarios for T2DM incidence rates were considered: *1*) constant incidence over time at 2002 levels (baseline scenario); *2*) a yearly 2.3% increase applied uniformly to all ages (0\u201319 years) for each of the 10 race-sex combinations. This increase represents the overall increase of T1DM registered in the Colorado youth population (18).\n\n# RESULTS\n\nOur estimates of the 2002 T1DM and T2DM incidence rates by age and race\/ethnicity from fitting the final models are presented in <a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<\/a>. The incidence of T1DM peaks around the age of 10 years and is highest among NHWs followed by NHBs, Hispanics, APIs, and AIANs (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>A<\/em><\/a>). The incidence of T2DM peaks around 14 years of age. AIAN youth have the highest incidence rate, followed by NHBs, Hispanics, APIs, and NHWs (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>B<\/em><\/a>).\n\nWe used these incidence rates in the projection model for each year from 2002 through 2050 in the baseline scenario, where we assume constant incidence rates over time.\n\n## Baseline scenario\n\n<a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a> shows the projected number of youth with T1DM and T2DM by race\/ethnicity for selected years. The model forecasts that the number of youth with T1DM will increase by 23%, from 166,018 in 2010 to 203,385 in 2050. Due to the absolute increases in the numbers of minority youths in the population as projected by the U.S. Census, this increase is primarily driven by these youths. In 2010, NHWs represented 71% of all youth with T1DM, but by 2050 this proportion will decrease to 55%. Over the 40-year period, the number of Hispanic youth with T1DM is projected to increase 2.5-fold. The overall prevalence of T1DM remains largely unchanged from 1.97\/1,000 in 2010 to 1.80\/1,000 in 2050, a decrease of 9%, reflecting the lower incidence of T1DM among youth from minority groups compared with NHW youth.\n\nProjections of the number of individuals aged \\<20 years with T1DM or T2DM and prevalence estimates\\* for selected years, by race\/ethnicity under baseline scenario\u2020\n\n![](2515tbl1)\n\nThe model estimated that in 2010 20,203 youth in the U.S. had T2DM. Youth of minority groups represented the majority of T2DM cases, while 25% were NHW. The number of youth with T2DM is projected to increase to 30,111 by 2050. As for T1DM, this increase is driven largely by the increase in the population of youth of minority groups. By 2050 Hispanics are estimated to represent 50% of U.S. youth aged \\<20 years with T2DM. Between 2010 and 2050, the overall prevalence of T2DM may increase by 13%, from 0.24\/1,000 to 0.27\/1,000.\n\n## Increased incidence scenario\n\n<a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a> presents the results of the sensitivity analyses in which the incidence rates of T1DM and T2DM increase while the relative risk of death is equal to 1.0. Under this scenario, the model projects the number of youth with T1DM will increase 3.3-fold, from 179,388 in 2010 to 587,488 in 2050. The number of youth with T1DM will increase 6.6-fold in Hispanics, 5.4-fold in APIs, 4.4-fold in AIANs, 3.0-fold in NHBs, and 2.5-fold in NHWs. The prevalence will increase by 144%, from 2.13\/1,000 to 5.20\/1,000, with the highest estimate still among NHW youth (7.04\/1,000).\n\nProjections of the number of individuals aged \\<20 years with T1DM or T2DM and prevalence estimates\\* for selected years, by race\/ethnicity under increased incidence rate scenario\u2020\n\n![](2515tbl2)\n\nUnder the scenario of an annual increase of 2.3% in the incidence of T2DM, the model indicates that, in the U.S., in 2010 there were 22,820 youth aged \\<20 years with T2DM. By 2050, our model predicts that this number will almost quadruple, to 84,131, with Hispanics representing 50% and NHBs 27% of all youth with T2DM. On the basis of our projections, the prevalence will rise from 0.27\/1,000 in 2010 to 0.75\/1,000 in 2050, an increase of 178%. The prevalence will be highest in NHBs (1.63\/1,000) and lowest in NHWs (0.28\/1,000).\n\nFor both T1DM and T2DM, the differences in the numbers of youth with diabetes from the baseline scenario are due to the increasing incidence rates over baseline.\n\nUnder the increasing incidence scenario and the baseline scenario, when we set the relative risk of death at 1.5 for youth with either T1DM or T2DM diabetes, results were virtually identical (data not shown).\n\n# CONCLUSIONS\n\nWe have estimated the future burden of diabetes in youth by type in the major race\/ethnic groups in the U.S., using the most recent population-based estimates of diabetes incidence and prevalence and taking into account demographic changes over time. Our model projected that over the next 40 years, at the current incidence rates, the number of youth with T1DM and T2DM may increase by 23% and 49%, respectively. However, if the incidence of T1DM or T2DM increases, there may be more than a threefold increase in the number of youth with T1DM and about a fourfold increase in the number of youth with T2DM, especially among minority youth.\n\nVery little is known about effective prevention of T1DM, and more research is needed. However, T2DM can be prevented in high risk adults. Additional research is needed to examine the most effective methods for T2DM prevention in youth and should address strategies applicable to obesity prevention and control, as well as strategies for youth at high risk for T2DM. The projected increase in the prevalence of T2DM should serve as a call to action so that by 2050 the actual number of affected youth will fall markedly short of our projections.\n\nBecause of the early age of onset and longer diabetes duration, children and adolescents are at risk for developing diabetes-related complications at a younger age. As these youth age, this profoundly affects their productivity, quality of life, and life expectancy and increases health care costs. Even in childhood, the medical expenditures of youth with diabetes are approximately 6.2 times of those without diabetes (19). The health care system and society as a whole will need to plan and prepare for the delivery of quality health care to meet the needs of the growing number of youth with diabetes. This may need to include the training of additional health care professionals to treat and manage children and adolescents with T1DM and T2DM.\n\nStrengths of the current study include the use of contemporary population-based estimates of the prevalence and incidence of T1DM and T2DM from the SEARCH study for the major race\/ethnic groups in the U.S. This enabled us to quantify race\/ethnicity\u2013specific future diabetes burden. Prevalence and incidence estimates were based on physician's diagnosis of T1DM or T2DM, and case definitions met consistent eligibility criteria (2,3). Moreover, physician's diagnosis of diabetes type was in good agreement with the etiologic biochemical and clinical characteristics of the two major types of diabetes (20).\n\nThe projections have some limitations. First, the recent estimated increase in the incidence of T1DM is limited and only available in one U.S. study conducted in Colorado (18). The Colorado study found a slightly lower annual increase in T1DM incidence among youth than a large registry-based study conducted in 17 European countries (EURODIAB; overall yearly average increase 2.3% in Colorado vs. 3.9% in Europe) (18,21). However, the pattern of the increase in Colorado was similar to that observed in Europe, with children younger than 5 years old experiencing the greatest relative increase. If the actual rate of increase in the U.S. is more similar to that observed in Europe, then our projections may underestimate the future burden of T1DM in the U.S. However, it should be noted that EURODIAB included only children aged 0\u201315 years and the greatest relative increase in the incidence rate was observed in countries with low baseline incidence. Second, in our study we applied the same rate of increase across all race\/ethnic groups. The Colorado study population included only NHWs and Hispanics, and the overall rate of increase in Hispanics was slightly lower than that of NHWs (1.6 vs. 2.7% per year, respectively). The U.S. Census projections indicate that the proportion of the youth population of NHW race\/ethnicity will diminish from 62% in 2001 to 41% in 2050 (22). Because of this demographic shift and the possibility that youth of other races\/ethnicities than NHW may experience a lower increase in the incidence, it is possible that the number of youth with T1DM could be lower than that estimated by our study under the increasing incidence scenario. Third, we assumed constant increases in T1DM incidence over time and did not account for the possible effect of yet to be identified primary prevention strategies that may influence our predicted number of youth with T1DM. Given our current knowledge, the increased incidence scenario should be taken with caution. However, we would like to point out that recent findings from Europe indicated a constant linear trend over a 15-year period (21) or, starting in the early 1990s, even a steeper increase (23).\n\nFinally, because of the lack of population-based estimates of T2DM incidence trends in youth aged \\<20 years, we used a yearly increase of 2.3% in our increasing incidence scenario, based on the overall increase of T1DM in the Colorado study (18). In the Pima Indians, a population at very high risk of developing T2DM (24), among youth aged 5\u201314 years, between 1965 and 2003 the incidence of T2DM increased almost sixfold (25). In Finnish adolescents and young adults (aged 15\u201339 years) during a 10-year period from 1992 to 2002, the incidence of T2DM increased on average by 4.3% per year, while that of T1DM increased by 3.9% per year (26). Obesity is a major risk factor for the development of T2DM. Since the 1980s, obesity prevalence among U.S. children and adolescents tripled; however, recent national data indicate that during the last decade obesity prevalence may be leveling off at 17% (27). If obesity remains stable for the next 40 years, it is plausible that the current T2DM incidence rate will remain steady. However, even under this scenario, the number of youth with T2DM may increase by 49%. On the other hand, implementation of interventions for the prevention of childhood obesity at the individual or population level may result in decreasing T2DM incidence over time (28,29).\n\nIn both scenarios in our study, increasing the relative risk of death to 1.5 did not affect our estimates. This might be partially explained by the very low number of diabetes-related deaths in this age-group (1.15 per million youths) (30).\n\nOur projections suggest a shift in the proportional distribution of racial\/ethnic groups among youth with T1DM. By 2050, about half of T1DM youths will be of minority race\/ethnic groups. This change may influence potential trends in clinical presentation, treatment patterns, and quality of care. Minority youth are more likely to be overweight or obese (27) and this may lead to a misdiagnosis of T2DM. Among SEARCH study participants, minority youth with T1DM were significantly more likely to have poor glucose control (glycated hemoglobin \\>9%) than NHW youth (31). Minority youth with T1DM are also more likely to live in households with low income and parental education (7\u201311). This in turn may affect their access to and quality of health care (32,33). Because of the changing demographics of the youth population with T1DM, health care policies and delivery systems need to assure that less advantaged youth receive appropriate care.\n\nOur projections indicate a serious picture of the future national diabetes burden in youth. Even if the incidence remains at 2002 levels, because of the population growth projected by the U.S. Census the future numbers of youth with diabetes is projected to increase, resulting in increased health care needs and costs. Future planning should include strategies for implementing childhood obesity prevention programs and primary prevention programs for youth at risk for developing T2DM. Likewise, to prevent future human suffering and health care costs, effective interventions for the prevention of diabetes-related complications should be available to all youth with diabetes (34). At the same time, it is crucial to continuously monitor diabetes trends at the population level, as well as complications and quality of care among youth.\n\n# Supplementary Material\n\n###### Supplementary Data\n\n###### News Release\n\n###### Slide Set\n\n## Acknowledgments\n\nSEARCH for Diabetes in Youth is funded by the Centers for Disease Control and Prevention (PA 00097, DP-05-069, and DP-10-001) and supported by the National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases.\n\nJ.M.L. is employed by Kaiser Permanente Southern California. No other potential conflicts of interest relevant to this article were reported.\n\nG.I. conceived the study, participated in study design and coordination, and wrote a first draft of the manuscript. J.P.B. developed and programmed the multistate dynamic models, participated in study design and coordination, and helped draft the manuscript. T.J.T. developed and programmed the incidence projection model, participated in study design and coordination, and helped draft the manuscript. D.C. participated in study design and coordination, assembled the SEARCH prevalence and incidence data for analysis, and commented on drafts of the manuscript. D.D., R.F.H., J.M.L., A.D.L., L.L.L., E.J.M.-D., and D.S. reviewed and edited the manuscript and contributed to discussion. B.L.R. reviewed the manuscript and contributed to discussion. G.I. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.\n\nParts of this study were presented in poster form at the 71st\u00a0Scientific Sessions of the American Diabetes Association, San Diego, California, 24\u201328 June 2011.\n\nThe SEARCH for Diabetes in Youth study is indebted to the many youth and their families, and their health care providers, whose participation made this study possible.\n\n# References","meta":{"dup_signals":{"dup_doc_count":154,"dup_dump_count":71,"dup_details":{"curated_sources":2,"2023-14":2,"2022-40":2,"2022-21":4,"2021-49":1,"2021-43":2,"2021-39":2,"2021-31":2,"2021-25":2,"2021-21":3,"2021-17":4,"2021-10":2,"2021-04":1,"2020-45":1,"2020-24":3,"2020-10":2,"2019-51":1,"2019-43":2,"2019-39":2,"2019-35":1,"2019-30":2,"2019-26":3,"2019-22":1,"2019-18":1,"2019-13":2,"2018-51":2,"2018-47":1,"2018-30":2,"2018-13":2,"2018-05":4,"2017-47":2,"2017-43":1,"2017-39":2,"2017-34":1,"2017-30":2,"2017-26":2,"2017-22":2,"2017-17":4,"2017-09":3,"2017-04":3,"2016-50":2,"2016-44":3,"2016-40":3,"2016-36":2,"2016-30":3,"2016-26":2,"2016-22":3,"2016-18":2,"2016-07":2,"2015-48":1,"2015-40":2,"2015-35":3,"2015-32":1,"2015-27":3,"2015-22":3,"2015-14":2,"2014-52":2,"2014-49":3,"2014-42":5,"2014-41":3,"2014-35":4,"2014-23":2,"2014-15":1,"2024-10":1,"2017-13":2,"2015-18":3,"2015-11":2,"2015-06":2,"2014-10":1,"2013-48":1,"2013-20":1,"2024-30":1}},"file":"PMC3507562"},"subset":"pubmed_central"}
{"text":"date: 2017\nreferences:\ntitle: Non-O blood groups associated with higher risk of heart attack\n\n# Introduction\n\nHaving a non-O blood group is associated with a higher risk of heart attack, according to research presented recently at Heart Failure 2017 and the 4th World Congress on Acute Heart Failure.\n\nLead author Tessa Kole, a Master's degree student at the University Medical Centre Groningen, the Netherlands, said: 'It has been suggested that people with non-O blood groups (A, B, AB) are at higher risk for heart attacks and overall cardiovascular mortality, but this suggestion comes from case\u2013control studies, which have a low level of evidence. If this was confirmed, it could have important implications for personalised medicine.'\n\nThe current study was a meta-analysis of prospective studies reporting on O and non-O blood groups, and incident cardiovascular events, including myocardial infarction (heart attack), coronary artery disease, ischaemic heart disease, heart failure, cardiovascular events and cardiovascular mortality.\n\nThe study included 1 362 569 subjects from 11 prospective cohorts, described in nine articles. There were a total of 23 154 cardiovascular events. The researchers analysed the association between blood group and all coronary events, combined cardiovascular events and fatal coronary events.\n\nThe analysis of all coronary events included 771 113 people with a non-O blood group and 519 743 people with an O blood group, of whom 11 437(1.5%) and 7 220 (1.4%) suffered a coronary event, respectively. The odds ratio (OR) for all coronary events was significantly higher in carriers of a non-O blood group, at 1.09 \\[95% confidence interval (CI) of 1.06\u20131.13\\].\n\nThe analysis of combined cardiovascular events included 708 276 people with a non-O blood group and 476 868 people with an O blood group, of whom 17 449 (2.5%) and 10 916 (2.3%) had an event, respectively. The OR for combined cardiovascular events was significantly higher in non-O blood group carriers, at 1.09 (95% CI 1.06\u20131.11).\n\nThe analysis of fatal coronary events did not show a significant difference between people with O and non-O blood groups.\n\n'We demonstrate that having a non-O blood group is associated with a 9% increased risk of coronary events and a 9% increased risk of cardiovascular events, especially myocardial infarction', said Ms Kole.\n\nThe mechanisms that might explain this risk are under study. The higher risk for cardiovascular events in non-O blood group carriers may be due to having greater concentrations of von Willebrand factor, a blood clotting protein which has been associated with thrombotic events. Further, non-O blood group carriers, specifically those with an A blood group, are known to have higher cholesterol. And galectin-3, which is linked to inflammation and worse outcomes in heart failure patients, is also higher in those with a non-O blood group.\n\nMs Kole said: 'More research is needed to identify the cause of the apparent increased cardiovascular risk in people with a non-O blood group. Obtaining more information about risk in each non-O blood group (A, B and AB) might provide further explanations of the causes.'\n\nShe concluded: 'In future, blood group should be considered in risk assessment for cardiovascular prevention, together with cholesterol, age, sex and systolic blood pressure. It could be that people with an A blood group should have a lower treatment threshold for dyslipidaemia or hypertension, for example. We need further studies to validate if the excess cardiovascular risk in non-O blood group carriers may be amenable to treatment.'\n\n# References","meta":{"dup_signals":{"dup_doc_count":108,"dup_dump_count":55,"dup_details":{"curated_sources":2,"2023-40":3,"2023-23":2,"2023-14":3,"2023-06":3,"2022-49":2,"2022-40":2,"2022-33":2,"2022-27":2,"2022-21":2,"2022-05":1,"2021-49":2,"2021-39":1,"2021-31":2,"2021-25":3,"2021-21":2,"2021-17":1,"2021-10":1,"2021-04":2,"2020-50":1,"2020-45":2,"2020-40":2,"2020-34":1,"2020-29":1,"2020-16":1,"2020-10":1,"2019-47":2,"2019-35":1,"2019-30":1,"2019-26":2,"2019-18":1,"2019-13":1,"2019-04":1,"2018-51":3,"2018-47":2,"2018-43":1,"2018-39":3,"2018-30":3,"2018-26":3,"2018-22":1,"2018-17":2,"2018-13":4,"2018-09":3,"2018-05":1,"2017-51":1,"2017-47":4,"2017-39":4,"2017-34":1,"2017-30":3,"2017-22":3,"2023-50":1,"2024-26":1,"2024-22":1,"2024-18":3,"2024-10":3,"2024-30":1}},"file":"PMC5558144"},"subset":"pubmed_central"}
{"text":"abstract: This study describes and validates a new method for metagenomic biomarker discovery by way of class comparison, tests of biological consistency and effect size estimation. This addresses the challenge of finding organisms, genes, or pathways that consistently explain the differences between two or more microbial communities, which is a central problem to the study of metagenomics. We extensively validate our method on several microbiomes and a convenient online interface for the method is provided at <http:\/\/huttenhower.sph.harvard.edu\/lefse\/>.\nauthor: Nicola Segata; Jacques Izard; Levi Waldron; Dirk Gevers; Larisa Miropolsky; Wendy S Garrett; Curtis Huttenhower\ndate: 2011\ninstitute: 1Department of Biostatistics, 677 Huntington Avenue, Harvard School of Public Health, Boston, MA 02115, USA; 2Department of Molecular Genetics, 245 First Street, The Forsyth Institute, Cambridge, MA 02142, USA; 3Department of Oral Medicine, Infection and Immunity, 188 Longwood Ave, Harvard School of Dental Medicine, Boston, MA 02115, USA; 4Microbial Sequencing Center, 7 Cambridge Center, The Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; 5Department of Immunology and Infectious Diseases, 665 Huntington Avenue, Harvard School of Public Health, Boston, MA 02115, USA; 6Department of Medicine, 75 Francis Street, Harvard Medical School, Boston, MA 02115, USA; 7Department of Medical Oncology, 44 Binney Street, Dana-Farber Cancer Institute, MA 02215, USA\nreferences:\ntitle: Metagenomic biomarker discovery and explanation\n\n# Background\n\nBiomarker discovery has proven to be one of the most broadly applicable and successful means of translating molecular and genomic data into clinical practice. Comparisons between healthy and diseased tissues have highlighted the importance of tasks such as class discovery (detecting novel subtypes of a disease) and class prediction (determining the subtype of a new sample) \\[1-4\\], and recent metagenomic assays have shown that human microbial communities can be used as biomarkers for host factors such as lifestyle \\[5-7\\] and disease \\[7-10\\]. As sequencing technology continues to develop and makes microbial biomarkers increasingly easily detected, this enables clinical diagnostic and microbiological applications through the comparison of microbial communities \\[11,12\\].\n\nThe human microbiome, consisting of the total microbial complement associated with human hosts, is an important emerging area for metagenomic biomarker discovery \\[13,14\\]. Changes in microbial abundances in the gut, oral cavity, and skin have been associated with disease states ranging from obesity \\[15-17\\] to psoriasis \\[18\\]. More generally, the metagenomic study of microbial communities is an effective approach for identifying the microorganisms or microbial metabolic characteristics of any uncultured sample \\[19,20\\]. Analyses of metagenomic data typically seek to identify the specific organisms, clades, operational taxonomic units, or pathways whose relative abundances differ between two or more groups of samples, and several features of microbial communities have been proposed as potential biomarkers for various disease states. For example, single pathogenic organisms can signal disease if present in a community \\[21,22\\], and increases and decreases in community complexity have been observed in bacterial vaginosis \\[23\\] and Crohn's disease \\[8\\]. Each of these different types of microbial biomarkers is correlated with disease phenotypes, but few bioinformatic methods exist to explain the class comparisons afforded by metagenomic data.\n\nIdentifying the most biologically informative features differentiating two or more phenotypes can be challenging in any genomics dataset, and this is particularly true for metagenomic biomarkers. Robust statistical tools are needed to ensure the reproducibility of conclusions drawn from metagenomic data, which is crucial for clinical application of the biological findings. Related challenges are associated with high-dimensional data regardless of the data type or experimental platform; the number of potential biomarkers, for example, is typically much higher than the number of samples \\[24-26\\]. Metagenomic analyses additionally present their own specific issues, including sequencing errors, chimeric reads \\[27,28\\], and complex underlying biology; many microbial communities have been found to show remarkably high inter-subject variability. For example, large differences are detected even among the gut microbiomes of twins \\[29\\], and both human microbiomes and environmental communities are thought to be characterized by the presence of a long tail of rare organisms \\[30-32\\]. Moreover, simply identifying potential biomarkers without elucidating their biological consistency and roles is only a precursor to understanding the underlying mechanisms of microbe-microbe or host-microbe interactions \\[33\\]. In many cases, it is necessary to explain not just how two biological samples differ, but why. This problem is referred to as class comparison: how can the differences between phenotypes such as tumor subtype or disease state be explained in terms of consistent biological pathways or molecular mechanisms?\n\nA number of methods have been proposed for class discovery or comparison in metagenomic data. MEGAN \\[34\\] is a metagenomic analysis tool with recent additions for phylogenetic comparisons \\[35\\] and statistical analyses \\[36\\]. MEGAN, however, can only compare single pairs of metagenomes, as is also the case with STAMP \\[37\\], which does introduce a concept of 'biological relevance' in the form of confidence intervals. UniFrac \\[38\\] compares sets of metagenomes at a strictly taxonomic level using phylogenetic distance, while MG-RAST \\[39\\], ShotgunFunctionalizeR \\[40\\], mothur \\[41\\], and METAREP \\[42\\] all process metagenomic data using standard statistical tests (mainly *t*-tests with some modifications). Most methods for community analysis from an ecological perspective rely on unsupervised cluster analyses based on principal component analysis \\[43\\] or principal coordinate analysis \\[44\\]. These can successfully detect groups of related samples, but they fail to include prior knowledge of phenotypes or environmental conditions associated with the groups, and they generally do not identify the biological features responsible for group relationships. Metastats \\[45\\] is the only current method that explicitly couples statistical analysis (to assess whether metagenomes differ) with biomarker discovery (to detect features characterizing the differences) based on repeated *t* statistics and Fisher's tests on random permutations. However, none of these methods, even those offering nuanced analyses of metagenomic data, provide biological class explanations to establish statistical significance, biological consistency, and effect size estimation of predicted biomarkers.\n\nIn this work, we present the linear discriminant analysis (LDA) effect size (LEfSe) method to support high-dimensional class comparisons with a particular focus on metagenomic analyses. LEfSe determines the features (organisms, clades, operational taxonomic units, genes, or functions) most likely to explain differences between classes by coupling standard tests for statistical significance with additional tests encoding biological consistency and effect relevance. Class comparison methods typically predict biomarkers consisting of features that violate a null hypothesis of no difference between classes; we additionally detect the subset of features with abundance patterns compatible with an algorithmically encoded biological hypothesis and estimate the sizes of the significant variations. In particular, effect size provides an estimation of the magnitude of the observed phenomenon due to each characterizing feature and it is thus a valuable tool for ranking the relevance of different biological aspects and for addressing further investigations and analyses. The introduction of prior biological knowledge in the method contributes to constrain the analysis and thus to address the challenges traditionally connected with high-dimensional data mining. LEfSe thus aims to support biologists by suggesting biomarkers that explain most of the effect differentiating phenotypes of interest (two or more) in biomarker discovery comparative and hypothesis-driven investigations. The visualization of the discovered biomarkers on taxonomic trees provides an effective means for summarizing the results in a biologically meaningful way, as this both statistically and visually captures the hierarchical relationships inherent in 16S-based taxonomies\/phylogenies or in ontologies of pathways and biomolecular functions.\n\nWe validated this approach using data from human microbiomes, a mouse model of ulcerative colitis, and environmental samples, in each case predicting groups of organisms or operational taxonomic units that concisely differentiate the classes being compared. We further evaluated LEfSe using synthetic data, observing that it achieves a substantially better false positive rate compared to standard statistical tests, at the price of a moderately increased false negative rate (that can be adjusted as needed by the user). An implementation of LEfSe including a convenient graphical interface incorporated in the Galaxy framework \\[46,47\\] is provided online at \\[48\\].\n\n# Results and discussion\n\nLEfSe is an algorithm for high-dimensional biomarker discovery and explanation that identifies genomic features (genes, pathways, or taxa) characterizing the differences between two or more biological conditions (or classes) (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>). It emphasizes statistical significance, biological consistency and effect relevance, allowing researchers to identify differentially abundant features that are also consistent with biologically meaningful categories (subclasses; see Materials and methods). LEfSe first robustly identifies features that are statistically different among biological classes. It then performs additional tests to assess whether these differences are consistent with respect to expected biological behavior; for example, given some known population structure within a set of input samples, is a feature more abundant in all population subclasses or in just one? Specifically, we first use the non-parametric factorial Kruskal-Wallis (KW) sum-rank test \\[49\\] to detect features with significant differential abundance with respect to the class of interest; biological consistency is subsequently investigated using a set of pairwise tests among subclasses using the (unpaired) Wilcoxon rank-sum test \\[50,51\\]. As a last step, LEfSe uses LDA \\[52\\] to estimate the effect size of each differentially abundant feature and, if desired by the investigator, to perform dimension reduction.\n\nWe have specifically designed LEfSe for biomarker discovery in metagenomic data. We thus summarize our results here from applying the tool to 16S rRNA gene and whole genome shotgun datasets to detect bacterial organisms and functional characteristics differentially abundant between two or more microbial environments. These include body sites within human microbiomes (mucosal surfaces and aerobic\/anaerobic environments), adult and infant microbiomes, inflammatory bowel disease status in a mouse model, bacterial and viral environmental communities, and synthetic data for quantitative computational evaluation.\n\n## Taxa characterizing body sites within the human microbiome\n\nMicrobial community organization at multiple human body sites is an area of active current research, since both low- and high-throughput methods have shown both differences and overlaps among the microbiota of multiple body sites \\[53,54\\]. We examined these differences in the 16S-based phylometagenomic dataset from 24 individuals enrolled in the Human Microbiome Project \\[13,55\\]. A minimum of 5,000 16S rRNA gene sequences were obtained for 301 samples from 24 healthy subjects (12 male, 12 female) covering 18 body sites, including 6 main body site categories: the oral cavity (9 sub-sites sampled), the vagina (3 sub-sites sampled), the skin (2 sub-sites sampled), the retroauricular crease (2 sub-sites sampled), the nasal cavity (1 sample) and the gut (1 sample). We validated LEfSe by contrasting mucosal versus non-mucosal body site classes and by comparing three levels of aerobic environments (anaerobic, microaerobic, and aerobic). In both cases, the sub-sites within each class of body site were used as a biological subclass.\n\n## Mucosal surfaces are colonized by diverse bacteria; non-mucosal microbiomes are strongly enriched for Actinobacteria\n\nOur first analysis focused on differences in microbiota composition between mucosal and non-mucosal body sites. The oral cavity, gut, and vaginal sites were classified as sources of mucosal communities and the anterior fossa (skin), nasal cavity, and retroauricular crease as non-mucosal. Mucosal environments differ greatly from the other body sites, characterized primarily by interaction with the human immune system, oxidative challenge, and hydration \\[56\\].\n\nLEfSe provides three main outputs (Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>), describing the effect sizes of differences observed among mucosal\/non-mucosal communities, the phylogenetic distribution of these differences based on the Ribosomal Database Project (RDP) bacterial taxonomy \\[57\\], and the raw data driving these effects. LEfSe detected 15 bacterial clades showing statistically significant and biologically consistent differences in non-mucosal body sites (Figure <a href=\"#F2\" data-ref-type=\"fig\">2a<\/a>).\n\nThe most differentially abundant bacterial taxa in non-mucosal body sites belong to phyla with prevalent aerobic members: Actinobacteria, Firmicutes, and Proteobacteria, including environmental organisms from the Betaproteobacteria and Gammaproteobacteria clades. Non-mucosal overrepresented genera include *Propionibacterium*, *Staphylococcus* (found exclusively in non-mucosal samples), *Corynebacterium*, and *Pseudomonas*. Also of note is the relevant representation of plastids from plant organisms (chloroplasts), for which the distribution of associated taxa varies, as some are limited to non-mucosal surfaces (environmental exposure and potentially cosmetic products) and others to the digestive track (ingested food). No clades are consistently present in all mucosal body sites, demonstrating the \u03b2-diversity of these communities (that is, differences among their population structure), but many taxa within Actinobacteria, Bacillales, and several other clades are relatively abundant at all non-mucosal sites. The within-subject \u03b2-diversity at all phylogenetic levels is highlighted in Additional file <a href=\"#S1\" data-ref-type=\"supplementary-material\">1<\/a>, quantifying the extent to which distances among different mucosal body sites are larger than the equivalent distances among non-mucosal sites. This leads to a lack of taxa common to all mucosal body sites, and therefore no taxa are determined by LEfSe to be characteristic of the mucosa as a whole.\n\nThe Actinomycetales are usually the most abundant phylogenetic unit (order level) in non-mucosal communities, with percentages higher than 90% in several skin samples and at most 20% in the great majority of the oral mucosal samples and substantially lower in the vagina and gut (Figure <a href=\"#F2\" data-ref-type=\"fig\">2c<\/a>). From a quantitative viewpoint, the taxonomic order Actinomycetales makes up essentially all of the detected members of the phylum Actinobacteria, except in the vaginal site, which reported a substantial Bifidobacteriales presence. Bifidobacteriales themselves are not detected as differentially abundant between mucosal and non-mucosal body sites, since this is a feature only of the vaginal samples and not of all mucosal body sites. The contrast of many clades' abundance versus distribution is striking; for example, the genera *Alloscardovia*, *Parascardovia* and *Scardovia* are present in all body sites at very low abundances, while *Gardnerella* is overrepresented only in vaginal samples, with over three orders of magnitude difference in abundance. A similar commonality of distribution was found for the Bacillales at an even lower abundance. At the genus level, *Propionibacterium*, *Staphylococcus*, *Corynebacterium* and *Pseudomonas* are differentiated by both distribution and abundance. The *Staphylococcus* genus in particular is detected by LEfSe with a very high LDA score (more than five orders of magnitude), reflecting marked abundance in non-mucosal sites (mean 10%, 18% and 21% in the skin, retroauricular crease and anterior nares body sites, respectively) and consistently low abundance in mucosal sites (mean less than 0.001%).\n\n## Classes with multiple levels: distinct aerobic, anaerobic, and microaerobic communities in the human microbiome\n\nThe roles of anaerobic metabolism in the commensal human microbiota have not yet been fully investigated due to the difficulty of studying these communities in culture. We thus further investigated the aerobicity characteristics of human microbial communities at a high level by grouping body sites into three classes with distinct levels of available molecular oxygen. The high-O~2~ exposure class includes body sites directly and permanently exposed to oxygen: skin, anterior nares and retroauricular crease. The mid-O~2~ exposure class includes the oral and vaginal body sites that can be directly, but not permanently, atmospherically exposed, and the low-O~2~ exposure class (the gut) is mainly anaerobic. The body sites included in the three classes may have other distinguishing features in addition to different oxygen exposure and, in general, these confounding factors can cause features unrelated with aerobiosis to be detected as biomarkers. However, the LEfSe biological consistency step assures that the detected biomarkers are characteristic of all the subclasses of a given class and with respect to all subclasses of the other classes. For example, the high-abundance of a bacterial clade in the mouth due to an oral-specific niche is not detected as a biomarker unless the same niche is also present in the vaginal samples (the other body site in the mid-O~2~ class) and not present in any high-O~2~ or low-O~2~ single body sites. So LEfSe will detect biomarkers more confidently connected with the aerobiosis characteristics than traditional methods that do not incorporate subclass information. Moreover, LEfSe is specifically able to analyze ordinal classes with multiple levels, and in agreement with established microbiology, we observed specific microbial clades ubiquitous within and characteristic to each of these three environments, detailed as follows (Figure <a href=\"#F2\" data-ref-type=\"fig\">2d<\/a>).\n\nLEfSe allows ordinal classes with more than two levels to be analyzed in two different stringencies. The first requires significant taxa to differ between every pair of class values (that is, aerobicity in this example; see Materials and methods); the discovered biomarkers must accurately distinguish all individual classes (high-, mid-, and low-O~2~). In this example (Figure <a href=\"#F2\" data-ref-type=\"fig\">2d<\/a>; strict version), we detected 13 clades with LDA scores above 2, showing three distinct abundance levels. Alternatively, LEfSe can determine significant taxa differing in at least one (and possibly multiple) class value(s) (non-strict version); in other words, biomarkers that distinguish at least one individual class. Using this method (Figure <a href=\"#F2\" data-ref-type=\"fig\">2e<\/a>), we find 60 clades with LDA scores of at least 2.\n\nUsing either approach, each oxygen level is broadly characterized by a specific clade. The overall abundances of the Actinobacteria phylum are higher in body sites directly exposed to molecular oxygen with several members of the Actinomycetales order that colonize the skin. Actinomycetales includes the *Propionibacterium* genus, which is highly abundant on the skin, low in moderate-O~2~ environments, and absent from the gut. The Lactobacillales (primarily Bacilli) are specific to moderate O~2~ exposure levels, with conversely lower presences in the high-O~2~ exposure class, and are again absent from the gut. The Bacteroidaceae (particularly *Bacteroides*) are ubiquitous in anaerobic samples; interestingly, however, members of this family are more abundant in high oxygen availability conditions (particularly in skin and retroauricular crease) than in medium oxygen availability, showing the niche diversity within the phylogenetic branching. This is in agreement with observations that the microenvironment of many microbial consortia shows extreme biogeographical variation with respect to nutrients, metabolites, and oxygen availability \\[58,59\\].\n\n## Bifidobacteria and additional clades are underrepresented in a mouse model of ulcerative colitis\n\nRodent models have been established to provide a uniquely accurate and tractable model for studying the gut microbiota, including the molecular and cellular mechanisms driving chronic intestinal inflammation \\[60-63\\]. In particular, mouse models of inflammatory bowel disease \\[63\\] facilitate a mechanistic evaluation of the contribution of the gut microbiota to the initiation and perpetuation of chronic intestinal inflammation, as occurs in human Crohn's disease and ulcerative colitis \\[64\\]. One host molecular mechanism known to maintain the balance between immune regulation and the commensal microflora is T-bet, a transcription factor expressed in many immune cell subsets. Its loss in the absence of an adaptive immune system results in a highly penetrant and aggressive form of ulcerative colitis \\[65\\] that is specifically dependent on and transmissible through the gut flora. We thus sought to investigate the characteristics of the fecal microbiota in a mouse model of spontaneous colitis that occurs in a colony of Balb\/c *T-bet*^-\/-^ \u00d7 *Rag2*^-\/-^ mice using 16S rRNA gene metagenomic data \\[66,67\\].\n\nLEfSe was applied to the microbiota data of 20 *T-bet*^-\/-^ \u00d7 *Rag2*^-\/-^ (case) and 10 *Rag2*^-\/-^ (control) mice (dataset provided in Additional File <a href=\"#S10\" data-ref-type=\"supplementary-material\">10<\/a>), finding 19 differentially abundant taxonomic clades (\u03b1 = 0.01) with an LDA score higher than 2.0 (Figure <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>). These differentially abundant clades were consonant with both our prior 16S rRNA-based sequence analysis using complete linkage hierarchical clustering and quantitative real time PCR-based experiments performed on the same fecal DNA samples \\[67\\]. More specifically, the marked loss in Bifidobacteriaceae and *Bifidobacterium* associated with *T-bet*^-\/-^ \u00d7 *Rag2*^-\/-^ we observed here may explain the positive responsiveness of this colitis to a *Bifidobacterium animalis* subsp. *lactis* fermented milk product validated with low-throughput approaches \\[67\\].\n\nAt the family level, the *Rag2*^-\/-^ enrichment of Bifidobacteriaceae, Porphyromonadaceae, Staphylococcaceae and the *T-bet*^-\/-^ \u00d7 *Rag2*^-\/-^ enrichment of Lachnospiraceae confirm our reports in \\[68\\] using culture-based and quantitative real time PCR techniques. LEfSe's LDA score more informatively reorders these taxa relative to the *P*-values found for these families in our previous work, highlighting the Bifidobacteria and, interestingly, several clades within the Clostridia. These include the *Rag2*^-\/-^-specific *Roseburia* and *Papillibacter* genera belonging to *T-bet*^-\/-^ \u00d7 *Rag2*^-\/-^-specific families (Lachnospiraceae and Ruminococcaceae). The significant presence of *Metascardovia* (Bifidobacteriaceae) in *Rag2*^-\/-^ mice is also interesting, as it may have a role similar to *Bifidobacterium* and because *Metascardovia* has been previously observed primarily in the oral cavity \\[68\\]. This analysis both highlights the agreement of LEfSe's effect size estimation with respect to low-throughput confirmations and suggests additional clades to be further investigated experimentally.\n\n## A comparison with current metagenomic analysis tools using viral and microbial pathways from environmental data\n\nWe applied LEfSe to the environmental data of \\[69\\], a dataset with the goal of characterizing the functional roles of viromes (that is, viral metagenomes) versus microbiomes (that is, bacterial metagenomes). This task was used in \\[45\\] to characterize the Metastats algorithm on the same raw data. Among the 29 high-level functional roles (including unclassified roles) in the subsystem hierarchy of the SEED \\[70\\] and NMPDR \\[71\\] frameworks, LEfSe identifies only the 'Nucleosides and nucleotides' subsystem to be strictly differentially abundant among all environmental subclasses, specifically with higher levels in viromes than microbiomes. This is an accurate characterization of exactly the protein function most commonly encoded in viral genomes, whereas bacterial genomes of course encode a wide range of less specifically enriched functionality. When LEfSe is relaxed to detect significant variations consistent for at least one, rather than all, environmental subclasses, we additionally determine the 'Respiration' subsystem to be significantly enriched in microbiomes with respect to viromes, likely reflecting the uniformly aerobic bacterial metabolism captured by these data.\n\nIn addition to the Nucleosides and nucleotides and Respiration subsystems, Metastats \\[45\\] reports five other high-level functional roles as differentially abundant (*P* = 0.001). However, when taking the subclass structure into account across the sampled environments, these additional differences show much less consistent variation. This is demonstrated in Figure <a href=\"#F4\" data-ref-type=\"fig\">4<\/a>, which reports histograms of raw data for these cases and the different results of LEfSe, Metastats and the KW test alone. Moreover, since the subsystem framework is hierarchical (three levels), LEfSe's results include a cladogram showing the significant differences on each level (see Figure <a href=\"#F4\" data-ref-type=\"fig\">4<\/a> for a two-level cladogram, and Additional file <a href=\"#S2\" data-ref-type=\"supplementary-material\">2<\/a> for a three-level cladogram).\n\nConsidering all three levels of SEED functional specificity, LEfSe reports 59 subsystems to be more abundant in microbial metagenomes and only 7 that are more abundant in viral metagenomes (Additional file <a href=\"#S3\" data-ref-type=\"supplementary-material\">3<\/a>). Bacterial genomes encode a much greater quantity and diversity of biomolecular functionality than most viral genomes, and these differences are thus to be expected. However, they also highlight a consideration specific to most metagenomic (and, more generally, ecological) analyses, which typically analyze relative abundances. A few very common subsystems in viromes (that is, Nucleosides and nucleotides) will force the relative abundance of all other subsystems to decrease, resulting in apparent under-abundance. The subsystems detected to be virus-specific may thus show this trend in part due to the normalization of abundances in each sample. This issue is specific to neither LEfSe nor Metastats, however, and must be taken into account during interpretation of any relative abundance data, metagenomic or otherwise \\[72\\].\n\n## Functional activity within the infant and adult microbiota indicates post-weaning microbial specialization\n\nJust as LEfSe can determine whether organisms or pathways are differentially abundant among several metagenomic samples, it can also focus on individual enzymes or orthologous groups. Kurokawa *et al*. \\[73\\] analyzed 13 gut metagenomes from nine adults and four unweaned infants in terms of the functions of orthologous gene families. They originally did this by comparing the COGs \\[74,75\\] found in each metagenome to a reference database; later, White *et al*. \\[45\\] applied the Metastats algorithm to directly detect differences between infant and adult microbiomes. Using significance \u03b1 values of 0.01 due to the low cardinality of the classes (in particular the infant class), LEfSe detected 366 COGs to be enriched in either adult or infant metagenomes, 17 of which have a LDA score higher than 3 (Additional file <a href=\"#S4\" data-ref-type=\"supplementary-material\">4<\/a>).\n\nAmong the 17 COG profiles with LEfSe scores higher than 3, 11 are also detected by Metastats. The six COGs not detected by Metastats (Additional file <a href=\"#S5\" data-ref-type=\"supplementary-material\">5<\/a>) are Outer membrane protein (COG1538) and Na^+^-driven multidrug efflux pump (COG0534), enriched in adults, and Transposase and inactivated derivatives (COG2801, COG2963), Transcriptional regulator\/sugar kinase (COG1940) and Transcriptional regulator (COG1309), enriched in infants. All six COGs possess abundance profiles that are completely non-overlapping between infant and adult individuals (apart from COG1538, in which the lowest level in adults is slightly lower than the highest in infants) and are thus nominally quite discriminative. On the other hand, among the 192 COGs found by Metastats, 9 are not detected by LEfSe even at the lowest LDA score threshold (Additional file <a href=\"#S6\" data-ref-type=\"supplementary-material\">6<\/a>). All possess overlapping abundance values between infant and adult classes (at least two, and often more, of the highest samples in the less abundant class overlap the putatively more abundant class). This lack of discriminatory power precludes LEfSe from highlighting the differences as significant between adults and infants, particularly given the low number of infant samples.\n\nIntriguingly, LEfSe's distinct list of functional activities in the core infant and adult microbiomes is suggestive of 'generalist' microbial activity during early life and specialization over time \\[76\\]. In fact, inspecting the five differentially abundant COGs with the highest effect sizes for each class, we find for infants very high-level functional groups related to broad transcriptional regulation (COG1609, COG1940, COG1309 and COG3711). In adults, all five represent more specialized orthologous groups, including COG1629 (Outer membrane receptor proteins, mostly Fe transport), COG1595 (DNA-directed RNA polymerase specialized sigma subunit, sigma24 homolog), and COG4771 (Outer membrane receptor for ferrienterochelin and colicins). Since the number of differentially abundant COGs is very high (366), this observation was only highlighted at the top of the candidate biomarker list due to LEfSe's effect size quantification, which allows the most characteristic differences among classes to emerge. For the same reason, we can easily confirm that sugar metabolism plays a crucial role in the infant gut and iron metabolism in adults, as already stated in \\[45,73\\]; the COGs with the highest LDA scores indeed possess sugar and glucose functional activities for infants and iron-related functionality for adults.\n\n## LEfSe achieves a very low false positive rate in synthetic data\n\nWe further investigated the ability of LEfSe to detect biomarkers using synthetic high-dimensional data (see Materials and methods for the description of the dataset) in comparison with the KW test alone (a non-parametric adaptation of the analysis of variance (ANOVA)) and with Metastats \\[45\\]. The LDA effect size step of LEfSe is not considered here for simplicity, and the artificial data are detailed in Figure <a href=\"#F5\" data-ref-type=\"fig\">5<\/a>.\n\nTheoretically, the settings of the first two experiments (Figure <a href=\"#F5\" data-ref-type=\"fig\">5a,b<\/a>) exactly match the application conditions for the KW test. The false positive rate (mean 2.5%, regardless of the distance between feature means and of the standard deviation of the normal distribution) is in fact consistent with the \u03b1 value of 0.05, given that the negative features are half of the total. LEfSe behaved qualitatively very similar to KW, but with a considerably lower false positive rate (less than 0.5% in the great majority of the cases against a mean value of 2.5%) and a higher false negative rate. In biology, false positives are often perceived as more dramatic than false negatives \\[77-79\\]; this is often attributable to the fact that it is undesirable to invest in expensive experimental follow-up of false positives, whereas in high-throughput settings, a few true positives outweigh the false negatives that are left uninvestigated. With this motivation for minimizing false positives, we conclude that LEfSe performs at least as well as KW when no meaningful subclass structure is available. On the other hand, when subclasses can be identified internally to the classes and some of them do not agree with the trend among classes, LEfSe performs qualitatively and quantitatively much better than KW (Figure <a href=\"#F5\" data-ref-type=\"fig\">5c<\/a>). The false positives are in fact always substantially lower than KW, whereas the false negatives are higher only for very noisy features. Metastats \\[45\\] seems to achieve results very similar to KW (Additional file <a href=\"#S7\" data-ref-type=\"supplementary-material\">7<\/a>) with the same disadvantages with respect to LEfSe.\n\n# Conclusions\n\nGaining insight into the structure, organization, and function of microbial communities has been proposed as one of the major research challenges of the current decade \\[80\\], and it will be enabled by both experimental and computational metagenomic analyses. To this end, we have developed the LEfSe algorithm for comparative metagenomic studies, permitting the characterization of microbial taxa specific to an experimental or environmental condition, the detection of pathways and biological mechanisms over- or under-represented in different communities, and the identification of metagenomic biomarkers in mammalian microbiomes. LEfSe is shown here to be effective in detecting differentially abundant features in the human microbiome (characteristically mucosal or aerobic taxa) and in a mouse model of colitis. A comparison with existing statistical methods and state-of-the-art metagenomic analyses of environmental, infant gut microbiome, and synthetic data shows that LEfSe consistently provides lower false positive rates and can effectively aid in explaining the biology underlying differences in microbial communities.\n\nThese findings demonstrate that a concept of class explanation including both statistical and biological significance is highly beneficial in tackling the statistical challenges associated with high-dimensional biomarker discovery \\[28,81,82\\]. Specifically, LEfSe determines features potentially able to explain the differences among conditions rather than the features that simply possess uneven distributions among classes. This is distinct from most current statistical approaches \\[45\\] and akin to the incorporation of biological prior knowledge that has proven highly successful in recent genome-wide association studies \\[83-85\\]. Moreover, particularly in (often noisy) metagenomic datasets, effect size can serve as an orthogonal measure to complement ranking biomarkers based on *P*-values alone. Differences between classes can be very statistically significant (low *P*-value) but so small that they are unlikely to be biologically responsible for phenotypic differences. On the other hand, a biomarker with a relatively large *P*-value (for example, 0.01) may correspond to a large effect size, with statistical significance diminished by technical noise. LEfSe investigates both aspects computationally by testing both the consistency and the effect size of differences in feature abundance among classes with respect to the structure of the problem. This is performed subsequently to standard statistical significance tests and is integrated in LEfSe by assessing biologically meaningful groups of samples among subclasses within each condition. This coupling of statistical approaches with biological consistency and effect size estimation alleviates possible artifacts or statistical inhomogeneity known to be common in metagenomic data, for example, extreme variability among subjects or the presence of a long tail of rare organisms \\[32,86\\]. Similarly, while multiple hypothesis corrected statistical significance speaks to the potential reproducibility of a result, estimation of effect size in high-dimensional settings is crucial for addressing biological consistency and interpretability.\n\nThe biology highlighted by these investigations speaks to the potential of metagenomics for both microbial ecology and translational applications. For example, certain bacterial clades are frequently detected as biomarkers even in diverse environments, suggesting that some species can adapt in surprisingly condition-specific manners. *Staphylococcus* and the Bacillales, for example, are discriminative for mucosal tissues, aerobic conditions, and murine colitis, whereas no Proteobacteria consistently characterize any of these conditions, even though they always represent a substantial portion of the communities. These observations may reflect extensive microenvironmental heterogeneity and the coexistence of generalist and specialist bacteria \\[87-89\\].\n\nIn addition to these insights into microbiology, metagenomic biomarkers, including the abundances of specific organisms, abundances of entire clades, or the presence\/absence of specific organisms, can serve to describe host phenotypes, lifestyle, diet, and disease as well \\[5-10\\]. If the depletion of *Bifidobacterium* species in ulcerative colitis proves to occur early in human disease etiology, this and comparable shifts in the microbiota have potential applications in the detection of human disorders \\[90,91\\], especially as shifts in some bacterial consortia can be detected easily and inexpensively. Oral microbial biomarkers, for example, can be easily acquired and analyzed with microarray chips targeted for bacterial profiling \\[92\\]. These appear particularly promising for clinical applications \\[11\\], as the microbial communities in the saliva seem to represent one potential proxy for other human microbiota \\[93\\]. Other important clinical applications of metagenomic analyses include probiotic treatments \\[94,95\\] and microbiome transplantation \\[96-99\\] for gastrointestinal diseases.\n\nLEfSe, the computational approach to biomarker class comparisons detailed here, thus contributes to the understanding of microbial communities and guides biologists in detecting novel metagenomic biomarkers. The algorithm's effectiveness on real and synthetic data has been highlighted by several experiments in which we successfully characterized both host-associated microbiota and environmental microbiomes in multiple contexts. To support ongoing metagenomic analyses, we have implemented LEfSe as a user-friendly web application that can provide both raw data and publication-ready graphical results, including reports of detected microbial variation on taxonomic trees for visual and biological summarization. LEfSe is freely available online in the Galaxy workflow framework \\[46,47\\] at the following link \\[48\\].\n\n# Materials and methods\n\nThe LEfSe algorithm is introduced in overview in the Results section, and Figure <a href=\"#F6\" data-ref-type=\"fig\">6<\/a> illustrates in detail the format of the input (a matrix with *n* rows and *m* columns) and the three steps performed by the computational tool: the KW rank sum test \\[49\\] on classes, the pairwise Wilcoxon test \\[50,51\\] between subclasses of different classes, and the LDA \\[52\\] on the relevant features.\n\nEach of the *n* features is represented with a positive-valued vector containing its abundances in the *m* samples, and each sample is associated with values describing its class and, optionally, subclass and\/or originating subject. The factorial KW rank sum test is applied to each feature with respect to the class factor; the subclass and subject information are used as stratifying subgroups when present. Features that, according to the KW rank sum test, do not violate the null hypothesis of identical value distribution among classes (with default *P*-value, \u03b1 = 0.05) are not further analyzed. The pairwise Wilcoxon test is applied to retained features belonging to subclasses of different classes. For each feature, the pairwise Wilcoxon test is not satisfied if at least one comparison between subclasses has a *P*-value higher than the chosen \u03b1 or if the sign of variation is not equal among all comparisons. For example, if a feature appears in samples from two classes with three subclasses each, all nine comparisons between subclasses in different classes must violate the null hypothesis, and all signs of the differences between medians must be consistent. The features that pass the pairwise Wilcoxon test are considered successful biomarkers. An LDA model is finally built with the class as dependent variable and the remaining feature values, subclass, and subject values as independent variables. This model is used to estimate their effect sizes, which are obtained by averaging the differences between class means (using unmodified feature values) with the differences between class means along the first linear discriminant axis, which equally weights features' variability and discriminatory power. The LDA score for each biomarker is obtained computing the logarithm (base 10) of this value after being scaled in the \\[1,10^6^\\] interval and, regardless of the absolute values of the LDA score, it induces the ranking of biomarker relevance. For robustness, LDA is additionally supported by bootstrapping (default 30-fold) and subsequent averaging.\n\nLEfSe's first two steps employ non-parametric tests because of the nature of metagenomic data. Relative abundances will, in most cases, violate the main assumption of typical parametric tests (normal population in each class), whereas non-parametric tests are much more robust to the underlying distribution of the data since they are distribution-free approaches. The only assumption of the Wilcoxon and KW tests is that the distributions in each class are identically shaped with possible differences in the medians. For example, the bimodal or multimodal abundance distribution of an organism violates the assumptions of parametric tests but not those of non-parametric tests, unless the number of peaks in the distribution (or, more generally, the shape of the distribution) also changes among classes. LDA is used for effect size estimation as our experiments determined it to more accurately estimate biological consistency compared to approaches like differences in group means\/medians or support vector machines (SVMs) \\[100\\]. A comparison between LDA and SVM approaches for effect size estimation on the murine model of ulcerative colitis (for which low-throughput biological validations of biomarkers are available in \\[67\\]) is reported in our supplemental material (Additional files <a href=\"#S8\" data-ref-type=\"supplementary-material\">8<\/a> and <a href=\"#S9\" data-ref-type=\"supplementary-material\">9<\/a>) and shows the advantages of LDA with respect to upranking features of potential biological interest. Theoretically, this is motivated by LDA's ability to find the axis of highest variance and SVM's focus on features' combined predictive power rather than single feature relevance. Note that as we are performing class comparison rather than class prediction, it is worth specifying that the effect size estimation accuracy of an algorithm is not directly connected with its predictive ability (for example, SVM approaches are generally considered more accurate than LDA for prediction).\n\n## Multiclass strategies\n\nComparisons with more than two classes require special strategies for applying the Wilcoxon and LDA steps, whereas the factorial KW test is already appropriate for this setting. Our multiclass strategy for the Wilcoxon test depends on the problem-specific strategy chosen by the user to define features differentially distributed among the *n* classes. In the most stringent strategy, we require that all *n* abundance profiles of a feature are statistically significantly distinct among all *n* classes. This strategy, called 'strict', is implemented by requiring that all Wilcoxon tests between classes are significant. A more permissive strategy, called 'non-strict', considers a feature as a biomarker if at least one class is significantly different from all others. The more permissive strategy thus needs to satisfy only a subset of the Wilcoxon tests. Regardless of the strategy, the LDA step always reports the highest score detected among all pairwise class comparisons.\n\n## Subclass structure variants encoding different biological hypotheses\n\nDifferent interpretations of the biomarker class comparison problem are implemented in LEfSe by modifying the requirements for pairwise Wilcoxon comparisons among subclasses. If classes contain subclasses that represent distinct strata, we test only comparisons within each identical subclass (Figure <a href=\"#F4\" data-ref-type=\"fig\">4<\/a>). For example, to assess the effect of a treatment on two sub-types of the same disease, we compare pre- and post-treatment levels within each subclass and require that the trend observed at the class level is significant independently for both subclasses. To implement this variant, LEfSe performs the Wilcoxon step only comparing subclasses with the same name. Alternatively, subclasses may represent covariates within which feature levels may vary but for which the problem does not dictate explicit stratification (Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>). In both settings, we explicitly require all the pairwise comparison to reject the null hypothesis for detecting the biomarker; thus, no multiple testing corrections are needed.\n\n## Subclasses containing few samples\n\nWhen few samples are available, non-parametric tests like the Wilcoxon have reduced power to detect differences. This can affect LEfSe when subclasses are very small, preventing the overall test from even rejecting the null hypothesis. For this reason, small subclasses should be avoided when possible, for example, by excluding them from the problem or by grouping together all subclasses with small cardinalities. For cases in which removing or grouping subclasses is not possible or disrupts the biological consistency of the analysis, LEfSe substitutes the Wilcoxon test with a test to compare whether subclass medians differ with the expected sign. The user can choose the subclass cardinality threshold at which this median comparison is substituted for the Wilcoxon test.\n\n## Parameter settings\n\nExcept as stated otherwise in Results, all experiments in this study were run with LEfSe's \u03b1 parameter for pairwise tests set to 0.05 for both class normality and subclass tests, and the threshold on the logarithmic score of LDA analysis was set to 2.0. The stringency of these parameters is easily tunable (also through the web interface) and allows the user to detect biomarkers with lower *P*-values and\/or higher effect size in order, for example, to prioritize additional biological experiments and validations. All LDA scores are determined by bootstrapping over 30 cycles, each sampling two-thirds of the data with replacement, with the maximum influence of the LDA coefficients in the LDA score of three orders of magnitude.\n\n## Data description\n\nExcept as stated otherwise, taxonomic abundances for 16S samples were generated from filtered sequence reads using the RDP classifier \\[101\\], with confidences below 80% rebinned to 'uncertain'. For all the datasets described below, the final input for LEfSe is a matrix of relative abundances obtained from the read counts with per-sample normalization to sum to one. Witten-Bell smoothing \\[102\\] was used to accommodate rare types, but due to LEfSe's non-parametric approach, this has minimal effect on the discovered biomarkers and on the LDA score. This also allows our biomarker discovery method to avoid most effects of sequence quality issues as long as any sequencing biases are homogeneous among different conditions, as no specific assumptions on the statistical distribution and noise model are made by the algorithm as is standard for non-parametric approaches.\n\n## Human microbiome data\n\nThe 16S rRNA-based phylometagenomic dataset of the normal (healthy) human microbiome was made available through the Human Microbiome Project \\[13\\], and consists of 454 FLX Titanium sequences spanning the V3 to V5 variable regions obtained for 301 samples from 24 healthy subjects (12 male, 12 female) enrolled at a single clinical site in Houston, TX. These samples cover 18 different body sites, including 6 main body site categories: the oral cavity (9 samples), the gut (1 sample), the vagina (3 samples), the retroauricular crease (2 samples), the nasal cavity (1 sample) and the skin (2 samples). Detailed protocols used for enrollment, sampling, DNA extraction, 16S amplification and sequencing are available on the Human Microbiome Project Data Analysis and Coordination Center website \\[103\\], and are also described elsewhere \\[55,56\\]. In brief, genomic DNA was isolated using the Mo Bio PowerSoil kit \\[104\\] and subjected to 16S amplifications using primers designed incorporating the FLX Titanium adapters and a sample barcode sequence, allowing directional sequencing covering variable regions V5 to partial V3 (primers: 357F 5'-CCTACGGGAGGCAGCAG-3' and 926R 5'-CCGTCAATTCMTTTRAGT-3'). Resulting sequences were processed using a data curation pipeline implemented in mothur \\[41\\], which reduces the sequencing error rate to less than 0.06% as validated on a mock community. As part of the pipeline parameters, to pass the initial quality control step, one unambiguous mismatch to the sample barcode and two mismatches to the PCR amplification primers were allowed. Sequences with an ambiguous base call or a homopolymer longer than eight nucleotides were removed from subsequent analyses, as suggested previously \\[105\\]. Based on the supplied quality scores, all sequences were trimmed when a base call with a score below 20 was encountered. All sequences were aligned using a NAST-based sequence aligner to a custom reference based on the SILVA alignment \\[106,107\\]. Sequences that were shorter than 200 bp or that did not align to the anticipated region of the reference alignment were removed from further analysis. Chimeric sequences were identified using the mothur implementation of the ChimeraSlayer algorithm \\[108\\]. Unique reads were classified with the MSU RDP classifier v2.2 \\[58\\] using the taxonomy proposed by \\[109\\], maintained at the RDP (RDP 10 database, version 6). The 16S rRNA reads are available in the Sequence Read Archive at \\[110\\].\n\n## *T-bet^-\/-^ \u00d7 Rag2^-\/-^* and *Rag2^-\/-^* mouse data\n\n*T-bet*^-\/-^ \u00d7 *Rag2*^-\/-^ and *Rag2*^-\/-^ mice, their husbandry, and their chow have been described in \\[67\\]. The animal studies and experiments were approved and carried out according to Harvard University's Standing Committee on Animals as well as National Institutes of Health guidelines. Collection, processing, and extraction of DNA from fecal samples were performed as described in \\[67\\]. The V5 and V6 regions of the 16S rRNA gene were targeted for amplification and multiplex pyrosequencing with error-correcting barcodes. Sequencing was performed using a Roche FLX Genome Sequencer at DNAVision (Charleroi, Belgium) and data were preprocessed to remove sequences with low-quality scores. There were 7,579 \u00b1 2,379 high-quality 16S reads per sample with a mean read length of 278 bp.\n\n## Viral and microbial environmental data\n\nWe retrieved from the online supplemental material of \\[69\\] the 80 available metagenomes (42 viromes, 38 microbiomes). We identified three environments containing at least seven samples and grouped them into coral, hyper-saline, and marine subclasses; the fourth subclass, other, groups all environments with few samples.\n\n## Infant and adult microbiome data\n\nThe COG profiles of the nine adult and four unweaned infant microbiomes were obtained from the supplemental material of \\[73\\] and used unmodified in this study.\n\n## Synthetic datasets\n\nWe built three collections of artificial datasets in order to compare LEfSe to KW and Metastats. All datasets have 1,000 features and 100 samples belonging evenly to two classes, and the values are sampled from a Gaussian normal distribution. The samples in the two classes are further organized in four subclasses (two per class) with equal cardinality. Of the 1,000 features, 500 features have different means across classes and should thus be detected as biomarkers (positive features), the other 500 features are evenly distributed among classes or among at least one subclass in both classes and should not be detected as discriminative (negative features). The methods are evaluated assessing the false positive rate (number of erroneously detected biomarkers with respect to the total number of features) and the false negative rate (number of correctly detected non-discriminant features with respect to the total number of features, that is, sensitivity). The three collections of datasets (graphically shown in Figure <a href=\"#F5\" data-ref-type=\"fig\">5<\/a>) differ in the distribution of values in the subclasses and in the mean\/standard deviation of the normal distribution. (a) The subclasses in the same class have the same parameters (thus the subclass organization is meaningless). Negative features all have \u03bc = 10,000 and \u03c3 = 100, whereas one class of the positive features has \u03bc = 10,000 - t (\u03c3 = 100) and the other \u03bc = 10,000 + t (\u03c3 = 100) where t is a parameter ranging from 1 to 150. The performances of all methods are assessed at regular steps of the t parameter. (b) Datasets in this collection are defined in the same way as collection (a) but with t = 1,000 for all datasets and \u03c3 ranging from 1,000 to 10,000. (c) The negative class in the third collection has different subclass distribution. In particular, the second subclass of the first class has the same mean of the first subclass of the second class. The other two subclasses have different means (\u03bc = 10,000 - t and \u03bc = 10,000 + t, t = 1,000), but the feature is not considered differential since the difference is not consistent between subclasses. The positive features are defined in the same way as dataset (b).\n\n## Implementation and availability of the method\n\nLEfSe is implemented in Python and makes use of R statistical functions in the coin \\[111\\] and MASS \\[112\\] libraries through the rpy2 library \\[113\\] and of the matplotlib \\[114\\] library for graphical output. LEfSe is provided with a graphical interface in the Galaxy framework \\[46,47\\], which allows the user to select parameters (the primary three stringency parameters, the multiclass setting, and other computational, statistical, and graphical preferences), to pipeline data between modules in a workflow framework, to generate publication-quality graphical outputs, and to combine these results with other statistical and metagenomic analyses. LEfSe is available at \\[48\\].\n\n# Abbreviations\n\nbp: base pair; KW: Kruskal-Wallis; LDA: linear discriminant analysis; LEfSe: linear discriminant analysis effect size; PCR: polymerase chain reaction; RDP: Ribosomal Database Project; SVM: support vector machines.\n\n# Authors' contributions\n\nNS and CH conceived the study; NS and LM implemented the methodology; NS: JI: LW: DG: WG: and CH analyzed the results; NS: JI: LW: DG: WG: and CH wrote the manuscript. All authors read and approved the manuscript in its final form.\n\n# Supplementary Material\n\n###### Additional file 1\n\n**Supplementary Figure S6**. Histogram of within-subject \u03b2-diversity (community dissimilarity) between different mucosal (red) and non-mucosal (green) body sites.\n\nClick here for file\n\n###### Additional file 2\n\n**Supplementary Figure S1**. Cladogram representing the differences between viromes and microbiomes on the subsystem framework.\n\nClick here for file\n\n###### Additional file 3\n\n**Supplementary Figure S2**. Histogram of LDA logarithmic scores of biomarkers found by LEfSe comparing microbiomes and viromes within the subsystem framework.\n\nClick here for file\n\n###### Additional file 4\n\n**Supplementary Figure S3**. Histogram of LDA logarithmic scores of COG biomarkers found by LEfSe comparing adult and infant microbiomes.\n\nClick here for file\n\n###### Additional file 5\n\n**Supplementary Figure S4**. Functional features (COGs) that are discrimantive for the comparison between adult and infant microbiomes according to LEfSe but not detected by Metastats among the discriminant features with LDA score higher than 3. If we consider all the discriminant features without threhold on LDA score, LEfSe identifies 366 COGs in total, 185 of which are not discriminant for Metastats.\n\nClick here for file\n\n###### Additional file 6\n\n**Supplementary Figure S5**. Functional features (COGs) that are discrimantive for the comparison between adult and infant microbiomes according to Metastats but not detected by LEfSe. Even if median and variance suggest the differences to be discriminative, there are always some microbiomes (at least two) that are overlapping between classes. This is due to the stringent \u03b1-value (0.01) set for the KW test in LEfSe and to the fact that we use non-parametric statistics (differently from Metastats). Notice, however, that even using a low \u03b1-value LEfSe detects many more biomarkers than metastats (366 versus 192).\n\nClick here for file\n\n###### Additional file 7\n\n**Supplementary Figure S9**. Comparison between LEfSe and Metastats using the synthetic data described in Figure <a href=\"#F5\" data-ref-type=\"fig\">5<\/a> and in the Materials and methods. LEfSe was applied as detailed in the paper; for Metastats we used the default settings (that is, \u03b1 = 0.05 and N~permutations~ = 1,000) and, as for LEfSe and KW, we disabled the per-sample normalization as the features are independent. **(a,b)** Metastats has a higher false positive rate (average 5%) than LEfSe (average below 0.5%) and lower false negative rate. **(c)** When the subclass information is meaningful (see Figure <a href=\"#F5\" data-ref-type=\"fig\">5<\/a> for the representation of the dataset), LEfSe performs substantially better than Metastats both in terms of false positive and false negatives. Overall, on these synthetic data, Metastats achieves very similar results compared to KW (Figure <a href=\"#F5\" data-ref-type=\"fig\">5<\/a>) and neither of them can make use of additional information regarding the within-class structure, thus achieving poor results compared to LEfSe when such kinds of information are available.\n\nClick here for file\n\n###### Additional file 8\n\n**Supplementary Figure S7**. SVM-based effect size estimation for the biomarkers found for the *Rag2^-\/-^* versus *T-bet^-\/-^xRag2^-\/-^* comparison reported in Figure <a href=\"#F3\" data-ref-type=\"fig\">3<\/a> of the manuscript. The LDA-based approach for assessing effect size (Figure <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>) is closer to the biological follow-up experiments and is more visually consistent. The reason for LDA superiority over SVM approaches for effect size estimation is theoretically connected with the ability of LDA to find the axis with the highest variance, and the SVM effort on evaluating the combined feature predictive power rather than single feature relevance. It is worth specifying that the effect size estimation accuracy of an algorithm is not directly connected with its predictive ability (SVM approaches are usually considered more accurate than LDA for prediction).\n\nClick here for file\n\n###### Additional file 9\n\n**Supplementary Figure S8**. Comparison between the features with the highest SVM-based effect size (*Papillibacter*, on the left), the highest LDA-based effect size (*Bifidobacterium*, in the center), and the Actinobacteria phylum (on the right). From a visual analysis, *Bifidobacerium* shows a larger effect size, which is also evident looking at the ratios between class means, suggesting LDA as a better option for effect size estimation than SVM approaches. As detailed in the manuscript, the relevance of *Bifidobacterium* has been experimentally validated. Moreover, the large difference in the score given by the SVM approach to Actinobacteria compared to *Bifidobacterium* and *Papillibacter* is not consistent.\n\nClick here for file\n\n###### Additional file 10\n\n***T-bet*^-\/-^ \u00d7 *Rag2*^-\/-^ - *Rag2*^-\/-^ dataset**. Input LEfSe file for the analysis of the ulcerative colitis phenotype in mice.\n\nClick here for file\n\n## Acknowledgements\n\nWe would like to thank the entire Human Microbiome Project consortium, including the four sequencing centers (the Broad Institute, Washington University, Baylor College of Medicine, and the J Craig Venter Institute), associated investigators from many additional institutions, and the NIH Office of the Director Roadmap Initiative. This work was supported in part by grant DE017106 from the National Institute of Dental and Craniofacial Research (JI), NIH grants AI078942 (WSG) and Burroughs Wellcome Fund (WSG), and was funded by NIH 1R01HG005969 to CH.","meta":{"dup_signals":{"dup_doc_count":222,"dup_dump_count":48,"dup_details":{"curated_sources":2,"2023-40":1,"2023-23":2,"2022-49":4,"2022-33":3,"2022-27":1,"2022-05":2,"2021-43":1,"2021-31":1,"2021-25":2,"2021-17":3,"2021-10":1,"2021-04":1,"2020-45":1,"2020-34":1,"2020-24":1,"2020-05":1,"2019-51":2,"2019-43":1,"2019-39":1,"2017-09":26,"2016-44":1,"2016-40":1,"2016-36":26,"2016-30":24,"2015-48":5,"2015-40":3,"2015-35":5,"2015-32":5,"2015-27":5,"2015-22":5,"2015-14":4,"2014-52":4,"2014-49":4,"2014-42":12,"2014-41":9,"2014-35":8,"2014-23":8,"2014-15":3,"2023-50":1,"2024-26":2,"2024-18":1,"2017-13":2,"2015-18":4,"2015-11":5,"2015-06":5,"2014-10":5,"2013-48":5,"2013-20":2}},"file":"PMC3218848"},"subset":"pubmed_central"}
{"text":"date: 2017-12\ntitle: Global response to malaria at crossroads\n\n**29 NOVEMBER 2017 \\| GENEVA -** After unprecedented global success in malaria control, progress has stalled, according to the World malaria report 2017. There were an estimated 5 million more malaria cases in 2016 than in 2015. Malaria deaths stood at around 445 000, a similar number to the previous year.\n\n\"In recent years, we have made major gains in the fight against malaria,\" said Dr Tedros Adhanom Ghebreyesus, Director-General of WHO. \"We are now at a turning point. Without urgent action, we risk going backwards, and missing the global malaria targets for 2020 and beyond.\"\n\nThe WHO Global Technical Strategy for Malaria calls for reductions of at least 40% in malaria case incidence and mortality rates by the year 2020. According to WHO's latest malaria report, the world is not on track to reach these critical milestones.\n\nA major problem is insufficient funding at both domestic and international levels, resulting in major gaps in coverage of insecticide-treated nets, medicines, and other life-saving tools.\n\n**Funding shortage**\n\nAn estimated US\\$ 2.7 billion was invested in malaria control and elimination efforts globally in 2016. That is well below the US \\$6.5 billion annual investment required by 2020 to meet the 2030 targets of the WHO global malaria strategy.\n\nIn 2016, governments of endemic countries provided US\\$ 800 million, representing 31% of total funding. The United States of America was the largest international funder of malaria control programmes in 2016, providing US\\$1 billion (38% of all malaria funding), followed by other major donors, including the United Kingdom of Great Britain and Northern Ireland, France, Germany and Japan.\n\n**The global figures**\n\nThe report shows that, in 2016, there were an estimated 216 million cases of malaria in 91 countries, up from 211 million cases in 2015. The estimated global tally of malaria deaths reached 445 000 in 2016 compared to 446 000 the previous year.\n\nWhile the rate of new cases of malaria had fallen overall, since 2014 the trend has levelled off and even reversed in some regions. Malaria mortality rates followed a similar pattern.\n\nThe African Region continues to bear an estimated 90% of all malaria cases and deaths worldwide. Fifteen countries \u2013 all but one in sub-Saharan Africa \u2013 carry 80% of the global malaria burden.\n\n\"Clearly, if we are to get the global malaria response back on track, supporting the most heavily affected countries in the African Region must be the primary focus,\" said Dr Tedros.\n\n**Controlling malaria**\n\nIn most malaria-affected countries, sleeping under an insecticide-treated bednet (ITN) is the most common and most effective way to prevent infection. In 2016, an estimated 54% of people at risk of malaria in sub-Saharan Africa slept under an ITN compared to 30% in 2010. However, the rate of increase in ITN coverage has slowed since 2014, the report finds.\n\nSpraying the inside walls of homes with insecticides is another effective way to prevent malaria. The report reveals a steep drop in the number of people protected from malaria by this method \u2013 from an estimated 180 million in 2010 to 100 million in 2016 \u2013 with the largest reductions seen in the African Region.\n\nThe African Region has seen a major increase in diagnostic testing in the public health sector: from 36% of suspected cases in 2010 to 87% in 2016. A majority of patients (70%) who sought treatment for malaria in the public health sector received artemisinin-based combination therapies (ACTs) \u2013 the most effective antimalarial medicines.\n\nHowever, in many areas, access to the public health system remains low. National-level surveys in the African Region show that only about one third (34%) of children with a fever are taken to a medical provider in the public health sector.\n\nTackling malaria in complex settings\n\nThe report also outlines additional challenges in the global malaria response, including the risks posed by conflict and crises in malaria endemic zones. WHO is currently supporting malaria responses in Nigeria, South Sudan, Venezuela (Bolivarian Republic of) and Yemen, where ongoing humanitarian crises pose serious health risks. In Nigeria's Borno State, for example, WHO supported the launch of a mass antimalarial drug administration campaign this year that reached an estimated 1.2 million children aged under 5 years in targeted areas. Early results point to a reduction in malaria cases and deaths in this state.\n\n**A wake-up call**\n\n\"We are at a crossroads in the response to malaria,\" said Dr Pedro Alonso, Director of the Global Malaria Programme, commenting on the findings of this year's report. \"We hope this report serves as a wake-up call for the global health community. Meeting the global malaria targets will only be possible through greater investment and expanded coverage of core tools that prevent, diagnose and treat malaria. Robust financing for the research and development of new tools is equally critical.\"\n\nAvailable from: <http:\/\/www.who.int\/mediacentre\/news\/releases\/2017\/malaria-report-response\/en\/>","meta":{"dup_signals":{"dup_doc_count":145,"dup_dump_count":51,"dup_details":{"curated_sources":2,"2023-50":2,"2023-23":2,"2023-14":3,"2023-06":2,"2022-40":2,"2022-33":3,"2022-27":1,"2022-21":2,"2021-49":1,"2021-43":4,"2021-39":2,"2021-31":1,"2021-25":3,"2021-21":1,"2021-17":2,"2021-10":4,"2021-04":1,"2020-50":3,"2020-40":3,"2020-34":3,"2020-29":1,"2020-24":3,"2020-16":2,"2020-10":2,"2020-05":3,"2019-51":2,"2019-47":2,"2019-43":2,"2019-39":4,"2019-35":3,"2019-30":5,"2019-26":3,"2019-22":2,"2019-18":4,"2019-13":2,"2019-09":3,"2019-04":1,"2018-51":3,"2018-47":3,"2018-43":4,"2018-39":5,"2018-34":1,"2018-30":4,"2018-26":3,"2018-22":1,"2018-17":3,"2018-13":8,"2018-09":6,"2018-05":6,"2017-51":6,"2024-26":1}},"file":"PMC5787647"},"subset":"pubmed_central"}
{"text":"date: 2018\nreferences:\ntitle: An egg a day could significantly reduce CVD risk\n\nPeople who consume an egg a day could significantly reduce their risk of cardiovascular disease (CVD) compared with eating no eggs, suggests a study carried out in China. CVD is the leading cause of death and disability worldwide, including China, mostly due to ischaemic heart disease and stroke (including both haemorrhagic and ischaemic stroke).\n\nUnlike ischaemic heart disease, which is the leading cause of premature death in most Western countries, stroke is the most responsible cause in China, followed by heart disease. Although ischaemic stroke accounted for the majority of strokes, the proportion of haemorrhagic stroke in China is still higher than that in high-income countries.\n\nEggs are a prominent source of dietary cholesterol, but they also contain high-quality protein, many vitamins and bioactive components such as phospholipids and carotenoids.\n\nPrevious studies looking at associations between eating eggs and impact on health have been inconsistent, and most of them found insignificant associations between egg consumption and coronary heart disease or stroke. Therefore, a team of researchers from China and the UK led by Prof Liming Li and Dr Canqing Yu from the School of Public Health, Peking University Health Science Centre, set out to examine the associations between egg consumption and cardiovascular disease, ischaemic heart disease, major coronary events, haemorrhagic stroke and ischaemic stroke.\n\nThey used data from the China Kadoorie Biobank (CKB) study, an ongoing prospective study of around half a million (512 891) adults aged 30 to 79 years from 10 different geographical areas in China. The participants were recruited between 2004 and 2008 and were asked about the frequency of their egg consumption. They were followed up to determine their morbidity and mortality.\n\nFor the new study, the researchers focused on 416 213 participants who were free of prior cancer, CVD and diabetes. From that group at a median follow up of 8.9 years, a total of 83 977 cases of CVD and 9 985 CVD deaths were documented, as well as 5 103 major coronary events. At the start of the study period, 13.1% of participants reported daily consumption of eggs (usual amount 0.76 eggs\/day) and 9.1% reported never or very rare consumption of eggs (usual amount 0.29 eggs\/day).\n\nAnalysis of the results showed that compared with people not consuming eggs, daily egg consumption was associated with a lower risk of CVD overall. In particular, daily egg consumers (up to one egg\/day) had a 26% lower risk of haemorrhagic stroke, a 28% lower risk of haemorrhagic stroke death and an 18% lower risk of CVD death.\n\nIn addition, there was a 12% reduction in risk of ischaemic heart disease observed for people consuming eggs daily (estimated amount 5.32 eggs\/week), when compared with the 'never\/rarely' consumption category (2.03 eggs\/week).\n\nThis was an observational study, so no firm conclusions can be drawn about cause and effect, but the authors said their study had a large sample size and took into account established and potential risk factors for CVD.\n\nThe authors concluded: 'The present study finds that there is an association between moderate level of egg consumption (up to 1 egg\/day) and a lower cardiac event rate. Our findings contribute scientific evidence to the dietary guidelines with regard to egg consumption for the healthy Chinese adult.'\n\n# References","meta":{"dup_signals":{"dup_doc_count":185,"dup_dump_count":52,"dup_details":{"curated_sources":2,"2023-50":3,"2023-40":3,"2023-23":4,"2023-14":1,"2023-06":3,"2022-49":1,"2022-40":1,"2022-33":1,"2022-27":3,"2022-21":3,"2022-05":1,"2021-49":3,"2021-43":2,"2021-39":2,"2021-31":2,"2021-25":2,"2021-21":6,"2021-17":2,"2021-10":2,"2021-04":3,"2020-50":2,"2020-45":1,"2020-40":5,"2020-34":1,"2020-29":4,"2020-24":2,"2020-16":3,"2020-10":2,"2020-05":3,"2019-51":3,"2019-47":2,"2019-43":4,"2019-39":5,"2019-35":3,"2019-30":9,"2019-26":3,"2019-22":6,"2019-18":4,"2019-13":6,"2019-09":9,"2019-04":3,"2018-51":9,"2018-47":4,"2018-43":8,"2018-39":5,"2018-34":9,"2018-30":3,"2018-26":7,"2024-22":3,"2024-18":2,"2024-10":4,"2024-30":1}},"file":"PMC6107805"},"subset":"pubmed_central"}
{"text":"abstract: Plant chloroplast originated, through endosymbiosis, from a cyanobacterium, but the genomic legacy of cyanobacterial ancestry extends far beyond the chloroplast itself, and persists in organisms that have lost chloroplasts completely.\nauthor: John A Raven; John F Allen\ndate: 2003\ninstitute: 1Division of Environmental and Applied Biology, University of Dundee, Dundee DD1 4HN, UK; 2Department of Plant Biochemistry, Center for Chemistry and Chemical Engineering, Box 124, Lund University, SE-221 00 Lund, Sweden; Correspondence: John A Raven. E-mail: email@example.com\nreferences:\ntitle: Genomics and chloroplast evolution: what did cyanobacteria do for plants?\n\n# The endosymbiont hypothesis is mainstream\n\nChloroplasts, the sites of photosynthesis within plant cells, comprise a prominent and well-known class of plastids, subcellular organelles with diverse, specialist functions in plant and algal cells. Mereschkowsky \\[1,2\\] is widely recognized as having written the first clear exposition of the hypothesis that plastids are derived from endosymbiotic cyanobacteria, then known as blue-green algae. Initially greeted with skepticism or even derision, Mereschkowsky's 1905 hypothesis gained support from electron microscopical and biochemical studies which showed that plastids contain DNA, RNA and ribosomes, supplying a structural and biochemical basis for non-Mendelian, cytoplasmic inheritance of plastid-related characters \\[3\\]. Subsequent molecular genetic studies have demonstrated the ubiquity of plastid genomes and confirmed that their replication, transcription and translation closely resemble those of (eu)bacteria.\n\nMolecular phylogenetic studies now make it abundantly clear that the closest bacterial homologs of plastids are indeed cyanobacteria \\[4\\], supporting earlier conclusions from the comparative biochemistry of photosynthesis. Only cyanobacteria and chloroplasts have two photosystems and split water, to make oxygen, as a source of reducing power. But it has long been clear that many of the proteins needed for plastid functions, including photosynthesis, are now encoded in the nuclear genome and arrived there during evolution by the wholesale uptake of cyanobacteria, including their genomes, followed by gene transfer into the nucleus \\[5\\]. Recent advances in genomics have greatly enhanced our understanding of the evolution of plastids, allowing us to address specific questions such as which genes were moved or retained, and why. It also becomes possible to see clearly the algal ancestry of cells that have vestigial and otherwise unrecognizable plastids, and even to discern the unmistakable genomic footprint of plastids long lost from organisms one might never imagine to have descended from plants.\n\nMolecular genetic studies of plastid genomes show that they encode only 60-200 proteins, while perhaps as many as 5,000 nuclear-coded gene products are targeted to plastids \\[6\\]. From complete sequences it is known that each cyanobacterial genome codes for at least 1,500 proteins, and they are therefore at least an order of magnitude larger than plastid genomes. It is perhaps surprising that the size of the proteome of a free-living cyanobacterium is not greatly different from that of a subcellular organelle. Genomic studies have been very important in showing the evolutionary fate of the cyanobacterial genes that originated from the endosymbiotic pre-plastids. The genes in pre-plastids were either retained, lost, or transferred to the nucleus. The process of transfer of genes to the nucleus would have involved duplication of each plastid gene, and a nuclear copy of the gene becoming able to produce a functional product in the cytosol or, with appropriate targeting sequences, in other compartments.\n\n# The fates of endosymbiont genes\n\nAn important recent analysis by Martin *et al.* \\[6\\] has put limits on the number of genes in the nucleus of *Arabidopsis thaliana* that derive from the plastid ancestor. Previous analyses of limited portions of the *A. thaliana* nuclear genome suggested that 800-2000 genes from the plastid ancestor were transferred to the nucleus. The analysis by Martin *et al.* \\[6\\] was based on comparison of the whole nuclear genome of *A. thaliana* with whole genomes of three cyanobacteria (*Nostoc punctiforme, Prochlorococcus marinus* and *Synechocystis* sp. PCC 6803), 16 other prokaryotes, and *Saccharomyces cerevisiae* (yeast). The analysis was restricted to the 9,368 *A. thaliana* gene products that are sufficiently conserved for the comparison of primary sequences. Of these, the greatest number of similarities were detected with the yeast nuclear genome; these common genes were presumably inherited by *Arabidopsis* from the host cell that acquired the plastid(s) \\[6\\]. The second most numerous class of genes in the *Arabidopsis* nuclear genome are those directly homologous to cyanobacterial genes. A decreasing number of similarities is found in Gram-positive bacteria, non-proteobacterial Gram-negative bacteria, proteobacteria, and least of all in archaebacteria \\[6\\]. Extrapolating the data from the 9,368 conserved proteins to the total of 24,990 non-redundant nuclear genes of *Arabidopsis* gives a total of some 4,500 genes, or 18% of the nuclear genes, that came from the cyanobacterial ancestor of the plastids. More than half of these are not targeted back to the plastids but to other cell compartments (including the secretory pathway) \\[6\\]. The protein products of many nuclear genes that were not acquired from the plastid ancestor are now targeted to the plastid. The genes within the nuclear genomes that originated from the plastid ancestor cover all of the functional categories defined by The *Arabidopsis* Genome Initiative \\[7\\].\n\nThe cyanobacterial ancestor of the plastids was, relative to the three cyanobacteria with completed genome sequences that were examined by Martin *et al.* \\[6\\], closer to *N. punctiforme* than to *P. marinus* or *Synechocystis* sp. Although three genomes is not a large sample size, it is of interest that *N. punctiforme* is a diazotroph, so the plastid ancestor could also have been a nitrogen-fixer. Were early plastids perhaps also able to fix atmospheric nitrogen?\n\n# Losing chloroplasts but keeping cyanobacterial genes\n\nThe work of Brinkman *et al.* \\[8\\] re-examines the processes that have led to the high proportion of proteins of a bacterial human pathogen, *Chlamydia*, that are similar to those of plants. This similarity was formerly attributed to horizontal gene transfer from plants, or plant-like host organisms, to the bacterium. Brinkman *et al.* \\[8\\] point out that such gene transfer is unlikely since all extant Chlamydiaceae are obligate intracellular parasites of animals. Instead, the analysis by Brinkman *et al.* \\[8\\] shows that the majority of the plant-like genes in *Chlamydia* are, in plant cells, targeted to the chloroplast. But the conclusion that this targeting of proteins to chloroplasts is necessarily a function of their origin from a plastid ancestor is not always sound. Furthermore, Martin *et al.* \\[6\\] did not find much similarity between *Chlamydia* and *Arabidopsis* (see Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a> in \\[6\\]). Clearly, further investigation is needed.\n\nFigure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a> illustrates the various endosymbiotic events described here. Amongst eukaryotes, the apicomplexan parasitic pathogens *Toxoplasma* and *Plasmodium* have curious cytoplasmic organelles bounded by three membranes, namely 'apicoplasts', which genome sequencing has established as *bona fide* plastids complete with a characteristic inverted repeat within the plastid genome \\[9\\]. The presence of three membranes, as is found around chloroplasts of dinoflagellates and euglenoids, betrays an ancestry from a secondary symbiosis, as does the presence of four membranes surrounding the plastids of, for example, photosynthetic heterokonts (a diverse group, some of which are algae) such as diatoms and brown algae. The function of the apicoplast is not clearly understood, but one suggestion is that it is indispensable for the synthesis of iron-sulfur proteins. The function of the residual plastid genome is even less clear, and it provides a test case for any theory for the function of organellar genes. Although *Plasmodium* has a plastid genome that some think is on the way out, trypanosomes, which are also non-photosynthetic, have no plastid or plastid genome at all, but are now clearly seen to be former euglenoids because of the remaining genes for a variety of plant-like enzymes, including sedoheptulose-1,7-bisphosphatase (otherwise found only in the Benson-Calvin cycle) \\[10,11\\].\n\n# *Arabidopsis* is not the only plant\n\nThe article by Martin *et al.* \\[6\\] uses chloroplast genomics to infer plastid phylogeny, as well as gene loss and gene transfer, for 16 sequenced plastid genomes. An important conclusion from this analysis is that two secondary endosymbiotic events involving a red alga are needed to explain the occurrence of plastids in cryptophytes (algae with phycobilin pigments in the thylakoid lumen rather than in particles on the thylakoid membrane as in cyanobacterial and red algae; an example is *Guillardia*) and heterokonts (the diatom *Odontella*). This contrasts with the arguments of Cavalier-Smith (recently set out in \\[12\\]) for a single endosymbiotic event, based on evidence such as the replacement of the glyceraldehyde-3-phosphate dehydrogenase gene derived from the red algal plastid with one of host origin in both cases.\n\nAnother recent article \\[13\\] deals with genome-based phylogenies of plastids; 19 complete chloroplast genomes are studied using a new computational method, and broadly similar conclusions are reached to those of Martin and co-workers \\[6\\]. This work also allows novel functional assignments to a number of chloroplast open reading frames. The functional implications of chloroplast genomics, with special reference to experimental opportunities and 'directional genetics' in *Arabidopsis thaliana*, have recently been reviewed by Leister \\[14\\].\n\nAn important question relating to the evolution of plastid genomes in higher plants is the timing of the changes in the plastid genome in the streptophyte clade (made up of charophytes, a group of green algae or chlorophytes, plus embryophytes, or higher plants), which evolved more than 500 million years ago. From the unicellular flagellate *Mesostigma*, which is either a basal chlorophyte or lies at the split between Chlorophyta and Streptophyta, to the embryophytes, of which the liverwort *Marchantia* is the most basal to have been sequenced, the changes are gene losses, including transfer to the nucleus, scrambling of gene order, and intron insertion \\[15\\].\n\nAn important contribution to bridging the evolutionary gap between *Mesostigma* and *Marchantia* is the work of Turmel *et al.* \\[15\\] on a member of the charophytes *sensu stricto* (that is, excluding *Mesostigma*) *Chaetosphaeridium globosum*. Before the work of Turmel *et al.* \\[15\\] only fragmentary data addressed the issue of gene content and organization of the eight charophytes *sensu stricto*. The complete plastid genome sequence of *Chaetosphaeridium globosum* \\[15\\] shows that most of the embryophyte characteristics were present in the charophyte alga, so that the major changes had occurred between the branch to *Mesostigma* and that to *Chaetosphaeridium.* The common features shared by the plastid DNA of *Chaetosphaeridium* and of embryophytes include the gene content, the intron composition, and the gene order. Thus, the *Chaetosphaeridium* chloroplast genome has 124 genes (compared to 136 in *Mesostigma* and 110-120 in embryophytes), one Group I intron (there are none in *Mesostigma* and one in embryophytes), 16 *cis*-spliced Group II introns (none in *Mesostigma* and 18-19 in embryophytes) and one *trans*-spliced Group II intron (none in *Mesostigma*, one in embryophytes). Genome size (118-155 kilobases) is relatively constant among *Mesostigma, Chaetosphaeridium* and higher plant plastids. By contrast, the mitochondrial genome of *Chaetosphaeridium* is closely similar to that of *Mesostigma* in terms of size (57 kb and 42 kb, respectively), gene content and, perhaps, intron content. *Chaetosphaeridium* has a much smaller genome size than the obese mitochondrial genomes of *Marchantia* (187 kb) or *Arabidopsis* (367 kb), and many more *cis*-spliced Group II introns (18-25 rather than two). The apparently different tempo of evolution in mitochondria and plastids of the charophytes deserves further investigation. An important point about the functional genomics of the plastid is the determinant of which genes essential for plastid function are retained in the plastid genome. Higher plant plastid genomes have slightly fewer genes than in the plastids of the charophytes *sensu lato* (that is, the charophytes *sensu stricto* plus *Mesostigma*).\n\n# Cells within cells\n\nOne requirement of the endosymbiont hypothesis is whole-scale gene transfer from the chloroplast to the nucleus. Long thought to be either impossible or, at best, highly problematical, its difficulties are often thought to relate to the failure of some genes to move at all. Gene transfer from chloroplast to nucleus is now estimated to occur naturally in tobacco at a frequency of one transposition in 16,000 pollen grains \\[16\\]. In natural populations and over evolutionary time, this frequency represents a massive informational onslaught and highlights the urgency of the question of why chloroplasts have genomes at all. There must be some crucial, over-riding, selective advantage in retaining certain genes in chloroplasts but not others. Evidence is now accruing for the ten-year-old proposal that gene expression in the chloroplast is regulated by the function of a core of chloroplast gene products in photosynthesis and electron transport \\[17,18\\].\n\nIt is clear that genomics, in the sense of whole-genome analyses, is making very important contributions to our understanding of the evolution of plastids, and is complementing, and to a significant extent supplanting, 'single gene' phylogenies. Genomics is revolutionizing our understanding of the changes involved in the primary endosymbiosis that produced the plastids of red, green and glaucophyte algae, and in the subsequent genetic changes in green (charophycean) plastids with the evolution of higher plants. Genomics is also indispensable for understanding how red and green algae yielded the plastids derived from secondary endosymbiosis.\n\nThe endosymbiont hypothesis took a long time to graduate from wild and untestable speculation to an accepted view of plastid origins and evolution. In contrast, comparative genomics has quickly elevated the kinship of chloroplasts and cyanobacteria to a keystone of our understanding of the most abundant of cells, the primary producers on which life now depends, not to mention some vicious and enterprising pathogens whose exploits are a global burden to human health. The title of this article asks what the cyanobacteria have done for plants. \"What have they not done?\" is a question perhaps more easily addressed.\n\n### Acknowledgements\n\nWe thank W. Martin for comments and NERC (JAR) and NFR (JFA) for related research grants.\n\n## Figures and Tables","meta":{"dup_signals":{"dup_doc_count":139,"dup_dump_count":49,"dup_details":{"curated_sources":4,"2023-23":1,"2022-21":1,"2019-30":1,"2019-18":1,"2019-09":1,"2019-04":1,"2018-51":1,"2018-47":1,"2018-43":1,"2018-39":1,"2018-34":2,"2018-17":1,"2017-51":1,"2017-39":1,"2017-34":1,"2017-26":1,"2017-17":1,"2017-09":10,"2017-04":1,"2016-44":1,"2016-40":1,"2016-36":11,"2016-30":9,"2016-22":1,"2016-18":1,"2016-07":4,"2015-48":3,"2015-40":1,"2015-35":2,"2015-32":4,"2015-27":3,"2015-22":4,"2015-14":2,"2014-52":3,"2014-49":4,"2014-42":8,"2014-41":4,"2014-35":6,"2014-23":7,"2014-15":3,"2023-40":1,"2017-13":1,"2015-18":4,"2015-11":3,"2015-06":4,"2014-10":3,"2013-48":4,"2013-20":3,"2024-18":1}},"file":"PMC153454"},"subset":"pubmed_central"}
{"text":"author: Melissa Hawkins\ndate: 2020-08-13\ntitle: Quarantine Bubbles \u2013 When Done Right \u2013 Limit Coronavirus Risk and Help Fight Loneliness\n\nAfter three months of lockdowns, many people in the U.S. and around the world are turning to quarantine bubbles, pandemic pods or quaranteams in an effort to balance the risks of the pandemic with the emotional and social needs of life.\n\nI am an epidemiologist and a mother of four, three of whom are teenagers in the throes of their risk-taking years. As the country grapples with how to navigate new risks in the world, my kids and I are doing the same.\n\nWhen done carefully, the research shows that quarantine bubbles can effectively limit the risk of contracting SARS-CoV-2 while allowing people to have much needed social interactions with their friends and family.\n\nQuaranteams are founded on the idea that people can interact freely within a group, but that group stays isolated from other people as much as possible.\n\n# Reduce risk if you can't eliminate it\n\nA quaranteam is a small group of people who form their own social circle to quarantine together \u2013 and a perfect example of a harm reduction strategy.\n\nHarm reduction is a pragmatic public health concept that explicitly acknowledges that all risk cannot be eliminated, so it encourages the reduction of risk. Harm reduction approaches also take into consideration the intersection of biological, psychological and social factors that influence both health and behavior.\n\nFor example, abstinence-only education doesn't work all that well. Safe-sex education, on the other hand, seeks to limit risk, not eliminate it, and is better at reducing teen pregnancy and sexually transmitted infection.\n\nQuarantine bubbles are a way to limit the risk of getting or transmitting SARS-CoV-2 while expanding social interaction.\n\n# Mental health matters too\n\nStaying indoors, avoiding all contact with friends or family and having food and groceries delivered would be the best way to limit your risk of catching SARS-CoV-2. But the risks of the pandemic extend beyond the harm from infection. Health encompasses mental as well as physical well-being.\n\nThe negative mental health impacts of the pandemic are already starting to become evident. A recent survey of U.S. adults found that 13.6% reported symptoms of serious psychological distress, up from 3.9% in 2018. A quarter of people 18 to 29 years old reported serious psychological distress, the highest levels of all ages groups. Many people are experiencing anxiety and depression due to the pandemic or were already living with these challenges. Loneliness certainly doesn't help.\n\nLoneliness and social isolation increase the risk for depression and anxiety and can also lead to increases in the risk for serious physical diseases like coronary heart disease, stroke and premature death.\n\nQuaranteams, therefore, are not simply a convenient idea because they let people see their friends and family. Isolation poses serious health risks \u2013 both physically and mentally \u2013 that social bubbles can help alleviate while improving social well-being and quality of life.\n\nManaging a virus is all about managing human interactions, and quarantine bubbles work to insulate groups from risk.\n\n# Social network theory shows that quaranteams work\n\nSocial relationships enhance well-being and mental health but they also act as a vehicle for infection transmission. As people around the world emerge from lockdowns, this is the conundrum: How do we increase social interaction while limiting the risk of spread?\n\nA recent study used social network theory \u2013 how information spreads among groups of people \u2013 and infectious disease models to see if quaranteams would work in this pandemic.\n\nTo do that, the researchers built computer models of social interactions to measure how the virus spread. They built a model of typical behavior, of typical behavior but with only half the number of interactions and of three different social distancing approaches that also had half the number of interactions as normal.\n\nThe first social distancing scenario grouped people by characteristics \u2013 people would only see people of a similar age, for example. The second scenario grouped people by local communities and limited inter-community interaction. The last scenario limited interactions to small social groups of mixed characteristics from various locations \u2013 i.e. quarantine bubbles. These bubbles could have people of all ages and from various neighborhoods, but those people would only interact with each other.\n\nAll of the social distancing measures reduced the severity of the pandemic and were also better than simply reducing interactions at random, but the quaranteam approach was the most effective at flattening the curve. Compared to no social distancing, quarantine bubbles would delay the peak of infections by 37%, decrease the height of the peak by 60% and result in 30% fewer infected individuals overall.\n\nOther countries are starting to incorporate quaranteams in their prevention guidelines now that infection rates are low and contact tracing programs are in place. England is the latest country to announce quaranteam guidance with their support bubble policy.\n\nNew Zealand implemented a quarantine bubble strategy in early May and it seems to have worked. Additionally, a recent survey of 2,500 adults in England and New Zealand found a high degree of support for the policies and high degree of motivation to comply.\n\nPeople in a quarantine bubble need to agree on how much risk is acceptable and establish a set of rules.\n\n# How to build a quarantine bubble\n\nTo make an effective quaranteam, here's what you need to do.\n\nFirst, everyone must agree to follow the rules and be honest and open about their actions. Individual behavior can put the whole team at risk and the foundation of a quaranteam is trust. Teams should also talk in advance about what to do if someone breaks the rules or is exposed to an infected person. If someone starts to show symptoms, everyone should agree to self-isolate for 14 days.\n\nSecond, everyone must decide how much risk is acceptable and establish rules that reflect this decision. For example, some people might feel OK about having a close family member visit but others may not. Our family has agreed that we only visit with friends outside, not inside, and that everyone must wear masks at all times.\n\nFinally, people need to actually follow the rules, comply with physical distancing outside of the quaranteam and be forthcoming if they think they may have been exposed.\n\nAdditionally, communication should be ongoing and dynamic. The realities of the pandemic are changing at a rapid pace and what may be OK one day might be too risky for some the next.\n\n# The risks of joining a quaranteam\n\nAny increase in social contact is inherently more risky right now. There are two important ideas in particular that a person should consider when thinking about how much risk they're willing to take.\n\nYou must be smart and honest when determining how much risk you're willing to take and who is affected.\n\nThe first is asymptomatic spread. Current data suggests that at any given time, anywhere between 20% and 45% of people infected with SARS-CoV-2 are asymptomatic or pre-symptomatic and able to transmit the virus to others. The best way to know if someone is infected or not is to get tested, so some people might consider requiring testing before agreeing to join a quaranteam.\n\nThe second thing to consider is that consequences of getting sick are not the same for everyone. If you or someone you live with has another health condition \u2013 like asthma, diabetes, a heart condition or a compromised immune system \u2013 the assessment of risk and reward from a quaranteam should change. The consequences of a high-risk person developing COVID-19 are much more serious.\n\nOne of the greatest difficulties facing both scientists and the public alike is the uncertainty about this virus and what lies ahead. But some things are known. If individuals are informed and sincere in their quaranteam efforts and follow the regular guidance of social distancing, mask wearing and enthusiastic hand-washing, quaranteams can offer a robust and structured middle ground approach to manage risk while experiencing the joy and benefits of friends and family. These are things we could all benefit from these days, and for now, quaranteams may be the best step forward as we emerge from this pandemic together.\n\nThis article is republished from The Conversation under a Creative Commons license. Read the original article.","meta":{"dup_signals":{"dup_doc_count":171,"dup_dump_count":30,"dup_details":{"curated_sources":3,"2023-50":3,"2023-40":4,"2023-23":6,"2023-14":4,"2023-06":4,"2022-49":4,"2022-40":3,"2022-33":4,"2022-27":4,"2022-21":8,"2022-05":3,"2021-49":6,"2021-43":4,"2021-39":7,"2021-31":7,"2021-25":6,"2021-21":3,"2021-17":8,"2021-10":5,"2021-04":6,"2020-50":7,"2020-45":11,"2020-40":8,"2020-34":8,"2020-29":17,"2024-30":3,"2024-26":5,"2024-22":4,"2024-18":1,"2024-10":5}},"file":"PMC8389109"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  Conventional systematic review techniques have limitations when the aim of a review is to construct a critical analysis of a complex body of literature. This article offers a reflexive account of an attempt to conduct an interpretive review of the literature on access to healthcare by vulnerable groups in the UK\n  .\n  # Methods\n  .\n  This project involved the development and use of the method of Critical Interpretive Synthesis (CIS). This approach is sensitised to the processes of conventional systematic review methodology and draws on recent advances in methods for interpretive synthesis.\n  .\n  # Results\n  .\n  Many analyses of equity of access have rested on measures of utilisation of health services, but these are problematic both methodologically and conceptually. A more useful means of understanding access is offered by the synthetic construct of candidacy. Candidacy describes how people's eligibility for healthcare is determined between themselves and health services. It is a continually negotiated property of individuals, subject to multiple influences arising both from people and their social contexts and from macro-level influences on allocation of resources and configuration of services. Health services are continually constituting and seeking to define the appropriate objects of medical attention and intervention, while at the same time people are engaged in constituting and defining what they understand to be the appropriate objects of medical attention and intervention. Access represents a dynamic interplay between these simultaneous, iterative and mutually reinforcing processes. By attending to how vulnerabilities arise in relation to candidacy, the phenomenon of access can be better understood, and more appropriate recommendations made for policy, practice and future research.\n  .\n  # Discussion\n  .\n  By innovating with existing methods for interpretive synthesis, it was possible to produce not only new methods for conducting what we have termed critical interpretive synthesis, but also a new theoretical conceptualisation of access to healthcare. This theoretical account of access is distinct from models already extant in the literature, and is the result of combining diverse constructs and evidence into a coherent whole. Both the method and the model should be evaluated in other contexts.\nauthor: Mary Dixon-Woods; Debbie Cavers; Shona Agarwal; Ellen Annandale; Antony Arthur; Janet Harvey; Ron Hsu; Savita Katbamna; Richard Olsen; Lucy Smith; Richard Riley; Alex J Sutton\ndate: 2006\ninstitute: 1Department of Health Sciences, University of Leicester, 22\u201328 Princess Road West, Leicester LE1 6TP, UK; 2Division of Oncology\/General Practice, University of Edinburgh, Edinburgh Centre for Neuro-Oncology, Western General Hospital, Crewe Road South, Edinburgh EH4 2XU, UK; 3Department of Health Sciences, University of Leicester, Leicester General Hospital, Leicester LE5 4PW, UK; 4Department of Sociology, University of Leicester, Leicester LE1 7RH, UK; 5School of Nursing, University of Nottingham, Queens Medical Centre, Nottingham NG7 2HA, UK; 6Centre for Research in Social Policy, Loughborough University, Leicestershire LE11 3TU, UK; 7Nuffield Research Unit, Department of Health Sciences, University of Leicester, 22\u201328 Princess Road West, Leicester LE1 6TP, UK\nreferences:\ntitle: Conducting a critical interpretive synthesis of the literature on access to healthcare by vulnerable groups\n\n# Background\n\nLike many areas of healthcare practice and policy, the literature on access to healthcare is large, diverse, and complex. It includes empirical work using both qualitative and quantitative methods; editorial comment and theoretical work; case studies; evaluative, epidemiological, trial, descriptive, sociological, psychological, management, and economics papers, as well as policy documents and political statements. \"Access\" itself has not been consistently defined or operationalised across the field. There are substantial adjunct literatures, including those on quality in healthcare, priority-setting, and patient satisfaction. A review of the area would be of most benefit if it were to produce a \"mid-range\" theoretical account of the evidence and existing theory that is neither so abstract that it lacks empirical applicability nor so specific that its explanatory scope is limited.\n\nIn this paper, we suggest that conventional systematic review methodology is ill-suited to the challenges that conducting such a review would pose, and describe the development of a new form of review which we term \"Critical Interpretive Synthesis\" (CIS). This approach draws is sensitised to the range of issues involved in conducting reviews that conventional systematic review methodology has identified, but draws on a distinctive tradition of qualitative inquiry, including recent interpretive approaches to review \\[1\\]. We suggest that that using CIS to synthesise a diverse body of evidence enables the generation of theory with strong explanatory power. We illustrate this briefly using an example based on synthesis of the literature on access to healthcare in the UK by socio-economically disadvantaged people.\n\n## Aggregative and interpretive reviews\n\nConventional systematic review developed as a specific methodology for searching for, appraising, and synthesising findings of primary studies \\[2\\]. It offers a way of systematising, rationalising, and making more explicit the processes of review, and has demonstrated considerable benefits in synthesising certain forms of evidence where the aim is to *test* theories, perhaps especially about \"what works\". It is more limited when the aim, as here, is to include many different forms of evidence with the aim of *generating* theory \\[3\\]. Conventional systematic review methods are thus better suited to the production of *aggregative* rather than *interpretive* syntheses.\n\nThis distinction between aggregative and interpretive syntheses, noted by Noblit and Hare in their ground-breaking book on meta-ethnography\\[1\\] allows a useful (though necessarily crude) categorisation of two principal approaches to conducting reviews \\[4\\]. Aggregative reviews are concerned with assembling and pooling data, may use techniques such as meta-analysis, and require a basic comparability between phenomena so that the data can be *aggregated* for analysis. Their defining characteristics are a focus on *summarising data*, and an assumption that the concepts (or variables) under which those data are to be summarised are largely secure and well specified. Key concepts are defined at an early stage in the review and form the categories under which the data from empirical studies are to be summarised.\n\n*Interpretive* reviews, by contrast, see the essential tasks of synthesis as involving both induction and interpretation. Their primary concern is with the development of concepts and theories that integrate those concepts. An interpretive review will therefore avoid specifying concepts in advance of the synthesis. The interpretive analysis that yields the synthesis is conceptual in process and output. The product of the synthesis is not aggregations of data, but theory grounded in the studies included in the review. Although there is a tendency at present to conduct interpretive synthesis only of qualitative studies, it should in principle be possible and indeed desirable to conduct interpretive syntheses of all forms of evidence, since theory-building need not be based only on one form of evidence. Indeed, Glaser and Strauss \\[5\\] in their seminal text, included an (often forgotten) chapter on the use of quantitative data for theory-building.\n\nRecent years have seen the emergence of a range of methods that draw on a more interpretive tradition, but these also have limitations when attempting a synthesis of a large and complex body of evidence. In general, the use to date of interpretive approaches to synthesis has been confined to the synthesis of qualitative research only \\[6-8\\]. Meta-ethnography, an approach in which there has been recent significant activity and innovation, has similarly been used solely to synthesise qualitative studies, and has typically been used only with small samples \\[9-11\\]. Few approaches have attempted to apply an interpretive approach to the whole corpus of evidence (regardless of study type) included in a review, and few have treated the literature they examine as itself an object of scrutiny, for example by questioning the ways in which the literature constructs its problematics, the nature of the assumptions on the literature draw, or what has influenced proposed solutions.\n\nIn this paper we offer a reflexive account of our attempt to conduct an interpretive synthesis of all types of evidence relevant to access to National Health Service (NHS) healthcare in the UK by potentially vulnerable groups. These groups had been defined at the outset by the funders of the project (the UK Department of Health Service Delivery and Organisation R&D Programme) as children, older people, members of minority ethnicities, men\/women, and socio-economically disadvantaged people. We explain in particular our development of Critical Interpretive Synthesis as a method for conducting this review.\n\n# Methods\n\n## Formulating the review question\n\nConventional systematic review methodology \\[12,13\\] emphasises the need for review questions to be precisely formulated. A tightly focused research question allows the parameters of the review to be identified and the study selection criteria to be defined in advance, and in turn limits the amount of evidence required to address the review question.\n\nThis strategy is successful where the phenomenon of interest, the populations, interventions, and outcomes are all well specified \u2013 i.e. if the aim of the review is *aggregative*. For our project, it was neither possible nor desirable to specify in advance the precise review question, *a priori* definitions, or categories under which the data could be summarised, since one of its aims was to allow the definition of the phenomenon of access to emerge from our analysis of the literature \\[14\\]. This is not to say that we did not have a review question, only that it was not a specific hypothesis. Instead it was, as Greenhalgh and colleagues \\[15\\] describe, tentative, fuzzy and contested at the outset of the project. It did include a focus on equity and on how access, particularly for potentially vulnerable groups, can best be understood in the NHS, a health care system that is, unlike most in the world, free at the point of use.\n\nThe approach we used to further specify the review question was highly iterative, modifying the question in response to search results and findings from retrieved items. It treated, as Eakin and Mykhalovskiy \\[16\\] suggest, the question as a compass rather than an anchor, and as something that would not finally be settled until the end of the review. In the process of refining the question, we benefited from the multidisciplinary nature of our review team: this allowed a range of perspectives to be incorporated into the process, something that was also helpful and important in other elements of the review.\n\n## Searching the literature\n\nA defining characteristic of conventional systematic review methodology is its use of explicit searching strategies, and its requirement that reviewers be able to give a clear account of how they searched for relevant evidence, such that the search methods can be reproduced. \\[2\\] Searching normally involves a range of strategies, but relies heavily on electronic bibliographic databases.\n\nWe piloted the use of a highly structured search strategy using protocol-driven searches across a range of electronic databases but, like Greenhalgh and Peacock \\[17\\] found that was this unsatisfactory. In particular, it risked missing relevant materials by failing to pick up papers that, while not ostensibly about \"access\", were nonetheless important to the aim of the review. We then developed a more organic process that fitted better with the emergent and exploratory nature of the review questions. This combined a number of strategies, including searching of electronic databases; searching websites; reference chaining; and contacts with experts. Crucially, we also used expertise within the team to identify relevant literature from adjacent fields not immediately or obviously relevant to the question of \"access\".\n\nHowever, searching generated thousands of potentially relevant items \u2013 at one stage over 100,000 records. A literature of this size would clearly be unmanageable, and well exceed the capacity of the review team. We therefore redefined the aim of the searching phase. Rather than aiming for comprehensive identification and inclusion of all relevant literature, as would be required under conventional systematic review methodology, we saw the purpose of the searching phase as identifying potentially relevant papers to provide a sampling frame. Our sampling frame eventually totalled approximately 1,200 records.\n\n## Sampling\n\nConventional systematic review methodology limits the number of papers to be included in a review by having tightly specified inclusion criteria for papers. Effectively, this strategy constructs the field to be known as having specific boundaries, defined as research that has specifically addressed the review question, used particular study designs and fulfilled the procedural requirements for the proper execution of these. Interpretive reviews might construct the field to be known rather differently, seeing the boundaries as more diffuse and ill-defined, as potentially overlapping with other fields, and as shifting as the review progresses. Nonetheless, there is a need to limit the number of papers to be included in an interpretive synthesis not least for practical reasons, including the time available. Sampling is also warranted theoretically, in that the focus in interpretive synthesis is on the development of concepts and theory rather than on exhaustive summary of all data. A number of authors \\[18-20\\] suggest drawing on the sampling techniques of primary qualitative research, including principles of theoretical sampling and theoretical saturation, when conducting a synthesis of qualitative literature.\n\nFor purposes of our synthesis, we used purposive sampling initially to select papers that were clearly concerned with aspects of access to healthcare, partly informed by an earlier scoping study \\[21\\] and later used theoretical sampling to add, test and elaborate the emerging analysis. Sampling therefore involved a constant dialectic process conducted concurrently with theory generation.\n\n## Determination of quality\n\nConventional systematic review methodology uses assessment of study quality in a number of ways. First, as indicated above, studies included in a review may be limited to particular study designs, often using a \"hierarchy of evidence\" approach that sees some designs (e.g. randomized controlled trials) as being more robust than others (e.g. case-control studies). Second, it is usual to devise broad inclusion criteria \u2013 for example adequate randomisation for RCTs \u2013 and to exclude studies that fail to meet these. Third, an appraisal of included studies, perhaps using a structured quality checklist, may be undertaken to allow sensitivity analyses aimed at assessing the effects of weaker papers.\n\nUsing this approach when confronted with a complex literature, including qualitative research, poses several challenges. No hierarchy of study designs exists for qualitative research. How or whether to appraise papers for inclusion in an interpretive reviews has received a great deal of attention, but there is little sign of an emergent consensus \\[22\\]. Some argue that formal appraisals of quality may not be necessary, and some argue that there is a risk of discounting important studies for the sake of \"surface mistakes\" \\[23\\]. Others propose that weak papers should be excluded from the review altogether, and several published syntheses of qualitative research have indeed used quality criteria to make decisions about excluding papers. \\[10,24\\]\n\nWe aimed to prioritise papers that appeared to be relevant, rather than particular study types or papers that met particular methodological standards. We might therefore be said to be prioritising \"signal\" (likely relevance) over \"noise\" (the inverse of methodological quality) \\[25\\]. We felt it important, for purposes of an interpretive review, that a low threshold be applied to maximise the inclusion and contribution of a wide variety of papers at the level of *concepts*. We therefore took a two-pronged approach to quality. First, we decided that only papers that were deemed to be fatally flawed would be excluded. Second, once in the review, the synthesis itself crucially involved judgements and interpretations of credibility and contribution, as we discuss later.\n\nTo identify fatally flawed papers, we used the criteria in Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>, adapted from those proposed (at the time of our review) by the National Health Service (NHS) National Electronic Library for Health for the evaluation of qualitative research, to inform judgements on the quality of the papers. These criteria were used for assessing *all* empirical papers (but not those classified as 'reviews') regardless of study type. The final judgement about inclusion of the review rested both on an assessment of relevance as well as on the assessment of the quality of the individual papers. Decisions about relevance and quality were recorded, and a small sample of decisions about relevance and quality was reviewed. In the event, very few papers \u2013 approximately 20 \u2013 were excluded on grounds of being \"fatally flawed\", because even weak papers were often judged to have potentially high relevance. The value of deferring judgements of credibility and contribution until the synthesis became increasingly evident.\n\nAppraisal prompts for informing judgements about quality of papers\n\n<table>\n<tbody>\n<tr>\n<td style=\"text-align: left;\">Are the aims and objectives of the research clearly stated?<br \/>\nIs the research design clearly specified and appropriate for the aims and objectives of the research?<br \/>\nDo the researchers provide a clear account of the process by which their findings we reproduced?<br \/>\nDo the researchers display enough data to support their interpretations and conclusions?<br \/>\nIs the method of analysis appropriate and adequately explicated?<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\nMost fundamentally, as the review progressed, we became increasingly convinced that the assumption that all studies deemed to have satisfactorily fulfilled criteria of execution and reporting can contribute equally to a synthesis is flawed. As we discuss further below, one of the distinctive characteristics of a *critical* interpretive synthesis is its emphasis not only on summary of data reported in the literature but also on a more fundamental critique, which may involve questioning taken-for-granted assumptions.\n\n## Data extraction\n\nA data-extraction pro-forma was initially devised to assist in systematically identifying characteristics of research participants, methods of data collection, methods of data analysis and major findings of each paper. For both qualitative and quantitative papers, this involved extracting the titles of the categories and sub-categories using the terms used in the paper itself and a summary of the relevant material. Practically, however, it proved impossible to conduct this form of data extraction on all documents included in the review, including very large documents. We therefore summarised some documents more informally, for example using highlighter pen. More generally, the value of formal data extraction for purposes of this type of study will require further evaluation.\n\n## Conducting an interpretive synthesis\n\nWe had intended, at the outset of this project, to use meta-ethnography, a method for interpretive synthesis where there is currently an active programme of methodological research, \\[9-11\\] as our approach to synthesis. However, this had previously only been used to synthesise qualitative studies. Our experiences of working with a large sample of papers using multiple methods led us to refine and respecify some of the concepts and techniques of meta-ethnography in order to enable synthesis of a very large and methodologically diverse literature. Eventually we had made so many amendments and additions to the original methodology that we felt it was more appropriate, helpful and informative to deem it a new methodology with its own title and processes. It is this approach which we term critical interpretive synthesis (CIS). It is important to emphasise, however, that CIS is an approach to review and is not solely a method for synthesis.\n\nMeta-ethnography, as originally proposed \\[1\\], involves three major strategies.:\n\n1\\. **Reciprocal translational analysis** (RTA). The key metaphors, themes, or concepts in each study report are identified. An attempt is then made to translate the concepts into each other. Judgements about the ability of the concept of one study to capture concepts from others are based on attributes of the themes themselves, and the concept that is \"most adequate\" is chosen.\n\n2\\. **Refutational synthesis.** Contradictions between the study reports are characterised, and an attempt made to explain them.\n\n3\\. **Lines-of-argument synthesis** (LOA) involves building a general interpretation grounded in the findings of the separate studies. The themes or categories that are most powerful in representing the entire dataset are identified by constant comparisons between individual accounts.\n\n### Reciprocal translational analysis\n\nReciprocal translational analysis involves translating findings of one paper into another by systematically comparing findings from each study, using techniques such as maps. \\[9\\] We encountered considerable methodological and practical problems in trying to apply RTA across a large set of papers, in part because of the kinds of iterations we were conducting in refining the sample. These meant that there were difficulties in identifying a stable \"set\" of papers on which an RTA could be conducted. RTA appears to be most suitable for a well-defined, relatively small (fewer than 50) and complete set of papers, because substitution or deletion of papers causes problems with both identifying index concepts and showing which concepts from other papers translate into these. A further problem is that, when confronted with a very large and diverse literature such as ours, RTA tends to provide only a summary in terms that have already been used in the literature. Although this may be a useful strategy as a stage on the way to a more interpretive synthesis, its value may be more limited than is the case for smaller samples of qualitative study reports where its benefits have been more evident.\n\nBefore our review, RTA had previously only been used for synthesising interpretive research, not a large and diverse body of literature, so this may be one reason why it was unsuccessful for our purposes. It is important to distinguish between the doubtful value of RTA in our synthesis (particularly because of the size and diversity of the literature), and the doubtful use of RTA in general. The diversity of the literature would also have prevented us from undertaking an aggregative synthesis using meta-analysis, but this clearly could not be read as a criticism of meta-analysis itself, but of its limitations when applying it to a diverse literature.\n\n### Lines of argument synthesis\n\nRecent work \\[9-11\\] has innovated in the methodology of lines-of-argument (LOA) synthesis originally proposed by Noblit and Hare by building on Schutz's \\[26\\] notions of \"orders\" of constructs Schutz used the idea of \"first order construct\" to refer to the everyday understandings of ordinary people and \"second order construct\" to refer to the constructs of the social sciences. The explanations and theories used by authors in primary study reports could therefore be seen as second order interpretations. This recent work uses LOA synthesis to develop what are referred to as \"third order\" interpretations, which build on the explanations and interpretations of the constituent studies, and are simultaneously consistent with the original results while extending beyond them. Our experiences have led us to respecify some of this approach.\n\nWe suggest that the appropriate way of conceptualising the output of an LOA synthesis is as a *synthesising argument*. This argument integrates evidence from across the studies in the review into a coherent theoretical framework comprising a network of constructs and the relationships between them. Its function is to provide more insightful, formalised, and generalisable ways of understanding a phenomenon. A synthesising argument can be generated through detailed analysis of the evidence included in a review, analogous to the analysis undertaken in primary qualitative research. It may require the generation of what we call *synthetic constructs*, which are the result of a transformation of the underlying evidence into a new conceptual form. Synthetic constructs are grounded in the evidence, but result from an interpretation of the whole of that evidence, and allow the possibility of several disparate aspects of a phenomenon being unified in a more useful and explanatory way.\n\nWhat we have called a \"synthetic construct\" might also be seen as a \"third order construct\". We suggest that the term \"synthetic construct\" is a more useful term because it is more explicit, and also because we emphasise that a *synthesising argument* need not consist solely of synthetic constructs. Instead, synthesising arguments may explicitly link not only synthetic constructs, but also second order constructs already reported in the literature. In effect, therefore, our approach does not make this precise distinction between second and third order constructs.\n\n### Refutational syntheses\n\nWe further suggest that what Noblit and Hare \\[1\\] call \"refutational syntheses\" are best conducted as part of the analysis that produces the synthesising argument Few published meta-ethnographies have in fact reported a separate refutational synthesis. It is, we suggest, more productive instead to adopt a critical and reflexive approach to the literature, including consideration of contradictions and flaws in evidence and theory.\n\nAn important element of producing a synthesising argument is the need, when conducting the analysis, to consider and reflect on the credibility of the evidence, to make critical judgements about how it contributes to the development of the synthesising argument, and to root the synthesising argument appropriately in critique of existing evidence. Clearly, credibility depends on the quality of the research, its currency, and the robustness of its theoretical base. But more generally, a critical interpretive synthesis is critical in the broader sense of *critique* rather than this more limited sense of critical appraisal, in which each study is judged against the standards of its type. Critique may involve identification of the research traditions or meta-narratives that have guided particular fields of research \\[27\\] as well as critical analysis of particular forms of discourses. Its aim is therefore to treat the literature as warranting critical scrutiny in its own right.\n\n## Conducting the analysis\n\nOur analysis of the evidence, in order to produce a synthesising argument, was similar to that undertaken in primary qualitative research. We began with detailed inspection of the papers, gradually identifying recurring themes and developing a critique. We then generated themes that helped to explain the phenomena being described in the literature, constantly comparing the theoretical structures we were developing against the data in the papers, and attempting to specify the categories of our analysis and the relationships between them. To facilitate the process of identifying patterns, themes, and categories across the large volumes of text-based data in our study, we used QSR N5 software. However, it is important to note that, as with any qualitative analysis, full transparency is not possible because of the creative, interpretive processes involved. Nonetheless, the large multidisciplinary team involved in the review, and the continual dialogue made necessary by this, helped to introduce \"checks and balances\" that guarded against framing of the analysis according to a single perspective.\n\nA key feature of this process that distinguishes it from some other current approaches to interpretive synthesis (and indeed of much primary qualitative research) was its aim of being *critical*: its questioning of the ways in which the literature had constructed the problematics of access, the nature of the assumptions on which it drew, and what has influenced its choice of proposed solutions. Our critique of the literature was thus dynamic, recursive and reflexive, and, rather than being a stage in which individual papers are excluded or weighted, it formed a key part of the synthesis, informing the sampling and selection of material and playing a key role in theory generation.\n\n## Findings: access to healthcare by socio-economically disadvantaged people\n\nOur critical interpretive synthesis of the literature on access to healthcare by socio-economically disadvantaged people in the UK included 119 papers. Early analytic categories were tentative and contingent, but gradually became firmed up and more highly specified as our analysis continued. Our synthesis involved a critique of the tendency to use measures of utilisation as a means of assessing the extent to which access to healthcare is equitable. It further involved the generation of a synthesising argument that has the synthetic construct of *candidacy* at its core. For space reasons, we can report here only a brief illustrative summary.\n\n## Critique of utilisation as a measure of access\n\nMuch of the evidence on whether access to healthcare in the UK is equitable has relied on measuring utilisation of health services. This approach measures the units of healthcare (consultations, procedures, etc) that people have actually consumed. The literature suggests that different groups have identifiable patterns of use of services, but the significance of these is often difficult to interpret. General practice (GP) consultation rates among socio-economically disadvantaged people have generally been found to be higher \\[28,29\\] though some recent work has suggested that social class variables are generally insignificant in explaining health service use \\[30\\] Studies that have attempted to adjust for need, usually on the basis of estimates of morbidity, have generally suggested that the apparent excess of GP consultation can be explained by higher need \\[31\\].\n\nOur critique of the literature suggests that utilisation is a generally unhelpful measure of equity of access. Not only do the logistical and practical problems of conducting utilisation studies pose substantial threats to validity and reliability, these studies are problematic for other reasons. They rely on a largely untested set of normative (i.e. ideas about how the world ought to be) and somewhat questionable assumptions about the \"correct\" level of utilisation, and on a difficult-to-measure (or conceputalise) estimates of \"need\". They often invoke normative assumptions about need relative to some apparently privileged though often ill-defined reference group (such as \"affluent\" people), and therefore risk failing to identify problems in access for that reference group. Misleadingly reassuring results may be produced that indicate that \"need\" and use or receipt are proportionate. We argue that utilisation, or, more appropriately, *receipt* of healthcare is the outcome of many different complex processes, which all need to be recognised if access is to be properly understood.\n\nOur analysis suggested that a focus instead on *candidacy*, a synthetic construct that we generated during the course of our analysis, would demonstrate the vulnerabilities associated with socio-economic disadvantage, emphasise the highly dynamic, multi-dimensional and contingent character of access, and allow a more insightful interpretation of the evidence on receipt of healthcare.\n\n## Candidacy\n\nOur synthesising argument around access to healthcare by socio-economically disadvantaged people is organised around a set of central concepts and, in particular, the core synthetic category of \"candidacy\". Candidacy functions as a synthetic construct because it is the product of the transformation of the evidence into a new conceptual form. It is distinct from earlier uses of the term \"candidacy\", including its use in the lay epidemiology of heart disease \\[32\\].\n\nWe have defined candidacy as follows: candidacy describes the ways in which people's eligibility for medical attention and intervention is jointly negotiated between individuals and health services. Our synthesising argument runs as follows: candidacy is a dynamic and contingent process, constantly being defined and redefined through interactions between individuals and professionals, including how \"cases\" are constructed. Accomplishing access to healthcare requires considerable work on the part of users, and the amount, difficulty, and complexity of that work may operate as barriers to receipt of care. The social patterning of perceptions of health and health services, and a lack of alignment between the priorities and competencies of disadvantaged people and the organisation of health services, conspire to create vulnerabilities. Candidacy is managed in the context of operating conditions that are influenced by individuals, the setting and environment in which care takes place, situated activity, the dynamics of face-to-face activity, and aspects of self (such as gender), the typifications staff use in categorising people and diseases, availability of economic and other resources such as time, local pressures, and policy imperatives.\n\n## Identification of candidacy\n\nHow people recognise their symptoms as needing medical attention or intervention is clearly key to understanding how they assert a claim to candidacy. Our analysis suggests that people in more deprived circumstances are likely to manage health and to recognise candidacy as a series of crises. There is significant evidence of lower use of preventive services among more deprived groups, \\[33,34\\] as well as evidence of higher use of accident and emergency facilities, emergency admissions and out-of-hours use \\[35,35,37,38\\]. Among more deprived groups, there is a tendency to seek help in response to specific events that are seen as warranting candidacy. \"Warning signs\" may be downgraded in importance by socio-economically disadvantaged populations because of a lack of a positive conceptualisation of health, \\[39,40\\] the normalisation of symptoms within deprived communities \\[41-43\\], and fear of being \"blamed\" by health professionals \\[44\\].\n\n## Navigation\n\nUsing services requires considerable work on the part of people. First, people must be aware of the services on offer, and there has been persistent concern that more deprived people may lack awareness of some services \\[45,46\\]. Second, using health services requires the mobilisation of a range of practical resources that may be variably available in the population. A key practical resource that impacts on the ability to seek care for the socio-economically disadvantaged, for example, is transport \\[44,47,48\\]. Other practical resources that may impact on the ability of disadvantaged groups to negotiate health services include more rigid patterns of working life \\[47\\]. Goddard and Smith \\[49\\] summarise evidence suggesting that those from more deprived social groups face financial costs of attending health services which, though not sufficient to dissuade them from using services when they are ill (i.e. in response to a specific \"event\"), act as a barrier to attending \"optional\" services related to health promotion and health prevention\n\n## The permeability of services\n\nPatterns of use of health services reflect issues in the organisation of services as much as they reflect a tendency to manage health as a series of crises on the part of disadvantaged people. We generated the synthetic construct of \"permeability\" to refer to the ease with which people can use services. Porous services require few qualifications of candidacy to use them, and may require the mobilisation of relatively fewer resources. Such services might include Accident and Emergency departments. Services that are less permeable demand qualifications (such as a referral), and also demand a higher degree of cultural alignment between themselves and their users, particularly in respect of the extent to which people feel comfortable with the organisational values of the service. Such services might include out-patients clinics in hospitals.\n\nServices that are less permeable tend to have high levels of default by socio-economically disadvantaged people \\[50-53\\]. Appointments systems, for example, are a threat to permeability by socio-economically disadvantaged people because they require resources and competencies (including stable addresses, being able to read, and being able to present in particular places at particular times \\[33,50,54\\] In addition, the extent to which people feel alienated from the cultural values of health services and their satisfaction with services have important implications for which services they choose to use \\[41,55\\].\n\n## Appearances at health services\n\n*Appearing* at health services involves people in asserting a claim to candidacy for medical attention or intervention. Whatever the nature of the claim, making it clearly involves work that requires a set of competencies, including the ability to formulate and articulate the issue for which help is being sought, and the ability to present credibly. More deprived people are at risk in these situations: they may be less used to or less able to provide coherent abstracted explanations of need, and may feel intimidated by their social distance from health professionals. Sword \\[56\\] points out that people with low incomes may feel alienated by the power relations that often characterise encounters with professionals. Dixon et al \\[57\\] and, in the US, Cooper and Roter \\[58\\] suggest that middle class people may be more adept at using their \"voice\" to demand better and extensive services: they may be more articulate, more confident, and more persistent, while people from lower class backgrounds are less verbally active. Somerset et al \\[59\\] report that in making referral decisions, patients' social status and their ability to articulate verbally act as background (and unexpressed) influences that affect the likelihood of referral.\n\n## Adjudications\n\nOnce a patient has asserted their candidacy by presenting to health services, the professional judgements made about that candidacy strongly influence subsequent access to attention and interventions. We generated the synthetic construct of \"adjudication\" to refer to the judgements and decisions made by professionals which allow or inhibit continued progression of candidacy. May et al's \\[60\\] analysis suggests doctors' practices are often exercised through a repertoire of routine judgements about the possibilities presented by individual patients and the routinely available means of solving these. These typifications are, we suggest, strongly influenced by local conditions, including the operating conditions in which practitioners work and sensitivity to resource constraints. Candidacy of socially disadvantaged people appears to be at risk of being judged to be less eligible, at least for some types of interventions, although the evidence that this happens is not particularly strong.\n\nOur analysis suggests that it is likely that professionals' perceptions of patients who are likely to \"do well\" as a result of interventions may disadvantage people in more deprived circumstances. As Hughes and Griffiths \\[61\\] identify, clinical decisions may rest on often implicit social criteria about which patients \"ought\" to receive care People in disadvantaged groups are more likely to smoke, to be overweight and to have co-morbidities, and professional perceptions of the cultural and health capital required to *convert* a unit of health provision into a given unit of health gain may function as barriers to healthcare \\[34\\]. In addition, perceptions of social \"deservingness\" may play a role \\[61,62\\]. Goddard and Smith \\[49\\] summarise evidence suggesting that independent of the severity of the disease, some GPs are more likely to refer the economically active and those with dependants. Clearly, there is potential for socially disadvantaged people to be disfavoured in such decisions.\n\n## Offers and resistance\n\nMuch of the work on utilisation of healthcare explicitly or implicitly assumes that non-utilisation is a direct reflection of non-offer. However, this type of normative analysis fails to acknowledge that people may choose to refuse offers. There is some evidence of patterns of resistance to offers. Referral implies that a GP has identified particular features of candidacy and is seeking to match those to a service that deals with that form of candidacy, but patients can resist being referred \\[42,63\\] and can resist offers of medication \\[64,65\\].\n\n## Operating conditions and the local production of candidacy\n\nA small body of recent research has identified what might be called local influences on the production of candidacy, and in our analysis these are hugely important. These are the contingent and locally specific influences on interactions between practitioners and patients, which may be emergent over time through repeated encounters. Crucial to the local production of candidacy is the perceived or actual availability and suitability of resources to address that candidacy \\[60,63\\].\n\n# Discussion\n\nDemands from health policy-makers and managers for syntheses of evidence that are useful, rigorous and relevant are fuelling interest in the development of methods that can allow the integration of diverse types of evidence \\[66\\]. With the diversity of techniques for evidence synthesis now beginning to appear, those using existing, 'new' or evolving techniques need to produce critical reflexive accounts of their experiences of using the methods \\[3\\]. Our experience of conducting a review of access to healthcare, where there is a large, amorphous and complex body of literature, and a need to assemble the findings into a form that is useful in informing policy and that is empirically and theoretically grounded \\[67\\], has led us to propose a new method \u2013 Critical Interpretive Synthesis \u2013 which is sensitised to the kinds of processes involved in conventional systematic review while drawing on a distinctively qualitative tradition of inquiry.\n\nConventional systematic review methodology is well-suited to aggregative syntheses, where what is required is a summary of the findings of the literature under a set of categories which are largely pre-specified, secure, and well-defined. It has been important in drawing attention to the weaknesses of informal reviews, including perceived failures in their procedural specification and the possibility that the (thus) undisciplined reviewer might be chaotic or negligent in identifying the relevant evidence, or might construct idiosyncratic theories and marshall the evidence in support of these. It has thus revealed some of the pitfalls of informal literature review. Conventional systematic review methodology has demonstrated considerable benefits in synthesising certain forms of evidence where the aim is to *test* theories (in the form of hypotheses), perhaps especially about \"what works\". However, this approach is limited when the aim, confronted with a complex body of evidence, is to *generate* theory \\[15,27\\].\n\nCurrent methods for conducting an interpretive synthesis of the literature, (such as meta-ethnography) are also limited, in part because application of many interpretive methods for synthesis has remained confined to studies reporting qualitative research. Realist synthesis \\[68\\], which does include diverse forms of evidence, is oriented towards theory evaluation, in particular by focusing on theories of change. Methods for including qualitative and quantitative evidence in systematic reviews developed by the EPPI Centre at the Institute of Education, London, have involved refinements and extensions of conventional systematic review methodology \\[6-8\\], and have limited their application of interpretive techniques to synthesis of qualitative evidence.\n\nMore generally, many current approaches fail to be sufficiently *critical*, in the sense of offering a critique. There is rarely an attempt to reconceptualise the phenomenon of interest, to provide a more sweeping critique of the ways in which the literature in the area have chosen to represent it, or to question the epistemological and normative assumptions of the literature. With notable exceptions such as the recent approach of meta-narrative analysis \\[15\\], critique of papers in current approaches to review tends to be limited to appraisal of the methodological specificities of the individual papers.\n\nConducting an interpretive review of the literature on access to healthcare by vulnerable groups in the UK therefore required methodological innovation that would be alert to the issues raised by systematic review methodology but also move beyond both its limitations and those of other current interpretive methods. The methods for review that we developed in this project (Table <a href=\"#T2\" data-ref-type=\"table\">2<\/a>) built on conventional systematic review methodology in their sensitivity to the need for attentiveness to a range of methodological processes. Crucially, in doing so, we drew explicitly on traditions of qualitative research inquiry, and in particular on the principles of grounded theory \\[5\\].\n\nKey Processes in critical interpretive synthesis\n\n<table>\n<tbody>\n<tr>\n<td style=\"text-align: left;\">\u25aa A review question should be formulated at the outset, but should remain open to modification. Precise definitions of many constructs may be deferred until late in the review and may be a product of the review itself.<br \/>\n\u25aa Searching, sampling, critique and analysis proceed hand in hand, and should be seen as dynamic and mutually informative processes.<br \/>\n\u25aa Searching initially should use a broadly defined strategy, including purposive selection of material likely or known to be relevant.<br \/>\n\u25aa The analysis should be aimed towards the development of a synthesising argument: a critically informed integration of evidence from across the studies in the review. The synthesising argument takes the form of a coherent theoretical framework comprising a network of constructs and the relationships between them. The synthesising argument links synthetic constructs (new constructs generated through synthesis) and existing constructs in the literature.<br \/>\n\u25aa There is a need for constant reflexivity to inform the emerging theoretical notions, as these guide the other processes.<br \/>\n\u25aa Ongoing selection of potentially relevant literature should informed by the emerging theoretical framework. Literatures not directly or obviously relevant to the question under review may be accessed as part of this process.<br \/>\n\u25aa CIS encourages an ongoing critical orientation to the material to be included in the review. Some limited formal appraisal of methodological quality of individual papers is likely to be appropriate. Generally the aim will be to maximise relevance and theoretical contribution of the included papers.<br \/>\n\u25aa Formal data extraction procedures may be helpful, particularly at the outset of the review, but are unlikely to be an essential feature of the approach.<br \/>\n\u25aa CIS does not offer aim to offer a series of pre-specified procedures for the conduct of review. It explicitly acknowledges the \"authorial voice\"; that some aspects of its production of the account of the evidence will not be visible or auditable; and that its account may not be strictly reproducible. Its aim is to offer a theoretically sound and useful account that is demonstrably grounded in the evidence.<br \/>\n\u25aa CIS demands constant reflexivity on the part of authors of reviews. Authors are charged with making conscientious and thorough searches, with making fair and appropriate selections of materials, with seeking disconfirming evidence and other challenges to the emergent theory, and with ensuring that the theory they generate is, while critically informed, plausible given the available evidence.<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\nIn addition to its explicit orientation towards theory generation, perhaps what most distinguishes CIS from conventional systematic review methods is its rejection of a \"stage\" approach to review. Processes of question formulation, searching, selection, data extraction, critique and synthesis are characterised as iterative, interactive, dynamic and recursive rather than as fixed procedures to be accomplished in a pre-defined sequence. CIS recognises the need for flexibility in the conduct of review, and future work would need to assess how far formal methods of critical appraisal and data extraction will be essential elements of the method. Our experience suggests that while attention to scientific quality is required, more generally the emphasis should be on critique rather than critical appraisal, and an ongoing critical orientation to the material examined and to emerging theoretical ideas. Formal data extraction may also be an unnecessarily constraining and burdensome process.\n\nCIS emphasises the need for theoretical categories to be generated from the available evidence and for those categories to be submitted to rigorous scrutiny as the review progresses. Further, it emphasises a need for constant reflexivity to inform the emerging theoretical notions, and guides the sampling of articles. Although CIS demands attention to flaws in study design, execution and reporting in our judgements of the quality of individual papers, its critical approach goes beyond standard approaches. Thus, in our review, some methodologically weak papers were important in terms of their theoretical contribution, or in terms of demonstrating the breadth of evidence considered in the construction of particular categories, or in terms of providing a more comprehensive summary of the evidence, while a single strong paper might be pivotal in the development of the synthesis. Hughes and Griffiths' paper on micro-rationing of healthcare \\[61\\], for example, was a key paper in helping to generate the construct of candidacy that later came to unify the themes of our analysis. The critical interpretation in our analysis focused on how a synthesising argument could be fashioned from the available evidence, given the quality of the evidence and the kinds of critiques that could be offered of the theory and assumptions that lay behind particular approaches. In treating the literature as an object of scrutiny in its own right, CIS problematises the literature in ways that are quite distinctive from most current approaches to literature reviewing.\n\n## Access to healthcare\n\nThe CIS approaches we adopted deferred final definition of the phenomenon of access and the appropriate ways of conceptualising it until our analysis was complete. Our critique of the current literature focused on the inadequacies of studies of utilisation as a guide to explaining inequities in health care. The conceptual model of access that we developed emphasises candidacy as the core organising construct, and recasts access as highly dynamic and contingent, and subject to constant negotiation.\n\nIn this conceptual model of access to healthcare, health services are continually constituting and seeking to define the appropriate objects of medical attention and intervention, while at the same time people are engaged in constituting and defining what they understand to be the appropriate objects of medical attention and intervention. Candidacy describes how people's eligibility for healthcare is determined between themselves and health services. Candidacy is a continually negotiated property of individuals, subject to multiple influences arising both from people and their social contexts and from macro-level influences on allocation of resources and configuration of services. \"Access\" represents a dynamic interplay between these simultaneous, iterative and mutually reinforcing processes. By attending to how vulnerabilities arise in relation to candidacy, the phenomenon of access can be much better understood, and more appropriate recommendations made for policy, practice and future research. Although our review focused on the UK, we suggest that the construct of candidacy is transferable, and has useful explanatory value in other contexts.\n\nIn addition to the core construct of candidacy, our analysis required the production of a number of other linked synthetic constructs \u2013 constructs generated through an attempt to summarise and integrate diverse concepts and data \u2013 including \"adjudications\" and \"offers\". It was also possible to link existing \"second order\" constructs, for example relating to help-seeking as the identification of candidacy by patients, into the synthesising argument, and making these work as synthesising constructs. We feel that this approach allows maximum benefit to be gained from previous analyses as well as the new synthesis.\n\n## Reflections on the method\n\nClearly, questions can be raised about the validity and credibility of the CIS analysis we have presented here. Conventional systematic review methodology sets great store by the reproducibility of its protocols and findings. It would certainly have been possible to produce an account of the evidence that was more reproducible. For example, we could have used the evidence to produce a thematic summary that stuck largely to the terms and concepts used in the evidence itself. However, we felt it important that we produced an interpretation of the evidence that could produce new insights and fresh ways of understanding the phenomenon of access, and that the \"critical voice\" of our interpretation was maintained throughout the analysis. Simply to have produced a thematic summary of what the literature was saying would have run the risk of accepting that the accounts offered in the evidence-base were the only valid way of understanding the phenomenon of access to healthcare by vulnerable groups. We therefore make no claim to reproducibility, but wish to address some possible concerns. First, it could be argued that a different team using the same set of papers would have produced a different theoretical model. However, the same would be true for qualitative researchers working with primary qualitative data, who accept that other possible interpretations might be given to, say, the same set of transcripts. Clearly, the production of a synthesizing argument, as an interpretive process, produces one privileged reading of the evidence, and, as the product of an authorial voice, it cannot be defended as an inherently reproducible process or product. We would suggest, however, that our analysis can be defended on the grounds that it is demonstrably grounded in the evidence; that it is plausible; that it offers insights that are consistent with the available evidence; and that it can generate testable hypotheses and empirically valuable questions for future research.\n\nSecond, subjecting a question to continual review and refinement, as we did, may make it more difficult for those conducting critical interpretive reviews to demonstrate, as required by conventional systematic review methodology, the \"transparency\", comprehensiveness, and reproducibility of search strategies. This dilemma between the \"answerable\" question and the \"meaningful\" question has received little attention, but it underpins key tensions between the two ends of the academic\/pragmatic systematic review spectrum. On balance, faced with a large and amorphous body of evidence in an area such as access to healthcare, and given the aims of an interpretive synthesis, we feel that our decision not to limit the focus of the review at the outset, and our subsequent sampling strategies, were well justified. Our decision not to commit to a particular view of what access might be and how it should be assessed at the outset of the project was critical to our subsequent development of a more satisfactory understanding of access.\n\nThird, it could be argued that we have synthesized too small a sample of the available papers, or that the processes used to select the papers are not transparent. We recognize that we have analyzed and synthesized only a fraction of all relevant papers in the area of access to healthcare by vulnerable groups. However, a common strategy in conventional systematic review is to limit the study types to be included; this strategy also might result in only a proportion of the potentially relevant literature being synthesised. While we have described our methods for sampling as purposive, it is possible that another team using the same approach could have come up with a different sample, because, particularly in the later stages of our review, our sampling was highly intuitive and guided by the emerging theory.\n\nThe final version of the conceptual model of access to healthcare that we eventually developed did not emerge until quite late in the review process, and much of the later sampling was directed at testing and purposively challenging the theory as we began to develop it. Again, such forms of searching and sampling do not lend themselves easily to reproducibility or indeed auditability. Testing whether the interpretations change in response to different findings will be an important focus for future research, which will also need to evaluate whether apparently disconfirming evidence is the result of methodological flaws or poses a genuine challenge to theory.\n\n# Conclusion\n\nConducting interpretive reviews in challenging areas where there is a large body of diverse evidence demands an approach that can draw on the strengths of conventional systematic review methodology and on the recent advances in methods for interpretive synthesis. We have termed the approach we developed to this review \"critical interpretive synthesis\". We believe that this methodology offers the potential for insight, vividness, illumination, and reconceptualisation of research questions, particularly in challenging areas such as access to healthcare, and look forward to further evaluations of its application.\n\n# Competing interests\n\nThe author(s) declare that they have no competing interests.\n\n# Authors' contributions\n\nMDW designed the project, led and supervised its execution, and drafted the manuscript. EA, AA, JH, RH, SK, RO, LS, RR and AJS participated in the design of the study. All authors engaged in searching, screening, sampling, data extraction, and critical appraisal\/critique activities, and contributed to the thematic analysis. DC and SA managed the searching, maintained the databases and coded material using N6 software. All authors contributed to the draft of the manuscript.\n\n# Pre-publication history\n\nThe pre-publication history for this paper can be accessed here:\n\n<http:\/\/www.biomedcentral.com\/1471-2288\/6\/35\/prepub>\n\n### Acknowledgements\n\nThis project was funded by the NHS Service Delivery and Organisation (SDO) Research Programme.","meta":{"dup_signals":{"dup_doc_count":133,"dup_dump_count":73,"dup_details":{"curated_sources":2,"2023-50":3,"2023-40":3,"2023-14":2,"2022-40":1,"2022-33":1,"2022-27":4,"2022-05":2,"2021-49":1,"2021-43":1,"2021-39":3,"2021-21":1,"2021-17":1,"2021-10":1,"2021-04":3,"2020-50":1,"2020-40":3,"2020-29":2,"2020-24":1,"2020-16":2,"2020-05":4,"2019-51":2,"2019-43":1,"2019-35":2,"2019-26":2,"2019-18":1,"2019-09":1,"2018-51":1,"2018-47":1,"2018-43":1,"2018-34":2,"2018-22":1,"2018-13":1,"2018-09":1,"2017-51":1,"2017-43":1,"2017-39":1,"2017-34":1,"2017-26":2,"2017-22":2,"2017-17":2,"2017-09":2,"2017-04":2,"2016-50":2,"2016-44":2,"2016-40":3,"2016-36":2,"2016-30":3,"2016-26":1,"2016-22":1,"2016-18":1,"2016-07":1,"2015-48":1,"2015-35":1,"2015-32":1,"2015-27":2,"2015-22":2,"2015-14":2,"2014-52":2,"2014-49":4,"2014-42":6,"2014-41":4,"2014-35":3,"2014-23":1,"2014-15":1,"2024-10":1,"2017-13":1,"2015-18":3,"2015-11":1,"2015-06":1,"2014-10":2,"2013-48":2,"2013-20":2,"2024-26":1}},"file":"PMC1559637"},"subset":"pubmed_central"}
{"text":"abstract: # OBJECTIVE\n  .\n  To examine in obese young adults the influence of ethnicity and subcutaneous adipose tissue (SAT) inflammation on hepatic fat fraction (HFF), visceral adipose tissue (VAT) deposition, insulin sensitivity (SI), \u03b2-cell function, and SAT gene expression.\n  .\n  # RESEARCH DESIGN AND METHODS\n  .\n  SAT biopsies were obtained from 36 obese young adults (20 Hispanics, 16 African Americans) to measure crown-like structures (CLS), reflecting SAT inflammation. SAT, VAT, and HFF were measured by magnetic resonance imaging, and SI and \u03b2-cell function (disposition index \\[DI\\]) were measured by intravenous glucose tolerance test. SAT gene expression was assessed using Illumina microarrays.\n  .\n  # RESULTS\n  .\n  Participants with CLS in SAT (*n* = 16) were similar to those without CLS in terms of ethnicity, sex, and total body fat. Individuals with CLS had greater VAT (3.7 \u00b1 1.3 vs. 2.6 \u00b1 1.6 L; *P* = 0.04), HFF (9.9 \u00b1 7.3 vs. 5.8 \u00b1 4.4%; *P* = 0.03), tumor necrosis factor-\u03b1 (20.8 \u00b1 4.8 vs. 16.2 \u00b1 5.8 pg\/mL; *P* = 0.01), fasting insulin (20.9 \u00b1 10.6 vs. 9.7 \u00b1 6.6 mU\/mL; *P* \\< 0.001) and glucose (94.4 \u00b1 9.3 vs. 86.8 \u00b1 5.3 mg\/dL; *P* = 0.005), and lower DI (1,559 \u00b1 984 vs. 2,024 \u00b1 829 \u00d710^\u22124^ min^\u22121^; *P* = 0.03). Individuals with CLS in SAT exhibited upregulation of matrix metalloproteinase-9 and monocyte antigen CD14 genes, as well as several other genes belonging to the nuclear factor-\u03baB (NF-\u03baB) stress pathway.\n  .\n  # CONCLUSIONS\n  .\n  Adipose tissue inflammation was equally distributed between sexes and ethnicities. It was associated with partitioning of fat toward VAT and the liver and altered \u03b2-cell function, independent of total adiposity. Several genes belonging to the NF-\u03baB stress pathway were upregulated, suggesting stimulation of proinflammatory mediators.\nauthor: Kim-Anne L\u00ea; Swapna Mahurkar; Tanya L. Alderete; Rebecca E. Hasson; Tanja C. Adam; Joon Sung Kim; Elizabeth Beale; Chen Xie; Andrew S. Greenberg; Hooman Allayee; Michael I. GoranCorresponding author: Michael I. Goran, <email@example.com>.\ndate: 2011-11\nreferences:\ntitle: Subcutaneous Adipose Tissue Macrophage Infiltration Is Associated With Hepatic and Visceral Fat Deposition, Hyperinsulinemia, and Stimulation of NF-\u03baB Stress Pathway\n\nAdipose tissue inflammation is now recognized as an important mediating link that may help explain the relationship between obesity and several metabolic abnormalities, including insulin resistance (1,2), liver fat accumulation (1,2), and vascular dysfunction (3). This association, however, is not consistent across obese individuals. For example, despite a similar degree of obesity, some obese individuals develop insulin resistance, type 2 diabetes, and nonalcoholic fatty liver disease, whereas others remain protected. This has led to the description of metabolically healthy obese patients (4) who display low hepatic fat content and high insulin sensitivity (SI) together with a favorable inflammatory profile (5). One factor that may explain differences in metabolic risk between individuals with the same degree of body fat is adipose tissue inflammation.\n\nAdipose tissue has long been considered as an inactive tissue, in which its presumed primary role was to store energy excess as triglycerides (TG). It is now widely accepted that adipose tissue acts as an endocrine organ as well through secretion of various adipokines and cytokines and plays a role in regulation of metabolic pathways (6). In obese individuals, excessive storage of free fatty acids (FFA) as TGs may lead to subcutaneous adipose tissue (SAT) dysfunction, resulting in impaired TG storage and possibly diversion of FFA to other tissues, such as the liver or the visceral compartment (7). Such a condition has been associated with high adipose tissue inflammation, characterized by higher secretion of proinflammatory cytokines and macrophage recruitment. Previous research has demonstrated that in adipose tissue from obese mice and humans, such macrophages aggregate around dead adipocytes, forming characteristic ring patterns referred to as crown-like structures (CLS) (8). Furthermore, the macrophages within CLS have been shown to be proinflammatory, and their presence is associated with insulin resistance (9,10).\n\nHispanics are more prone to an ectopic fat pattern, such as visceral and liver fat accumulation (11), when compared with African Americans; this may be driven partly by impaired SAT storage function and associated with adipose macrophage infiltration (12). Conversely, African Americans are similarly prone to obesity but appear protected against visceral and hepatic fat accumulation.\n\nThe purpose of this study, therefore, was to investigate the effect of adipose tissue inflammation on visceral and hepatic fat deposition, SI, and adipose tissue gene expression in two different ethnic groups. We hypothesized that individual differences in adipose tissue inflammation, reflected by the presence of CLS, may explain metabolic abnormalities of obesity, such as hepatic fat deposition and insulin resistance. We therefore recruited participants of Hispanic and African American ethnicity, who are obese and at high risk for type 2 diabetes but show very distinct fat repartition patterns, and investigated whether histological and gene expression differences in adipose tissue contribute to poor metabolic outcomes, such as higher liver fat and insulin resistance.\n\n# RESEARCH DESIGN AND METHODS\n\n## Study participants.\n\nThis cross-sectional analysis includes 36 obese (BMI \u226530 kg\/m^2^) African American (7 men, 9 women) or Hispanic participants (9 men, 11 women) aged 18\u201325 years. Participants were excluded if they had taken medications known to affect body composition, been diagnosed with any major illness since birth, or had any diagnostic criteria for diabetes. Written informed consent and assent were received from all participants. This study was approved by the Institutional Review Board of the Keck School of Medicine, University of Southern California.\n\n## Fat quantification.\n\nWhole-body fat was measured by dual-energy X-ray absorptiometry using a Hologic QDR 4500 W (Hologic, Bedford, MA). Abdominal magnetic resonance imaging data were obtained by the Dixon method, with a sensitive three-point chemical-shift fat-water separation method using a 1.5 Tesla Siemens Symphony Maestro whole-body scanner (Siemens AG, Erlangen, Germany) with Numaris 4 software. A two-dimensional multislice breath-hold protocol previously reported by Hussain et al. (13) was adopted to obtain 19 axial images across the abdomen from the dome of the liver to the L2-L3 vertebrae. The slice thickness was 10 mm with no interslice gaps. The fat-only dataset was used in the subsequent quantification of SAT volume and visceral adipose tissue (VAT) volume, whereas the fat fraction dataset was used to assess percent hepatic fat content (hepatic fat fraction \\[HFF\\]). A commercially available image segmentation and quantification software (SliceOmatic; Tomovision, Inc.) was used. SAT and VAT volumes were computed across all 19 image slices in each participant. HFF was computed as the mean fat fraction of all imaging slices within which the liver was present.\n\n## SAT collection.\n\nAbdominal SAT biopsies were performed lateral to the umbilicus in the skin crease below the abdominal pannus in the lower abdomen using standard sterile techniques. The region was locally anesthetized with 2 mL of 2% lidocaine. Through a 0.5-cm skin incision, SAT was collected with a disposable 0.5-cm diameter biopsy punch using three passes. All tissue samples were stored in formalin or promptly frozen in liquid nitrogen (LN2) and stored at \u221280\u00b0C.\n\n## Adipose tissue immunohistochemistry.\n\nInfiltration of macrophage cell populations into adipose tissue was characterized using cell-specific stains against CD68, an established cell surface marker for macrophages (predilute antibodies from DakoCytomation Corporation). In brief, 5-\u00b5m-thick adipose tissue sections were fixed and loaded onto a Biogenex I-6000 machine for incubation with primary antibodies. Multilink biotinylated secondary antibody was then allowed to react for 30 min at room temperature. Slides were then washed with PBS and placed in diaminobenzidine solution and microscopically examined for a positive reaction and counterstained with hematoxylin. All samples were evaluated in a blinded fashion by the pathologist for the presence (+) or absence (\u2212) of macrophage CLS, indicating the presence of dead adipocytes (8). Tissue sections were observed with a Nikon Eclipse E800 light microscope using a \u00d720 objective, and digital images were captured with a DXM 1200 camera. CLS density (CLS per 400 adipocytes) was determined using a drawing tablet and the Nikon Lucia IMAGE version 4.61 morphometric program. Four representative tissue sections in each sample were observed, and participants were dichotomously categorized as being CLS+ if distinct adipose tissue macrophage clusters were present in any examined high-power field or CLS\u2212 if clusters were completely absent in all histological fields for a given participant. To further characterize macrophage type, we performed CD11c immunoreactivity in six CLS+ (four Hispanics, two African Americans) and four CLS\u2212 participants (three Hispanics, one African American) using a mouse monoclonal antibody against human CD11c, a specific cell surface marker for type 1 macrophages (Novacastra Mouse Monoclonal Antibody CD11c \\[Clone 5D11\\]).\n\n## Adipose tissue microarray analysis.\n\nTotal RNA was isolated from adipose tissue biopsies using RNeasy Mini kits (Qiagen, Valencia, CA). Biotinylated RNA for hybridization with the Illumina arrays was amplified using the TotalPrep RNA Amplification Kit. Global gene expression was determined for each sample using an Illumina (San Diego, CA) HumanHT-12 v3 expression bead chip through a service provided by the Southern California Genotyping Consortium (<http:\/\/scgc.genetics.ucla.edu\/>). Quality control was performed and fulfilled the criteria for array hybridization suggested by the Tumor Analysis Best Practices Working Group (14). Data analysis was performed using Genome studio and Partek Inc. (St. Louis, MO). Background corrected and quantile normalized signal intensity values were exported to Partek. Array data have been submitted to the Gene Expression Omnibus (accession: GSE23506). A fold change of 1.3 was used as a criterion for inclusion of genes in functional annotation and pathway analysis. Our study was 70% powered to detect a fold change of 1.3 (<http:\/\/bioinformatics.mdanderson.org\/MicroarraySampleSize\/MicroarraySampleSize.aspx>).\n\n## RT-PCR.\n\nGenes of interest were validated using quantitative RT-PCR. Reverse transcription was performed with 0.5 \u00b5g of total RNA and random hexamer primers (Applied Biosystems, Foster City, CA). Inventoried and validated TaqMan probes were used. RT-PCR amplification was performed using an ABI HT7900 instrument (Applied Biosystems). All values are expressed as the average relative expression normalized to GUS-B endogenous control subjects.\n\n## SI and intravenous glucose tolerance test.\n\nAn insulin-modified frequently sampled intravenous glucose tolerance test (IVGTT) (15) was performed after an overnight fast. Upon arrival, a catheter was inserted into both arms at the antecubital level. At time 0, glucose (25% dextrose, 0.3 g\/kg body wt) was administered intravenously. Insulin (0.02 units\/kg body wt, Humulin R \\[regular insulin for human injection\\]; Eli Lilly, Indianapolis, IN) was injected intravenously at 20 min. Blood samples of glucose and insulin were collected at time points \u221215, \u22125, 2, 4, 8, 19, 22, 30, 40, 50, 70, 100, and 180 min and of FFA, TG, cholesterol, and cytokines at time \u221215. Glucose and insulin values obtained from the IVGTT were entered into the MINMOD Millenium 2003 computer program (version 5.16, Bergman, USC) to determine SI, glucose effectiveness, acute insulin response (AIR), and disposition index (DI) (15). Homeostasis model assessment-insulin resistance (HOMA-IR) was calculated according to the following formula: fasting insulin \\[mU\/mL\\] \u00d7 fasting glucose \\[mmol\/L\\]\/22.5, while HOMA\u2013\u03b2-cell = 20 \u00d7 fasting insulin \\[mU\/mL\\]\/fasting glucose \\[mmol\/L\\] \u2212 3.5.\n\n## Blood analysis.\n\nBlood samples from all time points taken during the IVGTT were centrifuged immediately for 10 min at 2,500 revolutions per minute at 8\u201310\u00b0C and frozen at \u221270\u00b0C until analysis. Glucose was assayed in duplicate on a Yellow Springs Instrument 2700 Analyzer (Yellow Springs, OH) using the glucose oxidase method. Insulin was assayed in duplicate using a specific human insulin ELISA kit from Linco (St. Charles, MO), and FFA were quantified using a colorimetric kit (NEFA-HR(2); Wako Diagnostics, Richmond, VA). TGs and total, LDL, and HDL cholesterol were measured using the Kodak Ektachem DT slide assay. Circulating inflammatory mediators including plasminogen activator inhibitor 1, monocyte chemoattractant protein 1 (MCP-1), interleukin-8 (IL-8), tumor necrosis factor-\u03b1 (TNF-\u03b1), and hepatocyte growth factor were measured in batch using multiplex Luminex assays (Linco Research). High-sensitivity C-reactive protein was measured chemically using ADVIA 1800 Chemistry System (Siemens Healthcare Diagnostics, Deerfield, IL).\n\n## Statistical methods.\n\nAll data are means \u00b1 SD, unless otherwise specified. Statistical analyses were performed using STATA 11.0 (Stata Corporation, College Station, TX). *P* values \\< 0.05 were considered statistically significant. Values for high-sensitivity C-reactive protein were log-transformed to reach a normal distribution. Unadjusted comparisons for parameters between adipose inflammatory status were done using Student *t* tests. Adjustments of comparisons for sex, ethnicity, total fat, and VAT were performed using ANCOVA when appropriate. Because of the large SDs of HOMA\u2013\u03b2-cell, SI, AIR, and DI, these parameters were tested using the nonparametric Wilcoxon signed-rank test. \u03c7^2^ Tests were performed to assess the effect of ethnicity and sex on inflammatory status. Correlation analyses were done using Pearson correlation tests. Differentially expressed genes were determined using one-way ANOVA and exported to Ingenuity pathway analysis 8.6 (Ingenuity Systems, Redwood City, CA).\n\n# RESULTS\n\n## Clinical and histological data.\n\nA total of 36 obese participants with an average BMI of 35.6 \u00b1 3.9 kg\/m^2^ completed the study (mean age 21.2 \u00b1 2.3 years; 55% Hispanics, 55% women). We first carried out histological analyses of adipose biopsies to determine the presence of aggregated macrophages as CLS. Mean section area was 14.0 \u00b1 5.1 mm^2^; 16 participants showed presence of CLS (CLS+), whereas there were no signs of CLS in the remaining 20 participants (CLS\u2212). In the CLS+ participants, the mean number of CLS per 400 adipocytes was 12.9 \u00b1 17.9, with a median of 5.4 and an interquartile range of 2.4\u201314.2. The characteristics of the participants, stratified by CLS status, are shown in <a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>. Of note, CLS+ participants were equally distributed among men and women as well as Hispanics and African Americans (\u03c7^2^ tests: *P* \\> 0.05), even after adjusting for total fat and VAT. This suggests that major differences with respect to the presence of CLS are not driven by sex or ethnicity in our study population. In the whole group, CLS+ individuals had increased VAT (3.7 \u00b1 1.3 vs. 2.6 \u00b1 1.6 L; *P* = 0.04), HFF (9.9 \u00b1 7.3 vs. 5.8 \u00b1 4.4%; *P* = 0.03), fasting TNF-\u03b1 (20.8 \u00b1 4.8 vs. 16.2 \u00b1 5.8 pg\/mL; *P* = 0.01), and insulin concentrations (20.8 \u00b1 2.6 vs. 9.7 \u00b1 6.5 mU\/mL; *P* = 0.0007), independent of sex, ethnicity, total fat, and visceral fat volume (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<\/a>). Markers of insulin resistance, including fasting glucose, fasting insulin, HOMA-IR, and HOMA-\u03b2, were also significantly higher in the CLS+ group, compared with the CLS\u2212, and these comparisons remained significant after adjusting for covariates. DI, reflecting \u03b2-cell function, was significantly lower in the CLS+ group (1,559 \u00b1 984 vs. 2,024 \u00b1 829 \u00d710^\u22124^ min^\u22121^; *P* = 0.03), whereas SI, glucose effectiveness, and AIR were not significantly different (<a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>). HDL cholesterol concentrations tended to be lower in CLS+ participants (*P* = 0.09), but there were no differences in fasting TGs and total and LDL cholesterol concentrations. In a subset of participants, we performed further immunohistochemical studies to examine the presence of CD11c+ immunoreactivity, to detect the presence of dendritic cells. Of the six participants with CLS+, four of them showed positive CD11c immunoreactivity staining, whereas it was completely absent in all four CLS\u2212 participants (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<\/a>). CD11c+ cells are a subclass of macrophages, called dendritic cells, which have been demonstrated to be proinflammatory and linked to systemic insulin resistance (10,16).\n\nAnthropometric, body composition, SI, and plasma parameters stratified by adipose tissue inflammatory status\n\n|  | CLS\u2212 | CLS+ | *P* value |\n|----|----|----|----|\n| *n* | 20 | 16 |  |\n| Number of CLS (per 400 adipocyte) | 0 \u00b1 0 | 12.9 \u00b1 17.9 | 0.002 |\n| Age (years) | 21.6 \u00b1 2.2 | 21.1 \u00b1 2.4 | NS |\n| Sex (men\/women) | 8\/12 | 8\/8 | NS |\n| Ethnicity (Hispanic\/African American) | 10\/10 | 10\/6 | NS |\n| Body composition |  |  |  |\n| \u2003BMI (kg\/m^2^) | 35.1 \u00b1 3.3 | 36.3 \u00b1 4.1 | NS |\n| \u2003Total fat (%) | 37.7 \u00b1 7.4 | 37.3 \u00b1 6.3 | NS |\n| \u2003SAT (L) | 16.0 \u00b1 4.9 | 17.3 \u00b1 3.9 | NS |\n| \u2003VAT (L) | 2.6 \u00b1 1.6 | 3.7 \u00b1 1.3 | 0.04 |\n| \u2003HFF (%) | 5.8 \u00b1 4.4 | 9.9 \u00b1 7.3 | 0.03 |\n| SI |  |  |  |\n| \u2003Fasting glucose (mg\/dL) | 86.8 \u00b1 5.3 | 94.4 \u00b1 9.3 | 0.005 |\n| \u2003Fasting insulin (mU\/mL) | 9.7 \u00b1 6.6 | 20.9 \u00b1 10.6 | \\<0.001 |\n| \u2003HOMA-IR | 2.0 \u00b1 1.4 | 4.4 \u00b1 2.2 | \\<0.001 |\n| \u2003HOMA\u2013\u03b2-cell | 191.4 \u00b1 125.4 | 399 \u00b1 301 | 0.01 |\n| \u2003Acute insulin response (mU\/mL \u00d7 10 min) | 1,320 \u00b1 912 | 1,116 \u00b1 796 | NS |\n| \u2003SI (\u00d710^\u22124^ min^\u22121^\/mU\/mL) | 1.9 \u00b1 0.7 | 1.3 \u00b1 0.9 | NS |\n| \u2003DI (\u00d710^\u22124^ min^\u22121^) | 2,024 \u00b1 829 | 1,559 \u00b1 984 | 0.03 |\n| Lipid metabolism |  |  |  |\n| \u2003FFAs (mmol\/L) | 0.87 \u00b1 0.19 | 0.83 \u00b1 0.15 | NS |\n| \u2003TGs (mg\/dL) | 113 \u00b1 59 | 102 \u00b1 48 | NS |\n| \u2003Cholesterol (mg\/dL) |  |  |  |\n| \u2003\u2003Total | 155 \u00b1 31 | 158 \u00b1 35 | NS |\n| \u2003\u2003LDL | 81 \u00b1 26 | 94 \u00b1 39 | NS |\n| \u2003\u2003HDL | 50 \u00b1 11 | 43 \u00b1 13 | 0.09 |\n| Inflammation |  |  |  |\n| \u2003MCP-1 (pg\/mL) | 349 \u00b1 182 | 379 \u00b1 97 | NS |\n| \u2003IL-8 (pg\/mL) | 9.7 \u00b1 3.3 | 11.4 \u00b1 3.2 | NS |\n| \u2003TNF-\u03b1 (pg\/mL) | 16.2 \u00b1 5.8 | 20.8 \u00b1 4.8 | 0.01 |\n| \u2003HGF (ng\/mL) | 2.1 \u00b1 0.8 | 2.1 \u00b1 0.8 | NS |\n| \u2003PAI-1 (ng\/mL) | 104 \u00b1 44 | 121 \u00b1 43 | NS |\n| \u2003hs-CRP (mg\/L) | 7.7 \u00b1 16.2 | 6.7 \u00b1 8.3 | NS |\n| \u2003Leptin (ng\/mL) | 45 \u00b1 22 | 51 \u00b1 28 | NS |\n| \u2003Adiponectin (mg\/mL) | 17 \u00b1 6 | 14 \u00b1 5 | NS |\n\nData are means \u00b1 SD. HGF, hepatocyte growth factor; hs-CRP, high-sensitivity C-reactive protein; NS, not significant; PAI-1, plasminogen activator inhibitor 1.\n\nWe then investigated whether presence of CLS translated into the same phenotypes between Hispanics and African Americans. When analyses were stratified by ethnicity, presence of CLS was specifically associated with higher VAT (2.8 \u00b1 0.9 vs. 1.6 \u00b1 0.9 L; *P* = 0.02) and glucose (100.1 \u00b1 8.6 vs. 84.4 \u00b1 5.6 mg\/dL; *P* \\< 0.001) in African Americans. In Hispanics, CLS was associated with higher TNF-\u03b1 (22.6 \u00b1 3.8 vs. 16.5 \u00b1 5.8 pg\/mL; *P* = 0.01) and a trend for lower SI (1.6 \u00b1 0.4 vs. 2.1 \u00b1 0.2 \\[\u00d710^\u22124^ min^\u22121^\/mU\/mL\\]; *P* = 0.06).\n\n## Adipose tissue gene expression analysis.\n\nIn the same participants, we subsequently assessed gene expression in SAT biopsies. Of the 23,000 known annotated genes analyzed on the Illumina Human HT-12 chip, 375 genes (\u223c2%) were differentially expressed between the CLS+ and CLS\u2212 groups based on a detection *P* value \\< 0.05. <a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a> shows the top 15 differentially up- and downregulated genes in CLS+ compared with CLS\u2212 individuals. Based on Gene Ontology descriptions, molecules involved in inflammatory disease, such as matrix metalloproteinase-9 (*MMP9*; fold change: +4.8; *P* = 0.0004), interferon \u03b3-inducible protein 30 (fold change: +2.2; *P* = 0.003), and IL-1 receptor antagonist (fold change: +2.0; *P* = 0.03), were among the most upregulated genes in CLS+ subjects compared with CLS\u2212 individuals. Genes involved in response to inflammation were also upregulated, including lipopolysaccharide binding protein (fold change: +2.0; *P* = 0.004), TNF receptor superfamily, member 11b (fold change: +1.4; *P* = 0.01), *MCP-1* (fold change: +1.6; *P* = 0.02), and monocyte antigen *CD14* (fold change: +1.5; *P* = 0.01). By comparison, insulin receptor substrate (*IRS*)-*1* (fold change: \u22121.6; *P* = 0.02) and *IRS-2* (fold change: \u22121.5; *P* = 0.01), which play central roles in the insulin signaling cascade, were downregulated in CLS+ individuals. Importantly, the differential expression of these inflammatory and insulin signaling genes was observed in both Hispanics and African Americans. On the basis of their relevance to inflammatory pathways we selected the following genes for RT-PCR validation: *CD14*, *MMP9*, suppressor of cytokine signaling 3, and *IRS-1.* In agreement with our hypothesis and the microarrays results, CLS+ individuals showed upregulation of *CD14* (1.7 \u00b1 0.9 vs. 0.9 \u00b1 0.3; *P* = 0.02) and *MMP9* (1.3 \u00b1 1.1 vs. 0.3 \u00b1 0.2; *P* = 0.01) compared with CLS\u2212 individuals. The suppressor of cytokine signaling 3 was upregulated (1.5 \u00b1 1.9 vs. 0.8 \u00b1 0.5; *P* = 0.2) and *IRS1* was downregulated in CLS+ individuals (1.0 \u00b1 0.6 vs. 1.4 \u00b1 1.2; *P* = 0.3), but these results did not reach significance. <a href=\"#F3\" data-ref-type=\"fig\">Figure 3<\/a> shows a schematic representation of the differentially expressed molecules and their functions.\n\nTop 15 up- and downregulated genes in CLS+ compared with CLS\u2212 individuals\n\n| Upregulated in CLS+ individuals |  |  |  | Downregulated in CLS+ individuals |  |  |  |\n|----|----|----|----|----|----|----|----|\n| Unigene official gene symbol | Gene name | *P* value | Fold change | Unigene official gene symbol | Gene name | *P* value | Fold change |\n| *MMP9* | matrix metallopeptidase 9 | 0.0004 | +4.8 | *KRT15* | keratin 15 | 0.04 | \u22122.2 |\n| *SPP1* | secreted phosphoprotein 1 | 0.01 | +3.2 | *CIDEA* | cell death-inducing *DFFA*-like effector a | 0.04 | \u22121.9 |\n| *SLC2A5* | solute carrier family 2 (facilitated glucose\/fructose transporter), member 5 | 0.0001 | +2.7 | *COBL* | cordon-bleu homolog (mouse) | 0.01 | \u22121.9 |\n| *PLA2G7* | platelet-activating factor acetylhydrolase, plasma | 0.005 | +2.6 | *COL6A6* | collagen, type VI, \u03b1-6 | 0.004 | \u22121.8 |\n| *IFI30* | interferon, \u03b3-inducible protein 30 | 0.003 | +2.2 | *CEACAM6* | carcinoembryonic antigen-related cell adhesion molecule 6 | 0.04 | \u22121.8 |\n| *PLA2G2A* | phospholipase A2, group IIA | 0.003 | +2.1 | *S100P* | S100 calcium binding protein P | 0.02 | \u22121.6 |\n| *LBP* | lipopolysaccharide binding protein | 0.004 | +2.1 | *GSDMB* | gasdermin B | 0.02 | \u22121.6 |\n| *ITGAX* | integrin, \u03b1-X | 0.006 | +2.0 | *CISH* | cytokine inducible SH2-containing protein | 0.02 | \u22121.6 |\n| *IL1RN* | IL-1 receptor antagonist | 0.03 | +2.0 | *ALDH3B2* | aldehyde dehydrogenase 3 family, member B2 | 0.04 | \u22121.6 |\n| *CLIC6* | chloride intracellular channel 6 | 0.03 | +2.0 | *ADH1A* | alcohol dehydrogenase 1A (class I), \u03b1-polypeptide | 0.006 | \u22121.6 |\n| *CHI3L2* | chitinase 3-like 2 | 0.001 | +2.0 | *IRS1* | insulin receptor substrate 1 | 0.02 | \u22121.6 |\n| *HMOX1* | heme oxygenase 1 | 0.0001 | +2.0 | *FHOD3* | formin homology 2 domain containing 3 | 0.02 | \u22121.6 |\n| *ACP5* | acid phosphatase 5 | 0.007 | +2.0 | *IRS2* | insulin receptor substrate 2 | 0.01 | \u22121.5 |\n| *PRND* | prion protein 2 | 0.005 | +1.9 | *RAP1GAP* | RAP1 GTPase activating protein | 0.03 | \u22121.5 |\n| *HP* | haptoglobin | 0.009 | +1.8 | *ADH1B* | alcohol dehydrogenase 1B (class I), \u03b2-polypeptide | 0.02 | \u22121.5 |\n\nPositive fold change, upregulated in individuals with CLS (CLS+); negative fold change, downregulated in individuals with CLS (CLS\u2212).\n\nTo gain further insight into the role of these 375 differentially expressed genes, we performed a pathway analysis using Ingenuity Pathway Analysis Systems and identified 31 significantly differentially regulated pathways (some of which are listed in <a href=\"#T3\" data-ref-type=\"table\">Table 3<\/a>). The first few differentially regulated pathways were related to liver disease or injury, including: *1*) liver X receptor\/retinoid X receptor activation pathways, which play a major role in hepatic lipid synthesis; *2*) hepatic cholestasis, a metabolic disease resulting from abnormal bile flow; *3*) bile acid biosynthesis; *4*) xenobiotic detoxification by cytochrome P450 enzymes; and *5*) hepatic fibrosis and hepatic stellate cell activation. Of note, molecules found in these various pathways included *MMP9*; lipopolysaccharide binding protein; *CD14*, IL-1-receptor-antagonist; TNF receptor superfamily, member 11b; and *MCP1*, which all belong to the nuclear factor-\u03baB (NF-\u03baB) signaling pathway.\n\nDifferentially regulated pathways between CLS+ and CLS\u2212 individuals\n\n| Canonical pathway | No. of genes | *P* value | Symbol |\n|----|----|----|----|\n| Liver X receptor\/retinoid X receptor activation | 9 | 4.20 E-05 | CD14, CCL2, TNFRSF11B, APOC1, MSR1, MMP9, NGFR, IL1RN, LBP |\n| Hepatic cholestasis | 12 | 6.69 E-05 | CD14, CYP27A1, ABCC3, SLCO3A1, TNFRSF11B, IRAK1, ESR1, NGFR, RARA, IL1RN, LBP, HSD3B7 |\n| Bile acid biosynthesis | 7 | 9.26 E-05 | CYP27A1, ALDH1A3, ADHFE1, DHRS9, ADH1A, ADH1B, HSD3B7 |\n| Metabolism of xenobiotics by cytochrome P450 | 10 | 1.17 E-04 | AKR1C2, ALDH1A3, CYP2F1, ALDH3B2 (includes EG:222), ADHFE1, CYP2C9, CYP2S1, DHRS9, ADH1A, ADH1B |\n| Atherosclerosis signaling | 10 | 1.4 E-04 | PDGFA, CXCR4, CCL2, ITGB2, ALOX5, MSR1, MMP9, PLA2G2A, TNFRSF12A, IL1RN |\n| Hepatic fibrosis\/hepatic stellate cell activation | 11 | 2.7 E-04 | CD14, PDGFA, TIMP1, CCL2, EDNRA, TNFRSF11B, AGTR1, MMP9, NGFR, EGFR, LBP |\n| IL-10 signaling | 7 | 6.6 E-04 | CD14, FCGR2B, CCR1, IL1RN, LBP, FCGR2A, HMOX1 |\n| Fatty acid metabolism | 9 | 9.4 E-04 | ALDH1A3, CYP2F1, ADHFE1, AUH, CYP2C9, CYP2S1, DHRS9, ADH1A, ADH1B |\n| Complement system | 5 | 1.2 E-03 | C5AR1, C1QC, C2, C3AR1, CFB |\n| Fc\u03b3 receptor-mediated phagocytosis in macrophages and monocytes | 8 | 1.71 E-03 | LYN, FCGR1A, PLD3, FGR, SYK, FCGR2A, WAS, HMOX1 |\n| Lipopolysaccharide\/IL-1-mediated inhibition of retinoid X receptor function | 12 | 2.1 E-03 | CD14, ABCC3, IL4I1, TNFRSF11B, ALDH1A3, APOC1, IRAK1, ALDH3B2 (includes EG:222), CYP2C9, NGFR, RARA, LBP |\n| Acute phase response signaling | 11 | 2.8 E-03 | TNFRSF11B, IRAK1, SERPINA3, C2, NGFR, HAMP, IL1RN, HP, LBP, HMOX1, CFB |\n| Dendritic cell maturation | 10 | 2.9 E-03 | FCGR2B, CD86, TNFRSF11B, TYROBP, FCGR1A, NGFR, IL1RN, TREM2, FCGR2A, FCGR1B |\n| IL-9 signaling | 4 | 6.8 E-03 | IRS1, BCL3, IRS2, CISH |\n| PPAR signaling | 5 | 1.5 E-02 | PDGFA, TNFRSF11B, CITED2, NGFR, IL1RN, PPARGC1A |\n| IL-8 signaling | 9 | 2.01 E-02 | ITGB2, ITGAX, IRAK1, MMP9, PLD3, GNAI1, ANGPT2, EGFR, HMOX1 |\n| IL-6 signaling | 6 | 2.07 E-02 | CD14, TNFRSF11B, NGFR, IL1RN, LBP, TNFAIP6 |\n| Role of macrophages, fibroblasts, and endothelial cells in rheumatoid arthritis | 13 | 3.6 E-02 | PDGFA, F2RL1, TNFRSF11B, FCGR1A, CFB, C5AR1, CCL2, FZD2, IRAK1, NGFR, HP, IL1RN, TNFSF13B |\n| Type 2 diabetes signaling | 1.26E00 | 3.8E-02 | IRS1, TNFRSF11B, IRS2, PKM2, NGFR, SMPD1 |\n\nPPAR, peroxisome proliferator\u2013activated receptor.\n\n# DISCUSSION\n\nObesity is associated with inflammation, which may play an important role in fatty liver disease and insulin resistance (1,2,17,18). In this study, we showed that in a group of obese Hispanics and African Americans, 44% of the participants had adipose tissue inflammation, which was associated with higher amounts of VAT and liver fat, hyperinsulinemia, and reduced \u03b2-cell function. At the molecular level, individuals with CLS showed upregulation of several genes belonging to the NF-\u03baB stress pathway.\n\nIn obese individuals, inflammatory processes are thought to originate from the excessive accumulation of fat in the adipose tissue, where it translates into recruitment of macrophages around dead adipocytes, forming ring patterns known as CLS (12). Consistent with a role for CLS in adipose tissue inflammation, we demonstrated the presence of a subclass of proinflammatory macrophages, CD11c+ dendritic cells, which have been linked to systemic insulin resistance, only in adipose tissue of subjects with CLS (10,16). Moreover, we demonstrated that in obese Hispanics and African Americans, VAT, liver fat, and circulating TNF-\u03b1 were significantly higher in individuals with CLS in fat biopsies, independent of ethnicity, sex, total body fat, and SAT. Such accumulation of fat in the visceral compartment and the liver markedly increases the risk for type 2 diabetes, and these metabolic abnormalities have been recognized as independent features of the metabolic syndrome (19,20). Indeed, inflammation is also closely associated with insulin resistance, both in the liver and the adipose tissue (21\u201323). This may be because of decreased insulin signaling by inflammatory mediators such as IL-1 and TNF-\u03b1 (24). Consistent with these previous observations, we show that individuals with CLS (CLS+) had increased fasting glucose and insulin, as well as decreased DI, reflecting altered glucose homeostasis and \u03b2-cell function.\n\nWe further investigated the effect of inflammation separately in Hispanics and African Americans. The prevalence of individuals with inflammation was equally distributed among ethnicities, suggesting that there is no existing ethnic predisposition to adipose tissue inflammation. Previous reports have shown that Hispanics are more prone to accumulation of lipids in the VAT and the liver (11). Given the tight link between ectopic fat and inflammation, we therefore expected Hispanics to have a higher degree of inflammation. However, our results show that inflammation may occur in Hispanics regardless of VAT amount. Moreover, presence of CLS was associated with lower SI and increased plasma TNF-\u03b1 concentrations. In contrast, African Americans usually display lower amounts of VAT; in this ethnic group, we found that VAT was significantly increased only by the presence of adipose tissue inflammation. Although we cannot establish any causal relationship at this point, these ethnic discrepancies suggest that in Hispanics, presence of inflammation is not a major player in these adipose tissue depots accumulation, but is instead associated with the development of systemic inflammation and insulin resistance. By contrast, in African Americans, who are usually protected against VAT accumulation, presence of adipose tissue inflammation may reflect a generalized increase in adipocyte depot mass and activation of inflammatory pathways. This suggests that presence of adipose tissue inflammation may be linked to distinct metabolic outcomes, depending on ethnicity.\n\nBased on these clinical observations, we investigated how different gene expression patterns between CLS+ and CLS\u2212 individuals could provide a functional link between adipose tissue inflammation, hepatic fat accumulation, and insulin resistance. As a result, several molecules related to the NF-\u03baB pathway were upregulated in CLS+ individuals. NF-\u03baB pathway mediates important stress responses and activates several proinflammatory cascades (25). Classically, its activation requires the binding of proinflammatory stimuli such as TNF-\u03b1, IL-1, or bacterial lipopolysaccharide to their appropriate membrane receptors, which subsequently trigger the activation cascade (26). Activation of NF-\u03baB results in enhanced transcription of IL-6, IL-8, and TNF-\u03b1, which trigger stress and inflammatory pathways and further stimulate the NF-\u03baB pathway (21). NF-\u03baB also leads to increased MCP-1, which locally recruits macrophages, and MMP9, which plays a role in inflammation-mediated tissue remodeling, such as adipocyte size expansion (27) and fibrosis in the liver (28). Notably, all of these molecules were upregulated in CLS+ individuals in the current study, reflecting a higher degree of inflammation and macrophage activation (<a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<\/a>). In mononuclear cells from obese individuals, the NF-\u03baB pathway is also activated, contributing to higher blood concentrations of proinflammatory mediators such IL-6, TNF-\u03b1, and MMP9 (29). This suggests that CLS-associated macrophages may have similar roles in adipose tissue, thus contributing to local release of proinflammatory cytokines (27). Using pathway analysis, we also observed that the most differentially regulated pathways were related to liver functions\/diseases, such as liver X receptor\/retinoid X receptor activation, hepatic cholestasis, and hepatic fibrosis pathways, with activation of several genes related to inflammatory processes, and more specifically to the NF-\u03baB pathway. These pathways play pivotal roles in liver injury and may lead to increased hepatic lipid synthesis (30\u201332).\n\nOur present results show that CLS may be found in some but not all obese individuals, independently of their ethnic background or sex, and is associated with increased VAT, hepatic fat content, and insulin resistance (33). Although our results are based on associations, when evaluated together with existing literature, the findings collectively suggest that with increasing obesity, adipocytes enlarge, until reaching a threshold (33). This may promote adipocyte death, macrophage aggregation, and CLS formation (8). The ensuing inflammatory activity may trigger the NF-\u03baB stress pathway and additional remodeling mechanism, characterized by increased MMP9 protease expression (34,35). Excess TG accumulation in adipose tissue may subsequently spill over to the systemic circulation and in turn accumulate in ectopic tissues, such as VAT and the liver (7,36), which may both contribute to decreased SI. The fact that in Hispanics high amounts of VAT are not necessarily associated with adipose tissue inflammation suggests that such relationships may be more subtle and possibly depend on other environmental and\/or genetic factors.\n\nOne limitation of our study is the absence of nutritional data. Both high-fat or high-fructose diets induce insulin resistance and inflammation (37). It remains therefore possible that differences in dietary intakes may contribute to the inflammatory status of adipose tissue. Whether the nutritional status may affect adipose tissue inflammation and the presence of CLS over the long run remains to be investigated. Moreover, this study was limited to African Americans and Hispanics, and obesity duration was not collected. It remains therefore to determine how the onset and duration of obesity may affect adipose tissue inflammation in these populations. Finally, we did not separate adipocytes from other cell types. Further studies using cell sorting will be required to discriminate the expression profile between cell types within the adipose tissue.\n\nIn conclusion, this study demonstrates that macrophage infiltration in SAT from obese individuals is equally distributed between sexes and ethnicities. Presence of inflammation is associated with higher VAT and hepatic fat content, as well as higher fasting glucose and insulin and reduced \u03b2-cell function, independent of total fat. These phenotypes may be attributed to upregulation of major gene pathways involved in proinflammatory cascades, such as the NF-\u03baB stress pathway. Further intervention studies will be required to assess the time course of such metabolic alterations and establish causal relationship between adipose tissue inflammation, hepatic fat accumulation, and development of insulin resistance.\n\n## ACKNOWLEDGMENTS\n\nThis work was supported by grants from the American Diabetes Association; National Institutes of Health Grant R01-DK-082574 to A.S.G.; the Robert C. and Veronica Atkins Foundation Grant to A.S.G. and M.I.G.; and the U.S. Department of Agriculture, Agricultural Research Service, under agreement No. 58-1950-7-70 to A.S.G. K.-A.L. is supported by a grant from the Swiss National Science Foundation (PBLA33-122719).\n\nNo potential conflicts of interest relevant to this article were reported.\n\nK.-A.L. designed the study, contributed to data and sample collection, analyzed data, wrote the manuscript, and reviewed and edited the manuscript. S.M. analyzed data and reviewed and edited the manuscript. T.L.A., R.E.H., and T.C.A. contributed to data and sample collection and reviewed and edited the manuscript. J.S.K. reviewed and edited the manuscript. E.B., C.X., and A.S.G. contributed to data and sample collection and reviewed and edited the manuscript. H.A. designed the study and reviewed and edited the manuscript. M.I.G. designed the study, analyzed data, and reviewed and edited the manuscript.\n\n# REFERENCES","meta":{"dup_signals":{"dup_doc_count":141,"dup_dump_count":51,"dup_details":{"curated_sources":2,"2021-10":1,"2020-45":1,"2020-05":1,"2019-51":1,"2019-39":1,"2018-30":1,"2018-26":1,"2018-17":3,"2018-09":1,"2018-05":3,"2017-51":1,"2017-47":2,"2017-43":3,"2017-39":2,"2017-34":3,"2017-30":4,"2017-26":2,"2017-22":3,"2017-17":5,"2017-09":5,"2017-04":4,"2016-50":3,"2016-44":5,"2016-40":4,"2016-36":4,"2016-30":3,"2016-26":3,"2016-22":4,"2016-18":2,"2016-07":3,"2015-48":4,"2015-40":2,"2015-35":5,"2015-27":4,"2015-22":4,"2015-14":4,"2014-52":4,"2014-49":2,"2014-42":2,"2014-41":4,"2014-35":3,"2014-23":1,"2014-15":2,"2021-25":1,"2017-13":4,"2015-18":4,"2015-11":3,"2015-06":2,"2014-10":2,"2013-48":2,"2013-20":1}},"file":"PMC3198061"},"subset":"pubmed_central"}
{"text":"abstract: \u03b2-Cell function improves in patients with type 2 diabetes in response to an oral glucose stimulus after Roux-en-Y gastric bypass (RYGB) surgery. This has been linked to the exaggerated secretion of glucagon-like peptide 1 (GLP-1), but causality has not been established. The aim of this study was to investigate the role of GLP-1 in improving \u03b2-cell function and glucose tolerance and regulating glucagon release after RYGB using exendin(9-39) (Ex-9), a GLP-1 receptor (GLP-1R)\u2013specific antagonist. Nine patients with type 2 diabetes were examined before and 1 week and 3 months after surgery. Each visit consisted of two experimental days, allowing a meal test with randomized infusion of saline or Ex-9. After RYGB, glucose tolerance improved, \u03b2-cell glucose sensitivity (\u03b2-GS) doubled, the GLP-1 response greatly increased, and glucagon secretion was augmented. GLP-1R blockade did not affect \u03b2-cell function or meal-induced glucagon release before the operation but did impair glucose tolerance. After RYGB, \u03b2-GS decreased to preoperative levels, glucagon secretion increased, and glucose tolerance was impaired by Ex-9 infusion. Thus, the exaggerated effect of GLP-1 after RYGB is of major importance for the improvement in \u03b2-cell function, control of glucagon release, and glucose tolerance in patients with type 2 diabetes.\nauthor: Nils B. J\u00f8rgensen; Carsten Dirksen; Kirstine N. Bojsen-M\u00f8ller; Siv H. Jacobsen; Dorte Worm; Dorte L. Hansen; Viggo B. Kristiansen; Lars Naver; Sten Madsbad; Jens J. HolstCorresponding author: Nils B. J\u00f8rgensen, <firstname.lastname@example.com>.\ndate: 2013-09\nreferences:\ntitle: Exaggerated Glucagon-Like Peptide 1 Response Is Important for Improved \u03b2-Cell Function and Glucose Tolerance After Roux-en-Y Gastric Bypass in Patients With Type 2 Diabetes\n\nHyperglycemia in patients with type 2 diabetes is resolved shortly after Roux-en-Y gastric bypass (RYGB), suggesting that mechanisms independent of weight loss contribute to the improvement in glycemic control (1\u20134).\n\nWithin 1 month and as early as 5 days after RYGB, \u03b2-cell function in response to a meal improves in subjects with type 2 diabetes, and this is accompanied by an increased postprandial glucagon-like peptide (GLP)-1 response (3,5,6). In contrast, after intravenous infusion of glucose, which does not elicit the incretin effect, an improvement in \u03b2-cell function is absent (5,7,8). Therefore, it could be speculated that the early improvements in \u03b2-cell function after RYGB are due to the enhanced GLP-1 secretion related to eating a meal, but causality has not been established (9).\n\nIn patients with type 2 diabetes, energy restriction per se is known to result in improved hepatic insulin sensitivity and decreased hepatic glucose production and, as a result, lowered fasting plasma glucose concentrations (10\u201312). Similar metabolic changes are seen after RYGB, when energy intake is limited (13,14), and this has led to the proposal that caloric restriction with a subsequent reduction in glucotoxicity, rather than an increased effect of GLP-1, is responsible for the improved \u03b2-cell function (14,15).\n\nThe aim of this study was to investigate the role of GLP-1 in the improved \u03b2-cell function and glucose tolerance seen after RYGB in subjects with type 2 diabetes. This was accomplished by pharmacologically blocking the GLP-1 receptor (GLP-1R) during a liquid meal tolerance test before and after surgery using exendin(9-39) (Ex-9; Bachem AG, Bubendorf, Switzerland), a specific GLP-1R antagonist (16).\n\nPrevious studies have documented increased meal-related glucagon secretion after RYGB despite improvements in insulin secretion and sensitivity and exaggerated GLP-1 release (3,17,18). This observation is surprising given the glucagonostatic properties of GLP-1 and insulin (19,20). Therefore, a further aim of this study was to evaluate the interaction between GLP-1 and glucagon release after RYGB in both the fasting and postprandial states.\n\n# RESEARCH DESIGN AND METHODS\n\nPatients with type 2 diabetes were recruited from the Hvidovre Hospital's bariatric surgery program (Hvidovre, Denmark), met the criteria for bariatric surgery (age \\>25 years and BMI \\>35 kg\/m^2^), and had accomplished a mandatory preoperative, diet-induced loss of 8% of total body wt before inclusion. Patients were excluded if they had uncontrolled hypothyroidism, had been taking antithyroid medication or anorectic agents within 3 months before the experiments, or had a fasting C-peptide level \\<700 pmol\/L. To confirm the diagnosis of type 2 diabetes, an oral glucose tolerance test (OGTT) was performed \u22641 month before the first experiment.\n\nThe study was approved by the Municipal Ethical Committee of Copenhagen (reg. nr. H-A-2008-080-31742), was in accordance with the Declaration of Helsinki II, and was registered with [clinicaltrials.gov](http:\/\/clinicaltrials.gov) (NCT01579981) and the Danish Data Protection Agency. Written informed consent was obtained from all patients before entering the study. Incretin-based therapies were put on hold for at least 14 days and all other antidiabetic medications for at least 3 days before the first preoperative experiment. Insulin analogs were replaced with NPH insulin at least 2 weeks before the first experiment. RYBG was performed as previously described (18). Patients were examined at 3 visits: before, 1 week after, and 3 months after RYGB. Visits consisted of 2 days where the patients were examined during a liquid meal tolerance test with a concurrent patient-blinded, primed, continuous infusion of Ex-9 or isotonic saline in random order. On each study day, patients met at 0800 h after a 10-h overnight fast. Patients were weighed (Tanita Corp., Tokyo, Japan), a catheter was inserted into the antecubital vein of each arm (one for blood sampling and one for infusion), and three fasting blood samples were drawn (\u221240 to 30 min). A primed continuous infusion of either saline or Ex-9 was initiated at time \u221230 min using a precision infusion pump (P2000; IVAC Medical Systems, Hampshire, U.K.). Saline was infused at a rate corresponding to the Ex-9 infusion volumes. After infusion was started, participants maintained a fasting state for 30 min to allow the drug to reach target tissues and drug concentrations to stabilize before the meal. Three further baseline samples were drawn just before ingestion of the meal (\u221210 to 0 min). At time 0 min, a liquid meal (Fresubin Energy Drink, 200 mL, 300 kcal, carbohydrate \\[50% of energy\\], protein \\[15% of energy\\], fat \\[35% of energy\\]; Fresenius Kabi Deutschland, Bad Homburg, Germany) was provided. To estimate gastric emptying, 1 g of paracetamol (Pamol; Nycomed Danmark, Roskilde, Denmark) was crushed to a powder and added to the meal. To avoid dumping after surgery and to obtain a comparable stimulus before and after RYGB, meal ingestion was supervised to ensure even distribution of meal intake over a 30-min period.\n\nBlood was sampled before and frequently following the meal for a total of 4 h (\u221240, \u221235, \u221230, \u221210, \u22125, 0, 30, 45, 60, 90, 120, 180, and 240 min). During each test, patients were placed sitting in a reclined position in a hospital bed, and no strenuous activity was allowed.\n\n## Ex-9.\n\nEx-9 was purchased from Bachem AG. Ex-9 from the same lot was used for all experiments. The peptide was dissolved in sterilized water containing 1% human albumin (Plasma Product Unit PSU; Novo Nordisk A\/S, Bagsvaerd, Denmark) and subjected to sterile filtration. The dissolved peptide was dispensed in appropriate volumes and stored frozen (\u221220\u00b0C) under sterile conditions until the day of the experiment. The peptide was demonstrated to be 99.5% pure by high-performance liquid chromatography. The Ex-9 infusion rate of 900 pmol\/kg body wt\/min was chosen because this had previously been reported to block the GLP-1R by 95% (21,22), and the resulting steady-state concentration of Ex-9 was predicted to be 391 nmol\/L using previously published pharmacokinetic parameters (23). Likewise, bolus size was calculated to be 43.000 pmol\/kg body wt.\n\n## Sample collection and laboratory analyses.\n\nBlood was collected into clot activator tubes for insulin and C-peptide analysis and into prechilled EDTA tubes for analysis of GLP-1, glucose-dependent insulinotropic polypeptide (GIP), glucagon, glucose, paracetamol, and Ex-9.\n\nClot activator tubes were left to coagulate for 30 min, whereas EDTA tubes were placed on ice until centrifuged at 4\u00b0C. Plasma glucose was measured immediately using the glucose oxidase technique (YSI model 2300 STAT Plus; YSI, Yellow Springs, OH). Samples of GLP-1, GIP, glucagon, and Ex-9 were stored at \u221220\u00b0C. All other samples were frozen and stored at \u221280\u00b0C until batch analysis.\n\nSerum insulin and C-peptide concentrations were determined by AutoDELFIA fluoroimmunoassay (Wallac OY, Turku, Finland). Plasma samples were assayed for total GLP-1 immunoreactivity, as described previously (24). Total GIP was measured using a C-terminally directed antiserum 867 with characteristics similar to the previously employed R65 (25,26). Glucagon was measured with the LINCO assay (Millipore, Billerica, MA) because it does not cross-react with Ex-9 (27). Ex-9 was measured using antibody 3145 raised in rabbits immunized with exendin-4, which shows 100% cross-reactivity with Ex-9 but \\<0.01% cross-reactivity with GLP-1, glucagon, or GIP (27). Paracetamol was measured using a calorimetric assay (Roche Diagnostics GmbH, Mannheim, Germany), and HbA~1c~ was measured using high-performance liquid chromatography with a cation exchange column (Tosoh Bioscience, Tokyo, Japan).\n\n## Calculations and statistical analyses.\n\nFasting glucose and hormone values were calculated as the mean of the time points from the interval \u221240 to \u221230 min. Total area under the curve (AUC) was calculated using the trapezoidal model. Baseline values were the mean of the time points from the interval \u221210 to 0 min. Incremental AUC (I-AUC) was calculated as AUC above baseline.\n\nInsulin resistance was calculated using homeostasis model assessment of insulin resistance (HOMA-IR) using the following equation: Insulin~fasting~ \\[pmol\/L\\] \u00d7 Glu~fasting~ \\[mmol\/L\\] \/ (22.5 \u00d7 6.945), where Glu is glucose.\n\nPrehepatic insulin secretion rates (ISRs) were calculated by deconvolution of peripheral C-peptide concentrations and application of population-based parameters for C-peptide kinetics using the ISEC software (28,29). ISR is expressed as picomoles \u00d7 kilograms^\u22121^ \u00d7 minutes^\u22121^. The relationship between glucose concentrations and ISR during the meal, the \u03b2-cell glucose sensitivity (\u03b2-GS), was characterized as previously described (3). The time to peak paracetamol concentration was used as a marker of gastric emptying.\n\nData are expressed as means \u00b1 SE. Statistical analyses were carried out using Wilcoxon matched pairs signed rank test or Kruskal-Wallis rank sum test, as appropriate. *P* values \\<0.05 were considered significant. The analyses were carried out using the R 2.11.1 statistical software package.\n\n# RESULTS\n\n## Patient characteristics.\n\nEleven patients with type 2 diabetes were recruited for the study. Two patients were excluded from data analysis: one because intravenous cannulation could not be performed on the second experimental day and the other because RYGB was abandoned because of massive intestinal adherences on the basis of earlier gastrointestinal surgery and peritonitis.\n\nThus, a total of nine patients with type 2 diabetes (age 50 \u00b1 3 years; three females and six males; BMI 39.2 \u00b1 2.4 kg\/m^2^; diabetes duration 5.7 \u00b1 1.3 years; HbA~1c~ 6.5 \u00b1 0.3% \\[48 \u00b1 4 mmol\/mol\\]; 2-h OGTT plasma glucose 14.4 \u00b1 1.0 mmol\/L) were examined 9 \u00b1 2 days before and 7 \u00b1 1 days (1 week) and 96 \u00b1 2 days (3 months) after RYGB. Preoperatively, one patient was treated with diet alone and eight were treated with metformin either as monotherapy (*n* = 4) or in combination with glimepiride (1), vildagliptin (1), liraglutide (1), or liraglutide and insulin glargine (1). None of the patients received any antidiabetic medication after RYGB. Further comorbidities were hypertension (6), hypercholesterolemia (2), hypothyroidism (*n* = 1, well controlled, with thyroid-stimulating hormone within the normal range), and myocardial infarction 4 years earlier (1). None had records or symptoms of gastroparesis, neuropathy, retinopathy, or nephropathy.\n\nThe patients entered the bariatric surgery program 337 \u00b1 40 days before RYGB with a BMI of 42.9 \u00b1 2.8 kg\/m^2^. On the day of the OGTT (30 \u00b1 9 days before RYGB), BMI had decreased to 39.5 \u00b1 2.5 kg\/m^2^ (92 \u00b1 0.9% of initial BMI), which was not different from that measured on the first experimental day. One week after RYGB, patients had lost 3.9 \u00b1 0.4% (*P* \\< 0.01) of preoperative BMI, and after 3 months they had lost 13.5 \u00b1 1.1% (*P* \\< 0.01). HbA~1c~ decreased to 5.7 \u00b1 0.2% (39 \u00b1 2 mmol\/mol; *P* \\< 0.01) 3 months after surgery.\n\n## Ex-9 concentrations.\n\nMean Ex-9 concentrations from time 0 to 240 min were similar before (404 \u00b1 52 nmol\/L) and after RYGB (1 week: 444 \u00b1 65 nmol\/L; 3 months: 405 \u00b1 51 nmol\/L; *P* = 0.7) and were stable throughout the experiment (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<\/a>).\n\n## Gastric emptying.\n\nParacetamol profiles are shown in <a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<\/a>. Before the operation, peak paracetamol concentrations were reached 70 \u00b1 10 min after the start of the meal, but after RYGB they peaked at the first postprandial sample point (30 min) (1 week: 33 \u00b1 2 min \\[*P* \\< 0.01\\]; 3 months: 32 \u00b1 2 min \\[*P* \\< 0.01\\]). GLP-1R blockade had a minor insignificant effect on time to peak paracetamol concentration before the operation (before Ex-9: 60 \u00b1 10 min \\[*P* = 0.49\\]) but no detectable effect after RYGB (Ex-9 at 1 week: 30 \u00b1 0 min; at 3 months: 30 \u00b1 0 min).\n\n## Fasting glucose and hormone concentrations.\n\nData regarding fasting glucose and hormone concentrations are listed in <a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>. Following RYGB, fasting glucose, insulin, and C-peptide concentrations decreased, whereas fasting GLP-1, GIP, and glucagon concentrations did not substantially change.\n\nFasting glucose and hormone concentrations (\u221240, \u221235, and \u221230 min) before infusion of saline or Ex-9\n\n![](3044tbl1)\n\nChanges in glucose and hormone concentrations 30 min after start of infusion, that is, before the meal, are reported in <a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a>. Ex-9 infusion caused glucose concentrations to increase significantly at all study times, whereas ISR decreased most before and at least 3 months after the operation. Fasting GLP-1 and GIP concentrations were not affected by Ex-9 infusion, but glucagon concentrations increased significantly compared with the changes observed during saline infusion before and 3 months after RYGB.\n\nChanges in fasting glucose and hormone concentrations after a primed infusion of saline or Ex-9 but before the meal\n\n![](3044tbl2)\n\n## Insulin resistance.\n\nInsulin resistance decreased by \u223c50% after RYGB (HOMA-IR before RYGB: 5.72 \u00b1 0.74; 1 week after: 2.71 \u00b1 0.41 \\[*P* = 0.004\\]; 3 months after: 2.18 \u00b1 0.30 \\[*P* = 0.004\\]). HOMA-IR did not differ between the days of saline and Ex-9 infusion at any of the three visits (data not shown).\n\n## Postprandial glucose concentrations and insulin secretion.\n\nData for postprandial glucose concentrations and insulin secretion are reported in <a href=\"#T3\" data-ref-type=\"table\">Table 3<\/a>, and glucose and ISR profiles are shown in <a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<\/a>. Following RYGB, 2-h postprandial glucose concentrations were significantly decreased but peak glucose concentrations were unchanged. Blocking the GLP-1R with Ex-9 caused 2-h and peak glucose concentrations to significantly increase compared with saline at all visits.\n\nPostprandial glucose and hormone responses with saline or Ex-9 infusion\n\n![](3044tbl3)\n\nAfter the operation, insulin secretion was significantly increased in response to the meal as evidenced by both increased peak ISR and total meal-induced insulin secretion (I-AUC ISR). Ex-9 infusion resulted in significant decreases in peak ISR and I-AUC ISR 1 week and 3 months after RYGB but not before the operation.\n\n## \u03b2-Cell glucose sensitivity.\n\nData for \u03b2-GS are shown in <a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<\/a>. In response to the liquid meal, \u03b2-GS increased as a consequence of RYGB. One week after the operation, \u03b2-GS had almost doubled (*P* = 0.008), and it remained elevated at 3 months (*P* = 0.027). When the GLP-1R was blocked with Ex-9 before the operation, an insignificant decrease in \u03b2-GS (*P* = 0.07) was observed. After RYGB, Ex-9 infusion reduced \u03b2-GS to preoperative levels both at 1 week and 3 months after the operation, and these decreases were highly significant (both *P* = 0.004 vs. saline).\n\n## Postprandial GLP-1, GIP, and glucagon concentrations.\n\nAfter RYGB, I-AUC GLP-1 was increased eightfold, reflecting a greatly elevated meal-induced GLP-1 response (<a href=\"#F4\" data-ref-type=\"fig\">Fig. 4<em>A<\/em><\/a>). Ex-9 infusion resulted in a further augmentation of GLP-1 release both before and after RYGB. The GIP response (<a href=\"#F4\" data-ref-type=\"fig\">Fig. 4<em>B<\/em><\/a>) was not affected by RYGB or Ex-9 infusion.\n\nMeal-induced glucagon secretion (I-AUC glucagon) (<a href=\"#F4\" data-ref-type=\"fig\">Fig. 4<em>C<\/em><\/a> and <a href=\"#T3\" data-ref-type=\"table\">Table 3<\/a>) increased after RYGB. Before the operation, I-AUC glucagon was not affected by GLP-1R blockade (*P* = 0.3), but after RYGB, Ex-9 infusion resulted in a marked and significant increase in glucagon secretion upon stimulation by the meal compared with the corresponding day of saline infusion.\n\n# DISCUSSION\n\nOne week and 3 months after RYGB, glucose tolerance and \u03b2-GS are improved and the meal-related GLP-1 response and glucagon release are increased in subjects with type 2 diabetes, as we have previously demonstrated (3). The key finding in the current study is that pharmacological blockade of the GLP-1R after RYGB reverses the improvements in \u03b2-GS and glucose tolerance and increases postprandial glucagon release. These results strongly suggest that the increased endogenous GLP-1 secretion is of great importance for the acute effects of RYGB on \u03b2-cell function and for glucose tolerance in patients with type 2 diabetes. Furthermore, we demonstrate that glucagon secretion is inhibited by GLP-1 after RYGB.\n\nBefore RYGB, GLP-1R inhibition impaired glucose tolerance because of an increment in fasting and postprandial glucose concentrations. Fasting insulin secretion decreased but meal-induced secretion was unaltered, and \u03b2-GS was not significantly reduced when Ex-9 was infused. Fasting glucagon concentrations increased during GLP-1R blockade, but the meal-induced glucagon response was unaltered. The effects of Ex-9 infusion on insulin and glucagon secretion during fasting were similar to those reported in two recent studies of patients with type 2 diabetes (30,31), but contrary to our results, these studies found a pronounced effect of GLP-1R inhibition on insulin and glucagon secretion when endogenous GLP-1 secretion was stimulated. However, both studies used the hyperglycemic clamp technique to keep plasma glucose concentrations fixed, whereas our study allowed plasma glucose concentrations to vary; as a consequence, they were higher on the day of Ex-9 infusion. This greater glucose stimulus is likely to counteract the effects of GLP-1R antagonism on postprandial insulin and glucagon secretion in our study and may explain these seemingly contradictory results.\n\nOne week after RYGB, plasma glucose concentrations had improved in both the fasting and postprandial states. After the operation, insulin secretion increased, \u03b2-GS doubled, the GLP-1 response was greatly increased, and meal-induced glucagon release was elevated. These results confirm our previous report and demonstrate that RYGB improves \u03b2-cell function soon after surgery (3). GLP-1R blockade caused glucose tolerance to deteriorate and \u03b2-GS to decrease to preoperative levels. At the same time, postprandial GLP-1 and glucagon responses increased.\n\nThree months after the operation, glucose tolerance was further improved with enhanced insulin and GLP-1 secretion compared with before surgery, and overall glucose control was improved, as evidenced by a decreased HbA~1c~, despite patients not receiving antidiabetic medication. GLP-1R antagonism produced results similar to those observed 1 week after the operation, again returning \u03b2-GS to preoperative levels and augmenting the meal-related glucagon response, resulting in an impairment of glucose tolerance.\n\nBefore the operation, the weight of patients was stable, but after RYGB they experienced rapid weight loss, amounting to \u223c4% within a week. It has been suggested that the acute effects of RYGB on \u03b2-cell function in patients with type 2 diabetes are strictly related to reduced energy intake (14,15), although reports on the effects of such acute energy restriction are conflicting (12,32). However, while energy restriction has little or no effect on GLP-1 release, postprandial GLP-1 secretion is greatly increased after RYGB (3,5,14,33\u201335). Previous reports have shown that the incretin effect after RYGB is normalized in patients with type 2 diabetes and that this cannot be explained simply by weight loss (36,37). Furthermore, in patients with type 2 diabetes, \u03b2-cell function in response to a meal or an oral glucose load has consistently been reported to improve within the first month after RYGB but not after gastric restrictive surgery where patients undergo the same postoperative dietary regimen or when \u03b2-cell function is evaluated by intravenous glucose infusion (3,5\u20137,33,38,39). The present data illustrate that the exaggerated GLP-1 response in patients with type 2 diabetes seems to be the main explanation for the improvement in \u03b2-cell function after RYGB in response to a meal. Reduced glucotoxicity may play a role in the improved \u03b2-cell function after surgery, but it seems to be of minor importance compared with the actions of GLP-1 (40,41). Of note, HOMA-IR was not different between saline and Ex-9 infusion at any visit. Therefore, the changes we observed with GLP-1R blockade are unlikely to be a consequence of adaptation of \u03b2-cells to changes in insulin resistance (42). It has been suggested that the large GLP-1 response after RYGB is triggered by the accelerated delivery of nutrients to the enteroendocrine cells of the distal ileum (43,44). We recently reported the gastric emptying of liquids to be greatly accelerated in patients who have received RYGB as measured using a scintigraphic method, and this was associated with an increased release of GLP-1 (45). In agreement with this finding, paracetamol absorption in this study was accelerated after RYGB, supporting such a mechanism of action. GLP-1R blockade increased postprandial GLP-1 release by 60\u2013100% before and after RYGB, a finding that may be related to inhibition of GLP-1R on the enteroendocrine cells that work to feed negatively back on GLP-1 secretion (46). Although GLP-1 inhibits gastric emptying (19), paracetamol absorption kinetics were not significantly changed with Ex-9 infusion, making accelerated gastric emptying an unlikely mechanism by which GLP-1R blockade increases GLP-1 secretion in this study. It must be emphasized that estimation of gastric emptying using this method is limited by the blood sampling frequency, especially after RYGB. Previous studies have indicated that GLP-1 tonically suppresses fasting glucagon concentrations in patients with type 2 diabetes, and we were able to reproduce that finding here (31). The glucagonostatic properties of GLP-1 and the exaggerated response after RYGB would predict a reduced release of glucagon as induced by a meal (19); however, the glucagon response increases after the operation. Eliminating the effect of endogenous GLP-1 with Ex-9 further increased glucagon secretion, implying that meal-induced glucagon release continues to be under the inhibitory control of GLP-1. The mechanism underlying the increased meal-induced glucagon response remains to be elucidated.\n\nSalehi et al. (47) reported that Ex-9 infusion resulted in a more pronounced decrease of insulin secretion when a meal was ingested during a hyperglycemic clamp in nondiabetic patients who received RYGB compared with normal controls. Strengths of the current study include the prospective design, the inclusion of patients with type 2 diabetes, and the use of a physiological meal test, allowing us to demonstrate the effects of endogenous GLP-1 signaling before and after RYGB and to evaluate both \u03b2-cell function and glucose tolerance. Therefore, our results expand the observations of Salehi et al.\n\nWe used a primed infusion of Ex-9 at a rate of 900 pmol \u00d7 kg^\u22121^ \u00d7 min^\u22121^. This dose of Ex-9, which resulted in stable plasma concentrations of \u223c400 nmol\/L on all experimental days, has previously been reported to result in 95% inhibition of the GLP-1R even in the presence of very high GLP-1 concentrations, such as those expected in the portal vascular bed after RYGB upon stimulation by a meal (21,22). GLP-1R blockade did not influence GIP secretion in this study, and it has previously been shown that Ex-9 infusion does not interfere with GIP signaling on the \u03b2-cells (48). In our own studies of the effect of GIP in COS-7 cells transfected with the human GIP receptor, there was no effect of Ex-9 in nanomolar to micromolar concentrations (M. Rosenkilde, unpublished observations). Collectively, this shows that Ex-9 specifically inhibits the actions of GLP-1 with respect to the incretin effect.\n\nA limitation of this study is the small sample size of nine patients, which makes the detection of more subtle changes less likely. Despite this, we were able to demonstrate a highly significant effect of GLP-1R inhibition on insulin secretion and \u03b2-cell function after RYGB, underscoring the strength of the association.\n\nIn light of the marked inhibition of \u03b2-cell function, glucose tolerance was only moderately affected during Ex-9 infusion after the operation. This could reflect an increase in the relative importance of non-insulin-mediated glucose uptake for glucose tolerance (49\u201351), but the study was not designed to evaluate this.\n\nIn addition, we did not account for hepatic glucose production in this study, which could already be decreased 1 week after the operation, perhaps as a result of caloric restriction (9). Further studies are needed to investigate this early aspect of glucose metabolism after RYGB.\n\nIn vitro studies of the murine GLP-1R suggest that it is constitutively active and inhibited by Ex-9 even in the absence of GLP-1; i.e., Ex-9 is an inverse agonist (52). It is questionable whether this is a property of the human GLP-1R in vivo (53), but if so our results could be influenced by an increased GLP-1R sensitivity to the inverse agonism of Ex-9 after surgery. However, the effects of Ex-9 on basal insulin and glucagon secretion were greatest before the operation and decreased with time from surgery, which makes it highly unlikely that Ex-9 should more potently inhibit GLP-1R signaling after RYGB.\n\nWe conclude that an increased effect of GLP-1 as a consequence of hypersecretion is the main explanation for the improved \u03b2-cell function after RYGB in response to a meal in patients with type 2 diabetes. Furthermore, the effects of GLP-1 are important in controlling glucagon release and glucose tolerance in these patients after the operation.\n\n## ACKNOWLEDGMENTS\n\nThis work was carried out as a part of the program of the UNIK: Food, Fitness & Pharma for Health and Disease (see [www.foodfitnesspharma.ku.dk](http:\/\/www.foodfitnesspharma.ku.dk)). The UNIK project is supported by the Danish Ministry of Science, Technology and Innovation. Further support was received from the Danish Diabetes Association, The Novo Nordisk Foundation, and The Strategic Research Council for the Capital Area and the Danish Research Agency (Ministry of Science, Technology and Innovation). The purchase of Ex-9 was made possible through a donation from Desir\u00e9e & Niels Ydes Foundation.\n\nNo potential conflicts of interest relevant to this article were reported.\n\nN.B.J. planned and conducted experiments, researched data, and wrote the manuscript. C.D. conducted experiments, contributed to discussion, and reviewed the manuscript. K.N.B.-M., S.H.J., S.M., and J.J.H. contributed to discussion and reviewed the manuscript. D.W., D.L.H., V.B.K., and L.N. reviewed the manuscript. S.M. and J.J.H. planned experiments. N.B.J. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.\n\nParts of this study were presented in oral form at the 48th Annual Meeting of the European Association for the Study of Diabetes, Berlin, Germany, 30 September\u20131 October 2012, and in poster form at the 72nd Scientific Sessions of the American Diabetes Association, Philadelphia, Pennsylvania, 8\u201312 June 2012.\n\nThis work would not have been possible without the technical assistance of Alis Sloth Andersen and Dorthe Baunbjerg Nielsen (Department of Endocrinology, Hvidovre Hospital) and Lene Brus Alb\u00e6k (Department of Biomedical Sciences, Faculty of Health Sciences, University of Copenhagen).\n\n# REFERENCES","meta":{"dup_signals":{"dup_doc_count":175,"dup_dump_count":53,"dup_details":{"curated_sources":2,"2022-21":1,"2021-21":1,"2020-34":2,"2020-29":1,"2020-16":1,"2019-47":1,"2019-39":1,"2018-39":1,"2018-26":3,"2018-22":2,"2018-17":3,"2018-13":2,"2018-09":2,"2018-05":2,"2017-51":3,"2017-47":1,"2017-43":6,"2017-39":1,"2017-34":5,"2017-30":4,"2017-26":6,"2017-22":1,"2017-17":10,"2017-09":6,"2017-04":9,"2016-50":5,"2016-44":6,"2016-40":5,"2016-36":5,"2016-30":5,"2016-26":4,"2016-22":5,"2016-18":5,"2016-07":3,"2015-48":4,"2015-40":1,"2015-35":2,"2015-32":3,"2015-27":5,"2015-22":3,"2015-14":3,"2014-52":5,"2014-49":3,"2014-42":4,"2014-41":3,"2014-35":2,"2023-23":1,"2024-22":1,"2017-13":4,"2015-18":5,"2015-11":2,"2015-06":3,"2024-26":1}},"file":"PMC3749359"},"subset":"pubmed_central"}
{"text":"author: Nigel Irwin; Victor Alan GaultCorresponding author: Victor Alan Gault, <email@example.com>.\ndate: 2013-09\nreferences:\ntitle: Unraveling the Mechanisms Underlying Olanzapine-Induced Insulin Resistance\n\nAtypical antipsychotics (AAPs) are widely prescribed agents for treatment of schizophrenia and other related psychiatric disorders. Although AAPs were a major development in psychopharmacology, so-called second-generation agents such as olanzapine have exhibited unexpected and unfavorable metabolic side effects. These side effects include weight gain, glucose intolerance, and insulin resistance, all of which increase the likelihood of developing diabetes and cardiovascular disease (1). Nonetheless, how these adverse metabolic effects arise following AAP treatment remains unclear. A key question is whether AAP-associated metabolic impairments are because of the psychiatric illness itself or if they are merely secondary to weight gain.\n\nIn this issue, Teff et al. (2) examine the direct effects of second-generation AAPs on insulin resistance and postprandial gut hormone profiles following a mixed meal. The antipsychotic drugs used in these experiments\u2014olanzapine and aripiprazole\u2014were administered to healthy volunteers for 9 consecutive days to exclude potential confounding issues associated with psychiatric disease or potential weight gain. In the clinic, olanzapine therapy tends to result in weight gain and metabolic dysregulation (3), whereas aripiprazole is considered metabolically neutral (4). Thus, Teff et al. logically hypothesized that detrimental effects on meal-related metabolism would be limited to olanzapine. Consistent with this hypothesis, the authors showed that aripiprazole had no effect on body weight, blood pressure, and circulating levels of key blood parameters over the 9-day treatment period. Aripiprazole also did not result in significant changes in postprandial metabolism following a mixed meal. In contrast, olanzapine increased fasting plasma insulin by day 9. This corresponded with postprandial hyperinsulinemia, suggesting that olanzapine had detrimental effects on insulin sensitivity. Indeed, hyperinsulinemic-euglycemic clamps supported this theory. However, aripiprazole also induced insulin resistance despite it generally being considered lacking in metabolic effects.\n\nA unique aspect to the study by Teff et al. (2) was participant ingestion of a mixed meal that elicited both incretin responses (5) and neurally mediated insulin release (6), both of which could be critical for the metabolic effects of centrally acting drugs. Surprisingly, olanzapine administration increased postprandial glucagon-like peptide-1 (GLP-1) secretion and circulating glucagon levels. These responses were unexpected for two reasons. First, GLP-1 is believed to improve insulin sensitivity and reduce weight gain. Second, it directly inhibits glucagon release (7). The authors speculate that other unknown factors, such as glucose-dependent insulinotropic polypeptide (GIP) secretion or cholinergic vagally mediated actions, may mediate the altered postprandial metabolic profile following olanzapine administration. In this regard, it has recently been shown that biologic action of GIP can be potentiated by xenin-25, a peptide cosecreted with GIP from intestinal K cells, which is thought to indirectly activate muscarinic receptors (8). Olanzapine is a well-characterized muscarinic receptor antagonist (9). Thus, interactions between GIP and other vagal inputs could be of importance for the postprandial metabolic effects of AAPs (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<\/a>).\n\nThe new data are interesting but need to be corroborated in studies with larger sample sizes to increase power and also to be verified in psychiatric patients. A previous study has shown no effect of olanzapine on gut hormone secretion (10). The direct effect of AAPs on GIP secretion and action certainly needs to be considered. Although weight gain did not affect data interpretation, Teff et al. (2) clearly acknowledge that adiposity could be a confounding factor. In addition, the nutrient composition of the mixed meal could have important implications on GIP and GLP-1 secretion. Both incretin hormones are strongly released in response to carbohydrates, but GIP is released much more powerfully than GLP-1 in response to fat (11).\n\nTaken together, the data illustrate that olanzapine can induce insulin resistance and postprandial hormonal dysregulation independently of weight gain. Although the regulatory mechanisms involved remain to be fully elucidated, the well-characterized weight gain following prolonged olanzapine administration would likely exacerbate these effects. Thus, interventions to inhibit weight gain in patients receiving AAP therapy may only be partially effective in preventing metabolic disease. The report by Teff et al. (2) should stimulate continued efforts aimed at resolving the direct detrimental metabolic effects of AAPs that may ultimately lead to improved treatment options for these patients.\n\n## ACKNOWLEDGMENTS\n\nNo potential conflicts of interest relevant to this article were reported.\n\n# REFERENCES","meta":{"dup_signals":{"dup_doc_count":133,"dup_dump_count":49,"dup_details":{"curated_sources":2,"2022-27":2,"2022-21":1,"2022-05":1,"2021-17":1,"2020-29":1,"2018-26":1,"2018-22":3,"2018-13":2,"2018-09":1,"2018-05":1,"2017-51":2,"2017-47":2,"2017-43":5,"2017-39":2,"2017-34":2,"2017-30":7,"2017-26":3,"2017-22":2,"2017-17":9,"2017-09":4,"2017-04":8,"2016-50":3,"2016-44":3,"2016-40":3,"2016-36":3,"2016-30":3,"2016-26":3,"2016-22":3,"2016-18":2,"2016-07":2,"2015-48":2,"2015-40":3,"2015-35":2,"2015-32":3,"2015-27":2,"2015-22":2,"2015-14":2,"2014-52":3,"2014-49":4,"2014-42":2,"2014-41":3,"2014-35":4,"2014-23":2,"2023-50":1,"2017-13":3,"2015-18":3,"2015-11":2,"2015-06":2,"2024-30":1}},"file":"PMC3749356"},"subset":"pubmed_central"}
{"text":"date: 2017-03\ntitle: EARLY CANCER DIAGNOSIS SAVES LIVES, CUTS TREATMENT COSTS\n\n**3 FEBRUARY 2017 \\| GENEVA -** New guidance from WHO, launched ahead of World Cancer Day (4 February), aims to improve the chances of survival for people living with cancer by ensuring that health services can focus on diagnosing and treating the disease earlier.\n\nNew WHO figures released this week indicate that each year 8.8 million people die from cancer, mostly in low- and middle-income countries. One problem is that many cancer cases are diagnosed too late. Even in countries with optimal health systems and services, many cancer cases are diagnosed at an advanced stage, when they are harder to treat successfully.\n\n\"Diagnosing cancer in late stages, and the inability to provide treatment, condemns many people to unnecessary suffering and early death,\" says Dr Etienne Krug, Director of WHO's Department for the Management of Noncommunicable Diseases, Disability, Violence and Injury Prevention.\n\n\"By taking the steps to implement WHO's new guidance, healthcare planners can improve early diagnosis of cancer and ensure prompt treatment, especially for breast, cervical, and colorectal cancers. This will result in more people surviving cancer. It will also be less expensive to treat and cure cancer patients.\"\n\nAll countries can take steps to improve early diagnosis of cancer, according to WHO's new Guide to cancer early diagnosis.\n\n**The three steps to early diagnosis are:**\n\nImprove public awareness of different cancer symptoms and encourage people to seek care when these arise.\n\nInvest in strengthening and equipping health services and training health workers so they can conduct accurate and timely diagnostics.\n\nEnsure people living with cancer can access safe and effective treatment, including pain relief, without incurring prohibitive personal or financial hardship.\n\nChallenges are clearly greater in low- and middle-income countries, which have lower abilities to provide access to effective diagnostic services, including imaging, laboratory tests, and pathology \u2013 all key to helping detect cancers and plan treatment. Countries also currently have different capacities to refer cancer patients to the appropriate level of care.\n\nWHO encourages these countries to prioritize basic, high-impact and low-cost cancer diagnosis and treatment services. The Organization also recommends reducing the need for people to pay for care out of their own pockets, which prevents many from seeking help in the first place.\n\nDetecting cancer early also greatly reduces cancer's financial impact: not only is the cost of treatment much less in cancer's early stages, but people can also continue to work and support their families if they can access effective treatment in time. In 2010, the total annual economic cost of cancer through healthcare expenditure and loss of productivity was estimated at US\\$ 1.16 trillion.\n\nStrategies to improve early diagnosis can be readily built into health systems at a low cost. In turn, effective early diagnosis can help detect cancer in patients at an earlier stage, enabling treatment that is generally more effective, less complex, and less expensive. For example, studies in high-income countries have shown that treatment for cancer patients who have been diagnosed early are 2 to 4 times less expensive compared to treating people diagnosed with cancer at more advanced stages.\n\nDr Oleg Chestnov, WHO Assistant Director-General for Noncommunicable Diseases and Mental Health, notes: \"Accelerated government action to strengthen cancer early diagnosis is key to meet global health and development goals, including the Sustainable Development Goals (SDGs).\"\n\nSDG 3 aims to ensure healthy lives and promote well-being for all at all ages. Countries agreed to a target of reducing premature deaths from cancers and other noncommunicable diseases (NCDs) by one third by 2030. They also agreed to achieve universal health coverage, including financial risk protection, access to quality essential health-care services, and access to safe, effective, quality and affordable essential medicines and vaccines for all. At the same time, efforts to meet other SDG targets, such as improving environmental health and reducing social inequalities can also help reduce the cancer burden.\n\nCancer is now responsible for almost 1 in 6 deaths globally. More than 14 million people develop cancer every year, and this figure is projected to rise to over 21 million by 2030. Progress on strengthening early cancer diagnosis and providing basic treatment for all can help countries meet national targets tied to the SDGs.\n\nAvailable from: <http:\/\/www.who.int\/mediacentre\/news\/releases\/2017\/early-cancer-costs\/en\/>","meta":{"dup_signals":{"dup_doc_count":171,"dup_dump_count":59,"dup_details":{"curated_sources":2,"2023-50":2,"2023-40":1,"2023-23":2,"2023-06":3,"2022-49":2,"2022-40":1,"2022-33":2,"2022-27":2,"2022-21":2,"2022-05":2,"2021-43":3,"2021-31":2,"2021-25":2,"2021-21":2,"2021-17":1,"2021-10":2,"2020-50":2,"2020-45":1,"2020-34":1,"2020-29":3,"2020-24":5,"2020-16":2,"2020-10":2,"2019-51":3,"2019-47":3,"2019-43":1,"2019-39":2,"2019-30":3,"2019-26":1,"2019-22":2,"2019-13":4,"2019-09":1,"2019-04":3,"2018-51":1,"2018-47":3,"2018-43":3,"2018-39":1,"2018-34":3,"2018-30":3,"2018-26":3,"2018-22":6,"2018-17":3,"2018-13":6,"2018-09":5,"2018-05":4,"2017-51":5,"2017-47":6,"2017-43":6,"2017-39":4,"2017-34":5,"2017-30":4,"2017-26":5,"2017-22":6,"2017-17":6,"2017-09":6,"2024-26":1,"2024-10":2,"2017-13":1,"2024-30":1}},"file":"PMC5387916"},"subset":"pubmed_central"}
{"text":"abstract: A report on the 44th Annual *Drosophila* Research Conference, Chicago, USA, 5-9 March, 2003.\nauthor: Stephen Richards\ndate: 2003\ninstitute: 1Human Genome Sequencing Center, Department of Human and Molecular Genetics, Baylor College of Medicine, Houston, TX 77006, USA. E-mail: email@example.com\ntitle: How is the *Drosophila* research community making use of the genome sequence?\n\n*Drosophila* researchers have capitalized on the genome sequence in the three years since it was released. From identifying paralogs and orthologs to microarray analysis of specific cell types, it is clear that the genome sequence is changing approaches to *Drosophila* research. In this review of the 2003 *Drosophila* research conference, I have focused on talks illustrating the various ways in which the genome sequence is being used.\n\nThe Larry Sandler Award is presented annually to the graduate student with the best thesis that uses *Drosophila* as a model system. This year's recipient, Sinisa Urban (University of Cambridge, UK), gave perhaps the best presentation of the meeting. His thesis focused on how the cell controls the release of the Spitz epidermal growth factor (EGF) signal. In *Drosophila*, a single EGF receptor is used repeatedly in over 60 different contexts; control of how the signal is released is therefore critical. Spitz is a transmembrane protein, which must be cleaved by a protease to release the signaling portion for secretion. Firstly, Urban presented biochemical data showing that cleavage of the Spitz protein by Rhomboid occurs in the Golgi apparatus and is followed by glycosylation and secretion of the Spitz signaling portion. Secondly, using biochemical and mutagenic approaches, he demonstrated that Rhomboid is a serine protease: it contains the residues necessary for serine protease catalysis, and is inhibited by known serine protease inhibitors. He also identified a family of seven Rhomboid-like proteins in *Drosophila* and additional Rhomboid-like proteins in species as diverse as the Gram-negative bacteria *Pseudomonas aeruginosa* and *Providencia stuartii.* Finally, he discussed the cleavage site in the Spitz protein: a seven-amino-acid sequence, ASIASGA in the single-letter amino-acid code, in a transmembrane region of the Spitz protein. This motif is also present in the TGF\u03b1 and Delta signaling proteins, suggesting they may also be substrates for Rhomboid cleavage. The ASIASGA motif seems to have two functions that allow it to be cleaved by Rhomboid: it produces a kink in the transmembrane \u03b1 helix and it forms a hydrophilic pocket at the top of the helix allowing water, which is necessary for protease activity, to enter the cleavage site.\n\nMichelle Markstein (University of California, Berkeley, USA) used a computational method (Fly Enhancer <http:\/\/flyenhancer.org>) to search the genome sequence for clusters of enhancers that are targets of the Dorsal transcription factor. Looking for clusters of enhancer sequences appears to improve the sensitivity of such methods and has allowed the identification of approximately one third of the genes estimated to be directly affected by Dorsal. Besides known targets such as *zen, sog* and *brinker*, she found novel targets, including *Phm*, *Ady* and CG12443; these were confirmed by embryonic *in situ* hybridization and expression of *lacZ* under the control of the putative enhancer. Interestingly, it seems that clusters of different enhancer binding sequences may be more diagnostic for the identification of *cis*-control regions than clusters of a single binding site.\n\nA number of groups described research using microarray analysis. Ulrike Gaul (Rockefeller University, New York, USA) presented an analysis of glial cell transcription. Glial cells labeled with green fluorescent protein under the control of the *repo* promoter were chemically dissociated from embryos and sorted by fluorescence-activated cell sorting (FACS). Gene expression in glial and non-glial cell fractions was assessed using an Affymetrix gene array, and 255 strongly expressed genes were identified. CG11Q10 is expressed only in midline and longitudinal glia; reduction of the transcript level by RNA interference (RNAi) prevents midline glial cells from separating axon tracks in embryonic commissures. Other examples of new genes found in this screen include molecules affecting axon guidance, cell migration and shape, and axon wrapping. The combination of microarray analysis and RNAi provides a new paradigm for rapid screening.\n\nAmir Orian (Fred Hutchinson Cancer Center, Seattle, USA) has investigated the binding sites of the Myc-Max-Mad (MMM) transcription factor complex. Fusions of *Dam* methylase to these proteins were introduced into transgenic flies, then genomic DNA was digested with a methylation-sensitive restriction enzyme and the fragments were analyzed on a microarray. Interestingly, methylation of genes encoding synaptic-vesicle and mitochondrial proteins was observed, suggesting that the MMM complex may exert previously unknown influences on these processes.\n\nGreg Gibson (North Carolina State University, Raleigh, USA) used long-oligonucleotide arrays to study the inheritance of gene expression. Gene expression was measured in seven strains of *D. melanogaster* and all F1 progeny of crosses between those strains. His data show that approximately 10% of genes are differentially expressed between any two of the strains studied, and that 20% of genes are expressed differently in the F1 compared to the parental strains. It is possible to divide these differences into several classes: some are expected, such as additive, dominant and recessive patterns of inheritance of the expression level; in other cases, the level of gene expression in the F1 is significantly greater or less than can be explained by additive expression of both parental strains.\n\nMany researchers are making use of the expanding *Drosophila* gene collection. Mark Stapleton (Lawrence Berkeley National Laboratory, Berkeley, USA) identified RNA-editing substrates by comparing the high-quality cDNA and genomic sequences. He found 27 adenosine deaminase substrates, the majority of which are ion-channel transcripts. Pavel Tomancak (University of California, Berkeley, USA) presented a comparison of *D. melanogaster* and *D. pseudoobscura* embryonic expression patterns for a number of genes. The vast majority of the 176 genes investigated showed identical expression patterns in the two species. But two genes with different expression patterns were identified. The expression of the midline fasciclin transcript is moved from the neuroectoderm in *D. melanogaster* to the mesoderm in *D. pseudoobscura. Ecdysone-inducible gene E2* (described in a poster presented by Amy Beaton, University of California, Berkeley, USA) is expressed in the anterior of early embryos and in the developing foregut by stage 11 in *D. melanogaster*, but in *D. pseudoobscura* it is expressed in the posterior of early embryos and in the developing hindgut by stage 11.\n\nLaura Lee (Massachusetts Institute of Technology, Cambridge, USA) identified seven novel substrates for Pan gu, a protein kinase required early in the cell cycle during embryogenesis. Her biochemical screen made use of coupled transcription-translation of cDNA clones from the *Drosophila* gene collection to produce \\[^35^S\\]-labeled proteins in a 384-well format. Pools of 24 proteins were then screened in a variety of binding, degradation and enzymatic assays. Examples include screens for Disheveled-binding proteins, microtubule-binding proteins and the Pan gu kinase assay based on band shirts on electrophoretic gels.\n\nOne talk highlighted the imprecise art of gene prediction. Marc Hild (University of Heidelberg, Germany) presented a microarray constructed using a less stringent gene-prediction program and a possible 21,396 putative ORFs. Expression data aquired using this array suggests that there are 3,000 more *Drosophila* genes than were predicted in the Release 3 version of the genome. Some of these sequences produce phenotypes in S2 tissue culture cells when inhibited by RNAi. Once the data are made public and analyzed in detail, many of these 'novel' genes will no doubt be found to have exons that overlap those of previous predictions. Other differences may be 'philosophical': for example, should a gene prediction be considered if it has an open reading frame of less than 100 amino acids? It is clear that biological evidence is required to positively identify a gene.\n\nThe *D. pseudoobscura* sequence, available from the Drosophila Genome Project <http:\/\/hgsc.bcm.tmc.edu\/projects\/drosophila>, may be the surest way to identify the meaningful sequences of the *D. melanogaster* genome. Richard Gibbs (Baylor College of Medicine, Houston, USA) presented the initial release of the *D. pseudoobscura* sequence. A tBLASTn comparison of the two *Drosophila* genomes identified putative orthologs in *D. pseudoobscura* for 95% of *D. melanogaster* genes. Alignment of the two genomic sequences identified both large features, such as chromosomal inversions, and small ones, such as conserved non-coding regions. A comparative genomic approach using both sequences will improve gene prediction and allow the identification of *cis*-regulatory sequences for the majority of *Drosophila* genes. We can hope that the sequence of *D. pseudoobscura* will be as informative to *Drosophila* research as that of *D. melanogaster*, and many presentations on 'the other *Drosophila*' can be expected at future annual conferences.","meta":{"dup_signals":{"dup_doc_count":122,"dup_dump_count":44,"dup_details":{"curated_sources":3,"2023-14":1,"2020-40":1,"2019-51":1,"2019-47":1,"2019-09":1,"2019-04":1,"2018-51":1,"2018-43":1,"2018-34":1,"2018-22":1,"2018-05":1,"2017-39":1,"2017-30":1,"2017-22":1,"2017-09":10,"2016-44":1,"2016-40":1,"2016-36":10,"2016-30":9,"2016-22":1,"2016-18":1,"2016-07":5,"2015-48":3,"2015-40":2,"2015-35":3,"2015-32":3,"2015-27":3,"2015-22":3,"2015-14":2,"2014-52":3,"2014-49":4,"2014-42":8,"2014-41":4,"2014-35":4,"2014-23":6,"2014-15":2,"2023-50":1,"2015-18":4,"2015-11":2,"2015-06":3,"2014-10":2,"2013-48":2,"2013-20":2,"2024-26":1}},"file":"PMC156583"},"subset":"pubmed_central"}
{"text":"abstract: A report on the 13th International Conference on Systems Biology, held in Toronto, Ontario, Canada, 19-23 August 2012.\nauthor: Adam P Rosebrock; Amy A Caudy\ndate: 2012\ninstitute: 1Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 160 College St., Toronto, Ontario, Canada, M5S3E1; 2Department of Molecular Genetics, University of Toronto, 160 College St., Toronto, Ontario, Canada, M5S3E1\ntitle: The future of deciphering personal genomes? The flies (and yeast and worms) still have it\n\n# Introduction\n\nSydney Brenner said in his 2002 Nobel lecture that we are 'drowning in a sea of data, starving for knowledge'. Drafts of the human genome sequence were barely a year old at the time, and even optimistic projections placed the \\$1,000 genome beyond 2040. Just a decade after Brenner's lecture, technological revolutions in sequencing and protein analysis have changed the pace of genome biology. The reference genome has given way to individual patient sequences. Sydney's sea of data is becoming ever deeper.\n\nNew tools are necessary to extract biological meaning from these data. The diverse group of scientists gathered in systems biology is at the forefront of devising new ways to net knowledge out of a data sea that widens and deepens every day. The flood of data produced by modern 'omics technologies demands new methods that connect genotype to phenotypic consequence. With the \\$1,000 genome now predicted by the end of 2012, we are faced with the challenge of interpreting raw sequence into health-relevant information.\n\nHere, we cover some of the highlights of an inherently diverse meeting, focusing on the role of systems biology as relevant to genomic medicine (abstracts are freely available online: <http:\/\/www.icsb2012toronto.com\/schedule.php>).\n\n# Biological systems are dynamic; systems biologists are catching up\n\nTwenty-three pairs of chromosomes within a single cell give rise to hundreds of tissues with diverse functions, morphologies and biochemical activities. The humble budding yeast is born, has a fruitful replicative life on a variety of foods, and eventually succumbs to senescence. Life consists of a series of changing processes that cannot be explained by a snapshot view; models that accurately represent biological processes must be built from data that capture these dynamics.\n\nAdvances in mass spectrometry have enabled examination of rare and modified proteins; modified proteins are turning out to be not rare at all. Kirti Sharma in Matthias Mann's lab (Max Planck Institute of Biochemistry, Martinsreid, Germany) claims that 75% of the proteins in cancer cells are present as phosphorylated forms, and that this is likely to be a vast underestimate, with current methods capturing only one-tenth of the phosphopeptides in the cell.\n\nA major focus of systems biology is understanding the interaction between biomolecules. Genetic approaches, including systematic identification of synthetic lethal gene deletions (championed by Charley Boone at the University of Toronto, ON, Canada), are being supplemented by measurement of physical interactions. These efforts are focused on reducing the massive disconnect between genome sequence and physical function.\n\nMany key proteins in the cell are interaction hubs and form complexes with many different partners. Interactions are frequently mutually exclusive or separated in space, time, or by cellular state. Anne-Claude Gingras (Samuel Lunenfeld Research Institute, Toronto, ON, Canada) and Andrew Emili (University of Toronto, Toronto, ON, Canada) are working to move from a simple static representation of protein complexes to an understanding of these dynamic interactions. Gingras is interrogating protein interactions by affinity purification of different components of a single complex. Emili has taken a global approach by analyzing the entire proteome following multiple orthogonal separation methods and identifying complexes by common separation patterns. Michael Washburn (Stowers Institute, Kansas City, MO, USA) has been using mass spectrometry to address questions once reserved for structural biology. Washburn has used affinity purification and mass spectrometry to measure protein interactions in partially disassociated complexes. Interactions that survive are likely to occur between directly contacting molecules.\n\nWeak interactions are common and essential in biological systems. Mike Tyers (IRIC, Universit\u00e9 de Montr\u00e9al, QC, Canada) reminded listeners that without weak interactions, we would be stones. Tyers' work demonstrates that multiple weak sites provide a mechanism for buffering, redundancy and cooperative kinetic behavior. Disruption of individual weak-functioning residues can frequently be tolerated, confounding current mutation-counting sequencing efforts.\n\n# Model organisms drive understanding of genotype to phenotype\n\nStudies in model organisms can answer questions that have proved to be difficult to study in man. Missing heritability is one of the biggest problems in human genome-wide association studies. Joshua Bloom (Princeton University, Princeton, NJ, USA) mapped dozens of quantitative trait loci in yeast. They were able to account for more than 90% of the heritability of these traits, suggesting that the failure to map quantitative traits in human populations results from the diversity of alleles and the influence of environment rather than a problem with the approach of mapping.\n\nEnrolling more patients is a common way to increase the power of genome-wide association studies. Two talks at the meeting presented a systems-biology alternative. Chad Myers (University of Minnesota, Minneapolis, MN, USA) and Manuel Mattheisen (Brigham and Williams Hospital, Boston, MA, USA) demonstrated approaches that group genes prior to testing for significant variants. Both researchers used functional groupings of genes based largely on data from model systems. Model organisms continue to be a critical scaffold for understanding biology.\n\nBen Lehner (ICREA, Barcelona, Spain) highlighted how chance controls life. *Caenorhabditis elegans* harboring null alleles of Tbx-9, a transcription factor regulating development, have a fifty-fifty chance of death or completely normal life. Lehner's group has identified fluctuations in gene expression within this isogenic population that modulate this life and death coin-toss. In our world of Brownian motion and stochastic gene expression, deterministic sequence to phenotype analysis will remain the stuff of science fiction.\n\n# 'Personal genomes' are of more value to the community than the patient\n\nGeneticist Sewall Wright once said that natural variation is the worst kind of experiment to study. Wright, the father of genetic drift and the fitness landscape, had a prescient understanding of our predicament. Mike Snyder (Stanford University, Stanford, CA, USA) gave an update on the Snyderome, a longitudinal study of his transcriptome, proteome, metabolome and other physiological parameters in what may well be the most expensive case study in history. Next-generation sequencing has become Snyder's 'Ouiji board'. His risk allele for diabetes may have manifested itself in an episode of elevated blood glucose, which Snyder (possibly) corrected by a wholesale elimination of sweets. Fortunately for Snyder, none of his other disease risk alleles have been borne out or have required such radical life changes. Personal genome sequencing is here, but the interpretation is difficult, and as Snyder advises, not for worriers.\n\nAccording to Chris Saunder (Memorial Sloan Kettering Cancer Center, New York, NY, USA), genome medicine is sequence-rich and phenotype-poor. Saunder remarked that the erudite molecular profiling of the Cancer Genome Atlas and related efforts frequently lacks accompanying data on phenotype, cautioning that careful and deep phenotyping is as important as sequencing itself. George Church (Harvard University, Cambridge, MA, USA) highlighted efforts coordinated through the Personal Genomics Project to provide such rich phenotypic information. This dataset of genomes coupled with phenotype will be useful in understanding the scope of variation, but Church cautioned that the diagnostic value of genomes is limited. Like a stethoscope, sequencing is just one source of health information.\n\nIdentification of causative alleles is particularly critical in cancer; cells that appear in relapse are often from a different or earlier lineage than cells in the original tumor. John Dick (Princess Margaret Hospital, Toronto, ON, Canada) described work to identify causative mutations in genomes using xenografts. As in relapsing patients, many of the tumors that grew in mice are genetically different from the primary tumor. Dick's work questions the value of a 'tumor genome' derived from bulk cells, emphasizing the need to identify rare cells that can form new tumors. Personalized medicine may well become cell-, not patient-, specific.\n\nDave Hill (CCSB, Boston, MA, USA) reminded attendees that many proteins are multifunctional. Patient-specific mutations will affect some or many of a diverse array of protein interaction partners. To move from sequence to diagnosis and prognosis, one needs to understand these complex interactions. Hill cautioned that early successes with Mendelian traits will not translate into rapid understanding of complex genetic interactions.\n\nThe most remarkable personal genome, though, was that of only half a human. Thijn Brummelkamp (Netherlands Cancer Institute, Amsterdam, The Netherlands) presented a series of functional genetic screens using a haploid somatic human cell line. Brummelkamp used traditional insertional mutagenesis to identify essential human genes, bringing classic tools of model organism research to human genetic analysis.\n\n# In addition to new tools, systems biology requires new ways of training tomorrow's scientists\n\nRadical advances in technology have driven systems biology in the past decade. George Church highlighted the phenomenal growth of sequencing technology, noting that 44 commercial sequencing technologies are now available or soon will be. New sequencing technologies will be cheaper, faster and more accurate, and will permit new types of analyses.\n\nThe diverse skills required by systems biology require development of a new kind of scientist. David Botstein (Princeton University, Princeton, NJ, USA) accepted an award for excellence in quantitative education for his creation of a new teaching paradigm. Botstein's view is that students languish in prerequisite general courses while thirsting for access to current research and educational specialization. Contrary to the status quo, Botstein envisions research and teaching as organically connected tasks. This integrated training approach is necessary to bridge the significant cultural gap between biologists, mathematicians and computer scientists. Students should be taught current scientific practice, not scientific history. If the acceleration in systems biology continues, merely keeping up with the present will be a monumental task.","meta":{"dup_signals":{"dup_doc_count":108,"dup_dump_count":58,"dup_details":{"curated_sources":2,"2022-21":1,"2021-43":1,"2021-31":1,"2021-17":1,"2020-40":1,"2019-30":1,"2019-18":1,"2019-13":1,"2019-04":2,"2018-47":2,"2018-39":2,"2018-30":2,"2018-22":2,"2018-17":1,"2018-13":2,"2018-09":1,"2017-51":1,"2017-47":1,"2017-43":1,"2017-39":2,"2017-34":2,"2017-30":1,"2017-26":2,"2017-22":2,"2017-17":2,"2017-09":2,"2017-04":2,"2016-50":2,"2016-44":2,"2016-40":2,"2016-36":2,"2016-30":2,"2016-26":1,"2016-22":1,"2016-18":1,"2016-07":2,"2015-48":2,"2015-40":2,"2015-35":2,"2015-32":2,"2015-27":2,"2015-22":2,"2015-14":2,"2014-52":2,"2014-49":1,"2014-42":6,"2014-41":3,"2014-35":4,"2014-23":3,"2014-15":4,"2023-23":1,"2017-13":2,"2015-18":2,"2015-11":2,"2015-06":2,"2014-10":2,"2013-48":2,"2024-18":1}},"file":"PMC3580444"},"subset":"pubmed_central"}
{"text":"abstract: Detailed comprehensive molecular analysis using families and multiple matched tissues is essential to determine whether imprinted genes have a functional role in humans.\n  .\n  See research article: <http:\/\/genomebiology.com\/2011\/12\/3\/R25>\nauthor: Gudrun Moore; Rebecca Oakey\ndate: 2011\ninstitute: 1Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK; 2Department of Medical and Molecular Genetics, Kings College London, 8th Floor Tower Wing, London SE1 9RT, UK\nreferences:\ntitle: The role of imprinted genes in humans\n\n# Research highlight\n\nImprinted or parent-of-origin-dependent gene expression has over the past 25 years developed into an exciting and dynamic research field. Its functional or even evolutionary importance is considered most relevant in mammals and in flowering plants \\[1\\]. In mammals the link to the existence of the placenta and the differences between the two parental sexes in terms of resources and evolutionary drive through imprinting has been the focus of much debate. One fundamental question remains: has parent-of-origin gene expression evolved and been maintained because of the different needs of the mother and father in producing viable, strong offspring? The mother needs to survive the pregnancy but the father's drive is focused on the offspring being the fittest. Much of the functional relevance of the research in the imprinting field, particularly with its application to the human, has grown out of this 'resources for fittest' debate. A study in this issue of *Genome Biology* \\[2\\] starts to analyze more thoroughly which genes are truly imprinted in humans using genome-wide assessment.\n\nImprinting in the mouse is well understood. It was discovered separately by the Surani \\[3\\] and McGrath \\[4\\] groups in the early 1980 s, who found that gynogenetic embryos (which contain only maternal genomes) developed differently *in utero* and with emphasis on different tissues to the androgenotes (only paternal genomes). Interestingly, the androgenotes had a more developed placenta and the gynogenotes had a better developed embryo. Links were soon made between imprinted gene models in the mouse and human diseases, imprinted genes were implicated in many fetal growth syndromes, and they were shown to regulate maternal-fetal interactions, postnatal feeding behaviors and neurological development. Disturbance of the apparently rigorous mono-allelic imprinted gene expression was also linked to cancer, and alterations in imprinting methylation patterns or expression in peripheral blood leukocytes were considered as biomarkers for cancer \\[5\\].\n\nThe study by Morcos *et al.* \\[2\\] extends this human analysis comparison further. Here the authors \\[2\\] make a genome-wide assessment of imprinted expression in paired sets of samples of adult human tissue, comparing lymphoblastoid cell lines with primary fibroblasts. These two cell lines are both relatively easy to obtain from humans with ethical approval. Using families they could track parental-allele-specific expression, and using paired tissue samples they could study tissue-specific variation between lymphoblastoid cells and fibroblasts. To truly confirm whether a gene is imprinted, differential methylation, tissue-specific expression and parental allele origin must all be tracked in the same family. Observing differentiated methylated patterns in isolation, however, does not always totally reflect monoallelic expression \\[6\\]. These all-inclusive experiments can be done relatively easily in mouse but are ethically impossible to copy in humans. These authors \\[2\\] have achieved the best compromise by using matched tissues and by studying families. Their results are both interesting and intriguing.\n\nPrevious careful comparative analysis between the imprinted genes in mouse and humans showed that roughly half of the mouse imprinted genes are either not or never have been imprinted in humans. Of the about 140 imprinted genes identified so far in the mouse, only 60 are imprinted in humans and several are specific to humans. In addition, some have different tissue-specific expression profiles; for example, growth factor receptor binding protein 10 (*GRB10*) in humans is imprinted only in invasive trophoblasts (maternally expressed) and brain (paternally expressed) \\[7\\], whereas in mouse it is maternally expressed in most embryonic tissues and predominantly paternally expressed in brain \\[8\\]. If the regulation of gene dosage is so important, why is there not greater conservation of imprinted expression? Or maybe the genes still imprinted in humans have been selected and\/or maintained for important reasons. Epigenetics provides the mechanisms through which imprinting influences gene expression. These mechanisms affect the processes of cell differentiation and embryonic growth, although they are as yet not completely understood. When epigenetic mechanisms go awry, transcriptional activity may be perturbed and result in disorders and syndromes. This underscores the rationale for studies such as these \\[2\\], particularly on a genome-wide scale, for identifying imprinted genes and classifying their conservation across mammalian species.\n\nIn the study by Morcos *et al.* \\[2\\], of the 44 informative imprinted genes from the literature that were analyzed, 19 were validated as imprinted using this rigorous assessment. More importantly, only 1 in 13 candidate imprinted genes were confirmed. This demonstrates again that only over 50% of mouse imprinted genes are truly imprinted in humans in the adult tissues assayed and only 10% of candidates can be verified.\n\nOne caveat of this approach stems from the fact that human embryonic tissues are extremely difficult to access and thus the authors \\[2\\] used lymphoblastoid cells and fibroblasts instead; this has some limitations. It is known that imprinting is important in the developing embryo and fetus and typically occurs in a tissue-specific manner. So the use of transformed lymphoblastoid cells as a human tissue resource does not necessarily reflect the *in situ* state. It could be argued that the true role of imprinted genes is in fetal development but, even so, analysis of fetal tissues and placenta has also revealed much lower numbers of imprinted genes in humans than in mouse \\[9\\]. In humans there are fewer imprinted genes and these may be the ones that are most relevant for the 'resources for fittest' needs that are most important in human fetal growth.\n\nThis study \\[2\\] plus other work on human tissues in this dynamic field are all helping to clarify the numbers of imprinted genes in humans and lead towards an understanding of the role of imprinting in humans. There remains no doubt that gene dosage control in the developmental period is exquisitely sensitive and needs accurate control mechanisms. The future focus in humans needs to be on careful dissection of the function of those genes that are confirmed to be imprinted using methods similar to those in this study \\[2\\].","meta":{"dup_signals":{"dup_doc_count":107,"dup_dump_count":35,"dup_details":{"curated_sources":1,"2023-14":1,"2021-43":1,"2020-24":1,"2019-47":1,"2019-22":1,"2019-09":1,"2018-51":1,"2018-43":1,"2018-34":1,"2018-26":1,"2017-43":1,"2017-22":1,"2017-09":10,"2016-44":1,"2016-40":1,"2016-36":10,"2016-30":8,"2016-22":1,"2016-18":1,"2016-07":6,"2015-48":3,"2015-40":3,"2015-35":3,"2015-32":3,"2015-27":3,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":4,"2014-42":7,"2014-41":6,"2014-35":5,"2014-23":5,"2014-15":4,"2023-50":1}},"file":"PMC3129664"},"subset":"pubmed_central"}
{"text":"date: 2014-01-10\ntitle: Findings of research misconduct.\n\n# Findings of Research Misconduct\n\n**Notice Number:** NOT-OD-14-037\n\n**Key Dates  \nRelease Date:** January 10, 2014\n\n**Related Announcements  \n** None\n\nNone\n\n**Issued by  \n** Department of Health and Human Services ([DHHS](http:\/\/www.hhs.gov\/))\n\nPurpose\n\nNotice is hereby given that the Office of Research Integrity (ORI) has taken final action in the following case:\n\nDong-Pyou Han, Ph.D., Iowa State University of Science and Technology: Based on the report of an inquiry conducted by the Iowa State University of Science and Technology (ISU), a detailed admission by the Respondent, and additional analysis conducted by ORI, ORI and ISU found that Dr. Dong-Pyou Han, former Research Assistant Professor, Department of Biomedical Services, ISU, engaged in research misconduct in research supported by National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), grants P01 AI074286, R33 AI076083, and U19 AI091031.\n\nORI and ISU found that the Respondent falsified results in research to develop a vaccine against human immunodeficiency virus-1 (HIV-1) by intentionally spiking samples of rabbit sera with antibodies to provide the desired results. The falsification made it appear that rabbits immunized with the gp41-54 moiety of the HIV gp41 glycoprotein induced antibodies capable of neutralizing a broad range of HIV-1 strains, when the original sera were weakly or non-reactive in neutralization assays. Falsified neutralization assay results were widely reported in laboratory meetings, seven (7) national and international symposia between 2010 and 2012, and in grant applications and progress reports P01 AI074286-03, -04, -05, and -06; R33 AI076083-04; U19 AI091031-01 and -03; and R01 AI090921-01. Specifically:\n\na\\. Respondent falsified research materials when he provided collaborators with sera for neutralization assays from (i) rabbits immunized with peptides from HIV gp41-54Q (and related antigens HR1-54Q, gp41-54Q-OG, gp41-54Q-GHC, gp41-54Q-Cys and Cys-gp41-54Q) to assay HIV neutralizing activity, when Respondent had spiked the samples with human IgG known to contain broadly neutralizing antibodies to HIV-1; and (ii) rabbits immunized with HIV gp41-54Q to assay HIV neutralizing activity, when Respondent had spiked the samples with sera from rabbits immunized with HIV-1 gp120 that neutralized HIV.\n\nb\\. Respondent falsified data files for neutralization assays, and provided false data to his laboratory colleagues, to make it appear that rabbits immunized with gp41-54Q and recombinant Lactobacillus expressing gp41-64 (LAB gp41-64) produced broadly reactive neutralizing antibodies, by changing the numbers to show that samples with little or no neutralizing activity had high activity.\n\nDr. Han has entered into a Voluntary Exclusion Agreement and has voluntarily agreed for a period of three (3) years, beginning on November 25, 2013:\n\n\\(1\\) To exclude himself from any contracting or subcontracting with any agency of the United States Government and from eligibility or involvement in nonprocurement programs of the United States Government referred to as \\`\\`covered transactions' pursuant to HHS' Implementation (2 CFR Part 376 et seq) of OMB Guidelines to Agencies on Governmentwide Debarment and Suspension, 2 CFR Part 180 (collectively the \\`\\`Debarment Regulations'); and\n\n\\(2\\) To exclude himself voluntarily from serving in any advisory capacity to the U.S. Public Health Service (PHS) including, but not limited to, service on any PHS advisory committee, board, and\/or peer review committee, or as a consultant.\n\nInquiries\n\nPlease direct all inquiries to:**  \n  \n** David E. Wright, Ph.D.**  \n** Director**  \n** Office of Research Integrity**  \n** 1101 Wootton Parkway, Suite 750**  \n** Rockville, MD 20852**  \n** Telephone: 240-453-8800","meta":{"dup_signals":{"dup_doc_count":109,"dup_dump_count":41,"dup_details":{"curated_sources":2,"2021-21":1,"2020-45":1,"2019-35":1,"2019-30":1,"2018-51":1,"2018-30":1,"2018-17":1,"2018-05":1,"2017-30":1,"2017-22":1,"2017-17":1,"2017-09":2,"2017-04":1,"2016-50":1,"2016-44":2,"2016-40":2,"2016-36":2,"2016-30":3,"2016-26":3,"2016-22":2,"2016-18":3,"2016-07":5,"2015-48":5,"2015-40":4,"2015-35":6,"2015-32":3,"2015-27":5,"2015-22":5,"2015-14":4,"2014-52":3,"2014-49":2,"2014-42":2,"2014-41":6,"2014-35":2,"2014-23":3,"2014-15":1,"2021-31":1,"2015-18":5,"2015-11":5,"2015-06":4,"2014-10":4}},"file":"PMC4259752"},"subset":"pubmed_central"}
{"text":"abstract: For the first time in history, psychiatrists during the Nazi era sought to systematically exterminate their patients. However, little has been published from this dark period analyzing what may be learned for clinical and research psychiatry. At each stage in the murderous process lay a series of unethical and heinous practices, with many psychiatrists demonstrating a profound commitment to the atrocities, playing central, pivotal roles critical to the success of Nazi policy. Several misconceptions led to this misconduct, including allowing philosophical constructs to define clinical practice, focusing exclusively on preventative medicine, allowing political pressures to influence practice, blurring the roles of clinicians and researchers, and falsely believing that good science and good ethics always co-exist. Psychiatry during this period provides a most horrifying example of how science may be perverted by external forces. It thus becomes crucial to include the Nazi era psychiatry experience in ethics training as an example of proper practice gone awry.\nauthor: Rael D Strous\ndate: 2007\ninstitute: 1Department of Psychiatry, Beer Yaakov Mental Health Center, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel\nreferences:\ntitle: Psychiatry during the Nazi era: ethical lessons for the modern professional\n\n# Background\n\nDuring the Nazi era, for the first time in history, psychiatrists sought to systematically exterminate their patients. It has been acknowledged that the medical profession was profoundly involved in crimes against humanity during this period, with various publications describing this malevolent period of medical history. It is less known, however, that psychiatrists were among the worst transgressors. At each stage of the descent of the profession into the depths of criminal and genocidal clinical practice lay a series of unethical decisions and immoral professional judgments. Furthermore, very little has been published on lessons that may be learned from this dark period in the history of psychiatry and on ethical principles that may be extrapolated for the future practice of clinical and research psychiatry and for inclusion in educational programs. This paper reviews the role of psychiatrists in the Nazi era and analyzes the underlying misconceptions that led to the aberrant behavior. Finally, some recommendations for inclusion of the study of this period in ethics training are presented \\[26\\].\n\n# Role of psychiatrists in Nazi atrocities\n\nThe professional status of psychiatrists did not place any obstacle to their participation in Nazi crimes, and many demonstrated a profound commitment to the atrocities. Psychiatrists were instrumental in instituting a system of identifying, notifying, transporting, and killing hundreds of thousands of mentally ill and \"racially and cognitively compromised\" individuals in settings ranging from centralized psychiatric hospitals to prisons and death camps. Their role was *central* and *critical* to the success of Nazi policy, plans, and principles. Psychiatrists, along with many other physicians, facilitated the resolution of many of the regime's ideological and practical challenges, rather than taking a passive or even active stance of resistance \\[1\\]. Psychiatrists played a prominent and central role in two categories of the crimes against humanity, namely sterilization and euthanasia \\[2\\]. It was psychiatrists (many of whom were senior professors in academia) who sat on planning committees for both processes and who provided the theoretical backing for what transpired. It was psychiatrists who reported their patients to the authorities and coordinated their transfer from all over Germany to gas chambers situated on the premises of the six psychiatric institutions: Brandenburg, Grafeneck, Hartheim, Sonnenstein, Bernburg, and Hadamar \\[2,3\\]. It was psychiatrists who coordinated the \"channeling\" of patients on arrival into specially modified rooms where gassing took place. It was psychiatrists who saw to the killing of the patients (initially using carbon monoxide and later, starvation and injection). Finally, it was psychiatrists who faked causes of death on certificates sent to these patients' next of kin. It has been estimated that over 200,000 individuals with mental disorders of all subtypes were put to death in this manner \\[4-7\\]. Much of this process took place before the plan to annihilate the Jews, Gypsies and homosexuals of Europe. Hitler never gave the order to kill patients with mental illness. He only permitted it in a letter written in October 1939 and backdated to September 1, 1939 \\[2,6\\]. Psychiatrists were therefore never ordered to facilitate the process or carry out the murder of mentally ill...they were empowered to do so. Activity by psychiatrists and psychiatric institutions thus constituted the connection between euthanasia and the larger scale annihilation of Jews and other \"undesirables\" such as homosexuals in what came to be known as the Holocaust. Parenthetically, only one physician ever came to command an extermination camp. His name was Dr Imfried Eberl, a psychiatrist, who established Treblinka based on his experience as the Brandenburg Psychiatry Facility medical superintendent. He managed the camp for six months until he was fired for inefficiency in disposing of the thousands of bodies he succeeded in accumulating \\[2\\].\n\n# Attitude of mainstream psychiatry to Nazi psychiatry practice following the war\n\nWhile it would be expected that the involvement of psychiatrists in such a profound manner would be well-known in the field, this is not the case. Little has been published on the subject in mainstream psychiatry journals and even less is part of the formal education process for medical students and psychiatry residents. Several reasons may be proposed for this. First, it remains an embarrassment for the field that so many senior members \u2013 professors, department heads and internationally known figures \u2013 were so intimately involved. Second, many of those involved continued to practice and conduct research long after the war and were protected by colleagues. Third, and arguably most important, what psychiatrists did was based upon a paradigm shift in how patients and mental illness were viewed. Activities of psychiatrists became much of a value judgment in how they \"read\" the community and principles of neo-Darwinism with subsequent consideration of racial hygiene. In the absence of firm and unbending timeless ethical underpinnings to the practice of psychiatry, many felt that what they were doing was correct from a moral and scientific standpoint; therefore, they were not the demons and \"paradigms of evil\" that we perceive them to be. Their actions were a colossal misjudgment based on what today we may term \"pseudoscience\", but which at the time was deemed correct by many. Although actions based on \"scientific theories\" of mental illness in the past have led to patient deaths \u2013 one example being Henry Cotton and his belief that mental illness results from focal infection or chronic sepsis \\[8\\] \u2013 the extent and scale of the German psychiatrists' actions during the Nazi era remains unprecedented. These rationalizations based on faulty scientific theory and unethical medical practice were difficult to accept and therefore the nature and extent of these activities remained on the backbenches of the academic literature until more recently, when these issues have begun to be faced in an era of openness and transparency.\n\n# Common assumptions leading to gross ethical misconduct\n\nIn addition to resting on poor science, the atrocities of the German psychiatric establishment were based upon several fundamental errors of ethical, professional, and scientific conduct. While many may simply brush off any deeper consideration of the issues with the stance that \"they were just evil\", such an approach only deepens the risk that such events will be repeated. The truth cannot be more different: perversion of ethical medical practice due to theoretical misjudgment and fundamental error in approach to the patient are what led to these atrocities of catastrophic proportions. So where did they go wrong? Several misconceptions lay at the source:\n\n## 1) Medical ethics is ethnic, cultural, and time sensitive\n\nThe theory behind such a proposition is that much of medical ethics is time and culture bound \\[9\\]. Therefore what may be unethical now may not necessarily have been unethical then. This approach inculcates a relative attitude to the atrocities, minimizing the severity of the injustice and gross professional negligence so inherent in what transpired. Certain aspects of medical ethics transcend time and culture. Except under very specific and precise circumstances, such as when there is a serious and immediate risk to others, a physician should always respect autonomy, beneficence, and patients' confidentiality and dignity. Although it may be suggested that there is a major leap between disregard of these time-honored factors and the genocide of euthanasia, this is how it all began \u2013 it may even define the central thread of the atrocities to the mentally ill. While the form of ethical medical practice may depend on resources and cultural nuances (Tarasoff etc.), the basis for ethical behavior should remain constant, irrespective of time and place. Thus, while some maintain that for one generation a practice may be considered unethical but not for another \\[9\\], this is a misconception, as certain practices and concepts do not change with context. It is therefore never appropriate to kill one's patients *en masse* based on diagnosis and economic and racial-hygiene considerations for the community at large.\n\n## 2) Philosophical constructs and ideas should define clinical practice\n\nDuring the period of the Nazi regime, psychiatry supported compulsory sterilization and euthanasia of the physically and mentally ill, and subsequently, the killing of \"inferior\" races. They did this by applying scientifically invalid conclusions from evolutionary biology \\[10\\]. Aside from the fact that these philosophical constructs and scientific paradigms of evolutionary theory were flawed, they were also immoral and contravened basic tenets of medical ethics and clinical practice. Much of this approach was based on theories of neo-Darwinism. Furthermore, ever since Francis Galton in 1865 first published the idea of eugenics (a term rooted in the Greek \"good in birth\" or \"noble in heredity\"), individuals with mental illness had been targeted by eugenics programs, with psychiatrists intimately involved in the theoretical debate. The eugenics movement was not limited to Germany, and proponents of eugenics were prominent in several other countries including most notably Britain and the USA \\[11\\]. Interestingly, during the period in which euthanasia of the mentally ill was taking place in Germany, a fascinating debate transpired between two prominent American academics and was published in the *American Journal of Psychiatry* in 1942. Foster Kennedy, professor of neurology at Cornell University in New York, argued that all children with proven mental retardation (\"feeblemindedness\") over the age of five should be put to death. Leo Kanner, however, maintained that such individuals might still serve a purpose to society \u2013 garbage collection, postmen, etc. \u2013 as well as give meaning to their parents by virtue of having to care for them. Astoundingly, no one emphasized the unethical nature of putting individuals with disability to death. Instead, the editorial, published anonymously, appeared to side with Kennedy, and advised help for the parents in coming to terms with such a reality for their children and for the need for \"enabling legislation\" in order to facilitate the process legally (apparently in contrast to that of the German experience) \\[12\\]. The Nazi experience, which took much of the concept to fruition, was an extreme perversion of this movement, which existed already (at least at the conceptual level) in the minds of many psychiatrists supporting the idea.\n\n## 3) Preventative medicine is more important than curative medicine\n\nIn the interests of preserving the future quality and purity of the Aryan race, racial hygiene became the battle cry of the German nation with Nazi medicine attempting to prevent the proliferation of illness. Within this context, it became the role of physicians in general, and psychiatrists in particular, to define who should be eliminated in order to best preserve the German nation's uniqueness and \"higher-being\". Thus in place of managing mental illness with the available tools (which were minimal) or investing resources in research for more appropriate treatment, it became important for physicians and psychiatrists to prevent such forms of illness or defects through euthanasia \\[7\\]. A particular focus was placed on psychiatric patients in the racial-hygiene program because they were perceived as weakening the \"master-race\" with no known cure. Therefore these \"lives not worth living\" were deemed useless and dangerous to German society and, in order to prevent their dissemination, the process of eliminating them in the context of the sterilization and euthanasia program came about. This procedure of trying to prevent illness, while a noble concept, should never be instituted at the expense of (and complete exclusion of) treating illness, as the disastrous Nazi program proved \\[7\\]. Even if one accepts their reasoning \u2013 and in this case they were wrong \u2013 selective sterilization of the mentally-ill would never significantly reduce the frequency of mental illness based on the Hardy-Weinberg law of preservation of rare recessive genes in a population of phenotypically normal carriers \\[10\\]. The Nazis embraced an exclusionary biological and racial determinism that removed any reparative function from clinical psychiatry. What remained was prevention of mental illness. Psychiatrists lived up to the challenge.\n\n## 4) Psychiatrists have a particular role in channeling societal issues and public discussion\n\nMany psychiatrists maintain that they have an inherent responsibility more than other medical professions to be involved in community affairs. This is because psychiatry by nature advocates a holistic approach to the patient, which often includes taking into account societal factors and contemporary ideology. Thus while the unique role of psychiatry in the genocide may be overstated, since other areas of medicine were also involved, psychiatrists fitted in particularly well. The dangers inherent in such involvement, while not obvious, are, however, prominent when important boundaries become blurred. Clinical practice and political machinations need to be kept separate. Many psychiatrists during the Nazi era were state-controlled and this further facilitated their conforming to the program. The rights of individuals cannot be totally ignored in the interests of society. The dangers become particularly acute in psychiatry compared to other subspecialties in medicine since it may be suggested that the field of psychiatry is often used in order to remove undesirables from society and place them in asylums. It may be argued that labeling of mental disease and its classification is a means of controlling members of the community who do not comply with accepted norms; therefore their freedom should be taken away and replaced with hospitalization. However, while at times there may be a fine line separating mental health and illness, it becomes very clear that the extent to which Nazi psychiatry allowed the political and community atmosphere to influence and govern clinical practice was grossly unethical, murderous, and unacceptable to an extreme extent.\n\n## 5) Political and economic pressures may influence clinical practice\n\nThe management of patients must be dictated primarily by the patient's best interests and not by virtue of any ideology that may be prevalent at the time in society. This may include economic \"ideological\" considerations. Thus while pressures may exist \"encouraging\" the physician to make decisions one way or another based on the prevailing mood or tendency of the community at any time or place, this should be resisted and medical management should continue, unaffected by external considerations. The patient has to receive individual management and not be treated according to what is in vogue at the time. Psychiatrists should be wary of political and economic pressures that impinge upon medical decisions and health service provision. Nazism was supposed to be \"applied biology\" \\[13\\]. Science in general and psychiatry in particular needs to be independent from contemporary sociological and political contexts as well as protected from political abuse, even when embraced by the medical establishment. It has been proposed that the primary downfall of Nazi medicine was the failure of physicians to challenge the substantive core of Nazi values \"Too many physicians were willing to go with the political flow; too many were unwilling to resist, to 'deviate' from 'commonly accepted' practices\" \\[14\\]. Sound medical practice should be protected from the movement of political forces.\n\n## 6) Psychiatrists\/scientists have a responsibility to \"enhance\" mankind\n\nMuch of the early involvement by psychiatric clinicians and researchers in the process of \"racial purification\" arose from a genuine desire to improve mankind and not necessarily from the perspective of racist genocide. While no direct parallel can be drawn, today many continue in a sincere scientific effort towards the \"enhancement\" of man through molecular biology and genetic engineering \\[15\\]. Appropriate dialogue is required in order to ensure that the desire for \"improving man\", creating a \"better human\", does not come at the expense of the individual patient.\n\n## 7) The interests of science take priority over the interests of the individual patient\n\nClinical management and research participation may appear to be equivalent, but they are not. A clear distinction must be made between the two and the patient must be aware of this. Research is critically important for the future of good medical practice and is fundamental to the philosophy of medical ethics in psychiatry which would be reflected in the long-term striving for excellence in clinical management. However, it should always be made clear to the patient that participation is voluntary and that more conventional treatment regimes exist and are available if preferred. Particular issues such as scientific validity, favorable risk-benefit ratio, voluntaryism, and decisional capacity, while important in all aspects of clinical practice, become of acute importance with respect to individuals with mental illness \\[16,17\\]. The Nazi experience, which completely disregarded such factors in the interests of \"science\" and racial-hygiene, is a prime example of the dangers inherent when such factors are not respected. Ethical commitment to research safeguards needs to be reflected in appropriate standards, guaranteeing appropriate study participation \\[16\\]. Refusal to participate in a study should likewise never interfere with the doctor-patient relationship and in the case of a patient agreeing to participate in research, it remains the duty of the physician to protect the health of the individual.\n\n## 8) High-quality science and high-quality ethics always co-exist\n\nIt has become easy for those in the West to dismiss the depths of unethical medical practice of the Nazi physicians by categorizing it as bad science. This is easier to accept than the possibility that even within the context of good science, ethical behavior by physicians may go astray. In fact, the Nazi era in Germany was a time of remarkable scientific advances in several areas including cancer research and treatment, biochemistry, and quantum mechanics to name a few. In addition, the Nazis were pioneers of jet-propelled air flight, guided missiles, electronic computers, electron microscopes, and atomic fission \\[14\\]. Thus, scientific advancement does not necessarily go hand in hand with ethical advancement. It would be incorrect to brush off the ethical challenges that true scientific advancement in medicine may present, since the connection with true ethical practice is not necessarily a natural one.\n\n# Ethics in the training of future psychiatrists\n\nThe theory of \"medical ethics\" has become a requirement in residency training programs in several countries around the world \\[18\\]. This has become a particularly pressing issue considering the need to understand principles of research ethics and roles of psychiatrists as investigators and researchers. The teaching of medical ethics during residency is particularly well-timed because professional identity and ethical practices are in their formative stages. Such ethics training is important in order to define the critical role that the four principles of autonomy, beneficence, non-maleficence, and justice play in clinical practice \\[19\\]. Several reports have been published that extol the virtues of medical ethics training in psychiatry residency training programs \\[20\\].\n\nHowever, while the importance of such training programs is well recognized, so was the importance of medical ethics acknowledged in Germany in the 1930's. In fact, Germany possessed one of the most advanced and sophisticated codes of medical ethics in the world in existence from 1931. Some have even suggested that in certain aspects it was stricter than the subsequent Nuremberg Code or Helsinki Accord \\[14,21\\]. Doctors in general and psychiatrists in particular who were involved in the euthanasia program were not morally blind or devoid of the power of moral reflection. This belief would render the guilty parties not responsible for their actions. However, such codes did not help and when it came to bringing to fruition the ideology and plans of the wider society's beliefs, psychiatrists cooperated fully. They even took enthusiastic initiative in the process, allowing societal politics and ideas to interfere with clinical practice. Furthermore, although a broader consideration of potential abuse and malpractice in other totalitarian regimes would further strengthen the importance of the subject, a focus on Nazi psychiatric practice in particular brings to the fore clearly a most apt and recent example of how such interference can go awry. The example of Nazi psychiatry is a prime illustration of how ethics training without a focus on history is useless; where policy, even though existing, can be disregarded in the most grotesque fashion.\n\nFurthermore, Cowley \\[19\\] has argued that although many medical schools have now given medical ethics a secure place in the curriculum, they err in treating it like a scientific body of knowledge. Ethics is a unique subject precisely because of its widespread relevance in all areas of life, and any teaching has to start from this shared understanding and from the familiar ethical concepts of ordinary language. Ethical jargon obscures the essential integration of ethics with the personal and \"drives a wedge between ethical concepts and ethical conduct\". This may have accounted for some of the unethical conduct of \"Hitler's psychiatrists\" in their disregard of basic principles despite the existence of strong ethical policy. \"Ethical mantras\" have little value when they exist away from a context of a mature understanding and self-reflection that needs to precede good ethical judgment and professionalism \\[19\\].\n\nThe question remains: why did so many psychiatrists willingly participate in the process of mass murder of the mentally ill? Perhaps some light may be shed on this issue by consideration of similar behavior as reported by Browning \\[22\\] in the history of an \"ordinary\" and unremarkable battalion of the Order Police that participated in mass shootings and deportations. Browning describes how these ordinary men were not coerced to kill, but rather participated in a willing fashion due to peer pressure, government sanction and following of orders, and in order to advance their own interests (careerism). This is in contrast to Goldhagen \\[23\\], who suggested that the average German citizen either participated in or ignored genocidal actions during the Nazi Era due to ingrained anti-Semitism, which was an intrinsic part of German society and had built up over centuries.\n\nYet another approach proposes that psychiatrists during the Nazi era were at particularly high risk for moral and ethical breaches because of how society and they themselves defined their role and power. Inherent in their work lay the risk of dehumanizing the patients with whom they had daily involvement, individuals at the extremes of human behavior. Moreover, psychiatry by nature incorporates contemporary ideology in its approach to the individual and society, and psychiatrists during that period were in essence state-controlled. All of these factors may have led to their tendency to objectify patients \\[reviewed in \\[1\\]\\]. Thus, these psychiatrists were primed to become involved in furthering Nazi ideology.\n\nThese differing approaches and considerations need to be conveyed to students of psychiatry, emphasizing that merely explaining the actions of psychiatrists and other physicians during this period by saying \"they were evil\" is misleading and reductionistic \\[24\\]. Danger exists in such an approach since it would preclude consideration of one's own risk for involvement in such a process.\n\nDespite the wealth of ethics literature and the requirement for medical ethics in training programs, the experience of Nazi German psychiatry receives minimal mention, if at all, in contemporary medical student and resident ethics training courses. This is a serious oversight, since well-developed ethical principles did not stop the trespassing of political ideology into clinical practice and research in the 1930's. The result was equally devastating to the patients and to the practice of the profession. Every psychiatry resident needs to know about this. A knowledge and appreciation of Nazi psychiatry practices should become an important component of an integrated program of psychiatry ethics, with a focus on the human aspects of the psychiatric clinician and researcher, and a warning against the influences of political and community ideology impinging upon professional practice.\n\nThe content of ethics training for medical students and residents will remain a creative exercise for educators. Such a program should include an informative historical review of this period, including information on the unethical medical activities that transpired, case studies of psychiatrists at all levels of involvement, a consideration of various ethical frameworks for psychiatric care that were violated, as well as a consideration of why psychiatrists came to be so inextricably involved. Such a course would pave the way for a discussion about creating the most optimal framework of ethics for psychiatric practice. Several approaches may be considered, including the recent one by Bloch and Green \\[25\\], who proposed a model based on a \"complementarity of principalism\" (pragmatic focus on respect for autonomy, beneficence, non-maleficence and justice) and care ethics (highlighting character traits pertinent to caring for vulnerable psychiatric patients).\n\nThus, in addition to the usual formal medical ethics training in which the importance of autonomy, beneficence, confidentiality, and professionalism is emphasized, an in-depth appreciation of the actions of German psychiatrists during the Nazi years should be imparted. Since medical students and residents today are learning in an ethical environment that is unprecedented in its complexity \\[20\\], it becomes crucial to include the Nazi era medical experience as an example of proper practice gone awry, despite its being in the interest of science, and despite receiving the support of many of the foremost world leaders of the profession. The responsibility of psychiatrists to act as moral agents in the interests of patients, especially in the area of psychiatry, is thus of paramount value.\n\nThe professional burden of the memory of what transpired during the Nazi Era by the hands of members of the psychiatry profession is great. Those that were inextricably involved were colleagues, and this requires us to grapple with the intrinsic guilt of the profession, and to take responsibility to fix fundamental flaws in how we view patients and their management. A dark side to medicine exists: psychiatry, academia, and science played a key role in the establishment of National Socialism and all that ensued. The experience of psychiatry during the Nazi era provides an example of how science can be perverted by politics and therefore can become vulnerable to misuse and abuse. An exclusive focus on the monstrous aspects of Nazi medicine enables us to dismiss such events as aberrant and deviant, with a subsequent failure to internalize the inherent and very real dangers of the perversion of science and clinical management by outside political influences. Psychiatry cannot afford to turn a blind eye to such a past.\n\n# Competing interests\n\nThe author declares that he has no competing interests.","meta":{"dup_signals":{"dup_doc_count":101,"dup_dump_count":68,"dup_details":{"curated_sources":4,"2023-40":2,"2023-23":1,"2022-40":1,"2022-27":1,"2021-43":1,"2021-39":1,"2021-25":1,"2021-10":1,"2021-04":1,"2020-50":1,"2020-45":1,"2020-40":1,"2020-29":1,"2019-51":1,"2019-43":2,"2019-35":1,"2019-30":1,"2019-26":1,"2019-18":2,"2019-04":1,"2018-47":1,"2018-43":1,"2018-39":1,"2018-34":1,"2018-22":3,"2018-17":1,"2018-13":1,"2018-09":1,"2018-05":1,"2017-51":1,"2017-47":1,"2017-43":1,"2017-39":3,"2017-34":3,"2017-30":3,"2017-26":2,"2017-22":2,"2017-17":2,"2017-09":2,"2017-04":3,"2016-50":2,"2016-44":2,"2016-40":1,"2016-36":2,"2016-30":2,"2016-26":1,"2016-22":1,"2016-18":1,"2015-48":1,"2015-35":1,"2015-32":1,"2015-27":1,"2015-22":1,"2015-14":1,"2014-52":1,"2014-49":2,"2014-42":4,"2014-41":2,"2014-23":2,"2014-15":2,"2017-13":2,"2015-18":1,"2015-11":1,"2015-06":1,"2014-10":1,"2013-48":1,"2013-20":1,"2024-18":1}},"file":"PMC1828151"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  Alternate day modified fasting (ADMF) is an effective strategy for weight loss in obese adults.\n  .\n  # Objective\n  .\n  The objective of this study was to examine the dietary and physical activity adaptations that occur during short-term ADMF, and to determine how these modulations affect rate of weight loss.\n  .\n  # Methods\n  .\n  Sixteen obese subjects (12 women\/4 men) completed a 10-week trial consisting of 3 phases: 1) 2-week control phase, 2) 4-week ADMF controlled feeding phase, and 3) 4-week ADMF self-selected feeding phase.\n  .\n  # Results\n  .\n  Body weight decreased (*P* \\< 0.001) by 5.6 \u00b1 1.0 kg post-treatment. Energy intake on the fast day was 26 \u00b1 3% of baseline needs (501 \u00b1 28 kcal\/d). No hyperphagic response occurred on the feed day (95 \u00b1 6% of baseline needs consumed, 1801 \u00b1 226 kcal\/d). Daily energy restriction (37 \u00b1 7%) was correlated to rate of weight loss (*r* = 0.42, *P* = 0.01). Dietary fat intake decreased (36% to 33% of kcal, *P* \\< 0.05) with dietary counseling, and was related to rate of weight loss (*r* = 0.38, *P* = 0.03). Hunger on the fast day decreased (*P* \\< 0.05) by week 2, and remained low. Habitual physical activity was maintained throughout the study (fast day: 6416 \u00b1 851 steps\/d; feed day: 6569 \u00b1 910 steps\/d).\n  .\n  # Conclusion\n  .\n  These findings indicate that obese subjects quickly adapt to ADMF, and that changes in energy\/macronutrient intake, hunger, and maintenance of physical activity play a role in influencing rate of weight loss by ADMF.\nauthor: Monica C Klempel; Surabhi Bhutani; Marian Fitzgibbon; Sally Freels; Krista A Varady\ndate: 2010\ninstitute: 1Department of Kinesiology and Nutrition, University of Illinois at Chicago, Chicago, IL, USA; 2Department of Medicine, University of Illinois at Chicago, Chicago, IL, USA; 3Department of Biostatistics, University of Illinois at Chicago, Chicago, IL, USA\nreferences:\ntitle: Dietary and physical activity adaptations to alternate day modified fasting: implications for optimal weight loss\n\n# Introduction\n\nRates of obesity have dramatically increased over the past three decades. At present, 34% of adults in the United States are obese (body mass index (BMI) \\> 30 kg\/m^2^) \\[1\\]. According to the National Heart Blood and Lung Institute (NHLBI) Obesity Guidelines \\[2\\], dietary interventions should be implemented as the first line of treatment to help obese individuals lose weight. The most common diet therapy prescribed by practitioners is daily calorie restriction (CR). CR involves decreasing energy intake by 15 to 40% of baseline needs everyday. Evidence from short-term CR trials (8 to 24 weeks) demonstrate that CR is an effective means of decreasing body weight by 5 to 10% from baseline in obese patients \\[3-6\\].\n\nAlthough CR is the most frequent diet strategy implemented to facilitate weight loss \\[7\\], many obese patients find it difficult to adhere to CR since food intake must be limited *every day*\\[8-10\\]. Alternate day modified fasting (ADMF) was created as an alternative to CR to improve compliance with dietary restriction regimens \\[11\\]. ADMF includes a \"feed day\" where food is consumed ad-libitum over a 24-h period, alternated with a \"fast day\", where food intake is partially reduced for 24-h. ADMF only requires an individual to restrict food intake on *every other day*, and as such, greatly increases adherence to these protocols. To date, four ADMF human trials have been performed \\[12-15\\], two of which were weight loss studies \\[13,14\\]. In the first trial by Johnson et al. \\[13\\], 8 weeks of modified ADMF, which allowed for 20% of energy needs to be consumed on the fast day, decreased body weight by 8% from baseline in overweight adults. In the second study conducted by our group \\[14\\], 8 weeks of modified ADMF (i.e. 25% energy intake on the fast day, alternated with an ad libitum feed day) resulted in a 6% weight loss in obese individuals. Although these preliminary findings suggest that ADMF may be an effective weight loss strategy, \\[13,14,16\\] what has yet to be examined is the dietary and physical activity adaptations that contributed to this pronounced weight loss by ADMF. Key questions that remain unresolved include: Are obese subjects able to dramatically change their meal pattern and limit their energy intake to 25% of needs on the fast day? If this is the case, what degree of hyperphagia occurs on the feed day in response to this lack of food on the fast day, and how does this affect net energy restriction and rate of weight loss? Moreover, how long does it take for obese subjects to become habituated to ADMF (i.e. no longer feel hungry on the fast day)? Furthermore, what changes in habitual physical activity occur during ADMF, and how do these changes affect rate of weight loss?\n\nAccordingly, the objective of the present study was to examine the dietary and physical activity adaptations that occur during short-term ADMF, and to determine how these modulations affect rate of weight loss.\n\n# Methods\n\n## Subject selection\n\nThis study was approved by the Office for the Protection of Research Subjects at the University of Illinois, Chicago, and all volunteers gave their written informed consent prior to participation in the trial. As reported previously \\[14\\], participants were recruited by means of advertisements placed in community centers in the Chicago metropolitan area. Inclusion and exclusion criteria were assessed by an in-person interview. Participants meeting the following criteria were included in the study: age 35 to 65 y; body mass index between 30 and 39.9 kg\/m^2^; weight stable for 3 months prior to the beginning of the study (i.e. less than 5 kg weight loss or weight gain); non-diabetic; no history of cardiovascular disease; lightly active (i.e. \\< 3 h\/week of light intensity exercise at 2.5 to 4.0 metabolic equivalents (METs) for 3 months prior to the study); not participating in an exercise class; non-smoker; and not taking weight loss, lipid or glucose lowering medications. Peri-menopausal women were excluded from the study, and post-menopausal women (absence of menses for more than 2 y) were required to maintain their current hormone replacement therapy regimen for the duration of the study.\n\n## Experimental design\n\nObese participants were enrolled in the study as a single cohort. Subjects participated in a 10-week trial consisting of three consecutive dietary intervention phases: (1) 2-week pre-loss control phase, (2) 4-week weight loss\/ADMF controlled feeding phase, and (3) 4-week weight loss\/ADMF self-selected feeding phase. During *Phase 1*, each subject maintained their usual eating and exercise habits in order to maintain a stable body weight. During *Phase 2*, subjects participated in a 4-week controlled-feeding ADMF period. All subjects consumed 25% of their baseline energy needs on the fast day (24 h), and then ate ad libitum on each alternating feed day (24 h). Individual baseline energy requirement was determined by the Mifflin equation \\[17\\]. Subjects were provided with a calorie-restricted meal on each fast day, and ate ad libitum at home on the feed day. The provided fast day meal was formulated for each subject using Nutritionist Pro Software (version 4.3, Axxya Systems, Stafford, TX). All diets were prepared in the metabolic kitchen at the Human Nutrition Research Unit (HNRU) at the University of Illinois, Chicago, and were provided as a 3-day rotating menu consisting of typical American foods. Meals provided on fast days during the controlled feeding phase are displayed in Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>. Each feed\/fast day began at midnight, and all fast day meals were consumed between 12.00 pm and 2.00 pm to ensure that each subject was undergoing the same duration of fasting. During *Phase 3*, all subjects participated in a self-selected feeding ADMF period in conjunction with weekly dietary counseling. This phase was put in place to determine if subjects could maintain the ADMF regimen on their own at home. During this phase, subjects still consumed 25% of their baseline energy needs on the fast day (between 12.00 pm and 2.00 pm), and ate ad libitum on the feed day. No food was provided to the subjects during this phase. Instead, a Registered Dietician met with each subject each week (for approximately 30 min per session) to develop individualized fast day meal plans. These plans included menus, portion sizes, and food lists that were consistent with each subject's food preferences and prescribed calorie levels for the fast day. Subjects were also instructed how to make healthy food choices on the ad libitum feed days, by choosing low fat meat and dairy options, and increasing fruit and vegetable intake.\n\nMeal components of provided fast day meals during controlled feeding phase\n\n| Foods           | Fast day 1         | Fast day 2       | Fast day 3        |\n|-----------------|--------------------|------------------|-------------------|\n| Entree          | Chicken fettuccini | Vegetarian pizza | Chicken enchilada |\n| Fruit\/vegetable | Carrot sticks      | Apple            | Orange            |\n| Snack           | Cookie             | Peanuts          | Crackers          |\n\n## Weight loss assessment\n\nBody weight was measured weekly to the nearest 0.25 kg in the fasted state, without shoes, and in light clothing using a balance beam scale (HealthOMeter, Sunbeam Products, Boca Raton, FL).\n\n## Reported food intake on feed days\n\nEach participant completed a 3-day food record on 2 feed days during the week, and on 1 feed day during the weekend, at each week of the 10-week trial. Thus, a total of 30 feed day food records were collected for each subject. At baseline, the Research Dietician provided 15 min of instruction to each participant on how to complete the food records. These instructions included verbal information and detailed reference guides on how to estimate portion sizes and record food items in sufficient detail to obtain an accurate estimate of dietary intake. Subjects were instructed to record food items, in as much detail as possible, in the blank food diary provided. Any mixed foods were broken down to individual food items to be recorded one per line. Participants were not required to weigh foods but were asked to measure the volume of foods consumed with household measures (i.e. measuring cups and measuring spoons). When a commercial product was consumed, subjects were asked to indicate the weight of the product to assess portion size. Food records were collected at the weigh-in each week, and were reviewed by the Dietician for accuracy and completeness. All dietary information from the food records was entered into the food analysis program, Nutritionist Pro (Axxya Systems) by a single trained operator to alleviate inter-investigator bias. The program was used to calculate the total daily intake of energy, fat, protein, carbohydrate, cholesterol, and fiber.\n\n## Reported food intake on fast days\n\nDuring the ADMF controlled feeding phase, subjects were asked to report any additional food item consumed that was not included in the provided meal. During the ADMF self-selected feeding phase, each participant was asked to record his or her food intake on each fast day. At the beginning of this phase, the Research Dietician went over the food record instructions once again with each subject. Fast day food records were collected at the weigh-in each week, and all records were reviewed for accuracy and completeness by the Dietician. Dietary information from the fast day food records was analyzed by a single trained operator using Nutritionist Pro (Axxya Systems).\n\n## Hunger, satisfaction with diet, and fullness assessment\n\nSubjects completed a validated visual analog scale (VAS) on each fast day, in the evening, approximately 5 min before going to bed (reported bedtime ranged from 8.20 pm to 1.40 am) \\[18\\]. In brief, the VAS consisted of 100-mm lines, and subjects were asked to make a vertical mark across the line corresponding to their feelings from 0 (not at all) to 100 (extremely) for hunger, satisfaction with diet, or fullness. The VAS was collected at the weigh-in each week and reviewed for completeness. Quantification was performed by measuring the distance from the left end of the line to the vertical mark.\n\n## Physical activity assessment\n\nHabitual, free-living physical activity was assessed by a pedometer (Digiwalker SW-200, Yamax Corporation, Tokyo, Japan SW). Subjects wore the pedometer each day throughout the 10-week trial. The pedometer was worn attached to the participant's waistband during waking hours (except while bathing or swimming), and reset to zero each morning. Number of daily steps were recorded in a pedometer log provided, and the log was collected by study personnel at the weigh-in each week. No subjects were enrolled in an exercise class, and all participants were asked to refrain from joining any exercise programs during the course of the study. In this way, any changes in physical activity during the study could be estimated by the use of the pedometer.\n\n## Statistics\n\nResults are presented as means \u00b1 standard error of the mean (SEM). Tests for normality were included in the model. One-factor repeated measures analysis of variance was performed to determine an overall *P* value over time. The main variables tested included body weight, energy intake, nutrient intake, hunger, satisfaction and fullness. The Bonferroni correction was used to assess significance. Relations between continuous variables (i.e. body weight, energy intake, nutrient intake, hunger, satisfaction and fullness) were assessed by simple regression analyses as appropriate. Data were analyzed by using SPSS software (version 18.0 for Mac OS X; SPSS Inc., Chicago, IL).\n\n# Results\n\n## Subject characteristics at baseline\n\nOf the 52 participants screened, 20 were deemed eligible to participate in the study, and 16 (4 men\/12 women) completed the entire 10-week trial. Subjects who completed the study were middle age (46 \u00b1 3 y, 35-65 y), obese (BMI 34 \u00b1 1 kg\/m^2^, 30.2-39.9 kg\/m^2^), sedentary (2.4 \u00b1 0.3 h\/week of physical activity), and borderline hypercholesterolemic (LDL cholesterol level 106 \u00b1 10 mg\/dl). Eight participants were African-American, 2 were Caucasian, and 6 were Hispanic.\n\n## Changes in body weight in response to ADMF\n\nDuring the control phase, body weight remained stable (week 1: 96.4 \u00b1 5.3 kg, week 2: 96.5 \u00b1 5.2 kg). At the end of the ADMF controlled feeding phase (week 6), body weight decreased (*P* \\< 0.001) to 93.8 \u00b1 5.0 kg (feed day measurement) and 93.7 \u00b1 5.0 kg (fast day measurement). By the end of the ADMF self-selected feeding phase (week 10), body weight was further reduced (*P* \\< 0.001) to 92.8 \u00b1 4.8 kg (feed day measurement) and 90.8 \u00b1 5.0 kg (fast day measurement). Thus, a total weight loss of 5.6 \u00b1 1.0 kg (-0.7 \u00b1 1.0 kg per week) was attained after 8 weeks of ADMF.\n\n## Degree of energy restriction achieved with ADMF and relation to body weight changes\n\nEnergy intake and percent energy restriction were determined from food record data collected on feed and fast days. Mean completion rate of feed and fast day food records was 83 \u00b1 5%, and 86 \u00b1 4%, respectively. Energy intake on feed and fast days during each week of the trial is displayed in Figure <a href=\"#F1\" data-ref-type=\"fig\">1A<\/a>. During the control phase, mean energy intake was 1937 \u00b1 180 kcal. Mean feed day energy intake (1801 \u00b1 226 kcal) at each week of the trial was similar to that of the control phase, and did not differ between ADMF controlled-feeding and self-selected feeding phases. Mean energy intake on the fast day (501 \u00b1 28 kcal, 26 \u00b1 3% of baseline needs consumed) was lower (*P* \\< 0.001) than that of the feed day at each week of the trial. The ratio of energy consumed on the fast day versus the feed day during the controlled feeding phase (0.28 \u00b1 0.03) did not differ from that of the self-selected feeding phase (0.30 \u00b1 0.05). Percent energy restriction is reported in Figure <a href=\"#F1\" data-ref-type=\"fig\">1B<\/a>. Over the course of the trial, percent daily energy restriction remained high and stable (37 \u00b1 7%), and did not differ between the ADMF controlled-feeding and self-selected feeding phases. Degree of energy restriction achieved by ADMF was correlated to rate of weight loss (*r* = 0.42, *P* = 0.01) and absolute post-treatment weight loss (*r* = 0.48, *P* = 0.008).\n\n## Hyperphagic response\n\nHyperphagia on the feed day in response to the lack of food on the fast day is reported in Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>. We hypothesized that the participants would increase their energy intake on the feed day by approximately 125% of their baseline needs. However, no such hyperphagic response was observed, as mean feed day energy intake (1801 \u00b1 226 kcal) was similar to calculated requirements (1896 \u00b1 160 kcal) at each week of the trial. Thus, on average, subjects were only consuming 95 \u00b1 6% of their calculated energy needs on the feed day.\n\n## Changes in nutrient intake during ADMF and relation to body weight changes\n\nThe nutrient composition of feed and fast day meals during each phase of the trial is displayed in Table <a href=\"#T2\" data-ref-type=\"table\">2<\/a>. During the control phase, subjects were consuming a high fat (\\> 35% of kcal), high saturated fat (\\> 7% of kcal), high cholesterol (\\> 200 mg\/d), and low fiber diet (\\< 25 g\/d), as per the National Cholesterol Education Program (NCEP) dietary guidelines \\[19\\]. During the ADMF controlled feeding phase, the nutrient composition of feed day diet was similar to that of the control phase (i.e. high total fat, high saturated fat, high cholesterol and low fiber). During the ADMF self-selected feeding phase, total fat (33 \u00b1 4% kcal) and saturated fat (7 \u00b1 1% kcal) intake on the feed day decreased (*P* \\< 0.05), relative to the control phase. Dietary cholesterol, however, was still above the recommended daily allowance (223 \u00b1 27 mg\/d), and dietary fiber (15 \u00b1 1 g) was still below the recommended intake level on the feed day. Decrease in total fat intake was related to rate of weight loss (*r* = 0.38, *P* = 0.03).\n\nNutrient composition of feed day and fast day meals during each phase of the trial^1^\n\n|  | Pre-loss control phase^2^ | Weight loss\/ADMF controlled feeding phase |  | Weight loss\/ADMF self-selected feeding phase |  |\n|----|----|----|----|----|----|\n|  |  | **Feed day^2^** | **Fast day^3^** | **Feed day^2^** | **Fast day^2^** |\n|  |  |  |  |  |  |\n| Energy (kcal) | 1937 \u00b1 180 | 1792 \u00b1 228 | 413 \u00b1 20 | 1645 \u00b1 187 | 588 \u00b1 46 |\n| Protein (% kcal) | 18 \u00b1 1 | 18 \u00b1 1 | 23 \u00b1 1 | 19 \u00b1 1 | 20 \u00b1 1 |\n| Carbohydrate (% kcal) | 46 \u00b1 3 | 47 \u00b1 3 | 52 \u00b1 0 | 46 \u00b1 2 | 51 \u00b1 3 |\n| Total fat (% kcal) | 36 \u00b1 5^a^ | 36 \u00b1 6^a^ | 25 \u00b1 1^b^ | 33 \u00b1 4^b^ | 29 \u00b1 1^b^ |\n| \u2003Saturated fat (% kcal) | 11 \u00b1 1^a^ | 10 \u00b1 1^a^ | 6 \u00b1 1^b^ | 7 \u00b1 1^b^ | 9 \u00b1 1^a^ |\n| \u2003Monounsaturated fat (% kcal) | 11 \u00b1 1 | 12 \u00b1 1 | 11 \u00b1 1 | 13 \u00b1 1 | 8 \u00b1 1 |\n| \u2003Polyunsaturated fat (% kcal) | 10 \u00b1 2 | 11 \u00b1 1 | 8 \u00b1 1 | 10 \u00b1 1 | 9 \u00b1 1 |\n| \u2003Trans fat (% kcal) | 4 \u00b1 1^a^ | 3 \u00b1 1^a^ | 0^b^ | 3 \u00b1 1^a^ | 3 \u00b1 1^a^ |\n| Cholesterol (mg) | 249 \u00b1 46^a^ | 239 \u00b1 24^a^ | 68 \u00b1 3^b^ | 223 \u00b1 27^a^ | 73 \u00b1 9^b^ |\n| Cholesterol (mg\/kcal) | 0.13 \u00b1 0 | 0.13 \u00b1 0 | 0.17 \u00b1 0 | 0.14 \u00b1 0 | 0.12 \u00b1 0 |\n| Fiber (g) | 16 \u00b1 2^a^ | 12 \u00b1 2^a^ | 10 \u00b1 1^b^ | 15 \u00b1 1^a^ | 7 \u00b1 1^b^ |\n| Fiber (g\/kcal) | 0.008 \u00b1 0 | 0.008 \u00b1 0 | 0.02 \u00b1 0 | 0.009 \u00b1 0 | 0.01 \u00b1 0 |\n\n^1^ Values reported as mean \u00b1 SEM. Values in the same row with different superscript letters are significantly different, *P* \\<0.05 (One-factor ANOVA with Bonferroni analysis).\n\n^2^ Food intake self-reported each week using 3-d food record.\n\n^3^ Food was provided on the fast day during the controlled feeding phase.\n\n## Hunger, satisfaction with diet, and fullness\n\nChanges in hunger, satisfaction, and fullness during the trial are displayed in Figure <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>. During the first week of ADMF, hunger scores were elevated. However, after two weeks of ADMF, hunger scores decreased (*P* \\< 0.05) and remained low throughout the rest of the trial. Satisfaction with the ADMF diet was low during the first 4 weeks of the intervention, but gradually increased (*P* \\< 0.05) during the last 4 weeks of the study. Fullness scores remained low during the entire 8-week ADMF intervention.\n\n## Changes in physical activity habits\n\nAll subjects wore a pedometer each day throughout the entire trial to assess changes in physical activity habits. On average, subjects were very compliant with pedometer use, and steps were recorded on 87 \u00b1 4% of study days. We hypothesized that subjects would feel less energetic on the fast days, and would therefore take less steps\/d on fast days than feed days. Interestingly, no difference was noted when fast day values (6416 \u00b1 851 steps\/d) were compared to feed day values (6569 \u00b1 910 steps\/d) (Figure <a href=\"#F4\" data-ref-type=\"fig\">4<\/a>). Moreover, physical activity remained constant throughout the 10-week study, as steps\/d taken during the control phase was similar to that of the ADMF phases.\n\n# Discussion\n\nPreliminary reports indicate that ADMF may be an effective strategy to help obese individuals lose weight \\[13,14\\]. However, the dietary and physical activity adaptations that contributed to this pronounced weight loss by ADMF were not tested previously. We show here, for the first time, that weight loss by ADMF occurred due to change in meal pattern, i.e. obese subjects limited their energy intake to 25% of needs on the fast day with no hyperphagic response on the feed day. This change in meal pattern helped these subjects to achieve a marked degree of energy restriction (37% net daily) which was related to the pronounced weight loss attained (5.6 kg in 8 weeks). This study is also the first to demonstrate that subjects become habituated to the ADMF diet (i.e. feel very little hunger on the fast day) after approximately 2 weeks, and that physical activity habits are not affected by fasting on alternate days.\n\nA key objective of the present study was to examine the degree of energy restriction achieved by ADMF and to investigate how this relates to rate of weight loss. In order to measure energy intake and percent energy restriction, we asked obese participants to complete food records on feed and fast days throughout the trial. Results from the food record analysis reveal that obese subjects were able to consistently limit their energy intake to approximately 25% of needs (500 kcal) on the fast day. Our data also show that the ratio of energy consumed on the fast day versus the feed day did not differ between phases. However, it should be noted that there was a trend towards consuming less energy on the feed days, and more energy on the fast days over the course of the trial. It will therefore be of interest in long-term ADMF studies to examine whether restriction gradually diminishes on the fast day after several months of diet. The degree of hyperphagia that occurred on the feed day in response to the lack of food on the fast day was also assessed. Our data indicate that no hyperphagic response took place as subjects only consumed approximately 95% of their calculated energy needs on each feed day throughout the trial. These findings therefore suggest that obese subjects are able to drastically change their meal pattern in a way that conforms to the ADMF protocol. Nevertheless, there are several limitations to these data that must be discussed. First and foremost, it is well known that obese subjects underreport energy intake by 20 to 40% when completing food records \\[20,21\\]. The extent to which these subjects underreported energy intake became apparent when we tried to relate reported energy intake to the weight loss achieved. From the food record data, we calculated that, on average, subjects were restricted by 37% of calculated needs every day. If the subjects were indeed restricted by this amount, this would have resulted in a rate if weight loss of 1.2 kg\/week. In actuality, the rate of weight loss was 0.7 kg\/week. This disparity between reported intake and weight loss can be observed when examining the limited amount of weight lost during the 4-week self-selected feeding phase (body weight reduction of 93.8 kg to 92.8 on the feed day, equivalent to 1 kg of weight loss). The incongruity between self-reported energy intake and rate of weight loss therefore suggests that subjects were underreporting energy intake. In view of this, it will be important for future ADMF trials to assess energy intake and energy restriction by more robust methods, such as the doubly labeled water technique \\[22,23\\]. It should also be mentioned that assessing body weight changes by ADMF is difficult as weight measurements are drastically different from feed to fast day. This discrepancy in body weight is most likely due to the additional weight of food present in the gastrointestinal tract, and not changes in fat mass from day to day. As a potential solution, future trials of ADMF should average body weight measurements taken from consecutive feed and fast days to attain a more accurate assessment of weight.\n\nIn addition to energy intake, we also examined changes in dietary macronutrient composition throughout the course of the trial. We hypothesized that during the ADMF controlled feeding phase (weeks 3-6), when dietary counseling was not provided, subjects would instinctively choose higher fat\/more energy dense foods on the feed day to make up for the lack of energy consumed on the fast day. Interestingly, fat intake did not increase from the baseline period (36% of kcal) to the ADMF-controlled feeding period (36% of kcal). These preliminary data suggest that subjects are not likely to consume higher fat diets on the feed day when partaking in an ADMF regimen. We also hypothesized that the dietary counseling provided during the self-selected feeding phase (weeks 7-10), would help subjects decrease total fat, saturated fat, and cholesterol intake, while increasing fiber intake. Results reveal that counseling assisted these individuals in lowering their total fat and saturated fat intakes to levels that conform with NCEP dietary recommendations \\[19\\], and that these changes in fat intake were related to rate of weight loss. On the other hand, dietary counseling appeared to have no effect on cholesterol or fiber intake. This lack of effect of dietary counseling on the intakes of these nutrients has been reported previously \\[24\\]. It should also be noted that fiber intake on the fast day was particularly low (7-10 g\/d). Since quantity of food consumed on the fast day is limited, it would be difficult for individuals to meet fiber requirements \\[25\\]. As such, it is recommended that future trials in the ADMF field provide a fiber supplement on the fast day to help individuals meet recommendations \\[19,26\\].\n\nChanges in perceived hunger, satisfaction with diet, and fullness were also evaluated on each fast day throughout the trial. This study is the first to show that obese subjects become habituated with ADMF after approximately 2 weeks of diet (i.e. feel very little hunger on the fast day). Our data also demonstrate that subjects become more satisfied with ADMF after approximately 4 weeks of diet. Feelings of fullness, however, remained low across the course of the trial suggesting that subjects never felt \"full\" at any point while undergoing 8-weeks of ADMF. These findings may have important implications for long-term adherence to ADMF by obese men and women \\[27-29\\]. More specifically, since hunger virtually diminishes, and since satisfaction with diet considerably increases within a short amount of time (2-4 weeks), it is likely that obese participants would be able to follow the diet for longer periods of time. It is important to note, however, that the subjects only completed the VAS scales pre-bedtime. Thus, the data only reflects their feelings immediately before going to bed, and is not indicative of their feelings of hunger and satisfaction throughout the day. Future trials in this area should administer these VAS scales throughout the day to obtain a more complete data set for these variables. It should also be noted that hunger spiked at week 8. We speculate that this may have occurred because this study week corresponded to Memorial Day weekend, and subjects may have felt hungrier while attending food-related celebrations. Moreover, trials examining the ability of obese subjects to comply with ADMF for longer durations (i.e. 24 to 52 weeks), and in consequence, lose larger amounts of weight, will be an important focus of future research in this field.\n\nThe effects of ADMF on habitual physical activity was also assessed by having the subjects wear a pedometer on everyday of the study. We hypothesized that subjects would feel less energetic on the fast days, and would therefore be less physically active (i.e. take less steps\/d) on fast days than feed days. Surprisingly, physical activity level did not differ between feed and fast days. Moreover, there was no difference in activity level when steps\/d taken during the ADMF phase were compared to steps\/d taken during the control phase. Similar results have also been reported in normal weight individuals undergoing ADMF \\[12\\]. These data suggest that obese individuals are able to maintain their level of habitual physical activity despite decreases in energy intake on the fast day. This maintenance of physical activity while undergoing ADMF would thus allow obese individuals to lose weight consistently on feed and fast days as energy expenditure would stay constant.\n\nA key limitation of this study is that there was no true control group. Having a control arm run parallel to the treatment (ADMF) arm would have strengthened the study by allowing us to: 1) compare changes in the ADMF group to that of a non-restricted control group at each time point, and 2) identify events (such as holidays) that may have resulted in deviations from the prescribed diet. Future studies aiming to test similar objectives should employ a control group where possible.\n\nIn summary, these findings indicate that obese subjects quickly adapt to ADMF, and that changes in energy\/macronutrient intake, hunger level, and maintenance of physical activity play a role in influencing rate of weight loss by ADMF. These preliminarily data offer promise for the implementation of ADMF as a long-term weight loss strategy in obese populations.\n\n# Competing interests\n\nThe authors declare that they have no competing interests.\n\n# Authors' contributions\n\nMCK performed all the diet analyses and assisted with trial coordination and manuscript preparation. SB coordinated the human clinical trial. MF and SF assisted with data analysis and manuscript preparation. KAV designed the study and wrote the manuscript. All authors read and approved the final manuscript.\n\n## Acknowledgements\n\nWe are grateful for the help of Kathryn Tomaszewski and Kristin Hoody during the analysis phase of the trial. Funding source: University of Illinois at Chicago, Departmental funding.","meta":{"dup_signals":{"dup_doc_count":130,"dup_dump_count":38,"dup_details":{"curated_sources":2,"2023-40":2,"2023-14":1,"2022-40":1,"2022-21":1,"2021-49":1,"2021-39":1,"2021-31":1,"2021-17":1,"2021-04":1,"2020-40":1,"2020-29":1,"2020-16":1,"2020-10":1,"2019-39":1,"2019-30":1,"2015-48":5,"2015-40":2,"2015-35":5,"2015-32":3,"2015-27":3,"2015-22":5,"2015-14":5,"2014-52":4,"2014-49":7,"2014-42":14,"2014-41":8,"2014-35":6,"2014-23":7,"2014-15":7,"2024-26":1,"2024-18":1,"2015-18":4,"2015-11":5,"2015-06":5,"2014-10":5,"2013-48":5,"2013-20":4,"2024-30":1}},"file":"PMC2941474"},"subset":"pubmed_central"}
{"text":"abstract: The 'action' in genome-level evolution lies not in the large gene-containing segments that are conserved among related species, but in the breakpoint regions between these segments. Two recent papers in *BMC Genomics* detail the pattern of repetitive elements associated with breakpoints and the epigenetic conditions under which breakage occurs.\nauthor: David Sankoff\ndate: 2009\ninstitute: 1Department of Mathematics and Statistics, University of Ottawa, 585 King Edward Avenue, Ottawa K1N 6N5, Canada\nreferences:\ntitle: The where and wherefore of evolutionary breakpoints\n\nFor many years, dating back to well before the genomics era, there have been numerous observations and hypotheses of associations between the presence or absence of breakpoints of chromosomal evolution and prominent features of the genomic landscape: telomeres, centromeres, recombination hotspots, gene deserts or gene-rich regions, isochores, cytogenetically fragile sites, oncological rearrangements, segmental duplications, transposons and other repetitive elements. Two recent papers in *BMC Genomics* take somewhat different tacks on this subject. Longo *et al.* \\[1\\] capitalize on new sequencing resources for the tammar wallaby, *Macropus eugenii*, to substantiate the links between the rapid and complex patterns of evolution of centromeric sequence and recurrent rearrangement activity in marsupials, and to discover one evolutionary breakpoint region in humans that has repetitive element similarity to corresponding regions in marsupials. Lemaitre *et al.* \\[2\\] combine a high-resolution breakpoint localization procedure with specialized data that they have calculated or obtained on DNAse sensitivity, CG content, hypomethylation and replication origins \\[3\\] to dispel some of the most widespread folklore in the field. They show that propensity to breakage is not favored in gene deserts but, on the contrary, is closely related to transcriptional activity and DNA accessibility in a region, a conclusion that lends a decidedly epigenetic flavor to our understanding of rearrangement.\n\n# The ephemeral breakpoint\n\nA breakpoint or breakpoint region is not a tangible physical entity in a genome; it is an analytical construct arising only in the comparison of two genomes and, as such, exists or not, and has one set of characteristics or another, depending on the assumptions and methodology of this comparison. When we can identify two contiguous chromosomal segments in one genome, each of which seems orthologous to a different segment in another genome, and these latter segments are not contiguous, we can say that there is a breakpoint. When one of the segments is small (according to a threshold of anywhere from 10^2^ to 10^6^ base pairs), we might wish to consider the two breakpoints delimiting the segment as reflecting a single breakpoint. If the two segments are actually contiguous in the second genome but one is inverted compared with its orientation in the first genome, we might want to count the breakpoint or not. Normally, the DNA alignment of the two genomes will not be such that the breakpoint can be pinpointed as separating two specific adjacent base pairs, but rather there will be a more or less lengthy region in the middle of the segment on the first genome that does not align well to either of the two segments of the second genome or their flanking sequences. Instead of break 'point', we have a break 'region' with its own particular characteristics \\[4\\].\n\nTo complete the deconstruction of the breakpoint terminology, we can na\u00efvely imagine the free ends of two or more double-stranded breaks in DNA molecules flailing around inside the nucleus until they are repaired (incorrectly), resulting in a rearrangement within a chromo some or involving two chromosomes. This does indeed happen as a result of radiation, toxic or mechanical stress or, as is clearly demonstrated by Lemaitre *et al.* \\[2\\], following normal cellular activity that requires regions of open chromatin. It should be emphasized, however, especially where breakpoints are associated with repetitive elements, rearrangements do not derive from any actual DNA breakage, but from nonhomologous recombination caused by faulty alignment of repetitive elements during meiosis.\n\nThe Longo *et al.* article \\[1\\] contains a carefully executed and controlled analysis of the distribution of different kinds of repetitive elements in selected segments from three kinds of genomic region in the tammar wallaby: centromeric regions, breakpoint regions (actually three locations in one breakpoint region) and euchromatic regions not containing a breakpoint. They showed a dramatic enrichment in the breakpoint region of sequence characteristic of endogenous retroviruses (ERVs) and LINE1 transposable elements, and a deficiency of SINE and CR1 transposable element sequences, when compared with the euchromatic regions, with the centromeric regions falling in between the two other patterns. In addition, in a human genomic region containing parts homologous to the marsupial breakpoint region and parts homologous to one of the euchromatin selections, the pattern of repetitive elements makes a transition from ERVs and LINE1s to SINEs. This is suggestive of an association between neocentromeric tendencies, regional instabilities around evolutionary breakpoints and the incorporation of specific kinds of repetitive elements. Although the authors' \\[1\\] longstanding interest in marsupial evolution and the role of centromeres in genomic rearrangements, as well as the availability of new sequence resources on the tammar wallaby, are certainly sufficient motivation for the study of repetitive elements in this context, and given the very different patterns known for human and primate pericentromeric evolution, it will now be important to generalize this work to genomes for which sequencing is essentially complete and to undertake a more comprehensive survey of repetitive elements in regions of each kind.\n\n# Reuse and recurrence\n\nThe term 'breakpoint reuse' is used in the rearrangements literature to cover two rather different concepts. In its original algorithmic use \\[5\\], it denoted the excess of the number of rearrangements necessary to transform one genome into another compared with half the number of breakpoints induced by the comparison of two genomes (given that inversions and reciprocal translocations normally create two breakpoints each). This was accounted for by assuming that some breakpoints (without specifying which ones) were used more than once in the transformation. Soon afterwards, its most frequent meaning became the recurrence of the same breakpoint in two lineages but not their common ancestor with respect to an outgroup lineage \\[6\\]. Despite the attractiveness of these concepts to many authors (such as Longo *et al.* \\[1\\]), neither breakpoint reuse nor breakpoint recurrence is solidly established as a major evolutionary phenomenon, in contrast to well-known disease-causing somatic cell rearrangements. The original concept of reuse, which did not pertain to particular breakpoints but only their aggregates, has rarely if ever been systematically and quantitatively documented at the level of all the individual breakpoints induced by a pair of genomes. Indeed, the algorithmic results suggesting breakpoint reuse are not only wildly variable depending on how telomeric breakpoints are weighted \\[7\\], but are in any case predictable artifacts of highly constrained models of evolution through rearrangement \\[8\\] (models that permit no deletion of chromosome segments, no chromosome or chromosomal arm duplication, no segmental duplication, no transpositions, no jumping translocations and no deletion of paralogous syntenic blocks or interleaving deletions of duplicated blocks), and of the levels of resolution used in defining synteny blocks and breakpoint regions \\[9,10\\]. In the breakpoint definition above, if two breakpoints are collapsed when the small segment between them is below threshold size (a common practice), this mistakenly shows up as an increase in breakpoint reuse. As for the phylogenetic recurrence of breakpoints, the major source in this field \\[6\\] actually shows that 80% of the breakpoints in their mammalian phylogeny are not recurrent, and that almost all of the remaining ones affect the syntenically unstable rodent lineage. The tiny proportion of apparently recurrent breakpoints in the rest of the phylogeny would be hard to distinguish from coincidence, given the resolution of the synteny block construction.\n\nThe connection between the 'fragile sites' in traditional cyto genetics and evolutionary breakpoints is exceedingly weak \\[11\\] and, indeed, statistically insignificant except through a heuristically contrived categorization of the data. The same may be said for the oft-cited attempt \\[6\\] to associate cancer breakpoints with evolutionary breakpoints by selectively comparing only two of the reported frequency categories of neoplastic breakpoints.\n\n# Accident and selection\n\nAn e volutionary breakpoint is the product not only of some meiotic accident at a site predisposed to breakage or nonhomologous recombination. It is also a configuration that has managed to do all of the following: make it through steps of abnormal chromosome alignment and segregation to the gamete stage; participate in creating a viable heterokaryotypic zygote that eventually develops into reproductive maturity; endure generations of likely negative selection; and emerge through genetic drift as a homokaryotypic feature of some presumably small bottleneck population. Predisposition to breakage at the cellular level is just the first step on the road to fixation, and phenotypic selection operating at the meiotic, embryonic, adult and population levels has a more important role. Somatic cells presumably have many of the same predispositions to physical breakage, although not of course to nonhomologous recombination, but cancer cells do not have to survive meiosis or life outside the affected individual, and that may be a large part of the reason why the repertoire and quantitative distribution of rearrangements in tumor genomes are very different from those in evolution \\[12\\].\n\nGenetic deduction appealing to selection-based arguments at the gene expression level, together with indirect and anecdotal evidence, has recently prompted speculation about prohibition of rearrangement breakage in short inter genic regions in mammals \\[13\\]. These claims, however, have effectively been demolished by Lemaitre *et al.* \\[2\\], who measured directly and systematically, at a high level of resolution, the connections between both high rate of breakage and short intergenic distances and four strong correlates of transcriptional activity: GC content, proximity to origins of replication (as inferred from 'N-domains' \\[3\\]), hypomethylation (based on CpG ratios) and DNase sensitivity. This innovative and convincing work, to which the authors added support ranging from the classic Bernardi theory of isochores \\[14\\] to the more recent mammalian replicon model, overturns the conventional genetic wisdom and reopens evolutionary questions about mechanisms promoting neutral variation at the karyotypic level. It adds a weighty contribution to the accumulating body of results, such as those on the gibbon *Nomascus leucogenys leucogenys* \\[15\\] and those previously produced by the O'Neills-Graves collaboration on marsupials, cited in the Longo *et al.* article \\[1\\], on the epigenetic conditioning of evolutionary chromosome rearrangement.\n\n### Acknowledgements\n\nThis work was supported in part by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC). DS holds the Canada Research Chair in Mathematical Genomics.","meta":{"dup_signals":{"dup_doc_count":130,"dup_dump_count":41,"dup_details":{"curated_sources":2,"2020-40":1,"2020-10":1,"2019-51":1,"2019-43":1,"2019-22":1,"2019-09":1,"2018-51":1,"2018-13":1,"2017-47":1,"2017-34":1,"2017-22":1,"2017-09":11,"2016-50":1,"2016-44":1,"2016-40":1,"2016-36":11,"2016-30":11,"2016-22":1,"2016-18":1,"2016-07":10,"2015-48":2,"2015-40":3,"2015-35":3,"2015-32":3,"2015-27":3,"2015-22":2,"2015-14":3,"2014-52":3,"2014-49":1,"2014-42":8,"2014-41":5,"2014-35":6,"2014-23":4,"2014-15":4,"2022-05":1,"2015-18":3,"2015-11":3,"2015-06":3,"2014-10":3,"2013-48":3,"2013-20":3}},"file":"PMC2736669"},"subset":"pubmed_central"}
{"text":"abstract: The authors describe the development, implementation, and evaluation of their innovative social marketing campaign.\nauthor: Katherine Ahrens; Charlotte K Kent; Jorge A Montoya; Harlan Rotblatt; Jacque McCright; Peter Kerndt; Jeffrey D Klausner\\* To whom correspondence should be addressed. E-mail:<firstname.lastname@example.com>\ndate: 2006-12\nreferences:\ntitle: Healthy Penis: San Francisco's Social Marketing Campaign to Increase Syphilis Testing among Gay and Bisexual Men\n\n# The Problem: Sharp Increase in Syphilis among Men Who Have Sex with Men\n\nSan Francisco experienced a sharp rise in early syphilis between 1999 and 2002, with the number of cases rising from 44 to 494 per year (<a href=\"#pmed-0030474-g001\" data-ref-type=\"fig\">Figure 1<\/a>). Rates continued to rise through 2004. Since 1999, most syphilis cases have been among men who identified as gay or bisexual (88%), were white (60%), and were infected with HIV (61%) (<a href=\"#pmed-0030474-g001\" data-ref-type=\"fig\">Figure 1<\/a>). In June 2002, the San Francisco Department of Public Health (SFDPH), STD Prevention and Control Services launched a social marketing campaign, called Healthy Penis, designed to increase syphilis testing and awareness among gay and bisexual men.\n\nSocial marketing, like marketing in the private sector, is a research-driven approach to behavior change \\[1\\]. Unlike commercial marketing, however, it is truly consumer-centered and designed to increase the health of the consumer \\[2\\]. The success of any social marketing campaign can be attributed to the net effect of its five main components: branding, segmentation, price, placement, and promotion \\[1\u20133\\]. The first component, *branding*, focuses on the health behavior message so that the desired behavior, or product, appeals to the needs and values of the consumer through functional and emotional attributes.\n\nThe second component is concerned with the *segmentation* of the intended audience\u2014developing a campaign message that emphasizes the target population's values, attitudes, and beliefs and\/or capitalizes on their current stage of behavior change. Messages that are customized in this manner will ensure that the health behavior (product) is appealing and applicable to each subgroup.\n\nThe third component is *price*. In social marketing, price is the social, psychological, or physical cost the consumer associates with performing the health behavior. The fourth component is product *placement*. In public health, this involves delivering the resources that make the desired health behavior possible at a time when it will most likely be sought out. The fifth component is the *promotion* of the health behavior through communication media like print, television, radio, outdoor advertising, or face-to-face techniques that are determined by the information-consumption habits of the consumer. Notable successful social marketing campaigns include North Carolina's \"Click It or Ticket\" campaign aimed at increasing seatbelt use, Florida's \"truth\" anti-smoking campaign, and the United States National WIC (Women, Infants and Children) Breastfeeding Promotion project \\[4\\].\n\nThe impact of a social marketing campaign is evaluated based on the measurement of change in the targeted behavior, i.e., increased syphilis testing in the case of Healthy Penis, and the extent to which this change is associated with people who received the campaign messages. To that end, we initially evaluated the Healthy Penis campaign in 2002\u20132003 and found a higher rate of syphilis testing among those aware of the campaign than those unaware of the campaign \\[5\\]. In addition, syphilis knowledge was higher among those aware, a secondary goal of the campaign.\n\nIn this article we describe the development, implementation, and evaluations of the Healthy Penis campaign. We present a simple method to evaluate the immediate and long-term effectiveness of a public health media campaign designed to change behavior. We include our most recent evaluation, conducted in 2004\u20132005, which assessed the long-term effectiveness of the campaign \\[6\\] and determined if respondents aware of the campaign 2.5 years after it began displayed the same syphilis knowledge and testing behavior as those initially aware of the campaign in the first evaluation.\n\n# Healthy Penis Campaign Development\n\nThe Healthy Penis campaign was launched during the annual San Francisco Lesbian Gay Bisexual Transgender Parade in late June 2002 and continued through December 2005. A San Francisco\u2013based social marketing firm, Better World Advertising, created the campaign in collaboration with the SFDPH to target the gay and bisexual community. This campaign was part of a broad multifaceted effort begun in 1999 by the SFDPH in response to the syphilis epidemic \\[7\\].\n\nThe campaign was developed in collaboration with the Los Angeles\u2013based Stop the Sores syphilis prevention campaign, which allowed us to lower our start-up costs to \\$75,000 in 2002. The campaign was continued through the end of 2005 at an additional cost of \\$295,000 \\[7\\]. Three-quarters of the campaign's first-year funds were spent on campaign development, with the remaining quarter spent on displaying campaign materials \\[8\\].\n\nSyphilis is often initially asymptomatic and testing is readily available in San Francisco for easy diagnosis. Based on these characteristics and input from the community partners group, the SFDPH determined that the primary campaign health behavior message should be \"get tested,\" with the main objective of changing community norms around syphilis testing. Secondary objectives were to increase awareness about the syphilis epidemic in gay and bisexual men and to increase knowledge about syphilis (symptoms, routes of transmission, the link between syphilis and HIV transmission, and so on). The complete methodology for selection of campaign concepts and themes for this campaign has been described in a previous manuscript \\[5\\].\n\nThe Healthy Penis campaign was promoted in neighborhoods where the greatest concentration of gay or bisexual men lived and where there were businesses that catered to this population. The campaign incorporated the use of humorous cartoon strips that featured characters like Healthy Penis and Phil the Sore to: (1) promote syphilis testing, (2) publicize the rise of syphilis among gay and bisexual men, (3) provide information on syphilis transmission, symptoms, and prevention, and (4) delineate the connection between syphilis and HIV (<a href=\"#pmed-0030474-g002\" data-ref-type=\"fig\">Figure 2<\/a>). These cartoon strips were initially published semi-monthly in a popular gay Bay Area publication. After publication, poster-size reproductions were posted on the streets; in bars and commercial sex venues; on bus shelters and bus advertising; on palm cards; and on banner advertisements on one of the most popular Internet sites for meeting sex partners among gay men.\n\nTopics addressed in campaign cartoons initially emphasized all four areas described above and then after 1.5 years focused primarily on the message \"get tested.\" In addition to messages contained within the cartoon strip, brief text boxes were displayed on the bottom of the cartoon images describing modes of syphilis transmission (skin-to-skin contact, oral, anal, and vaginal sex), symptoms (painless sore and\/or rash), and explaining that syphilis is curable. These text boxes were displayed on cartoons from June 2002 to October 2003 with decreasing frequency.\n\n# Evaluation of the Campaign: What Impact Has It Had?\n\n## Survey methodology.\n\nTo evaluate the effectiveness of the campaign, we conducted two waves of surveys using the same instrument: one six months after the campaign began (December 2002\u2013February 2003; Evaluation I), and a second 2.5 years after the campaign began (September 2004\u2013March 2005; Evaluation II). For each survey, men were intercepted at coffee shops, bars, markets, laundromats, sex clubs, a clean and sober community center, on sidewalks, and in other venues located in campaign-targeted neighborhoods.\n\nRespondents were asked about basic demographic information; unaided (e.g., spontaneous mention) and aided (e.g., prompted response) awareness of the Healthy Penis campaign; perceived key messages of the campaign; syphilis knowledge (via open-ended questions); sexual practices in the past month; HIV status; and how many times they were tested for syphilis in the past six months.\n\nComparisons were made among those aware of the campaign between the first and second sample of respondents. Recent history of syphilis testing was compared with campaign awareness level for each evaluation separately. Analysis included Fisher's Exact and Cochran-Armitage tests, and logistic regression.\n\n## Survey population.\n\nTwo hundred and forty-four interviews were conducted with San Francisco residents and included in the first evaluation; 150 interviews were included in the second evaluation. For each evaluation, all respondents were men who have sex with men (MSM) and most were white and HIV negative. Our respondents were similar to the MSM population in San Francisco. More detailed characteristics of the two samples are described in <a href=\"#pmed-0030474-t001\" data-ref-type=\"table\">Table 1<\/a>. There were no significant demographic differences between Evaluation I and II participants.\n\n###### Demographic Characteristics of Respondents from First and Second Evaluations of the Healthy Penis Campaign\n\n![](pmed.0030474.t001)\n\n## Campaign awareness.\n\nCampaign awareness was high with 80% (*n* = 194\/244) and 85% (*n* = 127\/150) of respondents aware of the campaign in Evaluation I and II, respectively. Unaided and aided awareness was similar between the two evaluations; 33% versus 41% spontaneously mentioned the Healthy Penis campaign (unaided awareness) when asked to recall recent advertisements or public events about sexual health issues, and an additional 47% versus 44% recognized the campaign (aided awareness) when shown a campaign image (Evaluation I and II, respectively). There was no difference in overall campaign awareness, unaided awareness, or aided awareness between the two evaluations (*p* = 0.23, *p* = 0.13, *p* = 0.60, respectively). Among respondents aware of the campaign, campaign exposure was greater in the first evaluation; Evaluation I respondents reported a median of six exposures (mean, 18) over the previous three months, while Evaluation II respondents reported a median of five times (mean, six) in the previous three months (Wilcoxon two-sample test, *p* \\< 0.0001). Perceptions of key messages among respondents who were aware of the campaign did not change markedly between Evaluations I and II, with the most common message identified being \"get tested\" (53% versus 55%, respectively, *p* = 0.73).\n\n## Syphilis testing in the last 6 months.\n\nBoth evaluations found a positive association between campaign awareness and recent syphilis testing\u2014the primary objective of the social marketing campaign. An increasing proportion of respondents reported syphilis testing in the previous six months by campaign awareness level (none, aided awareness, and unaided awareness): Evaluation I: 26%, 40%, 55% (Cochran-Armitage trend test z = \u22123.303, *p* = 0.001); Evaluation II: 35%, 42%, 60% (Cochran-Armitage trend test z = \u22122.304, *p* = 0.02) (<a href=\"#pmed-0030474-g004\" data-ref-type=\"fig\">Figure 4<\/a>). After controlling for potential confounders (age, HIV status, casual sex partners in the last month) in a multivariable logistic regression model, each increase in campaign awareness level during Evaluation I was associated with a 90% increase in likelihood for having tested for syphilis in the past six months (odds ratio \\[OR\\] 1.9 \\[95% confidence interval (CI), 1.3\u20132.9\\]). Other variables significantly related to syphilis testing in Evaluation I included being HIV infected (OR 4.9 \\[95% CI, 2.3\u201310.5\\]), and having casual sex partners in the last month (OR 2.0 \\[95% CI, 1.1\u20133.9\\]). The same multivariable model applied to Evaluation II respondents found each increase in campaign awareness level to be associated with a 76% increase in likelihood for syphilis testing (OR 1.76 \\[95% CI, 1.01\u20133.1\\]), and no other factors were significant in the model, although HIV-infected respondents showed an increased likelihood of syphilis testing (OR 2.1 \\[95% CI, 0.9\u20135.2\\], *p* = 0.10). In both evaluations, a higher proportion of HIV-positive respondents reported syphilis testing than did HIV-negative respondents (Evaluation I 71% versus 34%, p \\< 0.05, Evaluation II 63% versus 44%, *p* = 0.07).\n\nOverall, reported syphilis testing in the previous six months did not change from the first evaluation to the second evaluation (42% versus 49%, *p* = 0.25).\n\n## Syphilis knowledge.\n\nThere were few significant differences between evaluations among respondents aware of the campaign in terms of knowledge of syphilis, including knowledge of symptoms; groups most affected by syphilis; how to find out if you have syphilis; health consequences of untreated syphilis; and the relationship between syphilis and HIV acquisition and transmission (<a href=\"#pmed-0030474-g003\" data-ref-type=\"fig\">Figure 3<\/a>).\n\n## Syphilis incidence.\n\nIn 2005, incidence of early syphilis was lower than in the previous three years, with decreases in cases in gay\/bisexual men accounting for the drop \\[9\\]. Although our campaign evaluations were cross-sectional and causality cannot be determined, it does appear that the Healthy Penis campaign, along with other SFDPH syphilis elimination efforts \\[7\\], may have led to this decrease in syphilis incidence.\n\n# Conclusions from Evaluations\n\nMSM who were aware of the Healthy Penis campaign were more likely than those unaware to have recently tested for syphilis and to have greater knowledge about syphilis. This effect was sustained for almost three years.\n\n# Next Steps for Healthy Penis\n\nAfter initial evaluation of the Healthy Penis campaign, elements have been used in Philadelphia, Seattle, Palm Springs, and Santa Clara County, California ([www.healthypenis.org](http:\/\/www.healthypenis.org)). Thus the campaign can be translated to other jurisdictions. Because much of the material is already developed, other jurisdictions may adapt and use the campaign at a lower cost than was originally incurred in San Francisco, although they will need to conduct evaluations to ensure the campaign is effective for their population. In addition, the principles used in the development, implementation, and evaluation of Healthy Penis are universally applicable for any social marketing campaign.\n\nBased on our experience with the Healthy Penis campaign, we believe spending more start-up resources on campaign development rather than campaign placement \\[8\\] was probably what led to our high level of campaign awareness, demonstrating that our campaign resonated with our target population. We also learned that partnering with another jurisdiction (Los Angeles) lowered start-up costs and creating a Community Partners Group helped us tailor our campaign messages to be most effective. And most importantly, we learned that careful, well-planned evaluations are critical in assessing the impact of a campaign, which then supported continuing the campaign and implementing it elsewhere. Our evaluations strongly suggest that the Healthy Penis social marketing campaign was effective in augmenting syphilis testing and increasing syphilis awareness and knowledge in the San Francisco gay and bisexual community. This effect might have contributed to decreased syphilis incidence in 2005.\n\nThe authors would like to thank Les Pappas from Better World Advertising, the STD Community Advisory Group for Syphilis Elimination, and the SFDPH employees who administered the evaluation surveys.\n\n**Author contributions.** CKK, JAM, HR, JM, PK, and JDK conceived and designed the experiments. KA, JAM, and HR analyzed the data. KA, CKK, JAM, HR, JM, PK, and JDK wrote the paper. JM worked closely with Better World Advertising in the planning and development of the campaign in 1999.\n\n# References\n\n### Abbreviations\n\nCI\n\n:   confidence interval\n\nMSM\n\n:   men who have sex with men\n\nOR\n\n:   odds ratio\n\nSFDPH\n\n:   San Francisco Department of Public Health","meta":{"dup_signals":{"dup_doc_count":114,"dup_dump_count":38,"dup_details":{"curated_sources":2,"2020-45":1,"2020-24":1,"2019-51":1,"2019-09":1,"2018-22":1,"2017-43":1,"2017-34":1,"2017-22":1,"2017-09":7,"2016-50":1,"2016-44":1,"2016-40":1,"2016-36":1,"2016-30":3,"2016-18":1,"2016-07":2,"2015-48":5,"2015-40":2,"2015-35":2,"2015-32":2,"2015-27":2,"2015-22":3,"2015-14":5,"2014-52":6,"2014-49":5,"2014-42":8,"2014-41":7,"2014-35":7,"2014-23":7,"2014-15":7,"2022-49":1,"2024-18":1,"2024-10":1,"2015-18":5,"2015-11":3,"2015-06":2,"2014-10":5,"2024-26":1}},"file":"PMC1762065"},"subset":"pubmed_central"}
{"text":"abstract: Until the advent of modern neuroscience, free will used to be a theological and a metaphysical concept, debated with little reference to brain function. Today, with ever increasing understanding of neurons, circuits and cognition, this concept has become outdated and any metaphysical account of free will is rightfully rejected. The consequence is not, however, that we become mindless automata responding predictably to external stimuli. On the contrary, accumulating evidence also from brains much smaller than ours points towards a general organization of brain function that incorporates flexible decision-making on the basis of complex computations negotiating internal and external processing. The adaptive value of such an organization consists of being unpredictable for competitors, prey or predators, as well as being able to explore the hidden resource deterministic automats would never find. At the same time, this organization allows all animals to respond efficiently with tried-and-tested behaviours to predictable and reliable stimuli. As has been the case so many times in the history of neuroscience, invertebrate model systems are spearheading these research efforts. This comparatively recent evidence indicates that one common ability of most if not all brains is to choose among different behavioural options even in the absence of differences in the environment and perform genuinely novel acts. Therefore, it seems a reasonable effort for any neurobiologist to join and support a rather illustrious list of scholars who are trying to wrestle the term 'free will' from its metaphysical ancestry. The goal is to arrive at a scientific concept of free will, starting from these recently discovered processes with a strong emphasis on the neurobiological mechanisms underlying them.\nauthor: Bj\u00f6rn Brembs\\*<email@example.com>\ndate: 2011-03-22\ninstitute: Freie Universit\u00e4t Berlin, Institute for Biology \u2013 Neurobiology, K\u00f6nigin-Luise-Strasse 28\/30, 14195 Berlin, Germany\nreferences:\ntitle: Towards a scientific concept of free will as a biological trait: spontaneous actions and decision-making in invertebrates\n\n# Introduction: the rejection of the metaphysical concept of free will\n\nWhat could possibly get a neurobiologist with no formal training in philosophy beyond a few introductory lectures, to publicly voice his opinion on free will? Even worse, why use empirical, neurobiological evidence mainly from invertebrates to make the case? Surely, the lowly worm, snail or fly cannot even be close to something as philosophical as free will? The main reason is this neurobiologist's opinion that free will is a biological trait and not a metaphysical entity. 'Free will is a biological property, not a gift or a mystery' \\[1\\]. Today, neurobiology has accumulated sufficient evidence that we can move on from speculating about the existence of free will towards plausible models of how brains have implemented it. On the surface, this statement seems to contradict public statements from many other neurobiologists who fervently deny free will. In fact, it appears that if neurobiologists feel compelled to write about free will, they do so only to declare that it is an illusion \\[2\u20135\\]. Of course, all of these neurobiologists are correct in that free will as a metaphysical entity indeed most probably is an illusion. Colloquial and historical use of the term 'free will' has been inextricably linked with one variant or another of dualism. There have been so many and thorough recounts of the free will debate, that I will only reference some, which can serve to introduce the concepts used here \\[6\u20139\\]. Psychologists and neurobiologists have rightfully pointed out for decades now that there is no empirical support for any form of dualism. The interactionism proposed by Popper & Eccles was probably one of the last prominent accounts of dualism \\[10\\]. Since then, these and related positions have largely fallen into irrelevance. Today, the metaphysical concept of free will is largely devoid of any support, empirical or intellectual.\n\n# The rejection of determinism\n\nThat said, it is an all too common misconception that the failure of dualism as a valid hypothesis automatically entails that brains are deterministic and all our actions are direct consequences of gene\u2013environment interactions, maybe with some random stochasticity added in here and there for good measure \\[2\\]. It is tempting to speculate that most, if not all, scholars declaring free will an illusion share this concept. However, our world is not deterministic, not even the macroscopic world. Quantum mechanics provides objective chance as a trace element of reality. In a very clear description of how keenly aware physicists are that Heisenberg's uncertainty principle indeed describes a property of our world rather than a failure of scientists to accurately measure it, Stephen Hawking has postulated that black holes emit the radiation named after him \\[11\\], a phenomenon based on the well-known formation of virtual particle\u2013antiparticle pairs in the vacuum of space. The process thought to underlie Hawking radiation has recently been observed in a laboratory analogue of the event horizon \\[12,13\\]. On the 'mesoscopic' scale, fullerenes have famously shown interference in a double-slit experiment \\[14\\]. Quantum effects have repeatedly been observed directly on the nano-scale \\[15,16\\], and superconductivity (e.g. \\[17\\]) or Bose\u2013Einstein condensates (e.g. \\[18\\]) are well-known phenomena. Quantum events such as radioactive decay or uncertainty in the photoelectric effect are used to create random-number generators for cryptography that cannot be broken into. Thus, quantum effects are being observed also on the macroscopic scale. Therefore, determinism can be rejected with at least as much empirical evidence and intellectual rigor as the metaphysical account of free will. 'The universe has an irreducibly random character. If it is a clockwork, its cogs, springs, and levers are not Swiss-made; they do not follow a predetermined path. Physical indeterminism rules in the world of the very small as well as in the world of the very large' \\[9\\].\n\n# Behavioural variability as an adaptive trait\n\nIf dualism is not an option and determinism is equally untenable, what other options are we left with? Some scholars have resorted to quantum uncertainty in the brain as the solution, providing the necessary discontinuity in the causal chain of events. This is not unrealistic, as there is evidence that biological organisms can evolve to take advantage of quantum effects. For instance, plants use quantum coherence when harvesting light in their photosynthetic complexes \\[19\u201322\\]. Until now, however, it has proved difficult to find direct empirical evidence in support of analogous phenomena in brains \\[9\\]. Moreover, and more importantly, the pure chance of quantum indeterminism alone is not what anyone would call 'freedom'. 'For surely my actions should be caused because I want them to happen for one or more reasons rather that they happen by chance' \\[9\\]. This is precisely where the biological mechanisms underlying the generation of behavioural variability can provide a viable concept of free will.\n\nBiologists need not resort to quantum mechanics to understand that deterministic behaviour can never be evolutionarily stable. Evolution is a competitive business and predictability is one thing that will make sure that a competitor will be out of business soon. There are many illuminating examples of selection pressures favouring unpredictability, but three recently published reports dealing with one of the most repeatable and hence best-studied class of behaviours are especially telling. These examples concern escape behaviours.\n\nOne of the most well-studied escape behaviours is the so-called C-start response in fishes. The response is called C-start because fishes that perceive sudden pressure changes on one side of their body bend in a C-shape away from the perceived stimulus to escape in the opposite direction. One of the largest neurons in vertebrate nervous systems is mediating this response, the Mauthner cell (e.g. \\[23\\]). Recently, Kenneth Catania and colleagues described the hunting technique of tentacled snakes (*Erpeton tentaculatus*) \\[24,25\\]. The snakes hunt for fishes by cunningly eliciting a C-start response in the potential prey animal with a more caudal part of their body, prompting the fish to C-start exactly into the mouth of the snake.\n\nSome of the most important predators of earthworms are moles. When moles dig through the ground, they produce a very distinctive sound. Earthworms have evolved to respond to this sound by crawling to the surface, where the moles will not follow them. Kenneth Catania recently reported that the technique of 'worm-grunting', employed in order to catch earthworms as fish bait, exploits this response. The worm grunters use a combination of wooden poles and metal rods to generate the sound and then collect the worms from the surface \\[26\\].\n\nIn the third example, another very well-studied escape response is exploited by birds. Under most circumstances, the highly sophisticated jump response of dipteran flies is perfectly sufficient to catapult the animals out of harm's way (e.g. \\[27\\]). However, painted redstarts (*Myioborus pictus*) are ground-hunting birds that flush out dipterans by eliciting their jump response with dedicated display behaviours. Once the otherwise well-camouflaged flies have jumped, they are highly visible against the bright sky and can be caught by the birds \\[28,29\\].\n\nIt is not a huge leap to generalize these insights from escape responses to other behaviours. Predictability can never be an evolutionarily stable strategy. Instead, animals need to balance the effectiveness and efficiency of their behaviours with just enough variability to spare them from being predictable. Efficient responses are controlled by the environment and thus vulnerable. Conversely, endogenously controlled variability reduces efficiency but increases vital unpredictability. Thus, in order to survive, every animal has to solve this dilemma. It is no coincidence that ecologists are very familiar with a prominent, analogous situation, the exploration\/exploitation dilemma (originally formulated by March \\[30\\]): every animal, every species continuously faces the choice between staying and efficiently exploiting a well-known, but finite resource and leaving to find a new, undiscovered, potentially much richer, but uncertain resource. Efficiency (or optimality) always has to be traded off with flexibility in evolution, on many, if not all, levels of organization.\n\nA great invertebrate example of the sort of Protean behaviour \\[31,32\\] selected for by these trade-offs is yet another escape behaviour, that of cockroaches. The cerci of these insects have evolved to detect minute air movements. Once perceived, these air movements trigger an escape response in the cockroach away from the side where the movement was detected. However, which angle with respect to the air movement is taken by the animal cannot be predicted precisely, because this component of the response is highly variable \\[33\\]. Therefore, in contrast to the three examples above, it is impossible for a predator to predict the trajectory of the escaping animal.\n\n# Brains are in control of variability\n\nCompetitive success and evolutionary fitness of all ambulatory organisms depend critically on intact behavioural variability as an adaptive function \\[34\\]. Behavioural variability is an adaptive trait and not 'noise'. Not only biologists are aware of the fitness advantages provided by unpredictable behaviour, but philosophers also realized the adaptive advantages of behavioural variability and their potential to serve as a model for a scientific account of free will, as long as 25 years ago (e.g. \\[6\\]). The ultimate causes of behavioural variability are thus well understood. The proximate causes, however, are much less studied. One of the few known properties is that the level of variability also can vary. Faced with novel situations, humans and most animals spontaneously increase their behavioural variability \\[35\u201338\\]. Animals even vary their behaviour when a more stereotyped behaviour would be more efficient \\[39\\].\n\nThese observations suggest that there must be mechanisms by which brains control the variability they inject into their motor output. Some components of these mechanisms have been studied. For instance, tethered flies can be trained to reduce the range of the variability in their turning manoeuvres \\[40\\]. For example, one such stationary flying fly may be trained to cease generating left-turning manoeuvres by heating the fly (with an infrared heat beam) whenever it initiates such actions and by not heating it during right-turning manoeuvres. Before such training, it would generate left- and right-turning manoeuvres in equal measure. Protein kinase C activity is required for such a reduction \\[41\\]. Interestingly, analogous to the exploration\u2013exploitation dilemma mentioned above, the mechanism by which the animals learn to decrease their behavioural variability ('self-learning') interacts with the learning mechanism by which the animals learn about external stimuli ('world learning'). Part of this interaction balances self- and world learning such that self-learning (i.e. the endogenous reduction in behavioural variability) is slowed down whenever the world-learning mechanism is engaged. This part of the interaction is mediated by a subpopulation of neurons in a part of the insect brain called the mushroom bodies \\[42,43\\]. This population of neurons ensures that animals preferentially learn from their environment and reduce their endogenous behavioural variability only when there are good reasons for doing so. Such an organization may underlie the need for practice in order to reduce our behavioural variability when learning new skills, e.g. the basketball free-throw or the golf swing. The parallel to the exploration\u2013exploitation dilemma lies in the balance between the endogenous and exogenous processing these interactions bestow upon the animal: learning about the world first allows the animal to keep its behaviour flexible in case the environment changes, while at the same time being able to efficiently solve the experimental task. If, however, it turns out that the environment does not change, then\u2014and only then\u2014is the circuitry controlling the behaviour itself modified, to more permanently alter the behaviour-generating process itself and thereby maximize on efficiency by reducing the endogenous variability.\n\nAnimals other than insects also learn to control their variability using feedback from the environment, such that levels of behavioural variability\u2014from highly predictable to random-like\u2014are directly influenced by reinforcement. For instance, consummatory feeding behaviour of the marine snail *Aplysia* is highly variable \\[44,45\\]. Recent evidence suggests that the seemingly rhythmic cycling of biting, swallowing and rejection movements of the animal's radula (a tongue-like organ) vary in order to be able to adapt to varying food sources \\[46\\]. In fact, much like the reduced variability in flies trained to avoid heat in the self-learning paradigm explained above, *Aplysia* can be trained to reduce the variability in their feeding behaviour and generate rhythmic, stereotyped movements \\[47\u201352\\]. It also takes practice for snails to become efficient and predictable. The default state is to behave variably and unpredictably.\n\nThe mechanisms to control behavioural variability are in place also in humans. For instance, depression and autism are characterized by abnormally stereotypic behaviours and a concomitant lack of behavioural variability. Patients suffering from such psychopathologies can learn to vary their behaviours when reinforced for doing so \\[53,54\\]. Also, the interactions between world- and self-learning seem to be present in vertebrates: extended training often leads to so-called habit formation, repetitive responses, controlled by environmental stimuli (e.g. \\[55,56\\]). It is intriguing that recent fMRI studies have discovered a so-called default-mode network in humans, the fluctuations in which can explain a large degree of the individual's behavioural variability \\[57\\], and that abnormalities in this default network are associated with most psychiatric disorders \\[58\u201360\\].\n\n# What are the neural mechanisms generating behavioural variability?\n\nIt thus appears that behavioural variability is a highly adaptive trait, under constant control of the brain balancing the need for variability with the need for efficiency. How do brains generate and control behavioural variability in this balance? These studies have only just begun. As was the case in much of neuroscience's history, be it ion channels, genes involved in learning and memory, electrical synapses or neurotransmitters, invertebrate model systems are leading the way in the study of the neural mechanisms underlying behavioural variability as well.\n\nTwo recent reports, concerned another highly reproducible (and therefore well-studied) behaviour, optomotor responses. Tethered flies respond to a moving grating in front of them with characteristic head movements in the same direction as the moving grating, aimed at stabilizing the image on the retina. By recording from motion-sensitive neurons in fly optic lobes, the authors found that the variability in these neurons did not suffice to explain the variability in the head movements \\[61,62\\]. Presumably, downstream neurons in the central brain inject additional variability, not present in the sensory input, which is reflected in the behaviour.\n\nA corresponding conclusion can be drawn from two earlier studies, which independently found that the temporal structure of the variability in spontaneous turning manoeuvres both in tethered and in free-flying fruitflies could not be explained by random system noise \\[63,64\\]. Instead, a nonlinear signature was found, suggesting that fly brains operate at criticality, meaning that they are mathematically unstable, which, in turn, implies an evolved mechanism rendering brains highly susceptible to the smallest differences in initial conditions and amplifying them exponentially \\[63\\]. Put differently, fly brains have evolved to generate unpredictable turning manoeuvres. The default state also of flies is to behave variably. Ongoing studies are trying to localize the brain circuits giving rise to this nonlinear signature.\n\nResults from studies in walking flies indicate that at least some component of variability in walking activity is under the control of a circuit in the so-called ellipsoid body, deep in the central brain \\[65\\]. The authors tested the temporal structure in spontaneous bouts of activity in flies walking back and forth individually in small tubes and found that the power law in their data disappeared if a subset of neurons in the ellipsoid body was experimentally silenced. Analogous experiments have recently been taken up independently by another group and the results are currently being evaluated \\[66\\]. The neurons of the ellipsoid body of the fly also exhibit spontaneous activity in live imaging experiments \\[67\\], suggesting a default-mode network also might exist in insects.\n\nEven what is often presented to students as 'the simplest behaviour', the spinal stretch reflex in vertebrates, contains adaptive variability. Via the cortico-spinal tract, the motor cortex injects variability into this reflex arc, making it variable enough for operant self-learning \\[68\u201372\\]. Jonathan Wolpaw and colleagues can train mice, rats, monkeys and humans to produce reflex magnitudes either larger or smaller than a previously determined baseline precisely because much of the deviations from this baseline are not noise but variability deliberately injected into the reflex. Thus, while invertebrates lead the way in the biological study of behavioural variability, the principles discovered there can be found in vertebrates as well.\n\nOne of the common observations of behavioural variability in all animals seems to be that it is not entirely random, yet unpredictable. The principle thought to underlie this observation is nonlinearity. Nonlinear systems are characterized by sensitive dependence on initial conditions. This means such systems can amplify tiny disturbances such that the states of two initially almost identical nonlinear systems can diverge exponentially from each other. Because of this nonlinearity, it does not matter (and it is currently unknown) whether the 'tiny disturbances' are objectively random as in quantum randomness or whether they can be attributed to system, or thermal noise. What can be said is that principled, quantum randomness is always some part of the phenomenon, whether it is necessary or not, simply because quantum fluctuations do occur. Other than that it must be a non-zero contribution, there is currently insufficient data to quantify the contribution of such quantum randomness. In effect, such nonlinearity may be imagined as an amplification system in the brain that can either increase or decrease the variability in behaviour by exploiting small, random fluctuations as a source for generating large-scale variability. A general account of such amplification effects had already been formulated as early as in the 1930s \\[73\\]. Interestingly, a neuronal amplification process was recently observed directly in the barrel cortex of rodents, opening up the intriguing perspective of a physiological mechanism dedicated to generating neural (and by consequence behavioural) variability \\[74\\].\n\n# Determinism versus indeterminism is a false dichotomy\n\nTogether with Hume, most would probably subscribe to the notion that 'tis impossible to admit of any medium betwixt chance and an absolute necessity' \\[75\\]. For example, Steven Pinker (1997, p. 54) concurs that 'A random event does not fit the concept of free will any more than a lawful one does, and could not serve as the long-sought locus of moral responsibility' \\[76\\]. However, to consider chance and lawfulness as the two mutually exclusive sides of our reality is only one way to look at the issue. The unstable nonlinearity, which makes brains exquisitely sensitive to small perturbations, may be the behavioural correlate of amplification mechanisms such as those described for the barrel cortex \\[74\\]. This nonlinear signature eliminates the two alternatives, which both would run counter to free will, namely complete (or quantum) randomness and pure, Laplacian determinism. These represent opposite and extreme endpoints in discussions of brain functioning, which hamper the scientific discussion of free will. Instead, much like evolution itself, a scientific concept of free will comes to lie between chance and necessity, with mechanisms incorporating both randomness and lawfulness. The Humean dichotomy of chance and necessity is invalid for complex processes such as evolution or brain functioning. Such phenomena incorporate multiple components that are both lawful and indeterminate. This breakdown of the determinism\/indeterminism dichotomy has long been appreciated in evolution and it is surprising to observe the lack of such an appreciation with regard to brain function among some thinkers of today (e.g. \\[2\\]). Stochasticity is not a nuisance, or a side effect of our reality. Evolution has shaped our brains to implement 'stochasticity' in a controlled way, injecting variability 'at will'. Without such an implementation, we would not exist.\n\nA scientific concept of free will cannot be a qualitative concept. The question is not any more 'do we have free will?'; the questions is now: 'how much free will do we have?'; 'how much does this or that animal have?'. Free will becomes a quantitative trait.\n\n# Initiating activity: actions versus responses\n\nAnother concept that springs automatically from acknowledging behavioural variability as an adaptive trait is the concept of actions. In contrast to responses, actions are behaviours where it is either impossible to find an eliciting stimulus or where the latency and\/or magnitude of the behaviour vary so widely, that the term 'response' becomes useless.\n\nA long history of experiments on flies provides accumulating evidence that the behaviour of these animals is much more variable than it would need to be, given the variability in the neurons mediating the stimulus-response chain (reviewed in \\[77\\]). For instance, in the study of the temporal dynamics of turning behaviours in tethered flies referenced above \\[63\\], one situation recorded fly behaviour in constant stimulus conditions, i.e. nothing in the exquisitely controlled environment of the animals changed while the turning movements were recorded. Yet, the flies kept producing turning movements throughout the experiment as if there had been stimuli in their environment. Indeed, the temporal structure in these movements was qualitatively the same compared with when there were stimuli to be perceived. This observation is only one of many demonstrating the endogenous character of behavioural variability. Even though there was nothing in the environment prompting the animals to change their behaviour, they kept initiating turning manoeuvres in all directions. Clearly, each of these manoeuvres was a self-initiated, spontaneous action and not a response to some triggering, external stimulus.\n\nIn fact, such self-initiated actions are a necessary prerequisite for the kind of self-learning described above \\[41\u201343\\]. At the start of the experiment, the fly cannot know that it is its own turning manoeuvres that cause the switch from cold to hot and vice versa. To find out, the fly has to activate the behavioural modules it has available in this restrained situation and has to register whether one of them might have an influence on the punishing heat beam. There is no appropriate sensory stimulus from outside to elicit the respective behaviour. The fly must have a way to initiate its behaviours itself, in order to correlate these actions with the changes in the environment. Clearly, the brain is built such that under certain circumstances the items of the behavioural repertoire can get released independent of sensory stimuli.\n\nThe fly cannot know the solutions to most real-life problems. Beyond behaving unpredictably to evade predators or outcompete a competitor, all animals must explore, must try out different solutions to unforeseen problems. Without behaving variably, without acting rather than passively responding, there can be no success in evolution. Those individuals who have found the best balance between flexible actions and efficient responses are the ones who have succeeded in evolution. It is this potential to behave variably, to initiate actions independently of the stimulus situation, which provides animals with choices.\n\n# Freedom of choice\n\nThe neurobiological basis of decision-making can also be studied very well in invertebrate models. For instance, isolated leech nervous systems chose either a swimming motor programme or a crawling motor programme to an invariant electrical stimulus \\[78\u201380\\]. Every time the stimulus is applied, a set of neurons in the leech ganglia goes through a so far poorly understood process of decision-making to arrive either at a swimming or at a crawling behaviour. The stimulus situation could not be more perfectly controlled than in an isolated nervous system, excluding any possible spurious stimuli reaching sensory receptors unnoticed by the experimenter. In fact, even hypothetical 'internal stimuli', generated somehow by the animal must in this case be coming from the nervous system itself, rendering the concept of 'stimulus' in this respect rather useless. Yet, under these 'carefully controlled experimental circumstances, the animal behaves as it damned well pleases' (Harvard Law of Animal Behaviour) \\[34\\].\n\nSeymour Benzer, one of the founders of Neurogenetics, captured this phenomenon in the description of his first phototaxis experiments in 1967: ' \u2026 if you put flies at one end of a tube and a light at the other end, the flies will run to the light. But I noticed that not every fly will run every time. If you separate the ones that ran or did not run and test them again, you find, again, the same percentage will run. But an individual fly will make its own decision'. (cited from Brown & Haglund (1994) *J. NIH Res.* **6**, 66\u201373). Not even 10 years later, Quinn *et al.* separated flies, conditioned to avoid one of two odours, into those that did avoid the odour and those that did not. In a subsequent second test, they found that both the avoiders and the non-avoiders separated along the same percentages as in the first test, prompting the authors to conclude: 'This result suggests that the expression of learning is probabilistic in every fly' \\[81\\]. Training shifted the initial 50\u201350 decision of the flies away from the punished odour, but the flies still made the decisions themselves\u2014only with a different probability than before training. Most recently, in the experiments described above, the tethered flies without any feedback made spontaneous decisions to turn one way or another \\[63\\]. These are only three examples from more than 40 years in which many behavioural manifestations of decision-making in the fly brain have been observed. Like heat, flies can control also odour intensity with their yaw torque \\[40\\]. They can control the angular velocity of a panorama surrounding them not only by yaw torque but also by forward thrust, body posture or abdomen bending \\[82\\]. In ambiguous sensory situations, they actively switch between different perceptual hypotheses, they modify their expectations about the consequences of their actions by learning and they can actively shift their focus of attention restricting their behavioural responses to parts of the visual field \\[83,84\\]. These latest studies prompted further research into the process of the endogenous direction of selective attention in flies \\[85\u201389\\]. Martin Heisenberg realized early on \\[90\\] that such active processes entail the sort of fundamental freedom required for a modern concept of free will and keeps prominently advocating this insight today \\[91\\].\n\nJohn Searle has described free will as the belief 'that we could often have done otherwise than we in fact did' \\[92\\]. Taylor & Dennett cite the maxim 'I could have done otherwise' \\[93\\]. Clearly, leeches and flies could and can behave differently in identical environments. While some argue that unpredictable (or random) choice does not qualify for their definition of free will \\[2\\], it is precisely the freedom from the chains of causality that most scholars see as a crucial prerequisite for free will. Importantly, this freedom is a necessary but not a sufficient component of free will. In order for this freedom to have any bearing on moral responsibility and culpability in humans, more than mere randomness is required. Surely, no one would hold a person responsible for any harm done by the random convulsions during an epileptic seizure. Probably because of such considerations, two-stage models of free will have been proposed already many decades ago, first by James \\[94\\], later also by Henri Poincar\u00e9, Arthur Holly Compton, Karl Popper, Henry Margenau, Daniel Dennett, Robert Kane, John Martin Fisher, Alfred Mele, Stephen Kosslyn, Bob Doyle and Martin Heisenberg (cited, reviewed and discussed in \\[7\\]), as well as Koch \\[9\\]: one stage generates behavioural options and the other one decides which of those actions will be initiated. Put simply, the first stage is 'free' and the second stage is 'willed'. This implies that not all chance events in the brain must manifest themselves immediately in behaviour. Some may be eliminated by deterministic 'selection' processes before they can exert any effects. Analogous to mutation and selection in evolution, the biological process underlying free will can be conceptualized as a creative, spontaneous, indeterministic process followed by an adequately determined process, selecting from the options generated by the first process. Freedom arises from the creative and indeterministic generation of alternative possibilities, which present themselves to the will for evaluation and selection. The will is adequately determined by our reasons, desires and motives\u2014by our character\u2014but it is not *pre*-determined. John Locke (1689, p. 148) already separated free from 'will', by attributing free to the agent and not the will: 'I think the question is not proper, whether the will be free, but whether a man be free' \\[95\\]. Despite the long tradition of two-stage models of free will, only now are the first, tangible scientific pieces of evidence being published. For instance, the independent discovery of nonlinear mechanisms in brains from different phyla is compatible with such two-stage models \\[63,74\\]. Essentially, the existence of neural circuits implementing a two-stage model of free will 'would mean that you can know everything about an organism's genes and environment yet still be unable to anticipate its caprices' \\[96\\]. Importantly, this inability is not due to inevitable stochasticity beyond control; it is due to dedicated brain processes that have evolved to generate unpredictable, spontaneous actions in the face of pursuit\u2013evasion contests, competition and problem-solving.\n\n# Consciousness and freedom\n\nIt thus is no coincidence that we all feel that we possess a certain degree of freedom of choice. It makes sense that depriving humans of such freedom is frequently used as punishment and the deprived do invariably perceive this limited freedom as undesirable. This experience of freedom is an important characteristic of what it is like to be human. It stems in part from our ability to behave variably. Voltaire expressed this intuition in saying 'Liberty then is only and can be only the power to do what one will'. The concept that we can decide to behave differently even under identical circumstances underlies not only our justice systems. Electoral systems, our educational systems, parenting and basically all other social systems also presuppose behavioural variability and at least a certain degree of freedom of choice. Games and sports would be predictable and boring without our ability of constantly changing our behaviour in always the same settings.\n\nThe data reviewed above make clear that the special property of our brain that provides us with this freedom surely is independent of consciousness. Consciousness is not a necessary prerequisite for a scientific concept of free will. Clearly, a prisoner is regarded as un-free, irrespective of whether he is aware of it or not. John Austin \\[97\\] provides another instructive example 'Consider the case where I miss a very short putt and kick myself because I could have holed it'. We sometimes have to work extremely hard to constrain our behavioural variability in order to behave as predictably as possible. Sports commentators often use 'like a machine' to describe very efficient athletes. Like practice, conscious efforts are able to control our freedom up to a certain degree. Compare, for instance, a line that you quickly drew on a piece of paper, with a line that was drawn with the conscious effort of making it as straight as possible. However, the neural principle underlying the process generating the variability is beyond total conscious control, requiring us to use rulers for perfectly straight lines. Therefore, the famous experiments of Benjamin Libet and others since then \\[2,4,5,98\u2013100\\] only serve to cement the rejection of the metaphysical concept of free will and are not relevant for the concept proposed here. Conscious reflection, meditation or discussion may help with difficult decisions, but this is not even necessarily the case. The degree to which our conscious efforts can affect our decisions is therefore central to any discussion about the degree of responsibility our freedom entails, but not to the freedom itself.\n\n# The self and agency\n\nIn contrast to consciousness, an important part of a scientific concept of free will is the concept of 'self'. It is important to realize that the organism generates an action itself, spontaneously. In chemistry, spontaneous reactions occur when there is a chemical imbalance. The system is said to be far from thermodynamic equilibrium. Biological organisms are constantly held far from equilibrium, they are considered open thermodynamic systems. However, in contrast to physical or chemical open systems, some of the spontaneous actions initiated by biological organisms help keep the organism away from equilibrium. Every action that promotes survival or acquires energy sustains the energy flow through the open system, prompting Georg Litsche to define biological organisms as a separate class of open systems (i.e. 'subjects'; \\[101\\]). Because of this constant supply of energy, it should not be surprising to scientists that actions can be initiated spontaneously and need not be released by external stimuli. In controlled situations where there cannot be sufficient causes outside the organism to make the organism release the particular action, the brain initiates behaviour from within, potentially using a two-stage process as described above. The boy ceases to play and jumps up. This sort of impulsivity is a characteristic of children every parent can attest to. We do not describe the boy's action with 'some hidden stimuli made him jump'\u2014he jumped of his own accord. The jump has all the qualities of a beginning. The inference of agency in ourselves, others and even inanimate objects is a central component of how we think. Assigning agency requires a concept of self. How does a brain know what is self?\n\nOne striking characteristic of actions is that an animal normally does not respond to the sensory stimuli it causes by its own actions. The best examples are that it is difficult to tickle oneself and that we do not perceive the motion stimuli caused by our own eye saccades or the darkness caused by our eye blinks. The basic distinction between *self*-induced (re-afferent) and externally generated (ex-afferent) sensory stimuli has been formalized by von Holst & Mittelstaedt \\[102\\]. The two physiologists studied hoverflies walking on a platform surrounded by a cylinder with black and white vertical stripes. As long as the cylinder was not rotated, the animals seemed to behave as if they were oblivious to the stripes. However, as soon as the cylinder was switched on to rotate around the flies, the animals started to turn in register with the moving stripes, in an attempt to stabilize their orientation with respect to the panorama. Clearly, when the animals turned themselves, their eyes perceived the same motion stimuli as when the cylinder was rotated. The two scientists concluded that the animals detect which of these otherwise very similar motion signals are generated by the flies and which are not and dubbed this the 'principle of reafference'. To test the possibility that the flies just blocked all visual input during self-initiated locomotion, the experimenters glued the heads of the animals rotated by 180\u00b0 such that the positions of the left and right eye were exchanged and the proboscis pointed upwards. Whenever these 'inverted' animals started walking in the stationary striped cylinder, they ran in constant, uncontrollable circles, showing that they did perceive the *relative* motion of the surround. From this experiment, von Holst and Mittelstaedt concluded that self-generated turning comes with the expectation of a visual motion signal in the opposite direction that is perceived but normally does not elicit a response. If the visual motion signal is not caused by the animal, on the other hand, it most probably requires compensatory action, as this motion was not intended and hence not expected. The principle of reafference is the mechanism by which we realize which portion of the incoming sensory stream is under our own control and which portion is not. This is how we distinguish between those sensory stimuli that are consequences of our own actions and those that are not. Distinguishing self from 'world' is the prerequisite for the evolution of separate learning mechanisms for self- and world learning, respectively \\[43\\], which is the central principle of how brains balance actions and responses. The self\/world distinction is thus the second important function of behavioural variability, besides making the organism harder to predict: by using the sensory feedback from our actions, we are constantly updating our model of how the environment responds to our actions. Animals and humans constantly ask: What happens if I do this? The experience of willing to do something and then successfully doing it is absolutely central to developing a sense of self and that we are in control (and not being controlled).\n\nThus, in order to understand actions, it is necessary to introduce the term self. The concept of self necessarily follows from the insight that animals and humans initiate behaviour by themselves. It would make no sense to assign a behaviour to an organism if any behavioural activity could, in principle, be traced back by a chain of causations to the origin of the universe. An animal or human being is the agent causing a behaviour, as long as no sufficient causes for this activity to occur are coming from outside the organism. Agency is assigned to entities who initiate actions themselves. Agency is crucial for moral responsibility. Behaviour can have good or bad consequences. It is the agent for whom the consequences matter the most and who can be held responsible for them.\n\n# Why still use the term free will today?\n\nBy providing empirical data from invertebrate model systems supporting a materialistic model of free will, I hope to at least start a thought process that abandoning the metaphysical concept of free will does not automatically entail that we are slaves of our genes and our environment, forced to always choose the same option when faced with the same situation. In fact, I am confident I have argued successfully that we would not exist if our brains were not able to make a different choice even in the face of identical circumstances and history. In this article, I suggest re-defining the familiar free will in scientific terms rather than giving it up, only because of the historical baggage all its connotations carry with them. One may argue that 'volition' would be a more suitable term, less fraught with baggage. However, the current connotations of volition as 'willpower' or the forceful, conscious decision to behave against certain motivations render it less useful and less general a term than free will. Finally, there may be a societal value in retaining free will as a valid concept, since encouraging a belief in determinism increases cheating \\[103\\]. I agree with the criticism that retention of the term may not be ideal, but in the absence of more suitable terms, free will; remains the best option.\n\nI no longer agree that ' ''free will'' is (like ''life'' and ''love'') one of those culturally useful notions that become meaningless when we try to make them ''scientific'' ' \\[96\\]. The scientific understanding of common concepts enrich our lives, they do not impoverish them, as some have argued \\[100\\]. This is why scientists have and will continue to try and understand these concepts scientifically or at least see where and how far such attempts will lead them. It is not uncommon in science to use common terms and later realize that the familiar, intuitive understanding of these terms may not be all that accurate. Initially, we thought atoms were indivisible. Today we do not know how far we can divide matter. Initially, we thought species were groups of organisms that could be distinguished from each other by anatomical traits. Today, biologists use a wide variety of species definitions. Initially, we thought free will was a metaphysical entity. Today, I am joining a growing list of colleagues who are suggesting it is a quantitative, biological trait, a natural product of physical laws and biological evolution, a function of brains, maybe their most important one.\n\n## Acknowledgements\n\nConcepts and ideas in several sections of this article have been adapted from a to-be-published presentation of Martin Heisenberg. I am very grateful for his sharing this presentation with me. I am also indebted to Christopher Harris, Bob Doyle, Matt Leifer, Sandeep Gautam, Andrew Lang, Julien Colomb and two anonymous referees for helpful comments on an earlier version of the manuscript.\n\n# References","meta":{"dup_signals":{"dup_doc_count":122,"dup_dump_count":53,"dup_details":{"curated_sources":2,"2022-33":1,"2021-21":1,"2020-34":1,"2020-24":1,"2019-13":1,"2018-47":2,"2018-43":3,"2018-39":2,"2018-34":1,"2018-30":3,"2018-26":2,"2018-22":2,"2018-17":1,"2018-13":2,"2018-09":2,"2018-05":2,"2017-51":3,"2017-47":2,"2017-43":2,"2017-39":1,"2017-34":3,"2017-30":1,"2017-26":4,"2017-22":1,"2017-17":3,"2017-09":3,"2017-04":3,"2016-50":2,"2016-44":4,"2016-40":3,"2016-36":3,"2016-30":3,"2016-26":2,"2016-22":3,"2016-18":3,"2016-07":3,"2015-48":3,"2015-40":3,"2015-35":2,"2015-32":3,"2015-27":2,"2015-22":3,"2015-14":2,"2014-52":3,"2014-49":3,"2023-23":1,"2017-13":4,"2015-18":3,"2015-11":3,"2015-06":3,"2013-48":1,"2013-20":1,"2024-30":1}},"file":"PMC3049057"},"subset":"pubmed_central"}
{"text":"abstract: Protocol design complexity has increased substantially during the past decade and this in turn has adversely impacted drug development economics and performance. This article reviews the results of two major Tufts Center for the Study of Drug Development studies quantifying the direct cost of conducting less essential and unnecessary protocol procedures and of implementing amendments to protocol designs. Indirect costs including personnel time, work load and cycle time delays associated with complex protocol designs are also discussed. The author concludes with an overview of steps that research sponsors are taking to improve protocol design feasibility.\nauthor: Kenneth Getz\ndate: 2014-05-12\ninstitute: Center for the Study of Drug Development, School of Medicine, Tufts University, 75 Kneeland Street, Suite 1100, Boston, MA 02111, USA; E-Mail: <email@example.com>; Tel.: +1-617-636-3487; Fax: +1-617-636-2425\nreferences:\ntitle: Improving Protocol Design Feasibility to Drive Drug Development Economics and Performance\n\n# 1. Introduction\n\nResearch and Development leaders widely agree that among the many factors that drive the high and rising cost of drug development, one of the most significant is protocol design \\[1\\]. As the blueprint articulating project strategy and directing project execution performed by both internal and external personnel, protocol design is uniquely positioned to fundamentally and directly impact\u2014positively or negatively\u2014drug development efficiency and economics.\n\nProtocol design has historically been the domain of clinical research scientists. Under a resource-rich R&D operating environment, the regulatory approval of safe and effective medical products was the primary test for assessing study design quality. In the current resource-constrained operating environment, a primary test of quality study design seeks to exclude any protocol that substantially wastes scarce resources, gathers extraneous data, and exposes study subjects to unnecessary risk.\n\nMaking matters worse, in the current environment, a rising number of stakeholders influence protocol design practice and quality. Not only are scientists, regulatory agencies, health authorities, operating managers and key opinion leaders informing study design decision making, but also patients\/patient organizations, investigative site personnel, health care providers, policymakers and payers are increasingly playing a role.\n\nInterviews conducted among drug development executives suggest a number of considerations and pressures that protocol design decision-making must accommodate: New scientific understanding about chronic disease mechanisms and how to measure their progression and economic impact requires collecting more clinical data. Crowded classes of experimental therapies and the ongoing movement to develop stratified medicines compel research sponsors to collect more genetic and biomarker data.\n\nClinical scientists and statisticians often add procedures to gather more contextual data to aid in their interpretation of the findings and to guide development decisions. Clinical teams are now collecting biomarker data, genetic material, data on economic and therapeutic outcomes, and companion diagnostic data.\n\nClinical teams routinely add procedures guided by the belief that the marginal cost of doing so, relative to the entire clinical study budget, is small when the risk of not doing so is high \\[1,2\\]. Additional clinical data is collected as a precautionary measure in the event that a study fails to meet its primary and key secondary objectives. Data from these procedures may prove valuable in *post-hoc* analyses that reveal new and useful information about the progression of disease, its treatment and new directions for future development activity. Clinical teams add procedures for fear that they may neglect to collect data requested by regulatory agencies and health authorities, purchasers and payers. Failure to collect requested data elements could potentially delay regulatory submission, product launch and product adoption. And medical writers and protocol authors also often permit outdated and unnecessary procedures into new study designs because they are routinely included in legacy protocol authoring templates and operating policies.\n\nCollectively, these factors have all contributed to the dramatic increase in protocol design complexity during the past decade. Research conducted by the Tufts Center for the Study of Drug Development (Tufts CSDD, Boston, MA, USA) documents this alarming trend (see <a href=\"#ijerph-11-05069-t001\" data-ref-type=\"table\">Table 1<\/a>) and characterizes rising levels of scientific complexity (e.g., number of endpoints; number of procedures performed; number of study volunteer eligibility criteria) and operating complexity (e.g., number of countries where clinical trials are conducted; number of investigative sites activated and monitored; number of patients screened and enrolled).\n\nIn 2012, to demonstrate safety and efficacy, a typical phase III protocol had 170 procedures on average performed on each study volunteer during the course of 11 visits across an average 230-day time span. Ten years ago, the typical phase III protocol had an average of 106 procedures, nine visits and an average 187-day time span. For the typical phase III protocol conducted in 2012, study volunteers came from an average of 34 countries and 196 research centers, up from 11 countries and 124 research centers ten years ago. And in 2012, to qualify to participate in a typical phase III protocol, each volunteer had to meet 50 eligibility criteria -- up from an average of 31 inclusion and exclusion criteria ten years ago \\[1\\].\n\nijerph-11-05069-t001_Table 1\n\nComparing Scientific and Logistical Complexity of a Typical Phase III Protocol Across Two Time Periods.\n\n<table>\n<thead>\n<tr>\n<th style=\"text-align: left;\">Design Characteristics<br \/>\n(All Values are Means)<\/th>\n<th style=\"text-align: center;\">2002<\/th>\n<th style=\"text-align: center;\">2012<\/th>\n<\/tr>\n<\/thead>\n<tbody>\n<tr>\n<td style=\"text-align: left;\">Total number of endpoints<\/td>\n<td style=\"text-align: center;\">7<\/td>\n<td style=\"text-align: center;\">13<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Total number of procedures<\/td>\n<td style=\"text-align: center;\">106<\/td>\n<td style=\"text-align: center;\">167<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Total number of eligibility criteria<\/td>\n<td style=\"text-align: center;\">31<\/td>\n<td style=\"text-align: center;\">50<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Total number of countries<\/td>\n<td style=\"text-align: center;\">11<\/td>\n<td style=\"text-align: center;\">34<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Total number of investigative sites<\/td>\n<td style=\"text-align: center;\">124<\/td>\n<td style=\"text-align: center;\">196<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Total number of patients randomized<\/td>\n<td style=\"text-align: center;\">729<\/td>\n<td style=\"text-align: center;\">597<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\nIn an effort to reign in rising drug development costs, a growing number of pharmaceutical and biotechnology companies are looking to identify protocol design practices that can be modified. This article reviews two major Tufts CSDD studies that have quantified the direct cost impact of protocol design practices. This article also summarizes growing knowledge of the impact of protocol design complexity on indirect costs. At the conclusion of the article, steps that drug developers are taking to simplify protocol design and lower overall study costs are discussed.\n\n# 2. Direct Costs Associated with Protocol Design Practices\n\n## 2.1. Protocol Procedures\n\nIn a study completed in 2012, Tufts CSDD demonstrated that the cost of adding multiple individual protocol procedures, in the aggregate, is substantial. Fifteen mid-sized and large pharmaceutical and biotechnology companies participated in the study. Each company provided data on their phase II and III protocols targeting diseases across multiple therapeutic areas and executed by investigative sites dispersed globally since 2009. To minimize unusual and atypical designs, pediatric, medical device, orphan drug and extension studies were excluded from the sampling frame. In all, 116 unique phase II and III protocols having at least one procedure tied to a primary endpoint were analyzed \\[2\\].\n\nParticipating companies classified each protocol procedure according to the objective and endpoint it supported as defined by the clinical study report (CSR) and the study's specific statistical analysis plan (SAP) along the following lines:\n\n1.  \"Core\" procedures\u2014supported primary and\/or secondary study objectives or primary or key secondary and safety endpoints.\n\n2.  \"Required\" procedures\u2014supported screening requirements and compliance-related activity including drug dispensing, informed consent form review, and study drug return.\n\n3.  \"Standard\" procedures\u2014are commonly performed during initial and routine study participant visits including medical history, height and weight measurement, adverse event assessment, and concomitant medication review.\n\n4.  \"Non-Core\" procedures\u2014supported supplemental secondary, tertiary and exploratory endpoints, and safety and efficacy procedures not associated with a study endpoint or objective.\n\nParticipating companies classified 25,103 individual phase II and III protocol procedures. Direct cost data for 16,607 procedures was analyzed using Medidata Solutions PICAS^\u00ae^ database. Overall, half of the total procedures per protocol were classified as \"Core\" to the study. Wide variability in the incidence of procedures supporting endpoint classifications was observed across therapeutic areas. Protocols targeting endocrine and central nervous system (CNS) disorders contained a higher relative average number of procedures supporting supplementary, tertiary, and exploratory procedures (e.g., \"Non-Core\") endpoints. Oncology protocols had the highest relative proportion of procedures supporting \"Core\" endpoints and objectives, and the lowest relative proportion supporting \"Non-Core\" endpoints.\n\nThe distribution of direct costs was similar to that of categorized procedures: Overall, 47.9% of the total study budget on average was spent on the direct costs to administer \"Core\" procedures in phase II and phase III protocols. The direct cost to administer \"Required\" (regulatory compliance) and \"Standard\" procedures for phase III protocols was 22.7% and 12.0% of the study budget respectively.\n\nFor phase III protocols alone, approximately half of the total direct cost (46.7%) was spent to administer \"Core\" procedures; 18.6% on average was spent to administer \"Non-Core\" procedures; 22.7% to administer procedures supporting screening requirements and regulatory compliance; and 12% supported \"Standard\" procedures (See <a href=\"#ijerph-11-05069-t002\" data-ref-type=\"table\">Table 2<\/a>).\n\nijerph-11-05069-t002_Table 2\n\nDistribution of Procedures and Direct Costs per Procedure by End Point Classification.\n\n| Endpoint Type | Phase II Procedures | Phase III Procedures | Phase II Procedure Costs | Phase III Procedure Costs |\n|----|----|----|----|----|\n| Core | 54.4% | 47.7% | 55.2% | 46.7% |\n| Required | 8.0% | 10.0% | 16.3% | 22.7% |\n| Standard | 19.7% | 17.6% | 15.4% | 12.0% |\n| Non-Core | 17.9% | 24.7% | 13.1% | 18.6% |\n| Total | 100% | 100% | 100% | 100% |\n\nOne out of five (22.1%) phase II and III protocol procedures, on average, supported tertiary and exploratory objectives and endpoints. The proportion of procedures collecting non-core data in phase III studies alone was even higher (24.7%). Non-core procedures consumed on average 19% of total direct costs for each phase III study, and 13% of total direct costs for each phase II study \\[2\\].\n\nThe estimated total cost to the pharmaceutical industry each year to perform procedures supporting \"Non-Core\" objectives and endpoints for all FDA-regulated phase II and III protocols is an estimated \\$4\u2013\\$6 billion USD. This estimate is very conservative as it excludes all indirect costs for personnel and infrastructure required to capture, monitor, clean, analyze, manage and store extraneous protocol data and it does not include any estimate for the unnecessary risk to which patients may be exposed.\n\n## 2.2. Protocol Amendments\n\nA second Tufts CSDD study showed that the incidence of protocol amendments rises with an increase in protocol design complexity. Amendments that are implemented after protocols have been approved are commonplace, but they are widely viewed as a nuisance, resulting in unplanned study delays and additional costs. Although clinical research professionals view protocol amendments as major problems, it is perhaps more accurate to view amendments as attempts to address underlying protocol design problems and external factors impacting design strategies.\n\nConducted in 2010, Tufts CSDD convened a group of seventeen mid-sized and large pharmaceutical and biotechnology companies each of whom provided protocol amendment data from 3,410 protocols approved between January 2006 and December 2008. The protocols were representative of multiple therapeutic areas. Protocols approved within the most recent 12-months were excluded from the study because these had not had enough time to accumulate amendments \\[3\\].\n\nAmendments were defined as any change to a protocol requiring internal approval followed by approval from the Institutional Review Board (IRB), Ethical Review Board (ERB) or regulatory authority. Detailed data on 3,596 amendments containing 19,345 total protocol modifications was analyzed. Companies participating in this study assigned specific changes made per amendment yielding a total of 6,855 changes classified. Companies also provided the top causes for amendments and rated each amendment in terms of whether it was \"Completely Avoidable\", \"Somewhat Avoidable\", \"Somewhat Unavoidable\" or \"Completely Unavoidable\".\n\nNearly all protocols required at least one amendment. Completed protocols across all phases had an average of 2.3 amendments, though later-stage phase II and III protocols averaged 2.7 and 3.5 amendments respectively. Each amendment required an average of 6.9 changes to the protocol. Therapeutic areas that had the highest incidence of amendments and changes per amendment included cardiovascular and GI protocols (See <a href=\"#ijerph-11-05069-t003\" data-ref-type=\"table\">Table 3<\/a>).\n\nijerph-11-05069-t003_Table 3\n\nMean Number of Amendments and Changes per Amendment per Protocol by Phase.\n\n| Research Phase | Number of Amendments | Protocol Changes per Amendment |\n|----------------|----------------------|--------------------------------|\n| Phase I        | 1.9                  | 5.6                            |\n| Phase II       | 2.7                  | 6.8                            |\n| Phase III      | 3.5                  | 8.5                            |\n| Phase IIIb\/IV  | 2.6                  | 8.3                            |\n| All phases     | 2.3                  | 6.9                            |\n\nOf the 6,855 changes categorized, 16% were modifications made to the description and eligibility criteria of the patient population under investigation; 12% were adjustments made in the number and types of safety assessment procedures; 10% were edits and revisions made to the general information contained in the protocol (e.g., protocol title and study staff contact information).\n\nNearly 40% of all amendments occurred before the first study volunteer received his or her first dose in the clinical trial. This was most pronounced in phase I studies where 52% of amendments occurred prior to beginning patient enrollment. In phase II, III, and IIIb\/IV studies, 37%, 30%, and 38% of amendments occurred before first patient first dose, respectively (see <a href=\"#ijerph-11-05069-f001\" data-ref-type=\"fig\">Figure 1<\/a>).\n\nThe most common cause of amendments was the availability of new safety information (20%), followed by requests from regulatory agencies to amend the study (19%) and changes in the study strategy (18%). Protocol design flaws and difficulties recruiting study volunteers were also top cited causes at 11% and 9% of categorized amendments, respectively.\n\nTwo-thirds (63%) of amendments had causes that sponsor companies considered unavoidable, including amendments that were the result of new safety information, new regulatory requests, changes in the standard of care or study objectives. Nearly one-out-of-four protocol amendments (37%) was considered partially or completely avoidable.\n\nBased on data provided by participating companies, on average the direct cost to implement (*i.e.*, first patient resubmitted under the revised protocol) a single protocol amendment is approximately \\$500,000 (USD) in unplanned expense and adds 61 days to the project timeline. This figure undercounts the full economic impact as it does not include the cost of internal staff time dedicated to implementing each amendment, costs or fees associated with protocol language translation, and costs associated with resubmission to the local authority.\n\nTufts CSDD estimates that the total cost for sponsors to implement \"avoidable\" protocol amendments in 2014 was approximately \\$2 billion (USD). This estimate is based on the incidence of amendments by phase for all active global FDA regulated clinical trials as reported by the agency; the direct cost to implement each amendment; and the proportion of amendments that is avoidable. This estimate does not include the billions of dollars realized annually in aggregate time-savings and earlier commercialization due to the elimination of each avoidable amendment \\[3\\].\n\nTo date, Tufts CSDD research on the direct cost impact of protocol design complexity has focused on non-core procedures. Tufts CSDD is currently conducting research looking at the direct cost of Core, Required and Standard procedures.\n\n## 2.3. Indirect Costs\n\nTufts CSDD research is only beginning to quantify the indirect costs associated with protocol design complexity. These costs are no doubt many magnitudes higher than their estimated direct costs and include increasing cycle time and delays; full- and part-time staffing costs associated with project management and oversight, protocol administration and the gathering, monitoring, collecting, cleaning, analyzing, maintaining and storing of clinical data.\n\n## 2.4. Cycle Time\n\nResearch published in peer-reviewed and trade literature shows a very clear relationship between study design complexity and performance: Study designs that include a relatively large number of eligibility requirements and unique procedures conducted frequently have lower study volunteer recruitment and retention rates, take longer, and generate lower quality clinical data than designs without such features \\[4\\].\n\nTufts CSDD research has demonstrated that the average overall duration of clinical trials\u2014from \"Protocol Ready\" to the last patient completing her last visit\u2014is 74% longer for complex clinical trials. And whereas three-out-of-four volunteers are randomized following screening and two-thirds of volunteers complete simpler protocols; 59% are randomized and less than half (48%) complete complex protocols \\[4\\].\n\nOther studies in the literature corroborate these findings and provide insights into causes of cycle time delays \\[5,6,7,8,9\\]. Clark found, for example, that the collection of excessive and unnecessary clinical data is driving longer study durations. The author warned that data collection and regulatory agency submission delays may ultimately harm regulatory approval rates \\[5\\].\n\nRoss and colleagues conducted a comprehensive analysis of peer-reviewed academic studies and found that health professionals were less likely to refer, and patients less likely to participate in, more complex clinical trials \\[6\\]. Madsen showed that patients are significantly less likely to sign the informed consent form when facing a more demanding protocol design \\[7\\].\n\nIn a study conducted by Boericke and Gwinn, the higher the number of study eligibility criteria, the more frequent and longer were the delays in completing clinical studies \\[8\\]. Andersen and colleagues showed that volunteer drop-out rates are much higher among patients participating in more complex clinical trials. The authors cautioned that when volunteers terminate their participation early and are lost to follow-up, the validity of the study results may be compromised \\[9\\].\n\n## 2.5. Investigative Site Work Burden\n\nRising protocol design complexity also places additional execution burden on study staff. Work burden to administer protocol procedures was assessed using an approach developed by Tufts CSDD in 2008 and adapted from Medicare's Relative Value Unit (RVU) methodology \\[4\\]. The RVU scale was created by the Federal government in 1982 to determine reimbursement payment levels for physicians' relative costs instead of prevailing charges for medical procedures. Tufts CSDD mapped medical procedure RVUs to clinical trial procedures to derive Work Effort Units (WEU) per protocol procedure. For procedures that were not already assigned a Medicare RVU, a panel of 10 physicians at the Tufts University School of Medicine was convened to estimate the time spent per protocol procedure. \"Investigative Site Work Burden\" is the product of WEUs per procedure and the frequency of the procedures that were conducted over the course of the protocol.\n\nTotal investigative site work burden to administer phase II protocol procedures grew the fastest\u201473%\u2014during the ten year period 2002 to 2012 outpacing growth in the total number of procedures performed per protocol. The work effort required of site personnel to administer procedures supporting phase I, III and IV protocols increased 48%, 56% and 57% during the same ten year period respectively (see <a href=\"#ijerph-11-05069-f002\" data-ref-type=\"fig\">Figure 2<\/a>). Protocols targeting diseases associated with immunomodulation, oncology, CNS, and cardiovascular required the highest work effort from investigative site personnel to administer.\n\n## 2.6. Study Monitoring, Data Management and Logistics Costs\n\nTufts CSDD studies have only just started to develop methodologies to measure the incremental indirect costs supporting study monitoring activity, data management and study logistics associated with complex protocol designs. In a preliminary analysis of regulatory submissions from nine major pharmaceutical companies, Tufts CSDD found that the average NDA in 1997 was nearly 70,000 pages in length and required 2 gigabytes of memory to store electronically. In 2012, the average NDA was almost 3.5 million pages and required 100 gigabytes of digital memory \\[10\\]. According to research from Medidata Solutions, a typical Phase III study collected nearly 1 million clinical data points in 2012, up from an estimated half that level 10 years ago \\[1\\].\n\nStudies in the literature describe the impact of collecting too much data. Friedman and colleagues report that the high volume of data collected in today's protocols distracts research scientists, compromises the data analysis process and ultimately harms data quality \\[11\\]. As more data is collected during protocol administration, error rates increase, according to Nahm *et al*. \\[12\\]. Barrett found that more procedures per protocol were associated with a higher incidence of unused data in NDA (New Drug Application) submissions \\[13\\]. Abrams *et al.* found that unused clinical data compromises the data analysis process \\[14\\].\n\nPreliminary results from a 2014 Tufts CSDD study suggest that study monitors perform 100% source data verification on clinical data gathered from all non-core procedures and that one-third of all queries per case report form are associated with these less essential procedures.\n\nAs mentioned earlier, Tufts CSDD research indicates that the number of countries and investigative sites where clinical trials are conducted has grown considerably. As clinical trials have become more globally dispersed, research sponsors have introduced numerous logistical complexities including delivering clinical supplies and collecting lab data from more remote locations; interacting with multiple regulatory agencies and health authorities; monitoring and overseeing sites in remote locations where infrastructure is more variable.\n\n## 2.7. Steps to Optimize Study Design\n\nDuring the past several years, a growing number of organizations have taken the initial steps of diagnosing their protocol design practices and comparing them with industry benchmarks. Sponsor organizations have referred to published data in peer-reviewed papers to compare internal study design practices with benchmarks. Commercially available software and consulting services are also available to assist sponsor companies in assessing the problem and implementing new practices to streamline and simplify study design \\[1\\].\n\nIn addition to diagnosing the problem, sponsor organizations are looking for ways to ensure that protocol feasibility is conducted more effectively. Whereas the scientific objectives solely dictated study design elements in the past, in an environment where resources and capital are far more limited, operating objectives now carry substantially more influence over design decisions. As such, the primary objective in optimizing protocol design is to perform great science that can be feasibly and cost-effectively executed.\n\nMany sponsor organizations now solicit feedback from principal investigators, study coordinators and from patients to identify areas where study design feasibility can be improved prior to final approval of the protocol. Some of these feedback mechanisms are conducted as in-person meetings and focus groups while others are conducted online using social and digital media communities \\[15\\].\n\nProtocol authoring practices received a valuable new reference resource in 2013. A group of international study design stakeholders issued a collection of checklists and guidelines designed to ensure that the purpose of protocol procedures are transparent and tied to core objectives and endpoints. The SPIRIT checklist was developed with input from 115 multi-disciplinary contributors from medical writing, journal editors, regulatory agencies, ethics committees, clinical research and health care professionals.\n\nThe SPIRIT 2013 checklist and guidelines call for simple, minimalist designs that are clearly tied to core endpoints and objectives as defined by the clinical study report. Formatting conventions for various protocol elements (e.g., table of contents, glossary of terms, abbreviated terms) are also provided. The SPIRIT checklist has been pilot tested and can be downloaded for free \\[16\\].\n\nA 2013 Tufts CSDD study documented the creation of internal governance committees charged with challenging clinical teams to improve protocol feasibility \\[1\\]. Pharmaceutical and biotechnology companies began establishing these committees 24 months ago in an effort to adopt a more systematic and long-term approach to optimizing study design. Committees are positioned within their respective organizations as objective governance and assessment mechanisms, offering guidance and input into the existing protocol review process without requiring organizations to alter legacy study design practices and procedures.\n\nThe committees raise clinical team awareness of the impact that design decisions have on study budgets and on study execution feasibility. Committees typically provide input into the study design just prior to final protocol approval and they routinely offer insight into how protocol designs can be streamlined and better \"fit to purpose\". Long term, internal facilitation committees may assist organizations in fundamentally improving their study design practices.\n\nNearly all of the facilitation committees are comprised of cross-functional representatives who volunteer their time. Committees representatives come from a variety of functions including clinical development; clinical operations; statistics; data management; medical writing; clinical pharmacology; regulatory; safety; and pharmacovigilance in addition to finance and\/or procurement. Although some committees have more expansive goals than do others, all committees are charged with simplifying and streamlining protocols by reducing unnecessary procedures and the number of amendments. To meet these goals, committees assess the direct cost to perform non-core procedures and core procedures that may be conducted more frequently than necessary.\n\nAdaptive trial designs represent an optimization opportunity that has received limited attention from research sponsors to date. Adaptive trial designs are preplanned, typically through the use of trial simulations and scenario planning where one or more specified clinical trial design elements are modified and adjusted\u2014while the trial is underway\u2014based on an analysis of interim data.\n\nTufts CSDD estimates that approximately one out of five (20%) late stage clinical trials are using a simple adaptive design approach. A much lower percentage\u20145%\u2014is using more sophisticated adaptations (adaptive dose range studies and seamless phase II-III studies). Sponsor companies report that they expect the adoption of adaptive trial designs in earlier exploratory phase clinical trials to increase significantly over the next several years \\[17\\].\n\nStudy terminations due to safety and\/or efficacy futility are the most common simple adaptive design used and they are more widely used at this time. Sponsor companies have found that early terminations due to futility are relatively easy to implement and a growing number of companies similarly view studies employing sample size re-estimation adaptations.\n\nAlthough the concept of adaptive trial designs has been widely discussed for more than ten years, adoption has been slow for a variety of reasons. Internal organizational resistance appears to be the primary factor limiting more widespread adoption \\[17\\]. Based on interviews with company executives, regulatory agency receptivity to the use of adaptive trial designs does not appear to be as significant a barrier to adoption though agency clarity with regard to its position on the use of adaptive designs appears to be lacking.\n\nClinical teams and operating functions perceive enrollment and logistical factors\u2014specifically delays and disruptions in trial execution, patient participation and distribution of clinical supplies\u2014as major barriers to adoption. Sponsors are also concerned about introducing bias following interim analyses; the lack of adaptive trial design experience among both internal development teams and external contract research organizations; gaps in infrastructure and technology to implement more sophisticated adaptive designs; and the limited capacity of independent data monitoring committees.\n\nAdaptive trial designs hold promise in optimizing study design. Early study terminations due to futility and sample size re-estimation could save up to a hundred million dollars (USD) in direct and indirect costs annually per pharmaceutical company depending on clinical trial scope, when the trial is actually terminated, and on the sponsor's overall implementation of this adaptive approach across the development portfolio. Perhaps the greatest impact from adaptive trial designs will come from improvements in late stage success rates. Even modest improvements in success rates for new molecular entities (NME) and new biologic entities (BME) represent billions of dollars in increased revenue potential for research sponsors.\n\nIn the immediate term, adaptive trial designs are offering cross-functional teams direct insights into study design through scenario planning and trial simulation prior to finalizing the protocol. Rigorous upfront planning\u2014similar to optimization practices for traditional study designs\u2014is forcing organizations to challenge protocol feasibility prior to placing the protocol in the clinic.\n\n# 3. Conclusions\n\nProtocol design holds the key to fundamentally and sustainably transforming drug development performance, cost and success. Research conducted by Tufts CSDD and others contributes to our growing understanding of specific opportunities that can be implemented to optimize protocol design. There is no question that pharmaceutical and biotechnology companies will adopt and implement new approaches to test and modify study design feasibility with the goal of best executing excellent science. More flexible and adaptive trial designs are expected to play a growing role in helping to optimize study design by compelling sponsor companies to perform more rigorous upfront planning and simulation and to implement preplanned adaptions that may lower fixed operating costs and ultimately improve program success rates.\n\n## Acknowledgments\n\nThe author wishes to thank his colleagues at the Tufts Center for the Study of Drug Development and participating companies for their support of the Center's research studies and Medidata Solutions for providing an unrestricted study grant to quantify the direct cost of procedures by endpoint classification.\n\n## Conflicts of Interest\n\nThe author declares no conflict of interest.\n\n# References","meta":{"dup_signals":{"dup_doc_count":113,"dup_dump_count":37,"dup_details":{"curated_sources":4,"2018-39":1,"2018-26":1,"2018-17":1,"2018-09":1,"2018-05":1,"2017-47":1,"2017-39":1,"2017-30":3,"2017-22":1,"2017-17":1,"2017-09":1,"2017-04":2,"2016-50":3,"2016-44":4,"2016-40":4,"2016-36":4,"2016-30":4,"2016-26":4,"2016-22":2,"2016-18":2,"2016-07":3,"2015-48":2,"2015-40":3,"2015-35":4,"2015-32":4,"2015-27":4,"2015-22":4,"2015-14":4,"2014-52":4,"2014-49":5,"2014-42":12,"2014-41":5,"2018-51":1,"2015-18":3,"2015-11":4,"2015-06":4,"2017-13":1}},"file":"PMC4053871"},"subset":"pubmed_central"}
{"text":"author: James E Ferrell\ndate: 2009\ninstitute: 1Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA 94305-5174, USA\ntitle: Q&A: Systems biology\n\n# What is systems biology?\n\nSystems biology is the study of complex gene networks, protein networks, metabolic networks and so on. The goal is to understand the design principles of living systems.\n\n# How complex are the systems that systems biologists study?\n\nThat depends. Some people focus on networks at the 'omics'-scale: whole genomes, proteomes, or metabolomes. These systems can be represented by graphs with thousands of nodes and edges (see Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>). Others focus on small subcircuits of the network; say a circuit composed of a few proteins that functions as an amplifier, a switch or a logic gate. Typically, the graphs of these systems possess fewer than a dozen (or so) nodes. Both the large-scale and small-scale approaches have been fruitful.\n\n# Why is systems biology important?\n\nStas Shvartsman at Princeton tells a story that provides a good answer to this question. He likens biology's current status to that of planetary astronomy in the pre-Keplerian era. For millennia people had watched planets wander through the nighttime sky. They named them, gave them symbols, and charted their complicated comings and goings. This era of descriptive planetary astronomy culminated in Tycho Brahe's careful quantitative studies of planetary motion at the end of the 16th century. At this point planetary motion had been described but not understood.\n\nThen came Johannes Kepler, who came up with simple theories (elliptical heliocentric orbits; equal areas in equal times) that empirically accounted for Brahe's data. Fifty years later, Newton's law of universal gravitation provided a further abstraction and simplification, with Kepler's laws following as simple consequences. At that point one could argue that the motions of the planets were understood.\n\nSystems biology begins with complex biological phenomena and aims to provide a simpler and more abstract framework that explains why these events occur the way they do. Systems biology can be carried out in a 'Keplerian' fashion \u2013 look for correlations and empirical relationships that account for data \u2013 but the ultimate hope is to arrive at a 'Newtonian' understanding of the simple principles that give rise to the complicated behaviors of complex biological systems.\n\nNote that Kepler postulated other less-enduring mathematical models of planetary dynamics. His *Mysterium Cosmographicum* showed that if you nest spheres and Platonic polyhedra in the right order (sphere-octahedron-sphere-icosahedron-sphere-dodecahedron-sphere-tetrahedron-sphere-cube-sphere), the sizes of the spheres correspond to the relative sizes of the first six planets' orbits. This simple, abstract way of accounting for empirical data was probably just a happy coincidence. Happy coincidences are a potential danger in systems biology as well.\n\n# Is systems biology the antithesis of reductionism?\n\nIn a limited sense, yes. Some 'emerging properties', as discussed below, disappear when you reduce a system to its individual components.\n\nHowever, systems biology stands to gain a lot from reductionism, and in this sense systems biology is anything but the antithesis of reductionism. Just as you can build up to an understanding of complex digital circuits by studying individual electronic components, then modular logic gates, and then higher-order combinations of gates, one may well be able to achieve an understanding of complex biological systems by studying proteins and genes, then motifs (see below), and then higher-order combinations of motifs.\n\n# What are emergent properties?\n\nSystems of two proteins or genes can do things that individual proteins\/genes cannot. Systems of ten proteins or genes can do things that systems of two proteins\/genes cannot. Those things that become possible once a system reaches some level of complexity are termed emergent properties.\n\n# Can you give a concrete example of an emergent property?\n\nThree proteins connected in a simple negative-feedback loop (A \u2192 B \u2192 C -\\| A) can function as an oscillator; two proteins (A \u2192 B-\\|A)can not. Two proteins connected in a simple negative-feedback loop can convert constant inputs into pulsatile outputs; a one-protein loop (A -\\| A) cannot. So pulse generation emerges at the level of a two-protein system and oscillations emerge at the level of a three-protein system.\n\n# In systems biology there is a lot of talk about nodes and edges. What is a node? An edge?\n\nBiological networks are often depicted graphically: for example, you could draw a circle for protein A, a circle for protein B, and a line between them if A regulates B or vice versa. The circles are the nodes in the graph of the A\/B system. Nodes can represent genes, proteins, protein complexes, individual states of a protein, and so on.\n\nA line connecting two nodes is an edge. The edge can be directed: for example, if A regulates B, we write an arrow \u2013 a directed edge \u2013 from A to B, whereas if B regulates A we write an arrow from B to A. Or the edge can be undirected; for example, it represents a physical interaction between A and B.\n\n# Staying with graphs, what's a motif?\n\nAs defined by Uri Alon, a motif is a statistically over-represented subgraph of a graphical representation of a network. Motifs include things like negative feedback loops, positive feedback loops, and feed-forward systems.\n\n# Isn't positive feedback the same thing as feed-forward regulation?\n\nNo. They are completely different. In a positive-feedback system, A activates B and B turns around to activate A. A transitory stimulus that activates A could lock the system into a self-perpetuating state where both A and B are active. In this way, the positive-feedback loop can act like a toggle switch or a flip-flop. A positive-feedback loop behaves much like a double-negative feedback loop, where A and B mutually inhibit each other. That system can act like a toggle switch too, except that it toggles between A on\/B off and A off\/B on states, rather than between A off\/B off and A on\/B on states. Good examples of this type of system include the famous lambda phage lysis\/lysogeny toggle switch, and the CDK1\/Cdc25\/Wee1 mitotic trigger.\n\nIn a feed-forward system, A impinges upon C directly, but A also regulates B, which regulates C. A feed-forward system can be either 'coherent' or 'incoherent', depending upon whether the route through B does the same thing to C as the direct route does. There is no feedback \u2013 A affects C, but C does not affect A \u2013 and the system cannot function as a toggle switch. A good example of feed-forward regulation is the activation of the protein kinase Akt by the lipid second messanger PIP3 (PIP3 binds Akt, which promotes Akt activation, and PIP3 also stimulates the kinase PDK1, which phosphorylates Akt and further contributes to Akt activation). Since both routes contribute to Akt activation, this is an example of coherent feed-forward regulation. Uri Alon's classic analysis of motifs in *Escherichia coli* gene regulation identified numerous coherent feed-forward circuits in that system.\n\n# In high school I hated physics and math, but I loved biology. Should I go into systems biology?\n\nNo.\n\n# What kind of physics and math is most useful for understanding biological systems?\n\nSome level of comfort in doing simple algebra and calculus is a must. Beyond that, probably the most useful math is nonlinear dynamics. The Strogatz textbook mentioned below is a great introduction to nonlinear dynamics.\n\n# Do I need to understand differential equations?\n\nSystems biologists often model biological processes with ordinary differential equations (ODEs), but the fact is that almost none of them can be solved exactly. (The one that can be solved exactly describes exponential approach to a steady state, and it's something every biologist should work out at some point in his or her training.) Most often, systems biologists solve their ODEs numerically, often with canned software packages like Matlab or Mathematica.\n\nIdeally, a model should not only reproduce known biology and predict unknown biology, it should also be 'robust' in important respects.\n\n# What is robustness, and why is it important to systems biologists?\n\nRobustness is the imperviousness of some performance characteristic of a system in the face of some sort of insult \u2013 such as stochastic fluctuations, environmental insults, or deletion of nodes from the system. For example, the period of the circadian oscillator is robust with respect to changes in the temperature of the environment. Robustness can be quantitatively defined as the inverse of sensitivity, which itself can be defined a few ways \u2013 often sensitivity is taken to be:\n\n$$\\frac{d\\ln Response}{d\\ln Pertubation}$$\n\nso that robustness becomes\n\n$$\\frac{d\\ln Pertubation}{d\\ln Response}$$\n\nRobustness is important to systems biologists because of the attractiveness of the idea that a biological system must function reliably in the face of myriad uncertainties. Maybe robustness, more than efficiency or speed, is what evolution must optimize to create successful biological systems.\n\nModeling can provide some insight into the robustness of particular networks and circuits. Just as a biological system must be robust with respect to insults the system is likely to encounter, a successful model should also be robust with respect to parameter choice. If a model 'works', but only for a precisely chosen set of parameters, the system it depicts may be too finicky to be biologically useful, or to have been 'found' in evolution.\n\n# What other types of models are useful in systems biology?\n\nODE models assume that each dynamical species in the model \u2013 each protein, protein complex, RNA, or whatever \u2013 is present in large numbers. This is sometimes true in biological systems. For example, regulatory proteins are often present at concentrations of 10 to 1,000 nM. For a four picoliter eukaryotic cell, this corresponds to 24,000 to 2,400,000 molecules per cell. This is probably large enough to warrant ODE modeling. However, genes and some mRNAs are present at concentrations of one or two molecules per cell. At such low numbers, each individual transcriptional event or mRNA degradation event becomes a big deal, and the appropriate type of modeling is stochastic modeling.\n\nSometimes systems are too complicated, or have too many unknown parameters to warrant ODE modeling. In these cases, Boolean models and probabilistic Bayesian models can be particularly useful.\n\nSometimes it is important to see how dynamical behaviors propagate through space, in which case either partial differential equation (PDE) models or stochastic reaction\/diffusion models may be just the ticket.\n\n# Where can I go for more information?\n\n## Review articles\n\nHartwell LH, Hopfield JJ, Leibler S, Murray AW: **From molecular to modular biology**. *Nature* 2005, **402(Suppl):**C47\u2013C52.\n\nKirschner M: **The meaning of systems biology**. *Cell* 2005, **121:**503\u2013504.\n\nKitano H: **Systems biology: a brief overview**. *Science* 2002, **295:**1662\u20131664.\n\n## Textbooks\n\nAlon U: *An Introduction to Systems Biology: Design Principles of Biological Circuits*. Boca Raton, FL: Chapman & Hall\/CRC; 2006.\n\nHeinrich R, Schuster S: *The Regulation of Cellular Systems*. Berlin: Springer; 1996.\n\nKlipp E, Herwig R, Kowald A, Wierling C, Lehrach H: *Systems Biology in Practice: Concepts, Implementation and Application*. Weinheim, Germany: Wiley-VCH; 2005.\n\nPalsson B: *Systems Biology: Properties of Reconstructed Networks*. Cambridge University Press; 2006.\n\nStrogatz SH: *Nonlinear Dynamics and Chaos: With Applications to Physics, Biology, Chemistry and Engineering*. Boulder, CO: Westview Press; 2001.","meta":{"dup_signals":{"dup_doc_count":113,"dup_dump_count":41,"dup_details":{"curated_sources":2,"2022-05":1,"2020-40":1,"2020-10":1,"2019-47":1,"2019-22":1,"2018-05":1,"2017-39":1,"2017-34":1,"2017-26":1,"2017-22":3,"2017-17":1,"2017-09":9,"2017-04":1,"2016-50":1,"2016-44":2,"2016-40":2,"2016-36":7,"2016-30":5,"2016-22":1,"2016-18":1,"2015-48":3,"2015-40":3,"2015-35":3,"2015-32":3,"2015-27":3,"2015-22":4,"2015-14":3,"2014-52":3,"2014-49":3,"2014-42":8,"2014-41":3,"2014-35":6,"2014-23":5,"2014-15":6,"2022-27":1,"2015-18":2,"2015-11":2,"2015-06":2,"2014-10":2,"2013-48":2,"2013-20":2}},"file":"PMC2656213"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  Chondrosarcoma (Chs) is the third most frequent primary malignant tumour of bone and can be primary or secondary, the latter results mainly from the malignant transformation of a benign pre-existing tumour.\n  .\n  # Methods\n  .\n  All the cases diagnosed as Chs (primary tumours, recurrences and\/or metastasis and xenotransplanted Chs) from the files of our Department were collected. Only cases with paraffin blocks available were selected (Total 32 cases). Six Tissue Microarrays (TMAs) were performed and all the cases and biopsies were distributed into the following groups: a) only paraffin block available from primary and\/or metastatic tumours (3 TMAs), b) paraffin block available from primary and\/or metastatic tumours as well as from the corresponding Nude mice xenotransplant (2 TMAs), c) only paraffin block available from xenotransplanted Chs (1 TMA). A reclassification of all the cases was performed; in addition, conventional hematoxylin-eosin as well as immunohistochemistry staining (S100, SOX-9, Ki-67, BCL-2, p53, p16, CK, CD99, Survivin and Caveolin) was analyzed in all the TMA.\n  .\n  # Results\n  .\n  The distribution of the cases according to the histopathological pattern and the location of tumours were as follows: fourteen Grade I Chs (all primaries), two primary Grade II Chs, ten Grade III Chs (all primaries), five dedifferentiated Chs (four primaries and one primary with metastasis), and two Chs from cell cultures (Ch grade III). One recurrent extraskeletal myxoid Chs was included as a control in the TMA. Although there was heterogeneity in immunohistochemistry results of the different material analyzed, S100, SOX-9, Caveolin and Survivin were more expressed. The number of passages in xenotransplants fluctuated between 1 and 13. Curiously, in Grade I Chs, these implanted tumours hardly grew, and the number of passages did not exceed one.\n  .\n  # Conclusion\n  .\n  The study of Chs by means of TMA techniques is very important because it will improve the assessment of different antibodies applied in the immunohistochemical assays. Xenotransplanted tumours in TMA improve knowledge concerning the variability in the morphological pattern shown by these tumours during the evolution in nudes.\nauthor: Isidro Machado; Francisco Giner; Empar Mayordomo; Carmen Carda; Samuel Navarro; Antonio Llombart-Bosch\ndate: 2008\ninstitute: 1Department of Pathology, University of Valencia, Valencia, Spain\nreferences:\ntitle: Tissue microarrays analysis in chondrosarcomas: light microscopy, immunohistochemistry and xenograft study\n\n# Introduction\n\nThe development of Tissue Microarray (TMA) technology offers the advantage to screening large tissue cohorts for biomarker expression and to examine serial sections obtained from the same tumour specimen and xenograft tumours \\[1-8\\]. TMAs are especially suitable for the reproduction of morphological and immunohistochemical (IHC) results in different laboratories \\[2,4,5\\]. Chondrosarcoma (Chs) is the third most frequent primary malignant tumour of bone exceeded only by myeloma and osteosarcoma. It is characterised by the production of cartilage and can be primary or secondary, the latter results mainly from the malignant transformation of benign pre-existing tumours \\[9,10\\]. Several clinical and histopathological subtypes are recognized and the final diagnoses involve a large group of physicians including pathologists, radiologists and surgeons \\[10-12\\]. The conventional Chs can be classified according the location in bone in two groups: central Chs; located inside the medullar cavity and the peripheral Chs; located in the surface of the bone \\[9\\]. The histopathology is quite similar in both locations and is classified in three grades: Grade I, II and III. Grade I Chs are characterized by cells with small densely straining nuclei and chondroid or myxoid background, in Grade II Chs the nucleus are of moderate size with increased cellularity and low mitotic rate, Grade III Chs reveal large nuclei in areas with increased cellularity with moderate mitotic rate and scant chondroid or myxoid matrix\\[9\\]. Mesenchymal Ch, dedifferentiated Ch and clear cell Ch are described as infrequent variants of Chs and the clinical presentation, radiological findings and histopathological features are different to conventional forms \\[9-12\\]. The differential diagnosis is quite difficult between low grade Chs and benign chondral conditions, but usually the clinical data, radiography and morphological picture defines a definitive diagnosis \\[9,10\\].\n\nCytogenetic studies show heterogeneity related to karyotypic complexity, nevertheless, alterations in p16 tumour suppressor gene and p53 are related with Ch progression \\[9,10,13,14\\].\n\nXenograft models in bone tumour are of great importance because nude tumours constitute an easy source of material for tumour characterization using histopathological, IHC, electron microscopy, cytogenetic and molecular biology criteria \\[6,7,15\\]. Usually the histology of the tumours is preserved and they can also be followed over subsequent generations in various tumours passages \\[7,15\\]. Xenograft models in Chs are performed infrequently and being followed for successive generations is even more sporadic. Few publications exist on TMA study in Chs and the combination with a xenograft study is even more unusual. Therefore, the aims of this study are firstly the analysis of heterogeneity in Chondrosarcomas by means of TMA technology using histopathological and IHC criteria and secondly to analyze their successive xenografts after xenotransplantation into nude mice followed for several passages to describe the histopathological and IHC pattern variations occurring in the original tumour and their successive xenotransplants.\n\n# Materials and methods\n\n## Samples sources\n\nAll the cases diagnosed as Chs (primary tumours, recurrences and\/or metastasis and xenotransplanted Chs) from the files of our Department were collected. Only cases with paraffin blocks available were selected. All the clinical data of the patients were reviewed. (Total: 32 cases). A reclassification of all the cases according to the new criteria for Chs diagnosis (WHO) was performed.\n\n## Assembly of TMA\n\nSix TMAs were performed and all the cases and biopsies were distributed into the following groups: a) only paraffin block available from primary and\/or metastatic tumours (3 TMA), b) paraffin block available from primary and\/or metastatic tumours as well as from the corresponding Nude mice xenotransplant (2 TMA), c) only paraffin block available from xenotransplanted Chs (1 TMA). A hematoxylin and eosin stained (H\/E) section from each primary tumour and xenograft was prepared and areas of representative non-necrotic neoplasm circled on coverslip. The TMAs were assembled using a manual tissue arrayer (Beecher Instruments, Sun Prairie, WI). Normally, two cores (1 mm in thickness) of each biopsy were performed; nevertheless, more than 2 cores were made if the biopsy revealed a different pattern. All TMAs included two cores of normal kidney or liver as control tissues. Following TMA construction, H\/E stained section of the TMA recipient block was prepared and reviewed to confirm the presence of intact neoplasm. Several sections of 5 \u03bc were prepared in order to perform H\/E stain as well as different IHC staining.\n\n## Immunohistochemical analysis\n\nIHC analysis was performed using anti-CD99 antibody (clone 12E7, DakoCytomation) at a 1:50 dilution, anti-S100 polyclonal antibody (DakoCytomation) at a 1:200 dilution, anti-SOX-9 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:100 dilution, anti-survivin polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at 1: 50 dilution, anti-p16 antibody (clone F12, Santa Cruz Biotechnology, Santa Cruz, CA) at 1:100 dilution, anti-p53 antibody (clone DO7, Novocastra) at 1:50 dilution, pan-CK (AE1\/AE3) antibody (DakoCytomation) at 1:50 dilution, anti-Ki-67 antibody (MIB-1, DakoCytomation) at 1:50 dilution, anti-Caveolin (CAV) polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:200 dilution, anti-Bcl-2 antibody (clone 124, Novocastra) at 1:50 dilution. Antigen retrieval was performed by pressure cooker boiling for 3 minutes in 10 mmol\/L of citrate buffer (pH 6.0). The LSAB method (DakoCytomation) was used, followed by revelation with 3,3'-diaminobenzidine. Cytoplasmic and\/or membrane staining was considered positive for CD99, S100, CK, CAV and Bcl-2 antibodies, nuclear staining was considered positive for SOX-9, Survivin, p53, p16 and Ki-67 antibodies. Sections were examined and immunoreactivity was defined as follow: negative, fewer than 5% of tumour cells stained; poorly positive (+), 5% to 10% of tumours cells stained; moderately positive (++), 10% to 50% of tumours cells stained and strongly positive (+++), more than 50% of the tumours cells were stained. All sections were evaluated independently by 3 pathologists (IM, SN and ALLB). The agreement of staining intensity scoring by all was recorded, and in cases of disagreement, intensity and score was determined by consensus.\n\n## Xenotransplant\n\nMale nude mice, were purchase from IFFA-CREDO (Lyon, France), kept under specific pathogen-free conditions throughout the experiment, and provided with vinyl isolates plus sterilized food, water, cage and bedding. The specimens for xenotransplant were obtained at surgery (OT) and placed in a culture medium (RPMI 1640) plus antibiotic at 37 \u2103 until transplantation, usually 6 hours after surgery. Fragments of non-necrotic tumour, about 3 to 5 mm in size, were transplanted into the subcutaneous tissue in the backs of two nude mice. The new tumour transfers were made by following the same procedure as in the initial xenotransplant and always under highly sterile conditions. Material from different passages was obtained in order to perform all TMAs. Additional material was obtained for electron microscopy, culture, and frozen sections.\n\n# Results\n\nGrade I and Grade III Chs were the most frequent histopathological patterns, the distribution of the cases were as follows: fourteen Grade I Chs (all primaries), two primary Grade II Chs, ten Grade III Chs (all primaries) (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>), five dedifferentiated Chs (four primaries and one primary with metastasis) and two Chs from cell cultures (Ch grade III). One recurrent extraskeletal myxoid Chs was included as a control in the TMA. Most of the tumours were located in bone and\/or soft tissue. The results of IHC study are given in Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>. Although there was heterogeneity in IHC results of the different material analyzed, S100, SOX-9, Caveolin and Survivin were more expressed (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>).\n\nInmunohistochemical profile. N\/A = Not assessable\n\n| **Antibodies** | **N\/A** | **Negative (-)** | **Poorly positive (+)** | **Moderately positive (++)** | **Strongly positive (+++)** |\n|----|----|----|----|----|----|\n| **S100** | 3 | 0 | 0 | 1 | 28 |\n| **SOX-9** | 1 | 2 | 7 | 5 | 17 |\n| **Survivin** | 2 | 2 | 4 | 3 | 21 |\n| **Caveolin** | 1 | 1 | 6 | 12 | 12 |\n| **CD99** | 2 | 8 | 12 | 5 | 5 |\n| **p16** | 5 | 10 | 8 | 3 | 6 |\n| **p53** | 2 | 16 | 6 | 6 | 2 |\n| **Ki-67** | 4 | 7 | 5 | 9 | 7 |\n| **Bcl-2** | 3 | 16 | 7 | 3 | 3 |\n| **CK** | 1 | 28 | 2 | 1 | 0 |\n\nConcerning xenografts, the number of passages in xenotransplants fluctuated between 1 and 13. Curiously, in low grade \u2013 Grade I Chs, these implanted tumours hardly grew, and the number of passages did not exceed one. Nevertheless, in Grade II or Grade III Chs the number of passages were greater. An example of dedifferentiated Chs evolution in nudes is described in Table <a href=\"#T2\" data-ref-type=\"table\">2<\/a> and (Figures: <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>, <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>, <a href=\"#F4\" data-ref-type=\"fig\">4<\/a>, <a href=\"#F5\" data-ref-type=\"fig\">5<\/a>, <a href=\"#F6\" data-ref-type=\"fig\">6<\/a>).\n\nModel of Xenograft in Ch. Evolution in Nude mice\n\n| **Material** | **De-differentiation** | **S100** | **SOX-9** | **Ki-67** | **p53** |\n|----|----|----|----|----|----|\n| **Original Tumour** | \\+ | ++ | +++ | \\+ | \\+ |\n| **Nu385-P0** | ++ | ++ | ++ | ++ | ++ |\n| **Nu385-P1** | +++ | \\+ | \\+ | ++ | +++ |\n| **Nu385-P2** | +++ | \\+ | \\+ | +++ | +++ |\n| **Nu385-P3** | +++ Osteoid | \\+ | \\+ | +++ | +++ |\n\n# Discussion\n\nTMA-based morphological and IHC evaluation of Chs primary tumour and xenograft proved to be a high throughput source in demonstrating the phenotypic variability of original and xenograft Chs. Grading Chs has proven prognostic value, nevertheless the differential diagnosis between low grade Chs and benign chondroid lesions is quite difficult and requires an integration of clinical, radiology and histopathology information \\[9-12\\]. Grade I Ch was the most frequent in this cohort, as reported in the literature and most of the tumours appeared in bone and soft tissue \\[16\\]. At the histopathological level, the distinction between Grade I, II, III and dedifferentiated Chs was relatively easy using the updated criteria described \\[9,10\\]. S100 immunoreactivity in chondroid foci and the transcription factor SOX-9 (regulator of chondrogenesis) has been reported as the most sensitive and confirmatory markers in Chs diagnosis, both were the most expressed in the study. SOX-9 staining differentiates Chs from various small cells round tumour such as small cell osteosarcomas, non-Hodgkin lymphomas and Ewing\/PNET family tumours \\[17\\]. Recently, the expression of Bcl-2 and parathyroid-like hormone (PTHLH) as two antibodies that supported Ch diagnosis and differentiating them from other benign conditions as osteochondromas has been described \\[18\\], although in the present study, Bcl-2 expression was infrequent in the original tumours and in their corresponding xenograft. Curiously, CAV and Survivin were overexpressed in Chs, therefore apoptosis and cell interactions could be related to tumour chondrogenesis. Further studies with larger series could provide new insight into the pathogenesis of Chs.\n\nXenograft materials are widely used by researchers although in the case of Chs tumours; few studies have been published using TMA technology \\[19\\]. As many Chs tumours reveal different patterns in the same biopsy, it is not surprising that the xenograft tumour showed different histopathological pictures in some cases compared with the original tumour. Usually, Grade I Chs showed no variation in histology and IHC between the primary tumours and their xenograft. Intriguingly, in Grade I Chs, these implanted tumours hardly grew, and the number of passages did not exceed one, whereas in Grade III; dedifferentiated Chs as well as in Chs from culture, the numbers of successive passages were higher, and light microscopy as well as IHC study revealed differences between primary tumour and their xenograft. Tumour cells in Chs culture could acquire newer genetic and\/or epigenetic alterations that increase the aggressive phenotype; in addition, these cells in culture grow quite fast and replace stromal and necrotic cells from the original tumour. Original tumours (Grade III or dedifferentiated Ch) developed morphological transformation to a poorly differentiated subtype in some of the successive xenotransplanted tumours, in addition differences between IHC expression of chondrogenic differentiation markers (S100, SOX-9), cell cycle regulators (p53, p16) and proliferation marker (Ki-67) were detected. The dedifferentiated Ch xenograft model displayed a morphological transition over subsequent passages in nude mice, initially the tumour showed a biphasic pattern characterized by areas with typical chondroblastic differentiation surrounding by areas with marked dedifferentiation. Over the successive passages, the tumour acquired more dedifferentiation and the last tumours passages revealed osteoid material similar to osteosarcoma. The approximate period between passages was three months. In addition, S100 and SOX-9 expression showed relatively decreased expression and Ki-67, p53 and p16 revealed increased expression in the successive xenograft. Some of these changes may represent newer genetic and\/or epigenetic alterations developed in more aggressive subclones or could be induced by the establishment of the xenograft as occurs in tumour progression\\[6,7\\]. TMAs in xenografted tumours offer an invaluable tool for performing further research in order to study newer markers of cell differentiation, activation, genetics and cell signalling in Chs. Despite the increasing use of TMAs, limitations remain, particularly in the case of tumours, due to intratumoral heterogeneity of protein expression \\[1,5\\]. Therefore in order to ensure TMA representativeness, more representative areas of sections of the original donor block should be selected, as well as increasing the number of cores collected and the size of the single cores \\[5\\]. Nowadays, TMAs are becoming an indispensable tool in the study of cancer progression and provide newer insights concerning the biology of several tumours.\n\n# Conclusion\n\nThe analysis in TMA of Chs xenograft followed for successive generations provides new information concerning the biology and morphology of these tumours. The histopathology remained similar to the original tumour in most of the cases, but occasionally, as in the model displayed, the tumours acquire more dedifferentiation after several transfer. IHC studies using TMA techniques are useful for the assessment of the antibodies related to Chs.\n\n### Acknowledgements\n\nThis work was supported by grants PI06\/1576 , RD06-0020\/0102 from the Instituto Carlos III de Madrid, Spain and Contract n\u00b0: 018814 (EuroBoNet) from the 6thFP of the EC. We would like to acknowledge Elisa Alonso for her collaboration and technical assistance with the pathology.\n\nThis article has been published as part of *Diagnostic Pathology* Volume 3 Supplement 1, 2008: New trends in digital pathology: Proceedings of the 9th European Congress on Telepathology and 3rd International Congress on Virtual Microscopy. The full contents of the supplement are available online at <http:\/\/www.diagnosticpathology.org\/supplements\/3\/S1>","meta":{"dup_signals":{"dup_doc_count":114,"dup_dump_count":51,"dup_details":{"curated_sources":3,"2022-49":1,"2022-21":1,"2021-39":1,"2020-40":1,"2020-29":1,"2020-16":1,"2019-30":1,"2019-22":1,"2019-13":1,"2019-04":1,"2018-47":1,"2018-39":1,"2018-30":1,"2018-17":1,"2018-05":2,"2017-39":1,"2017-30":1,"2017-22":1,"2017-17":1,"2017-09":18,"2017-04":1,"2016-50":1,"2016-44":1,"2016-40":1,"2016-36":1,"2016-22":1,"2016-18":1,"2016-07":18,"2015-48":2,"2015-40":2,"2015-35":1,"2015-32":2,"2015-27":2,"2015-22":2,"2015-14":2,"2014-52":2,"2014-49":1,"2014-42":6,"2014-41":2,"2014-35":3,"2014-23":3,"2014-15":3,"2023-14":1,"2024-10":1,"2015-18":2,"2015-11":2,"2015-06":2,"2014-10":2,"2013-48":2,"2013-20":2,"2024-26":1}},"file":"PMC2500106"},"subset":"pubmed_central"}
{"text":"author: Jelle T. Prins\\*To whom correspondence should be addressed: Jelle T. Prins, University of Groningen, Groningen, Netherlands, E-mail: <firstname.lastname@example.com>\ndate: 2010-04-22\ninstitute: 1University of Groningen, Groningen, Netherlands\ntitle: PhD Thesis: burnout among Dutch medical residents\n\n# Introduction\n\nMedical residents (postgraduate trainees in a medical specialty) fulfil an important role in the Dutch health care system. They take their share of the responsibility for efficient patient care in hospitals, mental health care centres, rehabilitation centres and other medical institutions. Medical residents see the period of postgraduate training as a phase in which finding a balance between training, work and private life plays an important part. Not much was known about the extent to which the stress felt by residents causes them to develop symptoms of burnout. It was also unknown which factors determine the actual development of burnout. The research which was carried out therefore revolved around the incidence of burnout among medical residents in the Netherlands. It also looked into the potential risk-heightening or risk-lowering effects of a number of demographic and work-related factors. The aims of the studies were the following:\n\n-   to determine the prevalence of burnout among medical residents;\n\n-   to study the effect of individual and work-related factors on burnout;\n\n-   to examine the relationship between burnout and the quality of care.\n\nIn order to answer the research questions, two cross-sectional studies were carried out. The first study involved medical residents at University Medical Centre Groningen; the second all 5245 medical residents in the Netherlands.\n\n# First Study\n\nFor the first study on the prevalence of burnout, medical residents at University Medical Centre Groningen in the Netherlands were approached. The Utrecht BurnOut Scale (UBOS\/MBI-HHS) was used. This self-assessment form was sent to 292 medical residents. The response rate was 54%. The results show that 13% of the respondents claimed to experience moderate to severe burnout. The highest percentage of burnout was found among residents in psychiatry. Looking into the effects on burnout of emotional, informative and appreciative support experienced by residents from supervisors, fellow residents, nurses and patients showed that medical residents appeared to be less satisfied with the perceived emotional support from supervisors than with the support they received from colleagues and nurses. A significant relationship was established between dissatisfaction with emotional and appreciative support from supervisors and emotional exhaustion felt by residents. A link was also established between dissatisfaction with emotional support from supervisors and increased feelings of depersonalization. Medical residents also reported on the degree of reciprocity (the balance between pain and gain in relationships) that medical residents experienced in their working relationships with for example their supervisors. Only 13% of the residents experienced over-benefit in their relationships with their supervisors, 41% claimed under-benefit and 46% reported a good balance between give and take in the relationship (reciprocity). Medical residents who experienced under-benefit in the relationship with their supervisors reported significantly more emotional exhaustion and depersonalization than residents who experienced reciprocity in the relationship. Contrary to what we assumed, there did not appear to be any significant link between number of years in training and the perceived reciprocity in relationships.\n\n# National study\n\nFor the national study, all medical residents registered with the Medische Registratie Commissie (Medical Registration Committee) in 2005 were approached (N=5245); 41% responded. Of the respondents, 20.6% were classified as having burnout and 14.6% and 6% of these had moderate and severe symptoms respectively. Eleven per cent of the respondents appeared to be highly engaged, 23.2% of the residents scored above the cut-off point on vigour, 36.4% on dedication, and 27.8% on absorption. Medical residents with a partner and\/or children scored significantly lower on depersonalization than residents who did not have partners and\/or children. The percentage of residents with symptoms of burnout was lowest in the group of residents in general surgery, followed by residents in obstetrics & gynaecology and in the supportive specialties (such as radiology, pathology). Residents in general surgery were much more engaged and vigorous than residents in other surgical specialties, internal medicine, other medical specialties, supportive specialties and psychiatry. It also appeared that residents in general surgery were more dedicated and more absorbed in their work than residents in the supportive specialties.\n\nIn the national study 94% of the residents admitted to having made one or more errors which had no negative consequences for their patients, 71% claimed to have performed procedures that they were not actually competent to carry out and 56% admitted to having made one or more errors that had negative consequences for the patient **during their training so far**. Seventy five per cent of the respondents felt that the quality of the treatment they had given was inadequate. Male residents reported more errors than female residents. The group of residents in general surgery reported the highest numbers of errors in procedures compared with residents in other specialties. Medical residents in internal medicine also reported more errors than their colleagues in a number of other specialties. Medical residents in psychiatry reported the highest number of errors relating to time problems. Medical residents with symptoms of burnout appeared to report significantly more errors than residents who did not satisfy the criteria for burnout. Highly engaged residents reported fewer errors than their less engaged colleagues.\n\n# Conclusions\n\nThe two empirical studies included in the dissertation revealed that burnout among medical residents is no exception. The last and most comprehensive study made clear that nationwide 20.6% of medical residents experienced burnout. The Central Bureau for Statistics (CBS) in the Netherlands reported that 8-11% of the Dutch labour force was burned out. When the CBS criteria for assessing burnout were applied to the residents in the national study, the burnout percentage jumped to 41% \u2013 a prevalence that is four times higher than that in the national labour force. Although interesting from a research point of view, these percentages are troublesome from a health care perspective, especially since burnout was not only found to influence personal distress but also the quality of delivered care.\n\nThe conclusion that many residents experience an unbalanced relationship with their supervisor, which affects their well being in a negative way, is worrisome. A relationship with a supervisor is usually seen as a resource which should have positive connotations with well-being. Medical residents experience the relationship with their supervisors not only as one in which they under-benefit but they are also dissatisfied with the social support they receive from their supervisors. These factors contribute directly to the development of burnout in medical residents.\n\nMedical residents, like other young 'high potentials', are believed to excel in different areas at the same time: work, training and private life. It could be that in the present culture medical residents are overburdened. Balancing work, training and private life can lead to unintended effects for medical residents. One might question if the responsibility for keeping in balance during residency should rest on medical residents only. In the new competency driven training programmes medical residents have to be able to organize a balance between patient care and self development. However it can be questioned if today's residents are being taught the art of balancing during their training.\n\n# The author\n\nPrins J.T. Burnout among Dutch medical residents, University of Groningen, juni 17th 2009 (see Figure 1 <a href=\"#F1\" data-ref-type=\"fig\">(Fig. 1)<\/a> and 2 <a href=\"#F2\" data-ref-type=\"fig\">(Fig. 2)<\/a>), Promotor: prof. dr. H.B.M. van de Wiel, copromotor dr. J.E.H.M. Hoekstra-Weebers. 143 pages","meta":{"dup_signals":{"dup_doc_count":107,"dup_dump_count":37,"dup_details":{"curated_sources":2,"2018-09":1,"2018-05":1,"2017-30":3,"2017-22":2,"2017-09":2,"2017-04":2,"2016-50":2,"2016-44":3,"2016-40":2,"2016-36":3,"2016-30":3,"2016-26":2,"2016-22":2,"2016-18":2,"2016-07":3,"2015-48":3,"2015-40":2,"2015-35":3,"2015-32":3,"2015-27":3,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":5,"2014-42":8,"2014-41":4,"2014-35":5,"2014-23":6,"2014-15":4,"2018-26":1,"2015-18":3,"2015-11":2,"2015-06":2,"2014-10":3,"2013-48":3,"2013-20":2,"2024-30":1}},"file":"PMC3140360"},"subset":"pubmed_central"}
{"text":"author: M Khoury; V Escriou; A Galy; R Yao; C Largeau; D Scherman; C Jorgensen; F Apparailly\ndate: 2007\ninstitute: 1Inserm, U 844, INM, H\u00f4pital Saint Eloi, Montpellier, France; 2Universit\u00e9 Montpellier1, UFR de M\u00e9decine, Montpellier, France; 3Inserm, U 640, Paris, France; 4CNRS, UMR8151, Paris, France; 5Universit\u00e9 Paris Descartes, Facult\u00e9 de Pharmacie, Paris, France; 6Ecole Nationale Sup\u00e9rieure de Chimie de Paris, Paris, France; 7Inserm, U 790, Genethon, Evry, France; 8Universit\u00e9 Paris-Sud 11, Orsay, France; 9CHU Lapeyronie, service Immuno-Rhumatologie, Montpellier, France\ntitle: Combined anti-inflammatory tritherapy using a novel small interfering RNA lipoplex successfully prevents and cures mice of arthritis\n\n# Background\n\nTNF\u03b1 is a key cytokine in rheumatoid arthritis (RA) physiopathology. We recently demonstrated that a new cationic liposome formulation allowed intravenous delivery of a small interfering RNA (siRNA) targeting TNF\u03b1 and efficiently restoring the immunological balance in an experiment model of RA. Since 30% of patients do not respond to anti-TNF biotherapies, however, there is a need to develop alternative therapeutic approaches.\n\n# Objective\n\nStrong association of other proinflammatory cytokines with the pathogenesis of RA prompted us to investigate which cytokine other than TNF\u03b1 could be targeted for therapeutic benefit using RNA interference.\n\n# Methods\n\nTwo siRNA sequences were designed for IL-1\u03b2, IL-6 and IL-18 proinflammatory cytokines, and their efficacy and specificity were validated *in vitro* on J774.1 mouse macrophage cells, measuring both mRNA and protein levels following a lipopolysaccharide challenge. For *in vivo* administration, siRNAs were formulated as lipoplexes with the RPR209120\/DOPE liposome and a carrier DNA, and were injected intravenously in DBA\/1 mice having collagen-induced arthritis. The clinical course of the disease was assessed by paw thickness over time, and radiological and histological scores were obtained at euthanasia. The cytokine profiles were measured by ELISA in sera and knee-conditioned media. The immunological balance was assessed using antitype II collagen assays. The distribution of siRNAs was evaluated by fluorometry in GFP transgenic mice over time after anti-GFP siRNA lipoplex injections.\n\n# Results\n\nThe designed siRNA sequences silenced 70\u201375% of the lipopolysaccharide-induced IL-1\u03b2, IL-6 and IL-18 mRNA expression in macrophages compared with a control siRNA. Each siRNA affected the targeted cytokine specifically, without modifying other proinflammatory cytokine mRNAs. In the collagen-induced arthritis model, weekly injections of siRNA lipoplexes significantly reduced the incidence and severity of arthritis, abrogating joint swelling, and destruction of cartilage and bone, in both preventive and curative settings. The most striking therapeutic effect was observed when combining the three siRNAs targeting IL-1\u03b2\/IL-6\/IL-18 at once. Such tritherapy was associated with downregulation of both inflammatory and autoimmune components of the disease, and overall parameters were improved compared with the TNF\u03b1 siRNA lipoplex-based treatment. The siRNA formulation was widely distributed, delivering the siRNA to several organs with a strong efficacy in the liver and spleen.\n\n# Conclusion\n\nTritherapy targeting IL-1\u03b2\/IL-6\/IL-18 seems highly effective to reduce all pathological features of RA including inflammation, joint destruction and Th1 response. These data show that cytokines other than TNF\u03b1 can be targeted to improve symptoms of RA and reveal novel potential drug development targets. The systemic administration of anticytokine siRNA cocktails as a lipoplex could represent a novel and promising anti-inflammatory alternative therapy in RA.","meta":{"dup_signals":{"dup_doc_count":126,"dup_dump_count":38,"dup_details":{"curated_sources":2,"2019-35":1,"2019-26":1,"2019-18":1,"2019-09":1,"2018-51":1,"2018-43":1,"2018-34":1,"2018-22":1,"2017-47":1,"2017-34":1,"2017-22":1,"2017-09":8,"2016-44":1,"2016-40":1,"2016-36":7,"2016-30":6,"2015-48":4,"2015-40":4,"2015-35":3,"2015-32":3,"2015-27":3,"2015-22":4,"2015-14":3,"2014-52":4,"2014-49":5,"2014-42":10,"2014-41":7,"2014-35":4,"2014-23":7,"2014-15":4,"2019-43":1,"2015-18":4,"2015-11":4,"2015-06":4,"2014-10":3,"2013-48":4,"2013-20":4,"2024-10":1}},"file":"PMC4061928"},"subset":"pubmed_central"}
{"text":"author: Philip E. Bourne; Virginia Barbour\\* E-mail: <firstname.lastname@example.com>[^1]\ndate: 2011-06\ninstitute: 1Skaggs School of Pharmacy and Pharmaceutical Science, University of California San Diego, La Jolla, California, United States of America; 2Public Library of Science, Cambridge, United Kingdom\ntitle: Ten Simple Rules for Building and Maintaining a Scientific Reputation\n\n> *While we cannot articulate exactly what defines the less quantitative side of a scientific reputation, we might be able to seed a discussion. We invite you to crowd source a better description and path to achieving such a reputation by using the comments feature associated with this article. Consider yourself challenged to contribute.*\n\nAt a recent Public Library of Science (PLoS) journal editors' meeting, we were having a discussion about the work of the Committee on Publication Ethics (COPE; <http:\/\/www.publicationethics.org\/>), a forum for editors to discuss research and publication misconduct. Part of the discussion centered on the impact such cases have on the scientific reputation of those involved. We began musing: What on earth is a scientific reputation anyway? Not coming up with a satisfactory answer, we turned to a source of endless brainpower\u2014students and other editors. Having posed the question to a group of graduate students, PLoS, and other editors, we got almost as many different answers as people asked, albeit with some common themes. They all mentioned the explicit elements of a reputation that relate to measurables such as number of publications, H factor, overall number of citations etc., but they also alluded to a variety of different, qualitative, factors that somehow add up to the overall sense of reputation that one scientist has for another.\n\nWhat these students and editors identified en masse is one important side of a scientific reputation that is defined by data; but they also identified a much more nebulous side, that, while ill-defined, is a vital element to nurture during one's career. A side defined to include such terms as fair play, integrity, honesty, and caring. It is building and maintaining this kind of less tangible reputation that forms the basis for these Ten Simple Rules. You might be wondering, how can you define rules for developing and maintaining something you cannot well describe in the first place? We do not have a good answer, but we would say a reputation plays on that human characteristic of not appreciating the value of something until you do not have it any more.\n\nA scientific reputation is not immediate, it is acquired over a lifetime and is akin to compound interest\u2014the more you have the more you can acquire. It is also very easy to lose, and once gone, nearly impossible to recover. Why is this so? The scientific grapevine is extensive and constantly in use. Happenings go viral on social networks now, but science has had a professional and social network for centuries; a network of people who meet each other fairly regularly and, like everyone else, like to gossip. So whether it is a relatively new medium or a centuries-old medium, good and bad happenings travel quickly to a broad audience. Given this pervasiveness, here are some rules, some intuitive, for how to build and maintain a scientific reputation.\n\n# Rule 1: Think Before You Act\n\nScience is full of occasions whereupon you get upset\u2014a perceived poor review of a paper, a criticism of your work during a seminar, etc. It is so easy to immediately respond in a dismissive or impolite way, particularly in e-mail or some other impersonal online medium. Don't. Think it through, sleep on it, and get back to the offending party (but not a broader audience as it is so easy to do nowadays with, for example, an e-mail cc) the next day with a professional and thoughtful response, whatever the circumstances. In other words, always take the high road whatever the temptation. It will pay off over time, particularly in an era when every word you commit to a digital form is instantly conveyed, permanently archived somewhere, and can be retrieved at any time.\n\n# Rule 2: Do Not Ignore Criticism\n\nWhether in your eyes, criticism is deserved or not, do not ignore it, but respond with the knowledge of Rule 1. Failure to respond to criticism is perceived either as an acknowledgement of that criticism or as a lack of respect for the critic. Neither is good.\n\n# Rule 3: Do Not Ignore People\n\nIt is all too easy to respond to people in a way that is proportional to their perceived value to you. Students in particular can be subject to poor treatment. One day a number of those students will likely have some influence over your career. Think about that when responding (or not responding). As hard as it is, try to personally respond to mail and telephone calls from students and others, whether it is a question about your work or a request for a job. Even if for no other reason, you give that person a sense of worth just by responding. Ignoring people can take other serious forms, for example in leaving deserving people off as paper authors. Whether perceived or real, this can appear that you are trying to raise your contribution to the paper at the expense of others\u2014definitely not good for your reputation.\n\n# Rule 4: Diligently Check Everything You Publish and Take Publishing Seriously\n\nScience does not progress in certainties\u2014that is one of its joys but also what makes it such a hard profession. Though you cannot guarantee that everything you publish will, in 50 years' time, be shown to be correct, you can ensure that you did the work to the accepted standards of the time and that, whether you were the most junior or senior author, you diligently checked it (and checked it again\u2026) before you submitted it for publication. As a first author you may well be the only one who appreciates the accuracy of the work being undertaken, but all authors have a responsibility for the paper. So, however small or big your contribution, always be upfront with your co-authors as to the quality and accuracy of the data you have generated. When you come to be a senior author, it is so easy to take a draft manuscript at face value and madly publish it and move on. Both actions can come back to haunt you and lead to a perception of sloppy work, or worse, deception. As first author, this mainly lets down your other authors and has a subtle impact on your growing reputation. As the senior author of an error-prone study, it can have a more direct and long-lasting impact on your reputation. In short, take publication seriously. Never accept or give undeserved authorship and in addition never leave anyone out who should be an author, however lowly. Authorship is not a gift\u2014it must be earned and being a guest or gift author trivializes the importance of authorship. Never agree to be an author on a ghostwritten paper. At best these papers have undeclared conflicts of interest; at worst potential malpractice.\n\n# Rule 5: Always Declare Conflicts of Interest\n\nEveryone has conflicts of interest, whether they are financial, professional, or personal. It is impossible for anyone to judge for himself or herself how their own conflict will be perceived. Problems occur when conflicts are hidden or mismanaged. Thus, when embarking on a new scientific endeavor, ranging from such tasks as being a grant reviewer, or a member of a scientific advisory board, or a reviewer of a paper, carefully evaluate what others will perceive you will gain from the process. Imagine how your actions would be perceived if read on the front page of a daily newspaper. For example, we often agree to review a paper because we imagine we will learn from the experience. That is fine. Where it crosses the line is when it could be perceived by someone that you are competing with the person whose work you are reviewing and have more to gain than just general knowledge from reviewing the work. There is a gray area here of course, so better to turn down a review if not sure. Failure to properly handle conflicts will eventually impact your reputation.\n\n# Rule 6: Do Your Share for the Community\n\nThere is often unspoken criticism of scientists who appear to take more than they give back. For example, those who rarely review papers, but are always the first to ask when the review of their paper will be complete; scientists who are avid users of public data, but are very slow to put their own data into the public domain; scientists who attend meetings, but refuse to get involved in organizing them; and so on. Eventually people notice and your reputation is negatively impacted.\n\n# Rule 7: Do Not Commit to Tasks You Cannot Complete\n\nIt tends to be the same scientists over and over who fail to deliver in a timely way. Over an extended period, this becomes widely known and can be perceived negatively. It is human nature for high achievers to take on too much, but for the sake of your reputation learn how to say no.\n\n# Rule 8: Do Not Write Poor Reviews of Grants and Papers\n\nWho is a good reviewer or editor is more than just perception. Be polite, timely, constructive, and considerate and, ideally, sign your review. But also be honest\u2014the most valued reviewers are those who are not afraid to provide honest feedback, even to the most established authors. Editors of journals rapidly develop a sense of who does a good job and who does not. Likewise for program officers and grant reviews. Such perceptions will impact your reputation in subtle ways. The short term gain may be fewer papers or grants sent to you to review, but in the longer term, being a trusted reviewer will reflect your perceived knowledge of the field. Although the impact of a review is small relative to writing a good paper in the field yourself, it all adds up towards your overall reputation.\n\n# Rule 9: Do Not Write References for People Who Do Not Deserve It\n\nIt is difficult to turn down writing a reference for someone who asks for one, even if you are not inclined to be their advocate; yet, this is what you should do. The alternative is to write a reference that (a) does not put them in a good light, or (b) over exalts their virtues. The former will lead to resentment; the latter can impact your reputation, as once this person is hired and comes up short, the hirer may question aspects of your own abilities or motives.\n\n# Rule 10: Never Plagiarize or Doctor Your Data\n\nThis goes without saying, yet it needs to be said because it happens, and it is happening more frequently. The electronic age has given us tools for handling data, images, and words that were unimaginable even 20 years ago, and students and postdocs are especially adept in using these tools. However, the fundamental principle of the integrity of data, images, and text remains the same as it was 100 years ago. If you fiddle with any of these elements beyond what is explicitly stated as acceptable (many journals have guidelines for images, for example), you will be guilty of data manipulation, image manipulation, or plagiarism, respectively. And what is more, you will likely be found out. The tools for finding all these unacceptable practices are now sophisticated and are being applied widely. Sometimes the changes were done in good faith, for example, the idea of changing the contrast on a digital image to highlight your point, but one always needs to think how such a change will be perceived and in fact whether it might, even worse, give the average reader a false sense of the quality of that data. Unfortunately, even if done in good faith, if any of these practices are found out, or even raised as a suspicion, the impact on one's career can be catastrophic.\n\nIn summary, there are a number of dos and don'ts for establishing a good reputation\u2014whatever that might be. Do not hesitate in giving us your thoughts on what it means to be a reputable scientist.\n\n[^1]: Philip E. Bourne is Editor-in-Chief of *PLoS Computational Biology*. Virginia Barbour is Chief Editor of *PLoS Medicine* and Secretary of COPE.","meta":{"dup_signals":{"dup_doc_count":102,"dup_dump_count":43,"dup_details":{"curated_sources":2,"2023-06":1,"2022-49":1,"2022-05":2,"2021-10":1,"2020-10":2,"2019-18":1,"2019-13":2,"2018-47":1,"2018-34":1,"2017-51":1,"2017-43":1,"2017-39":1,"2017-26":1,"2017-22":1,"2017-17":1,"2017-09":2,"2016-50":1,"2016-30":2,"2016-26":1,"2016-22":1,"2016-18":1,"2016-07":1,"2015-48":3,"2015-40":3,"2015-35":4,"2015-32":5,"2015-27":3,"2015-22":3,"2015-14":2,"2014-52":3,"2014-49":5,"2014-42":9,"2014-41":3,"2014-35":3,"2014-23":7,"2014-15":5,"2023-40":1,"2024-30":2,"2024-22":2,"2017-13":1,"2015-18":3,"2015-11":3,"2014-10":3}},"file":"PMC3127799"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  Abel and Trevors have delineated three aspects of sequence complexity, Random Sequence Complexity (RSC), Ordered Sequence Complexity (OSC) and Functional Sequence Complexity (FSC) observed in biosequences such as proteins. In this paper, we provide a method to measure functional sequence complexity.\n  .\n  # Methods and Results\n  .\n  We have extended Shannon uncertainty by incorporating the data variable with a functionality variable. The resulting measured unit, which we call Functional bit (Fit), is calculated from the sequence data jointly with the defined functionality variable. To demonstrate the relevance to functional bioinformatics, a method to measure functional sequence complexity was developed and applied to 35 protein families. Considerations were made in determining how the measure can be used to correlate functionality when relating to the whole molecule and sub-molecule. In the experiment, we show that when the proposed measure is applied to the aligned protein sequences of ubiquitin, 6 of the 7 highest value sites correlate with the binding domain.\n  .\n  # Conclusion\n  .\n  For future extensions, measures of functional bioinformatics may provide a means to evaluate potential evolving pathways from effects such as mutations, as well as analyzing the internal structural and functional relationships within the 3-D structure of proteins.\nauthor: Kirk K Durston; David KY Chiu; David L Abel; Jack T Trevors\ndate: 2007\ninstitute: 1Department of Biophysics, University of Guelph, Guelph, ON, N1G 2W1, Canada; 2Department of Computing and Information Science, University of Guelph, Guelph, ON, N1G 2W1, Canada; 3Program Director, The Gene Emergence Project, The Origin-of-Life Foundation, Inc., 113 Hedgewood Drive Greenbelt, MD 20770-1610, USA; 4Department of Environmental Biology, University of Guelph, Guelph, ON, N1G 2W1, Canada\nreferences:\ntitle: Measuring the functional sequence complexity of proteins\n\n# Background\n\nThere has been increasing recognition that genes deal with information processing. They have been referred to as \"subroutines within a much larger operating system\". For this reason, approaches previously reserved for computer science are now increasingly being applied to computational biology \\[1\\]. If genes can be thought of as information-processing subroutines, then proteins can be analyzed in terms of the products of information interacting with laws of physics. It may be possible to advance our knowledge of proteins, such as their structure and functions, by examining the patterns of functional information when studying a protein family.\n\nOur proposed method is based on mathematical and computational concepts (e.g., measures). We show here that, at least in some cases in sequence analysis, the proposed measure is useful in analyzing protein families with interpretable experimental results.\n\nAbel and Trevors have delineated three qualitative aspects of linear digital sequence complexity \\[2,3\\], Random Sequence Complexity (RSC), Ordered Sequence Complexity (OSC) and Functional Sequence Complexity (FSC). RSC corresponds to stochastic ensembles with minimal physicochemical bias and little or no tendency toward functional free-energy binding. OSC is usually patterned either by the natural regularities described by physical laws or by statistically weighted means. For example, a physico-chemical self-ordering tendency creates redundant patterns such as highly-patterned polysaccharides and the polyadenosines adsorbed onto montmorillonite \\[4\\]. Repeating motifs, with or without biofunction, result in observed OSC in nucleic acid sequences. The redundancy in OSC can, in principle, be compressed by an algorithm shorter than the sequence itself. As Abel and Trevors have pointed out, neither RSC nor OSC, or any combination of the two, is sufficient to describe the functional complexity observed in living organisms, for neither includes the additional dimension of functionality, which is essential for life \\[5\\]. FSC includes the dimension of functionality \\[2,3\\]. Szostak \\[6\\] argued that neither Shannon's original measure of uncertainty \\[7\\] nor the measure of algorithmic complexity \\[8\\] are sufficient. Shannon's classical information theory does not consider the meaning, or function, of a message. Algorithmic complexity fails to account for the observation that 'different molecular structures may be functionally equivalent'. For this reason, Szostak suggested that a new measure of information\u2013functional information\u2013is required \\[6\\]. Chiu, Wong, and Cheung also discussed the insufficiency of Shannon uncertainty \\[9,10\\] when applied to measuring outcomes of variables. The differences between RSC, OSC and FSC in living organisms are necessary and useful in describing biosequences of living organisms.\n\nConsider two main uses for the proposed method for measuring FSC, which incorporates functionality: 1) comparative analysis of biosequence subgroups when explicit time lag is known; and 2) typicality analysis between biosequence subgroups when there is no explicit time lag. In the first case, such as an evolutionary time scale, an increase or decrease in FSC between an earlier gene or protein and a later gene or protein can be measured, evaluating its possible degradations and\/or functional effects due to various changes such as insertions, deletions, mutations and rearrangements. In the second case, a large set of aligned sequences representing a protein family can be subdivided according to phylogenetic relationships derived from typicality of species groupings and the FSC for each subgroup measured. This is important when evaluating the emergence or evolution of viral or microbial strains with novel functions such as in the comparisons of the Chlamydia family genomes \\[11\\]. An analysis may reveal the extent of the difference in FSC between one functional group and the other, as well as the modular interactions of the internal relationship structure of the sequences \\[12\\].\n\nThe ability to measure FSC would be a significant advance in the ability to identify, analyze, compare, and predict the metabolic utility of biopolymeric sequences. Mutational drift, emerging pathogenic viral and microbial species\/strains, generated mutations, acquired heritable diseases and mutagenic effects could all be evaluated quantitatively. Furthermore, *In vitro* experiments using SELEX \\[13-15\\] to study transitions in possible early ribozyme family growth could then be evaluated in a quantitative, as well as qualitative and intuitive fashion. Evolutionary changes, both actual and theoretical, can also be evaluated using FSC.\n\nIt is known that the variability of data can be measured using Shannon uncertainty \\[16\\]. However, Shannon's original formulation when applied to biological sequences does not express variations related to biological functionality such as metabolic utility. Shannon uncertainty, however, can be extended to measure *the joint variable* (*X, F*), where *X* represents the variability of data, and *F* functionality. This explicitly incorporates empirical knowledge of metabolic function into the measure that is usually important for evaluating sequence complexity. This measure of both the observed data and a conceptual variable of function jointly can be called *Functional Uncertainty* (*H*~*f*~) \\[17\\], and is defined by the equation:\n\nH\n\n(\n\nX\n\nf\n\n(\n\nt\n\n)) = -\u2211\n\nP\n\n(\n\nX\n\nf\n\n(\n\nt\n\n)) log\n\nP\n\n(\n\nX\n\nf\n\n(\n\nt\n\n))\n\nwhere *X*~f~ denotes the conditional variable of the given sequence data (*X*) on the described biological function *f* which is an outcome of the variable (*F*). For example, a set of 2,442 aligned sequences of proteins belonging to the ubiquitin protein family (used in the experiment later) can be assumed to satisfy the same specified function *f*, where *f* might represent the known 3-D structure of the ubiquitin protein family, or some other function common to ubiquitin. The entire set of aligned sequences that satisfies that function, therefore, constitutes the outcomes of *X*~f~. Here, functionality relates to the whole protein family which can be inputted from a database. The advantage of using *H*(*X*~f~(*t*)) is that changes in the functionality characteristics can be incorporated and analyzed. Furthermore, the data can be a single monomer, or a biosequence, or an entire set of aligned sequences all having the same common function. The significance of the statistical variations can then be evaluated if necessary \\[8\\]. The state variable *t*, representing time or a sequence of ordered events, can be fixed, discrete, or continuous. Discrete changes may be represented as discrete time states.\n\nFunctional bioinformatics is emerging as an important area of research \\[18-20\\]. Even though the term 'biological function' has been freely used for specific experimentation, there is no generally consistent usage of the term. According to Karp \\[21\\], biological functionality can refer to biochemical specified reactions, cellular responses, and structural properties of proteins and nucleic acids. It can be defined or specified at the global level (i.e., the entire organism), locally at the sub-molecular level, or applicable to the whole molecule. Hence confusion exists in interpreting its meaning. Karp \\[21\\] recognized that biological function is a complex concept. Using Webster, he refers to 'specially fitted' action or 'normal and specific contribution' of a part to the economy of the whole. Function can be related to cellular components (e.g., macromolecules, proteins or small molecules) that interact and catalyze biochemical transformations. In specific applications, it can be a local function of an enzyme such as the substrate that is acted on, or the ligands that activate or inhibit the enzyme. In more systemic integrated functions, it may refer to pathways, single or multiple, in a hierarchical, nested scope \\[22,23\\]. In general, it is a challenge 'to define a single best set of biologically acceptable rules for performing this decomposition' \\[21\\].\n\nIn our approach, we leave the specific defined meaning of functionality as an input to the application, in reference to the whole sequence family. It may represent a particular domain, or the whole protein structure, or any specified function with respect to the cell. Mathematically, it is defined precisely as an outcome of a discrete-valued variable, denoted as *F=*{*f*}. The set of outcomes can be thought of as specified biological states. They are presumed non-overlapping, but can be extended to be fuzzy elements. In order to get a meaningful calculation in measuring FSC, the measure should be statistically significant in practice \\[7,10\\], much larger than zero when relating to the sequences. When sequences are chosen that are unrelated to the function to be analyzed, or are simply arbitrarily ordered or randomly generated sequences, then the measure of FSC will be small and statistically not significant. For example, if many sequences that do not share the same function *f*, are mistakenly included within an aligned set representing some particular function, we should expect the measure of FSC of that set to be degraded, possibly even to a very small value. However, when the specified functionality is chosen meaningfully (even in part), then FSC can be interpreted.\n\nConsider further, when a biosequence is mutated, the mutating sequence can be compared at two different time states going from *t*~i~ to *t*~j~. For example, *t*~i~ could represent an ancestral gene and *t*~j~ a current mutant allele. Different sequences sharing the same function *f* (as outcomes of the variables denoted respectively as *X*~f~, *Y*~f~) can also be compared at the same time *t*. Within a sequence, any change of a monomer in the sequence represents a single step change that may or may not affect the overall function. Sequence reversals, gene splits, lateral transfers, and multiple point mutations can also be quantified between the two states *t*~i~, *t*~j~. The limits of the change in functional uncertainty between the two states can then be evaluated at *t* = *t*~i~ and *t* = *t*~*j*~.\n\nThe change in functional uncertainty (denoted as \u0394*H*~f~) between two states can be defined as\n\n\u0394\n\nH\n\n(\n\nX\n\ng\n\n(\n\nt\n\ni\n\n),\n\nX\n\nf\n\n(\n\nt\n\nj\n\n)) =\n\nH\n\n(\n\nX\n\ng\n\n(\n\nt\n\nj\n\n)) -\n\nH\n\n(\n\nX\n\nf\n\n(\n\nt\n\ni\n\n))\n\nwhere *X*~f~ (*t*~i~) and *X*~g~ (*t*~j~) can be applied to the same sequence at two different times or to two different sequences at the same time. \u0394*H*~f~ *can then quantify the change in functional uncertainty between two biopolymeric states with regard to biological functionality*. Unrelated biomolecules with the same function or the same sequence evolving a new or additional function through genetic drift can be compared and analyzed. A measure of \u0394*H*~f~ can increase, decrease, or remain unchanged.\n\nBiological function is mostly, though not entirely determined by the organism's genetic instructions \\[24-26\\]. The function could theoretically arise stochastically through mutational changes coupled with selection pressure, or through human experimenter involvement \\[13-15\\]. A time limit can be set in some situations to evaluate what changes to *X*~f~(*t*~i~) might be possible within that limit. For example, an estimation of the evolutionary limits projected over the next 10 years could be computed in this approach for any particular strain of the HIV virus. The specifics of the function (as an outcome of the function variable) can remain constant, or it can be permitted to vary within a range of efficiency. The limit may be determined by what is permitted metabolically. There is often a minimum limit of catalytic efficiency required by the organism for a given function.\n\nThe *ground state g* (an outcome of *F*) of a system is the state of presumed highest uncertainty (not necessarily equally probable) permitted by the constraints of the physical system, when no specified biological function is required or present. Certain physical systems may constrain the number of options in the ground state so that not all possible sequences are equally probable \\[27\\]. An example of a highly constrained ground state resulting in a highly ordered sequence occurs when the phosphorimidazolide of adenosine is added daily to a decameric primer bound to montmorillonite clay, producing a perfectly ordered, 50-mer sequence of polyadenosine \\[3\\]. In this case, the ground state permits only one single possible sequence. Since the ground state represents the state of presumed highest uncertainty permitted by the physical constraints of the system, the set of functional options, if there are any, will therefore be a subset of the permitted options, assuming the constraints for the physical system remain constant. If the ground state permits only one sequence, then there is no possibility of change in the functional uncertainty of the system.\n\nThe *null state*, a possible outcome of *F* denoted as \u00f8, is defined here as a special case of the ground state of highest uncertainly when the physical system imposes *no constraints at all*, *resulting in the equi-probability of all possible sequences or options*. Such sequencing has been called \"dynamically inert, dynamically decoupled, or dynamically incoherent\" \\[28,29\\]. For example, the ground state of a 300 amino acid protein family can be represented by a completely random 300 amino acid sequence where functional constraints have been loosened such that any of the 20 amino acids will suffice at any of the 300 sites. From Eqn. (1) the functional uncertainty of the null state is represented as\n\nH\n\n(\n\nX\n\n\u00f8\n\n(\n\nt\n\ni\n\n))= - \u2211\n\nP\n\n(\n\nX\n\n\u00f8\n\n(\n\nt\n\ni\n\n)) log\n\nP\n\n(\n\nX\n\n\u00f8\n\n(\n\nt\n\ni\n\n))\n\nwhere (*X*~\u00f8~(*t*~i~)) is the conditional variable for all possible equiprobable sequences. Consider the number of all possible sequences is denoted by *W*. Letting the length of each sequence be denoted by *N* and the number of possible options at each site in the sequence be denoted by *m*, *W* = *m*^*N*^. For example, for a protein of length *N* = 257 and assuming that the number of possible options at each site is *m* = 20, *W* = 20^257^. Since, for the null state, we are requiring that there are no constraints and all possible sequences are equally probable, *P*(*X*~\u00f8~(*t*~i~)) = 1\/*W* and\n\nH\n\n(\n\nX\n\n\u00f8\n\n(\n\nt\n\ni\n\n))= - \u2211(1\/\n\nW\n\n) log (1\/\n\nW\n\n) = log\n\nW\n\n.\n\nThe change in functional uncertainty from the null state is, therefore,\n\n\u0394\n\nH\n\n(\n\nX\n\n\u00f8\n\n(\n\nt\n\ni\n\n),\n\nX\n\nf\n\n(\n\nt\n\nj\n\n)) = log (\n\nW\n\n) -\n\nH\n\n(\n\nX\n\nf\n\n(\n\nt\n\ni\n\n)).\n\nPhysical constraints increase order and change the ground state away from the null state, restricting freedom of selection and reducing functional sequencing possibilities, as mentioned earlier. The genetic code, for example, makes the synthesis and use of certain amino acids more probable than others, which could influence the ground state for proteins. However, for proteins, the data indicates that, although amino acids may naturally form a nonrandom sequence when polymerized in a dilute solution of amino acids \\[30\\], actual dipeptide frequencies and single nucleotide frequencies in proteins are closer to random than ordered \\[31\\]. For this reason, the ground state for biosequences can be approximated by the null state. The value for the measured FSC of protein motifs can be calculated by relating the joint (*X*, *F*) pattern to a stochastic ensemble, the null state in the case of biopolymers that includes any random string from the sequence space.\n\n## A. Functional uncertainty as a measure of FSC\n\nThe measure of Functional Sequence Complexity, denoted as \u03b6, is defined as the change in functional uncertainty from the ground state *H*(*X*~g~(*t*~i~)) to the functional state *H*(*X*~f~(*t*~i~)), or\n\n\u03b6 = \u0394\n\nH\n\n(\n\nX\n\ng\n\n(\n\nt\n\ni\n\n),\n\nX\n\nf\n\n(\n\nt\n\nj\n\n)).\n\nThe resulting unit of measure is defined on the joint data and functionality variable, which we call *Fits (*or *F*unctional *bits)*. The unit Fit thus defined is related to the intuitive concept of *functional* information, including genetic instruction and, thus, provides an important distinction between functional information and Shannon information \\[6,32\\].\n\nEqn. (6) describes a measure to calculate the functional information of the whole molecule, that is, with respect to the functionality of the protein considered. The functionality of the protein can be known and is consistent with the whole protein family, given as inputs from the database. However, the functionality of a sub-sequence or particular sites of a molecule can be substantially different \\[12\\]. The functionality of a sub-molecule, though clearly extremely important, has to be identified and discovered. This problem of estimating the functionality as well as where it is expressed at the sub-molecular level is currently an active area of research in our group.\n\nTo avoid the complication of considering functionality at the sub-molecular level, we crudely assume that each site in a molecule, when calculated to have a high measure of FSC, correlates with the functionality of the whole molecule. The measure of FSC of the whole molecule, is then the total sum of the measured FSC for each site in the aligned sequences.\n\nConsider that there are usually only 20 different amino acids possible per site for proteins, Eqn. (6) can be used to calculate a maximum Fit value\/protein amino acid site of 4.32 Fits\/site. We use the formula log (*20*) - *H*(*X*~f~) to calculate the functional information at a site specified by the variable *X*~f~ such that *X*~f~ corresponds to the aligned amino acids of each sequence with the same molecular function *f*. The measured FSC for the whole protein is then calculated as the summation of that for all aligned sites. The number of Fits quantifies the degree of algorithmic challenge, in terms of probability, in achieving needed metabolic function. For example, if we find that the Ribosomal S12 protein family has a Fit value of 379, we can use the equations presented thus far to predict that there are about 10^49^ different 121-residue sequences that could fall into the Ribsomal S12 family of proteins, resulting in an evolutionary search target of approximately 10^-106^ percent of 121-residue sequence space. In general, the higher the Fit value, the more functional information is required to encode the particular function in order to find it in sequence space. A high Fit value for individual sites within a protein indicates sites that require a high degree of functional information. High Fit values may also point to the key structural or binding sites within the overall 3-D structure. Since the functional uncertainty, as defined by Eqn. (1) is proportional to the -log of the probability, we can see that the cost of a linear increase in FSC is an exponential decrease in probability.\n\nFor the current approach, both equi-probability of monomer availability\/reactivity and independence of selection at each site within the strand can be assumed as a starting point, using the null state as our ground state. For the functional state, however, an *a posteriori* probability estimate based on the given aligned sequence ensemble must be made. Although there are a variety of methods to estimate *P*(*X*~f~(*t*)), the method we use here, as an approximation, is as follows. First, a set of aligned sequences with the same presumed function, is produced by methods such as CLUSTAL, downloaded from Pfam. Since real sequence data is used, the effect of the genetic code on amino acid frequency is already incorporated into the outcome. Let the total number of sequences with the specified function in the set be denoted by *M*. The data set can be represented by the N-tuple *X* = (*X*~1~, ... *X*~N~) where *N* denotes the aligned sequence length as mentioned earlier. The total number of occurrences, denoted by *d*, of a specific amino acid \"aa\" in a given site is computed. An estimate for the probability that the given amino acid will occur in that site *X*~*i*~, denoted by *P*(*X*~*i*~ = \"aa\") is then made by dividing the number of occurrences *d* by *M*, or,\n\nP\n\n(\n\nX\n\ni\n\n= \"aa\") =\n\nd\n\n\/\n\nM\n\n.\n\nFor example, if in a set of 2,134 aligned sequences, we observe that proline occurs 351 times at the third site, then *P* (\"proline\") = 351\/2,134. Note that *P* (\"proline\") is a conditional probability for that site variable on condition of the presumed function *f*. This is calculated for each amino acid for all sites. The functional uncertainty of the amino acids in a given site is then computed using Eqn. (1) using the estimated probabilities for each amino acid observed. The Fit value for that site is then obtained by subtracting the functional uncertainty of that site from the null state, in this case using Eqn. (4), log20. The individual Fit values for each site can be tabulated and analyzed. The summed total of the fitness values for each site can be used as an estimate for the overall FSC value for the entire protein and compared with other proteins.\n\n## B. Measuring changes in FSC\n\nIn principle, some proteins may change from a non-functional state to a functional state gradually as their sequences change. Furthermore, iso-enzymes in some cases may catalyze the same reaction, but have different sequences. Also, certain enzymes may demonstrate variations in both their sequence and their function. Finally, a single mutation in a functional sequence can sometimes render the sequence non-functional relative to the original function. An example of this effect has been observed in experiments with the ultrabiothorax (Ubx) protein \\[17,33\\].\n\nFrom Eqn. (6), the FSC of a biosequence can be measured as it changes with time, as shown in Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>. When measuring evolutionary change in terms of FSC, it is necessary to account for a change in function due to insertions, deletions, substitutions and shuffling. Evolutionary change that involves a change from a non-functional state that is not in the null state, or from an existing function *f*~a~ to a modified function *f*~b~ that is either different in terms of efficiency or function, is given by\n\n\u03b6\n\nE\n\n= \u0394\n\nH\n\n(\n\nX\n\nfa\n\n(\n\nt\n\ni\n\n),\n\nX\n\nfb\n\n(\n\nt\n\nj\n\n)).\n\nThe sequences (corresponding to *X*~fa~ with initial function *f*~*a*~) have two components to it, relative to that of *X*~fb~ (with resulting mutated function *f*~*b*~). The *static component* is that portion of the subsequence that must remain within the permitted sequence variation of the original biosequence with function *f*~a~ while, at the same time, enabling the new function *f*~b~. The *mutating component* is the portion of *X*~fa~ that must change to achieve either the new function *f*~b~, where the new function is to be understood as either a new level of efficiency for the existing function, or a novel function different from *f*~a~. This is a convenient simplification, assuming that the two components are separate according to the aligned sites. Currently we are also studying scenarios when the two components may be mixed, possibly at different times. The mutating component can be assumed to be in the null state relative to the resulting sequences of *X*~fb~. Since the mutating component is the only part that must change, we can ignore the static component *provided we include the probability of it remaining static* during the mutational changes of the mutating component.\n\nIn practice, the sequence space for possible novel functional states may not be known. However, by considering particular proteins, estimated mutation rates, population size, and time, an estimated value for the probability can be chosen and substituted into the relevant components of Eqn. (9) to limit search areas around known biosequences that are observed, such as protein structural domains, to see what other possible states within that range might have some selective advantage. In this way, possible evolutionary paths for the formation of certain protein families might be reconstructed. For example, using this method, it might be possible to predict, say, future viral strains within certain limits.\n\nIntuitively, the greater the reduction in FSC a mutation produces, the more likely the mutation is deleterious to the given function. This can be evaluated using known mutations introduced individually into a set of aligned, wild-type sequences to measure the change in FSC. The results could then be ranked. Operating under the hypothesis that mutations producing the greatest decrease in FSC are most likely to be deleterious, experimental investigations into certain genes with certain mutations could be prioritized according to how negatively they affect FSC.\n\n# Results and Discussion\n\nFor the 35 protein families analyzed, a measure of FSC in Fits for each site was computed from their aligned sequence data on PFAM. The results for the families, as well as an array of randomly (uniformly) generated sequences and an ordered 50-mer polyadenosine sequence are shown in Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>. They reveal significant aspects of FSC described below.\n\nFSC of Selected Proteins\n\n|  | **length (aa)** | **Number of Sequences** | **Null State (Bits)** | **FSC (Fits)** | **FSC Density Fits\/aa** |\n|----|----|----|----|----|----|\n| Ankyrin | 33 | 1,171 | 143 | 46 | 1.4 |\n| HTH 8 | 41 | 1,610 | 177 | 76 | 1.9 |\n| HTH 7 | 45 | 503 | 194 | 83 | 1.8 |\n| HTH 5 | 47 | 1,317 | 203 | 80 | 1.7 |\n| HTH 11 | 53 | 663 | 229 | 80 | 1.5 |\n| HTH 3 | 55 | 3,319 | 238 | 80 | 1.5 |\n| Insulin | 65 | 419 | 281 | 156 | 2.4 |\n| Ubiquitin | 65 | 2,442 | 281 | 174 | 2.7 |\n| Kringle domain | 75 | 601 | 324 | 173 | 2.3 |\n| Phage Integr N-dom | 80 | 785 | 346 | 123 | 1.5 |\n| VPR | 82 | 2,372 | 359 | 308 | 3.7 |\n| RVP | 95 | 51 | 411 | 172 | 1.8 |\n| Acyl-Coa dh N-dom | 103 | 1,684 | 445 | 174 | 1.7 |\n| MMR HSR1 | 119 | 792 | 514 | 179 | 1.5 |\n| Ribosomal S12 | 121 | 603 | 523 | 359 | 3.0 |\n| FtsH | 133 | 456 | 575 | 216 | 1.6 |\n| Ribosomal S7 | 149 | 535 | 644 | 359 | 2.4 |\n| P53 DNA domain | 157 | 156 | 679 | 525 | 3.3 |\n| Vif | 190 | 1,982 | 821 | 675 | 3.6 |\n| SRP54 | 196 | 835 | 847 | 445 | 2.3 |\n| Ribosomal S2 | 197 | 605 | 851 | 462 | 2.4 |\n| Viral helicase1 | 229 | 904 | 990 | 335 | 1.5 |\n| Beta-lactamase | 239 | 1,785 | 1,033 | 336 | 1.4 |\n| RecA | 240 | 1,553 | 1,037 | 832 | 3.5 |\n| Bac luciferase | 272 | 1,900 | 1,176 | 357 | 1.3 |\n| tRNA-synt 1b | 280 | 865 | 1,210 | 438 | 1.6 |\n| SecY | 342 | 469 | 1,478 | 688 | 2.0 |\n| EPSP Synthase | 372 | 1,001 | 1,608 | 688 | 1.9 |\n| FTHFS | 390 | 658 | 1,686 | 1,144 | 2.9 |\n| DctM | 407 | 682 | 1,759 | 724 | 1.8 |\n| Corona S2 | 445 | 836 | 1,923 | 1,285 | 2.9 |\n| Flu PB2 | 608 | 1,692 | 2,628 | 2,416 | 4.0 |\n| Usher | 724 | 316 | 3,129 | 1,296 | 1.8 |\n| Paramyx RNA Pol | 887 | 389 | 3,834 | 1,886 | 2.1 |\n| ACR Tran | 949 | 1,141 | 4,102 | 1,650 | 1.7 |\n| Random sequences | 1000 | 500 | 4,321 | 0 | 0 |\n| 50-mer polyadenosine | 50 | 1 | 0 | 0 | 0 |\n\n**Results for 35 protein families** Shown above are the 35 protein families analyzed, their sequence length (column 1), the number of sequences analyzed for each family (column 2), the Shannon uncertainty of the Null State *H*~\u00f8~ (Eqn. 4) for each protein (column 3), the FSC value \u03b6 in Fits for each protein (column 4), and the average Fit value\/site (FSC\/length, column 5). For comparison, the results for a set of uniformly random amino acid sequences (RSC) are shown in the second from last row, and a highly ordered, 50-mer polyadenosine sequence (OSC) in the last row. All values, except for the OSC example, which was calculated from the constrained ground state required to produce OSC, were computed from the null state. The Fit values obtained can be discussed as the measure of the change in functional uncertainty required to specify any functional sequence that falls into the given family being analyzed.\n\nFirst, as observed in Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>, although we might expect larger proteins to have a higher FSC, that is not always the case. For example, 342-residue SecY has a FSC of 688 Fits, but the smaller 240-residue RecA actually has a larger FSC of 832 Fits. The Fit density (Fits\/amino acid) is, therefore, lower in SecY than in RecA. This indicates that RecA is likely more functionally complex than SecY. The results for the array of random sequences and for a 50-mer polyadenosine sequence formed on Montmorillonite show that \u0394*H*~f~ distinguishes FSC from RSC and OSC. The results for the array of random sequences are shown in the second from the last row of Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>, and indicate that random sequences, which are an example of RSC, tend to have an FSC of approximately 0 Fits. The results of the highly ordered 50-mer polyadenosine, which is an example of OSC, are shown in the last row of Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>, and indicate an FSC of approximately 0 Fits. This is consistent with Abel and Trevors' prediction that neither OSC nor RSC can contain the functional sequence complexity observed in biosequences.\n\nA plot of FSC vs. protein length for the 35 selected protein families is shown in Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>. The x-intercept was found to be approximately 23 amino acids. For the 35 protein families analyzed, there were no points corresponding to any protein families in the lower right area of the plot. The right hand plot in Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a> shows the average number of Fits\/site for the 35 protein families analyzed. For our small sample of 35 protein families, we found no points between 0 and 1.3 Fits\/site.\n\nTo demonstrate some ways in which our approach can be applied to proteins, ubiquitin was chosen. A sample plot of FITs\/site and amino acid conservation for each site, is shown in Figure <a href=\"#F3\" data-ref-type=\"fig\">3<\/a> for Ubiquitin. Data for the first 5 sites in the aligned set was not available from PFAM. The conservation value was obtained by subtracting the total number of different options observed at a given site, from the total possible options. In this case, 20 different amino acids. If all 20 amino acids were permitted at a site, then the conservation value was 0. A maximum value of 19 would obtain if only 1 amino acid was observed at the site. From Figure <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>, it can be observed that a high conservation value usually corresponded to a high measured FSC value. The measurement is also affected by the number of amino acids observed which could be different for different sites. For example, site 37 shows 20 observed amino acids, but still a relatively high value of 2.9 Fits. Conservation of a site reflects the degree of variations which is affected by both the number of observed amino acids and their frequency of observation in the alignment \\[12\\]. For example, if a site is observed to be dominated by only a few amino acids even though all the amino acid types are observed, its measured FSC could still be high.\n\nFrom the plot in Figure <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>, one can observe which sites have higher measured FSC value. An arbitrary lower cutoff limit of 3.32 Fits\/site was chosen to indicate high measured FSC sites, presumed to be significantly associated with the functionality of the whole molecule. A more rigorous statistical analysis can also be used as well as other types of measures based on dependency \\[12\\]. All sites exceeding that lower cutoff value were examined. A total of seven sites were found that had a high value that was between 3.32 Fits\/site and the maximum of 4.32 Fits\/site. These sites were located on a 3-D model of ubiquitin (1AAR.pdb) as shown in Figure <a href=\"#F4\" data-ref-type=\"fig\">4<\/a>. Out of the 7 sites, 6 where located on the binding domain containing the binding site Lys-48 \\[34\\]. The site with the maximum FIT value was site 47, immediately next to the binding site. Surprisingly, the binding site itself was poorly conserved, with an amino acid conservation value of only 1, albeit a relatively high value of 2.91 Fits. Five of the remaining 6 sites were found to be clustered in the area of the binding site, with bonds between Leu-50 and Leu-43, as well as between Gly-41, Lys-27, and Asp-52, as shown in Figure <a href=\"#F5\" data-ref-type=\"fig\">5<\/a>. Since these sites had the highest FSC values in ubiquitin, we infer that they play a critical role in either binding, or in the structure of the binding site domain for that protein. The fact that of the top seven sites, one was immediately adjacent to the binding site, and five others were located on the structure supporting the binding site lends support for our hypothesis that high FIT values can be used to locate key functional components of a protein family.\n\nIn this paper, we have presented an important advance in the measurement of the FSC of biopolymers. It was assumed that aligned sequences from the same Pfam family, could be assigned the same functionality label. Even though the same functionality may not be applicable to individual sites, site independence and significance was assumed and the measured FSC of each site was summed. However, further extension of the method should be considered \\[12,35\\]. For example, if dependency of joint occurrences is detected between the outcomes of two variables *X*~3~ and *X*~4~ in the aligned sequences, then the N-tuple representation of the sequences could be transformed into a new R-tuple *Y*~R~ where these outcomes of *X*~3~ and *X*~4~ are represented as outcome by a single variable *Y*~3~ as shown in Figure <a href=\"#F6\" data-ref-type=\"fig\">6<\/a>. An outcome of the two variables in *X*~3~ and *X*~4~ correspond to a hypercell in Y~R~. A more accurate estimate of FSC could then be calculated. We are currently considering this more general scenario.\n\nThe measurement in Fits of the FSC provides significant information about how specific each monomer in the sequence must be to provide the needed\/normal biofunction. The functional information measures the degree of challenge involved in searching the sequence space for a sequence capable of performing the function. In addition, Fits can be summed for every sequence required to achieve a complete functional biochemical pathway and integrated cellular metabolism, including regulatory proteins. In principle, it may be possible to estimate a FSC value for an entire prokaryotic cell where the genome has been sequenced and all translated proteins are known. Simpler genomes in viruses may be an excellent example for this kind of analysis. That is, further analysis of the FSC values will provide a starting point to reveal important information about the processes for an entire organism such as the virus.\n\n# Conclusion\n\nA mathematical measure of functional information, in units of Fits, of the functional sequence complexity observed in protein family biosequences has been designed and evaluated. This measure has been applied to diverse protein families to obtain estimates of their FSC. The Fit values we calculated ranged from 0, which describes no functional sequence complexity, to as high as 2,400 that described the transition to functional complexity. This method successfully distinguishes between FSC and OSC, RSC, thus, distinguishing between order, randomness, and biological function.\n\n# Methods\n\nThe following is a brief summary of the methods used (additional file <a href=\"#S1\" data-ref-type=\"supplementary-material\">1<\/a>). A more detailed description is available as an online supplement. Eqn. (6) was applied to 35 protein families or protein domain families, to estimate the value of the FSC for any protein included within that family. A program was written, using Python, to analyze the two-dimensional array of aligned sequences for a protein family and is available online (additional files <a href=\"#S2\" data-ref-type=\"supplementary-material\">2<\/a>, <a href=\"#S3\" data-ref-type=\"supplementary-material\">3<\/a>, <a href=\"#S4\" data-ref-type=\"supplementary-material\">4<\/a>, <a href=\"#S5\" data-ref-type=\"supplementary-material\">5<\/a>, <a href=\"#S6\" data-ref-type=\"supplementary-material\">6<\/a>, <a href=\"#S7\" data-ref-type=\"supplementary-material\">7<\/a>, <a href=\"#S8\" data-ref-type=\"supplementary-material\">8<\/a>, and <a href=\"#S9\" data-ref-type=\"supplementary-material\">9<\/a>). The data for the arrays was obtained from the Pfam database \\[36\\]. Eqn. (7) was used to estimate the probability for each amino acid at each site.\n\n# Competing interests\n\nThe author(s) declare that they have no competing interests.\n\n# Authors' contributions\n\nKD helped develop the formulation and measure of FSC, wrote the software, carried out the analysis and drafted the manuscript. DC developed, together with KD, the basic formula of functional entropy and evaluated the different mathematical forms. DC provided suggestions to the experimental design and its interpretations. DLA's contributions included defining\/delineating the three subsets of sequence complexity and their relevance to biopolymeric information, contributing to the first draft of the paper, critiquing KD's quantification methodology, contributing references, and coining the term \"fits\" for \"functional bits\" as the unit of measure of Functional Sequence Complexity (FSC). JT participated in the design and coordination of the study. All authors read and approved the final manuscript.\n\n# Supplementary Material\n\n###### Additional File 1\n\nMethods. Additional details of the methods used in this project\n\nClick here for file\n\n###### Additional File 2\n\nMain Program. A copy of the coding for the main program used in this paper\n\nClick here for file\n\n###### Additional File 3\n\nAminoFreq. A required module for the main program\n\nClick here for file\n\n###### Additional File 4\n\nColTot. A required module for the main program\n\nClick here for file\n\n###### Additional File 5\n\nConvert. A required module for the main program\n\nClick here for file\n\n###### Additional File 6\n\nDistEnt. A required module for the main program\n\nClick here for file\n\n###### Additional File 7\n\nFormArray. A required module for the main program\n\nClick here for file\n\n###### Additional File 8\n\nStripName. A required module for the main program\n\nClick here for file\n\n###### Additional File 9\n\nP53DNADom. A sample data set for the reader\n\nClick here for file\n\n### Acknowledgements\n\nThe research is supported by the National Sciences and Engineering research Council (NSERC) of Canada, Discovery Grant, the Korean Research Foundation Grant (KRF-2004-042-C00020) of one of the authors.","meta":{"dup_signals":{"dup_doc_count":136,"dup_dump_count":70,"dup_details":{"curated_sources":2,"2023-14":1,"2022-40":1,"2022-33":1,"2022-21":1,"2021-49":1,"2021-39":2,"2021-17":1,"2021-04":1,"2020-40":2,"2020-29":1,"2020-16":1,"2020-10":1,"2020-05":1,"2019-47":1,"2019-39":1,"2019-35":1,"2019-26":1,"2019-18":1,"2019-09":1,"2019-04":2,"2018-47":1,"2018-43":1,"2018-34":2,"2018-26":2,"2018-22":1,"2018-17":1,"2018-13":1,"2018-09":1,"2018-05":3,"2017-51":3,"2017-47":1,"2017-43":2,"2017-39":2,"2017-34":2,"2017-30":2,"2017-26":2,"2017-22":2,"2017-17":2,"2017-09":3,"2017-04":2,"2016-50":2,"2016-44":4,"2016-40":5,"2016-36":4,"2016-30":4,"2016-26":1,"2016-22":1,"2016-18":1,"2015-48":2,"2015-40":2,"2015-35":2,"2015-32":2,"2015-27":2,"2015-22":2,"2015-14":2,"2014-52":2,"2014-49":3,"2014-42":6,"2014-41":4,"2014-35":3,"2014-23":4,"2014-15":4,"2023-40":1,"2015-18":2,"2015-11":1,"2015-06":2,"2014-10":2,"2013-48":2,"2013-20":2,"2024-18":1}},"file":"PMC2217542"},"subset":"pubmed_central"}
{"text":"author: Neil Hall\ndate: 2012\ninstitute: 1Centre for Genomic Research, University of Liverpool, Crown St, Liverpool L69 7ZB, UK\ntitle: Why science and synchronized swimming should not be Olympic sports\n\nThe brief, for my intermittent comment column for *Genome Biology*, was to \"give a UK perspective\" while \"keeping it interesting for an international audience\". That's a tough brief. There is little reason for people to take an interest in events taking place in a rainy windswept island, famous mostly for bad food, greedy bankers and reality shows. Recently, though, because of the London 2012 Olympics, the focus of the world's attention has briefly flitted in our direction. So, while basking in our reflected Olympic glory, I will start with sport.\n\nIn the UK we can be rightly proud of the fact that (as the head of the International Olympic Committee pointed out) we as a nation have done much to codify many Olympic sports. But this probably points to a national failing: we love rules, we love measuring performance and hence we love inventing sports. That's why we invented cricket; it has loads of obscure rules and loads of complex performance statistics. A game that can take 5 days and has to stop for rain or bad light (in England!) was not invented for the drama or spectacle. Our obsession with performance metrics is not just limited to sport; the government has taken to measuring and publishing the performance of everything from schools and hospitals to police forces and train operators. They have even tried to quantify our national happiness (<http:\/\/www.ons.gov.uk\/ons\/rel\/wellbeing\/measuring-national-well-being\/summary-of-proposed-domains-and-measures\/summary-of-proposed-domains-and-measures-of-national-well-being.html>).\n\nSportsmen and -women can obsess about measuring their performance and gauging it against their past performance and the performance of others, but researchers are quite different. In academia we don't like other people judging what we do (or even defining what we do) and we tend not to like metrics designed to measure our performance. But many years ago, UK government decided to ignore these protestations from our ivory towers and created a mechanism for measuring research quality called the Research Excellence Framework (or REF for short). REF is the reason that some readers may have noticed UK-based collaborators acting increasingly strangely, maybe looking stressed and distant, obsessing about impact factors and questioning the value of everything they do against impenetrable metrics.\n\n# REF 101\n\nFor the benefit of non-UK-based readers I will need to give some background. In the UK we have only recently taken to crucifying our youth with a lifetime of debt to fund their education. Hence, there are still large sums of money coming from central government to universities. Each year, \u00a31billion will be paid directly to institutions based on research quality. The sum of money each university receives is decided on by the REF, and here, in brief, are the rules.\n\n## 1. Publish rarely but well\n\nThe REF occurs every 7 years, although it changes its name more than The Artist Formerly Known As Prince. It used to be called the Research Assessment Exercise and before that the Research Selectivity Exercise. The nuances of the process have evolved, but a key metric in each assessment is the quality of the research outputs (papers). Each academic can submit just four papers published in the last 7 years to be judged for their 'excellence in originality, significance and rigor', against these murky definitions:\n\n\u2022 4 star - Quality that is world leading\n\n\u2022 3 star - Quality that is internationally excellent\n\n\u2022 2 star - Quality that is recognized internationally\n\n\u2022 1 star - Quality that is recognized nationally\n\nTo me, these read like color descriptions on paint tins (linen white, antique white, foam white, cloud white, and so on) and if you are trying to work out what the difference is between 'world leading' and 'internationally excellent', you are not alone. Most UK academics are currently kept awake with such thoughts. This is probably a futile exercise as the star ratings are assigned by REF panels, each with about 20 members, and each member will have to read hundreds of papers, many of which are outside their area of expertise. It is hard to imagine that this review process is not guided by journal impact factors or the esteem with which the submitting academic is held in by the panel. However, these ratings are important as the stars for all the papers submitted by each department will be added up to evaluate their research quality.\n\nMost of you will also be thinking that four papers in 7 years is not a lot... and it isn't. So, the reason your UK collaborators are not publishing the stuff they presented at conferences last year is because they are hoping to roll it in with more data into a more prestigious journal later.\n\n## 2. Hire for the short term\n\nInexplicably, although the process is designed to measure the performance of universities, the star ratings for the papers go to the investigator, regardless of where they were working at the time of publication. This means that you can buy in researchers with a good set of publications simply to use them in your REF audit. This has created what amounts to a 'transfer deadline' and a highly skewed academic job market in the UK in which universities hire on a 7-yearly cycle, and increases in professorial salaries have outstripped those for junior faculty. This is reminiscent of sports stars; a few individuals will be paid extremely well but very little cash goes to the grass roots.\n\nI should acknowledge that I have been a beneficiary of this system. When I realized that if I lived in the USA any longer my son would soon complete his metamorphosis into Bart Simpson, I started to look for work in the UK. It turned out that this was just before the cyclical REF (then called the RAE). And, as I had been working as a project leader at genome centers during a period when *Nature* would essentially publish raw electropherograms (or as I call it, 'The good old days'), I was nailed-on 'Four Star'. Therefore, my prospective employer could hire me on a healthy professorial salary and provide me with a good start up in the knowledge that the government would refund the cost in return for my papers.\n\nAs a scientist, I was delighted; as a taxpayer, I should have been outraged.\n\n## 3. Spend, spend, spend\n\nAt this point I know some of you think I am making this up, but the other metric that decides how much money you receive is how much money you have spent. This does sound like a futile positive feedback loop that could only have been invented by a Lehman Brothers investment banker, but it genuinely is a measure used to score the 'Research Environment', which is a key metric in the REF. According to how this metric is measured, a department that has a large research spend is considered to be a better environment than those with a lower spend. As I run a genomics lab, I am obviously thrilled with this metric because I can certainly generate a good research environment as judged by the REF, but some people may argue that a great research environment is instead one that produces good papers without spending huge sums of money.\n\n# Measuring the unmeasurable\n\nI would like to think that anyone reading this would conclude that the REF is a shockingly bad way to judge research performance. And it's fair to say I have paraphrased the rules (there is a 106 page document on just the panel working methods for those wanting more detail: <http:\/\/www.ref.ac.uk\/pubs\/2012-01\/>). I have also highlighted the easy targets for ridicule. I can totally understand that governments who fund research would like to measure performance, but I question their ability to do so and I would challenge anyone to come up with a better system.\n\nAthletics is sport in its purest form and has basic measurements (height, length and speed) that can be used to define who is the best. Usain Bolt has a single, very simple KPI (that's a Key Performance Indicator for those not on university management committees). But there are other 'sports' such as gymnastics, diving, dressage and synchronized swimming that are much less clear cut. I personally think that anything that uses a judging panel to score artistic interpretation should not be a sport. Now imagine what would happen if, instead of judging panels, the synchronized swimmers scored each other anonymously. I expect that things could get quite divisive.\n\nLike many others, I really enjoyed my 4-yearly dose of synchronized swimming during the Olympics. It was dramatic, entertaining and thrilling, but making it a sport is missing the point. You may as well make ballet and stand-up comedy Olympic sports as well. Unlike a 100 m sprint, the measurement of performance is far too subjective. At some point one has to accept that some things can have intrinsic value but they can't be objectively measured.\n\nScience quality is difficult to measure, yet we all know when we see something amazing or dramatic, whether it is a Higgs boson or a Neanderthal genome, and choosing which of these is best is pointless. In science, as with ballet, comedy or synchronized swimming, there should be no winners; because you can't measure who won.","meta":{"dup_signals":{"dup_doc_count":123,"dup_dump_count":72,"dup_details":{"curated_sources":2,"2022-27":1,"2021-49":1,"2020-45":1,"2020-34":1,"2020-24":1,"2020-16":1,"2020-10":1,"2020-05":1,"2019-51":1,"2019-47":1,"2019-43":1,"2019-39":1,"2019-35":1,"2019-30":1,"2019-26":1,"2019-22":1,"2019-18":1,"2019-13":1,"2019-09":1,"2019-04":1,"2018-51":1,"2018-47":1,"2018-43":1,"2018-39":1,"2018-34":1,"2018-30":1,"2018-26":1,"2018-22":1,"2018-17":1,"2018-13":1,"2018-09":1,"2018-05":2,"2017-51":2,"2017-47":2,"2017-43":1,"2017-39":2,"2017-34":2,"2017-30":1,"2017-26":1,"2017-22":1,"2017-17":2,"2017-09":7,"2017-04":1,"2016-50":1,"2016-44":1,"2016-40":1,"2016-36":8,"2016-30":7,"2016-22":1,"2016-18":1,"2016-07":3,"2015-48":2,"2015-40":2,"2015-35":2,"2015-32":2,"2015-27":2,"2015-22":2,"2014-52":2,"2014-49":4,"2014-42":4,"2014-41":3,"2014-35":2,"2014-23":3,"2014-15":2,"2023-23":1,"2017-13":1,"2015-18":2,"2015-11":2,"2015-06":2,"2014-10":1,"2013-48":1,"2024-10":1}},"file":"PMC3491389"},"subset":"pubmed_central"}
{"text":"abstract: A report from the Keystone Symposium on Molecular and Cellular Biology, 'Deregulation of transcription in cancer: controlling cell fate decisions', Killarney, Ireland, 21-26 July 2009.\nauthor: Aisling M Redmond; Jason S Carroll\ndate: 2009\ninstitute: 1Endocrine Oncology Research Group, Department of Surgery, Royal College of Surgeons in Ireland, St Stephens Green, Dublin 2, Ireland; 2Cancer Research UK, Cambridge Research Institute, Robinson Way, Cambridge CB2 0RE, UK\ntitle: Defining and targeting transcription factors in cancer\n\nThis Keystone meeting focused on mechanisms of transcriptional regulation, the effects of transcriptional deregulation and the consequences on cancer, and the possibilities for inhibiting transcription factors as potential therapeutic targets. Greg Verdine (Harvard University, Cambridge, USA) suggested that, until now, only 20% of the genome was targetable with drugs (druggable). Transcription factors have traditionally been considered too difficult to target, but with increased understanding of transcription factor biology and technological advances, targeting them is becoming a realistic option.\n\nThe role of polycomb complexes in regulating transcriptional activity was an important theme to the meeting. Kristian Helin (University of Copenhagen, Denmark) summarized the role of polycomb repressor complexes (involving EZH2) in controlling senescence, in particular their role in the regulation of the p16^INK4A^ and p14^ARF^ tumor suppressor proteins. Competitive binding between polycomb repressor complexes and the histone demethylase JMJD3 determines transcriptional activity, suggesting that JMJD3 can act as a tumor suppressor. Maarten Van Lohuizen (Netherlands Cancer Institute, Amsterdam, The Netherlands) presented work on BMI1, a member of the polycomb repressor complex PRC1, which is crucial in the maintenance of adult stem cells. Loss of BMI1 in mouse models causes a dramatic reduction in proliferation in the mammary gland. Levels of BMI1 and EZH2 and interactions between them influence tumor progression. Qiang Yu (Genome Institute of Singapore, Singapore) focused on the possibility of targeting EZH2 in cancer. Their compound, 3-deazaneplanocin A (DZNep), an *S*-adenosylhomocysteine hydrolase inhibitor, depletes EZH2 in breast cancer cells. In colorectal cancer, DZNep reactivates the tumor suppressor microRNA *miR449*, leading to cell cycle arrest. He suggested that DZNep, in combination with 5-azacytidine, may be an effective cancer treatment.\n\nSeveral talks used contemporary genomics technologies to define transcription factor binding sites, regulatory regions and changes in chromatin structure, all of which can contribute to altered cellular growth. Manel Esteller (Catalan Institute of Oncology, Barcelona, Spain) has assessed global DNA methylation in normal and cancer cells and found significant changes in DNA methylation at promoters. He discussed the DNA epigenome project, an ambitious project that will study the methylation state of 10,000 promoters in tumors from more than 1,000 patients. Bing Ren (University of California, San Diego, USA) discussed global DNA methylation and histone modification data generated by chromatin immunoprecipitation microarrays (ChIP-chip) or ChIP-sequencing (ChIP-seq). In human embryonic stem cells that were induced to differentiate, he showed that 30,000 putative enhancers exist in pre- and post-differentiation states, but only 8,000 of these were common between the two states. Within the regions shown to have altered chromatin structure following differentiation, he could show enrichments of motifs for various transcription factors. Both positive and inverse correlations were found when the histone maps were combined with DNA methylation data. Importantly, he could show that in specific cell types, non-CG methylation could occur and this was usually depleted from promoters of actively transcribed genes.\n\nTwo talks from members of the Genome Institute of Singapore highlighted new data on estrogen receptor (ER) transcription and chromatin dynamics. Yijun Ruan presented data on a novel technique called 'whole genome chromatin interaction analysis using paired-end ditagging' (ChIA-PET), which is a global method for identifying chromatin loops that form during transcription. By applying this to estrogen-induced gene transcription in breast cancer cells, his group found many hundreds of estrogen-induced intrachromosomal chromatin loops that form over distances as great at 1 Mb, representing *cis*-regulatory components that physically interact. As a follow-up to this presentation, Ed Liu presented data from recent genome-wide mapping of ER binding sites. He showed that only small subsets of predicted motifs are actual binding sites *in vivo* and that the ER binding sites occur in gene-rich areas. Within the list of ER binding sites, the strongest (that is, those most enriched by ChIP-seq) were more likely to contain responsive motifs and to be adjacent to genes that are differentially regulated by estrogen. This suggests that there is a hierarchy of ER binding sites, in which the strongest sites are more likely to be functional, possibly as a result of superior ER-DNA interactions.\n\nMaintaining the theme of nuclear receptor transcriptional activity, Ralf Kittler (University of Chicago, USA) has engineered bacterial artificial chromosomes of more than 20 different nuclear receptor (NR) genes to include an enhanced green fluorescent protein tag, and these have subsequently been used for genome-wide mapping using ChIP-chip. By correlating the binding profiles of all NRs, they found profile similarities and common binding sites between ER and retinoic acid receptor \u03b1; they hypothesized that this was an antagonistic interaction.\n\nArul Chinnaiyan (University of Michigan, Ann Arbor, USA) presented data characterizing gene fusions in prostate cancer, including his group's original discovery of fusions between the *TMPRSS2* gene (which encodes a transmembrane serine protease) and the *ERG* ETS-family oncogene in prostate cancers. He showed the results from large-scale genomic screens, which have resulted in more than 100 validated gene fusions. Interestingly, all of the fusions contain an ETS factor as the transcriptionally active partner and the other half of the fusion is usually an androgen-regulated gene target. He showed that fusions of various kinds are found in more than half of all prostate cancers and that their specificity to cancer and not normal tissue has led to a urine-based test for detecting fusion genes that may function as a prognostic indicator of prostate cancer. One of the major issues that researchers in the field face is defining which fusions are driving factors and which ones are passenger events.\n\nOne of the key themes of the meeting was the ability to target transcription factors using drugs. Traditionally, transcription factors were generally considered too difficult to target, and kinase pathways or cell surface proteins have instead been popular therapeutic targets. However, transcription factors are the downstream effectors of many pathways and this, coupled with technological advances, has made them attractive and realistic drug targets. Greg Verdine presented data on 'stapled peptides'. Normally, peptides used to block or inhibit transcription factors are easily degraded or have poor solubility. Verdine's approach provides a stabilizing backbone (or 'staple') to the short peptides or proteins, thereby generating proteins that are stable and maintain correct conformation. His group has successfully developed stapled peptides that target Bcl-2, Bax and BID (proapoptotic proteins containing only the BH-3 motif). One of their inhibitors is about to enter clinical trials, and the approach provides a realistic option for targeting a specific transcription factor in cancer.\n\nJohn Rossi (Beckman Research Institute of the City of Hope, Duarte, USA) discussed his group's RNA interference method as a potential therapeutic approach. He suggested that the delivery of small interfering RNAs (siRNAs) to target cells has improved considerably, but one of the major problems is producing sufficient quantities of any specific siRNA. He also showed that they can generate siRNAs that target two different transcripts simultaneously, allowing increased effectiveness from a single siRNA. Lyubomir Vassilev (Roche Pharmaceuticals, Nutley, USA) showed data on Nutlin, an inhibitor of the interaction between the p53 tumor suppressor and its regulator Mdm2. This inhibitor binds selectively to the pocket of Mdm2, resulting in increased p53 levels. Nutlin was an effective inhibitor in cell lines and in mice with various types of cancer and was bioavailable when administered orally. These data confirm that protein-protein interactions between transcription factors and regulatory proteins can be blocked successfully with chemical inhibitors. Sandra Dunn (University of British Columbia, Vancouver, Canada) provided compelling evidence for an important role for the transcription factor YB-1, which is expressed in 40% of breast cancers but not normal breast tissue. YB-1 was shown to correlate with poor prognosis in patients and in mouse models, and YB-1 transgenic mice readily generated tumors. YB-1 activity is dependent on phosphorylation, and Dunn's group has shown that the ribosomal S6 kinase (Rsk) complex is involved. They are currently testing the effectiveness of peptides that target YB-1.\n\nRen\u00e9 Bernards (Netherlands Cancer Institute) stressed the need to be able to stratify breast cancer patients, firstly in order to restrict chemotherapeutic drug administration only to those who will benefit from it, and secondly to allow informed decisions regarding the choice of drug for each patient. As stated by Joe Nevins (Duke University Medical Center, Durham, USA), by finding and focusing on the specific subset of patients that will probably benefit from an individual therapy, a potential failed drug can become a blockbuster drug. Nevins presented gene expression profiles resulting from exposure to the major cancer drugs; from these, his group could generate signatures that represent a likely response or lack of response to individual therapies. They could use this approach to predict patients who would benefit from particular drug regimes. They are currently using these genomic screening tools in trials. Similarly, Bernards discussed gene expression signatures that predict outcome in women with breast cancer. He also showed data from a 159 gene signature of activated phosphatidylinositol 3-kinase, which was used to predict outcome in colon cancers. Bernards also showed data from a short hairpin RNA (shRNA) library screen to find genes involved in resistance to trastuzumab (known as Herceptin) in a BT474 breast cancer cell line model. By simultaneously screening 24,000 human shRNAs against 8,000 genes, his group could identify genes required for trastuzumab effectiveness. They identified and validated the phosphatase pTEN as an essential component in the trastuzumab response.\n\nClearly, the single gene or single protein approach is rapidly becoming redundant. The use of screens allows researchers to simultaneously assess all genes, identify thousands of regulatory sites, test a multitude of compounds and combine these different screens in multifactorial ways. By distilling this information we can progress more rapidly towards personalized treatments.","meta":{"dup_signals":{"dup_doc_count":122,"dup_dump_count":52,"dup_details":{"curated_sources":2,"2023-23":1,"2022-33":1,"2022-05":1,"2021-39":1,"2021-10":1,"2021-04":1,"2020-40":1,"2020-24":1,"2020-05":1,"2019-51":1,"2019-47":2,"2019-22":1,"2019-09":1,"2019-04":1,"2018-51":1,"2018-47":2,"2018-39":1,"2018-09":1,"2017-47":1,"2017-34":1,"2017-22":1,"2017-17":2,"2017-09":7,"2016-44":1,"2016-40":1,"2016-36":9,"2016-30":5,"2016-22":1,"2016-18":1,"2016-07":4,"2015-48":3,"2015-40":1,"2015-35":3,"2015-32":2,"2015-27":3,"2015-22":3,"2015-14":2,"2014-52":2,"2014-49":3,"2014-42":7,"2014-41":5,"2014-35":5,"2014-23":4,"2014-15":3,"2023-50":1,"2015-18":3,"2015-11":3,"2015-06":3,"2014-10":3,"2013-48":3,"2013-20":3,"2024-30":1}},"file":"PMC2728525"},"subset":"pubmed_central"}
{"text":"date: 2010-04-16\ntitle: Findings of research misconduct.\n\n# Findings of Research Misconduct\n\nNotice Number: NOT-OD-10-085\n\nKey Dates**  \n** Release Date: April 16, 2010\n\nIssued by**  \n** Department of Health and Human Services\n\nNotice is hereby given that the Office of Research Integrity (ORI) and the Assistant Secretary for Health have taken final action in the following case:\n\nEmily M. Horvath, Indiana University: Based on the Respondent's own admissions in sworn testimony and as set forth below, Indiana University (IU) and the U.S. Public Health Service (PHS) found that Ms. Emily M. Horvath, former graduate student, IU, engaged in research misconduct in research supported by National Center for Complementary and Alternative Medicine (NCCAM), National Institutes of Health (NIH), grant R01 AT001846 and Predoctoral Fellowship Award F31 AT003977-01, and National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), NIH, grant R01 DK082773-01.\n\nSpecifically, the Respondent admitted to falsifying the original research data when entering values into computer programs for statistical analysis with the goal of reducing the magnitude of errors within groups, thereby gaining greater statistical power. The Respondent, IU, and ORI agree that the figures identified below in specific grant applications and published papers are false and that these falsifications rise to the level of research misconduct:\n\nRespondent admitted to falsifying Figures 6B, 18, 22, 23B, and 24 in NCCAM, NIH, grant application R01 AT001846-06, \\`\\`Chromium Enhanced Insulin & GLUT4 Action via Lipid Rafts,' Jeffery S. Elmendorf, P.I. (07\/01\/04-05\/31\/20) (application was withdrawn in May 2009).\n\nRespondent admitted to falsifying Figures 6B, 8, 9D, 16D, and 21 in NIDDK, NIH, grant application R01 DK082773-01, \\`\\`Mechanisms of Membrane-Based Insulin Resistance & Therapeutic Reversal Strategies,' Jeffrey S. Elmendork, P.I. (3\/15\/09-01\/31\/13).\n\nRespondent admitted to falsifying Figures 2C, 5, 6D, and 11 in the publication: Horvath, E.M., Tacket, L., McCarthy, A.M., Raman, P., Brozinick, J.T., & Elmendorf, J.S. \\`\\`Antidiabetogenic Effects of Chromium Mitigate Hyperinsulinemia-induced Cellular Insulin Resistance via Correction of Plasma Membrane Cholesterol Imbalance.' Molecular Endocrinology 22:937-950, 2008.\n\nRespondent admitted to falsifying Figure 2C in the publication: Bhonagiri, P., Patter, G.R., Horvath, E.M., Habegger, K.M., McCarthy, A.M., Elmendorf, J.S. \\`\\`Hexosamine biosysthesis pathway flux contributes to insulin resistance via altering membrane PIP 2 and cortical F-actin.' Endocrinology 150(4):1636-1645, 2009.\n\nRespondent also admitted to falsifying Figures 2C, 5, 6D, 11, 13C, 15A, 16A, 17A, 18, 19C, and 20A, which are included in her thesis, \\`\\`Cholesterol-dependent mechanism(s) of insulin-sensitizing therapeutics.' The Ph.D. was awarded to the Respondent on December 31, 2008. Respondent was supported by a Predoctoral Fellowship Award F31 AT003977 from 09\/30\/2006 to 09\/29\/2009.\n\nMs. Horvath has entered into a Voluntary Settlement Agreement in which she has voluntarily agreed, for a period of three (3) years, beginning on March 22, 2010:\n\n\\(1\\) To exclude herself from serving in any advisory capacity to PHS, including but not limited to service on any PHS advisory committee, board, and\/or peer review committee, or as a consultant;\n\n\\(2\\) That any institution that submits an application for PHS support for a research project on which the Respondent's participation is proposed or that uses her in any capacity on PHS-supported research, or that submits a report of PHS-funded research in which she is involved, must concurrently submit a plan for supervision of her duties to the funding agency for approval; the supervisory plan must be designed to ensure the scientific integrity of her research contribution; respondent agreed that she will not participate in any PHS-supported research until such a supervisory plan is submitted to ORI;\n\n\\(3\\) That any institution employing her submits, in conjunction with each application for PHS funds or report, manuscript, or abstract of PHS-funded research in which the Respondent is involved, a certification that the data provided by the Respondent are based on actual experiments or are otherwise legitimately derived and that the data, procedures, analyses, and methodology are accurately reported in the application, report, manuscript, or abstract; the Respondent must ensure that the institution sends a copy of the certification to ORI; and\n\n\\(4\\) That she will write letters, approved by ORI, to relevant journal editors of the published papers cited above to state what she falsified\/fabricated and to provide corrections if she has not already done so. These letters should state that her falsifications\/fabrications were the underlying reason for the retraction\/corrections.\n\nInquiries\n\nFOR FURTHER INFORMATION CONTACT:**  \n  \n** Director**  \n** Division of Investigative Oversight**  \n** Office of Research Integrity**  \n** 1101 Wootton Parkway, Suite 750**  \n** Rockville, MD 20852**  \n** (240) 453-8800","meta":{"dup_signals":{"dup_doc_count":107,"dup_dump_count":42,"dup_details":{"curated_sources":2,"2020-34":1,"2019-51":1,"2018-51":1,"2018-30":1,"2018-17":1,"2018-05":1,"2017-30":1,"2017-22":1,"2017-17":1,"2017-09":2,"2017-04":1,"2016-50":1,"2016-44":1,"2016-40":1,"2016-36":1,"2016-30":1,"2016-26":1,"2016-22":1,"2016-18":1,"2016-07":3,"2015-48":2,"2015-40":2,"2015-35":4,"2015-32":3,"2015-27":3,"2015-22":4,"2015-14":3,"2014-52":3,"2014-49":5,"2014-42":5,"2014-41":5,"2014-35":6,"2014-23":8,"2014-15":4,"2022-33":1,"2015-18":3,"2015-11":4,"2015-06":4,"2014-10":4,"2013-48":4,"2013-20":4,"2024-30":1}},"file":"PMC4259714"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  Many clinicians depend solely on journal abstracts to guide clinical decisions.\n  .\n  # Objectives\n  .\n  This study aims to determine if there are differences in the accuracy of responses to simulated cases between resident physicians provided with an abstract only and those with full-text articles. It also attempts to describe their information-seeking behaviour.\n  .\n  # Methods\n  .\n  Seventy-seven resident physicians from four specialty departments of a tertiary care hospital completed a paper-based questionnaire with clinical simulation cases, then randomly assigned to two intervention groups\u2014access to abstracts-only and access to both abstracts and full-text. While having access to medical literature, they completed an online version of the same questionnaire.\n  .\n  # Findings\n  .\n  The average improvement across departments was not significantly different between the abstracts-only group and the full-text group (p=0.44), but when accounting for an interaction between intervention and department, the effect was significant (p=0.049) with improvement greater with full-text in the surgery department. Overall, the accuracy of responses was greater after the provision of either abstracts-only or full-text (p\\<0.0001). Although some residents indicated that 'accumulated knowledge' was sufficient to respond to the patient management questions, in most instances (83% of cases) they still sought medical literature.\n  .\n  # Conclusions\n  .\n  Our findings support studies that doctors will use evidence when convenient and current evidence improved clinical decisions. The accuracy of decisions improved after the provision of evidence. Clinical decisions guided by full-text articles were more accurate than those guided by abstracts alone, but the results seem to be driven by a significant difference in one department.\nauthor: Alvin Marcelo; Alex Gavino; Iris Thiele Isip-Tan; Leilanie Apostol-Nicodemus; Faith Joan Mesa-Gaerlan; Paul Nimrod Firaza; John Francis Faustorilla; Fiona M Callaghan; Paul FonteloCorrespondence to: **Dr Alex Gavino**, Office of High Performance Computing and Communications, Lister Hill National Center for Biomedical Communications, National Library of Medicine, 8600 Rockville Pike, Bethesda, MD 20894, USA; <email@example.com>\ndate: 2013-04\ninstitute: 1National Telehealth Center, University of the Philippines, Manila, Philippines; 2Department of Surgery, Philippine General Hospital, Manila, Philippines; 3National Library of Medicine, National Institutes of Health, Bethesda, Maryland, USA; 4Department of Medicine, Philippine General Hospital, Manila, Philippines; 5Department of Family and Community Medicine, Philippine General Hospital, Manila, Philippines; 6Department of Emergency Medicine, Philippine General Hospital, Manila, Philippines\nreferences:\ntitle: A comparison of the accuracy of clinical decisions based on full-text articles and on journal abstracts alone: a study among residents in a tertiary care hospital\n\n# Introduction\n\n## Background\n\nBroad estimates show that a specialist would need almost two million pieces of data to practice good medicine.1 To keep updated and apply evidence-based medicine (EBM) in practice, a physician must critically appraise full-text articles to guide their clinical decision making.2 However, owing to limited access to full-text articles, inadequate critical appraisal skills or lack of time to read the entire article, many clinicians depend solely on journal abstracts to answer clinical questions.3\u20138 Journal abstracts may have become the de facto resource for health professionals wanting to practice EBM because they are easy to read and are easily accessible anywhere.2 5 7 9\n\nAlthough abstracts are commonly utilised for clinical decisions, caution should be made in using them because they may not completely reflect the entire article.7 Studies by Pitkin *et al*10 11 and Peacock *et al*12 identified abstracts that contained data which were different or missing in the full-text. High-impact factor journals had abstracts that failed to include harm despite being mentioned in the main article.13 A study by Berwanger *et al*8 found that the abstracts of randomised controlled trials from major journals were reported with suboptimal quality. Moreover, abstracts are also subject to authors' biases which may mislead the readers.14\n\nEfforts have been made to improve the quality and accuracy of journal abstracts since they are often the most commonly read part of an article\u2014if not the only part read.10 11 15 In 1987, the Ad Hoc Working Group for Critical Appraisal of the Medical Literature introduced a seven-heading format (Objectives, Design, Setting, Patients, Interventions, Measurements and Conclusion) for structured abstracts.16 Variations in structured abstracts include the eight-heading format proposed by Haynes *et al*,17 IMRAD18\u201320 (Introduction, Methods, Results and Discussion), and more recently, *BMJ*'s pico format21 (Patient, Intervention, Comparison and Outcome). Structured abstracts tend to be longer than traditional ones but they also tend to have better content, readability, recall and retrieval.14 18 22\u201325 Aside from structuring, 'quality criteria' and guidelines have been developed to assist authors in preparing abstracts.26 27\n\nMost of the research on journal abstracts focus on their quality compared to the full-text,10 11 13 22 23 or based on their structure.14 20 24 Given the tendency of physicians to use abstracts for evidence, there is a need to evaluate their reliability in clinical decision making. A study by Barry *et al*5 looked at the effect of abstract format on physicians' management decisions. However, we are unable to find studies that compare clinical decisions between those with access to abstract-only or full-text.\n\nThe primary objective of this study was to determine whether there is a significant difference in the accuracy of the clinical decisions made on simulated cases by residents with access to full-text articles and those with access to abstract-only. The specific objectives were: (1) to compare the effect of access to abstracts-only or full-text articles on the clinical decision-making of residents; (2) to determine whether providing either the abstract or full-text article increased the accuracy of clinical decisions and (3) to characterise the information-seeking behaviour and use of information resources by residents of four departments in a tertiary care hospital.\n\n# Methods\n\n## Ethics review\n\nThe research protocol was submitted for technical review to the Research Grants Administration Office of the University of the Philippines Manila and for ethical evaluation to the Institutional Review Board, both of which approved the study.\n\n## Prestudy clinical case development\n\nA physician consultant from each of four clinical departments (Surgery, Internal Medicine, Emergency Medicine and Family and Community Medicine) prepared five simulated clinical cases of varying complexity and the corresponding clinical questions to assess the residents' management decisions. They searched PubMed for at least three recent (from 2007 onwards) journal articles that were deemed relevant for each case. 'Gold standard' answers to the clinical questions were based on the journal articles and other relevant information (applicability and appropriateness to local conditions, available resources and practice environments). A paper-based questionnaire was used for the preintervention assessment while an online version was used during the intervention phase to allow access to journal abstracts or full-text articles.\n\n## Study participants and setting\n\nSeventy-seven resident physicians from the four clinical departments (above) at the Philippine General Hospital participated in the study. The Philippine General Hospital is a 1500-bed tertiary care, state-owned, referral center and teaching hospital of the University of the Philippines College of Medicine, College of Nursing, College of Dentistry and allied colleges. It is the largest government hospital with a yearly patient load of 600\u2005000, mostly indigent patients. Fourteen clinical departments offer residency and fellowship training.\n\n## Study design\n\nDuring the prestudy briefing, the residents were informed that they were to answer questions related to the case simulations and that they could access reference articles if needed during the online phase of the study. Written consent was obtained and paper-based case simulations were given to each resident to replicate the hospital scenario of paper patient records. After reading the case simulations, they were asked to respond to five clinical questions and indicate whether they considered a literature search was needed to answer the questions or accumulated knowledge28 was adequate. Accumulated knowledge was defined in this study as the residents' personal knowledge base accumulated through years of formal education, training, research of the medical literature and clinical experience. Immediately after the preintervention phase, the residents were randomly assigned to one of two groups\u2014access to 'full-text' or 'abstracts-only,' stratified by department. The same clinical cases and questions in the preintervention phase were presented to the residents using the online version of the questionnaire to simulate real-time access to medical literature. A 20-min time limit was allotted for each question both for the paper-based and online questionnaire. The journal material provided, whether abstracts-only or full-text, was dependent on their assigned group. If the resident assigned to the abstracts-only group clicked on the link to the full-text, a prompt saying, 'Sorry, full-text is unavailable' appeared. Although access to either journal abstracts or full-text articles on the online version was available to all residents, they had the option of not using any resource at all. The residents' actions regarding the use or non-use of medical literature were recorded. Mouse clicks related to the residents' request for the articles' abstracts or full-text were logged in the server. The accuracy of response was a measure of correctness of residents' answer when compared with the answers ('gold standard') provided by the consultants. The same consultants who prepared the clinical cases and questions evaluated the accuracy of the residents' answers. A correct response was scored as '1' and an incorrect response scored '0'. Incomplete responses were rated as inaccurate and scored '0'. Resident responses were anonymised in both the paper and online versions.\n\n## Data analysis\n\nIn order to account for the repeated measures nature of the data (physicians answered multiple questions), we fit mixed effects logistic regression models with department type, intervention, and the interaction between department and intervention as independent variables, and accuracy of the response as the dependent variable. Unless otherwise stated, all results were based on this model. Resident year level was also considered as a predictor in the model but was not found to be significant and was dropped. We also fit a model with an interaction between intervention and department. For univariate analysis, we used the nonparametric Wilcoxon two-sample tests and Fisher exact tests, as appropriate.29 All analyses were performed using R Statistical Software.30\n\n# Results\n\n## Participant profile\n\nSeventy-seven residents from the departments of Surgery (n=20), Internal Medicine (n=20), Emergency Medicine (n=20) and Family and Community Medicine (n=17) participated in this study. <a href=\"#EBMED2012100537TB1\" data-ref-type=\"table\">Table\u00a01<\/a> shows the description of the study participants by department.\n\nCharacteristics of participating resident trainees by department\n\n| Characteristics | Surgery, n=20 | Internal medicine, n=20 | Emergency medicine, n=20 | Family and community medicine, n=17 |\n|:---|----|----|----|----|\n| Mean age, years (SD\\*) | 29.2 (2.7) | 28.1 (1.6) | 30.8 (3.3) | 30.5 (2.4) |\n| Gender, n (%) |  |  |  |  |\n| \u2003Female | 5 (25) | 3 (15) | 6 (30) | 13 (76) |\n| \u2003Male | 15 (75) | 17 (85) | 14 (70) | 4 (24) |\n| Years in residency training, n (%) |  |  |  |  |\n| \u20031 | 6 (30) | 7 (35) | 7 (35) | 11 (65) |\n| \u20032 | 2 (10) | 6 (30) | 7 (35) | 1 (6) |\n| \u2003\\>3 | 12 (60) | 7 (35) | 6 (30) | 5 (29) |\n\n\\*Standard deviation.\n\n## Comparing the effect of abstract-only and full-text access on the accuracy of clinical decision making\n\nThe first objective was to answer the question: Is there a significant difference in the accuracy of responses of residents in the abstract-only group and full-text group? Overall, there was no significant difference between the interventions (p*\u2009*=\u20090.44). Post-hoc power of the experiment to detect an overall difference between the interventions was low and varied from approximately 44\u201358%,31 depending on the level of correlation between the answers within each department. In a model fit to include an interaction between intervention and department, the interaction was significant suggesting that intervention effects differed by department (p*\u2009*=\u20090.03). In that model, access to full-text was significantly better than access to abstracts-only (p=0.049).\n\nWe then compared the effect of the interventions within departments in order to investigate which departments seemed to respond differently to the others with respect to the effect of the interventions. We found no significant difference between the interventions for the Internal Medicine, Emergency Medicine and Family Medicine departments (p=0.73, 0.13 and 0.37, respectively), but there was a difference between the interventions for the Surgery department (p=0.02). The OR for each department is given in <a href=\"#EBMED2012100537TB2\" data-ref-type=\"table\">table 2<\/a>. The full-text group had 3.6 times the probability of getting a correct answer on a case simulation compared to the abstract-only group. Note that the CI for Surgery does not include 1.0, which indicates a significant difference. There were no differences found between the interventions for any other department. Power to detect a difference between the interventions in a specific department was low (approx. 17%) because of the reduced sample size in each group (n=10). We also investigated whether resident year was a significant predictor of clinical decision accuracy, but it was not significant in any model.\n\nEstimates of the odds of getting an accurate response after full-text intervention compared to abstract-only intervention\n\n| Department         | OR (95% CI)       |\n|:-------------------|:------------------|\n| Surgery            | 3.6 (1.3 to 10.3) |\n| Internal medicine  | 0.8 (0.3 to 2.2)  |\n| Emergency medicine | 0.5 (0.2 to 1.3)  |\n| Family medicine    | 1.6 (0.6 to 4.7)  |\n\n## Accuracy of clinical decisions before and after access to literature search\n\nWe calculated the mean percentage of accurate responses to the simulated clinical questions before and after each intervention. Overall, mean accuracy increased from 42% to 68% for the abstract-only intervention, and 48\u201375% for the full-text intervention. The differences between the scores before and after the two interventions were significant (p\\<0.0001). <a href=\"#EBMED2012100537TB3\" data-ref-type=\"table\">Table\u00a03<\/a> shows the comparison of these percentages by department and the tests of significance.\n\nComparison of the average percentage of accurate of responses before and after interventions and tests of significance\n\n|  | N | Average % of accurate responses before intervention (SD) | Average % of accurate responses after intervention (SD) | Difference in % before and after (95% CI)\\* | Test of significance for each department and overall (p value)\u2020 | Does the amount of improvement differ by department? (p value)\u2020 |\n|:---|----|:---|:---|----|----|:---|\n| Abstract-only (intervention 1) |  |  |  |  |  |  |\n| \u2003Department |  |  |  |  |  |  |\n| \u2003\u2003Surgery | 10 | 48 (27) | 62 (15) | 14 (0 to 40) | 0.16 |  |\n| \u2003\u2003Internal medicine | 10 | 36 (18) | 66 (16) | 30 (20 to 60) | 0.003 | 0.06 |\n| \u2003\u2003Emergency medicine | 10 | 54 (21) | 72 (14) | 18 (0 to 40) | 0.06 |  |\n| \u2003\u2003Family medicine | 8 | 25 (14) | 75 (14) | 50 (40 to 60) | \\<0.0001 |  |\n| \u2003\u2003Overall | 38 | 42 (23) | 68 (15) | 27 (20 to 40) | \\<0.0001 |  |\n| Full-text (intervention 2) |  |  |  |  |  |  |\n| \u2003Department |  |  |  |  |  |  |\n| \u2003\u2003Surgery | 10 | 60 (13) | 86 (0.16) | 26 (20 to 40) | 0.003 |  |\n| \u2003\u2003Internal medicine | 10 | 46 (16) | 68 (0.1) | 22 (0 to 40) | 0.03 | 0.0001 |\n| \u2003\u2003Emergency medicine | 10 | 64 (25) | 64 (0.23) | 0 (\u221220 to 20) | 1.0 |  |\n| \u2003\u2003Family medicine | 9 | 20 (14) | 82 (0.21) | 62 (40 to 80) | \\<0.0001 |  |\n| \u2003\u2003Overall | 39 | 48 (24) | 75 (0.2) | 27 (20 to 40) | \\<0.0001 |  |\n\n\\*95% CI based on Wilcoxon test.\n\n\u2020Based on values from the logistic regression mixed model.\n\nWhen given full-text articles, the departments of Surgery, Internal Medicine and Family Medicine showed significant improvements (p=0.003, 0.03 and \\<0.0001, respectively), while there was no change for the department of Emergency Medicine (p*\u2009*=\u20091.0). The differences among the departments were significant (p*\u2009*\\<\u20090.0001) for full-text intervention group. This suggests that full-text was more effective for Surgery, Internal Medicine and Family Medicine, but not in the Emergency Medicine department. However, the sample size was small (n=10 or less) at this level. The effect of the abstract-only intervention seems to have been in a similar direction for all the departments, and no significant difference in effects across departments was detected.\n\n## Information-seeking trends of the residents\n\nThe majority of the residents (86%) indicated that the articles provided in the online version were adequate to answer their questions and 77% indicated that they had actually read the articles. When asked whether they used abstracts-only or full-text articles to answer clinical questions in actual practice, 53 of the 77 residents (69%) indicated that they relied on abstracts most of the time, while only 24 (31%) said they would read the full-text article.\n\nResidents were asked whether or not they felt they needed extra information in order to answer the question correctly. We recorded whether they clicked on the links for the abstract or the full-text. We wanted to answer the question: does a perceived need for more information correlate with how often the physicians actually accessed the links for abstract-only or full-text? For the 157 cases where the resident indicated that they did not require additional information, there were 131 (83%) instances where literature was actually accessed (95% CI 77% to 88%). In contrast, out of the 228 cases where residents indicated that they needed additional information, there were only 12 (5%) cases where they did not actually access literature (95% CI 3% to 9%). <a href=\"#EBMED2012100537TB4\" data-ref-type=\"table\">Table\u00a04<\/a> shows a summary of whether the resident requested additional information and whether they actually accessed literature.\n\nResidents' perceived need for additional information and actual access of literature\n\n|  |  | Was literature actually accessed? |  |\n|:---|:---|:---|:---|\n|  |  | No | Yes |\n| Perceived need for more information? | No | 26 (17%) | 131 (83%) |\n|  | Yes | 12 (5%) | 216 (95%) |\n\nRow percentages are displayed.\n\n# Discussion\n\nThe main question we wanted to address in this study was whether there is a significant difference in the clinical decisions between residents who have access to abstracts-only and those with access to full-text articles. Overall, our results seem to demonstrate no difference in the accuracy of responses between residents provided with full-text articles and those with abstracts-only (p=0.4415). When we consider the clustering of physicians by department, we found a difference between the two interventions (p=0.0494) but further analysis showed that this difference was observed only in the department of Surgery (p=0.016). The effects of abstracts-only and full-text were not significantly different for the Internal Medicine, Emergency Medicine and Family Medicine departments. However, the study had low power to detect differences between the interventions within a department.\n\nOur study provides preliminary but useful information related to the use of journal abstracts in evidence-based practice. We believe this to be the first report involving physicians, that attempted to evaluate how abstracts measure up to full-text articles in guiding clinical decisions. This finding offers support for using 'consensus abstracts' (concurring and corroborating abstracts from independently conducted randomised clinical studies and systematic research from meta-analysis and systematic reviews that form the basis of clinical evidence) as a possible alternative when access to full-text is limited or in other circumstances when it is not feasible.2 However, clinicians who want to practice EBM will also find online many summaries, reviews and preappraised free resources (TRIP Database, ACP Journal Club, Cochrane Library, etc) or by subscription (UpToDate, 5-Minute Clinical Consult, etc). EBM websites will have links to these. Many of these resources will have applications for mobile devices like the iPhone or Android devices. Our observations set the stage for further research on the role of using abstracts in evidence-based practice. Future studies may include randomised controlled trials with real-time clinical decision-making encountered at the bedside.\n\nEBM encourages the use of timely and relevant information to complement the clinical acumen of clinicians.32 We found that the average improvement in the accuracy of responses across all the departments when either abstracts or full-text articles were provided was significant (p\\<0.0001 for both interventions). This finding supports previous research regarding the role of medical literature in improving clinical decisions.33\u201336 However, when individual departments were considered, there seems to be a significant difference between the departments in the full-text intervention group (p=0.0001). This difference in the effect of full-text between the departments appears to be due to the fact that there was no change in the accuracy of responses of Emergency Medicine residents compared to the increase in scores for the other residents when full-text was provided. This may mean that full-text articles were beneficial to Surgery, Family Medicine and Internal Medicine residents but did not benefit Emergency Medicine residents. A possible explanation for this is that the Emergency Medicine department is fast paced and residents may not have the time to read the full-text article. This hypothesis was further supported by data for the abstract-only group where we found no significant difference between the departments on how the intervention improved the accuracy of the responses by residents.\n\nOur study also demonstrated some trends in information-seeking and utilisation of evidence by residents when presented with clinical questions. We observed that although residents indicated that accumulated knowledge was sufficient to answer the questions, in most instances (83.4%), they still accessed the medical literature provided. This observation supports earlier studies that health professionals will use evidence from the literature when they are easily accessible at the time the question arises.37\n\nMore than a third of the residents (68.8%) who participated in this study claimed that they commonly used abstracts in seeking answers to their clinical dilemma. Other studies have reported similar observations. A study by Haynes *et al*3 found that two-thirds of clinical decisions were influenced by literature even if the full-text was not read. Moreover, internists reported that in 63% of the articles they come across, only the abstracts were read.4 These findings may even be higher among physicians in low- and middle-income countries because of even more limited availability of full-text articles.\n\n## Limitations\n\nThe small sample of residents from a tertiary government hospital in the Philippines limits the generalisability of the study to the larger medical community. Simulated clinical cases were used as surrogate to actual clinical encounters that a resident may be presented with. The clinical questions were specific within the realm of the disciplines and are not necessarily comparable to each other. The residents only answered five questions which reduced the variation in the study. A 'learning effect' was considered to explain the higher score during the intervention phase but was deemed unlikely because of the short interval period\u2014the residents took the online version questions immediately after the preintervention session. Furthermore, this study does not address whether access to full-text would have more impact than access to the abstract in a complex case, a case in which the details of a treatment or outcome or magnitude or significance might affect practice. It also does not address the impact on standard or routine or long-term practice. Finally, although there was reasonable power to detect a difference between the interventions overall, there was low power to detect differences within a department. It is possible that there were differences between the interventions for each department but our study did not have enough sample size to investigate the effect of the intervention at the department level.\n\n# Conclusion\n\nIn this study, we demonstrated that clinical decisions made by residents improved when evidence, either abstracts or full-text articles were provided. However, this study also indicates that some clinical questions may be simple enough; answered quickly using accumulated knowledge, but accumulated knowledge was enhanced by the use of appropriate medical information. The residents, in spite of initially stating that accumulated knowledge was adequate to answer clinical questions, accessed evidence anyway. This confirms previous findings that easy availability of evidence encourages the practice of evidence-based medicine. When clustered by department, clinical decisions guided by full-text articles were more accurate than those guided by abstracts alone, but this difference can be largely attributed to a significant difference in Surgery. It may be less or not at all in the other three departments but the analysis is not conclusive because of the limited power of this study. Without departmental clustering, the findings seem to show that they may not be significantly different.\n\n# References","meta":{"dup_signals":{"dup_doc_count":144,"dup_dump_count":60,"dup_details":{"curated_sources":2,"2023-50":2,"2023-40":1,"2023-06":2,"2022-40":2,"2022-21":2,"2021-49":1,"2021-43":2,"2021-31":1,"2021-17":3,"2021-10":2,"2019-43":1,"2018-51":1,"2018-34":1,"2018-26":1,"2018-22":2,"2018-17":1,"2018-13":3,"2018-09":1,"2018-05":1,"2017-51":2,"2017-47":1,"2017-43":3,"2017-39":1,"2017-34":2,"2017-22":1,"2017-17":2,"2017-09":2,"2017-04":2,"2016-50":3,"2016-44":3,"2016-40":2,"2016-36":3,"2016-30":4,"2016-26":1,"2016-22":3,"2016-18":3,"2016-07":2,"2015-48":2,"2015-40":3,"2015-35":3,"2015-32":2,"2015-27":3,"2015-22":4,"2015-14":2,"2014-52":3,"2014-49":5,"2014-42":9,"2014-41":6,"2014-35":4,"2014-23":5,"2014-15":2,"2024-10":1,"2017-13":3,"2015-18":3,"2015-11":3,"2015-06":2,"2014-10":2,"2013-48":2,"2013-20":2,"2024-30":1}},"file":"PMC3607116"},"subset":"pubmed_central"}
{"text":"author: Alexandre B. Hardy; Emma M. Allister; Michael B. WheelerCorresponding author: Michael B. Wheeler, <firstname.lastname@example.com>.\ndate: 2013-08\nreferences:\ntitle: Response to Comment on: Allister et al. UCP2 Regulates the Glucagon Response to Fasting and Starvation. Diabetes 2013;62:1623\u20131633\n\nWe thank Dr. Gylfe (1) for his interest in our work showing a role for uncoupling protein 2 (UCP2) in regulating \u03b1-cell glucagon secretion and suggesting that this is an interesting and potentially important finding. We agree that the role and mechanism of glucose sensing in \u03b1-cells is still highly controversial and that two opposing models are promoted in the literature. Our data suggests that the presence of UCP2 in \u03b1-cells may play a role in glucose sensing because the absence of UCP2 impaired the release of glucagon, and this was accompanied by reduced mitochondrial membrane potential hyperpolarization, increased reactive oxygen species production, more depolarized plasma membrane potential, and lower intracellular calcium levels. So although the objective of this study was not to define the mechanisms of glucose-regulated glucagon secretion in control cells, but rather to investigate the impact of UCP2 on \u03b1-cell function, we have attempted to fit our data into the published models. The first model was suggested by Dr. Gylfe and is based on the role of glucose as an activator of Ca^2+^ sequestration in the endoplasmic reticulum, which inhibits glucagon secretion (2,3). This model also suggests a depolarizing effect of low glucose concentration on \u03b1-cell plasma membrane potential (2,4,5). The second model (by Rorsman and colleagues \\[6\\]) describes regulation of glucagon secretion by an ATP-sensitive potassium channel\u2013dependent pathway. This model predicts that glucose metabolism increases intracellular ATP, closing ATP-sensitive potassium channels. The channel closure depolarizes \u03b1-cell membrane potential to a level that inactivates Na^+^ and Ca^2+^ ion channels, thereby reducing glucagon secretion (7). In our study, we present data that fits with both models. The glucose-induced changes in membrane potential recorded in isolated dispersed \u03b1-cells fit with the first model; we show that low glucose concentration caused depolarization and increased intracellular calcium levels along with enhanced secretion. However, the \u03b1-cell\u2013specific UCP2 knockout mouse \u03b1-cells were more depolarized under both high and low glucose concentrations and secreted less glucagon, which fits with the model by Rorsman and colleagues. In addition, our data show that low-dose diazoxide (1\u00b5mol\/L), which should hyperpolarize the membrane, increased glucagon secretion under high glucose conditions in control \u03b1-cells and could enhance glucagon secretion to control levels in the absence of UCP2. Again these data are in line with the second model of glucagon secretion and perhaps point to the \u03b1-cell being secretory within a narrow range of plasma membrane potentials. There may be differences in the data based on the use of dispersed versus whole islets, which were used for the electrophysiological and secretion experiments, respectively. Complex factors such as release of paracrine molecules can regulate glucagon secretion and may play a role in the whole islets studies. In addition, it cannot be ignored that the elevated reactive oxygen species levels (even under low glucose conditions) in the \u03b1-cell\u2013specific UCP2 knockout mouse islets could potentially be affecting secretion via a channel-independent mechanism, and this area deserves more investigation. However, as pointed out by Dr. Gylfe, in the context of normal \u03b1-cells, low glucose in our hands caused depolarization.\n\n## ACKNOWLEDGMENTS\n\nNo potential conflicts of interest relevant to this article were reported.\n\n# REFERENCES","meta":{"dup_signals":{"dup_doc_count":128,"dup_dump_count":46,"dup_details":{"curated_sources":2,"2020-10":1,"2019-04":1,"2018-43":1,"2018-30":1,"2018-26":2,"2018-17":4,"2018-09":2,"2018-05":1,"2017-51":1,"2017-47":3,"2017-43":5,"2017-39":2,"2017-34":1,"2017-30":8,"2017-26":2,"2017-22":2,"2017-17":8,"2017-09":4,"2017-04":8,"2016-50":3,"2016-44":4,"2016-40":3,"2016-36":2,"2016-30":3,"2016-26":3,"2016-22":3,"2016-18":2,"2016-07":3,"2015-48":2,"2015-40":3,"2015-35":3,"2015-32":2,"2015-27":2,"2015-22":2,"2015-14":2,"2014-52":3,"2014-49":1,"2014-42":4,"2014-41":2,"2014-35":4,"2014-23":3,"2023-06":1,"2017-13":2,"2015-18":3,"2015-11":2,"2015-06":2}},"file":"PMC3717857"},"subset":"pubmed_central"}
{"text":"abstract: Meta-analysis is a popular way to try to get the most out of different studies linking genes and disease. But there are cons as well as pros.\nauthor: Gregory A Petsko\ndate: 2008\ninstitute: 1Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02454-9110, USA\ntitle: Meta-morphosis\n\nThere's an old chemistry joke, based on the *ortho, meta* and *para* nomenclature for substituents attached to a benzene ring, that goes like this:\n\nOK, I know it's not very funny, but how many funny chemistry jokes are there? Anyway, someone reminded me of this joke the other day and while I was busy not laughing I was also thinking about something else. Because these days, for most people in the life sciences, and I bet nearly everybody in genome biology, the prefix *meta* doesn't conjure up images of di-substituted benzene. It calls to mind the meta-analysis of data.\n\nMeta-analysis - sometimes contracted to metanalysis - is one of those Sudoku-like fads that seem to pop up overnight and sweep the entire country in about as little time. 'Metanalysis' doesn't mean the same thing to scientists as it does to other academics. In linguistics, metanalysis is the act of breaking down a word or phrase into segments or meanings not original to it. The term was coined by the linguist Otto Jespersen, and comes from the Greek for 'a change of breakdown'. Here's an example, courtesy of Wikipedia: in the phrase \"God rest ye merry gentlemen\", originally merry was a complement with rest (as in \"God rest ye merry, gentlemen\" - note the position of the comma - that is, \"\\[may God\\] give you gentlemen a pleasant repose\"). But now, by a process of metanalysis, merry is frequently construed as an ordinary adjective modifying gentlemen (\"God rest ye, merry gentlemen\") - and in all probability it can be relexicalized with the current sense of merry, that is, cheerful, jolly, though that is harder to be certain of. (The expression \"to rest merry\" and the like was once generally current, by the way.) Incidentally, I love this sort of thing, but it isn't what 'meta-analysis' means in biomedical research.\n\nMeta-analysis for us is a technique whereby all data from all available studies of something are combined, often regardless of the relative quality of the data. The method is used by researchers to get a maximum amount of statistical information from a set of studies that might not have large enough individual sample sizes, or whose results may be of marginal statistical significance.\n\nIn a typical meta-analysis, the results of, say, four different clinical trials of a drug, or the data from five independent studies of the association of a genetic variation with a particular disease, are merged into a single statistical sample. The sample is then analyzed for the same correlation, or lack thereof.\n\nI don't know where it the practice came from but it's of relatively recent origin - the late 1970s or so. Certainly when I was a student there wasn't an entire subfield devoted to pooling studies and reinterpreting the results. But the medical literature, and the literature of human genetics, is awash with it now.\n\nImagine the excitement of the person who thought this up. \"Omigod! I can take other people's results, throw them together, and come up with a conclusion that in some cases will horrify them, and I can get it published in good journals -sometimes with quite high visibility if the data concern an important human disease or a dietary substance that's popular - and I don't have to do any of the really hard work! I don't have to actually do the studies. I don't have to find the patients, or collect the questionnaires, or analyze the genomes, or any of that difficult, real science. They'll do it for me, and then I can use their data for my own benefit. This is like making money with other people's money! This is how the first banker must have felt!!\"\n\nI am not a professional statistician, but I have taught statistics, and as a protein crystallographer I have to be familiar with most of the rudimentary forms of statistical analysis of data. And everything I know about the subject suggests to me that meta-analysis could lead to all kinds of trouble. One of the things I've learned is that you don't improve bad data by combining it with good data. In fact, exactly the opposite occurs: the bad data degrade the better. \"When in doubt, leave it out\" is what I usually tell my students - occasionally modified to \"when in doubt, weight it down\". But most meta-analyses don't apply relative weights to the studies they combine (it's pretty hard to see where they would get the weights anyway), so studies of all sorts of varying quality are sometimes pooled as though they were equally reliable.\n\nAnd even if we allow that the people who do this may get better statistical precision this way, owing to an enlargement of the sample size, I don't think statistical precision is the issue here. Adding more data may reduce the random errors, but I believe that in most biomedical and genomics studies - especially the latter - the real importance may lie in the systematic errors, or, more precisely (pun intended), the systematic differences.\n\nMeta-analysts justify combining data from different studies in part because doing the same experiment in different ways has long been a way to avoid problems caused by non-random or systematic errors. And it is true that using multiple studies that involve different questions or different experimental techniques, with different patient populations and other variations, may allow global trends to emerge from underneath spikes of systematic differences. But there is a danger there. The first meta-analysis I ever encountered, some years ago, concerned a gene I was interested in. It had been reported that a particular polymorphism in this gene was associated with a reduction in risk for certain diseases in the Han Chinese population. Several other studies, including some with much smaller sample sizes, had looked for a disease association in other ethnic groups but had failed to find any. The meta-analysis pooled all of these studies and concluded, rather definitively, that the particular variant did not confer a change in risk for any of the diseases in question. And their overall statistics certainly bore that out.\n\nThere was just one problem: I had read the original Han Chinese study rather carefully, and the work was well done, and the statistical correlation with disease risk was absolutely significant. But if you only read the meta-analysis -and I've seen that referred to repeatedly since and even quoted from at meetings - you wouldn't know that.\n\nBecause in the age of genomics, it's not about the general population anymore. Personalized medicine is coming, and our studies of haplotypes and other natural variations make it clear that, even if we can't quite get down to the level of the individual yet in all studies, the genetic (and environmental) background of the population being evaluated can make a huge difference. Ethnic and geographical differences aren't not systematic errors to be averaged out; they're essential components of the ways genes (or drugs, or nutrients) interact with the human body. The right question to be asking in the case of the study I referred to is not whether the polymorphism alters disease risk in the general population, but rather why it does so in the Han Chinese population. What are the particular combinations of other genetic and environmental factors that cause this variant to become associated with a set of diseases? That's where the really interesting science, and medicine, lies. Average different studies together willy-nilly and you run the risk that sometimes you may average out precisely the those variations that provide the clues we are looking for as to how human health really works.\n\nNow you may think that this column is all about trashing meta-analysis, and, to be honest, it started out that way. But then I had a discussion with David Altshuler, a human geneticist at Harvard Medical School and the Broad Institute at MIT, that made me rethink what was about to become a blanket condemnation. He pointed out to me another goal of meta-analysis in current human genetic studies. The key issues, as he put it, are first, the different statistical thresholds needed in discovery science with a low prior (as compared with hypothesis testing in a well-established field), and second, in human genetics the ability to use the rest of the genome as independent tests to assess the matching of cases and controls in human genetics.\n\nHere's how he explained the first issue: in genetic mapping with the goal of initially determining which genes might actually play a role in human disease, there is a very low *a priori* probability of any variant being associated with any risk (on the order of one in a million), and - that is, with an initial goal of determining which genes might actually play a role in human disease risk - one can't say that a *p* value of 0.01 or even 0.0001 is 'absolutely significant'. He's right: most findings of that magnitude turn out to be entirely irreproducible, and are likely to be false positives. But finding consistent results by meta-analysis increases evidence that the null hypothesis is wrong - and confidence that there is a relationship between the variant and risk. Without that confidence, he asserts, the field was awash with wishful thinking about lots of candidate genes that reductionist biologists had found in cells and in animal models, and wanted to believe were 'genetically validated in people' - but that turned out to be noise.\n\nIf you look into it, you will find that there is a long history in genetic mapping of setting the right threshold based on the number of tests. In linkage mapping, the LOD score that indicated linkage between a gene variant and a trait was 3.0 -not *p* \\< 0.05 (LOD stands for logarithm to the base 10 of odds; a LOD score of 3.0 means the likelihood of observing the given pedigree if two loci are not linked is less than 1 in 1000). By contrast, in genome-wide human genetic studies, a *p* of less than or equal to 0.0000001 is typically required for proof of association of a gene with a trait. And while biologists often like to say that for 'candidate genes' like the one I mentioned above it is more like *p* \\< 0.05, that threshold has a history of not always supporting reproducible discoveries. It is in gathering enough data to really get the confidence level to where it needs to be that meta-analysis has proven proved valuable in many cases.\n\nThe second issue is the quality of the data that goes into meta-analysis, and the ability to compare and align it. In most studies, Altshuler agrees with me that you can't know if you are washing out good data with bad. But he makes a strong case, with which I am forced to agree, that in the current wave of genome-wide association studies, the studies often use the same phenotype definition, or the same microarray protocols, and so the data may well be comparable (clearly, it is important to see whether that is the case, if you want to evaluate the meta-analysis critically). When done properly, information from the rest of the genome can be used to assess the properties of the data, matching of cases and controls, and so on, which, as he puts it, \"can result in valid combination of lots of good data, rather than sloppy mashing up of lots of bad data\". Finally, sometimes you actually may want to test the hypothesis across ethnicity or age or other variables. In those instances, meta-analysis allows you to put the data together in a valid way - a way that is probably better than just reading the papers and trying to compare them.\n\nDr Altshuler went on to make a very valuable point, which I think belongs in this column: he feels there that is currently a big and unfortunate divide between cell and molecular biologists and people using human genetics to find disease genes. The tone of the first part of my column just reinforced his concern, I'm sorry to say. For disease research, it's certainly true that knowing up front that a gene influences the disease in humans is invaluable, and I think he's right that meta-analysis can be a powerful tool for getting that correct. (As he asked me: \"Would you want to do functional work on a gene that turns out to be a false positive?\")\n\nA final point: meta-analysis is currently being used in two ways in human genetics, and I may have given a false impression that there is only one. One method is when there is a pre-existing hypothesis that needs to be tested. A good example comes from Altshuler's own work (Altshuler D, *et al.: Nat Genet* 2000, **26:**76-80). The data the authors had collected gave a positive result, but others had claimed the opposite. The studies were comparable enough that meta-analysis was able to show that the data were actually consistent and reinforced one another. In such cases meta-analysis serves as a reality check, and helps avoid possible bias in the selection of data that might support one hypothesis or another.\n\nBut careful meta-analysis can also in some cases be hypothesis generating, because enough studies might, as I mentioned above, allow previously undetected signals to rise above the noise. These can then be tested experimentally, and of course, need to be.\n\nDr Altshuler closed our discussion with a nice comment: \"Meta-analysis,\" he pointed out, \"is just a method. Garbage in, garbage out. But in genetic mapping of common complex diseases, where it is clear there are many different variants contributing, and where studies are expensive, combining data to learn the most is well justified and valid.\"\n\nIt is true that it's often the original studies that matter, that data need to be examined carefully, and that a study ideally must be accepted or rejected on its own merits. People are fond of saying that the devil is in the details. But God is in the details too. In the age of genomics, the details are ultimately what matter: the complex interplay of individual gene and genetic background, diet, environment, perhaps even state of mind, is what determines whether we are prone to this disease or that, react poorly or well to this drug or that, age well or badly. Meta-analysis can hide those details, but used properly, it can also be revealing. I guess you could say that writing this column has led to a meta-morphosis in the way I think about this subject, and that I have to back away from my meta-phor about making money with other people's money. In the right hands, meta-analysis can be a valuable tool, which is a pity, because it's so much more fun to write a column that completely trashes something. Meta-physically speaking, of course.","meta":{"dup_signals":{"dup_doc_count":118,"dup_dump_count":40,"dup_details":{"curated_sources":2,"2021-49":1,"2019-39":1,"2019-26":1,"2019-13":1,"2018-51":1,"2018-39":1,"2018-30":1,"2018-17":1,"2018-05":1,"2017-39":1,"2017-22":1,"2017-09":10,"2016-44":1,"2016-40":1,"2016-36":11,"2016-30":8,"2016-22":1,"2016-18":1,"2016-07":5,"2015-48":3,"2015-40":2,"2015-35":3,"2015-32":3,"2015-27":2,"2015-22":3,"2015-14":2,"2014-52":3,"2014-49":2,"2014-42":8,"2014-41":6,"2014-35":6,"2014-23":4,"2014-15":2,"2023-50":1,"2015-18":3,"2015-11":3,"2015-06":3,"2014-10":3,"2013-48":3,"2013-20":2}},"file":"PMC2760864"},"subset":"pubmed_central"}
{"text":"author: T Pap; A Cinski; A Drynda; A Wille; A Baier; S Gay; I Meinecke\ndate: 2005\ninstitute: 1Division of Molecular Medicine of Musculoskeletal Tissue, University Hospital, Munster, Germany; 2Division of Experimental Rheumatology, University Hospital Magdeburg, Germany; 3Center of Experimental Rheumatology, Department of Rheumatology, University Hospital, Zurich, Switzerland; 4Department of Traumatology, Hand and Reconstructive Surgery, University Hospital, Munster, Germany\ntitle: Regulating apoptosis in fibroblasts\n\nApoptosis constitutes a highly selective way of eliminating aged and injured cells and is a key mechanism for the balanced growth and regeneration of tissues. In addition to genotoxic stress and the withdrawal of growth factors, apoptosis can be induced by death receptors. Such receptors trigger apoptosis through an approximately 90 amino acid death domain. Prominent and best-characterized members of the death domain receptor family are Fas (CD-95\/Apo-1) and the p55 TNF receptor (TNFRI). Alterations in receptor-mediated apoptosis may result in changes of tissue homeostasis and have been found in a variety of malignancies as well as in the inflamed and hyperplastic synovium in rheumatoid arthritis. Accumulating evidence suggests that the activation of rheumatoid arthritis synovial fibroblasts (RASF) is associated with alterations in apoptosis, especially at sites of invasion into cartilage and bone. Specifically, it has been demonstrated that RASF are less susceptible to Fas-induced cell death than osteoarthritis synovial fibroblasts but show higher expression levels of Fas.\n\nRecent studies have shown that both cytokine-dependent and cytokine-independent mechanisms contribute to the resistance of RASF against Fas-induced apoptosis. Thus, tumor necrosis factor alpha (TNF-\u03b1) \u2013 a major inflammatory cytokine in the rheumatoid synovium \u2013 fails to induce apoptosis in RASF, but reduces the susceptibility of these cells to Fas-mediated cell death through the induction of the transcription factor NF-\u03baB. Providing a link between altered apoptosis and cartilage destruction, we have shown that overexpression of TIMP-3 through gene transfer not only reduces the invasiveness of RASF but also modulates the apoptosis-inhibiting effects of TNF-\u03b1. RASF overexpressing TIMP-3 are sensitized strongly to Fas\/CD95-mediated cell death by TNF-\u03b1, and gene transfer of TIMP-3 inhibits the TNF-\u03b1-induced activation of NF-\u03baB. While these effects of TNF-\u03b1 on apoptosis can be found in different fibroblasts, several anti-apoptotic molecules have been demonstrated to be upregulated specifically in RASF.\n\nIn this context, we have investigated the role of the small ubiquitin-like modifier SUMO-1 in the regulation of apoptosis. We found that increased levels of SUMO-1 in RASF but not in osteoarthritis synovial fibroblasts were associated with a reduced susceptibility of RASF to Fas-induced apoptosis. Using small interfering (si)RNA to knock-down the expression of SUMO-1 as well as retroviral gene transfer of SUMO-1, we could establish a functional relationship between the expression of SUMO-1 and the resistance of RASF against apoptosis. Moreover, it was demonstrated by gene transfer of the nuclear SUMO-protease SENP1 that, rather than by interacting directly with the Fas-associated death domain, SUMO-1 inhibits apoptosis through recruiting pro-apoptotic molecules such as DAXX into nuclear PML bodies, where they cannot exert their pro-apoptotic effects.\n\nTaken together, there is growing evidence that in activated fibroblasts, such as in RASF, there is a close association between anti-apoptotic and destructive pathways. In addition to cytokine-dependent inflammatory mechanisms, the intrinsic upregulation of anti-apoptotic molecules contributes to the resistance of RASF against apoptosis. Post-translational modification of nuclear proteins through SUMO-1 appears to constitute an important mechanism of apoptosis regulation. Therefore, the inhibition of molecules that confer the resistance of RASF to apoptosis constitutes a most interesting therapeutic target.","meta":{"dup_signals":{"dup_doc_count":117,"dup_dump_count":42,"dup_details":{"curated_sources":2,"2022-21":1,"2021-43":1,"2021-17":1,"2020-45":1,"2020-29":1,"2020-05":1,"2019-39":1,"2018-30":1,"2018-09":1,"2017-39":1,"2017-30":1,"2017-22":1,"2017-09":7,"2016-44":2,"2016-40":1,"2016-36":7,"2016-30":4,"2016-22":1,"2016-18":1,"2016-07":7,"2015-48":3,"2015-40":2,"2015-35":3,"2015-32":2,"2015-27":3,"2015-22":3,"2015-14":2,"2014-52":3,"2014-49":4,"2014-42":10,"2014-41":3,"2014-35":5,"2014-23":5,"2014-15":5,"2022-40":1,"2015-18":3,"2015-11":2,"2015-06":3,"2014-10":3,"2013-48":3,"2013-20":4,"2024-26":1}},"file":"PMC2833993"},"subset":"pubmed_central"}
{"text":"abstract: The primary associations of the HLA class II genes, *HLA-DRB1* and *HLA-DQB1*, and the class I genes, *HLA-A* and *HLA-B*, with type 1 diabetes (T1D) are well established. However, the role of polymorphism at the *HLA-DRB3, HLA-DRB4*, and *HLA-DRB5* loci remains unclear. In two separate studies, one of 500 subjects and 500 control subjects and one of 366 DRB1\\*03:01\u2013positive samples from selected multiplex T1D families, we used Roche 454 sequencing with Conexio Genomics ASSIGN ATF 454 HLA genotyping software analysis to analyze sequence variation at these three *HLA-DRB* loci. Association analyses were performed on the two HLA-DRB loci haplotypes (*DRB1*-*DRB3*, -*DRB4*, or -*DRB5*). Three common *HLA-DRB3* alleles (\\*01:01, \\*02:02, \\*03:01) were observed. DRB1\\*03:01 haplotypes carrying DRB3\\*02:02 conferred a higher T1D risk than did DRB1\\*03:01 haplotypes carrying DRB3\\*01:01 in DRB1\\*03:01\/\\*03:01 homozygotes with two DRB3\\*01:01 alleles (odds ratio \\[OR\\] 3.4 \\[95% CI 1.46\u20138.09\\]), compared with those carrying one or two DRB3\\*02:02 alleles (OR 25.5 \\[3.43\u2013189.2\\]) (*P* = 0.033). For DRB1\\*03:01\/\\*04:01 heterozygotes, however, the *HLA-DRB3* allele did not significantly modify the T1D risk of the DRB1\\*03:01 haplotype (OR 7.7 for \\*02:02; 6.8 for \\*01:01). These observations were confirmed by sequence analysis of *HLA-DRB3* exon 2 in a targeted replication study of 281 informative T1D family members and 86 affected family-based association control (AFBAC) haplotypes. The frequency of DRB3\\*02:02 was 42.9% in the DRB1\\*03:01\/\\*03:01 patients and 27.6% in the DRB1\\*03:01\/\\*04 (*P* = 0.005) compared with 22.6% in AFBAC DRB1\\*03:01 chromosomes (*P* = 0.001). Analysis of T1D-associated alleles at other HLA loci (*HLA-A, HLA-B*, and *HLA-DPB1*) on DRB1\\*03:01 haplotypes suggests that DRB3\\*02:02 on the DRB1\\*03:01 haplotype can contribute to T1D risk.\nauthor: Henry A. Erlich; Ana Maria Valdes; Shana L. McDevitt; Birgitte B. Simen; Lisbeth A. Blake; Kim R. McGowan; John A. Todd; Stephen S. Rich; Janelle A. Noble; Corresponding author: Henry A. Erlich, <email@example.com>.\ndate: 2013-07\nreferences:\ntitle: Next Generation Sequencing Reveals the Association of DRB3\\*02:02 With Type 1 Diabetes\n\nThe Type 1 Diabetes Genetics Consortium (T1DGC) (1) is an international collaboration that has collected thousands of multiplex family samples, as well as case and control samples, and has carried out linkage and association analysis for genome-wide single nucleotide polymorphisms (SNPs), candidate gene SNPs, and major histocompatibility complex (MHC) SNPs, as well as for alleles and haplotypes at the highly polymorphic HLA class I and class II genes (2\u20137). More than 40 different loci have been associated with T1D (3); however, linkage and association analyses have identified the HLA region as the major genetic determinant of T1D risk. The most strongly T1D-associated MHC markers are defined by the HLA-DRB1, -DQA1, and -DQB1 haplotypes (4), although alleles at other HLA loci, notably *HLA-A* and *HLA-B*, as well as *HLA DPB1* (5\u201311) and other non-HLA loci across the genome, also contribute to T1D risk (3).\n\nAlthough the association of *HLA-DRB1* alleles with T1D is well established, the role of polymorphism at the *HLA-DRB3*, *HLA-DRB4*, and *HLA-DRB5* loci has been studied less frequently, partly due to technical issues in genotyping. Most high-resolution genotyping strategies for *HLA-DRB1* depend on DRB1-specific PCR primers to minimize confounding signals from coamplified secondary DRB loci. All copies of chromosome 6 have a DRB1 locus, and most, but not all, have a functional second DRB locus. This second DRB locus is DRB3 for DRB1\\*03, \\*11, \\*12, \\*13, and \\*14 haplotypes, DRB4 for DRB1\\*04, \\*07, and \\*09 haplotypes, and DRB5 for DRB1\\*15 and \\*16 haplotypes. DRB1\\*01, DRB1\\*08, and DRB1\\*10 haplotypes do not have a second DRB locus.\n\nThe clonal sequencing property of next generation sequencing systems, such as the Roche 454 GS FLX and GS Junior Systems, allows the use of generic DRB primers to coamplify and sequence exon 2 from DRB1, DRB3, DRB4, and DRB5 loci (12\u201314). We have used the Roche 454 amplicon sequencing system with \"fusion primers\" containing the 454 adaptor (A and B) sequences and 10 base multiplex ID tags (MIDs) to amplify and sequence exon 2 from different individuals (13,14). The genotyping software consolidates these clonal sequence readings, sorts them to individual DRB loci and to individual samples, and compares them to the IMGT\/HLA Sequence Database to assign specific genotypes at the *HLA-DRB1*, *HLA-DRB3*, *HLA-DRB4*, and *HLA-DRB5* loci.\n\nTo assess the potential role of these secondary HLA-DRB loci in T1D, association analyses were carried out at the two DRB locus haplotype levels, focusing on the DRB1\\*03:01 haplotypes bearing DRB3\\*01:01 or DRB3\\*02:02. The role of *HLA-DRB3* polymorphism in T1D risk was initially evaluated in a case\/control study and then examined in a targeted replication study of informative patients from multiplex HLA-genotyped families, allowing analysis of HLA haplotypes and the potential role of T1D-associated alleles at other HLA loci.\n\n# RESEARCH DESIGN AND METHODS\n\n## Subjects.\n\nThe initial set of DNA samples from 500 case and 500 control subjects was provided by the JDRF\/Wellcome Trust Diabetes and Inflammation Laboratory (case subjects) and the British 1958 Birth Cohort (control subjects) from a study described previously (15).\n\nTo test the hypothesis generated in the case\/control study, DNA samples from the T1DGC and the Human Biological Data Interchange (HBDI) multiplex family collections were used. Samples from 280 T1D-affected family members were selected if they had a DRB1\\*03 haplotype. Samples were selected based on the known T1D risk DRB1 genotypes, DRB1\\*03:01\/DRB1\\*03:01 (*n* = 54 T1D subjects; 105 independent DRB1\\*03:01 haplotypes), DRB1\\*03:01\/DRB1\\*04:01 (*n* = 154 DRB1\\*03:01 haplotypes), and DRB1\\*03:01\/DRB1\\*04:04 (*n* = 73 DRB1\\*03:01 haplotypes), with the aim of testing the hypothesis that DRB3\\*02:02 and \\*01:01 alleles may confer differential T1D risk. Parent samples with a DRB1\\*03:01 allele known not to be transmitted to an affected T1D patient were also selected as control subjects from the T1DGC and the HBDI collections (affected family-based association controls \\[AFBACs\\], *n* = 115 haplotypes) (16). These AFBAC control haplotypes were used to determine population frequencies of DRB3\\*02:02 and DRB3\\*01:01 alleles on DRB1\\*03:01 haplotypes.\n\n## HLA genotyping using next generation sequencing (Roche 454).\n\nHLA sequence data were generated on the Roche 454 GS FLX and GS Junior Systems and analyzed using Conexio Genomics HLA ASSIGN ATF genotyping software to interpret the sequence files as HLA genotypes (13,14). Amplicons were generated from genomic DNA using DRB generic exon 2 454 fusion primers. The 454 fusion primers consist of a locus-specific sequence on the 3\u2032 end, a 10-bp MID tag, and an \"A\" or \"B\" 454-specific primer sequence on the 5\u2032 end. The MID tag serves as a sample barcode recognized by Conexio ASSIGN ATF genotyping software.\n\nAmplicons were purified with AMPure beads (Becton Dickinson, Franklin Lakes, NJ), quantified using the Quant-iT PicoGreen dsDNA reagent (Life Technologies, Foster City, CA), and mixed with capture beads after dilution. Individual DRB exon 2 amplicon molecules captured by these beads were amplified in an emulsion PCR amplification and DNA-containing beads subsequently analyzed by pyrosequencing to obtain sequence readings originating from a single molecule (12\u201314). Sequencing on the GS FLX and GS Junior System was performed, as described (13,14). HLA genotypes were assigned to samples using Conexio ASSIGN ATF genotyping software, as described (13,14).\n\n## Statistical analysis.\n\nDRB3 allele frequencies on DR3 haplotypes in DR3 homozygotes, DR3\/DR4 heterozygotes, and AFBACs were compared using a Fisher exact test where the overall sample size was less than 50 or a Pearson \u03c7^2^ statistic.\n\n# RESULTS\n\n## Case\/control study.\n\nA total of 500 case and 500 control DNA samples from the Wellcome Trust\/JDRF Diabetes Inflammation Laboratory collection were amplified with 454 DRB fusion primers containing 32 MID tags and sequenced in two GS FLX System runs using PicoTiterPlates fitted with 16-region gaskets. The long read lengths of \\>400 bp spanned the amplicon in both directions and allowed setting phase (haplotyping) for the polymorphisms within exon 2. This provided, in most cases, unambiguous genotype assignments for *HLA-DRB1*, *HLA-DRB3*, and *HLA-DRB5*. For *HLA-DRB4*, however, several different genotypes were consistent with the observed sequence reads (ambiguity string). (The *HLA-DRB4* exon 2 sequence reads were identical for the DRB4\\*01:01, DRB4\\*01:03, and DRB4\\*01:06 alleles.) This DRB4 exon 2 sequence was present on all DRB1\\*04, \\*07, and \\*08 haplotypes; thus, the role of DRB4 alleles for association with T1D or for defining specific linkage disequilibrium (LD) patterns could not be evaluated.\n\nFor *HLA-DRB3* and *HLA-DRB5*, the allele assignments were unambiguous and, as expected, a pattern of very strong LD between *HLA-DRB1* and the secondary DRB locus was observed. The T1D association analyses were performed on the two-locus haplotypes (<a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>). In these data, DRB1\\*15:01 was linked exclusively to DRB5\\*01:01, and although the numbers were small (*n* = 6), DRB1\\*16:01 was linked exclusively to DRB5\\*02:02. Three common DRB3 alleles (\\*01:01, \\*02:02, \\*03:01) were observed. Of those haplotypes carrying a DRB3 locus, most DRB1 alleles were linked to a unique DRB3 allele in both case and control subjects (<a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>). The DRB1\\*03:01 and the DRB1\\*13:01 haplotypes, however, were linked to one of two different DRB3 alleles (\\*01:01 or \\*02:02).\n\nDRB1-DRB3 and DRB1-DRB5 haplotype frequencies in T1D case and control subjects\n\n![](2618tbl1)\n\nThus, the role of DRB3\\*01:01 versus DRB3\\*02:02 on DRB1\\*03:01 haplotypes could be evaluated. DRB1\\*03:01 haplotypes carrying DRB3\\*02:02 had a nominally higher risk for T1D; this difference in T1D risk was observed in the DRB1\\*03:01\/\\*03:01 homozygotes with two DRB3\\*01:01 alleles (odds ratio \\[OR\\] 3.4 \\[95% CI 1.46\u20138.09\\]) compared with those with one or two DRB3\\*02:02 alleles (25.5 \\[3.43\u2013189.2\\]; *P* = 0.033; <a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a>). For DRB1\\*03:01\/\\*04:01 heterozygotes, however, there was no difference in the T1D risk between DRB1\\*03:01 haplotypes distinguished by the DRB3 allele (OR 7.7 vs. 6.8; *P* = 0.29; <a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a>). That the apparent difference in risk for DRB1\\*03:01 haplotypes bearing DRB3\\*02:02 versus DRB3\\*01:01 is not evident in DRB1\\*03:01\/DRB1\\*04 heterozygotes may reflect the very high risk associated with this genotype and attributed, in part, to the *trans*-complementing DQ-\u03b1 (\\*05:01) or DQ-\u03b2 (\\*03:02) heterodimer (4).\n\nAssociation of DRB1\\*03:01\/03:01 and DRB1\\*03:01\/04:01 genotypes with T1D stratified by DRB3 genotype\n\n![](2618tbl2)\n\nAlthough many samples were sequenced in this case\/control study, the number of informative haplotypes (DRB1\\*03:01) and genotypes was limited; therefore, the statistical power of the association with DRB3:02:02 was very modest. Access to the T1DGC collection of HLA-genotyped families allowed selective targeting of informative genotypes to directly address the hypothesis of an effect of DRB3\\*02:02 in DRB1\\*03:01\/\\*03:01 homozygotes and replicate the results of the case\/control study.\n\n## Targeted replication study of informative samples from the T1DGC family collection.\n\nTo further evaluate the putative role of *HLA-DRB3* polymorphism on DRB1\\*03:01 haplotype risk, a panel of DRB1\\*03:01\/\\*03:01 homozygotes (*n* = 54 T1D family members, 105 nonshared chromosomes) and DRB1\\*03:01\/DRB1\\*04-DQB1\\*03:02 (*n* = 227) heterozygous T1D members were selected from the T1DGC families. Of the DRB1\\*03:01\/DRB1\\*04 subjects, 154 were \\*04:01 and 73 were \\*04:04. The distribution of DRB3 alleles in these T1D subjects is reported in <a href=\"#T3\" data-ref-type=\"table\">Table 3<\/a>. The frequency of the DRB3\\*02:02 allele in the DRB1\\*03:01\/\\*03:01 T1D members was significantly greater than in those with DRB1\\*03:01\/\\*04-DQB1\\*03:02 (42.9 vs. 27.6%, *P* \\< 0.005), consistent with the observations in the previous case\/control study (<a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a>). The distribution of DRB3 alleles on control DRB1\\*03:01 chromosomes was evaluated by examining the nontransmitted AFBAC (16) DRB1\\*03:01 chromosomes from heterozygous parents. The frequency of DRB3\\*02:02 in those chromosomes not transmitted to a T1D-affected child was 22.6%, significantly less than the 42.9% (*P* = 0.001) observed in the DRB1\\*03:01\/DRB1\\*03:01 homozygous patients but not significantly different from the frequency in DRB1\\*03:01\/DRB1\\*04:01 heterozygous T1D patients (27.3%).\n\nComparison of distribution of DRB3 alleles on DR3 haplotypes in control subjects (AFBAC), DRB1\\*03:01 homozygous patients, and DRB1\\*03:01\/DRB1\\*04 heterozygous patients\n\n![](2618tbl3)\n\n## Does DRB3\\*02:02 allele modify risk or is it simply a marker for a high-risk DRB1\\*03:01 haplotype?\n\nOn the basis of these two studies, the DRB3\\*02:02 allele appears to mark a higher-risk DRB1\\*03:01 haplotype than the DRB1\\*03:01-DRB3\\*01:01 haplotype, and the effect of this high-risk haplotype can be discerned primarily in the DRB1\\*03:01\/\\*03:01 homozygotes. T1D risk heterogeneity in DRB1\\*03:01 haplotypes has been previously reported with HLA-A\\*30:02, HLA-B\\*18:01, and DPB1\\*02:02 alleles distinguishing the higher-risk from the lower-risk DRB1\\*03:01 haplotypes (17). To investigate whether the DRB3\\*02:02 allele might contribute to T1D risk or simply mark a high-risk DRB1\\*03:01 haplotype in which HLA class I (or other alleles) was a modifying risk, we examined the distribution of these and other T1D-associated alleles at other HLA loci on these two (DRB3\\*01:01 and DRB3\\*02:02) DRB1\\*03:01 haplotypes. The increase of DRB3\\*02:02 on DRB1\\*03:01 haplotypes in DRB1\\*03:01\/\\*03:01 case subjects versus DRB1\\*03:01\/DRB1\\*04 case subjects and AFBAC control subjects could reflect LD with alleles at other high-risk loci. The analysis of the case subjects selected from T1DGC families with informative HLA-DRB1 genotypes included parents; thus, haplotypes across the HLA region could be determined.\n\nEight locus haplotypes were phased and assigned based on familial inheritance. In the absence of parental genotyping data for the DRB3 locus, only a fraction of these could be assigned phase unambiguously with regard to the remaining eight loci. In total, 327 DRB1\\*03:01 haplotypes were used for this analysis of extended HLA haplotypes. The DRB1\\*03:01 haplotype counts in the three groups (AFBAC, DRB1\\*03:01\/DRB1\\*04, DRB1\\*03:01\/DRB1\\*03:01) for selected DPB1, HLA-B, -C, and -A alleles are reported in <a href=\"#T4\" data-ref-type=\"table\">Table 4<\/a> and [Supplementary Table 1](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1387\/-\/DC1). These alleles were selected based on previously published reports of T1D association. <a href=\"#T4\" data-ref-type=\"table\">Table 4<\/a> and [Supplementary Table 1](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1387\/-\/DC1) compare the distribution of DRB3\\*01:01 and \\*02:02 alleles on DRB1\\*03:01 haplotypes bearing various high-risk alleles at other HLA loci in DRB1\\*03:01\/\\*03:01 versus DRB1\\*03:01\/\\*04 patients.\n\nDistribution of DRB3\\*02:02 and \\*01:01 alleles on the extended DRB1\\*03:01-DPB1\\*03:01 haplotype\n\n![](2618tbl4)\n\nWe note that the alleles A\\*01:01, B\\*08:01, C\\*07:02, and DPB1\\*01:01 are in very strong LD with DRB3\\*01:01. These alleles on this extended haplotype mark a \"lower-risk\" DRB1\\*03:01 haplotype. In addition, the LD between alleles on the extended DRB1\\*03:01 haplotype, A\\*30:02-B\\*18:01-DRB1\\*03:01-DRB3\\*02:02-DPB1\\*02:02 is sufficiently strong that it does not permit evaluation of the independent contribution of DRB3\\*02:02. One haplotype, the DRB1\\*03:01-DPB1\\*03:01, however, is informative; DRB1\\*03:01 haplotypes carrying both DPB1\\*03:01 and DRB3\\*02:02 are significantly increased (*P* = 0.0006) in DRB1\\*03:01\/\\*03:01 versus DRB1\\*03:01\/\\*04 case subjects compared with those who carry DRB3\\*01:01 (<a href=\"#T4\" data-ref-type=\"table\">Table 4<\/a>). This effect cannot be explained by the presence of A\\*30:02 or B\\*18:01, which are also markers of high-risk DRB1\\*03:01 haplotypes on these 16 DPB1\\*03:01-DRB3\\*02:02 haplotypes. Only 4 of these 16 haplotypes carry A\\*30:02, and of the 9 DPB1\\*03:01-DRB3\\*02:02 haplotypes that also carry B\\*18:01, 5 are found in DR3\/DR4 and 4 in DR3\/DR3 case subjects. These observations, taken together, suggest that the DRB3\\*02:02 allele may contribute to T1D risk rather than simply marking a high-risk DRB1\\*03:01 haplotype.\n\nDRB1\\*13:01 is the only other DRB1 haplotype that can carry DRB3\\*01:01 or DRB3\\*02:02 (<a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>). We note that the OR of the DRB1\\*13:01-DRB3\\*02:02 haplotype is nominally higher than the DRB1\\*13:01-DRB3\\*01:01 haplotype (0.29 vs. 0.15; <a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>). However, the CIs overlap, indicating that much larger sample sizes will be necessary to evaluate the risk of these two haplotypes.\n\n# DISCUSSION\n\nLong-read next generation clonal sequencing allows genotyping the secondary HLA-DRB loci (*HLA-DRB3*, *HLA-DRB4*, and *HLA-DRB5*) as well as *HLA-DRB1* by using DRB generic 454 fusion primers for exon 2. Using the Roche 454 GS FLX and GS Junior Systems, we have used this capability to examine the role of the secondary HLA-DRB loci in T1D susceptibility. Most *HLA-DRB1* alleles are linked exclusively to a specific allele at the secondary DRB locus (<a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>); however, DRB1\\*03:01 and DRB1\\*13:01 can carry DRB3\\*01:01 or \\*02:02. DRB1\\*03:01 haplotypes carrying DRB3\\*02:02 appear to confer higher risk than those carrying DRB3\\*01:01. The difference in T1D risk is observed in DRB1\\*03:01\/\\*03:01 homozygotes but not in DRB1\\*03:01\/DRB1\\*04-DQB1\\*03:02 heterozygotes. The very high risk for T1D associated with this heterozygous genotype has been attributed to the *trans*-complementing DQ heterodimer formed by the DQ-\u03b1 chain encoded by the DQA1\\*05:01 allele on the DRB1\\*03:01 haplotype and the DQ-\u03b2 chain encoded by the DQB1\\*03:02 on the DRB1\\*04-DQB1\\*03:02 haplotype (4). One possible explanation of the different effect of *HLA-DRB3* in this heterozygote and in the DRB1\\*03:01\/DRB1\\*03:01 homozygote is that the putative risk conferred by the *trans-*complementing DQ heterodimers in the heterozygote is sufficiently large so that the risk difference between the \"higher-risk\" and \"lower-risk\" DRB1\\*03:01 haplotypes has a minimal effect on the overall T1D risk for the heterozygote. A recent study on the effect of other MHC markers (*HLA-B*, *HLA-A*, *HLA-DPB1*, and *TNF-\u03b1*) on DRB1\\*03:01 haplotype risk reported a similar observation\u2014these markers were associated with differential risk in DRB1\\*03:01\/DRB1\\*03:01 homozygotes but not in the DRB1\\*03:01\/DRB1\\*04-DQB1\\*03:02 heterozygotes (17).\n\nDoes DRB3\\*02:02 play a role in T1D risk or does it only *mark* the higher-risk DRB1\\*03:01 haplotype? To address this question, eight locus DRB1\\*03:01 haplotypes were phased and assigned based on familial inheritance, and the distribution of the DRB3\\*01:01 and \\*02:02 alleles on extended haplotypes bearing high-risk alleles at other HLA loci was compared (<a href=\"#T4\" data-ref-type=\"table\">Table 4<\/a> and [Supplementary Table 1](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1387\/-\/DC1)). The linkage disequilibrium between A\\*30:02, B\\*18:01, DPB1\\*02:02, and DRB3\\*02:02 was so great (virtually 100%) that the effect of these alleles on this high-risk DRB1\\*03:01 haplotype could not be distinguished. Some DRB1\\*03:01-DRB3\\*02:02 haplotypes, however, *do not* carry these alleles, associated with the high-risk DRB1\\*03:01 haplotype, but still demonstrate a significant increase in DRB1\\*03:01\/\\*03:01 case subjects versus DRB1\\*03:01\/\\*04. In particular, DRB1\\*03:01 haplotypes bearing DPB1\\*03:01 can carry DRB3\\*01:01 or DRB3\\*02:02; those that carry DRB3\\*02:02 are dramatically increased among the DRB1\\*03:01\/\\*03:01 case subjects (*P* = 0.0006). These observations are consistent with the hypothesis that DRB3\\*02:02 is not simply a marker of high-risk haplotypes but, in fact, increases the risk of DRB1\\*03:01 haplotypes, particularly in DRB1\\*03:01\/\\*03:01 homozygotes.\n\nThe amino acid sequence differences between DRB3\\*01:01 and DRB3\\*02:02 are substantial (10 differences). Consequently, DRB3\\*02:02 is likely to have a different repertoire of peptide binding and presentation and may thus confer greater T1D risk than DRB3\\*01:01 in certain genotype contexts (i.e., DRB1\\*03:01\/\\*03:01 homozygotes) through altered peptide binding and T-cell repertoire. The crystallographic structure of the DRB3\\*01:01 (DR52a) and the DRB3\\*03:01 (DR52c) molecules have been reported recently (18,19). The P9 pocket of the DRB3\\*02:02 (DR52b) differs substantially from that of DRB3\\*01:01 (DR52a); in particular, it contains Tyr-37, Ala-38, Asp-57, and Tyr-60, making the pocket more accommodating to smaller, polar, or charged peptide residues. A structural model with bound peptide of the DRB3\\*02:02 molecule with the differences highlighted is shown in [Supplementary Fig. 1](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1387\/-\/DC1).\n\nThe results presented here support the conclusion that the T1D risk of a given HLA haplotype is determined by specific combinations of alleles at a variety of HLA loci with genotype-dependent effects and support a role for the DRB3 locus in T1D susceptibility conferred by the DRB1\\*03:01 haplotype.\n\n# Supplementary Material\n\n###### Supplementary Data\n\n## ACKNOWLEDGMENTS\n\nThis research used resources provided by the T1DGC, a collaborative clinical study sponsored by the National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Allergy and Infectious Diseases, National Human Genome Research Institute, National Institute of Child Health and Human Development, and JDRF and was supported by U01 DK-062418. This work also was supported in part by National Institutes of Health Grant DK-61722 (J.A.N.).\n\nNo potential conflicts of interest relevant to this article were reported.\n\nH.A.E. designed the initial study, designed the second study with the support of the T1DGC Steering Committee, and drafted the manuscript. A.M.V. provided statistical analyses and edited the manuscript. S.L.M. contributed to the manuscript and with L.A.B. and K.R.M., they all contributed to generating sequence data. B.B.S. contributed to generating sequence data and edited the manuscript. J.A.T. provided samples for the first study and edited the manuscript. S.S.R. edited the manuscript. J.A.N. designed the second study with the support of the T1DGC Steering Committee and edited the manuscript. H.A.E. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.\n\nThe authors are grateful to the Wellcome Trust and JDRF support for J.A.T., and also to Neil Walker and Helen Stevens, of the JDRF\/Wellcome Trust Diabetes and Inflammation Laboratory, for providing DNA samples and HLA typing information.\n\n# REFERENCES","meta":{"dup_signals":{"dup_doc_count":120,"dup_dump_count":47,"dup_details":{"curated_sources":2,"2021-10":1,"2020-16":1,"2019-43":1,"2019-39":1,"2019-30":1,"2018-30":1,"2018-26":2,"2018-17":3,"2018-09":1,"2018-05":3,"2017-47":2,"2017-43":4,"2017-39":2,"2017-34":2,"2017-30":5,"2017-26":3,"2017-22":2,"2017-17":8,"2017-09":4,"2017-04":6,"2016-50":3,"2016-44":4,"2016-40":3,"2016-36":3,"2016-30":3,"2016-26":3,"2016-22":3,"2016-18":3,"2016-07":2,"2015-48":2,"2015-35":1,"2015-32":2,"2015-27":2,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":3,"2014-42":2,"2014-41":2,"2014-35":1,"2014-23":1,"2023-40":1,"2017-13":4,"2015-18":3,"2015-11":2,"2015-06":2,"2024-30":1}},"file":"PMC3712046"},"subset":"pubmed_central"}
{"text":"abstract: The \"Spanish\" influenza pandemic of 1918\u20131919, which caused \u224850 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tissues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.\nauthor: Jeffery K. Taubenberger; David M. MorensJeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: <firstname.lastname@example.com>\ndate: 2006-01\nreferences:\ntitle: 1918 Influenza: the Mother of All Pandemics\n\n> \"Curiouser and curiouser!\" cried Alice\n>\n> Lewis Carroll, Alice's Adventures in Wonderland, 1865\n\nAn estimated one third of the world's population (or \u2248500 million persons) were infected and had clinically apparent illnesses (*1**,**2*) during the 1918\u20131919 influenza pandemic. The disease was exceptionally severe. Case-fatality rates were \\>2.5%, compared to \\<0.1% in other influenza pandemics (*3**,**4*). Total deaths were estimated at \u224850 million (*5**\u2013**7*) and were arguably as high as 100 million (*7*).\n\nThe impact of this pandemic was not limited to 1918\u20131919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide (excepting human infections from avian viruses such as H5N1 and H7N7), have been caused by descendants of the 1918 virus, including \"drifted\" H1N1 viruses and reassorted H2N2 and H3N2 viruses. The latter are composed of key genes from the 1918 virus, updated by subsequently incorporated avian influenza genes that code for novel surface proteins, making the 1918 virus indeed the \"mother\" of all pandemics.\n\nIn 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza viruses (now known to be H1N1 viruses) were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic (*8*). Subsequent research indicates that descendants of the 1918 virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 (\"Asian flu\"), the direct H1N1 viral descendants of the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 viruses suddenly \"reemerged\" from a laboratory freezer (*9*). They continue to circulate endemically and epidemically.\n\nThus in 2006, 2 major descendant lineages of the 1918 H1N1 virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic\/endemic H1N1 lineage, a porcine enzootic H1N1 lineage (so-called classic swine flu), and the reassorted human H3N2 virus lineage, which like the human H1N1 virus, has led to a porcine H3N2 lineage. None of these viral descendants, however, approaches the pathogenicity of the 1918 parent virus. Apparently, the porcine H1N1 and H3N2 lineages uncommonly infect humans, and the human H1N1 and H3N2 lineages have both been associated with substantially lower rates of illness and death than the virus of 1918. In fact, current H1N1 death rates are even lower than those for H3N2 lineage strains (prevalent from 1968 until the present). H1N1 viruses descended from the 1918 strain, as well as H3N2 viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction.\n\n# Trying To Understand What Happened\n\nBy the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and substantially lowered death rates. Did some critical viral genetic event produce a 1918 virus of remarkable pathogenicity and then other critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 virus?\n\nIn 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small viral RNA fragments to determine the genomic structure of the causative influenza virus (*10*). These efforts have now determined the complete genomic sequence of 1 virus and partial sequences from 4 others. The primary data from the above studies (*11**\u2013**17*) and a number of reviews covering different aspects of the 1918 pandemic have recently been published (*18**\u2013**20*) and confirm that the 1918 virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the \"extinct\" H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza viruses have been found in the 1918 genome, but ongoing studies to map virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the origin of the virus (*19*) and about the epidemiology of the pandemic.\n\n# When and Where Did the 1918 Influenza Pandemic Arise?\n\nBefore and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an \u224812-month period in 1918\u20131919, in Europe, Asia, and North America (the first wave was best described in the United States in March 1918). Historical and epidemiologic data are inadequate to identify the geographic origin of the virus (*21*), and recent phylogenetic analysis of the 1918 viral genome does not place the virus in any geographic context (*19*).\n\nAlthough in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 (*22*). Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succession by much more fatal second and third waves in the fall and winter of 1918\u20131919, respectively (<a href=\"#F1\" data-ref-type=\"fig\">Figure 1<\/a>). Is it possible that a poorly-adapted H1N1 virus was already beginning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consistent with this possibility were reported at the time from European military camps (*23*), but a counter argument is that if a strain with a new hemagglutinin (HA) was causing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partially immunoprotected. \"Herald\" events in 1915, 1916, and possibly even in early 1918, if they occurred, would be difficult to identify.\n\nThe 1918 influenza pandemic had another unique feature, the simultaneous (or nearly simultaneous) infection of humans and swine. The virus of the 1918 pandemic likely expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 (*12**,**20*). Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neuraminidase (NA) surface proteins of the 1918 virus were derived from an avianlike influenza virus shortly before the start of the pandemic and that the precursor virus had not circulated widely in humans or swine in the few decades before (*12**,**15**,**24*). More recent analyses of the other gene segments of the virus also support this conclusion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the initial circulation of the 1918 precursor virus in humans at approximately 1915\u20131918 (*20*). Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date (*19*). In summary, its origin remains puzzling.\n\n# Were the 3 Waves in 1918\u20131919 Caused by the Same Virus? If So, How and Why?\n\nHistorical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza viruses behave differently when they find a universal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immunity, these pandemic viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recurrences caused by the drifted virus variants.\n\nIn the 1918\u20131919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months (<a href=\"#F1\" data-ref-type=\"fig\">Figure 1<\/a>). Illness rates were high, but death rates in most locales were not appreciably above normal. A second or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 (*21*). Clinical similarities led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandemic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infections to fatal pneumonia, a hallmark of the 1918\u20131919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher frequency of complicated, severe, and fatal cases in the last 2 waves.\n\nBut 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recurrent annual outbreaks, are driven by viral antigenic drift, with an antigenic variant virus emerging to become dominant approximately every 2 to 3 years. Without such drift, circulating human influenza viruses would presumably disappear once herd immunity had reached a critical threshold at which further virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting virus spread in all but the most favorable circumstances, which include lower environmental temperatures and human nasal temperatures (beneficial to thermolabile viruses such as influenza), optimal humidity, increased crowding indoors, and imperfect ventilation due to closed windows and suboptimal airflow.\n\nHowever, such factors cannot explain the 3 pandemic waves of 1918\u20131919, which occurred in the spring-summer, summer-fall, and winter (of the Northern Hemisphere), respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave periods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could susceptible persons be too few to sustain transmission at 1 point and yet enough to start a new explosive pandemic wave a few weeks later? Could the virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And having occurred, such mutated viruses normally take months to spread around the world.\n\nAt the beginning of other \"off season\" influenza pandemics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 (more than a year after the first pandemic appearance) and the third in early 1892 (*21*). As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recurrences in 1889\u20131892, however, were spread over \\>3 years, in contrast to 1918\u20131919, when the sequential waves seen in individual countries were typically compressed into \u22488\u20139 months.\n\nWhat gave the 1918 virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic virus samples we have yet identified are from second-wave patients (*16*), nothing can yet be said about whether the first (spring) wave, or for that matter, the third wave, represented circulation of the same virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been protected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do little to resolve the question of whether the first wave was caused by the same virus or whether major genetic evolutionary events were occurring even as the pandemic exploded and progressed. Only influenza RNA\u2013positive human samples from before 1918, and from all 3 waves, can answer this question.\n\n# What Was the Animal Host Origin of the Pandemic Virus?\n\nViral sequence data now suggest that the entire 1918 virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the contrary, the 1918 virus appears to be an avianlike influenza virus derived in toto from an unknown source (*17**,**19*), as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza virus gene sequences from a number of fixed specimens of wild birds collected circa 1918 show little difference from avian viruses isolated today, indicating that avian viruses likely undergo little antigenic change in their natural hosts even over long periods (*24**,**25*).\n\nFor example, the 1918 nucleoprotein (NP) gene sequence is similar to that of viruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains (*13**,**19*). One way of looking at the evolutionary distance of genes is to compare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a viral gene subjected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance.\n\nBecause the 1918 gene segments have more synonymous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid differences from those of wild-bird strains to have spent many years adapting only in a human or swine intermediate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 virus come from?\n\nIn contrast to the genetic makeup of the 1918 pandemic virus, the novel gene segments of the reassorted 1957 and 1968 pandemic viruses all originated in Eurasian avian viruses (*26*); both human viruses arose by the same mechanism\u2014reassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences.\n\n# What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?\n\nSequence analysis alone does not offer clues to the pathogenicity of the 1918 virus. A series of experiments are under way to model virulence in vitro and in animal models by using viral constructs containing 1918 genes produced by reverse genetics.\n\nInfluenza virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza viruses adapted to infect birds and those adapted to infect humans. Influenza virus strains adapted to birds preferentially bind sialic acid receptors with \u03b1 (2\u20133) linked sugars (*27**\u2013**29*). Human-adapted influenza viruses are thought to preferentially bind receptors with \u03b1 (2\u20136) linkages. The switch from this avian receptor configuration requires of the virus only 1 amino acid change (*30*), and the HAs of all 5 sequenced 1918 viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments virus binding to the human receptor may also occur, but only 3 of 5 1918 HA sequences have it (*16*).\n\nThis means that at least 2 H1N1 receptor-binding variants cocirculated in 1918: 1 with high-affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chronologic indication exists to suggest that one of these variants was the precursor of the other, nor are there consistent differences between the case histories or histopathologic features of the 5 patients infected with them. Whether the viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown.\n\nIn a series of in vivo experiments, recombinant influenza viruses containing between 1 and 5 gene segments of the 1918 virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice (*31*). Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA\/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage (*32*). These findings are unexpected because the viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human viruses showed little disease and limited viral replication. The lungs of animals infected with the 1918 HA\/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA (and possibly the NA) contain virulence factors for mice. The viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 proteins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of virulence of the 1918 virus in various animal models are planned. These experiments may help define the viral component to the unusual pathogenicity of the 1918 virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns.\n\n# Why Did the 1918 Virus Kill So Many Healthy Young Adults?\n\nThe curve of influenza deaths by age at death has historically, for at least 150 years, been U-shaped (<a href=\"#F2\" data-ref-type=\"fig\">Figure 2<\/a>), exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a \"W-shaped\" curve, similar to the familiar U-shaped curve but with the addition of a third (middle) distinct peak of deaths in young adults \u224820\u201340 years of age. Influenza and pneumonia death rates for those 15\u201334 years of age in 1918\u20131919, for example, were \\>20 times higher than in previous years (*35*). Overall, nearly half of the influenza-related deaths in the 1918 pandemic were in young adults 20\u201340 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those \\<65 years of age than in those \\>65; persons \\<65 years of age accounted for \\>99% of all excess influenza-related deaths in 1918\u20131919. In comparison, the \\<65-year age group accounted for 36% of all excess influenza-related deaths in the 1957 H2N2 pandemic and 48% in the 1968 H3N2 pandemic (*33*).\n\nA sharper perspective emerges when 1918 age-specific influenza morbidity rates (*21*) are used to adjust the W-shaped mortality curve (<a href=\"#F3\" data-ref-type=\"fig\">Figure 3<\/a>, panels, A, B, and C \\[35,37\\]). Persons \\<35 years of age in 1918 had a disproportionately high influenza incidence (<a href=\"#F3\" data-ref-type=\"fig\">Figure 3<\/a>, panel A). But even after adjusting age-specific deaths by age-specific clinical attack rates (<a href=\"#F3\" data-ref-type=\"fig\">Figure 3<\/a>, panel B), a W-shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case-fatality curves typically seen in other influenza years, e.g., 1928\u20131929 (<a href=\"#F3\" data-ref-type=\"fig\">Figure 3<\/a>, panel C). Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the virus to host and environmental factors, possibly including immunopathology (e.g., antibody-dependent infection enhancement associated with prior virus exposures \\[38\\]) and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents.\n\nOne theory that may partially explain these findings is that the 1918 virus had an intrinsically high virulence, tempered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating virus capable of providing partial immunoprotection against the 1918 virus strain only in persons old enough (\\>35 years) to have been infected during that prior era (*35*). But this theory would present an additional paradox: an obscure precursor virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later.\n\nEpidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evidence for the emergence of an antigenically novel influenza virus in 1918 (*21*). Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the \\>65-year age group accounted for 6% of influenza cases. But in 1918, cases in the 5- to 15-year-old group jumped to 25% of influenza cases (compatible with exposure to an antigenically novel virus strain), while the \\>65 age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related viral protein to which older persons had once been exposed. Mortality data are in accord. In 1918, persons \\>75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 1911\u20131917. At the other end of the age spectrum (<a href=\"#F2\" data-ref-type=\"fig\">Figure 2<\/a>), a high proportion of deaths in infancy and early childhood in 1918 mimics the age pattern, if not the mortality rate, of other influenza pandemics.\n\n# Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It?\n\nIn its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordinary number of global deaths, most influenza cases in 1918 (\\>95% in most locales in industrialized nations) were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombinant influenza viruses containing genes from the 1918 virus suggest that the 1918 and 1918-like viruses would be as sensitive as other typical virus strains to the Food and Drug Administration\u2013approved antiinfluenza drugs rimantadine and oseltamivir.\n\nHowever, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5\u201320 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year's time (<a href=\"#F1\" data-ref-type=\"fig\">Figure 1<\/a>). Each of these unique characteristics may reflect genetic features of the 1918 virus, but understanding them will also require examination of host and environmental factors.\n\nUntil we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.\n\nLike the 1918 virus, H5N1 is an avian virus (*39*), though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the current situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic \"precursor\" virus caused a highly pathogenic outbreak in domestic poultry, and no highly pathogenic avian influenza (HPAI) virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza virus human cell adaptation (e.g., receptor binding) are beginning to be understood at the molecular level, the basis for viral adaptation to efficient human-to-human spread, the chief prerequisite for pandemic emergence, is unknown for any influenza virus. The 1918 virus acquired this trait, but we do not know how, and we currently have no way of knowing whether H5N1 viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 \"swine flu\" emergence in 1976.\n\nEven with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pandemic virus equivalent in pathogenicity to the virus of 1918 would likely kill \\>100 million people worldwide. A pandemic virus with the (alleged) pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.\n\nWhether because of viral, host or environmental factors, the 1918 virus causing the first or 'spring' wave was not associated with the exceptional pathogenicity of the second (fall) and third (winter) waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for virulence by allowing differences in viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 virus. Surveillance and genomic sequencing of large numbers of animal influenza viruses will help us understand the genetic basis of host adaptation and the extent of the natural reservoir of influenza viruses. Understanding influenza pandemics in general requires understanding the 1918 pandemic in all its historical, epidemiologic, and biologic aspects.\n\nDr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular pathophysiology and evolution of influenza viruses.\n\nDr Morens is an epidemiologist with a long-standing interest in emerging infectious diseases, virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases.\n\n# References","meta":{"dup_signals":{"dup_doc_count":113,"dup_dump_count":64,"dup_details":{"curated_sources":1,"2023-23":1,"2023-14":1,"2023-06":2,"2022-49":2,"2022-40":2,"2022-27":2,"2021-49":2,"2021-43":3,"2021-31":3,"2021-21":1,"2021-17":3,"2021-10":1,"2021-04":1,"2020-50":1,"2020-40":1,"2020-29":1,"2020-16":1,"2020-05":2,"2019-43":1,"2019-30":2,"2019-26":1,"2019-22":1,"2019-09":2,"2018-51":1,"2018-47":1,"2018-39":1,"2018-26":1,"2018-22":3,"2018-17":2,"2018-13":1,"2018-09":1,"2018-05":3,"2017-51":2,"2017-47":2,"2017-43":3,"2017-39":3,"2017-34":2,"2017-30":2,"2017-26":3,"2017-22":1,"2017-17":1,"2017-09":2,"2017-04":1,"2016-50":1,"2016-44":2,"2016-40":3,"2016-36":1,"2016-30":2,"2016-26":1,"2016-22":1,"2015-48":2,"2015-40":1,"2015-35":2,"2015-32":1,"2015-27":2,"2015-22":4,"2015-14":2,"2014-52":2,"2014-42":1,"2014-41":5,"2014-35":1,"2014-23":3,"2014-15":1,"2023-40":1}},"file":"PMC3291398"},"subset":"pubmed_central"}
{"text":"abstract: # Objective\n  .\n  To evaluate the long-term safety (up to 3\u2005years) of treatment with pegloticase in patients with refractory chronic gout.\n  .\n  # Methods\n  .\n  This open-label extension (OLE) study was conducted at 46 sites in the USA, Canada and Mexico. Patients completing either of two replicate randomised placebo-controlled 6-month trials received pegloticase 8\u2005mg every 2\u2005weeks (biweekly) or every 4\u2005weeks (monthly). Safety was evaluated as the primary outcome, with special interest in gout flares and infusion-related reactions (IRs). Secondary outcomes included urate-lowering and clinical efficacy.\n  .\n  # Results\n  .\n  Patients (n=149) received a mean\u00b1SD of 28\u00b118 pegloticase infusions and were followed for a mean of 25\u00b111\u2005months. Gout flares and IRs were the most frequently reported adverse events; these were least common in patients with a sustained urate-lowering response to treatment and those receiving biweekly treatment. In 10 of the 11 patients with a serious IR, the event occurred when uric acid exceeded 6\u2005mg\/dl. Plasma and serum uric acid levels remained \\<6\u2005mg\/dl in most randomised controlled trial (RCT)-defined pegloticase responders throughout the OLE study and were accompanied by sustained and progressive improvements in tophus resolution and flare incidence.\n  .\n  # Conclusions\n  .\n  The safety profile of long-term pegloticase treatment was consistent with that observed during 6\u2005months of RCT treatment; no new safety signals were identified. Improvements in clinical status, in the form of flare and tophus reduction initiated during RCT pegloticase treatment in patients maintaining goal range urate-lowering responses were sustained or advanced during up to 2.5\u2005years of additional treatment.\nauthor: Michael A Becker; Herbert S B Baraf; Robert A Yood; Aileen Dillon; Janitzia V\u00e1zquez-Mellado; Faith D Ottery; Dinesh Khanna; John S Sundy[^1]Correspondence to Dr Michael A Becker, 237 East Delaware Pl, Chicago, IL 60611-1713, USA; <email@example.com>\ndate: 2013-09\ninstitute: 1Rheumatology Section, The University of Chicago, Chicago, Illinois, USA; 2Center for Rheumatology and Bone Research, Wheaton, Maryland, USA; 3Reliant Medical Group, Worcester, Massachusetts, USA; 4Rheumatology Section, Kaiser Permanente Medical Center, San Francisco, California, USA; 5Department of Rheumatology, Hospital General de Mexico, Mexico City, Mexico; 6Savient Pharmaceuticals, Inc., East Brunswick, New Jersey, USA; 7Department of Medicine, University of Michigan, Ann Arbor, Michigan, USA; 8Duke Clinical Research Unit, Duke University Medical Center, Durham, North Carolina, USA\nreferences:\ntitle: Long-term safety of pegloticase in chronic gout refractory to conventional treatment\n\n# Introduction\n\nUrate-lowering therapy is the mainstay of chronic gout management and aims at achieving and maintaining sub-saturating serum uric acid (SUA) concentrations, most often recommended as \u22640.36\u2005mmol\/l (\u22646\u2005mg\/dl).1 2 Long-term maintenance of goal range SUA is associated with depletion of urate crystals in synovial fluid3 4 and reductions in tophus size and flare frequency.5\u20137 Because the rates of achieving these improvements are related to the degree of urate-lowering,4 6 8 the optimal therapeutic target may be substantially lower than 6\u2005mg\/dl in patients with severe manifestations of chronic gout,5 prompting one recommendation for a target SUA of \\<5\u2005mg\/dl.9\n\nUp to 3% of the estimated eight million patients with gout in the USA10 fail urate-lowering management with standard-of-care therapies (oral xanthine oxidase inhibitors) because of drug intolerance\/contraindication or treatment refractoriness.11\u201313 Such patients are at risk of progression to increasing flare recurrences, gouty arthropathy, destructive and deforming tophi and chronic pain, frequently accompanied by impaired physical function and poor health-related quality of life. Until recently these patients had few or no effective urate-lowering options to prevent or reverse gout progression. Pegloticase, a mammalian recombinant uricase conjugated to monomethoxypoly(ethylene glycol) (mPEG), was developed as an enzymatic alternative for the treatment of patients with gout refractory to conventional oral therapies and was approved in the USA in 2010.14\u201316 When administered intravenously, pegloticase reduces the urate concentration in the intravascular space to below the limit of solubility (6.8\u2005mg\/dl). The resulting reduction in extracellular soluble urate concentration is hypothesised to favour dissolution of deposited urate crystals, resulting in progressive normalisation of body urate pools and improvements in clinical signs and symptoms of gout.17 18\n\nThe tolerability and efficacy of pegloticase treatment in patients with refractory chronic gout were demonstrated in replicate 6-month randomised double-blind placebo-controlled trials (RCTs).17 Pegloticase administered every 2\u2005weeks (biweekly) or every 4\u2005weeks (monthly) produced treatment responses (plasma uric acid (PUA)\\<6\u2005mg\/dl for \u226580% of the time during months 3 and 6) in 42% and 35% of patients, respectively, compared with 0% for patients receiving placebo. PUA normalised within 24\u2005h of the first pegloticase infusion in all patients, but in non-responders the urate-lowering response was lost over time. Infusion-related reactions (IRs) were the most common reason for discontinuations in the RCTs (10% of biweekly treated patients and 13% of monthly treated patients).17 This report focuses on the long-term open-label extension (OLE) of the RCTs which provided an additional 2.5\u2005years of safety data.\n\n# Methods\n\n## Study design and patients\n\nThe OLE (NCT01356498) enrolled patients at 46 centres in the USA, Canada and Mexico who had completed either of the two RCTs (NCT01356498, NCT00325195).17 The OLE was conducted from December 2006 to July 2009; compliance and permission information is provided in the online supplementary material. Protocol amendments extended the OLE from 12\u2005months to a maximum of 30\u2005months. <a href=\"#ANNRHEUMDIS2012201795F1\" data-ref-type=\"fig\">Figure\u00a01<\/a> summarises the OLE study design, inclusion criteria (exclusion criteria are presented in the online supplementary material), end points and evaluations. Delays in treatment between the randomised trials and the OLE resulted from administrative requirements, such as time for Institutional Review Board approvals and site implementation. Except where specifically indicated, patients were categorised for analysis purposes according to the first dosing regimen administered during the OLE study.\n\nDetails of safety evaluations and prophylaxis regimens for IRs and flares are provided in the online supplementary material. As IRs were one of the events of interest, physical examinations and monitoring of vital signs were performed at the time of all IRs and medical intervention was provided as appropriate. For IRs occurring during infusions, the infusion could be discontinued, slowed by half, or interrupted and later restarted at a slower rate.\n\nImmunogenicity was assessed from serum collected every 12\u2005weeks using a validated ELISA to detect IgG and IgM pegloticase antibody. Antibody titres were categorised as low (\u22641\u2009:\u20092430) or high (\\>1\u2009:\u20092430), consistent with the RCTs.17 Tophus complete response was defined as 100% reduction in the measured area of at least one tophus of baseline diameter \u22655\u2005mm without growth of any other baseline tophus or appearance of any new tophus.\n\n## Statistical analysis\n\nSafety and efficacy end points were evaluated using descriptive statistics in the intent-to-treat population, which included all patients who received at least one dose of pegloticase during the OLE study and had follow-up data. Demographic and baseline clinical values are described in the online supplementary material.\n\n# Results\n\n## Patient disposition\n\nA total of 225 patients were enrolled in the two RCTs and 212 patients were included in the primary efficacy analysis. Of these, 157 patients (74%) completed the RCTs and 151 (96% of completers) entered the OLE study, including 57 (97%) patients from the biweekly pegloticase group, 55 (93%) from the monthly pegloticase group and all 39 patients from the placebo group (<a href=\"#ANNRHEUMDIS2012201795F2\" data-ref-type=\"fig\">figure 2<\/a>). All patients received pegloticase in the OLE study, except for two patients who chose observation only after receiving monthly pegloticase in the RCTs. For patients treated with pegloticase in the RCTs, a higher proportion of those defined as responders than those defined as non-responders completed the OLE study, as did a higher proportion of patients allocated to placebo in the RCTs who then received biweekly pegloticase compared with those who received monthly pegloticase (<a href=\"#ANNRHEUMDIS2012201795F2\" data-ref-type=\"fig\">figure 2<\/a>). The most common reasons for discontinuing treatment during the OLE study were adverse events (AEs) in 18% (27\/149) of patients and loss of urate-lowering response in 9% (13\/149).\n\n## Patients\n\nThe OLE study population had a mean age of 56.8\u2005years (range 30\u201389) and 79% were men. Most patients were white (69%), African-American (13%) or Hispanic\/Latino (11%). Risk factors were common; the most common were cardiovascular (94% of patients had at least one cardiovascular risk factor, most commonly hypertension, 72%), obesity (body mass index (BMI) \u226530\u2005kg\/m^2^, 65%) and dyslipidaemia (52%). Chronic kidney disease (creatinine clearance \\<1.0\u2005ml\/s) was present in 26% of patients. Less commonly occurring risk factors and concomitant medications are described in the online supplementary material.\n\n## Exposure to pegloticase\n\nPatients received a mean of 28 pegloticase infusions (median 26; range 1\u201359) during the OLE study, with RCT responders receiving more infusions than non-responders (mean 35 vs 26). When exposure to pegloticase was pooled from the RCT and OLE studies, patients received a mean of 35 infusions (range 1\u201370). Thirty of 67 patients (45%) who started on monthly pegloticase switched to biweekly treatment at some point during the OLE study; only 12% (10\/82) switched from biweekly to monthly treatment. Overall, patients remained in the OLE study for a mean of 25\u2005months (median 29\u2005months; range 0\u221237\u2005months, including a mandated end-of-study observation period (no treatment) for a maximum of 6\u2005months).\n\n## Safety\n\n### Adverse events (AEs)\n\nNearly all patients (98%) had at least one AE during the OLE study (<a href=\"#ANNRHEUMDIS2012201795TB1\" data-ref-type=\"table\">table 1<\/a>). The overall incidence of AEs did not differ between responders and non-responders from the RCTs or for patients initially on placebo who started pegloticase biweekly versus monthly. Both gout flares and IRs occurred at a lower rate with the biweekly regimen during the OLE study (2.7 per patient per year vs 4.7 with the monthly regimen and 1.3 per patient per year vs 2.1, respectively).\n\nSummary of AEs during the OLE study\n\n|  | All treated patients (N=149) |\n|:---|----|\n| AEs in the OLE study | N (%) |\n| Subjects with any AE | 146 (98) |\n| Subjects with any serious AE | 51 (34) |\n| Subjects with serious AEs considered related to pegloticase | 13 (9) |\n| Discontinuations due to AE | 11 (7) |\n| Most common AEs (incidence \\>10%) |  |\n| \u2003Gout flare | 106 (71) |\n| \u2003Infusion-related reaction | 65 (44) |\n| \u2003Arthralgia | 29 (20) |\n| \u2003Upper respiratory tract infection | 27 (18) |\n| \u2003Pain in extremity | 26 (17) |\n| \u2003Back pain | 25 (17) |\n| \u2003Diarrhoea | 22 (15) |\n| \u2003Peripheral oedema | 21 (14) |\n| \u2003Urinary tract infection | 20 (13) |\n| \u2003Nausea | 17 (11) |\n| \u2003Headache | 16 (11) |\n| \u2003Fatigue | 15 (10) |\n| \u2003Sinusitis | 15 (10) |\n| \u2003Nasopharyngitis | 15 (10) |\n\nAE, adverse event; OLE, open-label extension.\n\nThe incidence of gout flares during the OLE study was lowest for responders who had received biweekly pegloticase in the RCTs and highest for RCT non-responders to monthly pegloticase and patients treated with placebo in the RCTs. The incidence of IRs in the OLE study was lowest for RCT responders regardless of pegloticase schedule and highest for RCT non-responders to monthly pegloticase and RCT placebo patients starting monthly pegloticase. Eleven patients (7%) discontinued pegloticase due to AEs, including seven patients with IRs; six of these seven patients were RCT pegloticase non-responders or RCT placebo-treated patients. Among all patients with AEs judged as treatment-related by the investigators (66%, 99\/149), only gout flares and IRs were reported in \\>5% of patients.\n\nMost AEs (53% of patients) were investigator-rated as moderate in intensity. Overall, 36% (54\/149) of patients had severe AEs, of which 17% were treatment-related, most commonly IRs (7%) or gout flares (7%). Of note, no RCT pegloticase responder had a severe treatment-related IR or a severe gout flare. The highest rates of severe IRs and flares (26% for both; 6\/23) were reported in patients treated with biweekly pegloticase during the OLE study who had received placebo in the RCTs. Assessments of haematology, clinical chemistry and urinalysis identified no significant change from baseline (except for UA) in any of the subgroups defined by response to pegloticase or pegloticase administration schedule.\n\nTwenty-four adjudicated cardiovascular events in 21 patients were identified in the OLE study by the independent expert committee. These events occurred in responders and non-responders with no apparent relationship to the duration of treatment or time since the last pegloticase infusion. Eight of these events occurred in patients who were being observed and had not received pegloticase for \\>30\u2005days.\n\n### Serious AEs and deaths\n\nApproximately one-third of patients (34%, 51\/149) experienced a total of 106 serious AEs during the OLE study. Thirteen patients who reported serious AEs discontinued treatment. Among the 13 serious AEs considered possibly related to study drug, there were 11 IRs (one serious AE of IR was judged unlikely to be related to the study drug), one skin necrosis (severe) and one nephrolithiasis (moderate). Among the 11 serious AEs of IR, all but one occurred when the UA values exceeded 6 mg\/dl. Four deaths occurred during the OLE study, all of which were judged as unlikely to be related to the study drug by the investigator (see online supplementary material).\n\n### IRs\n\nDuring the RCTs, IRs were the second most common AE and were reported in 26% of patients receiving biweekly pegloticase and 42% of patients receiving monthly pegloticase. In the OLE, IRs were reported in 44% (60\/149) of patients. The rate of IRs was lower in RCT responders (17%, 10\/60) than in non-responders (52%, 26\/50) and highest among patients who received placebo in the RCT (62%, 24\/39). IRs were rated mild in 27% (16\/60) of patients, moderate in 55% (33\/60) and severe in 18% (11\/60). IRs rated as severe in the OLE occurred in four patients, none of whom sustained goal range urate lowering, and in seven patients who received placebo in the RCT. IRs were the reason cited for withdrawal from the study for 6% (9\/149) of patients.\n\nExcept for three patients with IRs manifesting in the 2\u2005h period after infusion, all IRs occurred during infusion. The most common signs and symptoms associated with IRs were musculoskeletal pain\/discomfort, flushing, erythema, nausea\/vomiting, dyspnoea, sweating, headache, blood pressure changes, urticaria and pruritus. All IRs resolved with supportive measures and no patient required intubation, mechanical ventilatory support, pressors or hospitalisation. Three patients were found to have anaphylaxis based on a retrospective analysis. Symptoms included red, itching or swollen eyes, throat irritation, musculoskeletal symptoms, skin flushing, hypotension, dizziness and vomiting.\n\n### Immunogenicity\n\nA total of 31% (52\/169) of patients treated with pegloticase had high pegloticase antibody titres (\\>1\u2009:\u20092430) during the RCTs. High-titre antibodies were identified in 53% (67\/127) of patients at the week 13 assessment of the OLE; this number increased by the end of the study (antibody titres \\>1\u2009:\u20092430 in 60%; 90\/149). Only one patient had evidence of in vitro neutralising antibodies against uricase activity; this was measurable at only one time point. Consistent with findings from the RCTs,17 low titres (\u22641\u2009:\u20092430) of anti-pegloticase antibodies were less likely to be associated with loss of SUA response (see online supplementary material for supporting data).\n\n## Efficacy\n\n### Serum uric acid response\n\nThe concordance of PUA and SUA (collected for all patients at the same time points) for values above and below the target of 6\u2005mg\/dl was 95% in samples collected during the OLE study. The efficacy data reported here focus on SUA because of its ready accessibility in clinical settings. The overall mean\u00b1SD SUA at baseline was 10.1\u00b11.4\u2005mg\/dl. Most responders to biweekly and monthly pegloticase in the RCTs maintained SUA\\<6\u2005mg\/dl throughout the OLE study (<a href=\"#ANNRHEUMDIS2012201795F3\" data-ref-type=\"fig\">figure 3<\/a>A). Among patients randomised to placebo in the RCTs, biweekly pegloticase administration in the OLE study produced greater reductions in mean SUA and a greater proportion of patients maintaining SUA\\<6\u2005mg\/dl than monthly pegloticase (<a href=\"#ANNRHEUMDIS2012201795F3\" data-ref-type=\"fig\">figure 3<\/a>B).\n\n### Tophus response\n\nTophus burden continued to decrease during ongoing pegloticase therapy. At the end of the RCTs, a complete response in at least one tophus was reported for 40% (21\/52) of patients with tophi treated with biweekly pegloticase, 21% (11\/52) of those treated with monthly pegloticase and 7% (2\/27) of those treated with placebo. By OLE week 13 a complete response in at least one tophus was reported for 45% (36\/80) of patients with tophi at RCT inception. At the final OLE visit (week 125), 60% (56\/94) of patients with evaluable tophi had a complete response. Patients with a sustained urate-lowering response to treatment were more likely to experience complete tophus resolution. A complete response was seen in 87% (32\/37) of patients in the responder group and in 31% (11\/36) designated non-responders on the basis of SUA measurements in the RCTs.\n\nAs prespecified in the protocol, each patient could have up to five measurable (and two additional) target or index tophi. Responses based on the total number of tophi were assessed to complement information on the proportion of patients with tophus complete response. After 1\u2005year of pegloticase treatment in the OLE study, 61% (185\/302) of all target tophi had completely resolved. Among patients who qualified as responders in the RCTs, a complete response of 70% (102\/145) of all target tophi was achieved after 1\u2005year of open-label treatment.\n\n### Gout flares\n\nGout flares occurred in 71% of patients (106\/149) during the OLE study; the mean number of flares per patient per 3-month period was 0.5 over the duration of the trial. The highest flare rates occurred in 52% of patients (78\/149; 1.1 flares\/patient) during the first 3-month period. Flare rates diminished with continued treatment in the OLE among patients who were urate-lowering responders during the RCT compared with patients who were RCT non-responders (<a href=\"#ANNRHEUMDIS2012201795F4\" data-ref-type=\"fig\">figure 4<\/a>A,B). For example, in the cohort of patients sustaining goal urate-lowering on the biweekly pegloticase OLE dose schedule, flares diminished substantially (occurring in 26% (9\/35) during months 1\u20133 of the OLE study, 9% (3\/32) during months 10\u201312 and 3% (1\/29) during months 22\u201324).\n\n# Discussion\n\nPegloticase is a PEGylated mammalian recombinant uricase that was developed to control hyperuricaemia and its clinical manifestations in patients with refractory chronic gout and no other urate-lowering options. In the present OLE of two RCTs,17 long-term treatment with pegloticase was safe and generally well tolerated, especially in patients who had experienced sustained goal range urate-lowering responses during blinded treatment. In addition to maintaining markedly sub-saturating urate levels, these patients showed progressive clinical benefit in gout flares and tophus reduction during pegloticase treatment for an average of 2\u2005years in the OLE study.\n\nTarget range SUA was achieved for 55% of all patients at week 25 of the OLE study. This is consistent with findings from the RCTs in which 42% of patients treated with biweekly pegloticase showed sustained urate lowering for 6\u2005months.17 Conversely, patients losing urate-lowering efficacy did so within the first few months of treatment in the RCTs, and urate levels for this cohort remained above target for the duration of the OLE study. About one-third of patients who did not sustain SUA\\<6\u2005mg\/dl in the RCTs retained clinical benefit during the OLE study with at least one tophus remaining resolved. However, this finding could be explained by an initial response followed by a protracted period needed for sufficient reaccumulation of crystal deposits to become clinically detectable.\n\nA post hoc analysis of the RCTs revealed relationships between loss of urate-lowering efficacy, risk of IRs and development of high-titre pegloticase antibodies.17 Because of blinding of urate levels during the RCTs, the relationship between loss of urate-lowering efficacy and the risk of pegloticase infusion reactions was not appreciated until several months after initiation of the OLE study. As a result, some patients who were RCT pegloticase non-responders continued receiving pegloticase during the OLE study, providing additional information with regard to these relationships. Among patients entering the OLE study, 71 had at least one IR during the randomised trials or the OLE study; 85% of these patients (60\/71) experienced their first IR when SUA was \\>6\u2005mg\/dl. Overall, these data support the view that the great majority of IRs can be avoided if patients discontinue treatment when, with routine preinfusion monitoring of SUA, levels in excess of 6\u2005mg\/dl indicate a sustained loss of pegloticase urate-lowering efficacy.\n\nThere are limitations to this study. First, the open-label study design carries both well-documented value and some bias as a result of being uncontrolled.19 Second, the RCT protocol called for stratification of patients based on treatment response and dosing schedule. Although this provided the opportunity to follow safety and efficacy outcomes during the OLE study for patients with specific treatment histories, it also resulted in six distinct cohorts, each containing small numbers of patients. Nevertheless, the analyses of OLE study data indicate that patients who achieve treatment success throughout the first 6\u2005months of treatment are likely to benefit from sustained long-term urate-lowering by pegloticase with favourable clinical responses. Conversely, patients not sustaining urate-lowering treatment responses to pegloticase infusions should not be expected to have treatment benefits (extending substantially beyond the period of urate-lowering initially achieved) and incur an increased risk of IRs. We recommend discontinuation of pegloticase therapy in such patients.\n\nA final limitation is that patients were identified by their initial allocation to biweekly or monthly treatment in the RCTs but were allowed to change dosing frequency at two time points upon entering the OLE study. A higher proportion of patients switched from monthly to biweekly treatment than vice versa, however, indicating that most patients were receiving the US FDA-approved pegloticase biweekly regimen**.**\n\nIn summary, the safety profile of pegloticase in the OLE study was consistent with that observed in the RCTs17 with no evidence of new safety concerns related to long-term exposure. Efficacy findings further demonstrated that clinical improvements were durable and probably progressive during long-term therapy in patients with persistent goal range urate-lowering responses to pegloticase.\n\n# Supplementary Material\n\n###### Web supplement\n\nThe authors thank all the investigators and patients who participated in the pegloticase trials. Members of the Cardiovascular Event Adjudication Committee were William B White (chair), Glen E Cooke and Philip Gorelick who were compensated for their participation. The authors wish to acknowledge writing and editorial support from the fm*P* group of Fallon Medica LLC, funded by Savient Pharmaceuticals, in the preparation of this manuscript.\n\n# References\n\n[^1]: **Handling editor** Tore K Kvien","meta":{"dup_signals":{"dup_doc_count":102,"dup_dump_count":52,"dup_details":{"curated_sources":2,"2022-40":1,"2021-43":1,"2021-17":1,"2020-50":1,"2020-40":1,"2020-05":1,"2019-30":1,"2018-22":3,"2018-13":2,"2018-05":4,"2017-51":1,"2017-47":2,"2017-43":1,"2017-39":2,"2017-34":3,"2017-30":2,"2017-26":1,"2017-22":2,"2017-17":2,"2017-09":2,"2017-04":2,"2016-50":2,"2016-44":2,"2016-40":2,"2016-36":2,"2016-30":2,"2016-26":2,"2016-22":2,"2016-18":1,"2016-07":1,"2015-48":2,"2015-40":2,"2015-35":1,"2015-32":2,"2015-27":1,"2015-22":2,"2015-14":2,"2014-52":2,"2014-49":2,"2014-42":6,"2014-41":3,"2014-35":3,"2014-23":3,"2014-15":4,"2023-40":1,"2015-18":2,"2015-11":1,"2015-06":2,"2014-10":2,"2013-48":2,"2013-20":2,"2017-13":1}},"file":"PMC3756467"},"subset":"pubmed_central"}
{"text":"date: 2020-05\ntitle: GLOBAL LEADERS UNITE TO ENSURE EVERYONE EVERYWHERE CAN ACCESS NEW VACCINES, TESTS AND TREATMENTS FOR COVID-19: Unprecedented gathering of heads of government, institutions and industry cements commitment to accelerate development and delivery for all populations\n\n**24 April 2020** - Heads of state and global health leaders today made an unprecedented commitment to work together to accelerate the development and production of new vaccines, tests and treatments for COVID-19 and assure equitable access worldwide.\n\nThe COVID-19 pandemic has already affected more than 2.4 million people, killing over 160,000. It is taking a huge toll on families, societies, health systems and economies around the world, and for as long as this virus threatens any country, the entire world is at risk.\n\nThere is an urgent need, therefore, while following existing measures to keep people physically distanced and to test and track all contacts of people who test positive, for innovative COVID-19 vaccines, diagnostics and treatments.\n\n\"We will only halt COVID-19 through solidarity,\" said Dr Tedros Adhanom Ghebreyesus, WHO Director-General. \"Countries, health partners, manufacturers, and the private sector must act together and ensure that the fruits of science and research can benefit everybody.\"\n\nWork has already started. Since January, WHO has been working with researchers from hundreds of institutions to develop and test vaccines, standardize assays and standardize regulatory approaches on innovative trial designs and define criteria to prioritize vaccine candidates. The Organization has prequalified diagnostics that are being used all over the world, and more are in the pipeline. And it is coordinating a global trial to assess the safety and efficacy of four therapeutics against COVID-19.\n\nThe challenge is to speed up and harmonize processes to ensure that once products are deemed safe and effective, they can be brought to the billions of people in the world who need them. Past experience, in the early days of HIV treatment, for example, and in the deployment of vaccines against the H1N1 outbreak in 2009, shows that even when tools are available, they have not been equally available to all.\n\nSo today leaders came together at a virtual event, co-hosted by the World Health Organization, the President of France, the President of the European Commission, and the Bill & Melinda Gates Foundation. The event was joined by the UN Secretary General, the AU Commission Chairperson, the G20 President, heads of state of France, South Africa, Germany, Vietnam, Costa Rica, Italy, Rwanda, Norway, Spain, Malaysia and the UK (represented by the First Secretary of State).\n\nHealth leaders from the Coalition for Epidemic Preparedness Innovations (CEPI), GAVI-the Vaccine Alliance, the Global Fund, UNITAID, the Wellcome Trust, the International Red Cross and Red Crescent Movement (IFRC), the International Federation of Pharmaceutical Manufacturers (IFPMA), the Developing Countries Vaccine Manufacturers' Network (DCVMN), and the International Generic and Biosimilar Medicines Association (IGBA) committed to come together, guided by a common vision of a planet protected from human suffering and the devastating social and economic consequences of COVID-19, to launch this groundbreaking collaboration. They are joined by two Special Envoys: Ngozi Okonjo-Iweala, Gavi Board Chair and Sir Andrew Witty, former CEO of GlaxoSmithKline.\n\nThey pledged to work towards equitable global access based on an unprecedented level of partnership. They agreed to create a strong unified voice, to build on past experience and to be accountable to the world, to communities and to one another.\n\n\"Our shared commitment is to ensure all people have access to all the tools to prevent, detect, treat and defeat COVID-19,\" said Dr Tedros. \"No country and no organization can do this alone. The Access to COVID-19 Tools Accelerator brings together the combined power of several organizations to work with speed and scale.\"\n\nHealth leaders called on the global community and political leaders to support this landmark collaboration and for donors to provide the necessary resources to accelerate achievement of its objectives, capitalizing on the opportunity provided by a forthcoming pledging initiative that starts on 4 May 2020. This initiative, spearheaded by the European Union, aims to mobilize the significant resources needed to accelerate the work towards protecting the world from COVID-19.\n\nAvailable from: <https:\/\/www.who.int\/news-room\/detail\/24-04-2020-global-leaders-unite-to-ensure-everyone-everywhere-can-access-new-vaccines-tests-and-treatments-for-covid-19>","meta":{"dup_signals":{"dup_doc_count":130,"dup_dump_count":31,"dup_details":{"curated_sources":2,"2023-40":1,"2023-23":3,"2023-14":1,"2023-06":3,"2022-49":2,"2022-40":5,"2022-33":3,"2022-27":4,"2022-21":4,"2022-05":3,"2021-49":5,"2021-43":2,"2021-39":8,"2021-31":5,"2021-25":5,"2021-21":7,"2021-17":4,"2021-10":4,"2021-04":5,"2020-50":11,"2020-45":7,"2020-40":5,"2020-34":6,"2020-29":4,"2020-24":11,"2023-50":1,"2024-30":2,"2024-26":2,"2024-22":3,"2024-18":1,"2024-10":1}},"file":"PMC7253839"},"subset":"pubmed_central"}
{"text":"author: J Yazdany; C Feldman; J Liu; MM Ward; MA Fischer; KH Costenbader\ndate: 2012\ninstitute: 1University of California, San Francisco, CA, USA; 2Brigham and Women's Hospital, Boston, MA, USA; 3National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, MD, USA\ntitle: Usual source of care and geographic region are largest predictors of healthcare quality for incident lupus nephritis in US Medicaid recipients\n\n# Background\n\nLittle is known about the quality of healthcare delivered to patients with lupus nephritis in the United States and the major determinants of quality remain unknown. We aimed to examine the sociodemographic, geographic, and healthcare system factors associated with performance on a healthcare quality measure in a nationwide cohort of Medicaid recipients with incident lupus nephritis.\n\n# Methods\n\nWe used US Medicaid analytic extract (MAX) data from 2000 to 2004 containing person-level files on Medicaid eligibility, utilization and payments. We identified patients meeting a validated administrative data definition of incident lupus nephritis, and used this group as the denominator population for the quality metric (QM). The QM numerator assessed receipt of **i**nduction therapy with glucocorticoids and another immunosuppressant (azathioprine, mycophenolate mofetil, mycophenolic acid, cyclophosphamide, cyclosporine A, or tacrolimus) within 90 days of lupus nephritis onset. Patients with end-stage renal disease were excluded. We used multivariable logistic regression models to examine sociodemographic (age, sex, race\/ethnicity), geographic (US region), and healthcare (health professional shortage areas, HPSAs, from the Area Resource File) predictors of higher performance. We also examined the restrictiveness of Medicaid benefits in each state, defined by less generous medication coverage policies (mandatory generic substitution, requirements for prior authorization and drug co-payments), and whether the patient's usual source of care was the emergency department or the ambulatory setting (\\>50% visits).\n\n# Results\n\nA total of 974 Medicaid recipients met the definition of incident lupus nephritis. The mean age was 39 years (SD 12), 93% were female, and most were African American (African American 48%, White 27%, Hispanic 13%, Asian 6%). Individuals were geographically dispersed (20% Midwest, 22% Northeast, 34% South, 24% West), and 95% resided in partial or complete HPSAs. One hundred and sixty-four individuals resided in states with more restrictive Medicaid benefits. At 90 days, only 16% of patients met all numerator components of QM1; 45% of individuals received only steroids (mean prednisone dose 28 mg\/day), and 3% received an immunosuppressant alone. Among those treated with an immunosuppressant, 31% received azathioprine, 47% received mycophenolate, 14% received cyclophosphamide, and 11% received a calcineurin inhibitor. For 20% (*n* = 192) of patients, the usual source of care was in the emergency setting. In multivariable logistic regression models, younger individuals were more likely to receive optimal treatment (OR for 18 to 34 years vs. 51 to 64 years = 3.5, CI = 1.6 to 7.6), while those living in the South and Midwest were less likely (OR = 0.49, CI = 0.24 to 0.67 and OR = 0.30 CI = 0.15 to 0.61, respectively). Those whose usual source of care was the emergency department were less likely to receive optimal treatment (OR = 0.47, CI = 0.28 to 0.81). In this adjusted analysis, we did not find significant associations for race\/ethnicity, HPSA or Medicaid restrictiveness with QM performance.\n\n# Conclusion\n\nMost US Medicaid recipients with incident lupus nephritis in our study did not receive timely induction therapy, and many were treated with high-dose steroids alone. We found significant geographic variation in performance, with the South and Midwest having lower performance than other regions. A substantial number of Medicaid patients with lupus nephritis used the emergency department as a usual source of care and performance on the QM is lower in this setting. These data suggest a need for targeted quality improvement interventions, including increasing access to appropriate ambulatory care for Medicaid recipients with lupus nephritis.","meta":{"dup_signals":{"dup_doc_count":129,"dup_dump_count":30,"dup_details":{"curated_sources":2,"2017-47":1,"2017-34":1,"2017-26":1,"2017-17":1,"2017-09":1,"2016-50":1,"2016-40":1,"2016-22":1,"2016-18":1,"2015-48":5,"2015-40":4,"2015-35":5,"2015-32":5,"2015-27":4,"2015-22":5,"2015-14":5,"2014-52":5,"2014-49":5,"2014-42":14,"2014-41":8,"2014-35":9,"2014-23":7,"2014-15":6,"2020-05":1,"2015-18":6,"2015-11":4,"2015-06":5,"2014-10":5,"2013-48":5,"2013-20":5}},"file":"PMC3467494"},"subset":"pubmed_central"}
{"text":"author: Linda S. Birnbaum[^1]\ndate: 2012-08-01\nreferences:\ntitle: NIEHS's New Strategic Plan\n\nI am pleased to present \"our\" new strategic plan for the National Institute of Environmental Health Sciences (NIEHS 2012). I say that it's \"our\" plan because the entire document reflects tremendous thought, discussion, and sharing of ideas by hundreds of scientists and community stakeholders, and it speaks to the entire field of environmental health research. This plan is about what we will strive to accomplish together as we devote ourselves to research that, in my opinion, has the greatest chance for preventing disease and for improving health throughout the world.\n\nAs reflected in the new strategic plan (NIEHS 2012), the NIEHS has a fresh vision, not because our values have changed but because our research has been so successful\u2014and many of you have made a huge contribution to that progress.\n\nNew technologies and increasing knowledge bring exciting new opportunities each and every year. The NIEHS's new strategic plan builds upon the accomplishments and vision that came before.\n\nThe NIEHS has come a long way in making environmental health research responsive to the needs and concerns of the American people\u2014\nto make environmental health part of the public health debate. This continues to be a source of motivation and purpose for NIEHS staff and our research partners. Environmental justice is an everlasting core value for NIEHS research.\n\nIn the past few years, we at the NIEHS have made some important progress in exposure science, supporting new technologies for sensor devices and bioinformatics. We have been embracing new science such as epigenetics and exposure phenotyping, focusing on interdisciplinary research and translational research. In addition, our clinical research unit is now up and running.\n\nAs the NIEHS moves forward, our overall goal is to make the institute, including the National Toxicology Program (NTP), the foremost trusted source of environmental health knowledge, leading the field in innovation and the application of research to solve health problems.\n\nOur new vision statement (NIEHS 2012) captures our collective dreams and aspirations, and reflects our strong commitment to making a real difference:\n\n> The vision of the National Institute of Environmental Health Sciences is to provide global leadership for innovative research that improves public health by preventing disease and disability from our environment.\n\nWhat this means in practical terms is that we are pursuing some of the \"big influences\" that have been understudied, all of which interact with traditional environmental exposures: the microbiome, for example, and inflammation pathways, immunological pathways, nutrition, and epigenetic processes. We also want to lead the process of defining the \"exposome,\" which is the totality of exposure encountered by humans.\n\nWe have elevated the NTP to the divisional level within the NIEHS. And we will continue to integrate our toxicology research with our excellent basic and translational science programs, not because I am a toxicologist but because the NTP is a problem-solving program\u2014a truly translational component of the NIEHS. For example, the NTP is part of our consortium on bisphenol A, contributing to the scientific deliberations, right along with our intramural scientists and our grantees.\n\nThe NTP is leading the Tox21 initiative along with the National Institute of Health's National Center for Comparative Genomics, the U.S. Environmental Protection Agency, and the Food and Drug Administration. This high throughput testing program shows great promise not only as a new and faster method but also for moving toxicology into a predictive science.\n\nThe NTP is moving beyond the traditional approaches of testing one chemical at a time and are taking on the significant challenge of evaluating mixtures. We are also looking at the effects of exposures throughout the life span, expanding our research and testing to include prenatal exposures and how they may link to adult disease. It is clear that there are multiple windows of susceptibility and that exposures early in life may have long-lasting consequences to both health and disease.\n\nFinally, the antiquated idea that the dose makes the poison is overly simplistic. The newest research clearly shows that biology is affected by low doses of chemicals, often within the range of general population exposure, and that these biological changes can be harmful, especially during periods of development. Therefore, low-dose research must go hand in hand with our life-span approach.\n\nThe NIEHS's job doesn't stop with the publication of scientific results. We also have an obligation to help translate the nation's research investment into public health intervention, new policy, and preventive clinical practice.\n\nTo be successful, we at the NIEHS need to conduct and support the best science, whether it is led by an individual researcher or a multidisiplinary team, and this means working together with all of our partners. We thank everyone who joined us in our strategic planning process. We appreciate your commitment and support.\n\n# Reference\n\n[^1]: Linda S. Birnbaum, director of the NIEHS and the NTP, oversees a budget that funds multidisciplinary biomedical research programs and prevention and intervention efforts that encompass training, education, technology transfer, and community outreach. She recently received an honorary Doctor of Science from the University of Rochester, the distinguished alumna award from the University of Illinois, and was elected to the Institute of Medicine. She is the author of \\> 900 peer-reviewed publications, book chapters, abstracts, and reports. Birnbaum received her M.S. and Ph.D. in microbiology from the University of Illinois, Urbana. A board-certified toxicologist, she has served as a federal scientist for \\> 32 years, 19 with the U.S. EPA Office of Research and Development, preceded by 10 years at the NIEHS as a senior staff fellow, a principal investigator, a research microbiologist, and a group leader for the institute's Chemical Disposition Group.","meta":{"dup_signals":{"dup_doc_count":229,"dup_dump_count":23,"dup_details":{"curated_sources":4,"2016-18":9,"2016-07":9,"2015-48":10,"2015-40":7,"2015-35":10,"2015-32":10,"2015-27":10,"2015-22":9,"2015-14":9,"2014-52":10,"2014-49":10,"2014-42":17,"2014-41":15,"2014-35":15,"2014-23":12,"2014-15":14,"2021-49":1,"2015-18":10,"2015-11":10,"2015-06":9,"2014-10":8,"2013-48":10,"2013-20":1}},"file":"PMC3440102"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  We have previously shown that irregular lifestyle of young Japanese female students are significantly related to their desire to be thinner. In the present study, we examined the nutritional knowledge and food habits of Chinese university students and compared them with those of other Asian populations.\n  .\n  # Methods\n  .\n  A self-reported questionnaire was administered to 540 students, ranging in age from 19-24 years. Medical students from Beijing University (135 men and 150 women) in Northern China and Kunming Medical College in southern China (95 men and 160 women) participated in this study. The parametric variables were analyzed using the Student's *t*-test. Chi-square analyses were conducted for non-parametric variables\n  .\n  # Results\n  .\n  Our results showed that 80.5% of students had a normal BMI and 16.6 % of students were underweight with the prevalence of BMI\\>30 obesity being very low in this study sample. Young Chinese female students had a greater desire to be thinner (62.0%) than males (47.4%). Habits involving regular eating patterns and vegetable intake were reported and represent practices that ought to be encouraged.\n  .\n  # Conclusions\n  .\n  The university and college arenas represent the final opportunity for the health and nutritional education of a large number of students from the educator's perspective. Our findings suggest the need for strategies designed to improve competence in the area of nutrition.\nauthor: Ruka Sakamaki; Kenji Toyama; Rie Amamoto; Chuan-Jun Liu; Naotaka Shinfuku\ndate: 2005\ninstitute: 1International Center for Medical Research. Kobe University Graduate School of Medicine, Kobe, 650-0017, Japan; 2Seinan Jo Gakuin University Faculty of Health and Welfare, Department of Nutritional Sciences, Kitakyusyu, 803-0835, Japan; 3Department of Plastic Surgery, Kobe University Graduate School of Medicine, Kobe, 650-0017, Japan\nreferences:\ntitle: Nutritional knowledge, food habits and health attitude of Chinese university students \u2013a cross sectional study\u2013\n\n# Background\n\nThe increasing problem of obesity has been observed in many lower-income countries during the last decades. China has adopted an open-market policy and experienced explosive economic growth, which has led to less food scarcity at the national level and to a remarkable transition in the structure of the diet of Chinese \\[1\\]. The composition of the Chinese diet has been shifting towards a diet higher in fat and meat, and lower in carbohydrates and fiber \\[2\\]. Additionally, decreased levels of physical activity and leisure are linked to increases in the prevalence of an overweight condition, obesity and diet-related non-communicable diseases \\[3\\].\n\nIn previous reports, we examined eating habits and dietary knowledge of female students in Japan. Our results showed that irregular lifestyle was significantly related to indefinite complaint, with the majority of students having a desire to be thinner although the prevalence of students who were overweight was very low in this study sample \\[4\\]. Universities and colleges are potentially important targets for the promotion of healthy lifestyles of the adult population. However, little is known concerning the body mass index (BMI) distribution and nutritional and health-related behavior of Chinese university students. The purpose of this study was to obtain a preliminary understanding of the relative level of BMI distribution of Chinese university students and to determine the nutritional knowledge and body-shape perceptions.\n\n# Material and Methods\n\nThis study was carried out between February 2001 and April 2002. Medical students from Beijing University (135 men and 150 women) in Northern China and Kunming Medical College in southern China (95 men and 160 women) participated in this study. A sample of 540 students aged 19 \u2013 24 years were administered a self-reported questionnaire. The questionnaire consisted of 21 questions regarding eating, drinking and smoking habits (19 questions), with 2 questions related to dieting (trying to lose weight). Self-reported height and weight were used to calculate BMI (kg\/m^2^). The questionnaire was designed by the authors and based on a national dietary survey held by the Health and Labor Ministry of Japan. Some of the authors also traveled to China to investigate the dietary life of Chinese to facilitate questionnaire design. The questionnaire was first written in Japanese and then translated to Chinese utilizing fluent bilingual linguistic services. The translated Chinese version was back-translated to insure the original meaning was not lost. Informed consent was obtained from all participants of this study according to the Declaration of Helsinki. The statistical software package SPSS 10.0 was used for the analysis of data \\[5\\]. In this study, parametric variables were analyzed using the Student's *t*-test. Chi-square analyses were conducted for non-parametric variables. All analyses were two-tailed, and a 'p' value less than 0.05 was considered statistically significant.\n\n# Results\n\n## Characteristics of the sample and BMI categories\n\nThe response rate was 96% (512 \/ 540). The characteristics of the subjects are shown in Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>. A total of 212 men and 300 women, with a mean age of 20 \u00b1 1.9 years, participated in this study. The average height was 165.8 \u00b1 7.8 cm, while the average weight was 56.9 \u00b1 9.2 kg. Mean BMI was 20.6 \u00b1 2.2. To analyze the distribution of BMI and health-related behavior, BMI was categorized into 4 groups according to mean BMI of \u00b1 1 standard deviation (SD) (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>). The average BMI for male students was 21.4 \u00b1 2.5 and was highest in the categories 18.9\u2264BMI\\<21.4 (37.7%) and 21.4\u2264BMI\\<23.9 (32.5%). The average BMI for female students was 20.0 \u00b1 1.8, with the categories 18.2\u2264BMI\\<20.0 (37.5%) and 20.0\u2264BMI\\<21.8 (31.4%) displaying high values. According to WHO BMI classifications \\[6\\], 97.1% of students were classified into the underweight or normal weight categories. 2.5% (13\/512) students were overweight (BMI\\>25) and 0.4% (2\/512) of students were obese (BMI\\>30). BMI values of deviations from the average sample show the presence of few extreme values.\n\n## Eating habit\n\nThe life style practices were compared by gender (see <a href=\"#S1\" data-ref-type=\"supplementary-material\">additional file 1<\/a>). The majority of students (83.6 %) reported taking meals regularly, with 79.0% eating meals 3 times per day; there were no gender differences. However, a significant gender difference was found in the response relating to breakfast intake, with 66.8% of males and 82.3% of females reporting eating breakfast regularly (p \\< 0.0006). The frequency of snacking rate was significantly higher in females (31.1%) than in males (11.5%; p \\< 0.0001). The present sample demonstrated high consumption of vegetable and fruits. A total of 47.9% of students reported the consumption of colored vegetables such as spinach and carrots, and 32.5% of subjects reported eating fruit daily. Female students tend to eat more fruit than males (p \\< 0.0001). In addition, female students tend to eat with friends and family more frequently than males (p \\< 0.01). Few subjects smoke or drink alcohol. When the students eat out, female students are more likely to consider the calorie content of the menu than males (data not shown). Although 85.6% of students are aware of the concept of nutritionally balanced food, only a small number of students (7%) apply this concept when selecting food from a menu. Moreover, only 51% of students showed a desire to learn about healthy diets.\n\n## Body image and health consciousness\n\nWhen subjects were asked about their history of dieting, 22.7% of respondents reported that they had dieted (see <a href=\"#S2\" data-ref-type=\"supplementary-material\">additional file 2<\/a>). The proportion of female students having a dieting experience (29.8%) was more than twice as great as that of male subjects (12.7%; p \\< 0.0006). In total, 56% of the students selected 'thin or slim is beautiful'. The percentage by gender was, 47.4% for male and 62.0% for female students. Female students have a significantly greater desire to be thinner than males (p \\< 0.001). More than half of the respondents reported a desire to adopt healthier dietary habits. Moreover, a question regarding the degree of consciousness pertaining to health and diet was asked; 45.2% of male students and 48.3% of female students wish to learn about health and diet. Among female subjects, BMI\\<18.2 strongly showed their consciousness of health and diet (p \\< 0.03).\n\n# Discussion\n\nThis study aimed to determine the health, nutritional knowledge and dietary behavior of university students in China. As a result, we recorded the distribution of BMI among Chinese students and found a low prevalence of obesity, a finding that is consistent with a study of Japanese female students (BMI\u226525 overweight was 5.8%, BMI\\>30 of obesity was 0%) \\[4\\]. In the United States, 35% of the college students are reported to be overweight or obese (BMI\u226525) \\[7\\]. According to the WHO definition of obesity, BMI\\>30 is the cut-off point \\[6\\]. The definition is based on research of Caucasian populations. Asian populations are reported to have a higher body fat (%) at a lower BMI compared to Caucasians \\[8\\]. The WHO expert consultation reported that BMI in Asian populations is related to disease at a lower level \\[9\\]. In order to compare obesity prevalence between ethnic groups, BMI cut-off points for Asians need to be considered by well constructed and standardized body composition studies. It is notable that in China, the prevalence of overweight individuals increased from 1991 to 1997, with the increasing rate changing from 6.4 to 7.7 \\[10\\]. The proportion of energy derived from the fat of both vegetable and animal sources increased each year. A recent study revealed that energy derived from dietary fat accounted for more than 30% of the total energy \\[11\\]. Changes in dietary composition, which correspond to socioeconomic growth, may accelerate the prevalence of obesity in China.\n\nThe results of our study show that the majority of students regularly eat three times per day, and almost 80% of students eat vegetables and fruit twice per day. These eating habits ought to be encouraged. The traditional Chinese diet contains plenty of vegetables and is rice-based. The present study reported a high proportion of Chinese students eat breakfast daily. In contrast, a dietary survey of young Japanese subjects revealed a low rate of individuals engaged in regular eating patterns \\[12\\]. The skipping of breakfast has been associated with lower nutritional status and the risk of cardiovascular diseases \\[13\\]. It has also been reported that less adequate breakfast habits may contribute to the appearance and further development of obesity \\[14\\]. Therefore the importance of regular eating patterns cannot be overemphasized in nutritional education.\n\nOur results showed that body figure perception was significantly different between female and male students. A number of researchers have investigated the relationship of body image and gender role. Women tend to desire a thinner figure, express more anxiety about becoming fat, and are more likely to diet than men \\[15,16\\]. In contrast, men have reported a desire for a heavier physique and muscularity \\[17\\]. In recent years, eating disorders have been increasing dramatically among young women. The results of our study did not confirm this suggestion to the level of statistical significance; however, it is worth pointing out that 65.0% of female students with BMI\\<20, which is under to normal weight range, indicated a desire to be thin. Dissatisfaction with body figure and eating disorders are closely related \\[18-20\\]. Being young, female, and dieting are identified risk factors that have been reliably linked to the development of eating disorders \\[21\\]. It was speculated that some of the students who were preoccupied with a thin body may develop eating disturbances. Thus, the promotion of healthy weight management practices should be considered when developing health education programs.\n\n# Conclusions\n\nIn conclusion, our findings reveal that the majority of students were classified into the normal BMI group, with the prevalence of BMI \\>30 obesity being very low in this study sample. Young female students had a greater desire to be thinner than male students. Habits involving regular eating patterns and vegetable intake were found and represent practices that ought to be encouraged. The meal and snack patterns in Chinese students were very similar to the traditional eating pattern model, although diets are changing rapidly in China and other low-income countries. The university and college arenas represent the final opportunity for nutritional education of a large number of students from the educator's perspective. Our findings suggest the need for strategies designed to improve competence in the area of nutrition, especially with respect to information relating to sources of nutrition and healthy weight management. Furthermore, public demand for health and nutritional information should be taken into consideration when implementing strategies aimed at improving the nutritional well-being of individuals.\n\n# Authors' contributions\n\nR.S carried out questionnaire design, manuscript drafting and total coordination of the study. K.T has been involved in drafting and revision of the article. R.A contributed to the data entry and its analysis. L.CJ contributed to the questionnaire design, data collection and language translations. N.S contributed to final approval of the manuscript.\n\n# Supplementary Material\n\n###### Additional File 1\n\nTable 2 containing the results of questions related to lifestyle practices with special reference to food habit. The meal patterns, consumption of fruits and vegetables, consumption of fried foods, consumption of alcohol were assessed in male and female students. The Chi-square analyses were employed to compare the behavioral differences by gender. The evaluations of statistical significance were made at the p \\< 0.05.\n\nClick here for file\n\n###### Additional File 2\n\nTable 3 contains the results of body shape perception and health consciousness of male and female students. Male and female respondents were categorized in to 4 groups respectively, according to mean BMI of \u00b1 1 standard deviation (SD). Analyses were made between BMI groups using Chi-square analysis. The evaluations of statistical significance were made at the p \\< 0.05.\n\nClick here for file\n\n### Acknowledgements\n\nThe authors express their appreciation for the invaluable partnership and support of Dr. Wang of Beijing University, Dr Zhao of Kunming Medical College and all the study participants of both institutes. We also thank Dr. Shigeki Minakami for valuable comments on the manuscript.\n\n## Figures and Tables\n\nCharacteristics of Participants\n\n|                   | **Total**   | **Male**    | **Female**  |\n|-------------------|-------------|-------------|-------------|\n| **Variable**      | n = 512     | n = 212     | n = 300     |\n| **Age (y)**       | 20.4 \u00b1 1.9  | 20.3 \u00b1 1.7  | 20.4 \u00b1 2.0  |\n| **weight (kg)**   | 56.9 \u00b1 9.2  | 63.7 \u00b1 8.8  | 52.1 \u00b1 5.9  |\n| **height (cm)**   | 165.8 \u00b1 7.8 | 172.3 \u00b1 5.5 | 161.2 \u00b1 5.6 |\n| **BMI (kg\/m^2^)** | 20.6 \u00b1 2.2  | 21.4 \u00b1 2.5  | 20.0 \u00b1 1.8  |\n\nGender distribution in the sampled population. BMI is based on self-reported height and weight. BMI = weight \\[kg\\] \/ height \\[m\\]^2^","meta":{"dup_signals":{"dup_doc_count":191,"dup_dump_count":48,"dup_details":{"curated_sources":6,"2022-05":1,"2020-45":1,"2020-34":1,"2020-24":1,"2019-47":1,"2019-35":2,"2019-18":1,"2019-09":1,"2018-51":1,"2018-47":1,"2018-39":1,"2018-30":1,"2018-22":1,"2018-09":1,"2018-05":1,"2017-51":1,"2017-47":1,"2017-39":1,"2017-34":1,"2017-26":1,"2017-17":1,"2017-09":18,"2016-36":1,"2016-22":1,"2016-18":1,"2016-07":22,"2015-48":5,"2015-40":6,"2015-35":5,"2015-32":4,"2015-27":5,"2015-22":6,"2015-14":5,"2014-52":5,"2014-49":7,"2014-42":12,"2014-41":7,"2014-35":8,"2014-23":10,"2014-15":5,"2023-50":1,"2015-18":5,"2015-11":5,"2015-06":4,"2014-10":5,"2013-48":4,"2013-20":5,"2024-26":1}},"file":"PMC553986"},"subset":"pubmed_central"}
{"text":"date: 2016-12\ntitle: OVER 1 MILLION TREATED WITH HIGHLY EFFECTIVE HEPATITIS C MEDICINES\n\n2 SEPTEMBER 2016 \\| GENEVA - 27 OCTOBER 2016 \\| GENEVA - Over one million people in low- and middle-income countries have been treated with a revolutionary new cure for hepatitis C since its introduction two years ago.\n\nWhen Direct Acting Antivirals (DAAs) were first approved for hepatitis C treatment in 2013, there were widespread fears that their high price would put them out of reach for the more than 80 million people with chronic hepatitis C infections worldwide.\n\nThe new medicines have a cure rate of over 95%, fewer side effects than previously available therapies, and can completely cure the disease within three months. But at an initial estimated price of some US\\$85 000 they were unaffordable even in high-income countries.\n\nCountries show hepatitis C treatment is achievable\n\nThanks to a series of access strategies supported by the World Health Organization (WHO) and other partners, a range of low- and middle-income countries, including Argentina, Brazil, Egypt, Georgia, Indonesia, Morocco, Nigeria, Pakistan, Philippines, Romania, Rwanda, Thailand and Ukraine \u2013 are beginning to succeed in getting drugs to people who need them. Strategies include competition from generic medicines through licensing agreements, local production and price negotiations.\n\n\"Maximizing access to lifesaving hepatitis C treatment is a priority for WHO,\" says Dr Gottfried Hirnschall, Director of WHO's Department of HIV and Global Hepatitis Programme. \"It is encouraging to see countries starting to make important progress. However, access still remains beyond the reach for most people.\"\n\nA new WHO report, Global Report on Access to Hepatitis C Treatment: Focus on Overcoming Barriers, released today shows how political will, civil society advocacy and pricing negotiations are helping address hepatitis C, a disease which kills almost 700 000 people annually and places a heavy burden on health systems' capacities and resources.\n\n\"Licensing agreements and local production in some countries have gone a long way to make these treatments more affordable,\" says Dr Suzanne Hill, WHO Director for Essential Medicines and Health Products. For example, the price of a three-month treatment in Egypt dropped from US\\$900 in 2014 to less than US\\$200 in 2016.\n\n\"But there are still huge differences between what countries are paying. Some middle-income countries, which bear the largest burden of hepatitis C, are still paying very high prices. WHO is working on new pricing models for these, and other expensive medicines, in order to increase access to all essential medicines in all countries,\" says Dr Hill.\n\n80% of people in need still face challenges\n\nAmong middle-income countries, the price for a three-month treatment of sofosbuvir and daclatasvir varies greatly. Costs range from US\\$9 400 in Brazil to US\\$79 900 in Romania.\n\nHigh costs have led to treatment rationing in some countries, including in the European Union, where price agreements have not accounted for the full cost of treating the whole affected population.\n\n\"Today's report on access, prices, patents and registration of hepatitis C medicines will help create the much needed market transparency which should support country efforts to increase access to DAAs,\" said Dr Hirnschall. \"We hope countries will update their hepatitis treatment guidelines, work to remove barriers to access, and make these medicines available promptly for everyone in need.\"\n\nIn May 2016, at the World Health Assembly, 194 countries adopted the first-ever Global Health Sector Strategy on Viral Hepatitis, agreeing to eliminate hepatitis as a public health threat by 2030. The strategy includes a target to treat 80% of people in need by this date.\n\nWHO issued guidelines recommending the use of DAAs in 2014 and 2016 and included DAAs on its Essential Medicines List \u2013 which is compiled to address the priority healthcare needs of populations; to make needed essential medicines available at all times in adequate amounts, at a price the health system and community can afford.\n\nAvailable from: <http:\/\/www.who.int\/mediacentre\/news\/releases\/2016\/hepatitis-c-medicines\/en\/>","meta":{"dup_signals":{"dup_doc_count":104,"dup_dump_count":46,"dup_details":{"curated_sources":4,"2022-27":1,"2022-21":1,"2021-49":2,"2021-31":2,"2021-21":1,"2021-17":2,"2021-04":3,"2020-45":4,"2020-34":1,"2020-29":2,"2020-24":2,"2020-16":2,"2020-10":1,"2019-51":2,"2019-43":2,"2019-35":1,"2019-30":3,"2019-22":1,"2019-18":2,"2019-13":1,"2019-09":4,"2019-04":1,"2018-51":4,"2018-47":2,"2018-43":4,"2018-39":2,"2018-34":3,"2018-30":3,"2018-26":1,"2018-22":1,"2018-17":4,"2018-13":2,"2018-09":1,"2018-05":3,"2017-51":3,"2017-47":1,"2017-43":3,"2017-39":4,"2017-34":2,"2017-30":2,"2017-26":3,"2017-22":3,"2017-17":2,"2023-14":1,"2017-13":4,"2024-10":1}},"file":"PMC5303791"},"subset":"pubmed_central"}
{"text":"abstract: Hypoglycemia stimulates counterregulatory hormone release to restore euglycemia. This protective response is diminished by recurrent hypoglycemia, limiting the benefits of intensive insulin treatment in patients with diabetes. We previously reported that EphA5 receptor-ephrinA5 interactions within the ventromedial hypothalamus (VMH) influence counterregulatory hormone responses during acute hypoglycemia in nondiabetic rats. In this study, we examined whether recurrent hypoglycemia alters the capacity of the ephrinA5 ligand to activate VMH EphA5 receptors, and if so, whether these changes could contribute to pathogenesis of defective glucose counterregulation in response to a standard hypoglycemic stimulus. The expression of ephrinA5, but not EphA5 receptors within the VMH, was reduced by antecedent recurrent hypoglycemia. In addition, the number of synaptic connections was increased and astroglial synaptic coverage was reduced. Activation of VMH EphA5 receptors via targeted microinjection of ephrinA5-Fc before a hyperinsulinemic hypoglycemic clamp study caused a reduction in the glucose infusion rate in nondiabetic rats exposed to recurrent hypoglycemia. The increase in the counterregulatory response to insulin-induced hypoglycemia was associated with a 150% increase in glucagon release (*P* \\< 0.001). These data suggest that changes in ephrinA5\/EphA5 interactions and synaptic plasticity within the VMH, a key glucose-sensing region in the brain, may contribute to the impairment in glucagon secretion and counterregulatory responses caused by recurrent hypoglycemia.\nauthor: Barbara Szepietowska; Tamas L. Horvath; Robert S. SherwinCorresponding author: Robert S. Sherwin, <email@example.com>.\ndate: 2014-03\nreferences:\ntitle: Role of Synaptic Plasticity and EphA5-EphrinA5 Interaction Within the Ventromedial Hypothalamus in Response to Recurrent Hypoglycemia\n\nFrequent episodes of acute hypoglycemia represent the principal obstacle to achieving optimal glycemic control during insulin treatment in patients with type 1 diabetes and long-standing type 2 diabetes (1,2). This problem is further magnified by the loss of an appropriate counterregulatory response to hypoglycemia that results as a consequence of frequent episodes iatrogenic insulin-induced hypoglycemia (3,4). The molecular mechanisms underlying this phenomenon remain uncertain but are likely to involve the key brain glucose\u2013sensing region, the ventromedial hypothalamus (VMH) (5,6).\n\nWe have previously reported that local stimulation of VMH EphA5 receptors by microinjection of ephrinA5-Fc or ephrinA5 overexpression increased, whereas knockdown of VMH ephrinA5 reduced, counterregulatory responses to hypoglycemia. Furthermore, overexpression of VMH ephrinA5 transiently increased local glutamate concentrations, whereas ephrinA5 knockdown produced profound suppression of VMH interstitial fluid glutamine concentrations in the basal state and during hypoglycemia. These data suggest that the activation of VMH EphA5 receptors by ephrinA5 may act in concert with \u03b2-cell Eph receptor forward signaling to restore glucose homeostasis during acute hypoglycemia via alterations in glutamate\/glutamine cycling (7,8).\n\nWithin the central nervous system, Eph receptors and their ligands, the ephrins, play a key role in cell-to-cell communication as well as in synaptic structure and function. Eph receptors function as transmembrane receptor tyrosine kinases and are divided by sequence similarity and ligand affinity into an A and a B subclass. Their ligands, the ephrins, are also divided into an A and a B subclass: the A subclass is tethered to the cell membrane by a glycosylphosphatidylinositol anchor, and members of the B subclass have a transmembrane domain and as a short cytoplasmic region. For the most part, A-type receptors bind to most or all A-type ligands, and B-type receptors bind to most or all B-type ligands (9). As for many other receptor tyrosine kinases, ligand-binding induces \"forward signaling,\" mostly through phosphotyrosine-mediated pathways. However, ephrins can also signal into their host cell\u2014referred to as \"reverse signaling'\"(10). Eph receptors and their ephrin ligands are present in the adult brain and are specifically enriched in glutamate excitatory synapses (11). Moreover, Eph receptor tyrosine kinases are mainly expressed in synaptic terminals where they influence synaptic plasticity via binding to ephrins found on astrocytic processes that surround the synapse or on neuronal synapses (12,13).\n\nSeveral observations suggest that changes in hypothalamic synaptic plasticity may play a significant role in the regulation of energy balance. For example, peripheral signals, such as leptin, ghrelin, and estrogen, induce synaptic adaptations that serve as dynamic regulators of neuronal activity in the arcuate nucleus, the hypothalamic center for feeding control (14,15). Whether hypoglycemia per se induces local changes in the VMH affecting both neuronal synapses and\/or surrounding glia cells is unknown, but such alterations could potentially modulate neurotransmission within VMH, thereby altering brain glucose sensing. This study tests whether recurrent hypoglycemia alters EphA5 receptor\u2013ephrinA5 interactions within the VMH, which might contribute to diminished activation of counterregulatory responses to acute hypoglycemia.\n\n# Research Design and Methods\n\n## Animals\n\nMale Sprague-Dawley rats (Charles River, Wilmington, MA) weighing 300\u2013350 g were individually housed in the Yale Animal Resource Center in temperature- (22\u201323\u00b0C) and humidity-controlled rooms. Animals were fed rat chow (Prolab 3000; Agway, Syracuse, NY) and water ad libitum and were acclimatized to a 12-h light-dark cycle. The Yale University Institutional Animal Care and Use Committee approved the experimental protocols. Different sets of animals were used for the clamp and electron microscopy (EM) experiments described below.\n\n## Vascular and Stereotaxic Surgery\n\nAnimals were anesthetized \u223c7\u201310 days before study and underwent aseptic surgery in which vascular catheters were implanted into the left carotid artery for blood sampling and into the right jugular vein for infusion, as previously described (8). These catheters were tunneled subcutaneously and exteriorized at the back of the neck between the scapulae. Wound staples were used to close the incision. For stereotaxic surgery, animals were placed into a stereotaxic frame (David Kopf Instruments, Tujunga, CA), and stainless steel guide cannulas (Plastics One, Roanoke, VA) were inserted into the brain and secured in place with screws and dental acrylic (coordinates from Bregma: anteroposterior \u22122.6 mm, mediolateral \u00b1 0.8 mm, and dorsoventral \u22128.0) for microinjection.\n\n## Recurrent Hypoglycemia Protocol\n\nRats received intraperitoneal injections of insulin (10 units\/kg) for 3 consecutive days. After each injection, food was withheld to allow plasma glucose to fall into the hypoglycemic range (30\u201350 mg\/dL); throughout, animals were monitored with tail vein glucose measurements every 30 min using the AlphaTRAK rodent glucometer (Abbott Animal Health, Chicago, IL) to ensure sustained hypoglycemia and to avoid glucose reduction sufficient to cause seizure activity. At the end of this period, the rats were given free access to food again. Control rats received an injection of 0.9% saline under the same conditions.\n\n## Microinjection of EphrinA5-Fc or Control-Fc\n\nOn the morning of the study, awake, overnight-fasted rats were connected to infusion pumps \u223c90 min before the start of the experiment and then left undisturbed to recover from handling stress. After the recovery period, 22-gauge microinjection needles (Plastics One), designed to extend 1 mm beyond the tip of the guide cannula, were inserted bilaterally through the guide cannula into each VMH region. Rats then received a microinjection of recombinant human ephrinA5-Fc or recombinant human IgG1-Fc (control-Fc protein; both R&D Systems, Minneapolis, MN; catalog number: 374-EA-200) in a concentration of 0.3 \u03bcg\/\u03bcL dissolved in artificial extracellular fluid delivered at a rate of (0.1 \u00b5L\/min) over 60 min (dose: 1.8 \u03bcg for each side). After the microinjection, needles were left in place for 30 min before being removed. Immediately thereafter, a hyperinsulinemic hypoglycemic clamp study was performed. These compounds have been previously administered into the central nervous system in in vivo studies (16) and in vitro in brain slices (17), without adverse effects. In addition, ephrinA5-Fc has been shown to specifically bind EphA5 receptors (18).\n\n## Hyperinsulinemic-Hypoglycemic Clamp\n\nA primed continuous infusion of 20 mU \u22c5 kg^\u20131^ \u22c5 min^\u20131^ insulin (Humulin R; Eli Lilly, Indianapolis, IN) was given, and a variable infusion of 20% dextrose was adjusted at 5- to 10-min intervals based on glucose measurements (Analox Instruments, Lunenburg, MA) designed to maintain plasma glucose at 50 mg\/dL from 30 to 90 min (19). Additional blood was drawn at baseline and at 30, 60, and 90 min for measurement of insulin, glucagon, epinephrine, and norepinephrine. Rats were killed at study termination, and the probe position was confirmed histologically.\n\n## Hormone and Neurotransmitter Analyses\n\nPlasma catecholamine measurements were analyzed by high-performance liquid chromatography using electrochemical detection, and plasma insulin and glucagon concentrations were determined by radioimmunoassay (Linco, St. Charles, MO).\n\n## Immunoblot Analysis\n\nFrozen tissue micropunches from VMH and control regions were homogenized in buffer containing 1% NP40, 150 mmol\/L NaCl, 50 mmol\/L Tris (pH 7.4), 1 mmol\/L Na~3~VO~4~, 1 mmol\/L phenylmethylsulfonyl fluoride, and protease inhibitor (Roche Diagnostics) using a plastic pestle and ultrasonicator. Protein content was assessed with the Bradford protein assay. Protein samples were fractioned under reducing conditions on SDS-9% PAGE (Bio-Rad). After electrophoresis, proteins were electroblotted onto nitrocellulose membranes, blocked with 5% nonfat dry milk in PBS, probed with first antibody (\u03b1-tubulin; Cell Signaling, cat. 2125S; ephrin-A5; R&D Systems, cat. AF3743) and EpA5 receptor (EphA5; Sigma-Aldrich, cat. P8651), and incubated with the appropriate secondary antibody conjugated to peroxidase by horseradish peroxidase\u2013linked protein A (1:2000; Sigma-Aldrich). The immunoblots were developed using an enhanced chemiluminescence detection system (Amersham Biosciences).\n\n## Electron Microscopy\n\nBriefly, animals were perfused with paraformaldehyde fixative and ultrathin brain sections were cut on a Leica ultramicrotome, collected on Formvar-coated single-slot grids, and analyzed with a Tecnai 12 Biotwin EM (FEI). Glia coverage of the cell membrane of random VMH cells was performed using ImageJ software. EM photographs (original magnification \u00d711,500) were used to first measure the perimeter of each VMH neuron analyzed, followed by determination of the amount of membrane covered by glia (in nanometers). Results are reported as glia coverage\/perimeter of the VMH neurons.\n\nCharacteristics of synaptic contacts were defined, as previously described (20). They were collected from serial sections of the cell membrane of random VMH neurons. Synapse characterization was performed at original magnification \u00d720,000, and quantitative measurements were performed at original magnification \u00d711,500. Results are reported as synapses number\/perimeter of the VMH neurons (21).\n\n## Statistics\n\nData are expressed as the means \u00b1 SEM. Analysis was performed by one-way ANOVA or the Student *t* test, as appropriate. Statistical analysis was then performed by two-way ANOVA for repeated measures, followed by post hoc analysis using GraphPad Prism 4.0 software (GraphPad Software, Inc., San Diego, CA). *P* \\< 0.05 was considered statistically significant.\n\n# Results\n\n## Recurrent Hypoglycemia and EphrinA5 Expression\n\nAs shown in <a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<\/a>, rats exposed to recurrent hypoglycemia for 3 days exhibit a 25% reduction in ephrinA5 expression in the VMH (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>A<\/em> and <em>B<\/em><\/a>). In contrast, no significant change in the expression of the EphA5 receptor in the VMH was detected (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>C<\/em> and <em>D<\/em><\/a>).\n\n## Stimulation of VMH EphA5 Receptors in Rats Exposed to Recurrent Hypoglycemia\n\nTo assess the biological consequences of the reduction of ephrinA5 expression, we microinjected the Eph receptor agonist, ephrinA5-Fc, into the VMH before conducting a hyperinsulinemic-hypoglycemic clamp study in rats previously exposed to three episodes of insulin-induced hypoglycemia. Schematic representation of the experimental protocol is presented in <a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<em>A<\/em><\/a>. Body weight and plasma levels of glucose, insulin, glucagon, epinephrine, and norepinephrine were indistinguishable at baseline and immediately after completion of the VMH microinjection of ephrinA5-Fc or control-Fc (<a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>). Subsequently, during the hypoglycemic clamp study, plasma glucose (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<em>B<\/em><\/a>) and insulin (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<em>C<\/em><\/a>) were indistinguishable between the two groups. EphrinA5-Fc delivery, however, significantly reduced within 15 min the glucose infusion rate required to maintain hypoglycemia (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<em>D<\/em><\/a>). This was accompanied by a rapid 150% (*P* \\< 0.001) increase in glucagon release (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<em>E<\/em><\/a>). As was observed in our previous study (8) in rats not exposed to antecedent hypoglycemia, neither plasma epinephrine (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<em>F<\/em><\/a>) nor norepinephrine (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<em>G<\/em><\/a>) responses to hypoglycemia were significantly altered by VMH delivery of ephrinA5-Fc compared with the control-Fc microinjection.\n\nCharacteristics for the rats with recurrent hypoglycemia at baseline and after microinjection of ephrinA5-Fc in the VMH\n\n![](1140tbl1)\n\n## Effect of Recurrent Hypoglycemia on Glia Ensheetment and Synaptic Input Organization\n\nNext, we assessed if recurrent hypoglycemia affected the VMH synaptic organization and glia ensheetment in rats exposed for 3 days to recurrent hypoglycemia compared with controls, a model we have previously shown suppresses hypoglycemic counterregulation (22). <a href=\"#F3\" data-ref-type=\"fig\">Figure 3<em>A<\/em> and <em>B<\/em><\/a> compares representative EMs of glia ensheetment in random VMH neuron perikarya in control and recurrent hypoglycemic rats. The rats exposed to recurrent hypoglycemia exhibited reduced glial coverage of neurons (*P* \\< 0.001) (<a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<em>C<\/em><\/a>) and, as a result, more total synaptic contacts in random VMH neurons (*P* \\< 0.05) (<a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<em>D<\/em><\/a>).\n\n# Discussion\n\nThe current study demonstrates that exposure of nondiabetic rats to recurrent hypoglycemia for 3 consecutive days diminishes ephrinA5 expression within the VMH in association with reduced glial coverage of VMH neurons and synaptic remodeling. In addition, targeted VMH delivery of the EphA5 receptor ligand ephrinA5-Fc enhances glucose counterregulation and glucagon release in rats exposed to recurrent hypoglycemia. These findings are consistent with the possibility that recurrent hypoglycemia diminishes EphA5 receptor forward signaling in the VMH, which in turn reduces the magnitude of glucagon secretion.\n\nSignaling via the EphA\/ephrinA receptor system has been reported to regulate neuron-astrocyte interactions that cause rapid changes in synaptic structural and functional plasticity (17,23). It has been proposed that the loss of ephrinA alters astrocytic-neuronal contacts (24), whereas application of ephrinA3-Fc or endogenous ephrin induces rapid growth of the astrocytes processes and growth of new filopodia (17). In addition, activation of EphA4 by ephrinA3 has been shown to induce spine retraction (25). The current finding that recurrent hypoglycemia decreases ephrinA5 expression and produces diminished glia coverage and more synaptic connections within the VMH raises the question of a possible relationship. The fact that bypassing ephrinA5 using a targeted VMH ephrinA5-Fc microinjection can increase counterregulatory responses in animals exposed to recurrent hypoglycemia is in keeping with the hypothesis that reduced VMH ephrinA5 expression might induce alterations in VMH synaptic plasticity that in turn contribute to the development of disordered glucose counterregulation. However, a direct link between the observed changes in ephrinA5 and in glia coverage as well as synaptic connection rearrangements remains to be established in future studies.\n\nPrevious studies have reported rapid changes in synaptic network connectivity and glia morphology in the VMH in response to alterations in energy substrate bioavailability (15,26\u201330). Acute hypoglycemia has been shown to alter synaptophysin expression, findings consistent with a rapid alternation in synaptic morphology (31,32), whereas insulin-deficient diabetic rats display a decrease in the number of hypothalamic astrocytes as a consequence of increased death and decreased proliferation (33). Given that diabetes and recurrent glucose deprivation are both accompanied by impaired counterregulation (22), these observations are consistent with the hypothesis that synaptic connectivity and the function of glia in the VMH play a significant role in supporting the neurotransmission required for proper counterregulatory responses to acute hypoglycemia.\n\nIt is noteworthy that the principal effect of VMH microinjection of the ephrinA5 ligand on hypoglycemia-induced counterregulatory hormone release was on glucagon, whereas recurrent hypoglycemia normally leads to a suppression of glucagon and epinephrine levels in nondiabetic animals. These findings are consistent with previous studies in mice showing that knockdown of VGLUT2 (vesicular glutamate transporter) selectively in SF1 VMH neurons predominately inhibited the secretion of glucagon in response to acute hypoglycemia (34). It should be noted, however, that alterations in EphA receptor\/ephrinA signaling appear to influence epinephrine responses as well. Using a targeted gene expression manipulation approach to chronically alter VMH ephrinA5 expression, we observed that overexpression of ephrinA5 in the VMH stimulates, whereas targeted VMH knockdown of ephrinA5 inhibits, epinephrine as well as glucagon responses to acute hypoglycemia. These effects also appear to be mediated by alterations in glutamate\u2013glutamine cycling (8). In this study, we did not include a control group not subjected to recurrent hypoglycemia, and thus, the extent that acute delivery of the EphA receptor agonist restored the glucagon response to hypoglycemia in rats exposed to recurrent hypoglycemia cannot be determined. However, results from previous studies from our laboratory using a similar hypoglycemic clamp protocol show the effect of the EphA receptor agonist on glucagon responses appears to be at least as great as the suppressive effect of recurrent hypoglycemia on glucagon levels (8).\n\nGiven that the EphA5\/ephrinA5 system is mainly localized on glutamatergic synapses (12,25), it is intriguing to speculate that the stimulation of glucagon produced by the EphA5 receptor agonist in the current study is most likely mediated by the augmented VMH glutamatergic neurotransmission. The maintenance of VMH glutamate neurotransmission during acute hypoglycemia is supported by the transport of glutamate into astrocytes, resulting in the production of glutamine for delivery to neurons and glutamate\u2013glutamine cycle activation (35). Previous studies have shown that astrocyte synaptic coverage is linked to glutamate clearance and the activation of metabotropic glutamate receptors (36), and this has been proposed to alter synaptic and astroglia organization, and in turn, neurotransmission (37,38). Thus, the reduced glia coverage of the VMH neurons observed in the current study in rats exposed to recurrent hypoglycemia may have produced a deficit in astroglial function to support proper glutamate neurotransmission during hypoglycemia. Interestingly, this was associated with more neuronal synaptic contacts in the VMH (<a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<em>D<\/em><\/a>), which appeared to be in large part symmetric and thus potentially \u03b3-aminobutyric acid (GABA) inhibitory in nature (21). An increase in the VMH GABA tone has been shown to be an important contributor to the development of impaired glucose counterregulation in response to recurrent hypoglycemia (39).\n\nTaken together, our data demonstrate that recurrent hypoglycemia alters neuron-glia plasticity in VMH nuclei and diminishes ephrinA5 ligand expression within the VMH. It is thus possible decreased ephrin-induced activation of Eph receptors in VMH glutamate neurons may contribute to the impairment in glucose counterregulation in response to recurrent antecedent hypoglycemia.\n\n## Article Information\n\n**Acknowledgments.** The authors are grateful to Wanling Zhu, from R.S.S.'s laboratory, for performing animal surgeries; Aida Groszman, Maria Batsu, Codruta Todeasa, Ralph J. Jacob, from the Yale Center for Clinical Investigation Core Laboratory of the Yale School of Medicine, and Jan Czyzyk, from the University of Rochester Medical Center School of Medicine and Dentistry, for assisting with the protein expression experiments; and Erzsebet Borok, from the Yale Program in Integrative Cell Signaling and Neurobiology of Metabolism, for excellent technical support and assistance.\n\n**Funding.** This work was supported by research grants from National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (20495), the Juvenile Diabetes Research Foundation, and the NIDDK-supported Diabetes Endocrinology Research Center.\n\n**Duality of Interest.** No potential conflicts of interest relevant to this article were reported.\n\n**Author Contributions.** B.S. designed the study, performed animal surgery and studies, analyzed data, and wrote the manuscript. T.L.H. designed EM experiments. R.S.S. designed the study, reviewed data, and revised the manuscript. B.S. and R.S.S. are the guarantors of this work, and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.\n\n**Prior Presentation.** This study was presented at the 70th Scientific Sessions of the American Diabetes Association, Orlando, FL, 25\u201329 June 2010.\n\n# References","meta":{"dup_signals":{"dup_doc_count":122,"dup_dump_count":47,"dup_details":{"curated_sources":2,"2020-40":1,"2020-05":1,"2019-04":1,"2018-43":1,"2018-34":1,"2018-30":1,"2018-22":3,"2018-13":3,"2018-09":1,"2018-05":1,"2017-51":2,"2017-47":2,"2017-43":4,"2017-39":2,"2017-34":2,"2017-30":6,"2017-26":3,"2017-22":2,"2017-17":8,"2017-09":4,"2017-04":9,"2016-50":3,"2016-44":4,"2016-40":3,"2016-36":3,"2016-30":2,"2016-26":3,"2016-22":3,"2016-18":2,"2016-07":2,"2015-48":1,"2015-40":1,"2015-35":2,"2015-27":3,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":2,"2014-42":1,"2014-41":3,"2014-35":1,"2023-40":1,"2017-13":4,"2015-18":3,"2015-11":2,"2015-06":3,"2024-26":1}},"file":"PMC3931406"},"subset":"pubmed_central"}
{"text":"date: 2017-08\ntitle: WHO urges action against HIV drug resistance threat\n\n20 JULY 2017 \\| GENEVA - WHO alerts countries to the increasing trend of resistance to HIV drugs detailed in a report based on national surveys conducted in several countries. The Organization warns that this growing threat could undermine global progress in treating and preventing HIV infection if early and effective action is not taken.\n\nThe WHO HIV drug resistance report 2017 shows that in 6 of the 11 countries surveyed in Africa, Asia and Latin America, over 10% of people starting antiretroviral therapy had a strain of HIV that was resistant to some of the most widely used HIV medicines. Once the threshold of 10% has been reached, WHO recommends those countries urgently review their HIV treatment programmes.\n\n# HIV drug resistance report 2017\n\n\"Antimicrobial drug resistance is a growing challenge to global health and sustainable development,\" said Dr Tedros Adhanom Ghebreyesus, WHO Director-General. \"We need to proactively address the rising levels of resistance to HIV drugs if we are to achieve the global target of ending AIDS by 2030.\"\n\nHIV drug resistance develops when people do not adhere to a prescribed treatment plan, often because they do not have consistent access to quality HIV treatment and care. Individuals with HIV drug resistance will start to fail therapy and may also transmit drug-resistant viruses to others. The level of HIV in their blood will increase, unless they change to a different treatment regimen, which could be more expensive \u2013 and, in many countries, still harder to obtain.\n\nOf the 36.7 million people living with HIV worldwide, 19.5 million people were accessing antiretroviral therapy in 2016. The majority of these people are doing well, with treatment proving highly effective in suppressing the HIV virus. But a growing number are experiencing the consequences of drug resistance.\n\nWHO is therefore issuing new guidelines to help countries address HIV drug resistance. These recommend that countries monitor the quality of their treatment programmes and take action as soon as treatment failure is detected.\n\n\"We need to ensure that people who start treatment can stay on effective treatment, to prevent the emergence of HIV drug resistance,\" said Dr Gottfried Hirnschall, Director of WHO's HIV Department and Global Hepatitis Programme. \"When levels of HIV drug resistance become high we recommend that countries shift to an alternative first-line therapy for those who are starting treatment.\"\n\nIncreasing HIV drug resistance trends could lead to more infections and deaths. Mathematical modelling shows an additional 135 000 deaths and 105 000 new infections could follow in the next five years if no action is taken, and HIV treatment costs could increase by an additional US\\$ 650 million during this time.\n\nTackling HIV drug resistance will require the active involvement of a broad range of partners. A new five-year Global Action Plan calls on all countries and partners to join efforts to prevent, monitor and respond to HIV drug resistance and to protect the ongoing progress towards the Sustainable Development Goal of ending the AIDS epidemic by 2030. In addition, WHO has developed new tools to help countries monitor HIV drug resistance, improve the quality of treatment programmes and transition to new HIV treatments, if needed.\n\nThe WHO HIV drug resistance report 2017 was co-authored by the Global Fund to Fight AIDS, Tuberculosis and Malaria, and the Centers for Disease Control and Prevention, USA.\n\n\"This new report shows a worrying picture of increasing levels of HIV drug resistance and, if unchecked, it will be a major risk to program impact,\" said Dr Marijke Wijnroks, Interim Executive Director of the Global Fund. \"We strongly recommend implementing WHO recommendations for early warning indicators and HIV drug resistance surveys in every national plan for antiretroviral therapy, and to consider funding them through Global Fund grants or reprogramming.\"\n\nDr Shannon Hader, Director of CDC's Division of Global HIV and Tuberculosis, US Centers for Disease Control and Prevention, added: \"The new report pulls together key HIV drug resistance survey findings from across the globe that, taken together with other national-level data, confirm we must be forward-thinking in our efforts to combat resistance: scaling up viral load testing, improving the quality of treatment programs, and transitioning to new drugs like dolutegravir.\"\n\nDr. Hader continued, stating that \"Overall high rates of viral suppression across three recent national Population-based HIV Impact Assessments showed that present first-line regimens remain largely effective. However, special attention to populations at risk for higher resistance, such as pediatrics, adolescents, pregnant women and key populations, will be critical to target more urgent interventions. We call on the global community for continued vigilance and responsiveness.\"\n\nAvailable from: <http:\/\/www.who.int\/mediacentre\/news\/releases\/2017\/hiv-drug-resistance\/en\/>","meta":{"dup_signals":{"dup_doc_count":130,"dup_dump_count":51,"dup_details":{"curated_sources":2,"2023-40":3,"2023-14":1,"2023-06":2,"2022-49":2,"2022-40":2,"2022-33":2,"2022-27":1,"2022-21":1,"2022-05":1,"2021-43":1,"2021-39":3,"2021-31":1,"2021-21":3,"2021-10":1,"2021-04":1,"2020-45":2,"2020-40":3,"2020-29":1,"2020-24":1,"2020-16":2,"2020-05":2,"2019-51":1,"2019-47":1,"2019-39":1,"2019-35":2,"2019-30":3,"2019-22":2,"2019-13":3,"2019-09":2,"2019-04":3,"2018-51":2,"2018-47":4,"2018-43":3,"2018-39":2,"2018-34":5,"2018-30":7,"2018-26":4,"2018-22":4,"2018-17":6,"2018-13":1,"2018-09":7,"2018-05":3,"2017-51":4,"2017-47":5,"2017-43":5,"2017-39":1,"2017-34":5,"2024-30":2,"2024-26":1,"2024-22":2,"2024-18":1}},"file":"PMC5654036"},"subset":"pubmed_central"}
{"text":"abstract: Decades of research in rodent models has shown that early postnatal overnutrition induces excess adiposity and other components of metabolic syndrome that persist into adulthood. The specific biologic mechanisms explaining the persistence of these effects, however, remain unknown. On postnatal day 1 (P1), mice were fostered in control (C) or small litters (SL). SL mice had increased body weight and adiposity at weaning (P21), which persisted to adulthood (P180). Detailed metabolic studies indicated that female adult SL mice have decreased physical activity and energy expenditure but not increased food intake. Genome-scale DNA methylation profiling identified extensive changes in hypothalamic DNA methylation during the suckling period, suggesting that it is a critical period for developmental epigenetics in the mouse hypothalamus. Indeed, SL mice exhibited subtle and sex-specific changes in hypothalamic DNA methylation that persisted from early life to adulthood, providing a potential mechanistic basis for the sustained physiological effects. Expression profiling in adult hypothalamus likewise provided evidence of widespread sex-specific alterations in gene expression. Together, our data indicate that early postnatal overnutrition leads to a reduction in spontaneous physical activity and energy expenditure in females and suggest that early postnatal life is a critical period during which nutrition can affect hypothalamic developmental epigenetics.\nauthor: Ge Li; John J. Kohorst; Wenjuan Zhang; Eleonora Laritsky; Govindarajan Kunde-Ramamoorthy; Maria S. Baker; Marta L. Fiorotto; Robert A. WaterlandCorresponding author: Robert A. Waterland, <firstname.lastname@example.com>.[^1]\ndate: 2013-08\nreferences:\ntitle: Early Postnatal Nutrition Determines Adult Physical Activity and Energy Expenditure in Female Mice\n\nEnvironmental influences on the development of body weight regulatory mechanisms may be an important factor in the worldwide obesity epidemic (1,2). Evidence in humans indicates that overnutrition during early postnatal life can permanently alter body weight regulation, increasing susceptibility to obesity throughout life (3,4). Accordingly, various animal models have been developed to explore the effects of infant overnutrition on lifelong obesity risk. Artificial feeding of rodent pups by intragastric cannula provides clear evidence for sustained effects of early postnatal overnutrition (5) but requires raising newborn rodents in isolation, which itself has long-term consequences. Overfeeding dams during lactation, with a high-fat diet, for example, could indirectly overnourish pups. Indeed, two recent rodent studies (6,7) report that the obesogenic effect of maternal high-fat diet occurs specifically during the suckling period. Pups from high-fat-fed dams are not consistently heavier at weaning, however (8,9), indicating that maternal overnutrition does not reliably induce early postnatal overnutrition.\n\nIn the rodent small litter model of early postnatal overnutrition (10), offspring from several litters born on the same day are randomized and fostered to either normal size (control \\[C\\]) or small litters (SL). Suckling in SL is naturalistic, easy to implement, and consistently induces early postnatal overnutrition, providing an apt model in which to study potential long-term effects of infantile overnutrition by excessive formula feeding (11). The early postnatal exposure induces elevated body weight and adiposity that persists to adulthood (10\u201312), with concomitant increases in plasma insulin (11,13) and leptin concentrations (13) and impaired glucose tolerance (11,13,14). It remains unresolved, however, whether the sustained increase in adiposity of adult SL rodents results from increased energy intake or decreased energy expenditure (13,15). Moreover, the fundamental mechanisms by which the metabolic effects of SL exposure persist to adulthood are unknown.\n\nEnvironmental influences on developmental epigenetics (16,17) provide a likely mechanism. Epigenetic mechanisms regulate mitotically heritable alterations in gene expression potential that are not caused by changes in DNA sequence (18) and are known to play key roles in brain development (19). DNA methylation, the most stable epigenetic modification (20), is a likely mechanism to explain effects that persist for a lifetime (2). Given its central role in regulating food intake and energy expenditure (21), the hypothalamus is an obvious tissue in which to explore a potential epigenetic basis for induced alterations in body weight regulation. We therefore set out to determine *1*) whether the persistently elevated adiposity of SL mice is caused by increased food intake or decreased energy expenditure, and *2*) whether early postnatal overnutrition causes persistent changes in hypothalamic epigenetic regulation that may perpetuate altered body weight regulation.\n\n# RESEARCH DESIGN AND METHODS\n\nFor the litter size studies, virgin FVB\/NJ females (The Jackson Laboratory) were mated with FVB\/NJ males at age 8 weeks. In each batch, 14\u201315 mating pairs were set up on the same day; four independent batches of mice were studied over the course of 2 years. On postnatal day 1 (P1), pups from all litters born on the same day (P0) were weighed, sexed, and pooled randomly. Only pups from a birth litter size of 6\u201312 were included. Foster dams received either four (SL) or nine (C) pups. There were two females and two males in each SL and four to five females and males in each C litter. Litter assignment was performed systematically to balance body weight at P1. At P21, offspring from both groups were weaned onto a fixed-formula, soy protein\u2013free diet (2020X; Harlan Teklad); females were housed two to five per cage, and males were housed individually. Body composition, food intake, energy expenditure, and physical activity were measured at P21\u2013P25 and approximately P180. The P21 vs. P0 methylation-specific amplification and microarray hybridization (MSAM) comparisons used female C57BL\/6J mice, and the pyrosequencing validation studies were performed in C57BL\/6J and FVB\/NJ mice of both sexes (The Jackson Laboratory). Sex was confirmed by PCR amplification of *Sry*. All applicable institutional and governmental regulations concerning the ethical use of animals were followed during this research. The protocol was approved by the Institutional Animal Care and Use Committee of Baylor College of Medicine. All mice were housed in a temperature-controlled facility (22\u00b0C), provided free access to food and water, and maintained on a 12-h light cycle.\n\n## Body composition.\n\nBody composition was determined by quantitative magnetic resonance (EchoMRI-100; Echo Medical Systems) according to the manufacturer's instructions.\n\n## Food intake, energy expenditure, and physical activity.\n\nPrior to metabolic study, mice were acclimatized to single housing in Comprehensive Laboratory Animal Monitoring System (CLAMS) cages (Columbus Instruments, Columbus, OH) for 3 days. Mice were subsequently transferred to calorimetry cages for 4 days, during which food intake, energy expenditure (by indirect calorimetry), and physical activity were monitored in real time (22). Only data from the last 3 days (6:00 <span class=\"smallcaps\">a.m.<\/span> to 6:00 <span class=\"smallcaps\">a.m.<\/span>) were used. Upon weaning (P21), mice were housed in a normal cage for 1 day before being introduced to the CLAMS cages. Hence, the metabolic measurements did not commence until P25.\n\n## Genome-scale DNA methylation profiling.\n\nMSAM was performed as previously described (23,24), using a starting quantity of 0.5 \u00b5g genomic DNA. In both MSAM experiments, two independent cohybridizations (biological replicates) were performed on a custom 2\u00d7105k array (Agilent Technologies). The array includes 90,694 probes covering 86% (23,742) of the 27,675 potentially informative *Sma*I*\/Xma*I intervals between 200 bp and 2 kb in the mouse genome (average 3.8 probes per interval) (25). Fold change and *P* value were calculated as mean and median, respectively, of all probes in each interval; \"hits\" were called for fold change \\>2 or \\<0.5 and *P* \\< 0.0001 in both cohybridizations. Gene Ontology analysis was performed using the Gene Ontology enRIchment anaLysis and visuaLizAtion tool (GOrilla) (26), in each case using the potentially informative genes on the array as the background set. *Sma*I*\/Xma*I cut sites were annotated by RefGene according to NCBI36\/mm8 mouse genome (Feb 2006 release), according to the schema in [Supplementary Fig. 2](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1306\/-\/DC1). Relevant details of the MSAM experiments and the hybridization data are available in the GEO database: P21 vs. P0, [www.ncbi.nlm.nih.gov\/geo\/query\/acc.cgi?acc=GSE32475](http:\/\/www.ncbi.nlm.nih.gov\/geo\/query\/acc.cgi?acc=GSE32475); and SL vs. C, P180, [www.ncbi.nlm.nih.gov\/geo\/query\/acc.cgi?acc=GSE32477](http:\/\/www.ncbi.nlm.nih.gov\/geo\/query\/acc.cgi?acc=GSE32477).\n\n## Quantitative analysis of DNA methylation.\n\nSite-specific analysis of CpG methylation was performed by bisulfite pyrosequencing (23,24). For validation of MSAM hits, pyrosequencing assays were designed to cover both informative *Sma*I*\/Xma*I sites when possible. A hit was considered validated if either assay showed a substantial methylation difference in the same direction as in MSAM. Sensitivity and linearity of each pyrosequencing assay were confirmed by running methylation standards (27). Pyrosequencing primers and annotations of the CpG sites examined are listed in [Supplementary Tables 3 and 4](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1306\/-\/DC1), respectively.\n\n## Gene expression profiling and analysis.\n\nTotal hypothalamic RNA was isolated by the RNeasy kit (Qiagen). RNA quality control, cRNA preparation, labeling, and microarray hybridization were conducted as previously described (28). cRNA was hybridized onto Illumina MouseWG6 v2 Expression BeadChips (Illumina) following the manufacturer's protocol. Signal intensities extracted from Illumina Genome Studio software were preprocessed using LUMI (29) and the R statistical package (<http:\/\/www.r-project.org\/>), including probes with a detection *P* value \u22640.05 in at least half of the samples. These 20,791 probe signals were then quantile normalized. To test for effects of group, sex, and group \u00d7 sex, we performed two-way ANOVA using Genomics Suite Software, version 6.6 \u03b2 (Partek). Contrasts were applied to all groups to identify differentially expressed transcripts, using an \u03b1 level of 0.05 after Benjamini-Hochberg false discovery rate adjustment. Network analyses were performed using IPA (Ingenuity Systems). Relevant details of the expression microarray experiment and the hybridization data are available in the GEO database: [www.ncbi.nlm.nih.gov\/geo\/query\/acc.cgi?acc=GSE40616](http:\/\/www.ncbi.nlm.nih.gov\/geo\/query\/acc.cgi?acc=GSE40616).\n\n## Quantitative analysis of gene expression.\n\nExpression levels of specific genes were determined by quantitative PCR. Total hypothalamic RNA was isolated by the RNeasy kit (Qiagen), and cDNA was synthesized using M-MLV Reverse Transcriptase (Promega) with random primers (Life Technologies). Gene expression levels were determined with either TaqMan (Life Technologies) or Sybr Green (Life Technologies), using the 2^\u2212\u0394\u0394Ct^ method (assay details provided in [Supplementary Table 6](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1306\/-\/DC1)). In all cases, *ActB* was used as an endogenous control.\n\n## General statistical methods.\n\nEnrichment of *Sma*I*\/Xma*I cut sites relative to genomic regions was analyzed by \u03c7^2^ test. Group differences in body weight at P1 were analyzed by two-tailed *t* test. Body composition data at P25 and P180 were analyzed by ANOVA (SAS Proc Mixed). Body weight data at P21, P60, P120, and P180 were analyzed by repeated-measures ANOVA (SAS Proc Mixed, compound symmetry covariance structure) with age in the repeated effect. For the CLAMS studies, hourly measurements of food intake, energy expenditure, and activity counts for three consecutive days were averaged into one 24-h record for each mouse. Repeated-measures ANOVA used the full power of these time-series data while recognizing the nonindependence of the 24 multiple measures within each mouse. Analysis of food intake, energy expenditure, and physical activity were performed both with and without lean mass and fat mass included as independent variables to adjust for group differences in body size and composition (30). Group differences in DNA methylation by pyrosequencing were analyzed by repeated-measures ANOVA, with CpG site as the repeated effect ([Supplementary Table 2](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1306\/-\/DC1)). Loci that showed significant group effects on methylation but no significant group \u00d7 age interaction were considered persistently altered by SL suckling. Requiring the same group difference, in the same direction, at both ages (in independent sets of mice) affords substantial protection against type 1 error; these analyses were therefore not otherwise adjusted for multiple testing. Akaike information criterion (AIC) model selection by adjusted *R*^2^ (SAS Proc Reg) was performed based on individual average dark-period energy expenditure and physical activity data.\n\n# RESULTS\n\n## Early postnatal overnutrition reduces adult energy expenditure in females.\n\nWe used the SL mouse model (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>A<\/em><\/a>) to study persistent effects of overnutrition during the suckling period. We studied four independent batches (groups of litters cross-fostered at one time) over 2 years, including offspring from 24 C and 26 SL litters total. Consistent with previous studies, SL mice were heavier at P21 and remained so into adulthood (*P* \\< 0.0001 in both females and males) (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>B<\/em><\/a>). Although the increase in adult body weight was modest, effects on body composition were substantial. Both male and female SL adults had 50% higher fat mass and percent body fat compared with C mice (*P* \\< 0.005 in all comparisons) (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>C<\/em><\/a>). There were no group differences in lean mass. Clearly, suckling in a small litter induces persistent changes in regulatory mechanisms that affect adult body composition.\n\nTo determine whether these changes involve alterations in food intake and\/or energy expenditure, we used metabolic cages to simultaneously monitor food intake, energy expenditure, and voluntary physical activity. In an attempt to identify persistent metabolic differences that might explain the sustained group differences in adiposity, we performed the metabolic measurements shortly after weaning (P25) and in adulthood (P180). (Again, these data represent four batches of mice studied over the course of 2 years.) After appropriate least squares normalization for lean mass and fat mass (30), food intake of SL mice tended, surprisingly, to be slightly lower than that of C mice at both P25 and P180 (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<em>A<\/em><\/a>), but these differences were not statistically significant. Energy expenditure (normalized for lean mass and fat mass \\[30\\]) was nearly identical between SL and C mice at P25 (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<em>B<\/em><\/a>). At P180, however, energy expenditure of SL females was significantly lower than that of C females (*P* = 0.002); this group difference was significant during both the light and dark periods. Resting metabolic rate was estimated as the lowest average energy expenditure within 1 h for each mouse. After least squares normalization for lean mass and fat mass, female mice showed no group differences in resting metabolic rate at either age. Resting metabolic rate of SL males, however, was higher at P25 (*P* = 0.02) and lower at P180 (*P* = 0.03) relative to C males. There were no group differences in respiratory exchange ratio. Group differences in voluntary physical activity, again normalized for lean mass and fat mass, were consistent with those in energy expenditure: none were found at P25, but SL females were significantly less active than C females at P180, specifically during the dark period (group \u00d7 light interaction *P* = 0.0009) (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<em>C<\/em><\/a>). It is noteworthy that adult SL females were less physically active even after including body weight and body composition in the model; hence, their lower activity was not caused by their excess adiposity. (For comparison, unnormalized data on food intake, energy expenditure, and physical activity are shown in [Supplementary Fig. 1](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1306\/-\/DC1).) Including physical activity in the model for energy expenditure of P180 females drastically reduced the significance of the SL effect (from *P* = 0.002 to *P* = 0.01), suggesting that physical activity explains much of the group difference in energy expenditure. Together, these data indicate that the persistent alterations in energy balance of female SL mice are due not to excess food intake but, rather, to reduced energy expenditure. The sex specificity of this effect may be related to the male-specific decline of physical activity with age ([Supplementary Fig. 1*C*](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1306\/-\/DC1)).\n\n## Extensive epigenetic development occurs in the early postnatal hypothalamus.\n\nOntogenic periods when epigenetic mechanisms are being established or undergoing maturation constitute critical periods during which environmental influences can cause persistent changes in epigenetic regulation (31,32). To determine whether the suckling period might be a critical period for developmental epigenetics in the hypothalamus, we tested for changes in hypothalamic DNA methylation. We used MSAM, which is based on sequential digestion of genomic DNA with the methylation-sensitive and -insensitive isoschizomers *Sma*I and *Xma*I (23,33). Two independent P21 vs. P0 MSAM cohybridizations (incorporating a dye swap) were performed.\n\nUsing stringent criteria validated in our previous studies (24,33), 868 *Sma*I*\/Xma*I intervals changed methylation from P0 to P21. Only 31 intervals lost methylation (<a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<em>A<\/em><\/a>), and the genomic distribution of associated *Sma*I*\/Xma*I cut sites was not different from that on the array (<a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<em>B<\/em><\/a> and [Supplementary Fig. 2](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1306\/-\/DC1)). Methylation increased at 837 intervals (<a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<em>A<\/em><\/a>); associated cut sites were significantly underrepresented at promoters (*P* \\< 0.0001) and overrepresented in introns (*P* \\< 0.0001) (<a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<em>B<\/em><\/a>). In a larger number of P0 and P21 mice, we used bisulfite pyrosequencing (23,33) to measure P0\u2013P21 changes in hypothalamic CpG methylation at 10 intervals identified by MSAM; all 10 validated (100%) (7 are shown in <a href=\"#F4\" data-ref-type=\"fig\">Fig. 4<\/a>). Additionally, since the P21 vs. P0 MSAM studies were performed in C57BL\/6J mice, we confirmed (at a subset of loci) that these methylation changes also occur in both sexes of FVB\/NJ mice (the strain used for the litter size studies) ([Supplementary Fig. 3](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1306\/-\/DC1)).\n\nFunctional significance of the developmental changes in DNA methylation was evaluated by gene ontology analysis. Relative to all potentially informative genes on the array, no enriched ontologies were found for the few genes associated with intervals that lost methylation. Genes that gained methylation, however, were significantly enriched for 20 biologic process categories (<a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<em>C<\/em><\/a>); of these, 17 are explicitly related to development, including neurodevelopmental processes such as axon guidance and neuron differentiation. Postnatal development of the mouse hypothalamus clearly involves functionally important epigenetic changes. This may be a critical period during which environment can influence these processes, with long-term consequences.\n\n## Early postnatal overnutrition causes persistent and sex-specific alterations in hypothalamic DNA methylation and gene expression.\n\nWe therefore examined DNA methylation differences among SL and C hypothalami. Intrigued by the large group differences in energy expenditure and physical activity in P180 females, we used MSAM to compare hypothalamic DNA methylation of SL and C females at P180. Two independent SL vs. C cohybridizations were performed, with each hypothalamic DNA sample pooled from five females drawn from different foster litters. The results, however, provided no evidence of persistent group differences in hypothalamic DNA methylation.\n\nReasoning that DNA methylation changes in SL mice might be too subtle to detect by MSAM, we used bisulfite pyrosequencing to examine a panel of candidate genes. Since genomic regions undergoing methylation change from P0 to P21 are most likely to show persistent effects of overnutrition during this period (2), most of the genomic regions that we selected were those identified in our P21 vs. P0 MSAM experiments. In addition to 10 of those already validated (<a href=\"#F4\" data-ref-type=\"fig\">Fig. 4<\/a>), we examined six hits near genes previously reported to change expression in hypothalamus from P0 to P21 (34) and two showing interindividual variation in DNA methylation. Additionally, promoters of a few genes critical to hypothalamic function and development (*Agrp, Fto*, and *Pomc*) were included. In total, 24 loci (named according to the nearest gene) were selected ([Supplementary Table 1](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1306\/-\/DC1)).\n\nOf these, 15 showed no DNA methylation differences between SL and C hypothalamus at P25 ([Supplementary Fig. 4](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1306\/-\/DC1) and [Supplementary Table 2](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1306\/-\/DC1)) and were not examined at P180. The remaining nine loci were examined at both P25 and P180 ([Supplementary Fig. 5](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1306\/-\/DC1) and [Supplementary Table 2](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1306\/-\/DC1)). Considering this an exploratory data analysis, we set an \u03b1-level of 0.1 for main effects and 0.2 for interactions. The initial model including both sexes showed significant main effects for age but not group (SL vs. C); interestingly, however, four loci showed significant group \u00d7 sex interactions ([Supplementary Table 2](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1306\/-\/DC1)). We therefore performed sex-specific analyses. In females, *AK145544*, *Aqp4*, and *Nolz1* and in males *Gadd45b* showed main effects of group that did not differ by age (no group \u00d7 age interaction). Plots of average site-specific methylation at these loci (<a href=\"#F5\" data-ref-type=\"fig\">Fig. 5<\/a>) illustrate subtle but persistent differences in DNA methylation. Since most of these changes were found in females, we used quantitative real-time RT-PCR to measure gene expression of *AK145544*, *Aqp4*, and *Nolz1* in hypothalamus of P180 females but found no significant group differences. To test whether differences in methylation and expression at these loci could explain individual variation in adult physical activity or energy expenditure, we applied the AIC (35) to identify the best model for each. In addition to methylation and expression of the three genes, SL group membership, lean mass, and fat mass were included as potential explanatory variables. Physical activity was not significantly predicted by any model. Remarkably, however, the best model for energy expenditure ([Supplementary Fig. 6](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1306\/-\/DC1)) included expression of all three genes and methylation at *Nolz1* and *Aqp4* and predicted 55% of the variation in P180 energy expenditure in females (*P* = 0.025). Hence, group differences in DNA methylation and gene expression at these loci, though subtle, may explain some of the observed alterations in energy balance.\n\nAs a complementary approach to identify genes with persistent alterations in epigenetic regulation, we profiled gene expression in P180 hypothalamus of SL and C mice. Three males and three females were studied in each group (12 arrays total). The results showed strong effects of sex (<a href=\"#F6\" data-ref-type=\"fig\">Fig. 6<em>A<\/em><\/a>), with 342 transcripts showing significant differences (false discovery rate \\<0.05). Although none of the group or group \u00d7 sex effects survived multiple testing correction, there was an enrichment of low *P* value probes in the group \u00d7 sex analysis (<a href=\"#F6\" data-ref-type=\"fig\">Fig. 6<em>A<\/em><\/a>), suggesting subtle sex-specific effects. We therefore performed gene ontology analysis on the 37 and 732 transcripts with at least a 20% group difference (*P* \\< 0.05) in females and males, respectively. (The reference list comprised the 14,628 transcripts significantly expressed in hypothalamus.) No enriched gene ontology terms were found in females. In males, however, for both the 381 genes upregulated and the 351 downregulated in SL hypothalamus, the foremost biological process related to formation of neuronal projections. Examination of the gene ontology terms associated with the genes comprising these enrichments (<a href=\"#F6\" data-ref-type=\"fig\">Fig. 6<em>B<\/em><\/a> and [Supplementary Table 5](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1306\/-\/DC1)) suggests a subtle shift in expression profile that may favor neuronal remodeling in the hypothalamus of adult SL males. Additionally, in an analysis of gene networks associated with the expression changes in male hypothalamus ([Supplementary Fig. 7](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1306\/-\/DC1)), two of the top three networks were related to cellular development and nervous system development. These networks are centered on Atn1 and dynein, respectively (both regulators of neurodegeneration), again supporting potential alterations of neuronal remodeling in SL males.\n\n# DISCUSSION\n\nHere we showed that early postnatal overnutrition, known to permanently increase body weight and adiposity, also reduces voluntary physical activity and energy expenditure in adult females. These physiological changes were associated with persistent alterations in hypothalamic DNA methylation at specific loci. Overall, these findings provide support for the hypothesis that early postnatal overnutrition causes subtle but widespread changes in hypothalamic epigenetic regulation that persist to influence adult energy balance.\n\nOur study addresses a key outstanding question: whether the persistently altered energy balance of SL rodents is due to increased food intake or decreased physical activity. Previous studies reported increased food intake (13,36) and energy expenditure (37) in adult SL rodents. Those conclusions, however, were based on nonnormalized data, disregarding the altered weight and body composition of SL rodents. Here, we used least squares means to appropriately adjust expenditure and intake data for body weight and composition (30). Compared with C mice of the same weight and body composition, adult SL mice were not hyperphagic (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<em>A<\/em><\/a>). Their energy expenditure, however, again compared with C mice of the same weight and body composition, was lower (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<em>B<\/em><\/a>), significantly so in females. Hence, our data provide strong evidence that reduced energy expenditure, not increased food intake, explains the increased adiposity of female adult SL mice.\n\nIn addition to food intake and energy expenditure, however, there are other determinants of energy balance, such as nutrient absorption, which were not measured in this study. Also, it is possible that group differences in central regulation of food intake may have been unmasked if mice were provided a highly palatable diet (38). Other than physical activity, we did not measure additional determinants of energy expenditure, such as brown adipose tissue activity. These shortcomings may explain why the excess adiposity of male SL mice occurred without measurable differences in physical activity or energy expenditure. (Notably, a recent study found age-associated alterations in thermogenic capacity of brown adipose tissue in male SL rats \\[39\\], consistent with our finding that resting metabolic rate is increased at P21 but decreased at P180 in male SL mice.) In females, the reduced energy expenditure was largely explained by reduced physical activity (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<em>C<\/em><\/a>). In an earlier report in rats, prenatal undernutrition likewise caused persistent reductions in locomotor activity, most prominently in females (40). Given the worldwide trends of decreasing physical activity (41), it is crucial to determine whether, in humans as in rodents, nutrition during early life modulates voluntary physical activity for a lifetime.\n\nDespite its importance in central regulation of food intake and energy expenditure (21), our understanding of the molecular mechanisms driving functional development of the hypothalamus remains limited. Mouse hypothalamic development continues into early postnatal life, a critical period for formation of leptin-sensitive neuroanatomic projections that function in energy homeostasis (42) and major alterations of hypothalamic gene expression (34). Here, we have shown for the first time that during this same period widespread changes in DNA methylation\u2014mostly increases\u2014are underway. The association of these methylation increases with genes involved in neural development (<a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<em>C<\/em><\/a>) suggests a process of postnatal epigenetic maturation. Because projections from the arcuate nucleus of the hypothalamus to other brain regions form prenatally in primates but postnatally in rodents (43), it is often proposed that hypothalamic development during the suckling period in the mouse is comparable with that in a third-trimester human. It is currently unknown, however, whether and when the epigenetic maturation we have documented in the postnatal mouse occurs in humans. Moreover, our findings that postnatal overnutrition leads to a decrease in physical activity in female mice raises the question of whether the mouse is a good model for physical activity in humans. Although we currently have only a rudimentary understanding of the neurobiological regulation of spontaneous physical activity (44), the hypothalamus and other brain regions are known to be involved, as are several highly conserved neuropeptides including cholecystokinin, corticotrophin-releasing hormone, leptin, and orexins.\n\nIt was recently reported that SL rats have alterations in hypothalamic DNA methylation (45,46). Those studies, however, assessed DNA methylation only at P21. Our data therefore provide the first evidence that early postnatal overnutrition induces persistent epigenetic changes in the hypothalamus. Additionally, unlike previous studies on related models (45\u201348), rather than focus on single CpG sites we performed integrated analysis of all CpG sites represented in each assay because *1*) regional changes in DNA methylation are more likely to affect gene expression and *2*) concordant changes at multiple adjacent sites are less likely to occur by chance. Notably, contrary to the previous report of increased DNA methylation (at 2 of 20 CpG sites measured) at the *Pomc* promoter in the hypothalamus of P21 SL rats (45), our methylation assay spanning five nearby CpG sites found no SL vs. C differences at P25 ([Supplementary Fig. 4](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1306\/-\/DC1)).\n\nWe developed the strategy of examining genomic regions undergoing DNA methylation changes from P0 to P21, based on the conjecture that these changes may be susceptible to environmental influences. Indeed, 4 of 21 regions undergoing P0\u2013P21 DNA methylation change showed evidence of persistent methylation differences between SL and C mice, supporting the utility of this approach. Hence, the \u223c900 loci that we report that undergo postnatal methylation changes may provide useful candidate regions for future studies of environmental influences on hypothalamic developmental epigenetics.\n\nWith the potential exception of *AK145544*, all four genes with persistent changes in hypothalamic DNA methylation in SL hypothalamus play important roles in neural development or function (49\u201351). At each of these genes, the methylation change in SL mice was modest (<a href=\"#F5\" data-ref-type=\"fig\">Fig. 5<\/a>); the cumulative effect of such changes at hundreds or thousands of genes, however, could be considerable. This interpretation is supported by the AIC model selection ([Supplementary Fig. 6](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1306\/-\/DC1)), which included as significant predictors of adult energy expenditure expression of three and methylation at two of the genes that we identified, in most cases with *F* values comparable with that of lean body mass.\n\nThe results of transcriptional profiling in P180 hypothalamus mirrored our DNA methylation analyses in detecting subtle, widespread, and sex-specific alterations in gene expression. Analyzing the corpus of genes with potentially altered expression in male SL hypothalamus identified highly significant gene ontology enrichments pertaining to regulation of neuronal projections (<a href=\"#F6\" data-ref-type=\"fig\">Fig. 6<\/a>). The adult rodent hypothalamus maintains significant synaptic plasticity (52); our data suggest that early postnatal overnutrition in males may persistently augment this capability. Adult mice that become obese owing to a high-fat diet, conversely, appear to have reduced hypothalamic neurogenesis (53).\n\nAll the potential explanatory effects we found\u2014changes in energy expenditure, physical activity, DNA methylation, and gene expression\u2014were sex specific. The long-term consequences of early life exposures have long been recognized to differ by sex (16). Our findings of sexual dimorphism in the epigenetic responses to early postnatal environment suggest that nutrition may interact with the epigenetic mechanisms regulating hormone-dependent sexualization of the neonatal hypothalamus (54). In fact, the sex differences found here might provide an answer as to why the lower physical activity in SL females arose only in adulthood. In male mice, physical activity declined with age in both groups, but in females this decline was seen only in SL mice ([Supplementary Fig. 1*C*](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1306\/-\/DC1)). Our results may suggest, therefore, that postnatal overnutrition is leading to masculinization of the CNS pathways that regulate age-related changes in physical activity.\n\nEncouraged by earlier studies that gained insights into hypothalamic developmental epigenetics (55), we too studied DNA methylation in whole hypothalamus. The interpretation of our data is therefore complicated by the heterogeneity of the hypothalamus at both the regional and the cellular level. The hypothalamus is comprised of distinct regions, or \"nuclei,\" with specialized functions, gene expression patterns (21), and epigenetic regulation (56). Additionally, the nervous system includes diverse cell types; the simplest classification distinguishes neurons and glia, which are epigenetically distinct (57,58). To improve our understanding of how early postnatal overnutrition causes persistent changes in regulation of body weight and body composition, it will be advantageous to characterize epigenetic effects within specific hypothalamic nuclei and specific cell types. For example, based on our current data we cannot exclude the possibility that the persistent alterations in DNA methylation that we identified represent a shift in the proportion of hypothalamic cell types rather than induced alterations in epigenetic regulation within specific cell types. Moreover, since early postnatal life is a critical period for not only epigenetic but also neuroanatomic development (42), studying these processes in an integrated fashion will likely be necessary to gain a clear understanding of how early postnatal nutrition affects the establishment of hypothalamic body weight regulation.\n\n## ACKNOWLEDGMENTS\n\nThis work was supported by grants from the National Institutes of Health\/National Institute of Diabetes and Digestive and Kidney Diseases (1R01DK081557) and the U.S. Department of Agriculture (USDA) (CRIS 6250-51000-055) (to R.A.W.). Body composition and CLAMS studied were performed in the Mouse Metabolic Research Unit at the USDA\/Agricultural Research Service (ARS) Children's Nutrition Research Center, which is supported by funds from the USDA ARS.\n\nNo potential conflicts of interest relevant to this article were reported.\n\nG.L. and J.J.K. performed experiments and wrote the manuscript. W.Z. and E.L. performed experiments. G.K.-R. performed bioinformatic analyses. M.S.B. performed experiments. M.L.F. provided critical guidance on experimental procedures and edited the manuscript. R.A.W. designed the study and wrote the manuscript. R.A.W. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.\n\nThe authors thank Adam Gillum (USDA\/ARS Children's Nutritional Research Center \\[CNRC\\]) for assistance with the figures and Firoz Vohra (USDA\/ARS CNRC) for assistance with the CLAMS studies.\n\n# REFERENCES\n\n[^1]: G.L. and J.J.K. contributed equally to this study.","meta":{"dup_signals":{"dup_doc_count":114,"dup_dump_count":51,"dup_details":{"curated_sources":2,"2022-05":1,"2020-16":1,"2019-47":1,"2018-30":1,"2018-26":1,"2018-22":1,"2018-17":1,"2018-13":2,"2018-09":1,"2018-05":1,"2017-51":2,"2017-47":1,"2017-43":6,"2017-39":1,"2017-34":2,"2017-30":9,"2017-26":3,"2017-22":1,"2017-17":10,"2017-09":3,"2017-04":10,"2016-50":2,"2016-44":3,"2016-40":3,"2016-36":3,"2016-30":3,"2016-26":2,"2016-22":3,"2016-18":1,"2016-07":1,"2015-48":2,"2015-40":2,"2015-32":1,"2015-27":1,"2015-22":2,"2015-14":2,"2014-52":2,"2014-49":1,"2014-42":3,"2014-41":3,"2014-35":1,"2014-23":1,"2014-15":2,"2023-06":1,"2024-26":1,"2017-13":1,"2015-18":2,"2015-11":1,"2015-06":1,"2014-10":1,"2024-30":1}},"file":"PMC3717861"},"subset":"pubmed_central"}
{"text":"abstract: Cardiovascular autonomic neuropathy (CAN) is associated with increased mortality in diabetes. Since CAN often develops in parallel with diabetic nephropathy as a confounder, we aimed to investigate the isolated impact of CAN on cardiovascular disease in normoalbuminuric patients. Fifty-six normoalbuminuric, type 1 diabetic patients were divided into 26 with (+) and 30 without (\u2212) CAN according to tests of their autonomic nerve function. Coronary artery plaque burden and coronary artery calcium score (CACS) were evaluated using computed tomography. Left ventricular function was evaluated using echocardiography. Blood pressure and electrocardiography were recorded through 24 h to evaluate nocturnal drop in blood pressure (dipping) and pulse pressure. In patients +CAN compared with \u2212CAN, the CACS was higher, and only patients +CAN had a CACS \\>400. A trend toward a higher prevalence of coronary plaques and flow-limiting stenosis in patients +CAN was nonsignificant. In patients +CAN, left ventricular function was decreased in both diastole and systole, nondipping was more prevalent, and pulse pressure was increased compared with \u2212CAN. In multivariable analysis, CAN was independently associated with increased CACS, subclinical left ventricular dysfunction, and increased pulse pressure. In conclusion, CAN in normoalbuminuric type 1 diabetic patients is associated with distinct signs of subclinical cardiovascular disease.\nauthor: Ulrik Madvig Mogensen; Tonny Jensen; Lars K\u00f8ber; Henning Kelb\u00e6k; Anne Sophie Mathiesen; Ulrik Dixen; Peter Rossing; Jannik Hilsted; Klaus Fuglsang KofoedCorresponding author: Ulrik Madvig Mogensen, <email@example.com>.\ndate: 2012-07\nreferences:\ntitle: Cardiovascular Autonomic Neuropathy and Subclinical Cardiovascular Disease in Normoalbuminuric Type 1 Diabetic Patients\n\nCardiovascular autonomic neuropathy (CAN) is a severe complication of diabetes associated with increased morbidity and mortality (1,2). CAN results from damage to the autonomic nerve fibers to the heart, and an early manifestation is a decrease in heart rate variation (HRV) during deep breathing (3). CAN is present in 25% of type 1 diabetic patients but is often overlooked (1,3).\n\nThe increased mortality associated with CAN appears to be partly explained by coexistence with other long-term complications of diabetes (4), and since especially nephropathy and CAN generally develop in parallel, the relative contribution of CAN on mortality has been difficult to identify (5). However, some studies have found CAN to be associated with sudden cardiac death (6) and to be an independent predictor of mortality (7,8). Different mechanisms through which CAN may promote mortality have been suggested including silent myocardial ischemia, silent myocardial infarction, impaired respiratory response to hypoxia, intraoperative cardiovascular lability, and fatal arrhythmia due to QT prolongation (2,5,9\u201311). However, the mechanism remains unclear (12,13).\n\nA noninvasive evaluation of coronary artery stenosis can be performed with high diagnostic accuracy using multislice computed tomography (MSCT) coronary angiography (14). MSCT also allows assessment of coronary artery calcification, which can be quantified as a coronary artery calcium score (CACS). Coronary artery calcium deposit has been shown to be a strong predictor of future cardiovascular events (15\u201317). Other markers of increased cardiovascular risk have recently been introduced: left ventricular dysfunction evaluated with tissue Doppler imaging (TDI) (18,19), a reduction in nighttime blood pressure drop (20,21), and increased arterial pulse pressure (22) are all independently associated with increased cardiovascular risk.\n\nThe aim of this study was to investigate the potential association between CAN and subclinical coronary atherosclerosis in addition to other prognostic markers of cardiovascular disease. For avoidance of the confounding effect of diabetic nephropathy, only patients with normal albumin excretion rates (albuminuria) were included.\n\n# RESEARCH DESIGN AND METHODS\n\nPatients with long-lasting type 1 diabetes, persistent normoalbuminuria, and no history or symptoms of cardiac disease (23) were recruited from the outpatient clinic cohort of type 1 diabetic patients at Steno Diabetes Center and the Diabetes Unit, Rigshospitalet. Inclusion criteria were type 1 diabetes according to American Diabetes Association criteria of at least 10 years' duration, age between 18 and 75 years, and HbA~1C~ \\<10%. Exclusion criteria were albuminuria (urinary albumin-to-creatinine ratio \\>30 mg\/g; elevated *S*-creatinine \\>120 \u03bcmol\/L), untreated hypertension (\\>140\/85 mmHg), and electrocardiographic signs or clinical symptoms of heart disease.\n\nIn the study population registry of 3,000 type 1 diabetic patients, autonomic testing had previously been performed in 350 patients owing to clinical suspicion of autonomic neuropathy, and 123 of these patients were eligible according to inclusion\/exclusion criteria. CAN testing was repeated in all patients included in the current study.\n\nFrom this group, informed consent to participate in the study was obtained in 60 randomly selected patients during their visit to the outpatient clinic. Patients were divided into two groups according to the outcome of four autonomic function tests: HRV during deep breathing, Valsalva ratio, lying-to-standing test, and blood pressure response to standing up. CAN was defined as two or more abnormal tests (24), and age-normative values were used to define abnormality (25,26). Of the included 60 patients, 26 had CAN, 30 were without CAN, and 4 had inconclusive tests.\n\nAll patients underwent MSCT and transthoracic echocardiography (TTE). In order to decrease heart rate for optimized image quality, all patients were given 5 mg ivabradine orally on the evening before and on the morning of cardiac imaging. Ivabradine was selected to reduce heart rate because \u03b2-adrenoceptor blockade was considered relative contraindicated in this population. MSCT and TTE were performed on the same day, and 24-h blood pressure and Holter monitoring was performed simultaneously after a few days. Information on retinopathy was retrieved from the records of the patients, as retinopathy status is registered at least once a year. Vibratory perception threshold (V) was evaluated using biothesiometry (Bio-Medical Instrument Company, Newbury, OH) as an assessment of distal neuropathy. Blood and urine samples were collected and analyzed with standardized clinical chemistry methods including measurements of N-terminal pro-brain natriuretic peptide (NT-proBNP), high-sensitivity C-reactive protein, and cystatin in addition to routine blood tests to exclude signs of other diseases or neuropathies.\n\nCoronary artery disease evaluated with MSCT was the primary outcome measure. Other parameters were included as confounding factors in the analysis.\n\nAll measurements and analysis were performed with the investigator blinded to the CAN status of the patients. The study was conducted in accordance with the Declaration of Helsinki II and approved by the local ethics committee (protocol number H-4-2009-091). All patients gave written informed consent.\n\n## Cardiovascular autonomic neuropathy tests.\n\nAge-normative values were used to define abnormality in all tests (25,26). HRV was assessed in previously trained patients who were asked to breathe deeply at a rate of six breaths per minute while being monitored on a (50 mm\/s) 12-lead electrocardiography. The maximum and minimum heart rates during each breathing cycle were measured, and the mean difference of six cycles was calculated.\n\nThe lying-to-standing heart rate ratio was determined after at least 5 min rest in the supine position, and HRV was determined by calculating the maximal-to-minimal heart rate ratio: the longest R-R interval, measured around the 30th beat after standing up, to the shortest R-R interval, measured around the 15th beat after standing up.\n\nThe Valsalva test consisted of forced exhalation into a mouthpiece with a pressure of 40 mmHg for 15 s, and the ratio of the maximum R-R after the maneuver to the minimum R-R during the maneuver was calculated. The test was performed three times, and the mean value of the ratios was used.\n\nOrthostatic hypotension was defined as a decrease in systolic blood pressure (SBP) of 30 mmHg when changing from supine to the upright position. Measurements were taken every minute for at least 3 min.\n\n## MSCT.\n\nAll examinations were performed using a Toshiba Aquillon One 320 Volume scanner (Toshiba Medical Systems, Tokyo, Japan) according to the recommendations of the vendor and analyzed on dedicated software (Vitrea 2; Vital Images).\n\nFirst, a prospective low-dose calcium scan without contrast enhancement was performed followed by coronary angiography using a prospective protocol. For the calcium score, slices of 3 mm were acquired using prospective electrocardiogram-gated axial scanning (27). We infused 80\u2013100 mL intravenous contrast agent (Visipaque 320; GE Healthcare) (according to body weight) with a flow rate of 5 mL\/s, followed by a saline chaser (50 mL). Image acquisition was initiated automatically at a density threshold of 180 Hounsfield units (HU) in the descending aorta. The scan parameters were 320 \u00d7 0.5 mm detector collimation and 100\u2013120 kV tube voltage depending on BMI (threshold 30 kg\/m^2^). Rotation time was between 350 and 375 ms depending on the heart rate. Owing to the relatively high heart rate (25 patients with heart rate \\>65 bpm), the scanners (sureCardio software) frequently required two gantry rotations. Median radiation dose for nonconstrast and for angiography scans was 1.2 mSv (range 0.9\u20132.6) and 4.3 mSv (1.9\u201315.3), respectively. For the coronary computed tomography angiography, 0.5-mm slices were obtained. The images were reconstructed at 0.5-mm slices with an increment of 0.25 mm, thereby giving true isotropic voxels of 0.5 mm (28).\n\n## Coronary calcium scoring.\n\nCoronary calcium deposit was identified as a dense area in the coronary artery exceeding the threshold of 130 HU. Areas were automatically registered by the software and were manually assigned the corresponding coronary arteries. Total mass of the coronary calcium deposit and the Agatston score were determined for each patient. CACS was compared with reference values on an individual level according to age and sex (29,30).\n\n## Coronary stenosis and plaque composition.\n\nBased on computed tomography coronary angiography recordings, the presence of coronary atherosclerosis was evaluated by visual inspection of all coronary arteries. Degree of coronary stenosis was classified according to previously published guidelines (31): absence of plaque; minimal, plaque with \\<25% stenosis; mild, 25\u201349% stenosis; moderate, 50\u201369% stenosis; severe, 70\u201399% stenosis; and occluded.\n\n## Echocardiography.\n\nEach patient underwent examination with a Philips IE with a S5 transducer with two-dimensonal and TDI in the left lateral decubitus using standard parasternal short axis and apical four-chamber, two-chamber, and long-axis views. Data were analyzed with commercially available software (Excelera; Philips).\n\nEvaluation of the left ventricular ejection fraction was performed by the modified Simpsons biplane method. Left ventricular mass index was calculated as the anatomic mass divided by body surface area of the patient.\n\nPulsed-wave Doppler at the apical position was used to record mitral inflow between the tips of the mitral leaflets. Peak velocities of early (E) and atrial (A) transmitral flow velocities and deceleration time of the early transmitral flow were measured, and the E-to-A ratio was calculated. All valves were examined to exclude significant valvular disease.\n\nWith TDI, peak systolic (s\u2032), early diastolic (e\u2032), and late diastolic (a\u2032) velocities were measured in all six mitral annular positions. Ratios of E to e\u2032, e\u2032 to a\u2032, and e\u2032 to (a\u2032 \u00d7 s\u2032) (eas index), were calculated as measures of left ventricular filling pressures, diastolic performance, and combined systolic and diastolic performance (19).\n\n## Ambulatory 24-h blood pressure recording.\n\nMeasurements were performed on the nondominant arm with a properly calibrated Blood Pressure Monitor System 90217 from Space Laboratories (Washington, DC). Blood pressure measurements were validated with an automatic oscillometric device. The examination was not considered valid if \\>30% of the measurements were missing. The SBP, diastolic blood pressure (DBP), and heart rate were measured automatically every 20 min during daytime (between 0600 and 2200 h) and once every hour during nighttime (between 2200 and 0600 h) for 24 consecutive hours. Participants were instructed to go about their normal daily conditions; however they were advised not to rest in bed during the day or exercise heavily and to abstain from caffeine beverages and tobacco.\n\nAnalyses were performed with dedicated software (Spacelabs ABP 92506 report management system). Blood pressure dipping was defined as an average reduction in SBP and DBP \u226510% from day to night (32).\n\n## Ambulatory 24-h electrocardiography.\n\nAmbulatory electrocardiography recordings were obtained with a 12-lead Rozinn RZ153+. The electrocardiography recordings were analyzed with dedicated software and reviewed by highly trained observers. Time domain analysis was performed in order to verify the difference in autonomic function in patients with CAN (26,33). Measures included SD of R-R intervals during a 22-h period, SD of the average R-R interval in all 5-min recordings, and mean of the SD of all filtered R-R intervals for all 5-min segments over 22 h.\n\n## Statistics.\n\nAll analyses were performed with SAS 9.2 (SAS Institute, Cary, NC). The \u03c7^2^ or Fisher exact test was used for dichotomous variables and the Wilcoxon rank-sum test for continuous variables. Dichotomous variables are listed as percentages. Data of continuous variables are presented as median (range).\n\nA multivariable logistic regression model was created with CAN status as the dependent variable and including sex, age, diabetes duration, HbA~1C~, total cholesterol, and smoking as variables considered important to coronary artery disease, and the corresponding *P* values for the independent association of CAN with a given variable are presented in all tables. Univariate linear regression models were created to identify independent predictors of increased calcium score, and these variables were included stepwise in a multiple logistic regression model.\n\nTo further compare groups of similar age, we performed a sensitivity analysis on 22 patients in each group. Excluded from this analysis were the four oldest patients with CAN (+CAN) and the eight youngest without CAN (\u2212CAN) to obtain 22 matched pairs.\n\n# RESULTS\n\n## Patient characteristics.\n\nPatients +CAN had a higher mean age and longer diabetes duration compared with patients \u2212CAN (<a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>). The proportions of all other cardiovascular risk factors and recorded diabetes complications were similar in the two groups.\n\nClinical characteristics and laboratory results\n\n![](1822tbl1)\n\nWhereas +CAN tended to be associated with higher levels of high-sensitivity C-reactive protein than \u2212CAN (median 1.1 mg\/L \\[range 0.2\u201313\\] vs. 0.7 mg\/L \\[0.4\u201314.4\\], respectively, *P* = 0.06), levels of NTproBNP and cystatin C were similar in +CAN and \u2212CAN patients (10.3 \u03bcmol\/L \\[6.1\u201328.9\\] vs. 18.6 \u03bcmol\/L \\[7.8\u201356.5\\], *P* = 0.18, and 0.64 mg\/L \\[0.5\u20130.72\\] vs. 0.67 mg\/L \\[0.45\u20131.2\\], *P* = 0.16, respectively). Peripheral vibration threshold was significantly higher in patients +CAN compared with that in patients \u2212CAN (23 V \\[8\u201350\\] vs. 17 V \\[6\u201335\\], *P* \\< 0.01).\n\n## MSCT.\n\nThe mean CACS was significantly higher in +CAN than in \u2212CAN patients (<a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a>). Categories of increasing CACS according to CAN status are illustrated in <a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>A<\/em><\/a>. The proportions of patients having a CACS \u2265400 were significantly higher in patients +CAN than in those \u2212CAN. While nine patients +CAN were found in categories of CACS \\>400, the highest CACS in \u2212CAN was 312. Furthermore, a CACS \\<10 was less common in patients +CAN than in patients \u2212CAN.\n\nMultislice computed tomography calcium scoring\n\n![](1822tbl2)\n\nWith use of the CACS of each individual patient to find the corresponding percentile in a background population according to age and sex, the median percentile was significantly higher in patients +CAN compared with that in patients \u2212CAN (median 92 \\[range 0\u201399\\] vs. 39 \\[0\u201398\\], respectively, *P* = 0.0205). Similarly, the proportion of patients with a CACS above the 95th percentile according to age and sex was significantly higher in patients +CAN compared with \u2212CAN (13 \\[72%\\] vs. 5 \\[17%\\], *P* = 0.0077).\n\nCoronary computed tomography angiography revealed focal coronary plaques in 38 (69%) patients. Obstructive lesions were found in 9 (16%) and occlusions in 1 (2%) patient.\n\nPatients +CAN had a higher median number of plaque lesions than \u2212CAN patients (three vs. one, respectively, *P* = 0.039) and a higher proportion of patients with more than seven plaque lesions (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>B<\/em><\/a>) in univariate analysis. However, the prevalence of obstructive stenosis was not significantly higher (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>C<\/em><\/a>).\n\nAbsence of elevated CACS did not exclude obstructive stenosis, as three patients had coronary atherosclerosis with a CACS of zero. Two of these patients had minimal plaques (\\<25%), but one patient (+CAN) had a 70\u201399% noncalcified stenosis.\n\n## Echocardiography.\n\nAll patients had a normal left ventricular ejection fraction. CAN status had no significant impact on left ventricular systolic function as measured by conventional echocardiography, but TDI measures of longitudinal systolic function (s\u2032) were significantly lower in +CAN than in \u2212CAN (<a href=\"#T3\" data-ref-type=\"table\">Table 3<\/a>).\n\nEchocardiography findings\n\n![](1822tbl3)\n\nTDI measures of left ventricular diastolic function indicated higher filling pressures (E-to-e\u2032 ratio) and impaired diastolic performance according to both e\u2032 and e\u2032-to-a\u2032 ratio in +CAN compared with \u2212CAN, whereas the eas index was not significantly different in the two groups (<a href=\"#T3\" data-ref-type=\"table\">Table 3<\/a>).\n\n## Ambulatory blood pressure measurements.\n\nPatients +CAN had higher SBP and lower DBP but mean arterial blood pressure similar to that in \u2212CAN patients. Accordingly, the pulse pressure was significantly higher in patients +CAN compared with that in \u2212CAN patients (<a href=\"#T4\" data-ref-type=\"table\">Table 4<\/a>).\n\nTwenty-four-hour blood pressure and Holter measurements\n\n![](1822tbl4)\n\nThe nocturnal drop in blood pressure was lower in +CAN patients compared with that in \u2212CAN patients with respect to both SBP and DBP. The number of patients with abnormal dipping was significantly higher in +CAN patients compared with \u2212CAN patients.\n\n## Ambulatory electrocardiography.\n\nMean 24-h heart rate was independent of CAN status, but the maximal increase and decrease in heart rate were lower in +CAN compared with \u2212CAN patients (<a href=\"#T4\" data-ref-type=\"table\">Table 4<\/a>).\n\nThe total number of ventricular extrasystoles (VES) was significantly higher in +CAN compared with \u2212CAN patients. The majority of the VES were isolated, and the overall prevalence of consecutive VES was low and without difference between the groups. The number of patients having \\>30 VES\/h, which has been defined as frequent (32), were not significantly higher in +CAN than in the \u2212CAN patients.\n\nIn the time domain analysis of HRV patients +CAN compared with \u2212CAN had significantly lower SD of R-R intervals during a 22-h period (median 99 \\[range 47\u2013185\\] vs. 152 \\[99\u2013208\\], *P* = 0.0005) as well as SD of the average R-R interval in all 5-min recordings and mean of the SD of all filtered R-R intervals for all 5-min segments over 22 h (data not shown).\n\n## Statistical analysis.\n\nVariables independently associated with increased CACS from univariate analysis were DBP, pulse pressure, and measures of diastolic function (E-to-e\u2032 ratio, interventricular septum diameter \\[IVSD\\], E, A, and left atrial volume). When all significant variables were included stepwise in a multiple logistic regression model, CAN remained an independent predictor of CACS (*P* = 0.0009) together with age (*P* = 0.0136), diabetes duration (*P* = 0.0010), and daytime DBP (*P* = 0.0269). Including BMI, pulse pressure, and\/or urine albumin excretion in the model did not change the significance of any of the presented results.\n\n## Sensitivity analysis.\n\nA sensitivity analysis of 22 matched patient pairs (+CAN and \u2212CAN) is presented in <a href=\"#T5\" data-ref-type=\"table\">Table 5<\/a>. In this analysis, there was no significant difference in age, sex, diabetes duration, HbA~1C~, or other cardiovascular risk factors between +CAN and \u2212CAN patients. In this subset of patients, results regarding CACS, echocardiography, ambulatory blood pressure, and Holter monitoring did not differ from those of the entire study population (<a href=\"#T5\" data-ref-type=\"table\">Table 5<\/a>).\n\nFindings of analysis of 22 patients with and without CAN of comparable mean age and diabetes duration\n\n![](1822tbl5)\n\n## Coexistence of CAN and markers of increased cardiovascular risk.\n\nA higher proportion of patients +CAN had markers associated with increased cardiovascular mortality, including a CACS \u2265400 (hazard ratio 2.99\u20133.24 for mortality \\[15\\]), abnormal dipping (2.16 for mortality \\[21\\]), a pulse pressure \u226562 mmHg (1.8 for mortality \\[34\\]), and a trend toward a higher proportion of patients with coronary artery stenosis \u226550% (41 for a composite end point of death, nonfatal myocardial infarction, and revascularization \\[35\\]) (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<\/a>).\n\n# DISCUSSION\n\nThe current study demonstrates an association between the presence of CAN and several distinct signs of subclinical cardiovascular disease in type 1 diabetic patients with normal urinary albumin excretion rate. Patients with CAN were characterized by increased coronary calcium deposit, impaired left ventricular function, increased arterial pulse pressure, a higher prevalence of nondipping, and increased ventricular ectopia compared with patients without CAN.\n\nLong-term type 1 diabetes carries an excess cardiovascular mortality (36), and nephropathy is a major contributing factor (37,38). Thus, even in asymptomatic patients a greater atherosclerotic plaque burden was demonstrated by magnetic resonance imaging in diabetic patients with albuminuria compared with patients with normal albumin excretion rate (39).\n\nCAN has also been associated with high cardiovascular mortality (2). However, since nephropathy and CAN generally develop in parallel in type 1 diabetic patients, the relative contribution of CAN to cardiovascular disease has been difficult to identify (5). In an attempt to overcome this important confounder, the present CAN population was identified in a subset of type 1 diabetic patients characterized by long duration of diabetes yet with absence of nephropathy (i.e., normal albumin excretion rate).\n\nCACS is a powerful predictor of clinical coronary artery disease (17). An association between CACS and reduced cardiac autonomic function has recently been reported in both type 1 (40\u201342) and type 2 (43) diabetes. Colhoun et al. (40) observed an association between CACS and CAN independent of age and triglycerides but not independent of BMI and SBP. By contrast, Rodrigues et al. (42) found an association between CAN and progression of CACS, independent of all confounders tested, including inflammatory markers. In the current study, we extended the evaluation of the coronary arteries with a combination of CACS and computed tomography angiography and included measurements of several other potential confounders, including left ventricular function and 24-h blood pressure.\n\nCACS was markedly higher in patients +CAN, with a median CACS of 196 compared with a median CACS of 5 in patients \u2212CAN. With use of MESA data as reference (29,30), 72% of patients +CAN had CACS similar to that in subjects in the upper 95th percentile according to age and sex, whereas this was only the case for 17% of patients \u2212CAN. Likewise, the estimated arterial age for a person with CACS of 5 is 52 years (95% CI 48\u201356 years) (30,44), in agreement with the median age of patients \u2013CAN. By contrast, a CACS of 196 corresponds to an estimated arterial age of 77 years (75\u201380), which is almost 20 years more than the actual median age of patients +CAN.\n\nDespite a higher CACS among +CAN patients, computed tomography angiography did not reveal significant differences in the prevalence of obstructive stenoses. It could be speculated whether the increased calcification without increased luminal coronary plaque burden is due to a different localization of the calcified area relative to the intima and media. Surgical sympathectomy leads to arterial media calcification, a condition frequently found in the lower extremities in patients with diabetic neuropathy (45), and it has been suggested that arterial media calcification could be the result of autonomic denervation (40,46). Sympathetic denervation may cause dedifferentiation of vascular smooth muscle cells, and these alterations are associated with extracellular matrix production and migration to the intima\u2014changes that are seen in atherosclerosis (40,47). Another suggested mechanism of how CAN could result in increased calcification is through loss of neuropeptides, involving biochemical pathways similar to those observed in studies on calcification processes in bone metabolism (46).\n\nData from the current study could be interpreted as supportive of the concept of increased media sclerosis. However, the relationship between CACS and luminal stenoses is known to be only modest (48), and a nonsignificant difference in the prevalence of stenoses does not necessarily imply that the calcification is differently located in the arterial wall. Furthermore, the existence of media sclerosis has been questioned (49).\n\nRegardless of the location of the calcification, the difference in CACS could be caused by other confounding factors. Many cardiovascular parameters are interrelated, and associations could merely reflect that these conditions share common risk factors.\n\nThe presence of CAN was associated with a decreased systolic and diastolic function of the left ventricle. We found a decrease in s\u2032 of approximately 1 cm\/s in patients +CAN compared with patients \u2212CAN. A similar decrease in s\u2032 in a study of the background population corresponded to a hazard ratio of 1.35 for 5-year mortality (19), suggesting that the observed left ventricular dysfunction might have prognostic importance. Several echocardiographic measures of diastolic function were univariately associated with CACS. Blood pressure variables are other factors associated with CAN, CACS, and left ventricular function. However, CAN remained independently associated with CACS, even when these variables were included in multivariable models.\n\nAn association between CAN and nondipping has been attributed to impaired vagal activity during nighttime in patients with CAN (50). In the current study, nondipping was more prevalent in patients with CAN, but it was not an isolated phenomenon in these patients, since 37% of patients without CAN were also nondippers. Likewise, a recent study did not find CAN to be the main causal factor for nondipping in type 1 diabetes (51). Nonetheless, blood pressure levels at night were significantly higher in +CAN compared with patients \u2212CAN, suggesting a relationship between CAN and nocturnal blood pressure regulation.\n\nCAN was also found to be associated with increased pulse pressure. In addition to being associated with increased cardiovascular risk, arterial pulse pressure is considered an indirect marker of arterial stiffness (22). With use of other methods, arterial stiffness has been shown to contribute to left ventricular diastolic dysfunction (52) and has been suggested as a link between CAN and cardiovascular disease (53).\n\nWe did not observe a significant difference in mean corrected QT or in the prevalence of QTc prolongation. Very few studies have reported on ventricular ectopia associated with CAN. We found no signs of pathological arrhythmias, but CAN was associated with an increased number of isolated premature ventricular beats.\n\nThough interrelationships are difficult to exclude, CAN remained independently associated with CACS even when differences in cardiovascular parameters were adjusted for in multivariable analysis and sensitivity analysis was applied, and CAN was found to be associated with markers of increased cardiovascular risk (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<\/a>).\n\nThis study had limitations. First, owing to stringent inclusion criteria and since autonomic dysfunction is a rare isolated complication in long-term diabetes (10), it was only possible to match patients for age, sex, and diabetes duration in a limited number of patients, and several of the variables measured are age dependent. However, both multivariable logistic regression models and sensitivity analysis of smaller groups of patients with no differences in demographics and cardiovascular risk factors were performed. In these analyses, all reported findings remained similar and significant. Second, investigations were only carried out on diabetic patients; reference values on the different measurements from a nondiabetic control group were therefore not available. Third, we did not have information on HRV on all patients in our registry. Many patients had HRV measured in previous studies, but some of the patients had HRV measured owing to clinical suspicion of CAN. We cannot exclude having captured a particular subgroup of patients being free of long-term diabetes complications not similar to the most common phenotype of diabetic patients, and our findings can only to a limited extent be extrapolated to the entire diabetic population.\n\nIn the analysis of ambulatory blood pressure measurements, a fixed method was used and not diary time. This could potentially have biased the results if differences in the sleep patterns between the two groups exist.\n\nIn the comparison between CACS and individual reference values from the MESA study, diabetes duration was a parameter that could not be accounted for. The longer diabetes duration in +CAN patients must be remembered when evaluating these data.\n\nConfounding effects from differences in antihypertensive treatment and use of diuretics cannot be excluded, even though the differences were nonsignificant in demographics.\n\nIn conclusion, CAN was associated with several distinct signs of subclinical cardiovascular disease in type 1 diabetic patients with normoalbuminuria. These included increased coronary calcium deposit, subtle impairment of left ventricle systolic and diastolic function, increased arterial pulse pressure, a higher prevalence of nondipping, and marginally increased ventricular ectopia, and CAN was independently associated with CACS even with adjustment for confounding factors.\n\n## ACKNOWLEDGMENTS\n\nThis study was supported by a grant from Arvid Nilssons Foundation. The MSCT Cardiac Research Unit, Rigshospitalet, is supported by a grant from the A.P. M\u00f8ller og Hustru Chastine McKinney M\u00f8llers Fond til almene Formaal.\n\nU.M.M. was supported by a scholarship from Novo Nordisk. No other potential conflicts of interest relevant to this article were reported.\n\nU.M.M. researched data and wrote the manuscript. T.J. included patients, contributed to discussion, and reviewed the manuscript. L.K. researched data and reviewed the manuscript. H.K. reviewed the manuscript. A.S.M. screened patients. U.D. and P.R. reviewed the manuscript. J.H. contributed to discussion and reviewed the manuscript. K.F.K. researched data and reviewed the manuscript. U.M.M. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.\n\nParts of this study were presented in abstract form at the European Society of Cardiology Congress 2011, Paris, France, 27\u201331 August 2011, and at the 47th European Association for the Study of Diabetes Annual Meeting, Lisbon, Portugal, 12\u201316 September 2011.\n\nThe authors thank Tina Bock-Pedersen, MSCT Cardiac Research Unit, Rigshospitalet, for excellent technical assistance.\n\n# REFERENCES","meta":{"dup_signals":{"dup_doc_count":138,"dup_dump_count":56,"dup_details":{"curated_sources":2,"2021-04":1,"2020-40":1,"2020-16":1,"2020-05":1,"2019-51":1,"2019-47":1,"2019-43":1,"2019-39":1,"2019-26":1,"2019-13":1,"2018-43":1,"2018-30":2,"2018-22":1,"2018-17":1,"2018-13":3,"2018-09":1,"2018-05":2,"2017-51":1,"2017-47":3,"2017-43":6,"2017-39":2,"2017-34":2,"2017-30":7,"2017-26":3,"2017-22":1,"2017-17":9,"2017-09":4,"2017-04":9,"2016-50":3,"2016-44":4,"2016-40":3,"2016-36":3,"2016-30":3,"2016-26":3,"2016-22":3,"2016-18":2,"2016-07":2,"2015-48":3,"2015-40":2,"2015-35":2,"2015-32":1,"2015-27":2,"2015-22":2,"2015-14":3,"2014-52":2,"2014-49":3,"2014-42":1,"2014-41":4,"2014-35":2,"2014-23":2,"2023-14":1,"2017-13":4,"2015-18":3,"2015-11":2,"2015-06":2,"2024-10":1}},"file":"PMC3379682"},"subset":"pubmed_central"}
{"text":"abstract: Several aspects of *Journal of Biology* seem to have caught readers' attention. Some of the questions asked have arisen sufficiently often to be worth addressing here.\nauthor: Martin Raff; Theodora Bloom; Peter Newmark\ndate: 2002\ninstitute: 1Editor-in-Chief, *Journal of Biology*; 2Editor, *Journal of Biology*; 3Editorial Director, BioMed Central\ntitle: Editorial\n\nSeveral aspects of *Journal of Biology* seem to have caught readers' attention since issue 1 appeared this summer. Some of the questions asked have arisen sufficiently often to be worth addressing here. In summary, the journal differs from other top-tier journals in four main ways. First, and most importantly, no fee will ever be charged to readers of the research articles, and the authors retain full copyright, so that the articles can be freely read and distributed by anyone, from the day of publication onwards, in perpetuity. This is the 'open access' policy of all of the journals published by BioMed Central, which is currently the only publisher that is wholly committed to the principles of open-access publishing.\n\nWhy is the immediate free use and distribution of the entire article so important? Not only is it possible and desirable, but it benefits both scientists and science; restrictions on use and distribution serve publishers, not scientists or readers. Open access also allows full archiving and retrieval. Extensive efforts are being made to create public archives of the scientific literature, containing complete copies of all scientific papers. (PubMed Central is one example of this, and all research articles published in all BioMed Central journals are deposited there in full.) In time, these freely accessible archives will greatly facilitate the practice and dissemination of science, and will lead to exciting and sophisticated ways of using full-text information, much as GenBank has done for DNA sequences. The current restrictions on access, use, and distribution put in place by most journals - even those that offer copyright to authors while in fact denying them permission to distribute their article - will seriously impede the development and comprehensiveness of central archives.\n\n*Journal of Biology* differs in a second way from some of the best-known high-profile journals with which it aims to compete. Its reviewing process is designed to be as fast, fair, and constructive as possible. Decisions are made jointly by a scientist as editor-in-chief and a professional editor. No submitted article is rejected without advice from a relevant scientist, and fashion is not a consideration. And at least one of three peer-reviewers for each article is chosen from a list supplied by the authors.\n\nThe third difference comes from our commitment to maximize the impact of the research we publish. Each research article is accompanied by commissioned commentaries. We aim for the most effective presentation of data, both online and in print, and the print issue is distributed free of charge to over 80,000 life scientists.\n\nFinally, we do not wait for a threshold number of papers of sufficient quality before publishing an issue. Instead, an issue appears whenever a research article of suitable caliber is ready for publication. For this reason, the first two issues of *Journal of Biology* each contain only one keynote research article and its associated commentaries.\n\nTo date, *Journal of Biology* has declined tens of articles for every one it has published. This is because we are committed to publishing only the most significant research. The challenge is to convince scientists with an important story to tell to try *Journal of Biology* instead of their usual preferred journal. We have met enthusiasm for open-access publishing, and for *Journal of Biology*, across the board, from students to Nobel laureates, and at all levels in between. The merits of open access, the wide dissemination of each article, and the usefulness of the associated commentaries, result in each paper published in *Journal of Biology* having unparalleled impact. Why not make 2003 the year you discover the benefits of publishing in *Journal of Biology* for yourself?\n\n**Editor's note:** Martin Raff has recently joined the Scientific Advisory Board of Curis, the company responsible for the research article in this issue. He was not involved in the refereeing of this article or in the decision to publish it.","meta":{"dup_signals":{"dup_doc_count":135,"dup_dump_count":44,"dup_details":{"curated_sources":2,"2020-40":1,"2020-10":1,"2019-22":1,"2019-09":1,"2018-51":1,"2018-43":1,"2018-30":1,"2018-13":1,"2017-47":1,"2017-34":1,"2017-26":1,"2017-22":1,"2017-17":1,"2017-09":11,"2016-44":1,"2016-40":1,"2016-36":11,"2016-30":10,"2016-22":1,"2016-18":1,"2016-07":8,"2015-48":3,"2015-40":2,"2015-35":3,"2015-32":3,"2015-27":3,"2015-22":3,"2015-14":2,"2014-52":3,"2014-49":3,"2014-42":11,"2014-41":5,"2014-35":6,"2014-23":5,"2014-15":5,"2022-21":1,"2017-13":1,"2015-18":3,"2015-11":2,"2015-06":3,"2014-10":2,"2013-48":3,"2013-20":3,"2024-18":1}},"file":"PMC137066"},"subset":"pubmed_central"}
{"text":"abstract: A major goal in diabetes research is to find ways to enhance the mass and function of insulin secreting \u03b2-cells in the endocrine pancreas to prevent and\/or delay the onset or even reverse overt diabetes. In this *Perspectives in Diabetes* article, we highlight the contrast between the relatively large body of information that is available in regard to signaling pathways, proteins, and mechanisms that together provide a road map for efforts to regenerate \u03b2-cells in rodents versus the scant information in human \u03b2-cells. To reverse the state of ignorance regarding human \u03b2-cell signaling, we suggest a series of questions for consideration by the scientific community to construct a human \u03b2-cell proliferation road map. The hope is that the knowledge from the new studies will allow the community to move faster towards developing therapeutic approaches to enhance human \u03b2-cell mass in the long-term goal of preventing and\/or curing type 1 and type 2 diabetes.\nauthor: Rohit N. Kulkarni; Ernesto-Bernal Mizrachi; Adolfo Garcia Ocana; Andrew F. StewartCorresponding authors: Rohit N. Kulkarni, <firstname.lastname@example.com>, and Andrew F. Stewart, <email@example.com>.\ndate: 2012-09\nreferences:\nsubtitle: : Driving in the Dark Without a Road Map\ntitle: Human \u03b2-Cell Proliferation and Intracellular Signaling\n\n# The Challenge of Inducing Human \u03b2-Cell Replication\n\nA principal goal of the National Institutes of Health\/National Institute of Diabetes and Digestive and Kidney Diseases, Juvenile Diabetes Research Foundation, American Diabetes Association, and their European and Asian equivalents is to develop viable therapeutic approaches to induce adult human \u03b2-cell replication\/expansion for regeneration therapies in patients with type 1 diabetes and\/or type 2 diabetes mellitus (T2DM). Unfortunately, all observers of adult human \u03b2-cell replication find that it is very limited (\u223c0.2% of \u03b2-cells\/24 h) and poorly responsive or unresponsive to the many mitogens, growth factors, and nutrients that have been shown to induce expansion in rodent models. For example, glucagon-like peptide-1 (GLP-1) and its analog, exendin-4, hepatocyte growth factor (HGF), lactogens, insulin, IGF-I, and many other molecules have been shown to increase rat and mouse \u03b2-cell proliferation and mass expansion, but have shown limited effects in human \u03b2-cells.\n\n# Species- and Age-Related Failure of \u03b2-Cell Replicative Capacity\n\nSome of this refractoriness to proliferation in both rodents and humans is age-related. For example, whereas \u03b2-cell replication is easy to induce using exendin-4 or partial pancreatectomy in young mice, it is markedly attenuated in older mice (1). Similarly, whereas \u03b2-cell replication has been difficult to induce in adult human \u03b2-cells, reports of human embryonic and neonatal \u03b2-cell proliferation do indicate that proliferation can occur in juvenile human \u03b2-cells (2,3). But even in this study, the proliferation is very limited (\u223c2\u20134% in embryonic human \u03b2-cells by Ki-67 staining) as compared with other fetal and adult tissues (e.g., spleen, bone marrow, gastrointestinal crypts, basal keratinocytes) where proliferation is often 10-fold higher.\n\nIn contrast, there are also clear species differences, because rodent \u03b2-cell models can display relatively large proliferative responses (e.g., 10\u201315%), whereas this type of proliferation is never seen in human \u03b2-cells under physiological conditions, even in embryonic life. Perhaps more germane to human diabetes, whatever the underlying reasons, these issues are a major hurdle to driving therapeutic human \u03b2-cell expansion, because the major source of human \u03b2-cells is adult cadaveric donors, and the major therapeutic target for expansion of endogenous human \u03b2-cells, at least initially, will be adults with type 1 diabetes mellitus. Thus, there is an urgent need to understand why adult \u03b2-cells are refractory to replication, and, at the end of this *Perspectives in Diabetes* article, we suggest a series of questions to be addressed by the scientific community to reverse this state of ignorance.\n\n# The Rodent \u03b2-Cell Receptor\u2013Nutrient\u2013Signaling\u2013Cell-Cycle Pathway Road map\n\nIn rodent \u03b2-cells, we have an impressive and expanding intracellular signaling road map, or \"wiring diagram,\" that reveals how proliferation normally occurs, how it intersects with downstream cell-cycle machinery, and how it can be manipulated for therapeutic purposes. Space prevents a detailed discussion of every pathway, but several oversimplified examples are shown in <a href=\"#F1\" data-ref-type=\"fig\">Figs. 1<\/a>\u2013<a href=\"#F3\" data-ref-type=\"fig\">3<\/a> and described briefly below.\n\n# Signaling Pathways\n\n## Insulin receptor substrate\/phosphatidylinositol-3 kinase\/Akt signaling.\n\nInsulin and IGF-I constitute two primary members of the growth factor family for which receptors are expressed ubiquitously and mediate the growth and metabolic effects of the hormones in virtually all mammalian tissues. Insulin and IGF-I classically bind to their own receptors, but can also cross-react and activate common downstream proteins. Receptor activation transmits signals by phosphorylating insulin receptor substrates (IRS), including the four IRS proteins\u2014Shc, Gab-1, focal adhesion kinase, and Cbl\u2014and others, leading to activation of phosphatidylinositol-3 kinase (PI3K)\/Akt (see below). Several recent reviews provide an excellent resource for interested readers (4,5). Mouse \u03b2-cells express both the insulin and IGF-I receptors, and most components of their signaling pathways. Recent studies on insulin receptor (IR) signaling in \u03b2-cells have provided cumulative evidence for an autocrine role of insulin on its own receptor. Two early mouse models that provided direct genetic evidence for a role for insulin\/IGF-I signaling in the regulation of \u03b2-cell biology include the \u03b2-cell\u2013specific knockout of the IR (\u03b2IRKO) (6) and the global knockout of IRS-2 (7). Both \u03b2IRKO and IRS2KO mice failed to maintain their \u03b2-cell mass and manifested a phenotype most resembling human T2DM. Following these two studies, multiple laboratories have reported the creation and characterization of transgenics\/KOs complemented by in vitro and ex vivo approaches to indicate the significance of proteins in the insulin\/IGF-I cascade for the regulation of \u03b2-cells (5).\n\nContrary to traditional thought, we and others have used genetic approaches to directly demonstrate that the insulin\/IGF-1 signaling pathway is not critical for early development of \u03b2-cells (5,8). However, whereas both \u03b2IRKO and \u03b2-cell\u2013specific IGF-1 receptor KO mice exhibit impaired glucose tolerance and secretory defects, only \u03b2IRKO mice show an age-dependent decrease in \u03b2-cell mass and an increased susceptibility to develop overt diabetes, suggesting a dominant role for insulin signaling in the regulation of adult \u03b2-cell mass (9). It has been observed for several decades that mouse models of diabetes and obesity exhibit a remarkable ability to compensate for the increase in insulin demand in response to insulin resistance. One such model, the liver-specific IR KO mouse, develops severe insulin resistance and glucose intolerance, but the mice do not become overtly diabetic due, in part, to an \u223c30-fold increase in \u03b2-cell mass as a compensatory mechanism to counter the ambient insulin resistance (9,10).\n\nThe IRSs and Akt, important downstream signaling molecules in the IR\/IGF-I receptor signaling pathway, have been reported to play a dominant role in \u03b2-cell growth. Indeed, global KO of IRS-1 in mice leads to postnatal growth retardation and hyperplastic and dysfunctional islets, but the mice do not develop overt diabetes due to \u03b2-cell compensation (11\u201313). In contrast, IRS-2 global KOs develop mild growth retardation and, depending on their genetic background, develop either mild glucose intolerance or \u03b2-cell hypoplasia and overt diabetes (7,14). \u03b2-cell\u2013specific deletion of IRS-2 also leads to mild diabetes (15). Importantly, IRS-2 mediates the effects of the incretin hormone, GLP-1, to promote survival and\/or proliferation of rodent \u03b2-cells (16). Thus, IRS-2 appears to be a positive regulator of \u03b2-cell compensation, whereas IRS-1 predominantly regulates insulin secretion.\n\nA similar scenario is observed in the context of the Akt isoforms. Thus, global KOs for Akt2 develop overt diabetes largely due to insulin resistance in peripheral tissues and \u03b2-cell failure, despite islet hyperplasia and hyperinsulinemia (17,18). Transgenic mice expressing a kinase-dead mutant of Akt1 under control of the rat insulin I promoter showed increased susceptibility to diabetes following fat feeding (19). Islet hyperplasia, \u03b2-cell hypertrophy, and hyperinsulinemia are observed in \u03b2-cell\u2013specific transgenic mice expressing constitutively active Akt1 (20,21). Akt regulates proliferation by modulation of multiple downstream targets including glycogen synthase kinase-3 (GSK3) (see below), FoxO1, and tuberous sclerosis proteins (TSC)\/mammalian target of rapamycin (mTOR) among others. Recent experiments have also linked the cyclin\/cyclin-dependent kinase (CDK) 4 complex to Akt in \u03b2-cell proliferation, showing that Akt1 upregulates cyclins D1 and D2, p21Cip1 levels (but not p27Kip1), and CDK4 activity (22). Collectively, these in vivo data suggest a dual role for the Akts in the regulation of \u03b2-cell mass. In addition to regulating proliferation, class Ia PI3K also modulates \u03b2-cell function by multiple mechanisms (23).\n\n## GSK3 and liver kinase B1 signaling.\n\nGSK3 is a ubiquitously expressed serine\/threonine protein kinase originally identified as a regulator of glycogen metabolism. It is now well-established that GSK3 acts as a downstream regulatory switch for numerous signaling pathways and is involved in cell-cycle regulation and cell proliferation. There are two mammalian GSK3 isoforms encoded by distinct genes: GSK3\u03b1 and GSK3\u03b2. Activated Akt phosphorylates and inactivates GSK3. GSK3 phosphorylation was observed in mice overexpressing a constitutively active form of Akt1 in \u03b2-cells (22), which correlated with an increase in cyclin D1 levels in islets, suggesting that GSK3 is an important negative regulator of \u03b2-cell cycle progression in mice. Indeed, a decrease in GSK3\u03b2 expression can correct diabetes in mouse models of insulin resistance (24,25). More recently, the GSK3\u03b2\/\u03b2-catenin\/T-cell factor pathway has been shown to act in concert with cAMP-responsive element\u2013binding protein (CREB) to downregulate cyclin D2 expression by phosphatase and tensin homolog (PTEN), suggesting a convergence of the PI3K\/PTEN and Wnt pathways. Furthermore, phosphorylation and activation of TSC2 by GSK3\u03b2 inhibits mTOR signaling (26). However, whether this occurs in \u03b2-cells is unknown.\n\nAnother kinase that physically associates in vivo with GSK3\u03b2 is the tumor suppressor liver kinase B1 (LKB1). Loss of LKB1 in adult \u03b2-cells increases \u03b2-cell proliferation, size, and mass and enhances glucose tolerance in mice (27,28), suggesting that this kinase is a regulator of \u03b2-cell growth in vivo in rodents. LKB1 is also known to associate with protein kinase C\u03b6 (PKC\u03b6), as described below. Whether there is any interaction among these three different kinases in the \u03b2-cell is currently unclear.\n\n## TSC, mTOR, S6 kinase, and 4E-BP1 signaling.\n\nmTOR is essential for cell growth and proliferation and is part of two mTOR complexes (mTORC), mTORC1 and 2 \\[recently reviewed (29)\\]. mTORC1 activity is negatively regulated by TSC1, TSC2, and the small G protein Rheb. TSC2 phosphorylation and inactivation by Akt and extracellular signal\u2013related kinase, among other kinases, releases the inhibition of Rheb, leading to activation of mTOR (29). In contrast, phosphorylation and activation of TSC2 by several kinases inhibits mTOR signaling (29). mTORC1 constitutes the rapamycin-sensitive arm of mTOR signaling and contains at least three proteins: raptor, mLst\/G\u03b2L, and PRAS40 (29). This pathway integrates signals from growth factors and nutrients and controls growth (cell size), proliferation (cell number), and metabolism by directly modulating 4E-BP and S6 kinases (S6K) and by indirectly attenuating Akt signaling via an mTORC1\/S6K-mediated negative-feedback loop on IRS signaling. S6K phosphorylates downstream substrates, such as ribosomal S6 protein and eukaryotic translation initiation factor 4B, to promote mRNA translation and synthesis of ribosomes. Phosphorylation of 4E-BPs triggers their release from eukaryotic translation initiation factor 4E and initiates cap-dependent translation. Thus, mTORC1 substrates regulate cell growth, proliferation, and mRNA translation initiation and progression, thereby controlling the rate of protein synthesis.\n\nAs relates to \u03b2-cells, evidence from mouse genetic models demonstrates that mice with conditional deletion of TSC2 (leading to mTORC1 activation) in \u03b2-cells exhibit increases in \u03b2-cell mass, proliferation, and cell size (30). Separate studies demonstrated that conditional deletion of TSC2 or TSC1 in \u03b2-cells causes a similar phenotype, but these mice developed diabetes and \u03b2-cell failure after 40 weeks (31,32). Further confirmation of the effect of mTORC1 activation to increase \u03b2-cell mass comes from mice overexpressing Rheb in \u03b2-cells (33). However, proliferation in \u03b2-cells with mTORC1 activation has not been demonstrated in all studies: thus, precisely how mTORC1 acting upon 4E-BPs and S6K modulates \u03b2-cell mass, proliferation, and function is unclear. Evidence for a role of S6K in \u03b2-cell proliferation was demonstrated in mice with activation of Akt signaling in an S6K1-deficient background (34). However, S6K1 gain of function by transgenic overexpression failed to induce proliferation, in part due to negative feedback on IRS-1 and -2 signaling (35). The importance of 4E-BPs on regulation of \u03b2-cell proliferation is hard to decipher at present because of alterations in insulin sensitivity in global KO models.\n\nFurther evidence for a role of mTORC1 in \u03b2-cell proliferation comes from studies with its inhibitor, rapamycin. These studies demonstrate that: *1*) rapamycin treatment blocks \u03b2-cell expansion, cell size, and proliferation induced by activation of Akt in \u03b2-cells (36). The inhibition of \u03b2-cell proliferation by rapamycin results from decreases in cyclin D2 and D3 and in cyclin-dependent kinase (cdk) 4 activity. *2*) Rapamycin treatment results in reduced proliferation in \u03b2-cells in pregnant mice and causes antiproliferative effects on transplanted rat \u03b2-cells in vivo (37,38). *3*) Rapamycin also attenuates \u03b2-cell expansion in a model of insulin resistance and \u03b2-cell regeneration (39,40) and reduces proliferation of rodent islets in vitro (41). These studies support the concept that mTORC1 pays an important role in the regulation of \u03b2-cell mass and cell cycle and can mediate adaptation of \u03b2-cells to insulin resistance.\n\nThe mTORC2 complex, containing rictor, mSin, and protor, is insensitive to rapamycin. This complex phosphorylates Akt on Ser473, suggesting that this pathway indirectly could be linked to proliferation. However, the only evidence for a role for mTORC2 in \u03b2-cells comes from mice with conditional deletion of rictor in \u03b2-cells (42). These mice exhibit mild hyperglycemia resulting, in part, from decreased \u03b2-cell proliferation, mass, and insulin secretion.\n\n## Other pathways: PKC\u03b6 signaling.\n\nPKC\u03b6 is a member of the atypical PKC subfamily activated by PI3K\/3\u2032-PI-dependent protein kinase-1 (PDK-1). Multiple studies during the 1990s showed that PKC\u03b6 is a critical kinase for mitogenic signal transduction in a variety of cell types, including fibroblasts, brown adipocytes, endothelial cells, and oocytes (43). More recently, the importance of PKC\u03b6 for \u03b2-cell replication has also been elucidated. We and others have shown that growth factors such as GLP-1, parathyroid hormone-related protein, HGF, and nutrients such as glucose increase PKC\u03b6 phosphorylation and activity in insulinoma cells and rodent islets (44\u201346). Interestingly, activation of PKC\u03b6 using a constitutively active form of this kinase (CA-PKC\u03b6) markedly increases \u03b2-cell proliferation in insulinoma and primary mouse \u03b2-cells in vitro, suggesting that PKC\u03b6 activation could be involved in growth factor-induced rodent \u03b2-cell proliferation (45,46). Indeed, small interfering RNA-based downregulation of PKC\u03b6 or the use of a dominant-negative form of PKC\u03b6 completely abolished growth factor-induced \u03b2-cell proliferation (45,46). Conversely, transgenic mice with expression of CA-PKC\u03b6 in \u03b2-cells display increased \u03b2-cell proliferation, size, and mass with a concomitant enhancement in insulin secretion and improved glucose tolerance (47). Downstream, activation of mTOR and upregulation of cyclin Ds and A are essential for PKC\u03b6-mediated mitogenic effects in rodent \u03b2-cells (47). Collectively, these results indicate that PKC\u03b6 is a critical kinase for mitogenic signal transduction in rodent \u03b2-cells in vitro and in vivo.\n\nPKC\u03b6 activation in rodent \u03b2-cells also leads to phosphorylation and inactivation of GSK3\u03b2 (47), suggesting that PKC\u03b6-mediated \u03b2-cell mitogenic effects might also be favored by GSK3\u03b2 inactivation. GSK3\u03b2 is one of the kinases known to phosphorylate D-cyclins and to regulate their degradation (48). Because PKC\u03b6 activation results in increased GSK3\u03b2 phosphorylation\/inactivation and increased expression of <span class=\"smallcaps\">D<\/span>-cyclins, it is possible that PKC\u03b6 increases the accumulation of <span class=\"smallcaps\">D<\/span>-cyclins in \u03b2-cells through inactivation of GSK3\u03b2.\n\n## Lactogen and Janus kinase\u2013signal transducer and activation of transcription signaling.\n\nIn rodent \u03b2-cells, pregnancy-associated lactogens bind to the prolactin receptor, which then activates the Janus kinase JAK2, which results in phosphorylation\/activation of the transcription factor signal transducer and activation of transcription 5 (STAT5) and its nuclear translocation. Phospho-STAT5 dimers bind to the promoter of the cyclin D2 gene and upregulate transcription of cyclin D2 mRNA, with resultant induction of \u03b2-cell proliferation. This pathway was demonstrated to exist in rodent \u03b2-cells (49). More recently, two reports have added depth and definition to this pathway. First, Kim and colleagues (50) has demonstrated that lactogenic signaling during pregnancy upregulates the transcription factor Bcl6, with resulting repression of the epigenetic histone methylase complex, trithorax component, menin. Through the trithorax complex, menin acts as a transcriptional repressor of the CDK inhibitors p18INK4 and p27cip, and their repression is therefore permissive for \u03b2-cell replication in pregnancy. Most recently, the German group (Kim et al.) (51) has demonstrated that lactogenic signaling during pregnancy also is associated with a dramatic, 1,000\u00d7, increase in serotonin production by \u03b2-cells, a reflection of the induction of \u03b2-cell tryptophan hydroxylases by lactogens. Unanswered questions still remain, such as which signaling pathways does 5HT2b activate in the \u03b2-cell? How exactly do these downstream signaling molecules affect the cell-cycle symphony?\n\n## Additional growth factor signaling pathways.\n\nIn this brief *Perspectives in Diabetes*, only a few rodent \u03b2-cell signaling pathways can be described. It should be clear, however, that multiple other signaling pathways linking growth factors to rodent \u03b2-cell replication exist. Examples include: a glucose\u2013glut2\u2013ChREBP\u2013cMyc pathway (52); a glucose\u2013AMP-activated protein kinase\u2013LKB1\u2013calcineurin\u2013nuclear factor of activated T-cell pathway (27,28,53); an epidermal growth factor (EGF) ligand family (EGF, betacellulin, heparin-binding EGF, amphiregulin, and trefoil factor family 3)\u2013EGF receptor\u2013ras\/raf\u2013mitogen-activated protein kinase (MAPK)\u2013cdk\u2013cyclin pathway (54,55); a cAMP\u2013cAMP-dependent protein kinase\u2013CREB\u2013cAMP response element modulator\u2013cyclin A pathway (56,57); parathyroid hormone-related protein induction of proliferation (5); a leptin\u2013IRS-2 pathway (58); a transforming growth factor-\u03b2 activin family\u2013SMAD pathway (59); sex hormone signaling pathways (60); cannabinoid receptors (CB1) that cross-talk with insulin receptors and IRS-2 (61) and calpains (62); and recently, a platelet-derived growth factor (PDGF) receptor linked to MAPK signaling pathway, driving the polycomb repressive complex family member Ezh2, with repression of p16INK4, thereby permitting activation of downstream cell-cycle molecules such as the pRb family pocket proteins and E2Fs (63).\n\n# Cell-Cycle Control of Proliferation in the Rodent \u03b2-Cell\n\nThis is an important and rapidly moving area. It is complex and well beyond the scope of a brief review, but detailed reviews are available (64\u201366). Briefly, downstream of every developmental program and nutrient- or growth factor-driven signaling pathway that is able to drive rodent \u03b2-cell proliferation, intracellular signaling pathways converge on the molecules that control the G~1~\/S checkpoint. This checkpoint is shown schematically in <a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<\/a>, and examples of interactions with upstream signaling pathways are shown in <a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<\/a>. Briefly, there are a number of cell cycle\u2013activating molecules: the <span class=\"smallcaps\">D<\/span>-cyclins and their cognate cdks and the E\/A-cyclins and their cognate cdks, cdk1 and -2. Overexpression of all of these in various combinations is able to drive rodent \u03b2-cell proliferation, either when delivered adenovirally (67) or transgenically, and in some cases (but not all), disruption of their genes leads to \u03b2-cell hypoplasia, hypoinsulinemia, and diabetes (64\u201366). Thus, these cell-cycle activators are key targets of upstream pathways, and they have been shown to be regulated by these signaling pathways, growth factors (as described above), and glucose and glucokinase activators (68).\n\nThese cell-cycle activators are balanced by a series of cell-cycle inhibitors, which include the so-called \"pocket proteins\" (pRb, p107, and p130), the INK4 family (p15, p16, p18, and p19), the CIP\/KIP family (p21, p27, and p57), menin, and p53. The details of their actions and interactions with the cyclins and cdks are too complex for a brief discussion, but detailed reviews are available. The key points in the current context are that they, too, are essential regulators of \u03b2-cell cycle progression (e.g., p21 and p27 are regulated by mitogens and p16 and p18 by free fatty acids), and genetic disruption of the genes encoding some of these molecules leads to \u03b2-cell hyperplasia and hyperfunction (69,70).\n\nIn this context, it is important to emphasize that these G~1~\/S molecules have short half-lives and frequently, if not most often, are regulated at the level of protein stability. This is important because changes seen at the mRNA level frequently correlate poorly with those observed at the protein level.\n\nThus, we know an enormous amount about cell-cycle control in the rodent \u03b2-cell and how key signaling pathways regulate these molecules.\n\n# Cell-Cycle Control in the Human \u03b2-Cell\n\nAs in the rodent \u03b2-cell, the G~1~\/S molecules controlling human \u03b2-cell proliferation have also been studied, and much is now known. For example, we have a relatively complete understanding of which of the \u223c30 G~1~\/S molecules are present in the human islet, and they can be arranged in a working wiring diagram or road map, which looks very similar to the rodent version in <a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<\/a> (71). In addition, we know that adenoviral overexpression of certain cell-cycle regulatory molecules (e.g., cMyc, the cdks, or the cyclins D or E) in the human \u03b2-cell activates proliferation robustly. For example, with adenoviral overexpression of cell-cycle molecules, as many as 10\u201315% of adult human \u03b2-cells are able to replicate as assessed using Ki-67 immunohistochemistry or bromodeoxyuridine incorporation (71\u201374).\n\nAlthough the human \u03b2-cell G~1~\/S road map is similar to that in the rodent \u03b2-cell, there are two principal differences, indicated by the black numbers in <a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<\/a>. First, cdk6 is absent in rodent \u03b2-cells but abundant in human \u03b2-cells (71). This is relevant because genetic loss of cdk4 in mice leads to \u03b2-cell hypoplasia and diabetes, presumably because there is no cdk6 to compensate for this loss. Second, although all three <span class=\"smallcaps\">D<\/span>-cyclins are present in the mouse \u03b2-cell, genetic loss of cyclin D2 (but not cyclins D1 or D3) in mice leads to \u03b2-cell hypoplasia and diabetes, indicating that this is the key <span class=\"smallcaps\">D<\/span>-cyclin in the rodent \u03b2-cell (75,76). In contrast, human islets contain little or no cyclin D2 (71), suggesting, paradoxically, either that cyclin D2 is irrelevant in the human \u03b2-cell or that it is absolutely essential, with its paucity being precisely the reason that human \u03b2-cells do not replicate.\n\n## Failure to signal from cell surface to cell-cycle machinery.\n\nMost importantly in the current context, because adult human \u03b2-cells do not replicate in response to the long list of growth factors, nutrients, and maneuvers that induce rodent \u03b2-cells to replicate, but clearly can replicate when cyclins and cdks are overexpressed (71\u201373), it is clear that failure or inadequacy of the cell-cycle machinery in human \u03b2-cells is not the reason they do not replicate; the machinery is there and waiting to be activated. Thus, the likely missing link in adult human \u03b2-cell replication is not failure to express key cell-cycle molecules, but rather a failure to activate them in response to what would appear to be appropriate upstream signals. More specifically, the blockade(s) would appear to be somewhere among the levels of the upstream growth factors, their receptors, signaling pathways that should, but do not, alter these cell-cycle molecules.\n\n# There is No Human \u03b2-Cell Receptor\u2013Nutrient\u2013Signaling\u2013Cell-Cycle Pathway Road map\n\nIf human \u03b2-cells possess the requisite G~1~\/S molecules to drive \u03b2-cell replication, and their upregulation can drive human \u03b2-cell replication, it is then axiomatic that signaling events that should be reaching the cell-cycle machinery are not. It therefore would be particularly useful to have a human \u03b2-cell signaling road map that describes these intracellular signaling pathways, with their upstream ligands and receptors and downstream cell-cycle targets. Unfortunately, in contrast to the rich and complex road map for growth factor and nutrient induction of rodent \u03b2-cell replication, no such road map exists for the human \u03b2-cell: we remain very much in the dark regarding receptors, nutrient transporters\/sensors, their downstream signaling pathways, and their putative connections to the ultimate downstream cell-cycle regulatory molecules. Simply said, the human \u03b2-cell signaling road map is almost a blank slate, a tabula rasa, as shown in the light gray lines and text in the upper portion of <a href=\"#F4\" data-ref-type=\"fig\">Fig. 4<\/a>, which should be contrasted to their counterparts in <a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<\/a>. The following sections briefly summarize the little that we do know.\n\n## IRS-PI3K-Akt signaling.\n\nHuman islet cells express insulin and IGF-I receptors and components of these signaling pathways (5,77). In functional terms, several studies support the concept of a direct insulin action in the \u03b2-cell that promotes a positive-feedback effect in vivo in healthy humans (78) that is blunted in patients with T2DM (79,80). Ex vivo examination of human islets and pancreas sections from T2DM patients (77,81) supports a role for the insulin-signaling network in the regulation of cell-cycle proteins. Although an increase in \u03b2-cell volume has been reported in humans with obesity (82), it is unclear whether this occurs as a compensatory response to insulin resistance, as occurs in animal models, or is genetically predetermined. Additional studies that directly address this possibility are warranted.\n\n## mTORC1 and regulation of human \u03b2-cell proliferation.\n\nThe expression of some mTOR signaling components has been demonstrated in human islets (83) exemplified by its induction by glucose, amino acids, and insulin (84). Most of our understanding of mTOR signaling in human islets is derived from studies using rapamycin derivatives as immunosuppressants or as treatment of patients with insulinomas and other neuroendocrine tumors. Rapamycin treatment has been shown to inhibit human \u03b2-cell proliferation in vitro (41). Moreover, *TSC2* and *PTEN* expression is decreased in insulinomas and pancreatic endocrine tumors, suggesting that activation of mTORC1 and Akt signaling plays a role in the proliferative changes observed in these tumors (85). However, insulinomas are rare in patients with tuberous sclerosis. In contrast, mTORC1 inhibition by everolimus (a rapamycin analog) improves glycemic control in patients with insulinoma and prolongs progression-free survival in patients with pancreatic neuroendocrine tumors (86,87). The extent to which reduced \u03b2-cell mass or inhibition of insulin secretion contributes to the responses to mTORC1 inhibition is unclear, but, based on the current evidence, it is likely that inhibition of \u03b2-cell proliferation plays a major role.\n\n## PKC\u03b6 and GSK3 signaling in the human \u03b2-cell.\n\nPharmacological inhibition of GSK3\u03b2 has been shown to enhance (approximately fourfold) glucose-mediated human \u03b2-cell proliferation (although from very low to low basal rates) (41), and adenoviral transduction of human islets with CA-PKC\u03b6 in vitro enhances (approximately fourfold) human \u03b2-cell proliferation (45). However, whether PKC\u03b6-induced human \u03b2-cell proliferation is mediated or potentiated by GSK3\u03b2 inactivation is unknown.\n\nIn summary, it is important to emphasize that at present, although many examples exist in rodent \u03b2-cells, we do not have a single example of a complete pathway in the human \u03b2-cell that links a growth factor or nutrient, via a cell-surface receptor through a complete signaling pathway, to the downstream cell-cycle machinery that translates to proliferation. In this regard, the recent report of a PDGF\u2013PDGF receptor\u2013MAPK\u2013Ezh2\u2013p16 pathway (63) is exciting and comes the closest to filling this void.\n\n# Why We Need a Human \u03b2-Cell Mitogenic Signaling Pathway Road map\n\nComparing <a href=\"#F1\" data-ref-type=\"fig\">Figs. 1<\/a> and <a href=\"#F4\" data-ref-type=\"fig\">4<\/a> makes it clear that informed efforts to drive therapeutic human \u03b2-cell proliferation are severely hampered by the essentially complete lack of a human \u03b2-cell road map. Further, the fact that direct cell-cycle activation by cyclin\/cdk overexpression can markedly activate human \u03b2-cell proliferation places at least one key obstacle to human \u03b2-cell proliferation within these upstream signaling pathways, about which we know so little. As noted below, it would be informative to investigate the upstream signaling pathways and identify proteins that link the cell-surface receptors with key molecules in the cell-cycle machinery in human \u03b2-cells.\n\nExamples of key questions about which we have no answers are: *1*) do the gray molecules and lines in <a href=\"#F4\" data-ref-type=\"fig\">Fig. 4<\/a> even exist in adult human \u03b2-cells?; *2*) are there key signaling pathways in human islets that we have completely overlooked? \\[e.g., erythropoietin, insulin, serotonin, and osteocalcin have all been proposed as physiologic drivers of rodent \u03b2-cell replication (51,88,89), but have not been comprehensively tested in human \u03b2-cells\\]; *3*) do adult human \u03b2-cells require engagement of receptors or nutrients distinct from those on rodent \u03b2-cells; *4*) what are the intracellular signaling networks and cell-cycle targets regulated by the kinases in <a href=\"#F4\" data-ref-type=\"fig\">Fig. 4<\/a> that *are* present in human \u03b2-cells?; *5*) are these kinases activated by growth factors and nutrients, and, if not, what is preventing them from being activated?; *6*) can coordinate activation of combinations of pathways synergize to enhance proliferation (e.g., if overexpression of PKC\u03b6 and inhibition of GSK3\u03b2 can increase human \u03b2-cell replication from 0.2 to 1.0%, would coordinate activation of multiple pathways further increase replication?)?; *7*) are the cell-cycle inhibitors that are so abundant in human \u03b2-cells restraining proliferation, and how are they regulated?; *8*) are key cell-cycle regulatory elements epigenetically regulated (in this study, for example, the epigenetic methylases and demethylases menin, Ezh2, and Bmi1 have all been shown to participate in restraining rodent \u03b2-cell replication)?; *9*) in this era of high-throughput screening, which are the optimal small-molecule targets in the human \u03b2-cell to drive proliferation?; and *10*) why and how do \u03b2-cells in young humans proliferate at a higher rate compared with those from older individuals?\n\nThe answers to these critical questions are simply that we know very little: we have no road map from which to hypothesize, model, and integrate data from multiple systems. We believe that obtaining the information required to create such a road map is essential. Without this information, we are driving at night without headlights, without a road map, and without global positioning system navigation. It is time to develop such a high-content signaling road map that connects the cell surface to cell-cycle machinery in the human \u03b2-cell.\n\n## ACKNOWLEDGMENTS\n\nThis work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)\/National Institutes of Health (NIH) grants R01-DK-67536 and R21-RR-24905 and Juvenile Diabetes Research Foundation (JDRF) Grant 17-2011-644 (to R.N.K.), NIDDK\/NIH grants R01-DK-73716 and R01-DK-84236, JDRF Grant 46-2010-758, and Career Development Award 7-06-CD-02 from the American Diabetes Association (ADA) (to E.-B.M.), NIDDK\/NIH grants R01-DK-77096 and R01-DK-67351 and Research Award 1-10-BS-59 from the ADA (to A.G.O.), NIDDK and the Beta Cell Biology Consortium through NIH grants U01-DK-089538 and R01-DK-55023, and JDRF grants 1-2008-39 and 34-2008-630 (to A.F.S.).\n\nNo potential conflicts of interest relevant to this article were reported.\n\nR.N.K., E.-B.M., A.G.O., and A.F.S. wrote the manuscript, reviewed and edited the manuscript, and contributed to the discussion. R.N.K., E.-B.M., A.G.O., and A.F.S. are the guarantors of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.\n\nThe authors thank the innumerable authors who have contributed to the field of \u03b2-cell intracellular signaling and apologize for not being able to discuss and\/or cite all of the important contributions because of editorial limitations on the text and numbers of references allowed.\n\n# REFERENCES","meta":{"dup_signals":{"dup_doc_count":177,"dup_dump_count":64,"dup_details":{"curated_sources":2,"2023-40":1,"2022-21":1,"2021-49":2,"2021-39":1,"2021-31":1,"2021-25":1,"2021-21":1,"2020-40":1,"2020-16":1,"2019-51":1,"2019-47":1,"2019-43":1,"2019-26":2,"2019-09":1,"2018-51":1,"2018-47":1,"2018-34":1,"2018-30":1,"2018-26":2,"2018-22":1,"2018-17":2,"2018-13":1,"2018-05":2,"2017-51":1,"2017-47":2,"2017-43":7,"2017-39":2,"2017-34":2,"2017-30":7,"2017-26":4,"2017-22":4,"2017-17":9,"2017-09":5,"2017-04":10,"2016-50":3,"2016-44":5,"2016-40":4,"2016-36":5,"2016-30":5,"2016-26":3,"2016-22":4,"2016-18":4,"2016-07":4,"2015-48":4,"2015-40":3,"2015-35":2,"2015-27":3,"2015-22":5,"2015-14":2,"2014-52":3,"2014-49":3,"2014-42":5,"2014-41":4,"2014-35":4,"2014-23":3,"2023-50":1,"2017-13":3,"2015-18":3,"2015-11":2,"2015-06":3,"2014-10":1,"2013-48":1,"2013-20":1,"2024-22":1}},"file":"PMC3425429"},"subset":"pubmed_central"}
{"text":"abstract: The individual 'validation' experiments typically included in papers reporting genome-scale studies often do not reflect the overall merits of the work.\nauthor: Timothy R Hughes\ndate: 2009\ninstitute: 1Banting and Best Department of Medical Research, Department of Molecular Genetics, and Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON M5S 3E1, Canada\nreferences:\ntitle: 'Validation' in genome-scale research\n\nFollowing the advent of genome sequencing, the past decade has seen an explosion in genome-scale research projects. Major goals of this type of work include gaining an overview of how biological systems work, generation of useful reagents and reference datasets, and demonstration of the efficacy of new techniques. The typical structure of these studies, and of the resulting manuscripts, is similar to that of a traditional genetic screen. The major steps often include development of reagents and\/or an assay, systematic implementation of the assay, and analysis and interpretation of the resulting data. The analyses are usually centered on identifying patterns or groups in the data, which can lead to predictions regarding previously unknown or unanticipated properties of individual genes or proteins.\n\nSo that the work is not purely descriptive \u2013 anathema in the molecular biology literature \u2013 there is frequently some follow-up or 'validation', for example, application of independent assays to confirm the initial data, an illustration of how the results obtained apply to some specific cellular process, or the testing of some predicted gene functions. As the first few display items are often schematics, example data, clustering diagrams, networks, tables of *P*-values and the like, these validation experiments usually appear *circa* Figure 5 or 6 in a longer-format paper. This format is sufficiently predominant that my colleague Charlie Boone refers to it as \"applying the formula\". I have successfully used the formula myself for many papers.\n\nMy motivation for writing this opinion piece is that, in my own experience, as both an author and a reviewer, the focal point of the review process \u2013 and of the editorial decision \u2013 seems too often to rest on the quality of the validation, which is usually not what the papers are really about. While it is customary for authors to complain about the review process in general (and for reviewers to complain about the papers they review), as a reader of such papers and a user of the datasets, I do think there are several legitimate reasons why our preoccupation with validation in genomic studies deserves reconsideration.\n\nFirst, single-gene experiments are a poor demonstration that a large-scale assay is accurate. To show that an assay is consistent with previous results requires testing a sufficiently large collection of gold-standard examples to be able to assess standard measures such as sensitivity, false-positive rate and false-discovery rate. A decade ago, there were many fewer tools and resources available; for example, Gene Ontology (GO) did not exist before the year 2000 \\[1\\], and many of the data analysis techniques now in common use were unfamiliar to most biologists. Proving that one could make accurate predictions actually required doing the laboratory analyses. But today, many tools are in place to make the same arguments by cross-validation, which produces all of the standard statistics. It is also (gradually) becoming less fashionable for molecular biologists to be statistical Luddites.\n\nSecond, and similarly, single-gene experiments, or illustrations relating to a specific process, do not describe the general utility of a dataset. Many studies have shown (even if they did not emphasize) that specific data types and reagents are more valuable for the study of some things than others. Validation experiments tend to focus on the low-hanging fruit, for instance, functional categories that seem to be yielding the best examples, and the largest numbers. To minimize the ire of my colleagues, I will give an example from my own work. Our first efforts at systematically predicting yeast gene functions from gene-expression data \\[2\\] resulted in more predictions relating to RNA processing than to any other category, and Northern blots are something even my lab can do, so these were the ones we tested. Although we would like to think that the success at validating predictions from other processes will also be as high as our cross-validation predicted, laboratory validation of predictions from only one category does not show that. Moreover, if one is engaged in high-throughput data collection, it is possible to perform a large number of validations, and show only those that work. It is also possible to choose the validation experiments from other screens already in progress, or already done, or even from other labs. I suspect this practice may be widespread.\n\nA third issue is that focus on the validation is often at the expense of a thorough evaluation of the key points of the remainder of the paper. I may be further ruffling the fur of my colleagues here, but I think it is fair to say that a hallmark of the functional genomics\/systems biology\/network analysis literature is an emphasis on artwork and *P*-values, and perhaps not enough consideration of questions such as the positive predictive value of the large-scale data. David Botstein has described certain findings as \"significant, but not important\" \u2013 if one is making millions of measurements, an astronomically significant statistical relationship can be obtained between two variables that barely correlate, and an overlap of only one or a few percent in a Venn diagram can be very significant by the widely used hypergeometric test. A good yarn seems to distract us from a thorough assessment of whether statistical significance equates to biological significance, and even whether the main dataset actually contains everything that is claimed.\n\nI'm writing for an issue of *Journal of Biology* that is about how to make the peer review process easier, but I do believe that papers in our field would be better if referees were allowed and expected (and given time) to look at the primary data, have a copy of the software, use the same annotation indices, and so on, and see whether they can verify the claims and be confident in conclusions that are reached from computational analyses. Even simple reality checks such as comparing replicates (when there are some) are often ignored by both authors and reviewers. I bring this up because one of the major frustrations expressed by a group of around 30 participants at the Computational and Statistical Genomics workshop I attended at the Banff International Research Station last June was the difficulty of reproducing computational analyses in the functional genomics literature. Often, the trail from the primary data to the published dataset is untraceable, let alone the downstream analyses.\n\nFourth, and finally, the individual validation experiments may not garner much attention, unless they are mentioned in the title, or have appropriate keywords in the abstract. They are rarely as useful as they would be in a paper in which they were explored in more depth and in which the individual hypothesis-driven experiments could be summarized. For instance, a paper we published in *Journal of Biology* in 2004 \\[3\\] described an atlas of gene expression in 55 mouse tissues and cell types. Using SVM (Support Vector Machine) cross-validation scores, we found that, for many GO annotation categories, it was possible to predict which genes were in the category, to a degree that is orders of magnitude better than random guessing, although usually still far from perfect. The most interesting aspect of the study to me was the observation that there is a quantitative relationship between gene expression and gene function; not that this was completely unexpected, but it is nice to have experimental evidence to support the generality of one's assumptions. The SVM scores were used mainly to prove the general point, and whether any individual predictions were correct was not the key finding \u2013 we knew ahead of time (from the cross-validation results) that most of the individual predictions would not be correct; this is the nature of the business. Nonetheless, final acceptance of the manuscript hinged on our being able to show that the predictions are accurate, so at the request of reviewers and editors, we showed that Pwp1 is involved in rRNA biogenesis, as predicted. According to Google Scholar, this paper now has 139 citations, and my perusal of all of them suggests that neither Pwp1 nor ribosome biogenesis is the topic of any of the citing papers. The vast majority of citations are bioinformatics analyses, reviews, and other genomics and proteomics papers, many of them concerning tissue-specific gene expression. Thus, the initial impact appears primarily to have been the proof-of-principle demonstration of the relationship between gene function and gene expression across organs and cell types, and the microarray data themselves. It is the use of genome-scale data and cross-validation that proves the point, not the individual follow-up experiments.\n\nA small survey of my colleagues suggests that many such examples would be found in a more extensive analysis of the literature in functional genomics and systems biology.\n\nFor instance, Jason Moffat explained that in the reviews of his 2006 *Cell* paper describing the RNAi Consortium lentivirus collection \\[4\\], which already contained a screen for alteration of the mitotic index in cultured cells, a major objection was that more work was needed to validate the reagents by demonstrating that the screen would also work in primary cell cultures \u2013 which may be true, but so far, even the mitotic index screen seems to have served primarily as an example of what one can do with the collection. The paper has clearly had a major impact: it has 161 citations according to Google Scholar, the vast majority of which relate to use of the RNAi reagents, not any of the individual findings in this paper.\n\nTo conclude, I would propose that, as authors, reviewers and editors, we should re-evaluate our notion of what parts of genome-scale studies really are interesting to a general audience, and consider carefully which parts of papers prove the points that are being made. It is, of course, important that papers are interesting to read, have some level of independent validation, and a clear connection to biology. But it seems likely that pioneering reagent and data collections, technological advances, and studies proving or refuting common perceptions will continue to be influential and of general interest, judging by citation rates. As erroneous data or poorly founded conclusions could have a proportionally detrimental influence, we should be making an effort to scrutinize more deeply what is really in the primary data, rather than waiting to work with it once it is published. Conversely, the individual 'validation' studies that occupy the nethermost figures, although contributing some human interest, may be a poor investment of resources, making papers unnecessarily long, delaying the entry of valuable reagents and datasets into the public domain, and possibly distracting from the main message of the manuscript.","meta":{"dup_signals":{"dup_doc_count":109,"dup_dump_count":45,"dup_details":{"curated_sources":2,"2022-05":1,"2020-40":1,"2020-10":1,"2019-47":1,"2018-51":2,"2018-34":1,"2018-30":1,"2018-13":1,"2017-47":2,"2017-39":1,"2017-34":2,"2017-26":2,"2017-22":2,"2017-17":1,"2017-09":9,"2017-04":1,"2016-50":1,"2016-44":2,"2016-40":1,"2016-36":8,"2016-30":8,"2016-22":1,"2016-18":1,"2016-07":9,"2015-48":2,"2015-40":1,"2015-35":2,"2015-32":2,"2015-27":2,"2015-22":2,"2015-14":1,"2014-52":2,"2014-49":1,"2014-42":7,"2014-41":3,"2014-35":2,"2014-23":4,"2014-15":4,"2022-27":1,"2015-18":2,"2015-11":2,"2015-06":2,"2014-10":2,"2013-48":2,"2013-20":1}},"file":"PMC2656219"},"subset":"pubmed_central"}
{"text":"abstract: Through its adoption of the biomedical model of disease which promotes medical individualism and its reliance on the individual-based anthropology, mainstream bioethics has predominantly focused on respect for autonomy in the clinical setting and respect for person in the research site, emphasizing self-determination and freedom of choice. However, the emphasis on the individual has often led to moral vacuum, exaggeration of human agency, and a thin (liberal?) conception of justice. Applied to resource-poor countries and communities within developed countries, autonomy-based bioethics fails to address the root causes of diseases and public health crises with which individuals or communities are confronted. A sociological explanation of disease causation is needed to broaden principles of biomedical ethics and provides a renewed understanding of disease, freedom, medical practice, patient-physician relationship, risk and benefit of research and treatment, research priorities, and health policy.\nauthor: Jacquineau Az\u00e9tsop; Stuart Rennie\ndate: 2010\ninstitute: 1Facult\u00e9 de M\u00e9d\u00e9cine Teilhard de Chardin, Complexe M\u00e9dical le Bon Samaritain, N'djam\u00e9na, BP 456, Chad; 2Department of Philosophy, University of Cape Town, Cape Town, Private Bag X3 Rondebosch7701, South Africa; 3Department of Social Medicine, University of North Carolina School of Medicine Chapel Hill, 333 S Columbia Street MacNider Hall, Room 348, CB 7240, Chapel Hill, NC 27599-7240, USA\nreferences:\ntitle: Principlism, medical individualism, and health promotion in resource-poor countries: can autonomy-based bioethics promote social justice and population health?\n\n# Introduction\n\nRespect for autonomy or respect for persons has tended to be the leading principle of biomedical ethics or research ethics, respectively. This principle historically has its roots in the liberal moral and political tradition of the Enlightenment in Western Europe. Within this tradition, the ethical justification of actions or practices strongly depends on the free decisions of individuals, i.e. an action or practice can only be ethically justified when undertaken without any coercive influence and entered by free and informed agreement. While there have always been disagreements on the details, all theories of autonomy agree on two essential conditions: the first is liberty, specifying the independence from controlling influences; the second is agency, referring to the capacity for intentional action\\[1\\]. Used in clinical ethics, autonomy functions primarily to examine decision-making in health care and serves to identify actions that are protected by the rules of informed consent, informed refusal, truth telling, and confidentiality\\[1\\]. Autonomy-based approaches are strongly expressed in Tom Beauchamp and James Childress' classic text *Principles of Biomedical Ethics* for clinical bioethics and, for research ethics, the influential *Belmont Report*\\[1,2\\].\n\nMany criticisms of autonomy-based bioethics have appeared over the past thirty years from a number of different angles, such as feminism, casuistry, disability rights, multiculturalism, cultural studies, and ethnography. In this article, we take a different approach by exploring what we will call the 'medical individualism' that autonomy-based bioethics largely assumes, and by raising questions about the relevance and impact of autonomy-based bioethics in developing countries (and communities within developed equitable ones), especially in light of initiatives to 'build capacity' in research sites and to ensure access to healthcare in resource-poor settings. This paper argues that the medical individualism underlying autonomy-based bioethics renders the latter incapable of addressing some of the most pressing bioethical issues in resource-poor settings, which have to do with social justice. The first section of this paper considers some of the limitations of principlism. The second section examines the inability of this approach to address social justice concerns in resource-poor countries. Finally, the third section attempts to offer an alternative approach by exploring the contribution of the sociological model of disease causation to research ethics, health justice and health policy.\n\n# A brief anatomy of autonomy-based bioethics\n\nOne of the major defenders of the centrality of autonomy in bioethics, the British medical ethicist and pediatrician, Raanan Gillon argues that respect for autonomy should hold a primary place among the four principles of biomedical ethics\\[3\\]. Other proponents of autonomy, Beauchamp and Childress, define autonomy as a form of personal liberty of action where the individual determines his or her own course of action in accordance with a plan chosen by himself or herself\\[1\\]. In application to clinical medicine, respect for autonomy dictates that patients with decision-making ability have a right to voice their medical treatment preferences, and physicians have the concomitant duty to respect those preferences\\[4\\]. Like Beauchamp and Childress, Gillon embraces a Millian understanding of autonomy, understanding it as deliberated self rule; the ability and tendency to think for oneself, to make decisions for oneself about the way one wishes to lead one's life based on that thinking, and then to enact those decisions--is what makes morality--any sort of morality--possible\\[3\\]. Given its supreme ethical importance, autonomy is not merely a value to be respected, but a virtue or trait that ought to be actively developed, nurtured and promoted.\n\nAccording to Gillon, other ethical principles (beneficence, non-maleficence, and justice) presuppose (and can be reduced to) respect for autonomy. Beneficence and non-maleficence toward autonomous moral agents presuppose respect for the autonomy of these agents even when they choose to refuse medical interventions which are life-saving. Gillon also takes an autonomy-centered approach to justice, arguing that responding to people's needs justly will require respect for those people's autonomous views, including autonomous rejection of offers to meet their needs; and, more importantly, because providing for people's needs requires resources, including other people's resources\\[3\\]. To conclude his praise for autonomy, Gillon writes that respect for autonomy contingently builds in a *prima facie* moral requirement to respect both individual and cultural moral variability\\[3\\]. While it is true that not all autonomy-based approaches in bioethics take the explicit and extreme form expressed by Gillon, autonomy continues to be treated implicitly as a primary value in many controversial clinical and research debates, from end of life issues (such as the Terri Shiavo case) to questions of exploitation of research subjects in international health research. When ethical principles conflict, it is often thought that the conflict can be resolved in an ideally impartial way by asking, for example, what the patient wants (or would have wanted) or whether the research subject really understood and freely consented to the procedures described in the research protocol. In this way, the multifarious values involved in the practice of medicine and biomedical research tend to be reduced to the principle of respect for persons, itself narrowly understood as respect for autonomy. Furthermore, the preeminence of autonomy as an ethical value within bioethics is deeply related to the increasing commoditization of medicine in developed countries. For the more that medical practices are justified by reference to patient choice, the more that patients will be viewed as 'clients' and health care professionals perceived as 'service providers'. This model of patient as 'client', which is prevalent in the United States of America and some parts of the western world, assumes affluence and power: the (literate) patient has to be capable of understanding and rationally weighing his\/her options--possibly even in disagreement with the physician--and be in a position to pay in exchange for services chosen.\n\n## Autonomy, exaggeration of human agency, and ethical pluralism\n\nAn autonomy-based ethics places the responsibility for medical decision-making largely in the hands of the patient. This raises the descriptive question of whether this conception accurately depicts how clinical decisions are actually made, as well as the normative question about whether such a conception of responsibility should (or should not) function as a universal ideal. In regard to the descriptive issue, patients in resource-poor settings are often not concerned with their ability to determine and shape the course of cure. Their arrival at the local health center is the outcome of a long family discussion that led to the collection of money. Sometimes, the patient arrives at the dispensary when the disease has reached its critical stage because the cost of care is too high. The primary expectation of both patient and family is to get the medicine or undergo a medical procedure they need and go back to their workplace. Spending time at the hospital means loss of earnings for them and their families or the diminishment of financial resources. When people can barely afford the cost of care or satisfy the nutritional requirements for a good recovery, the ethics of medical encounter should be understood differently and expressed in different terms than patient choice. Instead of developing a highly-organized medical bureaucracy that cares for the enforcement of patients' rights and protects medical professionals from accusations of malpractice, it would be more helpful to develop new sets of values that guide medical practice and promote patient participation in the healing relationship. The framing of these values may encourage and foster a non-confrontational relationship between health professionals and patients in the clinical setting, and include social challenges that influence health in the bioethics agenda. The role of bioethics will then consist in identifying social values and laws that may guide clinical work, restore the social dimension of medicine, connect the macro-determinants of health to medical practice and health system delivery, avoid the fragmentation of healthcare, and advocate for good health policies.\n\nThe challenge facing bioethics in resource-poor settings is not then to mislead people with unrealistic promises of autonomy that very few people can indeed achieve, but to articulate moral principles and societal values that are oriented around the promotion of equitable access to care and which broaden the goals of medicine and public health. The goals of medicine cannot be confined to the alleviation of suffering within the clinical setting. Medicine needs to be concerned with the determinants of good and bad health outside the clinical context in order to contribute to evidence-based clinical and public health interventions and education. The major bioethical questions prevalent in resource-poor countries do not essentially revolve around the provision of informed consent at the individual level, but rather around the burning social questions of access to care, commodification and quality of medical care, the relationship between income disparities and health inequities, the impact of poverty and underdevelopment on population health, priorities in biomedical research, and impacts of gender discrimination on women's health\\[5,6\\]. Once the focus is shifted away from the individualistic 'patient as client' paradigm, the social problems connected with the domination of medicine by market forces become apparent. If the goal of medicine is to restore health functioning, bioethics should avoid adopting a conception of autonomy that can be used to justify the domination of healthcare delivery by market forces alone and (wittingly or unwittingly) legitimizing health care systems that exclude the needy sick because the latter are unable to pay (or co-pay) for services or afford hefty medical insurance premiums. Even those bioethicists who promote market-driven medicine based on a libertarian anthropology\\[7,8\\] ought to carefully articulate alternative ethical values for health care and biomedical research, if they not to be lured into a 'self-defeating' conception of medicine. As an example of the latter tendency, Robert Sade considers medicine as a market commodity and understands medical practice as sets of skills that physicians are entitled to sell on the marketplace to make as much money as possible. Even the cries of the destitute sick or government regulatory function cannot restrict the physicians' appetite for greater financial reward. Sade's anthropology and approach to medicine is based on the assumption that individuals have the right to select the values that they deem necessary to sustain one's own life. They are also entitled to exercise their judgment to take the best course of action to achieve chosen values. Finally, they have the right to dispose of those values, once gained, in any way one chooses, without coercion by other men\\[7\\]. Similarly, Tristram Engelhardt protects human freedom to the point of ignoring the fact that the concern that we have for each other makes life in society possible. For him, as long as freedom functions as a side constraint, and as long as the moral community is based on respect for freedom and not force, individual persons will have the possibility of holding entitlements\\[8\\], Engelhardt's suggestion is paradoxical because, in trying to protect freedom of individuals to use their resources to access health care and other goods, he does not ensure that those with few resources have the freedom to obtain health care. Realistically, a genuine affirmation of autonomy cannot result in action informed or motivated by the desire to avoid being a responsible member of one's moral community\\[9\\]. Here, responsibility means that one should not exploit others by using autonomy as a warrant to market-driven medicine or profit-seeking attitudes. Once medicine is understood as a commoditized product like any other, those who cannot afford services are merely unfortunate consumers. In this way, a strong emphasis on autonomy can contribute to a culture in which healing and health promotion are no longer at the center of clinical practice and biomedical research.\n\nOne can hardly refute the fact that complex social and economic forces have placed patient autonomy at the center of medical ethics, and thereby undermined the age-old ethic of physician beneficence\\[10\\]. This change is sustained by waning trust in the traditional patient-physician relationship. With the control of medicine by the forces of the market, patients have become consumers of a market commodity called medical care. As a result of this change, the clinical relationship between the patient and physician begins to be seen as a contract and not as a covenant of care as it was in the past. Autonomy-based bioethics has a tendency to distort the relationship between individuals and the world. On the one hand, it exaggerates the power and range of individual agency; furthermore, it underestimates the impact of society, culture and environment, both on individual decision-making and on health. If persons are regarded as atomistic, certain defensive notions of individualistic rights-based autonomy prevail. If a relational construction of personal identity is employed instead, then respect for autonomy becomes part of a wider morality of relationship and care\\[1\\]. 'Atomistic autonomy' is divisive and lacks social rootedness while relational autonomy brings about trust and communality. The second version of autonomy, which reveals our true self in society, presents the possibility of placing trust and partnership at the center of the patient-physician relationship. With such an understanding of personhood, bioethics can better balance its concerns over choices and actions with those of relationship and responsibility. A more plausible philosophical anthropology would conceive individuals as entangled in the world, both capable of acting on it and subject to being affected by it.\n\nReflection on the notion of disease, both infectious and chronic, can contribute to a more plausible philosophical anthropology for bioethics. Infectious diseases question our understanding of autonomous agency in two important ways. First, as both a victim and a vector, a patient cannot be simply seen as a rational agent who has the final ethical word on his own decisions. Both vulnerability to infection and threat of transmission to others should shape our understanding of patient agency. Second, the concept of choice that shapes our conception of agency in bioethics can no longer be understood in isolation from society. Risk of acquiring and transmitting infectious diseases reflects the patient's interconnectedness with others and the biological environment, an interconnectedness which is always there even when infectious disease is not present\\[11\\]. Although the values and desires of the patient obviously need to be considered, the ideal of the autonomous agent will remain a fiction unless the social context of the patient's vulnerability is also considered. For other reasons, chronic disease also challenges our understanding of autonomy, especially when the patient finds it hard to manage his or her chronic condition. Family or friends stand as important resources for decision-making and long-term daily care for chronic diseases. We should then recognize that the family and community, which may play an important role in patient care, are part of the resource needed by the patient to exercise agency\\[12\\]. More and more, it is becoming obvious that the promotion of patients' agency requires serious consideration of patients' best interests in a broader way. Against the backdrop of contemporary institutional medicine, family solidarity is more important than ever to help maintain patient's dignity and agency throughout stressful time\\[13\\]. Exclusion of family and relatives from the sphere of decision-making on account of respect for individual autonomy does not necessarily serve patients' best interest. Furthermore, primary care, because of its focus on treatment and prevention of chronic and infectious diseases, is the domain of medicine that goes beyond techno-medical solutions to consider patients as persons with their stories, relationships, and social environment in which they live. Consequently, primary care should essentially rely on socially-grounded values rather than on desocialized principles\\[14\\].\n\nFamily and social relationships are important in the context of clinical medicine. However, we cannot undermine the importance of individual freedom. We simply reject strong claims that do not have any social rootedness. It would be almost unsound and socially untrue to radically endorse autonomy to the detriment of an ethic of responsibility and socially-based care because they are mutually interdependent, and a complete account of medicine's moral axis requires that they be integrated. This reorientation is crucial for reasserting the ethos of clinical medicine, whose fundamental mandate remains the care of others\\[10\\].\n\n## Autonomy ethics and the 'moral vacuum'\n\nFor Immanuel Kant, respect for persons never refers to the freedom to be left alone. Kant's understanding of respect for autonomy provides the ground for the categorical imperative, which he formulated in five different ways. The third formulation, \".act so that you treat humanity whether in your own person or in that of another, always as an end and never as means only\" \\[15\\] cannot be reduced to the respect for autonomy often found in the bioethics literature. The view of autonomy commonly found among individuals and in some of the bioethics literature in North America or Western culture is more in tune with John Stuart Mill's formulation of liberty: do not intrude on the freedom of any person by an invasion foreign to his or her own wishes and values. When Kant talks about autonomy, he does not imply that one should act according to one's own desires, unconstrained by a balanced consideration of one's situation as a being-among-others\\[9\\] Instead, he refers to the dignity of humans who are capable of making for themselves and others universal law. Hence, autonomy, rightly construed... results in action informed and motivated by the desire to be responsible member of one's moral community (the ground of one's being-among-others)\\[9\\]. Kantian autonomy is tied the moral agent's search for the truth and respectable conduct. The autonomous subject does not act in accordance to his or her primary inclination. Kantian autonomy is applied to actions performed when the will is freed from any selfish determination. When humans treat each other as ends and never as means merely, there arises a systematic union of rational beings under common objective laws. Physician and patient, each with their own needs, desires, capabilities, must find those principles that allow them to coalesce into a helping alliance to achieve a common goal.\n\nContemporary readings often accept a Millian version of autonomy that is associated with self-seeking attitudes. This approach to respect for autonomy refers to the capacity to act on needs, wants, or wishes; a capacity shared by many creatures. Since the person's action is informed by instrumental reasoning, it constricts the scope of reason so that it is subject to any desire or disposition that one happens to endorse at the time one acts\\[9\\]. Focusing essentially on individual choices sets up a false and pernicious opposition between persons and the community to which they belong. It is reasonable, on both conceptual and empirical grounds, to suppose that individuals acquire their values through engagement with a concrete moral tradition, rather than through a private and self-directed process. Instead of providing ethical decision-making with an objective and rational process, the obsession with individual autonomy tends to create what McCormick calls a 'moral vacuum', i.e. the disappearance of the network of shared and established goods and values that make the choices of individuals right or wrong, moral or immoral\\[16\\].\n\n## Balancing autonomy and community in ethical decision-making\n\nIt is hard to undermine the influence of social, cultural and environmental factors on moral decision making. We have to take these factors into account in order to fully appreciate the moral dilemmas and health challenges in settings and traditions where individualism does not prevail. Writing from their Jewish background, Barth-Rogers and Jotkowitz note that within Jewish tradition, the idea of unlimited human autonomy is not a defining value; Judaism deems the intrinsic human value of each individual's life to take precedence over patient autonomy\\[12\\]. Similarly, the Confucian culture from East Asia understands the person not only as a rational, autonomous being but also as a relational and altruistic entity whose self-actualization involves participating in and promoting the welfare of fellow persons\\[4\\]. In the same line of thought, African traditions present a view of the human person that is essentially relational; it is within the social network that the individual lives and acts as a free person. The Jewish, Confucian, and African cultures convey an understanding of the human person and society which is different from individualism operative in some cultures.\n\nThis is where the shortcomings of Gillon's autonomy-centered conception of bioethics become the most obvious. Gillon does not reject the view that particular cultures should be respected, instead he theorizes that the *prima facie* nature of autonomy requires that both the individual and cultural moral variability be respected\\[3\\]. But this sense of respect for culture does not adequately reflect the social rootedness of the human person. Despite making 'concessions' to culture, Gillon continues to view societal relationships, determinants and influences to be peripheral to human reason and, because of the danger of ethical relativism, something to be transcended by a universal ethic. Hence, the four principles (with autonomy as supreme among them) can account for all our moral worries and being applied straightforwardly to all situations and contexts\\[17\\]. Gillon contends that any other moral principle or value can be explained by one or some combination of the four principles. In fact, however, Gillon's quest for a universal discourse is nothing more than the promotion of one approach to ethics among others, one which reflects specific cultural assumptions concerning individual choice and future-oriented action that are associated with class position and social opportunities and foreign to the lived reality of the poor, the marginalized, and people of color in a multicultural society like the United States\\[18\\]. Any attempt to universalize an ethnic particularity fails the test of respect for pluralism in bioethics and in our ever-globalizing world.\n\nIn resource-poor countries where medical paternalism prevails on account of patient beneficence and shared-responsibility for health promotion\\[19\\], the necessity to create the conditions that improve, for example, patient-physician communication in ways that favor patient agency needs to be acknowledged. Very often, the physician does not even tell the patient what is going on with his or her health. However, the one-sided view of the human person which prevails in autonomy-based bioethics should not be adopted as a model to correct paternalism; a more fruitful alternative would be a combination between a community- and tradition-oriented view and autonomy that conceives decision-making as guided by important human values such as partnership, trust and solidarity, in addition to autonomy. This view would acknowledge the embedded and relational nature of human choices, behavior, ways of expressing emotions and feelings, patterns of thinking, and conceptions of disease and healing.\n\n# Autonomy, biomedical individualism, and social justice\n\nSome criticisms of autonomy-centered bioethics have been purely conceptual. Others have emerged from reflections on its limitations in dealing with collective macro-problems including social, sanitary and environmental problems that mark everyday life in poor countries. Autonomy-based bioethics fails to engage the lived worlds of diversely constituted and situated social groups, particularly those that are marginalized\\[18\\]. Similarly, in clinical medicine, broad issues such as the common good, distributive justice and the spirituality of the patient are ignored for the sake of the primacy of secular business concerns. To guide clinical practice, laws have been developed to reduce risk for malpractice and protect patients. However, emphasis placed on the principle of autonomy has led to an excessive control of clinical practice by judicial institutions. Consequently, this obsession with the law has led to the elimination of a wide range of moral concerns from public consideration\\[16\\]. To emphasize this point, McCormick criticizes clinical ethics for being preoccupied with cost control that focuses narrowly on matters of financial efficiency, thus exiling the more basic ethical questions (ends of medicine, the meaning of life, death, illness and health)\\[16\\]. Furthermore, any public health intervention that adopts the biomedical model fails to address issues of wider social injustices that are responsible for health-related vulnerability and risk.\n\n## Autonomy ethics and medical individualism\n\nThe biomedical model is premised on individualism, because it adopts an abstract view of the body and mind of an individual person from a liberal model of economy and politics\\[20\\]. In this model, individuals choose health behaviors. Thus, poor health is largely due to exposures to health risks that the individuals have decided not to avoid. This approach to health risks disregards the role of social structures in structuring the array of risk factors that individuals are supposed to avoid\\[18\\], and fails to explain how social inequalities can be embodied in poor-health outcomes\\[21\\]. Thus, autonomy-focused bioethics, rather than presenting an objective perspective, deprives itself of theoretical tools to adequately address non-pathological causes of ill-health. Similarly, in research sites, much effort is often invested in securing the informed consent of individual participants while often ignoring the broader issues of justice in places where research takes place\\[22\\]. Consequently, the absolutization of autonomy with the unreal and distorted picture of the person helps explain why so much bioethical writing is concerned with procedures that protect choice, rather than more substantive issues, with consent itself rather than what is consented to\\[16\\]. This tendency to make the social causes of poor health (and the broader ethical problems related to health improvement) invisible can even be seen among those working in public health to the extent that they subscribe to the biomedical model\\[20\\].\n\n## Biomedical model and the social gradient in health\n\nHealth differentials between individuals cannot be explained simply by their health behavior or lifestyles, but also by their social position and economic status, the social networks to which they belong, and the levels of education that provide them with the means to avoid health risks, deal with adversity, and have access to life-protecting information. The pervasiveness of the social gradient in health remains even when well-designed public health interventions are implemented. Even when these public health interventions may reduce health risks and mortality, they do not eliminate the social gradient because individuals in the lower socioeconomic groups take less advantage of health interventions than those who are better off.\n\nWhen we compare the health statistics between poor and rich within countries or between countries, the differentials are striking. HIV\/AIDS statistics provide us with striking examples of the impacts of socioeconomic status on risk differentials and chances of survival between groups within countries and between countries. Even in developed countries, the geography of HIV\/AIDS challenges us to investigate the social causes of its distribution. Risks and survival differentials prompt us to consider a view that places political-economic critiques of global resource distributions, and criticism based on the higher and qualitatively different disease burdens in poor countries within a common framework of international and internal socio-economic structure\\[23\\]. At the local level, income inequality in poor countries affects health and can be an indicator of life expectancy\\[24,25\\]. Poverty affects individuals' ability to have access to goods which are instrumental for well-being. At the country level, poverty limits government's ability to found social programs and provide people with basic social goods such as safe drinking water, electricity, good public health coverage, healthcare institutions, schools, social services, and economic opportunities. These structural causes are steady and they include access to basic resources that can be used to avoid all sorts of health risks or reduce the negatives outcomes of diseases when they occur\\[26\\].\n\nMost public health interventions focus on individual risk factors and behavior. To lessen vulnerability and risk, health professionals will need to address income differences between individuals and population groups. Otherwise, they will only address the symptoms and not the root-causes of poor health. As public health practitioners and other health professions 'resocialize' their conceptions of health and disease, bioethicists should join and inform their efforts. A sociological approach to disease can increase the social relevance of bioethics because it provides an acute perception of disease etiology and pathology that includes the social and material conditions in which people live.\n\n# Sociological model and autonomy-based bioethics\n\nTo underscore the difference between Western and non-Western conception of illness, Bowman writes that most non-Western cultures tend to perceive illness in a much broader and far less tangible manner. Illness is often viewed as being linked to social, spiritual, and environmental determinants\\[27\\]. The sociological model of disease explanation shares some important connections with many non-Western cultures in which disease representation and explanation is not primarily understood in biomedical terms, but in social ones. Autonomy-based bioethics is premised on the view that disease is located in the individual. The focus on the individual person often reduces the scope of justice in clinical medicine and health research to an equal treatment of individuals involved and a fair distribution of available resources and burden regardless of people's social status, age, race, gender or religion. In the clinics, for example, justice requires that patients whose circumstances are the same deserve the same level and quality of care.\n\nConversely, the sociological model perceives the disease as an integrated social-physiological process which includes the person's relation to the environment. In addition to its bio-physiological dimension, a disease is a relational phenomenon; as a subjective and socially-constructed reality, a disease develops out of the omnipresence of symptoms and bodily feelings in everyday life. The sociological model allows us to develop a socially-relevant approach to health justice, a new set of principles that may guide research as well as an approach to health policy based on the features of the site where research is done. Thus, this model points to the fact that there are two reminders of our embeddedness in the world relevant to bioethics: first, biological embeddedness and infectious disease and second, social embeddedness, particularly (but not exclusively) in contexts where people are obviously dependent on one another and traditional behavior and customs are strong.\n\n## Contribution of medical sociology: Sociological model and social justice\n\nThe current formulation of ethical principles as they are applied to medical research in poor countries is inappropriate for capturing some crucial implications of medical research since they ignore the roots of health crises with which these countries are confronted\\[28\\]. Analyzing the health crises in African countries in the late 1980s, the Cameroonian sociologist Jean-Marc Ela argues that disease and malnutrition never exist by themselves; rather they come from a system characterized by violence, by a pattern of impoverishment of the majority, and by the monopoly by a minority of the means to live with dignity\\[29\\]. Health interventions should not merely address the symptoms of a disease-producing society, but also its structures. Social structures not only shape distribution of disease across population, but they also determine societal and individual responses to suffering. When the major determinants of health are far from being addressed by a conceptual framework that prioritizes individual problems and morality, there is a need to call its relevance into question. The high rates of infectious diseases in poor countries are linked to poor living conditions and structural problems. These primary sources of exposure and vulnerability to health hazards should necessarily be considered in any attempt to develop bioethical standards for research or any bioethical agenda. The poverty that permeates all spheres of society should be studied because poverty never exists in isolation from societal influences, but rather is integrally a product of the inner workings of each society's political economy. Minimizing the contribution of poverty to the production of disease and disability in poor countries makes suffering invisible and limits our understanding of the etiology of disease.\n\nMedical sociology scrutinizes patterns of diseases and pathways through which social inequalities are embodied in individual vulnerabilities and major epidemics. Thus, the model of disease causation that comes from sociological investigations challenges us to move beyond the clinics or research sites to broaden the scope of justice. Similarly, the prevalence of infectious diseases in resource-poor countries challenges the way justice is understood in research sites. If we consider the patient as a potential victim and vector, we need to shift our gaze from the healthcare that might be most desirable for the individual patient to broader social concerns and the worldview distribution of care that might enable all to achieve opportunities over a reasonable life span\\[11\\]. The extension of care to all not only aims at serving individual needs for care, but more importantly it addresses infectious diseases as a threat to population health. Opting out from an intervention of this kind would simply mean that the individual remains a threat to the entire population\\[3\\].\n\nThe sociological explanation of disease incorporates a distinctive view of etiology, prevention, pathology, treatment, and justice. This approach to disease explanation tacitly promotes a conception of responsibility for infection or disease causation which is not only individual. This approach questions the uses of individualism as methodology and framework for analyzing disease occurrence, and thus criticizes the one-sidedness of the anthropology that sustains the biomedical model.\n\n## Sociological model and justice in current biomedical research\n\nDocuments such as the *Declaration of Helsinki* issued by the World Medical Association and the *International ethical guidelines for biomedical research involving human subjects* (CIOMS) as well as the work of the National Council on Bioethics in 2002 and that of the National Bioethics Advisory Commission (NBAC) in 2001 all take material poverty as the main reason for developing bioethical standards that apply to medical research conducted in poor countries. Surprisingly, the bioethics standards they promote hardly reflect the physical, social, and cultural environment of poor countries. This is another important area for revision\\[28\\].\n\nGiven the substantial differences in individual exposure to health risks and the availability of health protective resources as well as differences in the disease burden and mortality and morbidity at the population level, it is clear that illness in poor countries can be better understood using a 'social causation of illness' perspective. The principles of respect for persons, beneficence, and justice that shape the *Belmont Report* are all built on the biomedical model. The principle of respect for persons reinforces individual agency and protection in the research setting by ensuring that participants are properly informed about the research or the course of care that will be taken to restore normal functioning. The principle of beneficence extends the latter by insisting that research protocols should maximize potential benefits and minimize harm. Finally, the principle of justice ensures that those with diminished autonomy are protected and that participants share in the benefits of the research. Agency, benefit, participation, risk, and vulnerability are all understood from the standpoint of the individually-focused disease management whether in the clinical setting or the research site. To be of broader global significance, ethical principles of biomedical research should be responsive to the context of poverty and social inequities, since these structural factors can lead to increased vulnerability and exploitation. For example, the incapacity of poor people to satisfy their basic needs can lead to increased participation in clinical trials without true understanding of risk and benefit at least in part due to financial incentives. Thus, even if these people 'consent' to participation in a trial, is that decision truly autonomous? It is then clear that 'research protections' cannot be ensured solely through the use of the consent form and the provision of information to the subject. A formal provision of consent by the research subject can simply mask the misery that inhibits his or her ability to consent freely.\n\nSimilarly, what counts as 'benefits' can be tied to different levels of poverty and disease burden in different resource-poor countries. Ethical principles and guidelines that oversee biomedical research can be defined in terms of public good rather than merely as an improvement in individual health status because public good and social policy transcend the framework of individual-based ethics\\[28\\]. In resource-poor countries, death-rates are high and infectious diseases contribute significantly to the burden of disease--as opposed to richer countries, where cardiovascular disease and cancer are the leading causes of mortality--the difference in exposure, health risk, mortality, and morbidity between poor and rich countries challenges us to develop a new approach to the concept of benefit in biomedical research. We need to think of 'benefits' as running to the whole community in which research takes place, and not just to single research subjects. Therefore, the availability of and access to modern health services is a substantial issue for evaluating the impact of biomedical research benefits in poor countries since the outcomes of health initiatives are largely determined by some structural arrangements that transcend the benefits of research subjects. These arrangements are based upon national and international patterns of control over society's resources.\n\nCurrent ethical guidelines continue to be inappropriate because they do not address the international context of exploitation within which research is done. People's health status cannot be separated from the capitalist system of resource distribution and exchanges which favors the rich countries or high socioeconomic groups and reinforces the impoverishment of the poor ones. The economic exploitation that prevails in the capitalist system shapes the global and local distribution of resources and diseases as well as the health risks and vulnerability of those who live on the margins of the global market. The concepts of 'benefit' and 'justice' have been inadequately extended to biomedical research in poor countries because the possibility of exploiting the underprivileged is more complex than an exploitative relationship with vulnerable populations in developed countries, where at least the rule of law and the respect due to every citizen have already been institutionalized. Furthermore, the number of research studies conducted in poor countries is increasing because regulatory measures are often less strict; this situation may facilitate the exploitation of the poor, non-respect for basic ethical standards, and unlimited search for benefit.\n\nBioethics scholarship that focuses on the sociological model considers local as well as global issues of social inequality, because this model is premised on the intimate connection that exists between social inequality and health inequality. The distribution of illness is likely to reflect the geography of inequality. A social approach to bioethics emphasizes distributive justice and benefits at both the population and individual level. Three important principles flow from this analysis. The first one can be called principle of public benefits (community-based approach to benefits); it is a context-based principle which derives from factors that contribute to ill-health and vulnerability to preventable diseases in poor countries. It states that risks, benefits, and equity can no longer be defined in terms of individual health, but also in relation to the international, national and local contexts\\[23\\]. Such a principle challenges the individualistic understanding of benefits in places where exploitation and inequality are at the center of research. Consequently, a community-based understanding of benefits calls for a large-scale distribution of the benefits of research as an important requirement of justice. This principle is relevant for political and socioeconomic critiques of the ethics of carrying on research in poor countries, given well-established patterns of exploitation and oppression of the underprivileged. Reliance on the sociological model brings out the fact that the health conditions under study originate in socioeconomic conditions that need to be treated to have an impact on the health status of research participants\\[28\\]. Thus, the notion of population or community-based benefits is related to that of health as a public good which is, in turn, linked to the global-capitalistic system that significantly contributes to the health conditions found in poor countries.\n\nThe second principle, the principle of social justice, is rooted in a broad approach to justice that places poor health at the center of public and research policy and seeks to correct systemic injustices. This principle is related to the principle of public benefit since it states that the distribution of benefits should take into account the poverty of local healthcare systems and people's disempowerment as a function of social structures\\[23\\]. Here, the challenge is that the distribution of benefits should address the root-causes of poor health and not only its symptoms. The third principle underscores the need for building local capacity. This principle states that building capacity to promote healthcare sustainability will have a lasting effect on people's health. This principle emphasizes the need for building local capacity and improving human capital to reduce the burden of preventable diseases. For example, research on AIDS vaccine often uses existing facilities or new ones built by funding agencies to conduct research or administrate the vaccine on trial. Building capacity may involve researchers and funding agencies improving the training of local medical professionals and reinforcing existing facilities to reduce the burden of disease; and, if a new medical facility has been built for the research study, local communities can still use it even after the research project comes to an end.\n\nTo avoid exploiting the underprivileged and reinforcing an existing system of oppression, the distribution of benefits should be determined by the context within which diseases occur, the state of the healthcare system, and available resources. Therefore, research institutions and their financial sponsors are morally obligated to contribute to the development of a healthcare system and the improvement of human resources that can benefit the whole population. Carrying on research in impoverished parts of the world where people have been enduring a systemic marginalization would not be ethical if our understanding of benefit will not address the root causes of poor health. Thus, it is no longer enough to avoid not doing harm; addressing health challenges that prevail in the research site is consistent with a broader view of justice\\[28\\].\n\n## Sociological model, bioethics, and health policy\n\nAn autonomy-centered ethics places the burden of prevention and access to healthcare on the moral agent. In doing so, it frames disease within a model that limits political intervention in the health domain strictly to biomedical solutions or behavior change. This leads to the perpetuation of the social *status quo* within which risks for poor health are greater, and lends legitimacy to the social forces that increase health risks. This failure to promote social justice contrasts with John Lynch's understanding of public health intervention. Lynch believes that elements of the social fabric should shape the conception, framework, and implementation of public health intervention. Discussing the influence of socioeconomic status on behavioral and psychosocial risk factors for cardiovascular disease, he argues that the public health community should consider the potential for a broad array of social, educational, and economic policies as effective public health interventions to reduce the unequal distribution of risk factors and the unequal burden of disease\\[30\\]. Similarly, bioethicists need to study health-promoting effects of structural interventions to determine which ones are ethically acceptable and justified. Such a move requires bioethicists to look at broad issues of social equity and advocate for a shift in public policymaking.\n\nIn a population-based study examining the associations between socioeconomic status measures (education, income, and occupation) reflecting different stages of the lifecourse of 2674 middle-aged Finnish men, health behaviors, and psychosocial characteristics in adulthood, Lynch et al. conclude that: understanding that adult health behavior and psychosocial health orientations are associated with socioeconomic conditions throughout the lifecourse implies that efforts to reduce socioeconomic inequalities in health must recognize that economic policy is public health policy\\[31\\]. The sociological model within which Lynch's understanding of public health intervention is built challenges us to advocate for a shift in policymaking mindset because health is not a sphere of justice which is separate from other aspects of human life. Since disease is a social process, a policy vision that focuses on the individual and individual risk factors fails to promote social justice and to address structural elements that create conditions favorable to the production of disease. Hence, we need to move from healthcare policy to health policy, or rather, a healthcare policy that is responsive to facts explaining why (certain) people with (certain) diseases from (certain) communities require medical care. Health policy should embrace healthcare policies but include considerations regarding welfare, work, occupational, economic development, employment, and educational policies.\n\n# Conclusion\n\nSociologists and social epidemiologists challenge bioethicists, especially those working in developing countries, to be socially and culturally relevant. The sociological theory of disease explanation starts with a concrete analysis of the social setting within which illness occurs or research is carried on. Since societal factors shape patterns of mortality and morbidity, principles of biomedical and research ethics need to be framed within the context of the social inequalities that shape vulnerability to illness. Aligning bioethics to perspectives, concerns and information in the fields of public health, health policy and medical sociology could vastly improve its global significance. Thus, bioethicists should be challenged to develop a philosophical anthropology that goes beyond radical affirmations of the individuality to acknowledge both the communal and the individual dimension of the human person.\n\n# Competing interests\n\nThe authors declare that they have no competing interests.\n\n# Authors' contributions\n\nJA originated the article, did the research and wrote a first rough draft of the manuscript. SR read the first version and contributed editorial and critical suggestions. After the first peer-review, JA made substantial revisions to the earlier draft in close collaboration with SR. They have both read and approved the final version of the manuscript.\n\n# Authors' informations\n\nJacquineau Az\u00e9tsop obtained his PhD in theological and social ethics at Boston College, Massachusetts\/USA and a Masters in Public Health (MPH) at the Bloomberg School of Public Health from Johns Hopkins University in Baltimore, USA. Currently, he is lecturer in health policy and bioethics at Facult\u00e9 de M\u00e9d\u00e9cine Teilhard de Chardin in N'djam\u00e9na, Chad.\n\nStuart Rennie (PhD, Philosophy) is currently Lecturer in the Department of Philosophy at the University of Cape Town and Research Assistant Professor in the Department of Social Medicine at the University of North Carolina (USA). He is co-Principal Investigator of UNC's Fogarty International Center bioethics capacity building project in the Democratic Republic of Congo, and ethics consultant for CDC\/Global AIDS Projects in the DR Congo and Madagascar. He is co-chair of the UNC's Behavioral Institutional Review Board (IRB) and is also currently lead author of research ethics guidelines for the HIV Prevention Trials Network (HPTN). Dr. Rennie has published on research ethics and bioethics topics in PLoS Medicine, Science, the Hastings Center Report, Developing World Bioethics and the Journal of Medical Ethics, as well as writing for his own Global Bioethics Blog.\n\n## Acknowledgements\n\nIn thinking about writing this paper we have benefitted tremendously from conversations with and comments from John Paris, the Wash professor of Bioethics at Boston College. The authors would also like to thank the anonymous reviewers for their extensive and challenging comments. They have contributed to the strength of this paper.","meta":{"dup_signals":{"dup_doc_count":101,"dup_dump_count":51,"dup_details":{"curated_sources":4,"2022-40":1,"2022-33":1,"2022-05":2,"2021-39":1,"2021-21":1,"2021-10":2,"2020-45":1,"2020-40":1,"2020-34":1,"2019-47":1,"2019-04":1,"2018-13":1,"2017-47":1,"2017-43":1,"2017-34":1,"2017-26":1,"2017-17":1,"2017-09":3,"2017-04":2,"2016-50":2,"2016-44":2,"2016-40":2,"2016-36":2,"2016-30":2,"2016-26":1,"2016-22":2,"2016-18":2,"2015-48":7,"2015-40":1,"2015-35":2,"2015-32":2,"2015-27":2,"2015-22":1,"2015-14":2,"2014-52":2,"2014-49":1,"2014-42":6,"2014-41":3,"2014-35":4,"2014-23":4,"2014-15":4,"2023-23":1,"2024-18":1,"2017-13":2,"2015-18":2,"2015-11":2,"2015-06":2,"2014-10":2,"2013-48":2,"2013-20":2,"2024-30":1}},"file":"PMC2828974"},"subset":"pubmed_central"}
{"text":"abstract: Semiconductor nanoparticles (quantum dots) are promising fluorescent markers, but it is very little known about interaction of quantum dots with biological molecules. In this study, interaction of CdTe quantum dots coated with thioglycolic acid (TGA) with bovine serum albumin was investigated. Steady state spectroscopy, atomic force microscopy, electron microscopy and dynamic light scattering methods were used. It was explored how bovine serum albumin affects stability and spectral properties of quantum dots in aqueous media. CdTe\u2013TGA quantum dots in aqueous solution appeared to be not stable and precipitated. Interaction with bovine serum albumin significantly enhanced stability and photoluminescence quantum yield of quantum dots and prevented quantum dots from aggregating.\nauthor: Vilius Poderys; Marija Matulionyte; Algirdas Selskis; Ricardas Rotomskis\ndate: 2011\ninstitute: 1Laboratory of Biomedical Physics, Vilnius University Institute of Oncology, Vilnius, Lithuania; 2Biophotonics Laboratory, Quantum Electronics Department, Physics Faculty, Vilnius University, Vilnius, Lithuania; 3Department of Material Structure, Institute of Chemistry, Vilnius, Lithuania\nreferences:\ntitle: Interaction of Water-Soluble CdTe Quantum Dots with Bovine Serum Albumin\n\n# Introduction\n\nSince the first time fluorescent semiconductor nanoparticles (quantum dots) were synthesized, they are widely explored due to their possible applications in many fields, including medicine. Tunable emission wavelength, broad absorption and sharp emission spectra, high quantum yield (QY), resistance to chemical degradation and photo bleaching and versatility in surface modification make quantum dots very promising fluorescent markers \\[1\\].\n\nQuantum dots can be used for live cell labeling ex vivo, detection and imaging of cancer cells ex vivo \\[2\\], as a specific marker for healthy and diseased tissues labeling \\[3\\], for labeling healthy and cancerous cells in vivo \\[4\\] and for treatment of cancer using photodynamic therapy \\[5\\]. Despite all unique photo physical properties, some problems must be solved before quantum dots can be successfully applied in medicine. Quantum dots usually are water insoluble and made of materials that are toxic for biological objects (Cd, Se). To make them suitable for application in medicine, surface of quantum dots has to be modified to make them water-soluble and resistant to biological media. After injection of quantum dots to live organisms, they are exposed to various biomolecules (ions, proteins, blood cells, etc.). This could lead to degradation of quantum dot coating or quantum dot itself. In this case, toxic Cd^2+^ ions are released and can cause damage to cells or even cell death.\n\nA lot of research is done to better understand quantum dots synthesis \\[6\\] growth \\[7\\] and modification \\[1\\]. Recently, the interaction of quantum dots with biomolecules attracted much interest and is studied using various methods, such as atomic force microscopy, gel electrophoresis, dynamic light scattering, size-exclusion high-performance liquid chromatography, circular dichroism spectroscopy and fluorescence correlation spectroscopy \\[7-11\\]. It was shown that interaction of quantum dots with biological molecules can enhance optical properties and stability of quantum dots \\[12-14\\] or it may oppositely lead to their degradation \\[15\\]. Serum albumin is one of the most studied proteins. It is the most abundant protein in blood plasma and plays a key role in the transport of a large number of metabolites, endogenous ligands, fatty acids, bilirubin, hormones, anesthetics and other commonly used drugs.\n\nIn this study, we investigated effect of interaction between bovine serum albumin (BSA) and water-soluble CdTe quantum dots in aqueous solutions using microscopy and spectroscopy methods.\n\n# Materials and Methods\n\nQuantum dots solutions were prepared by dissolving CdTe quantum dots coated with thioglycolic acid (*\u03bb*~PL~ = 550 \u00b1 5 nm, PlasmaChem GmbH, Germany) in deionized water (pH\u22486) or saline (0.9% NaCl solution, pH\u22485.6). Experiments of CdTe quantum dots solution with protein were performed by adding a small amount of concentrated bovine serum albumin (BSA) (BSA, V fraction, *M* = 69,000 g\/mol, Sigma, Germany) solution in saline to the quantum dots solution.\n\nSpectral measurements were performed immediately after preparation of solutions. Absorbance spectra were measured with Varian Cary Win UV (Varian Inc., Australia) absorption spectrometer. Photoluminescence spectra were measured with Varian Cary Eclipse (Varian Inc., Australia) and PerkinElmer LS 50B (PerkinElmer, USA) fluorimeters. Photoluminescence excitation wavelength was 405 nm, excitation slits were 5 nm and emission slits 5 and 4 nm for Varian Cary Eclipse and PerkinElmer LS 50B, respectively. Measurements were taken in 1-cm path length quartz cells (Hellma, Germany). Samples for atomic force microscopy measurements were prepared by casting a drop (40 \u03bcl) of solution on freshly cleaved V-1 grade muscovite mica (SPI supplies, USA) spinning at 1,000 rpm. Atomic force microscope (AFM) diInnova (Veeco instruments inc., USA) was used to take 3-dimensional (3-D) images of quantum dots. Measurements were performed in tapping mode in air; RTESP7 cantilevers (Veeco instruments inc., USA) were used. Samples for scanning transmission electron microscopy (STEM) measurements were prepared by casting a drop of solution on TEM grid and drying it in ambient air. STEM images were obtained with HITACHI SU8000 microscope (Hitachi High-Technologies Corporation, Japan). Malvern Zetasizer Nano S (Malvern Instruments Ltd., England) was used to determine particles size distributions in investigated solutions.\n\n# Results\n\nNormalized photoluminescence and absorption spectra of BSA and CdTe quantum dots coated with thioglycolic acid is presented in Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>. BSA has absorption band in UV region at 280 nm, and fluorescence band peak is at 338 nm. CdTe\u2013TGA quantum dots absorb light in wide spectral region and have excitonic absorption band at 508 nm, and photoluminescence band peak of quantum dots solution is at 550 nm. Titration of freshly prepared quantum dots solution with BSA showed that addition of protein to CdTe quantum dots solution increases photoluminescence intensity of quantum dots (simultaneously a slight (\\~4 nm) bathochromic shift of quantum dots excitonic absorption band is observed). This effect was observed until 10^-5^ mol\/l BSA concentration was reached. Further increase of BSA concentration in quantum dots solution induced slight decrease in photoluminescence intensity (Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>, curve A). Constant decrease in CdTe quantum dots solution photoluminescence intensity was observed, when CdTe quantum dots solution was titrated with saline (Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>, curve B). This constant decrease in photoluminescence intensity was caused by decreasing concentration of quantum dots (dilution effect). Curve C (Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>) shows CdTe quantum dots photoluminescence intensity change caused by CdTe\u2013BSA interaction (dilution effect is eliminated). The biggest increase in CdTe quantum dots photoluminescence intensity (120% of initial value) was observed when ratio of BSA\/quantum dot was 1.75:1.\n\nDynamics of quantum dots photoluminescence properties (photoluminescence intensity and photoluminescence band peak position) in solutions with BSA and without BSA are presented in Figure <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>. Photoluminescence intensity of CdTe\u2013TGA quantum dots solution (*c* = 6 \u00d7 10^-6^ mol\/l) without bovine serum albumin was increasing for the first 144 h (Figure <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>, curve A). Photoluminescence band maximum position and width stayed intact. After 144-h photoluminescence intensity started to decrease, band started to narrow and shift to longer wavelength region. Simultaneously absorption slightly decreased (Figure <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>, curve B). Decrease in quantum dots photoluminescence intensity and bathochromic shift of photoluminescence band indicates aggregation of quantum dots. After 9 days, precipitate of large aggregates appeared in quantum dots solution.\n\nA sudden increase in photoluminescence intensity (by 27%) was observed after protein was added to the CdTe quantum dots solution in saline (Figure <a href=\"#F3\" data-ref-type=\"fig\">3a<\/a>). Photoluminescence intensity further increased for approximately 40 h. Later photoluminescence intensity started decreasing, but decrease in intensity was quite slow and at longer time scale became negligible (even after 6 months no precipitate was observed). Photoluminescence band width and maximum position remained constant, and absorption intensity slightly increased. This indicates that core of quantum dot remained intact.\n\nInvestigation of quantum dot size with atomic force microscope (AFM) and scanning electron transmission microscope (STEM) showed that in solution without protein quantum dots aggregate (Figure <a href=\"#F4\" data-ref-type=\"fig\">4a\u2013d<\/a>). AFM image of quantum dots, deposited from solution that was kept for 40 min, is presented in Figure <a href=\"#F4\" data-ref-type=\"fig\">4a<\/a>. A lot of small round structures were present on the surface. These structures were \\~2.5 nm in height and \\~25 nm in width. Shape of colloidal quantum dots should be close to spherical (width of quantum dot should be approximately equal to height). Height of these structures is approximately equal to a height of single quantum dot, but width was much bigger. This could be explained by AFM imaging artifact called \"tip imaging\". It is also possible that these small structures are not single quantum dots but few quantum dots attached to each other. AFM image of quantum dots deposited from solution that was kept for 5 h shows larger structures (Figure <a href=\"#F4\" data-ref-type=\"fig\">4b<\/a>). Height and width of these structures varied in broader range. Some small structures (height\u20132.5 nm, width\u201320 nm) could be seen, but bigger structures (up to 9 nm in height and up to 70 nm in width) were also present. Image of sample prepared from solution that was kept for 24 h (Figure <a href=\"#F4\" data-ref-type=\"fig\">4c<\/a>) showed that sizes of the structures increased even more (height\u2013up to 13 nm, width\u2013up to 150 nm). In STEM images (Figure <a href=\"#F4\" data-ref-type=\"fig\">4d<\/a>), obtained 2 days after solution preparation, various size structures (much larger than single quantum dots) were seen. This shows that CdTe\u2013TGA quantum dots dissolved in aqueous solution are not stable, and aggregates and forms large clusters of quantum dots.\n\nAFM image (Figure <a href=\"#F4\" data-ref-type=\"fig\">4e<\/a>) of sample prepared from CdTe quantum dots solution in saline with BSA (solution was kept for 2 months) showed that there were no large structures that could form precipitate, but there were plenty of round structures that were 9\u201320 nm in height and 40\u201360 nm in width. Height of structures seen in image (9\u201320 nm) was bigger than height of single quantum dot (\\~2.5 nm). BSA is heart-shaped molecule; its approximate size is 8 nm \u00d7 8 nm \u00d7 3 nm \\[14\\]. Structures observed in AFM image were a bit bigger than BSA molecules. Structures observed in AFM image could be CdTe quantum dots coated with BSA. In STEM image, only small structures (single quantum dot) \\~3 nm in diameter are seen. In bigger collections, quantum dots are separated one from another by \\~3 nm (Figure <a href=\"#F4\" data-ref-type=\"fig\">4f<\/a>). Interaction of quantum dots with BSA could lead to the formation of additional quantum dot coating layer that prevents quantum dots from aggregation. Additional coating layer is not visible in STEM image because BSA is formed of light atoms that are not visible in STEM images.\n\nParticle size distributions in BSA solution, CdTe\u2013TGA quantum dots solution and CdTe\u2013TGA quantum dots solution with BSA are presented in Figure <a href=\"#F5\" data-ref-type=\"fig\">5<\/a> (solutions were kept for 1 week). Average diameter of particles in BSA solution is 8.7 nm. This result very well coincides with dimensions of BSA molecule presented in literature \\[16\\]. Sizes of particles present in CdTe quantum dots solution are bigger than 50 nm in diameter, much bigger than size of single quantum dot (that should be approximately 2\u20133 nm). This shows that quantum dots formed aggregates and confirms results obtained with AFM and STEM. Particle size distribution in CdTe\u2013TGA with BSA solution shows that in this solution average particle size is slightly bigger (diameter \\~12.5 nm) than in BSA solution (diameter \\~8.7 nm). This shows that CdTe\u2013TGA quantum dots interact with BSA and form quantum dot\u2013protein complex whose size is approximately 12.5 nm.\n\n# Discussion\n\nOur proposed model explaining spectral dynamics of CdTe\u2013TGA quantum dots in aqueous solution with and without BSA is presented in Figure <a href=\"#F6\" data-ref-type=\"fig\">6<\/a>.\n\nDynamics of photoluminescence properties of investigated solutions (presented in Figure <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>) show two phases\u2014growth of photoluminescence and decrease of photoluminescence. In the first phase, photoluminescence of quantum dots increased in both investigated solutions (quantum dots without protein and quantum dots with protein). Despite quite large increase in photoluminescence spectra, changes in absorption spectrum were very small. During this phase, photoluminescence band peak position and photoluminescence band width remained constant. These changes indicate that core of quantum dot remains intact. Core degradation would cause blue shift of photoluminescence band; aggregation of quantum dots would cause a red shift. Change in photoluminescence intensity indicates that properties of quantum dot coating (or coating itself) are changing: molecules coating core of quantum dot are rearranging, being replaced by other molecules of being washed-out. Theoretically, increase in quantum dots photoluminescence intensity is explained by decrease in non-radiative transitions or their speeds. Decrease in defects on quantum dots surface would cause this effect \\[17\\]. Another process that can change intensity of quantum dots photoluminescence is aggregation. Aggregation of quantum dots decreases photoluminescence quantum yield. Slow dissolution (monomerization) of quantum dots powder (aggregates) could cause increasing photoluminescence intensity due to increased photoluminescence quantum yield of single quantum dots compared with aggregated form. More detailed investigation into absorption spectrum dynamics during first day after preparation of solution contradicts to this explanation. Absorption of quantum dots dissolved in deionized water decreases during first day. This decrease can be explained by aggregation of quantum dots. Aggregation of quantum dots leads to decrease in absorption intensity, red shift, broadening and photoluminescence band intensity decrease. But in first phase, width and wavelength of photoluminescence band do not change, whereas photoluminescence intensity increases. So these changes are caused not by aggregation of quantum dots but by changes in quantum dot coating. CdTe\u2013TGA quantum dots are fluorescent nanoparticles composed of CdTe core and TGA coating. Rearrangement of quantum dot coating can lead to decrease in defects on quantum dot surface and increase in photoluminescence quantum yield. Sudden increase in quantum dots photoluminescence band intensity, after adding BSA to solution, shows that interaction of quantum dots with BSA strongly increases photoluminescence quantum yield. Photoluminescence decay measurements presented in literature \\[18\\] confirm this result. Photoluminescence decay of quantum dots with BSA is tri-exponential, while photoluminescence decay of quantum dots is described with four exponents. This shows that addition of protein eliminates one excitation relaxation path. Photoluminescence lifetime analysis shows that fastest relaxation component (\u03c4~1~ = 3.4 ns) disappears \\[18\\]. Fastest relaxation component is caused by defects of quantum dots \\[19\\]. Elimination of this component leads to increase in quantum dots photoluminescence quantum yield. So increase in photoluminescence intensity at the first phase is caused by rearrangement of TGA molecules (Figure <a href=\"#F6\" data-ref-type=\"fig\">6IA, IB<\/a>).\n\nIn the second phase, photoluminescence of quantum dots starts to decrease. TGA molecules are not covalently bound to CdTe core (they are attached to it by coordinating bonds \\[20\\]) and probably are washing out slowly (Figure <a href=\"#F6\" data-ref-type=\"fig\">6IIA, IIIB<\/a>). This process increases number of defects on quantum dots surface and leads to decrease in photoluminescence quantum yield. AFM and STEM images (Figure <a href=\"#F4\" data-ref-type=\"fig\">4a\u2013d<\/a>) show that quantum dots in aqueous media aggregate. TGA coating makes CdTe quantum dots water soluble. Washing out of coating decreases water solubility of quantum dots, increases aggregation speed (Figure <a href=\"#F6\" data-ref-type=\"fig\">6IIIA<\/a>) and leads to formation of precipitate (Figure <a href=\"#F6\" data-ref-type=\"fig\">6IVA<\/a>). In the second phase, effects of aggregation (decrease in photoluminescence intensity and red shift of photoluminescence band) are seen in quantum dots solution without protein (Figure <a href=\"#F3\" data-ref-type=\"fig\">3a<\/a>).\n\nSecond phase is different for quantum dots solution with protein. In this case, photoluminescence decreases slowly and after some time stabilizes. Position of photoluminescence band does not change during this phase. This shows that quantum dots in the presence of protein do not aggregate, and protein prevents the degradation of quantum dot coating and aggregation of quantum dots.\n\n# Conclusions\n\nThis study showed that water-soluble CdTe\u2013TGA quantum dots in aqueous solutions are not stable. Spectroscopic and atomic force microscopy measurements showed that quantum dots aggregate in solution, and 9 days after preparation of solution, precipitate was observed. BSA interacts with CdTe\u2013TGA quantum dots, prevents them from aggregating, increases photoluminescence quantum yield and makes them stable. This effect is achieved by forming a new layer of quantum dot coating.\n\n## Acknowledgements\n\nThis work was supported by the project \"Multifunctional nanoparticles for specific non-invasive early diagnostics and treatment of cancer\" (No. 2004-LT0036-IP-1NOR).","meta":{"dup_signals":{"dup_doc_count":107,"dup_dump_count":22,"dup_details":{"curated_sources":3,"2022-21":1,"2015-48":5,"2015-40":5,"2015-35":5,"2015-32":1,"2015-27":5,"2015-22":5,"2015-14":4,"2014-52":5,"2014-49":4,"2014-42":10,"2014-41":7,"2014-35":4,"2014-23":9,"2014-15":7,"2022-40":1,"2015-18":5,"2015-11":3,"2015-06":4,"2014-10":5,"2013-48":5,"2013-20":4}},"file":"PMC3212239"},"subset":"pubmed_central"}
{"text":"abstract: The complete sequence of the 1,267,782 bp genome of *Wolbachia pipientis w*Mel, an obligate intracellular bacteria of Drosophila melanogaster, has been determined. *Wolbachia*, which are found in a variety of invertebrate species, are of great interest due to their diverse interactions with different hosts, which range from many forms of reproductive parasitism to mutualistic symbioses. Analysis of the *w*Mel genome, in particular phylogenomic comparisons with other intracellular bacteria, has revealed many insights into the biology and evolution of *w*Mel and *Wolbachia* in general. For example, the *w*Mel genome is unique among sequenced obligate intracellular species in both being highly streamlined and containing very high levels of repetitive DNA and mobile DNA elements. This observation, coupled with multiple evolutionary reconstructions, suggests that natural selection is somewhat inefficient in *w*Mel, most likely owing to the occurrence of repeated population bottlenecks. Genome analysis predicts many metabolic differences with the closely related *Rickettsia* species, including the presence of intact glycolysis and purine synthesis, which may compensate for an inability to obtain ATP directly from its host, as *Rickettsia* can. Other discoveries include the apparent inability of *w*Mel to synthesize lipopolysaccharide and the presence of the most genes encoding proteins with ankyrin repeat domains of any prokaryotic genome yet sequenced. Despite the ability of *w*Mel to infect the germline of its host, we find no evidence for either recent lateral gene transfer between *w*Mel and D. melanogaster or older transfers between *Wolbachia* and any host. Evolutionary analysis further supports the hypothesis that mitochondria share a common ancestor with the \u03b1-Proteobacteria, but shows little support for the grouping of mitochondria with species in the order Rickettsiales. With the availability of the complete genomes of both species and excellent genetic tools for the host, the *w*Mel\u2013D. melanogaster symbiosis is now an ideal system for studying the biology and evolution of *Wolbachia* infections.\nauthor: Martin Wu; Ling V Sun; Jessica Vamathevan; Markus Riegler; Robert Deboy; Jeremy C Brownlie; Elizabeth A McGraw; William Martin; Christian Esser; Nahal Ahmadinejad; Christian Wiegand; Ramana Madupu; Maureen J Beanan; Lauren M Brinkac; Sean C Daugherty; A. Scott Durkin; James F Kolonay; William C Nelson; Yasmin Mohamoud; Perris Lee; Kristi Berry; M. Brook Young; Teresa Utterback; Janice Weidman; William C Nierman; Ian T Paulsen; Karen E Nelson; Herv\u00e9 Tettelin; Scott L O'Neill; Jonathan A Eisen\ndate: 2004-03\ninstitute: **1**The Institute for Genomic Research, RockvilleMarylandUnited States of America; **2**Department of Epidemiology and Public Health, Yale University School of MedicineNew Haven, ConnecticutUnited States of America; **3**Department of Zoology and Entomology, School of Life SciencesThe University of Queensland, St Lucia, QueenslandAustralia; **4**Institut f\u00fcr Botanik III, Heinrich-Heine Universit\u00e4tD\u00fcsseldorfGermany\nreferences:\ntitle: Phylogenomics of the Reproductive Parasite *Wolbachia pipientis w*Mel: A Streamlined Genome Overrun by Mobile Genetic Elements\n\n# Introduction\n\n*Wolbachia* are intracellular gram-negative bacteria that are found in association with a variety of invertebrate species, including insects, mites, spiders, terrestrial crustaceans, and nematodes. *Wolbachia* are transovarialy transmitted from females to their offspring and are extremely widespread, having been found to infect 20%\u201375% of invertebrate species sampled (Jeyaprakash and Hoy 2000; Werren and Windsor 2000). *Wolbachia* are members of the Rickettsiales order of the \u03b1-subdivision of the Proteobacteria phyla and belong to the Anaplasmataceae family, with members of the genera *Anaplasma*, *Ehrlichia*, *Cowdria*, and *Neorickettsia* (Dumler et al. 2001). Six major clades (A\u2013F) of *Wolbachia* have been identified to date (Lo et al. 2002): A, B, E, and F have been reported from insects, arachnids, and crustaceans; C and D from filarial nematodes.\n\n*Wolbachia\u2013*host interactions are complex and range from mutualistic to pathogenic, depending on the combination of host and *Wolbachia* involved. Most striking are the various forms of \"reproductive parasitism\" that serve to alter host reproduction in order to enhance the transmission of this maternally inherited agent. These include parthenogenesis (infected females reproducing in the absence of mating to produce infected female offspring), feminization (infected males being converted into functional phenotypic females), male-killing (infected male embryos being selectively killed), and cytoplasmic incompatibility (in its simplest form, the developmental arrest of offspring of uninfected females when mated to infected males) (O'Neill et al. 1997a).\n\n*Wolbachia* have been hypothesized to play a role in host speciation through the reproductive isolation they generate in infected hosts (Werren 1998). They also provide an intriguing array of evolutionary solutions to the genetic conflict that arises from their uniparental inheritance. These solutions represent alternatives to classical mutualism and are often of more benefit to the symbiont than the host that is infected (Werren and O'Neill 1997). From an applied perspective, it has been proposed that *Wolbachia* could be utilized to either suppress pest insect populations or sweep desirable traits into pest populations (e.g., the inability to transmit disease-causing pathogens) (Sinkins and O'Neill 2000). Moreover, they may provide a new approach to the control of human and animal filariasis. Since the nematode worms that cause filariasis have an obligate symbiosis with mutualistic *Wolbachia*, treatment of filariasis with simple antibiotics that target *Wolbachia* has been shown to eliminate microfilaria production as well as ultimately killing the adult worm (Taylor et al. 2000; Taylor and Hoerauf 2001).\n\nDespite their common occurrence and major effects on host biology, little is currently known about the molecular mechanisms that mediate the interactions between *Wolbachia* and their invertebrate hosts. This is partly due to the difficulty of working with an obligate intracellular organism that is difficult to culture and hard to obtain in quantity. Here we report the completion and analysis of the genome sequence of Wolbachia pipientis *w*Mel, a strain from the A supergroup that naturally infects Drosophila melanogaster (Zhou et al. 1998).\n\n# Results\/Discussion\n\n## Genome Properties\n\nThe *w*Mel genome is determined to be a single circular molecule of 1,267,782 bp with a G+C content of 35.2%. This assembly is very similar to the genetic and physical map of the closely related strain *w*MelPop (Sun et al., 2003). The genome does not exhibit the GC skew pattern typical of some prokaryotic genomes (<a href=\"#pbio-0020069-g001\" data-ref-type=\"fig\">Figure 1<\/a>) that have two major shifts, one near the origin and one near the terminus of replication. Therefore, identification of a putative origin of replication and the assignment of basepair 1 were based on the location of the *dnaA* gene. Major features of the genome and of the annotation are summarized in <a href=\"#pbio-0020069-t001\" data-ref-type=\"table\">Table 1<\/a> and <a href=\"#pbio-0020069-g001\" data-ref-type=\"fig\">Figure 1<\/a>.\n\n###### *w*Mel Genome Features\n\n![](pbio.0020069.t001)\n\n## Repetitive and Mobile DNA\n\nThe most striking feature of the *w*Mel genome is the presence of very large amounts of repetitive DNA and DNA corresponding to mobile genetic elements, which is unique for an intracellular species. In total, 714 repeats of greater than 50 bp in length, which can be divided into 158 distinct families (<a href=\"#st001\" data-ref-type=\"supplementary-material\">Table S1<\/a>), were identified. Most of the repeats are present in only two copies in the genome, although 39 are present in three or more copies, with the most abundant repeat being found in 89 copies. We focused our analysis on the 138 repeats of greater than 200 bp (<a href=\"#pbio-0020069-t002\" data-ref-type=\"table\">Table 2<\/a>). These were divided into 19 families based upon sequence similarity to each other. These repeats were found to make up 14.2 % of the *w*Mel genome. Of these repeat families, 15 correspond to likely mobile elements, including seven types of insertion sequence (IS) elements, four likely retrotransposons, and four families without detectible similarity to known elements but with many hallmarks of mobile elements (flanked by inverted repeats, present in multiple copies) (<a href=\"#pbio-0020069-t002\" data-ref-type=\"table\">Table 2<\/a>). One of these new elements (repeat family 8) is present in 45 copies in the genome. It is likely that many of these elements are not able to autonomously transpose since many of the transposase genes are apparently inactivated by mutations or the insertion of other transposons (<a href=\"#st002\" data-ref-type=\"supplementary-material\">Table S2<\/a>). However, some are apparently recently active since there are transposons inserted into at least nine genes (<a href=\"#st002\" data-ref-type=\"supplementary-material\">Table S2<\/a>), and the copy number of some repeats appears to be variable between *Wolbachia* strains (M. Riegler et al., personal communication). Thus, many of these repetitive elements may be useful markers for strain discrimination. In addition, the mobile elements likely contribute to generating the diversity of phenotypically distinct *Wolbachia* strains (e.g., mod^\u2212^ strains \\[McGraw et al. 2001\\]) by altering or disrupting gene function (<a href=\"#st002\" data-ref-type=\"supplementary-material\">Table S2<\/a>).\n\n###### *w*Mel DNA Repeats of Greater than 200 bp\n\n![](pbio.0020069.t002)\n\nThree prophage elements are present in the genome. One is a small pyocin-like element made up of nine genes (WD00565\u2013WD00575). The other two are closely related to and exhibit extensive gene order conservation with the WO phage described from *Wolbachia* sp. *w*Kue (Masui et al. 2001) (<a href=\"#pbio-0020069-g002\" data-ref-type=\"fig\">Figure 2<\/a>). Thus, we have named them *w*Mel WO-A and WO-B, based upon their location in the genome. *w*Mel WO-B has undergone a major rearrangement and translocation, suggesting it is inactive. Phylogenetic analysis indicates that *w*Mel WO-B is more closely related to the *w*Kue WO than to *w*Mel WO-A (<a href=\"#sg001\" data-ref-type=\"supplementary-material\">Figure S1<\/a>). Thus, *w*Mel WO-A likely represents either a separate insertion event in the *Wolbachia* lineage or a duplication that occurred prior to the separation of the *w*Mel and *w*Kue lineages. Phylogenetic analysis also confirms the proposed mosaic nature of the WO phage (Masui et al. 2001), with one block being closely related to lambdoid phage and another to P2 phage (data not shown).\n\n## Genome Structure: Rearrangements, Duplications, and Deletions\n\nThe irregular pattern of GC skew in *w*Mel is likely due in part to intragenomic rearrangements associated with the many DNA repeat elements. Comparison with a large contig from a *Wolbachia* species that infects Brugia malayi is consistent with this (Ware et al. 2002) (<a href=\"#pbio-0020069-g003\" data-ref-type=\"fig\">Figure 3<\/a>). While only translocations are seen in this plot, genetic comparisons reveal that inversions also occur between strains (Sun et al., 2003), which is consistent with previous studies of prokaryotic genomes that have found that the most common large-scale rearrangements are inversions that are symmetric around the origin of DNA replication (Eisen et al. 2000). The occurrence of frequent rearrangement events during *Wolbachia* evolution is supported by the absence of any large-scale conserved gene order with *Rickettsia* genomes. The rearrangements in *Wolbachia* likely correspond with the introduction and massive expansion of the repeat element families that could serve as sites for intragenomic recombination, as has been shown to occur for some other bacterial species (Parkhill et al. 2003). The rearrangements in *w*Mel may have fitness consequences since several classes of genes often found in clusters are generally scattered throughout the *w*Mel genome (e.g., ABC transporter subunits, Sec secretion genes, rRNA genes, F-type ATPase genes).\n\nAlthough the common ancestor of *Wolbachia* and *Rickettsia* likely already had a reduced, streamlined genome, *w*Mel has lost additional genes since that time (<a href=\"#st003\" data-ref-type=\"supplementary-material\">Table S3<\/a>). Many of these recent losses are of genes involved in cell envelope biogenesis in other species, including most of the machinery for producing lipopolysaccharide (LPS) components and the alanine racemase that supplies D-alanine for cell wall synthesis. In addition, some other genes that may have once been involved in this process are present in the genome, but defective (e.g., mannose-1-phosphate guanylyltransferase, which is split into two coding sequences \\[CDSs\\], WD1224 and WD1227, by an IS5 element) and are likely in the process of being eliminated. The loss of cell envelope biogenesis genes has also occurred during the evolution of the *Buchnera* endosymbionts of aphids (Shigenobu et al. 2000; Moran and Mira 2001). Thus, *w*Mel and *Buchnera* have lost some of the same genes separately during their reductive evolution. Such convergence means that attempts to use gene content to infer evolutionary relatedness needs to be interpreted with caution. In addition, since *Anaplasma* and *Ehrlichia* also apparently lack genes for LPS production (Lin and Rikihisha 2003), it is likely that the common ancestor of *Wolbachia*, *Ehrlichia*, and *Anaplasma* was unable to synthesize LPS. Thus, the reports that *Wolbachia*-derived LPS-like compounds is involved in the immunopathology of filarial nematode disease in mammals (Taylor 2002) either indicate that these *Wolbachia* have acquired genes for LPS synthesis or that the reported LPS-like compounds are not homologous to LPS.\n\nDespite evident genome reduction in *w*Mel and in contrast to most small-genomed intracellular species, gene duplication appears to have continued, as over 50 gene families have apparently expanded in the *w*Mel lineage relative to that of all other species (<a href=\"#st004\" data-ref-type=\"supplementary-material\">Table S4<\/a>). Many of the pairs of duplicated genes are encoded next to each other in the genome, suggesting that they arose by tandem duplication events and may simply reflect transient duplications in evolution (deletion is common when there are tandem arrays of genes). Many others are components of mobile genetic elements, indicating that these elements have expanded significantly after entering the *Wolbachia* evolutionary lineage. Other duplications that could contribute to the unique biological properties of *w*Mel include that of the mismatch repair gene *mutL* (see below) and that of many hypothetical and conserved hypothetical proteins.\n\nOne duplication of particular interest is that of *wsp*, which is a standard gene for strain identification and phylogenetic reconstruction in *Wolbachia* (Zhou et al. 1998). In addition to the previously described *wsp* (WD0159), *w*Mel encodes two *wsp* paralogs (WD0009 and WD0489), which we designate as *wspB* and *wspC*, respectively. While these paralogs are highly divergent from *wsp* (protein identities of 19.7% and 23.5%, respectively) and do not amplify using the standard *wsp* PCR primers (Braig et al. 1998; Zhou et al. 1998), their presence could lead to some confusion in classification and identification of *Wolbachia* strains. This has apparently occurred in one study of *Wolbachia* strain *w*KueYO, for which the reported *wsp* gene (gbAB045235) is actually an ortholog of *wspB* (99.8% sequence identity and located at the end of the *virB* operon \\[Masui et al. 2000\\]) and not an ortholog of the *wsp* gene. Considering that the *wsp* gene has been extremely informative for discriminating between strains of *Wolbachia*, we designed PCR primers to the *w*Mel *wspB* gene to amplify and then sequence the orthologs from the related *w*Ri and *w*AlbB *Wolbachia* strains from Drosophila simulans and Aedes albopictus, respectively, as well as the *Wolbachia* strain that infects the filarial nematode Dirofilaria immitis to determine the potential utility of this locus for strain discrimination. A comparison of genetic distances between the *wsp* and *wspB* genes for these different taxa indicates that overall the *wspB* gene appears to be evolving at a faster rate than *wsp* and, as such, may be a useful additional marker for discriminating between closely related *Wolbachia* strains (<a href=\"#st005\" data-ref-type=\"supplementary-material\">Table S5<\/a>).\n\n## Inefficiency of Selection in *w*Mel\n\nThe fraction of the genome that is repetitive DNA and the fraction that corresponds to mobile genetic elements are among the highest for any prokaryotic genome. This is particularly striking compared to the genomes of other obligate intracellular species such as *Buchnera*, *Rickettsia*, *Chlamydia*, and *Wigglesworthia*, that all have very low levels of repetitive DNA and mobile elements. The recently sequenced genomes of the intracellular pathogen Coxiella burnetti (Seshadri et al. 2003) has both a streamlined genome and moderate amounts of repetitive DNA, although much less than *w*Mel. The paucity of repetitive DNA in these and other intracellular species is thought to be due to a combination of lack of exposure to other species, thereby limiting introduction of mobile elements, and genome streamlining (Mira et al. 2001; Moran and Mira 2001; Frank et al. 2002). We examined the *w*Mel genome to try to understand the origin of the repetitive and mobile DNA and to explain why such repetitive\/mobile DNA is present in *w*Mel, but not other streamlined intracellular species.\n\nWe propose that the mobile DNA in *w*Mel was acquired some time after the separation of the *Wolbachia* and *Rickettsia* lineages but before the radiation of the *Wolbachia* group*.* The acquisition of these elements after the separation of the *Wolbachia* and *Rickettsia* lineages is suggested by the fact that most do not have any obvious homologous sequences in the genomes of other \u03b1-Proteobacteria, including the closely related *Rickettsia* spp. Additional evidence for some acqui-sition of foreign DNA after the *Wolbachia\u2013Rickettsia* split comes from phylogenetic analysis of those genes present in *w*Mel, but not in the two sequenced rickettsial genomes (see <a href=\"#st003\" data-ref-type=\"supplementary-material\">Table S3<\/a>; unpublished data). The acquisition prior to the radiation of *Wolbachia* is suggested by two lines of evidence. First, many of the elements are found in the genome of the distantly related *Wolbachia* of the nematode B. malayi (see <a href=\"#pbio-0020069-g003\" data-ref-type=\"fig\">Figure 3<\/a>; unpublished data). In addition, genome analysis reveals that these elements do not have significantly anomalous nucleotide composition or codon usage compared to the rest of the genome. In fact, there are only four regions of the genome with significantly anomalous composition, comprising in total only approximately 17 kbp of DNA (<a href=\"#pbio-0020069-t003\" data-ref-type=\"table\">Table 3<\/a>). The lack of anomalous composition suggests either that any foreign DNA in *w*Mel was acquired long enough ago to allow it to \"ameliorate\" and become compositionally similar to endogenous *Wolbachia* DNA (Lawrence and Ochman 1997, 1998) or that any foreign DNA that is present was acquired from organisms with similar composition to endogenous *w*Mel genes. Owing to their potential effects on genome evolution (insertional mutagenesis, catalyzing genome rearrangements), we propose that the acquisition and maintenance of these repetitive and mobile elements by *w*Mel have played a key role in shaping the evolution of *Wolbachia*.\n\n###### Regions of Anomalous Nucleotide Composition in the *wMel* Genome\n\n![](pbio.0020069.t003)\n\nIt is likely that much of the mobile\/repetitive DNA was introduced via phage, given that three prophage elements are present; experimental studies have shown active phage in some *Wolbachia* (Masui et al. 2001) and *Wolbachia* superinfections occur in many hosts (e.g., Jamnongluk et al. 2002), which would allow phage to move between strains. Whatever the mechanism of introduction, the persistence of the repetitive elements in *w*Mel in the face of apparently strong pressures for streamlining is intriguing. One expla-nation is that *w*Mel may be getting a steady infusion of mobile elements from other *Wolbachia* strains to counteract the elimination of elements by selection for genome streamlining. This would explain the absence of anomalous nucleotide composition of the elements. However, we believe that a major contributing factor to the presence of all the repetitive\/mobile DNA in *w*Mel is that *w*Mel and possibly *Wolbachia* in general have general inefficiency of natural selection relative to other species. This inefficiency would limit the ability to eliminate repetitive DNA. A general inefficiency of natural selection (especially purifying selection) has been suggested previously for intracellular bacteria, based in part on observations that these bacteria have higher evolutionary rates than free-living bacteria (e.g., Moran 1996). We also find a higher evolutionary rate for *w*Mel than that of the closely related intracellular *Rickettsia*, which themselves have higher rates than free-living \u03b1-Proteobacteria (<a href=\"#pbio-0020069-g004\" data-ref-type=\"fig\">Figure 4<\/a>). Additionally, codon bias in *w*Mel appears to be driven more by mutation or drift than selection (<a href=\"#sg002\" data-ref-type=\"supplementary-material\">Figure S2<\/a>), as has been reported for *Buchnera* species and was suggested to be due to inefficient purifying selection (Wernegreen and Moran 1999). Such inefficiencies of natural selection are generally due to an increase in the relative contribution of genetic drift and mutation as compared to natural selection (Eiglmeier et al. 2001; Lawrence 2001; Parkhill et al. 2001). Below we discuss different possible explanations for the inefficiency of selection in *w*Mel, especially in comparison to other intracellular bacteria.\n\nLow rates of recombination, such as occur in centromeres and the human Y chromosome, can lead to inefficient selection because of the linkage among genes. This has been suggested to be occurring in *Buchnera* species because these species do not encode homologs of RecA, which is the key protein in homologous recombination in most species (Shigenobu et al. 2000). The absence of recombination in *Buchnera* is supported by the lack of genome rearrangements in their recent evolution (Tamas et al. 2002). Additionally, there is apparently little or no gene flow into *Buchnera* strains. In contrast, *w*Mel encodes the necessary machinery for recombination, including RecA (<a href=\"#st006\" data-ref-type=\"supplementary-material\">Table S6<\/a>), and has experienced both extensive intragenomic homologous recombination and introduction of foreign DNA. Therefore, the unusual genome features of *w*Mel are unlikely to be due to low levels of recombination.\n\nAnother possible explanation for inefficient selection is high mutation rates. It has been suggested that the higher evolutionary rates in intracellular bacteria are the result of high mutation rates that are in turn due to the loss of genes for DNA repair processes (e.g., Itoh et al. 2002). This is likely not the case in *w*Mel since its genome encodes proteins corresponding to a broad suite of DNA repair pathways including mismatch repair, nucleotide excision repair, base excision repair, and homologous recombination (<a href=\"#st006\" data-ref-type=\"supplementary-material\">Table S6<\/a>). The only noteworthy DNA repair gene absent from *w*Mel and present in the more slowly evolving *Rickettsia* is *mfd,* which is involved in targeting DNA repair to the transcribed strand of actively transcribing genes in other species (Selby et al. 1991). However, this absence is unlikely to contribute significantly to the increased evolutionary rate in *w*Mel, since defects in *mfd* do not lead to large increases in mutation rates in other species (Witkin 1994). The presence of mismatch repair genes (homologs of *mutS* and *mutL*) in *w*Mel is particularly relevant since this pathway is one of the key steps in regulating mutation rates in other species. In fact, *w*Mel is the first bacterial species to be found with two *mutL* homologs. Overall, examination of the predicted DNA repair capabilities of bacteria (Eisen and Hanawalt 1999) suggests that the connection between evolutionary rates in intracellular species and the loss of DNA repair processes is spurious. While many intracellular species have lost DNA repair genes in their recent evolution, different species have lost different genes and some, such as *w*Mel and *Buchnera* spp., have kept the genes that likely regulate mutation rates. In addition, some free-living species without high evolutionary rates have lost some of the same pathways lost in intracellular species, while many free-living species have lost key pathways resulting in high mutation rates (e.g., Helicobacter pylori has apparently lost mismatch repair \\[Eisen 1997, Eisen 1998b; Bjorkholm et al. 2001\\]). Given that intracellular species tend to have small genomes and have lost genes from every type of biological process, it is not surprising that many of them have lost DNA repair genes as well.\n\nWe believe that the most likely explanations for the inefficiency of selection in *w*Mel involve population-size related factors, such as genetic drift and the occurrence of population bottlenecks. Such factors have also been shown to likely explain the high evolutionary rates in other intracellular species (Moran 1996; Moran and Mira 2001; van Ham et al. 2003). *Wolbachia* likely experience frequent population bottlenecks both during transovarial transmission (Boyle et al. 1993) and during cytoplasmic incompatibility mediated sweeps through host populations. The extent of these bottlenecks may be greater than in other intracellular bacteria, which would explain why *w*Mel has both more repetitive and mobile DNA than other such species and a higher evolutionary rate than even the related *Rickettsia spp.* Additional genome sequences from other *Wolbachia* will reveal whether this is a feature of all *Wolbachia* or only certain strains.\n\n## Mitochondrial Evolution\n\nThere is a general consensus in the evolutionary biology literature that the mitochondria evolved from bacteria in the \u03b1-subgroup of the Proteobacteria phyla (e.g., Lang et al. 1999). Analysis of complete mitochondrial and bacterial genomes has very strongly supported this hypothesis (Andersson et al. 1998, 2003; Muller and Martin 1999; Ogata et al. 2001). However, the exact position of the mitochondria within the \u03b1-Proteobacteria is still debated. Many studies have placed them in or near the Rickettsiales order (Viale and Arakaki 1994; Gupta 1995; Sicheritz-Ponten et al. 1998; Lang et al. 1999; Bazinet and Rollins 2003). Some studies have further suggested that mitochondria are a sister taxa to the *Rickettsia* genus within the Rickettsiaceae family and thus more closely related to *Rickettsia* spp. than to species in the Anaplasmataceae family such as *Wolbachia* (Karlin and Brocchieri 2000; Emelyanov 2001a, 2001b, 2003a, 2003b).\n\nIn our analysis of complete genomes, including that of *w*Mel, the first non-*Rickettsia* member of the Rickettsiales order to have its genome completed, we find support for a grouping of *Wolbachia* and *Rickettsia* to the exclusion of the mitochondria, but not for placing the mitochondria within the Rickettsiales order (<a href=\"#pbio-0020069-g005\" data-ref-type=\"fig\">Figure 5<\/a>A and <a href=\"#pbio-0020069-g005\" data-ref-type=\"fig\">5<\/a>B; <a href=\"#st007\" data-ref-type=\"supplementary-material\">Table S7<\/a>; <a href=\"#st008\" data-ref-type=\"supplementary-material\">Table S8<\/a>). Specifically, phylogenetic trees of a concatenated alignment of 32 proteins show strong support with all methods (see <a href=\"#st007\" data-ref-type=\"supplementary-material\">Table S7<\/a>) for common branching of: (i) mitochondria, (ii) *Rickettsia* with *Wolbachia*, (iii) the free-living \u03b1-Proteobacteria, and (iv) mitochondria within \u03b1-Proteobacteria. Since amino acid content bias was very severe in these datasets, protein LogDet analyses, which can correct for the bias, were also performed. In LogDet analyses of the concatenated protein alignment, both including and excluding highly biased positions, mitochondria usually branched basal to the *Wolbachia\u2013Rickettsia* clade, but never specifically with *Rickettsia* (see <a href=\"#st007\" data-ref-type=\"supplementary-material\">Table S7<\/a>). In addition, in phylogenetic studies of individual genes, there was no consistent phylogenetic position of mitochondrial proteins with any particular species or group within the \u03b1-Proteobacteria (see <a href=\"#st008\" data-ref-type=\"supplementary-material\">Table S8<\/a>), although support for a specific branch uniting the two *Rickettsia* species with *Wolbachia* was quite strong. Eight of the proteins from mitochondrial genomes (YejW, SecY, Rps8, Rps2, Rps10, RpoA, Rpl15, Rpl32) do not even branch within the \u03b1-Proteobacteria, although these genes almost certainly were encoded in the ancestral mitochondrial genome (Lang et al. 1997).\n\nThis analysis of mitochondrial and \u03b1-Proteobacterial genes reinforces the view that ancient protein phylogenies are inherently prone to error, most likely because current models of phylogenetic inference do not accurately reflect the true evolutionary processes underlying the differences observed in contemporary amino acid sequences (Penny et al. 2001). These conflicting results regarding the precise position of mitochondria within the \u03b1-Proteobacteria can be seen in the high amount of networking in the Neighbor-Net graph of the analyses of the concatenated alignment shown in <a href=\"#pbio-0020069-g005\" data-ref-type=\"fig\">Figure 5<\/a>. An important complication in studies of mitochondrial evolution lies in identifying \"\u03b1-Proteobacterial\" genes for comparison (Martin 1999). For example, in our analyses, proteins from *Magnetococcus* branched with other \u03b1-Proteobacterial homologs in only 17 of the 49 proteins studied, and in five cases they assumed a position basal to \u03b1-, \u03b2-, and \u03b3-Proteobacterial homologs.\n\n## Host\u2013Symbiont Gene Transfers\n\nMany genes that were once encoded in mitochondrial genomes have been transferred into the host nuclear genomes. Searching for such genes has been complicated by the fact that many of the transfer events happened early in eukaryotic evolution and that there are frequently extreme amino acid and nucleotide composition biases in mitochondrial genomes (see above). We used the *w*Mel genome to search for additional possible mitochondrial-derived genes in eukaryotic nuclear genomes. Specifically, we constructed phylogenetic trees for *w*Mel genes that are not in either *Rickettsia* genomes. Five new eukaryotic genes of possible mitochondrial origin were identified: three genes involved in de novo nucleotide biosynthesis (*purD*, *purM*, *pyrD*) and two conserved hypothetical proteins (WD1005, WD0724). The \u03b1-Proteobacterial origin of these genes suggests that at least some of the genes of the de novo nucleotide synthesis pathway in eukaryotes might have been laterally acquired from bacteria via the mitochondria. The presence of such genes in other Proteobacteria suggests that their absence from *Rickettsia* is due to gene loss (Gray et al. 2001). This finding supports the need for additional \u03b1-Proteobacterial genomes to identify mitochondrion-derived genes in eukaryotes.\n\nWhile organelle to nuclear gene transfers are generally accepted, there is a great deal of controversy over whether other gene transfers have occurred from bacteria into animals. In particular, claims of transfer from bacteria into the human genome (Lander et al. 2001) were later shown to be false (Roelofs and Van Haastert 2001; Salzberg et al. 2001; Stanhope et al. 2001). *Wolbachia* are excellent candidates for such transfer events since they live inside the germ cells, which would allow lateral transfers to the host to be transmitted to subsequent host generations. Consistent with this, a recent study has shown some evidence for the presence of *Wolbachia-*like genes in a beetle genome (Kondo et al. 2002). The symbiosis between *w*Mel and D. melanogaster provides an ideal case to search for such transfers since we have the complete genomes of both the host and symbiont. Using BLASTN searches and MUMmer alignments, we did not find any examples of highly similar stretches of DNA shared between the two species. In addition, protein-level searches and phylogenetic trees did not identify any specific relationships between *w*Mel and D. melanogaster for any genes. Thus, at least for this host\u2013symbiont association, we do not find any likely cases of recent gene exchange, with genes being maintained in both host and symbiont. In addition, in our phylogenetic analyses, we did not find any examples of *w*Mel proteins branching specifically with proteins from any invertebrate to the exclusion of other eukaryotes. Therefore, at least for the genes in *w*Mel, we do not find evidence for transfer of *Wolbachia* genes into any invertebrate genome.\n\n## Metabolism and Transport\n\n*w*Mel is predicted to have very limited capabilities for membrane transport, for substrate utilization, and for the biosynthesis of metabolic intermediates (<a href=\"#sg003\" data-ref-type=\"supplementary-material\">Figure S3<\/a>), similar to what has been seen in other intracellular symbionts and pathogens (Paulsen et al. 2000). Almost all of the identifiable uptake systems for organic nutrients in *w*Mel are for amino acids, including predicted transporters for proline, asparate\/glutamate, and alanine. This pattern of transporters, coupled with the presence of pathways for the metabolism of the amino acids cysteine, glutamate, glutamine, proline, serine, and threonine, suggests that *w*Mel may obtain much of its energy from amino acids. These amino acids could also serve as material for the production of other amino acids. In contrast, carbohydrate metabolism in *w*Mel appears to be limited. The only pathways that appear to be complete are the tricarboxylic acid cycle, the nonoxidative pentose phosphate pathway, and glycolysis, starting with fructose-1,6-biphosphate. The limited carbohydrate metabolism is consistent with the presence of only one sugar phosphate transporter. *w*Mel can also apparently transport a range of inorganic ions, although two of these systems, for potassium uptake and sodium ion\/proton exchange, are frameshifted. In the latter case, two other sodium ion\/proton exchangers may be able to compensate for this defect.\n\nMany of the predicted metabolic properties of *w*Mel, such as the focus on amino acid transport and the presence of limited carbohydrate metabolism, are similar to those found in *Rickettsia.* A major difference with the *Rickettsia* spp. is the absence of the ADP\u2013ATP exchanger protein in *w*Mel. In *Rickettsia* this protein is used to import ATP from the host, thus allowing these species to be direct energy scavengers (Andersson et al. 1998). This likely explains the presence of glycolysis in *w*Mel but not *Rickettsia.* An inability to obtain ATP from its host also helps explain the presence of pathways for the synthesis of the purines AMP, IMP, XMP, and GMP in *w*Mel but not *Rickettsia.* Other pathways present in *w*Mel but not *Rickettsia* include threonine degradation (described above), riboflavin biosynthesis, pyrimidine metabolism (i.e., from PRPP to UMP), and chelated iron uptake (using a single ABC transporter). The two *Rickettsia* species have a relatively large complement of predicted transporters for osmoprotectants, such as proline and glycine betaine, whereas *w*Mel possesses only two of these systems.\n\n## Regulatory Responses\n\nThe *w*Mel genome is predicted to encode few proteins for regulatory responses. Three genes encoding two-component system subunits are present: two sensor histidine kinases (WD1216 and WD1284) and one response regulator (WD0221). Only six strong candidates for transcription regulators were identified: a homolog of arginine repressors (WD0453), two members of the TenA family of transcription activator proteins (WD0139 and WD0140), a homolog of *ctrA*, a transcription regulator for two component systems in other \u03b1-Proteobacteria (WD0732), and two \u03c3 factors (RpoH\/WD1064 and RpoD\/WD1298). There are also seven members of one paralogous family of proteins that are distantly related to phage repressors (see above), although if they have any role in transcription, it is likely only for phage genes. Such a limited repertoire of regulatory systems has also been reported in other endosymbionts and has been explained by the apparent highly predictable and stable environment in which these species live (Andersson et al. 1998; Read et al. 2000; Shigenobu et al. 2000; Moran and Mira 2001; Akman et al. 2002; Seshadri et al. 2003).\n\n## Host\u2013Symbiont Interactions\n\nThe mechanisms by which *Wolbachia* infect host cells and by which they cause the diverse phenotypic effects on host reproduction and fitness are poorly understood, and the *w*Mel genome helps identify potential contributing factors. A complete Type IV secretion system, portions of which have been reported in earlier studies, is present. The complete genome sequence shows that in addition to the five *vir* genes previously described from Wolbachia wKueYO (Masui et al. 2001), an additional four are present in *w*Mel. Of the nine *w*Mel *vir* ORFs, eight are arranged into two separate operons. Similar to the single operon identified in *w*Tai and *w*KueYO, the *w*Mel *virB8*, *virB9*, *virB10*, *virB11*, and *virD4* CDSs are adjacent to *wspB*, forming a 7 kb operon (WD0004\u2013WD0009). The second operon contains *virB3*, *virB4*, and *virB6* as well as four additional non-*vir* CDSs, including three putative membrane-spanning proteins, that form part of a 15.7 kb operon (WD0859\u2013WD0853). Examination of the Rickettsia conorii genome shows a similar orga-nization (<a href=\"#pbio-0020069-g006\" data-ref-type=\"fig\">Figure 6<\/a>A). The observed conserved gene order for these genes between these two genomes suggests that the putative membrane-spanning proteins could form a novel and, possibly, integral part of a functioning Type IV secretion system within these bacteria. Moreover, reverse transcription (RT)-PCRs have confirmed that *wspB* and WD0853\u2013WD0856 are each expressed as part of the two *vir* operons and further indicate that these additional encoded proteins are novel components of the *Wolbachia* Type IV secretion system (<a href=\"#pbio-0020069-g006\" data-ref-type=\"fig\">Figure 6<\/a>B).\n\nIn addition to the two major *vir* clusters, a paralog of *virB8* (WD0817) is also present in the *w*Mel genome. WD0818 is quite divergent from *virB8* and, as such, does not appear to have resulted from a recent gene duplication event. RT-PCR experiments have failed to show expression of this CDS in *w*Mel-infected *Drosophila* (data not shown). PCR primers were designed to all CDSs of the *w*Mel Type IV secretion system and used to successfully amplify orthologs from the divergent *Wolbachia* strains *w*Ri and *w*AlbB (data not shown). We were able to detect orthologs to all of the *w*Mel Type IV secretion system components as well as most of the adjacent non-*vir* CDSs, suggesting that this system is conserved across a range of A- and B-group *Wolbachia*. An increasing body of evidence has highlighted the importance of Type IV secretion systems for the successful infection, invasion, and persistence of intracellular bacteria within their hosts (Christie 2001; Sexton and Vogel 2002). It is likely that the Type IV system in *Wolbachia* plays a role in the establishment and maintenance of infection and possibly in the generation of reproductive phenotypes.\n\nGenes involved in pathogenicity in bacteria have been found to be frequently associated with regions of anomalous nucleotide composition, possibly owing to transfer from other species or insertion into the genome from plasmids or phage. In the four such regions in *w*Mel (see above; see <a href=\"#pbio-0020069-t003\" data-ref-type=\"table\">Table 3<\/a>), some additional candidates for pathogenicity-related activities are present including a putative penicillin-binding protein (WD0719), genes predicted to be involved in cell wall synthesis (WD0095\u2013WD0098, including D-alanine-D-alanine ligase, a putative FtsQ, and D-alanyl-D-alanine carboxy peptidase) and a multidrug resistance protein (WD0099). In addition, we have identified a cluster of genes in one of the phage regions that may also have some role in host\u2013symbiont interactions. This cluster (WD0611\u2013WD0621) is embedded within the WO-B phage region of the genome (see <a href=\"#pbio-0020069-g002\" data-ref-type=\"fig\">Figure 2<\/a>) and contains many genes that encode proteins with putative roles in the synthesis and degradation of surface polysaccharides, including a UDP-glucose 6-dehydrogenase (WD0620). Since this cluster appears to be normal in terms of phylogeny relative to other genes in the genome (i.e., the genes in this region have normal *w*Mel nucleotide composition and branch in phylogenetic trees with genes from other \u03b1-Proteobacteria), it is not likely to have been acquired from other species. However, it is possible that these genes can be transferred among *Wolbachia* strains via the phage, which in turn could lead to some variation in host\u2013symbiont interactions between *Wolbachia* strains.\n\nOf particular interest for host-interaction functions are the large number of genes that encode proteins that contain ankyrin repeats (<a href=\"#pbio-0020069-t003\" data-ref-type=\"table\">Table 4<\/a>). Ankyrin repeats, a tandem motif of around 33 amino acids, are found mainly in eukaryotic proteins, where they are known to mediate protein\u2013protein interactions (Caturegli et al. 2000). While they have been found in bacteria before, they are usually present in only a few copies per species. *w*Mel has 23 ankyrin repeat-containing genes, the most currently described for a prokaryote, with C. burnetti being next with 13. This is particularly striking given *w*Mel's relatively small genome size. The functions of the ankyrin repeat-containing proteins in *w*Mel are difficult to predict since most have no sequence similarity outside the ankyrin domains to any proteins of known function. Many lines of evidence suggest that the *w*Mel ankyrin domain proteins are involved in regulating host cell-cycle or cell division or interacting with the host cytoskeleton: (i) many ankyrin-containing proteins in eukaryotes are thought to be involved in linking membrane proteins to the cytoskeleton (Hryniewicz-Jankowska et al. 2002); (ii) an ankyrin-repeat protein of Ehrlichia phagocytophila binds condensed chromatin of host cells and may be involved in host cell-cycle regulation (Caturegli et al. 2000); (iii) some of the proteins that modify the activity of cell-cycle-regulating proteins in D. melanogaster contain ankyrin repeats (Elfring et al. 1997); and (iv) the *Wolbachia* strain that infects the wasp Nasonia vitripennis induces cytoplasmic incompatibility, likely by interacting with these same cell-cycle proteins (Tram and Sullivan 2002). Of the ankyrin-containing proteins in *w*Mel, those worth exploring in more detail include the several that are predicted to be surface targeted or secreted (<a href=\"#pbio-0020069-t004\" data-ref-type=\"table\">Table 4<\/a>) and thus could be targeted to the host nucleus. It is also possible that some of the other ankyrin-containing proteins are secreted via the Type IV secretion system in a targeting signal independent pathway. We call particular attention to three of the ankyrin-containing proteins (WD0285, WD0636, and WD0637), which are among the very few genes, other than those encoding components of the translation apparatus, that have significantly biased codon usage relative to what is expected based on GC content, suggesting they may be highly expressed.\n\n###### **Table 4.** Ankyrin-Domain Containing Proteins Encoded by the *w*Mel Genome\n\n![](pbio.0020069.t004)\n\n## Conclusions\n\nAnalysis of the *w*Mel genome reveals that it is unique among sequenced genomes of intracellular organisms in that it is both streamlined and massively infected with mobile genetic elements. The persistence of these elements in the genome for apparently long periods of time suggests that *w*Mel is inefficient at getting rid of them, likely a result of experiencing severe population bottlenecks during every cycle of transovarial transmission as well as during sweeps through host populations. Integration of evolutionary reconstructions and genome analysis (phylogenomics) has provided insights into the biology of *Wolbachia*, helped identify genes that likely play roles in the unusual effects *Wolbachia* have on their host, and revealed many new details about the evolution of *Wolbachia* and mitochondria. Perhaps most importantly, future studies of *Wolbachia* will benefit both from this genome sequence and from the ability to study host\u2013symbiont interactions in a host (D. melanogaster) well-suited for experimental studies.\n\n# Materials and Methods\n\n### Purification\/source of DNA\n\n*w*Mel DNA was obtained from *D. melanogaster yw* ^67c23^ flies that naturally carry the *w*Mel infection. *w*Mel was purified from young adult flies on pulsed-field gels as described previously (Sun et al. 2001). Plugs were digested with the restriction enzyme AscI (GG\\^CGCGCC), which cuts the bacterial chromosome twice (Sun et al. 2001), aiding in the entry of the DNA into agarose gels. After electrophoresis, the resulting two bands were recovered from the gel and stored in 0.5 M EDTA (pH 8.0). DNA was extracted from the gel slices by first washing in TE (Tris\u2013HCl and EDTA) buffer six times for 30 min each to dilute EDTA followed by two 1-h washes in \u03b2-agarase buffer (New England Biolabs, Beverly, Massachusetts, United States). Buffer was then removed and the blocks melted at 70\u00b0C for 7 min. The molten agarose was cooled to 40\u00b0C and then incubated in \u03b2-agarase (1 U\/100 \u03bcl of molten agarose) for 1 h. The digest was cooled to 4\u00b0C for 1 h and then centrifuged at 4,100 \u00d7 *g* ~max~ for 30 min at 4\u00b0C to remove undigested agarose. The supernatant was concentrated on a Centricon YM-100 microconcentrator (Millipore, Bedford, Massachusetts, United States) after prerinsing with 70% ethanol followed by TE buffer and, after concentration, rinsed with TE. The retentate was incubated with proteinase K at 56\u00b0C for 2 h and then stored at 4\u00b0C. *w*Mel DNA for gap closure was prepared from approximately 1,000 *Drosophila* adults using the Holmes\u2013Bonner urea\/phenol:chloroform protocol (Holmes and Bonner 1973) to prepare total fly DNA.\n\n### Library construction\/sequencing\/closure\n\nThe complete genome sequence was determined using the whole-genome shotgun method (Venter et al. 1996). For the random shotgun-sequencing phase, libraries of average size 1.5\u20132.0 kb and 4.0\u20138.0 kb were used. After assembly using the TIGR Assembler (Sutton et al. 1995), there were 78 contigs greater than 5000 bp, 186 contigs greater than 3000 bp, and 373 contigs greater than 1500 bp. This number of contigs was unusually high for a 1.27 Mb genome. An initial screen using BLASTN searches against the nonredundant database in GenBank and the Berkeley *Drosophila* Genome Project site (<http:\/\/www.fruitfly.org\/blast\/>) showed that 3,912 of the 10,642 contigs were likely contaminants from the *Drosophila* genome. To aid in closure, the assemblies were rerun with all sequences of likely host origin excluded. Closure, which was made very difficult by the presence of a large amount of repetitive DNA (see below), was done using a mix of primer walking, generation, and sequencing of transposon-tagged libraries of large insert clones and multiplex PCR (Tettelin et al. 1999). The final sequence showed little evidence for polymorphism within the population of *Wolbachia* DNA. In addition, to obtain sequence across the AscI-cut sites, PCR was performed on undigested DNA. It is important to point out that the reason significant host contamination does not significantly affect symbiont genome assembly is that most of the *Drosophila* contigs were small due to the approximately 100-fold difference in genome sizes between host (approximately 180 Mb) and *w*Mel (1.2 Mb).\n\nSince it has been suggested that *Wolbachia* and their hosts may undergo lateral gene transfer events (Kondo et al. 2002), genome assemblies were rerun using all of the shotgun and closure reads without excluding any sequences that appeared to be of host origin. Only five assemblies were found to match both the D. melanogaster genome and the *w*Mel assembly. Primers were designed to match these assemblies and PCR attempted from total DNA of *w*Mel infected D. melanogaster. In each case, PCR was unsuccessful, and we therefore presume that these assemblies are the result of chimeric cloning artifacts. The complete sequence has been given GenBank accession ID AE017196 and is available at <http:\/\/www.tigr.org\/tdb>.\n\n### Repeats\n\nRepeats were identified using RepeatFinder (Volfovsky et al. 2001), which makes use of the REPuter algorithm (Kurtz and Schleiermacher 1999) to find maximal-length repeats. Some manual curation and BLASTN and BLASTX searches were used to divide repeat families into different classes.\n\n### Annotation\n\nIdentification of putative protein-encoding genes and annotation of the genome was done as described previously (Eisen et al. 2002). An initial set of ORFs likely to encode proteins (CDS) was identified with GLIMMER (Salzberg et al. 1998). Putative proteins encoded by the CDS were examined to identify frameshifts or premature stop codons compared to other species. The sequence traces for each were reexamined and, for some, new sequences were generated. Those for which the frameshift or premature stops were of high quality were annotated as \"authentic\" mutations. Functional assignment, identification of membrane-spanning domains, determination of paralogous gene families, and identification of regions of unusual nucleotide composition were performed as described previously (Tettelin et al. 2001). Phylogenomic analysis (Eisen 1998a; Eisen and Fraser 2003) was used to aid in functional predictions. Alignments and phylogenetic trees were generated as described (Salzberg et al. 2001).\n\n### Comparative genomics\n\nAll putative *w*Mel proteins were searched using BLASTP against the predicted proteomes of published complete organismal genomes and a set of complete plastid, mitochondrial, plasmid, and viral genomes. The results of these searches were used (i) to analyze the phylogenetic profile (Pellegrini et al. 1999; Eisen and Wu 2002), (ii) to identify putative lineage-specific duplications (those proteins with a top *E*-value score to another protein from *w*Mel), and (iii) to determine the presence of homologs in different species. Orthologs between the *w*Mel genome and that of the two *Rickettsia* species were identified by requiring mutual best-hit relationships among all possible pairwise BLASTP comparisons, with some manual correction. Those genes present in both *Rickettsia* genomes as well as other bacterial species, but not *w*Mel, were considered to have been lost in the *w*Mel branch (see <a href=\"#st003\" data-ref-type=\"supplementary-material\">Table S3<\/a>). Genes present in only one or two of the three species were considered candidates for gene loss or lateral transfer and were also used to identify possible biological differences between these species (see <a href=\"#st003\" data-ref-type=\"supplementary-material\">Table S3<\/a>). For the *w*Mel genes not in the *Rickettsia* genomes, proteins were searched with BLASTP against the TIGR NRAA database. Protein sequences of their homologs were aligned with CLUSTALW and manually curated. Neighbor-joining trees were constructed using the PHYLIP package.\n\n### Phylogenetic analysis of mitochondrial proteins\n\nFor phylogenetic analysis, the set of all 38 proteins encoded in both the Marchantia polymorpha and Reclinomonas americana (Lang et al. 1997) mitochondrial genomes were collected. Acanthamoeba castellanii was excluded due to high divergence and extremely long evolutionary branches. Six genes were excluded from further analysis because they were too poorly conserved for alignment and phylogenetic analysis (*nad7*, *rps10*, *sdh3*, *sdh4*, *tatC*, and *yejV*), leaving 32 genes for investigation: *atp6*, *atp9*, *atpA*, *cob*, *cox1*, *cox2*, *cox3*, *nad1*, *nad2*, *nad3*, *nad4*, *nad4L*, *nad5*, *nad6*, *nad9*, *rpl16*, *rpl2*, *rpl5*, *rpl6*, *rps1*, *rps11*, *rps12*, *rps13*, *rps14*, *rps19*, *rps2*, *rps3*, *rps4*, *rps7*, *rps8*, *yejR*, and *yejU*. Using FASTA with the mitochondrial proteins as a query, homologs were identified from the genomes of seven \u03b1-Proteobacteria: two intracellular symbionts (*W. pipientis w*Mel and Rickettsia prowazekii) and five free-living forms (Sinorhozobium loti, Agrobacterium tumefaciens, Brucella melitensis, Mesorhizobium loti, and *Rhodopseudomonas* sp.). Escherichia coli and Neisseria meningitidis were used as outgroups. Caulobacter crescentus was excluded from analysis because homologs of some of the 32 genes were not found in the current annotation. In the event that more than one homolog was identified per genome, the one with the greatest sequence identity to the mitochondrial query was retrieved. Proteins were aligned using CLUSTALW (Thompson et al. 1994) and concatenated. To reduce the influence of poorly aligned regions, all sites that contained a gap at any position were excluded from analysis, leaving 6,776 positions per genome for analysis. The data contained extreme amino acid bias: all sequences failed the \u03c7^2^ test at *p* = 0.95 for deviation from amino acid frequency distribution assumed under either the JTT or mtREV24 models as determined with PUZZLE (Strimmer and von Haeseler 1996). When the data were iteratively purged of highly variable sites using the method described (Hansmann and Martin 2000), amino acid composition gradually came into better agreement with acid frequency distribution assumed by the model. The longest dataset in which all sequences passed the \u03c7^2^ test at *p* = 0.95 consisted of the 3,100 least polymorphic sites. PROTML (Adachi and Hasegawa 1996) analyses of the 3,100-site data using the JTT model detected mitochondria as sisters of the five free-living \u03b1-Proteobacteria with low (72%) support, whereas PUZZLE, using the same data, detected mitochondria as sisters of the two intracellular symbionts, also with low (85%) support. This suggested the presence of conflicting signal in the less-biased subset of the data. Therefore, protein log determinants (LogDet) were used to infer distances from the 6,776-site data, since the method can correct for amino acid bias (Lockhart et al. 1994), and Neighbor-Net (Bryant and Moulton 2003) was used to display the resulting matrix, because it can detect and display conflicting signal. The result (see <a href=\"#pbio-0020069-g005\" data-ref-type=\"fig\">Figure 5<\/a>A) shows both signals. In no analysis was a sister relationship between *Rickettsia* and mitochondria detected.\n\nFor analyses of individual genes, the 63 proteins encoded in the *Reclinomonas* mitochondrial genome were compared with FASTA to the proteins from 49 sequenced eubacterial genomes, which included the \u03b1-Proteobacteria shown in <a href=\"#pbio-0020069-g005\" data-ref-type=\"fig\">Figure 5<\/a>, R. conorii, and *Magnetococcus* MC1, one of the more divergent \u03b1-Proteobacteria. Of those proteins, 50 had sufficiently well-conserved homologs to perform phylogenetic analyses. Homologs were aligned and subjected to phylogenetic analysis with PROTML (Adachi and Hasegawa 1996).\n\n### Analysis of *wspB* sequences\n\nTo compare *wspB* sequences from different *Wolbachia* strains, PCR was done on total DNA extracted from the following sources: *w*Ri was obtained from infected adult D. simulans, Riverside strain; *w*AlbB was obtained from the infected Aa23 cell line (O'Neill et al. 1997b), and *D. immitis Wolbachia* was extracted from adult worm tissue. DNA extraction and PCR were done as previously described (Zhou et al. 1998) with *wspB*-specific primers (*wspB*-F, 5\u2032-TTTGCAAGTGAAACAGAAGG and *wspB*-R, 5\u2032-GCTTTGCTGGCAAAATGG). PCR products were cloned into pGem-T vector (Promega, Madison, Wisconsin, United States) as previously described (Zhou et al. 1998) and sequenced (Genbank accession numbers AJ580921\u2013AJ508923). These sequences were compared to previously sequenced *wsp* genes for the same *Wolbachia* strains (Genbank accession numbers AF020070, AF020059, and AJ252062). The four partial *wsp* sequences were aligned using CLUSTALV (Higgins et al. 1992) based on the amino acid translation of each gene and similarly with the *wspB* sequences. Genetic distances were calculated using the Kimura 2 parameter method and are reported in <a href=\"#st005\" data-ref-type=\"supplementary-material\">Table S5<\/a>.\n\n### Type IV secretion system\n\nTo determine whether the *vir*-like CDSs, as well as adjacent ORFs, were actively expressed within *w*Mel as two polycistronic operons, RT-PCR was used. Total RNA was isolated from infected *D. melanogaster yw* ^67c23^ adults using Trizol reagent (Invitrogen, Carlsbad, California, United States) and cDNA synthesized using SuperScript III RT (Invitrogen) using primers *wspB*R, WD0817R, WD0853R, and WD0852R. RNA isolation and RT were done according to manufacturer's protocols, with the exception that suggested initial incubation of RNA template and primers at 65\u00b0C for 5 min and final heat denaturation of RT-enzyme at 70\u00b0C for 15 min were not done. PCR was done using r*Taq* (Takara, Kyoto, Japan), and several primer sets were used to amplify regions spanning adjacent CDSs for most of the two operons. For operon virB3-WD0853, the following primers were used: (*virB3*-*virB4*)F, (*virB3*-*virB4*)R, (*virB6*-WD0856)F, (*virB6*-WD0856)R, (WD0856-WD0855)F, (WD0856-WD0855)R, (WD0854-WD0853)F, (WD0854-WD0853)R. For operon *virB8*-*wspB*, the following primers were used: (*virB8*-*virB9*)F, (*virB8*-*virB9*)R, (*virB9*-*virB11*)F, (*virB9*-*virB11*)R, (*virB11*-*virD4*)F, (*virB11*-*virD4*)R, (*virD4*-*wspB*)F, and (*virD4*-*wspB*)R. The coexpression of *virB4* and *virB6*, as well as WD0855 and WD0854, was confirmed within the putative *virB3*-WD0853 operon using nested PCR with the following primers: (*virB4*-*virB6*)F1, (*virB4*-*virB6*)R1, (*virB4*-*virB6*)F2, (*virB4*-*virB6*)R2, (WD0855-WD0854)F1, (WD0855-WD0854)R1, (WD0855-WD0854)F2, and (WD0855-WD0854)R2. All ORFs within the putative *virB8*-*wspB* operon were shown to be coexpressed and are thus considered to be a genuine operon. All products were amplified only from RT-positive reactions (see <a href=\"#pbio-0020069-g006\" data-ref-type=\"fig\">Figure 6<\/a>). Primer sequences are given in <a href=\"#st009\" data-ref-type=\"supplementary-material\">Table S9<\/a>.\n\n# Supporting Information\n\n###### Phage Trees\n\nPhylogenetic tree showing the relationship between WO-A and WO-B phage from *w*Mel with reported phage from *w*Kue and *w*Tai. The tree was generated from a CLUSTALW multiple sequence alignment (Thompson et al. 1994) using the PROTDIST and NEIGHBOR programs of PHYLIP (Felsenstein 1989).\n\n(60 KB PDF).\n\nClick here for additional data file.\n\n###### Plot of the Effective Number of Codons against GC Content at the Third Codon Position (GC3)\n\nProteins with fewer than 100 residues are excluded from this analysis because their effective number of codon (ENc) values are unreliable. The curve shows the expected ENc values if codon usage bias is caused by GC variation alone. Colors: yellow, hypothetical; purple, mobile element; blue, others. Most of the variation in codon bias can be traced to variation in GC, indicating that the mutation forces dominate the *w*Mel codon usage. Multivariate analysis of codon usage was performed using the CODONW package (available from <http:\/\/www.molbiol.ox.ac.uk\/cu\/codonW.html>).\n\n(289 KB PDF).\n\nClick here for additional data file.\n\n###### Predicted Metabolism and Transport in *w*Mel\n\nOverview of the predicted metabolism (energy production and organic compounds) and transport in *w*Mel*.* Transporters are grouped by predicted substrate specificity: inorganic cations (green), inorganic anions (pink), carbohydrates (yellow), and amino acids\/peptides\/amines\/purines and pyrimidines (red). Transporters in the drug-efflux family (labeled as \"drugs\") and those of unknown specificity are colored black. Arrows indicate the direction of transport. Energy-coupling mechanisms are also shown: solutes transported by channel proteins (double-headed arrow); secondary transporters (two-arrowed lines, indicating both the solute and the coupling ion); ATP-driven transporters (ATP hydrolysis reaction); unknown energy-coupling mechanism (single arrow). Transporter predictions are based upon a phylogenetic classification of transporter proteins (Paulsen et al. 1998).\n\n(167 KB PDF).\n\nClick here for additional data file.\n\n###### Repeats of Greater Than 50 bp in the *w*Mel Genome (with Coordinates)\n\n(649 KB DOC).\n\nClick here for additional data file.\n\n###### Inactivated Genes in the *w*Mel Genome\n\n(147 KB DOC).\n\nClick here for additional data file.\n\n###### Ortholog Comparison with *Rickettsia* spp\n\n(718 KB XLS).\n\nClick here for additional data file.\n\n###### Putative Lineage-Specific Gene Duplications in *w*Mel\n\n(116 KB DOC).\n\nClick here for additional data file.\n\n###### Genetic Distances as Calculated for Alignments of *wsp* and *wspB* Gene Sequences from the Same *Wolbachia* Strains\n\n(24 KB DOC).\n\nClick here for additional data file.\n\n###### Putative DNA Repair and Recombination Genes in the *w*Mel Genome\n\n(26 KB DOC).\n\nClick here for additional data file.\n\n###### Phylogenetic Results for Concatenated Data of 32 Mitochondrial Proteins\n\n(34 KB DOC).\n\nClick here for additional data file.\n\n###### Individual Phylogenetic Results for *Reclinomonas* Mitochondrial DNA-Encoded Proteins\n\n(117 KB DOC).\n\nClick here for additional data file.\n\n###### PCR Primers\n\n(47 KB DOC).\n\nClick here for additional data file.\n\n## Accession Numbers\n\nThe complete sequence for *w*Mel has been given GenBank (<http:\/\/www.ncbi.nlm.nih.gov\/Genbank\/>) accession ID number AE017196 and is available through the TIGR Comprehensive Microbial Resourceat <http:\/\/www.tigr.org\/tigr-scripts\/CMR2\/GenomePage3.spl?database=dmg>\n\nThe GenBank accession numbers for other sequences discussed in this paper are AF020059 (*Wolbachia* sp. *w*AlbB outer surface protein precursor *wsp* gene), AF020070 (*Wolbachia* sp. *w*Ri outer surface protein precursor *wsp* gene), AJ252062 (*Wolbachia* endosymbiont of D. immitis sp. gene for surface protein), AJ580921 (*Wolbachia* endosymbiont of D. immitis partial *wspB* gene for *Wolbachia* surface protein B), AJ580922 (*Wolbachia* endosymbiont of A. albopictus partial *wspB* gene for *Wolbachia* surface protein B), and AJ580923 (*Wolbachia* endosymbiont of D. simulans partial *wspB* gene for *Wolbachia* surface protein B).\n\nWe acknowledge Barton Slatko, Jeremy Foster, New England Biolabs, and Mark Blaxter for helping inspire this project; Rehka Seshadri for help in examining pathogenicity factors and reading the manuscript; Derek Fouts for examination of group II introns; Susan Lo, Michael Heaney, Vadim Sapiro, and Billy Lee for IT support; Maria-Ines Benito, Naomi Ward, Michael Eisen, Howard Ochman, and Vincent Daubin for helpful discussions; Steven Salzberg and Mihai Pop for help in comparing *w*Mel with the D. melanogaster genome; Elodie Ghedin for access to the *B. malayi Wolbachia* sequence data; Maria Ermolaeva for assistance with analysis of operons; Dan Haft for designing protein family hidden Markov models for annotation; Owen White for general bioinformatics support; four anonymous reviewers for very helpful comments and suggestions; and Claire M. Fraser for continuing support of TIGR's scientific research. This project was supported by grant UO1-AI47409\u201301 to Scott O'Neill and Jonathan A. Eisen from the National Institutes of Allergy and Infectious Diseases.\n\n# References\n\n## Abbreviations\n\nCDS\n\n:   coding sequence\n\nENc\n\n:   effective number of codons\n\nIS\n\n:   insertion sequence\n\nLPS\n\n:   lipopolysaccharide\n\nRT\n\n:   reverse transcription\n\nTIGR\n\n:   The Institute for Genomic Research","meta":{"dup_signals":{"dup_doc_count":104,"dup_dump_count":44,"dup_details":{"curated_sources":2,"2022-49":1,"2022-27":1,"2021-49":1,"2021-43":1,"2020-24":1,"2019-51":1,"2019-26":1,"2019-09":2,"2018-47":1,"2018-39":1,"2018-34":1,"2018-30":1,"2018-22":1,"2018-13":1,"2018-05":1,"2017-47":1,"2017-39":1,"2017-30":1,"2017-22":1,"2017-09":3,"2016-44":1,"2016-30":2,"2016-26":1,"2016-22":1,"2015-40":3,"2015-35":3,"2015-32":3,"2015-27":4,"2015-14":2,"2014-52":4,"2014-49":3,"2014-42":10,"2014-41":5,"2014-35":6,"2014-23":6,"2014-15":8,"2023-06":1,"2024-18":2,"2024-10":2,"2015-18":4,"2015-11":3,"2015-06":1,"2014-10":3,"2024-26":1}},"file":"PMC368164"},"subset":"pubmed_central"}
{"text":"author: Naomi Attar\ndate: 2012\ninstitute: 1Genome Biology, BioMed Central, 236 Gray's Inn Road, London WC1X 8HB, UK\ntitle: The Research Works Act: a comment\n\nIt's been a tale of two 'opens'; recent events in US political life suggest a reconciliation with the concept of open marriage but a relationship with open access to scientific research that may be headed for splitsville.\n\nIn a rare feat of bipartisanship during a Congress famed for partisan pettiness, the Research Works Act (RWA) has received cross-party sponsorship in the House and is now being heard in Committee. The Act, in its own words, seeks to prevent Federal agencies from requiring that authors 'assent to network dissemination of a private-sector research work'. Translation? It will be illegal for Federally funded research grants to have open access strings attached. This includes a bar on policies insisting on deposition in public repositories; in fact, it will be illegal to 'cause', 'permit' or 'authorize' such a deposition.\n\nRWA has not attracted the same volume of headlines as the equally controversial SOPA (Stop Online Piracy Act), which was another recent bill pertaining to internet freedoms. But although the opponents of RWA cannot rival the political and global reach of the likes of Google and Wikipedia, a spirited, academia-led campaign against RWA is underway on the blogosphere and Twitter. As with many grassroots protests in the social media era, the RWA rebutters have mixed a sense of genuine outrage with mischievous and irreverent humor.\n\nOne notable line of campaigning has been to instigate a boycott of publishers deemed to be supportive of RWA. The boycott includes refusing to peer review manuscripts, in addition to submitting manuscripts only to publishers opposed or indifferent to RWA. Particular ire has been reserved for publishing houses thought to be proactive in their RWA support, either through lobbying or in terms of financial backing provided to Washington politicos. As one company singled out for approbation is a competitor of this journal's publisher, there is an undoubted temptation to indulge in a little schadenfreude and jump on the boycott bandwagon. However, while researchers are of course at liberty to submit to and peer review for whichever journals they choose (and on whatever grounds they choose), it seems that the focus on publishers is misplaced. Companies should be expected to make representations, by legal means, on behalf of what they perceive to be their best interests (admittedly, a boycott may influence these perceptions); instead, the war for open access must be waged against the Congressmen, accountable to the People, who are driving RWA forward.\n\nLooking into the US from the other side of the Atlantic, Federal funding for scientific research can only be viewed through a green mist of envy. The contribution of these funds to global science is remarkable (just take a look at a list of Nobel Prize winners, and note how many institutional affiliations are in the US), and is a record of which US tax payers should be justly proud. This contribution is not paralleled by any other government. Furthermore, efforts by funding agencies to make the benefits of research as widely available as possible - including to those outside the US - are laudable. It seems to be a truism that maximizing access to the results of scientific endeavor is in the best interests of further scientific progress, and so offers the best value to the taxpayer. So why would the representatives of the very same taxpayer seek to restrict access to this research, and by the same measure subsidize the publishing industry with money diverted away from scientists? It defies belief to imagine that these Congressmen are arguing for their constituents to pay exorbitant prices simply to read articles that they themselves have paid for with their tax dollars.\n\nSo what is the defense? Supporters of RWA pitch it as a battle for the free market; in the words of the Association of American Publishers, its motivation is the 'freedom from regulatory interference for \\[the\\] private sector'. Of course, this is quite the opposite of what RWA actually represents, which is *additional* government regulation contrary to the spirit of the free market. In fact, market forces scare traditional publishing models, because left to their own devices they will arrive at the most efficient use of capital, which is undoubtedly, for the funding agencies, open access publishing. Given that Federal funding ultimately pays for both access to publications and publishing costs, the best value option is an open access model. This is because the cost of publication should not vary according to access level, only the size of the audience able to access the material. To prevent Federal agencies from pursuing what is therefore a no-brainer option, RWA is designed to skew the market; it leaves the decision of which model to publish manuscripts under to individual authors, thereby creating a disconnect between the source of capital and the choice of how it is spent. No freely operating market would tolerate those paying for the product (the taxpayers) being barred from access to its benefits.\n\nOne component of the defense put forward for RWA is particularly provocative, and warrants more detailed attention. Here's the Association of American Publishers again: RWA will protect 'millions of dollars invested by publishers in... operational funding of independent peer review by specialized experts.' Publishers would do well to remember that academics offer a peer review service free of charge, and so to focus on peer review as a publisher-added value, even though there are undoubtedly publisher-incurred costs to this process, is unnecessarily antagonistic.\n\nCongress must judge RWA in the true spirit of the free market, the taxpayer and the great tradition of American science. Open access publishing is an economic inevitability. It's time to get on board.\n\n# Competing interests\n\nAs *Genome Biology* publishes research under an exclusively open access model, the journal is unashamed to oppose any legislation aiming to restrict the fruits of scientific research to a limited audience.\n\n# Your views\n\n*Genome Biology* would be interested to hear any dissenting views on this Editorial, or indeed any positive feedback. Tell us your own perspective either by posting a comment on this article or by including our @GenomeBiology handle in a tweet.","meta":{"dup_signals":{"dup_doc_count":101,"dup_dump_count":44,"dup_details":{"curated_sources":2,"2021-43":1,"2020-50":1,"2020-40":1,"2020-24":1,"2019-30":1,"2019-04":1,"2018-43":1,"2018-34":1,"2018-26":1,"2018-05":1,"2017-39":1,"2017-26":1,"2017-17":1,"2017-09":9,"2016-44":1,"2016-40":1,"2016-36":10,"2016-30":7,"2016-22":1,"2016-18":1,"2016-07":6,"2015-48":2,"2015-40":1,"2015-35":2,"2015-32":2,"2015-27":2,"2015-22":2,"2015-14":2,"2014-52":1,"2014-49":3,"2014-42":6,"2014-41":3,"2014-35":3,"2014-23":2,"2014-15":3,"2022-33":1,"2017-13":1,"2015-18":2,"2015-11":2,"2015-06":2,"2014-10":2,"2013-48":2,"2013-20":3,"2024-18":1}},"file":"PMC3334566"},"subset":"pubmed_central"}
{"text":"abstract: DNA microarrays (representing approximately 30,000 human genes) were used to analyze gene expression in six different human eye compartments, revealing candidate genes for diseases affecting the cornea, lens and retina.\nauthor: Jennifer J Diehn; Maximilian Diehn; Michael F Marmor; Patrick O Brown\ndate: 2005\ninstitute: 1Department of Ophthalmology, Stanford University School of Medicine, Stanford, CA 94305, USA; 2Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305, USA; 3Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305, USA; 4Department of Ophthalmology, University of California, San Francisco, San Francisco, CA 94143, USA\nreferences:\ntitle: Differential gene expression in anatomical compartments of the human eye\n\n# Background\n\nThe human eye is composed of multiple substructures of diverse form, function, and even embryologic origin that work in concert to provide us with our sense of sight. Identifying the global patterns of gene expression that specify the distinctive characteristics of each of the various compartments of the eye is an important step towards understanding how these complex normal tissues function, and how dysfunction leads to disease. The Human Genome sequence \\[1,2\\] provides a basis for examining gene expression on a genomic scale, and cDNA microarrays provide an efficient method for analyzing the expression of thousands of genes in parallel. Previous studies have used microarrays to investigate gene expression within normal eye tissues, including cornea \\[3\\] and retina \\[4\\], as well as within pathological tissues such as glaucomatous optic nerve heads \\[5\\], uveal melanomas \\[6\\], and aging retina \\[7\\].\n\nAnalysis of gene expression in the eye has been notoriously difficult because of the technical obstacles associated with extracting sufficient quantities of high quality RNA from the tissues. This is especially true for the lens and cornea, which have relatively few RNA-producing cells when compared to a highly cellular tissue such as retina. Furthermore, pigmented ocular tissues contain melanin, which often co-purifies with RNA and inhibits subsequent enzymatic reactions \\[8\\]. Any delay between the patient's death and the harvesting of ocular tissues can also compromise RNA quality and yield. To date, many experiments examining the gene expression profile of particular eye compartments have relied on pooled samples or cell culture in order to obtain adequate amounts of RNA. In contrast to these studies, the experiments described in this paper were performed using a linear amplification procedure \\[9\\], which made it possible to examine individual specimens using DNA microarrays, thereby eliminating the potentially confounding effects of pooling multiple donor samples or culturing cells, which can elicit dramatic changes in gene expression based on the cell culture media \\[10\\]. We chose an *in vitro* transcription-based, linear amplification approach because this has previously been shown to reproducibly generate microarray gene expression results that are extremely similar to data generated using unamplified RNA \\[9,11,12\\]. Additionally, the amplification process has been shown to selectively and reproducibly 'over-amplify' some low-copy number transcripts, resulting in a larger fraction of the expressed genome that can be reliably measured on DNA microarrays. Importantly, by analyzing individual donor samples on arrays, we can detect variation in the eye compartments of different donors, which will be critical for future studies that examine how gene expression varies between individuals at baseline and also in disease states.\n\nA major goal of this study was to discover how the various eye compartments differ from one another on a molecular level by identifying clusters of differentially expressed genes, or 'gene signatures', characteristic of each eye compartment. We also wanted to investigate how gene expression varies between geographical regions of the retina. Because certain retinal diseases such as retinitis pigmentosa (RP) and age-related macular degeneration (ARMD) preferentially affect a specific retinal region, identification of genes that are differentially expressed in the macula versus peripheral retina may provide valuable clues to the molecular mechanisms underlying these diseases. Recent work using serial analysis of gene expression (SAGE), a method that involves sequencing thousands of transcripts from a given RNA sample, identified several genes that were significantly enriched in either the macula or the periphery \\[13\\]. Our cDNA microarray studies confirmed some of these genes, but also significantly added to the catalog of macula-enriched genes. Lastly, because many ophthalmologic diseases preferentially affect a particular eye compartment, our study demonstrates that gene signatures can be combined with gene linkage studies in order to identify candidate disease genes.\n\n# Results\n\nTo explore relationships among the different eye compartments and among genes expressed in these compartments, we performed hierarchical cluster analysis of both genes and samples \\[14\\] using genes that met our selection criteria (see Materials and methods). The display generated through hierarchical clustering analysis is shown in Figure <a href=\"#F1\" data-ref-type=\"fig\">1a<\/a>. In this display, relatively high expression levels are indicated by a red color, and relatively low expression levels are represented by a green color; each column represents data from a single tissue sample, and each row represents the series of measurements for a single gene. Tissue samples with similar gene expression patterns are clustered adjacent to one another, and genes with similar expression patterns are clustered together. In our experiments, samples of the same eye compartment from different donors clustered in discrete groups (for example, cornea with cornea, retina with retina), with the only exception being an intermingling of the ciliary body and iris specimens (Figure <a href=\"#F1\" data-ref-type=\"fig\">1a<\/a>). The lack of a clear distinction between the expression patterns of the ciliary body and iris may be due to both their shared embryological origin and their close anatomical approximation, resulting in sub-optimal separation during dissection. The division between the retinal samples and all other samples was the most striking. Furthermore, there was a distinct grouping of the various macula specimens, which formed a tightly clustered subgroup among the retinal samples. The expression patterns of the optic nerve samples were most similar to those of the three brain specimens.\n\nEach anatomical compartment of the eye expressed a distinct set of genes that were not expressed, or expressed at much lower levels, in the other eye compartments (Figure <a href=\"#F1\" data-ref-type=\"fig\">1b<\/a>). The repertoire of genes specifically expressed in the retina was especially large and diverse (3,727 genes), but we also found a surprisingly large number of transcripts (1,777 genes) expressed predominantly in the lens. To explore the connections between these compartment-enriched genes and phenotypic features of the compartments in which they were expressed, we considered each group of compartment-enriched genes in detail.\n\n## Corneal signature\n\nThe cornea is a multi-layered structure consisting of an epithelium of stratified squamous cells, a thick stroma of layered collagen fibrils, and an underlying endothelial layer. To provide an effective physical barrier to the outside world, the corneal epithelial cells bind to one another and to the underlying connective tissue through a series of linked structures known collectively as the 'adhesion complex'. As shown in Figure <a href=\"#F2\" data-ref-type=\"fig\">2a<\/a>, many genes enriched in the corneal signature encoded proteins that stabilize epithelial sheets and promote cell-cell adhesion, including keratins (KRT5, KRT6B, KRT13, KRT15, KRT16, KRT17, KRT19), laminins (LAMB3, LAMC2), and desmosomal components (DSG1, DSC3, BPAG1).\n\nOther genes highly expressed in the cornea signature encoded proteins that help maintain the shape, transparency, or integrity of the cornea, which serves as the primary refractive element in the eye. Some of the genes encoded proteins specifically expressed by either squamous epithelial cells or fibroblasts, reflecting the histological composition of corneal tissue. For example, the signature included numerous genes that encode collagens (COL5A2, COL6A3, COL12A1, COL17A1), along with the gene for lysyl oxidase (LOX), an enzyme that promotes collagen cross-linking. The gene encoding keratocan (KERA), a proteoglycan involved in maintaining corneal shape in mice knock-out studies \\[15\\], and linked to abnormal corneal morphology (keratoconus and cornea plana) in humans, was selectively expressed in corneal tissue, as were the genes encoding lumican (LUM), a keratan sulfate-containing proteoglycan that has been shown to be important for mouse corneal transparency \\[16\\], and aquaporin 3 (AQP3), which encodes a water\/small solute-transporting molecule. Immunolabeling studies performed on corneas with pseudophakic bullous keratopathy demonstrated increased AQP3 in the superficial epithelial cells, suggesting that AQP3 may be associated with increased fluid accumulation, resulting in the decrease in corneal transparency seen in pseudophakic bullous keratopathy corneas \\[17\\]. Modulating genes or proteins involved in corneal shape and transparency could potentially lead to non-invasive treatments for some corneal diseases, which are often only remediable through corneal transplantation.\n\nAn intriguing subset of genes in the cornea signature has been studied in tumor metastasis models because these genes encode proteins that regulate cell-cell or cell-matrix interactions (TWIST, MMP10, SERPINB5, THBS1, CEACAM1, C4.4A). For example, TWIST encodes a transcription factor shown to promote metastasis in a murine breast tumor model through the loss of cadherin-mediated cell-cell adhesion \\[18\\]. Another corneal signature gene encodes matrix metalloproteinase 10 (MMP10), a protein capable of degrading extracellular matrix components. Overexpression of MMP10 in transfected lymphoma cells has been shown to stimulate invasive activity *in vitro* and promote thymic lymphoma growth in an *in vivo* murine model \\[19\\]. Various matrix metalloproteinases have been examined for their roles in corneal wound healing (reviewed in \\[20\\]), including MMP10, which was identified in migrating epithelial cells in cultured human cornea tissues that were experimentally wounded \\[21\\], which may suggest that the process of corneal wound healing may mimic some aspects of tumor biology. Certainly, in both wound healing and cancer, cells undergo rapid proliferation, invade and remodel the extracellular matrix, and migrate to other areas.\n\nRecent microarray investigations identified a gene expression signature related to a wound response in the expression profiles of several common carcinomas, and the presence of this wound healing gene signature predicted an increased risk of metastasis and death in breast, lung, and gastric carcinomas \\[22,23\\]. Further research into corneal wound healing may also provide us with a model for better understanding the pathophysiology underlying tumor metastasis because the cornea is exceptionally efficient among human tissues at degrading and remodeling its extracellular matrix, allowing it to heal superficial wounds within hours.\n\n## Ciliary body\/iris signature\n\nThe ciliary body and iris are components of the eye's highly pigmented and vascular layer known as the uveal tract. As might be expected, genes related to pigmentation were a feature of the distinctive expression pattern of these tissues (Figure <a href=\"#F2\" data-ref-type=\"fig\">2b<\/a>). These genes encoded enzymes involved in melanogenesis, including tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1), and dopachrome tautomerase (DCT), as well as melanosomal matrix proteins such as SILV and MLANA. Several of the ciliary body\/iris signature genes were noteworthy in that their mutation can lead to albinism or hypopigmentation phenotypes, including OA1 (ocular albinism type 1), TYR and TYRP1 (oculocutaneous albinism 1A and 3, respectively), and MLPH (Griscelli syndrome). Investigation of the numerous uncharacterized genes with similar expression patterns to those of pigmentation genes may expand our knowledge about the pigmentation process in eyes and the molecular mechanisms behind hypopigmentation syndromes.\n\nThe ciliary body is also responsible for aqueous humor formation and lens accommodation, while the contiguous iris filters light entering the eye by constricting and dilating the muscles around the pupillary opening. Histologically, the ciliary body consists predominately of smooth muscle, but also contains striated muscle (reviewed in \\[24\\]). Previous work has demonstrated that contractility of both the ciliary body and the trabecular meshwork is critical in modulating aqueous humor outflow (reviewed in \\[25\\]), one of the key determinants of intraocular pressure, along with aqueous humor production and episcleral venous pressure. Muscle-related proteins encoded by genes in the ciliary body\/iris cluster included smooth muscle actin (ACTG2), and actin cross-linking proteins such as filamin (FLNC), tropomyosin (TPM2), and tensin (TNS). Other iris\/ciliary body signature genes have known roles in myosin phosphorylation (PPP1R12B), sarcolemmal calcium homeostasis (CASQ2), and ATP availability (CKMT2), all of which may contribute to ciliary body\/trabecular meshwork contractility.\n\nBoth ciliary body and trabecular meshwork contractility, as well as aqueous humor production, have been linked to changes in membrane potential, and membrane channels have been studied extensively in the ciliary body \\[25-27\\]. Of note, transcripts encoding an inward-rectifying potassium channel (KCNJ8), not previously identified in the ciliary body, were highly enriched in the ciliary body\/iris signature and may warrant further study. The signature also included the gene for adrenergic receptor 2\u03b1 (ADRA2A), a regulator of aqueous humor production and outflow, and the molecular target of the ocular hypotensive agent brimonidine. Identification of other genes that facilitate aqueous production and outflow may provide additional molecular targets for future glaucoma therapeutics aimed at lowering intraocular pressure, the only modifiable risk factor for the development and progression of glaucoma.\n\n## Immune system genes expressed within anterior segment tissues\n\nGenes related to immune defense mechanisms were prominent among the large set of genes selectively expressed in both the ciliary body\/iris and corneal tissues. These included genes encoding proteins involved in intracellular antigen processing and transport for eventual surface presentation to immune cells (PSMB8, TAP1), antigen presentation proteins, including HLA class I molecules (HLA-A, HLA-C, HLA-F, and HLA-G) and HLA class II molecules (HLA-DRB1, DRB4, DRB5, DPA1, and DPB1), cytokines involved in the recruitment of monocytes (SCYA3, SCYA4, CD14), and cytokine receptors (IL1R2, IL4R, and IL6R). Several anterior segment-enriched genes encoded proteins with intrinsic antibiotic activity, including defensin (DEFB1) and lysozyme (LYZ), which may protect epithelial surfaces from microbial colonization.\n\nGenes encoding components of the complement cascade, a major arm of the innate immune system, were a particularly prominent feature of the anterior segment signature. Most of the early classical pathway complement genes, including C1 components (C1S, C1QA, C1QG, C1R), C2, and C4b, as well as a component of the late complement cascade (C7), were selectively expressed in both the corneal and ciliary body\/iris tissues. In addition, the gene encoding the trigger for the alternative complement pathway, properdin (BF), was highly expressed in these tissues.\n\nTo prevent the destructive reactions that could ensue from the daily bombardment of the eye with potentially antigenic stimuli, regulatory mechanisms must counteract the multitude of pro-inflammatory mediators found in the eye. A study by Sohn *et al*. \\[28\\] that examined a number of complement and complement-regulating components in rat eyes suggested that the complement system is continuously active at a low level in the normal eye and is kept in check by regulatory proteins. Indeed, we found that the anterior segment selectively expressed many critical negative regulators of the immune system, especially of the complement cascade. These included SERPING1 and DAF, two genes that encode proteins that limit the production of early complement components, and CD59, which encodes a protein that inhibits the assembly of complement subunits into the membrane attack complex.\n\nThe presence of complement activation products in the human eye during infection or inflammation has been previously described \\[29\\]. Studies have suggested that the complement pathway contributes to the pathophysiology of uveitis, an inflammatory disease of the uveal tract that is often idiopathic in etiology \\[30\\]. In support of this theory, Bardenstein *et al*. \\[31\\] showed that blocking the complement regulator CD59 in the rat eye precipitated massive inflammation in the anterior eye, including intense conjunctival inflammation and iritis. Our evidence that complement pathway components and regulators are highly expressed in anterior segment tissues provides further impetus for investigating their links to ocular disease.\n\nA caution to bear in mind in interpreting these results is that all of our ocular specimens were obtained post-mortem. The expression of the inflammatory genes could therefore reflect, at least in part, changes in the eye that occur after death. Future studies examining gene expression in fresh tissue samples obtained at surgery, such as peripheral iridectomy specimens, should help to further address this issue.\n\n## Lens signature\n\nThe distinctive features of the lens are its transparency, precisely crafted shape, and deformability, all of which are critical for proper light refraction. Elucidating the molecular mechanisms that maintain or disrupt lens transparency is fundamental in preventing cataract, the leading cause of world blindness. Our studies showed that lens gene expression is very distinct from the other eye compartments (Figure <a href=\"#F2\" data-ref-type=\"fig\">2c<\/a>), perhaps reflecting the extraordinary specialization of the lens as an isolated, avascular structure within the eye. We found more than a thousand genes selectively expressed in the lens; clearly, diverse RNA populations are still present in the adult lens, even though its population of active epithelial cells is outnumbered by the mature fiber cells that have lost their organelles, including nuclei.\n\nGenes encoding the subunits of crystallins, the predominant structural proteins in the lens, were prominent in the lens signature, including subunits for crystallin alpha (CRYAA), beta (CRYBA1, CRYBA4), and gamma (CRYGA, CRYGC). Work by Horwitz and colleagues \\[32,33\\] on alpha-crystallins, which are structurally similar to small heat shock proteins, showed these crystallins may preserve lens transparency by serving as molecular chaperones that protect other lens proteins from irreversible denaturation and aggregation. Of the other heat shock proteins highly enriched in the lens signature (HSPA6, HSPA8, HSPB1), HSPB1 may be of particular interest because it is a protein with an alpha-crystallin domain that may have a role in lens differentiation \\[34\\]. The lens signature also included genes encoding subunits of the proteasome complex (PSMA6, PSMA7, PSMB6, PSMB7, PSMB9, PSMD13), a multicatalytic proteinase structure that is responsible for degrading intracellular proteins. Previous studies have demonstrated the significance of the proteasome pathway in removing oxidatively damaged proteins within the lens \\[35\\].\n\nBesides the crystallin genes, other genes encoding previously described structural components of the lens, including lens intrinsic membrane (LIM2), beaded filament structural protein (BFSP2), spectrin (SPTBN2), and actin binding protein (ABLIM) were included in the lens signature. More interestingly, the signature also contained intermediate filament genes, such as those encoding erythrocyte membrane band 4.9 and 4.1 (EPB49 and EPB41L1, EPB41L4), that are characteristically expressed in erythrocytes, another cell whose highly stereotyped shape is critical to its function. Previous studies have shown that protein 4.1 helps stabilize the spectrin-actin cytoskeleton, which is present in both erythrocytes and lenticular tissue \\[36\\]. Further investigations comparing erythroid and lens cells may reveal other similarities in their cytoskeletons, both of which define a distinctive and stereotyped cell shape that must endure substantial amounts of mechanical stress.\n\nAnother notable feature of the lens signature was the enrichment of genes encoding proteins involved in endocytosis, including clathrin (CLTCL1, PICALM) and caveolin (CAV1). Currently, intercellular transport within the lens is thought to occur predominately by diffusion through gap junctions, but several investigators have proposed the uptake of nutrients must be supplemented by mechanisms other than gap junctions because of the paucity of gap junctions identified in microscopy studies and the confirmed presence of clathrin-coated vesicles in freeze-fracture studies \\[37,38\\].\n\nOxidative stress mediated by free radical production has been associated with cataract formation (reviewed in \\[39\\]). Therefore, we looked for genes involved in scavenging free radicals in the lens signature. Two of these genes encode enzymes, glutathione synthetase (GSS) and glutathione reductase (GSR), that facilitate the production of glutathione, a potent anti-oxidant and essential cofactor for redox enzymes. Superoxide dismutase (SOD1) and anti-oxidant protein 2 (AOP2), two proteins responsible for reducing free oxygen radicals and hydrogen peroxide species, respectively, were also selectively expressed in lens tissue. Drugs or environmental agents that modulate the expression or activity of these proteins could have a significant impact on cataract progression or prevention.\n\n## Optic nerve signature\n\nThe gene expression pattern in the optic nerve was overall quite similar to that seen in brain tissue (Figure <a href=\"#F2\" data-ref-type=\"fig\">2d<\/a>), very likely reflecting the preponderance of glial cells present in both tissues. Both signatures included a number of genes (MBP, MOBP, MAG, OLIG1, and OLIG2) previously found in glial cells, several of which have been linked to neurological diseases. For example, myelin-associated oligodendrocyte basic protein (MOBP) is implicated as an antigen stimulus for multiple sclerosis, a disease that also can present with optic neuritis (reviewed in \\[40\\]). Interestingly, the optic nerves in MOBP knock-out mice lacked the radial component of myelination \\[41\\]. In another study, transgenic mice with T-cell receptors specific to myelin associated glycoprotein (MAG) spontaneously presented with optic neuritis \\[42\\]. The majority of the genes in the brain and optic nerve signatures encoded proteins of unknown function; our results, showing that these genes may have specialized roles in these tissues, may be a step toward discovering the biological role(s) for these uncharacterized proteins.\n\n## Retina signature\n\nThe retina, a complex tissue composed of neuronal and glial elements, is essentially an extension of the central nervous system, and the genes found in the retina signature appear to reflect its distinctive histology and embryology (Figure <a href=\"#F3\" data-ref-type=\"fig\">3a<\/a>). For example, the signature included the receptors for known retinal neurotransmitters, including gamma-aminobutyric acid (GABRA1, GABRG2, GABRB3), glutamate (GRIA1, GRIN2D), glycine (GLRB), and dopamine (DRD2). Retinal neurotransmitters are packaged into small vesicles in the pre-synaptic regions of photoreceptors. Many retinal signature genes encoded proteins associated with synaptic vesicle docking and fusion (SNAP25, VAMP2, SYP, SNPH), as well as vesicle exocytosis and neurotransmitter release (SYN2, SYT4). One of the retinal signature genes with a role in synaptic transmission, human retinal gene 4 (HRG4\/UNC119), has been linked to late-onset cone-rod dystrophy in humans and marked synaptic degeneration in a transgenic mouse model \\[43\\].\n\nThe retina protects the integrity of its neuronal layers by regulating its extracellular environment through a blood-retina barrier consisting of vessel tight junctions and cell basement membranes. The exchange of nutrients and metabolites across these barriers likely requires diverse, specialized transporters. Indeed, over 30 different genes encoding small molecule transporters were found within the retina signature, including carriers of glucose (SLC2A1, SLC2A3), glutamate (SLC1A7), and other amino acids (SLC7A5, SLC38A1, SLC6A6). Of note, severe retinal degeneration was observed in mice mutated in *SLC6A6*, a gene encoding a transporter of the amino acid taurine \\[44\\]. Several genes encoding ABC transporters (ABCA3, ABCA4, ABCA5, ABCA7), which use ATP energy to transport various molecules across cell membranes, were contained in the retinal signature. The most notable of these, ABCA4, is involved in vitamin A transport in photoreceptor cells; mutations in the gene encoding ABCA4 can result in a spectrum of retinopathies, including retinitis pigmentosa, Stargardt's disease, cone-rod dystrophy, and ARMD.\n\nThe retinal signature was also enriched in transcripts encoding vitamin and mineral transporters. The inclusion of a vitamin C transporter (SLC23A1) and a zinc transporter (SLC39A3) within the signature was of particular interest, in light of the Age-Related Eye Disease Research Group study that demonstrated supplementation with zinc and anti-oxidants, including vitamin C, lowered the probability of developing neovascular ARMD in some high-risk patient subgroups \\[45\\]. The presence of transferrin (TF), an iron transport molecule, and its receptor (TFRC), in the retina signature may also be noteworthy because a higher accumulation of iron has been observed in some ARMD-affected maculas \\[46\\].\n\nSomewhat unexpectedly, the retina signature contained the gene encoding thyroid releasing hormone (TRH) and numerous thyroid hormone receptor-related genes (THRA, TRIP8, TRIP15, TRAP100). TRH expression was previously observed in the retinal amacrine cells of amphibians \\[47\\]. Previous work has demonstrated the importance of thyroid hormone in the developing rat retina \\[48\\], and thyroid hormone receptors are required for green cone photoreceptor development in rodents \\[49\\]. Further studies of these genes may uncover additional roles of thyroid hormone and its receptors in the human retina.\n\nThe retina is ultimately responsible for executing the visual cycle, the process by which a photon signal is translated into an electrical impulse. This complex cycle is initiated when photoreceptor pigments activate G-proteins. G-proteins in turn activate phosphodiesterases to break down cyclic GMP (cGMP) to GMP, thereby influencing cell polarization via the downstream modulation of ion channel efflux. The retina signature incorporated many genes encoding known visual cycle elements, including the photopigment rhodopsin (RHO), G-proteins from rods and cones (GNAT1, GNAT2, GNB5), subunits of rod and cone phosphodiesterases (PDE6A, PDE6B, PDE6G, PDE6H), and cGMP-sensitive channels (CNGB1, CNGA1). Genes responsible for visual cycle recovery, such as arrestins (SAG, ARR3), were also present. Intriguingly, transcripts encoding other G-proteins (GNB1, GNAZ) and several phosphodiesterases (PDE8B, PDE7A, PDE4A) with no established roles in the visual cycle were enriched in the retinal signature. Additionally, the signature contained CDS1, which, though it has no clear function in humans, is homologous to the phototransduction gene CDS that has been linked to light-induced retinal degeneration in *Drosophila* mutants \\[50\\]. Perhaps further in-depth study of the many uncharacterized genes in the retinal signature will reveal roles in phototransduction for these genes, which may expand our current concept of the visual cycle pathway.\n\n## Macula signature\n\nWe used the statistical analysis of microarrays (SAM) algorithm to select genes whose expression differed significantly between the central and peripheral retinal tissues (Figure <a href=\"#F3\" data-ref-type=\"fig\">3b<\/a>). The large set of genes that we identified as selectively expressed in macula tissues included a subset of genes involved in lipid biosynthesis. The majority of these genes are regulated by sterol response element-binding protein (SREBP), a transcription factor that has emerged as a master regulator of cholesterol and fatty acid metabolic pathways \\[51\\]. Previous studies by Fliesler *et al*. \\[52\\] have provided evidence for rapid *de novo* synthesis of cholesterol in the rat retina *in vivo*, and our findings strongly suggested the human retina also contains the enzymes needed for cholesterol biogenesis. Transcripts encoding the enzymes that catalyze multiple steps in cholesterol synthesis were enriched in the macula, including stearoyl-CoA desaturase (SCD), mevalonate decarboxylase (MVD), hydroxy-3-methylglutaryl-coenzyme A synthase 1 (HMGCS1), and HMG-coenzyme A reductase (HMGCR), the rate-limiting enzyme in cholesterol synthesis and the target of the 'statin' class of drugs for patients with dyslipidemia. Other macula signature genes encoded enzymes that act later in cholesterol biosynthesis, such as lanosterol synthase (LSS) and squalene epoxide (SQLE). In addition, the macula-enriched cluster included the gene for low-density lipoprotein receptor (LDLR), known for its role in binding low-density lipoprotein (LDL), the major cholesterol-carrying lipoprotein of plasma. LDL receptors and LDL-like receptors have been previously identified in retinal pigment epithelium and retinal muller cells \\[53,54\\], but their function in cholesterol transport within the retina has been minimally explored.\n\nThe genes represented in the macula cluster at least partially reflect cell types present in a higher density in the macula than in the peripheral retina, such as ganglion cells and photoreceptors. For example, a substantial number of genes in the macula signature have previously been characterized in ganglion cells (THY1, POU4F1, L1CAML1, NRN1). Interestingly, cholesterol is involved in the physiology of both retinal ganglion cells and photoreceptors. Cholesterol has been identified in rod outer segments in a wide variety of animal species (reviewed in \\[55\\]), as well as in oil droplets isolated from chicken cone photoreceptors \\[56\\]. *In vitro*, cholesterol has the capacity to modulate phototransduction in rods by altering the rod outer segment membrane structure \\[57\\], as well as by directly binding to rhodopsin itself \\[58\\]. Histological studies on retinas from patients with abetalipoproteinemia and familial hypobetalipoproteinemia, (serum LDL-cholesterol levels \\<5% of normal) demonstrated a profound absence of photoreceptors throughout most of the posterior retina \\[59,60\\]. In addition, patients with Smith-Lemli-Opitz Syndrome, a disease of abnormal cholesterol metabolism caused by a defect in 7-dehydrocholesterol reductase (DHCR7), another enzyme encoded by a gene selectively expressed in macula tissues, exhibited slower activation and recovery kinetics of their rod photoreceptors \\[61\\].\n\n*In vitro* studies by Mauch *et al*. \\[62\\] have demonstrated that retinal ganglion cells require cholesterol in order to form mature, functioning synapses. The retinal ganglion cells in their experiments produced enough cholesterol to survive and grow, but effective synaptogenesis demanded additional cholesterol supplied by glial cells. Other work by Hayashi *et al*. \\[63\\] showed that exposure to lipoproteins containing cholesterol and apolipoprotein E stimulated retinal ganglion cell axons to extend, and that this effect was mediated by receptors of the LDL receptor family present on distal axons. Studying the role of cholesterol in synaptogenesis may lead to insights useful in the development of protective or restorative therapeutics for neurodegenerative disease, as well as for ocular diseases that affect ganglion cells.\n\nIn view of epidemiological studies that have suggested connections among atherosclerosis, serum cholesterol levels, and ARMD \\[64-66\\], the enrichment of cholesterol biosynthesis genes within the macula warrants further investigation. The presence of cholesterol in drusen, the extracellular deposits of ARMD, has been confirmed \\[67,68\\], although the origin of this cholesterol remains unclear. Disregulation of lipid metabolism and transport, either on a local and\/or systemic level, may contribute to macular diseases, such as ARMD. Studies have associated statin use with a decreased rate of ARMD \\[69,70\\], but randomized, prospective studies have yet to be completed.\n\n## Identifying candidate disease genes\n\nOne direct application of the gene expression patterns we have defined is the identification of candidate genes for genetic diseases that differentially affect the various eye compartments. This strategy relies on the hypothesis that if mutations in a gene cause physiological aberrations specifically in a particular tissue, the gene is more likely to be selectively expressed in that tissue. We therefore used the literature, RetNet \\[71\\], and the Online Mendelian Inheritance in Man \\[72\\] databases, to collate lists of genetic diseases affecting the lens, cornea, and retina, along with the genetic intervals to which the disease loci have been mapped. Next, we identified genes that were relatively selectively expressed in each of the three compartments. Briefly, we standardized the Cy5 intensity data for each array and calculated the average intensity for every gene across all samples from each compartment. We then empirically identified an intensity cut-off that resulted in selection of greater than 85% of genes included in the retinal compartment signature from Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>, but also included highly expressed genes that were expressed in more than one compartment. Using this cut-off, we identified separate compartment gene lists for the three compartments and identified the subset of these genes that were located in the appropriate cytogenetic intervals for each compartment-specific disease (see Additional data files 4, 5, 6 and Materials and methods).\n\nTo assess the potential of this approach, we analyzed the subset of diseases for which candidate intervals were listed in our sources but for which the causative gene is now known. The density of affected-tissue-expressed genes located in the candidate intervals was similar to that for the unknown diseases, and thus this subset served as a reliable positive control. The disease gene for a remarkable 50% to 70% of the diseases of known genetic cause was selectively expressed in the cognate compartment (Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>). We tested the statistical significance of this result by comparing the number of disease genes identified by the compartment gene expression lists with the aggregate list of all genes detectably expressed in any of the samples shown in Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>. We found that for all three groups of diseases, the compartment signatures were significantly enriched for candidate disease genes (lens, p \\< 0.002; cornea, p \\< 0.005; retina, p \\< 0.0004, by the hypergeometric distribution). By focusing on the genes expressed within the compartment displaying the disease phenotype, we could enrich for potential candidate genes by an average of 2 to 2.5-fold.\n\nAs an example of this approach, we more closely examined Retinitis Pigmentosa 29 (RP 29), an autosomal recessive form of RP that was mapped to chromosomal region 4q32-q34 in a consanguineous Pakistani family \\[73\\]. At least two genes within this interval (*WDR17*, *GPM6A*), and one gene near the interval (*CCN3*), were previously examined by sequencing and were excluded as candidates \\[74\\]. In our data, only one gene, *KIAA1712*, was both located within the mapped interval and selectively expressed in our retinal samples. Little is currently known about this gene, except that it appears in expressed sequence tags (EST) and SAGE libraries from several tissues, including brain. Our analysis suggests that *KIAA1712* is a strong candidate gene for RP 29, and deserves further study. We expect our candidate gene lists to be highly enriched for the causative genes for a large fraction of the diseases we analyzed, and thus should prove useful in accelerating identification of genes important in various aspects of ocular pathology.\n\n# Discussion\n\nOur microarray studies identified distinct molecular signatures for each compartment of the human eye. As we predicted, many of the genes differentially expressed in each tissue could be related to the histology and embryology of the cognate structure in the eye; more usefully, each signature uncovered numerous genes whose expression or function in the eye had not been previously characterized and for which their expression pattern now provides a new clue to their roles. Through a comparative analysis of gene expression among eye compartments, we can also gain insight into the pathophysiology of diseases that afflict specific eye tissues. Furthermore, our data may help anticipate or understand drug effects and side-effects, when the molecular targets of the drugs are preferentially expressed in particular ocular tissues.\n\nThe extensive set of genes selectively expressed in the macula demonstrates that there is significant regional variation in gene expression programs in the human retina. The macula-enriched expression pattern may provide clues to the pathogenesis of retinal diseases that preferentially affect the macula, such as ARMD. Because no ophthalmologic clinical data accompanied the autopsy globe samples used in our experiments and because of our limited sample sizes, we were unable to correlate our gene expression data with clinical exam findings or disease course. The techniques used in these experiments did, however, allow us to examine tissues from individual donors rather than requiring us to rely on either pooled tissue samples or cultured cells. Thus, our results show that future experiments examining individual diseased samples will be possible.\n\nBy analyzing our global gene expression data together with previous genetic mapping data, we were able to greatly refine sets of candidate genes for many corneal, lenticular, and retinal diseases whose genetic basis is still undefined. When we used a control set of diseases with known causative genes, the candidate gene lists we generated included 50% to 70% of the causative genes for this control set. One explanation for why we did not identify all the causative genes for the control disease set was that some causative genes did not meet our intensity threshold, and thus were not included in the compartment expression lists. Furthermore, we could not have identified those causative genes that are only expressed in the diseased state (but not in normal tissues), because we limited our microarray analyses to tissues with no known ocular pathology. Other reasons why our approach may have missed causative genes include expression of causative genes only at certain points in development and not in adult tissues, technical problems with the array element(s) representing these genes, and possible loss of transcripts in the RNA isolation or amplification process. Future investigation of these potential problems and comparison of our candidate gene lists with genome-scale gene expression data from diseased tissues will result in further refinement of the approach presented here.\n\nFinally, our studies were designed to provide an open resource for all investigators interested in ocular physiology and disease. The tissue signature data, as well as the diseases, genetic intervals, and candidate genes for all the diseases we examined, and the complete set of data from our studies is freely available without restriction from the Authors' Web Supplement accompanying this manuscript \\[75\\].\n\n# Materials and methods\n\n## Tissue specimens\n\nEight whole globes (G1 to G8) were harvested from autopsy donors (age range 30 to 85 years old) within 24 h of death, and the tissues were immediately stored at 4\u00b0C in RNAlater (Ambion, Austin, TX, USA). Four of the globes were from female donors (G3, G6 to G8) and four were from male donors (G1, G2, G4, G5). Globes 4 and 5 were harvested as a set from a single donor, as were globes 6 and 7. No ophthalmologic clinical records were available for any of the globes at the time of harvest. Seven of the globes (G1 to G7) were dissected into the following components: cornea, lens, iris, ciliary body, retina, and optic nerve, while only retinal tissue was available from G8. The maculas and the peripheral retinal tissues were further dissected from several of the retinal samples. The macula was defined as the visible xanthophyll-containing tissue temporal to the optic nerve, which encompassed an approximate area of 4 mm^2^. For comparison purposes, three post-mortem brain specimens were analyzed.\n\n## RNA extraction and amplification\n\nSpecimens were disrupted in TRIZOL (Gibco, Carlsbad, CA, USA) solution using a tissue homogenizer. Samples were processed according to the manufacturer's protocol until the aqueous supernatant was retrieved. The supernatant was mixed with 1 volume of 70% ethanol, applied to an RNeasy column (Qiagen, Valencia, CA, USA), and purified according to the manufacturer's protocol. RNA quality and quantity were assessed by gel electrophoresis and spectrophotometer measurements. Total RNA was amplified using a single round, linear amplification method \\[9\\] (also see Additional data files 1 and 2). Tissue samples that yielded inadequate amounts of RNA were excluded from any further analysis. A reference mixture of mRNAs derived from 10 different cell lines (Universal Human Reference RNA, Stratagene, La Jolla, CA, USA) was used in all experiments as an internal standard for comparative two-color fluorescence hybridization.\n\n## Microarray procedures\n\nHuman cDNA microarray construction and hybridization were as previously described \\[76\\]. The microarrays contained 43,198 elements, representing approximately 30,000 genes (estimated by UniGene clusters) and were manufactured by the Stanford Functional Genomics Facility \\[77\\]. In each analysis, amplified RNA from an eye tissue sample was labeled with Cy5, and amplified reference RNA was labeled with Cy3. The two labeled samples were combined, and the mixture was hybridized to a microarray. Arrays were scanned using a GenePix 4000B scanner (Axon Instruments Inc., Sunnyvale, CA, USA). The array images were processed using GenePix Pro 3.0, and the resulting data were indexed in the Stanford Microarray Database and normalized using their default total intensity normalization algorithm (more detailed methods are available in Additional data file 3). Searchable figures and all raw microarray data can be found at \\[75\\]. The complete microarray dataset is also accessible through the Gene Expression Omnibus \\[78\\] (accession number GSE3023).\n\n## Bioinformatic analyses\n\nFor the data shown in Figures <a href=\"#F1\" data-ref-type=\"fig\">1<\/a> and <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>, only elements for which at least 50% of the measurements across all samples had fluorescence intensity in either channel at least 3.25-fold over background intensity were included. The logarithm of the ratio of background-subtracted Cy5 fluorescence to background-subtracted Cy3 fluorescence was calculated. Then values for each array and each gene were median centered, and only cDNA array elements for which at least two measurements differed by more than 2.5-fold from the median were included in subsequent analyses. For the data in Figures <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>, we employed the Statistical Analysis of Microarrays (SAM) package \\[79\\]. Only elements for which the intensity to background ratio was at least 3.25 in at least 35% of the retina samples were considered. Only genes whose expression significantly differed between the macula and peripheral retina (false discovery rate \\<0.05 with 500 permutations) were selected. Finally, to focus on genes with the largest absolute difference in expression between the two regions, we selected genes whose expression differed by at least four-fold from the median in at least two samples.\n\n## Candidate disease gene analysis\n\nTo identify the gene sets expressed in each compartment, background-subtracted Cy5 intensities from each microarray were standardized to an array-median of 1,500, and genes exhibiting an average intensity of at least 2,500 in a compartment were identified (see Additional data file 4). This threshold was chosen empirically because it resulted in greater than 85% of the retinal signature from Figures <a href=\"#F1\" data-ref-type=\"fig\">1<\/a> to be included in the retina set, while less than 5% of these genes were contained in any of the other compartment sets. Genetic diseases affecting the lens, cornea, or retina were collated from the Online Mendelian Inheritance in Man database \\[72\\] and the Retinal Information Network \\[71\\], along with the genetic intervals to which they have been mapped (see Additional data file 5). Using Perl scripts, we mapped every sequence on our arrays to the human genome using data from the UCSC genome browser \\[80\\]. Genes in the corresponding compartment expression set that were located in the genetic interval associated with each compartment-specific disease were identified. For the benchmark analysis of diseases that were associated with known genes, we also identified all genes in the human genome that fell into the genetic interval associated with each disease. The compartment expression sets and our lists of candidate genes for the 147 diseases we analyzed can be found in Additional data file 6.\n\n# Additional data files\n\nThe following additional data are available with the online version of this paper. Additional data file <a href=\"#S1\" data-ref-type=\"supplementary-material\">1<\/a> contains the step-by-step amplification protocol used in this work. Additional data file <a href=\"#S2\" data-ref-type=\"supplementary-material\">2<\/a> is a table detailing RNA isolation and amplification yields. Additional data file <a href=\"#S3\" data-ref-type=\"supplementary-material\">3<\/a> contains more detailed supplemental materials and methods. Additional data file <a href=\"#S4\" data-ref-type=\"supplementary-material\">4<\/a> contains the compartment gene lists used in the disease gene analysis. Additional data file <a href=\"#S5\" data-ref-type=\"supplementary-material\">5<\/a> contains the list of diseases for each compartment with their mapped genetic intervals. Additional data file <a href=\"#S6\" data-ref-type=\"supplementary-material\">6<\/a> contains the results of the disease gene analysis, including the list of candidate genes for each disease.\n\n# Supplementary Material\n\n###### Additional data file 1\n\nStep-by-step amplification protocol.\n\nClick here for file\n\n###### Additional data file 2\n\nA table detailing RNA isolation and amplification yields.\n\nClick here for file\n\n###### Additional data file 3\n\nDetailed supplemental materials and methods.\n\nClick here for file\n\n###### Additional data file 4\n\nCompartment gene lists used in the disease gene analysis.\n\nClick here for file\n\n###### Additional data file 5\n\nThe list of diseases for each compartment with their mapped genetic intervals.\n\nClick here for file\n\n###### Additional data file 6\n\nResults of the disease gene analysis, including the list of candidate genes for each disease.\n\nClick here for file\n\n### Acknowledgements\n\nWe wish to thank members of the Brown laboratory for helpful advice and discussions, M van de Rijn and Stanford pathology for help with tissue acquisition, and T Hernandez-Boussard for computational assistance. This work was supported by the Howard Hughes Medical Institute, NCI grant CA77097, the Stanford Medical Scholars Program (J.D.), and by NIGMS training grant GM07365 (MD). P.O.B. is an investigator of the HHMI.\n\n## Figures and Tables\n\nCompartment gene sets are enriched for candidate genes of ocular diseases\n\n|  | Disease-associated genes on array | Disease-associated genes expressed in affected compartment | Percentage of known disease-associated genes identified | Average fold enrichment compared to total number of genes in interval | *P*-value |\n|----|----|----|----|----|----|\n| Lens | 15 | 8 | 53 | 2.4 | 0.002 |\n| Cornea | 13 | 9 | 69 | 2.0 | 0.005 |\n| Retina | 42 | 23 | 55 | 2.3 | 0.0004 |\n\nArrays were standardized to the same median intensity and genes exhibiting minimum intensities of 2,500 in any compartment were identified. Genetic diseases affecting the lens, cornea, or retina were collated from the RetNet \\[71\\] and Online Mendelian Inheritance in Man \\[72\\] databases, along with their cytogenetic map positions. The table indicates the number of cloned disease genes on the arrays, the number contained in a given compartment gene set, the percentage of known disease genes included in the signatures, the average fold enrichment compared to the total number of genes in each cytogenetic interval, and the statistical significance of this enrichment (using the hypergeometric distribution).","meta":{"dup_signals":{"dup_doc_count":154,"dup_dump_count":46,"dup_details":{"curated_sources":2,"2022-27":1,"2021-43":1,"2020-34":1,"2020-24":1,"2019-47":1,"2019-22":1,"2019-09":1,"2018-51":1,"2018-39":1,"2018-30":1,"2018-22":1,"2018-13":1,"2017-51":1,"2017-43":1,"2017-34":1,"2017-26":1,"2017-17":4,"2017-09":17,"2017-04":1,"2016-50":1,"2016-44":2,"2016-40":2,"2016-36":19,"2016-30":16,"2015-48":2,"2015-40":1,"2015-35":3,"2015-32":3,"2015-27":1,"2015-22":4,"2015-14":3,"2014-52":3,"2014-49":5,"2014-42":8,"2014-41":5,"2014-35":4,"2014-23":6,"2014-15":5,"2023-23":1,"2015-18":4,"2015-11":2,"2015-06":3,"2014-10":3,"2013-48":4,"2013-20":3,"2024-18":1}},"file":"PMC1242209"},"subset":"pubmed_central"}
{"text":"abstract: Connections have been revealed between very different human diseases using phenotype associations in other species\nauthor: Bolan Linghu; Charles DeLisi\ndate: 2010\ninstitute: 1Translational Sciences Department, Novartis Institutes for BioMedical Research, Cambridge, MA 02139, USA; 2Departments of Biomedical Engineering and Physics, Program in Bioinformatics and Systems Biology, Boston University, Boston, MA 02215, USA\nreferences:\ntitle: Phenotypic connections in surprising places\n\n# The human disease landscape\n\nThe past few years have witnessed a growing number of well documented connections between and among human disease phenotypes, whose relationship would not have been obvious within the current disease classification framework. The evidence stems from a variety of sources, spanning clinical epidemiology, computational genomics and various model systems \\[1-5\\]. The implications are potentially so fundamental to disease etiology, drug development and the general diagnostic paradigm that there have been calls for an NIH Roadmap (large trans-institute transformational grants) focused on delineating the human disease landscape - a quantitative bipartite correlation map relating disease phenotypes and their genetic structures. A new study of phenotype correlations \\[6\\] now takes this idea further.\n\nOne of the earliest comprehensive studies of human phenotype correlations was by Rzhetsky *et al*. \\[5\\], who established a correlation network between 161 diseases and disorders using evidence of comorbidities obtained from some 1.5 million patient records. Among other results, they found suggestive evidence for genetic relations between autism, which manifests in childhood, and several late onset diseases, including bipolar disorder and schizophrenia.\n\nA different approach was taken by Goh *et al*. \\[1\\], who constructed a human disease network by linking genetic disorders that are known to share causative genes. The network was based on an analysis of the Online Mendelian Inheritance in Man (OMIM) database, the most comprehensive compendium of well established associations between human disorders and their associated genes \\[7\\]. Among their findings was a large subnet formed by 516 of the 1,284 disorders studied, clearly showing many-to-many relationships between phenotypes and genes. For example, *KRAS* (encoding a small GTPase), *BRCA1* and *BRCA2* (both encoding tumor suppressors) are all involved in breast cancer, but *KRAS* is also implicated in pancreatic cancer, whereas *BRCA1* and *BRCA2* are associated with papillary carcinoma and prostate cancer, respectively \\[7\\]. The results of these and other studies support the concept that genes underlying a disorder tend to be functionally related; for example, they could be part of a particular protein complex, a particular pathway or process, or a particular set of coexpressed genes. A disorder can then be viewed as a phenotype that emerges from dysfunction of one or more components of a functionally coherent gene module. An important aspect of modularization is that any gene in the module that was not previously identified with the phenotype is a candidate for association with it \\[8\\]. Moreover, a particular module can, to varying extents, underlie related phenotypes, opening up the possibility of linking phenotypes not only on the basis of shared genes, but also on the basis of functionally related genes that are not shared.\n\nInferring disease connectivity through functional linkage starts with a network of genes whose functions are correlated; that is, each pair of nodes (genes or proteins) is connected by one or more sources of evidence supporting its functional coherence, such as physical interaction, correlated expression, adjacency in the same metabolic pathway or genetic interaction \\[2-4\\]. Connections can then be inferred between the diseases whose associated genes are linked in the gene network. For instance, we and our colleagues \\[3\\] constructed a network for the human genome by integration of diverse types of evidence using a Bayesian model, and annotated it with all OMIM disease genes for diseases known to be associated with five or more genes. Genes that are most tightly linked to those known to be associated with a given disease are immediate candidates for association with that disease. Furthermore, connections between disease pairs can be quantitatively identified on the basis of the magnitude of the functional linkage between their disease-associated genes. Thus, two diseases can be linked even if they do not share disease-associated genes. The associations found ranged from phenotypically disparate disease pairs, such as multiple sclerosis with malaria, to phenotypically similar pairs, such as muscular dystrophy with myopathy. Such results suggest that the current disease classification may be much less informative than is commonly believed.\n\nRecognition of molecular connections between disease phenotypes provides immediate insight into the molecular mechanisms underlying different diseases and can therefore generate novel hypotheses for therapeutic strategies. This is especially valuable if one disease is well studied and the other is not; it might also be valuable if viable drug targets have been found for one of the diseases but not the other. This prospect of drug repositioning could accelerate the introduction of therapies by years \\[9\\].\n\n# Human disease models and phenotypic connections in surprising places\n\nRecent work from Edward Marcotte's laboratory by McGary *et al*. \\[6\\] takes these concepts, and indeed the entire field of phenomics, to an entirely new level by developing, establishing and exploring a quantitative method to find non-obvious phenotypic connections, not just within a species, but across species. Such cross-species phenotypic connections stem from the evolutionary conservation of the underlying associated gene modules \\[8,10\\]. The importance of the work reported by McGary *et al*. \\[6\\] can be glimpsed by recalling that whereas OMIM, which is the most extensive database of well established human gene-phenotype associations, contains approximately 5,400 unique associations \\[7\\], the numbers for some model organisms are 1 to 2 orders of magnitude higher \\[6\\]. Some of these relations - such as between obesity and its implicated genes in mice - have obvious equivalences in humans, and when they do, the laboratory model can serve as a useful surrogate to study the disease or disorder. Other phenotypic pairs are entirely non-obvious, such as the fact that retinal cancer in humans and ectopic vulvae in the nematode can both be caused by disruption of the human retinoblastoma 1 (*RB1*) gene or its nematode ortholog \\[6\\]. Because such non-obvious relations can occur with surprising frequency, a method that can rapidly and reliably link human genes to non-obviously related phenotypes in model systems offers the prospect of radically accelerating the rate at which we can explore disease landscapes in both humans and model organisms.\n\nMcGary *et al*. \\[6\\] provide more than a glimpse at the possibilities. They begin by defining 'phenologs' as cross-species mutant phenotypes that share a significant number of orthologous genes. The statistical significance of phenologs, whose emergence results from disruption of orthologous genes, can be estimated as the probability that the observed number of orthologues common to the two phenotypes would be found by chance and correcting for multiple hypotheses. In this way, using approximately 300 human diseases and over 6,000 phenotypes in model organisms, including mouse, worm, yeast and *Arabidopsis*, they identify 4,390 significant phenologs. As one of the positive controls, the authors note that the 3,755 mouse-human phenologs identified contain many of the known disease models - including cataracts, deafness and retinal disease - all at *P*-values well below 10-8. Given that phenologs map gene-phenotype associations across the phyla, an association known in one species can be used to find non-established relations in another. Cross-validations presented by McGary *et al*. \\[6\\] show that phenologs can predict genes associated with about a third to a half of tested human diseases.\n\nThe work \\[6\\] offers tantalizing evidence for several counterintuitive mammalian disease models, including reduced growth rate of yeast deletion strains in medium enriched with the cholesterol-lowering drug lovastatin as a model for abnormal angiogenesis in mice, and negative gravitropism defects in *Arabidopsis* as a model for human Waardenburg syndrome (which causes deafness with defects in neural-crest-derived tissues). Furthermore, using the yeast model, they demonstrate that *SOX13* (encoding a transcription factor related to the sex-determining gene *SRY*) is a new gene that regulates angiogenesis, and using the *Arabidopsis* model they show that *SEC23IP* (encoding a protein that interacts with SEC23, a component of the COPII complex that controls endoplasmic reticulum-to-Golgi trafficking) is probably a new Waardenburg syndrome gene. Notwithstanding the fact that many functionally coherent gene modules are conserved across different species \\[10\\], such demonstrations - especially the identification of phenologs that predate kingdom divergence - seem to mark one of those uncommon occasions in science in which intuition built on years of experience fails completely.\n\n# Additional connections\n\nAs staggering as these results \\[6\\] are, it seems possible that many phenologs have been missed, because the method is confined to connections based only on phenotype pairs sharing known orthologous genes. Gene-phenotype association data may, however, be far from complete; if so, many orthologous phenotypes will be missed. One possible way to increase the discovery rate would be to consider the functional relatedness for the genes associated with each phenotype. As described earlier, gene-gene functional relatedness has been successfully used to identify phenotypic connections within the same species \\[2-4\\], and it is possible that the same principle can be applied to identify phenotype connections between different species.\n\nA deeper and far more important connection relates to the impact of McGary *et al*. \\[6\\] on efforts to develop a more complete picture of the topology of the human phenome. It is evident that the way phenomics looks today, the amount of information and the incredible interconnectivities, could not have been imagined even 3 years ago. It seems likely that the methods developed and demonstrated by McGary *et al*. \\[6\\], and their inevitable extensions, will add unprecedented knowledge and quantitative detail to the interrelated landscapes of mutant phenotypes for humans and other species, and this will offer many more surprising correlations between and among human diseases. As our picture of the human disease landscape continues to take shape, perhaps the one thing that we should not be surprised about will be the need to fundamentally rethink the current disease classification and its associated diagnostic paradigm.","meta":{"dup_signals":{"dup_doc_count":124,"dup_dump_count":48,"dup_details":{"curated_sources":2,"2022-21":1,"2021-49":1,"2021-10":1,"2020-45":1,"2020-24":1,"2019-51":1,"2019-35":1,"2019-22":1,"2019-13":2,"2019-09":1,"2018-51":1,"2018-39":1,"2018-30":1,"2018-17":1,"2017-51":1,"2017-47":1,"2017-34":1,"2017-22":1,"2017-09":11,"2016-44":1,"2016-40":1,"2016-36":10,"2016-30":7,"2016-22":1,"2016-18":1,"2016-07":6,"2015-48":3,"2015-40":1,"2015-35":3,"2015-32":3,"2015-27":3,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":2,"2014-42":7,"2014-41":4,"2014-35":2,"2014-23":3,"2014-15":4,"2023-23":1,"2015-18":3,"2015-11":3,"2015-06":3,"2014-10":4,"2013-48":3,"2013-20":3,"2024-10":1}},"file":"PMC2884535"},"subset":"pubmed_central"}
{"text":"author: Mary R. LoekenCorresponding author: Mary R. Loeken, <email@example.com>.\ndate: 2012-07\nreferences:\ntitle: A New Role for Pancreatic Insulin in the Male Reproductive Axis\n\nThe recent discovery that insulin is expressed by the rat testis (1) raises questions about whether and how locally produced insulin regulates testicular function. It also raises the question whether pancreatic insulin regulates testicular function, and whether fertility might be impaired as a consequence of insulin deficiency in type 1 diabetes. As reported in this issue of *Diabetes*, Schoeller et al. (2) have found that pancreatic insulin regulates the male hypothalamic-pituitary-gonadal axis and is essential for fertility. In contrast, insulin produced in the testes may not be essential for testicular function. The article by Schoeller et al. provides an important advance toward understanding why fertility may be diminished in men with type 1 diabetes.\n\nIt has only recently been recognized that diabetes can reduce sperm quality and that the female partners of diabetic men have lower pregnancy rates (3,4). It is not well understood whether hyperglycemia or abnormal insulin signaling is responsible, and what stage(s) of spermatogenesis is affected. Increased nuclear and mitochondrial DNA damage in sperm samples from diabetic men suggests that hyperglycemia-induced oxidative stress may be responsible (3,5). However, it is also possible that the adverse effects of diabetes may be due to abnormal insulin signaling in the testis, systemic effects of insulin, or both, and may be separate from insulin's effects on blood glucose levels.\n\nSchoeller et al. (2) have taken advantage of the Akita mouse model of insulin-deficient diabetes to cleverly sort out these possibilities. The dominant Akita phenotype is caused by a mutation in the *Ins2* allele. The resulting misfolded protein product causes endoplasmic reticulum (ER) stress, leading to pancreatic \u03b2-cell death (6). Akita mice develop hyperglycemia, although the age at onset and severity of hyperglycemia depends on the sex, strain background, and whether the mice are heterozygous or homozygous for the mutant *Ins2* allele (M.R.L., personal observations; Schoeller et al. \\[2\\]). Notably, whereas primates carry only one insulin gene, rodents carry two functional insulin genes, *Ins1* and *Ins2*. *Ins1* arose from a duplication of the ancestral *Ins2* gene approximately 20 million years ago (7); therefore, the mouse *Ins2* gene is orthologous to the human insulin gene. Thus, if *Ins2* is expressed in the mouse testis, it is very likely that the human testis expresses insulin as well. The beauty of using the Akita model is that it has the potential to distinguish between the roles of testicular and pancreatic insulin on male reproductive function. This is not possible with other models, such as streptozotocin-induced diabetes or the NOD mouse, in which testicular insulin production would not be interrupted.\n\nThe Moley laboratory previously observed that sperm from homozygous Akita males fertilizes fewer oocytes and that the resulting blastocysts are developmentally impaired (8). In this study (2), they used males that were either heterozygous or homozygous for the Akita allele; heterozygous Akita males develop hyperglycemia (\\>300 mg\/dL) by 5 weeks of age, whereas homozygous Akita males develop hyperglycemia by 3 weeks of age (i.e., prior to puberty) and die by 8\u201312 weeks of age unless treated with insulin. Fertility diminishes by 4\u20136 months of age in heterozygous Akita males, whereas homozygous Akita males are completely infertile. RT-PCR demonstrated that, unlike the pancreas in which both *Ins1* and *Ins2* are transcribed, only *Ins2* was transcribed in the testis. Immunolocalization showed that insulin was detected predominantly in Sertoli cells. However, there was no associated ER stress. Thus, while the resulting Ins2 protein may be hypomorphic or nonfunctional in the testis, infertility in homozygous Akita males does not appear to be due to ER stress-induced apoptosis of testicular cells.\n\nAlthough treatment of the homozygous Akita males with exogenous insulin using subcutaneous insulin implants restored spermatogenesis and fertility, this was not due to restoration of insulin within the testes because insulin did not cross the blood-testis barrier. There could be effects on Leydig cells, which contain insulin receptors and are located outside the blood-testis barrier (9). Therefore, they would presumably not be responsive to insulin produced by Sertoli cells. However, because circulating levels of luteinizing hormone and testosterone, which were significantly reduced in Akita homozygotes, were restored by insulin treatment, the primary effects of exogenous insulin appear to be on the hypothalamic-pituitary axis.\n\nThis study by Schoeller et al. (2) is of clinical relevance to men with type 1 diabetes because it demonstrates that pancreatic insulin is crucial for the male reproductive axis. The infertility in homozygous Akita males appears to be due to insulin deficiency, not hyperglycemia. This is because heterozygous Akita males become as severely hyperglycemic as the homozygotes at only a slightly older age, and they are fertile at least until they are 4\u20136 months of age. On the other hand, it is possible that severe hyperglycemia before puberty interferes with the function of the hypothalamic-pituitary-testicular axis, whereas hyperglycemia occurring during or after puberty only interferes with the function of the reproductive axis after several months of chronic exposure. However, the ability to restore fertility in homozygous Akita males with exogenous insulin suggests that if prepubescent hyperglycemia interferes with the function of the reproductive axis, it is not irreversible.\n\nThis article (2) opens the door to future studies that aim to understand how insulin regulates the male reproductive axis, and whether insulin is regulating the pituitary, the hypothalamus, or higher central nervous system nuclei. Knockout of the insulin receptor gene in the central nervous system impairs luteinizing hormone production and spermatogenesis (10). There was no significant difference in follicle-stimulating hormone levels between wild-type, heterozygous, and homozygous Akita males, suggesting that the effects of insulin are on the hypothalamus or on the responsiveness of the pituitary gonadotropes to the hypothalamic gonadotropin\u2013releasing hormone.\n\nIt should be noted that insulin has long been recognized to play a role in the ovary. Insulin is detectable in ovarian follicular fluid and synergizes with gonadotropins for oogenesis, ovulation, and luteinization (11,12). Because the ovarian follicle is permeable to circulating hormones, it has been thought that follicular fluid insulin is derived from the pancreas. However, in light of this current study, whether insulin (and *Ins2* in particular) is expressed by ovarian cells ought to be examined. Although this study did not find an essential role for testicular insulin, additional experimentation, perhaps with testis-specific *Ins1* or *Ins2* knockout or transgenic strains, is necessary to further understand the function of testis-derived insulin. It will also be important to study the effects of insulin and insulin deficiency that are conserved, as well as those that are distinct, between the hypothalamic-pituitary-gonadal axes of females and males.\n\n## ACKNOWLEDGMENTS\n\nM.R.L. is supported by grants from the National Institutes of Health (RO1 DK52865 and RO1 DK58300).\n\nNo potential conflicts of interest relevant to this article were reported.\n\n# REFERENCES","meta":{"dup_signals":{"dup_doc_count":135,"dup_dump_count":49,"dup_details":{"curated_sources":2,"2022-33":2,"2019-51":1,"2019-47":1,"2019-43":2,"2019-39":1,"2019-26":1,"2019-09":1,"2018-47":1,"2018-39":1,"2018-30":2,"2018-26":2,"2018-17":2,"2018-09":3,"2018-05":1,"2017-51":2,"2017-47":2,"2017-43":4,"2017-39":2,"2017-34":2,"2017-30":5,"2017-26":3,"2017-22":2,"2017-17":8,"2017-09":4,"2017-04":7,"2016-50":4,"2016-44":4,"2016-40":4,"2016-36":4,"2016-30":4,"2016-26":3,"2016-22":3,"2016-18":2,"2016-07":2,"2015-48":3,"2015-40":2,"2015-35":2,"2015-32":3,"2015-27":2,"2015-22":3,"2015-14":3,"2014-52":3,"2014-42":4,"2014-35":4,"2014-23":3,"2017-13":2,"2015-18":3,"2015-11":3,"2015-06":1}},"file":"PMC3379657"},"subset":"pubmed_central"}
{"text":"author: JH Anolik; A Palanichamy; J Bauer; J Barnard; J Biear; R Dedrick; I Sanz; J Liesveld; E Baechler\ndate: 2012\ninstitute: 1University of Rochester Medical Center, Rochester, NY, USA; 2University of Minnesota, Minneapolis, MN, USA; 3XDx, Brisbane, CA, USA; 4Emory University, Atlanta, GA, USA\ntitle: B cells at the adaptive-innate immune system interface in SLE\n\n# Background\n\nAccumulating data indicate that inappropriate activation of type I interferon (IFN) plays a key role in the pathogenesis of systemic lupus erythematosus (SLE). Given that IFN can influence B-cell lymphopoiesis in murine bone marrow (BM), we explored the hypothesis that IFN activation in SLE BM has direct effects on B-cell development. Additionally, the impact of B cells on pDC production of IFN was examined.\n\n# Methods\n\nPeripheral blood mononuclear cells (PBMC) and bone marrow mononuclear cells (BMMC) were isolated from 28 patients fulfilling ACR criteria for SLE and from 20 healthy controls. RNA isolates were analyzed for the expression of three to five IFN-regulated genes (IFIT1, IRF7, G1P2, CEB1, C1orf29) by quantitative PCR and IFN-regulated chemokines CXCL10 (IP-10), CCL2 (MCP-1), and CCL19 (MIP-3\u03b2) defined in serum and BM supernatant. B-cell subsets were delineated in single-cell suspensions of PBMC and BMMC by multi-parameter flow cytometry. BM and PB pDCs were stimulated with TLR9 (CpG ODN 2006 or 2216) or TLR7 (R848) agonists and intracellular IFN and TNF measured by flow cytometry.\n\n# Results\n\nThe majority of SLE patients had an IFN signature in the BM (57%), which was even more pronounced than the paired PB. Notably, the early B-cell compartment (consisting of pro B cells, pre B cells, immature B cells and early transitional T1 and T2 B cells) in SLE BM with an IFN signature was associated with a reduction in the fraction of pro\/pre B cells, suggesting an inhibition in early B-cell development. However, at the transitional B-cell stage this inhibition was reversed with enhanced selection of B cells into the transitional compartment. The composition of the mature B-cell compartment in IFN-activated SLE BM was notable for an expansion of CD27^+^, IgD^-^ switch memory B cells. SLE patients with a BM IFN signature were enriched for a high number of autoantibody specificities (*P* = 0.003 compared with IFN low), and the degree of IFN activation in the BM correlated with peripheral lymphopenia (*P* = 0.019) and disease activity (*P* = 0.05). In order to understand the etiology of IFN activation in SLE BM, we examined the production of IFN by pDCs. CpG induced IFN production in pDCs in a dose-dependent fashion. Notably, a higher proportion of BM pDCs produced IFN compared with paired PB. Moreover, pDCs produced 59% more IFN in the presence of B cells.\n\n# Conclusion\n\nThis is the first demonstration of an IFN signature in SLE BM. These results suggest that the BM is an important but previously unrecognized target organ in SLE with critical implications for B-cell ontogeny and selection. We postulate that in the setting of IFN activation the stringency of negative selection of autoreactive B cells in the BM may be reduced. Circulating immune complexes and apoptotic fragments in SLE BM may serve as ligands for Toll-like receptors on pDCs contributing to aberrant IFN production and, in turn, B cells may be critical regulators of pDC function.\n\n## Acknowledgements\n\nWork supported by grants from the NIH (R01 AI077674 and P01 AI078907) and the Rochester Autoimmunity Center of Excellence.","meta":{"dup_signals":{"dup_doc_count":107,"dup_dump_count":34,"dup_details":{"curated_sources":2,"2019-09":1,"2018-47":1,"2018-39":1,"2018-30":2,"2018-13":1,"2017-47":1,"2017-34":1,"2017-22":1,"2017-09":8,"2016-44":1,"2016-40":1,"2016-36":7,"2016-30":8,"2015-48":3,"2015-40":2,"2015-35":3,"2015-32":3,"2015-27":2,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":6,"2014-42":8,"2014-41":3,"2014-35":3,"2014-23":5,"2014-15":5,"2020-05":1,"2015-18":3,"2015-11":2,"2015-06":3,"2014-10":4,"2013-48":3,"2013-20":3}},"file":"PMC3467501"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  The main goal of the whole transcriptome analysis is to correctly identify all expressed transcripts within a specific cell\/tissue - at a particular stage and condition - to determine their structures and to measure their abundances. RNA-seq data promise to allow identification and quantification of transcriptome at unprecedented level of resolution, accuracy and low cost. Several computational methods have been proposed to achieve such purposes. However, it is still not clear which promises are already met and which challenges are still open and require further methodological developments.\n  .\n  # Results\n  .\n  We carried out a simulation study to assess the performance of 5 widely used tools, such as: CEM, Cufflinks, iReckon, RSEM, and SLIDE. All of them have been used with default parameters. In particular, we considered the effect of the following three different scenarios: the availability of complete annotation, incomplete annotation, and no annotation at all. Moreover, comparisons were carried out using the methods in three different modes of action. In the first mode, the methods were forced to only deal with those isoforms that are present in the annotation; in the second mode, they were allowed to detect novel isoforms using the annotation as guide; in the third mode, they were operating in fully data driven way (although with the support of the alignment on the reference genome). In the latter modality, precision and recall are quite poor. On the contrary, results are better with the support of the annotation, even though it is not complete. Finally, abundance estimation error often shows a very skewed distribution. The performance strongly depends on the true real abundance of the isoforms. Lowly (and sometimes also moderately) expressed isoforms are poorly detected and estimated. In particular, lowly expressed isoforms are identified mainly if they are provided in the original annotation as potential isoforms.\n  .\n  # Conclusions\n  .\n  Both detection and quantification of all isoforms from RNA-seq data are still hard problems and they are affected by many factors. Overall, the performance significantly changes since it depends on the modes of action and on the type of available annotation. Results obtained using complete or partial annotation are able to detect most of the expressed isoforms, even though the number of false positives is often high. Fully data driven approaches require more attention, at least for complex eucaryotic genomes. Improvements are desirable especially for isoform quantification and for isoform detection with low abundance.\nauthor: Claudia Angelini; Daniela De Canditiis; Italia De Feis\ndate: 2014\ninstitute: 1Istituto per le Applicazioni del Calcolo, CNR, Naples, Italy; 2Istituto per le Applicazioni del Calcolo, CNR, Rome, Italy\nreferences:\ntitle: Computational approaches for isoform detection and estimation: good and bad news\n\n# Background\n\nGene transcription represents a key step in the biology of living organisms. Several recent studies, including \\[1,2\\], have shown that, at least in eukaryotes, a large fraction of the genome is transcribed and almost all the genes (more than 90% of human genes) undergo alternative splicing. The discovery of the pervasive nature of eukaryotic transcription, its unexpected level of complexity - particularly in humans - and its accurate quantification are helping to have a deep insight into biological pathways and molecular mechanisms that regulate disease predisposition and progression \\[3\\].\n\nThe main goal of the whole transcriptome analysis is to identify, measure, characterize and catalogue all expressed transcripts within a specific cell\/tissue - at a particular stage and condition - in particular to determine the precise structure of genes and transcripts, the correct splicing patterns, their abundances, and to quantify the differential expressions in both physiological and pathological conditions.\n\nThanks to pioneer works of \\[4-6\\] that showed, among others, the potential of high-throughput mRNA sequencing (RNA-seq) and the development of efficient computational tools \\[7-9\\] to analyse such a data, RNA-seq has quickly become one of the preferred and most widely used approaches for discovering new genes and transcripts and for measuring transcript abundance from a single experiment (see \\[10,11\\] for reviews). To date, RNA-seq experiments have been successfully used in a wide spectrum of researches, offering tremendous benefits with respect to those previous approaches, such as microarrays, and also creating many challenges from both experimental and data analysis perspective \\[12\\].\n\nIn particular, to fully benefit of RNA-seq data, the following (strongly connected) computational challenges must be faced: **i)** Transcriptome reconstruction or isoform identification **ii)** Gene and Isoform detection (on\/off) **iii)** Gene and Isoform quantification (expression level in terms of either FPKM or read-count) **iv)** Gene and Isoform differential expression\n\nPoints i)\u2013iii) are aimed to provide a full characterization of the transcriptome of a given sample, with ii) and iii) often combined into a simultaneous step, where some parsimonious strategies are employed to deal with the high number of candidate isoforms. Point iv) is carried out to compare samples across different physiological and pathological conditions. To face these challenges, several computational methods have been proposed \\[13,14\\] and open-source software packages are available. However, despite the connection among the previous points, most of the available computational methods attempt to face each point independently. Therefore, sophisticated pipelines are built in order to provide a comprehensive answer (see the Tuxedo pipeline \\[15\\] as a remarkable example). Anyway, the choice of the best method to use for a specific dataset, the best parameter tuning and the expected performance are not clear to a beginner user. In particular, methods often require several additional parameters that are not easy to understand and choose. Assessing the best combination is very difficult and time consuming. In most cases the choice is done in a subjective way, partially driven by prior knowledge of the structure of the genome under analysis and by some heuristic considerations, rather than using an objective and general approach. Therefore, most users are often confused and tend to use default values.\n\nRecently, few independent studies have been devoted to compare the performance of computational methods for detecting differential expression under a wide type of settings, see for example \\[16-18\\]. For what concerns points i)\u2013iii) limited comparisons were carried out within the same paper that describes the proposed method \\[19-23\\]. However, to the best of our knowledge, no independent comparison was available until the recent study of \\[24\\], conducted almost simultaneously to our study.\n\nGoals of the present paper are to illustrate the results of a detailed comparison of five widely used tools, namely *CEM*\\[23\\], *Cufflinks*\\[20\\], *iReckon*\\[22\\], *RSEM*\\[19\\] and *SLIDE*\\[21\\], to provide a discussion about expected results, and to assess which promises are already met and which challenges are still open and require further methodological developments. Even though, at least for data driven approaches, our conclusions are similar to those achieved by \\[24\\], the way to carry out our analysis and the way to compare the methods are different. Therefore, this study can be viewed as a complement to \\[24\\]. Specifically, we assessed the particular improvements that may be obtained by using annotation.\n\nIn the following section, we briefly review some of the most widely used tools for isoform reconstruction and quantification in organisms for which a reference genome is available. When the reference genome is not available (or the user does not want to use it) such methods cannot be used and more computational demanding assembly strategies have to be taken into account instead, see for example \\[25,26\\] or more in general \\[24\\]. Subsequently, in Methods section we describe the approach considered to build the comparisons and provide the rationale about the compared methods, their parameters and modes of usage. Comparisons are mainly carried out for simulated paired-end reads (PE) with different through-put and read-lengths, since they represent the state of the art of most current experiments. However, for completeness we also implemented a limited simulation study using single-end reads (SE) at the same depth. All methods were mostly used with their default parameters, without attempting any internal parameter optimization to improve their performances, mimicking the expected usage of a non expert scientist in the analysis of RNA-seq data.\n\nMethods were compared under different experimental scenarios, assuming the availability of complete annotation, incomplete annotation and absence of annotation. Moreover, whenever possible, such scenarios were combined with three different modes of action that account for different strategies in the considered algorithms. In the first mode, the inference is limited only to those isoforms that are present in the annotation. In the second mode, the annotation is used as a guide to identify other possible transcripts. In the third mode, all inference is fully data driven. We observe that the case of complete annotation (combined with inference limited to handle only transcripts contained therein) represents an ideal case, that allows us to evaluate the performance of each method in detecting presence of isoforms and to quantify their expression when everything else in known. This situation is rarely met given our current knowledge of Biology. However, it can be considered as a limit case since, in the near future, it will be possible to work with almost complete annotations, thanks to the output of large international projects such as ENCODE (Encyclopedia of DNA Elements) \\[27\\], at least for widely studied organisms such as the Human one. The case of incomplete annotation (combined with an inference driven by the provided annotation) illustrates the realistic case in current studies, where previous projects have disclosed most information. However, data emerging from the literature show that such information represents only partial knowledge. Finally, the full data driven approach is necessary when studying novel sequenced organisms for which no previous information is available (or the user does not want to use the annotation explicitly). All comparisons in \\[24\\], except iReckon and SLIDE, were carried out fully data driven.\n\nIn Results and discussions section, we illustrate the results of our comparative study on two different experimental set-ups. The first models small genomes, while the second models large genomes. In particular, we compare recall and precision given a set of truly expressed isoforms and we evaluate the quality of the abundance estimates. Also with respect to this point, our method differs from \\[24\\]. In our case the truth is based on simulated data, about which we have a complete knowledge in terms of truly expressed isoforms and expression levels. Conversely, in \\[24\\] the comparison was carried out on a real dataset for which the same information was not available. In that case the aim was to benchmark data driven approaches in recovering the gene annotation, without taking into account whether the retrieved isoforms were present in the sample or not. Finally, Conclusions section summarizes all our evaluations.\n\n## An overview on computational methods for isoform identification and quantification\n\nThe classical pipeline for isoform detection and estimation consists of the following three logical steps. First, the reads are aligned to the reference genome. Subsequently, candidate isoforms are either identified or are directly provided by the user through an annotation file. Finally, the presence and the abundance of each isoform are (either independently or simultaneously) estimated. We refer to \\[13,14\\] for detailed reviews of the existing algorithms and software. Alternatively, it is also possible to use methods, such as \\[26\\], that assemble reads in longer fragments that constitute the transcriptome, and then use methods for quantifying the abundance of inferred transcripts. Assembly methods are based on local alignment and graph theory and are similar in spirit to those methods used to assemble genomes. Such methods are potential very interesting for detecting de-novo isoforms. However, the comparison of such approaches with aligned based algorithms is out of the scope of the current work.\n\nRNA-seq alignment can be performed by a series of devoted tools such a \\[28-32\\], that allow to map both reads to the reference genome without large gap (i.e., exon-body reads) and reads with large gap in terms of genomic coordinates that span exon-exon junctions (i.e., splice-junctions reads). Since the aim of this paper is to compare isoform estimation\/detection procedures, we chose for the alignment step Tophat2 \\[29\\] (version 2.0.7) and we refer to \\[18,31,33,34\\] for comparisons on different algorithms. The choice of Tophat2 is motivated by the fact that the analysed tools suggest it, or its previous version \\[28\\], as aligner. Nevertheless, in general these methods only require the user to provide an alignment file. Therefore, any of the existing RNA-seq mappers can be used. The ability of an aligner to properly map the junction reads is important since false negative junctions may prevent the possibility of reconstructing some isoforms, while false positive junctions can lead to false isoform identification. We also note that some methods, for example \\[35\\], align reads to the transcriptome to better map the (known) splice junctions. Others, such as \\[29\\], implement hybrid approaches using both transcriptome and genome.\n\nOnce the read alignment has been performed, the inference can be carried out at different biological levels. Quantification of multiple isoforms is more complicated than the single event one (i.e., exons, junctions or genes), since different isoforms of the same gene (or that insist on the same genomic locus) share great part of the sequences from common exons and junctions. Moreover, identification and quantification problems are affected by both positional and sequence content biases present in RNA-seq data and by several other -still not fully understood- sources of experimental biases. The differences among the methods mostly depend on the way they model reads and the way they account for the different sources of biases.\n\nIn principle RNA-Seq data (i.e. observed coverage and splice-junction) can be modeled as a linear combination of isoforms. Therefore, the problem can be seen as a deconvolution problem \\[36,37\\] with expression levels as weights and isoforms as convolution kernels. Under such formalism, the isoform expression can be estimated either by using the \"maximum likelihood principle\" or by using similar statistical optimizations. Unfortunately, the design matrix that describes the isoform structures is unknown (or at least not completely known) and potentially very large. Therefore, the problem can be treated as a two steps procedure where, first, a set of candidate isoforms is identified, then the inference is made on such a set. The isoform identification step is crucial since the rest of inference is carried out on the basis of this result. On the other hand, it is also possible to perform the two steps simultaneously, see \\[38\\]. Moreover, because of the large number of candidate isoforms, the problem becomes ill-posed. Therefore, some penalties have to be used to encourage sparse solutions and avoid data over-fitting.\n\nFor instance, the very famous Lasso-type penalty was used in \\[21,39\\]. This penalty is sub-optimal since it does not take into account that all abundances are non negatives and their sum is constrained. The penalty in \\[22\\] is one attempt to explicitly use such constraints and reinforce the sparsity. In other cases, see for example \\[20\\] the sparseness is achieved using post-filtering steps to reduce the number of candidate isoforms.\n\nIt should be noted that several methods proposed for studying isoforms \\[36,40,41\\] do not perform the identification step explicitly. Indeed, they require the user to provide such a-priori knowledge. This is usually done either in terms of annotation file (in.GTF or.BED format) that can be downloaded from some database (e.g., \\[42\\]) or as a preliminary result of some tools for transcript reconstruction, such as in \\[43\\]. In this context, the inference is often limited to the easier problem of quantifying only those isoforms that are contained in the annotation rather than identifying novel isoform structures. Despite the availability of several methods that allow both isoform reconstruction and quantification, we consider useful to consider those approaches since, when the annotation will become complete, they can turn back in competition. Moreover, providing them a list of candidate isoforms obtained from some assembly procedures, such methods claim to return accurate quantification. An example of such idea is given by RSEM \\[19\\] that is now used as quantification step combined with Trinity \\[26\\].\n\nMore in general, for methods performing the identification step, isoform reconstruction can be carried out by using two other philosophical approaches. In the first one, the algorithm is driven by an annotation (that represents the available information at state of the art). In the second case, all isoforms are reconstructed ab initio (or fully data driven), mainly using graph theory. These models must be used in combination with some (heuristic) approaches in order to make the graph optimization feasible due to the large number of potential transcripts coming from a splicing graph. Moreover, it often occurs that a same method can use different rationales according to the way it is used, see \\[20,23,43\\].\n\n# Methods\n\nIn order to evaluate and compare the performances of the proposed methodologies, we used simulated data for which the true isoform structures and abundances are known.\n\nFew RNA-Seq simulators have been proposed in the last years (BEERS Simulator \\[31\\], RSEM Read Simulator \\[35\\], RNASeqReadSimulator \\[44\\]). In this work we used Flux Simulator \\[45\\] (available at <http:\/\/sammeth.net\/confluence\/display\/SIM\/Home>), which is a tool able to model most of the experimental steps. Indeed, it takes into account reverse transcription, fragmentation, adapter ligation, PCR amplification, gel segregation and sequencing.\n\nWe ran Flux Simulator with the default file of parameters suggested for H. Sapiens (see Section 5.2 of the user manual at <http:\/\/sammeth.net\/confluence\/pages\/viewpage.action?pageId=786691>) where we only changed the number of molecules (*NB*\\_*MOLECULES*), the number of reads (*READ*\\_*NUMBER*) and the read length (*READ*\\_*LENGTH*) to achieve the desired sparsity. We used the error model for reads of length 76 bp provided in the software, as suggested in the user manual, because the simulator scales the error profile to the chosen read length not explicitly supported by the model.\n\nAs output Flux Simulator returns a.pro file containing for each transcript the number of simulated reads originating from it and its length in bp. Therefore, for each transcript the \"true\" abundance was evaluated in terms of FPKM (Fragments Per Kilobase of transcript per Million of mapped fragments). Transcripts not originating any simulated read were considered as not expressed.\n\n## Simulation scheme\n\nHuman genome (Hg19, UCSC) was considered as reference organism and release 69 annotation file was downloaded from Ensembl database \\[46\\]. For simplicity, we took into account only transcripts coding for proteins (i.e., 142692 potential transcripts in total).\n\nTwo experimental set-ups were simulated using Flux Simulator: **Set-up 1**, in which all transcripts from chromosome 1 were considered as Complete Annotation (CA), i.e., CA contains 13123 transcripts; **Set-up 2**, in which we considered a subsample of 85615 transcripts uniformly sampled from the list of all protein-coding transcripts as CA (i.e., CA contains 85615 transcripts). These scenarios were used to investigate the capability of the compared methodologies to deal with \"small\" and \"large\" genomes.\n\nFor **Set-up 1** Flux Simulator generated a large dataset of (strand-specific) PE reads of 100 bp per side and a set of 3726 transcripts with positive FPKM; for **Set-up 2** it generated a dataset of (strand-specific) PE reads of 75 bp per side and a set of 17032 transcripts with positive FPKM. Fastq files of reads underwent to a filtering process to remove those pairs that had one of the two sides smaller than 100 bp in **Set-up 1**, and one of the two sides smaller than 75 bp in **Set-up 2**, leading to a number of 31177152 PE fragments for **Set-up 1** and to a number of 74365564 PE fragments for **Set-up 2**.\n\nTo investigate the depth effect, in **Set-up 1** the simulated fragments were sub-sampled to obtain six subsets of cardinality 20M, 10M, 5M, 1M, 0.5M and 0.25M, where M stands for 10^6^ reads. To study the read-length effect, for each of the six subsets the reads were trimmed to obtain analogous sets of PE fragments of length 75 bp and 50 bp per side. Finally, to account for the library type effect, for each of the six sets of PE of 100 bp per side, only the left-mate reads were retained to obtain analogous sets of SE reads of length 100 bp. Analogously, in **Set-up 2** the dataset of PE reads was sub-sampled to obtain a subset of cardinality 60M, from which a second set was generated by trimming the reads at 50 bp.\n\nSummarizing, overall eighteen PE datasets and six SE datasets were obtained under **Set-up 1** and two datasets under **Set-up 2**. Set-up 2 was also analyzed for depths of 40M and 20M (both at 75 bp and 50 bp). However, such results are not showed here for the sake of brevity.\n\nTo investigate the ability of different methods for transcript identification at different abundance levels, isoforms were divided in high, medium and low expression classes, where (analogously to iReckon) the low class is given by the isoforms whose true expression belong to the lower 5% of the FPKM distribution, the high class by isoforms with expression larger than the 74% of the FPKM distribution, the remaining ones representing the medium class.\n\nFor the sake of completeness, we also generated an Incomplete Annotation (IA). IA was obained from the corresponding CA selecting 70% of the annotated transcripts (i.e. 9186 in **Set-up 1** and 59930 in **Set-up 2**). In **Set-up 1**, IA was aimed to mimic a normal condition where most of the not annotated isoforms are present at low abundance in the RNA sample. Therefore, IA contains about 70*%* of the non expressed transcripts and the remaining 30*%* of expressed transcripts. In particular, the highly expressed transcripts represent 93% of the true high class, the moderately expressed transcripts represent the 64% of the true medium class and the lowly expressed transcripts represent only the 30% of the true low class. On the contrary, in **Set-up 2**, IA was obtained by randomly sampling the 70% of isoforms from the corresponding CA, regardless of their expression, to mimic the situation where tissue specific conditions or pathologies can alter the normal expression profile and produce novel transcripts that are present at any expression level.\n\n## Read alignment\n\nPE reads of each dataset were aligned to the human reference genome by using TopHat2 \\[29\\] with option **\u2013library-type** fr-secondstrand turned on to benefit of the strand information of the simulated reads.\n\nIn particular, TopHat2 can be used with option **-G** turned on (i.e., by adding -G annotation.gtf to the command line). In this case, first, TopHat2 extracts the transcript sequences and uses Bowtie2 to align reads to this virtual transcriptome. Then, only the reads that do not fully map to the transcriptome are mapped to the reference genome where potential novel splice sites are identified. The reads mapped on the transcriptome are converted to genomic mappings (spliced as needed) and merged with the novel mappings and junctions in the final TopHat2 output. By contrast, if the option is turned off (i.e., -G is not used), TopHat2 aligns the reads directly to the genome and it searches for junctions with a data dependent approach \\[28\\]. It is clear that providing the annotation file allows TopHat2 to better map the splice junctions and cover the entire exon body, and to reduce the number of FP junctions, see \\[29\\] for a more detailed discussion.\n\nFor the scope of our analysis, when the option -G was turned on, we ran TopHat2 alignment on each set of PE reads twice. The first time, we provided CA. The second time, we provided IA. We also ran TopHat2 with the option -G turned off. For each set of the SE reads in **Set-up 1**, we repeated the alignment with TopHat2 using the same scheme.\n\n## Modes of action\n\nIn our study, the problem of isoform detection and quantification is analyzed under different modes of action: **Mode 1)** The method assumes that annotation is available and the algorithm is forced to quantify only those isoforms in the given annotation. Those isoforms that are not present in the annotation are set to zero. **Mode 2)** The method assumes that annotation is available, but it could be incomplete. Therefore, the algorithm uses the provided annotation as a guide in order to find potentially new isoforms. After that all potential isoforms are identified, their expressions are quantified. **Mode 3)** The method assumes that no information is available. Therefore, all potential isoforms are computed from the data. Then, their expressions are quantified.\n\nIt is important to note that the modes of action can be combined with the different type of available annotation. In particular, under Mode 1 and 2 we can further distinguish the case that the available annotation is CA or IA. The case of Mode 1 with CA represents an ideal situation, when everything else is known. Such scenario is rarely met given our current knowledge of Biology, but can be considered as limit case for studies in the near future. The case of Mode 2 with IA represents a realistic scenario in current studies. Indeed, it is true that previous projects have disclosed most information, but still data emerging from the literature shows that such information represents only partial knowledge. Therefore, IA in Mode 2 represents a more realistic situation. For the sake of completeness, we observe that the usage of the methods under Mode 1 with IA will not allow to recover the not-given transcripts. Analogously, all novel transcripts detected by any method in Mode 2 with CA will be false positives. In both cases we are aimed to evaluate how such drawbacks can affect the estimation of other isoforms. Finally, Mode 3 is considered to illustrate the expected results that one can obtain when studying novel sequenced organisms for which no previous information is available (or when the user does not want to use it) and all inference has to be carried out from the experimental data. Moreover, comparing our simulation scheme with the analyses carried out in \\[24\\], we observe that their results correspond to Mode 3 without annotation, except for iReckon and SLIDE that were used similar to our Mode 2.\n\nFigure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a> illustrates the simulation pipeline built for the two experimental set-ups.\n\n## Compared algorithms\n\nIn this paper, we assess the performance of five different methods: CEM, Cufflinks, iReckon, RSEM and SLIDE. All of them were used with mostly their default values and with modes of action illustrated in Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>. We observe that Cufflinks and CEM can perform all modes of action, while the other methods only some of them. All methods where compared in **Set-up 1** for PE reads. All methods, except iReckon, were compared in **Set-up 1** for SE reads. All methods, except SLIDE, were compared in **Set-up 2**.\n\nSoftware used in the comparison\n\n| **Name** | **Version** | **Modes of action** | **Link** | **Publication** |\n|:---|:--:|:--:|:--:|:--:|\n| TopHat2 | 2.07 | CA\/IA, no annotation | <http:\/\/tophat.cbcb.umd.edu\/> | \\[28,29\\] |\n| RSEM | 1.2.3 | 1 | <http:\/\/deweylab.biostat.wisc.edu\/rsem\/> | \\[19,35\\] |\n| Cufflinks | 2.0.2 | 1,2,3 | <http:\/\/cufflinks.cbcb.umd.edu\/> | \\[20\\] |\n| SLIDE | May 7th, 2012 | 1,2 | <https:\/\/sites.google.com\/site\/jingyijli\/> | \\[21\\] |\n| CEM | 0.9.1 | 1,2,3 | <http:\/\/alumni.cs.ucr.edu\/~liw\/cem.html> | \\[23\\] |\n| iReckon | 1.0.7 | 2 | <http:\/\/compbio.cs.toronto.edu\/ireckon\/> | \\[22\\] |\n\nTable <a href=\"#T1\" data-ref-type=\"table\">1<\/a> shows detailed information for the software used during the study.\n\n### CEM\n\nCEM \\[23\\] is a recent command line program written in C++ and Python developed by the authors of IsoLASSO \\[39\\] of which it constitutes a significant improvement. Its logic is very similar to the one of Cufflinks. Indeed the only required argument is the sam\/bam alignment file. In this case, it executes Mode 3. The assembly problem is solved via a *connectivity graph*, which is more general than the *overlap graph* implemented in Cufflinks. By using optional parameter **-x**, the user can specify the annotation file (in BED format) and execute Mode 1 or Mode 2. If **\u2013forceref** is turned on (i.e., -x annotation.bed \u2013 forceref), CEM will run in Mode 1. If the option **\u2013forceref** is turned off (i.e., -x annotation.bed), the existing gene annotation will be incorporated into the estimation process as a guide from which CEM assembles new isoforms. Regardless of the action modes, the estimation of transcript abundance is carried out by minimizing a lasso penalized squared data-fit loss, where data-fit is given modeling the coverage in each segment as a Poisson distribution whose intensity is proportional to the mixture of abundances of the isoforms that insist on the same segment. With respect to this point, the main difference between CEM and its parent, IsoLASSO, consists in the algorithm used to perform minimization: CEM uses the Expectation-Maximization (EM) algorithm instead of quadratic programming. As a consequence CEM results by far more efficient than IsoLASSO, and overall one of the most efficient algorithm in terms of computational cost. No explicit parameter is available for strand specificity. CEM supports both SE and PE dataset.\n\n### Cufflinks\n\nCufflinks \\[20\\] is a popular software developed by the authors of TopHat and Bowtie and is part of the Tuxedo pipeline \\[15\\]. It is a command line tool, written in C++, where the only required argument is the sam\/bam alignment file. In this case, it executes Mode 3. In particular, Cufflinks reduces the comparative assembly problem to a maximum matching problem in bipartite graphs and solves it by using the so call *overlap graph* approach. On the contrary, when using the optional parameters **-G** (i.e., -G annotation.gtf) or **-g** (i.e., -g annotation.gtf) the user can execute Mode 1 and Mode 2, respectively. Cufflinks was also used with the option **-u** turned on that allows an initial estimation procedure to assign more accurately those reads that mapped to multiple locations in the genome. Given the set of newly identified or annotated transcripts, for all modes of action, the transcript abundance is estimated via a Maximum Likelihood approach, where the probability of observing each fragment is modeled as a linear function of the transcript abundance that can originate the fragments. Because of linearity, the likelihood function has a unique maximum value that Cufflinks finds via a numerical optimization algorithm. Finally, we also observe that when the reads are aligned by TopHat by using strand-specific mode, Cufflinks will automatically treat data as strand-specific (otherwise library type has to be explicitly specified by the user). Cufflinks supports both SE and PE dataset.\n\n### iReckon\n\niReckon is a java software which implements the method presented in \\[22\\]. The input of iReckon are aligned reads, genome, annotation and reads themselves. Genome and annotation have to be provided in a house format obtained from Fasta and BED format after conversion in Savant \\[47\\]. The original reads and the genome files are necessary since after the construction of an enlarged transcriptome (i.e., all possible isoforms including pre-mRNA and isoforms with retained intron) from the mapped reads, the estimation is done by re-aligning the reads (using BWA 0.6.2, \\[48\\]) on the sequences of all possible isoforms. In principle iReckon could execute both in Mode 1 and Mode 2. Nevertheless, we used only Mode 2 (which is the iReckon's default approach), due to a potential bug appearing when executing version 1.07 with the option **-novel 0** that was forcing the method in quantifying only transcripts in the annotation.\n\nThe main advantage of iReckon is that it directly models (and to date is the only one to have this feature) multiple biological and technical phenomena, including novel isoforms, intron retention, unspliced pre-mRNA, PCR amplification biases, and multi-mapped reads. iReckon utilizes the EM algorithm with a new non linear regularization penalty to accurately estimate the abundance of known and novel isoforms. The reason is that abundances are very similar to frequencies being non negative and summing to a normalization constant.\n\nFor large datasets and genomes, iReckon requires a large memory space and execution time. Most of the running time is due to the re-alignment step. No additional parameter is available for strand specificity. iReckon only supports PE dataset.\n\n### RSEM\n\nRSEM \\[19\\] is a software package which implements the method originally presented in \\[35\\]. It is a command line program, written mainly in C++, with contributions in Perl and R. In contrast with previous methods, RSEM can only perform the estimation of isoform abundance given an annotation file, i.e. it can only work under Mode 1. As input data, RSEM requires either both the genome sequence and the annotation (in fasta and.GTF format) or the transcript sequences directly, and the read file. As first step, the reads are aligned directly to the transcript sequences using Bowtie (function **rsem-prepare-reference**); then abundances are estimated via an EM algorithm based on a generative statistical model that handle read mapping uncertainty (function **rsem-calculate-expression**). In particular, it uses an iterative process to fractionally assign reads to each transcript considering the probabilities of the reads being derived from each transcript and taking into account positional biases created by RNA-seq library-generating protocols. The interest in RSEM is that, although it can quantify only the known transcripts contained in the annotation file, if the annotation file includes potential novel transcripts or ab-initio estimates transcripts, the quantification can be performed as well. In this context it comes bundled with the Trinity software \\[26\\] where it is used to quantify the novel assembled transcripts. We used RSEM software with the **\u2013strand-specific** option activated. RSEM supports both SE and PE dataset.\n\n### SLIDE\n\nSLIDE (Sparse Linear modeling for Isoform Discovery and abundance Estimation) is a software mainly written in Python (with some R scripts) that implements the statistical method described in \\[21\\]. It can be used either in Mode 1 (**\u2013mode estimation**) and in Mode 2 (**\u2013mode discovery**). In particular, in Mode 2, SLIDE defines all the possible 2 ^*n*^ \u22121 transcripts obtained by enumerating the *n* sub-exons of each gene.\n\nIn contrast to the other methods, SLIDE does not work on empirical read coverage, but it associates to each PE fragment four genomic locations corresponding to the starting and ending position of its 5' and 3' reads, respectively, and converts such positions into four sub-exon indexes. Then, it computes the fraction of pairs whose genomic locations span a given bin (i.e., a combination of four given sub-exons, for all sub-exon combination). Subsequently, it uses a linear model to approximate the observed bin proportions in terms of isoform proportions, which represent the parameters to be estimated. SLIDE estimates isoform abundance with a non negative least squares solution of a linear model. The design matrix models the conditional probability of sampling a PE read in a bin given that it comes from an isoform. A modified lasso type approach is used to limit the number of non null isoforms as well as to favor longer isoforms.\n\nFinally, we observe that SLIDE has been mainly designed for relative small genomes. Therefore, the code has not been optimized. For this reason we compared its performance only on a small scale comparison (i.e., **Set-up 1**). In this case we considered both the higher confident output denoted, as \"less\", and the larger one, denoted by \"more\", similar to the original annotation. No explicit parameter is available for strand specificity. SLIDE supports both SE and PE dataset.\n\n## Novel isoform matching\n\nAll methods used in Mode 1 (i.e. Cufflinks -G; RSEM; CEM - forceref and SLIDE - mode estimation) directly estimate FPKM of each isoform given in the annotation (CA or IA). Therefore, the association between the estimated value and the true value is straightforward. On the contrary, methods used in Mode 2 or Mode 3 allow to discover (new) isoforms. Therefore, their output need to be further processed in order to properly associate the inferred isoforms with the true ones. To this purpose, Cuffcompare v2.1.1 (which is part of the Cufflinks suite) was used to associate the output of any considered method (usually a gtf file) with the true CA. In particular, a 'true' isoform name was associated to an assembled isoform whenever a complete match of the intron chain was observed (i.e, Class Code ' =' in \\<cuff_in\\>.tmap file, see user manual of \\[20\\] for details). Such level of transcript matching is quite stringent and could be relaxed, since in some cases we noticed that the match in Mode 2 and 3 was achieved at lower level of stringency (for example transcripts were contained in the true ones or other Class Codes were returned by Cuffcompare). However, the choice of using stringent match does not change the conclusions, but only the actual values of the performance indexes. The assembled transcripts which did not match an annotated one were classified as novel. In few cases, more assembled isoforms were associated to the same annotated isoform. Then, the estimated FPKM of the isoform was evaluated as the sum of all estimated FPKMs. Similarly to \\[24\\], for iReckon, transcripts with intron retention or unspliced events were not considered.\n\n## Measures of performance\n\nIn order to measure the performance of the considered methods, we first evaluated their capability in isoform detection in terms of true positives (TP), false positives (FP) and false negatives (FN), then their accuracy in isoform estimation in terms of estimation error.\n\nFor isoform detection, the following indicators were computed:\n\n\u25cf\u2003**Recall** (aimed at measuring the fraction of truly expressed isoforms that is retrieved) defined as\n\n$$\\text{recall} = \\frac{\\textit{TP}}{\\textit{TP} + \\textit{FN}} = \\frac{\\left| {\\left\\{ {\\overset{\\hat{}}{\\textit{FPKM}} > 0} \\right\\} \\cap \\left\\{ \\textit{FPKM} > 0 \\right\\}} \\right|}{\\left| \\left\\{ \\textit{FPKM} > 0 \\right\\} \\right|}.$$\n\n\u25cf\u2003**Precision** (aimed at measuring the fraction of predicted expressed isoforms that are truly expressed) defined as\n\n$$\\mspace{-2700mu}\\text{precision} = \\frac{\\textit{TP}}{\\textit{TP} + \\textit{FP}} = \\frac{\\left| {\\left\\{ {\\overset{\\hat{}}{\\textit{FPKM}} > 0} \\right\\} \\cap \\left\\{ \\textit{FPKM} > 0 \\right\\}} \\right|}{\\left| \\left\\{ {\\overset{\\hat{}}{\\textit{FPKM}} > 0} \\right\\} \\right|}.$$\n\nwhere $\\overset{\\hat{}}{\\textit{FPKM}}$ represents the estimated abundance of the isoform, *FPKM* is the true abundance and \\|*S*\\| stands for cardinality of set S. If *FPKM*\\>0 the isoform is truly expressed, while if *FPKM*=0 the isoform is not expressed, analogously for the estimated values. Obviously, recall is a measure of completeness and precision is a measure of accuracy. Recall was also evaluated on abundance classes (low, medium and high) defined as described in Section Simulation scheme.\n\nAs a global measure of performance we also considered the following\n\n\u25cf\u2003**F-Measure** defined as\n\n$$F = \\frac{2 \\ast \\text{precision} \\ast \\text{recall}}{\\text{precision} + \\text{recall}}.$$\n\nFor evaluating the accuracy in abundance estimation, we distinguished three cases and considered\n\n\u25cf\u2003**Estimation Error** (aimed at quantifying the FPKM retrieval accuracy) defined as\n\n$$\\mspace{-3060mu}\\textit{error}\\; = \\;\\left\\{ {\\;\\;\\begin{array}{lll}\n{E_{1} = \\frac{\\overset{\\hat{}}{\\textit{FPKM}} - \\textit{FPKM}}{\\textit{FPKM}}} & \\textit{if} & {\\textit{FPKM} > 0\\;\\quad\\text{and}\\;\\quad\\overset{\\hat{}}{\\textit{FPKM}} > 0} \\\\\n{E_{2} = \\overset{\\hat{}}{\\textit{FPKM}}} & \\textit{if} & {\\textit{FPKM} = 0\\quad\\;\\text{and}\\quad\\;\\overset{\\hat{}}{\\textit{FPKM}} > 0} \\\\\n{E_{3} = \\textit{FPKM}} & \\textit{if} & {\\textit{FPKM} > 0\\;\\quad\\text{and}\\;\\quad\\overset{\\hat{}}{\\textit{FPKM}} = 0}\n\\end{array}} \\right)\\;\\;,$$\n\nwhere *E*~1~ quantifies the (relative) accuracy in estimating the expressed isoforms that the method is able to identify, *E*~2~ quantifies the abundances assigned to FP isoforms and *E*~3~ quantifies loss of expression for FN isoforms.\n\nThe last, but not the least, important thing to be considered is the computational cost. Since algorithms are implemented in different languages and can be used on different computational architectures that can benefit or not of parallelism, we believe that any precise quantification of computational cost would be not fair. Therefore, this point will be only discussed from qualitative point of view in Section Results and discussions.\n\n# Results and discussions\n\nIn the following, we first compare the methods in terms of their capability in isoform detection, then in terms of their accuracy in isoform estimation. We stress that the goal of the comparison is not to make a rank list of the considered methods, but to underline global positive aspects, common weaknesses and open problems that might lead to over-optimistic conclusions about the performance of current methodology.\n\n## Isoform detection\n\nHere, we illustrate the results in terms of recall, precision and F-measure considering the following effects: type of alignment, modes of action, type of annotation, type of library, abundance level, read length and sequencing depth. In order to investigate such effects, the same figures have to be inspected several times evaluating different aspects each time. To facilitate such comparison, we first describe the general structure of the figures, then we focus the attention on some specific comparison.\n\nFigures <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>, <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>, <a href=\"#F4\" data-ref-type=\"fig\">4<\/a> and <a href=\"#F5\" data-ref-type=\"fig\">5<\/a> illustrate results for precision and recall obtained in **Set-up 1** for libraries 100 bp-PE, 75 bp-PE, 50 bp-PE and 100 bp-SE, respectively. For each of these cases, recall is further expanded with respect to the level of abundance of the true isoforms and results are reported in Figures <a href=\"#F6\" data-ref-type=\"fig\">6<\/a>, <a href=\"#F7\" data-ref-type=\"fig\">7<\/a>, <a href=\"#F8\" data-ref-type=\"fig\">8<\/a> and <a href=\"#F9\" data-ref-type=\"fig\">9<\/a> in the same order. Finally, F-measure is illustrated in Additional file <a href=\"#S1\" data-ref-type=\"supplementary-material\">1<\/a>: Figure S1, Additional file <a href=\"#S2\" data-ref-type=\"supplementary-material\">2<\/a>: Figure S2, Additional file <a href=\"#S3\" data-ref-type=\"supplementary-material\">3<\/a>: Figure S3 and Additional file <a href=\"#S4\" data-ref-type=\"supplementary-material\">4<\/a>: Figure S4 in the same order for each of the four cases.\n\nIn Figures <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>, <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>, <a href=\"#F4\" data-ref-type=\"fig\">4<\/a> and <a href=\"#F5\" data-ref-type=\"fig\">5<\/a> results are visually depicted into four panels, A (upper left), B (upper right), C (bottom left) and D (bottom right). Panels A and B refer to the annotation driven alignment; panels C and D to the data driven alignment. Precision is depicted in panels A and C, while recall in panels B and D. Within each panel, the plot is divided in six sub-blocks according to the modes of action and the type of annotation. In particular, on the left blocks (Mode 1) there are RSEM, Cufflinks with the -G option turned on, CEM with the -forceref option turned on, SLIDE with -mode estimation. In the central blocks (Mode 2) there are iReckon, Cufflinks with the option -g turned on, CEM with the option -forceref turned off, SLIDE with -mode discovery. Finally, in the right blocks (Mode 3) there are only Cufflinks and CEM with all default options. Results obtained using CA\/IA are depicted in the first\/second row of each panel, respectively. In **Set-up 1** different bars for the same method and mode of action correspond to the different sequencing depth (0.25M, 0.5M, 1M, 5M, 10M and 20M, respectively). Dashed horizontal lines are added to facilitate comparisons among different cases. Since RSEM does not depend on the alignment strategy, for comparative purposes, panels A and C report the same precision and panels B and D the same recall, in correspondence of the same type of annotation for RSEM. Analogously, within each panel, methods in Mode 3 show the same precision and recall (within the upper and bottom rows of each panel), not depending on the provided annotation when the alignment is data driven.\n\nFigures <a href=\"#F6\" data-ref-type=\"fig\">6<\/a>, <a href=\"#F7\" data-ref-type=\"fig\">7<\/a>, <a href=\"#F8\" data-ref-type=\"fig\">8<\/a> and <a href=\"#F9\" data-ref-type=\"fig\">9<\/a> have a similar organisation. In particular, CA is used in panels A and C, IA in panels B and D. Within each panel, the plot is divided in nine sub-blocks. Along the horizontal direction the organization is analogous to that of Figures <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>, <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>, <a href=\"#F4\" data-ref-type=\"fig\">4<\/a> and <a href=\"#F5\" data-ref-type=\"fig\">5<\/a>, in the vertical direction recall is reported for the low, medium and high abundance classes, respectively.\n\nAdditional file <a href=\"#S1\" data-ref-type=\"supplementary-material\">1<\/a>: Figure S1, Additional file <a href=\"#S2\" data-ref-type=\"supplementary-material\">2<\/a>: Figure S2, Additional file <a href=\"#S3\" data-ref-type=\"supplementary-material\">3<\/a>: Figure S3 and Additional file <a href=\"#S4\" data-ref-type=\"supplementary-material\">4<\/a>: Figure S4 are also organized in four panels and illustrate the F-measure with respect the sequencing depth. Panels A and B refer to the annotation driven alignment; panels C and D to the data driven alignment. CA is used in panels A and C, IA in panels B and D. To better distinguish results among different modes of action, results in Mode 1 are shown with continuous lines, those in Mode 2 are reported in dashed lines, those in Mode 3 are reported in dotted lines.\n\nFor the sake of completeness, we also provide Additional file <a href=\"#S5\" data-ref-type=\"supplementary-material\">5<\/a>: Figure S5, Additional file <a href=\"#S6\" data-ref-type=\"supplementary-material\">6<\/a>: Figure S6, Additional file <a href=\"#S7\" data-ref-type=\"supplementary-material\">7<\/a>: Figure S7 and Additional file <a href=\"#S8\" data-ref-type=\"supplementary-material\">8<\/a>: Figure S8 showing the number of TP and FP obtained under **Set-up 1**, for the most extreme conditions PE vs SE, 20M vs 0.25M. The figures are again organized in four panels where panels A and B refer to the annotation driven alignment; panels C and D to the data driven alignment. CA is used in panels A and C, IA in panels B and D. Within each panel, the right bars (i.e., the one depicted in coral) show the number of FP and the left ones (depicted in aquamarine) the number of FN. An horizontal dashed line is added representing the number of truly expressed isoforms. Therefore, the difference between the number of FP and this line represents the number of FN.\n\nTo provide a better insight into the capability of methods in Mode 2 (with IA) to identify those isoforms that are not provided in the annotation, Figures <a href=\"#F10\" data-ref-type=\"fig\">10<\/a> and <a href=\"#F11\" data-ref-type=\"fig\">11<\/a> show the number of TP (with the annotation driven alignment and with data driven alignment, respectively). In these figures, the number of TP is divided in those already present in IA (denoted IA) and those not contained in IA (denoted No IA). The latter are further divided according to the true expression level. Finally, Figure <a href=\"#F12\" data-ref-type=\"fig\">12<\/a> illustrates the performance of Cufflinks and CEM (Mode 1) when a suitable threshold is applied to set to zero isoforms with very low estimated FPKM. The figure compares precision, recall and F-measure with the corresponding indexes observed for the same methods in Mode 2.\n\nAdditional file <a href=\"#S9\" data-ref-type=\"supplementary-material\">9<\/a>: Figure S9, Additional file <a href=\"#S10\" data-ref-type=\"supplementary-material\">10<\/a>: Figure S10, Additional file <a href=\"#S11\" data-ref-type=\"supplementary-material\">11<\/a>: Figure S11, Additional file <a href=\"#S12\" data-ref-type=\"supplementary-material\">12<\/a>: Figure S12, Additional file <a href=\"#S13\" data-ref-type=\"supplementary-material\">13<\/a>: Figure S13 and Additional file <a href=\"#S14\" data-ref-type=\"supplementary-material\">14<\/a>: Figure S14 show results for **Set-up 2**. In particular, Additional file <a href=\"#S9\" data-ref-type=\"supplementary-material\">9<\/a>: Figure S9, Additional file 10: Figure S10, Additional file <a href=\"#S11\" data-ref-type=\"supplementary-material\">11<\/a>: Figure S11, Additional file <a href=\"#S12\" data-ref-type=\"supplementary-material\">12<\/a>: Figure S12 are devoted to precision and recall. Additional file <a href=\"#S13\" data-ref-type=\"supplementary-material\">13<\/a>: Figure S13 is the analogous of Figure <a href=\"#F12\" data-ref-type=\"fig\">12<\/a> and Additional file <a href=\"#S14\" data-ref-type=\"supplementary-material\">14<\/a>: Figure S14 is the analogous of Additional file <a href=\"#S5\" data-ref-type=\"supplementary-material\">5<\/a>: Figure S5, Additional file <a href=\"#S6\" data-ref-type=\"supplementary-material\">6<\/a>: Figure S6, Additional file <a href=\"#S7\" data-ref-type=\"supplementary-material\">7<\/a>: Figure S7 and Additional file <a href=\"#S8\" data-ref-type=\"supplementary-material\">8<\/a>: Figure S8.\n\nAll methods have been evaluated under the two set-ups, except SLIDE, that for the high computational cost has been evaluated only under **Set-up 1**, and iReckon, that was evaluated only for PE reads, since it does not support SE reads. In Figures <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>, <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>, <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>, <a href=\"#F4\" data-ref-type=\"fig\">4<\/a>, <a href=\"#F5\" data-ref-type=\"fig\">5<\/a>, <a href=\"#F6\" data-ref-type=\"fig\">6<\/a>, <a href=\"#F7\" data-ref-type=\"fig\">7<\/a>, <a href=\"#F8\" data-ref-type=\"fig\">8<\/a> and <a href=\"#F9\" data-ref-type=\"fig\">9<\/a>, Additional file <a href=\"#S1\" data-ref-type=\"supplementary-material\">1<\/a>: Figure S1, Additional file <a href=\"#S2\" data-ref-type=\"supplementary-material\">2<\/a>: Figure S2, Additional file <a href=\"#S3\" data-ref-type=\"supplementary-material\">3<\/a>: Figure S3 and Additional file <a href=\"#S4\" data-ref-type=\"supplementary-material\">4<\/a>: Figure S4 and Additional file <a href=\"#S9\" data-ref-type=\"supplementary-material\">9<\/a>: Figure S9, Additional file 10: Figure S10, Additional file <a href=\"#S11\" data-ref-type=\"supplementary-material\">11<\/a>: Figure S11 and Additional file <a href=\"#S12\" data-ref-type=\"supplementary-material\">12<\/a>: Figure S12, Cufflinks is coloured in red, CEM is coloured in blue, SLIDE is coloured in orange (when present), RSEM is coloured in green and iReckon is coloured in brown (when present).\n\nRSEM shows relatively good performance when CA is provided, but its inference is limited to annotated transcripts (or to a list of potential transcripts that the user has to provide). On the other side, SLIDE shows worse performance than the others (in particular, in Mode 2) suggesting that it could be better suited for genome with lower complexity in terms of isoform structures.\n\nOverall Set-ups 1 and 2 show similar qualitative results. The best F-measure in **Set-up 1** was about 0.78, in **Set-up 2** was 0.75. In both cases, the best performance was achieved by RSEM in the CA case.\n\nLooking at Figures <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>, <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>, <a href=\"#F4\" data-ref-type=\"fig\">4<\/a> and <a href=\"#F5\" data-ref-type=\"fig\">5<\/a>, precision rarely exceeds 0.6\u20130.7 and it is often below such values, meaning that all methods produce a quite large number of FP isoforms (remarkably, even when the true CA is provided and the methods are forced to work in Mode 1). Such drawback is also confirmed by the inspection of Additional file <a href=\"#S5\" data-ref-type=\"supplementary-material\">5<\/a>: Figure S5, Additional file <a href=\"#S6\" data-ref-type=\"supplementary-material\">6<\/a>: Figure S6, Additional file <a href=\"#S7\" data-ref-type=\"supplementary-material\">7<\/a>: Figure S7 and Additional file <a href=\"#S8\" data-ref-type=\"supplementary-material\">8<\/a>: Figure S8, where we often observe a high number of FP (compared to TP) for the same set-up. These poor results can be due to a non sufficiently strong penalty term (or to a not sufficient strong post-filtering step) that should keep to zero not significant isoforms.\n\nThe best precision is achieved by Cufflinks in Mode 2 with CA (at a price of lower power). Surprisingly, Cufflinks (Mode 1) shows a worse precision. However, we noticed that most of the isoforms detected as presents by Cufflinks (Mode 1) were estimated with extremely low expression. Therefore, if we filter out these isoforms, precision in Mode 1 and Mode 2 becomes comparable (see Figure <a href=\"#F12\" data-ref-type=\"fig\">12<\/a>, left panel), as expected. For example, by setting to zero all isoforms with estimated $\\overset{\\hat{}}{\\textit{FPKM}} < 10^{- 5}$ the Cufflinks (Mode 1) precision increases to 0.59, without significantly reducing the recall (analogous behaviour with 10^\u22121^as threshold). Indeed, the F-measure of Cufflinks (Mode 1) with a threshold on the expressed isoforms becomes better than the Cufflinks (Mode 2) one, as expected. The tendency to produce a high number of FP with very low expression level was observed also for CEM (Mode 1), but with a much less pronounced effect (see Figure <a href=\"#F12\" data-ref-type=\"fig\">12<\/a>, right panel). The same effect occurs for the other conditions of depth, read length and library type (data not showed).\n\nOverall recall is above 0.80 when methods are used in Mode 1 with CA (and above 0.6 with IA), while recall does not exceed 0.3 in Mode 3. Recalls in Mode 1 are mostly satisfactory (except for SLIDE). The actual observed recall values for Mode 2 and Mode 3 depend on the stringency of the match between newly identified isoforms and existing ones. However, the low recall values in Mode 3 show that the performance of methods in recovering the precise isoform structure is still not satisfactory.\n\nFigures <a href=\"#F6\" data-ref-type=\"fig\">6<\/a>, <a href=\"#F7\" data-ref-type=\"fig\">7<\/a> and <a href=\"#F8\" data-ref-type=\"fig\">8<\/a> for PE and Figure <a href=\"#F9\" data-ref-type=\"fig\">9<\/a> for SE datasets illustrate how well methods are able to detect highly expressed isoforms (remarkably, recall is mostly higher than 0.50 also for methods in Mode 3). At the same time they underline that the major problem arises in identifying lowly expressed isoforms (recall rarely exceeds 0.2 either in Mode 2 and Mode 3). In particular, we observe that lowly expressed isoforms are identified mainly in Mode 1 with CA. Methods in Mode 2 with IA are able to identify lowly expressed isoforms mainly if they were already present in IA. On the contrary, for moderately and highly expressed isoforms they are capable of detecting isoforms not provided in IA, see Figures <a href=\"#F10\" data-ref-type=\"fig\">10<\/a> and <a href=\"#F11\" data-ref-type=\"fig\">11<\/a>. The figures show good results for Cufflinks and iReckon for medium and high expression classes, at high depth and regardless the alignment type.\n\nThe performance clearly drops down when decreasing the depth. In that case, it becomes almost impossible to recover isoforms that have not been provided in the annotation at the beginnig.\n\nAs mentioned above, **Set-up 2** bring us to analogous overall considerations, see Additional file <a href=\"#S9\" data-ref-type=\"supplementary-material\">9<\/a>: Figure S9, Additional file 10: Figure S10, Additional file <a href=\"#S11\" data-ref-type=\"supplementary-material\">11<\/a>: Figure S11, Additional file <a href=\"#S12\" data-ref-type=\"supplementary-material\">12<\/a>: Figure S12, Additional file <a href=\"#S13\" data-ref-type=\"supplementary-material\">13<\/a>: Figure S13 and Additional file <a href=\"#S14\" data-ref-type=\"supplementary-material\">14<\/a>: Figure S14.\n\n### Effect of alignment\n\nIn order to evaluate the effect of the alignment, we compared results obtained by the same method and mode of action across different alignment strategies, i.e., we investigate its effect on precision by comparing (in Figures <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>, <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>, <a href=\"#F4\" data-ref-type=\"fig\">4<\/a> and <a href=\"#F5\" data-ref-type=\"fig\">5<\/a>) panel A versus panel C, and on recall by comparing panel B versus panel D, for **Set-up 1**. To evaluate the global effect on the F-measure, it is sufficient to carry out the analogous comparison for Additional file <a href=\"#S1\" data-ref-type=\"supplementary-material\">1<\/a>: Figure S1, Additional file <a href=\"#S2\" data-ref-type=\"supplementary-material\">2<\/a>: Figure S2, Additional file <a href=\"#S3\" data-ref-type=\"supplementary-material\">3<\/a>: Figure S3, and Additional file <a href=\"#S4\" data-ref-type=\"supplementary-material\">4<\/a>: Figure S4. Clearly, RSEM is not affected by the alignment, since it maps the reads directly to the transcriptome. Therefore, its results differ only with respect to CA and IA within panels A and B.\n\nOverall comparisons show no appreciable difference in precision for Mode 1, a slight difference in Mode 2, where the precision increases along with the quality of mapping, and more remarkable improvements in Mode 3. Analogous results can be observed for recall. Moreover, both differences in precision and recall decrease when the sequencing depth increases. As global measure, we observe that F-measure for 20M 100 bp-PE is about 0.35 and 0.27, respectively, for Cuffinks and CEM in Mode 3 with reads aligned without any annotation; it becomes about 0.40 and 0.34, respectively, when the reads are aligned providing either IA or CA, see Additional file <a href=\"#S1\" data-ref-type=\"supplementary-material\">1<\/a>: Figure S1. To comment this effect, we inspected the alignment files and we observed that the main differences are in the number of mapped junctions. As an example, we observed that the dataset 20M 100 bp-PE identified 14500 junctions when CA was provided and 14479 junctions without annotation, with a very negligible loss due to the alignment. But, with 0.25M 100 bp-PE the number of mapped junctions was 9173 with CA and dropped down to 8047 without annotation. Analogously, for 20M 50 bp-PE, 14290 junctions were detected when CA was provided and only 12643 junctions without annotation; with 0.25M 50 bp-PE, the number of mapped junctions was 8241 with CA and dropped down to 5990 without annotation. The improved performance in Mode 3 can be explained by the fact that data driven methods can deeply benefit from the presence of informative junctions, even though current performances cannot be considered overall satisfactory.\n\nThe same conclusions apply for **Set-up 2**, comparing Additional file <a href=\"#S9\" data-ref-type=\"supplementary-material\">9<\/a>: Figure S9 and Additional file 10: Figure S10. In particular, in Mode 3, the F-measure increases from 0.18 of both Cufflinks and CEM in the case of data driven alignment up to 0.24 and 0.31 respectively in the case of CA based alignment (data not showed).\n\n### Effect of modes of action\n\nIn order to evaluate the effect of the modes of action, results have to be compared across the horizontal blocks of each panel of each figure, see Figures <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>, <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>, <a href=\"#F4\" data-ref-type=\"fig\">4<\/a>, <a href=\"#F5\" data-ref-type=\"fig\">5<\/a>, <a href=\"#F6\" data-ref-type=\"fig\">6<\/a>, <a href=\"#F7\" data-ref-type=\"fig\">7<\/a>, <a href=\"#F8\" data-ref-type=\"fig\">8<\/a> and <a href=\"#F9\" data-ref-type=\"fig\">9<\/a> for **Set-up 1** and Additional file <a href=\"#S9\" data-ref-type=\"supplementary-material\">9<\/a>: Figure S9, Additional file 10: Figure S10, Additional file <a href=\"#S11\" data-ref-type=\"supplementary-material\">11<\/a>: Figure S11 and Additional file <a href=\"#S12\" data-ref-type=\"supplementary-material\">12<\/a>: Figure S12 for **Set-up 2**. Moreover, we have to compare the continuous lines with respect to the dashed lines and the dotted lines in Additional file <a href=\"#S1\" data-ref-type=\"supplementary-material\">1<\/a>: Figure S1, Additional file <a href=\"#S2\" data-ref-type=\"supplementary-material\">2<\/a>: Figure S2, Additional file <a href=\"#S3\" data-ref-type=\"supplementary-material\">3<\/a>: Figure S3 and Additional file <a href=\"#S4\" data-ref-type=\"supplementary-material\">4<\/a>: Figure S4.\n\nAs expected, all methods in Mode 1 have better global performance than those in Mode 2 and perform significantly better than the methods in Mode 3. The latter ones are still a big challenge. Moreover, performance of methods in Mode 1 are much less affected by the depth and the read length, while still benefit of PE reads, if the coverage is not sufficiently high.\n\nAs previously mentioned, Figures <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>, <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>, <a href=\"#F4\" data-ref-type=\"fig\">4<\/a> and <a href=\"#F5\" data-ref-type=\"fig\">5<\/a> show that the precision cannot be considered fully satisfactory and the number of FP is high across all modes of action. In particular, Cufflinks (Mode 1) shows still poor precision. However, in this case, we already noted that most of the FP isoforms were estimated with a very low expression value and filtering out the low $\\overset{\\hat{}}{\\textit{FPKM}}$s restores the performance, see Figure <a href=\"#F12\" data-ref-type=\"fig\">12<\/a>. From Figures <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>, <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>, <a href=\"#F4\" data-ref-type=\"fig\">4<\/a> and <a href=\"#F5\" data-ref-type=\"fig\">5<\/a>, we observe that all methods in Mode 1 using CA have a very good recall, independently from alignment option. Methods in Mode 2 reach good\/sufficient recall, except for SLIDE that shows the worst behaviour. In particular, it seems that SLIDE (Mode 2) does not take advantage of the annotation and behaves similarly to methods in Mode 3.\n\nTo fully understand the action mode effect, it is important to inspect Figures <a href=\"#F6\" data-ref-type=\"fig\">6<\/a>, <a href=\"#F7\" data-ref-type=\"fig\">7<\/a>, <a href=\"#F8\" data-ref-type=\"fig\">8<\/a> and <a href=\"#F9\" data-ref-type=\"fig\">9<\/a>. Here, one can immediately observe that highly expressed isoforms are detectable in all action modes (in particular, for high sequencing depth), the scenario changes dramatically for moderately and lowly expressed ones. The latter have been recognized only in Mode 1 with CA and with sufficient sequencing depth.\n\nThe same conclusions apply for **Set-up 2**, comparing Additional file <a href=\"#S9\" data-ref-type=\"supplementary-material\">9<\/a>: Figure S9, Additional file 10: Figure S10, Additional file <a href=\"#S11\" data-ref-type=\"supplementary-material\">11<\/a>: Figure S11 and Additional file <a href=\"#S12\" data-ref-type=\"supplementary-material\">12<\/a>: Figure S12.\n\nIt follows that, while it is intriguing to promise to reconstruct the whole transcriptome using only RNA-seq data without any information about annotation, researchers have to be very careful in faithfully believe to the results obtained in Mode 3 (using current experimental protocols and computational methodologies analogues to those considered in the present work). As a consequence, for complex genomes, they should consider to complement the results with other sources of either experimental or computational evidence.\n\n### Effect of the annotation\n\nIn order to evaluate the effect of the annotation, in Figures <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>, <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>, <a href=\"#F4\" data-ref-type=\"fig\">4<\/a> and <a href=\"#F5\" data-ref-type=\"fig\">5<\/a> precision and recall have to be compared between the two horizontal blocks in each panel A,B,C e D of the same figure (where the upper row refers to CA and the lower row refers to IA) limiting the attention to Mode 1 and Mode 2. While, in Figures <a href=\"#F6\" data-ref-type=\"fig\">6<\/a>, <a href=\"#F7\" data-ref-type=\"fig\">7<\/a>, <a href=\"#F8\" data-ref-type=\"fig\">8<\/a> and <a href=\"#F9\" data-ref-type=\"fig\">9<\/a> and in Additional file <a href=\"#S1\" data-ref-type=\"supplementary-material\">1<\/a>: Figure S1, Additional file <a href=\"#S2\" data-ref-type=\"supplementary-material\">2<\/a>: Figure S2, Additional file <a href=\"#S3\" data-ref-type=\"supplementary-material\">3<\/a>: Figure S3 and Additional file <a href=\"#S4\" data-ref-type=\"supplementary-material\">4<\/a>: Figure S4, panels A have to be compared with the corresponding panels B; and panels C with the corresponding panels D.\n\nThis effect is very important considering that CA is usually unknown in a real experiment. Therefore, results obtained with CA represent a sort of optimal performance. Results obtained under IA can be seen as a measure of what one can reach with the current knowledge of the Biology.\n\nAs expected, moving from CA to IA, regardless the type of alignment, the performances degrade, with a more evident loss in Mode 1 with respect to Mode 2. The observed difference in the two modes of action allows us to illustrate the discovery power of methods in Mode 2. To this purpose, Figures <a href=\"#F10\" data-ref-type=\"fig\">10<\/a> and <a href=\"#F11\" data-ref-type=\"fig\">11<\/a> show the number of transcripts that were not present in IA, but are still recovered by methods in Mode 2 driven by IA. As mentioned before, the \"recovering\" effect is mainly concentrated on isoforms with high or moderate expression.\n\nIn general, the best F-measure observed in Mode 1 with CA is about 0.8 for the higher depth at 100 bp PE and it drops to about 0.6 when IA is used; while in Mode 2 the best F-measure is above 0.6 and drops down to 0.6 when IA is used, compare panel A and B of Additional file <a href=\"#S1\" data-ref-type=\"supplementary-material\">1<\/a>: Figure S1. Obviously, the present difference depends on the closeness between CA and IA.\n\nInspecting Figures <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>, <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>, <a href=\"#F4\" data-ref-type=\"fig\">4<\/a> and <a href=\"#F5\" data-ref-type=\"fig\">5<\/a>, it is possible to observe that the loss of performance occurs both in terms of precision and recall. While the loss in detection capability of true isoforms is expected with IA, the loss in precision is more devious. It can be justified by the fact that the methods tend to explain all the reads by assigning them to an isoform. Therefore, other isoforms are used to accommodate the differences in fitting and isoforms with few reads (and low expression) are often formed in absence of a sufficiently strong penalty term or of a post-filtering procedure.\n\nIssues apply for **Set-up 2** comparing Additional file <a href=\"#S9\" data-ref-type=\"supplementary-material\">9<\/a>: Figure S9, Additional file 10: Figure S10, Additional file <a href=\"#S11\" data-ref-type=\"supplementary-material\">11<\/a>: Figure S11 and Additional file <a href=\"#S12\" data-ref-type=\"supplementary-material\">12<\/a>: Figure S12, where IA was randomly generated from CA. Therefore, the main conclusion does not depend strongly on the precise content of IA. As a conclusion, we want to stress that the main limit of methods in Mode 2 is their inadequacy in recovering lowly expressed isoforms, unless already annotated in IA. On the contrary, such methods are able in detecting both moderately and highly expressed isoforms in absence of their annotation as well.\n\n### Effect of library type\n\nIn order to evaluate the effect of the library type, we have to compare PE results with SE results. Such comparison was carried out in **Set-up 1**, for the read length 100 bp and for all depths. Therefore, for precision and recall we have to compare the corresponding panels of Figures <a href=\"#F2\" data-ref-type=\"fig\">2<\/a> and <a href=\"#F6\" data-ref-type=\"fig\">6<\/a> with Figures <a href=\"#F5\" data-ref-type=\"fig\">5<\/a> and <a href=\"#F10\" data-ref-type=\"fig\">10<\/a>, respectively; for F-measure we have to compare Additional file <a href=\"#S1\" data-ref-type=\"supplementary-material\">1<\/a>: Figure S1 with Additional file <a href=\"#S4\" data-ref-type=\"supplementary-material\">4<\/a>: Figure S4; for number of TP and TN we have to compare Additional file <a href=\"#S5\" data-ref-type=\"supplementary-material\">5<\/a>: Figure S5 with Additional file <a href=\"#S6\" data-ref-type=\"supplementary-material\">6<\/a>: Figure S6 for the highest depth (20M) and Additional file <a href=\"#S7\" data-ref-type=\"supplementary-material\">7<\/a>: Figure S7 with Additional file <a href=\"#S8\" data-ref-type=\"supplementary-material\">8<\/a>: Figure S8 for the lowest depth (0.25M).\n\nWe observe that PE reads show better indexes with respect to SE reads at the same depth (at the price of higher experimental cost, not evaluated here). However, the differences are almost negligible in Mode 1 (in particular, at high coverage), they are relatively small for methods in Mode 2 when CA is provided. The gap increases in Mode 2 when IA is provided. In this case, Figures <a href=\"#F10\" data-ref-type=\"fig\">10<\/a> and <a href=\"#F11\" data-ref-type=\"fig\">11<\/a> show that PE reads allow to correctly detect more isoforms that were not present in IA. The gain is small for high depth, it becomes more evident for low depth. Advantages of using PE is also evident in Mode 3. To better evaluate the differences, we observe that 20M 100 bp-SE allows to map 13918 junctions when CA is provided and 13564 junctions in absence of annotation; 0.25M 100 bp-SE allows to map 8086 junctions when CA is provided and 6851 in absence of annotation.\n\nAs a conclusion, we can underline that the main advantage of using PE with respect to SE is in the better capability to recover novel isoforms when not provided in the annotation. On the other hand, we also observe that 100 bp-SE are quite long reads, with short SE reads the advantages of PE are more pronounced.\n\n### Effect of isoform abundance\n\nIn order to evaluate the effect of isoform abundance, we have to inspect Figures <a href=\"#F6\" data-ref-type=\"fig\">6<\/a>, <a href=\"#F7\" data-ref-type=\"fig\">7<\/a>, <a href=\"#F8\" data-ref-type=\"fig\">8<\/a> and <a href=\"#F9\" data-ref-type=\"fig\">9<\/a> that provide a deeper insight about the recall illustrated in Figures <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>, <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>, <a href=\"#F4\" data-ref-type=\"fig\">4<\/a> and <a href=\"#F5\" data-ref-type=\"fig\">5<\/a>. The index is now expanded into three rows depending on the level of expression of the corresponding true isoforms. From the figures, we can see that the capability in isoform detection strongly depends on their expression levels. In general, highly expressed isoforms are easily detected by methods in Mode 1, while methods in Mode 2 and Mode 3 show a lower (but still acceptable) detection capabilities. On the contrary, moderately and lowly expressed isoforms are detected well, or at least with an acceptable rate, in Mode 1. However, they are not well identified in Mode 2, and often completely lost in Mode 3. Integrating such observation with Figures <a href=\"#F10\" data-ref-type=\"fig\">10<\/a> and <a href=\"#F11\" data-ref-type=\"fig\">11<\/a>, we observe that lowly expressed isoforms are mostly detected if they are provided in the annotation.\n\nAdditional file <a href=\"#S11\" data-ref-type=\"supplementary-material\">11<\/a>: Figure S11 and Additional file <a href=\"#S12\" data-ref-type=\"supplementary-material\">12<\/a>: Figure S12 illustrate the recall with respect to the isoform abundance class in **Set-up 2**, providing analogous conclusions.\n\n### Effect of sequencing depth\n\nIn order to evaluate the effect of sequencing depth, we have to compare different bars of the same colour in each block and methods of panels of Figures <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>, <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>, <a href=\"#F4\" data-ref-type=\"fig\">4<\/a>, <a href=\"#F5\" data-ref-type=\"fig\">5<\/a>, <a href=\"#F6\" data-ref-type=\"fig\">6<\/a>, <a href=\"#F7\" data-ref-type=\"fig\">7<\/a>, <a href=\"#F8\" data-ref-type=\"fig\">8<\/a> and <a href=\"#F9\" data-ref-type=\"fig\">9<\/a>, and the behaviour of each coloured line in the F-measure reported in Additional file <a href=\"#S1\" data-ref-type=\"supplementary-material\">1<\/a>: Figure S1, Additional file <a href=\"#S2\" data-ref-type=\"supplementary-material\">2<\/a>: Figure S2, Additional file <a href=\"#S3\" data-ref-type=\"supplementary-material\">3<\/a>: Figure S3 and Additional file <a href=\"#S4\" data-ref-type=\"supplementary-material\">4<\/a>: Figure S4.\n\nIn most cases, the performance gets worse with the decrease in sequencing depth, but the loss is less evident than what one can expect. In particular, it is almost negligible for methods in Mode 1 with CA and it appears more evident for methods in Mode 2 or 3. The gap increases for data driven alignment and in absence of CA. Indeed, when the depth increases we observed less precision and simultaneously a higher recall. The loss in precision can be explained by the large number of FP isoforms, often with low expression values. More in general, we noticed that as far as a minimum level of depth is reached (in the case of **Set-up 1** such level is estimated in about 1M for PE) then further increases of the depth only play a trade-off role between the observed precision and recall without impacting the overall global performance. Conversely, below the saturation level the global performance drops down.\n\nIn a similar way, comparing Additional file <a href=\"#S5\" data-ref-type=\"supplementary-material\">5<\/a>: Figure S5, Additional file <a href=\"#S6\" data-ref-type=\"supplementary-material\">6<\/a>: Figure S6 and Additional file <a href=\"#S7\" data-ref-type=\"supplementary-material\">7<\/a>: Figure S7 and Additional file <a href=\"#S6\" data-ref-type=\"supplementary-material\">6<\/a>: Figure S6, Additional file <a href=\"#S7\" data-ref-type=\"supplementary-material\">7<\/a>: Figure S7 and Additional file <a href=\"#S8\" data-ref-type=\"supplementary-material\">8<\/a>: Figure S8, it is possible to see the benefit in the total number of correctly identified isoforms when increasing the depth from the extreme conditions of 0.25M to 20M.\n\n### Effect of read length\n\nIn order to evaluate the effect of read length, we have to compare precision and recall in Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a> (100 bp-PE) with Figures <a href=\"#F3\" data-ref-type=\"fig\">3<\/a> and <a href=\"#F4\" data-ref-type=\"fig\">4<\/a> (75 bp-PE and 50 bp-PE, respectively) and Additional file <a href=\"#S1\" data-ref-type=\"supplementary-material\">1<\/a>: Figure S1 with Additional file <a href=\"#S2\" data-ref-type=\"supplementary-material\">2<\/a>: Figure S2 and Additional file <a href=\"#S3\" data-ref-type=\"supplementary-material\">3<\/a>: Figure S3 in terms of F-measure. We found, as expected, that long reads are preferable to short ones. In particular, we observed an overall loss of performance both in terms of recall and precision. We quantified in about 5% the loss in performance in term of F-measure for methods in Mode 1 (the best performance achieved by RSEM with CA is about 0.78 for 100 bp-PE and becomes 0.75 for 75 bp-PE and 0.73 for 50 bp-PE). A more significant loss was observed when executing methods in Modes 2\/3, especially at low depth.\n\nWe also observe that in our experimental design short reads are obtained by trimming the long ones. Therefore, short reads generated in this way have a slightly better quality with respect to those (of the same length) generated following the error profile. As a consequence, we expect that the real difference between short and long reads could be slightly larger than the one we have reported.\n\nAdditional file <a href=\"#S9\" data-ref-type=\"supplementary-material\">9<\/a>: Figure S9 and Additional file 10: Figure S10 provide analogous insights for **Set-up 2**.\n\n## Isoform estimation\n\nHere, we illustrate the results for estimation error indexes *E*~1~, *E*~2~ and *E*~3~. Additional file <a href=\"#S15\" data-ref-type=\"supplementary-material\">15<\/a>: Table S1, Additional file <a href=\"#S16\" data-ref-type=\"supplementary-material\">16<\/a>: Table S2, Additional file <a href=\"#S17\" data-ref-type=\"supplementary-material\">17<\/a>: Table S3, Additional file <a href=\"#S18\" data-ref-type=\"supplementary-material\">18<\/a>: Table S4, Additional file <a href=\"#S19\" data-ref-type=\"supplementary-material\">19<\/a>: Table S5, Additional file <a href=\"#S20\" data-ref-type=\"supplementary-material\">20<\/a>: Table S6, Additional file <a href=\"#S21\" data-ref-type=\"supplementary-material\">21<\/a>: Table S7, Additional file <a href=\"#S22\" data-ref-type=\"supplementary-material\">22<\/a>: Table S8, Additional file <a href=\"#S23\" data-ref-type=\"supplementary-material\">23<\/a>: Table S9, Additional file <a href=\"#S24\" data-ref-type=\"supplementary-material\">24<\/a>: Table S10, Additional file <a href=\"#S25\" data-ref-type=\"supplementary-material\">25<\/a>: Table S11, Additional file <a href=\"#S26\" data-ref-type=\"supplementary-material\">26<\/a>: Table S12, Additional file <a href=\"#S27\" data-ref-type=\"supplementary-material\">27<\/a>: Table S13, Additional file <a href=\"#S28\" data-ref-type=\"supplementary-material\">28<\/a>: Table S14 Additional file <a href=\"#S29\" data-ref-type=\"supplementary-material\">29<\/a>: Table S15 and Additional file 30: Table S16 show errors statistics (median, 3rd quartile and maximum value) for the indexes in **Set-up 1**. In particular, the group of tables in Additional file <a href=\"#S15\" data-ref-type=\"supplementary-material\">15<\/a>: Table S1, Additional file <a href=\"#S16\" data-ref-type=\"supplementary-material\">16<\/a>: Table S2, Additional file <a href=\"#S17\" data-ref-type=\"supplementary-material\">17<\/a>: Table S3, Additional file <a href=\"#S18\" data-ref-type=\"supplementary-material\">18<\/a>: Table S4, Additional file <a href=\"#S19\" data-ref-type=\"supplementary-material\">19<\/a>: Table S5, Additional file 20: Table S6, Additional file <a href=\"#S21\" data-ref-type=\"supplementary-material\">21<\/a>: Table S7 and Additional file <a href=\"#S22\" data-ref-type=\"supplementary-material\">22<\/a>: Table S8 illustrate the results for PE, and those in Additional file <a href=\"#S23\" data-ref-type=\"supplementary-material\">23<\/a>: Table S9, Additional file <a href=\"#S24\" data-ref-type=\"supplementary-material\">24<\/a>: Table S10, Additional file <a href=\"#S25\" data-ref-type=\"supplementary-material\">25<\/a>: Table S11, Additional file <a href=\"#S26\" data-ref-type=\"supplementary-material\">26<\/a>: Table S12, Additional file <a href=\"#S27\" data-ref-type=\"supplementary-material\">27<\/a>: Table S13, Additional file <a href=\"#S28\" data-ref-type=\"supplementary-material\">28<\/a>: Table S14 Additional file <a href=\"#S29\" data-ref-type=\"supplementary-material\">29<\/a>: Table S15 and Additional file <a href=\"#S30\" data-ref-type=\"supplementary-material\">30<\/a>: Table S16 for SE. Within each group, the two most extreme depths are shown (i.e., 20M and 0.25M). Furthermore, the tables provide results either for annotation driven alignment (CA and IA) and for data driven alignment. Additional file <a href=\"#S31\" data-ref-type=\"supplementary-material\">31<\/a>: Table S17, Additional file <a href=\"#S32\" data-ref-type=\"supplementary-material\">32<\/a>: Table S18, Additional file <a href=\"#S33\" data-ref-type=\"supplementary-material\">33<\/a>: Table S19 and Additional file <a href=\"#S34\" data-ref-type=\"supplementary-material\">34<\/a>: Table S20 refer to **Set-up 2**. In particular, Additional file <a href=\"#S31\" data-ref-type=\"supplementary-material\">31<\/a>: Table S17 and Additional file <a href=\"#S32\" data-ref-type=\"supplementary-material\">32<\/a>: Table S18 describe the results for 60M 75 bp-PE (annotation driven alignment). The analogous cases for data driven alignment are shown in Additional file <a href=\"#S33\" data-ref-type=\"supplementary-material\">33<\/a>: Table S19 and Additional file <a href=\"#S34\" data-ref-type=\"supplementary-material\">34<\/a>: Table S20. In order to investigate the performance of the methods in correctly estimating the isoform abundances, the attention have to be mainly focused on the qualitative aspects related to error distributions rather that on the actual values shown in the tables.\n\nOverall, both set-ups show a similar behaviour for all error types. In fact, all methods produce errors that have a strongly asymmetric skewed distribution with a long right tail. This means that they fail to estimate a (significant) fraction of isoforms (see results for the 3rd quartile and the maximum observed value). Moreover, within the same table, the same index is also different (sometimes of order of magnitude) with respect to methods and mode of actions. Differences are observed, not only with respect to the most extreme value, but also with respect to the median and the 3rd quartile. This means that the methods may provide very different results on a large fraction of isoforms. In brief, all tables indicate that the problem of obtaining reliable estimates is still open. The loss of performance is not completely surprising, since estimation is carried out after isoform detection, without explicitly consider the uncertainty due to the identification step. The latter statement is clear when we compare the same method under different modes of action. In such cases, each method uses an analogous statistical procedure for estimating the abundances. However, the inference is carried out on different sets of isoforms and can produce different estimates. To mitigate such drawbacks confidence intervals for the estimates could be more reliable than point estimates.\n\nMore in detail, looking at the 3rd quartile in Additional file <a href=\"#S15\" data-ref-type=\"supplementary-material\">15<\/a>: Table S1 (i.e., 20M 100 bp-PE CA driven alignment), the best result for *E*~1~ oscillates between 0.27\u20130.29 (CEM and Cufflinks Mode 3, RSEM), for *E*~2~ is about 0.005 (Cufflinks, Mode 1), and for *E*~3~ is between 0.22\u20130.25 (RSEM, CEM Mode 1).\n\nThe low 3rd quartiles observed for *E*~3~ and *E*~2~ confirm that problems arise mainly with lowly expressed isoforms, that are either not detected or not set to zero by filtering or penalization procedures. On the contrary, the large extreme values observed for *E*~2~ and *E*~3~ indicate that the corresponding methods can completely fail (of several order of magnitude) some estimates. However, such failures are limited to few units when the corresponding 3rd quartile is low, being larger when the right tail of the distribution becomes heavier.\n\nMore attention requires the (relative) error *E*~1~, whose 3rd quartile is at least about 0.27\u20130.29 in Additional file <a href=\"#S15\" data-ref-type=\"supplementary-material\">15<\/a>: Table S1, (with the median value of about 0.07\u20130.15). This means that, even when we are able to correctly detect the presence of the isoforms, in more than 50% of the cases the estimation error is about 10% of the true value. Larger values for the median or the 3rd quartile indicate a worse performance.\n\nWe stress that Additional file <a href=\"#S15\" data-ref-type=\"supplementary-material\">15<\/a>: Table S1 illustrates (in principle) the most favourable condition. Comparing it with Additional file <a href=\"#S17\" data-ref-type=\"supplementary-material\">17<\/a>: Table S3 (i.e., 0.25M 100 bp-PE CA driven alignment), it is possible to evaluate the effect of the depth when the annotation is CA. Whereas precision and recall for methods in Mode 1 were not significantly influenced, the error indexes did. In particular, the largest differences are observed for *E*~2~ and *E*~3~. In general, at low depth the quality of the estimates is quite poor. Analogous considerations hold considering SE instead of PE, see Additional file <a href=\"#S23\" data-ref-type=\"supplementary-material\">23<\/a>: Table S9 and Additional file <a href=\"#S15\" data-ref-type=\"supplementary-material\">15<\/a>: Table S11, for the cases 20M SE and 0.25M SE, respectively.\n\nThe influence of annotation can be deduced comparing Additional file <a href=\"#S15\" data-ref-type=\"supplementary-material\">15<\/a>: Table S1 and Additional file <a href=\"#S17\" data-ref-type=\"supplementary-material\">17<\/a>: Table S2 (obtained using IA). Analogous comparison can be carried out for all pairs of corresponding tables (CA vs IA). In such cases the differences are larger, due to the fact that methods are not able to identify all isoforms (in particular those not provided in IA).\n\nAdditional file <a href=\"#S31\" data-ref-type=\"supplementary-material\">31<\/a>: Table S17, Additional file <a href=\"#S32\" data-ref-type=\"supplementary-material\">32<\/a>: Table S18, Additional file <a href=\"#S33\" data-ref-type=\"supplementary-material\">33<\/a>: Table S19 and Additional file <a href=\"#S34\" data-ref-type=\"supplementary-material\">34<\/a>: Table S20 drive to similar considerations for **Set-up 2** with more skewed error distributions.\n\n## Considerations about the computational cost\n\nSince the methods were implemented in different languages and ran in different environments, we found that a technical comparison of execution time was not fair. Therefore, we briefly reports only qualitative considerations. RSEM and Cufflinks (in all modes of action) are sufficiently fast, CEM (in all modes of action) is the fastest. On the contrary, iReckon is quite slow with respect to the others and also produces very large temporary files. Current implementation of SLIDE is too slow to be completed in **Set-up 2** in a reasonable amount of time, and therefore was considered only in **Set-up 1**.\n\n# Conclusions\n\nOur results show that algorithms have good or acceptable performance in detecting the presence of the isoforms (recall) when the annotation is provided, even if incomplete (Mode 1 and Mode 2, with both CA and IA). On the contrary, the data driven methods (Mode 3) are still not satisfactory, also when the reads are carefully aligned using the annotation during the mapping phase. Results obtained in Mode 3 are in agreement with what observed in \\[24\\].\n\nThe performance of all methods is strongly influenced by the strength of the isoforms. Highly and moderately expressed isoforms are identified with good accuracy. On the contrary, lowly expressed isoforms are still problematic, also when the exact annotation is provided. In particular, we observed that lowly expressed isoforms that are not present in the annotation, are not recovered using methods in Mode 2. Conversely, Mode 2 approaches are able to recover part of moderately and most of highly expressed transcripts also when not provided in the annotation.\n\nResults improve by increasing the sequencing depth and the read-length. However, for the depth there is a saturation limit -in current computational methodologies- that does not allow to achieve optimal reconstruction even at high coverage. In fact, when the coverage increases, the number of TP increase as expected, but also the number of FP increases. In particular, we noticed that some methods tend to identify several (novel and annotated) isoforms as present at very low expression levels. In most cases such isoforms are FP, as a consequence precision is often not satisfactory.\n\nRealistic estimation of isoforms abundance is also very problematic. Similar to \\[24\\] our results show that the estimation error is very skewed. The error distribution up to the third quartile is acceptable at least for E2 (connected to FP) and E3 (connected to FN), with a very long right tail. On the contrary, the distribution of the (relative) error E1 shows that more than 50% of isoforms are estimated with more than 10% of error. To this purpose, we should consider that estimation of isoform abundances is not easier than isoform identification. In fact, the estimates are often obtained for the set of isoforms that have been previously identified. However, methods usually do not take into account such uncertainty. As a consequence, the performance can be much worse. Therefore, we conclude that the estimation of the correctly identified isoforms is still challenging.\n\nAs observed by \\[24\\], the complexity of higher eukaryotic genomes, such as the human one, imposes severe limitations to the performance of all quantification and estimation methods, that are likely to remain limiting factors for the analysis of current-generation RNA-seq experiments. Such limitations can be partially solved providing existing annotations, but more in general require the development of further research and techniques from both methodological and experimental point of views.\n\nFinally, it should be noted that all methods considered here and in \\[24\\] work with a single RNA-seq sample. Recent works \\[49,50\\] propose to use a multiple-sample approach to achieve a more precise identification and estimation of isoform expression. The availability of such type of approaches, whose performances have to be further validated, seems to indicate that future studies have to investigate a larger variety of (homogeneous) samples at a lower depth per sample to obtain more confident transcript predictions, see \\[50\\].\n\n# Competing interests\n\nThe authors declare that they have no competing interests.\n\n# Authors' contributions\n\nAll authors participated in conceiving the analysis and setting-up the code for the comparisons and writing the manuscript. All authors also read and approved the submitted manuscript.\n\n# Supplementary Material\n\n###### Additional file 1\n\n**Figure S1.** F-measure in Set-up 1 for 100 bp-PE. Panels **A** (upper left) and **B** (upper right) depict F-measure versus the sequencing depth for each compared method when the alignment is annotation driven using CA and IA, respectively. Panels **C** (bottom left) and **D** (bottom right) are analogous to Panels **A** and **B**, when the alignment is data driven. The figure refers to Set-up 1 and 100 bp-PE. Within each panel, methods in Mode 1 are depicted with continuous line, methods in Mode 2 with dashed line, methods in Mode 3 with dotted line. When the alignment is annotation driven, the same annotation provided during the alignment was used for Mode 1 and 2.\n\nClick here for file\n\n###### Additional file 2\n\n**Figure S2.** F-measure in Set-up 1 for 75 bp-PE. Analogous to Additional file <a href=\"#S1\" data-ref-type=\"supplementary-material\">1<\/a>: Figure S1, but for Set-up 1 and 75 bp-PE.\n\nClick here for file\n\n###### Additional file 3\n\n**Figure S3.** F-measure in Set-up 1 for 50 bp-PE. Analogous to Additional file <a href=\"#S1\" data-ref-type=\"supplementary-material\">1<\/a>: Figure S1, but for Set-up 1 and 50 bp-PE.\n\nClick here for file\n\n###### Additional file 4\n\n**Figure S4.** F-measure in Set-up 1 for 100 bp-SE. Analogous to Additional file <a href=\"#S1\" data-ref-type=\"supplementary-material\">1<\/a>: Figure S1, but for Set-up 1 and 100 bp-SE.\n\nClick here for file\n\n###### Additional file 5\n\n**Figure S5.** True Positives and False Positives in Set-up 1 for 20M 100 bp-PE. Panels **A** (upper left) and **B** (upper right) depict TP (coral) and FP (aquamarine) bars for the compared methods when the alignment is annotation driven (CA and IA, respectively). Panels **C** (bottom left) and **D** (bottom right) are analogous to Panels **A** and **B**, when the alignment is data driven. The figure refers to Set-up 1 and 20M 100 bp-PE. The true number of expressed transcripts (i.e., 3726) is added as dashed horizontal line to each panel. The difference between the TP and the horizontal line represents the FN.\n\nClick here for file\n\n###### Additional file 6\n\n**Figure S6.** True Positives and False Positives in Set-up 1 for 20M 100 bp-SE. Analogous to Additional file <a href=\"#S5\" data-ref-type=\"supplementary-material\">5<\/a>: Figure S5, but for Set-up 1 and 20M 100 bp-SE.\n\nClick here for file\n\n###### Additional file 7\n\n**Figure S7.** True Positives and False Positives in Set-up 1 for 0.25M 100 bp-PE. Analogous to Additional file <a href=\"#S5\" data-ref-type=\"supplementary-material\">5<\/a>: Figure S5, but for Set-up 1 and 0.25M 100 bp-PE.\n\nClick here for file\n\n###### Additional file 8\n\n**Figure S8.** True Positives and False Positives in Set-up 1 for 0.25M 100 bp-SE. Analogous to Additional file <a href=\"#S5\" data-ref-type=\"supplementary-material\">5<\/a>: Figure S5, but for Set-up 1 and 0.25M 100 bp-PE.\n\nClick here for file\n\n###### Additional file 9\n\n**Figure S9.** Precision and Recall bar-plot in Set-up 2 for 75 bp-PE. Analogous to Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>, but for Set-up 2 for 60M 75 bp-PE.\n\nClick here for file\n\n###### Additional file 10\n\n**Figure 10.** Precision and Recall bar-plot in Set-up 2 for 50 bp-PE. Analogous to Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>, but for Set-up 2 for 60M 50 bp-PE.\n\nClick here for file\n\n###### Additional file 11\n\n**Figure S11.** Recall bar-plot versus isoform abundance in Set-up 2 for 60M 75 bp-PE. Analogous to Figure <a href=\"#F5\" data-ref-type=\"fig\">5<\/a>, but for Set-up 2 for 60M 75 bp-PE.\n\nClick here for file\n\n###### Additional file 12\n\n**Figure S12.** Recall bar-plot versus isoform abundance in Set-up 2 for 60M 50 bp-PE. Analogous to Figure <a href=\"#F5\" data-ref-type=\"fig\">5<\/a>, but for Set-up 2 for 60M 50 bp-PE.\n\nClick here for file\n\n###### Additional file 13\n\n**Figure S13.** Precision, Recall and F-measure when introducing thresholds (Set-up 2). Analogous to Figure <a href=\"#F12\" data-ref-type=\"fig\">12<\/a>, but for Set-up 2, 60M 75 bp-PE and the alignment with CA.\n\nClick here for file\n\n###### Additional file 14\n\n**Figure S14.** True Positives and False Positives in Set-up 2 for 60M 75 bp-PE. Panels **A** (upper left) and **B** (upper right) depict TP (coral) and FP (aquamarine) bars for the compared methods when the alignment is annotation driven (CA and IA, respectively). Panels **C** (bottom left) and **D** (bottom right) are analogous to Panels **A** and **B**, when the alignment is data driven. The figure refers to Set-up 2 and 60M 75 bp-PE. The true number of expressed transcripts (i.e., 17032) is added as dashed horizontal line to each panel. The difference between the TP and the horizontal line represents the FN.\n\nClick here for file\n\n###### Additional file 15\n\n**Table S1.** E1, E2 and E3 in Set-up 1 with annotation driven alignment (CA) for 20M 100 bp-PE. Median, 3rd Quartile and maximum value observed for error indexes E1, E2 and E3 in Set-up 1, 20M PE reads of 100 bp. Tables are divided in blocks, where the left block is for methods used in Mode 1, middle block is for methods used in Mode 2, right block is for methods used in Mode 3. Upper rows refer to E1, middle ones refer to E2, bottom ones refer to E3. CA was provided during the alignment.\n\nClick here for file\n\n###### Additional file 16\n\n**Table S2.** E1, E2 and E3 in Set-up 1 with annotation driven alignment (IA) for 20M 100 bp-PE. Analogous to Additional file <a href=\"#S15\" data-ref-type=\"supplementary-material\">15<\/a>: Table S1, but with IA provided during the alignment.\n\nClick here for file\n\n###### Additional file 17\n\n**Table S3.** E1, E2 and E3 in Set-up 1 with annotation driven alignment (CA) for 0.25M 100 bp-PE. Analogous to Additional file <a href=\"#S15\" data-ref-type=\"supplementary-material\">15<\/a>: Table S1, but for 0.25M PE reads.\n\nClick here for file\n\n###### Additional file 18\n\n**Table S4.** E1, E2 and E3 in Set-up 1 with annotation driven alignment (IA) for 0.25M 100 bp-PE. Analogous to Additional file <a href=\"#S15\" data-ref-type=\"supplementary-material\">15<\/a>: Table S1, but for 0.25M PE reads and with IA provided during the alignment.\n\nClick here for file\n\n###### Additional file 19\n\n**Table S5.** E1, E2 and E3 in Set-up 1, with data driven alignment and CA for 20M 100 bp-PE. Analogous to Additional file <a href=\"#S15\" data-ref-type=\"supplementary-material\">15<\/a>: Table S1, but with data driven alignment. CA was provided in Mode 1 and Mode 2.\n\nClick here for file\n\n###### Additional file 20\n\n**Table S6.** E1, E2 and E3 in Set-up 1, with data driven alignment and IA for 20M 100 bp-PE. Analogous to Additional file <a href=\"#S15\" data-ref-type=\"supplementary-material\">15<\/a>: Table S1, but with data driven alignment. IA was provided in Mode 1 and Mode 2.\n\nClick here for file\n\n###### Additional file 21\n\n**Table S7.** E1, E2 and E3 in Set-up 1, with data driven alignment and CA for 0.25M 100 bp-PE. Analogous to Additional file <a href=\"#S19\" data-ref-type=\"supplementary-material\">19<\/a>: Table S5, but for 0.25M PE reads.\n\nClick here for file\n\n###### Additional file 22\n\n**Table S8.** E1, E2 and E3 in Set-up 1, with data driven alignment and IA for 0.25M 100 bp-PE. Analogous to Additional file 20: Table S6, but for 0.25M PE reads.\n\nClick here for file\n\n###### Additional file 23\n\n**Table S9.** E1, E2 and E3 in Set-up 1 with annotation driven alignment (CA) for 20M 100 bp-SE. Analogous to Additional file <a href=\"#S15\" data-ref-type=\"supplementary-material\">15<\/a>: Table S1, but for 20M SE reads.\n\nClick here for file\n\n###### Additional file 24\n\n**Table S10.** E1, E2 and E3 in Set-up 1 with annotation driven alignment (IA) for 20M 100 bp-SE. Analogous to Additional file <a href=\"#S17\" data-ref-type=\"supplementary-material\">17<\/a>: Table S2, but for 20M SE reads.\n\nClick here for file\n\n###### Additional file 25\n\n**Table S11.** E1, E2 and E3 in Set-up 1 with annotation driven alignment (CA) for 0.25M 100 bp-SE. Analogous to Additional file <a href=\"#S17\" data-ref-type=\"supplementary-material\">17<\/a>: Table S3, but for 0.25M SE reads.\n\nClick here for file\n\n###### Additional file 26\n\n**Table S12.** E1, E2 and E3 in Set-up 1 with annotation driven alignment (IA) for 0.25M 100 bp-SE. Analogous to Additional file <a href=\"#S18\" data-ref-type=\"supplementary-material\">18<\/a>: Table S4, but for 0.25M SE reads.\n\nClick here for file\n\n###### Additional file 27\n\n**Table S13.** E1, E2 and E3 in Set-up 1, with data driven alignment and CA for 20M 100 bp-SE. Analogous to Additional file <a href=\"#S19\" data-ref-type=\"supplementary-material\">19<\/a>: Table S5, but for 20M SE reads.\n\nClick here for file\n\n###### Additional file 28\n\n**Table S14.** E1, E2 and E3 in Set-up 1, with data driven alignment and IA for 20M 100 bp-SE. Analogous to Additional file 20: Table S6, but for 20M SE reads.\n\nClick here for file\n\n###### Additional file 29\n\n**Table S15.** E1, E2 and E3 in Set-up 1, with data driven alignment and CA for 0.25M 100 bp-SE. Analogous to Additional file <a href=\"#S21\" data-ref-type=\"supplementary-material\">21<\/a>: Table S7, but for 025M SE reads.\n\nClick here for file\n\n###### Additional file 30\n\n**Table S16.** E1, E2 and E3 in Set-up 1, with data driven alignment and IA for 0.25M 100 bp-SE. Analogous to Additional file <a href=\"#S22\" data-ref-type=\"supplementary-material\">22<\/a>: Table S8, but for 025M SE reads.\n\nClick here for file\n\n###### Additional file 31\n\n**Table S17.** Statistics for E1, E2 and E3 in Set-up 2 with annotation driven alignment (CA) for 60M 75 bp-PE. Analogous to Additional file <a href=\"#S15\" data-ref-type=\"supplementary-material\">15<\/a>: Table S1, but for Set-up 2, 60M 75 bp-PE.\n\nClick here for file\n\n###### Additional file 32\n\n**Table S18.** Statistics for E1, E2 and E3 in Set-up 2 with annotation driven alignment (IA) for 60M 75 bp-PE. Analogous to Additional file <a href=\"#S17\" data-ref-type=\"supplementary-material\">17<\/a>: Table S2, but for Set-up 2, 60M 75 bp-PE.\n\nClick here for file\n\n###### Additional file 33\n\n**Table S19.** Statistics for E1, E2 and E3 in Set-up 2 with data driven alignment and CA for 60M 75 bp-PE. Analogous to Additional file <a href=\"#S19\" data-ref-type=\"supplementary-material\">19<\/a>: Table S5, but for Set-up 2, 60M 75 bp-PE.\n\nClick here for file\n\n###### Additional file 34\n\n**Table S20.** Statistics for E1, E2 and E3 in Set-up 2 with data driven alignment and IA for 60M 75 bp-PE. Analogous to Additional file 20: Table S6, but for Set-up 2, 60M 75 bp-PE.\n\nClick here for file\n\n## Acknowledgments\n\nThis research was supported by \"Italian Flagship Project Epigenomic\" of the Italian Ministry of Education, University and Research and the National Research Council (<http:\/\/www.epigen.it\/>). The authors would like to thank the anonymous reviewers for their valuable comments and suggestions to improve the quality of the paper, and Francesco Russo for reading the manuscript.","meta":{"dup_signals":{"dup_doc_count":102,"dup_dump_count":26,"dup_details":{"curated_sources":2,"2022-33":1,"2021-31":1,"2020-16":1,"2019-13":2,"2019-04":1,"2018-47":1,"2018-39":1,"2018-30":1,"2018-22":1,"2018-13":1,"2017-47":1,"2017-34":1,"2017-26":7,"2017-22":21,"2017-17":13,"2017-09":7,"2017-04":1,"2016-44":1,"2016-40":2,"2016-36":12,"2016-30":8,"2016-22":1,"2016-18":1,"2023-40":1,"2017-13":11,"2024-22":1}},"file":"PMC4098781"},"subset":"pubmed_central"}
{"text":"abstract: This paper distinguishes between two different scales of medium range order, MRO, in non-crystalline SiO~2~: (1) the first is \\~0.4 to 0.5 nm and is obtained from the position of the first sharp diffraction peak, FSDP, in the X-ray diffraction structure factor, S(*Q*), and (2) the second is \\~1 nm and is calculated from the FSDP full-width-at-half-maximum FWHM. Many-electron calculations yield Si\u2013O third- and O\u2013O fourth-nearest-neighbor bonding distances in the same 0.4\u20130.5 nm MRO regime. These derive from the availability of empty Si d\u03c0 orbitals for back-donation from occupied O p\u03c0 orbitals yielding narrow symmetry determined distributions of third neighbor Si\u2013O, and fourth neighbor O\u2013O distances. These are segments of six member rings contributing to connected six-member rings with \\~1 nm length scale within the MRO regime. The unique properties of non-crystalline SiO~2~ are explained by the encapsulation of six-member ring clusters by five- and seven-member rings on average in a compliant hard-soft nano-scaled inhomogeneous network. This network structure minimizes macroscopic strain, reducing intrinsic bonding defects as well as defect precursors. This inhomogeneous CRN is enabling for applications including thermally grown \\~1.5 nm SiO~2~ layers for Si field effect transistor devices to optical components with centimeter dimensions. There are qualitatively similar length scales in nano-crystalline HfO~2~ and phase separated Hf silicates based on the primitive unit cell, rather than a ring structure. Hf oxide dielectrics have recently been used as replacement dielectrics for a new generation of Si and Si\/Ge devices heralding a transition into nano-scale circuits and systems on a Si chip.\nauthor: Gerald Lucovsky; James C Phillips\ndate: 2010\ninstitute: 1Department of Physics, North Carolina State University, Raleigh, NC, 27695-8202, USA; 2Department of Physics and Astronomy, Rutgers University, Piscataway, NJ, 08854, USA\nreferences:\ntitle: Nano-regime Length Scales Extracted from the First Sharp Diffraction Peak in Non-crystalline SiO~2~ and Related Materials: Device Applications\n\n# Introduction\n\nThere have been many models proposed for the unique properties of non-crystalline SiO~2~. These are based on the concept of the continuous random network, CRN, structure as first proposed by Zachariasen \\[1,2\\]. CRN models assume the short range order, SRO, of SiO~2~ is comprised of fourfold coordinated Si in tetrahedral environments through corner-connected twofold coordinated O bridging two Si atoms in a bent geometry. The random character of the network has generally been attributed to a wide distribution of Si\u2013O\u2013Si bond angles, 150 \u00b1 30\u00b0 as determined by X-ray diffraction \\[3\\], as well as a random distribution of dihedral angles. These combine to give a distribution of ring geometries that defines a compliant and strain free CRN structure \\[2\\].\n\nMore recently, a semi-empirical bond constraint theory (SE-BCT) was proposed by one of the authors (JCP) to correlate the ease of glass formation in SiO~2~ and chalcogenide glass with local bonding constraints associated with two-body bond-stretching and three-body bond-bending forces \\[4,5\\]. The criterion for ease of glass formation was a mean-field relation equating the average number of stretching and bending constraints\/atom with the network dimensionality of three. When applied to SiO~2~, satisfaction of this criterion was met by assuming a broken bond bending constraint for the bridging O-atoms. This was inferred from the large bond angle distribution of the Si\u2013O\u2013Si bonding group, and the weak bonding force constant. The same local mean-field approach for the ease of glass formation has been applied with good success to other non-crystalline network glasses in the Ge\u2013Se and As\u2013Se alloy systems.\n\nSE-BCT makes no connection with medium MRO that is a priori deemed to be important with other properties. As such, SE-BCT cannot identify MRO bonding that has been associated with the FSDP \\[6,7\\]. Based on these references, the position and width of the FSDP identify two different MRO length scales. It will be demonstrated in this paper that these length scales provide a basis for explaining some of the unique nano-scale related properties of non-crystalline SiO~2~ that are enabling for device applications.\n\nThe FSDP in the structure factor, S(*Q*), has been determined from X-ray and neutron diffraction studies of oxide, silicate, germanate, borate and chalcogenide glasses \\[7\\]. There is a consensus that the position and width of this feature derive from MRO \\[6-8\\]. This is defined as order extending beyond the nearest- and next-nearest neighbor distances extracted from diffraction studies, and displayed in radial distribution function plots \\[2\\]. There has been much speculation and empirical modeling addressing the microscopic nature of bonding arrangements in the MRO regime including rings of bonded atoms \\[9\\], distances between layer-like ordering \\[10\\], and\/or void clustering that are responsible for the FSDP \\[11\\]. First-principle molecular dynamics calculations have been applied to the FSDP \\[7,12\\]. One of these papers ruled out models based on layer-like nano-structures, and nano-scale voids as the MRO responsible for the FSDP \\[12\\]. References \\[7\\] and \\[12\\] did not offered alternative explanations for the FSDP based a microscopic understanding of the relationship between atomic pair correlations in the MRO regime and constraints imposed by fundamental electronic structure at the atomic and molecular levels.\n\nMoss and Price in \\[7\\], building on the 1974 deNeufville et al. \\[6\\] observation and interpretation of the FSDP proposed that the position this feature, *Q*~1~(\u00c5^\u22121^), \"can be related, via an approximate reciprocal relation, to a distance *R* in real space by the expression *R* = 2\u03c0\/*Q*\". It is important to note the MRO-scale bonding structures previously proposed in \\[9\\] and \\[10\\], and ruled out in \\[12\\], could not explain why a large number of oxide and chalcogenide glasses exhibit FSDP's in a relative narrow regime of *Q*-values, \\~1 to 1.6 \u00c5^\u22121^. Nor could they account for the systematic differences among these *Q*-values, \\~1.5 for oxides, and 1.0\u20131.25 for chalcogenides. It has been shown in \\[13\\] that this is a result of an scaling relationship between the position of the FSDP in *Q*-space and the nearest neighbor bond length.\n\nBased on \\[7,8,13,14\\], the position, *Q*~1~(\u00c5^\u22121^), and full-width at half-maximum, FWHM, \u0394*Q*~1~(\u00c5^\u22121^) of the FSDP have been used to identify a second length scale within the MRO regime for a representative set of oxide and chalcogenide glasses \\[7,11\\]. The first length scale has been designated as a correlation length, *R* = 2\u03c0\/*Q*~1~(\u00c5^\u22121^), and is determined from the *Q*-space position of the spectral peak as suggested in \\[1\\], and the second has been designated as a coherence length, *L* = 2\u03c0\/\u0394*Q*~1~(\u00c5^\u22121^), and is determined from the FWHM \\[7\\]. This interpretation of the FSDP position and line-shape is consistent with the interpretation of diffraction peaks or local maxima in S(*Q*) for non-crystalline and crystalline solids \\[2\\]. These are interpreted as inter-atomic distances or equivalently atomic pair correlations that are repeated throughout a significant volume of the sample within the X-ray beam, but not in a periodic manner characteristic of long range crystalline order. Like other diffraction features, e.g., the width of the Si\u2013Si pair correlation length in SiO~2~ as determined in \\[3\\], is also associated with a characteristic real space distance, e.g., of a MRO-scale cluster of atoms.\n\nIn referring to the FSDP, Moss and Prince in \\[7\\] noted that \"such a diffraction feature thus represents the build up of correlation whose basic period is well beyond the first few neighbor distances\"; this basic period is within the MRO domain. It was also pointed out by them that \"In fact, the width of this feature can be used to estimate a correlation range over which the period in question survives\", or persists.\n\nReturning to paper published in 1974 by deNeufville, Moss and Ovshinsky; this article addressed photo-darkening in As~2~(S, Se)~3~ in a way that anticipated the quantitative definitions for *R* and *L* in subsequent publications \\[7\\]. This is of historical interest since FSDPs were observed for the first time in each compound and\/or alloy studied, and these were associated with a real space distance of \\~5.5 A in the MRO regime. It was also noted that the width of this feature in reciprocal (*Q*) space identified a larger scale of order over which these MRO-regime correlations persisted; this has subsequently been defined as the coherence length, *L*.\n\n# Experimental Results for SiO~2~\n\nThe position and width of the FSDP in glassy SiO~2~ have received considerable attention, and are well-characterized \\[7,8,14\\]. Based on these references and others as well, *Q*~1~(\u00c5^\u22121^) is equal to 1.52 \u00b1 0.03 \u00c5^\u22121^, and \u0394*Q*~1~(\u00c5^\u22121^) to 0.66 \u00b1 0.03 \u00c5^\u22121^. The calculated values of the correlation length, *R* = 2\u03c0\/*Q*~1~(\u00c5^\u22121^), and the coherence length, *L* = 2\u03c0\/\u0394*Q*~1~(\u00c5^\u22121^), are, respectively, *R* = 4.13 \u00b1 0.08 \u00c5, and *L* = 9.95 \u00b1 0.05 \u00c5. *R* gives rise to features in the RDF in a regime associated with rings of bonded atoms; these are a universal aspect of the CRN description of non-crystalline oxides and chalcogenides which include twofold coordinate atoms \\[2\\].\n\nIn a continuous random network, CRN, such as SiO~2~, the primitive ring size is defined by the number of Si atoms connected through bridging O atoms to form the smallest high symmetry ring structure. This primitive ring is the non-crystalline analog of the primitive unit cell (PUC) in crystalline solids and this provides an important connection between the properties of non-crystalline and nano-crystalline thin films.\n\nIt has been first demonstrated in the Bell and Dean model \\[15,16\\], and later by computer generated modeling \\[17-19\\], and molecular dynamic simulations as well \\[8\\], that the ring size distribution for SiO~2~ is dominated by six-member rings with six silicon and six oxygen atoms.\n\nThe contributions to the partial structure factor, S~*ij*~^*N*^ (*Q*) associated with Si\u2013O, O\u2013O and Si\u2013Si pair correlation distances have been determined using classical molecular dynamics simulations as addressed in \\[8\\]. Combined with RDFs from the Bell and Dean model \\[16\\], and computer modeling \\[17,18,20\\], these studies identify inter-atomic pair correlations in the regime of 4\u20135 \u00c5 that contribute to the position of FSDP. Figure 3 of \\[16\\], is a pair distribution histogram that indicates a (1) a Si\u2013O pair correlation, or third nearest neighbor distance of 4.1 \u00b1 0.5 \u00c5, and (2) an O\u2013O pair correlation, or fourth nearest neighbor distance of 4.5 \u00b1 0.3 \u00c5. These features are evident in the computed and experimental radial distribution function plots for X-ray diffraction in Fig. 4, and neutron diffraction in Fig. 5, also of \\[16\\]. As indicated in Fig. <a href=\"#F1\" data-ref-type=\"fig\">1<\/a> of this paper, the 4.1 \u00c5 feature is assigned with Si\u2013O third nearest-neighbor distances, and the 4.5 \u00c5 feature is assigned to fourth nearest-neighbor O\u2013O distances. Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a> is a schematic representation of local cluster that has been used to determine the Si\u2013O\u2013Si bond angle using many-electron ab initio quantum chemistry calculations in \\[18\\].\n\nThe importance of Si atom d-state symmetries in calculations of the electronic structure of non-crystalline SiO~2~ was recognized in \\[18\\], published in 2002. These symmetries, coupled with the O 2p\u03c0 states play a significant role in narrowing the two pair distribution distances identified above. The cluster displayed in Fig. <a href=\"#F1\" data-ref-type=\"fig\">1<\/a> is large enough to include the correlation length, *R* in the MRO regime. The calculations of \\[18\\] demonstrated that Si d-state basis Gaussian functions when included into a many-electron, ab initio calculation play a determinant role in generating a stable minimum for a Si\u2013O\u2013Si bond angle, \u0398, that is smaller than the ionic bonding value 180\u00b0. In addition these values of \u0398, and the bond angle distribution, \u0394\u0398 (1) were different from what had been determined by the X-ray diffraction studies of Mozzi and Warren in \\[3\\], but (2) were in excellent agreement with more recent studies that employed a larger range of *k* or *Q*\\[19\\]. The values obtained by Mozzi and Warren \\[3\\] are \u0398 \\~ 144\u00b0, and \u0394\u0398\\]FWHM \\~ 30\u00b0, whereas the studies in \\[19\\] obtained values of *Q* \\~ 148\u00b0 and \u0394\u0398\\]FWHM \\~ 13\u201315\u00b0 that were essentially the same as those calculated in \\[16\\]. The Bell and Dean model of \\[15\\] in Fig. 2 gave a Si\u2013O\u2013Si bond angle of 152\u00b0, and also wide bond angle distribution with a FWHM \\~ 15\u00b0. Of particular significance is the significantly narrower Si\u2013O\u2013Si bond angle distribution of the calculations in \\[18\\], and the X-ray diffraction studies of \\[19\\]. The bond angles and bond distributions of \\[16,18,19\\] have important implications for the existence of high symmetry six member Si\u2013O rings their importance as the primitive ring structure in both \u03b1-quartz and \u03b2-quartz, as well as non-crystalline SiO~2~.\n\nThe identification of the specific MRO regime features obtained from S(*Q*) rely heavily on the pair correlation functions derived from the Bell and Dean model \\[16\\], as well as from computer modeling of the Gaskell group \\[21\\] and Tadros et al. \\[17,20\\]. Combined with \\[16\\], The Si d\u03c0-O 2p\u03c0-Si d\u03c0 symmetry determined overlap and charge transfer from occupied O \u03c0-states into otherwise empty Si d\u03c0 states, plays the determinant role in forcing the narrowness of this MRO length scale feature. Stated differently, pairs of Si atoms connected through an intervening O atom as in Fig. <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>, are strongly correlated by the local symmetries forced on these Si d\u03c0-states. This correlation reflects the even symmetry of the respective Si d-states, and the odd symmetry of the O p-states. In contrast, the coherence length, *L*, as determined from the FWHM of the FSDP cannot be assigned to a specific inter-atomic repeat distance identified in any of the models addressed above, but instead is an average cluster dimension, in the spirit of the definitions in \\[6\\] and \\[7\\].\n\nThe coherence length, *L* in SiO~2~, as computed from the FWHM of the FSDP, is 9.5 \u00b1 0.5 \u00c5, and this identifies the cluster associated with this length scale. Based on a simple extension of the schematic diagram in Fig. <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>, this cluster includes a coupling of at least two, and no more than three symmetric six-member primitive rings. If this cluster is extended well beyond two to three rings in all directions, it would eventually generate the crystal structure of \u03b1-quartz. This helical aspect of this structure gives rise to a right- or left-handed optical rotary property of \u03b1-quartz \\[22\\]. The helical structure of \u03b1-quartz has its parentage in trigonal Se, which is comprised of right or left-handed helical chains with three Se atoms per turn of the helix. The two-atom helix analog is the cinnabar phase of HgS with six atoms\/turn, three Hg and three S. \u03b1-quartz is the three-atom analog with nine atoms\/turn, three Si and six O \\[22\\]. Returning to non-crystalline SiO~2~, the coupling of two to three-six-member rings is consist with the relative fraction of six member rings, \\~50% in the Bell and Dean \\[16\\] construction as well as other estimates of the ring fraction.\n\nMoreover, this two to three ring clustered structure is an example of the MRO structures addressed in \\[7\\]. With respect the FSDP, Moss and Price noted that \"such a diffraction feature thus represents the build up of correlation whose basic period is well beyond the first few neighbor distances\"; it therefore in the MRO regime. They also pointed out that: \"In fact, the width of this feature (the, FSDP) can be used to estimate a correlation range over which the period in question survives\". This incoherent coupling associated with less symmetric five- and seven-member rings than determines the correlation, or coherence range over which this period survives.\n\n# Revisiting the CRN in Context of Correlation and Coherence Length Determinations\n\nThe pair correlation assignments made for *R* and *L* are consistent with the global concept of a CRN, but the length scales for correlation, *R*, and coherence. *L*, are quantitatively different that what was proposed originally in \\[1\\], and discussed at length \\[3\\]. Each of these envisioned the CRN randomness to be associated with the relative widths of bond lengths and bond angles, as in Fig. 2 in the Bell and Dean \\[16\\]. Based on this model the Si\u2013O pair correlation has a width \\<0.05 \u00c5, and the Si\u2013O\u2013Si bond angle displays a 30\u00b0 width, corresponding to a Si\u2013Si pair correlation width at least two-to-three larger. In these conventional descriptions of the CRN, any dihedral angle correlations, or four-atom correlations, are removed by bond-angle widths.\n\nThe identification of the MRO length scales, *R* and *L*, also has important implications for the use of semi-empirical bond constraint theory (SE-BCT) for identifying and\/or describing ideal glass formers. This theory is a mean-field theory based on average properties that are determined by constraints restricted to SRO bonding arrangements \\[4,5,23\\]. The identification and interpretation of the two MRO length scales discussed above indicates that this emphasis on SRO is not sufficient for identifying the important nano-scale properties of SiO~2~. Indeed MRO is deemed crucial for establishing the unique and technologically important character of non-crystalline SiO~2~ over a dimensional scale from 1 to 2 nm thick gate dielectrics to centimeter dimensions for high-quality optically homogeneous components, e.g., lenses.\n\nThe FSDP has been observed, and studied in other non-crystalline oxide glasses, e.g., B~2~O~3~, GeO~2~, as well chalcogenide glasses including sulfides, GeS~2~ and As~2~S~3~, and selenides, GeSe~2~, As~2~Se~3~ and SiSe~2~\\[6,7\\]. The values of *R* and *L* have been calculated, and display anion, O, S and Se and cation coordination specific behaviors. For example, the values of the correlation length *R*, and the coherence length *L*, have been obtained from the position, and FWHM of the S(*Q*) FSDP peak for (a) SiO~2~: *R* = 4.1 \u00b1 0.2 \u00c5, and *L* = 9.5 \u00b1 0.5 \u00c5; (b) B~2~O~3~: *R* = 4.0 \u00b1 0.2 \u00c5, and *L* = 11 \u00b1 1 \u00c5; and (c) GeSe~2~: *R* = 6.3 \u00b1 0.3 \u00c5, and *L* = 24 \u00b1 4 \u00c5.\n\nIt has been noted previously elsewhere \\[7,13\\], that quantitative differences between the position of the FSDPs in SiO~2~ and GeSe~2~ can be correlated directly with differences between the respective (1) Si\u2013O and Ge\u2013Se bond-lengths, 1.65 and 2.39 \u00c5, and (2) Si\u2013Si and Ge\u2013Ge next neighbor features as determined by the respective Si\u2013O\u2013Si and Ge\u2013Se\u2013Ge bond angles, \\~148\u00b0 and \\~105\u00b0. This was addressed in \\[1\\] and \\[24\\], where it was shown that the products of nearest neighbor bond length (in \u00c5) and positions of the FSDP (*Q*(\u00c5^\u22121^) are approximately the same, \\~2.5 \u00b1 0.4 for the oxide and chalcogenide glasses \\[1,24\\]. Based on this scaling, the value *R* for GeSe~2~ (*x* = 0.33), is estimated to be 6.2 \u00b1 0.2 \u00c5, compared with the averaged experimental value of *R* = 6.30 \u00b1 0.07 \u00c5.\n\nThis values of *Q*~1~(\u00c5^\u22121^) show interesting correlations with the nature of the CRNs. For the three oxide glasses in Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>*Q*~1~(\u00c5^\u22121^) \\~ 1.55 \u00b1 0.03, and is independent of the network coordination, i.e., 3\u20132 for B~2~O~3~ and 4\u20132 SiO~2~ and GeO~2~. In contrast, the value of *Q*~1~(\u00c5^\u22121^) decreases to \\~1.05 for 4\u20132 selenides, and then increases to \\~1.25 for the 3\u20132 chalcogenides. This indicates a longer correlation length in the 3\u20132 alloys that is presumed to be associated with repulsions between lone pairs on As, and either the Se or S atoms of the particular alloy for the 3\u20132 chalcogenides.\n\nComparisons and scaling for R \\[1\\]\n\n| Glass     | *Q*~1~(\u00c5^\u22121^) | *R*(\u00c5) | *r*~1~(\u00c5) | *r*~1~*Q*~1~ |\n|:----------|---------------|--------|-----------|--------------|\n| SiO~2~    | 1.55          | 4.1    | 1.61      | 2.48         |\n| GeO~2~    | 1.55          | 4.1    | 1.74      | 2.70         |\n| B~2~O~3~  | 1.57          | 4.0    | 1.36      | 2.14         |\n| SiSe~2~   | 1.02          | 6.2    | 2.30      | 2.35         |\n| GeS~2~    | 1.00          | 6.3    | 2.37      | 2.37         |\n| GeS~2~    | 1.04          | 6.0    | 2.22      | 2.30         |\n| As~2~S~2~ | 1.27          | 4.9    | 2.28      | 3.10         |\n| As~2~S~2~ | 1.26          | 5.0    | 2.44      | 2.87         |\n\n*r*~1~ = bond length\n\nIt is significant to note that the scaling relationship based on SRO, breaks down for the coherence length *L* for GeSe~2~. The scaled ratio for *L* is estimated to be 15 \u00c5 compared with the higher average experimental value of *L* = 24 \u00b1 4 \u00c5 \\[11,25\\]. The comparisons based on scaling are consistent with *R* being determined by the extension of a local pair correlation determined by the ring structures in the SiO~2~ and GeSe~2~ CRNs. The microscopic basis for *L* in SiO~2~, and B~2~O~3~ as well, is determined by characteristic inter-ring bonding arrangements with a cluster size that related to coupling of two, two or three rings, respectively. These determine the period of the cluster repetition, and the encapsulation of these more symmetric rings by less symmetric rings of bonded atoms; i.e., five- and seven-member rings in SiO~2~. The inter-ring coupling in SiO~2~ is direct result of the softness of the Si\u2013O\u2013Si bonding force constant in SiO~2~\\[4,5\\]. For the case of the GeSe~2~ CRN because of the smaller Ge\u2013Se\u2013Ge bond angle and repulsive effects between the Se lone pair electrons and the bonding electrons localized in the more covalent Ge\u2013Se bonds, the coherence length is not attributed to rings of bonded atoms, but rather to a hard soft cluster mixture. The hard soft structure in GeSe alloys is determined by compositionally dependent constraints imposed by local bonding, e.g., locally rigid groups with Ge atoms separated by one bridging Se atom, Ge\u2013Se\u2013Se, and locally compliant groups associated with two bridging Se atoms, Ge\u2013Se\u2013Se\u2013Ge \\[23\\]. Similar considerations apply to the period of the hard-component of a hard-soft structure that have been proposed as the driving force for glass formation, and the associated low densities of defect and defect precursors which are associated with either broken and strained-bonds, respectively. The criterion is SiO~2~ and B~2~O~3~ is determined by a nano-structures that includes a multiplicity of different ring sizes, whereas the criterion is a volume percolation threshold that applies in chalcogenides glasses, and is consistent with locally rigid, and locally compliant groups been phase-separated into hard-soft mixtures \\[26\\]. The same considerations apply in As-chalcogenides, and for the compound As~2~Se~3~ and GeSe~2~ compositions that include local small discrete molecules that add compliance to the otherwise locally rigid CNRs that includes As\u2013Se\u2013As and Ge\u2013Se\u2013Ge bonding, respectively \\[23\\].\n\nThe conclusion is that SE-BCT, even with local modifications for symmetry-associated broken bending constraints, and additional constraints due to lone pair and terminal atom repulsions \\[23\\], has limited value in accounting the elimination of macroscopic strain reduction for technology applications. This property depends on MRO, as embodied in hard-soft mixtures, and\/or percolation of short-range order ground that exceeds a volume percolation threshold \\[23,27\\].\n\n# Nano-crystalline and Nano-crystalline\/Non-crystalline Alloys\n\nExtension of the MRO concepts of the previous sections from CRNS to nano-crystalline and nano-crystalline\/non-crystalline composites of technological importance is addressed in this section. One way to formulate this issue is to determine conditions that promote hard-soft mixtures in materials that are (1) chemically homogeneous, but inhomogeneous on a nano-meter length scale, or (2) both chemically inhomogeneous and phase-separated. The first of these is addressed in homogeneous HfO~2~ thin films, and the second for phase separated Hf silicates, as well as other phase separated materials in which SiO~2~ in a chemical constituent \\[28\\].\n\n## Nano-grain HfO~2~ Films\n\nThe nano-grain morphology of deposited and subsequently high temperature, \\>700\u00b0C, annealed HfO~2~ thin films is typically a mixture of monoclinic (m-) and tetragonal (t-) grains differentiated by Hf 5d features in combination with O 2p \u03c0 states that comprise local symmetry adapted linear combinations (SALCs) of atomic states into molecular orbitals (MO) \\[28,29\\]. These MOs are essentially one-electron states, in contrast to occupied Hf states that must by treated in a many-electron theory \\[30\\]. Of particular importance are the \u03c0-bonded MOs that contribute to the lowest conduction band features in O K edge XAS spectra \\[28,29\\]. Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a> indicates differences in these band edge features for nano-grain t-HfO~2~ and m-HfO~2~ thin films in which the grain-morphology has been controlled by interfacial bonding. The t-HfO~2~ films display a single asymmetric band edge feature, whereas m-HfO~2~ films display two band edge features. Figure <a href=\"#F3\" data-ref-type=\"fig\">3<\/a> is for films that have with a mixed t-\/m- nano-grain morphology, and a thickness that is increased from 2 to 3 nm, and then to 4 nm. Based of features in these spectra, and 2nd derivative spectra as well, the 2 nm film displays neither a t-, nor a m-nano-grain morphology, while the thicker films display a doublet structure indicative of a mixed nano-grain morphology.\n\nThe band edge 5d E~g~ splittings in Figs. <a href=\"#F2\" data-ref-type=\"fig\">2<\/a> and <a href=\"#F3\" data-ref-type=\"fig\">3<\/a> indicate a cooperative Jahn\u2013Teller (J\u2013T) distortion \\[28\\]. The theoretical model in \\[31\\] indicates that an electronic unit cell comprised of seven PUCs, each \\~0.5 to 0.55 nm is necessary for a cooperative J\u2013T effect, and this requires a nano-grain dimensions of \\~3 to 3.5 nm. This indicates a dimensional constraint in the 2 nm thick film. This film is simply too thin to support a high concentration of randomly oriented nano-grains with an electronic unit cell large enough to support a J\u2013T distortion. These 2 nm films are generally characterized as X-ray amorphous. As-deposited 3 and 4 nm thick films also display no J\u2013T, but when subjected to the same 900\u00b0C anneal as the 2 nm thick film, the dimensional constraint is relaxed and J\u2013T distortions are stabilized and are observed in O K edge XAS.\n\nThese differences in nano-scale morphology identify several scales of MRO for HfO~2~, as well as other TM d^0^ oxides, TiO~2~ and ZrO~2~. The first is the PUC \\~ 0.5 to 0.55 nm, and the second and third are for coupling of unit cells. The first coupling is manifest in 1.5\u20132.0 nm grains that are analogues of the SiO~2~ clusters comprised of 2\u20133 symmetric six-member rings. The second length scale is 3\u20133.5 nm and is sufficient to promote J\u2013T distortion which persist in thicker annealed film and bulk crystals as well. The PUC of HfO~2~ then plays the same role as the symmetric or regular six-member ring of non-crystalline SiO~2~ and in crystalline a-quartz.\n\nDifferences in nano-grain order have a profound effect on intrinsic bonding defects in HfO~2~. In films thicker than 3 nm they contribute to high densities of vacancy defects (\\~10^12^ cm^\u22122^, or equivalently 10^18^ cm^\u22123^), clustered on internal grain boundaries of nano-grains large enough to display J\u2013T term splittings \\[28\\]. These are indicated in Fig. <a href=\"#F4\" data-ref-type=\"fig\">4<\/a>.\n\nNano-grain HfO~2~ in the MRO size regime of 1.5\u20132 nm can also formed in phase-separated Hf silicates (HfO~2~)~*x*~(SiO~2~)~1\u2212*x*~, alloys in two narrow compositional regimes: 0.15 \\<*x* \\< 0.3, and 0.75 \\<*x* \\< 0.85. For the lower *x*-regime, the phase separation of an as-deposited homogeneous silicate yields a compliant hard-soft structure. This is comprised of X-ray amorphous nano-grains with \\<3 nm dimensions that are encapsulated by non-crystalline SiO~2~. For the higher *x*-regime. The phase separated silicates include X-ray amorphous nano-grains \\<3 nm in size, whose growth is frustrated by a random incorporation of 2 nm clusters of compliant non-crystalline SiO~2~. The concentration of these 2 nm clusters exceeds a volume percolation threshold accounting for the frustration of larger nano-grain growth \\[27\\].\n\nEach of these phase-separated silicate regimes exhibits low densities of defects and defect precursors. However, these diphasic silicates have not studied with respect to radiation stressing, so it would be ill-advised and inappropriate to call then SiO~2~-look-alikes, a label that has been attached to the homogeneous Hf Si oxynitride alloys in the next sub-section based on radiation stressing \\[32\\].\n\n## Homogeneous Hf Si Oxynitride Alloys\n\nThere is a unique composition (HfO~2~)~0.3~(SiO~2~)~0.3~(Si~3~N~4~)~0.4~(concentrations \u00b1 0.025) hereafter HfSiON~334~, which is stable to annealing temperatures \\>1,000\u00b0C, and whose electrical response after X-ray and \u03b3-ray stressing is essentially the same as SiO~2~\\[32\\].This similarity is with respect to (1) the linear dependence on dosing, (2) the sign of the fixed charge, always positive, and (3) the magnitude of the defect generation. The unique properties are attributed to a fourfold coordinated Hf substitute onto 16.7% of the possible fourfold coordinated Si bonding sites. This concentration is at the percolation threshold for connectivity of compliant local bonding arrangements \\[27\\]. Larger concentrations of (Si~3~N~4~) for the same or different combinations of HfO~2~ and SiO~2~ bonding leads to chemical phase separation with loss of bonded N, and therefore qualitatively different thin films.\n\n## Other Diphasic Materials with 20% SiO~2~\n\nThere are at least two other diphasic materials with a dimensionally stabilized symmetric nano-crystalline phase, and a 20% compliant non-crystalline phase, 2 nm clusters of SiO~2~. This includes a 20% mixture of non-crystalline SiO~2~ with (1) nano-crystalline zincblende-structured ZnS grains, or (2) a fine nano-grain ceramic as in Corning cookware \\[33\\]. In each of these thin materials, TEM imaging indicates that the 20% SiO~2~ is distributed uniformly in compliant clusters with an average size of \\~2\u20133 nm. These encapsulated nano-clusters reduce macroscopic strain, but equally important suppress the formation of more asymmetric crystal structures, e.g., wurtzite ZnS, which would lead to anisotropic optical properties, and make these films in unusable for use as protective layers in optical memory stacks for digital video disks (DVD) for information storage and retrieval. In the second application, the SiO~2~ makes these ceramics macroscopically strain free, and capable on being moved from the \"oven to the refrigerator\" without cracking \\[33\\].\n\n## (Si~3~N~4~)~*x*~(SiO~2~)~1\u2212*x*~ Gate Dielectrics\n\nSi oxynitride pseudo-binary alloys (Si~3~N~4~)~*x*~(SiO~2~)~1\u2212*x*~, have emerged in the late 1990s as replacement dielectrics \\[34\\]. These alloys have been used with small and high concentrations of Si~3~N~4~ with different objectives. At low concentration levels \\<5% Si~3~N~4~, for blocking Boron transported from B-doped poly-Si gate dielectrics \\[24\\], and at significantly higher concentrations, \\~50 to 60% Si~3~N~4~, as required for a significant increase in the dielectric constant from \\~3.9 to \\~5.4 to 5.8 \\[35\\].\n\nThe mid-gap interface state density, *D*~it~, and the flat-band voltage *V*~fb~ were obtained from a conventional C\u2013V analysis of metal\u2013oxide\u2013semiconductor capacitors on p-type Si substrates with \\~10^17^ cm^\u22123^ doping, *p*-MOSCAPs, with Al gate metal layers deposited after a post metal anneal in forming gas. Both *D*~it~ and and *V*~fb~ display qualitatively similar behavior as function of *x* for both as-deposited and Si-dielectric layers annealed at 900\u00b0C in Ar for 1 min \\[34\\]. The annealed dielectrics are processed at temperatures that validate comparisons with *p*-MOSCAPs with thermally grown SiO~2~ and similarly processed Al gates. *D*~it~ decreases from \\~10^11^ cm^\u22122^ eV^\u22121^ for Si~3~N~4~ (*x* = 1), to \\~10^10^ cm^\u22122^ eV^\u22121^ for *x* \\~ 0.7 to a value comparable to state of the art SiO~2~ MOSCAPs. The value of *D*~it~ is relatively constant, 1.1 \u00b1 0.2 \u00d7 10^\u221210^ cm^\u22122^ eV^\u22121^, for values of *x* from 0.65 to 0.0 (SiO~2~). In a complementary manner, *V*~fb~ increases from \u22121.3 eV for Si~3~N~4~ (*x* = 1), to \u22120.9 eV at *x* \\~ 0.7, and then remains relatively constant, \u22120.8 \u00b1 0.1 eV for values of *x* from 0.65 to 0.0 (SiO~2~). The values of *D*~it~ and *V*~fb~ are comparable to those for thermally grown SiO~2~, and therefore have been the basis for use of these Si oxynitrides in commercial devices \\[34\\].\n\nThe electrical measurements are consistent with significant decreases in macroscopic strain for Si oxnitride alloys with SiO~2~ concentrations exceeding about 35% or *x* = 0.65. This suggests a hard-soft mechanism in this regime similar to that in Hf silicates. At concentrations \\<0.35, i.e., SiO~2~ = 65%, the roles of the hard and soft components are assumed to be reversed. However, strain reduction over such an extensive composition regime suggests a more complicated nano-scale structure that has a mixed hard-soft character over a significant composition region, The proposed mixed phase is comprised of equal concentrations of Si~3~N~4~ encapsulating SiO~2~ at high Si~3~N~4~ concentrations, and an inverted hard-soft character with SiO~2~ encapsulating Si~3~N~4~ at lower Si~3~N~4~ concentrations. If this is indeed the case, it represents a rather interesting example of a double percolation process \\[26,36\\].\n\n# Summary and Conclusions\n\nThis will be displayed in a bulleted format.\n\n1\\. The spectral position of the FSDP for glasses, and its FWHM are associated with real space distances as obtained from the structure factor S(*Q*) derived from X-ray or neutron diffraction\\are in the MRO regime. The first length scale has been designated as a correlation length, *R* = 2\u03c0\/*Q*~1~(\u00c5^\u22121^), and the second length scale has been designated as a coherence length, *L* = 2\u03c0\/\u0394*Q*~1~(\u00c5^\u22121^) where *Q*~1~(\u00c5^\u22121^) and \u0394*Q*~1~(\u00c5^\u22121^) are, respectively, the position and FWHM of S(*Q*).\n\n2\\. The values of the correlation length *R*, and the coherence length *L*, obtained in this way are for: (a) SiO~2~:*R* = 4.1 \u00b1 0.2 \u00c5, and *L* = 9.5 \u00b1 0.5 \u00c5; (b) B~2~O~3~:*R* = 4.0 \u00b1 0.2 \u00c5, and *L* = 11 \u00b1 1 \u00c5; and (c) GeSe~2~: *R* = 6.3 \u00b1 0.3 \u00c5, and *L* = 24 \u00b1 4 \u00c5.\n\n3\\. Based on molecular dynamics calculations and modeling, the values of *R* correspond to third neighbor Si\u2013O, and associated with segments of six-member rings in SiO~2~. The larger value of *R* in GeSe~2~ is consistent with scaling based on Ge\u2013Se bond lengths and therefore has a similar origin.\n\n4\\. Based on molecular dynamics calculations and modeling, the coherence length features are not a direct result of inter-atomic pair correlations. This is supported by the analysis of X-ray diffraction data as well, where the coherence length is determined by the width of the FSDP rather than by an additional peak in S(*Q*).\n\n5\\. The ring clusters contributing to the coherence lengths for SiO~2~ are comprised of two, or at most three symmetric six-member rings, that are stabilized by back donation of electrons from occupied 2p \u03c0 states on O atoms to empty \u03c0 orbitals on the Si atoms. These rings are encapsulated by more compliant structures with lower symmetry irregular five- and seven-member rings to form a compliant hard-soft system.\n\n6\\. The coherence length in Ge~*x*~Se~1\u2212*x*~ alloys is different in Se-rich and Ge-rich composition regimes, and is significantly larger in each of these regimes than at the compound composition, GeSe~2~ which they bracket. It is determined in each alloy regime, and at the compound composition by minimization of macroscopic strain by a chemical bonding self-organization as in which site percolation dominates. There is a compliant alloy regime which extends from *x* = 0.2 to 0.26 in which locally compliant bonding arrangements, Ge\u2013Se\u2013Se\u2013Ge, completely encapsulate a more rigid cluster comprised of locally rigid Ge\u2013Se\u2013Ge bonding. For compositions greater than *x* = 0.26 and extending to *x* = 0.4, macroscopic compliance results form a diphasic mixture which includes small molecules with Ge\u2013Se, and Ge\u2013Ge bonding.\n\n7\\. The hard-soft mix in non-crystalline SiO~2~ with a length scale of at most 1 nm establishes the unique properties of gate dielectrics \\>1\u20131.5 nm thick, and for cm glasses with cm-dimensions as well.\n\n8\\. There is an analog between the properties of nano-crystalline HfO~2~, and phase separated HfO~2~-SiO~2~ silicate alloys, ZnS-SiO~2~ alloys and ceramic-SiO~2~ alloys that establishes their unique properties in device applications as diverse as gate dielectrics for aggressively scaled dielectrics, protective layers for stacks in with rewritable optical information storage, and for temperature compliance in ceramic cookware.\n\n9\\. *p*-MOSCAPs with Si oxynitride pseudo-binary alloys (Si~3~N~4~)~*x*~(SiO~2~)~1\u2212*x*~, gate dielectrics display an defect densities for interface trapping, *D*~it~, and fixed positive charge that determines the flat-band voltage, *V*~fb~, comparable to those of thermally grown SiO~2~ dielectrics for a range of concentrations extending for \\~70%, *x* = 0.7, Si~3~N~4~ to SiO~2~. The electrical measurements are consistent with significant decreases in macroscopic strain, suggesting a hard-soft mechanism in this regime similar to that in Hf silicates. However, strain reduction over such an extensive composition regime suggests a more complicated nano-scale structure that has a mixed hard-soft character over a significant composition region, The proposed mixed phase is comprised of equal concentrations of Si~3~N~4~ encapsulating SiO~2~ at high Si~3~N~4~ concentrations, and an inverted hard-soft character with SiO~2~ encapsulating Si~3~N~4~ at lower Si~3~N~4~ concentrations. If this is indeed the case, it represents a rather interesting example of a double percolation process.\n\n10\\. The properties of the films and bulk materials identified above are underpinned by the real-space correlation and coherence lengths, *R* and *L*, obtained from analysis of the SiO~2~ structure factor derived from X-ray or neutron diffraction. The real space interpretation relies of the application of many-electron theory to the structural, optical and defect properties on non-crystalline SiO~2~.\n\n## Acknowledgments\n\nOne of the authors (G. L.) acknowledges support from the AFOSR, SRC, DTRA and NSF.\n\n### Open Access\n\nThis article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.","meta":{"dup_signals":{"dup_doc_count":147,"dup_dump_count":46,"dup_details":{"curated_sources":2,"2022-33":1,"2021-10":1,"2020-05":1,"2019-47":1,"2019-39":1,"2019-26":1,"2019-18":1,"2019-04":1,"2018-43":1,"2018-34":1,"2018-26":1,"2017-51":1,"2017-39":1,"2017-30":2,"2017-22":1,"2017-17":1,"2017-09":16,"2017-04":1,"2016-50":1,"2016-44":1,"2016-36":1,"2016-30":2,"2016-26":1,"2016-22":2,"2016-18":2,"2015-48":5,"2015-40":5,"2015-35":5,"2015-32":3,"2015-27":3,"2015-22":5,"2015-14":4,"2014-52":5,"2014-49":5,"2014-42":6,"2014-41":7,"2014-35":6,"2014-23":8,"2014-15":5,"2023-40":1,"2015-18":5,"2015-11":3,"2015-06":5,"2014-10":5,"2013-48":5,"2013-20":5}},"file":"PMC2894147"},"subset":"pubmed_central"}
{"text":"author:\ndate: 2011-01\nreferences:\ntitle: Standards of Medical Care in Diabetes\u20142011\n\n# CONTENTS\n\n1.  CLASSIFICATION AND DIAGNOSIS OF DIABETES, p. S12\n\n    1.  Classification of diabetes\n\n    2.  Diagnosis of diabetes\n\n    3.  Categories of increased risk for diabetes (prediabetes)\n\n2.  TESTING FOR DIABETES IN ASYMPTOMATIC PATIENTS, p. S13\n\n    1.  Testing for type 2 diabetes and risk of future diabetes in adults\n\n    2.  Testing for type 2 diabetes in children\n\n    3.  Screening for type 1 diabetes\n\n3.  DETECTION AND DIAGNOSIS OF GESTATIONAL DIABETES MELLITUS, p. S15\n\n4.  PREVENTION\/DELAY OF TYPE 2 DIABETES, p. S16\n\n5.  DIABETES CARE, p. S16\n\n    1.  Initial evaluation\n\n    2.  Management\n\n    3.  Glycemic control\n\n        1.  Assessment of glycemic control\n\n            1.  Glucose monitoring\n\n            2.  A1C\n\n        2.  Glycemic goals in adults\n\n    4.  Pharmacologic and overall approaches to treatment\n\n        1.  Therapy for type 1 diabetes\n\n        2.  Therapy for type 2 diabetes\n\n    5.  Diabetes self-management education\n\n    6.  Medical nutrition therapy\n\n    7.  Physical activity\n\n    8.  Psychosocial assessment and care\n\n    9.  When treatment goals are not met\n\n    10. Hypoglycemia\n\n    11. Intercurrent illness\n\n    12. Bariatric surgery\n\n    13. Immunization\n\n6.  PREVENTION AND MANAGEMENT OF DIABETES COMPLICATIONS, p. S27\n\n    1.  Cardiovascular disease\n\n        1.  Hypertension\/blood pressure control\n\n        2.  Dyslipidemia\/lipid management\n\n        3.  Antiplatelet agents\n\n        4.  Smoking cessation\n\n        5.  Coronary heart disease screening and treatment\n\n    2.  Nephropathy screening and treatment\n\n    3.  Retinopathy screening and treatment\n\n    4.  Neuropathy screening and treatment\n\n    5.  Foot care\n\n7.  DIABETES CARE IN SPECIFIC POPULATIONS, p. S38\n\n    1.  Children and adolescents\n\n        1.  Type 1 diabetes Glycemic control\n\n            1.  Screening and management of chronic complications in children and adolescents with type 1 diabetes\n\n                1.  Nephropathy\n\n                2.  Hypertension\n\n                3.  Dyslipidemia\n\n                4.  Retinopathy\n\n                5.  Celiac disease\n\n                6.  Hypothyroidism\n\n            2.  Self-management\n\n            3.  School and day care\n\n            4.  Transition from pediatric to adult care\n\n        2.  Type 2 diabetes\n\n        3.  Monogenic diabetes syndromes\n\n    2.  Preconception care\n\n    3.  Older adults\n\n    4.  Cystic fibrosis\u2013related diabetes\n\n8.  DIABETES CARE IN SPECIFIC SETTINGS, p. S43\n\n    1.  Diabetes care in the hospital\n\n        1.  Glycemic targets in hospitalized patients\n\n        2.  Anti-hyperglycemic agents in hospitalized patients\n\n        3.  Preventing hypoglycemia\n\n        4.  Diabetes care providers in the hospital\n\n        5.  Self-management in the hospital\n\n        6.  Diabetes self-management education in the hospital\n\n        7.  Medical nutrition therapy in the hospital\n\n        8.  Bedside blood glucose monitoring\n\n        9.  Discharge planning\n\n9.  STRATEGIES FOR IMPROVING DIABETES CARE, p. S46\n\nDiabetes is a chronic illness that requires continuing medical care and ongoing patient self-management education and support to prevent acute complications and to reduce the risk of long-term complications. Diabetes care is complex and requires that many issues, beyond glycemic control, be addressed. A large body of evidence exists that supports a range of interventions to improve diabetes outcomes.\n\nThese standards of care are intended to provide clinicians, patients, researchers, payors, and other interested individuals with the components of diabetes care, general treatment goals, and tools to evaluate the quality of care. While individual preferences, comorbidities, and other patient factors may require modification of goals, targets that are desirable for most patients with diabetes are provided. These standards are not intended to preclude clinical judgment or more extensive evaluation and management of the patient by other specialists as needed. For more detailed information about management of diabetes, refer to references 1\u20133.\n\nThe recommendations included are screening, diagnostic, and therapeutic actions that are known or believed to favorably affect health outcomes of patients with diabetes. A grading system (<a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>), developed by the American Diabetes Association (ADA) and modeled after existing methods, was utilized to clarify and codify the evidence that forms the basis for the recommendations. The level of evidence that supports each recommendation is listed after each recommendation using the letters A, B, C, or E.\n\nADA evidence grading system for clinical practice recommendations\n\n<table>\n<thead>\n<tr>\n<th style=\"text-align: left;\">Level of evidence<\/th>\n<th style=\"text-align: center;\">Description<\/th>\n<\/tr>\n<\/thead>\n<tbody>\n<tr>\n<td style=\"text-align: left;\">A<\/td>\n<td style=\"text-align: left;\">Clear evidence from well-conducted, generalizable, randomized controlled trials that are adequately powered, including:\n<ul>\n<li><p>Evidence from a well-conducted multicenter trial<\/p><\/li>\n<li><p>Evidence from a meta-analysis that incorporated quality ratings in the analysis<\/p><\/li>\n<\/ul><\/td>\n<\/tr>\n<tr>\n<td><\/td>\n<td style=\"text-align: left;\">Compelling nonexperimental evidence, i.e., \"all or none\" rule developed by Center for Evidence Based Medicine at Oxford<\/td>\n<\/tr>\n<tr>\n<td><\/td>\n<td style=\"text-align: left;\">Supportive evidence from well-conducted randomized controlled trials that are adequately powered, including:\n<ul>\n<li><p>Evidence from a well-conducted trial at one or more institutions<\/p><\/li>\n<li><p>Evidence from a meta-analysis that incorporated quality ratings in the analysis<\/p><\/li>\n<\/ul><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">B<\/td>\n<td style=\"text-align: left;\">Supportive evidence from well-conducted cohort studies\n<ul>\n<li><p>Evidence from a well-conducted prospective cohort study or registry<\/p><\/li>\n<li><p>Evidence from a well-conducted meta-analysis of cohort studies<\/p><\/li>\n<\/ul><\/td>\n<\/tr>\n<tr>\n<td><\/td>\n<td style=\"text-align: left;\">Supportive evidence from a well-conducted case-control study<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">C<\/td>\n<td style=\"text-align: left;\">Supportive evidence from poorly controlled or uncontrolled studies\n<ul>\n<li><p>Evidence from randomized clinical trials with one or more major or three or more minor methodological flaws that could invalidate the results<\/p><\/li>\n<li><p>Evidence from observational studies with high potential for bias (such as case series with comparison to historical controls)<\/p><\/li>\n<li><p>Evidence from case series or case reports<\/p><\/li>\n<\/ul><\/td>\n<\/tr>\n<tr>\n<td><\/td>\n<td style=\"text-align: left;\">Conflicting evidence with the weight of evidence supporting the recommendation<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">E<\/td>\n<td style=\"text-align: left;\">Expert consensus or clinical experience<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\nThese standards of care are revised annually by the ADA's multidisciplinary Professional Practice Committee, incorporating new evidence. Members of the Professional Practice Committee and their disclosed conflicts of interest are listed on page S97. Subsequently, as with all Position Statements, the standards of care are reviewed and approved by the Executive Committee of ADA's Board of Directors.\n\n# I. CLASSIFICATION AND DIAGNOSIS OF DIABETES\n\n## A. Classification of diabetes\n\nThe classification of diabetes includes four clinical classes:\n\n-   Type 1 diabetes (results from \u03b2-cell destruction, usually leading to absolute insulin deficiency)\n\n-   Type 2 diabetes (results from a progressive insulin secretory defect on the background of insulin resistance)\n\n-   Other specific types of diabetes due to other causes, e.g., genetic defects in \u03b2-cell function, genetic defects in insulin action, diseases of the exocrine pancreas (such as cystic fibrosis), and drug- or chemical-induced (such as in the treatment of HIV\/AIDS or after organ transplantation)\n\n-   Gestational diabetes mellitus (GDM) (diabetes diagnosed during pregnancy that is not clearly overt diabetes)\n\nSome patients cannot be clearly classified as having type 1 or type 2 diabetes. Clinical presentation and disease progression vary considerably in both types of diabetes. Occasionally, patients who otherwise have type 2 diabetes may present with ketoacidosis. Similarly, patients with type 1 diabetes may have a late onset and slow (but relentless) progression of disease despite having features of autoimmune disease. Such difficulties in diagnosis may occur in children, adolescents, and adults. The true diagnosis may become more obvious over time.\n\n## B. Diagnosis of diabetes\n\nFor decades, the diagnosis of diabetes was based on plasma glucose criteria, either the fasting plasma glucose (FPG) or the 2-h value in the 75-g oral glucose tolerance test (OGTT) (4).\n\nIn 2009, an International Expert Committee that included representatives of the ADA, the International Diabetes Federation (IDF), and the European Association for the Study of Diabetes (EASD) recommended the use of the A1C test to diagnose diabetes, with a threshold of \u22656.5% (5), and ADA adopted this criterion in 2010 (4). The diagnostic test should be performed using a method that is certified by the National Glycohemoglobin Standardization Program (NGSP) and standardized or traceable to the Diabetes Control and Complications Trial (DCCT) reference assay. Point-of-care A1C assays are not sufficiently accurate at this time to use for diagnostic purposes.\n\nEpidemiologic datasets show a similar relationship between A1C and risk of retinopathy as has been shown for the corresponding FPG and 2-h plasma glucose thresholds. The A1C has several advantages to the FPG and OGTT, including greater convenience, since fasting is not required; evidence to suggest greater preanalytical stability; and less day-to-day perturbations during periods of stress and illness. These advantages must be balanced by greater cost, the limited availability of A1C testing in certain regions of the developing world, and the incomplete correlation between A1C and average glucose in certain individuals. In addition, A1C levels can vary with patients' ethnicity (6) as well as with certain anemias and hemoglobinopathies. For patients with an abnormal hemoglobin but normal red cell turnover, such as sickle cell trait, an A1C assay without interference from abnormal hemoglobins should be used (an updated list is available at [www.ngsp.org\/interf.asp](http:\/\/www.ngsp.org\/interf.asp)). For conditions with abnormal red cell turnover, such as pregnancy, recent blood loss or transfusion, or some anemias, the diagnosis of diabetes must employ glucose criteria exclusively.\n\nThe established glucose criteria for the diagnosis of diabetes (FPG and 2-h PG) remain valid as well (<a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a>). Just as there is less than 100% concordance between the FPG and 2-h PG tests, there is not perfect concordance between A1C and either glucose-based test. Analyses of National Health and Nutrition Examination Survey (NHANES) data indicate that, assuming universal screening of the undiagnosed, the A1C cut point of \u22656.5% identifies one-third fewer cases of undiagnosed diabetes than a fasting glucose cut point of \u2265126 mg\/dl (7.0 mmol\/l) (7). However, in practice, a large portion of the diabetic population remains unaware of their condition. Thus, the lower sensitivity of A1C at the designated cut point may well be offset by the test's greater practicality, and wider application of a more convenient test (A1C) may actually increase the number of diagnoses made.\n\nCriteria for the diagnosis of diabetes\n\n|  |\n|----|\n| A1C \u22656.5%. The test should be performed in a laboratory using a method that is NGSP certified and standardized to the DCCT assay.<a href=\"#TF2-1\" data-ref-type=\"table-fn\">*<\/a> |\n| or |\n| FPG \u2265126 mg\/dl (7.0 mmol\/l). Fasting is defined as no caloric intake for at least 8 h.<a href=\"#TF2-1\" data-ref-type=\"table-fn\">*<\/a> |\n| or |\n| 2-h plasma glucose \u2265200 mg\/dl (11.1 mmol\/l) during an OGTT. The test should be performed as described by the World Health Organization, using a glucose load containing the equivalent of 75 g anhydrous glucose dissolved in water.<a href=\"#TF2-1\" data-ref-type=\"table-fn\">*<\/a> |\n| or |\n| In a patient with classic symptoms of hyperglycemia or hyperglycemic crisis, a random plasma glucose \u2265200 mg\/dl (11.1 mmol\/l) |\n\n\\*In the absence of unequivocal hyperglycemia, result should be confirmed by repeat testing.\n\nAs with most diagnostic tests, a test result diagnostic of diabetes should be repeated to rule out laboratory error, unless the diagnosis is clear on clinical grounds, such as a patient with a hyperglycemic crisis or classic symptoms of hyperglycemia and a random plasma glucose \u2265200 mg\/dl. It is preferable that the same test be repeated for confirmation, since there will be a greater likelihood of concurrence in this case. For example, if the A1C is 7.0% and a repeat result is 6.8%, the diagnosis of diabetes is confirmed. However, if two different tests (such as A1C and FPG) are both above the diagnostic thresholds, the diagnosis of diabetes is also confirmed.\n\nOn the other hand, if two different tests are available in an individual and the results are discordant, the test whose result is above the diagnostic cut point should be repeated, and the diagnosis is made on the basis of the confirmed test. That is, if a patient meets the diabetes criterion of the A1C (two results \u22656.5%) but not the FPG (\\<126 mg\/dl or 7.0 mmol\/l), or vice versa, that person should be considered to have diabetes.\n\nSince there is preanalytic and analytic variability of all the tests, it is also possible that when a test whose result was above the diagnostic threshold is repeated, the second value will be below the diagnostic cut point. This is least likely for A1C, somewhat more likely for FPG, and most likely for the 2-h PG. Barring a laboratory error, such patients are likely to have test results near the margins of the threshold for a diagnosis. The healthcare professional might opt to follow the patient closely and repeat the testing in 3\u20136 months.\n\nThe current diagnostic criteria for diabetes are summarized in <a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a>.\n\n## C. Categories of increased risk for diabetes (prediabetes)\n\nIn 1997 and 2003, The Expert Committee on Diagnosis and Classification of Diabetes Mellitus (8,9) recognized an intermediate group of individuals whose glucose levels, although not meeting criteria for diabetes, are nevertheless too high to be considered normal. These persons were defined as having impaired fasting glucose (IFG) (FPG levels 100\u2013125 mg\/dl \\[5.6\u20136.9 mmol\/l\\]) or impaired glucose tolerance (IGT) (2-h PG values in the OGTT of 140\u2013199 mg\/dl \\[7.8\u201311.0 mmol\/l\\]). It should be noted that the World Health Organization (WHO) and a number of other diabetes organizations define the cutoff for IFG at 110 mg\/dl (6.1 mmol\/l).\n\nIndividuals with IFG and\/or IGT have been referred to as having prediabetes, indicating the relatively high risk for the future development of diabetes. IFG and IGT should not be viewed as clinical entities in their own right but rather risk factors for diabetes as well as cardiovascular disease (CVD). IFG and IGT are associated with obesity (especially abdominal or visceral obesity), dyslipidemia with high triglycerides and\/or low HDL cholesterol, and hypertension.\n\nAs is the case with the glucose measures, several prospective studies that used A1C to predict the progression to diabetes demonstrated a strong, continuous association between A1C and subsequent diabetes. In a systematic review of 44,203 individuals from 16 cohort studies with a follow-up interval averaging 5.6 years (range 2.8\u201312 years), those with an A1C between 5.5 and 6.0% had a substantially increased risk of diabetes with 5-year incidences ranging from 9\u201325%. An A1C range of 6.0\u20136.5% had a 5-year risk of developing diabetes between 25\u201350% and relative risk 20 times higher compared with an A1C of 5.0% (10). In a community-based study of black and white adults without diabetes, baseline A1C was a stronger predictor of subsequent diabetes and cardiovascular events than fasting glucose (11). Other analyses suggest that an A1C of 5.7% is associated with diabetes risk similar to that of the high-risk participants in the Diabetes Prevention Program (DPP).\n\nHence, it is reasonable to consider an A1C range of 5.7\u20136.4% as identifying individuals with high risk for future diabetes, a state that may be referred to as prediabetes (4). As is the case for individuals found to have IFG and IGT, individuals with an A1C of 5.7\u20136.4% should be informed of their increased risk for diabetes as well as CVD and counseled about effective strategies to lower their risks (see <span class=\"smallcaps\">iv. prevention\/delay of type 2 diabetes<\/span>). As with glucose measurements, the continuum of risk is curvilinear\u2014as A1C rises, the risk of diabetes rises disproportionately (10). Accordingly, interventions should be most intensive and follow-up particularly vigilant for those with A1Cs above 6.0%, who should be considered to be at very high risk.\n\n<a href=\"#T3\" data-ref-type=\"table\">Table 3<\/a> summarizes the categories of increased risk for diabetes.\n\nCategories of increased risk for diabetes (prediabetes)<a href=\"#TF3-1\" data-ref-type=\"table-fn\">*<\/a>\n\n|                                                                          |\n|--------------------------------------------------------------------------|\n| FPG 100\u2013125 mg\/dl (5.6\u20136.9 mmol\/l): IFG                                  |\n| or                                                                       |\n| 2-h plasma glucose in the 75-g OGTT 140\u2013199 mg\/dl (7.8\u201311.0 mmol\/l): IGT |\n| or                                                                       |\n| A1C 5.7\u20136.4%                                                             |\n\n\\*For all three tests, risk is continuous, extending below the lower limit of the range and becoming disproportionately greater at higher ends of the range.\n\n# II. TESTING FOR DIABETES IN ASYMPTOMATIC PATIENTS\n\n## Recommendations\n\n-   Testing to detect type 2 diabetes and assess risk for future diabetes in asymptomatic people should be considered in adults of any age who are overweight or obese (BMI \u226525 kg\/m^2^) and who have one or more additional risk factors for diabetes (<a href=\"#T4\" data-ref-type=\"table\">Table 4<\/a>). In those without these risk factors, testing should begin at age 45 years. (B)\n\n-   If tests are normal, repeat testing carried out at least at 3-year intervals is reasonable. (E)\n\n-   To test for diabetes or to assess risk of future diabetes, A1C, FPG, or 2-h 75-g OGTT is appropriate. (B)\n\n-   In those identified with increased risk for future diabetes, identify and, if appropriate, treat other CVD risk factors. (B)\n\nFor many illnesses, there is a major distinction between screening and diagnostic testing. However, for diabetes, the same tests would be used for \"screening\" as for diagnosis. Diabetes may be identified anywhere along a spectrum of clinical scenarios ranging from a seemingly low-risk individual who happens to have glucose testing, to a higher-risk individual whom the provider tests because of high suspicion of diabetes, to the symptomatic patient. The discussion herein is primarily framed as testing for diabetes in those without symptoms. Testing for diabetes will also detect individuals at increased future risk for diabetes, herein referred to as having prediabetes.\n\nCriteria for testing for diabetes in asymptomatic adult individuals\n\n<table>\n<tbody>\n<tr>\n<td style=\"text-align: left;\"><ol>\n<li><p>Testing should be considered in all adults who are overweight (BMI \u226525 kg\/m<sup>2<\/sup><a href=\"#TF4-1\" data-ref-type=\"table-fn\">*<\/a>) and have additional risk factors:<\/p>\n<ul>\n<li><p>physical inactivity<\/p><\/li>\n<li><p>first-degree relative with diabetes<\/p><\/li>\n<li><p>high-risk race\/ethnicity (e.g., African American, Latino, Native American, Asian American, Pacific Islander)<\/p><\/li>\n<li><p>women who delivered a baby weighing &gt;9 lb or were diagnosed with GDM<\/p><\/li>\n<li><p>hypertension (\u2265140\/90 mmHg or on therapy for hypertension)<\/p><\/li>\n<li><p>HDL cholesterol level &lt;35 mg\/dl (0.90 mmol\/l) and\/or a triglyceride level &gt;250 mg\/dl (2.82 mmol\/l)<\/p><\/li>\n<li><p>women with polycystic ovarian syndrome (PCOS)<\/p><\/li>\n<li><p>A1C \u22655.7%, IGT, or IFG on previous testing<\/p><\/li>\n<li><p>other clinical conditions associated with insulin resistance (e.g., severe obesity, acanthosis nigricans)<\/p><\/li>\n<li><p>history of CVD<\/p><\/li>\n<\/ul><\/li>\n<li><p>In the absence of the above criteria, testing for diabetes should begin at age 45 years.<\/p><\/li>\n<li><p>If results are normal, testing should be repeated at least at 3-year intervals, with consideration of more frequent testing depending on initial results and risk status.<\/p><\/li>\n<\/ol><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\\*At-risk BMI may be lower in some ethnic groups.\n\n## A. Testing for type 2 diabetes and risk of future diabetes in adults\n\nType 2 diabetes is frequently not diagnosed until complications appear, and approximately one-fourth of all people with diabetes in the U.S. may be undiagnosed. The effectiveness of early identification of prediabetes and diabetes through mass testing of asymptomatic individuals has not been proven definitively, and rigorous trials to provide such proof are unlikely to occur. However, mathematical modeling studies suggest that screening independent of risk factors beginning at age 30 or 45 years is highly cost-effective (\\<\\$11,000 per quality-adjusted life-year gained) (12). Prediabetes and diabetes meet established criteria for conditions in which early detection is appropriate. Both conditions are common and increasing in prevalence and impose significant public health burdens. There is a long presymptomatic phase before the diagnosis of type 2 diabetes is usually made. Relatively simple tests are available to detect preclinical disease. Additionally, the duration of glycemic burden is a strong predictor of adverse outcomes, and effective interventions exist to prevent progression of prediabetes to diabetes (see <span class=\"smallcaps\">iv. prevention\/delay of type 2 diabetes<\/span>) and to reduce risk of complications of diabetes (see <span class=\"smallcaps\">vi. prevention and management of diabetes complications<\/span>).\n\nRecommendations for testing for diabetes in asymptomatic, undiagnosed adults are listed in <a href=\"#T4\" data-ref-type=\"table\">Table 4<\/a>. Testing should be considered in adults of any age with BMI \u226525 kg\/m^2^ and one or more of the known risk factors for diabetes. Because age is a major risk factor for diabetes, testing of those without other risk factors should begin no later than age 45 years.\n\nEither A1C, FPG, or the 2-h OGTT is appropriate for testing. The 2-h OGTT identifies people with either IFG or IGT and thus more people at increased risk for the development of diabetes and CVD. It should be noted that the two tests do not necessarily detect the same individuals. The efficacy of interventions for primary prevention of type 2 diabetes (13\u201319) have primarily been demonstrated among individuals with IGT, not for individuals with IFG (who do not also have IGT) or for individuals with specific A1C levels.\n\nThe appropriate interval between tests is not known (20). The rationale for the 3-year interval is that false negatives will be repeated before substantial time elapses, and there is little likelihood that an individual will develop significant complications of diabetes within 3 years of a negative test result. In the modeling study, repeat screening every 3 or 5 years was cost-effective (12).\n\nBecause of the need for follow-up and discussion of abnormal results, testing should be carried out within the health care setting. Community screening outside a health care setting is not recommended because people with positive tests may not seek, or have access to, appropriate follow-up testing and care. Conversely, there may be failure to ensure appropriate repeat testing for individuals who test negative. Community screening may also be poorly targeted, i.e., it may fail to reach the groups most at risk and inappropriately test those at low risk (the worried well) or even those already diagnosed.\n\n## B. Testing for type 2 diabetes in children\n\nThe incidence of type 2 diabetes in adolescents has increased dramatically in the last decade, especially in minority populations (21), although the disease remains rare in the general pediatric population (22). Consistent with recommendations for adults, children and youth at increased risk for the presence or the development of type 2 diabetes should be tested within the health care setting. The recommendations of the ADA Consensus Statement on Type 2 Diabetes in Children and Youth (23), with some modifications, are summarized in <a href=\"#T5\" data-ref-type=\"table\">Table 5<\/a>.\n\nTesting for type 2 diabetes in asymptomatic children\n\n<table>\n<tbody>\n<tr>\n<td style=\"text-align: left;\">Criteria\n<ul>\n<li><p>Overweight (BMI &gt;85th percentile for age and sex, weight for height &gt;85th percentile, or weight &gt;120% of ideal for height)<\/p><\/li>\n<\/ul><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Plus any two of the following risk factors:\n<ul>\n<li><p>Family history of type 2 diabetes in first- or second-degree relative<\/p><\/li>\n<li><p>Race\/ethnicity (Native American, African American, Latino, Asian American, Pacific Islander)<\/p><\/li>\n<li><p>Signs of insulin resistance or conditions associated with insulin resistance (acanthosis nigricans, hypertension, dyslipidemia, PCOS, or small-for-gestational-age birth weight)<\/p><\/li>\n<li><p>Maternal history of diabetes or GDM during the child's gestation<\/p><\/li>\n<\/ul><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Age of initiation: age 10 years or at onset of puberty, if puberty occurs at a younger age<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Frequency: every 3 years<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n## C. Screening for type 1 diabetes\n\nGenerally, people with type 1 diabetes present with acute symptoms of diabetes and markedly elevated blood glucose levels, and most cases are diagnosed soon after the onset of hyperglycemia. However, evidence from type 1 prevention studies suggests that measurement of islet autoantibodies identifies individuals who are at risk for developing type 1 diabetes. Such testing may be appropriate in high-risk individuals, such as those with prior transient hyperglycemia or those who have relatives with type 1 diabetes, in the context of clinical research studies (see, for example, <http:\/\/www2.diabetestrialnet.org>). Widespread clinical testing of asymptomatic low-risk individuals cannot currently be recommended, as it would identify very few individuals in the general population who are at risk. Individuals who screen positive should be counseled about their risk of developing diabetes. Clinical studies are being conducted to test various methods of preventing type 1 diabetes, or reversing early type 1 diabetes, in those with evidence of autoimmunity.\n\n# III. DETECTION AND DIAGNOSIS OF GESTATIONAL DIABETES MELLITUS\n\n## Recommendations\n\n-   Screen for undiagnosed type 2 diabetes at the first prenatal visit in those with risk factors, using standard diagnostic criteria. (B)\n\n-   In pregnant women not known to have diabetes, screen for GDM at 24\u201328 weeks of gestation, using a 75-g 2-h OGTT and the diagnostic cut points in <a href=\"#T6\" data-ref-type=\"table\">Table 6<\/a>. (B)\n\n-   Screen women with GDM for persistent diabetes 6\u201312 weeks postpartum. (E)\n\n-   Women with a history of GDM should have lifelong screening for the development of diabetes or prediabetes at least every 3 years. (E)\n\nFor many years, GDM was defined as any degree of glucose intolerance with onset or first recognition during pregnancy (8), whether or not the condition persisted after pregnancy, and not excluding the possibility that unrecognized glucose intolerance may have antedated or begun concomitantly with the pregnancy. This definition facilitated a uniform strategy for detection and classification of GDM, but its limitations were recognized for many years. As the ongoing epidemic of obesity and diabetes has led to more type 2 diabetes in women of childbearing age, the number of pregnant women with undiagnosed type 2 diabetes has increased (24). Because of this, it is reasonable to screen women with risk factors for type 2 diabetes (<a href=\"#T4\" data-ref-type=\"table\">Table 4<\/a>) for diabetes at their initial prenatal visit, using standard diagnostic criteria (<a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a>). Women with diabetes found at this visit should receive a diagnosis of overt, not gestational, diabetes.\n\nScreening for and diagnosis of GDM\n\n<table>\n<tbody>\n<tr>\n<td style=\"text-align: left;\">Perform a 75-g OGTT, with plasma glucose measurement fasting and at 1 and 2 h, at 24\u201328 weeks of gestation in women not previously diagnosed with overt diabetes.<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">The OGTT should be performed in the morning after an overnight fast of at least 8 h.<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">The diagnosis of GDM is made when any of the following plasma glucose values are exceeded:\n<ul>\n<li><p>Fasting \u226592 mg\/dl (5.1 mmol\/l)<\/p><\/li>\n<li><p>1 h \u2265180 mg\/dl (10.0 mmol\/l)<\/p><\/li>\n<li><p>2 h \u2265153 mg\/dl (8.5 mmol\/l)<\/p><\/li>\n<\/ul><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\nGDM carries risks for the mother and neonate. The Hyperglycemia and Adverse Pregnancy Outcomes (HAPO) study (25), a large-scale (\u223c25,000 pregnant women) multinational epidemiologic study, demonstrated that risk of adverse maternal, fetal, and neonatal outcomes continuously increased as a function of maternal glycemia at 24\u201328 weeks, even within ranges previously considered normal for pregnancy. For most complications, there was no threshold for risk. These results have led to careful reconsideration of the diagnostic criteria for GDM. After deliberations in 2008\u20132009, the International Association of Diabetes and Pregnancy Study Groups (IADPSG), an international consensus group with representatives from multiple obstetrical and diabetes organizations, including ADA, developed revised recommendations for diagnosing GDM. The group recommended that all women not known to have diabetes undergo a 75-g OGTT at 24\u201328 weeks of gestation. Additionally, the group developed diagnostic cut points for the fasting, 1-h, and 2-h plasma glucose measurements that conveyed an odds ratio for adverse outcomes of at least 1.75 compared with the mean glucose levels in the HAPO study. Current screening and diagnostic strategies, based on the IADPSG statement (26), are outlined in <a href=\"#T6\" data-ref-type=\"table\">Table 6<\/a>.\n\nThese new criteria will significantly increase the prevalence of GDM, primarily because only one abnormal value, not two, is sufficient to make the diagnosis. The ADA recognizes the anticipated significant increase in the incidence of GDM to be diagnosed by these criteria and is sensitive to concerns about the \"medicalization\" of pregnancies previously categorized as normal. These diagnostic criteria changes are being made in the context of worrisome worldwide increases in obesity and diabetes rates, with the intent of optimizing gestational outcomes for women and their babies.\n\nAdmittedly, there are few data from randomized clinical trials regarding therapeutic interventions in women who will now be diagnosed with GDM based on only one blood glucose value above the specified cut points (in contrast to the older criteria that stipulated at least two abnormal values.) Expected benefits to their pregnancies and offspring is inferred from intervention trials that focused on women with more mild hyperglycemia than identified using older GDM diagnostic criteria and that found modest benefits (27,28). The frequency of their follow-up and blood glucose monitoring is not yet clear, but likely to be less intensive than women diagnosed by the older criteria. Additional well-designed clinical studies are needed to determine the optimal intensity of monitoring and treatment of women with GDM diagnosed by the new criteria (that would not have met the prior definition of GDM). It is important to note that 80\u201390% of women in both of the mild GDM studies (whose glucose values overlapped with the thresholds recommended herein) could be managed with lifestyle therapy alone.\n\nBecause some cases of GDM may represent preexisting undiagnosed type 2 diabetes, women with a history of GDM should be screened for diabetes 6\u201312 weeks postpartum, using nonpregnant OGTT criteria. Women with a history of GDM have a greatly increased subsequent risk for diabetes (29) and should be followed up with subsequent screening for the development of diabetes or prediabetes, as outlined in <span class=\"smallcaps\">ii<\/span>. <span class=\"smallcaps\">testing for diabetes in asymptomatic patients<\/span>.\n\n# IV. PREVENTION\/DELAY OF TYPE 2 DIABETES\n\n## Recommendations\n\n-   Patients with IGT (A), IFG (E), or an A1C of 5.7\u20136.4% (E) should be referred to an effective ongoing support program targeting weight loss of 7% of body weight and increasing physical activity to at least 150 min\/week of moderate activity such as walking.\n\n-   Follow-up counseling appears to be important for success. (B)\n\n-   Based on potential cost savings of diabetes prevention, such programs should be covered by third-party payors. (E)\n\n-   Metformin therapy for prevention of type 2 diabetes may be considered in those at the highest risk for developing diabetes, such as those with multiple risk factors, especially if they demonstrate progression of hyperglycemia (e.g., A1C \u22656%) despite lifestyle interventions. (B)\n\n-   Monitoring for the development of diabetes in those with prediabetes should be performed every year. (E)\n\nRandomized controlled trials have shown that individuals at high risk for developing diabetes (those with IFG, IGT, or both) can be given interventions that significantly decrease the rate of onset of diabetes (13\u201319). These interventions include intensive lifestyle modification programs that have been shown to be very effective (58% reduction after 3 years) and use of the pharmacologic agents metformin, \u03b1-glucosidase inhibitors, orlistat, and thiazolidinediones (TZDs), each of which has been shown to decrease incident diabetes to various degrees. A summary of major diabetes prevention trials is shown in <a href=\"#T7\" data-ref-type=\"table\">Table 7<\/a>.\n\nTherapies proven effective in diabetes prevention trials\n\n| Study (ref.) | *n* | Population | Mean age (years) | Duration (years) | Intervention (daily dose) | Incidence in control subjects (%\/year) | Relative risk reduction (%) (95% CI) | 3-Year number needed to treat<a href=\"#TF7-1\" data-ref-type=\"table-fn\">\u03b4<\/a> |\n|----|----|----|----|----|----|----|----|----|\n| Lifestyle |  |  |  |  |  |  |  |  |\n| \u2003\u2003\u2003\u2003Finnish DPS (14) | 522 | IGT, BMI \u226525 kg\/m^2^ | 55 | 3.2 | I-D&E | 6 | 58 (30\u201370) | 8.5 |\n| \u2003\u2003\u2003\u2003DPP (13) | 2,161<a href=\"#TF7-2\" data-ref-type=\"table-fn\">*<\/a> | IGT, BMI \u226524 kg\/m^2^, FPG \\>5.3 mmol\/l | 51 | 3 | I-D&E | 10.4 | 58 (48\u201366) | 6.9 |\n| \u2003\u2003\u2003\u2003Da Qing (15) | 259<a href=\"#TF7-2\" data-ref-type=\"table-fn\">*<\/a> | IGT (randomized groups) | 45 | 6 | G-D&E | 14.5 | 38 (14\u201356) | 7.9 |\n| \u2003\u2003\u2003\u2003Toranomon Study (35) | 458 | IGT (men), BMI = 24 kg\/m^2^ | \u223c55 | 4 | I-D&E | 2.4 | 67 (*P* \\< 0.043)<a href=\"#TF7-3\" data-ref-type=\"table-fn\">\u2020<\/a> | 20.6 |\n| \u2003\u2003\u2003\u2003Indian DPP (19) | 269<a href=\"#TF7-2\" data-ref-type=\"table-fn\">*<\/a> | IGT | 46 | 2.5 | I-D&E | 23 | 29 (21\u201337) | 6.4 |\n| Medications |  |  |  |  |  |  |  |  |\n| \u2003\u2003\u2003\u2003DPP (13) | 2,155<a href=\"#TF7-2\" data-ref-type=\"table-fn\">*<\/a> | IGT, BMI \\>24 kg\/m^2^, FPG \\>5.3 mmol\/l | 51 | 2.8 | Metformin (1,700 mg) | 10.4 | 31 (17\u201343) | 13.9 |\n| \u2003\u2003\u2003\u2003Indian DPP (19) | 269<a href=\"#TF7-2\" data-ref-type=\"table-fn\">*<\/a> | IGT | 46 | 2.5 | Metformin (500 mg) | 23 | 26 (19\u201335) | 6.9 |\n| \u2003\u2003\u2003\u2003STOP-NIDDM (17) | 1,419 | IGT, FPG \\>5.6 mmol\/l | 54 | 3.2 | Acarbose (300 mg) | 12.4 | 25 (10\u201337) | 9.6 |\n| \u2003\u2003\u2003\u2003XENDOS (36) | 3,277 | BMI \\>30 kg\/m^2^ | 43 | 4 | Orlistat (360 mg) | 2.4 | 37 (14\u201354) | 45.5 |\n| \u2003\u2003\u2003\u2003DREAM (18) | 5,269 | IGT or IFG | 55 | 3.0 | Rosiglitazone (8 mg) | 9.1 | 60 (54\u201365) | 6.9 |\n| \u2003\u2003\u2003\u2003Voglibose Ph-3 (37) | 1,780 | IGT | 56 | 3.0 (1-year Rx) | Vogliobose (0.2 mg) | 12.0 | 40 (18\u201357) | 21 (1-year Rx) |\n\nModified and reprinted with permission (38).\n\nPercentage points: \u03b4Number needed to treat to prevent 1 case of diabetes, standardized for a 3-year period to improve comparisons across studies.\n\n\\*Number of participants in the indicated comparisons, not necessarily in entire study.\n\n\u2020Calculated from information in the article. DPP, Diabetes Prevention Program; DPS, Diabetes Prevention Study; DREAM, Diabetes Reduction Assessment with Ramipril and Rosiglitazone Medication; STOP-NIDDM, Study to Prevent Non-Insulin Dependent Diabetes; XENDOS, Xenical in the prevention of Diabetes in Obese Subjects. I, individual; G, group; D&E, diet and exercise.\n\nFollow-up of all three large studies of lifestyle intervention has shown sustained reduction in the rate of conversion to type 2 diabetes, with 43% reduction at 20 years in the Da Qing study (30), 43% reduction at 7 years in the Finnish Diabetes Prevention Study (DPS) (31) and 34% reduction at 10 years in the U.S. Diabetes Prevention Program Outcomes Study (DPPOS) (32). A cost-effectiveness analysis suggested that lifestyle interventions as delivered in the DPP are cost-effective (33). Group delivery of the DPP intervention in community settings has the potential to be significantly less expensive while still achieving similar weight loss (34).\n\nBased on the results of clinical trials and the known risks of progression of prediabetes to diabetes, persons with an A1C of 5.7\u20136.4%, IGT, or IFG should be counseled on lifestyle changes with goals similar to those of the DPP (7% weight loss and moderate physical activity of at least 150 min\/week). Regarding the more difficult issue of drug therapy for diabetes prevention, a consensus panel felt that metformin should be the only drug considered (39). For other drugs, the issues of cost, side effects, and lack of persistence of effect in some studies led the panel to not recommend their use for diabetes prevention. Metformin, which was significantly less effective than lifestyle in the DPP and DPPOS, reasonably may be recommended for very-high-risk individuals (those with risk factors for diabetes and\/or those with more severe or progressive hyperglycemia). Of note, in the DPP metformin was most effective compared to lifestyle in those with BMI of at least 35 kg\/m^2^ and was not significantly better than placebo in those over age 60 years.\n\n# V. DIABETES CARE\n\n## A. Initial evaluation\n\nA complete medical evaluation should be performed to classify the diabetes, detect the presence of diabetes complications, review previous treatment and glycemic control in patients with established diabetes, assist in formulating a management plan, and provide a basis for continuing care. Laboratory tests appropriate to the evaluation of each patient's medical condition should be performed. A focus on the components of comprehensive care (<a href=\"#T8\" data-ref-type=\"table\">Table 8<\/a>) will assist the health care team to ensure optimal management of the patient with diabetes.\n\nComponents of the comprehensive diabetes evaluation\n\n<table>\n<tbody>\n<tr>\n<td style=\"text-align: left;\">Medical history\n<ul>\n<li><p>Age and characteristics of onset of diabetes (e.g., DKA, asymptomatic laboratory finding)<\/p><\/li>\n<li><p>Eating patterns, physical activity habits, nutritional status, and weight history; growth and development in children and adolescents<\/p><\/li>\n<li><p>Diabetes education history<\/p><\/li>\n<li><p>Review of previous treatment regimens and response to therapy (A1C records)<\/p><\/li>\n<li><p>Current treatment of diabetes, including medications, meal plan, physical activity patterns, and results of glucose monitoring and patient's use of data<\/p><\/li>\n<li><p>DKA frequency, severity, and cause<\/p><\/li>\n<li><p>Hypoglycemic episodes<\/p>\n<ul>\n<li><p>Hypoglycemia awareness<\/p><\/li>\n<li><p>Any severe hypoglycemia: frequency and cause<\/p><\/li>\n<\/ul><\/li>\n<li><p>History of diabetes-related complications<\/p>\n<ul>\n<li><p>Microvascular: retinopathy, nephropathy, neuropathy (sensory, including history of foot lesions; autonomic, including sexual dysfunction and gastroparesis)<\/p><\/li>\n<li><p>Macrovascular: CHD, cerebrovascular disease, PAD<\/p><\/li>\n<li><p>Other: psychosocial problems<a href=\"#TF8-1\" data-ref-type=\"table-fn\">*<\/a>, dental disease<a href=\"#TF8-1\" data-ref-type=\"table-fn\">*<\/a><\/p><\/li>\n<\/ul><\/li>\n<\/ul><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Physical examination\n<ul>\n<li><p>Height, weight, BMI<\/p><\/li>\n<li><p>Blood pressure determination, including orthostatic measurements when indicated<\/p><\/li>\n<li><p>Fundoscopic examination<a href=\"#TF8-1\" data-ref-type=\"table-fn\">*<\/a><\/p><\/li>\n<li><p>Thyroid palpation<\/p><\/li>\n<li><p>Skin examination (for acanthosis nigricans and insulin injection sites)<\/p><\/li>\n<li><p>Comprehensive foot examination:<\/p>\n<ul>\n<li><p>Inspection<\/p><\/li>\n<li><p>Palpation of dorsalis pedis and posterior tibial pulses<\/p><\/li>\n<li><p>Presence\/absence of patellar and Achilles reflexes<\/p><\/li>\n<li><p>Determination of proprioception, vibration, and monofilament sensation<\/p><\/li>\n<\/ul><\/li>\n<\/ul><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Laboratory evaluation\n<ul>\n<li><p>A1C, if results not available within past 2\u20133 months<\/p><\/li>\n<li><p>If not performed\/available within past year:<\/p>\n<ul>\n<li><p>Fasting lipid profile, including total, LDL and HDL cholesterol and triglycerides<\/p><\/li>\n<li><p>Liver function tests<\/p><\/li>\n<li><p>Test for urine albumin excretion with spot urine albumin-to-creatinine ratio<\/p><\/li>\n<li><p>Serum creatinine and calculated GFR<\/p><\/li>\n<li><p>Thyroid-stimulating hormone in type 1 diabetes, dyslipidemia, or women over age 50 years<\/p><\/li>\n<\/ul><\/li>\n<\/ul><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Referrals\n<ul>\n<li><p>Annual dilated eye exam<\/p><\/li>\n<li><p>Family planning for women of reproductive age<\/p><\/li>\n<li><p>Registered dietitian for MNT<\/p><\/li>\n<li><p>DSME<\/p><\/li>\n<li><p>Dental examination<\/p><\/li>\n<li><p>Mental health professional, if needed<\/p><\/li>\n<\/ul><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\\*See appropriate referrals for these categories.\n\n## B. Management\n\nPeople with diabetes should receive medical care from a physician-coordinated team. Such teams may include, but are not limited to, physicians, nurse practitioners, physician's assistants, nurses, dietitians, pharmacists, and mental health professionals with expertise and a special interest in diabetes. It is essential in this collaborative and integrated team approach that individuals with diabetes assume an active role in their care.\n\nThe management plan should be formulated as a collaborative therapeutic alliance among the patient and family, the physician, and other members of the health care team. A variety of strategies and techniques should be used to provide adequate education and development of problem-solving skills in the various aspects of diabetes management. Implementation of the management plan requires that each aspect is understood and agreed to by the patient and the care providers and that the goals and treatment plan are reasonable. Any plan should recognize diabetes self-management education (DSME) and ongoing diabetes support as an integral component of care. In developing the plan, consideration should be given to the patient's age, school or work schedule and conditions, physical activity, eating patterns, social situation and cultural factors, and presence of complications of diabetes or other medical conditions.\n\n## C. Glycemic control\n\n## 1. Assessment of glycemic control\n\nTwo primary techniques are available for health providers and patients to assess the effectiveness of the management plan on glycemic control: patient self-monitoring of blood glucose (SMBG) or interstitial glucose, and A1C.\n\n## a. Glucose monitoring\n\n## Recommendations\n\n-   SMBG should be carried out three or more times daily for patients using multiple insulin injections or insulin pump therapy. (A)\n\n-   For patients using less-frequent insulin injections, noninsulin therapies, or medical nutrition therapy (MNT) alone, SMBG may be useful as a guide to the success of therapy. (E)\n\n-   To achieve postprandial glucose targets, postprandial SMBG may be appropriate. (E)\n\n-   When prescribing SMBG, ensure that patients receive initial instruction in, and routine follow-up evaluation of, SMBG technique and their ability to use data to adjust therapy. (E)\n\n-   Continuous glucose monitoring (CGM) in conjunction with intensive insulin regimens can be a useful tool to lower A1C in selected adults (age \u226525 years) with type 1 diabetes. (A)\n\n-   Although the evidence for A1C-lowering is less strong in children, teens, and younger adults, CGM may be helpful in these groups. Success correlates with adherence to ongoing use of the device. (C)\n\n-   CGM may be a supplemental tool to SMBG in those with hypoglycemia unawareness and\/or frequent hypoglycemic episodes. (E)\n\nMajor clinical trials of insulin-treated patients that demonstrated the benefits of intensive glycemic control on diabetes complications have included SMBG as part of multifactorial interventions, suggesting that SMBG is a component of effective therapy. SMBG allows patients to evaluate their individual response to therapy and assess whether glycemic targets are being achieved. Results of SMBG can be useful in preventing hypoglycemia and adjusting medications (particularly prandial insulin doses), MNT, and physical activity.\n\nThe frequency and timing of SMBG should be dictated by the particular needs and goals of the patient. SMBG is especially important for patients treated with insulin to monitor for and prevent asymptomatic hypoglycemia and hyperglycemia. For most patients with type 1 diabetes and pregnant women taking insulin, SMBG is recommended three or more times daily. For these populations, significantly more frequent testing may be required to reach A1C targets safely without hypoglycemia. The optimal frequency and timing of SMBG for patients with type 2 diabetes on noninsulin therapy is unclear. A meta-analysis of SMBG in non\u2013insulin-treated patients with type 2 diabetes concluded that some regimen of SMBG was associated with a reduction in A1C of 0.4%. However, many of the studies in this analysis also included patient education with diet and exercise counseling and, in some cases, pharmacologic intervention, making it difficult to assess the contribution of SMBG alone to improved control (40). Several recent trials have called into question the clinical utility and cost-effectiveness of routine SMBG in non\u2013insulin-treated patients (41\u201343).\n\nBecause the accuracy of SMBG is instrument and user dependent (44), it is important to evaluate each patient's monitoring technique, both initially and at regular intervals thereafter. In addition, optimal use of SMBG requires proper interpretation of the data. Patients should be taught how to use the data to adjust food intake, exercise, or pharmacological therapy to achieve specific glycemic goals, and these skills should be reevaluated periodically.\n\nCGM through the measurement of interstitial glucose (which correlates well with plasma glucose) is available. These sensors require calibration with SMBG, and the latter are still recommended for making acute treatment decisions. CGM devices also have alarms for hypo- and hyperglycemic excursions. Small studies in selected patients with type 1 diabetes have suggested that CGM use reduces the time spent in hypo- and hyperglycemic ranges and may modestly improve glycemic control. A larger 26-week randomized trial of 322 type 1 patients showed that adults age 25 years and older using intensive insulin therapy and CGM experienced a 0.5% reduction in A1C (from \u223c7.6% to 7.1%) compared to usual intensive insulin therapy with SMBG (45). Sensor use in children, teens, and adults up to age 24 years did not result in significant A1C lowering, and there was no significant difference in hypoglycemia in any group. Importantly, the greatest predictor of A1C-lowering in this study for all age-groups was frequency of sensor use, which was lower in younger age-groups. In a smaller randomized controlled trial of 129 adults and children with baseline A1C \\<7.0%, outcomes combining A1C and hypoglycemia favored the group utilizing CGM, suggesting that CGM is also beneficial for individuals with type 1 diabetes who have already achieved excellent control with A1C \\<7.0 (46). Although CGM is an evolving technology, emerging data suggest that, in appropriately selected patients who are motivated to wear it most of the time, it may offer benefit. CGM may be particularly useful in those with hypoglycemia unawareness and\/or frequent episodes of hypoglycemia, and studies in this area are ongoing.\n\n## b. A1C\n\n## Recommendations\n\n-   Perform the A1C test at least two times a year in patients who are meeting treatment goals (and who have stable glycemic control). (E)\n\n-   Perform the A1C test quarterly in patients whose therapy has changed or who are not meeting glycemic goals. (E)\n\n-   Use of point-of-care testing for A1C allows for timely decisions on therapy changes, when needed. (E)\n\nBecause A1C is thought to reflect average glycemia over several months (44), and has strong predictive value for diabetes complications (47,48), A1C testing should be performed routinely in all patients with diabetes, at initial assessment and then as part of continuing care. Measurement approximately every 3 months determines whether a patient's glycemic targets have been reached and maintained. For any individual patient, the frequency of A1C testing should be dependent on the clinical situation, the treatment regimen used, and the judgment of the clinician. Some patients with stable glycemia well within target may do well with testing only twice per year, while unstable or highly intensively managed patients (e.g., pregnant type 1 women) may be tested more frequently than every 3 months. The availability of the A1C result at the time that the patient is seen (point-of-care testing) has been reported to result in increased intensification of therapy and improvement in glycemic control (49,50).\n\nThe A1C test is subject to certain limitations. Conditions that affect erythrocyte turnover (hemolysis, blood loss) and hemoglobin variants must be considered, particularly when the A1C result does not correlate with the patient's clinical situation (44). In addition, A1C does not provide a measure of glycemic variability or hypoglycemia. For patients prone to glycemic variability (especially type 1 patients, or type 2 patients with severe insulin deficiency), glycemic control is best judged by the combination of results of SMBG testing and the A1C. The A1C may also serve as a check on the accuracy of the patient's meter (or the patient's reported SMBG results) and the adequacy of the SMBG testing schedule.\n\n<a href=\"#T9\" data-ref-type=\"table\">Table 9<\/a> contains the correlation between A1C levels and mean plasma glucose levels based on data from the international A1C-Derived Average Glucose (ADAG) trial utilizing frequent SMBG and CGM in 507 adults (83% Caucasian) with type 1, type 2, and no diabetes (51). The American Diabetes Association and American Association of Clinical Chemists have determined that the correlation (*r* = 0.92) is strong enough to justify reporting both an A1C result and an estimated average glucose (eAG) result when a clinician orders the A1C test. The table in previous versions of the Standards of Medical Care in Diabetes describing the correlation between A1C and mean glucose was derived from relatively sparse data (one 7-point profile over 1 day per A1C reading) in the primarily Caucasian type 1 diabetic participants in the DCCT (52). Clinicians should note that the numbers in the table are now different, as they are based on \u223c2,800 readings per A1C in the ADAG trial.\n\nCorrelation of A1C with average glucose\n\n| A1C (%) | Mean plasma glucose |        |\n|---------|---------------------|--------|\n|         | mg\/dl               | mmol\/l |\n| 6       | 126                 | 7.0    |\n| 7       | 154                 | 8.6    |\n| 8       | 183                 | 10.2   |\n| 9       | 212                 | 11.8   |\n| 10      | 240                 | 13.4   |\n| 11      | 269                 | 14.9   |\n| 12      | 298                 | 16.5   |\n\nThese estimates are based on ADAG data of \u223c2,700 glucose measurements over 3 months per A1C measurement in 507 adults with type 1, type 2, and no diabetes. The correlation between A1C and average glucose was 0.92 (51). A calculator for converting A1C results into estimated average glucose (eAG), in either mg\/dl or mmol\/l, is available at <http:\/\/professional.diabetes.org\/eAG>.\n\nIn the ADAG trial, there were no significant differences among racial and ethnic groups in the regression lines between A1C and mean glucose, although there was a trend toward a difference between African\/African American participants and Caucasian ones that might have been significant had more African\/African American participants been studied. A recent study comparing A1C with CGM data in 48 type 1 diabetic children found a highly statistically significant correlation between A1C and mean blood glucose, although the correlation (*r* = 0.7) was significantly lower than in the ADAG trial (53). Whether there are significant differences in how A1C relates to average glucose in children or in African American patients is an area for further study. For the time being, the question has not led to different recommendations about testing A1C or to different interpretations of the clinical meaning of given levels of A1C in those populations.\n\nFor patients in whom A1C\/eAG and measured blood glucose appear discrepant, clinicians should consider the possibilities of hemoglobinopathy or altered red cell turnover, and the options of more frequent and\/or different timing of SMBG or use of CGM. Other measures of chronic glycemia such as fructosamine are available, but their linkage to average glucose and their prognostic significance are not as clear as is the case for A1C.\n\n## 2. Glycemic goals in adults\n\n## Recommendations\n\n-   Lowering A1C to below or around 7% has been shown to reduce microvascular and neuropathic complications of diabetes and, if implemented soon after the diagnosis of diabetes, is associated with long-term reduction in macrovascular disease. Therefore, a reasonable A1C goal for many nonpregnant adults is \\<7%. (B)\n\n-   Because additional analyses from several randomized trials suggest a small but incremental benefit in microvascular outcomes with A1C values closer to normal, providers might reasonably suggest more stringent A1C goals for selected individual patients, if this can be achieved without significant hypoglycemia or other adverse effects of treatment. Such patients might include those with short duration of diabetes, long life expectancy, and no significant CVD. (B)\n\n-   Conversely, less stringent A1C goals may be appropriate for patients with a history of severe hypoglycemia, limited life expectancy, advanced microvascular or macrovascular complications, extensive comorbid conditions, and those with longstanding diabetes in whom the general goal is difficult to attain despite DSME, appropriate glucose monitoring, and effective doses of multiple glucose-lowering agents including insulin. (C)\n\nGlycemic control is fundamental to the management of diabetes. The DCCT (47) (in patients with type 1 diabetes), the Kumamoto study (54), and the UK Prospective Diabetes Study (UKPDS) (55,56) (both in patients with type 2 diabetes) were prospective, randomized, controlled trials of intensive versus standard glycemic control in patients with relatively recently diagnosed diabetes. These trials showed definitively that improved glycemic control is associated with significantly decreased rates of microvascular (retinopathy and nephropathy) and neuropathic complications. Follow up of the DCCT cohorts in the Epidemiology of Diabetes Interventions and Complications (EDIC) study (57,58) and of the UKPDS cohort (59) has shown persistence of these microvascular benefits in previously intensively treated subjects, even though their glycemic control has been equivalent to that of previous standard arm subjects during follow-up.\n\nSubsequent trials in patients with more long-standing type 2 diabetes, designed primarily to look at the role of intensive glycemic control on cardiovascular outcomes also confirmed a benefit, although more modest, on onset or progression of microvascular complications. The Veterans Affairs Diabetes Trial (VADT) showed significant reductions in albuminuria with intensive (achieved median A1C 6.9%) compared to standard glycemic control, but no difference in retinopathy and neuropathy (60,61). The Action in Diabetes and Vascular Disease: Preterax and Diamicron Modified Release Controlled Evaluation (ADVANCE) study of intensive versus standard glycemic control in type 2 diabetes found a statistically significant reduction in albuminuria with an A1C target of \\<6.5% (achieved median A1C 6.3%) compared to standard therapy achieving a median A1C of 7.0% (62). Recent analyses from the Action to Control Cardiovascular Risk in Diabetes (ACCORD) trial have shown lower rates of measures of microvascular complications in the intensive glycemic control arm compared with the standard arm (63,64).\n\nEpidemiological analyses of the DCCT and UKPDS (47,48) demonstrate a curvilinear relationship between A1C and microvascular complications. Such analyses suggest that, on a population level, the greatest number of complications will be averted by taking patients from very poor control to fair or good control. These analyses also suggest that further lowering of A1C from 7 to 6% is associated with further reduction in the risk of microvascular complications, albeit the absolute risk reductions become much smaller. Given the substantially increased risk of hypoglycemia (particularly in those with type 1 diabetes, but also in the recent type 2 trials), the concerning mortality findings in the ACCORD trial (65), and the relatively much greater effort required to achieve near-normoglycemia, the risks of lower targets may outweigh the potential benefits on microvascular complications on a population level. However, selected individual patients, especially those with little comorbidity and long life expectancy (who may reap the benefits of further lowering of glycemia below 7%) may, at patient and provider judgment, adopt glycemic targets as close to normal as possible as long as significant hypoglycemia does not become a barrier.\n\nWhereas many epidemiologic studies and meta-analyses (66,67) have clearly shown a direct relationship between A1C and CVD, the potential of intensive glycemic control to reduce CVD has been less clearly defined. In the DCCT, there was a trend toward lower risk of CVD events with intensive control. However, 9-year post-DCCT follow-up of the cohort has shown that participants previously randomized to the intensive arm had a 42% reduction (*P* = 0.02) in CVD outcomes and a 57% reduction (*P* = 0.02) in the risk of nonfatal myocardial infarction (MI), stroke, or CVD death compared with those previously in the standard arm (68). The benefit of intensive glycemic control in this type 1 cohort has recently been shown to persist for several decades (69).\n\nThe UKPDS trial of type 2 diabetes observed a 16% reduction in cardiovascular complications (combined fatal or nonfatal MI and sudden death) in the intensive glycemic control arm, although this difference was not statistically significant (*P* = 0.052), and there was no suggestion of benefit on other CVD outcomes such as stroke. However, 10 years of follow-up of the UKPDS cohort demonstrated, for participants originally randomized to intensive glycemic control compared with those randomized to conventional glycemic control, long-term reductions in MI (15% with sulfonylurea or insulin as initial pharmacotherapy, 33% with metformin as initial pharmacotherapy, both statistically significant) and in all-cause mortality (13 and 27%, respectively, both statistically significant) (59).\n\nResults of three large trials (ACCORD, ADVANCE, and VADT) suggested no significant reduction in CVD outcomes with intensive glycemic control in these populations, who had more advanced diabetes than UKPDS participants. Details of these three studies are reviewed extensively in a recent ADA position statement (70).\n\nThe glycemic control arm of ACCORD was halted early due to the finding of an increased rate of mortality in the intensive arm compared with the standard arm (1.41% vs. 1.14% per year; HR 1.22 \\[95% CI 1.01 to 1.46\\]); with a similar increase in cardiovascular deaths. The primary outcome of ACCORD (MI, stroke, or cardiovascular death) was lower in the intensive glycemic control group, due to a reduction in nonfatal MI, but this reduction was not statistically significant when the study was terminated (65).\n\nThe potential cause of excess deaths in the intensive group of the ACCORD has been difficult to pinpoint. Exploratory analyses of the mortality findings of ACCORD (evaluating variables including weight gain, use of any specific drug or drug combination, and hypoglycemia) were reportedly unable to identify a clear explanation for the excess mortality in the intensive arm. The ACCORD investigators subsequently published additional analyses showing no increase in mortality in the intensive arm participants who achieved A1C levels \\<7% or in those who lowered their A1C quickly after trial enrollment. In fact, the converse was observed\u2014those at highest risk for mortality were participants in the intensive arm with the highest A1C levels (71).\n\nThe primary outcome of ADVANCE was a combination of microvascular events (nephropathy and retinopathy) and major adverse cardiovascular events (MI, stroke, and cardiovascular death). Intensive glycemic control significantly reduced the primary end point, although this was due to a significant reduction in the microvascular outcome, primarily development of macroalbuminuria, with no significant reduction in the macrovascular outcome. There was no difference in overall or cardiovascular mortality between the intensive compared with the standard glycemic control arms (62).\n\nThe VADT randomized participants with type 2 diabetes uncontrolled on insulin or maximal dose oral agents (median entry A1C 9.4%) to a strategy of intensive glycemic control (goal A1C \\<6.0%) or standard glycemic control, with a planned A1C separation of at least 1.5%. The primary outcome of the VADT was a composite of CVD events. The cumulative primary outcome was nonsignificantly lower in the intensive arm (60).\n\nUnlike the UKPDS, which was carried out in patients with newly diagnosed diabetes, all three of the recent type 2 cardiovascular trials were conducted in participants with established diabetes (mean duration 8\u201311 years) and either known CVD or multiple risk factors, suggesting the presence of established atherosclerosis. Subset analyses of the three trials suggested a significant benefit of intensive glycemic control on CVD in participants with shorter duration of diabetes, lower A1C at entry, and\/or or absence of known CVD. The DCCT-EDIC study and the long-term follow-up of the UKPDS cohort both suggest that intensive glycemic control initiated soon after diagnosis of diabetes in patients with a lower level of CVD risk may impart long-term protection from CVD events. As is the case with microvascular complications, it may be that glycemic control plays a greater role before macrovascular disease is well developed and minimal or no role when it is advanced. Consistent with this concept, data from an ancillary study of the VADT demonstrated that intensive glycemic control was quite effective in reducing CVD events in individuals with less atherosclerosis at baseline (assessed by coronary calcium) but not in persons with more extensive baseline atherosclerosis (72).\n\nThe evidence for a cardiovascular benefit of intensive glycemic control primarily rests on long-term follow-up of study cohorts treated early in the course of type 1 and type 2 diabetes and subset analyses of ACCORD, ADVANCE, and VADT. A recent group-level meta-analysis of the latter three trials suggests that glucose lowering has a modest (9%) but statistically significant reduction in major CVD outcomes, primarily nonfatal MI, with no significant effect on mortality. A prespecified subgroup analysis suggested that major CVD outcome reduction occurred in patients without known CVD at baseline (HR 0.84 \\[95% CI 0.74\u20130.94\\]) (73). Conversely, the mortality findings in ACCORD and subgroup analyses of VADT suggest that the potential risks of very intensive glycemic control may outweigh its benefits in some patients, such as those with very long duration of diabetes, known history of severe hypoglycemia, advanced atherosclerosis, and advanced age\/frailty. Certainly, providers should be vigilant in preventing severe hypoglycemia in patients with advanced disease and should not aggressively attempt to achieve near-normal A1C levels in patients in whom such a target cannot be reasonably easily and safely achieved.\n\nRecommended glycemic goals for many nonpregnant adults are shown in <a href=\"#T10\" data-ref-type=\"table\">Table 10<\/a>. The recommendations are based on those for A1C values, with listed blood glucose levels that appear to correlate with achievement of an A1C of \\<7%. Less-stringent treatment goals may be appropriate for adults with limited life expectancies or advanced vascular disease. Glycemic goals for children are provided in VII.A.1.a. Glycemic control. Severe or frequent hypoglycemia is an absolute indication for the modification of treatment regimens, including setting higher glycemic goals.\n\nSummary of glycemic recommendations for many nonpregnant adults with diabetes\n\n<table>\n<tbody>\n<tr>\n<td style=\"text-align: left;\">A1C<\/td>\n<td style=\"text-align: left;\">&lt;7.0%*<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Preprandial capillary plasma glucose<\/td>\n<td style=\"text-align: left;\">70\u2013130 mg\/dl* (3.9\u20137.2 mmol\/l)<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Peak postprandial capillary plasma glucose\u2020\n<ul>\n<li><p>Goals should be individualized based on*:<\/p>\n<ul>\n<li><p>duration of diabetes<\/p><\/li>\n<li><p>age\/life expectancy<\/p><\/li>\n<li><p>comorbid conditions<\/p><\/li>\n<li><p>known CVD or advanced microvascular complications<\/p><\/li>\n<li><p>hypoglycemia unawareness<\/p><\/li>\n<li><p>individual patient considerations<\/p><\/li>\n<\/ul><\/li>\n<li><p>More or less stringent glycemic goals may be appropriate for individual patients.<\/p><\/li>\n<li><p>Postprandial glucose may be targeted if A1C goals are not met despite reaching preprandial glucose goals.<\/p><\/li>\n<\/ul><\/td>\n<td style=\"text-align: left;\">&lt;180 mg\/dl* (&lt;10.0 mmol\/l)<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\nPostprandial glucose measurements should be made 1\u20132 h after the beginning of the meal, generally peak levels in patients with diabetes.\n\nThe issue of pre- versus postprandial SMBG targets is complex (74). Elevated postchallenge (2-h OGTT) glucose values have been associated with increased cardiovascular risk independent of FPG in some epidemiological studies. In diabetic subjects, some surrogate measures of vascular pathology, such as endothelial dysfunction, are negatively affected by postprandial hyperglycemia (75). It is clear that postprandial hyperglycemia, like preprandial hyperglycemia, contributes to elevated A1C levels, with its relative contribution being higher at A1C levels that are closer to 7%. However, outcome studies have clearly shown A1C to be the primary predictor of complications, and landmark glycemic control trials such as the DCCT and UKPDS relied overwhelmingly on preprandial SMBG. Additionally, a randomized controlled trial in patients with known CVD found no CVD benefit of insulin regimens targeting postprandial glucose compared with targeting preprandial glucose (76). A reasonable recommendation for postprandial testing and targets is that for individuals who have premeal glucose values within target but have A1C values above target, monitoring postprandial plasma glucose (PPG) 1\u20132 h after the start of the meal and treatment aimed at reducing PPG values to \\<180 mg\/dl may help lower A1C.\n\nAs regards goals for glycemic control for women with GDM, recommendations from the Fifth International Workshop-Conference on Gestational Diabetes (77) were to target maternal capillary glucose concentrations of:\n\n-   Preprandial \u226495 mg\/dl (5.3 mmol\/l) and either\n\n    -   1-h postmeal \u2264140 mg\/dl (7.8 mmol\/l)\n\n        or\n\n    -   2-h postmeal \u2264120 mg\/dl (6.7 mmol\/l)\n\nFor women with preexisting type 1 or type 2 diabetes who become pregnant, a recent consensus statement (78) recommended the following as optimal glycemic goals, if they can be achieved without excessive hypoglycemia:\n\n-   premeal, bedtime, and overnight glucose 60\u201399 mg\/dl (3.3\u20135.4 mmol\/l)\n\n-   peak postprandial glucose 100\u2013129 mg\/dl (5.4\u20137.1mmol\/l)\n\n-   A1C \\<6.0%\n\n## D. Pharmacologic and overall approaches to treatment\n\n## 1. Therapy for type 1 diabetes\n\nThe DCCT clearly showed that intensive insulin therapy (three or more injections per day of insulin, or continuous subcutaneous insulin infusion (CSII) (insulin pump therapy) was a key part of improved glycemia and better outcomes (47,68). At the time of the study, therapy was carried out with short- and intermediate-acting human insulins. Despite better microvascular outcomes, intensive insulin therapy was associated with a high rate in severe hypoglycemia (62 episodes per 100 patient-years of therapy). Since the time of the DCCT, a number of rapid-acting and long-acting insulin analogs have been developed. These analogs are associated with less hypoglycemia with equal A1C-lowering in type 1 diabetes (79,80).\n\nTherefore, recommended therapy for type 1 diabetes consists of the following components: *1*) use of multiple dose insulin injections (three to four injections per day of basal and prandial insulin) or CSII therapy; *2*) matching of prandial insulin to carbohydrate intake, premeal blood glucose, and anticipated activity; and *3*) for many patients (especially if hypoglycemia is a problem), use of insulin analogs. There are excellent reviews available that guide the initiation and management of insulin therapy to achieve desired glycemic goals (3,79,81).\n\nBecause of the increased frequency of other autoimmune diseases in type 1 diabetes, screening for thyroid dysfunction, vitamin B12 deficiency, or celiac disease should be considered based on signs and symptoms. Periodic screening in absence of symptoms has been recommended, but the effectiveness and optimal frequency are unclear.\n\n## 2. Therapy for type 2 diabetes\n\nThe ADA and the EASD published an expert consensus statement on the approach to management of hyperglycemia in individuals with type 2 diabetes (82). Highlights of this approach are: intervention at the time of diagnosis with metformin in combination with lifestyle changes (MNT and exercise) and continuing timely augmentation of therapy with additional agents (including early initiation of insulin therapy) as a means of achieving and maintaining recommended levels of glycemic control (i.e., A1C \\<7% for most patients). As A1C targets are not achieved, treatment intensification is based on the addition of another agent from a *different* class. The overall objective is to achieve and maintain glycemic control and to change interventions when therapeutic goals are not being met.\n\nThe algorithm took into account the evidence for A1C-lowering of the individual interventions, their additive effects, and their expense. The precise drugs used and their exact sequence may not be as important as achieving and maintaining glycemic targets safely. Medications not included in the consensus algorithm, owing to less glucose-lowering effectiveness, limited clinical data, and\/or relative expense, still may be appropriate choices in individual patients to achieve glycemic goals. Initiation of insulin at time of diagnosis is recommended for individuals presenting with weight loss or other severe hyperglycemic symptoms or signs.\n\n## E. Diabetes self-management education\n\n## Recommendations\n\n-   People with diabetes should receive diabetes self-management education (DSME) according to national standards when their diabetes is diagnosed and as needed thereafter. (B)\n\n-   Effective self-management and quality of life are the key outcomes of DSME and should be measured and monitored as part of care. (C)\n\n-   DSME should address psychosocial issues, since emotional well-being is associated with positive diabetes outcomes. (C)\n\n-   Because DSME can result in cost-savings and improved outcomes (B), DSME should be adequately reimbursed by third-party payors. (E)\n\nDSME is an essential element of diabetes care (83\u201388), and national standards for DSME (89) are based on evidence for its benefits. Education helps people with diabetes initiate effective self-management and cope with diabetes when they are first diagnosed. Ongoing DSME and support also help people with diabetes maintain effective self-management throughout a lifetime of diabetes as they face new challenges and treatment advances become available. DSME helps patients optimize metabolic control, prevent and manage complications, and maximize quality of life in a cost-effective manner (90).\n\nDSME is the ongoing process of facilitating the knowledge, skill, and ability necessary for diabetes self-care. This process incorporates the needs, goals, and life experiences of the person with diabetes. The overall objectives of DSME are to support informed decision-making, self-care behaviors, problem-solving, and active collaboration with the health care team to improve clinical outcomes, health status, and quality of life in a cost-effective manner (89).\n\nCurrent best practice of DSME is a skills-based approach that focuses on helping those with diabetes to make informed self-management choices. DSME has changed from a didactic approach focusing on providing information to more theoretically based empowerment models that focus on helping those with diabetes make informed self-management decisions. Care of diabetes has shifted to an approach that is more patient centered and places the person with diabetes and his or her family at the center of the care model working in collaboration with health care professionals. Patient-centered care is respectful of and responsive to individual patient preferences, needs, and values and ensures that patient values guide all decision making (91).\n\n## Evidence for the benefits of DSME\n\nMultiple studies have found that DSME is associated with improved diabetes knowledge and improved self-care behavior (83), improved clinical outcomes such as lower A1C (84,85,87,88,92), lower self-reported weight (83), improved quality of life (86,93), healthy coping (94), and lower costs (95). Better outcomes were reported for DSME interventions that were longer and included follow-up support (83,96\u201399), that were culturally (100,101) and age appropriate (102,103) and tailored to individual needs and preferences, and that addressed psychosocial issues and incorporated behavioral strategies (83,87,104\u2013106). Both individual and group approaches have been found effective (107\u2013110). There is growing evidence for the role of community health workers and peer (111,112) and lay leaders (113) in delivering DSME and support in addition to the core team (114).\n\nDiabetes education is associated with increased use of primary and preventive services and lower use of acute, inpatient hospital services (95). Patients who participate in diabetes education are more likely to follow best practice treatment recommendations, particularly among the Medicare population, and have lower Medicare and commercial claim costs (115).\n\n## National standards for DSME\n\nNational standards for DSME are designed to define quality DSME and to assist diabetes educators in a variety of settings to provide evidence-based education (89). The standards, most recently revised in 2007, are reviewed and updated every 5 years by a task force representing key organizations involved in the field of diabetes education and care.\n\n## Reimbursement for DSME\n\nDSME, when provided by a program that meets the national standards for DSME and is recognized by the ADA or other approval bodies, is reimbursed as partof the Medicare program as overseen by the Centers for Medicare and Medicaid Services (CMS) ([www.cms.hhs.gov\/DiabetesSelfManagement](http:\/\/www.cms.hhs.gov\/DiabetesSelfManagement)). DSME is also covered by a growing number of other health insurance plans.\n\n## F. Medical nutrition therapy\n\n## General recommendations\n\n-   Individuals who have prediabetes or diabetes should receive individualized medical nutrition therapy (MNT) as needed to achieve treatment goals, preferably provided by a registered dietitian familiar with the components of diabetes MNT. (A)\n\n-   Because MNT can result in cost-savings and improved outcomes (B), MNT should be adequately covered by insurance and other payors. (E)\n\n## Energy balance, overweight, and obesity\n\n-   In overweight and obese insulin-resistant individuals, modest weight loss has been shown to reduce insulin resistance. Thus, weight loss is recommended for all overweight or obese individuals who have or are at risk for diabetes. (A)\n\n-   For weight loss, either low-carbohydrate, low-fat calorie-restricted, or Mediterranean diets may be effective in the short-term (up to 2 years). (A)\n\n-   For patients on low-carbohydrate diets, monitor lipid profiles, renal function, and protein intake (in those with nephropathy), and adjust hypoglycemic therapy as needed. (E)\n\n-   Physical activity and behavior modification are important components of weight loss programs and are most helpful in maintenance of weight loss. (B)\n\n## Recommendations for primary prevention of diabetes\n\n-   Among individuals at high risk for developing type 2 diabetes, structured programs that emphasize lifestyle changes that include moderate weight loss (7% body weight) and regular physical activity (150 min\/week), with dietary strategies including reduced calories and reduced intake of dietary fat, can reduce the risk for developing diabetes and are therefore recommended. (A)\n\n-   Individuals at high risk for type 2 diabetes should be encouraged to achieve the U.S. Department of Agriculture (USDA) recommendation for dietary fiber (14 g fiber\/1,000 kcal) and foods containing whole grains (one-half of grain intake). (B)\n\n## Recommendations for management of diabetes\n\n## Macronutrients in diabetes management\n\n-   The best mix of carbohydrate, protein, and fat may be adjusted to meet the metabolic goals and individual preferences of the person with diabetes. (E)\n\n-   Monitoring carbohydrate, whether by carbohydrate counting, choices, or experience-based estimation, remains a key strategy in achieving glycemic control. (A)\n\n-   For individuals with diabetes, the use of the glycemic index and glycemic load may provide a modest additional benefit for glycemic control over that observed when total carbohydrate is considered alone. (B)\n\n-   Saturated fat intake should be \\<7% of total calories. (A)\n\n-   Reducing intake of *trans* fat lowers LDL cholesterol and increases HDL cholesterol (A), therefore intake of *trans* fat should be minimized. (E)\n\n## Other nutrition recommendations\n\n-   If adults with diabetes choose to use alcohol, daily intake should be limited to a moderate amount (one drink per day or less for adult women and two drinks per day or less for adult men). (E)\n\n-   Routine supplementation with antioxidants, such as vitamins E and C and carotene, is not advised because of lack of evidence of efficacy and concern related to long-term safety. (A)\n\n-   Individualized meal planning should include optimization of food choices to meet recommended dietary allowance (RDA)\/dietary reference intake (DRI) for all micronutrients. (E)\n\nMNT is an integral component of diabetes prevention, management, and self-management education. In addition to its role in preventing and controlling diabetes, ADA recognizes the importance of nutrition as an essential component of an overall healthy lifestyle. A full review of the evidence regarding nutrition in preventing and controlling diabetes and its complications and additional nutrition-related recommendations can be found in the ADA position statement, \"Nutrition Recommendations and Interventions for Diabetes,\" published in 2007 and updated for 2008 (116). Achieving nutrition-related goals requires a coordinated team effort that includes the active involvement of the person with prediabetes or diabetes. Because of the complexity of nutrition issues, it is recommended that a registered dietitian who is knowledgeable and skilled in implementing nutrition therapy into diabetes management and education be the team member who provides MNT.\n\nClinical trials\/outcome studies of MNT have reported decreases in A1C at 3\u20136 months ranging from 0.25% to 2.9% with higher reductions seen in type 2 diabetes of shorter duration. Multiple studies have demonstrated sustained improvements in A1C at 12 months and longer when an Registered Dietitian provided follow-up visits ranging from monthly to three sessions per year (117\u2013124). Studies in nondiabetic people suggest that MNT reduces LDL cholesterol by 15\u201325 mg\/dl up to 16% (125) and support a role for lifestyle modification in treating hypertension (125,126).\n\nBecause of the effects of obesity on insulin resistance, weight loss is an important therapeutic objective for overweight or obese individuals with prediabetes or diabetes (127). Short-term studies have demonstrated that moderate weight loss (5% of body weight) in subjects with type 2 diabetes is associated with decreased insulin resistance, improved measures of glycemia and lipemia, and reduced blood pressure (128); longer-term studies (52 weeks) showed mixed effects on A1C in adults with type 2 diabetes (129\u2013131), and in some studies results were confounded by pharmacologic weight loss therapy. A systematic review of 80 weight loss studies of \u22651 year in duration demonstrated that moderate weight loss achieved through diet alone, diet and exercise, and meal replacements can be achieved and maintained (4.8\u20138% weight loss at 12 months) (132). The multifactorial intensive lifestyle intervention employed in the DPP, which included reduced intake of fat and calories, led to weight loss averaging 7% at 6 months and maintenance of 5% weight loss at 3 years, associated with a 58% reduction in incidence of type 2 diabetes (13). A recent randomized controlled trial looking at high-risk individuals in Spain showed the Mediterranean dietary pattern reduced the incidence of diabetes in the absence of weight loss by 52% compared to the low-fat control group (133). Look AHEAD (Action for Health in Diabetes) is a large clinical trial designed to determine whether long-term weight loss will improve glycemia and prevent cardiovascular events in subjects with type 2 diabetes. One-year results of the intensive lifestyle intervention in this trial show an average 8.6% weight loss, significant reduction of A1C, and reduction in several CVD risk factors (134), with benefits sustained at 4 years (135). When completed, the Look AHEAD study should provide insight into the effects of long-term weight loss on important clinical outcomes.\n\nThe optimal macronutrient distribution of weight loss diets has not been established. Although low-fat diets have traditionally been promoted for weight loss, several randomized controlled trials found that subjects on low-carbohydrate diets (\\<130 g\/day of carbohydrate) lost more weight at 6 months than subjects on low-fat diets (136,137); however, at 1 year, the difference in weight loss between the low-carbohydrate and low-fat diets was not significant, and weight loss was modest with both diets. A study comparing low-fat to low-carbohydrate diets, both combined with a comprehensive lifestyle program, showed the same amount of weight loss (7%) at 2 years in both groups (138). Another study of overweight women randomized to one of four diets showed significantly more weight loss at 12 months with the Atkins low-carbohydrate diet than with higher-carbohydrate diets (139). Changes in serum triglyceride and HDL cholesterol were more favorable with the low-carbohydrate diets. In one study, those subjects with type 2 diabetes demonstrated a greater decrease in A1C with a low-carbohydrate diet than with a low-fat diet (137). A recent meta-analysis showed that at 6 months, low-carbohydrate diets were associated with greater improvements in triglyceride and HDL cholesterol concentrations than low-fat diets; however, LDL cholesterol was significantly higher on the low-carbohydrate diets (140). In a 2-year dietary intervention study, Mediterranean and low-carbohydrate diets were found to be effective and safe alternatives to a low-fat diet for weight reduction in moderately obese participants (141).\n\nThe RDA for digestible carbohydrate is 130 g\/day and is based on providing adequate glucose as the required fuel for the central nervous system without reliance on glucose production from ingested protein or fat. Although brain fuel needs can be met on lower-carbohydrate diets, long term metabolic effects of very-low-carbohydrate diets are unclear, and such diets eliminate many foods that are important sources of energy, fiber, vitamins, and minerals and are important in dietary palatability (142).\n\nAlthough numerous studies have attempted to identify the optimal mix of macronutrients for meal plans of people with diabetes, it is unlikely that one such combination of macronutrients exists. The best mix of carbohydrate, protein, and fat appears to vary depending on individual circumstances. It must be clearly recognized that regardless of the macronutrient mix, total caloric intake must be appropriate to weight management goal. Further, individualization of the macronutrient composition will depend on the metabolic status of the patient (e.g., lipid profile, renal function) and\/or food preferences. Plant-based diets (vegan or vegetarian) that are well planned and nutritionally adequate have also been shown to improve metabolic control (143,144).\n\nThe primary goal with respect to dietary fat in individuals with diabetes is to limit saturated fatty acids, *trans* fatty acids, and cholesterol intake so as to reduce risk for CVD. Saturated and *trans* fatty acids are the principal dietary determinants of plasma LDL cholesterol. There is a lack of evidence on the effects of specific fatty acids on people with diabetes, so the recommended goals are consistent with those for individuals with CVD (125,145).\n\n## Reimbursement for MNT\n\nMNT, when delivered by a registered dietitian according to nutrition practice guidelines, is reimbursed as part of the Medicare program as overseen by the Centers for Medicare and Medicaid Services (CMS) ([www.cms.hhs.gov\/medicalnutritiontherapy](http:\/\/www.cms.hhs.gov\/medicalnutritiontherapy)).\n\n## G. Physical activity\n\n## Recommendations\n\n-   People with diabetes should be advised to perform at least 150 min\/week of moderate-intensity aerobic physical activity (50\u201370% of maximum heart rate). (A)\n\n-   In the absence of contraindications, people with type 2 diabetes should be encouraged to perform resistance training three times per week. (A)\n\nExercise is an important part of the diabetes management plan. Regular exercise has been shown to improve blood glucose control, reduce cardiovascular risk factors, contribute to weight loss, and improve well-being. Furthermore, regular exercise may prevent type 2 diabetes in high-risk individuals (13\u201315). Structured exercise interventions of at least 8 weeks' duration have been shown to lower A1C by an average of 0.66% in people with type 2 diabetes, even with no significant change in BMI (146). Higher levels of exercise intensity are associated with greater improvements in A1C and in fitness (147). A new joint position statement of the American Diabetes Association and the American College of Sports Medicine summarizes the evidence for the benefits of exercise in people with type 2 diabetes (148).\n\n## Frequency and type of exercise\n\nThe U.S. Department of Health and Human Services' Physical Activity Guidelines for Americans (149) suggest that adults over age 18 years do 150 min\/week of moderate-intensity, or 75 min\/week of vigorous aerobic physical activity, or an equivalent combination of the two. In addition, the guidelines suggest that adults also do muscle-strengthening activities that involve all major muscle groups two or more days per week. The guidelines suggest that adults over age 65 years, or those with disabilities, follow the adult guidelines if possible or (if this is not possible) be as physically active as they are able. Studies included in the meta-analysis of effects of exercise interventions on glycemic control (146) had a mean number of sessions per week of 3.4, with a mean of 49 min\/session. The DPP lifestyle intervention, which included 150 min\/week of moderate intensity exercise, had a beneficial effect on glycemia in those with prediabetes. Therefore, it seems reasonable to recommend that people with diabetes try to follow the physical activity guidelines for the general population.\n\nProgressive resistance exercise improves insulin sensitivity in older men with type 2 diabetes to the same or even a greater extent as aerobic exercise (150). Clinical trials have provided strong evidence for the A1C-lowering value of resistance training in older adults with type 2 diabetes (151,152) and for an additive benefit of combined aerobic and resistance exercise in adults with type 2 diabetes (153).\n\n## Evaluation of the diabetic patient before recommending an exercise program\n\nPrior guidelines suggested that before recommending a program of physical activity, the provider should assess patients with multiple cardiovascular risk factors for coronary artery disease (CAD). As discussed more fully in VI.A.5. Coronary heart disease screening and treatment, the area of screening asymptomatic diabetic patients for CAD remains unclear, and a recent ADA consensus statement on this issue concluded that routine screening is not recommended (154). Providers should use clinical judgment in this area. Certainly, high risk patients should be encouraged to start with short periods of low intensity exercise and increase the intensity and duration slowly.\n\nProviders should assess patients for conditions that might contraindicate certain types of exercise or predispose to injury, such as uncontrolled hypertension, severe autonomic neuropathy, severe peripheral neuropathy or history of foot lesions, and unstable proliferative retinopathy. The patient's age and previous physical activity level should be considered.\n\n## Exercise in the presence of nonoptimal glycemic control\n\n### Hyperglycemia.\n\nWhen people with type 1 diabetes are deprived of insulin for 12\u201348 h and are ketotic, exercise can worsen hyperglycemia and ketosis (155); therefore, vigorous activity should be avoided in the presence of ketosis. However, it is not necessary to postpone exercise based simply on hyperglycemia, provided the patient feels well and urine and\/or blood ketones are negative.\n\n### Hypoglycemia.\n\nIn individuals taking insulin and\/or insulin secretagogues, physical activity can cause hypoglycemia if medication dose or carbohydrate consumption is not altered. For individuals on these therapies, added carbohydrate should be ingested if pre-exercise glucose levels are \\<100 mg\/dl (5.6 mmol\/l). Hypoglycemia is rare in diabetic individuals who are not treated with insulin or insulin secretagogues, and no preventive measures for hypoglycemia are usually advised in these cases.\n\n## Exercise in the presence of specific long-term complications of diabetes\n\n### Retinopathy.\n\nIn the presence of proliferative diabetic retinopathy (PDR) or severe nonproliferative diabetic retinopathy (NPDR), vigorous aerobic or resistance exercise may be contraindicated because of the risk of triggering vitreous hemorrhage or retinal detachment (156).\n\n### Peripheral neuropathy.\n\nDecreased pain sensation in the extremities results in increased risk of skin breakdown and infection and of Charcot joint destruction. Prior recommendations have advised non\u2013weight-bearing exercise for patients with severe peripheral neuropathy. However, studies have shown that moderate-intensity walking may not lead to increased risk of foot ulcers or re-ulceration in those with peripheral neuropathy (157). All individuals with peripheral neuropathy should wear proper footwear and examine their feet daily to detect lesions early. Anyone with a foot injury or open sore should be restricted to non\u2013weight-bearing activities.\n\n### Autonomic neuropathy.\n\nAutonomic neuropathy can increase the risk of exercise-induced injury or adverse event through decreased cardiac responsiveness to exercise, postural hypotension, impaired thermoregulation, impaired night vision due to impaired papillary reaction, and unpredictable carbohydrate delivery from gastroparesis predisposing to hypoglycemia (158). Autonomic neuropathy is also strongly associated with CVD in people with diabetes (159,160). People with diabetic autonomic neuropathy should undergo cardiac investigation before beginning physical activity more intense than that to which they are accustomed.\n\n### Albuminuria and nephropathy.\n\nPhysical activity can acutely increase urinary protein excretion. However, there is no evidence that vigorous exercise increases the rate of progression of diabetic kidney disease, and there is likely no need for any specific exercise restrictions for people with diabetic kidney disease (161).\n\n## H. Psychosocial assessment and care\n\n## Recommendations\n\n-   Assessment of psychological and social situation should be included as an ongoing part of the medical management of diabetes. (E)\n\n-   Psychosocial screening and follow-up should include, but is not limited to, attitudes about the illness, expectations for medical management and outcomes, affect\/mood, general and diabetes-related quality of life, resources (financial, social, and emotional), and psychiatric history. (E)\n\n-   Screen for psychosocial problems such as depression and diabetes-related distress, anxiety, eating disorders, and cognitive impairment when self-management is poor. (C)\n\nPsychological and social problems can impair the individual's (162\u2013165) or family's ability to carry out diabetes care tasks and therefore compromise health status. There are opportunities for the clinician to assess psychosocial status in a timely and efficient manner so that referral for appropriate services can be accomplished.\n\nKey opportunities for screening of psychosocial status occur at diagnosis, during regularly scheduled management visits, during hospitalizations, at discovery of complications, or when problems with glucose control, quality of life, or adherence are identified. Patients are likely to exhibit psychological vulnerability at diagnosis and when their medical status changes, e.g., the end of the honeymoon period, when the need for intensified treatment is evident, and when complications are discovered (164).\n\nIssues known to impact self-management and health outcomes include but are not limited to: attitudes about the illness, expectations for medical management and outcomes, affect\/mood, general and diabetes-related quality of life, diabetes-related distress (166), resources (financial, social, and emotional) (167), and psychiatric history (168\u2013170). Screening tools are available for a number of these areas (105). Indications for referral to a mental health specialist familiar with diabetes management may include: gross noncompliance with medical regimen (by self or others) (170), depression with the possibility of self-harm, debilitating anxiety (alone or with depression), indications of an eating disorder (171), or cognitive functioning that significantly impairs judgment. It is preferable to incorporate psychological assessment and treatment into routine care rather than waiting for identification of a specific problem or deterioration in psychological status (105). Although the clinician may not feel qualified to treat psychological problems, utilizing the patient-provider relationship as a foundation for further treatment can increase the likelihood that the patient will accept referral for other services. It is important to establish that emotional well-being is part of diabetes management.\n\n## I. When treatment goals are not met\n\nFor a variety of reasons, some people with diabetes and their health care providers do not achieve the desired goals of treatment (<a href=\"#T10\" data-ref-type=\"table\">Table 10<\/a>). Re-thinking the treatment regimen may require assessment of barriers including income, health literacy, diabetes distress, depression, and competing demands, including those related to family responsibilities and dynamics. Other strategies may include culturally appropriate and enhanced DSME, co-management with a diabetes team, referral to a medical social worker for assistance with insurance coverage, or change in pharmacological therapy. Initiation of or increase in SMBG, utilization of CGM, frequent contact with the patient, or referral to a mental health professional or physician with special expertise in diabetes may be useful. Providing patients with an algorithm for self-titration of insulin doses based on SMBG results may be helpful for type 2 patients who take insulin (172).\n\n## J. Hypoglycemia\n\n## Recommendations\n\n-   Glucose (15\u201320 g) is the preferred treatment for the conscious individual with hypoglycemia, although any form of carbohydrate that contains glucose may be used. If SMBG 15 min after treatment shows continued hypoglycemia, the treatment should be repeated. Once SMBG glucose returns to normal, the individual should consume a meal or snack to prevent recurrence of hypoglycemia. (E)\n\n-   Glucagon should be prescribed for all individuals at significant risk of severe hypoglycemia, and caregivers or family members of these individuals should be instructed in its administration. Glucagon administration is not limited to health care professionals. (E)\n\n-   Individuals with hypoglycemia unawareness or one or more episodes of severe hypoglycemia should be advised to raise their glycemic targets to strictly avoid further hypoglycemia for at least several weeks, to partially reverse hypoglycemia unawareness and reduce risk of future episodes. (B)\n\nHypoglycemia is the leading limiting factor in the glycemic management of type 1 and insulin-treated type 2 diabetes (173). Mild hypoglycemia may be inconvenient or frightening to patients with diabetes, and more severe hypoglycemia can cause acute harm to the person with diabetes or others, if it causes falls, motor vehicle accidents, or other injury. A large cohort study suggested that among older adults with type 2 diabetes, a history of severe hypoglycemia was associated with greater risk of dementia (174). Conversely, evidence from the DCCT\/EDIC trial, which involved younger type 1 patients, suggested no association of frequency of severe hypoglycemia with cognitive decline (175). Treatment of hypoglycemia (plasma glucose \\<70 mg\/dl) requires ingestion of glucose- or carbohydrate-containing foods. The acute glycemic response correlates better with the glucose content than with the carbohydrate content of the food. Although pure glucose is the preferred treatment, any form of carbohydrate that contains glucose will raise blood glucose. Added fat may retard and then prolong the acute glycemic response. Ongoing activity of insulin or insulin secretagogues may lead to recurrence of hypoglycemia unless further food is ingested after recovery.\n\nSevere hypoglycemia (where the individual requires the assistance of another person and cannot be treated with oral carbohydrate due to confusion or unconsciousness) should be treated using emergency glucagon kits, which require a prescription. Those in close contact with, or having custodial care of, people with hypoglycemia-prone diabetes (family members, roommates, school personnel, child care providers, correctional institution staff, or coworkers), should be instructed in use of such kits. An individual does not need to be a health care professional to safely administer glucagon. Care should be taken to ensure that unexpired glucagon kits are available.\n\nPrevention of hypoglycemia is a critical component of diabetes management. Teaching people with diabetes to balance insulin use, carbohydrate intake, and exercise is a necessary but not always sufficient strategy. In type 1 diabetes and severely insulin-deficient type 2 diabetes, the syndrome of hypoglycemia unawareness, or hypoglycemia-associated autonomic failure, can severely compromise stringent diabetes control and quality of life. The deficient counterregulatory hormone release and autonomic responses in this syndrome are both risk factors for, and caused by, hypoglycemia. A corollary to this \"vicious cycle\" is that several weeks of avoidance of hypoglycemia has been demonstrated to improve counterregulation and awareness to some extent in many patients (176). Hence, patients with one or more episodes of severe hypoglycemia may benefit from at least short-term relaxation of glycemic targets.\n\n## K. Intercurrent illness\n\nThe stress of illness, trauma, and\/or surgery frequently aggravates glycemic control and may precipitate diabetic ketoacidosis (DKA) or nonketotic hyperosmolar state, life-threatening conditions that require immediate medical care to prevent complications and death. Any condition leading to deterioration in glycemic control necessitates more frequent monitoring of blood glucose and (in ketosis-prone patients) urine or blood ketones. Marked hyperglycemia requires temporary adjustment of the treatment program and, if accompanied by ketosis, vomiting, or alteration in level of consciousness, immediate interaction with the diabetes care team. The patient treated with noninsulin therapies or MNT alone may temporarily require insulin. Adequate fluid and caloric intake must be assured. Infection or dehydration are more likely to necessitate hospitalization of the person with diabetes than the person without diabetes.\n\nThe hospitalized patient should be treated by a physician with expertise in the management of diabetes. For further information on management of patients with hyperglycemia in the hospital, see VIII.A. Diabetes care in the hospital. For further information on management of DKA or nonketotic hyperosmolar state, refer to the ADA consensus statement on hyperglycemic crises (172).\n\n## L. Bariatric surgery\n\n## Recommendations\n\n-   Bariatric surgery may be considered for adults with BMI \\>35 kg\/m^2^ and type 2 diabetes, especially if the diabetes or associated comorbidities are difficult to control with lifestyle and pharmacologic therapy. (B)\n\n-   Patients with type 2 diabetes who have undergone bariatric surgery need life-long lifestyle support and medical monitoring. (E)\n\n-   Although small trials have shown glycemic benefit of bariatric surgery in patients with type 2 diabetes and BMI of 30\u201335 kg\/m^2^, there is currently insufficient evidence to generally recommend surgery in patients with BMI \\<35 kg\/m^2^ outside of a research protocol. (E)\n\n-   The long-term benefits, cost-effectiveness, and risks of bariatric surgery in individuals with type 2 diabetes should be studied in well-designed controlled trials with optimal medical and lifestyle therapy as the comparator. (E)\n\nGastric reduction surgery, either gastric banding or procedures that involve bypassing, transposing, or resecting sections of the small intestine, when part of a comprehensive team approach, can be an effective weight loss treatment for severe obesity, and national guidelines support its consideration for people with type 2 diabetes who have BMI exceeding 35 kg\/m^2^. Bariatric surgery has been shown to lead to near- or complete normalization of glycemia in \u223c55\u201395% of patients with type 2 diabetes, depending on the surgical procedure. A meta-analysis of studies of bariatric surgery involving 3,188 patients with diabetes reported that 78% had remission of diabetes (normalization of blood glucose levels in the absence of medications), and that the remission rates were sustained in studies that had follow-up exceeding 2 years (177). Remission rates tend to be lower with procedures that only constrict the stomach, and higher with those that bypass portions of the small intestine. Additionally, there is a suggestion that intestinal bypass procedures may have glycemic effects that are independent of their effects on weight, perhaps involving the incretin axis.\n\nOne randomized controlled trial compared adjustable gastric banding to \"best available\" medical and lifestyle therapy in subjects with type 2 diabetes diagnosed less than 2 years before randomization and BMI 30\u201340 kg\/m^2^ (178). In this trial, 73% of surgically treated patients achieved \"remission\" of their diabetes, compared with 13% of those treated medically. The latter group lost only 1.7% of body weight, suggesting that their therapy was not optimal. Overall the trial had 60 subjects, and only 13 had a BMI under 35 kg\/m^2^, making it difficult to generalize these results widely to diabetic patients who are less severely obese or with longer duration of diabetes. In a more recent study involving 110 patients with type 2 diabetes and a mean BMI of 47 kg\/m^2^, Roux-en-Y gastric bypass resulted in a mean loss of excess weight of 63% at 1 year and 84% at 2 years (179).\n\nBariatric surgery is costly in the short term and has some risks. Rates of morbidity and mortality directly related to the surgery have been reduced considerably in recent years, with 30-day mortality rates now 0.28%, similar to those of laparoscopic cholecystectomy (180). Longer-term concerns include vitamin and mineral deficiencies, osteoporosis, and rare but often severe hypoglycemia from insulin hypersecretion. Cohort studies attempting to match subjects suggest that the procedure may reduce longer-term mortality rates (181), and it is reasonable to postulate that there may be recouping of costs over the long run. Recent retrospective analyses and modeling studies suggest that these procedures may be cost effective, when one considers reduction in subsequent health care costs (182\u2013184). However, studies of the mechanisms of glycemic improvement and long-term benefits and risks of bariatric surgery in individuals with type 2 diabetes, especially those who are not severely obese, will require well-designed clinical trials, with optimal medical and lifestyle therapy of diabetes and cardiovascular risk factors as the comparator.\n\n## M. Immunization\n\n## Recommendations\n\n-   Annually provide an influenza vaccine to all diabetic patients at least 6 months of age. (C)\n\n-   Administer pneumococcal polysaccharide vaccine to all diabetic patients \u22652 years of age. A one-time revaccination is recommended for individuals \\>64 years of age previously immunized when they were \\<65 years of age if the vaccine was administered \\>5 years ago. Other indications for repeat vaccination include nephrotic syndrome, chronic renal disease, and other immunocompromised states, such as after transplantation. (C)\n\nInfluenza and pneumonia are common, preventable infectious diseases associated with high mortality and morbidity in the elderly and in people with chronic diseases. Though there are limited studies reporting the morbidity and mortality of influenza and pneumococcal pneumonia specifically in people with diabetes, observational studies of patients with a variety of chronic illnesses, including diabetes, show that these conditions are associated with an increase in hospitalizations for influenza and its complications. People with diabetes may be at increased risk of the bacteremic form of pneumococcal infection and have been reported to have a high risk of nosocomial bacteremia, which has a mortality rate as high as 50% (185).\n\nSafe and effective vaccines are available that can greatly reduce the risk of serious complications from these diseases (186,187). In a case-control series, influenza vaccine was shown to reduce diabetes-related hospital admission by as much as 79% during flu epidemics (186). There is sufficient evidence to support that people with diabetes have appropriate serologic and clinical responses to these vaccinations. The Centers for Disease Control and Prevention (CDC) Advisory Committee on Immunization Practices recommends influenza and pneumococcal vaccines for all individuals with diabetes (<http:\/\/www.cdc.gov\/vaccines\/recs\/>).\n\n# VI. PREVENTION AND MANAGEMENT OF DIABETES COMPLICATIONS\n\n## A. CVD\n\nCVD is the major cause of morbidity and mortality for individuals with diabetes, and the largest contributor to the direct and indirect costs of diabetes. The common conditions coexisting with type 2 diabetes (e.g., hypertension and dyslipidemia) are clear risk factors for CVD, and diabetes itself confers independent risk. Numerous studies have shown the efficacy of controlling individual cardiovascular risk factors in preventing or slowing CVD in people with diabetes. Large benefits are seen when multiple risk factors are addressed globally (188,189). Risk for coronary heart disease (CHD) and for CVD in general can be estimated using multivariable risk factor approaches, and such a strategy may be desirable to undertake in adult patients prior to instituting preventive therapy.\n\n## 1. Hypertension\/blood pressure control\n\n## Recommendations\n\n## Screening and diagnosis\n\n-   Blood pressure should be measured at every routine diabetes visit. Patients found to have systolic blood pressure \u2265130 mmHg or diastolic blood pressure \u226580 mmHg should have blood pressure confirmed on a separate day. Repeat systolic blood pressure \u2265130 mmHg or diastolic blood pressure \u226580 mmHg confirms a diagnosis of hypertension. (C)\n\n## Goals\n\n-   A goal systolic blood pressure \\<130 mmHg is appropriate for most patients with diabetes. (C)\n\n-   Based on patient characteristics and response to therapy, higher or lower systolic blood pressure targets may be appropriate. (B)\n\n-   Patients with diabetes should be treated to a diastolic blood pressure \\<80 mmHg. (B)\n\n## Treatment\n\n-   Patients with a systolic blood pressure of 130\u2013139 mmHg or a diastolic blood pressure of 80\u201389 mmHg may be given lifestyle therapy alone for a maximum of 3 months and then, if targets are not achieved, be treated with addition of pharmacological agents. (E)\n\n-   Patients with more severe hypertension (systolic blood pressure \u2265140 or diastolic blood pressure \u226590 mmHg) at diagnosis or follow-up should receive pharmacologic therapy in addition to lifestyle therapy. (A)\n\n-   Lifestyle therapy for hypertension consists of: weight loss, if overweight; Dietary Approaches to Stop Hypertension (DASH)-style dietary pattern including reducing sodium and increasing potassium intake; moderation of alcohol intake; and increased physical activity. (B)\n\n-   Pharmacologic therapy for patients with diabetes and hypertension should be with a regimen that includes either an ACE inhibitor or an ARB. If one class is not tolerated, the other should be substituted. If needed to achieve blood pressure targets, a thiazide diuretic should be added to those with an estimated GFR (eGFR) (see below) \u226530 ml\/min\/1.73 m^2^ and a loop diuretic for those with an eGFR \\<30 ml\/min\/1.73 m^2^. (C)\n\n-   Multiple drug therapy (two or more agents at maximal doses) is generally required to achieve blood pressure targets. (B)\n\n-   If ACE inhibitors, ARBs, or diuretics are used, kidney function and serum potassium levels should be monitored. (E)\n\n-   In pregnant patients with diabetes and chronic hypertension, blood pressure target goals of 110\u2013129\/65\u201379 mmHg are suggested in the interest of long-term maternal health and minimizing impaired fetal growth. ACE inhibitors and ARBs are contraindicated during pregnancy. (E)\n\nHypertension is a common comorbidity of diabetes, affecting the majority of patients, with prevalence depending on type of diabetes, age, obesity, and ethnicity. Hypertension is a major risk factor for both CVD and microvascular complications. In type 1 diabetes, hypertension is often the result of underlying nephropathy, while in type 2 diabetes it usually coexists with other cardiometabolic risk factors.\n\n## Screening and diagnosis\n\nMeasurement of blood pressure in the office should be done by a trained individual and should follow the guidelines established for nondiabetic individuals: measurement in the seated position, with feet on the floor and arm supported at heart level, after 5 min of rest. Cuff size should be appropriate for the upper arm circumference. Elevated values should be confirmed on a separate day. Because of the clear synergistic risks of hypertension and diabetes, the diagnostic cut-off for a diagnosis of hypertension is lower in people with diabetes (blood pressure \u2265130\/80) than those without diabetes (blood pressure 140\/90 mmHg) (190).\n\nHome blood pressure self-monitoring and 24-h ambulatory blood pressure monitoring may provide additional evidence of \"white coat\" and masked hypertension and other discrepancies between office and \"true\" blood pressure, and in studies in nondiabetic populations, home measurements may better correlate with CVD risk than office measurements (191,192). However, the preponderance of the clear evidence of benefits of treatment of hypertension in people with diabetes is based on office measurements.\n\n## Treatment goals\n\nEpidemiologic analyses show that blood pressure values \\>115\/75 mmHg are associated with increased cardiovascular event rates and mortality in individuals with diabetes (190,193,194). Randomized clinical trials have demonstrated the benefit (reduction in CHD events, stroke, and nephropathy) of lowering blood pressure to \\<140 mmHg systolic and \\<80 mmHg diastolic in individuals with diabetes (190,195\u2013197). The ACCORD trial examined whether lowering blood pressure to a systolic \\<120 mmHg provides greater cardiovascular protection than a systolic blood pressure level of 130\u2013140 mmHg in patients with type 2 diabetes at high risk for CVD (198). The blood pressure achieved was 119\/64 mmHg in the intensive group and 133\/70 mmHg in the standard group; the difference achieved was attained with an average of 3.4 medications per participant in the intensive group and 2.1 in the standard therapy group. The primary outcome was a composite of nonfatal MI, nonfatal stroke, and CVD death; the hazard ratio for the primary end point in the intensive group was 0.88 (95% CI 0.73\u20131.06; *P* = 0.20). Of the prespecified secondary end points, only stroke and nonfatal stroke were statistically significantly reduced by intensive blood pressure treatment, with a hazard ratio of 0.59 (95% CI 0.39\u20130.89, *P* = 0.01) and 0.63 (95% CI 0.41\u20130.96, *P* = 0.03), respectively. If this finding is real, the number needed to treat to prevent one stroke over the course of 5 years with intensive blood pressure management is 89.\n\nIn predefined subgroup analyses, there was a suggestion of heterogeneity (*P* = 0.08) based on whether participants were randomized to standard or intensive glycemia intervention. In those randomized to standard glycemic control, the event rate for the primary end point was 1.89 per year in the intensive blood pressure arm and 2.47 in the standard blood pressure arm, while the respective rates in the intensive glycemia arm were 1.85 and 1.73. If this observation is true, it suggests that intensive management to a systolic blood pressure target of \\<120 mmHg may be of benefit in those who are not targeting an A1C of \\<6% and\/or that the benefit of intensive blood pressure management is diminished by more intensive glycemia management targeting an A1C of \\<6%.\n\nOther recent randomized trial data include those from ADVANCE, in which treatment with an angiotensin-converting enzyme inhibitor and a thiazide-type diuretic reduced the rate of death but not the composite macrovascular outcome. However, the ADVANCE trial had no specified targets for the randomized comparison, and the mean systolic blood pressure in the intensive group (135 mmHg) was not as low as the mean systolic blood pressure in the ACCORD standard therapy group (199). A post hoc analysis of blood pressure control in 6,400 patients with diabetes and CAD enrolled in the International Verapamil-Trandolapril (INVEST) trial demonstrated that \"tight control\" (\\<130 mmHg) was not associated with improved CV outcomes compared with \"usual care\" (130\u2013140 mmHg) (200).\n\nOnly the ACCORD blood pressure trial formally has examined treatment targets \\<130 mmHg in diabetes. It is possible that lowering systolic blood pressure from the low-130s to less than 120 mmHg does not further reduce coronary events or death, and that most of the benefit from lowering blood pressure is achieved by targeting a goal of \\<140 mmHg. However, this has not been formally assessed.\n\nThe absence of significant harm, the trends toward benefit in stroke, and the potential heterogeneity with respect to intensive glycemia management suggests that previously recommended targets are reasonable pending further analyses and results. Systolic blood pressure targets more or less stringent than \\<130 mmHg may be appropriate for individual patients, based on response to therapy, medication tolerance, and individual characteristics, keeping in mind that most analyses have suggested that outcomes are worse if the systolic blood pressure is \\>140 mmHg.\n\n## Treatment strategies\n\nAlthough there are no well-controlled studies of diet and exercise in the treatment of hypertension in individuals with diabetes, the Dietary Approaches to Stop Hypertension (DASH) study in nondiabetic individuals has shown anti-hypertensive effects similar to pharmacologic monotherapy. Lifestyle therapy consists of reducing sodium intake (to \\<1,500 mg\/day) and excess body weight; increasing consumption of fruits, vegetables (8\u201310 servings\/day), and low-fat dairy products (2\u20133 servings\/day); avoiding excessive alcohol consumption (no more than 2 servings\/day in men and no more than 1 serving\/day in women) (201); and increasing activity levels (190). These nonpharmacological strategies may also positively affect glycemia and lipid control. Their effects on cardiovascular events have not been established. An initial trial of nonpharmacologic therapy may be reasonable in diabetic individuals with mild hypertension (systolic blood pressure 130\u2013139 mmHg or diastolic blood pressure 80\u201389 mmHg). If systolic blood pressure is \u2265140 mmHg and\/or diastolic is \u226590 mmHg at the time of diagnosis, pharmacologic therapy should be initiated along with nonpharmacologic therapy (190).\n\nLowering of blood pressure with regimens based on a variety of antihypertensive drugs, including ACE inhibitors, ARBs, \u03b2-blockers, diuretics, and calcium channel blockers, has been shown to be effective in reducing cardiovascular events. Several studies suggested that ACE inhibitors may be superior to dihydropyridine calcium channel blockers in reducing cardiovascular events (202\u2013204). However, a variety of other studies have shown no specific advantage to ACE inhibitors as initial treatment of hypertension in the general hypertensive population, but rather an advantage on cardiovascular outcomes of initial therapy with low-dose thiazide diuretics (190,205,206).\n\nIn people with diabetes, inhibitors of the renin-angiotensin system (RAS) may have unique advantages for initial or early therapy of hypertension. In a nonhypertension trial of high-risk individuals, including a large subset with diabetes, an ACE inhibitor reduced CVD outcomes (207). In patients with congestive heart failure (CHF), including diabetic subgroups, ARBs have been shown to reduce major CVD outcomes (208\u2013211), and in type 2 patients with significant nephropathy, ARBs were superior to calcium channel blockers for reducing heart failure (212). Though evidence for distinct advantages of RAS inhibitors on CVD outcomes in diabetes remains conflicting (195,206), the high CVD risks associated with diabetes, and the high prevalence of undiagnosed CVD, may still favor recommendations for their use as first-line hypertension therapy in people with diabetes (190). Recently, the blood pressure arm of the ADVANCE trial demonstrated that routine administration of a fixed combination of the ACE inhibitor perindopril and the diuretic indapamide significantly reduced combined microvascular and macrovascular outcomes, as well as CVD and total mortality. The improved outcomes could also have been due to lower achieved blood pressure in the perindopril-indapamide arm (199). In addition, the Avoiding Cardiovascular Events through Combination Therapy in Patients Living with Systolic Hypertension (ACCOMPLISH) trial showed a decrease in morbidity and mortality in those receiving benazapril and amlodipine versus benazapril and hydrochlorothiazide. The compelling benefits of RAS inhibitors in diabetic patients with albuminuria or renal insufficiency provide additional rationale for use of these agents (see VI.B. Nephropathy screening and treatment).\n\nAn important caveat is that most patients with hypertension require multi-drug therapy to reach treatment goals, especially diabetic patients whose targets are lower. Many patients will require three or more drugs to reach target goals (190). If blood pressure is refractory to optimal doses of at least three antihypertensive agents of different classifications, one of which should be a diuretic, clinicians should consider an evaluation for secondary forms of hypertension.\n\nDuring pregnancy in diabetic women with chronic hypertension, target blood pressure goals of systolic blood pressure 110\u2013129 mmHg and diastolic blood pressure 65\u201379 mmHg are reasonable, as they contribute to long-term maternal health. Lower blood pressure levels may be associated with impaired fetal growth. During pregnancy, treatment with ACE inhibitors and ARBs is contraindicated, since they can cause fetal damage. Antihypertensive drugs known to be effective and safe in pregnancy include methyldopa, labetalol, diltiazem, clonidine, and prazosin. Chronic diuretic use during pregnancy has been associated with restricted maternal plasma volume, which might reduce uteroplacental perfusion (213).\n\n## 2. Dyslipidemia\/lipid management\n\n## Recommendations\n\n## Screening\n\n-   In most adult patients, measure fasting lipid profile at least annually. In adults with low-risk lipid values (LDL cholesterol \\<100 mg\/dl, HDL cholesterol \\>50 mg\/dl, and triglycerides \\<150 mg\/dl), lipid assessments may be repeated every 2 years. (E)\n\n## Treatment recommendations and goals\n\n-   Lifestyle modification focusing on the reduction of saturated fat, *trans* fat, and cholesterol intake; increase of omega-3 fatty acids, viscous fiber, and plant stanols\/sterols; weight loss (if indicated); and increased physical activity should be recommended to improve the lipid profile in patients with diabetes. (A)\n\n-   Statin therapy should be added to lifestyle therapy, regardless of baseline lipid levels, for diabetic patients:\n\n    -   with overt CVD. (A)\n\n    -   without CVD who are over age 40 years and have one or more other CVD risk factors. (A)\n\n-   For patients at lower risk than above (e.g., without overt CVD and under age 40 years), statin therapy should be considered in addition to lifestyle therapy if LDL cholesterol remains above 100 mg\/dl or in those with multiple CVD risk factors. (E)\n\n-   In individuals without overt CVD, the primary goal is an LDL cholesterol \\<100 mg\/dl (2.6 mmol\/l). (A)\n\n-   In individuals with overt CVD, a lower LDL cholesterol goal of \\<70 mg\/dl (1.8 mmol\/l), using a high dose of a statin, is an option. (B)\n\n-   If drug-treated patients do not reach the above targets on maximal tolerated statin therapy, a reduction in LDL cholesterol of \u223c30\u201340% from baseline is an alternative therapeutic goal. (A)\n\n-   Triglyceride levels \\<150 mg\/dl (1.7 mmol\/l) and HDL cholesterol \\>40 mg\/dl (1.0 mmol\/l) in men and \\>50 mg\/dl (1.3 mmol\/l) in women, are desirable. However, LDL cholesterol\u2013targeted statin therapy remains the preferred strategy. (C)\n\n-   If targets are not reached on maximally tolerated doses of statins, combination therapy using statins and other lipid-lowering agents may be considered to achieve lipid targets but has not been evaluated in outcome studies for either CVD outcomes or safety. (E)\n\n-   Statin therapy is contraindicated in pregnancy. (E)\n\n## Evidence for benefits of lipid-lowering therapy\n\nPatients with type 2 diabetes have an increased prevalence of lipid abnormalities, contributing to their high risk of CVD. For the past decade or more, multiple clinical trials demonstrated significant effects of pharmacologic (primarily statin) therapy on CVD outcomes in subjects with CHD and for primary CVD prevention (214). Sub-analyses of diabetic subgroups of larger trials (215\u2013219) and trials specifically in subjects with diabetes (220,221) showed significant primary and secondary prevention of CVD events \u00b1 CHD deaths in diabetic populations. As shown in <a href=\"#T11\" data-ref-type=\"table\">Table 11<\/a>, and similar to findings in nondiabetic subjects, reduction in \"hard\" CVD outcomes (CHD death and nonfatal MI) can be more clearly seen in diabetic subjects with high baseline CVD risk (known CVD and\/or very high LDL cholesterol levels), but overall the benefits of statin therapy in people with diabetes at moderate or high risk for CVD are convincing.\n\nReduction in 10-year risk of major CVD endpoints (CHD death\/non-fatal MI) in major statin trials, or substudies of major trials, in diabetic subjects (n = *16,032)*\n\n| Study (ref.) | CVD | Statin dose and comparator | Risk reduction (%) | Relative risk reduction (%) | Absolute risk reduction (%) | LDL cholesterol reduction (mg\/dl) | LDL cholesterol reduction (%) |\n|----|----|----|----|----|----|----|----|\n| 4S-DM (215) | 2\u00b0 | Simvastatin 20\u201340 mg vs. placebo | 85.7 to 43.2 | 50 | 42.5 | 186 to 119 | 36 |\n| ASPEN 2\u00b0 (220) | 2\u00b0 | Atorvastatin 10 mg vs. placebo | 39.5 to 24.5 | 34 | 15 | 112 to 79 | 29 |\n| HPS-DM (216) | 2\u00b0 | Simvastatin 40 mg vs. placebo | 43.8 to 36.3 | 17 | 7.5 | 123 to 84 | 31 |\n| CARE-DM (217) | 2\u00b0 | Pravastatin 40 mg vs. placebo | 40.8 to 35.4 | 13 | 5.4 | 136 to 99 | 27 |\n| TNT-DM (218) | 2\u00b0 | Atorvastatin 80 mg vs. 10 mg | 26.3 to 21.6 | 18 | 4.7 | 99 to 77 | 22 |\n| HPS-DM (216) | 1\u00b0 | Simvastatin 40 mg vs. placebo | 17.5 to 11.5 | 34 | 6.0 | 124 to 86 | 31 |\n| CARDS (221) | 1\u00b0 | Atorvastatin 10 mg vs. placebo | 11.5 to 7.5 | 35 | 4 | 118 to 71 | 40 |\n| ASPEN 1\u00b0 (220) | 1\u00b0 | Atorvastatin 10 mg vs. placebo | 9.8 to 7.9 | 19 | 1.9 | 114 to 80 | 30 |\n| ASCOT-DM (219) | 1\u00b0 | Atorvastatin 10 mg vs. placebo | 11.1 to 10.2 | 8 | 0.9 | 125 to 82 | 34 |\n\nStudies were of differing lengths (3.3\u20135.4 years) and used somewhat different outcomes, but all reported rates of CVD death and nonfatal MI. In this tabulation, results of the statin on 10-year risk of major CVD endpoints (CHD death\/nonfatal MI) are listed for comparison between studies. Correlation between 10-year CVD risk of the control group and the absolute risk reduction with statin therapy is highly significant (*P* = 0.0007). Analyses provided by Craig Williams, PharmD, Oregon Health & Science University, 2007.\n\nLow levels of HDL cholesterol, often associated with elevated triglyceride levels, are the most prevalent pattern of dyslipidemia in persons with type 2 diabetes. However, the evidence base for drugs that target these lipid fractions is significantly less robust than that for statin therapy (222). Nicotinic acid has been shown to reduce CVD outcomes (223), although the study was done in a nondiabetic cohort. Gemfibrozil has been shown to decrease rates of CVD events in subjects without diabetes (224,225) and in the diabetic subgroup in one of the larger trials (224). However, in a large trial specific to diabetic patients, fenofibrate failed to reduce overall cardiovascular outcomes (226).\n\n## Dyslipidemia treatment and target lipid levels\n\nFor most patients with diabetes, the first priority of dyslipidemia therapy (unless severe hypertriglyceridemia is the immediate issue) is to lower LDL cholesterol to a target goal of \\<100 mg\/dl (2.60 mmol\/l) (227). Lifestyle intervention, including MNT, increased physical activity, weight loss, and smoking cessation, may allow some patients to reach lipid goals. Nutrition intervention should be tailored according to each patient's age, type of diabetes, pharmacological treatment, lipid levels, and other medical conditions and should focus on the reduction of saturated fat, cholesterol, and *trans* unsaturated fat intake and increases in omega-3 fatty acids, viscous fiber (such as in oats, legumes, citrus), and plant stanols\/sterols. Glycemic control can also beneficially modify plasma lipid levels, particularly in patients with very high triglycerides and poor glycemic control.\n\nIn those with clinical CVD or over age 40 years with other CVD risk factors, pharmacological treatment should be added to lifestyle therapy regardless of baseline lipid levels. Statins are the drugs of choice for LDL cholesterol lowering.\n\nIn patients other than those described above, statin treatment should be considered if there is an inadequate LDL cholesterol response to lifestyle modifications and improved glucose control, or if the patient has increased cardiovascular risk (e.g., multiple cardiovascular risk factors or long duration of diabetes). Very little clinical trial evidence exists for type 2 diabetic patients under age 40 years or for type 1 patients of any age. In the Heart Protection Study (lower age limit 40 years), the subgroup of 600 patients with type 1 diabetes had a proportionately similar reduction in risk as patients with type 2 diabetes, although not statistically significant (216). Although the data are not definitive, consideration should be given to similar lipid-lowering goals in type 1 diabetic patients as in type 2 diabetic patients, particularly if they have other cardiovascular risk factors.\n\n## Alternative LDL cholesterol goals\n\nVirtually all trials of statins and CVD outcomes tested specific doses of statins against placebo, other doses of statin, or other statins, rather than aiming for specific LDL cholesterol goals (228). As can be seen in <a href=\"#T11\" data-ref-type=\"table\">Table 11<\/a>, placebo-controlled trials generally achieved LDL cholesterol reductions of 30\u201340% from baseline. Hence, LDL cholesterol lowering of this magnitude is an acceptable outcome for patients who cannot reach LDL cholesterol goals due to severe baseline elevations in LDL cholesterol and\/or intolerance of maximal, or any, statin doses. Additionally for those with baseline LDL cholesterol minimally above 100 mg\/dl, prescribing statin therapy to lower LDL cholesterol about 30\u201340% from baseline is probably more effective than prescribing just enough to get LDL cholesterol slightly below 100 mg\/dl.\n\nRecent clinical trials in high-risk patients, such as those with acute coronary syndromes or previous cardiovascular events (229\u2013231), have demonstrated that more aggressive therapy with high doses of statins to achieve an LDL cholesterol of \\<70 mg\/dl led to a significant reduction in further events. Therefore, a reduction in LDL cholesterol to a goal of \\<70 mg\/dl is an option in very-high-risk diabetic patients with overt CVD (232).\n\nIn individual patients, LDL cholesterol lowering with statins is highly variable, and this variable response is poorly understood (233). Reduction of CVD events with statins correlates very closely with LDL cholesterol lowering (214). When maximally tolerated doses of statins fail to significantly lower LDL cholesterol (\\<30% reduction from patients baseline), the primary aim of combination therapy should be to achieve additional LDL cholesterol lowering. Niacin, fenofibrate, ezetimibe, and bile acid sequestrants all offer additional LDL cholesterol lowering. The evidence that combination therapy provides a significant increment in CVD risk reduction over statin therapy alone is still elusive.\n\nIn 2008, a consensus panel convened by the American Diabetes Association and the American College of Cardiology recommended a greater focus on non-HDL cholesterol and apo lipoprotein B (apo B) in patients who are likely to have small LDL particles, such as people with diabetes (234). The consensus panel suggested that for statin-treated patients in whom the LDL cholesterol goal would be \\<70 mg\/dl (non-HDL cholesterol \\<100 mg\/dl), apo B should be measured and treated to \\<80 mg\/dl. For patients on statins with an LDL cholesterol goal of \\<100 mg\/dl (non-HDL cholesterol \\<130 mg\/dl), apo B should be measured and treated to \\<90 mg\/dl.\n\n## Treatment of other lipoprotein fractions or targets\n\nSevere hypertriglyceridemia may warrant immediate therapy of this abnormality with lifestyle and usually pharmacologic therapy (fibric acid derivative, niacin, or fish oil) to reduce the risk of acute pancreatitis. In the absence of severe hypertriglyceridemia, therapy targeting HDL cholesterol or triglycerides has intuitive appeal but lacks the evidence base of statin therapy. If the HDL cholesterol is \\<40 mg\/dl and the LDL cholesterol between 100 and 129 mg\/dl, gemfibrozil or niacin might be used, especially if a patient is intolerant to statins. Niacin is the most effective drug for raising HDL cholesterol. It can significantly increase blood glucose at high doses, but recent studies demonstrate that at modest doses (750\u20132,000 mg\/day), significant improvements in LDL cholesterol, HDL cholesterol, and triglyceride levels are accompanied by only modest changes in glucose that are generally amenable to adjustment of diabetes therapy (235,236).\n\n## Combination therapy\n\nCombination therapy, with a statin and a fibrate or statin and niacin, may be efficacious for treatment for all three lipid fractions, but this combination is associated with an increased risk for abnormal transaminase levels, myositis, or rhabdomyolysis. The risk of rhabdomyolysis is higher with higher doses of statins and with renal insufficiency and seems to be lower when statins are combined with fenofibrate than gemfibrozil (237). In the recent ACCORD study, the combination of fenofibrate and simvastatin did not reduce the rate of fatal cardiovascular events, nonfatal myocardial infarction, or nonfatal stroke, as compared with simvastatin alone, in patients with type 2 diabetes who were at high risk for CVD. However, prespecified subgroup analyses suggested heterogeneity in treatment effects according to sex, with a benefit for men and possible harm for women, and a possible benefit of combination therapy for patients with both triglyceride level \u2265204 mg\/dl and HDL cholesterol level \u226434 mg\/dl (238). Other ongoing trials may provide much-needed evidence for the effects of combination therapy on cardiovascular outcomes.\n\n<a href=\"#T12\" data-ref-type=\"table\">Table 12<\/a> summarizes common treatment goals for A1C, blood pressure, and HDL cholesterol.\n\nSummary of recommendations for glycemic blood pressure and lipid control for most adults with diabetes\n\n|  |  |\n|----|----|\n| A1C | \\<7.0%<a href=\"#TF12-1\" data-ref-type=\"table-fn\">*<\/a> |\n| Blood pressure | \\<130\/80 mmHg<a href=\"#TF12-2\" data-ref-type=\"table-fn\">\u2020<\/a> |\n| Lipids |  |\n| \u2003\u2003\u2003\u2003LDL cholesterol | \\<100 mg\/dl (\\<2.6 mmol\/l)<a href=\"#TF12-3\" data-ref-type=\"table-fn\">\u2021<\/a> |\n\n\\*More or less stringent glycemic goals may be appropriate for individual patients. Goals should be individualized based on: duration of diabetes, age\/life expectancy, comorbid conditions, known CVD or advanced microvascular complications, hypoglycemia unawareness, and individual patient considerations.\n\n\u2020Based on patient characteristics and response to therapy, higher or lower systolic blood pressure targets may be appropriate.\n\n\u2021In individuals with overt CVD, a lower LDL cholesterol goal of \\<70 mg\/dl (1.8 mmol\/l), using a high dose of a statin, is an option.\n\n## 3. Antiplatelet agents\n\n## Recommendations\n\n-   Consider aspirin therapy (75\u2013162 mg\/day) as a primary prevention strategy in those with type 1 or type 2 diabetes at increased cardiovascular risk (10-year risk \\>10%). This includes most men \\>50 years of age or women \\>60 years of age who have at least one additional major risk factor (family history of CVD, hypertension, smoking, dyslipidemia, or albuminuria). (C)\n\n-   Aspirin should not be recommended for CVD prevention for adults with diabetes at low CVD risk (10-year CVD risk \\<5%, such as in men \\<50 and women \\<60 years of age with no major additional CVD risk factors), since the potential adverse effects from bleeding likely offset the potential benefits. (C)\n\n-   In patients in these age-groups with multiple other risk factors (e.g., 10-year risk 5\u201310%), clinical judgment is required. (E)\n\n-   Use aspirin therapy (75\u2013162 mg\/day) as a secondary prevention strategy in those with diabetes with a history of CVD. (A)\n\n-   For patients with CVD and documented aspirin allergy, clopidogrel (75 mg\/day) should be used. (B)\n\n-   Combination therapy with ASA (75\u2013162 mg\/day) and clopidogrel (75 mg\/day) is reasonable for up to a year after an acute coronary syndrome. (B)\n\nADA and the American Heart Association (AHA) have, in the past, jointly recommended that low-dose aspirin therapy be used as a primary prevention strategy in those with diabetes at increased cardiovascular risk, including those who are over 40 years of age or those with additional risk factors (family history of CVD, hypertension, smoking, dyslipidemia, or albuminuria) (188). These recommendations were derived from several older trials that included small numbers of patients with diabetes.\n\nAspirin has been shown to be effective in reducing cardiovascular morbidity and mortality in high-risk patients with previous myocardial infarction or stroke (secondary prevention). Its net benefit in primary prevention among patients with no previous cardiovascular events is more controversial, both for patients with and without a history of diabetes (239). Two recent randomized controlled trials of aspirin specifically in patients with diabetes failed to show a significant reduction in CVD end points, raising further questions about the efficacy of aspirin for primary prevention in people with diabetes (240,241).\n\nThe Anti-thrombotic Trialists' (ATT) collaborators recently published an individual patient-level meta-analysis of the six large trials of aspirin for primary prevention in the general population. These trials collectively enrolled over 95,000 participants, including almost 4,000 with diabetes. Overall, they found that aspirin reduced the risk of vascular events by 12% (RR 0.88 \\[95% CI 0.82\u2013 0.94\\]). The largest reduction was for nonfatal myocardial infarction with little effect on CHD death (RR 0.95 \\[95% CI 0.78\u20131.15\\]) or total stroke. There was some evidence of a difference in aspirin effect by sex. Aspirin significantly reduced CHD events in men but not in women. Conversely, aspirin had no effect on stroke in men but significantly reduced stroke in women. Notably, sex differences in aspirin's effects have not been observed in studies of secondary prevention (239). In the six trials examined by the ATT collaborators, the effects of aspirin on major vascular events were similar for patients with or without diabetes: RR 0.88 (95% CI 0.67\u20131.15) and 0.87 (0.79\u20130.96), respectively. The CI was wider for those with diabetes because of their smaller number.\n\nBased on the currently available evidence, aspirin appears to have a modest effect on ischemic vascular events with the absolute decrease in events depending on the underlying CVD risk. The main adverse effects appear to be an increased risk of gastrointestinal bleeding. The excess risk may be as high as 1\u20135 per 1,000 per year in real-world settings. In adults with CVD risk greater than 1% per year, the number of CVD events prevented will be similar to or greater than the number of episodes of bleeding induced, although these complications do not have equal effects on long-term health (242).\n\nIn 2010, a position statement of the ADA, AHA, and American College of Cardiology Foundation (ACCF) updated prior joint recommendations for primary prevention (243). Low dose (75\u2013162 mg\/day) aspirin use for primary prevention is reasonable for adults with diabetes and no previous history of vascular disease who are at increased CVD risk (10-year risk of CVD events over 10%) and who are not at increased risk for bleeding. This generally includes most men over age 50 years and women over age 60 years who also have one or more of the following major risk factors: smoking, hypertension, dyslipidemia, family history of premature CVD, and albuminuria.\n\nHowever, aspirin is no longer recommended for those at low CVD risk (women under age 60 years and men under age 50 years with no major CVD risk factors; 10-year CVD risk under 5%) as the low benefit is likely to be outweighed by the risks of significant bleeding. Clinical judgment should be used for those at intermediate risk (younger patients with one or more risk factors, or older patients with no risk factors; those with 10-year CVD risk of 5\u201310%) until further research is available. Use of aspirin in patients under the age of 21 years is contraindicated due to the associated risk of Reye's syndrome.\n\nAverage daily dosages used in most clinical trials involving patients with diabetes ranged from 50 to 650 mg but were mostly in the range of 100 to 325 mg\/day. There is little evidence to support any specific dose, but using the lowest possible dosage may help reduce side effects (244). Although platelets from patients with diabetes have altered function, it is unclear what, if any, impact that finding has on the required dose of aspirin for cardioprotective effects in the patient with diabetes. Many alternate pathways for platelet activation exist that are independent of thromboxane A~2~ and thus not sensitive to the effects of aspirin (245). Therefore, while \"aspirin resistance\" appears higher in the diabetic patients when measured by a variety of ex vivo and in vitro methods (platelet aggrenometry, measurement of thromboxane B~2~), these observations alone are insufficient to empirically recommend higher doses of aspirin be used in the diabetic patient at this time.\n\nClopidogrel has been demonstrated to reduce CVD events in diabetic individuals (246). It is recommended as adjunctive therapy in the first year after an acute coronary syndrome or as alternative therapy in aspirin-intolerant patients.\n\n## 4. Smoking cessation\n\n## Recommendations\n\n-   Advise all patients not to smoke. (A)\n\n-   Include smoking cessation counseling and other forms of treatment as a routine component of diabetes care. (B)\n\nA large body of evidence from epidemiological, case-control, and cohort studies provides convincing documentation of the causal link between cigarette smoking and health risks. Much of the work documenting the impact of smoking on health did not separately discuss results on subsets of individuals with diabetes, but suggests that the identified risks are at least equivalent to those found in the general population. Other studies of individuals with diabetes consistently demonstrate that smokers have a heightened risk of CVD, premature death, and increased rate of microvascular complications of diabetes. Smoking may have a role in the development of type 2 diabetes.\n\nThe routine and thorough assessment of tobacco use is important as a means of preventing smoking or encouraging cessation. A number of large randomized clinical trials have demonstrated the efficacy and cost-effectiveness of brief counseling in smoking cessation, including the use of quit lines, in the reduction of tobacco use. For the patient motivated to quit, the addition of pharmacological therapy to counseling is more effective than either treatment alone. Special considerations should include assessment of level of nicotine dependence, which is associated with difficulty in quitting and relapse (247).\n\n## 5. CHD screening and treatment\n\n## Recommendations\n\n## Screening\n\n-   In asymptomatic patients, routine screening for CAD is not recommended, as it does not improve outcomes as long as CVD risk factors are treated. (A)\n\n## Treatment\n\n-   In patients with known CVD, ACE inhibitor (C) and aspirin and statin therapy (A) (if not contraindicated) should be used to reduce the risk of cardiovascular events.\n\n-   In patients with a prior myocardial infarction, \u03b2-blockers should be continued for at least 2 years after the event (B).\n\n-   Longer term use of \u03b2-blockers in the absence of hypertension is reasonable if well tolerated, but data are lacking. (E)\n\n-   Avoid TZD treatment in patients with symptomatic heart failure. (C)\n\n-   Metformin may be used in patients with stable CHF if renal function is normal. It should be avoided in unstable or hospitalized patients with CHF. (C)\n\nScreening for CAD is reviewed in a recently updated consensus statement (154). To identify the presence of CAD in diabetic patients without clear or suggestive symptoms, a risk factor\u2013based approach to the initial diagnostic evaluation and subsequent follow-up has intuitive appeal. However, recent studies concluded that using this approach fails to identify which patients with type 2 diabetes will have silent ischemia on screening tests (159,248).\n\nCandidates for cardiac testing include those with *1*) typical or atypical cardiac symptoms and *2*) an abnormal resting ECG. The screening of asymptomatic patients remains controversial, especially as intensive medical therapy, indicated in diabetic patients at high risk for CVD, has an increasing evidence base for providing equal outcomes to invasive revascularization, including in diabetic patients (249,250). There is also some evidence that silent myocardial ischemia may reverse over time, adding to the controversy concerning aggressive screening strategies (251). Finally, a recent randomized observational trial demonstrated no clinical benefit to routine screening of asymptomatic patients with type 2 diabetes and normal ECGs (252). Despite abnormal myocardial perfusion imaging in more than one in five patients, cardiac outcomes were essentially equal (and very low) in screened versus unscreened patients. Accordingly, the overall effectiveness, especially the cost-effectiveness, of such an indiscriminate screening strategy is now questioned.\n\nNewer noninvasive CAD screening methods, such as computed tomography (CT) and CT angiography have gained in popularity. These tests infer the presence of coronary atherosclerosis by measuring the amount of calcium in coronary arteries and, in some circumstances, by direct visualization of luminal stenoses. Although asymptomatic diabetic patients found to have a higher coronary disease burden have more future cardiac events (253\u2013255), the role of these tests beyond risk stratification is not clear. Their routine use leads to radiation exposure and may result in unnecessary invasive testing such as coronary angiography and revascularization procedures. The ultimate balance of benefit, cost, and risks of such an approach in asymptomatic patients remains controversial, particularly in the modern setting of aggressive CVD risk factor control.\n\nIn all patients with diabetes, cardiovascular risk factors should be assessed at least annually. These risk factors include dyslipidemia, hypertension, smoking, a positive family history of premature coronary disease, and the presence of micro- or macroalbuminuria. Abnormal risk factors should be treated as described elsewhere in these guidelines. Patients at increased CHD risk should receive aspirin and a statin and ACE inhibitor or ARB therapy if hypertensive, unless there are contraindications to a particular drug class. While clear benefit exists for ACE inhibitor and ARB therapy in patients with nephropathy or hypertension, the benefits in patients with CVD in the absence of these conditions is less clear, especially when LDL cholesterol is concomitantly controlled (256,257).\n\n## B. Nephropathy screening and treatment\n\n## Recommendations\n\n## General recommendations\n\n-   To reduce the risk or slow the progression of nephropathy, optimize glucose control. (A)\n\n-   To reduce the risk or slow the progression of nephropathy, optimize blood pressure control. (A)\n\n## Screening\n\n-   Perform an annual test to assess urine albumin excretion in type 1 diabetic patients with diabetes duration of 5 years and in all type 2 diabetic patients starting at diagnosis. (E)\n\n-   Measure serum creatinine at least annually in all adults with diabetes regardless of the degree of urine albumin excretion. The serum creatinine should be used to estimate GFR and stage the level of chronic kidney disease (CKD), if present. (E)\n\n## Treatment\n\n-   In the treatment of the nonpregnant patient with micro- or macroalbuminuria, either ACE inhibitors or ARBs should be used. (A)\n\n-   While there are no adequate head-to-head comparisons of ACE inhibitors and ARBs, there is clinical trial support for each of the following statements:\n\n    -   In patients with type 1 diabetes, with hypertension and any degree of albuminuria, ACE inhibitors have been shown to delay the progression of nephropathy. (A)\n\n    -   In patients with type 2 diabetes, hypertension, and microalbuminuria, both ACE inhibitors and ARBs have been shown to delay the progression to macroalbuminuria. (A)\n\n    -   In patients with type 2 diabetes, hypertension, macroalbuminuria, and renal insufficiency (serum creatinine \\>1.5 mg\/dl), ARBs have been shown to delay the progression of nephropathy. (A)\n\n-   If one class is not tolerated, the other should be substituted. (E)\n\n-   Reduction of protein intake to 0.8\u20131.0 g \u00b7 kg body wt^\u22121^ \u00b7 day^\u22121^ in individuals with diabetes and the earlier stages of CKD and to 0.8 g \u00b7 kg body wt^\u22121^ \u00b7 day^\u22121^ in the later stages of CKD may improve measures of renal function (urine albumin excretion rate, GFR) and is recommended. (B)\n\n-   When ACE inhibitors, ARBs, or diuretics are used, monitor serum creatinine and potassium levels for the development of acute kidney disease and hyperkalemia. (E)\n\n-   Continued monitoring of urine albumin excretion to assess both response to therapy and progression of disease is recommended. (E)\n\n-   When eGFR \\<60 ml\/min\/1.73 m^2,^ evaluate and manage potential complications of CKD. (E)\n\n-   Consider referral to a physician experienced in the care of kidney disease when there is uncertainty about the etiology of kidney disease (heavy proteinuria, active urine sediment, absence of retinopathy, rapid decline in GFR), difficult management issues, or advanced kidney disease. (B)\n\nDiabetic nephropathy occurs in 20\u201340% of patients with diabetes and is the single leading cause of end-stage renal disease (ESRD). Persistent albuminuria in the range of 30\u2013299 mg\/24 h (microalbuminuria) has been shown to be the earliest stage of diabetic nephropathy in type 1 diabetes and a marker for development of nephropathy in type 2 diabetes. Microalbuminuria is also a well-established marker of increased CVD risk (258,259). Patients with microalbuminuria who progress to macroalbuminuria (300 mg\/24 h) are likely to progress to ESRD (260,261). However, a number of interventions have been demonstrated to reduce the risk and slow the progression of renal disease.\n\nIntensive diabetes management with the goal of achieving near-normoglycemia has been shown in large prospective randomized studies to delay the onset of microalbuminuria and the progression of micro- to macroalbuminuria in patients with type 1 (262,263) and type 2 (55,56) diabetes. The UKPDS provided strong evidence that control of blood pressure can reduce the development of nephropathy (195). In addition, large prospective randomized studies in patients with type 1 diabetes have demonstrated that achievement of lower levels of systolic blood pressure (\\<140 mmHg) resulting from treatment using ACE inhibitors provides a selective benefit over other antihypertensive drug classes in delaying the progression from micro- to macroalbuminuria and can slow the decline in GFR in patients with macroalbuminuria (264\u2013266). In type 2 diabetes with hypertension and normoalbuminuria, RAS inhibition has been demonstrated to delay onset of microalbuminuria (267).\n\nIn addition, ACE inhibitors have been shown to reduce major CVD outcomes (i.e., myocardial infarction, stroke, death) in patients with diabetes (207), thus further supporting the use of these agents in patients with microalbuminuria, a CVD risk factor. ARBs do not prevent microalbuminuria in normotensive patients with type 1 or type 2 diabetes (268,269); however, ARBs have been shown to reduce the rate of progression from micro- to macroalbuminuria as well as ESRD in patients with type 2 diabetes (270\u2013272). Some evidence suggests that ARBs have a smaller magnitude of rise in potassium compared with ACE inhibitors in people with nephropathy (273,274). Combinations of drugs that block the renin-angiotensin-aldosterone system (e.g., an ACE inhibitor plus an ARB, a mineralocorticoid antagonist, or a direct renin inhibitor) have been shown to provide additional lowering of albuminuria (275\u2013278). However, the long-term effects of such combinations on renal or cardiovascular outcomes have not yet been evaluated in clinical trials, and they are associated with increased risk for hyperkalemia.\n\nOther drugs, such as diuretics, calcium channel blockers, and \u03b2-blockers should be used as additional therapy to further lower blood pressure in patients already treated with ACE inhibitors or ARBs (212), or as alternate therapy in the rare individual unable to tolerate ACE inhibitors or ARBs.\n\nStudies in patients with varying stages of nephropathy have shown that protein restriction of dietary protein helps slow the progression of albuminuria, GFR decline, and occurrence of ESRD (279\u2013282). Dietary protein restriction should be considered particularly in patients whose nephropathy seems to be progressing despite optimal glucose and blood pressure control and use of ACE inhibitor and\/or ARBs (282).\n\n## Assessment of albuminuria status and renal function\n\nScreening for microalbuminuria can be performed by measurement of the albumin-to-creatinine ratio in a random spot collection; 24-h or timed collections are more burdensome and add little to prediction or accuracy (283,284). Measurement of a spot urine for albumin only, whether by immunoassay or by using a dipstick test specific for microalbumin, without simultaneously measuring urine creatinine, is somewhat less expensive but susceptible to false-negative and -positive determinations as a result of variation in urine concentration due to hydration and other factors.\n\nAbnormalities of albumin excretion are defined in <a href=\"#T13\" data-ref-type=\"table\">Table 13<\/a>. Because of variability in urinary albumin excretion, two of three specimens collected within a 3- to 6-month period should be abnormal before considering a patient to have crossed one of these diagnostic thresholds. Exercise within 24 h, infection, fever, CHF, marked hyperglycemia, and marked hypertension may elevate urinary albumin excretion over baseline values.\n\nDefinitions of abnormalities in albumin excretion\n\n| Category                     | Spot collection (\u03bcg\/mg creatinine) |\n|------------------------------|------------------------------------|\n| Normal                       | \\<30                               |\n| Microalbuminuria             | 30\u2013299                             |\n| Macro (clinical)-albuminuria | \u2265300                               |\n\nInformation on presence of abnormal urine albumin excretion in addition to level of GFR may be used to stage CKD. The National Kidney Foundation classification (<a href=\"#T14\" data-ref-type=\"table\">Table 14<\/a>) is primarily based on GFR levels and therefore differs from other systems, in which staging is based primarily on urinary albumin excretion (285). Studies have found decreased GFR in the absence of increased urine albumin excretion in a substantial percentage of adults with diabetes (286). Serum creatinine should therefore be measured at least annually in all adults with diabetes, regardless of the degree of urine albumin excretion.\n\nStages of CKD\n\n| Stage | Description | GFR (ml\/min per 1.73 m^2^ body surface area) |\n|----|----|----|\n| 1 | Kidney damage<a href=\"#TF14-1\" data-ref-type=\"table-fn\">*<\/a> with normal or increased GFR | \u226590 |\n| 2 | Kidney damage<a href=\"#TF14-1\" data-ref-type=\"table-fn\">*<\/a> with mildly decreased GFR | 60\u201389 |\n| 3 | Moderately decreased GFR | 30\u201359 |\n| 4 | Severely decreased GFR | 15\u201329 |\n| 5 | Kidney failure | \\<15 or dialysis |\n\n\\*Kidney damage defined as abnormalities on pathologic, urine, blood, or imaging tests. Adapted from ref. 284.\n\nSerum creatinine should be used to estimate GFR and to stage the level of CKD, if present. eGFR is commonly co-reported by laboratories or can be estimated using formulae such as the Modification of Diet in Renal Disease (MDRD) study equation (287). Recent reports have indicated that the MDRD is more accurate for the diagnosis and stratification of CKD in patients with diabetes than the Cockcroft-Gault formula (288). GFR calculators are available at <http:\/\/www.nkdep.nih.gov>.\n\nThe role of continued annual quantitative assessment of albumin excretion after diagnosis of microalbuminuria and institution of ACE inhibitor or ARB therapy and blood pressure control is unclear. Continued surveillance can assess both response to therapy and progression of disease. Some suggest that reducing abnormal albuminuria (\\>30 mg\/g) to the normal or near-normal range may improve renal and cardiovascular prognosis, but this approach has not been formally evaluated in prospective trials.\n\nComplications of kidney disease correlate with level of kidney function. When the eGFR is less than 60 ml\/min\/1.73 m^2^, screening for complications of CKD is indicated (<a href=\"#T15\" data-ref-type=\"table\">Table 15<\/a>). Early vaccination against hepatitis B is indicated in patients likely to progress to end-stage kidney disease.\n\nManagement of CKD in diabetes\n\n| GFR (ml\/min\/1.73 m^2^) | Recommended |\n|----|----|\n| All patients | Yearly measurement of creatinine, urinary albumin excretion, potassium |\n| 45\u201360 | Referral to nephrology if possibility for nondiabetic kidney disease exists (duration type 1 diabetes \\<10 years, heavy proteinuria, abnormal findings on renal ultrasound, resistant hypertension, rapid fall in GFR, or active urinary sediment) |\n|  | Consider need for dose adjustment of medications |\n|  | Monitor eGFR every 6 months |\n|  | Monitor electrolytes, bicarbonate, hemoglobin, calcium, phosphorus, parathyroid hormone at least yearly |\n|  | Assure vitamin D sufficiency |\n|  | Consider bone density testing |\n|  | Referral for dietary counselling |\n| 30\u201344 | Monitor eGFR every 3 months |\n|  | Monitor electrolytes, bicarbonate, calcium, phosphorus, parathyroid hormone, hemoglobin, albumin, weight every 3\u20136 months |\n|  | Consider need for dose adjustment of medications |\n| \\<30 | Referral to nephrologist |\n\nAdapted from <http:\/\/www.kidney.org\/professionals\/KDOQI\/guideline_diabetes\/>.\n\nConsider referral to a physician experienced in the care of kidney disease when there is uncertainty about the etiology of kidney disease (heavy proteinuria, active urine sediment, absence of retinopathy, rapid decline in GFR, resistant hypertension), difficult management issues, or advanced kidney disease. The threshold for referral may vary depending on the frequency with which a provider encounters diabetic patients with significant kidney disease. Consultation with a nephrologist when stage 4 CKD develops has been found to reduce cost, improve quality of care, and keep people off dialysis longer (289). However, nonrenal specialists should not delay educating their patients about the progressive nature of diabetic kidney disease; the renal preservation benefits of aggressive treatment of blood pressure, blood glucose, and hyperlipidemia; and the potential need for renal replacement therapy.\n\n## C. Retinopathy screening and treatment\n\n## Recommendations\n\n## General recommendations\n\n-   To reduce the risk or slow the progression of retinopathy, optimize glycemic control. (A)\n\n-   To reduce the risk or slow the progression of retinopathy, optimize blood pressure control. (A)\n\n## Screening\n\n-   Adults and children aged 10 years or older with type 1 diabetes should have an initial dilated and comprehensive eye examination by an ophthalmologist or optometrist within 5 years after the onset of diabetes. (B)\n\n-   Patients with type 2 diabetes should have an initial dilated and comprehensive eye examination by an ophthalmologist or optometrist shortly after the diagnosis of diabetes. (B)\n\n-   Subsequent examinations for type 1 and type 2 diabetic patients should be repeated annually by an ophthalmologist or optometrist. Less frequent exams (every 2\u20133 years) may be considered following one or more normal eye exams. Examinations will be required more frequently if retinopathy is progressing. (B)\n\n-   High-quality fundus photographs can detect most clinically significant diabetic retinopathy. Interpretation of the images should be performed by a trained eye care provider. While retinal photography may serve as a screening tool for retinopathy, it is not a substitute for a comprehensive eye exam, which should be performed at least initially and at intervals thereafter as recommended by an eye care professional. (E)\n\n-   Women with preexisting diabetes who are planning a pregnancy or who have become pregnant should have a comprehensive eye examination and should be counseled on the risk of development and\/or progression of diabetic retinopathy. Eye examination should occur in the first trimester with close follow-up throughout pregnancy and for 1 year postpartum. (B)\n\n## Treatment\n\n-   Promptly refer patients with any level of macular edema, severe NPDR, or any PDR to an ophthalmologist who is knowledgeable and experienced in the management and treatment of diabetic retinopathy. (A)\n\n-   Laser photocoagulation therapy is indicated to reduce the risk of vision loss in patients with high-risk PDR, clinically significant macular edema, and in some cases of severe NPDR. (A)\n\n-   The presence of retinopathy is not a contraindication to aspirin therapy for cardioprotection, as this therapy does not increase the risk of retinal hemorrhage. (A)\n\nDiabetic retinopathy is a highly specific vascular complication of both type 1 and type 2 diabetes, with prevalence strongly related to the duration of diabetes. Diabetic retinopathy is the most frequent cause of new cases of blindness among adults aged 20\u201374 years. Glaucoma, cataracts, and other disorders of the eye occur earlier and more frequently in people with diabetes.\n\nIn addition to duration of diabetes, other factors that increase the risk of, or are associated with, retinopathy include chronic hyperglycemia (290), the presence of nephropathy (291), and hypertension (292). Intensive diabetes management with the goal of achieving near normoglycemia has been shown in large prospective randomized studies to prevent and\/or delay the onset and progression of diabetic retinopathy (47,55, 56,64). Lowering blood pressure has been shown to decrease the progression of retinopathy (195). Several case series and a controlled prospective study suggest that pregnancy in type 1 diabetic patients may aggravate retinopathy (293,294); laser photocoagulation surgery can minimize this risk (294).\n\nOne of the main motivations for screening for diabetic retinopathy is the established efficacy of laser photocoagulation surgery in preventing vision loss. Two large trials, the Diabetic Retinopathy Study (DRS) and the Early Treatment Diabetic Retinopathy Study (ETDRS), provide the strongest support for the therapeutic benefits of photocoagulation surgery.\n\nThe DRS (295) showed that panretinal photocoagulation surgery reduced the risk of severe vision loss from PDR from 15.9% in untreated eyes to 6.4% in treated eyes, with greatest risk-to-benefit ratio in those with baseline disease (disc neovascularization or vitreous hemorrhage).\n\nThe ETDRS (296) established the benefit of focal laser photocoagulation surgery in eyes with macular edema, particularly those with clinically significant macular edema, with reduction of doubling of the visual angle (e.g., 20\/50 to 20\/100) from 20% in untreated eyes to 8% in treated eyes. The ETDRS also verified the benefits of panretinal photocoagulation for high-risk PDR and in older-onset patients with severe NPDR or less-than-high-risk PDR.\n\nLaser photocoagulation surgery in both trials was beneficial in reducing the risk of further vision loss, but generally not beneficial in reversing already diminished acuity. This preventive effect and the fact that patients with PDR or macular edema may be asymptomatic provide strong support for a screening program to detect diabetic retinopathy.\n\nAs retinopathy is estimated to take at least 5 years to develop after the onset of hyperglycemia, patients with type 1 diabetes should have an initial dilated and comprehensive eye examination within 5 years after the onset of diabetes. Patients with type 2 diabetes, who generally have had years of undiagnosed diabetes and who have a significant risk of prevalent DR at time of diabetes diagnosis, should have an initial dilated and comprehensive eye examination soon after diagnosis. Examinations should be performed by an ophthalmologist or optometrist who is knowledgeable and experienced in diagnosing the presence of diabetic retinopathy and is aware of its management. Subsequent examinations for type 1 and type 2 diabetic patients are generally repeated annually. Less-frequent exams (every 2\u20133 years) may be cost effective after one or more normal eye exams, while examinations will be required more frequently if retinopathy is progressing (297).\n\nThe use of retinal photography with remote reading by experts has great potential in areas where qualified eye care professionals are not available, and may also enhance efficiency and reduce costs when the expertise of ophthalmologists can be utilized for more complex examinations and for therapy (298). In-person exams are still necessary when the photos are unacceptable and for follow-up of abnormalities detected. Photos are not a substitute for a comprehensive eye exam, which should be performed at least initially and at intervals thereafter as recommended by an eye care professional.\n\nResults of eye examinations should be documented and transmitted to the referring health care professional. For a detailed review of the evidence and further discussion of diabetic retinopathy, see the ADA's technical review and position statement on this subject (297,300).\n\n## D. Neuropathy screening and treatment (301)\n\n## Recommendations\n\n-   All patients should be screened for distal symmetric polyneuropathy (DPN) at diagnosis and at least annually thereafter, using simple clinical tests. (B)\n\n-   Electrophysiological testing is rarely needed, except in situations where the clinical features are atypical. (E)\n\n-   Screening for signs and symptoms of autonomic neuropathy should be instituted at diagnosis of type 2 diabetes and 5 years after the diagnosis of type 1 diabetes. Special testing is rarely needed and may not affect management or outcomes. (E)\n\n-   Medications for the relief of specific symptoms related to DPN and autonomic neuropathy are recommended, as they improve the quality of life of the patient. (E)\n\nThe diabetic neuropathies are heterogeneous with diverse clinical manifestations. They may be focal or diffuse. Most common among the neuropathies are chronic sensorimotor DPN and autonomic neuropathy. Although DPN is a diagnosis of exclusion, complex investigations to exclude other conditions are rarely needed.\n\nThe early recognition and appropriate management of neuropathy in the patient with diabetes is important for a number of reasons: *1*) nondiabetic neuropathies may be present in patients with diabetes and may be treatable, *2*) a number of treatment options exist for symptomatic diabetic neuropathy, *3*) up to 50% of DPN may be asymptomatic and patients are at risk of insensate injury to their feet, and *4*) autonomic neuropathy and particularly cardiovascular autonomic neuropathy is associated with substantial morbidity and even mortality. Specific treatment for the underlying nerve damage is currently not available, other than improved glycemic control, which may modestly slow progression (63) but not reverse neuronal loss. Effective symptomatic treatments are available for some manifestations of DPN and autonomic neuropathy.\n\n## Diagnosis of neuropathy\n\n### Distal symmetric polyneuropathy.\n\nPatients with diabetes should be screened annually for DPN using tests such as pinprick sensation, vibration perception (using a 128-Hz tuning fork), 10-g monofilament pressure sensation at the distal plantar aspect of both great toes and metatarsal joints, and assessment of ankle reflexes. Combinations of more than one test have \\>87% sensitivity in detecting DPN. Loss of 10-g monofilament perception and reduced vibration perception predict foot ulcers (301). Importantly, in patients with neuropathy, particularly when severe, causes other than diabetes should always be considered, such as neurotoxic mediations, heavy metal poisoning, alcohol abuse, vitamin B12 deficiency (especially in those taking metformin for prolonged periods (302), renal disease, chronic inflammatory demyelinating neuropathy, inherited neuropathies, and vasculitis (303).\n\n### Diabetic autonomic neuropathy (304).\n\nThe symptoms and signs of autonomic dysfunction should be elicited carefully during the history and physical examination. Major clinical manifestations of diabetic autonomic neuropathy include resting tachycardia, exercise intolerance, orthostatic hypotension, constipation, gastroparesis, erectile dysfunction, sudomotor dysfunction, impaired neurovascular function, and, potentially, autonomic failure in response to hypoglycemia.\n\nCardiovascular autonomic neuropathy, a CVD risk factor (93), is the most studied and clinically important form of diabetic autonomic neuropathy. Cardiovascular autonomic neuropathy may be indicated by resting tachycardia (\\>100 bpm) or orthostasis (a fall in systolic blood pressure \\>20 mmHg upon standing without an appropriate heart rate response); it is also associated with increased cardiac event rates.\n\nGastrointestinal neuropathies (e.g., esophageal enteropathy, gastroparesis, constipation, diarrhea, fecal incontinence) are common, and any section of the gastrointestinal tract may be affected. Gastroparesis should be suspected in individuals with erratic glucose control or with upper gastrointestinal symptoms without other identified cause. Evaluation of solid-phase gastric emptying using double-isotope scintigraphy may be done if symptoms are suggestive, but test results often correlate poorly with symptoms. Constipation is the most common lower-gastrointestinal symptom but can alternate with episodes of diarrhea.\n\nDiabetic autonomic neuropathy is also associated with genitourinary tract disturbances. In men, diabetic autonomic neuropathy may cause erectile dysfunction and\/or retrograde ejaculation. Evaluation of bladder dysfunction should be performed for individuals with diabetes who have recurrent urinary tract infections, pyelonephritis, incontinence, or a palpable bladder.\n\n## Symptomatic treatments\n\n### DPN.\n\nThe first step in management of patients with DPN should be to aim for stable and optimal glycemic control. Although controlled trial evidence is lacking, several observational studies suggest that neuropathic symptoms improve not only with optimization of control, but also with the avoidance of extreme blood glucose fluctuations. Patients with painful DPN may benefit from pharmacological treatment of their symptoms: many agents have efficacy confirmed in published randomized controlled trials, several of which are Food and Drug Administration (FDA)-approved for the management of painful DPN.\n\n## Treatment of autonomic neuropathy\n\nGastroparesis symptoms may improve with dietary changes and prokinetic agents such as metoclopramide or erythromycin. Treatments for erectile dysfunction may include phosphodiesterase type 5 inhibitors, intracorporeal or intraurethral prostaglandins, vacuum devices, or penile prostheses. Interventions for other manifestations of autonomic neuropathy are described in the ADA statement on neuropathy (301). As with DPN treatments, these interventions do not change the underlying pathology and natural history of the disease process, but may have a positive impact on the quality of life of the patient.\n\n## E. Foot care\n\n## Recommendations\n\n-   For all patients with diabetes, perform an annual comprehensive foot examination to identify risk factors predictive of ulcers and amputations. The foot examination should include inspection, assessment of foot pulses, and testing for loss of protective sensation (10-g monofilament plus testing any one of: vibration using 128-Hz tuning fork, pinprick sensation, ankle reflexes, or vibration perception threshold). (B)\n\n-   Provide general foot self-care education to all patients with diabetes. (B)\n\n-   A multidisciplinary approach is recommended for individuals with foot ulcers and high-risk feet, especially those with a history of prior ulcer or amputation. (B)\n\n-   Refer patients who smoke, have loss of protective sensation and structural abnormalities, or have history of prior lower-extremity complications to foot care specialists for ongoing preventive care and life-long surveillance. (C)\n\n-   Initial screening for peripheral arterial disease (PAD) should include a history for claudication and an assessment of the pedal pulses. Consider obtaining an ankle-brachial index (ABI), as many patients with PAD are asymptomatic. (C)\n\n-   Refer patients with significant claudication or a positive ABI for further vascular assessment and consider exercise, medications, and surgical options. (C)\n\nAmputation and foot ulceration, consequences of diabetic neuropathy and\/or PAD, are common and major causes of morbidity and disability in people with diabetes. Early recognition and management of risk factors can prevent or delay adverse outcomes.\n\nThe risk of ulcers or amputations is increased in people who have the following risk factors:\n\n-   Previous amputation\n\n-   Past foot ulcer history\n\n-   Peripheral neuropathy\n\n-   Foot deformity\n\n-   Peripheral vascular disease\n\n-   Visual impairment\n\n-   Diabetic nephropathy (especially patients on dialysis)\n\n-   Poor glycemic control\n\n-   Cigarette smoking\n\nMany studies have been published proposing a range of tests that might usefully identify patients at risk of foot ulceration, creating confusion among practitioners as to which screening tests should be adopted in clinical practice. An ADA task force was therefore assembled in 2008 to concisely summarize recent literature in this area and then recommend what should be included in the comprehensive foot exam for adult patients with diabetes. Their recommendations are summarized below, but clinicians should refer to the task force report (305) for further details and practical descriptions of how to perform components of the comprehensive foot examination.\n\nAt least annually, all adults with diabetes should undergo a comprehensive foot examination to identify high risk conditions. Clinicians should ask about history of previous foot ulceration or amputation, neuropathic or peripheral vascular symptoms, impaired vision, tobacco use, and foot care practices. A general inspection of skin integrity and musculoskeletal deformities should be done in a well lit room. Vascular assessment would include inspection and assessment of pedal pulses.\n\nThe neurologic exam recommended is designed to identify loss of protective sensation (LOPS) rather than early neuropathy. The clinical examination to identify LOPS is simple and requires no expensive equipment. Five simple clinical tests (use of a 10-g monofilament, vibration testing using a 128-Hz tuning fork, tests of pinprick sensation, ankle reflex assessment, and testing vibration perception threshold with a biothesiometer), each with evidence from well-conducted prospective clinical cohort studies, are considered useful in the diagnosis of LOPS in the diabetic foot. The task force agrees that any of the five tests listed could be used by clinicians to identify LOPS, although ideally two of these should be regularly performed during the screening exam\u2014normally the 10-g monofilament and one other test. One or more abnormal tests would suggest LOPS, while at least two normal tests (and no abnormal test) would rule out LOPS. The last test listed, vibration assessment using a biothesiometer or similar instrument, is widely used in the U.S.; however, identification of the patient with LOPS can easily be carried out without this or other expensive equipment.\n\nInitial screening for PAD should include a history for claudication and an assessment of the pedal pulses. A diagnostic ABI should be performed in any patient with symptoms of PAD. Due to the high estimated prevalence of PAD in patients with diabetes and the fact that many patients with PAD are asymptomatic, an ADA consensus statement on PAD (306) suggested that a screening ABI be performed in patients over 50 years of age and be considered in patients under 50 years of age who have other PAD risk factors (e.g., smoking, hypertension, hyperlipidemia, or duration of diabetes \\>10 years). Refer patients with significant symptoms or a positive ABI for further vascular assessment and consider exercise, medications, and surgical options (306).\n\nPatients with diabetes and high-risk foot conditions should be educated regarding their risk factors and appropriate management. Patients at risk should understand the implications of the LOPS, the importance of foot monitoring on a daily basis, the proper care of the foot, including nail and skin care, and the selection of appropriate footwear. Patients with LOPS should be educated on ways to substitute other sensory modalities (hand palpation, visual inspection) for surveillance of early foot problems. Patients' understanding of these issues and their physical ability to conduct proper foot surveillance and care should be assessed. Patients with visual difficulties, physical constraints preventing movement, or cognitive problems that impair their ability to assess the condition of the foot and to institute appropriate responses will need other people, such as family members, to assist in their care.\n\nPeople with neuropathy or evidence of increased plantar pressure (e.g., erythema, warmth, callus, or measured pressure) may be adequately managed with well-fitted walking shoes or athletic shoes that cushion the feet and redistribute pressure. Callus can be debrided with a scalpel by a foot care specialist or other health professional with experience and training in foot care. People with bony deformities (e.g., hammertoes, prominent metatarsal heads, bunions) may need extra-wide or -depth shoes. People with extreme bony deformities (e.g., Charcot foot) who cannot be accommodated with commercial therapeutic footwear may need custom-molded shoes.\n\nFoot ulcers and wound care may require care by a podiatrist, orthopedic or vascular surgeon, or rehabilitation specialist experienced in the management of individuals with diabetes.\n\n# VII. DIABETES CARE IN SPECIFIC POPULATIONS\n\n## A. Children and adolescents\n\n## 1. Type 1 diabetes\n\nThree-quarters of all cases of type 1 diabetes are diagnosed in individuals \\<18 years of age. It is appropriate to consider the unique aspects of care and management of children and adolescents with type 1 diabetes. Children with diabetes differ from adults in many respects, including changes in insulin sensitivity related to sexual maturity and physical growth, ability to provide self-care, supervision in child care and school, and unique neurological vulnerability to hypoglycemia and DKA. Attention to such issues as family dynamics, developmental stages, and physiological differences related to sexual maturity are all essential in developing and implementing an optimal diabetes regimen. Although recommendations for children and adolescents are less likely to be based on clinical trial evidence, expert opinion and a review of available and relevant experimental data are summarized in the ADA statement on care of children and adolescents with type 1 diabetes (307).\n\nIdeally, the care of a child or adolescent with type 1 diabetes should be provided by a multidisciplinary team of specialists trained in the care of children with pediatric diabetes. At the very least, education of the child and family should be provided by health care providers trained and experienced in childhood diabetes and sensitive to the challenges posed by diabetes in this age-group. At the time of initial diagnosis, it is essential that diabetes education be provided in a timely fashion, with the expectation that the balance between adult supervision and self-care should be defined by, and will evolve according to, physical, psychological, and emotional maturity. MNT and psychological support should be provided at diagnosis, and regularly thereafter, by individuals experienced with the nutritional and behavioral needs of the growing child and family.\n\n## a. Glycemic control\n\n## Recommendations\n\n-   Consider age when setting glycemic goals in children and adolescents with type 1 diabetes. (E)\n\nWhile current standards for diabetes management reflect the need to maintain glucose control as near to normal as safely possible, special consideration should be given to the unique risks of hypoglycemia in young children. Glycemic goals may need to be modified to take into account the fact that most children \\<6 or 7 years of age have a form of \"hypoglycemic unawareness,\" including immaturity of and a relative inability to recognize and respond to hypoglycemic symptoms, placing them at greater risk for severe hypoglycemia and its sequelae. In addition, and unlike the case in adults, young children under the age of 5 years may be at risk for permanent cognitive impairment after episodes of severe hypoglycemia (308\u2013310). Furthermore, findings from the DCCT demonstrated that near-normalization of blood glucose levels was more difficult to achieve in adolescents than adults. Nevertheless, the increased frequency of use of basal-bolus regimens and insulin pumps in youth from infancy through adolescence has been associated with more children reaching ADA blood glucose targets (311,312) in those families in which both parents and the child with diabetes participate jointly to perform the required diabetes-related tasks. Furthermore, recent studies documenting neurocognitive sequelae of hyperglycemia in children provide another compelling motivation for achieving glycemic targets (313,314).\n\nIn selecting glycemic goals, the benefits on long-term health outcomes of achieving a lower A1C should be balanced against the risks of hypoglycemia and the developmental burdens of intensive regimens in children and youth. Age-specific glycemic and A1C goals are presented in <a href=\"#T16\" data-ref-type=\"table\">Table 16<\/a>.\n\nPlasma blood glucose and A1C goals for type 1 diabetes by age-group\n\n<table>\n<thead>\n<tr>\n<th rowspan=\"2\"><\/th>\n<th colspan=\"2\" style=\"text-align: center;\">Plasma blood glucose goal range (mg\/dl)<\/th>\n<th rowspan=\"2\" style=\"text-align: center;\">A1C (%)<\/th>\n<th rowspan=\"2\" style=\"text-align: center;\">Rationale<\/th>\n<\/tr>\n<tr>\n<th style=\"text-align: center;\">Before meals<\/th>\n<th style=\"text-align: center;\">Bedtime\/overnight<\/th>\n<\/tr>\n<\/thead>\n<tbody>\n<tr>\n<td style=\"text-align: left;\">Toddlers and preschoolers (0\u20136 years)<\/td>\n<td style=\"text-align: center;\">100\u2013180<\/td>\n<td style=\"text-align: center;\">110\u2013200<\/td>\n<td style=\"text-align: center;\">&lt;8.5<\/td>\n<td style=\"text-align: left;\"><ul>\n<li><p>Vulnerability to hypoglycemia<\/p><\/li>\n<li><p>Insulin sensitivity<\/p><\/li>\n<li><p>Unpredictability in dietary intake and physical activity<\/p><\/li>\n<li><p>A lower goal (&lt;8.0%) is reasonable if it can be achieved without excessive hypoglycemia<\/p><\/li>\n<\/ul><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">School age (6\u201312 years)<\/td>\n<td style=\"text-align: center;\">90\u2013180<\/td>\n<td style=\"text-align: center;\">100\u2013180<\/td>\n<td style=\"text-align: center;\">&lt;8<\/td>\n<td style=\"text-align: left;\"><ul>\n<li><p>Vulnerability to hypoglycemia<\/p><\/li>\n<li><p>A lower goal (&lt;7.5%) is reasonable if it can be achieved without excessive hypoglycemia<\/p><\/li>\n<\/ul><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Adolescents and young adults (13\u201319 years)<\/td>\n<td style=\"text-align: center;\">90\u2013130<\/td>\n<td style=\"text-align: center;\">90\u2013150<\/td>\n<td style=\"text-align: center;\">&lt;7.5<\/td>\n<td style=\"text-align: left;\"><ul>\n<li><p>A lower goal (&lt;7.0%) is reasonable if it can be achieved without excessive hypoglycemia<\/p><\/li>\n<\/ul><\/td>\n<\/tr>\n<tr>\n<td colspan=\"4\" style=\"text-align: left;\"><strong>Key concepts in setting glycemic goals<\/strong>\n<ul>\n<li><p>Goals should be individualized and lower goals may be reasonable based on benefit-risk assessment.<\/p><\/li>\n<li><p>Blood glucose goals should be modified in children with frequent hypoglycemia or hypoglycemia unawareness.<\/p><\/li>\n<li><p>Postprandial blood glucose values should be measured when there is a discrepancy between pre-prandial blood glucose values and A1C levels and to help assess glycemia in those on basal\/bolus regimens.<\/p><\/li>\n<\/ul><\/td>\n<td><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n## b. Screening and management of chronic complications in children and adolescents with type 1 diabetes\n\n## i. Nephropathy\n\n## Recommendations\n\n-   Annual screening for microalbuminuria, with a random spot urine sample for albumin-to-creatinine (ACR) ratio, should be considered once the child is 10 years of age and has had diabetes for 5 years. (E)\n\n-   Confirmed, persistently elevated ACR on two additional urine specimens from different days should be treated with an ACE inhibitor, titrated to normalization of albumin excretion if possible. (E)\n\n## ii. Hypertension\n\n## Recommendations\n\n-   Treatment of high-normal blood pressure (systolic or diastolic blood pressure consistently above the 90th percentile for age, sex, and height) should include dietary intervention and exercise aimed at weight control and increased physical activity, if appropriate. If target blood pressure is not reached with 3\u20136 months of lifestyle intervention, pharmacologic treatment should be considered. (E)\n\n-   Pharmacologic treatment of hypertension (systolic or diastolic blood pressure consistently above the 95th percentile for age, sex, and height or consistently greater than 130\/80 mmHg, if 95% exceeds that value) should be initiated as soon as the diagnosis is confirmed. (E)\n\n-   ACE inhibitors should be considered for the initial treatment of hypertension, following appropriate reproductive counseling due to its potential teratogenic effects. (E)\n\n-   The goal of treatment is a blood pressure consistently \\<130\/80 or below the 90th percentile for age, sex, and height, whichever is lower. (E)\n\nIt is important that blood pressure measurements are determined correctly, using the appropriate size cuff, and with the child seated and relaxed. Hypertension should be confirmed on at least three separate days. Normal blood pressure levels for age, sex, and height and appropriate methods for determinations are available online at [www.nhlbi.nih.gov\/health\/prof\/heart\/hbp\/hbp_ped.pdf](http:\/\/www.nhlbi.nih.gov\/health\/prof\/heart\/hbp\/hbp_ped.pdf).\n\n## iii. Dyslipidemia\n\n## Recommendations\n\n## Screening\n\n-   If there is a family history of hypercholesterolemia (total cholesterol \\>240 mg\/dl) or a cardiovascular event before age 55 years, or if family history is unknown, then a fasting lipid profile should be performed on children \\>2 years of age soon after diagnosis (after glucose control has been established). If family history is not of concern, then the first lipid screening should be considered at puberty (\u226510 years). All children diagnosed with diabetes at or after puberty should have a fasting lipid profile performed soon after diagnosis (after glucose control has been established). (E)\n\n-   For both age-groups, if lipids are abnormal, annual monitoring is recommended. If LDL cholesterol values are within the accepted risk levels (\\<100 mg\/dl \\[2.6 mmol\/l\\]), a lipid profile should be repeated every 5 years. (E)\n\n## Treatment\n\n-   Initial therapy should consist of optimization of glucose control and MNT using a Step 2 AHA diet aimed at a decrease in the amount of saturated fat in the diet. (E)\n\n-   After the age of 10 years, the addition of a statin in patients who, after MNT and lifestyle changes, have LDL cholesterol \\>160 mg\/dl (4.1 mmol\/l), or LDL cholesterol \\>130 mg\/dl (3.4 mmol\/l) and one or more CVD risk factors, is reasonable. (E)\n\n-   The goal of therapy is an LDL cholesterol value \\<100 mg\/dl (2.6 mmol\/l). (E)\n\nPeople diagnosed with type 1 diabetes in childhood have a high risk of early subclinical (315\u2013317) and clinical (318) CVD. Although intervention data are lacking, the AHA categorizes children with type 1 diabetes in the highest tier for cardiovascular risk and recommends both lifestyle and pharmacologic treatment for those with elevated LDL cholesterol levels (319,320). Initial therapy should be with a Step 2 AHA diet, which restricts saturated fat to 7% of total calories and restricts dietary cholesterol to 200 mg\/day. Data from randomized clinical trials in children as young as 7 months of age indicate that this diet is safe and does not interfere with normal growth and development (321,322).\n\nNeither long-term safety nor cardiovascular outcome efficacy of statin therapy has been established for children. However, recent studies have shown short-term safety equivalent to that seen in adults, and efficacy in lowering LDL cholesterol levels, improving endothelial function, and causing regression of carotid intimal thickening (323\u2013325). No statin is approved for use under the age of 10 years, and statin treatment should generally not be used in children with type 1 diabetes prior to this age.\n\n## iv. Retinopathy\n\n## Recommendations\n\n-   The first ophthalmologic examination should be obtained once the child is 10 years of age and has had diabetes for 3\u20135 years. (E)\n\n-   After the initial examination, annual routine follow-up is generally recommended. Less frequent examinations may be acceptable on the advice of an eye care professional. (E)\n\nAlthough retinopathy most commonly occurs after the onset of puberty and after 5\u201310 years of diabetes duration, it has been reported in prepubertal children and with diabetes duration of only 1\u20132 years. Referrals should be made to eye care professionals with expertise in diabetic retinopathy, an understanding of the risk for retinopathy in the pediatric population, and experience in counseling the pediatric patient and family on the importance of early prevention\/intervention.\n\n## v. Celiac disease\n\n## Recommendations\n\n-   Children with type 1 diabetes should be screened for celiac disease by measuring tissue transglutaminase or anti-endomysial antibodies, with documentation of normal total serum IgA levels, soon after the diagnosis of diabetes. (E)\n\n-   Testing should be repeated in children with growth failure, failure to gain weight, weight loss, diarrhea, flatulence, abdominal pain, or signs of malabsorption, or in children with frequent unexplained hypoglycemia or deterioration in glycemic control. (E)\n\n-   Children with positive antibodies should be referred to a gastroenterologist for evaluation with endoscopy and biopsy. (E)\n\n-   Children with biopsy-confirmed celiac disease should be placed on a gluten-free diet and have consultation with a dietitian experienced in managing both diabetes and celiac disease. (E)\n\nCeliac disease is an immune-mediated disorder that occurs with increased frequency in patients with type 1 diabetes (1\u201316% of individuals compared with 0.3\u20131% in the general population) (326,327). Symptoms of celiac disease include diarrhea, weight loss or poor weight gain, growth failure, abdominal pain, chronic fatigue, malnutrition due to malabsorption, other gastrointestinal problems, and unexplained hypoglycemia or erratic blood glucose concentrations.\n\nThe advent of routine periodic screening has led to the diagnosis of celiac disease in asymptomatic children. While several studies have documented short-term benefits of gluten restriction on growth and bone mineral density in asymptomatic children diagnosed with celiac disease by routine screening, there is little literature available regarding the long-term benefit of gluten-free diets in this population.\n\n## vi. Hypothyroidism\n\n## Recommendations\n\n-   Children with type 1 diabetes should be screened for thyroid peroxidase and thyroglobulin antibodies at diagnosis. (E)\n\n-   TSH concentrations should be measured after metabolic control has been established. If normal, they should be re-checked every 1\u20132 years, or if the patient develops symptoms of thyroid dysfunction, thyromegaly, or an abnormal growth rate. (E)\n\nAuto-immune thyroid disease is the most common autoimmune disorder associated with diabetes, occurring in 17\u201330% of patients with type 1 diabetes (328). The presence of thyroid auto-antibodies is predictive of thyroid dysfunction, generally hypothyroidism but less commonly hyperthyroidism (329). Subclinical hypothyroidism may be associated with increased risk of symptomatic hypoglycemia (330) and with reduced linear growth (331). Hyperthyroidism alters glucose metabolism, potentially resulting in deterioration of metabolic control.\n\n## c. Self-management\n\nNo matter how sound the medical regimen, it can only be as good as the ability of the family and\/or individual to implement it. Family involvement in diabetes remains an important component of optimal diabetes management throughout childhood and into adolescence. Health care providers who care for children and adolescents, therefore, must be capable of evaluating the behavioral, emotional, and psychosocial factors that interfere with implementation and then must work with the individual and family to resolve problems that occur and\/or to modify goals as appropriate.\n\n## d. School and day care\n\nBecause a sizable portion of a child's day is spent in school, close communication with and cooperation of school or day care personnel is essential for optimal diabetes management, safety, and maximal academic opportunities. See the ADA position statement on Diabetes Care in the School and Day Care Setting (332) for further discussion.\n\n## e. Transition from pediatric to adult care\n\nAs they approach the young adult years, older adolescents are at increasing physical, behavioral, and other risks (333,334). As they leave both their home and their pediatric diabetes care providers, these older teens may become disengaged from the health care system, leading to lapses in medical care and deterioration in glycemic control (335). Though scientific evidence is limited to date, it is clear that early and ongoing attention be given to comprehensive and coordinated planning for seamless transition of all youth from pediatric to adult health care (336,337). The National Diabetes Education Program (NDEP) has materials available to facilitate this transition process (<http:\/\/ndep.nih.gov\/transitions\/>).\n\n## 2. Type 2 diabetes\n\nThe incidence of type 2 diabetes in adolescents is increasing, especially in ethnic minority populations (21). Distinction between type 1 and type 2 diabetes in children can be difficult, since the prevalence of overweight in children continues to rise and since autoantigens and ketosis may be present in a substantial number of patients with features of type 2 diabetes (including obesity and acanthosis nigricans). Such a distinction at the time of diagnosis is critical since treatment regimens, educational approaches, and dietary counsel will differ markedly between the two diagnoses.\n\nType 2 diabetes has a significant prevalence of comorbidities already present at the time of diagnosis (338). It is recommended that blood pressure measurement, a fasting lipid profile, microalbuminuria assessment, and dilated eye examination be performed at the time of diagnosis. Thereafter, screening guidelines and treatment recommendations for hypertension, dyslipidemia, microalbuminuria, and retinopathy in youth with type 2 diabetes are similar to those for youth with type 1 diabetes. Additional problems that may need to be addressed include polycystic ovary disease and the various comorbidities associated with pediatric obesity such as sleep apnea, hepatic steatosis, orthopedic complications, and psychosocial concerns. The ADA consensus statement on this subject (23) provides guidance on the prevention, screening, and treatment of type 2 diabetes and its comorbidities in young people.\n\n## 3. Monogenic diabetes syndromes\n\nMonogenic forms of diabetes (neonatal diabetes or maturity-onset diabetes of the young) represent a small fraction of children with diabetes (\\<5%), but the ready availability of commercial genetic testing is now enabling a true genetic diagnosis with increasing frequency. It is important to correctly diagnose one of the monogenic forms of diabetes, as these children may be incorrectly diagnosed with type 1 or type 2 diabetes, leading to nonoptimal treatment regimens and delays in diagnosing other family members.\n\nThe diagnosis of monogenic diabetes should be considered in the following settings: diabetes diagnosed within the first 6 months of life; in children with strong family history of diabetes but without typical features of type 2 diabetes (nonobese, low-risk ethnic group); in children with mild fasting hyperglycemia (100\u2013150 mg\/dl \\[5.5\u20138.5 mmol\\]), especially if young and nonobese; and in children with diabetes but with negative auto-antibodies without signs of obesity or insulin resistance. A recent international consensus document discusses in further detail the diagnosis and management of children with monogenic forms of diabetes (339).\n\n## B. Preconception care\n\n## Recommendations\n\n-   A1C levels should be as close to normal as possible (\\<7%) in an individual patient before conception is attempted. (B)\n\n-   Starting at puberty, preconception counseling should be incorporated in the routine diabetes clinic visit for all women of child-bearing potential. (C)\n\n-   Women with diabetes who are contemplating pregnancy should be evaluated and, if indicated, treated for diabetic retinopathy, nephropathy, neuropathy, and CVD. (E)\n\n-   Medications used by such women should be evaluated prior to conception, since drugs commonly used to treat diabetes and its complications may be contraindicated or not recommended in pregnancy, including statins, ACE inhibitors, ARBs, and most noninsulin therapies. (E)\n\n-   Since many pregnancies are unplanned, consider the potential risks and benefits of medications that are contraindicated in pregnancy in all women of childbearing potential, and counsel women using such medications accordingly. (E)\n\nMajor congenital malformations remain the leading cause of mortality and serious morbidity in infants of mothers with type 1 and type 2 diabetes. Observational studies indicate that the risk of malformations increases continuously with increasing maternal glycemia during the first 6\u20138 weeks of gestation, as defined by first-trimester A1C concentrations. There is no threshold for A1C values below which risk disappears entirely. However, malformation rates above the 1\u20132% background rate of nondiabetic pregnancies appear to be limited to pregnancies in which first-trimester A1C concentrations are \\>1% above the normal range for a nondiabetic pregnant woman.\n\nPreconception care of diabetes appears to reduce the risk of congenital malformations. Five nonrandomized studies compared rates of major malformations in infants between women who participated in preconception diabetes care programs and women who initiated intensive diabetes management after they were already pregnant. The preconception care programs were multidisciplinary and designed to train patients in diabetes self-management with diet, intensified insulin therapy, and SMBG. Goals were set to achieve normal blood glucose concentrations, and \\>80% of subjects achieved normal A1C concentrations before they became pregnant. In all five studies, the incidence of major congenital malformations in women who participated in preconception care (range 1.0\u20131.7% of infants) was much lower than the incidence in women who did not participate (range 1.4\u201310.9% of infants) (78). One limitation of these studies is that participation in preconception care was self-selected rather than randomized. Thus, it is impossible to be certain that the lower malformation rates resulted fully from improved diabetes care. Nonetheless, the evidence supports the concept that malformations can be reduced or prevented by careful management of diabetes before pregnancy.\n\nPlanned pregnancies greatly facilitate preconception diabetes care. Unfortunately, nearly two-thirds of pregnancies in women with diabetes are unplanned, leading to a persistent excess of malformations in infants of diabetic mothers. To minimize the occurrence of these devastating malformations, standard care for all women with diabetes who have child-bearing potential, beginning at the onset of puberty or at diagnosis, should include *1*) education about the risk of malformations associated with unplanned pregnancies and poor metabolic control; and *2*) use of effective contraception at all times, unless the patient has good metabolic control and is actively trying to conceive.\n\nWomen contemplating pregnancy need to be seen frequently by a multidisciplinary team experienced in the management of diabetes before and during pregnancy. The goals of preconception care are to *1*) involve and empower the patient in the management of her diabetes, *2*) achieve the lowest A1C test results possible without excessive hypoglycemia, *3*) assure effective contraception until stable and acceptable glycemia is achieved, and *4*) identify, evaluate, and treat long-term diabetes complications such as retinopathy, nephropathy, neuropathy, hypertension, and CHD (78).\n\nAmong the drugs commonly used in the treatment of patients with diabetes, a number may be relatively or absolutely contraindicated during pregnancy. Statins are category X (contraindicated for use in pregnancy) and should be discontinued before conception, as should ACE inhibitors (340). ARBs are category C (risk cannot be ruled out) in the first trimester but category D (positive evidence of risk) in later pregnancy and should generally be discontinued before pregnancy. Since many pregnancies are unplanned, health care professionals caring for any woman of childbearing potential should consider the potential risks and benefits of medications that are contraindicated in pregnancy. Women using medications such as statins or ACE inhibitors need ongoing family planning counseling. Among the oral antidiabetic agents, metformin and acarbose are classified as category B (no evidence of risk in humans) and all others as category C. Potential risks and benefits of oral antidiabetic agents in the preconception period must be carefully weighed, recognizing that data are insufficient to establish the safety of these agents in pregnancy.\n\nFor further discussion of preconception care, see the ADA's consensus statement on preexisting diabetes and pregnancy (78) and the position statement (341) on this subject.\n\n## C. Older adults\n\n## Recommendations\n\n-   Older adults who are functional, cognitively intact, and have significant life expectancy should receive diabetes care using goals developed for younger adults. (E)\n\n-   Glycemic goals for older adults not meeting the above criteria may be relaxed using individual criteria, but hyperglycemia leading to symptoms or risk of acute hyperglycemic complications should be avoided in all patients. (E)\n\n-   Other cardiovascular risk factors should be treated in older adults with consideration of the time frame of benefit and the individual patient. Treatment of hypertension is indicated in virtually all older adults, and lipid and aspirin therapy may benefit those with life expectancy at least equal to the time frame of primary or secondary prevention trials. (E)\n\n-   Screening for diabetes complications should be individualized in older adults, but particular attention should be paid to complications that would lead to functional impairment. (E)\n\nDiabetes is an important health condition for the aging population; at least 20% of patients over the age of 65 years have diabetes, and this number can be expected to grow rapidly in the coming decades. Older individuals with diabetes have higher rates of premature death, functional disability, and coexisting illnesses such as hypertension, CHD, and stroke than those without diabetes. Older adults with diabetes are also at greater risk than other older adults for several common geriatric syndromes, such as polypharmacy, depression, cognitive impairment, urinary incontinence, injurious falls, and persistent pain.\n\nThe American Geriatric Society's guidelines for improving the care of the older person with diabetes (342) have influenced the following discussion and recommendations. The care of older adults with diabetes is complicated by their clinical and functional heterogeneity. Some older individuals developed diabetes years earlier and may have significant complications; others who are newly diagnosed may have had years of undiagnosed diabetes with resultant complications or may have few complications from the disease. Some older adults with diabetes are frail and have other underlying chronic conditions, substantial diabetes-related comorbidity, or limited physical or cognitive functioning. Other older individuals with diabetes have little comorbidity and are active. Life expectancies are highly variable for this population, but often longer than clinicians realize. Providers caring for older adults with diabetes must take this heterogeneity into consideration when setting and prioritizing treatment goals.\n\nThere are few long-term studies in older adults demonstrating the benefits of intensive glycemic, blood pressure, and lipid control. Patients who can be expected to live long enough to reap the benefits of long-term intensive diabetes management and who are active, have good cognitive function, and are willing should be provided with the needed education and skills to do so and be treated using the goals for younger adults with diabetes.\n\nFor patients with advanced diabetes complications, life-limiting comorbid illness, or substantial cognitive or functional impairment, it is reasonable to set less-intensive glycemic target goals. These patients are less likely to benefit from reducing the risk of microvascular complications and more likely to suffer serious adverse effects from hypoglycemia. However, patients with poorly controlled diabetes may be subject to acute complications of diabetes, including dehydration, poor wound healing, and hyperglycemic hyperosmolar coma. Glycemic goals at a minimum should avoid these consequences.\n\nAlthough control of hyperglycemia may be important in older individuals with diabetes, greater reductions in morbidity and mortality may result from control of other cardiovascular risk factors rather than from tight glycemic control alone. There is strong evidence from clinical trials of the value of treating hypertension in the elderly (343,344). There is less evidence for lipid-lowering and aspirin therapy, although the benefits of these interventions for primary and secondary prevention are likely to apply to older adults whose life expectancies equal or exceed the time frames seen in clinical trials.\n\nSpecial care is required in prescribing and monitoring pharmacologic therapy in older adults. Metformin is often contraindicated because of renal insufficiency or significant heart failure. TZDs can cause fluid retention, which may exacerbate or lead to heart failure. They are contraindicated in patients with CHF (New York Heart Association class III and class IV) and if used at all should be used very cautiously in those with, or at risk for, milder degrees of CHF. Sulfonylureas, other insulin secretagogues, and insulin can cause hypoglycemia. Insulin use requires that patients or caregivers have good visual and motor skills and cognitive ability. Drugs should be started at the lowest dose and titrated up gradually until targets are reached or side effects develop.\n\nScreening for diabetes complications in older adults also should be individualized. Particular attention should be paid to complications that can develop over short periods of time and\/or that would significantly impair functional status, such as visual and lower-extremity complications.\n\n## D. Cystic fibrosis\u2013related diabetes\n\nCystic fibrosis\u2013related diabetes (CFRD) is the most common comorbidity in persons with CF, occurring in about 20% of adolescents and 40\u201350% of adults. The additional diagnosis of diabetes in this population is associated with worse nutritional status, more-severe inflammatory lung disease, and greater mortality from respiratory failure. For reasons that are not well understood, women with CFRD are particularly vulnerable to excess morbidity and mortality. Insulin insufficiency related to partial fibrotic destruction of the islet mass is the primary defect in CFRD. Genetically determined function of the remaining \u03b2-cells and insulin resistance associated with infection and inflammation may also play a role. Encouraging new data suggest that early detection and aggressive insulin therapy have narrowed the gap in mortality between CF patients with and without diabetes, and have eliminated the sex difference in mortality.\n\nA consensus conference on CFRD was co-sponsored in 2009 by the American Diabetes Association, the Cystic Fibrosis Foundation, and the Pediatric Endocrine Society. Recommendations for the clinical management of CFRD can be found in an ADA position statement (344a).\n\n# VIII. DIABETES CARE IN SPECIFIC SETTINGS\n\n## A. Diabetes care in the hospital\n\n## Recommendations\n\n-   All patients with diabetes admitted to the hospital should have their diabetes clearly identified in the medical record. (E)\n\n-   All patients with diabetes should have an order for blood glucose monitoring, with results available to all members of the health care team. (E)\n\n-   Goals for blood glucose levels:\n\n    -   Critically ill patients: Insulin therapy should be initiated for treatment of persistent hyperglycemia starting at a threshold of no greater than 180 mg\/dl (10 mmol\/l). Once insulin therapy is started, a glucose range of 140\u2013180 mg\/dl (7.8\u201310 mmol\/l) is recommended for the majority of critically ill patients. (A)\n\n    -   More stringent goals, such as 110\u2013140 mg\/dl (6.1\u20137.8 mmol\/l) may be appropriate for selected patients, as long as this can be achieved without significant hypoglycemia. (C)\n\n    -   Critically ill patients require an intravenous insulin protocol that has demonstrated efficacy and safety in achieving the desired glucose range without increasing risk for severe hypoglycemia. (E)\n\n    -   Non\u2013critically ill patients: There is no clear evidence for specific blood glucose goals. If treated with insulin, the premeal blood glucose target should generally be \\<140 mg\/dl (7.8 mmol\/l) with random blood glucose \\<180 mg\/dl (10.0 mmol\/l), provided these targets can be safely achieved. More stringent targets may be appropriate in stable patients with previous tight glycemic control. Less stringent targets may be appropriate in those with severe comorbidites. (E)\n\n-   Scheduled subcutaneous insulin with basal, nutritional, and correction components is the preferred method for achieving and maintaining glucose control in noncritically ill patients. (C) Using correction dose or \"supplemental\" insulin to correct premeal hyperglycemia in addition to scheduled prandial and basal insulin is recommended. (E)\n\n-   Glucose monitoring should be initiated in any patient not known to be diabetic who receives therapy associated with high risk for hyperglycemia, including high-dose glucocorticoid therapy, initiation of enteral or parenteral nutrition, or other medications such as octreotide or immunosuppressive medications. (B) If hyperglycemia is documented and persistent, treatment is necessary. Such patients should be treated to the same glycemic goals as patients with known diabetes. (E)\n\n-   A hypoglycemia management protocol should be adopted and implemented by each hospital or hospital system. A plan for treating hypoglycemia should be established for each patient. Episodes of hypoglycemia in the hospital should be documented in the medial record and tracked. (E)\n\n-   All patients with diabetes admitted to the hospital should have an A1C obtained if the result of testing in the previous 2\u20133 months is not available. (E)\n\n-   Patients with hyperglycemia in the hospital who do not have a diagnosis of diabetes should have appropriate plans for follow-up testing and care documented at discharge. (E)\n\nHyperglycemia in the hospital is extensively reviewed in an ADA technical review (345). A recent updated consensus statement by the American Association of Clinical Endocrinologists (AACE) and the ADA (346) forms the basis for the discussion and guidelines in this section.\n\nThe literature on hospitalized patients with hyperglycemia typically describes three categories:\n\n1.  Medical history of diabetes: diabetes has been previously diagnosed and acknowledged by the patient's treating physician.\n\n2.  Unrecognized diabetes: hyperglycemia (fasting blood glucose \u2265126 mg\/dl or random blood glucose \u2265200 mg\/dl) occurring during hospitalization and confirmed as diabetes after hospitalization by standard diagnostic criteria but unrecognized as diabetes by the treating physician during hospitalization.\n\n3.  Hospital-related hyperglycemia: hyperglycemia (fasting blood glucose \u2265126 mg\/dl or random blood glucose \u2265200 mg\/dl) occurring during the hospitalization that reverts to normal after hospital discharge.\n\nThe management of hyperglycemia in the hospital has often been considered secondary in importance to the condition that prompted admission (345). However, a body of literature now supports targeted glucose control in the hospital setting for potential improved clinical outcomes. Hyperglycemia in the hospital may result from stress, decompensation of type 1 or type 2 or other forms of diabetes, and\/or may be iatrogenic due to withholding of anti-hyperglycemic medications or administration of hyperglycemia-provoking agents such as glucocorticoids or vasopressors.\n\nPeople with diabetes are more likely to be hospitalized and to have longer lengths of stay than those without diabetes. A recent survey estimated that 22% of all hospital inpatient days were incurred by people with diabetes and that hospital inpatient care accounted for half of the \\$174 billion total U.S. medical expenditures for this disease (347). This is due, in part, to the continued expansion of the worldwide epidemic of type 2 diabetes. While the costs of illness-related stress hyperglycemia are not known, they are likely to be significant given the poor prognosis of such patients (348\u2013351).\n\nThere is substantial observational evidence linking hyperglycemia in hospitalized patients (with or without diabetes) to poor outcomes. Cohort studies as well as a few early randomized controlled trials (RCTs) suggested that intensive treatment of hyperglycemia improved hospital outcomes (345,350,351). In general, these studies were heterogeneous in terms of patient population, blood glucose targets and insulin protocols, provision of nutritional support, and the proportion of patients receiving insulin, which limits the ability to make meaningful comparisons among them. Recent trials in critically ill patients have failed to show a significant improvement in mortality with intensive glycemic control (352,353) or have even shown increased mortality risk (354). Moreover, these recent RCTs have highlighted the risk of severe hypoglycemia resulting from such efforts (352\u2013357).\n\nThe largest study to date, NICE-SUGAR, a multicenter, multinational RCT, compared the effect of intensive glycemic control (target 81\u2013108 mg\/dl, mean blood glucose attained 115 mg\/dl) to standard glycemic control (target 144\u2013180 mg\/dl, mean blood glucose attained 144 mg\/dl) on outcomes among 6,104 critically ill participants, the majority of whom (\\>95%) required mechanical ventilation (354). Ninety-day mortality was significantly higher in the intensive versus the conventional group (78 more deaths; 27.5% vs. 24.9%, *P* = 0.02) in both surgical and medical patients. Mortality from cardiovascular causes was more common in the intensive group (76 more deaths; 41.6% vs. 35.8%; *P* = 0.02). Severe hypoglycemia was also more common in the intensively treated group (6.8% vs. 0.5%; *P* \\< 0.001). The precise reason for the increased mortality in the tightly controlled group is unknown. The results of this study lie in stark contrast to a famous 2001 single-center study that reported a 42% relative reduction in intensive-care unit (ICU) mortality in critically ill surgical patients treated to a target blood glucose of 80\u2013110 mg\/dl (350). Importantly, the control group in NICE-SUGAR had reasonably good blood glucose management maintained at a mean glucose of 144 mg\/dl, only 29 mg\/dl above the intensively managed patients. Accordingly, this study's findings do not disprove the notion that glycemic control in the ICU is important. However, they do strongly suggest that it is not necessary to target blood glucose values \\<140 mg\/dl, and that a highly stringent target of \\<110 mg\/dl may actually be dangerous.\n\nIn a recent meta-analysis of 26 trials (*N* = 13,567), which included the NICE-SUGAR data, the pooled relative risk (RR) of death with intensive insulin therapy was 0.93 as compared with conventional therapy (95% CI 0.83\u20131.04) (357). Approximately half of these trials reported hypoglycemia, with a pooled RR of intensive therapy of 6.0 (95% CI 4.5\u20138.0). The specific ICU setting influenced the findings, with patients in surgical ICUs appearing to benefit from intensive insulin therapy (RR 0.63 \\[95% CI 0.44\u20130.91\\]), while those in other critical care settings did not (medical ICU, RR 1.0 \\[95% CI 0.78\u20131.28\\]; \"mixed\" ICU, RR 0.99 \\[95% CI 0.86\u20131.12\\]). It was concluded that overall, intensive insulin therapy increased the risk of hypoglycemia but provided no overall benefit on mortality in the critically ill, although a possible mortality benefit to patients admitted to the surgical ICU (RR 0.63 \\[95% CI 0.44\u20130.91\\]) was suggested.\n\n## 1. Glycemic targets in hospitalized patients\n\n## Definition of glucose abnormalities in the hospital setting\n\nHyperglycemia has been defined as any blood glucose level \\>140 mg\/dl (7.8 mmol\/l). Levels that are significantly and persistently above this may require treatment in hospitalized patients. In patients without a previous diagnosis of diabetes, elevated blood glucose may be due to \"stress hyperglycemia,\" a condition that can be established by a review of prior records or measurement of an A1C. A1C values \\>6.5% suggest that diabetes preceded hospitalization (358). Hypoglycemia has been defined as any blood glucose level \\<70 mg\/dl (3.9 mmol\/l). This is the standard definition in outpatients and correlates with the initial threshold for the release of counterregulatory hormones. Severe hypoglycemia in hospitalized patients has been defined by many as \\<40 mg\/dl (2.2 mmol\/l), although this is lower than the \u223c50 mg\/dl (2.8 mmol\/l) level at which cognitive impairment begins in normal individuals (359). As with hyperglycemia, hypoglycemia among inpatients is also associated with adverse short- and long-term outcomes. Early recognition and treatment of mild to moderate hypoglycemia (40 and 69 mg\/dl) (2.2 and 3.8 mmol\/l) can prevent deterioration to a more severe episode with potential adverse sequelae (346).\n\n## Critically ill patients\n\nBased on the weight of the available evidence, for the majority of critically ill patients in the ICU setting, insulin infusion should be used to control hyperglycemia, with a starting threshold of no higher than 180 mg\/dl (10.0 mmol\/l). Once intravenous insulin is started, the glucose level should be maintained between 140 and 180 mg\/dl (7.8 and 10.0 mmol\/l). Greater benefit maybe realized at the lower end of this range. Although strong evidence is lacking, somewhat lower glucose targets may be appropriate in selected patients. However, targets less than 110 mg\/dl (6.1 mmol\/l) are not recommended. Use of insulin infusion protocols with demonstrated safety and efficacy, resulting in low rates of hypoglycemia, are highly recommended (346).\n\n## Noncritically ill patients\n\nWith no prospective RCT data to inform specific glycemic targets in noncritically ill patients, recommendations are based on clinical experience and judgment. For the majority of noncritically ill patients treated with insulin, premeal glucose targets should generally be \\<140 mg\/dl (7.8 mmol\/l) with random blood glucose levels \\<180 mg\/dl (10.0 mmol\/l), as long as these targets can be safely achieved. To avoid hypoglycemia, consideration should be given to reassessing the insulin regimen if blood glucose levels fall below 100 mg\/dl (5.6 mmol\/l). Modification of the regimen is required when blood glucose values are \\<70 mg\/dl (3.9 mmol\/l), unless the event is easily explained by other factors (such as a missed meal, etc.)\n\nOccasional patients with a prior history of successful tight glycemic control in the outpatient setting who are clinically stable may be maintained with a glucose range below the above cut points. Conversely, higher glucose ranges may be acceptable in terminally ill patients or in patients with severe comorbidities, as well as in those in patient-care settings where frequent glucose monitoring or close nursing supervision is not feasible.\n\nClinical judgment, combined with ongoing assessment of the patient's clinical status, including changes in the trajectory of glucose measures, the severity of illness, nutritional status, or concurrent use of medications that might affect glucose levels (e.g., steroids, octreotide) must be incorporated into the day-to-day decisions regarding insulin dosing (346).\n\n## 2. Anti-hyperglycemic agents in hospitalized patients\n\nIn the hospital setting, insulin therapy is the preferred method of glycemic control in majority of clinical situations (346). In the ICU, intravenous infusion is the preferred route of insulin administration. When the patient is transitioned off intravenous insulin to subcutaneous therapy, precautions should be taken to prevent hyperglycemia escape (360,361). Outside of critical care units, scheduled subcutaneous insulin which delivers basal, nutritional, and correction (supplemental) components is preferred. Prolonged therapy with sliding scale insulin (SSI) as the sole regimen is ineffective in the majority of patients, increases risk of both hypoglycemia and hyperglycemia, and has recently been shown to be associated with adverse outcomes in general surgery patients with type 2 diabetes (362). SSI is potentially dangerous in type 1 diabetes (346). The reader is referred to several recent publications and reviews that describe currently available insulin preparations and protocols and provide guidance in use of insulin therapy in specific clinical settings including parenteral nutrition (363), enteral tube feedings, and with high-dose glucocorticoid therapy (346).\n\nThere are no data on the safety and efficacy of oral agents and injectable noninsulin therapies such as GLP1 analogs and pramlintide in the hospital. They are generally considered to have a limited role in the management of hyperglycemia in conjunction with acute illness. Continuation of these agents may be appropriate in selected stable patients who are expected to consume meals at regular intervals and they may be initiated or resumed in anticipation of discharge once the patient is clinically stable. Specific caution is required with metformin, due to the possibility that a contraindication may develop during the hospitalization, such as renal insufficiency, unstable hemodynamic status, or need for an imaging study that requires a radio-contrast dye.\n\n## 3. Preventing hypoglycemia\n\nHypoglycemia, especially in insulin-treated patients, is the leading limiting factor in the glycemic management of type 1 and type 2 diabetes (173). In the hospital, multiple additional risk factors for hypoglycemia are present. Patients with or without diabetes may experience hypoglycemia in the hospital in association with altered nutritional state, heart failure, renal or liver disease, malignancy, infection, or sepsis. Additional triggering events leading to iatrogenic hypoglycemia include sudden reduction of corticosteroid dose, altered ability of the patient to report symptoms, reduction of oral intake, emesis, new NPO status, inappropriate timing of short- or rapid-acting insulin in relation to meals, reduction of rate of administration of intravenous dextrose, and unexpected interruption of enteral feedings or parenteral nutrition.\n\nDespite the preventable nature of many inpatient episodes of hypoglycemia, institutions are more likely to have nursing protocols for the treatment of hypoglycemia than for its prevention. Tracking such episodes and analyzing their causes are important quality improvement activities (346).\n\n## 4. Diabetes care providers in the hospital\n\nInpatient diabetes management may be effectively championed and\/or provided by primary care physicians, endocrinologists, intensivists, or hospitalists. Involvement of appropriately trained specialists or specialty teams may reduce length of stay, improve glycemic control, and improve outcomes (346). In the care of diabetes, implementation of standardized order sets for scheduled and correction-dose insulin may reduce reliance on sliding-scale management. As hospitals move to comply with \"meaningful use\" regulations for electronic health records, as mandated by the Health Information Technology Act, efforts should be made to assure that all components of structured insulin order sets are incorporated into electronic insulin order sets (364,365).\n\nA team approach is needed to establish hospital pathways. To achieve glycemic targets associated with improved hospital outcomes, hospitals will need multidisciplinary support to develop insulin management protocols that effectively and safely enable achievement of glycemic targets (366).\n\n## 5. Self-management in the hospital\n\nSelf-management of diabetes in the hospital may be appropriate for competent adult patients who: have a stable level of consciousness, have reasonably stable daily insulin requirements, successfully conduct self-management of diabetes at home, have physical skills needed to successfully self-administer insulin and perform SMBG, have adequate oral intake, and are proficient in carbohydrate counting, use of multiple daily insulin injections or insulin pump therapy, and sick-day management. The patient and physician, in consultation with nursing staff, must agree that patient self-management is appropriate under the conditions of hospitalization.\n\nPatients who use CSII pump therapy in the outpatient setting can be candidates for diabetes self-management in the hospital, provided that they have the mental and physical capacity to do so (346). A hospital policy and procedures delineating inpatient guidelines for CSII pump therapy are advisable. The availability of hospital personnel with expertise in CSII therapy is essential. It is important that nursing personnel document basal rates and bolus doses taken on a regular basis (at least daily).\n\n## 6. DSME in the hospital\n\nTeaching diabetes self-management to patients in hospitals is a challenging task. Patients are ill, under increased stress related to their hospitalization and diagnosis, and in an environment not conducive to learning. Ideally, people with diabetes should be taught at a time and place conducive to learning\u2014as an outpatient in a recognized program of diabetes education.\n\nFor the hospitalized patient, diabetes \"survival skills\" education is generally a feasible approach. Patients and\/or family members receive sufficient information and training to enable safe care at home. Those newly diagnosed with diabetes or who are new to insulin and\/or blood glucose monitoring need to be instructed before discharge. Those patients hospitalized because of a crisis related to diabetes management or poor care at home need education to prevent subsequent episodes of hospitalization. An assessment of the need for a home health referral or referral to an outpatient diabetes education program should be part of discharge planning for all patients.\n\n## 7. MNT in the hospital\n\nThe goals of MNT are to optimize glycemic control, to provide adequate calories to meet metabolic demands, and to create a discharge plan for follow-up care (345,367). ADA does not endorse any single meal plan or specified percentages of macronutrients, and the term \"ADA diet\" should no longer be used. Current nutrition recommendations advise individualization based on treatment goals, physiologic parameters, and medication usage. Consistent carbohydrate meal plans are preferred by many hospitals since they facilitate matching the prandial insulin dose to the amount of carbohydrate consumed (368). Because of the complexity of nutrition issues in the hospital, a registered dietitian, knowledgeable and skilled in MNT, should serve as an inpatient team member. The dietitian is responsible for integrating information about the patient's clinical condition, eating, and lifestyle habits and for establishing treatment goals in order to determine a realistic plan for nutrition therapy (369,370).\n\n## 8. Bedside blood glucose monitoring\n\nPoint-of-care (POC) blood glucose monitoring performed at the bedside is used to guide insulin dosing. In the patient who is receiving nutrition, the timing of glucose monitoring should match carbohydrate exposure. In the patient who is not receiving nutrition, glucose monitoring is performed every 4 to 6 h (371,372). More frequent blood glucose testing ranging from every 30 min to every 2 h is required for patients on intravenous insulin infusions.\n\nSafety standards should be established for blood glucose monitoring, prohibiting sharing of fingerstick lancing devices, lancets, and needles to reduce the risk of transmission of blood borne diseases. Shared lancing devices carry essentially the same risk as shared syringes and needles (373).\n\nAccuracy of blood glucose measurements using POC meters has limitations that must be considered. Although the FDA allows a \u00b120% error for blood glucose meters, questions about the appropriateness of these criteria have been raised (388). Glucose measures differ significantly between plasma and whole blood, terms that are often used interchangeably and can lead to misinterpretation. Most commercially available capillary blood glucose meters introduce a correction factor of \u223c1.12 to report a \"plasma adjusted\" value (374).\n\nSignificant discrepancies between capillary, venous, and arterial plasma samples have been observed in patients with low or high hemoglobin concentrations, hypoperfusion, and the presence of interfering substances particularly maltose, as contained in immunoglobulins (375). Analytical variability has been described with several POC meters (376). Increasingly, newer generation POC blood glucose meters correct for variation in hematocrit and for interfering substances. Any glucose result that does not correlate with the patient's status should be confirmed through conventional laboratory sampling of plasma glucose. The FDA has become increasingly concerned about the use of POC blood glucose meters in the hospital and is presently reviewing matters related to their use.\n\n## 9. Discharge planning\n\nTransition from the acute care setting is a high-risk time for all patients, not just those with diabetes or new hyperglycemia. Although there is an extensive literature concerning safe transition within and from the hospital, little of it is specific to diabetes (377). It is important to remember that diabetes discharge planning is not a separate entity, but part of an overall discharge plan. As such, discharge planning begins at admission to the hospital and is updated as projected patient needs change.\n\nInpatients may be discharged to varied settings, including home (with or without visiting nurse services), assisted living, rehabilitation, or skilled nursing facilities. The latter two sites are generally staffed by health professionals; therefore diabetes discharge planning will be limited to communication of medication and diet orders. For the patient who is discharged to assisted living or to home, the optimal program will need to consider the type and severity of diabetes, the effects of the patient's illness on blood glucose levels, and the capacities and desires of the patient. Smooth transition to outpatient care should be ensured. The Agency for Healthcare Research and Quality recommends that at a minimum, discharge plans include:\n\n-   Medication reconciliation: The patient's medications must be cross-checked to ensure that no chronic medications were stopped and to ensure the safety of new prescriptions. Whenever possible, prescriptions for new or changed medication should be filled and reviewed with the patient and family at or before discharge\n\n-   Structured discharge communication: Information on medication changes, pending tests and studies, and follow-up needs must be accurately and promptly communicated to outpatient physicians, as soon as possible after discharge.\n\nIdeally the inpatient care providers or case managers\/discharge planners will schedule follow-up visit(s) with the appropriate professionals, including primary care provider, endocrinologist, and diabetes educator (378).\n\nAn outpatient follow-up visit with the primary care provider, endocrinologist, or diabetes educator within 1 month of discharge is advised for all patients having hyperglycemia in the hospital. Clear communication with outpatient providers either directly or via hospital discharge summaries facilitates safe transitions to outpatient care. Providing information regarding the cause or the plan for determining the cause of hyperglycemia, related complications and comorbidities, and recommended treatments can assist outpatient providers as they assume ongoing care.\n\nIt is important that patients be provided with appropriate durable medical equipment, medication, supplies, and prescriptions at the time of discharge in order to avoid a potentially dangerous hiatus in care. These supplies\/prescriptions should include:\n\n-   Insulin (vials or pens) (if needed)\n\n-   Syringes or pen needles (if needed)\n\n-   Oral medications (if needed)\n\n-   Blood glucose meter and strips\n\n-   Lancets and lancing device\n\n-   Urine ketone strips (type 1)\n\n-   Glucagon emergency kit (insulin-treated)\n\n-   Medical alert application\/charm\n\n# IX. STRATEGIES FOR IMPROVING DIABETES CARE\n\nThere has been steady improvement in the proportion of diabetic patients achieving recommended levels of A1C, blood pressure, and LDL cholesterol in the last 10 years, both in primary care settings and in endocrinology practices. Mean A1C nationally has declined from 7.82% in 1999\u20132000 to 7.18% in 2004 based on National Health and Nutrition Examination Survey (NHANES) data (379). This has been accompanied by improvements in lipids and blood pressure control and led to substantial reductions in end-stage microvascular complications in those with diabetes (380). Nevertheless, in some studies only 57.1% of adults with diagnosed diabetes achieved an A1C of \\<7%, only 45.5% had a blood pressure \\<130\/80 mmHg, and just 46.5% had a total cholesterol \\<200 mg\/dl, with only 12.2% of people with diabetes achieving all three treatment goals (381). Moreover, there is persistent variation in quality of diabetes care across providers and across practice settings even after adjusting for patient factors that indicates the potential for substantial further improvements in diabetes care.\n\nWhile numerous interventions to improve adherence to the recommended standards have been implemented, a major contributor to suboptimal care is a delivery system that too often is fragmented, lacks clinical information capabilities, often duplicates services, and is poorly designed for the delivery of chronic care. The Chronic Care Model (CCM) includes six core elements for the provision of optimal care of patients with chronic disease: *1*) delivery system design (moving from a *reactive* to a *proactive* care delivery system, where planned visits are coordinated through a team-based approach; *2*) self-management support; *3*) decision support (basing care on consistent, effective care guidelines); *4*) clinical information systems (using registries that can provide patient-specific and population-based support to the care team); *5*) community resources and policies (identifying or developing resources to support healthy lifestyles); and *6*) health systems (to create a quality-oriented culture). Alterations in reimbursement that reward the provision of quality care, as defined by the attainment of evidence-based quality measures, will also be required to achieve desired outcome goals. Redefinition of the roles of the clinic staff and promoting self-management on the part of the patient are fundamental to the successful implementation of the CCM (382). Collaborative, multidisciplinary teams are best suited to provide such care for people with chronic conditions like diabetes and to facilitate patients' performance of appropriate self-management.\n\nA rapidly evolving literature suggests that there are three major strategies to successfully improve the quality of diabetes care delivered by a team of providers. NDEP maintains an online resource ([www.betterdiabetescare.nih.gov](http:\/\/www.betterdiabetescare.nih.gov)) to help health care professionals design and implement more effective health care delivery systems for those with diabetes.\n\nThree specific objectives, with references to literature that outlines practical strategies to achieve each, are outlined below.\n\n## Objective 1\n\nProvider and team behavior change: Facilitate timely and appropriate intensification of lifestyle and\/or pharmaceutical therapy of patients who have not achieved beneficial levels of blood pressure, lipid, or glucose control.\n\n-   Clinical information systems including registries that can prospectively identify and track those requiring assessments and\/or treatment modifications by the team.\n\n-   Electronic medical record-based clinical decision support at the point of care, both personalize and standardize care and can be used by multiple providers (383).\n\n-   Use of checklists and\/or flow sheets that mirror guidelines.\n\n-   Detailed treatment algorithms enabling multiple team members to \"treat to target\" and appropriately intensify therapy.\n\n-   Availability of care or disease management services (384) by nurses, pharmacists, and other providers using detailed algorithms often catalyzing reduction in A1C, blood pressure, and LDL cholesterol (385,386).\n\n## Objective 2\n\nPatient behavior change: Implement a systematic approach to support patients' behavior change efforts as needed including *1*) healthy lifestyle (physical activity, healthy eating, nonuse of tobacco, weight management, effective coping, medication taking and management); *2*) prevention of diabetes complications (screening for eye, foot, and renal complications; immunizations); and *3*) achievement of appropriate blood pressure, lipid, and glucose goals.\n\n-   Delivery of high-quality DSME, which has been shown to improve patient self-management, satisfaction, and glucose control (115,387).\n\n-   Delivery of ongoing diabetes self-management support (DSMS) to ensure that gains achieved during DSME are sustained (128\u2013129). National DSME standards call for an integrated approach that includes clinical content and skills, behavioral strategies (goal-setting, problem solving), and addressing emotional concerns in each needed curriculum content area. Provision of continuing education and support (DSMS) improves maintenance of gains regardless of the educational methodology (89).\n\n-   Provision of automated reminders via multiple communication channels to various subgroups of diabetic patients (96).\n\n## Objective 3\n\nChange the system of care: Research on the comprehensive CCM suggests additional strategies to improve diabetes care, including the following:\n\n-   Basing care on consistent, evidence-based care guidelines\n\n-   Redefining and expanding the roles of the clinic staff (382)\n\n-   Collaborative, multidisciplinary teams to provide high-quality care and support patients' appropriate self-management\n\n-   Audit and feedback of process and outcome data to providers to encourage population-based care improvement strategies\n\n-   Care management, one of the most effective diabetes quality improvement strategies to improve glycemic control (384).\n\n-   Identifying and\/or developing community resources and public policy that support healthy lifestyles\n\n-   Alterations in reimbursement that reward the provision of appropriate and high-quality care and accommodate the need to personalize care goals, providing additional incentives to improve diabetes care (382,388\u2013392)\n\nThe most successful practices have an institutional priority for quality of care, expanding the role of teams and staff, redesigning their delivery system, activating and educating their patients, and using electronic health record tools (393,394). Recent initiatives such as the Patient Centered Medical Home show promise in improving outcomes through coordinated primary care and offer new opportunities for team-based chronic disease care (395).\n\nIt is clear that optimal diabetes management requires an organized, systematic approach and involvement of a coordinated team of dedicated health care professionals working in an environment where patient-centered high-quality care is a priority.\n\n# References","meta":{"dup_signals":{"dup_doc_count":218,"dup_dump_count":70,"dup_details":{"curated_sources":2,"2023-40":1,"2022-40":1,"2022-27":1,"2022-21":1,"2022-05":3,"2021-49":3,"2021-43":3,"2021-39":2,"2021-31":7,"2021-25":7,"2021-21":10,"2021-17":5,"2021-10":1,"2021-04":2,"2020-50":7,"2020-45":5,"2020-40":2,"2020-34":1,"2020-29":5,"2020-24":1,"2020-16":4,"2020-10":2,"2020-05":3,"2019-51":6,"2019-47":3,"2019-43":8,"2019-39":1,"2019-35":2,"2019-30":3,"2019-26":4,"2019-22":1,"2019-18":2,"2019-09":2,"2019-04":1,"2018-51":1,"2018-47":2,"2018-43":2,"2018-39":1,"2018-34":2,"2018-30":2,"2018-26":2,"2018-22":1,"2018-17":3,"2018-13":1,"2018-09":1,"2018-05":3,"2017-51":2,"2017-47":4,"2017-43":2,"2017-39":6,"2017-34":3,"2017-30":6,"2017-26":6,"2017-22":3,"2017-17":6,"2017-09":4,"2017-04":5,"2016-50":5,"2016-44":5,"2016-40":4,"2016-36":4,"2016-30":2,"2016-26":2,"2016-22":3,"2014-41":1,"2023-50":1,"2017-13":7,"2014-10":1,"2013-48":2,"2024-30":1}},"file":"PMC3006050"},"subset":"pubmed_central"}
{"text":"author: Ferid Murad\ndate: 2012\ninstitute: 1George Washington University, Washington, D.C., USA\ntitle: Discovery of nitric oxide and cyclic GMP in cell signaling and their role in drug development\n\nThe role of nitric oxide in cellular signaling in the past three decades has become one of the most rapidly growing areas in biology. Nitric oxide is a gas and a free radical with an unshared electron that can regulate an ever-growing list of biological processes. Nitric oxide is formed from L-arginine by a family of enzymes called nitric oxide synthases. These enzymes have a complex requirement for a number of cofactors and regulators including NADPH, tetrahydrobioterin, flavins, calmodulin and heme. The enzymes are present in most cells and tissues. In many instances, nitric oxide mediates its biological effects by activating the soluble isoform of guanylyl cyclase and increasing cyclic GMP synthesis from GTP. Cyclic GMP, in turn, can activate cyclic GMP-dependent protein kinase (PKG) and can cause smooth muscles and blood vessels to relax, decrease platelet aggregation, alter neuron function, etc. These effects can decrease blood pressure, increase blood flow to tissues, alter memory and behavior, decrease blood clotting, etc. The list of effects of nitric oxide that are independent of cyclic GMP formation is also growing at a rapid rate. For example, nitric oxide can interact with transition metals such as iron, thiol groups, other free radicals, oxygen, superoxide anion, unsaturated fatty acids, and other molecules. Some of these reactions result in the oxidation of nitric oxide to nitrite and nitrate to terminate the effect, while other reactions can lead to altered protein structure function and\/or catalytic capacity. These effects probably regulate bacterial infections, inflammation of tissues, tumor growth, and other disorders. These diverse effects of nitric oxide that are cyclic GMP dependent or independent can alter and regulate numerous important physiological events in cell regulation and function. Nitric oxide can function as an intracellular messenger, an antacoid, a paracrine substance, a neurotransmitter, or as a hormone that can be carried to distant sites for effects. Thus, it is a unique molecule with an array of signaling functions. However, with any messenger molecule, there can be too little or too much of the substance, resulting in pathological events. Some of the methods to regulate either nitric oxide formation metabolism, or function have been in clinical use for more than a century, as with the use of organic nitrates and nitroglycerin in angina pectoris that was initiated in the 1870s. Inhalation of low concentrations of nitric oxide can be beneficial in premature infants with pulmonary hypertension and increase survival rates. Ongoing clinical trials with nitric oxide synthase inhibitors and nitric oxide scavengers are examining the effects of these agents in septic shock, hypotension with dialysis, inflammatory disorders, cancer therapy, etc. Recognition of additional molecular targets in the areas of nitric oxide and cyclic GMP research will continue to promote drug discovery and development programs in the field. Current and future research will undoubtedly expand the clinician's therapeutic armamentarium to manage a number of important diseases by perturbing nitric oxide formation and metabolism. Such promise and expectations have obviously fueled the interests in nitric oxide research for a growing list of potential therapeutic applications. There have been and will continue to be many opportunities from nitric oxide and cyclic GMP march to develop novel and important therapeutic agents. There are presently more than 80,000 publications in the area of nitric oxide research. The lecture will discuss our discovery of the first biological effects of nitric oxide and how the field has evolved since our original reports in 1977. The possible utility of this signaling pathway to facilitate novel drug development and the creation of numerous projects in the Pharmaceutical and biotechnology industrials will also be discussed.","meta":{"dup_signals":{"dup_doc_count":168,"dup_dump_count":54,"dup_details":{"curated_sources":4,"2023-50":2,"2023-40":1,"2023-23":4,"2023-14":4,"2023-06":1,"2022-49":2,"2022-40":1,"2022-33":1,"2021-49":1,"2021-43":1,"2021-25":1,"2020-45":1,"2020-40":2,"2020-34":2,"2020-24":2,"2020-10":1,"2019-51":2,"2019-43":2,"2019-39":2,"2019-35":2,"2019-30":2,"2019-26":3,"2019-22":1,"2019-18":6,"2019-13":2,"2019-04":8,"2018-47":8,"2018-39":7,"2018-30":11,"2018-26":5,"2018-22":2,"2018-17":1,"2018-13":3,"2018-09":1,"2018-05":2,"2017-51":1,"2017-47":2,"2017-43":1,"2017-39":3,"2017-34":1,"2017-30":3,"2017-26":2,"2017-22":3,"2017-17":3,"2017-09":2,"2017-04":1,"2016-50":1,"2016-44":2,"2024-30":3,"2024-26":2,"2024-22":1,"2024-18":4,"2024-10":21,"2017-13":11}},"file":"PMC3287594"},"subset":"pubmed_central"}
{"text":"abstract: A report on work presented at the 39th Annual Meeting of the American Society for Cell Bilogy, Washington DC, December 11-15, 1999\n  .\n  Conference website: <http:\/\/www.faseb.org\/ascb\/meetings\/am99\/main99mtg.htm>\nauthor: Janet Rossant\ndate: 2000\ninstitute: 1Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, M5G 1X5, Canada\ntitle: How will knowing genome sequences affect the life of the average cell biologist?\n\nThe impact of 'the genome project' on all aspects of biomedical research in the next century cannot be underestimated. We will know the complete sequence of the human genome and of the genomes of an ever-increasing number of model organisms, providing masses of new raw genetic data to sustain the study of any particular cellular process or pathway. But perhaps more importantly, the tools and mind-set of the genomicist seem destined to alter the way we all think about and practise our science. Once we know the complete set of genes that exist in an organism, it will no longer be acceptable to focus on one gene at a time, to study its effects in terms of a read-out consisting of a few other related genes and cellular events, and then to conclude that we have understood its function. Conversely, if we want to understand a cellular process, we will need somehow to integrate all the molecular interactions that occur, rather than being satisfied with cataloguing the few major pathway components identified by genetic or biochemical screens. Genomics, and its newer siblings functional genomics, proteomics and bioinformatics, are teaching us all to think in a high-throughput, genome-wide manner and are providing the technologies to allow us to translate these thoughts into action.\n\nThe pace of technological progress in this area can be intimidating, to the established investigator and the new graduate student alike. No wonder, then, that there was a packed house at the opening symposium - entitled 'The Impact of Genome-wide Studies on Cell and Developmental Biology' - of the 39th American Society for Cell Biology meeting. David Botstein (Stanford University Medical School) began by emphasizing the importance of model organisms and comparative genomics in assigning meaning to the sequence of As, Ts, Cs and Gs that will make up the final product of the Human Genome Project. The complete sequence of the genome of the yeast *Saccharomyces cerevisiae* has been available since 1996, and that of the worm *Caenorhabditis elegans* since 1998. As detailed by Gerry Rubin (University of California Berkeley), 'version one' of the 185 Mb *Drosophila* genome is also complete, as a result of a collaboration between academic centers in the United States and Europe and the company Celera Genomics. A draft sequence of the human genome is expected to follow shortly, with the complete sequence due to be finished by 2003 at the latest.\n\nWith the sequence information in the databases to date, it is already possible to query the genome in interesting ways. Botstein pointed out that comparison of yeast and worm genomes has identified a set of conserved genes encoding core biological functions, such as metabolism, DNA replication and protein trafficking. These genes make up 40% of the yeast and 20% of the worm genome. Alongside this core set of proteins - which are expected to be common to single-cell and multicellular eukaryotes - worms, flies and humans have developed a complex set of genes involved in signal transduction and gene regulation, in order to accomplish their elaborate developmental programs. These pathways show less absolute conservation of gene sequence, but general classes of proteins and protein domains can be recognized across evolution.\n\nThe extent of cross-species conservation across the whole range of gene function is, in fact, remarkable. Of the human genes known to date, 74% have related sequences in the worm. Cori Bargmann (University of California, San Francisco) illustrated how inter- and intra-species sequence comparisons of gene families can confirm conserved functions and identify new ones, by scanning the *C. elegans* genome for genes encoding ion channels and G-protein-coupled receptors. There is no voltage-activated sodium channel identifiable in the *C. elegans* genome, consistent with the results of electrophysiological studies, but voltage-regulated calcium and potassium channels are found, some similar to vertebrate counterparts and some divergent. Clearly, some aspects of cell excitability are common and fundamental and some are subject to evolutionary pressure related to the adaptation of the organism to its particular environment. Despite the extensive prior mutational analysis of the worm, the genome sequence has thrown up many genes that were not identified by mutation. For example, Bargmann's lab cloned one odorant receptor gene, *Od-10*, through mutational analysis. It encodes a novel G-protein-coupled receptor. Analysis of the genome sequence revealed about 1,000 'orphan' G-protein-coupled receptors, many of which appear to be related to Od-10 and which probably represent more odorant receptors. Overnight, then, a lab working on one gene finds itself working on hundreds.\n\nThe need for systematic, genome-wide methods for assigning gene function to this deluge of sequence information was a common theme of the symposium. Annotation by sequence similarity is the usual first step, but it is difficult and can be misleading. Genes of similar sequence may have acquired new functions during evolution. For example, mutational analysis of a *Drosophila* gene whose sequence suggested that it could be a chitinase enzyme revealed instead that it was a novel growth factor (Rubin). Developing a common language for gene annotation across different databases is one of the steps needed to improve the situation. This is the goal of the Gene Ontology (GO) project, a collaborative effort between workers on the yeast, fly and mouse genome databases. But even a common language and more sophisticated sequence analysis tools are not likely yet to replace the need for the seasoned eye of the expert biologist when interpreting genome sequence. The publication of the *Drosophila* genome sequence depends on the completion of a major effort to annotate all the genes identified in the sequence to date, by a group of biologists expert in different gene families, working alongside bioinformatics experts.\n\nMuch more information about gene function can, of course, be gleaned from knowing expression patterns and the effects of loss or gain of function. Genome-wide initiatives in assessing expression and function are underway for all model organisms. For example, an assay of the function of all *C. elegans* genes, using RNA interference, for their effects on early cell division processes is under way at the European Molecular Biology Laboratory (poster presented by Pierre G\u00f6nczy). The Berkeley *Drosophila* Genome Project is surveying the expression of all *Drosophila* genes by whole-mount *in situ* hybridization in embryos and attempting a catalog of gene mutations by accumulating insertions of P elements or Gal4 activation domains into as many different sites in the genome as possible (Rubin). Major efforts like this in a few centers can provide enormous resources for the average cell or developmental biologist who wishes to pursue more detailed analysis of an individual gene or process of interest. Rubin noted that there are an estimated 15,000 *Drosophila* genes and 5,000 active *Drosophila* researchers, or three genes per biologist for future study. The general philosophy of the various academic genome initiatives - to share all information freely and rapidly via the internet - must surely apply to these next phases of large-scale functional genomics as well. A familiarity with database resources and the tools for database mining will therefore be essential in allowing biologists to gain the most from all the material made available by large-scale projects.\n\nThere are growing numbers of genome research applications, however, which the average biology lab will want to use directly for its own studies. None has generated more excitement than DNA microarrays or 'chips'. The cDNAs from all of the 6,000 or so yeast genes, a large proportion of the *Drosophila* and worm genes, and an increasing number of human and mouse genes, have been assembled onto glass microarrays in different labs world-wide. Hybridization analysis, combined with sophisticated clustering algorithms to analyse the results, allows analysis of gene expression changes in normal or abnormal states in unprecedented detail. Botstein has been a prime mover in validating this technology, along with his Stanford colleague, Pat Brown. Botstein described how yeast microarrays allow the identification of clusters of co-regulated genes whose expression changes together under a variety of different conditions. Because all the yeast genes are on a chip, complete sets of co-regulated genes can now be identified and studied. In larger organisms, microarrays are currently still sampling the genome, but a sample size of several thousand genes is sufficiently large to reveal unexpected patterns of co-regulated genes. Furthermore, microarrays are already finding utility in classifying tumor samples. 'Signature' patterns of gene expression can be discerned when many different tumor samples are compared, despite the potential heterogeneity imposed during sample collection (Botstein). These patterns can be used to define subclasses of tumors that are not apparent by more standard means. The possible applications of this technology to all aspects of biology seem bound to grow exponentially as the technology becomes more widely disseminated.\n\nWe are entering an exciting era, when genomes and genomics will provide the basis for a new form of truly integrative biological research. We cell and developmental biologists, who already think in integrative ways as a result of the questions our research addresses, need to be in the vanguard of this new biology.","meta":{"dup_signals":{"dup_doc_count":104,"dup_dump_count":47,"dup_details":{"curated_sources":2,"2023-14":1,"2022-21":1,"2021-49":1,"2021-04":1,"2020-34":1,"2020-24":1,"2019-47":2,"2019-26":1,"2019-13":1,"2019-04":2,"2018-47":2,"2018-39":2,"2018-34":1,"2018-30":2,"2018-22":1,"2018-17":1,"2018-09":2,"2018-05":1,"2017-39":1,"2017-30":1,"2017-22":1,"2017-09":1,"2017-04":1,"2016-07":1,"2015-48":3,"2015-40":3,"2015-35":3,"2015-32":3,"2015-27":3,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":3,"2014-42":7,"2014-41":5,"2014-35":4,"2014-23":4,"2014-15":4,"2023-50":1,"2017-13":1,"2015-18":3,"2015-11":3,"2015-06":3,"2014-10":3,"2013-48":3,"2013-20":3,"2024-22":1}},"file":"PMC138821"},"subset":"pubmed_central"}
{"text":"date: 2017-04\ntitle: WHO PUBLISHES LIST OF BACTERIA FOR WHICH NEW ANTIBIOTICS ARE URGENTLY NEEDED\n\n**27 FEBRUARY 2017 \\| GENEVA** - WHO today published its first ever list of antibiotic-resistant \"priority pathogens\" \u2013 a catalogue of 12 families of bacteria that pose the greatest threat to human health.\n\nThe list was drawn up in a bid to guide and promote research and development (R&D) of new antibiotics, as part of WHO's efforts to address growing global resistance to antimicrobial medicines.\n\nThe list highlights in particular the threat of gram-negative bacteria that are resistant to multiple antibiotics. These bacteria have built-in abilities to find new ways to resist treatment and can pass along genetic material that allows other bacteria to become drug-resistant as well.\n\n\"This list is a new tool to ensure R&D responds to urgent public health needs,\" says Dr Marie-Paule Kieny, WHO's Assistant Director-General for Health Systems and Innovation. \"Antibiotic resistance is growing, and we are fast running out of treatment options. If we leave it to market forces alone, the new antibiotics we most urgently need are not going to be developed in time.\"\n\nThe WHO list is divided into three categories according to the urgency of need for new antibiotics: critical, high and medium priority.\n\nThe most critical group of all includes multidrug resistant bacteria that pose a particular threat in hospitals, nursing homes, and among patients whose care requires devices such as ventilators and blood catheters. They include Acinetobacter, Pseudomonas and various Enterobacteriaceae (including Klebsiella, E. coli, Serratia, and Proteus). They can cause severe and often deadly infections such as bloodstream infections and pneumonia.\n\nThese bacteria have become resistant to a large number of antibiotics, including carbapenems and third generation cephalosporins \u2013 the best available antibiotics for treating multi-drug resistant bacteria.\n\nThe second and third tiers in the list \u2013 the high and medium priority categories \u2013 contain other increasingly drug-resistant bacteria that cause more common diseases such as gonorrhoea and food poisoning caused by salmonella.\n\nG20 health experts will meet this week in Berlin. Mr Hermann Gr\u00f6he, Federal Minister of Health, Germany says \"We need effective antibiotics for our health systems. We have to take joint action today for a healthier tomorrow. Therefore, we will discuss and bring the attention of the G20 to the fight against antimicrobial resistance. WHO's first global priority pathogen list is an important new tool to secure and guide research and development related to new antibiotics.\"\n\nThe list is intended to spur governments to put in place policies that incentivize basic science and advanced R&D by both publicly funded agencies and the private sector investing in new antibiotic discovery. It will provide guidance to new R&D initiatives such as the WHO\/Drugs for Neglected Diseases initiative (DNDi) Global Antibiotic R&D Partnership that is engaging in not-for-profit development of new antibiotics.\n\nTuberculosis \u2013 whose resistance to traditional treatment has been growing in recent years \u2013 was not included in the list because it is targeted by other, dedicated programmes. Other bacteria that were not included, such as streptococcus A and B and chlamydia, have low levels of resistance to existing treatments and do not currently pose a significant public health threat.\n\nThe list was developed in collaboration with the Division of Infectious Diseases at the University of T\u00fcbingen, Germany, using a multi-criteria decision analysis technique vetted by a group of international experts. The criteria for selecting pathogens on the list were: how deadly the infections they cause are; whether their treatment requires long hospital stays; how frequently they are resistant to existing antibiotics when people in communities catch them; how easily they spread between animals, from animals to humans, and from person to person; whether they can be prevented (e.g. through good hygiene and vaccination); how many treatment options remain; and whether new antibiotics to treat them are already in the R&D pipeline.\n\n\"New antibiotics targeting this priority list of pathogens will help to reduce deaths due to resistant infections around the world,\" says Prof Evelina Tacconelli, Head of the Division of Infectious Diseases at the University of T\u00fcbingen and a major contributor to the development of the list. \"Waiting any longer will cause further public health problems and dramatically impact on patient care.\"\n\nWhile more R&D is vital, alone, it cannot solve the problem. To address resistance, there must also be better prevention of infections and appropriate use of existing antibiotics in humans and animals, as well as rational use of any new antibiotics that are developed in future.\n\n**WHO priority pathogens list for R&D of new antibiotics**\n\n**Priority 1: CRITICAL**\n\n1.  Acinetobacter baumannii, carbapenem-resistant\n\n2.  Pseudomonas aeruginosa, carbapenem-resistant\n\n3.  Enterobacteriaceae, carbapenem-resistant, ESBL-producing\n\n**Priority 2: HIGH**\n\n1.  Enterococcus faecium, vancomycin-resistant\n\n2.  Staphylococcus aureus, methicillin-resistant, vancomycin-intermediate and resistant\n\n3.  Helicobacter pylori, clarithromycin-resistant\n\n4.  Campylobacter spp., fluoroquinolone-resistant\n\n5.  Salmonellae, fluoroquinolone-resistant\n\n6.  Neisseria gonorrhoeae, cephalosporin-resistant, fluoroquinolone-resistant\n\n**Priority 3: MEDIUM**\n\n1.  Streptococcus pneumoniae, penicillin-non-susceptible\n\n2.  Haemophilus influenzae, ampicillin-resistant\n\n3.  Shigella spp., fluoroquinolone-resistant\n\nAvailable from: <http:\/\/www.who.int\/mediacentre\/news\/releases\/2017\/bacteria-antibiotics-needed\/en\/>","meta":{"dup_signals":{"dup_doc_count":177,"dup_dump_count":60,"dup_details":{"curated_sources":4,"2023-50":5,"2023-40":2,"2023-14":5,"2023-06":1,"2022-49":3,"2022-40":1,"2022-33":3,"2022-21":1,"2022-05":2,"2021-43":2,"2021-39":2,"2021-31":1,"2021-25":2,"2021-17":3,"2021-10":1,"2021-04":2,"2020-45":2,"2020-40":3,"2020-34":2,"2020-29":3,"2020-24":1,"2020-16":3,"2020-10":1,"2020-05":2,"2019-51":1,"2019-43":1,"2019-39":2,"2019-35":1,"2019-30":2,"2019-26":2,"2019-18":3,"2019-13":5,"2019-09":2,"2019-04":4,"2018-51":3,"2018-47":4,"2018-43":4,"2018-39":4,"2018-34":3,"2018-30":3,"2018-26":2,"2018-22":3,"2018-17":6,"2018-13":4,"2018-09":2,"2018-05":6,"2017-51":2,"2017-47":4,"2017-43":3,"2017-39":3,"2017-34":3,"2017-30":3,"2017-26":5,"2017-22":4,"2017-17":4,"2024-30":3,"2024-26":3,"2024-18":4,"2024-10":4,"2017-13":8}},"file":"PMC5726826"},"subset":"pubmed_central"}
{"text":"abstract: With new technologies, concerns about gene patent claims regarding isolated DNA are becoming less relevant, but broad method claims could be more problematic.\nauthor: Allison W Dobson; James P Evans\ndate: 2012\ninstitute: 16628 Ridge Run Ct. Clemmons, NC 27012, USA; 2Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7264, USA\nreferences:\ntitle: Gene patents in the US - focusing on what really matters\n\nAs we move into the era of exome and whole-genome sequencing and witness a concurrent explosion in genomic data, concerns about related biotechnology patents must also shift accordingly. These are complex issues that are in a state of flux, and we find ourselves at an especially critical juncture given the US Supreme Court's recent treatment of several high-profile biotechnology cases. Here, we discuss recent developments in US patent law and the most pressing issues on the horizon, including pivotal issues surrounding the topic of patenting in genetics and biotechnology in the US. We particularly address some of the neglected or seemingly arcane points that nevertheless are critical to the researcher, medical practitioner, scientist, or indeed anyone seeking informed opinions about patent protection of new biotechnologies and genetic technologies. Striking the appropriate balance between intellectual property protection and public benefit is essential to promoting advances in the field.\n\nThe term 'gene patent' itself is ambiguous, and this term has been used loosely in the media to encompass a wide variety of patents related to genetics. Patents typically contain multiple claims in various forms, and each claim in a patent may stand or fall independently of the others. The subject matter claimed can be a tangible product (such as a DNA molecule) or a process indicated by gerunds (for example, 'constructing', 'connecting', or 'sequencing') that outline the steps of the method.\n\nBoth product and method claims are found in gene-related patents. Understanding the different claims encompassed by the imprecise term 'gene patent' is critical to the proper interpretation and application of patent law as we seek to optimally promote the development of genetic medicine. Because patent law is complicated (see Box 1 for a brief summary), the popular media understandably tends to avoid going into such differences in their attempts to simplify headlines about the topic \\[1,2\\]. The Supreme Court has recently cast serious doubt on broad method claims related to genes - but not on product claims.\n\nA claim directed to 'isolated DNA' covers a single type of molecule, such as a PCR product, usually referring to a specific DNA sequence. It does not cover the sequence itself, as many professionals seem to believe. Thus, the patent holder is legally entitled to exclude others from making or using only that isolated DNA, based on that particular claim. Depending on how the claim is worded, the court may interpret it to cover isolated molecules with additional sequences that flank the named sequence (such as in a purified plasmid or other vector), but under no circumstances should a claim to isolated DNA preempt others from using its corresponding sequence as it exists within genomic DNA. That is the whole point of using the word 'isolated' - to clarify that the natural occurrence of that sequence is not what is claimed in the patent, but rather the physical molecule.\n\nFor scientists and physicians, the critical question should be whether any given patent stands in the way of their work. Isolated DNA claims are becoming less relevant to medical diagnostics, because sequencing a patient's DNA with modern (next-generation) technology is unlikely to infringe any product claim directed to an isolated DNA molecule. Thus, in a new genomic era, it is appropriate to refocus on broad method claims that have greater potential to affect the practice of medicine and medical research.\n\n# The *Myriad* case: why it is important and where it stands today\n\nAs with many gene patents, the gene patent case *Association for Molecular Pathology et al. v. USTPO et al*. ('*Myriad*') \\[3\\] included both method and product claims, which are affected differently by recent court decisions. On 20 March 2012, a unanimous Supreme Court held in *Prometheus v. Mayo* ('*Prometheus*') \\[4\\] that Prometheus Laboratories' claims to methods of optimizing the dosage of drugs to treat gastrointestinal autoimmune diseases do not qualify as patentable subject matter under section 101 of the Patent Act (the section that specifically deals with what types of subject matter are eligible for patenting) \\[5\\].\n\n*Prometheus* itself is not a gene patent case but has important implications for gene patents, especially claims of the method type. Thus, shortly thereafter, the Supreme Court issued a 'GVR order' for the *Myriad* case \\[3\\] in light of their decision in *Prometheus*. A GVR order means that in one swift motion, the Supreme Court (1) granted review of the *Myriad* case, (2) vacated the decision of the court of appeals below, and (3) remanded the case back to the court of appeals for further review in light of the new Supreme Court decision in *Prometheus*. The justices apparently believe that the court of appeals might find something relevant to the *Myriad* case within their analysis of *Prometheus*. Unfortunately, the patent practitioner community is having a difficult time figuring out what that might be, and therefore a large number of biotechnology patents currently stand in limbo \\[6\\]. One reporter wrote, 'It is apparently possible to split legal hairs endlessly over whether DNA is or is not patentable, and these patent claims will certainly be fought to the bitter end, as they are a crucial test case for future decisions' \\[1\\].\n\nThe Court of Appeals for the Federal Circuit (CAFC) is the US court that hears first appeals on all US patent cases. In March 2010, in the *Myriad* case, a Federal District Court judge invalidated all of Myriad Genetics' challenged gene patents. Myriad appealed to the CAFC, and in July 2011 the CAFC announced its decision. That decision partially reversed that of the District Court on many of Myriad's challenged 'isolated DNA' product claims, finding them to encompass legitimately patentable subject matter under section 101 of the Patent Act.\n\nBut the plaintiffs (the patent challengers) actually won some very important aspects of the case at the CAFC level the first time. Although the CAFC decision upheld many of the product claims at issue, it also affirmed the lower court's invalidation of Myriad's broad method claims. This part of the decision has received little fanfare. A closer look suggests that disproportionately heightened public concern over isolated DNA product claims, although understandable, is misplaced and that we should keep our eye on the right ball - in this case, the outcome of deliberations regarding the eligibility of the method claims.\n\nA representative method claim from Myriad's US Patent No. 6,033,857 is:\n\nClaim 1: A method for identifying a mutant *BRCA2* nucleotide sequence in a suspected mutant *BRCA2* allele which comprises **comparing** the nucleotide sequence of the suspected mutant *BRCA2* allele with the wild-type *BRCA2* nucleotide sequence, wherein a difference between the suspected mutant and the wild-type sequences identifies a mutant *BRCA2* nucleotide sequence. \\[our emphasis\\]\n\nThe CAFC decision rejected these method claims to comparing *BRCA2* sequences on the basis that they recite 'nothing more than abstract mental steps'. The Supreme Court has previously said that the only recognized exceptions for patent eligibility, at the most basic level (section 101), are 'laws of nature, physical phenomena, and abstract ideas' \\[7\\]. Further guidance for determining when claims cover abstract ideas is scant, but methods comprising purely mental steps such as comparing have consistently been found to encompass abstract ideas \\[8\\].\n\nWhen the Supreme Court decided *Prometheus*, they said that the claims in dispute, which describe a process (or method) of adjusting drug dosage based on metabolite levels, essentially monopolized laws of nature. The relevant laws of nature, according to the court, are the 'relationships between concentrations of certain metabolites in the blood and the likelihood that a dosage of a thiopurine drug will prove ineffective or cause harm'. They asked, 'do the patent claims add enough to their statements of the correlations to allow the processes they describe to qualify as patent-eligible processes that apply natural laws?' \\[9\\].\n\nTheir unanimous answer was that in this case, the aspects of the claims related to application of the law of nature were 'not enough'. Supreme Court decisions are supposed to guide the lower judiciary and lawyers' expectations for subsequent cases, but it is unclear just how to determine whether similar method claims might pass muster under this analysis. That is, how can the Supreme Court's reasoning be applied to other patents to reasonably predict how much is enough? Without further guidance from the Supreme Court, method claims regarding specific genes are now in a tenuous position.\n\nMoreover, the Supreme Court's analysis discusses that the steps of the claimed process are 'well understood, routine, conventional activity'. This sounds to the patent community suspiciously close to a line of argument about novelty or obviousness rather than about proper subject matter eligibility \\[10\\]. Alternatively, it might suggest that the Supreme Court is insisting that novelty and obviousness have some relevance to subject matter eligibility. The CAFC has now asked the parties to submit supplemental briefs explaining how the *Prometheus* ruling applies to the *Myriad* claims.\n\n# Isolated DNA molecules\n\nAlthough broad method claims may have the potential for widespread effects on the genetic community, patent claims on any particular isolated DNA molecule, although common, are becoming less relevant in diagnostics because of advancing technology. Next-generation sequencing platforms generally avoid the need to isolate any particular DNA molecule. It is therefore unlikely that sequencing would infringe claims requiring DNA isolation, including a claim to an isolated DNA having any particular sequence. We are not the first to propose that whole-genome sequencing probably infringes patents involving genetic technologies to a lesser extent than widespread beliefs might suggest \\[11\\].\n\nThere is further reason to be less concerned about product claims that focus on isolated DNA. Owing to the dissemination of genetic knowledge and requirements of latter sections of the Patent Act (Box 1), it is becoming more difficult to receive allowance from the patent office on DNA claims. If the sequence has been published, such as in the reference human genome, the corresponding DNA molecules become far less novel and non-obvious; novelty and non-obviousness are both additional requirements for patenting. Moreover, the patents having DNA claims in the *Myriad* case will begin to expire in 2014, and many other patents covering DNA molecules with naturally occurring sequences will likewise expire in the next decade.\n\nThe Supreme Court has stated that Congress intended patentable subject matter to 'include anything under the sun that is made by man' \\[12\\], and it is indisputable that a PCR product fits that description. A gene as it exists in nature, by contrast, is certainly not 'made by man', and enforcement of the Myriad method claim discussed above could arguably monopolize the *BRCA2* gene to some extent. To be fair, holders of such gene patents have generally not used them against basic research activities. One only has to search 'BRCA' in literature databases to realize that Myriad has not asserted its patents against publicly funded basic research endeavors. Nevertheless, these claims have been asserted in the setting of clinical research for a fee by Myriad Genetics, and indiscriminate assertion of such patents is always a possibility.\n\nAlthough granting patent protection related to a naturally occurring gene is both fundamentally problematic to some and increasingly dubious from the standpoint of novelty and non-obviousness to others, the development of new, non-natural DNA sequences through synthetic biology is a field in which full public disclosure in patent applications is highly desirable and warrants rewarding the inventor with a monopoly. So for synthetic DNA technology, it should be permissible to claim a DNA having a truly novel sequence, because there is no DNA with that sequence found in nature.\n\n# More medical diagnostic claims: mental steps and abstract concepts\n\nThe *Myriad* case has received the most attention in recent popular media, and *Prometheus* is now running a close second. But there is a third relevant contemporary biotechnology case involving diagnostic methods claims in which the challenge is focused solely on whether the subject matter is patent eligible. The common thread among these cases is the primary issue argued - that the methods claims are 'abstract'. A judicially created doctrine forbids patenting of abstract principles \\[8\\].\n\nIt can be very challenging for the courts to determine where to draw the line between a purely abstract idea that effectively 'preempts broadly the use of any natural correlation' and a patentable application of that concept, such as a specific biochemical test \\[13\\]. For example, well-known methods used to administer prescription drugs and test blood have been found to involve 'transformations' of body chemistry, which moves the method out of the abstract category \\[14\\].\n\n*Classen Immunotherapies, Inc. v. Biogen Idec* ('*Classen*'), like *Prometheus*, is also not specifically about gene patents but involves conceptually similar method claims \\[13\\]. In this case, the *Classen* patents cover methods for correlating vaccination schedules with later development of immune-mediated diseases. Some of the claims are very broad. In fact, Judge Moore dissented from the other two judges in the ruling, and she read the claims to preempt the scientific method itself. The basic steps of the method are:\n\n\\(1\\) screening information on immunization schedules and the occurrence of chronic immune-mediated disease,\n\n\\(2\\) comparing the results from different vaccination schedules and identifying the lower risk schedule, and\n\n\\(3\\) administering the vaccine on that lower risk schedule.\n\nThe CAFC invalidated some claims that only include steps of screening and comparing vaccination schedules for correlations with later development of immune-mediated disorders. However, once again the 'administering' step takes the claims out of the abstract category according to the majority of judges. The court allowed claims that included an additional step of administering vaccinations, solely because the additional administering step transforms the method from an abstract concept to a patent-eligible application of that concept. The striking effect, as noted by the CAFC, is that a physician might infringe merely by reviewing the literature and then vaccinating on the schedule that seems best to him or her.\n\nA common theme in earlier higher court decisions is that a method claim solely comprising mental steps such as reviewing, comparing, analyzing, screening, selecting, and determining will not stand as eligible to be patented because it is abstract. And even if the claim includes additional non-abstract steps, such as in *Classen* and *Prometheus*, it may not stand if it does not meet the requirements of the rest of the Patent Act, such as novelty and non-obviousness.\n\n# Novelty and non-obviousness\n\nNovelty is a question of whether the technology is really new. Obviousness may be found where the 'subject matter as a whole... was obvious to one of ordinary skill in the art...' at the time of invention \\[15\\]. This is usually an inquiry that involves having experts from each party explain why their party is correct to advise the court on how obvious they think the invention was. This is also a difficult inquiry, but at least the courts are instructed to consider a number of better-defined criteria that come from precedential cases (called *Graham* factors).\n\nThe CAFC and many patent practitioners have expressed frustration over the recent trend of increased reliance on subject matter eligibility arguments (such as whether the patent claim is abstract) rather than novelty or obviousness arguments \\[16\\]. The patent community has long struggled to properly convey this important concept - the sections that follow section 101 (Box 1) are meant to provide the real teeth for eliminating objectionable patents. For example, according to a precedential patent office case (affirmed by the CAFC), a claim to the human DNA for protein \u00d7 is obvious if the mouse DNA for protein \u00d7 was already known in the literature and there was good reason to believe that homologous human DNA existed \\[17\\]. The Supreme Court analysis of *Prometheus* sounds disturbingly similar to an obviousness argument to many patent practitioners \\[10\\]. Thus, it is not clear that the present Supreme Court fully understands the distinctions between section 101 and the sections that follow it, which have historically been preferred over section 101 for finding problematic subject matter unpatentable.\n\nA general tenet of US law holds that, where multiple arguments are presented, the court focuses on the clearest and most determinative argument that can decide the case. In the interest of judiciary efficiency, the court does not consider other more difficult issues. It is much easier for judges to explain a decision that invalidates a claim based on arguments that the invention is obvious or because it has already been described in the literature than decisions based on other issues. The Supreme Court and CAFC both previously tended towards that strategy, to avoid addressing the more difficult and more broadly applicable section 101 subject matter question that is designated to Congress. And Congress does act on that responsibility occasionally - the 2011 America Invents Act includes a prohibition on patents covering human organisms \\[18\\].\n\nThe arguments available under the latter sections of the Patent Act are often much stronger and clearer. In fact, in *Classen* the CAFC took the unusual additional step of outlining exactly how all of the remaining 'administering' claims would have been successfully defeated under patent rules in other parts of the Act. The most glaring issue was that the literature had completely described a system for comparing vaccination schedules over a decade before Classen tried to patent the method, so the invention lacked novelty and would therefore be invalidated if challenged on that theory.\n\nIn addition, in *Classen* the CAFC panel specifically admonishes litigants over the lack of arguments based on other parts of the Patent Act, in three separate opinions. All three judges suggest to varying degrees that all of *Classen*'s claims would be invalid if properly challenged. Indeed, even in the *Myriad* decision the CAFC suggests that arguments under the more stringent sections of the Patent Act would have a much better chance of defeating even the isolated DNA claims.\n\n# Other important factors to consider\n\nEach claim in a patent may stand or fall independently of the other claims. Thus, each claim in a patent must be analyzed separately to determine what it encompasses. The examiners at the patent office are charged to do the same analysis before granting a patent, but as with all human activity, it is inevitable that examiners make mistakes. A safeguard lies in the fact that US patents can be challenged after they are granted on most of the same requirements as for examination.\n\nSecrecy is sometimes an alternative to patenting, but one that conveys less benefit to the public than the protection of technology investment through granting of patents. The public value of the patent system lies in the innovator's duty to fully disclose the patented discovery or invention to the public in return for the limited monopoly granted by a patent. In addition, patents serve to provide security to potential investors in new biotechnologies.\n\nThe possibility that privately funded ventures will increasingly resort to secrecy to protect new DNA technologies is substantial and could frustrate progress overall. Some types of technology will not be feasible to protect through secrecy, and those potential ventures may simply be abandoned for lack of confidence in the ability to protect investment. Thus, it can be dangerous to take an all-or-none approach to the patent system and its effects on progress in medical science. Licensing practices are also important in limiting or exacerbating negative effects of patents. In particular, those involved in technology transfer of federally funded university inventions should be careful in appropriate circumstances to maintain policies against exclusive licensing (\\[19\\] and Catherine Innes, personal communication).\n\nHistory is full of examples in which existing law could not keep up with what was happening on the ground. The Homestead Act of 1862 was an official acknowledgement by the US government of the pioneers' land claims, but people were moving west, staking those claims, and occupying the land before the Homestead Act anyway. Likewise, we are probably witnessing significant patent infringement (with regard to all of the now-tenuous method claims) owing to the reality of next-generation sequencing and its widespread adoption. Science forges ahead despite the more broadly drafted method claims, including the myriad large-scale sequencing efforts (pun intended) currently underway at multiple centers. Such efforts are potentially infringing many method patents that may or may not withstand legal challenge according to the Supreme Court's fuzzy analysis of *Prometheus*. Meanwhile, stakeholders will benefit from considering what activities are truly preempted by specific types of patent claims.\n\nFinally, it is important to remember the central intent of the US patent system, which is not primarily as a generator of revenue but rather to 'promote the progress of science and useful arts'. Broad patent claims covering diagnostic methods should be a greater concern for proponents of unencumbered scientific exploration and medicine than the 'isolated DNA' product claims more commonly associated with the term 'gene patent'. The medical and genetic communities should pay attention to what really matters.\n\n# Competing interests\n\nThe authors declare that they have no competing interests.\n\n# Box 1. A US patent law primer\n\nUS patent law requires that a number of different complex requirements be met before a patent is granted, and the validity of an issued patent may be challenged thereafter on any of the same grounds. The requirements are codified in sections 101, 102, 103, and 112 of USC Title 35, also known as the Patent Act. Section 101 addresses only what types of subject matter are eligible for patenting \\[5\\]; the other sections address issues of novelty, obviousness, and sufficiency of the accompanying descriptions for fully enabling others in the field to make and use the invention or discovery.\n\nHistorically, section 101 has been construed very broadly by the Supreme Court and the US Patent and Trademark Office to include 'anything under the sun made by man' and the other sections have been applied more stringently to filter out subject matter that is unsuitable for patenting (for example, products of nature and abstract concepts). The distinctions between the requirements of these four sections of the Patent Act are the source of much confusion for many non-patent professionals, including scientists, engineers, physicians, and even lawyers and judges who are not specialists in patent law.\n\nA US patent is enforceable only in its home country. However, international agreements such as the Patent Cooperation Treaty provide some degree of uniformity between the patent laws of various countries \\[20\\]. These agreements also provide for easier pursuit of patent protection on a new technology in multiple jurisdictions. Nevertheless, the courts of each country must interpret the particular law of their own jurisdiction, and all of these factors combined yield different end results for patent challenges across the world \\[21,22\\].\n\nThe Court of Appeals for the Federal Circuit (CAFC) is the highest patent specialist court in the US. In fact, this court of appeals has unique authority among all the US courts of appeals. Its decisions are binding precedents throughout the US on patent law matters, whereas the other courts of appeals are binding only for their own geographic jurisdictions. Thus, the only superseding authorities are the Supreme Court and Congress. Furthermore, because Supreme Court review is discretionary, CAFC decisions are often the final word on patent law matters \\[23\\].","meta":{"dup_signals":{"dup_doc_count":112,"dup_dump_count":58,"dup_details":{"curated_sources":4,"2022-40":1,"2021-39":1,"2021-21":1,"2020-45":1,"2020-40":1,"2020-24":1,"2019-47":1,"2019-35":1,"2019-26":1,"2019-13":1,"2019-04":1,"2018-51":1,"2018-47":1,"2018-43":1,"2018-39":1,"2018-34":1,"2018-05":2,"2017-51":2,"2017-47":2,"2017-43":2,"2017-39":2,"2017-34":1,"2017-30":1,"2017-26":1,"2017-22":1,"2017-17":1,"2017-09":8,"2017-04":1,"2016-50":1,"2016-44":1,"2016-40":1,"2016-36":7,"2016-30":6,"2016-22":1,"2016-18":1,"2016-07":6,"2015-48":2,"2015-40":1,"2015-35":2,"2015-32":2,"2015-27":1,"2015-22":2,"2015-14":1,"2014-52":2,"2014-49":2,"2014-42":4,"2014-41":3,"2014-35":3,"2014-23":2,"2014-15":3,"2023-40":1,"2017-13":1,"2015-18":2,"2015-11":2,"2015-06":2,"2014-10":3,"2013-48":2,"2024-22":1}},"file":"PMC3446309"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  Plan\u2013do\u2013study\u2013act (PDSA) cycles provide a structure for iterative testing of changes to improve quality of systems. The method is widely accepted in healthcare improvement; however there is little overarching evaluation of how the method is applied. This paper proposes a theoretical framework for assessing the quality of application of PDSA cycles and explores the consistency with which the method has been applied in peer-reviewed literature against this framework.\n  .\n  # Methods\n  .\n  NHS Evidence and Cochrane databases were searched by three independent reviewers. Empirical studies were included that reported application of the PDSA method in healthcare. Application of PDSA cycles was assessed against key features of the method, including documentation characteristics, use of iterative cycles, prediction-based testing of change, initial small-scale testing and use of data over time.\n  .\n  # Results\n  .\n  73 of 409 individual articles identified met the inclusion criteria. Of the 73 articles, 47 documented PDSA cycles in sufficient detail for full analysis against the whole framework. Many of these studies reported application of the PDSA method that failed to accord with primary features of the method. Less than 20% (14\/73) fully documented the application of a sequence of iterative cycles. Furthermore, a lack of adherence to the notion of small-scale change is apparent and only 15% (7\/47) reported the use of quantitative data at monthly or more frequent data intervals to inform progression of cycles.\n  .\n  # Discussion\n  .\n  To progress the development of the science of improvement, a greater understanding of the use of improvement methods, including PDSA, is essential to draw reliable conclusions about their effectiveness. This would be supported by the development of systematic and rigorous standards for the application and reporting of PDSAs.\nauthor: Michael J Taylor; Chris McNicholas; Chris Nicolay; Ara Darzi; Derek Bell; Julie E ReedCorrespondence to Michael J Taylor, Academic Surgical Unit, 10th Floor, QEQM building, St Mary's Hospital, Paddington, London W2 1NY, UK; <email@example.com>\ndate: 2014-04\ninstitute: 1Department of Surgery and Cancer, Imperial College London, London, UK; 2National Institute for Health Research (NIHR) Collaboration for Leadership in Applied Health Research and Care (CLAHRC) for North-West London, London, UK\nreferences:\ntitle: Systematic review of the application of the plan\u2013do\u2013study\u2013act method to improve quality in healthcare\n\n# Introduction\n\nDelivering improvements in the quality and safety of healthcare remains an international challenge. In recent years, quality improvement (QI) methods such as plan\u2013so\u2013study\u2013act (PDSA) cycles have been used in an attempt to drive such improvements. The method is widely used in healthcare improvement; however there is little overarching evaluation of how the method is applied. This paper proposes a theoretical framework for assessing the quality of application of PDSA cycles and explores the quality and consistency of PDSA cycle application against this framework as documented in peer-reviewed literature.\n\n## Use of PDSA cycles in healthcare\n\nDespite increased investment in research into the improvement of healthcare, evidence of effective QI interventions remains mixed, with many systematic reviews concluding that such interventions are only effective in specific settings.1\u20134 To make sense of these findings, it is necessary to understand that delivering improvements in healthcare requires the alteration of processes within complex social systems that change over time in predictable and unpredictable ways.5 Research findings highlight the influential effect that local context can have on the success of an intervention6 7 and, as such, 'single-bullet' interventions are not anticipated to deliver consistent improvements. Instead, effective interventions need to be complex and multi-faceted8\u201311 and developed iteratively to adapt to the local context and respond to unforeseen obstacles and unintended effects.12 13 Finding effective QI methods to support iterative development to test and evaluate interventions to care is essential for delivery of high-quality and high-value care in a financially constrained environment.\n\nPDSA cycles provide one such method for structuring iterative development of change, either as a standalone method or as part of wider QI approaches, such as the Model for Improvement (MFI), Total Quality Management, Continuous QI, Lean, Six Sigma or 'Quality Improvement Collaboratives'.3 4 14 Despite increased use of QI methods, the evidence base for their effectiveness is poor and under-theorised.15\u201317 PDSA cycles are often a central component of QI initiatives, however few formal objective evaluations of their effectiveness or application have been carried out.18 Some PDSA approaches have been demonstrated to result in significant improvements in care and patient outcomes,19 while others have demonstrated no improvement at all.20\u201322\n\nAlthough at the surface level these results appear disheartening for those involved in QI, there is a need to explore the extent to which the PDSA method has been successfully deployed to draw conclusions from these studies. Rather than see the PDSA method as a 'black box' of QI,23 it is important to understand that the use of PDSA cycles is, itself, a complex intervention made up of a series of interdependent steps and key principles that inform its application5 24 25 and that this application is also affected by local context.26 To interpret the results regarding the outcome(s) from the application of PDSA cycles (eg, whether processes or outcomes of care improved) and gauge the effectiveness of the method, it is necessary to understand how the method has been applied.\n\nNo formal criteria for evaluating the application or reporting of PDSA cycles currently exist. It is only in recent years, through SQUIRE guidelines, that frameworks for publication have been developed that explicitly consider description of PDSA application.27 28 We consider that such criteria are necessary to support and assess the effective application of PDSA cycles and to increase their legitimacy as a scientific method for improvement. We revisited the origins and theory of the method to develop a theoretical framework to evaluate the application of the method.\n\n## The origins and theory of PDSA cycles\n\nThe PDSA method originates from industry and Walter Shewhart and Edward Deming's articulation of iterative processes which eventually became known as the four stages of PDSA.25 PDCA (plan\u2013do\u2013check\u2013act) terminology was developed following Deming's early teaching in Japan.29 The terms PDSA and PDCA are often used interchangeably in reference to the method. This distinction is rarely referred to in the literature and for the purpose of this article we consider PDSA and PDCA but refer to the methodologies generally as 'PDSA' cycles unless otherwise stated.\n\nUsers of the PDSA method follow a prescribed four-stage cyclic learning approach to adapt changes aimed at improvement. In the 'plan' stage a change aimed at improvement is identified, the 'do' stage sees this change tested, the 'study' stage examines the success of the change and the 'act' stage identifies adaptations and next steps to inform a new cycle. The MFI30 and FOCUS31 (see <a href=\"#BMJQS2013001862F1\" data-ref-type=\"fig\">figure 1<\/a>) frameworks have been developed to precede the use of PDSA and PDCA cycles30 31 respectively (<a href=\"#BMJQS2013001862TB1\" data-ref-type=\"table\">table 1<\/a>).\n\nDescription of the plan\u2013do\u2013study\u2013act (PDSA) cycle method according to developers and commentators\n\n<table>\n<thead>\n<tr>\n<th style=\"text-align: left;\"><\/th>\n<th style=\"text-align: left;\">Deming (1986)<span class=\"citation\" data-cites=\"R25\">25<\/span>Original description of the method relating to manufacturing<\/th>\n<th style=\"text-align: left;\">Langley (1996)<span class=\"citation\" data-cites=\"R30\">30<\/span>How the PDSA method may be adapted for use in healthcare contexts<\/th>\n<th style=\"text-align: left;\">Speroff and O'Connor (2004)<span class=\"citation\" data-cites=\"R33\">33<\/span>How the PDSA method is analogous to scientific methodology<\/th>\n<\/tr>\n<\/thead>\n<tbody>\n<tr>\n<td style=\"text-align: left;\">Plan<\/td>\n<td style=\"text-align: left;\">Plan a change or test aimed at improvement<\/td>\n<td style=\"text-align: left;\"><ul>\n<li><p>Identify objective<\/p><\/li>\n<li><p>Identify questions and predictions<\/p><\/li>\n<li><p>Plan to carry out the cycle (who, when, where, when)<\/p><\/li>\n<\/ul><\/td>\n<td style=\"text-align: left;\">Formation of a hypothesis for improvement<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Do<\/td>\n<td style=\"text-align: left;\">Carry out the change or test (preferably on a small scale)<\/td>\n<td style=\"text-align: left;\"><ul>\n<li><p>Execute the plan<\/p><\/li>\n<li><p>Document problems and unexpected observations<\/p><\/li>\n<li><p>Begin data analysis<\/p><\/li>\n<\/ul><\/td>\n<td style=\"text-align: left;\">Conduct study protocol with collection of data<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Study<\/td>\n<td style=\"text-align: left;\">Examine the results. What did we learn? What went wrong?<\/td>\n<td style=\"text-align: left;\"><ul>\n<li><p>Complete the data analysis<\/p><\/li>\n<li><p>Compare data to predictions<\/p><\/li>\n<li><p>Summarise what was learnt<\/p><\/li>\n<\/ul><\/td>\n<td style=\"text-align: left;\">Analysis and interpretation of the results<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Act<\/td>\n<td style=\"text-align: left;\">Adopt the change, abandon it or run through cycle again<\/td>\n<td style=\"text-align: left;\"><ul>\n<li><p>What changes are to be made?<\/p><\/li>\n<li><p>What will the next cycle entail?<\/p><\/li>\n<\/ul><\/td>\n<td style=\"text-align: left;\">Iteration for what to do next<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\nIn comparison to more traditional healthcare research methods (such as randomised controlled trials in which the intervention is determined in advance and variation is attempted to be eliminated or controlled for), the PDSA cycle presents a pragmatic scientific method for testing changes in complex systems.32 The four stages mirror the scientific experimental method33 of formulating a hypothesis, collecting data to test this hypothesis, analysing and interpreting the results and making inferences to iterate the hypothesis.\n\nThe pragmatic principles of PDSA cycles promote the use of a small-scale, iterative approach to test interventions, as this enables rapid assessment and provides flexibility to adapt the change according to feedback to ensure fit-for-purpose solutions are developed.10 12 13 Starting with small-scale tests provides users with freedom to act and learn; minimising risk to patients, the organisation and resources required and providing the opportunity to build evidence for change and engage stakeholders as confidence in the intervention increases.\n\nIn line with the scientific experimental method, the PDSA cycle promotes prediction of the outcome of a test of change and subsequent measurement over time (quantitative or qualitative) to assess the impact of an intervention on the process or outcomes of interest. Thus, learning is primarily achieved through interventional experiments designed to test a change. In recognition of working in complex settings with inherent variability, measurement of data over time helps understand natural variation in a system, increase awareness of other factors influencing processes or outcomes, and understand the impact of an intervention.\n\nAs with all scientific methods, documentation of each stage of the PDSA cycle is important to support scientific quality, local learning and reflection and to ensure knowledge is captured to support organisational memory and transferability of learning to other settings.\n\nThis review examines the application of PDSA cycles as determined by these principle features of the PDSA method described above. We recognise that a number of health and research related contextual factors may affect application of the method but these factors are beyond the scope of this review. The review intends to improve the understanding of whether the PDSA method is being used and reported in line with the literature informed criteria and therefore inform the interpretation of studies that have used PDSA cycles to facilitate iterative development of an intervention.\n\n# Methods\n\nA systematic narrative review was conducted in adherence to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement.34\n\n## Search\n\nThe search was designed to identify peer-reviewed publications describing empirical studies that applied the PDSA method. Taking into account the development of the method and terminology, the search terms used were 'PDSA', 'PDCA', 'Deming Cycle', 'Deming Circle', 'Deming Wheel' and 'Shewhart Cycle'. No year of publication restrictions were imposed.\n\n## Information sources\n\nThe following databases were searched for articles: Allied and Complementary Medicine Database (AMED; 1985 to present), British Nursing Index (BNI; 1985 to present), Cumulative Index to Nursing and Allied Health Literature (CINAHL; 1981 to present), Embase (1980 to present), Health Business Elite (EMBESCO Publishing, Ipswich, Massachusetts, USA), the Health Management Information Consortium (HMIC), MEDLINE from PubMed (1950 to present) and PsychINFO (1806 to present) using the NHS Evidence online library (REF), and the Cochrane Database of Systematic Reviews. The last search date was 25 September 2012.\n\n## Data collection process and study selection\n\nData were collected and tabulated independently by MJT, CM and CN in a manner guided by the Cochrane Handbook. Eligibility was decided independently, in a standardised manner and disagreements were resolved by consensus. If an abstract was not available from the database, the full-text reference was accessed.\n\nInclusion criteria for articles were as follows: published in peer-reviewed journal; describes PDSA method being applied to improve quality in a healthcare setting; published in English. Editorial letters, conference abstracts, opinion and audit articles were excluded from the study selection.\n\n## Data items\n\nA theoretical framework was constructed by compartmentalising the key features of the PDSA method into observable variables for evaluation (<a href=\"#BMJQS2013001862TB2\" data-ref-type=\"table\">table 2<\/a>). This framework was developed in accordance with recommendations for PDSA use cited in the literature, describing the origins and theory of the method. Face validity of the framework was achieved through discussion among authors, with QI facilitators and at local research meetings.\n\nTheoretical framework based on key features of the plan\u2013do\u2013study\u2013act (PDSA) cycle method\n\n<table>\n<thead>\n<tr>\n<th style=\"text-align: left;\">Feature of PDSA<\/th>\n<th style=\"text-align: left;\">Description of feature<\/th>\n<th style=\"text-align: left;\">How this was measured<\/th>\n<\/tr>\n<\/thead>\n<tbody>\n<tr>\n<td style=\"text-align: left;\">Iterative cycles<\/td>\n<td style=\"text-align: left;\">To achieve an iterative approach, multiple PDSA cycles must occur. Lessons learned from one cycle link and inform cycles that follow. Depending on the knowledge gained from a PDSA cycle, the following cycle may seek to modify, expand, adopt or abandon a change that was tested<\/td>\n<td style=\"text-align: left;\"><ul>\n<li><p>Were multiple cycles used?<\/p><\/li>\n<li><p>Were multiple cycles linked to one another (ie, does the 'act' stage of one cycle inform the 'plan' stage of the cycle that follows)?<\/p><\/li>\n<li><p>When isolated cycles were used were future actions postulated in the 'act' stage?<\/p><\/li>\n<\/ul><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Prediction-based test of change<\/td>\n<td style=\"text-align: left;\">A prediction of the outcome of a change is developed in the 'plan' stage of a cycle. This change is then tested and examined by comparison of results with the prediction<\/td>\n<td style=\"text-align: left;\"><ul>\n<li><p>Was a change tested?<\/p><\/li>\n<li><p>Was an explicit prediction articulated?<\/p><\/li>\n<\/ul><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Small-scale testing<\/td>\n<td style=\"text-align: left;\">As certainty of success of a test of change is not guaranteed, PDSAs start small in scale and build in scale as confidence grows. This allows the change to be adapted according to feedback, minimises risk and facilitates rapid change and learning<\/td>\n<td style=\"text-align: left;\"><ul>\n<li><p>Sample size per cycle?<\/p><\/li>\n<li><p>Temporal duration of cycles?<\/p><\/li>\n<li><p>Number of changes tested per cycle?<\/p><\/li>\n<li><p>Did sequential cycles increase scale of testing?<\/p><\/li>\n<\/ul><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Use of data over time<\/td>\n<td style=\"text-align: left;\">Data over time increases understanding regarding the variation inherent in a complex healthcare system. Use of data over time is necessary to understand the impact of a change on the process or outcome of interest<\/td>\n<td style=\"text-align: left;\"><ul>\n<li><p>Was data collected over time?<\/p><\/li>\n<li><p>Were statistics used to test the effect of changes and\/or understand variation?<\/p><\/li>\n<\/ul><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Documentation<\/td>\n<td style=\"text-align: left;\">Documentation is crucial to support local learning and transferability of learning to other settings<\/td>\n<td style=\"text-align: left;\"><ul>\n<li><p>How thoroughly was the application of the PDSA method detailed in the reports?<\/p><\/li>\n<li><p>Was each stage of the PDSA cycles documented?<\/p><\/li>\n<\/ul><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\nData were collected regarding application of the PDSA method in line with the theoretical framework. Other data collected included first author, year of publication, country, area of healthcare, use of PDSA or PDCA terminology, and use of MFI or FOCUS as supporting frameworks. Ratios were used to analyse the results regarding the majority of variables, and mean scores regarding data associated with length of study, length of PDSA cycle and sample size were also used for analysis. Data were analysed independently by MJT and CM. Discrepancies (which occurred in less than 3% of data items) were resolved by consensus.\n\n## Risk of bias in individual studies\n\nThe present review aimed to assess the reported application of the PDSA method and the results of individual studies were not analysed in this review.\n\n## Risk of bias across studies\n\nDespite our review being focused on reported application, rather than success of interventions, it may still be possible that publication bias affected the results of this study. Research that used PDSA methodology, but did not yield successful results, may be less likely to get published than reports of successful PDSA interventions.\n\n# Results\n\n## Study selection\n\nA search of the databases yielded 942 articles. After removal of duplicates, 409 remained; 216 and 120 were further discarded following review of abstracts and full texts, respectively. Excluded articles did not apply the PDSA method as part of an empirical study or coincidently used the acronyms PDSA or PDCA for different terms, or were abstracts for conferences or poster presentations. A total of 73 articles met the inclusion criteria and were included in the review (see <a href=\"#BMJQS2013001862F2\" data-ref-type=\"fig\">figure 2<\/a>).\n\n## General study characteristics\n\n### Country of study\n\nThe retrieved articles describe studies conducted in the USA (n=46), the UK (n=13), Canada (n=3) Australia (n=3), the Netherlands (n=2) and one each from six other countries (see online supplementary appendix A for complete synthesis of results).\n\n### Healthcare discipline to which method was applied\n\nThis varied across acute and community care and clinical and organisational settings. The most common settings were those of pain management and surgery (six articles each).\n\n### Method terminology\n\nOf the 73 articles identified, 42 articles used 'PDSA' as terminology and 31 referred to the method as 'PDCA'. Eight of these reported using the MFI. Thirty-one articles used 'PDCA' terminology, with 20 using the preceding FOCUS framework. One article described use of FOCUS and MFI. Over time there was an increase in the prevalence of PDSA use with PDCA use diminishing (see online supplementary figure S1). The earliest reported use of PDCA and PDSA in healthcare was 1993 and 2000, respectively.\n\n## Documentation\n\nThe following four categories were used to describe the extent to which cycles were documented in articles (n=73): no detail of cycles (n=16); themes of cycles (but no additional details) (n=8); details of individual cycles, but not of stages within cycles (n=8); details of cycles including separated information on stages of cycles (n=41).\n\nAnalysis of articles against the developed framework was dependent on the extent to which the application of PDSA cycles was reported. Articles that provided no details of cycles or only themes of cycles were insufficient for full review and excluded for analysis against all features. Articles that provided further details of cycles completed (n=49) were included for analysis against the remaining four features of the framework. A full breakdown of findings can be viewed in online supplementary appendix B.\n\n## Application of method\n\n### Iterative cycles (n=49)\n\nFourteen articles described a sequence of iterative cycles (two or more cycles with lessons learned from one cycle linking and informing a subsequent cycle), 33 described isolated cycles that are not linked, and 2 articles described cycles that used PDSA stages in the incorrect order (in one article, one plan, one do, two checks and three acts were described, PDACACA35; a further study did not report use of a 'check' stage; PDA36) and are excluded from further review. Of the 33 articles that described non-iterative cycles, 29 reported a single cycle being used, and 4 described multiple, isolated (non-sequential) cycles. Although future actions are often suggested in articles that reported a single cycle, only three explicitly mentioned the possibility of further cycles taking place. A total of 13.6% (3\/22) of PDCA studies described the application of iterative cycles compared with 44% (11\/25) of PDSA studies describing the application of iterative cycles (see <a href=\"#BMJQS2013001862F3\" data-ref-type=\"fig\">figure 3<\/a>).\n\n### Prediction-based testing of change (n=47)\n\nThe aims of the cycles adhered to one of two themes: tests of a change; and collection or review of data without a change made. Of the 33 articles with single cycles, 30 aimed to test a change while 3 used the PDSA method to collect or review data. Of the 14 articles demonstrating sequential cycle use, 8 solely used their cycles to test change whilse5 began with a cycle collecting or reviewing data followed by cycles testing change. One article described a mixture of cycles testing changes and cycles that involved collection\/review of data. Four of the 47 studies contained an explicit prediction regarding the outcome of a change; all 4 aimed to test a change (see online supplementary table S1).\n\n### Small-scale testing (n=47)\n\nScale was assessed in three ways: sample size, duration and complexity. Sample size refers to quantity of observations used to measure the change; duration refers to the length of PDSA cycle application; and complexity refers to the quantity of changes administered per cycle.\n\n#### Sample size\n\nPatient data, staff data and case data were used as samples within PDSA cycles. Twenty-seven articles reported a sample size from at least one of their cycles. Twenty-one of these were isolated cycle studies with sample size ranging from 7 to 2079 (mean=323.33, SD=533.60). The remaining six studies reporting individual cycle sample sizes used iterative cycles; the sample size of the first cycles of these ranged from 1 to 34 (mean=16.75, SD=11.47). Two of these studies described the use of incremental sample sizes across cycles, three used non-incremental sample sizes across cycles, and one changed the type of sample. Of the eight iterative cycle articles that did not report individual cycle sample sizes, two did not differentiate sample sizes between cycles and instead gave an overall sample for the chain of cycles and six did not report sample size.\n\n#### Duration\n\nReported *s*tudy duration of isolated cycles ranged from 2\u2005weeks to 5\u2005years (mean=11.91\u2005months, SD=12.81). Only five articles describing iterative cycles explicitly reported individual cycle duration. Individual cycle duration could be estimated from the total duration of the PDSA cycle chain and the number of cycles conducted, resulting in approximate cycle lengths ranging from three cycles in 1\u2005day to one cycle in 16\u2005months (mean=5.41\u2005months, SD=4.80, see online supplementary figure S2). The total PDSA cycle duration for series of iterative cycles (first to last cycle of one chain) ranged from 1\u2005day to 4\u2005years (mean=20.38, SD=20.39\u2005months).\n\n#### Complexity\n\nTwenty-two articles reported more than one change being tested within a single cycle. Of the articles describing iterative cycles, 42% administered more than one change per cycle compared with 48% of the articles describing non-iterative PDSA cycles.\n\n### Data over time (n=47)\n\nAll studies used a form of qualitative and quantitative data to assess cycles. Studies were categorised according to four types of reporting quantitative data: regular (n=15), three or more data points with consistent time intervals; non-regular (n=16), before and after or per PDSA cycle; single data point (n=8), a single data point after PDSA cycle(s); and no quantitative data reported (n=8). Of the 15 articles that used regular data, only 7 used monthly or more frequent data intervals (see online supplementary figure S3 for full frequency of regular quantitative data reporting). No studies reported using statistical process control to analyse data collected from PDSA cycles. Eleven included analysis of data using inferential statistical tests (five of these studies collected isolated data, six involved continuous data collection).\n\nOf the eight articles that did not report any quantitative data, two reported that quantitative analyses had taken place but did not present the findings and six described the use of qualitative feedback only (one non-regular, five single data point). Qualitative data were gathered through a range of mechanisms from informal staff or patient feedback to structured focus groups.\n\n# Discussion\n\nPDSA cycles offer a supporting mechanism for iterative development and scientific testing of improvements in complex healthcare systems. A review of the historic development and rationale behind PDSA cycles has informed the development of a theoretical framework to guide the evaluation of PDSA cycles against use of iterative cycles, initial small-scale testing, prediction-based testing of change, use of data over time and documentation.\n\nUsing these criteria to assess peer-reviewed publications of PDSA cycles demonstrates an inconsistent approach to the application and reporting of PDSA cycles and a lack of adherence to key principals of the method. Only 2\/7337 38 articles demonstrated compliance with criteria in all five principles. Assessment of compliance was problematic due to the marked variation in reporting of this method, which reflects a lack of standardised reporting requirements for the PDSA method.\n\nFrom the articles that reported details of PDSA cycles it was possible to ascertain that variation is inherent not just in reporting standards, but in the conduct of the method, implying that the key principles of the PDSA method are frequently not followed. Less than 20% (14\/73) of reviewed articles reported the conduct of iterative cycles of change, and of these, only 15% (2\/14) used initial small-scale tests with increasing scale as confidence in the intervention developed. These results suggest that the full benefits of the PDSA method would probably not have been realised in these studies. Without an iterative approach, learning from one cycle is not used to inform the next cycle, and therefore it is unlikely that interventions will be adapted and optimised for use in a particular setting. Furthermore, large-scale cycles risk significant resource investment in an intervention that has not been tested and optimised within that environment and risk producing 'false' negatives.\n\nOnly 14% (7\/47) of articles reported use of regular data over time at monthly or more frequent intervals, indicating a lack of understanding around the use of the PDSA method to track change within a 'live' system, and limiting the ability to interpret the results from the study. Cycles that included an explicit prediction of outcomes were reported in only 9% (4\/47) of articles, suggesting that PDSA cycles were not used as learning cycles to test and revise theory-based predictions.\n\nOverall these results demonstrate poor compliance with key principles of the PDSA method, suggesting that it is not being used optimally. The increasing trend in using PDSA (as opposed to 'PDCA') cycles in recent years, however, does seem to have been accompanied by an increase in compliance with some key principles, such as use of iterative cycles. Deming was cautious over the use of the 'PDCA' terminology and warned it referred to an explicitly different process, referring to a quality control circle for dealing with faults within a system, rather than the PDSA process, which was intended for iterative learning and improvement of a product or a process.39 This subtle difference in terminologies may help to explain the better compliance with key methodological principles in studies that refer to the method as 'PDSA'.\n\nOne of the articles identified in the search included comments by the authors that the PDSA method should be 'more realistically represented',40 as ineffective cycles can be 'abandoned' early on, making it needless to go through all four stages in each iteration. These comments may provide insight into an important potential misunderstanding of the PDSA methodology. Ineffective changes will result in learning, which is a fundamental principle behind a PDSA cycle. However minor this abandoned trial may have been, it can still be usefully described as a PDSA cycle. A minor intervention may be planned (P) and put into practice (D). A barrier may be encountered (S), resulting in a decision being made to retract the intervention, and to do something differently (A).\n\nThe theoretical framework presented in this paper highlights the complexity of PDSA cycles and the underpinning knowledge required for correct application. The considerable variation in application observed in the reported literature suggests that caution should be taken in interpreting results from evaluations in which PDSAs are used in a controlled setting and as a 'black box' of QI. This review did not compare the effectiveness of use to reported outcomes and therefore this study does not conclude whether better application of the PDSA method results in better outcomes, but instead draws on theoretical principles of PDSAs to rationalise why this would be expected. Prospective mechanistic studies exploring the effective application of the method as well as study outcomes would be of greater use in drawing conclusions regarding the effectiveness of the method. The framework presented in this paper could act as a good starting point for such studies.\n\nThe fact that only peer-reviewed publications were assessed in this study means that results may be affected by publication bias. This is anticipated both in terms of what is accepted for publication but also the level and type of detail that is requested and allowed in typical publications (eg, before and after studies are more common than presenting data over time and this may make these types of studies easier to publish). Though QI work may be easier to publish now through recent changes in publication guidelines,27 possible publication outlets continue to be relatively limited.\n\nTo support systematic reporting and encourage appropriate usage, we suggest that reporting guidelines be produced for users of the PDSA method to increase transparency as to the issues that were encountered and how they were resolved. While PDSA is analogous to a scientific method, it appears to be rarely used or reported with scientific rigour, which in turn, inhibits perceptions of PDSA as a scientific method. Such guidelines are essential to increase the scientific legitimacy of the PDSA method as well as to improve scientific rigour or application and reporting. Although the SQUIRE guidelines make reference to the potential use of PDSA cycles, further support to users and teachers, and publication of this improvement method seems necessary. Consistent reporting of PDSA structure would allow meta-evaluation and systematic reviews to further build the knowledge of how to use such methods effectively and the principles to apply to increase chances of success.\n\nIt is clear from these findings that there is much room for improvement in the application and use of the PDSA method. Previous studies have discussed the influence of different context factors on the use of QI methods, such as motivation, data support infrastructure and leadership20 22 41\u201343 Understanding how high-quality usage can be promoted and supported needs to become the focus of further research if such QI methods are going to be used effectively in mainstream healthcare.\n\n# Conclusions\n\nThere is varied application and reporting of PDSAs and lack of compliance with the principles that underpin its design as a pragmatic scientific method. The varied practice compromises its effectiveness as a method for improvement and cautions against studies that view QI or PDSA as a 'black box' intervention.\n\nThere is an urgent need for greater scientific rigour in the application and reporting of these methods to advance the understanding of the science of improvement and efficacy of the PDSA method. The PDSA method should be applied with greater consistency and with greater accordance to guidelines provided by founders and commentators25 30 44 45\n\n# Supplementary Material\n\n###### Web appendix A\n\n###### Web appendix B\n\n###### Web figures\n\nThe authors would like to thank Dr Thomas Woodcock for his valuable input into the theoretical framework and data analysis.\n\n# References","meta":{"dup_signals":{"dup_doc_count":185,"dup_dump_count":56,"dup_details":{"curated_sources":2,"2023-50":4,"2023-40":4,"2023-23":7,"2023-14":2,"2023-06":9,"2022-49":5,"2022-40":4,"2022-33":8,"2022-27":5,"2022-21":4,"2022-05":4,"2021-49":9,"2021-43":11,"2021-39":4,"2021-31":1,"2021-25":3,"2021-21":1,"2021-17":1,"2021-10":1,"2021-04":3,"2020-50":3,"2020-45":6,"2020-40":3,"2020-34":5,"2020-29":1,"2020-24":3,"2020-16":1,"2020-10":3,"2020-05":3,"2019-51":3,"2019-47":2,"2019-43":5,"2019-35":1,"2019-30":5,"2019-26":4,"2019-22":3,"2019-13":1,"2019-04":1,"2018-51":2,"2018-43":1,"2018-34":1,"2018-30":1,"2018-26":3,"2018-22":2,"2018-09":2,"2018-05":2,"2017-47":2,"2017-39":1,"2017-30":1,"2017-26":1,"2017-22":2,"2024-30":3,"2024-26":3,"2024-22":5,"2024-18":6,"2024-10":2}},"file":"PMC3963536"},"subset":"pubmed_central"}
{"text":"date: 2005-03-25\ntitle: Findings of scientific misconduct.\n\n# Findings of Scientific Misconduct\n\n**Notice Number:** NOT-OD-05-040\n\n**Key Dates  \n**Release Date: March 22, 2005\n\n**Issued by  \n**Department of Health and Human Services (DHHS), ([http:\/\/www.hhs.gov](http:\/\/www.hhs.gov\/))\n\nDepartment of Health and Human Services (DHHS), ([http:\/\/www.hhs.gov](http:\/\/www.hhs.gov\/))\n\nNotice is hereby given that the Office of Research Integrity (ORI) and the Acting Assistant Secretary for Health have taken final action in the following case:\n\nEric T. Poehlman, Ph.D., University of Vermont: Based on the report of an investigation conducted by the University of Vermont (Report), admissions made by the respondent, and additional analysis conducted by ORI in its oversight review, the U.S. Public Health Service (PHS) found that Eric T. Poehlman, Ph.D., former Professor, Department of Medicine at the University of Vermont College of Medicine, engaged in scientific misconduct in research. The research was supported by National Institutes of Health (NIH) grants from the National Institute of Aging (NIA), the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), and the National Center for Research Resources (NCRR).\n\nSpecifically, PHS found that the respondent is responsible for scientific misconduct by engaging in the misleading and deceptive practices set forth herein below:\n\nGroup 1: Longitudinal study of aging; Protocol 678 and associated Excel spreadsheets\n\n**Proposing Research (Report, pp. 22-25)**\n\n1\\. That Respondent falsified preliminary data purportedly obtained in a longitudinal study of aging in NIH grant application 1 R01 AG17906-01, submitted May 27, 1999; specifically, the claim of 130 subjects at visit one (T1) and 70 subjects at visit two (T2), mean values for total energy expenditure (TEE) obtained with a doubly-labeled water technique were falsified; additional parameters such as physical activity energy expenditure (PAEE), resting metabolic rate (RMR), fat-free mass, appendicular skeletal muscle mass, and percent body fat were falsified to show significant trends during the aging process that were not reflective of the actual data (Abstract and pp. 19, 21, 22, 23, 27, 29, 34, 41, 42).\n\n2\\. That Respondent falsified preliminary data purportedly obtained in a longitudinal study of aging in NIH grant application 1 R01 AG17906-01A1, submitted February 2000, specifically, the claim of 130 subjects at visit one (T1) and 70 subjects at visit two (T2), mean values for total energy expenditure (TEE) obtained with a doubly-labeled water technique were falsified; additional parameters such as physical activity energy expenditure (PAEE), resting metabolic rate (RMR), fat-free mass, appendicular skeletal muscle mass, and percent body fat were falsified to show significant trends during the aging process that were not reflective of the actual data (Abstract and pp. 32, 34, 38, 39, 45, 46).\n\n**Conducting Research**\n\n3\\. That Respondent systematically falsified a number of metabolic and physical measures of subjects in the longitudinal study of aging; these falsifications of specific types of data in the Protocol 678 spreadsheet commenced immediately after he assigned responsibility for maintenance of the data to a young technician and simultaneously arranged to have personal access to the data; his widespread alteration of data in specific fields has been detected in a number of different versions, often with cumulative effect, and several were transmitted to different co-workers for specific reasons, as detailed in the following sub issues:\n\na\\. that in the spreadsheet labeled \"678data3.xls,\" produced during the late spring\/early summer of 2000, Respondent falsified and fabricated numerous values in the fields called underwater fat mass (UWFM), underwater fat-free mass (UWFFM), leisure time activity (LTA), and maximum oxygen consumption (VO 2 Max);\n\nb\\. that on July 16, 2000, Respondent transmitted a subset of the Protocol 678 spreadsheet to Witness 1 entitled \"RevisedTEE_s.xls;\" that had 135 values each for T1 and T2 for TEE; many values were fabricated and most of the remaining values had been falsified by reversing the original T1 and T2 values (Report, pp. 6-8);\n\nc\\. that Respondent falsified additional data fields in the version of the 678 data set called \"ExcelLongitudinal2.xls,\" on or about August 17, 2000; specifically values for total cholesterol, insulin, resting metabolic rate (RMR), and glucose values of the subjects with names in the second half of the alphabet were falsified (often by reversing T1 and T2) or fabricated (Report, p. 10);\n\nd\\. that Respondent gave falsified data to Witness 2 in August 2000 to provide him with data for a presentation to be given in September 2000 to UVM staff (initially postponed until February 2001); the spreadsheet given to Witness 2 contained the falsified and fabricated TEE and underwater body composition values of RevisedTEE_s.xls; the spreadsheet, when subsequently obtained by ORI, was labeled \"LongitudinalBodyCompWitness2.xls\";\n\ne\\. that Respondent falsified additional data in another version of \"ExcelLongitudinal2.xls\" that he sent to Witness 3 on or about August 22, 2000; specifically, this version contained the falsifications already described above (Issues 3a through 3c) and, in addition, the remainder of the glucose values, and individual lipid components (triglycerides, HDL, and LDL) were extensively falsified and fabricated; this spreadsheet was transmitted to Witness 3 with the expectation that he would write a paper describing the effect of aging on lipid metabolism (Report, pp. 8-10);\n\nf\\. that Respondent provided a falsified version of the Protocol 678 spreadsheet to Witness 4 in the fall of 2000 so that Witness 4 could write a review article;\n\ng\\. that Respondent, in late September\/early October 2000, extensively falsified body composition data (a number of parameters including but not limited to fat mass and fat-free mass) obtained with the DEXA method in a spreadsheet transmitted to Witness 5 so that Witness 5 could write a paper using the DEXA method to demonstrate body composition changes with age (Report, pp. 5 and 39);\n\n**Reporting Research**\n\nh\\. that Respondent reported falsified data from the longitudinal study of aging at the annual North American Association for the Study of Obesity (NAASO) meeting in October 2000, and to the Vermont community; the falsifications on his slides included falsified values for both the number of subjects tested at T1 and T2 for TEE and the claim of a significant difference between the means for TEE at T1 versus T2; values for RMR, PAEE, and body composition (fat mass and fat-free mass) were also falsely reported (Report, p. 34);\n\ni\\. From the falsified data set that Respondent provided him, Witness 4 developed a review article: Rawson, E. and Poehlman, E.T. \"Resting metabolic rate and aging.\" Recent Research Developments in Nutrition 4, 2001, coauthored by Respondent, that included falsified yet unpublished results about the decline in RMR upon aging (p. R1792). These results, ORI determined, are very similar to the falsified results that Respondent presented at NAASO, based on the falsified Protocol 678 data set;\n\n**Conducting Research**\n\nj\\. That on October 16, 2000, Respondent provided Witness 6 a version of the Protocol 678 data set entitled \"ExcelLongitudinal4.xls\" that included falsified cholesterol and individual lipid component data (as well as falsified parameters such as insulin, glucose (all subjects), TEE, RMR, PAEE, and underwater body composition data) so that Witness 6 could write a paper on the effect of aging on lipid composition (Report, pp. 8-10);\n\n**Other**\n\nk\\. That Respondent falsely testified to the University of Vermont Investigation Committee that he had never used data from the longitudinal study of aging in grant applications or in public presentations (Report, pp. 34 and 36).\n\nGroup 2: Muscle biopsy results\n\n**Proposing Research**\n\n4\\. That Respondent reported fabricated muscle biopsy data in NIH grant application 1 R01 AG17906-01A1 (p. 27), submitted in February 2000; specifically, he falsely claimed to have successfully tested five individuals on two occasions (1994 and 1999) when he had not (Report, pp. 25-26).\n\nGroup 3: Protocol 467, including the \"longitudinal menopause study\" and other falsifications\/fabrications\n\n**Reporting Research**\n\n5\\. That Respondent published falsified thyroid hormone results for women entered in a cross-sectional study (Protocol 467) (Figures 3A and 3B and related text and the portion of Table 2 related to T3 and free T3) in the following paper: Poehlman, E.T. Goran, M.I. Gardner, A.W., Ades, P.A., Arciero, P.J., Katzman-Rooks, S.M., Montgomery, S.M., Toth, M.J., and Sutherland, P.T. \"Determinants of decline in resting metabolic rate in aging females.\" American Journal of Physiology 264(Endrocrinol Metab. 27):E450-E455, March 1993 (Correction required);\n\n6\\. That Respondent published in November 1995 falsified and fabricated data from a longitudinal study of menopause in women in the following paper: Poehlman, E.T., Toth, M.J., and Gardner, A.W. \"Changes in energy balance and body composition at menopause: A controlled longitudinal study.\" Annals of Internal Medicine 123(9):673-675, November 1, 1995; Respondent has admitted that this longitudinal study was never conducted (the number of women seen at T1 was falsified, and there were at most 3, rather than 35, women seen at T2) (Report, pp. 27-32) (Retracted by editor; letter from Respondent required);\n\n**Proposing Research**\n\n7\\. That Respondent repeatedly reported this non-existent longitudinal menopause study and cited the 1995 Annals of Internal Medicine paper in NIH grant applications as proof that Respondent could conduct such longitudinal studies, and the falsified and fabricated data supported his proposed hypotheses:\n\na\\. Respondent provided for the annual report for the University of Vermont General Clinical Research Center (GCRC) grant (M01 RR00109) for the period 12\/1\/94-11\/30\/95, information about the falsified longitudinal menopause study, and the Annals of Internal Medicine paper was cited as having utilized the University of Vermont GCRC facilities;\n\nb\\. in application 5 K04 AG00564-05, submitted July 18, 1995, Respondent reported the results of a seven (7) year ^[1](#ftnref1)^ followup study of pre- and post-menopausal women, noting an article was in press in the Annals of Internal Medicine 1995 (unnumbered page 3);\n\nc\\. in application R01 AG13978-01, submitted in September 1995, Respondent reported falsified and fabricated data on menopause related changes in metabolism, body composition, and other variables in Preliminary Data (pp. 35, 41, and 42), and cited the published Annals of Internal Medicine 1995 paper;\n\nd\\. in application R01 AG13978-01A1, submitted in July 1996, Respondent reported falsified and fabricated data on menopause related changes in metabolism, body composition, and other variables in Preliminary Data (p. 33) and cited the published 1995 paper in the Annals of Internal Medicine and a submitted manuscript on the same topic (pp. 25, 29, 33, 40, 44, 49);\n\ne\\. in Project 1 of application P01 AG16782-01, submitted in June 1998, Respondent reported (p. 233) fabricated data showing that menopause led to significant changes in body composition (pp. 229, 230, 231, 232, 233, 246, 256) (Report, p. 32);\n\nf\\. in application 1 R01 AG 18238-01, submitted in April 1999, Respondent reported falsified and fabricated data from his longitudinal menopause study (RMR, leisure time physical activity, fat-free mass, fat mass, waist to hip ratio, and insulin (pp. 9, 18, 19, 20, 22, 23, 33, 37, 44);\n\ng\\. in application 1 R01 AG17906-01, submitted in May 1999, Respondent reported falsified and fabricated data in the description of his longitudinal menopause study (RMR, leisure time physical activity, and fat-free mass, p. 25);\n\nh\\. in Project 1 of application P01 AG16782-01A1, submitted in January 2000, Respondent reported the falsified and fabricated longitudinal menopause study (pp. 214, 220, 221, 228, 250) (Report, p. 32);\n\ni\\. in application 1 R01 AG17906-01A1, submitted in February 2000, Respondent reported the falsified and fabricated longitudinal menopause study (pp. 31 and 59);\n\nj\\. in application 1R01 AG19800-01, submitted in September 2000, Respondent reported the falsified and fabricated longitudinal menopause study (pp. 18 and 43).\n\n**Reporting Research**\n\n8\\. That Respondent continued to publish papers on the fictitious longitudinal menopause study, referring to the same cohort of 35 women, 18 of whom purportedly went through the menopause transition during the six year followup period; all or parts of the following additional papers [^2^](#ftnref2) reported this non-existent study and require correction or retraction:\n\na\\. Poehlman, E.T., Toth, M.J., Ades, P.A., and Rosen, C.J. \"Menopause-associated changes in plasma lipids, insulin-like growth factor I and blood pressure: A longitudinal study.\" European Journal of Clinical Investigation 27(4):322-326, April 1997 (Report, p. 30) (Retraction required);\n\nb\\. Tchernof, A., and Poehlman, E.T. \"Effects of the menopause transition on body fatness and body fat distribution.\" Obesity Research 6(3):246-254, May 1998 (pp. 249-251) (Correction required);\n\nc\\. Tchernof, A., Poehlman, E.T., and Despres, J.P. \"Body fat distribution, the menopause transition, and hormone replacement therapy.\" Diabetes and Metabolism 26(1):12-20, February 2000 (Report, p. 31) (p. 17 Correction required);\n\nd\\. Rawson, E. and Poehlman, E.T. \"Resting metabolic rate and aging.\" Recent Research Developments in Nutrition 4, 2001 (Correction required);\n\ne\\. Poehlman, E.T. \"Menopause, energy expenditure, and body composition.\" Acta Obstet. Gynecol. Scand. 81(7):603-611, July 2002 (Retraction required).\n\nf\\. Poehlman, E.T. and Tchernof, A. \"Traversing the menopause: Changes in energy expenditure and body composition.\" Coronary Artery Disease 9(12):799-803, 1998 (Correction\/retraction required).\n\n9\\. That Respondent reported falsified and fabricated longitudinal menopause data in a talk presented in October 2000 at the annual NAASO meeting and to the Vermont community; specifically he reported to the NAASO falsified RMR and fat mass data on 40 women followed over six years (17 pre-menopausal, 18 post-menopausal, and 5-peri-menopausal) and RMR, FM, F-FM, PAEE, WHR, and insulin (Vermont Community) (Report, pp. 33-34).\n\n**Other**\n\n10\\. That Respondent falsely wrote to the University of Vermont Investigation Committee that the subjects in the longitudinal menopause study had not stayed overnight in the GCRC for the second visit. In fact, no women were seen a second time at the GCRC on an in-patient or outpatient basis (Report, p. 29).\n\nGroup 4: Protocol 646 - Hormone replacement therapy and visceral fat and weight loss; the genetics of an obesity gene.\n\n**Proposing Research**\n\n11\\. That Respondent included Protocol 646 in grant application 2 M01 RR00109-33 (funding for the University of Vermont, GCRC), submitted in February-March 1996, in which he provided falsified and fabricated data on 40 women with and without the variant gene Trp64Arg; falsified parameters included body weight, body mass index, and percent body fat that were falsely claimed to be significantly different between the two groups.\n\n12\\. That Respondent reported falsified and fabricated preliminary data and results in application 1 R01 AG18238 on HRT and its preferential effect on abdominal fat content:\n\na\\. that Respondent, in grant application 1 R01 AG18238-01 (p. 24), submitted in April 1999, presented falsified data in Table 1, on a study of women who had reported to be on, or not on, hormone replacement therapy (HRT); specifically, he claimed that women on HRT had significantly lower intra-abdominal fat than non-users and that there was a significant difference in PAEE between the two groups;\n\nb\\. Respondent also falsely claimed to have evaluated the effect of HRT on intra-abdominal fat loss in a double blind placebo controlled study of 27 weeks duration (Figure 4); the actual study was not unblinded until 2002;\n\nc\\. Respondent also falsely claimed (pp. 36-37) to have completed a six month pilot study on the effect of exercise weight loss on postmenopausal women administered HRT, compared to women not on HRT.\n\n13\\. That Respondent, in grant application 1 P01 AG16782-01A1, submitted in January 2000, presented (p. 230) falsified data:\n\na\\. on a study of women reported to be on, or not on, HRT; specifically the number of subjects in Table 4 was 25 for HRT users and 23 for non-users, while seven of eight values for PAEE and intra-abdominal fat (3 means and 4 standard deviations) were unchanged from Table 1 of Application 1 R01 AG18328-01, where the number of subjects was 13 for each group;\n\nb\\. Respondent repeated the false claim in the April 1999 application to have evaluated the effect of HRT on intra-abdominal fat in a double blind placebo controlled study of 27 weeks duration; the actual study was not unblinded until 2002; Respondent admitted to falsifying the figure in this application relative to the version in the 1 R01 AG18328-01 application;\n\nc\\. Respondent falsely claimed (p. 231) to have studied 8 post-menopausal women on HRT and 7 women not on HRT in a six month weight loss program, when the average ages, standard deviations and certain mean values were unchanged from the smaller and purportedly different, groups described in the April 1999 application ( *see* PHS Issue 12c above).\n\n14\\. That Respondent, in grant application 2 R01 DK052752-05, submitted in June 2000:\n\na\\. falsified the number of subjects carrying or not carrying the Trp64Arg genotype in Tables 4, 5, and 6 (pp. 30-31); specifically in the application, he falsely claimed to have tested 40 in each group; Respondent admitted that the actual number tested varied from 8-13, depending on the group and parameter being measured;\n\nb\\. Respondent also falsely claimed that the number of women recruited to his funded grant on the menopause transition was 85 (p. 49).\n\n15\\. That Respondent, in grant application 1 R01 AG19800-01, submitted in September 2000:\n\na.-c. made the same three false claims with respect to HRT as in application 1 P01 AG16782-01A1 (Findings 13 a-c); in addition, Respondent falsely claimed in Table 5 that the number of subjects with and without HRT participating in the six-month weight loss program (see PHS Issue 13 c. above) was now 10 in each group rather than the group sizes of 8 and 7 claimed in Table 5 of the\n\n1 P01 AG16782-01A1 application; many of the means and standard deviations in these two tables match the values obtained in a 6 month weight loss pilot study described on pp. 36-37 of application 1 R01 AG18238-01, where the two groups were comprised of 3 and 4 individuals; (pp. 13, 15, 17, 20, 21 and Tables 4 and 5 and Figure 6);\n\nd\\. falsely claimed (Table 3, p. 19) to have weight-reduced 70 obese women in the genetic study.\n\n**Reporting Research**\n\n16\\. That in public presentations or material prepared for these fora, Respondent falsified or fabricated data and results of the effects of HRT and of the effects of the Trp64Arg genotype:\n\na\\. that Respondent, at talks given at the annual NAASO meeting in October 2000, and to the Vermont Community (October 17, 2000), presented false information on the effects of HRT on visceral fat loss and glucose disposal when the HRT users and non-users were on a six-month weight loss program;\n\nb\\. that Respondent, in both the NAASO and Vermont Community talks, falsely claimed that Trp64Arg carriers have significantly lower rates of glucose disposal than non-carriers.\n\n**Other**\n\n17\\. that Respondent falsely testified to the University of Vermont Investigation Committee that the slide shown at NAASO regarding the loss of visceral fat in women on or not on HRT during a six-month weight loss program (Issue 16a) had been labeled \"hypothesized.\" Respondent falsely labeled the NAASO slide \"hypothesized\" and submitted it to the University of Vermont Investigation Committee with the intention of misleading the committee (Report, pp. 34, 37).\n\nGroup 5: Alzheimer's disease\n\n18\\. that Respondent, in applications 2 R01 AG07857-06 and 7 R01 AG07857-07, submitted June 26, 1992, and March 28, 1994, respectively, falsified certain preliminary data (average ages, height, and fat-free weight values) to show that the Alzheimer's and control patients were more closely matched for age than shown in the original data;\n\n19\\. that Respondent, in application 5 R01 AG07857-09, submitted May 18, 1995, falsified preliminary data; specifically, compared to data in the preceding 5 R01 AG07857-08 application, where the number of Alzheimer's and control subjects was 7 and 13 respectively, the number of Alzheimer's and control subjects was doubled to 14 and 26 respectively, while many of the data values and standard deviations remained unchanged; in the latter application however, Respondent claimed that Alzheimer's patients had significantly lower fat-free mass and significantly higher fat mass than control patients, while no claim of significant differences had been made in the earlier application.\n\nGroup 6: Effect of endurance training on metabolism\n\n20\\. Respondent admitted to falsifying norepinephrine data (a measure of sympathetic nervous system activity) in two papers published in 1992 and 1994 and agreed to retraction of the papers. ^[3](#ftnref3)^ Specifically:\n\na\\. Respondent falsified norepinephrine data in Table 2 and Figure 4 of Poehlman, E.T., Gardner, A.W., and Goran, M.I. \"Influence of endurance training on energy intake, norepinephrine kinetics, and metabolic rate in older individuals.\" Metabolism 41(9):941-948, September 1992, in order to strengthen the relationship between endurance training and increased norepinephrine levels and rate of appearance (paper to be retracted);\n\nb\\. Respondent falsified norepinephrine data in Table 2 and associated text of Poehlman E.T., Gardner, A.W., Arciero, P.J., Goran, M.I., and Calles-Escandon, J. \"Effects of endurance training on total fat oxidation in elderly persons.\"\n\nJ. Appl. Physiol. 76(6):2281-2287, June 1994, in order to make the claims that norepinephrine concentration and norepinephrine appearance were significantly enhanced following endurance training (paper to be retracted).\n\nDr. Poehlman has entered into a Voluntary Exclusion Agreement (Agreement ) in which he has voluntarily agreed, beginning on March 9, 2005:\n\n\\(1\\) to exclude himself permanently from serving in any advisory capacity to PHS including but not limited to service on any PHS advisory committee, board, and\/or peer review committee, or as a consultant;\n\n\\(2\\) to exclude himself permanently from any contracting or subcontracting with any agency of the United States Government and from eligibility or involvement in nonprocurement programs of the United States Government referred to as \"covered transactions\" as defined in the debarment regulations at 45 C.F.R.\n\nPart 76; the respondent agrees that he will not petition HHS to reverse or reduce the scope of the permanent voluntary exclusion or administrative actions that are the subject of this Agreement; and\n\n\\(3\\) to execute and deliver letters requesting retraction or correction to the editors of the journals that published the ten papers named in the Agreement and cited above, and to sign the letters requesting the retraction or correction prepared for his signature by ORI without alteration or modification in any way.\n\nInquiries\n\nFor further information contact:\n\nDirector**  \n** Division of Investigative Oversight**  \n** Office of Research Integrity**  \n** 1101 Wootton Parkway, Suite 750**  \n** Rockville, MD 20852**  \n** Telephone: (301) 443-5330\n\nFootnotes\n\n^1^ All other reports of the \"longitudinal menopause study\" claimed an average of six (6) years of follow-up.\n\n^2^ The first paper describing the longitudinal menopause study, the 1995 Annals of Internal Medicine paper, was the subject of PHS Issue 6.\n\n^3^ Both the 1992 and 1994 papers were designed to reproduce, under more controlled conditions, an earlier result, published in Poehlman, E. and Danforth, E. \"Endurance training increases metabolic rate and norepinephrine appearance rate in older individuals.\" *Am. J. Physiol* . 261:E233-E239, 1991. These papers claimed that plasma levels of norepinephrine increased significantly in older individuals following endurance training. Because the norepinephrine results in the two carefully controlled studies conducted to verify this finding were falsified, it is apparent that this original report cannot be relied upon.","meta":{"dup_signals":{"dup_doc_count":115,"dup_dump_count":60,"dup_details":{"curated_sources":2,"2022-05":1,"2021-39":1,"2021-21":1,"2021-10":1,"2020-40":1,"2020-34":2,"2020-29":1,"2019-47":1,"2019-39":1,"2019-30":1,"2019-22":1,"2019-18":2,"2019-13":3,"2019-09":3,"2019-04":2,"2018-51":3,"2018-47":2,"2018-43":2,"2018-39":2,"2018-34":3,"2018-30":5,"2018-26":3,"2018-22":3,"2018-13":5,"2018-05":1,"2017-34":1,"2017-30":1,"2017-22":2,"2017-17":2,"2017-09":1,"2017-04":2,"2016-50":2,"2016-44":2,"2016-40":2,"2016-36":2,"2016-30":3,"2016-26":2,"2016-22":1,"2016-18":1,"2016-07":2,"2015-48":1,"2015-40":1,"2015-35":2,"2015-32":2,"2015-27":2,"2015-22":2,"2015-14":2,"2014-52":2,"2014-49":2,"2014-42":2,"2014-41":2,"2014-35":2,"2014-23":4,"2023-23":1,"2015-18":2,"2015-11":1,"2015-06":2,"2014-10":1,"2013-48":2,"2013-20":1}},"file":"PMC4259663"},"subset":"pubmed_central"}
{"text":"abstract: Geographic or ethnic differences in the occurrence of disease often provide insights into causes of disease and possible opportunities for disease prevention. Persons in China appear to have a consistently lower prevalence of rheumatoid arthritis and fibromyalgia than persons in the United States and Europe; reasons for these prevalence differences might include genetic differences, differences in environmental exposures or a combination of both. With increasing obesity, gout is becoming endemic in China. Finally, symptomatic knee osteoarthritis is extremely common in China and constitutes a major public health problem there.\nauthor: David T Felson\ndate: 2008\ninstitute: 1Clinical Epidemiology Research & Training Unit, 650 Albany Street, Suite X-200, Boston, MA 02118-2526, USA\nreferences:\ntitle: Comparing the prevalence of rheumatic diseases in China with the rest of the world\n\nSee related research by Zeng *et al.*, <http:\/\/arthritis-research.com\/content\/10\/1\/R17>\n\nIn the present issue of *Arthritis Research and Therapy* Zeng and colleagues comprehensively review studies evaluating the prevalence of rheumatic disease in China \\[1\\]. Their report suggests variations in the prevalence of symptomatic osteoarthritis throughout China, consistent prevalences for ankylosing spondylitis and rheumatoid arthritis, an absence of fibromyalgia, and a temporal increase in the prevalence of gout. What should we make of all of these estimates and why are they important to us outside China?\n\nEstimates of the prevalence of rheumatic disease provide information about the burden of disease and suggest a need for provision of health services. For knee osteoarthritis, where the prevalence in China is high and knee replacements are not widely available \\[2\\], there may be a need for additional provision of health services.\n\nComparing the prevalence of disease from one region with another often provides insights into disease etiology. For example, coronary heart disease rates increased as Japanese migrated from Japan to Hawaii to mainland United States, suggesting that diet and environment were major causes of coronary heart disease. For systemic lupus, which was not evaluated by Zeng and colleagues, a prevalence gradient exists with low prevalence of lupus occurring in Africans, moderate rates along the slave trade route among African descendents living in the Caribbean, and high rates in the United States \\[3\\]. Using data from the article by Zeng and colleagues, we can make inferences about differences in disease prevalence that might provide clues regarding rheumatic disease etiology.\n\nZeng and colleagues reviewed diverse studies, many of them using the Community Oriented Program for the Control of Rheumatic Diseases (COPCORD) protocol. The COPCORD has been a joint initiative by the World Health Organization and the International League of Associations for Rheumatology whose goal was to provide information about the epidemiology of rheumatic disease in developing countries. The COPCORD methods require comprehensive population-based sampling with a rigorous protocol translated into the language of interest, and then, for persons with rheumatic complaints, there is follow-up testing and often evaluation by a rheumatologist to determine how many persons have identifiable diseases.\n\nEstimates of rheumatic disease derived from the COPCORD and other studies have varied and, despite the impressive standardization of this protocol, variations in prevalence rates are probably still due, in part, to differences in how the studies are carried out from site to site. Also, for rheumatoid arthritis, many of these studies only detected a few cases in a community, so the estimate of prevalence is accompanied by wide confidence bounds, and there was substantial variation in prevalence from community to community. Another variation source is that crude prevalence estimates are usually presented, whereas age-standardized prevalence would often be more informative, especially since rheumatic diseases vary in prevalence by age. An additional source of variation is that some of the studies may not sample large numbers of older people who tend to have the highest prevalence of many rheumatic diseases, including rheumatoid arthritis. Finally, participation rates may differ in each study. While most COPCORD studies have reported participation rates exceeding 80% \\[4,5\\], some studies in Asia have not been so successful \\[6\\], and the failure to include persons with rheumatic diseases might seriously lower prevalence estimates.\n\nNotwithstanding these limitations, the estimates by Zeng and colleagues suggest rheumatic disease prevalence rates in China that are different from those in the western hemisphere or Europe. Among these different rates is a prevalence of rheumatoid arthritis in China of roughly 0.2\u20130.3% of the population versus a prevalence of 0.8% in the western world \\[7\\]. The genes predisposing to rheumatoid arthritis may differ among Han Chinese versus mixed populations from the western world. Viral infectious triggers also could predominate in the West or could infect persons at an age when their response to infections was most likely to cause rheumatoid arthritis in genetically susceptible individuals, whereas similar individuals might be infected at a less susceptible age in China. Certainly, studies of Asian migrants to the western world might help determine whether Asians retain a lower prevalence of rheumatoid arthritis, or whether their rate after migration rises to that of the surrounding population, suggesting that environmental risk factors are the main cause of disease.\n\nZeng and colleagues report a prevalence of ankylosing spondylitis that is similar in China to reported estimates from the western world. In general, the prevalence of HLA-B27 in the community determines its prevalence of spondyloarthropathies. In the Han ethnic population HLA-B27 is present in between 4% and 6% of persons, a figure lower than that in Caucasians in the United States. This would be consistent with a lower prevalence of ankylosing spondylitis in China than the United States, rather than a comparable prevalence. Further documentation of this absence of a prevalence gradient should be confirmed with studies using broader definitions of disease (for example, undifferentiated spondyloarthropathy).\n\nGout has become more prevalent in China, a phenomenon also seen the United States in recent years. Causes include increasing rates of obesity and of aging in both populations, both risk factors strongly associated with the occurrence of gout. The dramatic increase in gout in China probably reflects the rapid westernization and urbanization of the society.\n\nFinally, the absence of fibromyalgia in China could be explained by genetic differences in the processing of afferent nociceptors throughout the body or by the absence of central sensitization, a phenomenon thought to be closely tied to the development of fibromyalgia. Clearly some causes of fibro-myalgia are sociocultural, and it is possible that the different cultural environment in China might affect the acceptability of reporting chronic generalized pain. Investigation of the fibromyalgia prevalence in migrant Chinese communities might provide insights into which factors account for the exceedingly low prevalence of fibromyalgia in China. In addition, given the import of genetic variations in explaining racial and ethnic differences in chronic pain \\[8,9\\], an assessment of the prevalence of these genetic abnormalities in persons of Han ancestry might be of interest.\n\nIn summary, Zeng and colleagues have comprehensively summarized the prevalence of different rheumatic diseases in China. Their results present a mosaic of rheumatic disease prevalence, which differs from the rheumatic disease distributions in the western world. For those of us from the West interested in better understanding the etiology of rheumatic disease, the prevalence gradients that can be inferred from these data regarding rheumatoid arthritis and fibromyalgia, both of which may be considerably less common in China than in the western world, might provide important insights into what causes these diseases.\n\n# Abbreviations\n\nCOPCORD = Community Oriented Program for the Control of Rheumatic Diseases.\n\n# Competing interests\n\nThe authors declare that they have no competing interests.\n\n### Acknowledgements\n\nThe present work was supported by NIH AR47785.","meta":{"dup_signals":{"dup_doc_count":135,"dup_dump_count":49,"dup_details":{"curated_sources":4,"2023-14":1,"2022-21":1,"2021-43":1,"2021-31":1,"2021-17":1,"2021-10":1,"2020-45":1,"2020-24":1,"2020-10":1,"2020-05":1,"2019-51":1,"2019-47":1,"2019-43":1,"2019-39":1,"2019-35":1,"2017-17":1,"2017-09":9,"2017-04":1,"2016-50":1,"2016-44":1,"2016-40":1,"2016-36":10,"2016-30":8,"2016-22":1,"2016-18":1,"2016-07":11,"2015-48":3,"2015-40":4,"2015-35":3,"2015-32":3,"2015-27":3,"2015-22":3,"2015-14":2,"2014-52":2,"2014-49":5,"2014-42":5,"2014-41":5,"2014-35":3,"2014-23":5,"2014-15":3,"2023-50":1,"2017-13":1,"2015-18":4,"2015-11":3,"2015-06":3,"2014-10":3,"2013-48":3,"2013-20":3,"2024-30":1}},"file":"PMC2374465"},"subset":"pubmed_central"}
{"text":"date: 2014-01-10\ntitle: Findings of research misconduct.\n\n# Findings of Research Misconduct\n\n**Notice Number:** NOT-OD-14-038\n\n**Key Dates  \nRelease Date:** January 9, 2014\n\n**Related Announcements  \n** None\n\nNone\n\n**Issued by  \n**Department of Health and Human Services ([DHHS](http:\/\/www.hhs.gov\/))\n\nPurpose\n\nNotice is hereby given that the Office of Research Integrity (ORI) has taken final action in the following case:\n\nTimothy Sheehy, B.A., BSc., SAIC-Frederick, Inc.: Based on the report of an investigation conducted by SAIC-Frederick, Inc., and additional analysis conducted by ORI in its oversight review, ORI found that Mr. Timothy Sheehy, former Manager, DNA Extraction and Staging Laboratory (DESL), SAIC-Frederick, Inc., the Operations and Technical Services (OTC) Contractor for the Frederick National Laboratory for Cancer Research (FNLCR), Frederick, MD, engaged in research misconduct in research supported by National Cancer Institute (NCI), National Institutes of Health (NIH), contract HHSN2612008000001E awarded by FNLCR\/NCI, NIH, to SAIC-Frederick, Inc., and the intramural program at the Occupational and Environmental Epidemiology Branch, Division of Cancer Epidemiology and Genetics, NCI.\n\nORI found that the Respondent engaged in research misconduct by fabricating and\/or falsifying U.S. Public Health Service (PHS)-supported data in Table 1 included in Cancer Epidemiol Biomarkers Prev 19(4):973-977, 2010 (hereafter referred to as the \\`\\`CEBP paper').\n\nSpecifically, ORI found that Respondent fabricated the quantitative and qualitative data for RNA and DNA purportedly extracted from 900 formalin-fixed, paraffin-embedded (FFPE) colorectal tissue samples presented in Table 1 of the CEBP paper and falsely reported successful methodology to simultaneously recover nucleic acids from FFPE tissue specimens, when neither the extractions nor analyses of the FFPE samples were done. Thus, the main conclusions of the CEBP paper are based on fabricated data and are false.\n\nMr. Sheehy has entered into a Voluntary Settlement Agreement and has voluntarily agreed for a period of three (3) years, beginning on November 8, 2013:\n\n\\(1\\) To have his research supervised; Respondent agreed that prior to the submission of an application for U.S. Public Health Service (PHS) support for a research project on which the Respondent's participation is proposed and prior to Respondent's participation in any capacity on PHS-supported research, Respondent shall ensure that a plan for supervision of his duties is submitted to ORI for approval; the supervision plan must be designed to ensure the scientific integrity of Respondent's research contribution; Respondent agreed that he shall not participate in any PHS-supported research until such a supervision plan is submitted to and approved by ORI; Respondent agreed to maintain responsibility for compliance with the agreed-upon supervision plan;\n\n\\(2\\) That any institution employing him shall submit, in conjunction with each application for PHS funds, or report, manuscript, or abstract involving PHS-supported research in which Respondent is involved, a certification to ORI that the data provided by Respondent are based on actual experiments or are otherwise legitimately derived and that the data, procedures, and methodology are accurately reported in the application, report, manuscript, or abstract;\n\n\\(3\\) To exclude himself voluntarily from serving in any advisory capacity to PHS including, but not limited to, service on any PHS advisory committee, board, and\/or peer review committee, or as a consultant; and\n\n\\(4\\) That a letter will be submitted to the editors of CEBP requesting that the journal retract the publication.\n\nInquiries\n\nPlease direct all inquiries to:**  \n  \n** David E. Wright, Ph.D.**  \n** Director**  \n** Office of Research Integrity**  \n** 1101 Wootton Parkway, Suite 750**  \n** Rockville, MD 20852**  \n** Telephone: 240-453-8800","meta":{"dup_signals":{"dup_doc_count":101,"dup_dump_count":39,"dup_details":{"curated_sources":2,"2020-45":1,"2018-51":1,"2018-30":1,"2018-17":1,"2018-05":1,"2017-30":1,"2017-26":1,"2017-17":1,"2017-09":1,"2017-04":1,"2016-50":1,"2016-44":1,"2016-40":1,"2016-36":2,"2016-30":2,"2016-26":2,"2016-22":2,"2016-18":2,"2016-07":4,"2015-48":4,"2015-40":2,"2015-35":5,"2015-32":4,"2015-27":5,"2015-22":5,"2015-14":4,"2014-52":4,"2014-49":2,"2014-42":5,"2014-41":7,"2014-35":2,"2014-23":4,"2014-15":2,"2021-31":1,"2015-18":5,"2015-11":3,"2015-06":4,"2014-10":3,"2017-13":1}},"file":"PMC4259753"},"subset":"pubmed_central"}
{"text":"author: Jeffrey T Leek; Steven L Salzberg\ndate: 2013\ninstitute: 1Department of Biostatistics, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA; 2Center for Computational Biology, Johns Hopkins University, Baltimore, MD, USA; 3McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins School of Medicine, Baltimore, MD, USA\ntitle: Sequestration: inadvertently killing biomedical research to score political points\n\nThe US Congress just allowed the 'sequester' to happen, which means they cut the national budget for biomedical research by 8.2%. What happened to most people? Nothing. As many pundits have already written, the sky didn't fall, and the economy didn't collapse. 'We must cut spending!' they say, 'and look: nothing happened.'\n\nSo the budget cuts must be okay. We'll just trim some wasteful spending from all that research.\n\nCongress, here's what 'nothing' looks like: in 5, maybe 10 years from now, someone you care about gets sick, maybe from cancer. The only treatments are painful and not very effective, so the doctors decide the best option is to do nothing. Or maybe, 10 years from now, you have your first grandchild, but she is born with a life-threatening genetic disease. No treatment is known, so the doctors tell you and your children that nothing can be done.\n\nThat's what 'nothing' looks like. If we take only a short-term perspective, cutting the NIH and NSF research budgets won't seem to change anything outside of the scientific community. So the pundits are right: the sky won't fall, and the economy won't collapse. And in the long term, we'll have nothing.\n\nSo what will happen?\n\nWell, most university research in the US is funded by 3- to 5-year grants from the federal government. Even at 3 years, it is difficult for scientists to plan: it takes about a year to get a grant, which means that when you get a new 3-year grant you only have 2 years to generate results before you have to apply for another one. And when you get a grant, it often takes time before you can find someone qualified to do the work (someone who you may have to train).\n\nDue to uncertainty about sequestration, the NIH funded almost nothing new in the period from September until March, waiting to find out if the sequester would really happen. Literally thousands of biomedical researchers across the country have scrambled to keep funding their labs, waiting for decisions from NIH. As the cuts take effect, many of these labs will be forced to freeze hiring, cut back on experiments and reduce their scientific output.\n\nThe uncertainty is particularly devastating to young scientists. New PhD graduates continued to apply for jobs, but the jobs just haven't been there. 'Wait a while,' some of us said. 'We might have funding in the spring.' Some of the best young scientists won't wait: some have already gone to industry, and some might leave science entirely, discouraged over the sad state of fiscal uncertainty that now seems to be permanent. When highly skilled scientists leave the field, we've lost something that we will never get back.\n\nMeanwhile the cuts from sequestration paint an increasingly grim picture for new investigators just starting their independent careers. These scientists have already poured years into training to build up enough expertise, experience and ideas to build entirely new scientific groups. But this is the most precarious time for a young scientist as they are asked to obtain and maintain funding on their own for the first time. Already, due to the sequester, the NSF announced that over 1,000 new grants will not be awarded. At the same time, the NIH may fund as few as 6% of the grants that are submitted in 2013 (<http:\/\/www.einstein.yu.edu\/administration\/grant-support\/nih-paylines.aspx>) and ongoing grants may be reduced (<http:\/\/nexus.od.nih.gov\/all\/2013\/03\/04\/nih-operations-under-the-sequester\/>). The result is that it may go from being really hard to nearly impossible to get grants. If these young scientists can't support their work, we won't just lose the experiments they were going to perform this year. We may lose an entire generation of young scientists, leading to reduced American productivity in science that could last for decades (<http:\/\/www.theatlantic.com\/politics\/archive\/2013\/03\/the-sequester-is-going-to-devastate-us-science-research-for-decades\/273925\/>).\n\n# The worst part: they don't really mean it!\n\nOne of the more frustrating elements of the sequester fight is that no one in Congress seems to be in favor of pulling the rug out from under biomedical research. But due to their own inability to agree on larger budget issues, they are doing it anyway. And it's not just the sequester: the latest cuts come on top of a multi-year freeze in NIH funding, in which annual budgets have been flat or declining in real terms.\n\nScience doesn't operate on a short-term basis. Cures for major diseases take years, often decades, to develop. And within the field, we can see tremendous progress on many fronts. Our understanding of cancer, heart disease, infectious diseases, aging, genetic diseases and a host of other conditions is moving ahead in leaps and bounds. This is the time to invest more, not less.\n\nAlthough these cuts are devastating to NIH research, in the context of the US budget, they won't affect a thing. The entire NIH budget (<http:\/\/www.nih.gov\/about\/budget.htm>), supporting research on hundreds of diseases, is about \\$31 billion. The total US budget last year was \\$3.7 trillion. Thus an 8.2% cut from NIH saves just 0.07% of the federal budget. Or to put it another way: the Pentagon program to build the F-35 Joint Strike Fighter, which won't be ready until at least 2019, is already estimated to cost \\$400 billion (<http:\/\/www.gao.gov\/assets\/660\/652948.pdf>), equal to the entire NIH budget for 13 years. And that's just one plane.\n\nWhat's more, NIH funding is a terrific investment. A nonpartisan study in 2000 (<http:\/\/www.faseb.org\/portals\/0\/pdfs\/opa\/2008\/nih_research_benefits.pdf>) found that publicly funded research yields a return of 25% to 40% per year. Where else can you get that kind of return on anything? But because this is a long-term investment, private businesses won't fund it, although they too benefit from the stream of discoveries and new technologies supported by public research funds.\n\nWe recently bailed out Wall Street because it is 'too big to fail.' Our biomedical research enterprise is not too big to fail - financially, that is. But how sick do we have to get before we decide we shouldn't have cut our investment in biomedical research?\n\nCongress can do many things to show that they care about curing disease. If they really do care, they should first restore NIH's budget for this year. Then they should take action to insulate biomedical research from the endless, internecine battles between the parties. A good first step would be to approve multi-year funding plans for NIH and NSF, just as these agencies award multi-year grants to scientists. The default action should leave these multi-year plans in place. So the 'do nothing' option each year wouldn't cut the legs out from under active research projects, but instead would ensure at least level funding from year to year.\n\nBy the time scientists reach the point where they can lead productive research programs, we've already invested heavily in their training. Let's not kick them out on the street because someone in Congress wants to score political points. The health of the nation deserves better.","meta":{"dup_signals":{"dup_doc_count":143,"dup_dump_count":46,"dup_details":{"curated_sources":2,"2022-21":1,"2021-49":1,"2020-24":1,"2019-47":1,"2019-30":1,"2019-22":2,"2019-09":1,"2018-51":2,"2018-47":1,"2018-39":1,"2018-30":1,"2018-22":1,"2018-05":2,"2017-39":1,"2017-34":1,"2017-22":1,"2017-09":11,"2016-44":1,"2016-40":1,"2016-36":11,"2016-30":8,"2016-22":1,"2016-18":1,"2016-07":2,"2015-48":4,"2015-40":3,"2015-35":4,"2015-32":3,"2015-27":4,"2015-22":4,"2015-14":3,"2014-52":4,"2014-49":7,"2014-42":8,"2014-41":5,"2014-35":6,"2014-23":6,"2014-15":4,"2023-23":1,"2024-10":1,"2015-18":4,"2015-11":3,"2015-06":4,"2014-10":4,"2013-48":3,"2024-26":1}},"file":"PMC3663088"},"subset":"pubmed_central"}
{"text":"abstract: *Daphnia pulex* is the first crustacean to have its genome sequenced. Availability of the genome sequence will have implications for research in aquatic ecology and evolution in particular, as addressed by a series of papers published recently in *BMC Evolutionary Biology* and *BMC Genomics*.\n  .\n  See research articles <http:\/\/www.biomedcentral.com\/1471-2148\/9\/78>, <http:\/\/www.biomedcentral.com\/1471-2164\/10\/527>, <http:\/\/www.biomedcentral.com\/1471-2148\/9\/79>, <http:\/\/www.biomedcentral.com\/1471-2164\/10\/175>, <http:\/\/www.biomedcentral.com\/1471-2164\/10\/172>, <http:\/\/www.biomedcentral.com\/1471-2164\/10\/169>, <http:\/\/www.biomedcentral.com\/1471-2164\/10\/170> and <http:\/\/www.biomedcentral.com\/1471-2148\/9\/243>.\nauthor: Angelika Stollewerk\ndate: 2010\ninstitute: 1Queen Mary, University of London, School of Biological and Chemical Sciences, Mile End Road, London E1 4NS, UK\nreferences:\ntitle: The water flea *Daphnia* - a 'new' model system for ecology and evolution?\n\nOne of the major questions in evolutionary biology is to understand how species have adapted to different environments and how the underlying changes in morphology, physiology and behavior relate to modifications in the corresponding genes. The publication of the first crustacean genome sequence, that of *Daphnia pulex* \\[1,2\\], is part of an effort by the members of the *Daphnia* Genome Consortium to establish *Daphnia* as a model system for evolutionary environmental genomics. But can *Daphnia* rise to the challenge?\n\nThe vast number of publications on *Daphnia* in the literature prove that this animal is no newcomer to scientific research. *Daphnia* is most probably one of the best-studied subjects in ecology \\[3\\]. Populations can be found in freshwater environments ranging from huge lakes to small temporary pools and seasonally flooded depressions. The ecology of *Daphnia* has been studied from the point of view of its role as a primary consumer in aquatic food chains, its phenotypic plasticity, and its behavior, toxicology and the evolution of sexual and asexual reproduction. Extensive studies on the population genetics of *Daphnia* have addressed migration and gene flow, hybridization and inbreeding, among other topics. With the availability of the genome sequence, *Daphnia* research has now the potential to reach a new level. A number of papers on the *D. pulex* genome in relation to different aspects of *Daphnia* biology have been published in *BMC Evolutionary Biology* and *BMC Genomics* to accompany the genome release \\[4-11\\]. These constitute an initial exploration of the genome, and in this article I review some of the highlights and questions raised.\n\n# *Daphnia* ecology and life style\n\n*Daphnia* are filter feeders that direct small suspended particles into their mouth by a water current produced by their leaf-like legs (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>). *Daphnia*'s common name of 'water flea' comes from its jump-like movement, which results from the beat of the large antennae used for swimming (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>). In a normal growth season *Daphnia* generates diploid eggs by asexual reproduction (parthenogenesis). These eggs develop directly into larvae in the female brood chamber and are released into the water after about 3 days. In most species the larvae go through four to six larval stages before developing into sexually mature adults. However, the *Daphnia* life cycle is adapted to extreme environmental conditions such as cold winters or summer droughts. If triggered by external stimuli such as high population density and a scarcity of food, *Daphnia* can produce haploid resting eggs by meiosis; these require fertilization and a period of extended dormancy in order to develop \\[3\\]. Resting eggs are distributed by wind or animals and development is resumed in response to external stimuli (for example, rising temperature). Cyclic parthenogenesis, in which parthenogenesis and sexual reproduction alternate, is common in most *Daphnia* species, but lineages have been described that exclusively reproduce asexually (obligate parthenogenesis). Cyclic parthenogenetic *Daphnia* must contain the molecular tools for the production of both haploid gametes (by meiosis) and diploid eggs (by mitosis), the latter developing parthenogenetically into diploid zygotes. This makes *Daphnia* an ideal system to study the evolution of the molecular processes of parthenogenesis.\n\nIn this regard, Eads and co-workers (Schurko *et al*. \\[4\\]) suggest that differences between sexual and asexual reproduction most probably relate to mechanisms that differ between meiosis and mitosis, such as kinetochore orientation, DNA recombination and sister-chromatid cohesion, and have screened the *D. pulex* genome for genes associated with meiosis. The authors report an inventory of 130 *D. pulex* genes that are homologous with known genes in other organisms and which represent more than 40 distinct protein-coding genes with diverse roles in meiosis. The majority of these genes are present in multiple copies, and Schurko *et al*. \\[4\\] speculate that the extra copies may be partly responsible for changes to these meiotic processes that enable parthenogenesis. Parthenogenetic species are present in all major animal phyla and future comparison of the genomes of cyclic and obligate parthenogenetic lineages will shed light on the evolution of the underlying molecular processes.\n\nThe offspring produced in parthenogenetic cycles are genetic clones of their mother \\[3\\]. This includes the males, as sex is environmentally determined in *Daphnia*. The existence of clonal reproduction is a powerful tool for quantitative genetic studies because it facilitates the analysis of genetic variation within and between populations. Genetic variation has been reported in *Daphnia* for a vast number of traits such as size, aging, behavior (for example, vertical migration, fish-escape behavior), morphology (for example, defensive spines, helmets), and the immune system (for example, resistance against parasites, immune responses), and the great number of duplicated genes in *Daphnia* seems to correlate with *Daphnia*'s considerable phenotypic plasticity \\[3\\].\n\n# Predators and other enemies\n\nInterestingly, several of the above-mentioned traits can be induced by environmental cues. Changes in both the behavior and the morphology of *Daphnia* can, for example, be affected by predator-borne chemical cues (kairomones). In the presence of fish kairomones, *Daphnia magna* gives rise to smaller offspring, whereas chemical cues from the phantom midge *Chaoborus flavicans* induce the generation of larger progeny. This has been shown to be an adaptive phenotypic plasticity that helps avoid predation as fish and midges prefer different sizes of prey. These observations raise the question of the nature of the molecular response to kairomones. Schwarzenberger *et al*. \\[5\\] have addressed this question by comparing the expression levels of genes involved in protein biosynthesis and catabolism in *D. magna* in the presence or absence of kairomones. Interestingly, they found that expression of the cyclophilin gene, which encodes an enzyme involved in protein folding, is upregulated in the presence of fish kairomones but downregulated by *Chaoborus kairomones*, which correlates with the opposite effects of these kairomones on progeny size. The authors used the *D. magna* cyclophilin sequence to search the *D. pulex* genome and identified 16 paralogs, which showed a very high variability. Future research will show whether the differences in cyclophilin expression levels can be linked to the observed phenotypic variations and if additional paralogs are involved in the process.\n\nThe first step in kairomone-mediated adaptive changes in behavior and morphology is obviously the reception of the chemical signal by specialized sensory structures of the prey. Our knowledge about chemoreception in aquatic organisms is fragmentary, however. In insects, a conserved chemoreceptor superfamily has been identified which can be subdivided into the gustatory (taste) receptor family and the odorant (smell) receptor family. It is obvious that the sensing of odorants will be different in water than in air as aquatic odorants are hydrophilic molecules dissolved in water whereas airborne odorants are mainly hydrophobic volatile molecules in gaseous form. Penalva-Arana *et al.* \\[6\\] have identified 58 orthologs of the insect gustatory receptor family in the *D. pulex* genome. Interestingly, they found no evidence of genes homologous with insect odorant receptor genes and suggest that the odorant receptor family evolved concomitantly with the transition from sea to land in the lineage leading to the insects.\n\nPredators are not the only natural enemies of *Daphnia*. The study of parasites (viruses, bacteria and multicellular parasites) has also gained momentum as a result of their influence on *Daphnia* ecology and evolution \\[3\\]. Parasites can directly or indirectly affect host population dynamics, extinction, and maintenance of genetic diversity, among other features. It has been suggested that hosts continuously evolve to reduce parasite virulence, whereas parasites evolve to keep virulence as close as possible to an optimum level. Variation in resistance to infection has indeed been observed in natural fruit fly populations and has been associated with genetic polymorphisms \\[12\\].\n\nAll metazoans seem to have an innate immune system, and in insects, at least four different signaling pathways are involved in the immune response and mediate pathogen recognition, attack on the pathogen, and antiviral RNA interference, among other responses. McTaggart *etal.* \\[7\\] analyzed the *D. pulex* genome for genes related to the immune system and identified genes homologous with those in other arthropods. The authors found that the Toll signaling pathway, which is activated by the presence of pathogens, is conserved between insects and *Daphnia*. The activation of this pathway results in the production of antibacterial and antifungal proteins. These antimicrobial peptides could not be recovered from the *D. pulex* genome and thus seem to be less well conserved. In addition, McTaggart *et al.* \\[7\\] report considerable variation in gene family copy number in *Daphnia* and insects. These differences might reflect the evolutionary history of host-parasite interactions in the individual lineages. Further comparative studies are needed to uncover evolutionary changes in genes that mediate immune responses as well as taxon-specific expansions of gene families, which will contribute to our understanding of how host genes are evolving in response to parasites.\n\nAlthough a vast number of ecto-and endoparasites have been described for *Daphnia*, the non-parasitic symbionts have not been analyzed in detail. Ebert and co-workers (Qi *et al*. \\[8\\]) have used metagenomics to address this question. Metagenomes genetic material recovered directly from environmental samples are sequenced and compared to the databases in order to characterize the biological community of a given habitat. One of the advantages of this approach is the recovery of DNA sequences from organisms that cannot be cultured. Ebert and co-workers \\[8\\] searched the shotgun sequences of three clones of three different *Daphnia* species (*D. pulex, D. magna* and *D. pulicaria*) for indications of bacterial and plastid symbionts and found sequences representing a large number of bacterial species in each dataset. The majority of the sequences were from the Proteobacteria but many other taxa were also detected. No clear evidence was found for the presence of symbiotic cyanobacteria or of plastids. Interestingly, the composition of the bacterial communities was similar at genus and higher taxonomic levels in all three *Daphnia* clones, but different bacterial species were present in individual clones. The *D. pulex* and *D. pulicaria* DNA used in this study was isolated from clones cultured in North America, whereas the *D. magna* cultures originated from a laboratory in Switzerland. Since contamination of the *Daphnia* cultures by cross-Atlantic exchange is unlikely, the authors suggest that the similarities between the symbiont communities in European and North American *Daphnia* samples indicate a long-term stability of symbiotic associations.\n\n# Environmental challenges\n\n*Daphnia* species have been studied extensively because of their importance to aquatic ecosystems, and they show a striking ability to contend with environmental challenges. The availability of the *D. pulex* genome should now be able to provide insights into the adaptation to specific environmental conditions, from the ecological to the genetic level. On screening the *D. pulex* genome for genes involved in the biochemical response to toxicants, Baldwin *et al.* \\[9\\] identified 75 genes of the cytochrome P450 family, a protein family important in tolerance and resistance to environmental chemicals. The authors report that the same subgroups of cytochrome P450 genes are present in the *Daphnia* genome as in insects and nematodes, but they discovered distinct changes in the size and gene composition of each group. Dean and co-workers (Sturm *et al*. \\[10\\]) screened the *Daphnia* genome for the presence of members of the ABC transporter superfamily (ATP-binding cassette membrane transport proteins), which are also involved in biochemical defense against toxicants. They found that ABC family representation in *Daphnia* is as complex as in other metazoans, and that *Daphnia* most resembles the fruit fly in respect of its ABC transporter genes. Future studies on the expression and function of these genes will uncover their importance in the adaptation of *Daphnia* to environmental toxicants.\n\n# *Daphnia* and arthropod phylogeny\n\nThe *D. pulex* genome also has the potential to contribute to resolving long-standing debates on arthropod phylogeny. Current views of arthropod phylogenetic relationships are based mainly on two types of datasets - molecular genetic data and morphological characters - and this has led to partly contradictory concepts of arthropod phylogeny. There is now almost universal agreement that arthropods derive from a common ancestor, and that crustaceans and insects are sister groups \\[13\\]. However, some issues of arthropod relationships remain controversial, for example the question of whether insects, crustaceans and myriapods form a monophyletic group. Crustaceans show the greatest diversity of body organization and development among arthropods \\[14\\] and therefore the phylogenetic relationships within the crustaceans are far from being resolved. Several morphological and molecular studies have questioned the monophyly of crustaceans, and either Branchiopoda (such as *Daphnia*) or Malacostraca (lobster, shrimps) has been hypothesized to be the sister group to insects \\[15\\]. Some recent molecular analyses suggest a sister group relationship of myriapods (millipedes) and chelicerates (spiders) \\[16\\]. Interestingly, this suggestion is supported by recent morphological and molecular studies on the development of the nervous system that reveal a surprising degree of similarity between myriapods and chelicerates \\[17,18\\]. The morphological support for an insect-crustacean sister-group relationship is mainly based on the comparative analysis of neural characters in higher crustaceans (malacostracans) and insects. For example, in both insects and malacostracans, stem-cell-like neuroblasts have been detected that divide asymmetrically to generate the cells that contribute to the nervous system \\[14\\]. But are these neural characters representative of all crustacean groups? Are homologous genes required for the development and the function of the nervous system? With the availability of a branchiopod genome and the development of genetic tools for *Daphnia* these questions can now be addressed.\n\nFurthermore, using genome sequences of a wide range of organisms, the origin and evolution of neural signaling pathways can be traced, which will broaden our understanding of the evolution of nervous systems. The neurotrophin signaling pathway plays a role in neural development, regeneration and neural plasticity in mammals. Analyzing the *Daphnia* genome, Wilson \\[11\\] shows for the first time that a neurotrophin and both a tyrosine receptor kinase (Trk) and a p75-type neurotrophin receptor (p75NTR) are present in a protostome, indicating that this pathway existed in the last common ancestor of protostomes and deuterostomes.\n\nTo conclude, the initial exploration of the *D. pulex* genome outlined above proves that with the availability of the genome sequence *Daphnia* research has entered a new era. New and long-standing questions in ecology and evolution can be addressed and it may finally be possible to link evolutionary and environmental adaptations to the underlying genetic processes.\n\n## Acknowledgements\n\nI am grateful to Dieter Ebert for discussions and comments on the manuscript and I thank Petra Ungerer for the scanning electron micrograph of *Daphnia*. The work was supported by a grant to AS from the BBSRC.","meta":{"dup_signals":{"dup_doc_count":138,"dup_dump_count":48,"dup_details":{"curated_sources":2,"2021-10":1,"2020-50":1,"2020-40":1,"2020-29":1,"2020-16":1,"2020-05":1,"2019-47":1,"2019-43":1,"2019-35":1,"2019-18":1,"2019-09":1,"2018-51":1,"2018-30":1,"2018-22":1,"2018-09":1,"2018-05":1,"2017-39":1,"2017-34":1,"2017-30":1,"2017-22":1,"2017-17":1,"2017-09":11,"2016-44":1,"2016-40":1,"2016-36":13,"2016-30":12,"2015-48":3,"2015-40":3,"2015-35":3,"2015-32":3,"2015-27":3,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":4,"2014-42":10,"2014-41":5,"2014-35":5,"2014-23":5,"2014-15":4,"2021-21":1,"2015-18":3,"2015-11":3,"2015-06":3,"2014-10":4,"2013-48":3,"2013-20":2,"2024-22":1}},"file":"PMC2871515"},"subset":"pubmed_central"}
{"text":"abstract: Technical wizardry has made possible the combination of two powerful methodologies, optical trapping and single-molecule fluorescence, to measure precisely the force required to unzip DNA.\nauthor: Jonathan B Weitzman\ndate: 2003\nreferences:\ntitle: A marriage of techniques\n\nCombining scientific techniques that measure different parameters can create powerful and effective experimental methodologies. Indeed, many scientific advances are driven by the success of new technological achievements. But marrying techniques that might have very distinct technical requirements is often extremely challenging. The article by Matthew Lang and colleagues in this issue of the *Journal of Biology* \\[1\\] describes the first successful combination of **optical trap** and **single-molecule fluorescence** technologies in a single experiment and on a single molecule (see <a href=\"#F1\" data-ref-type=\"fig\">'The bottom line'<\/a> box for a summary of the work and the <a href=\"#F2\" data-ref-type=\"fig\">'Background'<\/a> box for definitions).\n\nThese two key technologies, optical traps and single-molecule fluorescence, have dominated the field of single-molecule research ever since they were independently developed over 15 years ago. Optical traps use the radiation pressure from a focused laser beam to trap and move single molecules. They can generate forces in the piconewton range, can measure displacements of nanometers, and have been creatively applied to the study of molecular motors such as kinesins moving along microtubules and to actin-myosin dynamics. Attaching fluorophores to biological macromolecules (protein or nucleic acids) allows investigators to image individual molecules by fluorescence microscopy. Single-molecule fluorescence has provided insights into many biological systems; for example, pioneering studies by Toshio Yanagida and colleagues in Osaka, Japan, investigated the kinetics of myosin binding to ATP using Cy3-labelled fluorescent ATP analogs \\[2\\]. Modern single-molecule fluorescence applications include **FRET (fluorescence resonance energy transfer)**, to measure nanometer distances between different fluorescent moieties, and fluorescence quenching.\n\nIt has been clear for some time that combining optical trapping and single-molecule fluorescence would offer a powerful approach to monitoring spatial or conformational changes in a temporal manner at the single-molecule level. But several inherent features of the two techniques have prevented this marriage. \"The infrared optical trapping light is extremely intense\", explains Steven Block of Stanford University, senior author of the *Journal of Biology* article. In contrast the single-molecule fluorophores have a relatively weak signal. The flux of the high intensity optical traps causes photobleaching of the fluorophores, and any leakage into the light channel drowns out the fluorescent signal. This probably explains why previous attempts to combine optical traps and single-molecule fluorescence failed. Some studies have circumvented these difficulties by sequential use of optical trap and single-molecule fluorescence, or the use of beads or microneedles to spatially separate the trap from the fluorophore \\[3\\]. But these solutions create their own constraints on experimental design and interpretation.\n\nLang *et al*. managed to overcome these technical problems and apply the two techniques simultaneously to measure the force required to prize apart two strands of DNA. They used complementary DNA strands labeled with rhodamine dye; one of the DNA strands was attached to a polystyrene bead and the other to a glass coverslip. The fluorescence of the dyes was quenched as a result of their proximity. The bead was trapped by the laser beam and the stage moved so as to 'unzip' the DNA strands. As the two strands are ripped apart the fluorophores separate, relieving the auto-quenching and releasing a short burst of light.\n\n\"It's a technical *tour de force*\", says Brandeis University's Jeff Gelles. \"This is an important technical development because it integrates two technologies that need to be done in very different conditions.\" Although there are lots of groups using optical traps and lots using single-molecule fluorescence, few have the technical know-how to overcome the challenge of combining them. \"The Block lab has always been a leader in showing us all how to do these things,\" says Gelles. Block explains that his group's success required bringing together expertise from several fields. He says \"It has all been made possible by extremely careful optical design, the use of special filters, a judicious choice of fluorescent dyes and the speed gained by automating many aspects of the experiment.\" (See the <a href=\"#F3\" data-ref-type=\"fig\">'Behind the scenes'<\/a> box for more of the background to the work.)\n\nNow that combined single-molecule fluorescence and optical trapping has been achieved, single-molecule researchers are predicting that it will find applications in a wide range of fields. In fact, it may be useful in any system that explores the conformational changes and biochemical steps involved in macromolecular function. Recent applications of optical trapping include studies of catalytic RNA functions, the chemical properties of DNA and RNA molecules, protein folding and the activities of DNA polymerases. And the combined optical trap and single-molecule fluorescence methodology will enhance the traditional single-molecule study of proteins such as the molecular motors kinesin and myosin. The marriage of optical trapping and single-molecule fluorescence opens up a wealth of new potential applications. Now that the two are finally together, the months ahead should prove to be an exciting honeymoon period for single-molecule research.\n\n## Figures and Tables","meta":{"dup_signals":{"dup_doc_count":102,"dup_dump_count":44,"dup_details":{"curated_sources":2,"2022-21":1,"2020-34":1,"2020-24":1,"2020-10":1,"2019-47":1,"2019-39":1,"2019-26":1,"2019-13":1,"2019-04":1,"2018-47":1,"2018-39":1,"2018-26":1,"2018-05":1,"2017-39":1,"2017-30":1,"2017-22":1,"2017-17":1,"2017-09":11,"2016-44":1,"2016-40":1,"2016-36":11,"2016-30":8,"2016-22":1,"2016-18":1,"2015-48":2,"2015-40":2,"2015-35":2,"2015-32":2,"2015-22":2,"2015-14":2,"2014-52":2,"2014-49":3,"2014-42":6,"2014-41":3,"2014-35":4,"2014-23":2,"2014-15":4,"2022-49":1,"2015-18":2,"2015-11":2,"2015-06":2,"2014-10":3,"2013-48":2,"2013-20":1}},"file":"PMC156593"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  There is considerable interest in the development of methods to efficiently identify all coding variants present in large sample sets of humans. There are three approaches possible: whole-genome sequencing, whole-exome sequencing using exon capture methods, and RNA-Seq. While whole-genome sequencing is the most complete, it remains sufficiently expensive that cost effective alternatives are important.\n  .\n  # Results\n  .\n  Here we provide a systematic exploration of how well RNA-Seq can identify human coding variants by comparing variants identified through high coverage whole-genome sequencing to those identified by high coverage RNA-Seq in the same individual. This comparison allowed us to directly evaluate the sensitivity and specificity of RNA-Seq in identifying coding variants, and to evaluate how key parameters such as the degree of coverage and the expression levels of genes interact to influence performance. We find that although only 40% of exonic variants identified by whole genome sequencing were captured using RNA-Seq; this number rose to 81% when concentrating on genes known to be well-expressed in the source tissue. We also find that a high false positive rate can be problematic when working with RNA-Seq data, especially at higher levels of coverage.\n  .\n  # Conclusions\n  .\n  We conclude that as long as a tissue relevant to the trait under study is available and suitable quality control screens are implemented, RNA-Seq is a fast and inexpensive alternative approach for finding coding variants in genes with sufficiently high expression levels.\nauthor: Elizabeth T Cirulli; Abanish Singh; Kevin V Shianna; Dongliang Ge; Jason P Smith; Jessica M Maia; Erin L Heinzen; James J Goedert; David B Goldstein\ndate: 2010\ninstitute: 1Center for Human Genome Variation, Duke University School of Medicine, Box 91009, Durham, NC 27708, USA; 2Infections and Immunoepidemiology Branch, Division of Cancer Epidemiology and Genetics, US National Cancer Institutes of Health, 6120 Executive Boulevard, Rockville, MD 20852, USA\nreferences:\ntitle: Screening the human exome: a comparison of whole genome and whole transcriptome sequencing\n\n# Background\n\nThe study of common human diseases is rapidly moving away from an exclusive focus on common variants using genome-wide association studies and toward sequencing approaches that represent most variants, including those that are rare in the general population.\n\nAlthough rapidly falling, the per base costs of next generation sequencing platforms still preclude the generation of large sample sizes of entirely sequenced genomes at high coverage. In addition to this economic constraint, it is widely appreciated that the very large number of variants identified in such studies will make it difficult to use association evidence alone to identify causal sites. For these reasons, there has been considerable interest in focusing attention on coding variants as a first step at complete representation of human variation. Part of the motivation for this approach stems from the experience with Mendelian diseases, in which 59% of the causal variants are either missense or nonsense mutations \\[1\\]. Although there has been considerable speculation on the topic, there are in fact no solid data showing that the picture is any different for common diseases, which may also be influenced by variants that are in or near protein coding sequence \\[1\\].\n\nThe most comprehensive approach for focusing on exons alone is clearly exome capture, where regions matching a defined set of coding exons are pulled from the genomic DNA (gDNA) using microarrays and then sequenced. However, this approach requires an initial and costly hybridization step. The cost of exome sequencing has contributed to the interest in sequencing the transcriptome (RNA-Seq) as an alternative, and possibly easier and less expensive strategy \\[2\\]. While this approach will clearly miss poorly expressed genes in whatever tissue is being studied, it does have the advantage of generating additional information, such as gene expression level and splicing patterns.\n\nAlthough exome capture was demonstrated to identify approximately 95% of genomic single nucleotide variants (SNVs) in curated and non-paralogous exons \\[3\\], it is not currently known to what extent SNVs identified by RNA-Seq capture the full set of exonic SNVs identified by genomic sequencing. If the ability to capture SNVs by RNA-Seq is highly dependent on expression level, then this method would be useful only when performed in the appropriate tissue type. If, on the other hand, RNA-Seq at high coverage allows SNVs to be captured even in genes that are not highly expressed, then both methods could be useful for opening up sequencing studies to larger datasets in more diverse scientific studies.\n\nHere, we have sequenced the entire genome and transcriptome of a single individual to high coverage. By comparing the SNVs identified in the transcriptome at different levels of coverage to those identified in the gDNA, we are able to directly evaluate how well RNA-Seq captures genomic variants.\n\n# Results\n\n## Alignment and coverage\n\nBoth DNA and RNA were extracted from peripheral blood mononuclear cells (PBMCs) from the same individual. Both the cDNA and gDNA were sequenced using the Illumina Genome Analyzer II. Sequencing of the gDNA produced 1,450 million reads, each 75 bp long. Ninety percent of these reads were aligned to the human reference genome by BWA \\[4\\], and after removing potential PCR duplicates, the remaining 980 million reads produced a coverage of at least 5\u00d7 for 94% of the bases in the genome (gaps in reference genome excluded), and the mean coverage for these bases was 24\u00d7.\n\nSequencing of the cDNA produced 280 million reads, half 75 bp long and half 68 bp long. TopHat \\[5\\] was used to align these reads to the reference genome, with exons and splice junctions restricted to the 45,455 protein-coding transcripts annotated in Ensembl version 50. Sixty-nine percent of these reads gave unique alignments, aligning to exactly one location in the specified transcriptome. Reads aligning to more than one location were discarded. After removing potential PCR duplicates, the remaining 81 million reads produced a mean coverage of above 5\u00d7 for 51% of exons: these exons had a median coverage of 51\u00d7.\n\n## Single nucleotide variants and overlap between datasets\n\nWe used SAMtools to call SNVs in our aligned gDNA and cDNA sequences. Indels and large structural variants were not analyzed. SAMtools called 51,055 SNVs in protein-coding exons in gDNA and 64,128 in cDNA. Of these, 48,740 in gDNA and 40,605 in cDNA passed quality control filters, and 19,054 of these overlapped between the two datasets in terms of position. When considering overlap between cDNA and genomic SNV calls, two measures were examined: sensitivity and specificity. Sensitivity was defined as the number of true positives (SNVs overlapping between the two datasets) divided by the number of true positives plus the number of false negatives (SNVs existing in the gDNA but not the cDNA). Specificity was defined as the number of true positives divided by the number of true positives plus the number of false positives (SNVs existing in the cDNA but not the gDNA). Quality control filters were optimized to maximize both the sensitivity and specificity in this study. In this dataset the sensitivity was 0.39 and the specificity was 0.47. If an exact match of the genotype was required as well as location, then the sensitivity fell to 0.35 and specificity to 0.42.\n\nSNVs called in the gDNA and cDNA were also compared with entries in dbSNP. It was found that 90% of the gDNA exonic SNVs corresponded to a dbSNP entry, while this was true of only 56% of the cDNA SNVs. However, a further breakdown revealed that 94% of the true positive cDNA SNVs corresponded to a dbSNP entry, while only 23% of the false positives did the same. The false negatives corresponded to dbSNP entries 89% of the time.\n\n## SNV identification at different levels of expression and coverage\n\nMany of the exons in Ensembl's transcript library are hypothetical and not confirmed to be expressed. A list of core exons as defined by the Affymetrix (Santa Clara, California, USA) Human Exon 1.0 ST Array was utilized to focus on exons with better curation. This list was further screened to only include exons present in Ensembl's list of canonical transcripts, resulting in 172,739 core exons. When focusing on just core exons, sensitivity and specificity rose to 0.44 and 0.55, respectively (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>), which coincided with the percentage of exons having at least 5\u00d7 coverage rising to 61% (median coverage for these exons was 57\u00d7).\n\nWe then evaluated how the number of reads and the level of expression affected the specificity and sensitivity of cDNA sequencing. Using data from previous studies on the level to which most of the transcripts containing these core exons are expressed in PBMCs \\[6\\], we defined expression level for each transcript as a percentage of the most highly expressed transcript in that tissue. For exons in our dataset the sensitivity and specificity both rise as known PBMC expression increases, until an expression level of 4% of the most highly expressed transcript, at which point both measures asymptote, with variants called about equally well for all expression levels above this (Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>). Ninety-four percent of exons from genes above this expression level, or 'PBMC-expressed genes', had at least 5\u00d7 coverage, and the median coverage for exons with at least 5\u00d7 coverage was 126\u00d7. The sensitivity also rose to 0.81 and the specificity to 0.67.\n\nWe also evaluated how the absolute number of true positive SNVs called depends on the amount of sequence data, in lanes, for all exons and for exons from PBMC-expressed genes (Figure <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>). Seventy-nine percent of the 6,434 true positive variants identified in PBMC-expressed genes were identified with even one lane of sequence data, which is approximately 35 million reads in this instance. The total number of variants identified in all genes, however, increased substantially as more lanes were added. For the approximately 4,500 PBMC-expressed genes (Figure <a href=\"#F4\" data-ref-type=\"fig\">4<\/a>), even a single lane can be expected to capture most of the coding variants present.\n\nWe also found that the percent overlap with dbSNP changed as expression level and coverage changed. Although the percentage of SNV calls with a corresponding dbSNP entry remained relatively stable at all expression and coverage levels for the true positives and false negatives in our dataset, this was not true of the false positives. The percentage of false positives that overlapped with dbSNP decreased as coverage increased (Supplemental figure S3 in Additional file <a href=\"#S1\" data-ref-type=\"supplementary-material\">1<\/a>) and increased as expression level increased (Supplemental figure S4 in Additional file <a href=\"#S1\" data-ref-type=\"supplementary-material\">1<\/a>).\n\n## SNV identification in genes with and without paralogs\n\nAn inspection of false positive SNVs identified in the cDNA revealed that some arose from alignment of a read to the wrong gene. In these cases the correct gene and the gene chosen for alignment always had very similar sequences. To determine if specificity would increase in a set of unrelated genes, SNVs were called separately for two groups of genes: those with paralogs, as annotated by Ensembl, and those without. When SNVs were restricted to exons in 12,124 transcripts from genes without annotated paralogs, the overall sensitivity rose from 0.39 to 0.42 and the specificity rose from 0.47 to 0.54. If restricted to PBMC-expressed genes without paralogs, the sensitivity actually dropped slightly from 0.81 to 0.80, but the specificity again rose from 0.67 to 0.72. In contrast, for SNVs in exons of 33,331 transcripts from genes with paralogs, the sensitivity was 0.38 and the specificity was 0.45 (sensitivity 0.81 and specificity 0.65 in PBMC-expressed genes with paralogs).\n\n## Single nucleotide variant identification at different read depths\n\nWe studied the effect of read depth on specificity by examining the read depth at individual SNV calls in our complete RNA-Seq dataset of eight lanes. We found that at a read depth of 3 (the minimum required for a variant to be called), the specificity for SNVs found in the complete set of exons was only 0.28, but that as read depth increased, so did specificity, until it reached a plateau of between 0.6 and 0.75 for read depths between 50 and 1,200 (Supplemental figure S1 in Additional file <a href=\"#S1\" data-ref-type=\"supplementary-material\">1<\/a>). The 13,892 SNVs called between these read depth levels had a specificity of 0.67 (sensitivity of 0.19). However, above a depth of 1,200, the specificity fell again, becoming as low as 0.05 for the 94 SNVs with read depths greater than 2,000. Similar trends were found for core exonic SNVs (sensitivity 0.22 and specificity 0.77), SNVs in PBMC-expressed genes (sensitivity 0.59 and specificity 0.84), and SNVs in PBMC-expressed genes without paralogs (sensitivity 0.58 and specificity 0.90) when restricting to SNVs with a read depth between 50 and 1,200 (Supplemental figure S1 in Additional file <a href=\"#S1\" data-ref-type=\"supplementary-material\">1<\/a>).\n\nWe also studied the effect of read depth on specificity as the dataset increased from one to eight lanes. We found that at only one lane of RNA-Seq data, the specificity was 0.5 for SNVs with a read depth of 3, which was far better than the value of 0.28 found when using all eight lanes of data. Again, the specificity increased as the read depth increased, but in this lower coverage dataset the specificity reached a plateau of between 0.6 and 0.75 at greater than 10 reads, far earlier than the 50 reads needed for specificity stability in the eight lane dataset. The specificity also decayed at a much lower read depth in this smaller dataset, becoming less than 0.6 when the read depth was greater than 150. As the dataset increased from one lane to all eight lanes, the overall specificity continually decreased (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>), and the minimum and maximum read depth value required for specificity to remain stable continually increased (Supplemental figure S2 in Additional file <a href=\"#S1\" data-ref-type=\"supplementary-material\">1<\/a>).\n\n# Discussion\n\nIf one simply considers all coding SNVs, our study suggests that about 40% can be identified by RNA-Seq using PBMCs as the RNA source. If we focus, however, on only PBMC-expressed genes, we find that 81% of coding variants are identified. This suggests that RNA-Seq may be a workable alternative for identifying exonic variants when performed in the appropriate tissue for the trait of interest.\n\nOne limiting factor in variant identification by RNA-Seq is the ability to uniquely align a given read. Although we were able to align 78% of our reads to a location in the transcriptome, only 69% aligned to exactly one location and could be kept in the analysis. Because exons are some of the most conserved regions of the genome, without the help of intervening and variable intron sequences it is much harder to align a read to the correct gene, and especially to have it align to only one location. This limits one's ability to identify variants in certain genomic locations, lowering the sensitivity of this method. Furthermore, if a read is uniquely aligned to the wrong gene, such as a paralog, then this can result in false positive SNVs being called in the cDNA. Restricting SNV calls to genes without paralogs did increase specificity from 0.47 to 0.54 and sensitivity from 0.39 to 0.42.\n\nAnother limiting factor is coverage. Because genes are expressed at different levels, in the random sampling of transcripts that are sequenced there will be gross imbalances. Some transcripts will have more than 1,000-fold coverage while other transcripts, although also expressed to some level in that tissue, will have coverage that is too low for variants to be accurately called. Given the diminishing returns of additional sequencing in terms of variants called (Figure <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>), it simply will not be possible to use RNA-Seq to capture all exonic variants in genes with low expression levels. RNA-Seq is most useful for identifying variants in a tissue type that highly expresses genes related to the trait under study. One interesting possibility to improve the proportion of SNVs that can be called would be to use more than one tissue type as a source for the RNA. For example, data available on expression of core exons in muscle shows that adding cDNA from this tissue to PBMC cDNA would increase the number of adequately expressed transcripts by 68% \\[6,7\\]. There are no expression data for a more easily accessible tissue such as skin for this exon array publicly available; however, one can extrapolate that adding cDNA from almost any tissue would be similarly beneficial to analysis.\n\nA large number of false positive SNVs were identified in this dataset: even when restricted to PBMC-expressed genes without paralogs the specificity was only 0.72. Many of these false positives, however, were due to the very high coverage produced in this study. At low levels of coverage, reads that could produce false positive calls are not yet abundant enough to pass the quality control filters used, and the specificity remains high. However, as more reads are added to the dataset, the number of incorrect alignments and sequencing mistakes increases, pushing more and more of these false positive calls over the quality control filters. Thus, the more coverage that is added in the search for true positives, the more false positives that will appear in the data. We found that the specificity for the dataset as a whole could be increased from 0.47 to 0.67 (and from 0.72 to 0.9 for PBMC-expressed genes without paralogs), at a substantial cost to sensitivity, by restricting the permissible read depth range for SNV calls. The specificity was low at low read depths, as is expected when a call is supported by less data; interestingly, the minimum read depth required for high specificity increased as coverage increased, supporting the view that high coverage introduces more noise and comes with a requirement for stricter quality control. An additional support to this view was the finding of low specificity at very high read depths. Both a minimum and a maximum read depth cutoff are advisable for increasing the specificity.\n\nSome of the false positives found in this dataset are certainly due to the incorrect alignment of reads, as even when a gene has no annotated paralog it can have sections with sequence similarities to other genes that permit errors during alignment. Another alignment problem that is unique to RNA-Seq is incorrect alignment at the very ends of a read due to splicing. Because reads that span exons require a certain number (four in our study) of bases to fall on both sides of the exon-exon boundary for proper alignment, reads that cross an exon boundary at the very edge of the read will have between one and three bases aligned to the wrong location. Our study removed most of this type of false positive SNV during the quality control process. Another scenario that would produce a seemingly false positive SNV would be sufficient coverage in the cDNA to call the variant but insufficient coverage in the gDNA sequencing to do the same: however, in our study such bases had a median coverage of 27\u00d7 in the gDNA, which does not support this explanation. Also, previous studies have shown that variants can be present in the RNA but not the gDNA due to RNA editing \\[2,8\\].\n\nThere are also likely to be disagreements between gDNA and cDNA sequencing stemming from expression differences. For example, some SNVs found in the genomic sequence may be missed in the cDNA due to expression balances, when an individual is heterozygous for a given SNV yet the reference allele is much more highly expressed. Also, an SNV may be called heterozygous in the gDNA but homozygous in the cDNA due to expression imbalances. In our dataset, 10% of the 19,054 SNVs that overlapped by location between gDNA and cDNA were heterozygous in gDNA and yet homozygous in cDNA. Differences in zygosity between the two methods can also result from insufficient coverage in one dataset or the other: for example, 1% of these 19,054 SNVs were homozygous in gDNA and yet heterozygous in cDNA, and the median coverage for these SNVs in gDNA was only 12\u00d7, compared to 28\u00d7 for SNVs that matched for zygosity. It is likely that many of the discrepancies where the SNV was heterozygous in gDNA and homozygous in cDNA also resulted from low coverage, as the median coverage for this group was only 8\u00d7 in the cDNA. It should also be noted that only 13 of the 19,054 SNVs that overlapped by location had completely mismatched alleles, such as the cDNA being homozygous for a G and the gDNA homozygous for C when the reference allele was A.\n\nOur study found that although the percent overlap of our false positive SNVs with dbSNP entries was far less than that of true positive (94%), false negative (89%), or all exonic gDNA (90%) SNVs, it was still substantially greater than zero (23%). Furthermore, we found that the percent of false positives corresponding to dbSNP entries increased as the PBMC expression level increased (Supplemental figure S4 in Additional file <a href=\"#S1\" data-ref-type=\"supplementary-material\">1<\/a>), implying that the 'false positives' seen at high expression levels may actually be true positives that were simply not seen in the gDNA due to issues with coverage or alignment or because of RNA editing. Additionally, we found that only 13% of the false positive SNVs found in dbSNP had been genotyped in HapMap, compared with 69% of the true positives found in dbSNP. This suggests that many of the dbSNP entries matching false positives are less well-curated, with less supporting evidence. A brief inspection of a subset of the SNVs showed that false positives were more likely to have cDNA evidence (as opposed to gDNA evidence) supporting their dbSNP entry than were true positives; this could be a reflection of either misalignment of the cDNA reads for dbSNP entries, or RNA edits that would not be seen in the gDNA. Finally, we found that the percent overlap of false positives with dbSNP decreased as the number of lanes of sequence data increased (Supplemental figure S3 in Additional file <a href=\"#S1\" data-ref-type=\"supplementary-material\">1<\/a>). This finding corresponds with the fact that the overall specificity decreased as coverage increased (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>); these phenomena are likely caused by the increasing ability of the random noise inherently present in the data to overcome quality control cutoffs as more and more reads were added, as described above.\n\nTwo previous studies have also looked at using RNA-Seq to identify SNVs. Chepelev *et al*. \\[2\\] used a dataset of 27 million uniquely aligned 30-bp RNA-Seq reads; while they detected 50% of known exons with at least 1\u00d7 coverage, and identified approximately 11,000 SNVs, they did not compare these data to gDNA sequence and thus could not calculate sensitivity or specificity of their methods. Shah *et al*. \\[8\\] used a dataset of 183 million RNA-Seq reads of 38.9 bp each, 55 million of which aligned to exons or exon junctions. They compared these data to 2.5 billion aligned gDNA reads of 48.2 bp to better understand the evolution of mutation in a lobular breast tumor. Although they showed that the number of SNVs called through RNA-Seq increased as the number of reads increased, they did not discuss the sensitivity or specificity of SNV calls when compared to whole genome sequencing, nor did they analyze how the number of SNVs changed as known expression level changed.\n\nAnother technology that has been used to sequence coding variants is exome capture. Because this method sequences reads that are from gDNA but enriched for the portions of interest, there are no complications with aligning splice junctions or being limited by expression level. A recent study showed that when restricted to the non-paralogous exons of 16,496 curated protein coding genes, exome capture utilizing 41 million 76-bp reads (one quarter the number of bases we aligned) captured SNVs with a sensitivity of 95% and a specificity of 90% \\[3\\]. Although this performance outshines the RNA-Seq data presented here, RNA-Seq does have some advantages beyond the ability to inexpensively genotype exonic SNVs. It can also be used to identify expression differences between individuals or even between alleles within an individual, which can lead to discovery of a nearby causal variant even if it is not exonic. RNA-Seq may also provide insight into novel exons, splice junctions or splice forms in the tissue or cell type being studied that might not be recognized as protein coding in genomic sequencing, or captured with targeted exomic sequencing.\n\nWhile it is most useful to perform RNA-Seq in a tissue relevant to the trait under study, other tissues can also be of some use. For example, data from Heinzen *et al*. \\[6\\] revealed an r^2^ of 0.23 between transcript expression in PBMCs and in brain, and that 63% of the transcripts highly expressed in brain were also expressed in blood to a level that allowed for consistent SNV detection by RNA-Seq (defined as expression level of at least 4% of the most highly expressed transcript in both).\n\n# Conclusions\n\nHere we show that RNA-Seq captured 81% of the exonic variants from genes that were well expressed in the source tissue. Although its usefulness is limited to these genes, the cheaper cost involved, as well as the extra information gained about expression and splice variants, may make this method a workable alternative to genomic sequencing or exome capture for groups that have access to the right types of tissue.\n\n# Materials and methods\n\n## Sample preparation and sequencing\n\nDNA was extracted from PBMCs using the QIAGEN Autopure LS (Venlo, The Netherlands). RNA was extracted from viable PBMCs using the Qiagen RNeasy kit. The DNA was prepared for sequencing according to Illumina's gDNA sample prep kit protocol: randomly fragment the DNA by nebulization, end repair, add a single A base, adaptor ligation, run a gel to isolate 300-bp fragments, and PCR amplification. The total RNA was prepared according to the Illumina RNA seq protocol: briefly, globin reduction, polyA enrichment, chemical fragmentation of the polyA RNA, cDNA synthesis, and size selection of 200-bp cDNA products. Next, the size-selected libraries were used for cluster generation on the flow cell. All prepared flow cells were run on the Genome Analyzer II using the paired-end module: nine flow cells (with eight lanes each) for the gDNA and one flow cell for the cDNA. The paired-end reads for the gDNA were each 75 bp long, although one flow cell only produced single reads of 75 bp each. Due to a machine error near the end of the read 1, the paired cDNA reads were not able to be matched to each other and read 1 was only 68 bp long while read 2 was 75 bp. The reads are available in the NCBI Sequence Read Archive \\[9\\], under study ID SRP001691. The Illumina GA Pipeline version was 1.4.0. This pipeline produced the quality score for each nucleotide in standard Illumina format where the base was 64 (Illumina quality score = Qphred +64, where Qphred = -10log10(e) and e = estimated probability of a nucleotide being wrong). We converted the quality scores for each nucleotide to standard Sanger fastq format, where the base was 33.\n\n## Alignment and single nucleotide variant identification\n\ngDNA was aligned to the reference genome (NCBI build 36 Ensembl release 50) using the BWA software (version 0.4.9) \\[4\\]. cDNA was aligned to the reference genome using TopHat \\[5\\]. The -GFF function utilized a transcript library downloaded from Ensembl to specify known protein-coding transcripts and splice junctions, and the library was screened to remove contigs and mitochondrial DNA. The --no-novel-juncs option was used to restrict alignment to those exons and splice junctions included in this transcript library. To assist in alignment to small exons, the 75-bp reads were broken down into three 25-bp segments (68-bp reads into two 34-bp segments), which were then joined back together after being individually aligned. Two mismatches were permitted per 25-bp (or 34-bp) segment, and no mismatches were permitted in the 4-bp anchor region on either side of a splice junction. Introns were permitted to range in size from 10 bp to 500 kb. Only unique alignments were kept: that is, reads that aligned to exactly one location. Reads mapping to multiple locations were excluded using in-house software.\n\nSAMtools (version 0.1.5c) was used to remove potential PCR duplicates via the rmdup (paired reads) and rmdupse (single reads) command \\[10\\]. It was also used for SNV identification, using the pileup command with the -c option and default settings. The SNVs were then filtered using SAMtool's variation filter with the default settings but removing the filter for a maximum allowed coverage per variant by setting it to 10 million for gDNA and 1 million for cDNA. SNVs lying outside exons as defined by the transcript library were removed. Indels were not considered. All SNVs were further screened for quality by only keeping those above a minimum SNP quality score: 30 for cDNA and 20 for gDNA. This score is calculated by SAMtools and is the Phred-scaled probability that the base at that location is identical to reference, with higher scores being less likely to be reference. SNVs were also excluded if there were fewer than three reads supporting the non-reference allele. cDNA SNVs were further screened to exclude all SNVs where more than 20% of the reads supporting the non-reference allele were from the first or last base of a sequence read.\n\n## Coverage\n\nCoverage of cDNA sequencing for each exon was calculating using in-house software. For each exon in the transcript library, coverage was calculated as the average number of reads covering each base within that exon.\n\n## Paralogous genes\n\nGenes were designated as paralogous using ENSG IDs as input in Genecards' paralog finder \\[11,12\\]. The list of paralogs from Ensembl was utilized, and genes were split into two groups: those with paralogs and those without. The 42 ENSG IDs not recognized by Genecards were individually examined for paralog status using Ensembl directly.\n\n# Abbreviations\n\nbp: base pair; gDNA: genomic DNA; PBMC: peripheral blood mononuclear cell; SNV: single nucleotide variant.\n\n# Authors' contributions\n\nETC participated in the design of the study, performed analyses, and drafted the paper. AS performed analyses and processed the cDNA reads. KVS supervised the sequencing of gDNA and cDNA. DG performed analyses and processed the gDNA reads. JPS sequenced the gDNA and cDNA. JMM performed analyses and processed the gDNA reads. ELH provided expression data. JJG collected the cohort, prepared the samples, and reviewed and edited the paper. DBG designed and supervised the study and helped to write the paper. All authors read and approved the final manuscript.\n\n# Supplementary Material\n\n###### Additional file 1\n\n**Supplemental figures S1 to S4**. Showing the specificity at different read depth levels and the overlap with dbSNP entries at different coverage levels and different expression levels.\n\nClick here for file\n\n## Acknowledgements\n\nFunding was provided by the NIAID Center for HIV\/AIDS Vaccine Immunology grant AI067854 and the Bill and Melinda Gates Foundation grant 157412. We also acknowledge C Gumbs, K Cronin and L Little for DNA and RNA extraction.","meta":{"dup_signals":{"dup_doc_count":169,"dup_dump_count":53,"dup_details":{"curated_sources":4,"2023-14":1,"2021-49":1,"2021-39":1,"2020-50":2,"2020-45":1,"2020-40":1,"2020-29":1,"2020-16":1,"2020-10":1,"2019-47":1,"2019-43":2,"2019-30":2,"2018-17":1,"2018-09":1,"2017-47":1,"2017-39":1,"2017-30":1,"2017-22":1,"2017-17":3,"2017-09":13,"2017-04":2,"2016-50":2,"2016-44":2,"2016-40":2,"2016-36":15,"2016-30":13,"2016-26":1,"2016-22":1,"2016-18":1,"2016-07":2,"2015-48":3,"2015-40":3,"2015-35":3,"2015-32":4,"2015-27":4,"2015-22":4,"2015-14":4,"2014-52":4,"2014-49":7,"2014-42":11,"2014-41":6,"2014-35":4,"2014-23":4,"2014-15":3,"2023-50":1,"2017-13":1,"2015-18":4,"2015-11":2,"2015-06":3,"2014-10":5,"2013-48":4,"2013-20":2,"2024-26":1}},"file":"PMC2898068"},"subset":"pubmed_central"}
{"text":"abstract: High quality protocols facilitate proper conduct, reporting, and external review of clinical trials. However, the completeness of trial protocols is often inadequate. To help improve the content and quality of protocols, an international group of stakeholders developed the SPIRIT 2013 Statement (Standard Protocol Items: Recommendations for Interventional Trials). The SPIRIT Statement provides guidance in the form of a checklist of recommended items to include in a clinical trial protocol.\n  .\n  This SPIRIT 2013 Explanation and Elaboration paper provides important information to promote full understanding of the checklist recommendations. For each checklist item, we provide a rationale and detailed description; a model example from an actual protocol; and relevant references supporting its importance. We strongly recommend that this explanatory paper be used in conjunction with the SPIRIT Statement. A website of resources is also available ([www.spirit-statement.org](http:\/\/www.spirit-statement.org\/)).\n  .\n  The SPIRIT 2013 Explanation and Elaboration paper, together with the Statement, should help with the drafting of trial protocols. Complete documentation of key trial elements can facilitate transparency and protocol review for the benefit of all stakeholders.\nauthor: An-Wen Chan; Jennifer M Tetzlaff; Peter C G\u00f8tzsche; Douglas G Altman; Howard Mann; Jesse A Berlin; Kay Dickersin; Asbj\u00f8rn Hr\u00f3bjartsson; Kenneth F Schulz; Wendy R Parulekar; Karmela Krle\u017ea-Jeri\u0107; Andreas Laupacis; David MoherCorrespondence to: A-W Chan <firstname.lastname@example.com>\ndate: 2013\nreferences:\ntitle: SPIRIT 2013 explanation and elaboration: guidance for protocols of clinical trials\n\nEvery clinical trial should be based on a protocol\u2014a document that details the study rationale, proposed methods, organisation, and ethical considerations.1 Trial investigators and staff use protocols to document plans for study conduct at all stages from participant recruitment to results dissemination. Funding agencies, research ethics committees\/institutional review boards, regulatory agencies, medical journals, systematic reviewers, and other groups rely on protocols to appraise the conduct and reporting of clinical trials.\n\nTo meet the needs of these diverse stakeholders, protocols should adequately address key trial elements. However, protocols often lack information on important concepts relating to study design and dissemination plans.2 3 4 5 6 7 8 9 10 11 12 Guidelines for writing protocols can help improve their completeness, but existing guidelines vary extensively in their content and have limitations, including non-systematic methods of development, limited stakeholder involvement, and lack of citation of empirical evidence to support their recommendations.13 As a result, there is also variation in the precise definition and scope of a trial protocol, particularly in terms of its relation to other documents such as procedure manuals.14\n\nGiven the importance of trial protocols, an international group of stakeholders launched the SPIRIT (Standard Protocol Items: Recommendations for Interventional Trials) Initiative in 2007 with the primary aim of improving the content of trial protocols. The main outputs are the SPIRIT 2013 Statement,14 consisting of a 33 item checklist of minimum recommended protocol items (table 1<a href=\"#tbl1\" data-ref-type=\"table\"><\/a>) plus a diagram (fig1<a href=\"#fig1\" data-ref-type=\"fig\"><\/a>); and this accompanying Explanation and Elaboration (E&E) paper. Additional information and resources are also available on the SPIRIT website ([www.spirit-statement.org](http:\/\/www.spirit-statement.org\/)).\n\nSPIRIT 2013 checklist: recommended items to address in a clinical trial protocol and related documents\\*\n\n| Section\/item | ItemNo | Description |\n|----|----|----|\n| Administrative information |  |  |\n| Title | 1 | Descriptive title identifying the study design, population, interventions, and, if applicable, trial acronym |\n| Trial registration | 2a | Trial identifier and registry name. If not yet registered, name of intended registry |\n|  | 2b | All items from the World Health Organization Trial Registration Data Set |\n| Protocol version | 3 | Date and version identifier |\n| Funding | 4 | Sources and types of financial, material, and other support |\n| Roles and responsibilities | 5a | Names, affiliations, and roles of protocol contributors |\n|  | 5b | Name and contact information for the trial sponsor |\n|  | 5c | Role of study sponsor and funders, if any, in study design; collection, management, analysis, and interpretation of data; writing of the report; and the decision to submit the report for publication, including whether they will have ultimate authority over any of these activities |\n|  | 5d | Composition, roles, and responsibilities of the coordinating centre, steering committee, endpoint adjudication committee, data management team, and other individuals or groups overseeing the trial, if applicable (see Item 21a for data monitoring committee) |\n| **Introduction** |  |  |\n| Background and rationale | 6a | Description of research question and justification for undertaking the trial, including summary of relevant studies (published and unpublished) examining benefits and harms for each intervention |\n|  | 6b | Explanation for choice of comparators |\n| Objectives | 7 | Specific objectives or hypotheses |\n| Trial design | 8 | Description of trial design including type of trial (eg, parallel group, crossover, factorial, single group), allocation ratio, and framework (eg, superiority, equivalence, noninferiority, exploratory) |\n| **Methods: Participants, interventions, and outcomes** |  |  |\n|  |  |  |\n| Study setting | 9 | Description of study settings (eg, community clinic, academic hospital) and list of countries where data will be collected. Reference to where list of study sites can be obtained |\n| Eligibility criteria | 10 | Inclusion and exclusion criteria for participants. If applicable, eligibility criteria for study centres and individuals who will perform the interventions (eg, surgeons, psychotherapists) |\n| Interventions | 11a | Interventions for each group with sufficient detail to allow replication, including how and when they will be administered |\n|  | 11b | Criteria for discontinuing or modifying allocated interventions for a given trial participant (eg, drug dose change in response to harms, participant request, or improving\/worsening disease) |\n|  | 11c | Strategies to improve adherence to intervention protocols, and any procedures for monitoring adherence (eg, drug tablet return, laboratory tests) |\n|  | 11d | Relevant concomitant care and interventions that are permitted or prohibited during the trial |\n| Outcomes | 12 | Primary, secondary, and other outcomes, including the specific measurement variable (eg, systolic blood pressure), analysis metric (eg, change from baseline, final value, time to event), method of aggregation (eg, median, proportion), and time point for each outcome. Explanation of the clinical relevance of chosen efficacy and harm outcomes is strongly recommended |\n| Participant timeline | 13 | Time schedule of enrolment, interventions (including any run-ins and washouts), assessments, and visits for participants. A schematic diagram is highly recommended (see fig 1\\[f1\\]) |\n| Sample size | 14 | Estimated number of participants needed to achieve study objectives and how it was determined, including clinical and statistical assumptions supporting any sample size calculations |\n| Recruitment | 15 | Strategies for achieving adequate participant enrolment to reach target sample size |\n| Methods: Assignment of interventions (for controlled trials) |  |  |\n| Allocation: |  |  |\n| Sequence generation | 16a | Method of generating the allocation sequence (eg, computer-generated random numbers) and list of any factors for stratification. To reduce predictability of a random sequence, details of any planned restriction (eg, blocking) should be provided in a separate document that is unavailable to those who enrol participants or assign interventions |\n| Allocation concealment mechanism | 16b | Mechanism of implementing the allocation sequence (eg, central telephone; sequentially numbered, opaque, sealed envelopes), describing any steps to conceal the sequence until interventions are assigned |\n| Implementation | 16c | Who will generate the allocation sequence, who will enrol participants, and who will assign participants to interventions |\n| Blinding (masking) | 17a | Who will be blinded after assignment to interventions (eg, trial participants, care providers, outcome assessors, data analysts) and how |\n|  | 17b | If blinded, circumstances under which unblinding is permissible and procedure for revealing a participant's allocated intervention during the trial |\n| Methods: Data collection, management, and analysis |  |  |\n| Data collection methods | 18a | Plans for assessment and collection of outcome, baseline, and other trial data, including any related processes to promote data quality (eg, duplicate measurements, training of assessors) and a description of study instruments (eg, questionnaires, laboratory tests) along with their reliability and validity, if known. Reference to where data collection forms can be found, if not in the protocol |\n|  | 18b | Plans to promote participant retention and complete follow-up, including list of any outcome data to be collected for participants who discontinue or deviate from intervention protocols |\n| Data management | 19 | Plans for data entry, coding, security, and storage, including any related processes to promote data quality (eg, double data entry; range checks for data values). Reference to where details of data management procedures can be found, if not in the protocol |\n| Statistical methods | 20a | Statistical methods for analysing primary and secondary outcomes. Reference to where other details of the statistical analysis plan can be found, if not in the protocol |\n|  | 20b | Methods for any additional analyses (eg, subgroup and adjusted analyses) |\n|  | 20c | Definition of analysis population relating to protocol non-adherence (eg, as randomised analysis), and any statistical methods to handle missing data (eg, multiple imputation) |\n| Methods: Monitoring |  |  |\n| Data monitoring | 21a | Composition of data monitoring committee (DMC); summary of its role and reporting structure; statement of whether it is independent from the sponsor and competing interests; and reference to where further details about its charter can be found, if not in the protocol. Alternatively, an explanation of why a DMC is not needed |\n|  | 21b | Description of any interim analyses and stopping guidelines, including who will have access to these interim results and make the final decision to terminate the trial |\n| Harms | 22 | Plans for collecting, assessing, reporting, and managing solicited and spontaneously reported adverse events and other unintended effects of trial interventions or trial conduct |\n| Auditing | 23 | Frequency and procedures for auditing trial conduct, if any, and whether the process will be independent from investigators and the sponsor |\n| **Ethics and dissemination** |  |  |\n| Research ethics approval | 24 | Plans for seeking research ethics committee\/institutional review board (REC\/IRB) approval |\n| Protocol amendments | 25 | Plans for communicating important protocol modifications (eg, changes to eligibility criteria, outcomes, analyses) to relevant parties (eg, investigators, REC\/IRBs, trial participants, trial registries, journals, regulators) |\n| Consent or assent | 26a | Who will obtain informed consent or assent from potential trial participants or authorised surrogates, and how (see Item 32) |\n|  | 26b | Additional consent provisions for collection and use of participant data and biological specimens in ancillary studies, if applicable |\n| Confidentiality | 27 | How personal information about potential and enrolled participants will be collected, shared, and maintained in order to protect confidentiality before, during, and after the trial |\n| Declaration of interests | 28 | Financial and other competing interests for principal investigators for the overall trial and each study site |\n| Access to data | 29 | Statement of who will have access to the final trial dataset, and disclosure of contractual agreements that limit such access for investigators |\n| Ancillary and post-trial care | 30 | Provisions, if any, for ancillary and post-trial care, and for compensation to those who suffer harm from trial participation |\n| Dissemination policy | 31a | Plans for investigators and sponsor to communicate trial results to participants, healthcare professionals, the public, and other relevant groups (eg, via publication, reporting in results databases, or other data sharing arrangements), including any publication restrictions |\n|  | 31b | Authorship eligibility guidelines and any intended use of professional writers |\n|  | 31c | Plans, if any, for granting public access to the full protocol, participant level dataset, and statistical code |\n| **Appendices** |  |  |\n| Informed consent materials | 32 | Model consent form and other related documentation given to participants and authorised surrogates |\n| Biological specimens | 33 | Plans for collection, laboratory evaluation, and storage of biological specimens for genetic or molecular analysis in the current trial and for future use in ancillary studies, if applicable |\n\nThe SPIRIT 2013 Statement and E&E paper reflect the collaboration and input of 115 contributors, including trial investigators, healthcare professionals, methodologists, statisticians, trial coordinators, journal editors, as well as representatives from research ethics committees, industry and non-industry funders, and regulatory agencies. Details of the scope and methods have been published elsewhere.13 14 15 Briefly, three complementary methods were specified beforehand*,* in line with current recommendations for development of reporting guidelines16: 1) a Delphi consensus survey15; 2) two systematic reviews to identify existing protocol guidelines and empirical evidence supporting the importance of specific checklist items; and 3) two face-to-face consensus meetings to finalise the SPIRIT 2013 checklist. Furthermore, the checklist was pilot tested by graduate course students, and an implementation strategy was developed at a stakeholder meeting.\n\nThe SPIRIT recommendations are intended as a guide for those preparing the full protocol for a clinical trial. A clinical trial is a prospective study in which one or more interventions are assigned to human participants in order to assess the effects on health related outcomes. The recommendations are not intended to prescribe how a trial should be designed or conducted. Rather, we call for a transparent and complete description of what is intended, regardless of the characteristics or quality of the plans. The SPIRIT 2013 Statement addresses the minimum content for interventional trials; additional concepts may be important to describe in protocols for trials of specific designs (eg, crossover trials) or in protocols intended for submission to specific groups (eg, funders, research ethics committees\/institutional review boards). If information for a recommended item is not yet available when the protocol is being finalised (eg, funding sources), this should be explicitly stated and the protocol updated as new information is obtained. Formatting conventions such as a table of contents, glossary of non-standard or ambiguous terms (eg, randomisation phase or off-protocol), and list of abbreviations and references will facilitate understanding of the protocol.\n\n# Purpose and development of explanation and elaboration paper\n\nModelled after other reporting guidelines,17 18 this E&E paper presents each checklist item with at least one model example from an actual protocol, followed by a full explanation of the rationale and main issues to address. This E&E paper provides important information to facilitate full understanding of each checklist item, and is intended to be used in conjunction with the SPIRIT 2013 Statement.14 These complementary tools serve to inform trial investigators about important issues to consider in the protocol as they relate to trial design, conduct, reporting, and organisation.\n\nTo identify examples for each checklist item, we obtained protocols from public websites, journals, trial investigators, and industry sponsors. Model examples were selected to reflect how key elements could be appropriately described in a trial protocol. Some examples illustrate a specific component of a checklist item, while others encompass all key recommendations for an item. Additional examples are also available on the SPIRIT website ([www.spirit-statement.org](http:\/\/www.spirit-statement.org\/)). The availability of examples for all checklist items indicates the feasibility of addressing each recommended item in the main protocol rather than in separate documents.\n\nExamples are quoted verbatim from the trial protocol. Proper names of trial personnel have been abbreviated with italicised initials, and any reference numbers cited in the original quoted text are denoted by \\[*Reference*\\] to distinguish them from references cited in this E&E paper.\n\nFor each checklist item we also strived to provide references to empirical data supporting its relevance, which we identified through a systematic review conducted to inform the content of the SPIRIT checklist. We searched MEDLINE, the Cochrane Methodology Register, and the Cochrane Database of Systematic Reviews (limited to methodology reviews) up to September 2009, and EMBASE up to August 2007. We searched reference lists, PubMed \"related articles,\" and citation searches using SCOPUS to identify additional relevant studies. We used piloted forms to screen and extract data relevant to specific checklist items.\n\nStudies were included if they provided empirical data to support or refute the importance of a given protocol concept. A summary of the relevant methodological articles was provided to each E&E author for use in preparing the initial draft text for up to six checklist items; each draft was also reviewed and revised by a second author. When citing empirical evidence in the E&E, we aimed to reference a systematic review when available. When no review was identified, we either cited all relevant individual studies, or if too numerous, a representative sample of the literature. Some items had little or no identified empirical evidence (eg, title) but their inclusion in the checklist is supported by a strong pragmatic or ethical rationale. Where relevant, we also provide references to non-empirical publications for further reading.\n\nTwo lead authors (AWC, JMT) collated and refined the content and format for all items, and then circulated three iterations of an overall draft to the coauthors for editing and final approval.\n\n# SPIRIT 2013 Explanation and Elaboration\n\n## Section 1: Administrative information\n\nItem 1: Descriptive title identifying the study design, population, interventions, and, if applicable, trial acronym\n\n### Example\n\n\"A multi-center, investigator-blinded, randomized, 12-month, parallel-group, non-inferiority study to compare the efficacy of 1.6 to 2.4 g Asacol\u00ae Therapy QD \\[once daily\\] versus divided dose (BID \\[twice daily\\]) in the maintenance of remission of ulcerative colitis.\"19\n\n### Explanation\n\nThe title provides an important means of trial identification. A succinct description that conveys the topic (study population, interventions), acronym (if any), and basic study design\u2014including the method of intervention allocation (eg, parallel group randomised trial; single-group trial)\u2014will facilitate retrieval from literature or internet searches and rapid judgment of relevance.20 It can also be helpful to include the trial framework (eg, superiority, non-inferiority), study objective or primary outcome, and if relevant, the study phase (eg, phase II).\n\n## Trial registration\u2014registry\n\nItem 2a: Trial identifier and registry name. If not yet registered, name of intended registry\n\n### Example\n\n\"EudraCT: 2010-019180-10\n\nClinicalTrials.gov: NCT01066572\n\nISRCTN: 54540667.\"21\n\n### Explanation\n\nThere are compelling ethical and scientific reasons for trial registration.22 23 24 Documentation of a trial's existence on a publicly accessible registry can help to increase transparency,24 25 decrease unnecessary duplication of research effort, facilitate identification of ongoing trials for prospective participants, and identify selective reporting of study results.26 27 28 As mandated by the International Committee of Medical Journal Editors (ICMJE) and jurisdictional legislation,29 30 31 registration of clinical trials should occur before recruitment of the first trial participant.\n\nWe recommend that registry names and trial identifiers assigned by the registries be prominently placed in the protocol, such as on the cover page. If the trial is not yet registered, the intended registry should be indicated and the protocol updated upon registration. When registration in multiple registries is required (eg, to meet local regulation), each identifier should be clearly listed in the protocol and each registry.\n\n## Trial registration\u2014data set\n\nItem 2b: All items from the World Health Organization trial registration data set\n\n**Example**<a href=\"#tbl2\" data-ref-type=\"table\"><\/a>\n\nExample of trial registration data\n\n<table>\n<thead>\n<tr>\n<th style=\"text-align: left;\">Data category<\/th>\n<th style=\"text-align: left;\">Information<sup>32<\/sup><\/th>\n<\/tr>\n<\/thead>\n<tbody>\n<tr>\n<td style=\"text-align: left;\">Primary registry and trial identifying number<\/td>\n<td style=\"text-align: left;\">ClinicalTrials.gov<br \/>\nNCT01143272<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Date of registration in primary registry<\/td>\n<td style=\"text-align: left;\">11 June, 2010<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Secondary identifying numbers<\/td>\n<td style=\"text-align: left;\">BNI-2009-01, 2009-017374-20, ISRCTN01005546, DRKS00000084<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Source(s) of monetary or material support<\/td>\n<td style=\"text-align: left;\">Bernhard Nocht Institute for Tropical Medicine<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Primary sponsor<\/td>\n<td style=\"text-align: left;\">Bernhard Nocht Institute for Tropical Medicine<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Secondary sponsor(s)<\/td>\n<td style=\"text-align: left;\">German Federal Ministry of Education and Research<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Contact for public queries<\/td>\n<td style=\"text-align: left;\">SE, MD, MPH [email address]<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Contact for scientific queries<\/td>\n<td style=\"text-align: left;\">SE, MD, MPH<br \/>\nBernhard Nocht Institute for Tropical Medicine, Hamburg, Germany<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Public title<\/td>\n<td style=\"text-align: left;\">Probiotic <em>Saccharomyces boulardii<\/em> for the prevention of antibiotic associated diarrhoea (SacBo)<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Scientific title<\/td>\n<td style=\"text-align: left;\"><em>S boulardii<\/em> for the prevention of antibiotic associated diarrhoea\u2014randomised, double blind, placebo controlled trial<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Countries of recruitment<\/td>\n<td style=\"text-align: left;\">Germany<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Health condition(s) or problem(s) studied<\/td>\n<td style=\"text-align: left;\">Antibiotic treatment, <em>Clostridium difficile,<\/em> diarrhoea<\/td>\n<\/tr>\n<tr>\n<td rowspan=\"2\" style=\"text-align: left;\">Intervention(s)<\/td>\n<td style=\"text-align: left;\">Active comparator: <em>S boulardii<\/em> (500 mg <em>S boulardii<\/em> per day)<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Placebo comparator: microcristallin cellulose (matching capsules containing no active ingredients)<\/td>\n<\/tr>\n<tr>\n<td rowspan=\"3\" style=\"text-align: left;\">Key inclusion and exclusion criteria<\/td>\n<td style=\"text-align: left;\">Ages eligible for study: \u226518 years<br \/>\nSexes eligible for study: both<br \/>\nAccepts healthy volunteers: no<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Inclusion criteria: adult patient (\u2265 18 years), patient hospitalised<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Exclusion criteria: allergy against yeast and\/or Perenterol forte and\/or placebos containing <em>S cerevisiae<\/em> HANSEN CBS 5926, lactose monohydrate, magnesium stearate, gelatine, sodium dodecyl sulfate, titan dioxide, microcrystalline cellulose<\/td>\n<\/tr>\n<tr>\n<td rowspan=\"4\" style=\"text-align: left;\">Study type<\/td>\n<td style=\"text-align: left;\">Interventional<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Allocation: randomized intervention model. Parallel assignment masking: double blind (subject, caregiver, investigator, outcomes assessor)<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Primary purpose: prevention<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Phase III<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Date of first enrolment<\/td>\n<td style=\"text-align: left;\">June 2010<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Target sample size<\/td>\n<td style=\"text-align: left;\">1520<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Recruitment status<\/td>\n<td style=\"text-align: left;\">Recruiting<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Primary outcome(s)<\/td>\n<td style=\"text-align: left;\">Cumulative incidence of any antibiotic associated diarrhoea (time frame: 2 years; not designated as safety issue)<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Key secondary outcomes<\/td>\n<td style=\"text-align: left;\">Cumulative incidence of <em>C difficile<\/em> associated diarrhoea (time frame: 2 years; not designated as safety issue)<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n### Explanation\n\nIn addition to a trial registration number, the World Health Organization (WHO) recommends a minimum standard list of items to be included in a trial registry in order for a trial to be considered fully registered ([www.who.int\/ictrp\/network\/trds\/en\/index.html](http:\/\/www.who.int\/ictrp\/network\/trds\/en\/index.html)). These standards are supported by ICMJE, other journal editors, and jurisdictional legislation.29 30 31 We recommend that the WHO Trial Registration Data Set be included in the protocol to serve as a brief structured summary of the trial. Its inclusion in the protocol can also signal updates for the registry when associated protocol sections are amended\u2014thereby promoting consistency between information in the protocol and registry.\n\n## Protocol version\n\nItem 3: Date and version identifier\n\n### Example\n\n1.  \"Issue date: 25 Jul 2005\n\n2.  Protocol amendment number: 05\n\n3.  Authors: *MD, JH*\n\n#### Revision chronology:\n\n1.  UM . . . 00, 2004-Jan-30 Original\n\n2.  UM . . . 01, 2004-Feb-7 Amendment 01.:\n\n    1.  Primary reason for amendment: changes in Section 7.1 regarding composition of comparator placebo\n\n    2.  Additional changes (these changes in and of themselves would not justify a protocol amendment): correction of typographical error in Section 3.3 . . .\n\n3.  UM . . . 05, 2005-Jul-25 Amendment No.5:\n\n    1.  At the request of US FDA statements were added to the protocol to better clarify and define the algorithm for determining clinical or microbiological failures prior to the follow-up visit.\"33\n\n### Explanation\n\nSequentially labelling and dating each protocol version helps to mitigate potential confusion over which document is the most recent. Explicitly listing the changes made relative to the previous protocol version is also important (see Item 25). Transparent tracking of versions and amendments facilitates trial conduct, review, and oversight.\n\n## Funding\n\nItem 4: Sources and types of financial, material, and other support\n\n### Example\n\n\"Tranexamic acid will be manufactured by Pharmacia (Pfizer, Sandwich, UK) and placebo by South Devon Healthcare NHS Trust, UK. The treatment packs will be prepared by an independent clinical trial supply company (Brecon Pharmaceuticals Limited, Hereford, UK) . . .\n\nLSHTM \\[London School of Hygiene and Tropical Medicine\\] is funding the run-in costs for the WOMAN trial and up to 2,000 patients' recruitment. The main phase is funded by the UK Department of Health and the Wellcome Trust. Funding for this trial covers meetings and central organisational costs only. Pfizer, the manufacturer of tranexamic acid, have provided the funding for the trial drug and placebo used for this trial. An educational grant, equipment and consumables for ROTEM \\[thromboelastometry procedure\\] analysis has been provided by Tem Innovations GmbH, M.-Kollar-Str. 13-15, 81829 Munich, Germany for use in the WOMAN-ETAC study. An application for funding to support local organisational costs has been made to University of Ibadan Senate Research Grant. The design, management, analysis and reporting of the study are entirely independent of the manufacturers of tranexamic acid and Tem Innovations GmbH.\"34\n\n### Explanation\n\nA description of the sources of financial and non-financial support provides relevant information to assess study feasibility and potential competing interests (Item 28). Although both industry funded and non-industry funded trials are susceptible to bias,4 35 the former are more likely to report trial results and conclusions that favour their own interventions.27 36 37 38 39 This tendency could be due to industry trials being more likely to select effective interventions for evaluation (Item 6a), to use less effective control interventions (Item 6b), or to selectively report outcomes (Item 12), analyses (Item 20) or full studies (Item 31).38 40 41 42 43 Non-financial support (eg, provision of drugs) from industry has not been shown to be associated with biased results, although few studies have examined this issue.44 45\n\nAt a minimum, the protocol should identify the sources of financial and non-financial support; the specific type (eg, funds, equipment, drugs, services) and time period of support; and any vested interest that the funder may have in the trial. If a trial is not yet funded when the protocol is first written, the proposed sources of support should be listed and updated as funders are confirmed.\n\nNo clear consensus exists regarding the level of additional funding details that should be provided in the trial protocol as opposed to trial contracts, although full disclosure of funding information in the protocol can help to better identify financial competing interests. Some jurisdictional guidelines require more detailed disclosure, including monetary amounts granted from each funder, the mechanism of providing financial support (eg, paid in fixed sum or per recruited participant), and the specific fund recipient (eg, trial investigator, department\/institute).46 Detailed disclosure allows research ethics committees\/institutional review boards (REC\/IRBs) to assess whether the reimbursement amount is reasonable in relation to the time and expenses incurred for trial conduct.\n\n## Roles and responsibilities\u2014contributorship\n\nItem 5a: Names, affiliations, and roles of protocol contributors\n\n### Example\n\n\"*RTL* \\[address\\], *EJM* \\[address\\], *AK* \\[address\\] . . .\n\n#### Authors' contributions\n\n*RTL* conceived of the study. *AK*, *EN*, *SB*, *PR*, *WJ*, *JH*, and *MC* initiated the study design and *JK* and *LG* helped with implementation. *RTL*, *JK*, *LG*, and *FP* are grant holders. *LT* and *EM* provided statistical expertise in clinical trial design and *RN* is conducting the primary statistical analysis. All authors contributed to refinement of the study protocol and approved the final manuscript.\"47\n\n### Explanation\n\nIndividuals who contribute substantively to protocol development and drafting should have their contributions reported. As with authorship of journal articles,48 listing the protocol contributors, their affiliations, and their roles in the protocol development process provides due recognition, accountability, and transparency. Naming of contributors can also help to identify competing interests and reduce ghost authorship (Items 28 and 31b).9 10 If professional medical writers are employed to draft the protocol, then this should be acknowledged as well.\n\nNaming of authors and statements of contributorship are standard for protocols published in journals such as *Trials*49 but are uncommon for unpublished protocols. Only five of 44 industry-initiated protocols approved in 1994-95 by a Danish research ethics committee explicitly identified the protocol authors.9\n\n## Roles and responsibilities\u2014sponsor contact information\n\nItem 5b: Name and contact information for the trial sponsor\n\n### Example\n\n1.  \"Trial Sponsor: University of Nottingham\n\n2.  Sponsor's Reference: RIS 8024 . . .\n\n3.  Contact name: Mr *PC*\n\n4.  Address: King's Meadow Campus . . .\n\n5.  Telephone: . . .\n\n6.  Email: . . .\"50\n\n### Explanation\n\nThe sponsor can be defined as the individual, company, institution, or organisation assuming overall responsibility for the initiation and management of the trial, and is not necessarily the main funder.51 52 In general, the company is the sponsor in industry initiated trials, while the funding agency or institution of the principal investigator is often the sponsor for investigator initiated trials. For some investigator initiated trials, the principal investigator can be considered to be a \"sponsor-investigator\" who assumes both sponsor and investigator roles.51 53\n\nIdentification of the trial sponsor provides transparency and accountability. The protocol should identify the name, contact information, and if applicable, the regulatory agency identifying number of the sponsor.\n\n## Roles and responsibilities\u2014sponsor and funder\n\nItem 5c: Role of study sponsor and funders, if any, in study design; collection, management, analysis, and interpretation of data; writing of the report; and the decision to submit the report for publication, including whether they will have ultimate authority over any of these activities\n\n### Example\n\n\"This funding source had no role in the design of this study and will not have any role during its execution, analyses, interpretation of the data, or decision to submit results.\"54\n\n### Explanation\n\nThere is potential for bias when the trial sponsor or funder (sometimes the same entity) has competing interests (Item 28) and substantial influence on the planning, conduct, or reporting of a trial. Empirical research indicates that specific forms of bias tend to be more prevalent in trials funded by industry compared to those funded by non-commercial sources.36 37 38 45 55 56 57 58 59 60 The design, analysis, interpretation, and reporting of most industry-initiated trials are controlled by the sponsor; this authority is often enforced by contractual agreements signed between the sponsor and trial investigators (Item 29).10 61\n\nThe protocol should explicitly outline the roles and responsibilities of the sponsor and any funders in study design, conduct, data analysis and interpretation, manuscript writing, and dissemination of results. It is also important to state whether the sponsor or funder controls the final decision regarding any of these aspects of the trial.\n\nDespite the importance of declaring the roles of the trial sponsor and funders, few protocols explicitly do so. Among 44 protocols for industry-initiated trials receiving ethics approval in Denmark from 1994-95, none stated explicitly who had contributed to the design of the trial.9\n\n## Roles and responsibilities\u2014committees\n\nItem 5d: Composition, roles, and responsibilities of the coordinating centre, steering committee, endpoint adjudication committee, data management team, and other individuals or groups overseeing the trial, if applicable (see Item 21a for data monitoring committee)\n\n### Example\n\n#### \"Principal investigator and research physician\n\n1.  Design and conduct of RITUXVAS\n\n2.  Preparation of protocol and revisions\n\n3.  Preparation of investigators brochure (IB) and CRFs \\[case report forms\\]\n\n4.  Organising steering committee meetings\n\n5.  Managing CTO \\[clinical trials office\\]\n\n6.  Publication of study reports\n\n7.  Members of TMC \\[Trial Management Committee\\]\n\n#### Steering committee (SC)\n\n1.  (see title page for members)\n\n2.  Agreement of final protocol\n\n3.  All lead investigators will be steering committee members. One lead investigator per country will be nominated as national coordinator.\n\n4.  Recruitment of patients and liaising with principle \\[sic\\] investigator\n\n5.  Reviewing progress of study and if necessary agreeing changes to the protocol and\/or investigators brochure to facilitate the smooth running of the study.\n\n#### Trial management committee (TMC)\n\n1.  (Principle \\[sic\\] investigator, research physician, administrator)\n\n2.  Study planning\n\n3.  Organisation of steering committee meetings\n\n4.  Provide annual risk report MHRA \\[Medicines and Healthcare Products Regulatory Agency\\] and ethics committee\n\n5.  SUSAR \\[Serious unexpected suspected adverse events\\] reporting to MHRA and Roche\n\n6.  Responsible for trial master file\n\n7.  Budget administration and contractual issues with individual centres\n\n8.  Advice for lead investigators\n\n9.  Audit of 6 monthly feedback forms and decide when site visit to occur.\n\n10. Assistance with international review, board\/independent ethics committee applications\n\n11. Data verification\n\n12. Randomisation\n\n13. Organisation of central serum sample collection\n\n#### Data manager\n\n1.  Maintenance of trial IT system and data entry\n\n2.  Data verification\n\n#### Lead investigators\n\nIn each participating centre a lead investigator (senior nephrologist\/rheumatologist\/ immunologist) will be identified, to be responsible for identification, recruitment, data collection and completion of CRFs, along with follow up of study patients and adherence to study protocol and investigators brochure. . . . Lead investigators will be steering committee members, with one investigator per country being nominated as national coordinator.\"62\n\n### Explanation\n\nThe protocol should outline the general membership of the various committees or groups involved in trial coordination and conduct; describe the roles and responsibilities of each; and (when known) identify the chairs and members. This information helps to ensure that roles and responsibilities are clearly understood at the trial onset, and facilitates communication from external parties regarding the trial. It also enables readers to understand the mandate and expertise of those responsible for overseeing participant safety, study design, database integrity, and study conduct. For example, empirical evidence supports the pivotal role of an epidemiologist or biostatistician in designing and conducting higher quality trials.63 64\n\n# Section 2: Introduction\n\n## Background and rationale\n\nItem 6a: Description of research question and justification for undertaking the trial, including summary of relevant studies (published and unpublished) examining benefits and harms for each intervention\n\n### Example\n\n#### \"Background\n\n*Introduction:* For people at ages 5 to 45 years, trauma is second only to HIV\/AIDS as a cause of death. . . .\n\n*Mechanisms*: The haemostatic system helps to maintain the integrity of the circulatory system after severe vascular injury, whether traumatic or surgical in origin.\\[reference\\] Major surgery and trauma trigger similar haemostatic responses . . . Antifibrinolytic agents have been shown to reduce blood loss in patients with both normal and exaggerated fibrinolytic responses to surgery, and do so without apparently increasing the risk of post-operative complications, . . .\n\n*Existing knowledge*: Systemic antifibrinolytic agents are widely used in major surgery to prevent fibrinolysis and thus reduce surgical blood loss. A recent systematic review \\[reference\\] of randomised controlled trials of antifibrinolytic agents (mainly aprotinin or tranexamic acid) in elective surgical patients identified 89 trials including 8,580 randomised patients (74 trials in cardiac, eight in orthopaedic, four in liver, and three in vascular surgery). The results showed that these treatments reduced the numbers needing transfusion by one third, reduced the volume needed per transfusion by one unit, and halved the need for further surgery to control bleeding. These differences were all highly statistically significant. There was also a statistically non-significant reduction in the risk of death (RR=0.85: 95% CI 0.63 to 1.14) in the antifibrinolytic treated group.\n\n. . .\n\n*Need for a trial*: A simple and widely practicable treatment that reduces blood loss following trauma might prevent thousands of premature trauma deaths each year and secondly could reduce exposure to the risks of blood transfusion. Blood is a scarce and expensive resource and major concerns remain about the risk of transfusion-transmitted infection. . . . A large randomised trial is therefore needed of the use of a simple, inexpensive, widely practicable antifibrinolytic treatment such as tranexamic acid . . . in a wide range of trauma patients who, when they reach hospital are thought to be at risk of major haemorrhage that could significantly affect their chances of survival.\n\n#### Dose selection\n\nThe systematic review of randomised controlled trials of antifibrinolytic agents in surgery showed that dose regimens of tranexamic acid vary widely.\\[reference\\] . . .\n\nIn this emergency situation, administration of a fixed dose would be more practicable as determining the weight of a patient would be impossible. Therefore a fixed dose within the dose range which has been shown to inhibit fibrinolysis and provide haemostatic benefit is being used for this trial. . . . The planned duration of administration allows for the full effect of tranexamic acid on the immediate risk of haemorrhage without extending too far into the acute phase response seen after surgery and trauma.\"65\n\n### Explanation\n\nThe value of a research question, as well as the ethical and scientific justification for a trial, depend to a large degree on the uncertainty of the comparative benefits or harms of the interventions, which depends in turn on the existing body of knowledge on the topic. The background section of a protocol should summarise the importance of the research question, justify the need for the trial in the context of available evidence, and present any available data regarding the potential effects of the interventions (efficacy and harms).66 67 This information is particularly important to the trial participants and personnel, as it provides motivation for contributing to the trial.68 69 It is also relevant to funders, REC\/IRBs, and other stakeholders who evaluate the scientific and ethical basis for trial conduct.\n\nTo place the trial in the context of available evidence, it is strongly recommended that an up-to-date systematic review of relevant studies be summarised and cited in the protocol.70 Several funders request this information in grant applications.71 72 Failure to review the cumulated evidence can lead to unnecessary duplication of research or to trial participants being deprived of effective, or exposed to harmful, interventions.73 74 75 76 A minority of published trial reports cite a systematic review of pre-existing evidence,77 78 and in one survey only half of trial investigators were aware of a relevant existing review when they had designed their trial.79 Given that about half of trials remain unpublished,80 81 82 and that published trials often represent a biased subset of all trials,80 83 it is important that systematic reviews include a search of online resources such as trial registries, results databases, and regulatory agency websites.84\n\n## Background and rationale\u2014choice of comparators\n\nItem 6b: Explanation for choice of comparators\n\n### Example\n\n#### \"Choice of comparator\n\nIn spite of the increasing numbers of resistant strains, chloroquine monotherapy is still recommended as standard blood-stage therapy for patients with *P* \\[*Plasmodium*\\] *vivax* malaria in the countries in which this trial will be conducted. Its selection as comparator is therefore justified. The adult dose of chloroquine will be 620 mg for 2 days followed by 310 mg on the third day and for children 10 mg\/kg for the first two days and 5 mg\/kg for the third day. Total dose is in accordance with the current practice in the countries where the study is conducted. The safety profile of chloroquine is well established and known. Although generally well tolerated, the following side-effects of chloroquine treatment have been described:\n\nGastro-intestinal disturbances, headache, hypotension, convulsions, visual disturbances, depigmentation or loss of hair, skin reactions (rashes, pruritus) and, rarely, bone-marrow suppression and hypersensitivity reactions such as urticaria and angioedema. Their occurrence during the present trial may however be unlikely given the short (3-day) duration of treatment.\"85\n\n### Explanation\n\nThe choice of control interventions has important implications for trial ethics, recruitment, results, and interpretation. In trials comparing an intervention to an active control or usual care, a clear description of the rationale for the comparator intervention will facilitate understanding of its appropriateness.86 87 For example, a trial in which the control group receives an inappropriately low dose of an active drug will overestimate the relative efficacy of the study intervention in clinical practice; conversely, an inappropriately high dose in the control group will lead to an underestimate of the relative harms of the study intervention.87 88\n\nThe appropriateness of using placebo-only control groups has been the subject of extensive debate and merits careful consideration of the existence of other effective treatments, the potential risks to trial participants, and the need for assay sensitivity\u2014that is, ability to distinguish an effective intervention from less effective or ineffective interventions.89 90 In addition, surveys have demonstrated that a potential barrier to trial participation is the possibility of being allocated a placebo-only or active control intervention that is perceived to be less desirable than the study intervention.68 69 91 92 Evidence also suggests that enrolled participants perceive the effect of a given intervention differently depending on whether the control group consists of an active comparator or only placebo.93 94 95 96\n\nFinally, studies suggest that some \"active\" comparators in head-to-head randomised trials are presumed by trial investigators to be effective despite having never previously been shown to be superior to placebo.74 97 In a systematic review of over 100 head-to-head antibiotic trials for mild to moderate chronic obstructive pulmonary disease,74 cumulative meta-analysis of preceding placebo controlled trials did not show a significant effect of antibiotics over placebo. Such studies again highlight the importance of providing a thorough background and rationale for a trial and the choice of comparators\u2014including data from an up-to-date systematic review\u2014to enable potential participants, physicians, REC\/IRBs, and funders to discern the merit of the trial.\n\n## Objectives\n\nItem 7: Specific objectives or hypotheses\n\n### Example\n\n#### \"1.1 Research hypothesis\n\nApixaban is noninferior to warfarin for prevention of stroke (hemorrhagic, ischemic or of unspecified type) or systemic embolism in subjects with atrial fibrillation (AF) and additional risk factor(s) for stroke.\n\n. . .\n\n### 2 STUDY OBJECTIVES\n\n#### 2.1 Primary objective\n\nTo determine if apixaban is noninferior to warfarin (INR \\[international normalized ratio\\] target range 2.0-3.0) in the combined endpoint of stroke (hemorrhagic, ischemic or of unspecified type) and systemic embolism, in subjects with AF and at least one additional risk factor for stroke.\n\n#### 2.2 Secondary objectives\n\n##### 2.2.1 Key secondary objectives\n\nThe key secondary objectives are to determine, in subjects with AF and at least one additional risk factor for stroke, if apixaban is superior to warfarin (INR target range 2.0 - 3.0) for,\n\n-   the combined endpoint of stroke (hemorrhagic, ischemic or of unspecified type) and systemic embolism\n\n-   major bleeding \\[International Society of Thrombosis and Hemostasis\\]\n\n-   all-cause death\n\n##### 2.2.2 Other secondary objectives\n\n-   To compare, in subjects with AF and at least one additional risk factor for stroke, apixaban and warfarin with respect to:\n\n    1.  The composite endpoint of stroke (ischemic, hemorrhagic, or of unspecified type), systemic embolism and major bleeding, in warfarin naive subjects\n\n. . .\n\n-   To assess the safety of apixaban in subjects with AF and at least one additional risk factor for stroke.\"98\n\n### Explanation\n\nThe study objectives reflect the scientific questions to be answered by the trial, and define its purpose and scope. They are closely tied to the trial design (Item 8) and analysis methods (Item 20). For example, the sample size calculation and statistical analyses for superiority trials will differ from those investigating non-inferiority.\n\nThe objectives are generally phrased using neutral wording (eg, \"to compare the effect of treatment A versus treatment B on outcome X\") rather than in terms of a particular direction of effect.99 A hypothesis states the predicted effect of the interventions on the trial outcomes. For multiarm trials, the objectives should clarify the way in which all the treatment groups will be compared (eg, A versus B; A versus C).\n\n## Trial design\n\nItem 8: Description of trial design including type of trial (eg, parallel group, crossover, factorial, single group), allocation ratio, and framework (eg, superiority, equivalence, non-inferiority, exploratory)\n\n### Example\n\n\"The PROUD trial is designed as a randomised, controlled, observer, surgeon and patient blinded multicenter superiority trial with two parallel groups and a primary endpoint of wound infection during 30 days after surgery . . . randomization will be performed as block randomization with a 1:1 allocation.\"100\n\n### Explanation\n\nThe most common design for published randomised trials is the parallel group, two arm, superiority trial with 1:1 allocation ratio.101 Other trial types include crossover, cluster, factorial, split body, and n of 1 randomised trials, as well as single group trials and non-randomised comparative trials.\n\nFor trials with more than one study group, the allocation ratio reflects the intended relative number of participants in each group (eg, 1:1 or 2:1). Unequal allocation ratios are used for a variety of reasons, including potential cost savings, allowance for learning curves, and ethical considerations when the balance of existing evidence appears to be in favour of one intervention over the other.102 Evidence also suggests a preference of some participants for enrolling in trials with an allocation ratio that favours allocation to an active treatment.92\n\nThe framework of a trial refers to its overall objective to test the superiority, non-inferiority, or equivalence of one intervention with another, or in the case of exploratory pilot trials, to gather preliminary information on the intervention (eg, harms, pharmacokinetics) and the feasibility of conducting a full-scale trial.\n\nIt is important to specify and explain the choice of study design because of its close relation to the trial objectives (Item 7) and its influence on the study methods, conduct, costs,103 results,104 105 106 and interpretation. For example, factorial and non-inferiority trials can involve more complex methods, analyses, and interpretations than parallel group superiority trials.107 108 In addition, the interpretation of trial results in published reports is not always consistent with the pre-specified trial framework,6 109 110 especially among reports claiming post hoc equivalence based on a failure to demonstrate superiority rather than a specific test of equivalence.109\n\nThere is increasing interest in adaptive designs for clinical trials, defined as the use of accumulating data to decide how to modify aspects of a study as it continues, without undermining the validity and integrity of the trial.111 112 Examples of potential adaptations include stopping the trial early, modifying the allocation ratio, re-estimating the sample size, and changing the eligibility criteria. The most valid adaptive designs are those in which the opportunity to make adaptations is based on prespecified decision rules that are fully documented in the protocol (Item 21b).\n\n# Section 3a: Methods\u2014participants, interventions, and outcomes\n\n## Study setting\n\nItem 9: Description of study settings (eg, community clinic, academic hospital) and list of countries where data will be collected. Reference to where list of study sites can be obtained\n\n### Example\n\n#### \"Selection of countries\n\n. . . To detect an intervention-related difference in HIV incidences with the desired power, the baseline incidences at the sites must be sufficiently high. We chose the participating sites so that the average baseline annual incidence across all communities in the study is likely to reach at least 3%. The various sites in sub-Saharan Africa met this criterion, but we also wanted sites in Asia to extend the generalizability of the intervention. The only location in Asia with sufficient incidence at the community level is in ethnic minority communities in Northern Thailand, where HIV incidence is currently in excess of 7%;\\[reference\\] thus they were invited to participate as well. Our final selection of sites combines rural (Tanzania, Zimbabwe, Thailand, and KwaZulu-Natal) and an urban (Soweto) location. The cultural circumstances between the sub-Saharan African sites vary widely.\n\n#### Definition of community\n\nEach of the three southern African sites (Harare, Zimbabwe; and Soweto and Vulindlela, South Africa) selected eight communities, the East African (Tanzanian) site selected 10 communities, and Thailand selected 14 communities . . . They are of a population size of approximately 10,000 . . . which fosters social familiarity and connectedness, and they are geographically distinct. Communities are defined primarily geographically for operational purposes for the study, taking into account these dimensions of social communality. The communities chosen within each country and site are selected to be sufficiently distant from each other so that there would be little cross-contamination or little possibility that individuals from a control community would benefit from the activities in the intervention community.\"113\n\n### Explanation\n\nA description of the environment in which a trial will be conducted provides important context in terms of the applicability of the study results; the existence and type of applicable local regulation and ethics oversight; and the type of healthcare and research infrastructure available. These considerations can vary substantially within and between countries.\n\nAt a minimum, the countries***,*** type of setting (eg, urban versus rural), and the likely number of study sites should be reported in the protocol. These factors have been associated with recruitment success and degree of attrition for some trials,68 91 92 114 115 116 117 but not for others.118 119 Trial location has also been associated with trial outcome,120 aspects of trial quality (eg, authenticity of randomisation121), and generalisability.122\n\n## Eligibility criteria\n\nItem 10: Inclusion and exclusion criteria for participants. If applicable, eligibility criteria for study centres and individuals who will perform the interventions (eg, surgeons, psychotherapists)\n\n### Examples\n\n\"Patients (or a representative) must provide written, informed consent before any study procedures occur (see Appendix 1 for sample Informed Consent Form) . . .\n\n#### 5.1. Inclusion Criteria\n\nPatients eligible for the trial must comply with all of the following **at randomization**:\n\n1\\. Age \u226516 years\n\n2\\. Current admission under the care of the heart-failure service at the site\n\n. . .\n\n#### 5.2. Exclusion Criteria\n\n1\\. Acute decompensation thought by the attending heart-failure physician to require or be likely to require PAC \\[pulmonary-artery catheter\\] during the next 24 hours. Such patients should be entered into the PAC Registry (see below).\n\n2\\. Inability to undergo PAC placement within the next 12 hours\n\n. . .\n\n**Patients enrolled in other investigational drug studies are potential candidates for ESCAPE**. As the ESCAPE protocol does not involve any investigational agents or techniques, patients would be eligible for dual randomization if they are on stable doses of the investigational drugs. . . .\n\n#### 13. Study Network, Training, and Responsibilities\n\n. . . To qualify, physicians responsible for PAC \\[pulmonary-artery catheter\\] placements will be required to show proof of insertion of \u226550 PACs in the previous year with a complication rate of \\<5%. Further, clinicians will need to show competence in the following areas to participate in the study: 1) insertion techniques and cardiovascular anatomy; 2) oxygen dynamics; . . . and 7) common PAC complications.\\[reference\\] . . . we will assume basic competence in these areas after satisfactory completion of the PACEP \\[PAC educational programme\\] module.\"123\n\n### \"Trial centre requirements\n\nA number of guidelines have stated thrombolysis should only be considered if the patient is admitted to a specialist centre with appropriate experience and expertise.\\[reference\\] Hospitals participating in IST-3 \\[third International Stroke Trial\\] should have an organized acute stroke service. The components of effective stroke unit care have been identified . . . In brief, the facilities (details of these requirements are specified in the separate operations manual) should include:\n\n-   Written protocol for the acute assessment of patients with suspected acute stroke to include interventions to reduce time from onset to treatment.\n\n-   Immediate access to CT \\[computed tomographic\\] or MR \\[magnetic resonance\\] brain scanning (preferably 24 hours a day).\n\nA treatment area where thrombolysis may be administered and the patient monitored according to trial protocol, preferably an acute stroke unit.\"124\n\n### Explanation\n\nEligibility criteria for potential trial participants define the study population. They can relate to demographic information; type or severity of the health condition; comorbidities; previous or current treatment; diagnostic procedures; pregnancy; or other relevant considerations.125 In trials of operator-dependent interventions such as surgery and psychotherapy, it is usually important to promote consistency of intervention delivery by also defining the eligibility criteria for care providers and centres where the intervention will be administered.126\n\nClear delineation of eligibility criteria serves several purposes. It enables study personnel to apply these criteria consistently throughout the trial.127 The choice of eligibility criteria can affect recruitment and attrition,67 114 115 117 118 128 129 130 as well as outcome event rates.39 131 In addition, the criteria convey key information related to external validity (generalisability or applicability).132 The importance of transparent documentation is highlighted by evidence that the eligibility criteria listed in publications are often different from those specified in the protocol.125 133 134\n\nCertain eligibility criteria warrant explicit justification in the protocol, particularly when they limit the trial sample to a narrow subset of the population.132 135 136 The appropriateness of restrictive participant selection depends on the trial objectives.137 When trial participants differ substantially from the overall population to whom the intervention will be applied, the trial results may not reflect the impact in real world practice settings.134 138 139 140 141 142 143 144\n\n## Interventions\n\nItem 11a: Interventions for each group with sufficient detail to allow replication, including how and when they will be administered\n\n### Example\n\n\"Eligible patients will be randomised in equal proportions between IL-1ra \\[interleukin-1 receptor antagonist\\] and placebo, receiving either a once daily, subcutaneous (s.c.) injection of IL-1ra (dose 100 mg per 24 h) for 14 days, or a daily s.c. injection of placebo for 14 days . . .\n\nThe study drug and placebo will be provided by Amgen Inc in its commercially available recombinant form . . . The study drug and placebo will be relabelled by Amgen, in collaboration with CTEU \\[Clinical Trials and Evaluation Unit\\] according to MHRA \\[Medicines and Healthcare Products Regulatory Agency\\] guidelines.\n\nThe first dose of IL-1ra will be given within 24 h +2 h of the positive Troponin. Injections will be given at a standardised time (24 \u00b1 2 h after the previous dose), immediately after blood sampling. IL-1ra or placebo will \\[be\\] administered to the patient by the research nurse while the patient is in hospital. During the hospital stay, the patient will be taught to self-administer the injection by the research nurse and on discharge will continue at home. This has proven possible in other ACS \\[acute coronary syndrome\\] trials that required self injection of subcutaneous heparin \\[reference\\]. Full written guidance on self injection will also be provided to patients. If self injection is found not to be possible in an individual patient for unexpected reasons, an alternative method will be sought (eg district nurse, or attending the hospital) to try and maintain full compliance with scheduled study drug regimen after discharge. Patients will also be asked to complete a daily injection diary. All personnel will be blinded to the identity of the syringe contents.\"145\n\n### Explanation\n\nStudies of trials and systematic reviews have shown that important elements of the interventions are not described in half of the publications.146 147 If such elements are also missing from the protocol, or if the protocol simply refers to other documents that are not freely accessible, then it can be impossible for healthcare providers, systematic reviewers, policymakers, and others to fully understand, implement, or evaluate the trial intervention.148 This principle applies to all types of interventions, but is particularly true for complex interventions (eg, health service delivery; psychotherapy), which consist of interconnected components that can vary between healthcare providers and settings.\n\nFor drugs, biological agents, or placebos, the protocol description should include the generic name, manufacturer, constituent components, route of administration, and dosing schedule (including titration and run-in periods, if applicable).149 150 The description of non-drug interventions\u2014such as devices, procedures, policies, models of care, or counselling\u2014is generally more complex and warrants additional details about the setting (Item 9) and individuals administering the interventions. For example, the level of pre-trial expertise (Item 10) and specific training of individuals administering these complex interventions are often relevant to describe (eg, for surgeons, psychologists, physiotherapists). When intervention delivery is subject to variation, it is important to state whether the same individuals will deliver the trial interventions in all study groups, or whether different individuals will manage each study group\u2014in which case it can be difficult to separate the effect of the intervention from that of the individual delivering it. Interventions that consist of \"usual care\" or \"standard of care\" require further elaboration in the protocol, as this care can vary substantially across centres and patients, as well as over the duration of the trial.\n\n## Interventions\u2014modifications\n\nItem 11b: Criteria for discontinuing or modifying allocated interventions for a given trial participant (eg, drug dose change in response to harms, participant request, or improving\/worsening disease)\n\n### Example\n\n#### \"Gastro-Intestinal Upset\n\nThe tablets may be taken in two equally divided doses, if necessary, to improve gastro-intestinal tolerance. Should it be necessary the daily dose may be reduced by one tablet at a time to improve gastro-intestinal tolerance.\n\n#### Renal Function Impairment\n\nSince sodium clodronate is excreted unchanged by the kidney its use is contra-indicated in patients with moderate to severe renal impairment (serum creatinine greater than 2 times upper limit of normal range of the centre). If renal function deteriorates to this extent the trial medication should be withdrawn from the patient. **This should be reported as an adverse event.** In patients with normal renal function or mild renal impairment (serum creatinine less than 2 times upper limit of normal range of the centre) serum creatinine should be monitored during therapy.\n\n#### Allergic Reactions\n\nAllergic skin reactions have been observed in rare cases. If this is suspected withdraw the trial medication from the patient. **This should be reported as an adverse event.**\n\n#### Biochemical Disturbances\n\nAsymptomatic hypocalcaemia has been noted rarely. Temporary suspension of the trial medication until the serum calcium returns into the normal range is recommended. The trial medication can be then restarted at half the previous dose. If the situation returns withdraw the trial medication from the patient. **This should be reported as an adverse event**.\"151\n\n### Explanation\n\nFor a given trial participant, the assigned study intervention may need to be modified or discontinued by trial investigators for various reasons, including harms, improved health status, lack of efficacy, and withdrawal of participant consent. Comparability across study groups can be improved, and subjectivity in care decisions reduced, by defining standard criteria for intervention modifications and discontinuations in the protocol. Regardless of any decision to modify or discontinue their assigned intervention, study participants should be retained in the trial whenever possible to enable follow-up data collection and prevent missing data (Item 18b).152\n\n## Interventions\u2014adherence\n\nItem 11c: Strategies to improve adherence to intervention protocols, and any procedures for monitoring adherence (eg, drug tablet return; laboratory tests)\n\n### Example\n\n#### \"Adherence reminder sessions\n\nFace-to-face adherence reminder sessions will take place at the initial product dispensing and each study visit thereafter. This session will include:\n\n-   The importance of following study guidelines for adherence to once daily study product\n\n-   Instructions about taking study pills including dose timing, storage, and importance of taking pills whole, and what to do in the event of a missed dose.\n\n-   Instructions about the purpose, use, and care of the MEMS\u00ae cap \\[medication event monitoring system\\] and bottle\n\n-   Notification that there will be a pill count at every study visit\n\n-   Reinforcement that study pills may be TDF \\[tenofovir disproxil fumarate\\] or placebo\n\n-   Importance of calling the clinic if experiencing problems possibly related to study product such as symptoms, lost pills or MEMS\u00ae cap.\n\nSubsequent sessions will occur at the follow-up visits. Participants will be asked about any problems they are having taking their study pills or using the MEMS\u00ae cap. There will be brief discussion of reasons for missed doses and simple strategies for enhancing adherence, eg, linking pill taking to meals or other daily activities. Participants will have an opportunity to ask questions and key messages from the initial session will be reviewed as needed . . .\n\n#### Adherence assessments\n\nTo enhance validity of data, multiple methods will be used to assess medication adherence including pill count; an electronic medication event monitoring system (MEMS\u00ae cap) \\[reference\\]; and ACASI \\[audio-computer administered interview\\] questionnaire items including a one month visual analogue scale,\\[reference\\] reasons for non-compliance, and use of the MEMS\u00ae cap. Participants will return the unused tablets and bottle at each follow-up visit. Unused tablets will be counted and recorded on the appropriate CRF \\[case report form\\]. Electronic data collected in the MEMS\u00ae cap will be downloaded into a designated, secure study computer.\"153\n\n### Explanation\n\nAdherence to intervention protocols refers to the degree to which the behaviour of trial participants corresponds to the intervention assigned to them.154 Distinct but related concepts include trial retention (Item 18b) and adherence to the follow-up protocol of procedures and assessments (Item 13).\n\nOn average, adherence to intervention protocols is higher in clinical trials than in non-research settings.155 Although there is no consensus on the acceptable minimum adherence level in clinical trials, low adherence can have a substantial effect on statistical power and interpretation of trial results.156 157 158 Since fewer participants are receiving the full intervention as intended, non-adherence can reduce the contrast between study groups\u2014leading to decreased study power and increased costs associated with recruiting larger sample sizes for evaluating superiority, or leading to potentially inappropriate conclusions of non-inferiority or equivalence. There is also the possibility of underestimating any efficacy and harms of the study intervention.\n\nFurthermore, if adherence is a marker for general healthy behaviour associated with better prognosis, then different rates of non-adherence between study groups can lead to a biased estimate of an intervention's effect. In support of this \"healthy adherer\" effect, non-adherers to placebo in clinical studies have been found to have poorer clinical outcomes than adherers.159\n\nTo help avoid these potential detrimental effects of non-adherence, many trials implement procedures and strategies for monitoring and improving adherence,67 156 157 158 and any such plans should be described in the protocol.160 Among applicable drug trials published in 1997-99, 47% reported monitoring the level of adherence.161 Although each of the many types of monitoring methods has its limitations,157 158 adherence data can help to inform the statistical analysis (Item 20c), trial interpretation, and choice of appropriate adherence strategies to implement in the trial as it progresses or in future trials and clinical practice.\n\nA variety of adherence strategies exist,156 157 158 and their use can be tailored to the specific type of trial design, intervention, and participant population. It may be desirable to select strategies that can be easily implemented in clinical practice, so that the level of adherence in the real world setting is comparable to that observed in the trial.158\n\n## Interventions\u2014concomitant care\n\nItem 11d: Relevant concomitant care and interventions that are permitted or prohibited during the trial\n\n### Example\n\n\"**2. Rescue Medication**\n\n**For weeks 0-3**, topical mometasone furoate 0.1% cream or ointment (30 g\/week) will be permitted with participants preferably using ointment. Participants will be instructed to apply the topical mometasone furoate to blisters\/lesions as required (not to areas of unaffected skin). If the participant is allergic to mometasone furoate or the hospital pharmacy does not stock it, then an alternative topical steroid may be prescribed but this must be in the potent class. In addition, participants will be advised that they can apply a light moisturiser to blisters\/lesions at any time during the study.\n\n**For weeks 3-6**, use of mometasone furoate (or other topical corticosteroids) is strongly discouraged to prevent potential systemic effects. Accidental use of mometasone furoate or other potent topical steroid during this period will be classified as a protocol deviation.\n\n**After week 6**, potent topical corticosteroids (up to 30 g\/week) may be used to treat symptoms and localised disease if they would have normally been used as part of normal clinical care by the physician in charge of that patient. This must be recorded on the trial treatment log.\n\nHowever, those patients who are on a dose reducing regime for oral steroids, 30 g\/week of a \"potent\" topical steroid will be allowed.\n\n3\\. Prohibited Concomitant Medications\n\nThe administration of live virus vaccines is not permitted for all participants during weeks 0-6 as the investigator is blinded to treatment allocation, and must therefore warn all participants to refrain for \\[sic\\] having a live virus vaccine. However, after week 6, once the investigator knows which medication the participant is on, only those taking prednisolone will not be allowed live virus vaccines.\n\nParticipants should continue to take medications for other conditions as normal. However, if it is anticipated that the participant will need a live virus vaccine during the intervention phase, they will be ineligible for entry into the study.\"50\n\n### Explanation\n\nIn a controlled trial, a key goal is to have comparable study groups that differ only by the intervention being evaluated, so that any difference in outcomes can be attributed to effects of the study intervention. Cointervention bias can arise when the study groups receive different concomitant care or interventions (in addition to the assigned trial interventions) that may affect trial outcomes.162 To promote comparability of study groups, the protocol should list the relevant concomitant care and interventions that are allowed (including rescue interventions), as well as any that are prohibited.\n\n## Outcomes\n\nItem 12: Primary, secondary, and other outcomes, including the specific measurement variable (eg, systolic blood pressure), analysis metric (eg, change from baseline, final value, time to event), method of aggregation (eg, median, proportion), and time point for each outcome. Explanation of the clinical relevance of chosen efficacy and harm outcomes is strongly recommended\n\n### Example\n\n#### \"1. Primary Outcome Measures\n\n-   Difference between the two treatment arms in the proportion of participants classed as treatment success at 6 weeks. Treatment success is defined as 3 or less significant blisters present on examination at 6 weeks. Significant blisters are defined as intact blisters containing fluid which are at least 5 mm in diameter. However, if the participant has popped a blister, or the blister is at a site that makes it susceptible to bursting such as the sole of the foot, it can be considered part of the blister count, providing there is a flexible (but not dry) roof present over a moist base. Mucosal blisters will be excluded from the count.\n\nA survey of the UK DCTN \\[Dermatology Clinical Trials Network\\] membership showed that a point estimate of 25% inferiority in effectiveness would be acceptable assuming a gain in the safety profile of at least 10%.\n\nThis measure of success was selected as it was considered to be more clinically relevant than a continuous measure of blister count. It would be less clinically relevant to perform an absolute blister count and report a percentage reduction. Instead, to state that treatment is considered a success if remission is achieved (ie the presence of three or less blisters on physical examination at 6 weeks) more closely reflects clinical practice. In addition, it is far less burdensome on investigators than including a full blister count, which would mean counting in the region of 50-60 blisters in many cases. This outcome measure will be performed as a single blind assessment.\n\n-   Difference between the two treatment arms in the proportion of participants reporting grade 3, 4 and 5 (mortality) adverse events which are possibly, probably or definitely related to BP \\[bullous pemphigoid\\] medication in the 52 weeks following randomisation. A modified version of The Common Terminology Criteria for Adverse Events (CTCAE v3.0) will be used to grade adverse events. At each study visit, participants will be questioned about adverse events they have experienced since the last study visit (using a standard list of known side effects of the two study drugs).\n\n#### 2. Secondary Outcome Measures\n\nFor the secondary and tertiary endpoints a participant will be classed as a treatment success if they have 3 or less significant blisters present on examination and have not had their treatment modified (changed or dose increased) on account of a poor response.\n\n-   Difference in the proportion of participants who are classed as a treatment success at 6 weeks.\n\n-   Difference in the proportion of participants in each treatment arm who are classed as treatment success at 6 weeks and are alive at 52 weeks. This measure will provide a good overall comparison of the two treatment arms.\"50\n\n### Explanation\n\nThe trial outcomes are fundamental to study design and interpretation of results. For a given intervention, an outcome can generally reflect efficacy (beneficial effect) or harm (adverse effect). The outcomes of main interest are designated as primary outcomes, which usually appear in the objectives (Item 7) and sample size calculation (Item 14). The remaining outcomes constitute secondary or other outcomes.\n\nFor each outcome, the trial protocol should define four components: the specific measurement variable, which corresponds to the data collected directly from trial participants (eg, Beck Depression Inventory score, all cause mortality); the participant-level analysis metric, which corresponds to the format of the outcome data that will be used from each trial participant for analysis (eg, change from baseline, final value, time to event); the method of aggregation, which refers to the summary measure format for each study group (eg, mean, proportion with score \\> 2); and the specific measurement time point of interest for analysis.163\n\nIt is also important to explain the rationale for the choice of trial outcomes. An ideal outcome is valid, reproducible, relevant to the target population (eg, patients), and responsive to changes in the health condition being studied.67 The use of a continuous versus dichotomous method of aggregation can affect study power and estimates of treatment effect,164 165 and subjective outcomes are more prone to bias from inadequate blinding (ascertainment bias) and allocation concealment (selection bias) than objective outcomes.166 167 Although composite outcomes increase event rates and statistical power, their relevance and interpretation can be unclear if the individual component outcomes vary greatly in event rates, importance to patients, or amount of missing data.168 169 170 171\n\nThe number of primary outcomes should be as small as possible. Although up to 38% of trials define multiple primary outcomes,4 35 163 this practice can introduce problems with multiplicity, selective reporting, and interpretation when there are inconsistent results across outcomes. Problems also arise when trial protocols do not designate any primary outcomes, as seen in half (28\/59) of protocols for a sample of trials published from 2002-2008,12 and in 25% of randomised trial protocols that received ethics approval in Denmark in 1994-95.4 Furthermore, major discrepancies in the primary outcomes designated in protocols\/registries\/regulatory submissions versus final trial publications are common; favour the reporting of statistically significant primary outcomes over non-significant ones; and are often not acknowledged in final publications.172 173 174 175 176 Such bias can only be identified and deterred if trial outcomes are clearly defined beforehand in the protocol and if protocol information is made public.177\n\nWhere possible, the development and adoption of a common set of key trial outcomes within a specialty can help to deter selective reporting of outcomes and to facilitate comparisons and pooling of results across trials in a meta-analysis.178 179 180 The COMET (Core Outcome Measures in Effectiveness Trials) Initiative aims to facilitate the development and application of such standardised sets of core outcomes for clinical trials of specific conditions ([www.comet-initiative.org](http:\/\/www.comet-initiative.org\/)). Trial investigators are encouraged to ascertain whether there is a core outcome set relevant to their trial and, if so, to include those outcomes in their trial. Existence of a common set of outcomes does not preclude inclusion of additional relevant outcomes for a given trial.\n\n## Participant timeline\n\n*Item 13: Time schedule of enrolment, interventions (including any run-ins and washouts), assessments, and visits for participants. A schematic diagram is highly recommended (see fig 1*<a href=\"#fig1\" data-ref-type=\"fig\"><\/a>*)*<a href=\"#fig2\" data-ref-type=\"fig\"><\/a>\n\n### Examples\n\n\"The main outcomes of interest are the drug and sex-related HIV and HCV \\[hepatitis C virus\\] risk behaviors . . . Clients will be assessed using the full battery of instruments from the Common Assessment Battery (CAB), along with the Self-Efficacy and Stages of Change questionnaires and a Urine Drug Screen after consenting . . . questionnaires will take place for all participants 14-30 days after randomization during which they will be given the Stages of Change and Self-Efficacy questionnaires, the Timeline Follow-Back, and a UA \\[urine analysis\\]. Follow-up interviews, using the full battery (CAB and questionnaires), will be collected at 2 months (56 days), 4 months (112 days) and 6 months (168 days) after the randomization date. A 14 day window, defined as 7 days before and 7 days after the due date, will be available to complete the 2 and 4 month follow-up interviews and a 28 day window, defined as 7 days before and 21 days after the due date, will be available to complete the 6 month follow up interview . . .\n\n#### 7.1.1 Common Assessment Battery (CAB)\n\n1.  A Demographic Questionnaire . . .\n\n2.  The Composite International Diagnostic Interview Version 2.1 . . .\n\n3.  The Addiction Severity Index-Lite (ASI-Lite) . . .\n\n4.  The Risk Behavior Survey (RBS), . . .\n\n#### 7.1.2 Additional Interviews\/Questionnaires\n\nTo assess drug use, urinalysis for morphine, cocaine, amphetamine, and methamphetamine will be performed at the 2-Week Interim Visit, and the 2-, 4-, and 6-month Follow-up visits . . .\n\n1.  Stage of change for quitting drug use will be measured using a modification of the\n\n2.  Motivation Scales\"<a href=\"#tbl3\" data-ref-type=\"table\"><\/a> 181\n\nHIV\/HCV risk reduction protocol schedule of forms and procedures (adapted from original table^181^)\n\n<table>\n<thead>\n<tr>\n<th style=\"text-align: left;\"><\/th>\n<th style=\"text-align: center;\"><\/th>\n<th style=\"text-align: center;\"><\/th>\n<th style=\"text-align: center;\"><\/th>\n<th colspan=\"2\" style=\"text-align: center;\">-1<\/th>\n<th style=\"text-align: center;\">0<\/th>\n<th style=\"text-align: center;\">T1<\/th>\n<th style=\"text-align: center;\">T2<\/th>\n<th style=\"text-align: center;\">F1<\/th>\n<th style=\"text-align: center;\">F2<\/th>\n<\/tr>\n<tr>\n<th style=\"text-align: left;\">Activity\/ assessment<\/th>\n<th style=\"text-align: center;\">CRF (Yes\/No)<\/th>\n<th style=\"text-align: center;\">Staff member<\/th>\n<th style=\"text-align: center;\">Approximate time to complete (min)<\/th>\n<th colspan=\"2\" style=\"text-align: center;\">Prestudy screening\/consent<\/th>\n<th style=\"text-align: center;\">Prestudy baseline\/ randomisation<\/th>\n<th style=\"text-align: center;\">Study visit 1<\/th>\n<th style=\"text-align: center;\">Study\/interim visit 2 and\/or 2 week interim<\/th>\n<th style=\"text-align: center;\">Follow-up 2 months<\/th>\n<th style=\"text-align: center;\">Follow-up 4-months<\/th>\n<\/tr>\n<\/thead>\n<tbody>\n<tr>\n<td style=\"text-align: left;\">Prescreening consent<\/td>\n<td style=\"text-align: center;\">No<\/td>\n<td style=\"text-align: left;\">Study coordinator<\/td>\n<td style=\"text-align: center;\">5<\/td>\n<td colspan=\"2\" style=\"text-align: center;\">X<\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Screening log<\/td>\n<td style=\"text-align: center;\">No<\/td>\n<td style=\"text-align: left;\">Study coordinator<\/td>\n<td style=\"text-align: center;\">5<\/td>\n<td colspan=\"2\" style=\"text-align: center;\">X<\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Consent form\/quiz<\/td>\n<td style=\"text-align: center;\">No<\/td>\n<td style=\"text-align: left;\">Study coordinator<\/td>\n<td style=\"text-align: center;\">45<\/td>\n<td colspan=\"2\" style=\"text-align: center;\">X<\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Inclusion\/<br \/>\nexclusion form<\/td>\n<td style=\"text-align: center;\">Yes<\/td>\n<td style=\"text-align: left;\">Study coordinator<\/td>\n<td style=\"text-align: center;\">N\/A<\/td>\n<td colspan=\"2\" style=\"text-align: center;\">X<\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Urine screen<\/td>\n<td style=\"text-align: center;\">Yes<\/td>\n<td style=\"text-align: left;\">Study coordinator<\/td>\n<td style=\"text-align: center;\">10<\/td>\n<td colspan=\"2\" style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\">X<\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\">X<\/td>\n<td style=\"text-align: center;\">X<\/td>\n<td style=\"text-align: center;\">X<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Locator form<\/td>\n<td style=\"text-align: center;\">No<\/td>\n<td style=\"text-align: left;\">Interviewer<\/td>\n<td style=\"text-align: center;\">10<\/td>\n<td colspan=\"2\" style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\">X<\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\">Update X<\/td>\n<td style=\"text-align: center;\">Update X<\/td>\n<td style=\"text-align: center;\">Update X<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Demographics questionnaire<\/td>\n<td style=\"text-align: center;\">Yes<\/td>\n<td style=\"text-align: left;\">Interviewer<\/td>\n<td style=\"text-align: center;\">10<\/td>\n<td colspan=\"2\" style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\">X<\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Addiction severity index (ASI) lite<\/td>\n<td style=\"text-align: center;\">Yes<\/td>\n<td style=\"text-align: left;\">Interviewer<\/td>\n<td style=\"text-align: center;\">45<\/td>\n<td colspan=\"2\" style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\">X<\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\">X<\/td>\n<td style=\"text-align: center;\">X<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Composite international diagnostic interview)<\/td>\n<td style=\"text-align: center;\">Yes<\/td>\n<td style=\"text-align: left;\">Interviewer<\/td>\n<td style=\"text-align: center;\">45<\/td>\n<td colspan=\"2\" style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\">X<\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">HIV risk behaviour survey<\/td>\n<td style=\"text-align: center;\">Yes<\/td>\n<td style=\"text-align: left;\">Interviewer<\/td>\n<td style=\"text-align: center;\">15<\/td>\n<td colspan=\"2\" style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\">X<\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\">X<\/td>\n<td style=\"text-align: center;\">X<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Timeline follow back<\/td>\n<td style=\"text-align: center;\">Yes<\/td>\n<td style=\"text-align: left;\">Interviewer<\/td>\n<td style=\"text-align: center;\">5<\/td>\n<td colspan=\"2\" style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\">X<\/td>\n<td style=\"text-align: center;\">X<\/td>\n<td style=\"text-align: center;\">X<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Self efficacy<\/td>\n<td style=\"text-align: center;\">Yes<\/td>\n<td style=\"text-align: left;\">Interviewer<\/td>\n<td style=\"text-align: center;\">5<\/td>\n<td colspan=\"2\" style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\">X<\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\">X<\/td>\n<td style=\"text-align: center;\">X<\/td>\n<td style=\"text-align: center;\">X<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Stage of change<\/td>\n<td style=\"text-align: center;\">Yes<\/td>\n<td style=\"text-align: left;\">Interviewer<\/td>\n<td style=\"text-align: center;\">5<\/td>\n<td colspan=\"2\" style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\">X<\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\">X<\/td>\n<td style=\"text-align: center;\">X<\/td>\n<td style=\"text-align: center;\">X<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Randomisation<\/td>\n<td style=\"text-align: center;\">Yes<\/td>\n<td style=\"text-align: left;\">Study coordinator<\/td>\n<td style=\"text-align: center;\">15<\/td>\n<td colspan=\"2\" style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\">X<\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Voluntary blood sample Counselling and education intervention (treatment group)<\/td>\n<td style=\"text-align: center;\">Yes<\/td>\n<td style=\"text-align: left;\">Study phlebotomist<\/td>\n<td style=\"text-align: center;\">15<\/td>\n<td colspan=\"2\" style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\">X<\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">All groups, optional blood sample at study close<\/td>\n<td style=\"text-align: center;\">Yes<\/td>\n<td style=\"text-align: left;\">Study phlebotomist<\/td>\n<td style=\"text-align: center;\">15<\/td>\n<td colspan=\"2\" style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Termination form<\/td>\n<td style=\"text-align: center;\">Yes<\/td>\n<td style=\"text-align: left;\">Study coordinator<\/td>\n<td style=\"text-align: center;\">N\/A<\/td>\n<td colspan=\"2\" style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Serious adverse event form<\/td>\n<td style=\"text-align: center;\">Yes<\/td>\n<td style=\"text-align: left;\">Study coordinator<\/td>\n<td style=\"text-align: center;\">N\/A<\/td>\n<td colspan=\"7\" style=\"text-align: left;\">As needed throughout protocol<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Progress notes<\/td>\n<td style=\"text-align: center;\">No<\/td>\n<td style=\"text-align: left;\">All team members<\/td>\n<td style=\"text-align: center;\">N\/A<\/td>\n<td style=\"text-align: center;\">X<\/td>\n<td colspan=\"2\" style=\"text-align: center;\">X<\/td>\n<td style=\"text-align: center;\">X<\/td>\n<td style=\"text-align: center;\">X<\/td>\n<td style=\"text-align: center;\">X<\/td>\n<td style=\"text-align: center;\">X<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Communication log<\/td>\n<td style=\"text-align: center;\">No<\/td>\n<td style=\"text-align: left;\">All team members<\/td>\n<td style=\"text-align: center;\">N\/A<\/td>\n<td colspan=\"7\" style=\"text-align: left;\">Every phone or in-person contact outside of a regular visit<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n### Explanation\n\nA clear and concise timeline of the study visits, enrolment process, interventions, and assessments performed on participants can help to guide trial conduct and enable external review of participant burden and feasibility. These factors can also affect the decision of potential investigators and participants to join the trial (Item 15).91\n\nA schematic diagram is highly recommended to efficiently present the overall schedule and time commitment for trial participants in each study group. Though various presentation formats exist, key information to convey includes the timing of each visit, starting from initial eligibility screening through to study close-out; time periods during which trial interventions will be administered; and the procedures and assessments performed at each visit (with reference to specific data collection forms, if relevant) (fig 1<a href=\"#fig1\" data-ref-type=\"fig\"><\/a>).\n\n## Sample size\n\nItem 14: Estimated number of participants needed to achieve study objectives and how it was determined, including clinical and statistical assumptions supporting any sample size calculations\n\n### Examples\n\n\"The sample size was calculated on the basis of the primary hypothesis. In the exploratory study,\\[reference\\] those referred to PEPS \\[psychoeducation with problem solving\\] had a greater improvement in social functioning at 6 month follow-up equivalent to 1.05 points on the SFQ \\[Social Functioning Questionnaire\\]. However, a number of people received PEPS who were not included in the trial (eg, the wait-list control) and, for this larger sample (N=93), the mean pre-post- treatment difference was 1.79 (pre-treatment mean=13.85, SD=4.21; post-treatment mean=12.06, SD=4.21). (Note: a lower SFQ score is more desirable). This difference of almost 2 points accords with other evidence that this is a clinically significant and important difference.\\[reference\\] A reduction of 2 points or more on the SFQ at 1 year follow-up in an RCT of cognitive behaviour therapy in health anxiety was associated with a halving of secondary care appointments (1.24.vs 0.65), a clinically significant reduction in the Hospital Anxiety and Depression Scale (HADS\\[reference\\]) Anxiety score of 2.5 (9.9 vs 7.45) and a reduction in health anxiety (the main outcome) of 5.6 points (17.8 vs 12.2) (11 is a normal population score and 18 is pathological).\\[reference\\] These findings suggest that improvements in social functioning may accrue over 1 year, hence we expect to find a greater magnitude of response at the 72 week follow-up than we did in the exploratory trial. Therefore, we have powered this trial to be able to detect a difference in SFQ score of 2 points. SFQ standard deviations vary between treatment, control, and the wait-list samples, ranging from 3.78 to 4.53. We have based our sample size estimate on the most conservative (ie, largest) SD \\[standard deviation\\]. To detect a mean difference in SFQ score of 2 point (SD = 4.53) at 72 weeks with a two-sided significance level of 1% and power of 80% with equal allocation to two arms would require 120 patients in each arm of the trial. To allow for 30% drop out, 170 will be recruited per arm, ie, 340 in total.\"183\n\n\"Superficial and deep incisional surgical site infection rates for patients in the PDS II\u00ae \\[polydioxanone suture\\] group are estimated to occur at a rate of 0.12.\\[reference\\] The trials by \\[reference\\] have shown a reduction of SSI \\[surgical site infections\\] of more than 50% (from 10.8% to 4.9% and from 9.2% to 4.3% respectively). Therefore, we estimate a rate of 0.06 for PDS Plus\u00ae \\[triclosan-coated continuous polydioxanone suture\\].\n\nFor a fixed sample size design, the sample size required to achieve a power of 1-\u03b2=0.80 for the one-sided chi-square test at level \u03b1=0.025 under these assumptions amounts to 2\u00d7356=712 (nQuery Advisor\u00ae, version 7.0). It can be expected that including covariates of prognostic importance in the logistic regression model as defined for the confirmatory analysis will increase the power as compared to the chi-square test. As the individual results for the primary endpoint are available within 30 days after surgery, the drop-out rate is expected to be small. Nevertheless, a potential dilution of the treatment effect due to drop-outs is taken into account (eg no photographs available, loss to follow up); it is assumed that this can be compensated by additional 5% of patients to be randomized, and therefore the total sample size required for a fixed sample size design amounts to n=712+38=750 patients.\n\n. . .\n\nAn adaptive interim analysis \\[reference\\] will be performed after availability of the results for the primary endpoint for a total of 375 randomized patients (ie, 50% of the number of patients required in a fixed sample size design). The following type I error rates and decision boundaries for the interim and the final analysis are specified:\n\n1.  \u2022 Overall one-sided type I error rate: 0.025\n\n2.  \u2022 Boundary for the one-sided p-value of the first stage for accepting the null-hypothesis within the interim analysis: \u03b1~0~=0.5\n\n3.  \u2022 One-sided local type I error rate for testing the null-hypothesis within the interim analysis: \u03b1~1~=0.0102\n\n4.  \u2022 Boundary for the product of the one-sided p-values of both stages for the rejection of the null-hypothesis in the final analysis: c\u03b1=0.0038\n\nIf the trial will be continued with a second stage after the interim analysis (this is possible if for the one-sided p-value p~1~ of the interim analysis p~1~\u2208\\]0.0102,0.5\\[ \\[ie 0.5\u2265P~1~\u22650.0102\\] holds true, the results of the interim analysis can be taken into account for a recalculation of the required sample size. If the sample size recalculation leads to the conclusion that more than 1200 patients are required, the study is stopped, because the related treatment group difference is judged to be of minor clinical importance.\n\n. . .\n\nThe actually achieved sample size is then not fixed but random, and a variety of scenarios can be considered. If the sample size is calculated under the same assumptions with respect to the SSI rates for the two groups, applying the same the overall significance level of \u03b1=0.025 (one-sided) but employing additionally the defined stopping boundaries and recalculating the sample size for the second stage at a conditional power of 80% on the basis of the SSI rates observed in the interim analysis results in an average total sample size of n=766 patients; the overall power of the study is then 90% (ADDPLAN\u00ae, version 5.0).\"100\n\n### Explanation\n\nThe planned number of trial participants is a key aspect of study design, budgeting, and feasibility that is usually determined using a formal sample size calculation. If the planned sample size is not derived statistically, then this should be explicitly stated along with a rationale for the intended sample size (eg, exploratory nature of pilot studies; pragmatic considerations for trials in rare diseases).17 184\n\nFor trials that involve a formal sample size calculation, the guiding principle is that the planned sample size should be large enough to have a high probability (power) of detecting a true effect of a given magnitude, should it exist. Sample size calculations are generally based on one primary outcome; however, it may also be worthwhile to plan for adequate study power or report the power that will be available (given the proposed sample size) for other important outcomes or analyses because trials are often underpowered to detect harms or subgroup effects.185 186\n\nAmong randomised trial protocols that describe a sample size calculation, 4-40% do not state all components of the calculation.6 11 The protocol should generally include the following: the outcome (Item 12); the values assumed for the outcome in each study group (eg, proportion with event, or mean and standard deviation) (table 4<a href=\"#tbl4\" data-ref-type=\"table\"><\/a>); the statistical test (Item 20a); alpha (type 1 error) level; power; and the calculated sample size per group\u2014both assuming no loss of data and, if relevant, after any inflation for anticipated missing data (Item 20c). Trial investigators are also encouraged to provide a rationale or reference for the outcome values assumed for each study group.187 The values of certain prespecified variables tend to be inappropriately inflated (eg, clinically important treatment effect size)188 189 or underestimated (eg, standard deviation for continuous outcomes),190 leading to trials having less power in the end than what was originally calculated. Finally, when uncertainty of a sample size estimate is acknowledged, methods exist for re-estimating sample size.191 The intended use of such an adaptive design approach should be stated in the protocol.\n\nOutcome values to report in sample size calculation\n\n|  | Type of summary outcome |  |  |\n|----|----|----|----|\n| Element | Binary | Continuous | Time to event |\n| Assumed result for each study group | Proportion (%) with event | Mean and standard deviation | Proportion (%) with event at a given time point |\n| Effect measure | Relative risk, odds ratio | Difference in means | Hazard ratio |\n\nNote: Although the sample size calculation uses the expected outcome value for each group, the corresponding contrast between groups (estimated effect) should also be reported.\n\nFor designs and frameworks other than parallel group superiority trials, additional elements are required in the sample size calculation. For example, an estimate of the standard deviation of within-person changes from baseline should be included for crossover trials192; the intracluster correlation coefficient for cluster randomised trials193; and the equivalence or non-inferiority margin for equivalence or non-inferiority trials respectively.108 194 Such elements are often not described in final trial reports,110 195 196 197 198 and it is unclear how often they are specified in the protocol.\n\nComplete description of sample size calculations in the protocol enables an assessment of whether the trial will be adequately powered to detect a clinically important difference.189 199 200 201 202 203 204 205 206 It also promotes transparency and discourages inappropriate post hoc revision that is intended to support a favourable interpretation of results or portray consistency between planned and achieved sample sizes.6 207\n\n## Recruitment\n\nItem 15: Strategies for achieving adequate participant enrolment to reach target sample size\n\n### Example\n\n\"Each center will screen subjects to achieve screening percentages of 50% women and 33% minority; screening will continue until the target population is achieved (12 subjects\/site). We recognize that, because of exclusion by genotype and genotypic variation among diverse populations,\\[reference\\], the enrolled cohort may not reflect the screened population. The enrollment period will extend over 12 months.\n\n#### Recruitment Strategy\n\nEach clinical center involved in the ACRN \\[Asthma Clinical Research Network\\] was chosen based on documentation for patient availability, among other things. It is, however, worthy to note the specific plans of each center.\n\n. . . The Asthma Clinical Research Center at the Brigham & Women's Hospital utilizes three primary resources for identifying and recruiting potential subjects as described below.\n\n1\\. Research Patient Database\n\nThe Asthma Clinical Research Center at the Brigham and Women's Hospital has a database of over 1,500 asthmatics . . .\n\n2\\. Asthma Patient Lists . . .\n\n3\\. Advertisements . . .\n\n. . . the Madison ACRN site has utilized some additional approaches to target minority recruitment. We have utilized a marketing expert to coordinate and oversee our overall efforts in recruiting and retaining minorities. . . . As a result of his efforts, we have advertised widely in newspapers and other publications that target ethnic minorities, established contacts with various ethnic community, university, church, and business groups, and conducted community-based asthma programs . . . For example, student groups such as AHANA (a pre-health careers organization focusing on minority concerns) will be contacted. . . . In addition, we will utilize published examples of successful retention strategies such as frequent payment of subject honoraria as study landmarks are achieved and study participant group social events. Study visits will be carefully planned and scheduled to avoid exam-time and university calendar breaks . . .\n\nThe Harlem Hospital Center Emergency Department (ED) sees an average of eight adult patients per day for asthma. Through the REACH (Reducing Emergency Asthma Care in Harlem) project, we have . . . successfully recruited and interviewed 380 patients from the ED . . .\n\nResponses to inquiries about participation in research studies are answered by a dedicated phone line that is manned during business hours and answered by voicemail at all other times. A research assistant responds to each inquiry immediately, using a screening instrument . . .\n\nPatients are recruited for clinical trials at the Jefferson Center through two primary mechanisms: (1) local advertising; and (2) identification in the asthma patient registry (database). Local advertising takes advantage of the printed as well as the audio-visual media. Printed media include . . . All advertising in the printed and audio-visual media has prior approval of the Institutional Review Board.\n\nThe Jefferson patient registry (database) has been maintained since 1992 and currently contains 3,100 patients . . . It is estimated that 300-400 new asthmatic patients are seen each year, while a smaller number become inactive due to relocation, change of health care provider, etc. Once identified in the database, patients potentially eligible for a specific study are contacted by the nurse coordinator who explains the study and ascertains the patient's interest. If interested, the patient is seen in the clinical research laboratories where more detailed evaluations are made . . .\n\nEach subject will receive financial compensation within FDA \\[Food and Drug Administration\\] guidelines for participation in an amount determined by the local center. For subjects who drop out, payments will be pro-rated for the length of time they stayed in the study, but payment will not be made until the study would have been completed had the subject not dropped out.\"208\n\n### Explanation\n\nThe main goal of recruitment is to meet the target sample size (Item 14). However, recruitment difficulties are commonly encountered in clinical trials.209 210 211 212 213 For example, reviews of government funded trials in the US and UK found that two thirds did not reach their recruitment targets.214 215 Low enrolment will reduce statistical power and can lead to early trial stoppage or to extensions with delayed results and greater costs.\n\nStrategies to promote adequate enrolment are thus important to consider during trial planning. Recruitment strategies can vary depending on the trial topic, context, and site. Different recruitment methods can substantially affect the number and type of trial participants recruited128 209 216 217 218 219 220 and can incur different costs.221 222 223 Design issues such as the number and stringency of eligibility criteria will also directly affect the number of eligible trial participants.\n\nProtocol descriptions of where participants will be recruited (eg, primary care clinic, community), by whom (eg, surgeon), when (eg, time after diagnosis), and how (eg, advertisements, review of health records) can be helpful for assessing the feasibility of achieving the target sample size and the applicability of the trial results in practice. Other relevant information to explicitly provide in the protocol includes expected recruitment rates, duration of the recruitment period, plans to monitor recruitment during the trial, and any financial or non-financial incentives provided to trial investigators or participants for enrolment (Item 4). If strategies differ by site in multicentre trials, these should be detailed to the extent possible.\n\n# Section 3b: Methods\u2014assignment of interventions (for controlled trials)\n\n## Allocation\u2014sequence generation\n\nItem 16a: Method of generating the allocation sequence (eg, computer-generated random numbers) and list of any factors for stratification. To reduce predictability of a random sequence, details of any planned restriction (eg, blocking) should be provided in a separate document that is unavailable to those who enrol participants or assign interventions\n\n### Example\n\n\"Participants will be randomly assigned to either control or experimental group with a 1:1 allocation as per a computer generated randomisation schedule stratified by site and the baseline score of the Action Arm Research Test (ARAT; \\<=21 versus \\>21) using permuted blocks of random sizes. The block sizes will not be disclosed, to ensure concealment.\"224\n\n### Explanation\n\nParticipants in a randomised trial should be assigned to study groups using a random (chance) process characterised by unpredictability of assignments. Randomisation decreases selection bias in allocation; helps to facilitate blinding\/masking after allocation; and enables the use of probability theory to test whether any difference in outcome between intervention groups reflects chance.17 225 226 227\n\nUse of terms such as \"randomisation\" without further elaboration is not sufficient to describe the allocation process, as these terms have been used inappropriately to describe non-random, deterministic allocation methods such as alternation or allocation by date of birth.121 In general, these non-random allocation methods introduce selection bias and biased estimates of an intervention's effect size,17 167 228 229 mainly due to the lack of allocation concealment (Item 16b). If non-random allocation is planned, then the specific method and rationale should be stated.\n\nBox 1 outlines the key elements of the random sequence that should be detailed in the protocol. Three quarters of randomised trial protocols approved by a research ethics committee in Denmark (1994-95) or conducted by a US cooperative cancer research group (1968-2006) did not describe the method of sequence generation.2 11\n\n#### Box 1: Key elements of random sequence to specify in trial protocols\n\n-   Method of sequence generation (eg, random number table or computerised random number generator)\n\n-   Allocation ratio (Item 8) (eg, whether participants are allocated with equal or unequal probabilities to interventions)\n\n-   Type of randomisation (box 2): simple versus restricted; fixed versus adaptive (eg, minimisation); and, where relevant, the reasons for such choices\n\n-   If applicable, the factors (eg, recruitment site, sex, disease stage) to be used for stratification (box 2), including categories and relevant cut-off boundaries\n\n#### Box 2: Randomisation and minimisation (adapted from CONSORT 2010 Explanation and Elaboration)17 230 231\n\n##### Simple randomisation\n\nRandomisation based solely on a single, constant allocation ratio is known as simple randomisation. Simple randomisation with a 1:1 allocation ratio is analogous to a coin toss, although tossing a coin is not recommended for sequence generation. No other allocation approach, regardless of its real or supposed sophistication, surpasses the bias prevention and unpredictability of simple randomisation.231\n\n##### Restricted randomisation\n\nAny randomised approach that is not simple randomisation is restricted. Blocked randomisation is the most common form. Other forms, used much less frequently, are methods such as replacement randomisation, biased coin, and urn randomisation.231\n\n##### Blocked randomisation\n\nBlocked randomisation (also called permuted block randomisation) assures that study groups of approximately the same size will be generated when an allocation ratio of 1:1 is used. Blocking can also ensure close balance of the numbers in each group at any time during the trial. After every block of eight participants, for example, four would have been allocated to each trial group.232 Improved balance comes at the cost of reducing the unpredictability of the sequence. Although the order of interventions varies randomly within each block, a person running the trial could deduce some of the next treatment allocations if they discovered the block size.233 Blinding the interventions, using larger block sizes, and randomly varying the block size will help to avoid this problem.\n\n##### Biased coin and urn randomisation\n\nBiased coin designs attain the similar objective as blocked designs without forcing strict equality. They therefore preserve much of the unpredictability associated with simple randomisation. Biased-coin designs alter the allocation ratio during the course of the trial to rectify imbalances that might be occurring.231 Adaptive biased-coin designs, such as the urn design, vary allocation ratios based on the magnitude of the imbalance. However, these approaches are used infrequently.\n\n##### Stratified randomisation\n\nStratification is used to ensure good balance of participant characteristics in each group. Without stratification, study groups may not be well matched for baseline characteristics, such as age and stage of disease, especially in small trials. Such imbalances can be avoided without sacrificing the advantages of randomisation. Stratified randomisation is achieved by performing a separate randomisation procedure within each of two or more strata of participants (eg, categories of age or baseline disease severity), ensuring that the numbers of participants receiving each intervention are closely balanced within each stratum. Stratification requires some form of restriction (eg, blocking within strata) in order to be effective. The number of strata should be limited to avoid over-stratification.234 Stratification by centre is common in multicentre trials.\n\n##### Minimisation\n\nMinimisation assures similar distribution of selected participant factors between study groups.230 235 Randomisation lists are not set up in advance. The first participant is truly randomly allocated; for each subsequent participant, the treatment allocation that minimises the imbalance on the selected factors between groups at that time is identified. That allocation may then be used, or a choice may be made at random with a heavy weighting in favour of the intervention that would minimise imbalance (for example, with a probability of 0.8). The use of a random component is generally preferable.236 Minimisation has the advantage of making small groups closely similar in terms of participant characteristics at all stages of the trial.\n\nMinimisation offers the only acceptable alternative to randomisation, and some have argued that it is superior.237 On the other hand, minimisation lacks the theoretical basis for eliminating bias on all known and unknown factors. Nevertheless, in general, trials that use minimisation are considered methodologically equivalent to randomised trials, even when a random element is not incorporated. For SPIRIT, minimisation is considered a restricted randomisation approach without any judgment as to whether it is superior or inferior compared to other restricted randomisation approaches.\n\nBox 2 defines the various types of randomisation, including minimisation. When restricted randomisation is used, certain details should not appear in the protocol in order to reduce predictability of the random sequence (box 3). The details should instead be described in a separate document that is unavailable to trial implementers. For blocked randomisation, this information would include details on how the blocks will be generated (eg, permuted blocks by a computer random number generator), the block size(s), and whether the block size will be fixed or randomly varied. Specific block size was provided in 14\/102 (14%) randomised trial protocols approved by a Danish research ethics committee in 1994-95, potentially compromising allocation concealment.2 For trials using minimisation, it is also important to state the details in a separate document, including whether random elements will be used.\n\n#### Box 3: Need for a separate document to describe restricted randomisation\n\nIf some type of restricted randomisation approach is to be used, in particular blocked randomisation or minimisation, then the knowledge of the specific details could lead to bias.238 239 For example, if the trial protocol for a two arm, parallel group trial with a 1:1 allocation ratio states that blocked randomisation will be used and the block size will be six, then trial implementers know that the intervention assignments will balance every six participants. Thus, if intervention assignments become known after assignment, knowing the block size will allow trial implementers to predict when equality of the sample sizes will arise. A sequence can be discerned from the pattern of past assignments and then some future assignments could be accurately predicted. For example, if part of a sequence contained two \"As\" and three \"Bs,\" trial implementers would know the last assignment in the sequence would be an \"A.\" If the first three assignments in a sequence contained three \"As,\" trial implementers would know the last three assignments in that sequence would be three \"Bs.\" Selection bias could result, regardless of the effectiveness of allocation concealment (Item 16b).\n\nOf course, this is mainly a problem in open label trials, where everyone becomes aware of the intervention after assignment. It can also be a problem in trials where everyone is supposedly blinded (masked), but the blinding is ineffective or the intervention harms provide clues such that treatments can be guessed.\n\nWe recommend that trial investigators do not provide full details of a restricted randomisation scheme (including minimisation) in the trial protocol. Knowledge of these details might undermine randomisation by facilitating deciphering of the allocation sequence. Instead, this specific information should be provided in a separate document with restricted access. However, simple randomisation procedures could be reported in detail in the protocol, because simple randomisation is totally unpredictable.\n\n## Allocation\u2014concealment mechanism\n\nItem 16b: Mechanism of implementing the allocation sequence (eg, central telephone; sequentially numbered, opaque, sealed envelopes), describing any steps to conceal the sequence until interventions are assigned\n\n### Example\n\n\"Participants will be randomised using TENALEA, which is an online, central randomisation service . . . Allocation concealment will be ensured, as the service will not release the randomisation code until the patient has been recruited into the trial, which takes place after all baseline measurements have been completed.\"240\n\n### Explanation\n\nSuccessful randomisation in practice depends on two interrelated aspects: 1) generation of an unpredictable allocation sequence (Item 16a) and 2) concealment of that sequence until assignment irreversibly occurs.233 241 The allocation concealment mechanism aims to prevent participants and recruiters from knowing the study group to which the next participant will be assigned. Allocation concealment helps to ensure that a participant's decision to provide informed consent, or a recruiter's decision to enrol a participant, is not influenced by knowledge of the group to which they will be allocated if they join the trial.242 Allocation concealment should not be confused with blinding (masking) (Item 17) (table 5<a href=\"#tbl5\" data-ref-type=\"table\"><\/a>).243\n\n\u2002Differences between allocation concealment and blinding (masking) for trials with individual randomisation\n\n<table>\n<thead>\n<tr>\n<th style=\"text-align: left;\"><\/th>\n<th style=\"text-align: center;\">Allocation concealment<\/th>\n<th style=\"text-align: center;\">Blinding<\/th>\n<\/tr>\n<\/thead>\n<tbody>\n<tr>\n<td style=\"text-align: left;\">Definition<\/td>\n<td style=\"text-align: left;\">Unawareness of the next study group assignment in the allocation sequence<\/td>\n<td style=\"text-align: left;\">Unawareness of the study group to which trial participants have already been assigned<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Purpose<\/td>\n<td style=\"text-align: left;\">Prevent selection bias by facilitating enrolment of comparable participants in each study group<\/td>\n<td style=\"text-align: left;\">Prevent ascertainment, performance, and attrition biases by facilitating comparable concomitant care (aside from trial interventions) and evaluation of participants in each study group<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Timing of implementation<\/td>\n<td style=\"text-align: left;\">Before study group assignment<\/td>\n<td style=\"text-align: left;\">Upon study group assignment and beyond<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Who is kept unaware<\/td>\n<td style=\"text-align: left;\">Trial participants and individuals enrolling them<\/td>\n<td style=\"text-align: left;\">One or more of the following: trial participants, investigators, care providers, outcome assessors.<br \/>\nOther groups: endpoint adjudication committee, data handlers, data analysts<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Always possible to implement?<\/td>\n<td style=\"text-align: left;\">Yes<\/td>\n<td style=\"text-align: left;\">No<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\nWithout adequate allocation concealment, even random, unpredictable assignment sequences can be subverted.233 241 For example, a common practice is to enclose assignments in sequentially numbered, sealed envelopes. However, if the envelopes are not opaque and contents are visible when held up to a light source, or if the envelopes can be unsealed and resealed, then this method of allocation concealment can be corrupted.\n\nProtocols should describe the planned allocation concealment mechanism in sufficient detail to enable assessment of its adequacy. In one study of randomised trial protocols in Denmark, over half did not adequately describe allocation concealment methods.2 In contrast, central randomisation was stated as the allocation concealment method in all phase III trial protocols initiated in 1968-2003 by a cooperative cancer research group that used extensive protocol review processes.11 Like sequence generation, inadequate reporting of allocation concealment in trial publications is common and has been associated with inflated effect size estimates.167 244 245\n\n## Allocation\u2014implementation\n\nItem 16c: Who will generate the allocation sequence, who will enrol participants, and who will assign participants to interventions\n\n### Example\n\n#### \"Randomization\n\nAll patients who give consent for participation and who fulfil the inclusion criteria will be randomized. Randomisation will be requested by the staff member responsible for recruitment and clinical interviews from CenTrial \\[Coordination Centre of Clinical Trials\\].\n\nIn return, CenTrial will send an answer form to the study therapist who is not involved in assessing outcome of the study. This form will include a randomisation number. In every centre closed envelopes with printed randomisation numbers on it are available. For every randomisation number the corresponding code for the therapy group of the randomisation list will be found inside the envelopes. The therapist will open the envelope and will find the treatment condition to be conducted in this patient. The therapist then gives the information about treatment allocation to the patient. Staff responsible for recruitment and symptom ratings is not allowed to receive information about the group allocation.\n\n. . .\n\nThe allocation sequence will be generated by the Institute for Medical Biometry (IMB) applying a permuted block design with random blocks stratified by study centre and medication compliance (favourable vs. unfavourable). . . . The block size will be concealed until the primary endpoint will be analysed. Throughout the study, the randomisation will be conducted by CenTrial in order to keep the data management and the statistician blind against the study condition as long as the data bank is open. The randomisation list remains with CenTrial for the whole duration of the study. Thus, randomisation will be conducted without any influence of the principal investigators, raters or therapists.\"246\n\n### Explanation\n\nBased on the risk of bias associated with some methods of sequence generation and inadequate allocation concealment, trial investigators should strive for complete separation of the individuals involved in the steps before enrolment (sequence generation process and allocation concealment mechanism) from those involved in the implementation of study group assignments. When this separation is not possible, it is important for the investigators to ensure that the assignment schedule is unpredictable and locked away from even the person who generated it. The protocol should specify who will implement the various stages of the randomisation process, how and where the allocation list will be stored, and mechanisms employed to minimise the possibility that those enrolling and assigning participants will obtain access to the list.\n\n## Blinding (masking)\n\nItem 17a: Who will be blinded after assignment to interventions (eg, trial participants, care providers, outcome assessors, data analysts) and how\n\n### Example\n\n\"Assessments regarding clinical recovery will be conducted by an assessor blind to treatment allocation. The assessor will go through a profound assessment training program . . . Due to the nature of the intervention neither participants nor staff can be blinded to allocation, but are strongly inculcated not to disclose the allocation status of the participant at the follow up assessments. An employee outside the research team will feed data into the computer in separate datasheets so that the researchers can analyse data without having access to information about the allocation.\"247\n\n### Explanation\n\nBlinding or masking (the process of keeping the study group assignment hidden after allocation) is commonly used to reduce the risk of bias in clinical trials with two or more study groups.166 248 Awareness of the intervention assigned to participants can introduce ascertainment bias in the measurement of outcomes, particularly subjective ones (eg, quality of life)166 167; performance bias in the decision to discontinue or modify study interventions (eg, dosing changes) (Item 11b), concomitant interventions, or other aspects of care (Item 11d)229; and exclusion\/attrition bias in the decision to withdraw from the trial or to exclude a participant from the analysis.249 250 We have elected to use the term \"blinding\" but acknowledge that others prefer the term \"masking\" because \"blind\" also relates to an ophthalmological condition and health outcome.251 252\n\nMany groups can be blinded: trial participants, care providers, data collectors, outcome assessors or committees (Item 5d), data analysts,253 and manuscript writers. Blinding of data monitoring committees is generally discouraged.254 255\n\nWhen blinding of trial participants and care providers is not possible because of obvious differences between the interventions,256 257 blinding of the outcome assessors can often still be implemented.17 It may also be possible to blind participants or trial personnel to the study hypothesis in terms of which intervention is considered active. For example, in a trial evaluating light therapy for depression, participants were informed that the study involved testing two different forms of light therapy, whereas the true hypothesis was that bright blue light was considered potentially effective and that dim red light was considered placebo.258\n\nDespite its importance, blinding is often poorly described in trial protocols.3 The protocol should explicitly state who will be blinded to intervention groups\u2014at a minimum, the blinding status of trial participants, care providers, and outcome assessors. Such a description is much preferred over the use of ambiguous terminology such as \"single blind\" or \"double blind.\"259 260 Protocols should also describe the comparability of blinded interventions (Item 11a)150\u2014for example, similarities in appearance, use of specific flavours to mask a distinctive taste\u2014and the timing of final unblinding of all trial participants (eg, after the creation of a locked analysis data set).3\n\nFurthermore, any strategies to reduce the potential for unblinding should be described in the protocol, such as pre-trial testing of blinding procedures.261 The use of a fixed code (versus a unique code for each participant) to denote each study group assignment (eg, A=Group 1; B=Group 2) can be problematic, as the unblinding of one participant will result in the inadvertent loss of blinding for all trial participants.\n\nSome have suggested that the success of blinding be formally tested by asking key trial persons to guess the study group assignment and comparing these responses to what would be expected by chance.262 However, it is unclear how best to interpret the results of such tests.263 264 If done, the planned testing methods should be described in the trial protocol.\n\n## Blinding (masking)\u2014emergency unblinding\n\nItem 17b: If blinded, circumstances under which unblinding is permissible and procedure for revealing a participant's allocated intervention during the trial\n\n### Example\n\n\"To maintain the overall quality and legitimacy of the clinical trial, code breaks should occur only in exceptional circumstances when knowledge of the actual treatment is absolutely essential for further management of the patient. Investigators are encouraged to discuss with the Medical Advisor or PHRI \\[Population Health Research Institute\\] physician if he\/she believes that unblinding is necessary.\n\nIf unblinding is deemed to be necessary, the investigator should use the system for emergency unblinding through the PHRI toll-free help line as the main system or through the local emergency number as the back-up system.\n\nThe Investigator is encouraged to maintain the blind as far as possible. The actual allocation must NOT be disclosed to the patient and\/or other study personnel including other site personnel, monitors, corporate sponsors or project office staff; nor should there be any written or verbal disclosure of the code in any of the corresponding patient documents.\n\nThe Investigator must report all code breaks (with reason) as they occur on the corresponding CRF \\[case report form\\] page.\n\nUnblinding should not necessarily be a reason for study drug discontinuation.\"265\n\n### Explanation\n\nAmong 58 blinded Danish trials approved in 1994-95, three quarters of protocols described emergency unblinding procedures.3 Such procedures to reveal the assigned intervention in certain circumstances are intended to increase the safety of trial participants by informing the clinical management of harms or other relevant conditions that arise. A clear protocol description of the conditions and procedures for emergency unblinding helps to prevent unnecessary unblinding; facilitates implementation by trial personnel when indicated; and enables evaluation of the appropriateness of the planned procedures. In some cases (eg, minor, reversible harms), stopping and then cautiously reintroducing the assigned intervention in the affected participant can avoid both unblinding and further harm.\n\n# Section 3c: Methods\u2014data collection, management, and analysis\n\n## Data collection methods\n\nItem 18a: Plans for assessment and collection of outcome, baseline, and other trial data, including any related processes to promote data quality (eg, duplicate measurements, training of assessors) and a description of study instruments (eg, questionnaires, laboratory tests) along with their reliability and validity, if known. Reference to where data collection forms can be found, if not in the protocol\n\n### Examples\n\n#### \"Primary outcome\n\n*Delirium recognition*: In accordance with national guidelines \\[reference\\], the study will identify delirium by using the RASS \\[Richmond Agitation-Sedation Scale\\] and the CAM-ICU \\[Confusion Assessment Method for the intensive care unit\\] on all patients who are admitted directly from the emergency room or transferred from other services to the ICU. Such assessment will be performed after 24 hours of ICU admission and twice daily until discharge from the hospital . . . RASS has excellent inter-rater reliability among adult medical and surgical ICU patients and has excellent validity when compared to a visual analogue scale and other selected sedation scales\\[reference\\] . . . The CAM-ICU was chosen because of its practical use in the ICU wards, its acceptable psychometric properties, and based on the recommendation of national guidelines\\[reference\\] . . . The CAM-ICU diagnosis of delirium was validated against the DSM-III-R \\[Diagnostic and Statistical Manual of Mental Disorders, Third Edition\u2014Revised\\] delirium criteria determined by a psychiatrist and found to have a sensitivity of 97% and a specificity of 92%.\\[reference\\] The CAM-ICU has been developed, validated and applied into ICU settings and multiple investigators have used the same method to identify patients with delirium.\\[reference\\]\n\n*Delirium severity*: Since the CAM-ICU does not evaluate delirium severity, we selected the Delirium Rating Scale revised-1998 (DRS-R-98)\\[reference\\] . . . The DRS-R-98 was designed to evaluate the breadth of delirium symptoms for phenomenological studies in addition to measuring symptom severity with high sensitivity and specificity . . . The DRS-R-98 is a 16-item clinician-rated scale with anchored items descriptions . . . The DRS-R-98 has excellent inter-rater reliability (intra-class correlation 0.97) and internal consistency (Cronbach**'**s alpha 0.94).\\[reference\\]\n\n#### Secondary outcomes\n\nThe study will collect demographic and baseline functional information from the patient**'**s legally authorized representative and\/or caregivers. Cognitive function status will be obtained by interviewing the patient**'**s legally authorized representative using the Informant Questionnaire on Cognitive Decline in the Elderly (IQCODE). IQCODE is a questionnaire that can be completed by a relative or other caregiver to determine whether that person has declined in cognitive functioning. The IQCODE lists 26 everyday situations . . . Each situation is rated by the informant for amount of change over the previous 10 years, using a Likert scale ranging from 1-much improved to 5-much worse. The IQCODE has a sensitivity between 69% to 100% and specificity of 80% to 96% for dementia.\\[reference\\]\n\nUtilizing the electronic medical record system (RMRS), we will collect several data points of interest at baseline and throughout the study period . . . We have previously defined hospital-related consequences to include: the number of patients with documented falls, use of physical restraints . . . These will be assessed using the RMRS, direct daily observation, and retrospective review of the electronic medical record. This definition of delirium related hospital complications has been previously used and published.\\[reference\\]\"266\n\n#### \"Training and certification plans\n\n. . . Each center's personnel will be trained centrally in the study requirements, standardized measurement of height, weight, and blood pressure, requirements for laboratory specimen collection including morning urine samples, counseling for adherence and the eliciting of information from study participants in a uniform reproducible manner.\n\n. . . The data to be collected and the procedures to be conducted at each visit will be reviewed in detail. Each of the data collection forms and the nature of the required information will be discussed in detail on an item by item basis. Coordinators will learn how to code medications using the WHODrug software and how to code symptoms using the MedDRA software. Entering data forms, responding to data discrepancy queries and general information about obtaining research quality data will also be covered during the training session.\n\n. . .\n\n#### 13.7. Quality Control of the Core Lab\n\nData from the Core Lab will be securely transmitted in batches and quality controlled in the same manner as Core Coordinating Center data; ie data will be entered and verified in the database on the Cleveland Clinic Foundation SUN with a subset later selected for additional quality control. Appropriate edit checks will be in place at the key entry (database) level.\n\nThe Core Lab is to have an internal quality control system established prior to analyzing any FSGS \\[focal segmental glomerulosclerosis\\] samples. This system will be outlined in the Manual of Operations for the Core Lab(s) which is prepared and submitted by the Core Lab to the DCC \\[data coordinating centre\\] prior to initiating of the study.\n\nAt a minimum this system must include:\n\n1\\) The inclusion of at least two known quality control samples; the reported measurements of the quality control samples must fall within specified ranges in order to be certified as acceptable.\n\n2\\) Calibration at FDA approved manufacturers' recommended schedules.\n\n#### 13.8. Quality Control of the Biopsy Committee\n\nThe chair of the pathology committee will circulate to all of the study pathologists . . . samples \\[sic\\] biopsy specimens for evaluation after criteria to establish diagnosis of FSGS has been agreed. This internal review process will serve to ensure common criteria and assessment of biopsy specimens for confirmation of diagnosis of FSGS.\"267\n\n### Explanation\n\nThe validity and reliability of trial data depend on the quality of the data collection methods. The processes of acquiring and recording data often benefit from attention to training of study personnel and use of standardised, pilot tested methods. These should be identical for all study groups, unless precluded by the nature of the intervention.\n\nThe choice of methods for outcome assessment can affect study conduct and results.268 269 270 271 272 273 Substantially different responses can be obtained for certain outcomes (eg, harms) depending on who answers the questions (eg, the participant or investigator) and how the questions are presented (eg, discrete options or open ended).269 274 275 276 Also, when compared to paper based data collection, the use of electronic handheld devices and internet websites has the potential to improve protocol adherence, data accuracy, user acceptability, and timeliness of receiving data.268 270 271 277\n\nThe quality of data also depends on the reliability, validity, and responsiveness of data collection instruments such as questionnaires278 or laboratory instruments. Instruments with low inter-rater reliability will reduce statistical power,272 while those with low validity will not accurately measure the intended outcome variable. One study found that only 35% (47\/133) of randomised trials in acute stroke used a measure with established reliability or validity.279 Modified versions of validated measurement tools may no longer be considered validated, and use of unpublished measurement scales can introduce bias and inflate treatment effect sizes.280\n\nStandard processes should be implemented by local study personnel to enhance data quality and reduce bias by detecting and reducing the amount of missing or incomplete data, inaccuracies, and excessive variability in measurements.281 282 283 284 285 Examples include standardised training and testing of outcome assessors to promote consistency; tests of the validity or reliability of study instruments; and duplicate data measurements.\n\nA clear protocol description of the data collection process\u2014including the personnel, methods, instruments, and measures to promote data quality\u2014can facilitate implementation and helps protocol reviewers to assess their appropriateness. Inclusion of data collection forms in the protocol (ie, as appendices) is highly recommended, as the way in which data are obtained can substantially affect the results. If not included in the protocol, then a reference to where the forms can be found should be provided. If performed, pilot testing and assessment of reliability and validity of the forms should also be described.\n\n## Data collection methods\u2014retention\n\nItem 18b: Plans to promote participant retention and complete follow-up, including list of any outcome data to be collected for participants who discontinue or deviate from intervention protocols\n\n### Examples\n\n#### \"5.2.2 Retention\n\n. . . As with recruitment, retention addresses all levels of participant.\n\nAt the parent and student level, study investigators and staff:\n\n-   Provide written feedback to all parents of participating students about the results of the \"health screenings\" . . .\n\n-   Maintain interest in the study through materials and mailings . . .\n\n-   Send letters to parents and students prior to the final data collection, reminding them of the upcoming data collection and the incentives the students will receive.\n\nAt the school level, study investigators and staff:\n\n-   Provide periodic communications via newsletters and presentations to inform the school officials\/staff, students, and parents about type 2 diabetes, the current status of the study, and plans for the next phase, as well as to acknowledge their support.\n\n-   . . .\n\n-   Become a presence in the intervention schools to monitor and maintain consistency in implementation, . . . be as flexible as possible with study schedule and proactive in resolving conflicts with schools.\n\n-   Provide school administration and faculty with the schedule or grid showing how the intervention fits into the school calendar . . .\n\n-   Solicit support from parents, school officials\/staff, and teachers . . .\n\n-   Provide periodic incentives for school staff and teachers.\n\n-   Provide monetary incentives for the schools that increase with each year of the study.<a href=\"#tbl6\" data-ref-type=\"table\"><\/a>\"286\n\nExcerpts from table showing compensation provided in study^286^\n\n| Who | What |  |\n|----|----|----|\n| **School** |  |  |\n| Intervention school | School program enhancement | \\$2000 in year 1, \\$3000 in year 2, \\$4000 in year 3 |\n|  | Physical education class equipment required to implement intervention | \\$15 000 over 3 years |\n|  | Food service department to defray costs of nutrition intervention | \\$3000\/year |\n| Control school | School program enhancement | \\$2000 in year 1, \\$4000 in year 2, \\$6000 in year 3 |\n| Student |  |  |\n| All | Return consent form (signed or not) | Gift item worth \\~ \\$5 |\n| Participation in health screening data collection measures and forms | \\$50 baseline (6th grade), \\$10 interim (7th grade), \\$60 end of study (8th grade) |  |\n| Family |  |  |\n| Intervention parents | Focus groups to provide input about family outreach events and activities | \\$35\/year per parent, up to two focus groups per field center, 6-10 participants per focus group |\n\n#### 5.4 Infant Evaluations in the Case of Treatment Discontinuation or Study Withdrawal\n\nAll randomized infants completing the 18-month evaluation schedule will have fulfilled the infant clinical and laboratory evaluation requirements for the study. . .\n\nAll randomized infants who are prematurely discontinued from study drug will be considered *off study drug\/on study* and will follow the same schedule of events as those infants who continue study treatment except adherence assessment. All of these infants will be followed through 18 months as scheduled.\n\nRandomized infants prematurely discontinued from the study before the 6-month evaluation will have the following clinical and laboratory evaluations performed, if possible: . . .\n\n-   Roche Amplicor HIV-1 DNA PCR \\[polymerase chain reaction\\] and cell pellet storage\n\n-   Plasma for storage (for NVP \\[nevirapine\\] resistance, HIV-1 RNA PCR and NVP concentration)\n\n. . .\n\nRandomized infants prematurely discontinued from the study at any time after the 6-month evaluation will have the following clinical and laboratory evaluations performed, if possible:\n\n. . .\n\n#### 5.5 Participant Retention\n\nOnce an infant is enrolled or randomized, the study site will make every reasonable effort to follow the infant for the entire study period . . . It is projected that the rate of loss-to-follow-up on an annual basis will be at most 5% . . . Study site staff are responsible for developing and implementing local standard operating procedures to achieve this level of follow-up.\n\n#### 5.6 Participant Withdrawal\n\nParticipants may withdraw from the study for any reason at any time. The investigator also may withdraw participants from the study in order to protect their safety and\/or if they are unwilling or unable to comply with required study procedures after consultation with the Protocol Chair, National Institutes of Health (NIH) Medical Officers, Statistical and Data Management Center (SDMC) Protocol Statistician, and Coordinating and Operations Center (CORE) Protocol Specialist.\n\nParticipants also may be withdrawn if the study sponsor or government or regulatory authorities terminate the study prior to its planned end date.\n\nNote: Early discontinuation of study product for any reason is not a reason for withdrawal from the study.\"287\n\n### Explanation\n\nTrial investigators must often seek a balance between achieving a sufficiently long follow-up for clinically relevant outcome measurement,122 288 and a sufficiently short follow-up to decrease attrition and maximise completeness of data collection. Non-retention refers to instances where participants are prematurely \"off-study\" (ie, consent withdrawn or lost to follow-up) and thus outcome data cannot be obtained from them. The majority of trials will have some degree of non-retention, and the number of these \"off-study\" participants usually increases with the length of follow-up.116\n\nIt is desirable to plan ahead for how retention will be promoted in order to prevent missing data and avoid the associated complexities in both the study analysis (Item 20c) and interpretation. Certain methods can improve participant retention,67 152 289 290 291 292 such as financial reimbursement; systematic methods and reminders for contacting patients, scheduling appointments, and monitoring retention; and limiting participant burden related to follow-up visits and procedures (Item 13). A participant who withdraws consent for follow-up assessment of one outcome may be willing to continue with assessments for other outcomes, if given the option.\n\nNon-retention should be distinguished from non-adherence.293 Non-adherence refers to deviation from intervention protocols (Item 11c) or from the follow-up schedule of assessments (Item 13), but does not mean that the participant is \"off-study\" and no longer in the trial. Because missing data can be a major threat to trial validity and statistical power, non-adherence should not be an automatic reason for ceasing to collect data from the trial participant prior to study completion. In particular for randomised trials, it is widely recommended that all participants be included in an intention to treat analysis, regardless of adherence (Item 20c).\n\nProtocols should describe any retention strategies and define which outcome data will be recorded from protocol non-adherers.152 Protocols should also detail any plans to record the reasons for non-adherence (eg, discontinuation of intervention due to harms versus lack of efficacy) and non-retention (ie, consent withdrawn; lost to follow-up), as this information can influence the handling of missing data and interpretation of results.152 294 295\n\n## Data management\n\nItem 19: Plans for data entry, coding, security, and storage, including any related processes to promote data quality (eg, double data entry; range checks for data values). Reference to where details of data management procedures can be found, if not in the protocol\n\n### Example\n\n#### \"13.9.2. Data Forms and Data Entry\n\nIn the FSGS-CT \\[focal segmental glomerulosclerosis\u2014clinical trial\\], all data will be entered electronically. This may be done at a Core Coordinating Center or at the participating site where the data originated. Original study forms will be entered and kept on file at the participating site. A subset will be requested later for quality control; when a form is selected, the participating site staff will pull that form, copy it, and sent \\[sic\\] the copy to the DCC \\[data coordinating center\\] for re-entry.\n\n. . . Participant files are to be stored in numerical order and stored in a secure and accessible place and manner. Participant files will be maintained in storage for a period of 3 years after completion of the study.\n\n#### 13.9.3. Data Transmission and Editing\n\nThe data entry screens will resemble the paper forms approved by the steering committee. Data integrity will be enforced through a variety of mechanisms. Referential data rules, valid values, range checks, and consistency checks against data already stored in the database (ie, longitudinal checks) will be supported. The option to chose \\[sic\\] a value from a list of valid codes and a description of what each code means will be available where applicable. Checks will be applied at the time of data entry into a specific field and\/or before the data is written (committed) to the database. Modifications to data written to the database will be documented through either the data change system or an inquiry system. Data entered into the database will be retrievable for viewing through the data entry applications. The type of activity that an individual user may undertake is regulated by the privileges associated with his\/her user identification code and password.\n\n#### 13.9.4. Data Discrepancy Inquiries and Reports to Core Coordinating Centers\n\nAdditional errors will be detected by programs designed to detect missing data or specific errors in the data. These errors will be summarized along with detailed descriptions for each specific problem in Data Query Reports, which will be sent to the Data Managers at the Core Coordinating Centers . . .\n\nThe Data Manager who receives the inquiry will respond by checking the original forms for inconsistency, checking other sources to determine the correction, modifying the original (paper) form entering a response to the query. Note that it will be necessary for Data Managers to respond to each inquiry received in order to obtain closure on the queried item.\n\nThe Core Coordinating Center and participating site personnel will be responsible for making appropriate corrections to the original paper forms whenever any data item is changed . . . Written documentation of changes will be available via electronic logs and audit trails.\n\n. . .\n\nBiopsy and biochemistry reports will be sent via e-mail when data are received from the Core Lab.\n\n. . .\n\n#### 13.9.5. Security and Back-Up of Data\n\n. . . All forms, diskettes and tapes related to study data will be kept in locked cabinets. Access to the study data will be restricted. In addition, Core Coordinating Centers will only have access to their own center's data. A password system will be utilized to control access . . . These passwords will be changed on a regular basis. All reports prepared by the DCC will be prepared such that no individual subject can be identified.\n\nA complete back up of the primary DCC database will be performed twice a month. These tapes will be stored off-site in a climate-controlled facility and will be retained indefinitely. Incremental data back-ups will be performed on a daily basis. These tapes will be retained for at least one week on-site. Back-ups of periodic data analysis files will also be kept. These tapes will be retained at the off-site location until the Study is completed and the database is on file with NIH \\[National Institutes of Health\\]. In addition to the system back-ups, additional measures will be taken to back-up and export the database on a regular basis at the database management level. . .\n\n#### 13.9.6. Study status reports\n\nThe DCC will send weekly email reports with information on missing data, missing forms, and missing visits. Personnel at the Core Coordinating Center and the Participating Sites should review these reports for accuracy and report any discrepancies to the DCC.\n\n. . .\n\n#### 13.9.8. Description of Hardware at DCC\n\nA SUN Workstation environment is maintained in the department with a SUN SPARCstation 10 model 41 as the server . . . Primary access to the departments \\[sic\\] computing facilities will be through the Internet . . . For maximum programming efficiency, the Oracle database management system and the SAS and BMDP statistical analysis systems will be employed for this study. . . .\n\nOracle facilitates sophisticated integrity checks through a variety of mechanisms including stored procedures, stored triggers, and declarative database integrity\u2014for between table verifications. Oracle allows data checks to be programmed once in the database rather than repeating the same checks among many applications . . . Security is enforced through passwords and may be assigned at different levels to groups and individuals.\"267\n\n### Explanation\n\nCareful planning of data management with appropriate personnel can help to prevent flaws that compromise data validity. The protocol should provide a full description of the data entry and coding processes, along with measures to promote their quality, or provide key elements and a reference to where full information can be found. These details are particularly important for the primary outcome data. The protocol should also document data security measures to prevent unauthorised access to or loss of participant data, as well as plans for data storage (including timeframe) during and after the trial. This information facilitates an assessment of adherence to applicable standards and regulations.\n\nDifferences in data entry methods can affect the trial in terms of data accuracy,268 cost, and efficiency.271 For example, when compared with paper case report forms, electronic data capture can reduce the time required for data entry, query resolution, and database release by combining data entry with data collection (Item 18a).271 277 When data are collected on paper forms, data entry can be performed locally or at a central site. Local data entry can enable fast correction of missing or inaccurate data, while central data entry facilitates blinding (masking), standardisation, and training of a core group of data entry personnel.\n\nRaw, non-numeric data are usually coded for ease of data storage, review, tabulation, and analysis. It is important to define standard coding practices to reduce errors and observer variation. When data entry and coding are performed by different individuals, it is particularly important that the personnel use unambiguous, standardised terminology and abbreviations to avoid misinterpretation.\n\nAs with data collection (Item 18a), standard processes are often implemented to improve the accuracy of data entry and coding.281 284 Common examples include double data entry296; verification that the data are in the proper format (eg, integer) or within an expected range of values; and independent source document verification of a random subset of data to identify missing or apparently erroneous values. Though widely performed to detect data entry errors, the time and costs of independent double data entry from paper forms need to be weighed against the magnitude of reduction in error rates compared to single-data entry.297 298 299\n\n# Statistical methods\n\nThe planned methods of statistical analysis should be fully described in the protocol. If certain aspects of the analysis plan cannot be prespecified (eg, the method of handling missing data is contingent on examining patterns of \"missingness\" before study unblinding), then the planned approach to making the final methodological choices should be outlined. Some trials have a separate document\u2014commonly called a statistical analysis plan (SAP)\u2014that fully details the planned analyses. Any SAP should be described in the protocol, including its key elements and where it can be found. As with the protocol, the SAP should be dated, amendments noted and dated, and the SAP authors provided.\n\n## Statistical methods\u2014outcomes\n\nItem 20a: Statistical methods for analysing primary and secondary outcomes. Reference to where other details of the statistical analysis plan can be found, if not in the protocol\n\n### Example\n\n\"The intervention arm (SMS \\[short message system (text message)\\]) will be compared against the control (SOC \\[standard of care\\]) for all primary analysis. We will use chi-squared test for binary outcomes, and T-test for continuous outcomes. For subgroup analyses, we will use regression methods with appropriate interaction terms (respective subgroup\u00d7treatment group). Multivariable analyses will be based on logistic regression . . . for binary outcomes and linear regression for continuous outcomes. We will examine the residual to assess model assumptions and goodness-of-fit. For timed endpoints such as mortality we will use the Kaplan-Meier survival analysis followed by multivariable Cox proportional hazards model for adjusting for baseline variables. We will calculate Relative Risk (RR) and RR Reductions (RRR) with corresponding 95% confidence intervals to compare dichotomous variables, and difference in means will be used for additional analysis of continuous variables. P-values will be reported to four decimal places with p-values less than 0.001 reported as p \\< 0.001. Up-to-date versions of SAS (Cary, NC) and SPSS (Chicago, IL) will be used to conduct analyses. For all tests, we will use 2-sided p-values with alpha\u22640.05 level of significance. We will use the Bonferroni method to appropriately adjust the overall level of significance for multiple primary outcomes, and secondary outcomes.\n\nTo assess the impact of potential clustering for patients cared by the same clinic, we will use generalized estimating equations \\[GEE\\] assuming an exchangeable correlation structure. Table \\[7<a href=\"#tbl7\" data-ref-type=\"table\"><\/a> provides a summary of methods of analysis for each variable. Professional academic statisticians (LT, RN) blinded to study groups will conduct all analyses.\"47\n\nVariables, measures, and methods of analysis (reproduced from original table^47^)\n\n<table>\n<thead>\n<tr>\n<th style=\"text-align: left;\">Variable\/outcome<\/th>\n<th style=\"text-align: left;\">Hypothesis<\/th>\n<th style=\"text-align: left;\">Outcome measure<\/th>\n<th style=\"text-align: left;\">Methods of analysis<\/th>\n<\/tr>\n<\/thead>\n<tbody>\n<tr>\n<td style=\"text-align: left;\">1) <strong>Primary<\/strong><\/td>\n<td style=\"text-align: left;\">Intervention improved outcome from baseline to 6 months<\/td>\n<td style=\"text-align: left;\"><\/td>\n<td style=\"text-align: left;\"><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">a) Adherence at 12 months<\/td>\n<td style=\"text-align: left;\"><\/td>\n<td style=\"text-align: left;\">Percent adherence in previous 30 days &gt;95% [binary]<\/td>\n<td style=\"text-align: left;\">Chi-squared test<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">b) Suppression of HIV viral load at 12 months<\/td>\n<td style=\"text-align: left;\"><\/td>\n<td style=\"text-align: left;\">Viral load \u2264400 copies\/ml [binary]<\/td>\n<td style=\"text-align: left;\">Chi-squared test<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">2) <strong>Secondary<\/strong><\/td>\n<td style=\"text-align: left;\">improvement occurred<\/td>\n<td style=\"text-align: left;\">Adherence % (&gt;95%) [binary]<\/td>\n<td style=\"text-align: left;\">Chi-squared test<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Adherence percentage at 12 months<\/td>\n<td style=\"text-align: left;\"><\/td>\n<td style=\"text-align: left;\"><\/td>\n<td style=\"text-align: left;\"><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">HIV viral load at 12 months<\/td>\n<td style=\"text-align: left;\">improvement occurred<\/td>\n<td style=\"text-align: left;\">Viral load (copies)<\/td>\n<td style=\"text-align: left;\">T-test<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Immune reconstitution (change in CD4 T cell<br \/>\ncount from baseline)<\/td>\n<td style=\"text-align: left;\">improvement occurred<\/td>\n<td style=\"text-align: left;\">CD4 T-cells\/mm<sup>3<\/sup> (continuous)<\/td>\n<td style=\"text-align: left;\">T-test<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Time to virological failure<\/td>\n<td style=\"text-align: left;\">improvement occurred<\/td>\n<td style=\"text-align: left;\">Virological failure after successful suppression<\/td>\n<td style=\"text-align: left;\">Kaplan-Meier survival<br \/>\nanalysis<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Weight gain [lbs] and BMI<\/td>\n<td style=\"text-align: left;\">improvement occurred<\/td>\n<td style=\"text-align: left;\">Change in weight (lbs) and BMI [continuous]<\/td>\n<td style=\"text-align: left;\">T-test<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Occurrence of opportunistic infections (OIs)<\/td>\n<td style=\"text-align: left;\">improvement occurred<\/td>\n<td style=\"text-align: left;\">Presence of AIDS defining<br \/>\nopportunistic infection [binary]<\/td>\n<td style=\"text-align: left;\">Chi-squared test<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Time to reporting of adverse drug events (ADEs)<\/td>\n<td style=\"text-align: left;\">improvement occurred<\/td>\n<td style=\"text-align: left;\">Presence of drug-related adverse event [time to event]<\/td>\n<td style=\"text-align: left;\">Kaplan-Meier survival<br \/>\nanalysis<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Deaths (all cause)<\/td>\n<td style=\"text-align: left;\">improvement occurred<\/td>\n<td style=\"text-align: left;\">All-cause mortality [binary]<\/td>\n<td style=\"text-align: left;\">Chi-squared test and Kaplan-Meier survival analysis<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">SF-12 [short form 12 adapted for regional application in Kiswahili]<\/td>\n<td style=\"text-align: left;\">improvement occurred<\/td>\n<td style=\"text-align: left;\">Quality pf [sic] life questionnaire<br \/>\n[continuous]<\/td>\n<td style=\"text-align: left;\">T-test<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Satisfaction with care provided<\/td>\n<td style=\"text-align: left;\">improvement occurred<\/td>\n<td style=\"text-align: left;\">Questionnaire [continuous]<\/td>\n<td style=\"text-align: left;\">T-test<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Level of disclosure of HIV status<\/td>\n<td style=\"text-align: left;\">improvement occurred<\/td>\n<td style=\"text-align: left;\">Disclosed to a family member [binary]<\/td>\n<td style=\"text-align: left;\">Chi-squared test<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Impression of stigma<\/td>\n<td style=\"text-align: left;\">improvement occurred<\/td>\n<td style=\"text-align: left;\">Questionnaire [continuous]<\/td>\n<td style=\"text-align: left;\">T-test<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Family dyamics [sic]<\/td>\n<td style=\"text-align: left;\">improvement occurred<\/td>\n<td style=\"text-align: left;\">Questionnaire [continuous]<\/td>\n<td style=\"text-align: left;\">T-test<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Employment attendance<\/td>\n<td style=\"text-align: left;\">improvement occurred<\/td>\n<td style=\"text-align: left;\">Questionnaire [continuous]<\/td>\n<td style=\"text-align: left;\">T-test<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Household member school attendance<\/td>\n<td style=\"text-align: left;\">improvement occurred<\/td>\n<td style=\"text-align: left;\">Questionnaire [continuous]<\/td>\n<td style=\"text-align: left;\">T-test<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Cell phones lost\/stolen<\/td>\n<td style=\"text-align: left;\">improvement occurred<\/td>\n<td style=\"text-align: left;\">Presence of cellphone [binary]<\/td>\n<td style=\"text-align: left;\">Poisson regression<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Stopped taking HAART [highly active antiretroviral therapy]<\/td>\n<td style=\"text-align: left;\">improvement occurred<\/td>\n<td style=\"text-align: left;\">Self-report [binary]<\/td>\n<td style=\"text-align: left;\">Chi-squared test<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Required active tracing for 12 month follow-up<\/td>\n<td style=\"text-align: left;\">improvement occurred<\/td>\n<td style=\"text-align: left;\">Field officers [binary]<\/td>\n<td style=\"text-align: left;\">Chi-squared test<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">3) <strong>Subgroup Analyses:<\/strong><\/td>\n<td style=\"text-align: left;\"><\/td>\n<td style=\"text-align: left;\"><\/td>\n<td style=\"text-align: left;\">Regression methods with appropriate interaction term<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Urban vs. rural<\/td>\n<td style=\"text-align: left;\">Distance affects<br \/>\nadherence<\/td>\n<td style=\"text-align: left;\"><\/td>\n<td style=\"text-align: left;\"><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Female vs. male<\/td>\n<td style=\"text-align: left;\">Sex affects adherence<\/td>\n<td style=\"text-align: left;\"><\/td>\n<td style=\"text-align: left;\"><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Phone ownership (owned vs. shared)<\/td>\n<td style=\"text-align: left;\">Ownership affects<br \/>\nadherence<\/td>\n<td style=\"text-align: left;\"><\/td>\n<td style=\"text-align: left;\"><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Level of education<\/td>\n<td style=\"text-align: left;\">Low education affects<br \/>\nadherence<\/td>\n<td style=\"text-align: left;\"><\/td>\n<td style=\"text-align: left;\"><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">4) <strong>Sensitivity Analyses:<\/strong><\/td>\n<td style=\"text-align: left;\">improvement occurred<\/td>\n<td style=\"text-align: left;\">All outcomes<\/td>\n<td style=\"text-align: left;\"><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">a) Per protocol analysis<\/td>\n<td style=\"text-align: left;\"><\/td>\n<td style=\"text-align: left;\"><\/td>\n<td style=\"text-align: left;\">a) Chi-squared\/T-test<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">b) Adjusting for baseline covariates<\/td>\n<td style=\"text-align: left;\"><\/td>\n<td style=\"text-align: left;\"><\/td>\n<td style=\"text-align: left;\">b) Multivariable regression<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">c) clustering among individuals within a clinic<\/td>\n<td style=\"text-align: left;\"><\/td>\n<td style=\"text-align: left;\"><\/td>\n<td style=\"text-align: left;\">c) GEE<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\nIMPORTANT REMARKS:\n\n\u2022 The GEE \\[generalised estimating equations\\] \\[reference\\] is a technique that allows to specify the correlation structure between patients within a hospital and this approach produces unbiased estimates under the assumption that missing observations will be missing at random. An amended approach of weighted GEE will be employed if missingness is found not to be at random \\[reference\\].\n\n\u2022 In all analyses results will be expressed as coefficient, standard errors, corresponding 95% and associated p-values.\n\n\u2022 Goodness-of-fit will be assessed by examining the residuals for model assumptions and chi-squared test of goodness-of-fit.\n\nBonferroni method will be used to adjust the overall level of significance for multiple secondary outcomes.\n\n### Explanation\n\nThe protocol should indicate explicitly each intended analysis comparing study groups. An unambiguous, complete, and transparent description of statistical methods facilitates execution, replication, critical appraisal, and the ability to track any changes from the original pre-specified methods.\n\nResults for the primary outcome can be substantially affected by the choice of analysis methods. When investigators apply more than one analysis strategy for a specific primary outcome, there is potential for inappropriate selective reporting of the most interesting result.6 The protocol should prespecify the main (\"primary\") analysis of the primary outcome (Item 12), including the analysis methods to be used for statistical comparisons (Items 20a and 20b); precisely which trial participants will be included (Item 20c); and how missing data will be handled (Item 20c). Additionally, it is helpful to indicate the effect measure (eg, relative risk) and significance level that will be used, as well as the intended use of confidence intervals when presenting results.\n\nThe same considerations will often apply equally to prespecified secondary and exploratory outcomes. In some instances, descriptive approaches to evaluating rare outcomes such as adverse events\u2014might be preferred over formal analysis given the lack of power.300 Adequately powered analyses may require preplanned meta-analyses with results from other studies.\n\nMost trials are affected to some extent by multiplicity issues.301 302 When multiple statistical comparisons are performed (eg, multiple study groups, outcomes, interim analyses), the risk of false positive (type 1) error is inflated and there is increased potential for selective reporting of favourable comparisons in the final trial report. For trials with more than two study groups, it is important to specify in the protocol which comparisons (of two or more study groups) will be performed and, if relevant, which will be the main comparison of interest. The same principle of specifying the main comparison also applies when there is more than one outcome, including when the same variable is measured at several time points (Item 12). Any statistical approaches to account for multiple comparisons and time points should also be described.\n\nFinally, different trial designs dictate the most appropriate analysis plan and any additional relevant information that should be included in the protocol. For example, cluster, factorial, crossover, and within-person randomised trials require specific statistical considerations, such as how clustering will be handled in a cluster randomised trial.\n\n## Statistical methods\u2014additional analyses\n\nItem 20b: Methods for any additional analyses (eg, subgroup and adjusted analyses)\n\n### Examples\n\n\"We plan to conduct two subgroup analyses, both with strong biological rationale and possible interaction effects. The first will compare hazard ratios of re-operation based upon the degree of soft tissue injury (Gustilo-Anderson Type I\/II open fractures vs. Gustilo-Anderson Type IIIA\/B open fractures). The second will compare hazard ratios of re-operation between fractures of the upper and lower extremity. We will test if the treatment effects differ with fracture types and extremities by putting their main effect and interaction terms in the Cox regression. For the comparison of pressure, we anticipate that the low\/gravity flow will be more effective in the Type IIIA-B open fracture than in the Type I\/II open fracture, and be more effective in the upper extremity than the lower extremity. For the comparison of solution, we anticipate that soap will do better in the Type IIIA-B open fracture than in the Type I\/II open fracture, and better in the upper extremity than the lower extremity.\"303\n\n\"A secondary analysis of the primary endpoint will adjust for those pre-randomization variables which might reasonably be expected to be predictive of favorable outcomes. Generalized linear models will be used to model the proportion of subjects with neurologically intact (MRS \u2264 3 \\[Modified Rankin Score\\]) survival to hospital discharge by ITD \\[impedance threshold device\\]\/sham device group adjusted for site (dummy variables modeling the 11 ROC \\[Resuscitation Outcomes Consortium\\] sites), patient sex, patient age (continuous variable), witness status (dummy variables modeling the three categories of unwitnessed arrest, non-EMS \\[emergency medical services\\] witnessed arrest, and EMS witnessed arrest), location of arrest (public versus non-public), time or response (continuous variable modeling minutes between call to 911 and arrival of EMS providers on scene), presenting rhythm (dummy variables modeling asystole, PEA \\[pulseless electrical activity\\], VT\/VF \\[ventricular tachycardia\/fibrillation\\], or unknown), and treatment assignment in the Analyze Late vs. Analyze Early intervention. The test statistic used to assess any benefit of the ITD relative to the sham device will be computed as the generalized linear model regression coefficient divided by the estimated \"robust\" standard error based on the Huber- White sandwich estimator\\[reference\\] in order to account for within group variability which might depart from the classical assumptions. Statistical inference will be based on one-sided P values and 95% confidence intervals which adjust for the stopping rule used for the primary analysis.\"304\n\n### Explanation\n\nSubgroup analysis\n\nSubgroup analyses explore whether estimated treatment effects vary significantly between subcategories of trial participants. As these data can help tailor healthcare decisions to individual patients, a modest number of prespecified subgroup analyses can be sensible.\n\nHowever, subgroup analyses are problematic if they are inappropriately conducted or selectively reported. Subgroup analyses described in protocols or grant applications do not match those reported in subsequent publications for more than two thirds of randomised trials, suggesting that subgroup analyses are often selectively reported or not prespecified.6 7 305 Post hoc (data driven) analyses have a high risk of spurious findings and are discouraged.306 Conducting a large number of subgroup comparisons leads to issues of multiplicity, even when all of the comparisons have been pre-specified. Furthermore, when subgroups are based on variables measured after randomisation, the analyses are particularly susceptible to bias.307\n\nPreplanned subgroup analyses should be clearly specified in the protocol with respect to the precise baseline variables to be examined, the definition of the subgroup categories (including cut-off boundaries for continuous or ordinal variables), the statistical method to be used, and the hypothesised direction of the subgroup effect based on plausibility.308 309\n\nAdjusted analysis\n\nSome trials prespecify adjusted analyses to account for imbalances between study groups (eg, chance imbalance across study groups in small trials), improve power, or account for a known prognostic variable. Adjustment is often recommended for any variables used in the allocation process (eg, in stratified randomisation), on the principle that the analysis strategy should match the design.310 Most trial protocols and publications do not adequately address issues of adjustment, particularly the description of variables.6 310\n\nIt is important that trial investigators indicate in the protocol if there is an intention to perform or consider adjusted analyses, explicitly specifying any variables for adjustment and how continuous variables will be handled. When both unadjusted and adjusted analyses are intended, the main analysis should be identified (Item 20a). It may not always be clear, in advance, which variables will be important for adjustment. In such situations, the objective criteria to be used to select variables should be prespecified. As with subgroup analyses, adjustment variables based on post-randomisation data rather than baseline data can introduce bias.311 312\n\n## Statistical methods\u2014analysis population and missing data\n\nItem 20c: Definition of analysis population relating to protocol non-adherence (eg, as randomised analysis), and any statistical methods to handle missing data (eg, multiple imputation)\n\n### Example\n\n\"Nevertheless, we propose to test non-inferiority using two analysis sets; the intention-to-treat set, considering all patients as randomized regardless of whether they received the randomized treatment, and the \"per protocol\" analysis set. Criteria for determining the \"per protocol\" group assignment would be established by the Steering Committee and approved by the PSMB \\[performance and safety monitoring board\\] before the trial begins. Given our expectation that very few patients will crossover or be lost to follow-up, these analyses should agree very closely. We propose declaring medical management non-inferior to interventional therapy, only if shown to be non-inferior using both the \"intention to treat\" and \"per protocol\" analysis sets.\n\n. . .\n\n#### 10.4.7 Imputation Procedure for Missing Data\n\nWhile the analysis of the primary endpoint (death or stroke) will be based on a log-rank test and, therefore, not affected by patient withdrawals (as they will be censored) provided that dropping out is unrelated to prognosis; other outcomes, such as the Rankin Score at five years post-randomization, could be missing for patients who withdraw from the trial. We will report reasons for withdrawal for each randomization group and compare the reasons qualitatively . . . The effect that any missing data might have on results will be assessed via sensitivity analysis of augmented data sets. Dropouts (essentially, participants who withdraw consent for continued follow-up) will be included in the analysis by modern imputation methods for missing data.\n\nThe main feature of the approach is the creation of a set of clinically reasonable imputations for the respective outcome for each dropout. This will be accomplished using a set of repeated imputations created by predictive models based on the majority of participants with complete data. The imputation models will reflect uncertainty in the modeling process and inherent variability in patient outcomes, as reflected in the complete data.\n\nAfter the imputations are completed, all of the data (complete and imputed) will be combined and the analysis performed for each imputed-and-completed dataset. Rubin's method of multiple (ie, repeated) imputation will be used to estimate treatment effect. We propose to use 15 datasets (an odd number to allow use of one of the datasets to represent the median analytic result).\n\nThese methods are preferable to simple mean imputation, or simple \"best-worst\" or \"worst-worst\" imputation, because the categorization of patients into clinically meaningful subgroups, and the imputation of their missing data by appropriately different models, accords well with best clinical judgment concerning the likely outcomes of the dropouts, and therefore will enhance the trial's results.\"313\n\n### Explanation\n\nIn order to preserve the unique benefit of randomisation as a mechanism to avoid selection bias, an \"as randomised\" analysis retains participants in the group to which they were originally allocated. To prevent attrition bias, outcome data obtained from all participants are included in the data analysis, regardless of protocol adherence (Items 11c and 18b).249 250 These two conditions (ie, all participants, as randomised) define an \"intention to treat\" analysis, which is widely recommended as the preferred analysis strategy.17\n\nSome trialists use other types of data analyses (commonly labelled as \"modified intention to treat\" or \"per protocol\") that exclude data from certain participants\u2014such as those who are found to be ineligible after randomisation or who deviate from the intervention or follow-up protocols. This exclusion of data from protocol non-adherers can introduce bias, particularly if the frequency of and the reasons for non-adherence vary between the study groups.314 315 In some trials, the participants to be included in the analysis will vary by outcome\u2014for example, analysis of harms (adverse events) is sometimes restricted to participants who received the intervention, so that absence or occurrence of harm is not attributed to a treatment that was never received.\n\nProtocols should explicitly describe which participants will be included in the main analyses (eg, all randomised participants, regardless of protocol adherence) and define the study group in which they will be analysed (eg, as randomised). In one cohort of randomised trials approved in 1994-5, this information was missing in half of the protocols.6 The ambiguous use of labels such as \"intention to treat\" or \"per protocol\" should be avoided unless they are fully defined in the protocol.6 314 Most analyses labelled as \"intention to treat\" do not actually adhere to its definition because of missing data or exclusion of participants who do not meet certain post-randomisation criteria (eg, specific level of adherence to intervention).6 316 Other ambiguous labels such as \"modified intention to treat\" are also variably defined from one trial to another.314\n\nIn addition to defining the analysis population, it is necessary to address the problem of missing data in the protocol. Most trials have some degree of missing data,317 318 which can introduce bias depending on the pattern of \"missingness\" (eg, not missing at random). Strategies to maximise follow-up and prevent missing data, as well as the recording of reasons for missing data, are thus important to develop and document (Item 18b).152\n\nThe protocol should also state how missing data will be handled in the analysis and detail any planned methods to impute (estimate) missing outcome data, including which variables will be used in the imputation process (if applicable).152 Different statistical approaches can lead to different results and conclusions,317 319 but one study found that only 23% of trial protocols specified the planned statistical methods to account for missing data.6\n\nImputation of missing data allows the analysis to conform to intention to treat analysis but requires strong assumptions that are untestable and may be hard to justify.152 318 320 321 Methods of multiple imputation are more complex but are widely preferred to single imputation methods (eg, last observation carried forward; baseline observation carried forward), as the latter introduce greater bias and produce confidence intervals that are too narrow.152 320 321 322 Specific issues arise when outcome data are missing for crossover or cluster randomised trials.323 Finally, sensitivity analyses are highly recommended to assess the robustness of trial results under different methods of handling missing data.152 324\n\n# Section 3d: Methods\u2014monitoring\n\n## Data monitoring\u2014formal committee\n\nItem 21a: Composition of data monitoring committee (DMC); summary of its role and reporting structure; statement of whether it is independent from the sponsor and competing interests; and reference to where further details about its charter can be found, if not in the protocol. Alternatively, an explanation of why a DMC is not needed\n\n### Example\n\n#### \"Appendix 3. Charter and responsibilities of the Data Monitoring Committee\n\nA Data Monitoring Committee (DMC) has been established. The DMC is independent of the study organisers. During the period of recruitment to the study, interim analyses will be supplied, in strict confidence, to the DMC, together with any other analyses that the committee may request. This may include analyses of data from other comparable trials. In the light of these interim analyses, the DMC will advise the TSC \\[trial steering committee\\] if, in its view:\n\na\\) the active intervention has been proved, beyond reasonable doubt\\*, to be different from the control (standard management) for all or some types of participants, and\n\nb\\) the evidence on the economic outcomes is sufficient to guide a decision from health care providers regarding recommendation of early lens extraction for PACG \\[primary angle closure glaucoma\\].\n\nThe TSC can then decide whether or not to modify intake to the trial. Unless this happens, however, the TSC, PMG \\[project management group\\], clinical collaborators and study office staff (except those who supply the confidential analyses) will remain ignorant of the interim results.\n\nThe frequency of interim analyses will depend on the judgement of the Chair of the DMC, in consultation with the TSC. However, we anticipate that there might be three interim analyses and one final analysis.\n\nThe Chair is Mr *D.G.-H.*, with Dr *D.C.*, and Professor *B.D.* Terms of reference for the DMC are available on request from the EAGLE \\[Effectiveness in Angle Closure Glaucoma of Lens Extraction\\] study office.\n\n\\*Appropriate criteria for proof beyond reasonable doubt cannot be specified precisely. A difference of at least three standard deviation \\[sic\\] in the interim analysis of a major endpoint may be needed to justify halting, or modifying, such a study prematurely.\\[reference\\]\"325\n\n### Explanation\n\nFor some trials, there are important reasons for periodic inspection of the accumulating outcome data by study group. In principle, a trial should be modified or discontinued when the accumulated data have sufficiently disturbed the clinical equipoise that justified the initiation of the trial. Data monitoring can also inform aspects of trial conduct, such as recruitment, and identify the need to make adjustments.\n\nThe decision to have a data monitoring committee (DMC) will be influenced by local standards. While certain trials warrant some form of data monitoring, many do not need a formal committee,326 such as trials with a short duration or known minimal risks. A DMC was described in 65% (98\/150) of cancer trial protocols with time-to-event outcomes in Italy in 2000-5,327 and in 17% (12\/70) of protocols for Danish randomised trials approved in 1994-5.6 About 40% of clinical trials registered on ClinicalTrials.gov from 2007-2010 reported having a DMC.328 The protocol should either state that there will be a DMC and provide further details, as discussed below, or indicate that there will not be a DMC, preferably with reasons.\n\nWhen formal data monitoring is performed, it is often done by a DMC consisting of members from a variety of disciplines.254 329 The primary role of a DMC is to periodically review the accumulating data and determine if a trial should be modified or discontinued. The DMC does not usually have executive power; rather, it communicates the outcome of its deliberations to the trial steering committee or sponsor.\n\nIndependence, in particular from the sponsor and trial investigators, is a key characteristic of the DMC and can be broadly defined as the committee comprising members who are \"completely uninvolved in the running of the trial and who cannot be unfairly influenced (either directly or indirectly) by people, or institutions, involved in the trial.\"254 DMC members are usually required to declare any competing interests (Item 28). Among the 12 trial protocols that described a DMC and were approved in Denmark in 1994-5,6 four explicitly stated that the DMC was independent from the sponsor and investigators; three had non-independent DMCs; and independence was unclear for the remaining five protocols.\n\nThe protocol should name the chair and members of the DMC. If the members are not yet known, the protocol can indicate the intended size and characteristics of the membership until further details are available. The protocol should also indicate the DMC's roles and responsibilities, planned method of functioning, and degree of independence from those conducting, sponsoring, or funding the trial.254 330 331 A charter is recommended for detailing this information331; if this charter is not appended to the protocol, the protocol should indicate whether a charter exists or will be developed, and if so, where it can be accessed.\n\n## Data monitoring\u2014interim analysis\n\nItem 21b: Description of any interim analyses and stopping guidelines, including who will have access to these interim results and make the final decision to terminate the trial\n\n### Example\n\n#### \"Premature termination of the study\n\nAn interim-analysis is performed on the primary endpoint when 50% of patients have been randomised and have completed the 6 months follow-up. The interim-analysis is performed by an independent statistician, blinded for the treatment allocation. The statistician will report to the independent DSMC \\[data and safety monitoring committee\\]. The DSMC will have unblinded access to all data and will discuss the results of the interim-analysis with the steering committee in a joint meeting. The steering committee decides on the continuation of the trial and will report to the central ethics committee. The Peto approach is used: the trial will be ended using symmetric stopping boundaries at P \\< 0.001 \\[reference\\]. The trial will not be stopped in case of futility, unless the DSMC during the course of safety monitoring advices \\[sic\\] otherwise. In this case DSMC will discuss potential stopping for futility with the trial steering committee.\"332\n\n### Explanation\n\nInterim analyses can be conducted as part of an adaptive trial design to formally monitor the accumulating data in clinical trials. They are generally performed in trials that have a DMC, longer duration of recruitment, and potentially serious outcomes. Interim analyses were described in 71% (106\/150) of cancer trial protocols with time-to-event outcomes in Italy in 2000-5,327 and in 19% (13\/70) of protocols for Danish randomised trials approved in 1994-5.6 The results of these analyses, along with non-statistical criteria, can be part of a stopping guideline that helps inform whether the trial should be continued, modified, or halted earlier than intended for benefit, harm, or futility. Criteria for stopping for harm are often different from those for benefit and might not employ a formal statistical criterion.333 Stopping for futility occurs in instances where, if the study were to continue, it is unlikely that an important effect would be seen (ie, low chance of rejecting null hypothesis). Multiple analyses of the accumulating data increase the risk of a false positive (type I) error, and various statistical strategies have been developed to compensate for this inflated risk.254 333 334 335 Aside from informing stopping guidelines, prespecified interim analyses can be used for other trial adaptations such as sample size re-estimation, alteration to the proportion of participants allocated to each study group, and changes to eligibility criteria.111\n\nA complete description of any interim analysis plan, even if it is only to be performed at the request of an oversight body (eg, DMC), should be provided in the protocol\u2014including the statistical methods, who will perform the analyses, and when they will be conducted (timing and indications). If applicable, details should also be provided about the decision criteria\u2014statistical or other\u2014that will be adopted to judge the interim results as part of a guideline for early stopping or other adaptations. Among 86 protocols for randomised trials with a time-to-event cancer outcome that proposed efficacy interim analyses, all stated the planned timing of the analyses, 91% specified the overall reason to be used for stopping (eg, superiority, futility), and 94% detailed the statistical approach.327\n\nIn addition, it is important to state who will see the outcome data while the trial is ongoing, whether these individuals will remain blinded (masked) to study groups, and how the integrity of the trial implementation will be protected (eg, maintaining blinding) when any adaptations to the trial are made. A third of protocols for industry initiated randomised trials receiving Danish ethics approval in 1994-95 stated that the sponsor had access to accumulating trial data, which can introduce potential bias due to competing interests.10 Finally, the protocol should specify who has the ultimate authority to stop or modify the trial\u2014eg, the principal investigator, trial steering committee, or sponsor.\n\n## Harms\n\nItem 22: Plans for collecting, assessing, reporting, and managing solicited and spontaneously reported adverse events and other unintended effects of trial interventions or trial conduct\n\n### Example\n\n\"**Secondary outcomes**\n\n. . . In our study an adverse event will be defined as any untoward medical occurrence in a subject without regard to the possibility of a causal relationship. Adverse events will be collected after the subject has provided consent and enrolled in the study. If a subject experiences an adverse event after the informed consent document is signed (entry) but the subject has not started to receive study intervention, the event will be reported as not related to study drug. All adverse events occurring after entry into the study and until hospital discharge will be recorded. An adverse event that meets the criteria for a serious adverse event (SAE) between study enrollment and hospital discharge will be reported to the local IRB \\[institutional review board\\] as an SAE. If haloperidol is discontinued as a result of an adverse event, study personnel will document the circumstances and data leading to discontinuation of treatment. A serious adverse event for this study is any untoward medical occurrence that is believed by the investigators to be causally related to study-drug and results in any of the following: Life-threatening condition (that is, immediate risk of death); severe or permanent disability, prolonged hospitalization, or a significant hazard as determined by the data safety monitoring board. Serious adverse events occurring after a subject is discontinued from the study will NOT be reported unless the investigators feels that the event may have been caused by the study drug or a protocol procedure. Investigators will determine relatedness of an event to study drug based on a temporal relationship to the study drug, as well as whether the event is unexpected or unexplained given the subject's clinical course, previous medical conditions, and concomitant medications.\n\n. . . The study will monitor for the following movement-related adverse effects daily through patient examination and chart review: dystonia, akathisia, pseudoparkinsonism, akinesia, and neuroleptic malignant syndrome. Study personnel will use the Simpson-Angus \\[reference\\] and Barnes Akathisia \\[reference\\] scales to monitor movement-related effects.\n\n. . .\n\nFor secondary outcomes, binary measures, eg mortality and complications, logistic regression will be used to test the intervention effect, controlling for covariates when appropriate.\"266\n\n### Explanation\n\nEvaluation of harms has a key role in monitoring the condition of participants during a trial and in enabling appropriate management of adverse events. Documentation of trial related adverse events also informs clinical practice and the conduct of ongoing and future studies. We use the term \"harms\" instead of \"safety\" to better reflect the negative effects of interventions.300 An adverse event refers to an untoward occurrence during the trial, which may or may not be causally related to the intervention or other aspects of trial participation.300 336 This definition includes unfavourable changes in symptoms, signs, laboratory values, or health conditions. In the context of clinical trials, it can be difficult to attribute causation for a given adverse event. An adverse effect is a type of adverse event that can be attributed to the intervention.\n\nHarms can be specified as primary or secondary outcomes (Item 12) or can be assessed as part of routine monitoring. To the extent possible, distinctions should be made between adverse events that are anticipated versus unanticipated, and solicited versus unsolicited, because expectation can influence the number and perceived severity of recorded events. For example, providing statements in the informed consent process about the possibility of a particular adverse effect or using structured, as opposed to open ended, questionnaires for data collection, can increase the reporting of specific events (\"priming\").269 337 338 339 The timeframe for recording adverse events can also affect the type of data obtained.340 341\n\nThe protocol should describe the procedures for and frequency of harms data collection, the overall surveillance timeframe, any instruments to be used, and their validity and reliability, if known. Substantial discrepancies have been observed between protocol specified plans for adverse event collection and reporting, and what is described in final publications.5 Although trials are often not powered to detect important differences in rates of uncommon adverse events, it is also important to describe plans for data analysis, including formal hypothesis testing or descriptive statistics.300 342\n\nFinally, the protocol should address the reporting of harms to relevant groups (eg, sponsor, research ethics committee\/institutional review board, data monitoring committee, regulatory agency), which is an important process that is subject to local regulation.343 Key considerations include the severity of the adverse event, determination of potential causality, and whether it represents an unexpected or anticipated event. For multicentre studies, procedures and timing should be outlined for central collection, evaluation, and reporting of pooled harms data.\n\n## Auditing\n\nItem 23: Frequency and procedures for auditing trial conduct, if any, and whether the process will be independent from investigators and the sponsor\n\n### Example\n\n#### \"11.4 Data Monitoring and Quality Assurance\n\nThrough the combination of our web-based, instantaneous electronic validation, the DCC's \\[data coordinating centre\\] daily visual cross-validation of the data for complex errors, and regular on-site monitoring, the quality and completeness of the data will be reflective of the state of the art in clinical trials.\n\nBoth the European and US DCCs will conduct monitoring of source documents via fax at all enrolling ARUBA \\[A Randomised trial of Unruptured Brain Arteriovenous malformations\\] sites and will conduct at least one on-site monitoring visit per year over the course of the study at 100% of clinical sites (with repeat visits to sites where performance is a concern). Monitoring of European study sites will be assured by the European Coordinating Center (Paris). The primary objectives of the DCC during the on-site visits are to educate, support and solve problems. The monitors will discuss the protocol in detail and identify and clarify any areas of weakness. At the start of the trial, the monitors will conduct a tutorial on the web-based data entry system. The coordinators will practice entering data so that the monitors can confirm that the coordinators are proficient in all aspects of data entry, query response, and communication with the DCC. They will audit the overall quality and completeness of the data, examine source documents, interview investigators and coordinators, and confirm that the clinical center has complied with the requirements of the protocol. The monitors will verify that all adverse events were documented in the correct format, and are consistent with protocol definition.\n\nThe monitors will review the source documents as needed, to determine whether the data reported in the Web-based system are complete and accurate. Source documents are defined as medical charts, associated reports and records including initial hospital admission report . . .\n\nThe monitors will confirm that the regulatory binder is complete and that all associated documents are up to date. The regulatory binder should include the protocol and informed consent (all revisions), IRB \\[institutional review board\\] approvals for all of the above documents, IRB correspondence, case report forms, investigator's agreements . . .\n\nScheduling monitoring visits will be a function of patient enrollment, site status and other commitments. The DCC will notify the site in writing at least three weeks prior to a scheduled visit. The investigators must be available to meet with the monitors. Although notification of the visits will include the list of patients scheduled to be reviewed, the monitors reserve the right to review additional ARUBA patients.\n\nIf a problem is identified during the visit (ie, poor communication with the DCC, inadequate or insufficient staff to conduct the study, missing study documents) the monitor will assist the site in resolving the issues. Some issues may require input from the Operations Committee, Steering Committee or one of the principal investigators.\n\nThe focus of the visit\/electronic monitoring will be on source document review and confirmation of adverse events. The monitor will verify the following variables for all patients: initials, date of birth, sex, signed informed consent, eligibility criteria, date of randomization, treatment assignment, adverse events, and endpoints.\"313\n\n### Explanation\n\nAuditing involves periodic independent review of core trial processes and documents. It is distinct from routine day-to-day measures to promote data quality (Items 18a and 19). Auditing is intended to preserve the integrity of the trial by independently verifying a variety of processes and prompting corrective action if necessary. The processes reviewed can relate to participant enrolment, consent, eligibility, and allocation to study groups; adherence to trial interventions and policies to protect participants, including reporting of harms (Item 22); and completeness, accuracy, and timeliness of data collection. In addition, an audit can verify adherence to applicable policies such as the International Conference on Harmonisation *Good Clinical Practice* and regulatory agency guidelines.160\n\nIn multicentre trials, auditing is usually considered both overall and for each recruiting centre. Audits can be done by exploring the trial dataset or performing site visits. Audits might be initially conducted across all sites, and subsequently conducted using a risk based approach that focuses, for example, on sites that have the highest enrolment rates, large numbers of withdrawals, or atypical (low or high) numbers of reported adverse events.\n\nIf auditing is planned, the procedures and anticipated frequency should be outlined in the protocol, including a description of the personnel involved and their degree of independence from the trial investigators and sponsor. If procedures are further detailed elsewhere (eg, audit manual), then the protocol should reference where the full details can be obtained.\n\n# Section 4: Ethics and dissemination\n\n## Research ethics approval\n\nItem 24: Plans for seeking research ethics committee\/institutional review board (REC\/IRB) approval\n\n### Example\n\n\"This protocol and the template informed consent forms contained in Appendix II will be reviewed and approved by the sponsor and the applicable IRBs\/ECs \\[institutional review boards\/ethical committees\\] with respect to scientific content and compliance with applicable research and human subjects regulations. . . .\n\nThe protocol, site-specific informed consent forms (local language and English versions), participant education and recruitment materials, and other requested documents\u2014and any subsequent modifications \u2014 also will be reviewed and approved by the ethical review bodies. . .\n\nSubsequent to initial review and approval, the responsible local Institutional Review Boards\/Ethical Committees (IRBs\/ECs) will review the protocol at least annually. The Investigator will make safety and progress reports to the IRBs\/ECs at least annually and within three months of study termination or completion at his\/her site. These reports will include the total number of participants enrolled . . . and summaries of each DSMB \\[data safety and monitoring board\\] review of safety and\/or efficacy.\"287\n\n### Explanation\n\nA universal requirement for the ethical conduct of clinical research is the review and approval of the research protocol by qualified individuals who are not associated with the research team and have no disqualifying competing interests as reviewers.1 The review is typically conducted by a formal REC\/IRB in accordance with jurisdictional policy. Despite the importance of ethics review, approval by a REC\/IRB is not always obtained. Among 767 trials published in leading general medical journals from 1993-95, 37 authors (5%) disclosed that such approval had not been sought for their trials.344 The protocol should document where approval has been obtained, or outline plans to seek such approval.\n\n## Protocol amendments\n\nItem 25: Plans for communicating important protocol modifications (eg, changes to eligibility criteria, outcomes, analyses) to relevant parties (eg, investigators, REC\/IRBs, trial participants, trial registries, journals, regulators)\n\n### Example\n\n#### \"13.10 Modification of the Protocol\n\nAny modifications to the protocol which may impact on the conduct of the study, potential benefit of the patient or may affect patient safety, including changes of study objectives, study design, patient population, sample sizes, study procedures, or significant administrative aspects will require a formal amendment to the protocol. Such amendment will be agreed upon by BCIRG \\[Breast Cancer International Research Group\\] and Aventis, and approved by the Ethics Committee\/IRB \\[institutional review board\\] prior to implementation and notified to the health authorities in accordance with local regulations.\n\nAdministrative changes of the protocol are minor corrections and\/or clarifications that have no effect on the way the study is to be conducted. These administrative changes will be agreed upon by BCIRG and Aventis, and will be documented in a memorandum. The Ethics Committee\/IRB may be notified of administrative changes at the discretion of BCIRG.\"345\n\n### Explanation\n\nAfter initial ethics approval, about half of trials have subsequent protocol amendments submitted to the REC\/IRB.125 346 347 While some amendments may be unavoidable, a study of pharmaceutical industry trials found that according to the sponsors, a third of amendments could have been prevented with greater attention to key issues during protocol development.346 Substantive amendments can generate challenges to data analysis and interpretation if they occur part way through the trial (eg, changes in eligibility criteria),348 and can introduce bias if the changes are made based on the trial data.173 174 175 176 The implementation and communication of amendments are also burdensome and potentially costly.346\n\nNumerous studies have revealed substantive changes between prespecified methods (eg, as stated in approved protocols, registries, or regulatory agency submissions) and those described in trial publications, including changes to primary outcomes,12 172 173 174 175 176 sample size calculations,6 eligibility criteria,125 133 134 as well as methods of allocation concealment,2 blinding,3 and statistical analysis.6 7 8 174 These substantive modifications are rarely acknowledged in the final trial reports, providing an inaccurate impression of trial integrity.\n\nIt is important that substantive protocol amendments be reviewed by an independent party, such as the REC\/IRB, and transparently described in trial reports. The notion of \"substantive\" is variably defined by authorities, but in general refers to a protocol amendment that can affect the safety of trial participants or the scientific validity, scope, or ethical rigour of the trial.349 350 To reflect the degree of oversight for the trial and adherence to applicable regulation, the protocol should describe the process for making amendments, including who will be responsible for the decision to amend the protocol and how substantive changes will be communicated to relevant stakeholders (eg, REC\/IRBs, trial registries, regulatory agencies). Version control using protocol identifiers and dates (Item 3), as well as a list of amendments, can help to track the history of amendments and identify the most recent protocol version.\n\n## Consent or assent\n\nItem 26a: Who will obtain informed consent or assent from potential trial participants or authorised surrogates, and how (see Item 32)\n\n### Example\n\n\" . . . Trained Research Nurses will introduce the trial to patients who will be shown a video regarding the main aspects of the trial. Patients will also receive information sheets. Research Nurses will discuss the trial with patients in light of the information provided in the video and information sheets. Patients will then be able to have an informed discussion with the participating consultant. Research Nurses will obtain written consent from patients willing to participate in the trial. Information sheets and consent forms are provided for all parents involved in the trial however these have been amended accordingly in order to provide separate information sheets and consent form \\[sic\\] which are suitable for children and teenagers. All information sheets, consent forms and the video transcript have been translated into Bengali, Punjabi, Gujarati, and Urdu. There are also separate information sheets and consent forms for the cohort group.\"351\n\n### Explanation\n\nThe notion of acquiring informed consent involves the presentation of comprehensible information about the research to potential participants, confirmation that they understand the research, and assurance that their agreement to participate is voluntary. The process typically involves discussion between the potential participant and an individual knowledgeable about the research; the presentation of written material (eg, information leaflet or consent document); and the opportunity for potential participants to ask questions. Surveys of trial investigators reveal that appropriate informed consent is not always obtained.344 352\n\nThe content, quantity, and mode of delivery of consent information can affect trial recruitment, participant comprehension, anxiety, retention rates, and recruitment costs.68 114 218 292 353 354 355 We recommend that a model consent or assent form be provided as a protocol appendix (Item 32). Assent represents a minor's affirmative agreement to participate in the trial, which typically involves signing a document that provides age appropriate information about the study.\n\nThe protocol should include details of the consent process as well as the status, experience, and training (if applicable) of the research team members who will conduct it. In paediatric research, regulations may stipulate obtaining affirmative assent for participation from children above a certain age.356 The protocol should then describe how pertinent information will be provided to potential participants and how their understanding and assent will be ascertained. When potential participants lack decisional capacity for reasons other than young age (eg, mental status), and proxy consent can be obtained from a legally-authorised representative, the protocol should describe who will determine an individual's decisional capacity, whether a formal capacity instrument will be utilised, and how the individual's informed agreement to continue participation will be secured should they regain decisional capacity. For certain trials, such as cluster randomised trials, it may not be possible to acquire individual informed consent from participants before randomisation, and the consent process may be modified or waived. An explanation should be provided in the protocol in these instances.357\n\n## Consent or assent\u2014ancillary studies\n\nItem 26b: Additional consent provisions for collection and use of participant data and biological specimens in ancillary studies, if applicable\n\n### Example\n\n#### \"6.4.1. Samples for Biorepositories\n\nAdditional biological samples will be obtained to be stored for use in future studies of the pathobiology of FSGS \\[focal segmental glomerulosclerosis\\]. A materials consent will be obtained to specifically address the collection of these . . . urine, serum and plasma specimens . . .\n\n#### 14.3.4. Instructions for Preparation of Requests for an Ancillary Study\n\n. . . A signed consent must be obtained from every participant in the ancillary study, if the data collection\/request is not covered in the original informed consent process for the main FSGS Clinical Trial.\n\n. . .\n\nA copy of the IRB \\[institutional review board\\] letter for the ancillary study should be sent to the DCC \\[data coordinating centre\\]. If a separate consent form is required for the ancillary study, a copy of the signed ancillary study consent form for each study participant must be included in the FSGS-CT \\[clinical trial\\] record. A data file tracking all signed ancillary consent forms must be maintained by the ancillary study and an electronic copy of that file must be delivered to the FSGS-CT DCC.\"267\n\n### Explanation\n\nAncillary studies involve the collection or derivation of data for purposes that are separate from the main trial. The acquisition and storage of data and biological specimens for ancillary studies is increasingly common in the context of clinical trials (Item 33). Specimens may be used for a specified subset of studies or for submission to biorepositories for future specified or unspecified research.\n\nAncillary studies have additional processes and considerations relating to consent, which should be detailed in the protocol. Guidance for the creation of a simplified informed consent document for biobanking is available.358 Participants can be given several options to consider with respect to their participation in ancillary research: consent for the use of their data and specimens in specified protocols; consent for use in future research unrelated to the clinical condition under study; consent for submission to an unrelated biorepository; and consent to be contacted by trial investigators for further informational and consent-related purposes. This is commonly referred to as tiered consent. Participants should also be informed about whether their withdrawal from the ancillary research is possible (eg, the data and specimens are coded and identifiable); what withdrawal means in this context (eg, used specimens and data derived from them cannot be withdrawn); and what information derived from the specimen related research will be provided to them, if any.\n\n## Confidentiality\n\nItem 27: How personal information about potential and enrolled participants will be collected, shared, and maintained in order to protect confidentiality before, during, and after the trial\n\n### Example\n\n#### \"8.5 Confidentiality\n\nAll study-related information will be stored securely at the study site. All participant information will be stored in locked file cabinets in areas with limited access. All laboratory specimens, reports, data collection, process, and administrative forms will be identified by a coded ID \\[identification\\] number only to maintain participant confidentiality. All records that contain names or other personal identifiers, such as locator forms and informed consent forms, will be stored separately from study records identified by code number. All local databases will be secured with password-protected access systems. Forms, lists, logbooks, appointment books, and any other listings that link participant ID numbers to other identifying information will be stored in a separate, locked file in an area with limited access.\n\nAll HIV test results will be kept strictly confidential, all counseling and blood draws will be conducted in private rooms, and study staff will be required to sign agreements to preserve the confidentiality of all participants. Study staff will never inform network members of the serostatus of other members of their group, but counselors will provide general messages about the prevalence of HIV in the study population in the interests of emphasizing harm reduction.\n\nParticipants' study information will not be released outside of the study without the written permission of the participant, except as necessary for monitoring by NIAID \\[National Institute of Allergy and Infectious Diseases\\] and\/or its contractors . . . representatives of the HPTN CORE \\[HIV Prevention Trials Network Coordinating and Operations Center\\] . . . and US or in-country government and regulatory authorities.\"359\n\n### Explanation\n\nPersonal information about participants is acquired during the process of trial recruitment, eligibility screening, and data collection. Much of this information consists of private details over which people customarily wish to maintain control, such as their health status, personal genotype, and social and family history.\n\nThe protocol should describe the means whereby personal information is collected, kept secure, and maintained. In general, this involves: 1) the creation of coded, depersonalised data where the participant's identifying information is replaced by an unrelated sequence of characters; 2) secure maintenance of the data and the linking code in separate locations using encrypted digital files within password protected folders and storage media; and 3) limiting access to the minimum number of individuals necessary for quality control, audit, and analysis. The protocol should also describe how the confidentiality of data will be preserved when the data are transmitted to sponsors and coinvestigators (eg, virtual private network internet transmission).\n\n## Declaration of interests\n\nItem 28: Financial and other competing interests for principal investigators for the overall trial and each study site\n\n### Example\n\n\"*PS***:**\n\n1.  1\\. Was the Principal Investigator of the second International Stroke Trial (IST-2) to evaluate a neuroprotective compound (619c89). . .\n\n2.  2\\. Has received lecture fees and travel expenses from Bayer and from Boehringer Ingelheim for lectures given at international conferences.\n\n3.  3\\. He serves on the Independent Data Monitoring and Safety Board of the RELY trial, funded by Boehringer Ingelheim and receives attendance fees and travel expenses for attending board meetings.\n\n4.  4\\. He does not have any paid consultancies with pharmaceutical companies, and is not a member of the Speaker's Panel of any company.\n\n*KBS*:\n\n1.  Received an honorarium for a lecture from Boehringer Ingelheim and had costs for participating in scientific meetings reimbursed\"124\n\n### Explanation\n\nCompeting interests, or conflicts of interest, exist when there is potential for divergence between an individual's or institution's private interests and their responsibilities to scientific and publishing activities.360 More positive outcomes, larger treatment effect sizes, and more favourable interpretation of results have been found in clinical trials with pharmaceutical industry sponsorship (Item 4)27 36 37 38 42 and investigators who have declared competing interests,57 60 compared to those without such interests. Although competing interests are most often associated with drug and device industries, they may exist with support from or affiliation with government agencies, charities, not for profit organisations, and professional and civic organisations.\n\nCompeting interests do not in themselves imply wrongdoing. Their disclosure and regular updating enables appropriate management plans to be developed and implemented, and facilitates transparent assessment of the potential for bias.\n\nMany trials and non-industry sponsors have a conflict of interest policy for their investigators, and checklists are available to guide potential interests that should be disclosed and regularly updated by trial investigators.361 362 Types of financial ties include salary support or grants; ownership of stock or options; honorariums (eg, for advice, authorship, or public speaking); paid consultancy or service on advisory boards and medical education companies; and receipt of patents or patents pending. Non-financial competing interests include academic commitments; personal or professional relationships; and political, religious, or other affiliations with special interests or advocacy positions.\n\n## Access to data\n\nItem 29: Statement of who will have access to the final trial dataset, and disclosure of contractual agreements that limit such access for investigators\n\n### Example\n\n#### \"12.10.1 Intra-Study Data Sharing\n\nThe Data Management Coordinating Center will oversee the intra-study data sharing process, with input from the Data Management Subcommittee.\n\nAll Principal Investigators (both US and host country) will be given access to the cleaned data sets. Project data sets will be housed on the Project Accept Web site and\/or the file transfer protocol site created for the study, and all data sets will be password protected. Project Principal Investigators will have direct access to their own site's data sets, and will have access to other sites data by request. To ensure confidentiality, data dispersed to project team members will be blinded of any identifying participant information.\"113\n\n### Explanation\n\nThe validity of results from interventional trials can be verified only by individuals who have full access to the complete final dataset. For some multicentre trials, only the steering group has access to the full trial dataset in order to ensure that the overall results are not disclosed by an individual study site prior to the main publication. Many of these trials will allow site investigators to access the full dataset if a formal request describing their plans is approved by the steering group. The World Medical Association supports the principle that trial investigators retain the right to access data.363 However, among protocols of industry initiated randomised trials published in 2008-9 in the *Lancet* or approved in 2004 by a Danish ethics committee, 30-39% stated that the sponsor owned the data while 0-3% stated that principal investigators had access to all trial data.10 364 Similar constraints were found in Danish trial protocols from 1994-5.10\n\nThe protocol should identify the individuals involved in the trial who will have access to the full dataset. Any restrictions in access for trial investigators should also be explicitly described.\n\n## Ancillary and post-trial care\n\nItem 30: Provisions, if any, for ancillary and post-trial care and for compensation to those who suffer harm from trial participation\n\n### Examples\n\n\"Patients that are enrolled into the study are covered by indemnity for negligent harm through the standard NHS \\[National Health Service\\] Indemnity arrangements. The University of Sheffield has insurance to cover for non-negligent harm associated with the protocol . . . This will include cover for additional health care, compensation or damages whether awarded voluntarily by the Sponsor, or by claims pursued through the courts. Incidences judged to arise from negligence (including those due to major protocol violations) will not be covered by study insurance policies. The liability of the manufacturer of IL1RA (Amgen Corporation) is strictly limited to those claims arising from faulty manufacturing of the commercial product and not to any aspects of the conduct of the study.\"145\n\n#### \"13.6 Access to Effective Products\n\nShould this study provide evidence of the effectiveness of TDF \\[tenofovir disoproxil fumarate\\], FTC \\[emtricitabine\\]\/TDF and\/or tenofovir 1% gel in preventing HIV infection, it will be critical to provide access to the effective product(s) to study participants, their communities, and the worldwide population at risk for HIV infection in a timely manner. In preparation for this study, discussions have begun with Gilead Sciences, Inc. and CONRAD \\[Contraceptive Research and Development Organization\\] to ensure such access. Considerations under discussion include licensing agreements and preferred pricing arrangements for the study communities and other resource-poor settings.\n\nWhile this study is ongoing, the MTN \\[Microbicide Trials Network\\] will continue these discussions. In addition, discussions will be initiated with other public and private funding sources such as the WHO, UNAIDS, Gates Foundation, and appropriate site government agencies that may be able to purchase product supplies in bulk and offer them at low or no cost to the study communities and other resource-poor communities most in need of the product(s). Operations and marketing research also may be conducted to determine how best to package and distribute the products, and maximize their acceptability and use, in at-risk populations.\"365\n\n### Explanation\n\nThe provision of ancillary care refers to the provision of care beyond that immediately required for the proper and safe conduct of the trial, and the treatment of immediate adverse events related to trial procedures. It is generally agreed that trial sponsors and investigators should plan to provide care for participants' healthcare needs that arise as a direct consequence of trial participation (eg, intervention related harms). It is also important to consider whether care should be provided for certain ancillary needs that may otherwise arise during trial participation. Provision of care for ancillary needs reflects the fact that participants implicitly, but unavoidably, entrust certain aspects of their health to the research team. The scope of entrustment will vary depending on the nature of the trial (eg, setting, health condition under study, investigations performed).366 Additional factors that influence the strength of the claim to ancillary care include participants' vulnerabilities; uncompensated burdens and harms; the intensity and duration of the participant-researcher relationship; and the degree to which participants are uniquely dependent on the research team for health care.367\n\nThe Declaration of Helsinki states that \"the protocol should describe arrangements for post-study access by study participants to interventions identified as beneficial in the study or access to other appropriate care or benefits.\"1 This principle is particularly applicable\u2014and controversial\u2014when research enabling the development and regulatory approval of interventions is performed in countries where subsequent access to the interventions is limited by cost or lack of availability.368\n\nThe protocol should describe any plans to provide or pay for ancillary care during the trial and identify any interventions, benefits, or other care that the sponsor will continue to provide to participants and host communities after the trial is completed.369 Any plans to compensate participants for trial related harms should also be outlined.\n\n## Dissemination policy\u2014trial results\n\nItem 31a: Plans for investigators and sponsor to communicate trial results to participants, healthcare professionals, the public, and other relevant groups (eg, via publication, reporting in results databases, or other data sharing arrangements), including any publication restrictions\n\n### Example\n\n#### \"XII. Publication Policy\n\nThe Publications subcommittee will review all publications following the guidelines given below and report its recommendations to the Steering Committee.\n\n#### A. Data analysis and release of results\n\nThe scientific integrity of the project requires that the data from all BEST \\[Beta-Blocker Evaluation of Survival Trial\\] sites be analyzed study-wide and reported as such. Thus, an individual center is not expected to report the data collected from its center alone . . . all presentations and publications are expected to protect the integrity of the major objective(s) of the study; data that break the blind will not be presented prior to the release of mainline results. Recommendations as to the timing of presentation of such endpoint data and the meetings at which they might be presented will be given by the Steering Committee.\n\n#### B. Review process\n\nEach paper or abstract, as described below, must be submitted to the appropriate Subcommittee for review of its appropriateness and scientific merit prior to submission. The Subcommittee may recommend changes to the authors and will finally submit its recommendations to the Steering Committee for approval.\n\n#### C. Primary outcome papers\n\nThe primary outcome papers of BEST are papers that present outcome data . . . The determination of whether or not a particular analysis represents a primary outcome will be made by the Steering Committee on the recommendation of the Publications Subcommittee . . .\n\n#### D. Other study papers, abstracts and presentations\n\nAll studies other than those designated as \"Primary Outcome\" fall within this category . . . All papers and abstracts must be approved by the Publications Committee before they are submitted.\n\nIt is possible that in certain instances BEST may be asked to contribute papers to workshops, symposia, volumes, etc. The individuals to work on such requests should be appointed by the Executive Committee, but where time permits, a proposal will be circulated soliciting other participants as in the case of other study papers as described in the Application Review Process.\n\n#### XIII. Close-out Procedures\n\nBEST may terminate at the planned target of 1.5 years after the last participant has been randomized, or at an earlier or later date if the circumstances warrant . . . Regardless of the timing and circumstances of the end of the study, close-out will proceed in two stages:\n\n-   Interim period for analysis and documentation of study results.\n\n-   Debriefing of participants and dissemination of study results.\n\n#### A. Interim\n\nEvery attempt will be made to reduce to an absolute minimum the interval between the completion of data collection and the release of the study results. We expect to take about 3 to 4 months to compile the final results paper for an appropriate journal.\n\n#### B. Reporting of study results\n\nThe study results will be released to the participating physicians, referring physicians, patients and the general medical community.\"370\n\n### Explanation\n\nA fundamental ethical principle in clinical trials is that the potential risks incurred by study participants should be balanced by the benefit of contributing to publicly available knowledge.371 Unfortunately, about half of clinical trials remain unpublished.80 83 Trials with statistically non-significant results or industry funding are more prone to non-publication,36 38 80 81 82 83 although government funded trials are also susceptible.81 When published, trials with non-significant results often have a longer delay to publication.80 83 Overall, the medical literature represents a biased subset of existing data, potentially leading to overestimation of benefits, underestimation of harms, and a detrimental impact on patient care and research.80 372 373 374 375 376 377\n\nAlthough peer reviewers can be biased in favour of positive findings,378 lack of publication appears to be primarily due to trial investigators or sponsors failing to submit negative or null results, rather than journals rejecting them.80 379 A plan to disseminate trial results to key stakeholders should be outlined in the protocol, including a process and timeframe for approving and submitting reports for dissemination (eg, via journal publication, trial registry, trial website), and an explicit statement that the results will be disseminated regardless of the magnitude or direction of effect.\n\nFurthermore, any conditions relating to the investigators' right to publish or present trial results should be explicitly described. Publication restrictions have been imposed by various groups, including industry sponsors or the trial steering group (eg, to maintain the integrity of the overall dataset).10 380 These restrictions are sometimes not described in the protocol but rather in separate publication agreements.10 However, as they can interfere with the ethical responsibility of investigators and sponsors to disseminate trial results in an unbiased and timely manner,38 381 382 383 384 any restrictions should be disclosed in the protocol for review by REC\/IRBs, funders, and other stakeholders. A review of industry initiated randomised trial protocols approved in Denmark in 1994-95 revealed that 91% had publication restrictions imposed by sponsors; similar constraints were noted for protocols approved in 2004.10\n\n## Dissemination policy\u2014authorship\n\nItem 31b: Authorship eligibility guidelines and any intended use of professional writers\n\n### Example\n\n#### \"17.4. Assignment of Writing Committees\n\nTopics suggested for presentation or publication will be circulated to the PIs \\[principal investigators\\] of the CCCs \\[core coordinating centers\\], the DCC \\[data coordinating centre\\], Core Lab and the NIH \\[National Institutes of Health\\]. These groups are requested to suggest and justify names for authors to be reviewed by the PC \\[publications committee\\]. . . If a topic is suggested by a participant of the FSGS-CT \\[focal segmental glomerulosclerosis\u2014clinical trial\\], the writing committee will be formed as just described except that the person making the suggestion may be considered as the lead author. The PI of an ancillary study should be considered for lead author of material derived from this study. Disputes regarding authorship will be settled by the Study Chair after consultation with the Chair of the PC . . .\n\n#### 17.5. Reports of the FSGS-CT: Classes of Reports\n\nThere are three classes of reports of the FSGS-CT:\n\n1.  A. Reports of the major outcomes of the Study.\n\n2.  B. Reports addressing in detail one aspect of the FSGS-CT, but in which the data are derived from the entire study.\n\n3.  C. Reports of data derived from a subset of centers by members of the FSGS-CT, (eg, sub-studies or ancillary studies), or reports of investigations initiated outside of the FSGS-CT, but using data or samples collected by the FSGS-CT. . .\n\n#### 17.6. Authorship Policy\n\nThe authors of FSGS publications will be listed as detailed below.\n\nType A publications:\n\n1.  abstracts: from the FSGS Clinical Trial Group^x^, presented by XXXX.\n\n2.  papers: from the FSGS Clinical Trial Group^x^, prepared by XXXX.\n\n^x^The FSGS participant box, detailed below, must be included in these papers. If a journal's publication policy does not allow authorship by a group, the authors will be listed first as in Type B publications.\n\nType B publications:\n\n. . .\n\n#### 17.7. Authorship: Professional Participants Listing in the FSGS Participant Box\n\nThe FSGS participant box will list all professionals that have participated in the FSGS-CT for a minimum of one year.\"267\n\n### Explanation\n\nSubstantive contributions to the design, conduct, interpretation, and reporting of a clinical trial are recognised through the granting of authorship on the final trial report. Authorship guidelines in the protocol are intended to help enhance transparency and avoid disputes or misunderstanding after trial completion. These guidelines should define criteria for individually named authors or group authorship.385\n\nIndividuals who fulfil authorship criteria should not remain hidden (ghost authorship) and should have final authority over manuscript content.9 386 387 Similarly, those who do not fulfil such criteria should not be granted authorship (guest authorship).386 388 The International Committee of Medical Journal Editors has defined authorship criteria for manuscripts submitted for publication,389 although these criteria have reportedly been open to abuse.390 If some protocol authors are not named authors of subsequent publications, their role in protocol design should at least be acknowledged in the published report. Among 44 protocols of industry initiated trials, 75% had evidence of ghost authorship when compared with corresponding journal publications.9\n\nProfessional medical writers are sometimes hired to improve clarity and structure in a trial report, and guidelines for ethical collaborative writing have been developed.391 392 Because the drafting of text can influence how the study results and conclusions are portrayed, plans for the employment of writers and their funding source should be acknowledged in both protocols and trial reports.\n\n## Dissemination policy\u2014reproducible research\n\nItem 31c: Plans, if any, for granting public access to the full protocol, participant level dataset, and statistical code\n\n### Example\n\n\"**Data sharing statement** No later than 3 years after the collection of the 1-year postrandomisation interviews, we will deliver a completely deidentified data set to an appropriate data archive for sharing purposes.\"393\n\n### Explanation\n\nGiven the central role of protocols in enhancing transparency, reproducibility, and interpretation of trial results, there is a strong ethical and scientific imperative to ensure that full protocols are made publicly available.24 394 395 High quality protocols contain relevant details on study design and conduct that are generally not available in journal publications or trial registries.84 396 It is also important to make available the full study report, such as the \"clinical study report\" submitted to regulatory agencies by industry sponsors.377 396 397 398 399 400 This detailed report provides the most comprehensive description of trial methods (including the full protocol) and all published and unpublished analyses. In addition, there have increasingly been calls to improve the availability of participant-level datasets and statistical code after journal publication to enable verification and replication of analyses, facilitate pooling with other studies, and accelerate research through open knowledge sharing.372 401 402 403 404 405 406\n\nAvenues for providing access to full protocols include journals,407 408 trial websites, and trial registries.163 Several journals and funders support the sharing of participant level data,405 409 410 411 while others routinely publish a statement regarding sharing of protocols, statistical codes, and datasets for all of their published research articles.412 413\n\nThe protocol should indicate whether the trial protocol, full study report, anonymised participant level dataset, and statistical code for generating the results will be made publicly available; and if so, describe the timeframe and any other conditions for access.\n\n# Section 5: Appendices\n\n## Informed consent materials\n\nItem 32: Model consent form and other related documentation given to participants and authorised surrogates\n\n### Example\n\n\"APPENDIX 7 SAMPLE PATIENT INFORMED CONSENT\n\nNote: . . . Each Ethics Committee or Institutional Review Board will revise and adapt according to their own institution's guidelines.\n\nMULTICENTER PHASE III RANDOMIZED TRIAL COMPARING DOXORUBICIN AND CYCLOPHOSPHAMIDE **. . .**\n\nStudy number: BCIRG 006 (TAX GMA 302)\n\nInvestigator name:\n\nAddress:\n\nConsent Form:\n\nThis consent form is part of the informed consent process. It is designed to give you an idea of what this research study is about and what will happen to you if you choose to be in the study.\"345\n\n### Explanation\n\nThe Declaration of Helsinki states that each potential trial participant must normally, at a minimum, be adequately informed about the purpose of the trial; potential benefits and risks; their right to refuse participation or to withdraw consent at any time; institutional affiliation and potential competing interests of the researcher; and sources of trial funding.1 There are rare exceptions where deferred consent can be acceptable, such as trials involving unconscious patients in emergency situations.\n\nSpecial attention is required to ensure that relevant information is provided and appropriate modes of delivery are used during the consent process (Item 26).414 Consent and participant information forms are often written at a much higher reading level than is acceptable for the general population.415 Depending on the nature of the trial, several different consent documents may be needed. For example, a paediatric trial may involve both parental permission and participant assent documents. For multicentre trials, a model or sample document is typically drafted for distribution to local investigators, who may then revise the document to comply with local requirements.\n\n## Biological specimens\n\nItem 33: Plans for collection, laboratory evaluation, and storage of biological specimens for genetic or molecular analysis in the current trial and for future use in ancillary studies, if applicable\n\n### Example\n\n#### \"White Blood Cell and Plasma Collection Procedures\n\n##### 1.0 Objectives\n\n1.1 To provide a resource for studies of early markers, etiology, and genetic risk factors for prostate cancer and other diseases.\n\n##### 2.0 Background\n\nThe Prostate Cancer Prevention Trial (PCPT) is a randomized double blind chemoprevention trial . . .\n\nInitial blood collection was specifically for the analysis of PSA \\[prostate specific antigen\\] and storage of serum . . . an additional blood collection will be carried out using anticoagulant so that plasma and white blood cells can be isolated. Plasma will allow the analysis of additional biomarkers . . . This DNA will be used (among other possible uses) for studies to investigate polymorphisms in genes which may influence prostate cancer risk . . .\n\nThe PCPT WBC \\[white blood cell\\] sample will be available to PCPT investigators as well as outside researchers who have important, timely hypotheses to test. Because the sample bank is a limited resource, proposals to use it will be evaluated in terms of scientific relevance, significance, and validity as well as the potential impact of the proposed study. The amount and type of material needed will also be considered and the efficient use of material will be required. Strict confidentiality will be exercised and the information provided to investigators will not contain personal identifiers.\n\nWhen specific uses of the WBC samples are approved, the SWOG-9217 protocol will be amended.\n\nParticipation in this research is not required for continued participation in the PCPT.\n\n#### 3.0 Methods\n\n3.1 Because the original model consent form did not specifically address genetic studies, participants will be asked to sign an additional consent form to document their consent to the collection and submission of additional blood samples for storage and future testing (including genetic analysis).\n\n3.2 Institutions will be asked to submit additional materials from participants who consent to the additional blood collection. The blood is to be collected, processed and shipped as described in the PCPT Study Manual.\n\n3.3 NCI-Frederick Cancer Research Development Center (FCRDC) in Frederick, Maryland will serve as the processing, aliquotting and storage facility.\n\n3.4 Upon arrival at FCRDC the blood will be pooled and centrifuged. Plasma will be separated into 5 x 1.8 ml aliquots and frozen . . .\n\n3.5 All samples will be logged in and aliquots will be bar coded with a unique storage ID. These data will be electronically transmitted to the Statistical Center for verification.\n\n3.6 The scientists who will carry out analyses on these materials will not have access to personal identifiers and will not be able to link the results of these tests to personal identifier information. No individual results will be presented in publications or other reports. . . .\n\n3.7 Participants will not be informed on an individual basis of any results from these studies . . .\n\n#### 4.0 Sample analysis\n\n4.1 Investigators planning to submit NIH \\[National Institutes of Health\\] grant applications must obtain approval for their study and specimen access from the PCPT Serum and Tissue Utilization Committee before submission of a grant proposal. Potential investigators will be required to submit a brief abstract and 1-4 page outline . . . This proposal will be circulated for review to members of the PCPT Serum and Tissue Utilization Committee and two ad hoc members having relevant expertise . . .\n\n4.2 It is anticipated that proposals will be reviewed once a year . . . Approval by this group as well as appropriate Institutional Review Board approval from the investigator's institution will be required before release of samples.\"416\n\n### Explanation\n\nBiological specimens (eg, biopsy tissue; blood for DNA extraction) obtained during the conduct of clinical trials can be stored in repositories\u2014often designated as biobanks\u2014for the current trial and future research. This process is usually governed by local regulation and has particular ethical considerations (Item 26b).\n\nIf the trial involves genetic or molecular analysis of biological specimens derived from humans, or if any specimens will be stored for future use (specified or unspecified), the protocol should describe details about specimen collection, storage, and evaluation, including the location of repositories. In addition, the protocol should state whether collected samples and associated participant related data will be de-identified or coded to protect participant confidentiality. If a repository is overseen by a named research ethics committee\/institutional review board, then this information should also be provided.\n\n# Discussion\n\nIt is critical that every clinical trial has a complete and transparent protocol, which can then facilitate trial conduct and appraisal by communicating relevant information to key stakeholders. In response to observed deficiencies in protocol content, the SPIRIT Initiative has produced recommendations for minimum relevant protocol items to include in a protocol, published in the form of the SPIRIT 2013 Statement and this Explanation and Elaboration (E&E) paper.14 The strengths that distinguish SPIRIT from other protocol guidance documents include its systematic and transparent development methods; participation of a wide range of key stakeholders; use of empirical evidence to support its recommendations; and availability of detailed guidance including model examples from protocols.\n\nThe overall aim of SPIRIT is to improve the completeness and transparency of trial protocols. The SPIRIT documents can serve as a practical resource for trial investigators and personnel to draft and understand the key elements of a protocol. In doing so, our vision is that the SPIRIT 2013 Statement and E&E paper will also facilitate and expedite the review of protocols by research ethics committees\/institutional review boards, scientific review groups, and funders\u2014for example, by reducing the number of avoidable queries to trial investigators regarding missing or unclear protocol information during the review process. Furthermore, improved protocol content would help facilitate the critical appraisal of final trial reports and results. Finally, several SPIRIT items correspond to items on the CONSORT 2010 checklist (Consolidated Standards of Reporting Trials),417 which should facilitate the transition from the protocol to the final study report.\n\nThe next steps for the SPIRIT Initiative include an implementation strategy to encourage uptake of the SPIRIT 2013 Statement. The SPIRIT website ([www.spirit-statement.org](http:\/\/www.spirit-statement.org\/)) will provide the latest resources and information on the initiative, including a list of supporters. We invite stakeholders to assist in the evaluation of the SPIRIT Statement and E&E paper by using the documents and providing feedback to inform future revisions. Through widespread uptake and support, the potential to improve the completeness and quality of trial protocols, as well as the efficiency of their review, can be fully realised.\n\nCite this as: *BMJ* 2013;346:e7586","meta":{"dup_signals":{"dup_doc_count":253,"dup_dump_count":75,"dup_details":{"curated_sources":2,"2023-50":4,"2023-40":4,"2023-23":3,"2023-14":6,"2023-06":9,"2022-49":5,"2022-40":5,"2022-33":8,"2022-27":4,"2022-21":8,"2022-05":3,"2021-49":5,"2021-43":7,"2021-39":4,"2021-31":4,"2021-25":7,"2021-21":4,"2021-17":7,"2021-10":5,"2021-04":1,"2020-50":5,"2020-45":4,"2020-40":1,"2020-34":2,"2020-29":1,"2020-24":6,"2020-16":4,"2020-10":4,"2020-05":3,"2019-51":4,"2019-47":3,"2019-39":4,"2019-35":2,"2019-30":2,"2019-26":1,"2019-22":5,"2019-18":2,"2019-13":1,"2019-04":1,"2018-51":2,"2018-47":1,"2018-43":1,"2018-39":1,"2018-26":3,"2018-13":3,"2018-05":1,"2017-51":2,"2017-47":2,"2017-43":4,"2017-39":1,"2017-34":2,"2017-30":1,"2017-26":4,"2016-30":1,"2016-18":2,"2016-07":3,"2015-48":2,"2015-40":2,"2015-35":3,"2015-32":4,"2015-27":3,"2015-22":4,"2015-14":3,"2014-52":3,"2014-49":4,"2024-30":4,"2024-26":2,"2024-22":5,"2024-18":8,"2024-10":1,"2015-18":3,"2015-11":2,"2015-06":2,"2013-48":3,"2013-20":1}},"file":"PMC3541470"},"subset":"pubmed_central"}
{"text":"abstract: Various entities have been designated keystone resources, but few tests have been attempted and we are unaware of any experimental manipulations of purported keystone resources. Mistletoes (Loranthaceae) provide structural and nutritional resources within canopies, and their pervasive influence on diversity led to their designation as keystone resources. We quantified the effect of mistletoe on diversity with a woodland-scale experiment, comparing bird diversities before and after all mistletoe plants were removed from 17 treatment sites, with those of 11 control sites and 12 sites in which mistletoe was naturally absent. Three years after mistletoe removal, treatment woodlands lost, on average, 20.9 per cent of their total species richness, 26.5 per cent of woodland-dependent bird species and 34.8 per cent of their woodland-dependent residents, compared with moderate increases in control sites and no significant changes in mistletoe-free sites. Treatment sites lost greater proportions of birds recorded nesting in mistletoe, but changes in species recorded feeding on mistletoe did not differ from control sites. Having confirmed the status of mistletoe as a keystone resource, we suggest that nutrient enrichment via litter-fall is the main mechanism promoting species richness, driving small-scale heterogeneity in productivity and food availability for woodland animals. This explanation applies to other parasitic plants with high turnover of enriched leaves, and the community-scale influence of these plants is most apparent in low productivity systems.\nauthor: David M. Watson; Matthew Herring\\*Author for correspondence (<firstname.lastname@example.com>).\ndate: 2012-09-22\ninstitute: Institute for Land, Water and Society, Charles Sturt University, PO Box 789, Albury, New South Wales 2640, Australia\nreferences:\ntitle: Mistletoe as a keystone resource: an experimental test\n\n# Introduction\n\nThe idea of an ecological keystone was first transferred from an individual species to a generalized resource derived from multiple species by Leighton & Leighton \\[1\\] (see also Terborgh \\[2\\]) in their work on palm seeds, figs and nectar as food for birds and mammals in a Bornean forest. Since then, lichens, saguaro cacti, mineral licks, water, honeydew, mistletoes, salmon, acorns and various fruiting trees *inter alia* have been proposed to represent keystone resources, either within a particular region or generally \\[3\u20135\\]. With one notable exception \\[6\\], there have been no manipulations to quantify the direct and indirect influence of purported keystone resources. With most resources, removal would be logistically difficult, if not impossible, and various procedural difficulties confound the design or interpretation of supplementation experiments. Although our understanding of keystone species has been informed by numerous experiments (natural and controlled; removal and addition), the lack of manipulative tests represents a major impediment to advancing our understanding of how purported keystone resources influence diversity.\n\nEngaged in a network of interdependencies with host plants, seed dispersers, pollinators and natural enemies, mistletoes are a group of highly interactive plants that have been proposed to represent keystone resources in forested ecosystems worldwide \\[7\\]. In addition to many regular and occasional consumers of their enriched foliage, abundant fruit and nectar resources offered when little else is available \\[8\u201310\\], mistletoe clumps are popular nest and roost sites \\[11\u201313\\] and numerous studies have reported positive relationships between mistletoe occurrence and species richness \\[10,14\\]. Mistletoe occurrence and density also affect several ecosystem processes: their abundant enriched litter has pronounced positive effects on nutrient dynamics and understorey composition \\[15,16\\] and changes to infected hosts increase colonization and functional diversity of mycorrhizal communities \\[17,18\\], increasing canopy complexity and changing fire behaviour and severity \\[19\\], thereby altering successional dynamics at the stand-scale \\[20\\].\n\nHaving called for removal experiments to test the keystone status of mistletoes, a series of predictions were outlined \\[7\\], detailing the short- and medium-term effects of mistletoe removal on community composition and structure. If mistletoe represents a keystone resource, areas where mistletoe has been removed would be expected to have:\n\n1.  lower abundances of mistletoe-obligate frugivores and folivores, with local populations declining towards local extinction;\n\n2.  lower abundances of regular mistletoe foragers (folivores, frugivores and nectarivores);\n\n3.  lower abundances of species that nest in mistletoe clumps and hollows;\n\n4.  lower richness of vertebrates generally; and\n\n5.  increased sensitivity to drought and other extreme events, supporting fewer residents and displaying increased seasonal and inter-annual variability in species richness, compared with control plots with typical numbers of mistletoe plants (after Watson \\[7\\]).\n\nThese predictions were provisionally tested by comparing two adjacent eucalypt woodlands, from one of which all mistletoe plants had been removed from one woodland five years prior to study \\[6\\]. Marked differences in avian species richness and incidence were detected, consistent with the predicted effects of mistletoe on resource availability, offering preliminary support for the keystone resource hypothesis. This study had an effective sample size of one and no pre-treatment data were collected, so the recorded effects may include pre-existing habitat differences and may not be representative of the influence of mistletoe more generally.\n\nHere, we report on a large-scale experiment evaluating the influence of mistletoe on community-level diversity and designed to test these five predictions. Pre-treatment bird diversities (estimated at the woodland scale) were compared explicitly with diversities three years post-treatment, contrasting changes in woodlands where all mistletoes were removed with those in control woodlands where mistletoe numbers were unmodified. As the first experimental test of a purported keystone resource, to our knowledge this 6-year study yielded a refined understanding of the direct and indirect effects of mistletoe on diversity and community composition. More broadly, this study contributes to a growing understanding of the role of parasitic plants as facilitators, affecting occurrence patterns of other plants and dependent animals through altered nutrient dynamics.\n\n# Methods\n\n## Study area and site selection\n\nThis study was conducted in the upper Billabong Creek catchment in southeastern New South Wales, Australia \\[21\\] (for a map, see electronic supplementary material, figure S1), located at the transition between xeric plains to the west and mesic highlands to the east \\[22\\]. Elevation ranges between 220 to 889 m a.s.l., and average annual rainfall increases from approximately 550 mm in the west to 900 mm in the east of the catchment. The study period (2003\u20132008) coincided with a prolonged drought affecting southeastern Australia, with annual rainfall totals for the nearby Albury weather station being approximately half of the long-term annual average of 770 mm.\n\nRather than individual trees or stands, entire woodland remnants were used as study units, isolated 80\u2013100 years ago as surrounding habitats were cleared for agriculture. Sites qualified for selection if: canopy cover was more than 5 per cent; fragment area was 1.5\u201325 ha (larger woodlands were not considered because complete mistletoe removal was deemed prohibitively difficult); vegetation was classed as either Dry Foothill Forest or Grassy Box Woodland; the woodland had 'hard' edges *sensu* \\[23\\] and was at least 500 m from the nearest study site. The ground layer of all woodlands was dominated by grasses (both native perennials, and native and exotic annuals) with occasional shrubs (*Acacia* spp., *Exocarpus* spp.) in low-to-very-low densities. Mistletoe plants occurred at medium-to-low densities in these woodlands\u2014primarily *Amyema miquelii* but with occasional *Muellerina eucalyptoides*, *Am. pendula* and *Am. miraculosa* (Loranthaceae). Except for the latter species (principally epiparasitic on *Am. miquelii* in this part of its range), these species are primarily eucalypt parasites, the plants forming dense pendulous clumps at the edge of the canopy \\[24\\].\n\n## Bird surveys\n\nTo estimate bird species richness in the 40 woodlands, inventories were compiled from eight patch-scale surveys that were conducted each season in 2003\/2004 prior to treatment and again in 2007\/2008, three years after treatment. Surveys were conducted using the standardized search \\[25,26\\] with sample duration set at 20 min, using a quantitative, results-based stopping rule to determine the number of samples per survey. The stopping rule applied\u2014stop sampling once observed richness of woodland-dependent species exceeds 80 per cent of predicted richness using the Chao 2 estimator \\[27\\]\u2014yielded surveys of between three and six samples (i.e. 60\u2013120 min of continuous sampling), with 260 of the 320 surveys tripping the stopping rule after three samples. Sampling involved walking throughout the woodland remnant and identifying all birds seen or heard within the woodland, including species flying beneath the canopy. Sampling was conducted only during favourable weather conditions, avoiding periods of heavy rain, strong wind or intense heat.\n\nIn addition to yielding richness estimates of uniform completeness, this approach generated incidence measures for each species in each woodland remnant, expressed as the proportion of samples in which it was detected \\[25\\]. Two incidence totals were calculated for each site in both years: summed incidence of all species that regularly feed on mistletoe nectar and\/or fruit using the list compiled by Reid \\[28\\], and summed incidence of all species that nest in mistletoe clumps using \\[12\\]. In addition to total richness and richness of woodland-dependent species (after Watson \\[6\\] and references therein), a third richness measure was used for analysis\u2014resident richness: those woodland-dependent species recorded in at least two seasons for the given year (excluding transients that were detected only in a single season). Analysis was restricted to changes in richness and incidence\u2014interactions with patch and landscape-scale factors and comparisons of the individual responses of particular species and functional groups will be explored in subsequent contributions.\n\n## Experimental design\n\nThe woodland remnants selected for study varied considerably in management history, area and degree of isolation from other remnant vegetation, potentially confounding the effects of mistletoe on diversity patterns. To guard against this, we ensured treatment and control groups were comparable, assigned treatment (mistletoe removal) using a blind stratified random approach and compared the effects of treatment in terms of proportional rather than absolute change. To assign treatment, woodland area was plotted against percentage tree cover, yielding a scatter plot of 40 points distinguishing larger and more densely wooded remnants from smaller, more open woodlands. Points closest to one another in graphical space were paired and treatment assigned by coin toss, yielding two groups of 20 woodland fragments with similar ranges of tree density and patch area (electronic supplementary material, table S1). A series of tests confirmed that these two groups exhibited comparable mean values for woodland area, percentage of tree cover in the surrounding 1 km radius buffer (an index of habitat openness), area of tree cover in the surrounding 1 km radius buffer (a measure of land-use intensity), mistletoe abundance and species richness of woodland-dependent birds prior to treatment. Some woodlands did not contain mistletoe, so, rather than a simple binary comparison, there were three groups: control woodlands with mistletoe (*n* = 11), treatment patches with mistletoe plants removed (*n* = 17) and woodlands from which mistletoe was naturally absent (*n* = 12); hereafter, they are referred as 'control', 'treatment' and 'mistletoe-free' woodlands, respectively.\n\n## Mistletoe removal\n\nMistletoe plants growing within treatment woodlands were systematically removed over a five-month period (winter and spring of 2004) by teams of volunteers using pole-mounted loppers and pruning saws. Unlike some groups of mistletoe (e.g. *Arceuthobium* spp., Viscaceae) that cause systemic infections throughout the host, these loranthaceous mistletoes can be removed by cutting proximal to the haustorium, the lack of cortical strands in these species precluding re-sprouting \\[24\\]. Hydraulic boom-lifts towed by four-wheel drive vehicles were used to access plants up to 18 m above the ground, the telescoping boom arm allowing access to most parts of the eucalypt canopies (electronic supplementary material, figure S2). Mistletoe plants and associated sections of host branches were left where they fell: beneath their former hosts. Sham removals were conducted in the control sites, driving within the woodland and removing branches from infected and non-infected trees, but leaving all mistletoe plants intact. Tree and branch selection was haphazard, determined partly by ease of access and partly to avoid any mistletoe clumps within the canopy. Comprehensive surveys conducted six months and three years after initial treatment revealed that not all mistletoe plants were removed, but less than four per cent of the original number of mistletoe plants remained or recolonized, which were subsequently removed in follow-up treatment in the summer of 2006\/2007.\n\n## Data manipulation and analysis\n\nResponses to mistletoe removal were measured in terms of net change between pre- and post-treatment inventories. In some cases, this was simply the datum for the 2003\/2004 subtracted from the equivalent datum for 2007\/2008. To make these values more meaningful, many of these differences were expressed as proportions of the original (pre-treatment) value. The only mistletoe-obligate frugivore detected was the mistletoebird *Dicaeum hirundinaceum*, so prediction 1 was tested by comparing mistletoebird incidence before and after treatment. Predictions 2 and 3 were tested using summed incidence of mistletoe-feeding and mistletoe-nesting species (respectively), comparing control and treatment woodlands in terms of the difference in the net change of summed incidence. Tests were conducted on the actual values, while plots correspond to proportions of total incidence (pre-treatment) to place the differences in biological context. Prediction 4 was tested by comparing the change in species richness in control versus treatment woodlands, expressed as a proportion of the pre-treatment value (i.e. proportional change). For prediction 5, the study period coincided with a prolonged drought, with many species responding by leaving the study area completely or only occupying some habitats seasonally. The change in numbers of residents between the two years was expressed as a proportion of the initial value (i.e. the proportion decreasing as the number of transient species increased). Except for prediction 1, which entailed a paired *t-*test applied to the 17 treatment woodlands, all treatment effects were tested using Mann\u2013Whitney\u2013Wilcoxon tests (one-tailed) comparing mean values for the 17 treatment sites (with mistletoe plants removed) with the 11 control sites (with mistletoe left intact), with exact probabilities of less than 0.05 deemed significant (using <span class=\"smallcaps\">spss<\/span> v. 17.0). Tests were conducted using all species ('total species') and the subset of species considered to depend on woodland as their primary habitat ('woodland species'; excluding most raptors, aerial foragers, open country and exotic species, after Watson \\[6\\]). Non-parametric tests were used, as sample sizes were uneven and the data were generally heteroscedastic, and one-tailed tests were appropriate as all comparisons were testing explicitly directional predictions \\[7\\]. In addition to comparing treatment and control sites, values for mistletoe-free woodlands are presented to contextualize the changes associated with experimental treatment and represent background variation between sites and years not associated with experimental manipulation.\n\n# Results\n\nMistletoe exhibited a highly irregular distribution across the 40 sites, being entirely absent from 12 sites and occurring in remaining sites at densities ranging from less than one per hectare to almost 200 per hectare. A total of 5493 mistletoe plants were removed: the great majority (5169; 94%) during the initial removal phase in mid-2004 and an additional 324 plants in follow-up removals in summer 2006\/2007, most of which were immature plants.\n\nOf the 75 woodland-dependent bird species recorded in the two years combined, 12 species were recorded only during pre-treatment surveys and two species were recorded only during post-treatment surveys\u2014i.e. there was a net loss of 10 species, or 14 per cent of the pre-treatment diversity of woodland-dependent species across all 40 sites. Turnover in the other 41 species was more symmetrical, with seven species recorded only pre-treatment (2003\/2004) and eight species recorded only post-treatment (2007\/2008)\u2014i.e. a net gain of one species (or 3% of the pre-treatment total). Additional detail on bird richness and mistletoe occurrence is summarized in the electronic supplementary material.\n\n## Prediction 1: specialist frugivores\n\nA significant difference in mistletoebird *D. hirundinaceum* incidence was detected in the 17 treatment sites (*t* = 1.825, *p* = 0.043, one-tailed), reflecting declines in the treatment woodlands following mistletoe removal\u2014incidence decreased in two sites and the species became locally extinct in a further four. By contrast, mistletoebird incidence increased in five of the 11 control woodlands, decreased in three others and they were not recorded in the remaining three.\n\n## Prediction 2: mistletoe foragers\n\nNo significant differences were detected between treatment and control sites in terms of net change in summed incidence of all 31 mistletoe foragers (*p* = 0.132, one-tailed), or for the 24 woodland-dependent mistletoe foragers (*p* = 0.373, one-tailed) for ranked data (<a href=\"#RSPB20120856F1\" data-ref-type=\"fig\">figure 1<\/a>*a*) \\[28,12\\]. There were decreases across all sites, with treatment sites losing an average of 8.7 per cent of summed incidence for total species and 10.6 per cent of woodland-dependent species (<a href=\"#RSPB20120856F1\" data-ref-type=\"fig\">figure 1<\/a>*a*), and control sites losing 2.9 per cent and 8 per cent, respectively. Note that these recorded losses were less than changes in summed incidence across all species in the treatment woodlands between years (decreases of 15.4% and 24.7%, respectively).\n\n## Prediction 3: mistletoe nesters\n\nClear differences in summed incidence of the 67 mistletoe nesters were detected between treatment and control woodlands (*p* = 0.016, one-tailed), equating to a mean loss of approximately 9.4 per cent of total incidence (s.e. = 3.4) in treatment woodlands, becoming more marked if just the 48 woodland-dependent species were considered (*p* = 0.008, one-tailed; mean loss of 17.6% of total incidence, s.e. = 4.26). Although significantly greater than decreases in control woodlands (<a href=\"#RSPB20120856F1\" data-ref-type=\"fig\">figure 1<\/a>*b*), these changes were less than change in incidence across all species in the treatment sites (decreases of 15.4% for all species; 24.7% for woodland-dependents).\n\n## Prediction 4: species richness\n\nTotal species richness decreased markedly in the treatment woodlands after experimental removal of mistletoe\u2014a significant difference compared with control woodlands (*p* = 0.004), reflecting mean losses of 20.9 per cent of their original richness (s.e. = 4.04). This pattern was more marked for those species dependent on woodlands as their principal habitat (*p* = 0.002, one-tailed; compared with control sites), equating to mean losses of 26.5 per cent (s.e. = 4.58) of their original woodland-dependent species richness (<a href=\"#RSPB20120856F2\" data-ref-type=\"fig\">figure 2<\/a>*a*). This change compares with a mean decrease in total richness of 10.3% (s.e. = 6.04) in mistletoe-free woodlands and an increase of 4.7 per cent (s.e. = 8.38) in the control woodlands (for total species richness); with woodland-dependent species richness decreasing by an average of 6.5 per cent in mistletoe-free woodlands (s.e. = 9.63), while control woodlands gained an average of 10.2 per cent of their pre-treatment richness (s.e. = 12.68).\n\n## Prediction 5: sensitivity to drought\n\nFor total species, clear-cut differences between control and treatment were detected (*p* = 0.008): treatment sites lost a mean proportion of 29.5 per cent (s.e. = 9.25) of their initial residents, while control sites exhibited an increase in the proportion of residents of 1.73 per cent (s.e. = 7.73). Differences in woodland-dependent species were greater (*p* = 0.002); control woodlands increased their proportion of residents (14.5%; s.e. = 12.34) while the proportion of residents in treatment woodlands decreased by 34.8 per cent (s.e. = 10.3), transient species becoming more dominant (<a href=\"#RSPB20120856F2\" data-ref-type=\"fig\">figure 2<\/a>*b*).\n\n# Discussion\n\nThe influence of mistletoe on diversity was evaluated directly with a woodland-scale removal experiment, and the hypothesized status of these hemiparasites as keystone resources was strongly supported. Removing mistletoe plants from entire woodlands resulted in average losses of more than a quarter of the woodland-dependent bird species, with the number of resident species decreasing by more than a third. Over the same period, control woodlands with variable mistletoe densities exhibited moderate increases in woodland-dependent species richness, while no marked changes to richness or incidence were detected in naturally mistletoe-free sites. Rather than affecting just those species that feed on mistletoe or nest preferentially in mistletoe clumps, these changes were apparent across the community, being more pronounced for woodland-dependent species and residents. The magnitude of these changes within three years of mistletoe removal provides clear evidence that mistletoes directly modify resource availability in woodlands and forests, consistent with previous descriptive work relating mistletoe density to community-level diversity in a range of other ecosystems \\[10,14\\].\n\nTwo aspects of this study probably underestimate the measured influence of mistletoe, so reported effect sizes should be regarded as conservative. The study period coincided with the final years of a prolonged drought affecting southern Australia, so the pre-treatment inventories yielded lower species richness estimates than anticipated. Rather than supporting comparable bird diversities in the two years, the 11 control woodlands gained an average of 10.2 per cent woodland-dependent species because numbers of drought-sensitive species rebounded in 2007. So the mean-recorded loss of 26.5 per cent woodland-dependent species in the treatment remnants may actually reflect net losses closer to 36.7 per cent species richness once the confounding effects of regional climatic conditions are removed, comparable to the 34.8 per cent mean-recorded loss of woodland-dependent residents. Second, mistletoe densities within the study sites were low compared with other habitats in the region, the hemiparasites being completely absent from 12 of the 40 woodlands. The same mistletoe species are two to five times more abundant in adjacent riparian and floodplain woodlands and nearby box-ironbark forests, probably having more pronounced effects on diversity and community composition.\n\n## Mistletoe as a direct nutritional resource\n\nOne of the main elements of the evidence base assembled to justify designation of mistletoe as a keystone resource \\[7\\] was dietary information for terrestrial vertebrates, with the popularity of mistletoe fruit, nectar and foliage deemed instrumental in explaining observed relationships with species richness. Although mistletoe specialists declined following mistletoe removal (in support of prediction 1), regular mistletoe foragers did not show significant reductions in occurrence or abundance following mistletoe removal (<a href=\"#RSPB20120856F2\" data-ref-type=\"fig\">figure 2<\/a>*a*). While it is likely that more of the bird species recorded in this study occasionally consume mistletoe fruit or nectar, Reid's \\[28\\] list corresponds closely with our own observations of nectarivory and frugivory, both within the study region and across southern Australia, and this negative result is considered robust.\n\nWe suggest that regular consumers of mistletoe fruit and nectar represent only a small component of the suite of species influenced by mistletoe occurrence in this system, and that direct nutritional effects comprise a relatively minor component of the community-level influence of mistletoe in general. Eucalypt forests are remarkable for their lack of fleshy fruited plants, and frugivorous species are only a minor component of the associated avifauna. Thus, changes in mistletoe diversity and abundance probably have more pronounced effects on frugivore occurrence in other systems, explaining temporal and spatial variation in frugivores numbers \\[8,29\\]. In terms of nectar, although 13 of the 75 recorded woodland-dependent species feed on mistletoe flowers and an additional seven visit mistletoe flowers opportunistically \\[28\\], none depends on mistletoes as their primary food source. Rather, eucalypts represent the dominant nectar-bearing plant in this region\u2014the canopy-dominant sclerophyllous trees exhibiting mass flowering events that are highly variable through space and time, driving large-scale movements as nectarivores track peak flowering across large distances \\[30\\]. Hence, the direct influence of mistletoe fruit and nectar availability on community-scale diversity may not be as consistently great as predicted, and the influence of more abundant fruit and nectar-bearing plants in the canopy or understorey is probably far greater in most systems \\[31,32\\].\n\n## Structural attributes\n\nIn addition to nutritional resources, mistletoes provide dense evergreen structures within forest canopies that are widely used by birds and mammals for nesting and roosting, and these structural attributes formed the second main justification for designating mistletoes as keystone resources. Accordingly, those species that use mistletoe for nest sites were expected to decrease in abundance following mistletoe removal (prediction 3). Although treatment sites displayed significant reductions in these species in terms of summed incidence, recorded changes were less than those exhibited across all woodland-dependent species\u2014i.e. mistletoe-nesting species were no more likely to undergo declines following mistletoe removal than any other woodland species. This conclusion is supported by the relatively small number of nests found during mistletoe removal\u2014fewer than one per cent of the plants removed had nests associated with them. So, although widely used as nesting and roosting sites, and preferentially selected by some species, we found no evidence that mistletoes were limiting as a nesting substrate, nor that woodlands with mistletoe can support greater abundances of mistletoe-nesting species.\n\nPrevious research has evaluated mistletoe nesting in more depth, and suggested that microclimatic factors may be important in explaining the widespread practice. Compared with nests elsewhere in the canopy, nests within mistletoe foliage experienced reduced fluctuation in both temperature and humidity, with the ambient climate ameliorated by the semi-succulent foliage of the hemiparasite \\[13\\]. The lack of signal detected here may reflect the temperate climate of the study region, and mistletoe is likely to have more pronounced effects in areas with more extreme climates. Indeed, research on wildlife use of dwarf mistletoe brooms in Arizona found evidence that mistletoes may be a limiting resource \\[33\\]. The influence of mistletoe on canopy structure may be better considered in terms of reproductive success rather than in terms of species richness, increasing the seasonal extent and geographical breadth within which successful fledging and recruitment can be achieved.\n\n## Facilitation via litter-fall and nutrient concentration\n\nRather than direct nutritional supplementation or increased structural complexity in infected host canopies, we propose that the marked influence of mistletoe on diversity is mediated primarily via the abundant enriched litter shed by these hemiparasites, increasing productivity and promoting coexistence through bottom-up trophic dynamics of woodland and forest food webs. Previous work on the contribution of mistletoes to litter-fall and nutrient dynamics within the study area estimated that individual *Amyema miquelii* plants contributed an average of 544 g of leaf litter annually, with 0.81 \u00b1 0.08 g of mistletoe litter produced per gram of mistletoe leaf biomass in the canopy \\[15\\]. The variation in mistletoe occurrence noted at the patch scale was even more pronounced within infected patches: mistletoe plants aggregated in discrete 'infection centres' and concentrated around the perimeter of remnants. Hence, rather than incremental changes in litter-fall across woodlands, mistletoe occurrence increases heterogeneity in litter-fall, effectively doubling or tripling total litter-fall in heavily infected stands. The effects of these qualitative changes in litter-fall are magnified by the enriched status of mistletoe litter compared with host litter, leading to marked increases in nutrient inputs \\[16\\] and pronounced spatial heterogeneity in nutrient returns.\n\nIn addition to affecting overall productivity and composition of understorey plant communities \\[15\\], these changes to the litter layer directly affect the litter-dwelling arthropods that form the principal food source for many woodland-dependent fauna. Previous work on the habitat preferences, dietary composition and foraging ecology of birds in eucalypt woodlands has consistently identified litter depth as a critical factor \\[34 and references therein\\], with deeper and more extensive litter beds considered to contain higher abundances of preferred prey \\[35\\]. Thus, we suggest that quantitative changes to litter-fall associated with mistletoe occurrence, plus qualitative changes in the chemical composition and associated rate of decomposition of resultant litter, result in fundamental changes to litter-dwelling arthropod communities, increasing and prolonging availability of prey for insectivores.\n\nRather than supplanting direct nutritional and structural factors, the facilitation explanation incorporates these pathways, with nutrient contributions by foraging and nesting animals adding to the inputs provided directly by mistletoes. Unlike litter-fall that represents reallocation of nutrients from the host to the litter-layer beneath the host, these animal-derived nutrients are drawn from a much larger pool, extending beyond the infected host and stand \\[36\\]. Similar mechanisms have been postulated to explain high concentrations of animal-derived elements near hollow trees, rock outcrops and copses of trees surrounded by grassland, summarized by Watson \\[37\\]. Rather than simply increased productivity, we suggest it is the heterogeneity of nutrient availability driven by the highly aggregated distribution of mistletoes that underpins their pervasive effects on diversity, boosting species richness by promoting species coexistence.\n\n## Effects of parasitic plants on diversity\n\nRather than being unique to this system, nutrient enrichment is an attribute of parasitic plants generally, and numerous studies (of other mistletoes and various root parasites) have reported findings consistent with facilitation via enriched litter-fall. These findings involve a range of vegetation types, from alpine tundra \\[38\\] and arctic shrubland \\[39\\] to coniferous forests \\[40\\]. In addition to direct effects of hemiparasite litter on decomposition and nutrient availability, these studies demonstrate various indirect effects of parasitic plants, including the boosting of species richness by preferentially parasitizing competitively dominant species \\[41,42\\]. Parallel research on dwarf mistletoes (Arceuthobium: Viscaceae) has detected increased colonization of infected *Pinus edulis* by ectomycorrhizae \\[17\\] and increased functional diversity of fungal communities beneath infected *Pinus contorta* \\[18\\], suggesting that the fundamental shifts in soil microbial communities detected by Bardgett *et al*. \\[43\\] in grasslands experimentally infected with *Rhinanthus minor* may apply to parasitic plants more generally.\n\n## Acknowledgments\n\nAll sampling was carried out under Scientific Research Permit S10921 issued by the Department of Environment and Climate Change (Parks and Wildlife Division) and the authority of Charles Sturt University Animal Care and Ethics Committee approval 05\/054.\n\nThis research was supported by the Australian Research Council (DP034259), the Winnifred Violet Scott Trust and Charles Sturt University. We are indebted to the 18 landowners in the upper Billabong Creek catchment for granting access to their properties, supporting our research and sharing their insights about woodland dynamics. This work would not have been possible without our dedicated band of assistants: chief mistletoe removalist David Webb, Hugh McGregor, Scott Hartvigsen and teams from Australian Trust for Conservation Volunteers and Greencorps. We are grateful to two anonymous referees for their considered input and constructive comments.\n\n# References","meta":{"dup_signals":{"dup_doc_count":109,"dup_dump_count":43,"dup_details":{"curated_sources":2,"2018-47":2,"2018-43":2,"2018-39":3,"2018-34":1,"2018-30":2,"2018-26":2,"2018-22":1,"2018-17":2,"2018-09":1,"2018-05":4,"2017-51":1,"2017-47":1,"2017-43":2,"2017-39":2,"2017-34":1,"2017-30":3,"2017-26":2,"2017-22":1,"2017-17":5,"2017-09":4,"2017-04":3,"2016-50":2,"2016-44":7,"2016-40":4,"2016-36":4,"2016-30":3,"2016-26":3,"2016-22":3,"2016-18":3,"2016-07":3,"2015-48":3,"2015-40":2,"2015-35":2,"2015-32":2,"2015-27":2,"2015-22":2,"2015-14":2,"2014-52":2,"2014-49":3,"2017-13":3,"2015-18":3,"2015-11":2,"2015-06":2}},"file":"PMC3415901"},"subset":"pubmed_central"}
{"text":"abstract: Writing a research manuscript is an intimidating process for many novice writers in the sciences. One of the stumbling blocks is the beginning of the process and creating the first draft. This paper presents guidelines on how to initiate the writing process and draft each section of a research manuscript. The paper discusses seven rules that allow the writer to prepare a well-structured and comprehensive manuscript for a publication submission. In addition, the author lists different strategies for successful revision. Each of those strategies represents a step in the revision process and should help the writer improve the quality of the manuscript. The paper could be considered a brief manual for publication.\nauthor: Elena D. KallestinovaTo whom all correspondence should be addressed: Elena D. Kallestinova, Graduate Writing Center, Yale Graduate School of Arts and Sciences, Yale University, New Haven, CT; E-mail: <firstname.lastname@example.com>.\ndate: 2011-09\nreferences:\ntitle: How to Write Your First Research Paper\n\nIt is late at night. You have been struggling with your project for a year. You generated an enormous amount of interesting data. Your pipette feels like an extension of your hand, and running western blots has become part of your daily routine, similar to brushing your teeth. Your colleagues think you are ready to write a paper, and your lab mates tease you about your \"slow\" writing progress. Yet days pass, and you cannot force yourself to sit down to write. You have not written anything for a while (lab reports do not count), and you feel you have lost your stamina. How does the writing process work? How can you fit your writing into a daily schedule packed with experiments? What section should you start with? What distinguishes a good research paper from a bad one? How should you revise your paper? These and many other questions buzz in your head and keep you stressed. As a result, you procrastinate. In this paper, I will discuss the issues related to the writing process of a scientific paper. Specifically, I will focus on the best approaches to start a scientific paper, tips for writing each section, and the best revision strategies.\n\n# 1. Schedule your writing time in Outlook\n\nWhether you have written 100 papers or you are struggling with your first, starting the process is the most difficult part unless you have a rigid writing schedule. Writing is hard. It is a very difficult process of intense concentration and brain work. As stated in Hayes' framework for the study of writing: \"It is a generative activity requiring motivation, and it is an intellectual activity requiring cognitive processes and memory\" \\[1\\]. In his book *How to Write a Lot: A Practical Guide to Productive Academic Writing*, Paul Silvia says that for some, \"it's easier to embalm the dead than to write an article about it\" \\[2\\]. Just as with any type of hard work, you will not succeed unless you practice regularly. If you have not done physical exercises for a year, only regular workouts can get you into good shape again. The same kind of regular exercises, or I call them \"writing sessions,\" are required to be a productive author. Choose from 1- to 2-hour blocks in your daily work schedule and consider them as non-cancellable appointments. When figuring out which blocks of time will be set for writing, you should select the time that works best for this type of work. For many people, mornings are more productive. One Yale University graduate student spent a semester writing from 8 a.m. to 9 a.m. when her lab was empty. At the end of the semester, she was amazed at how much she accomplished without even interrupting her regular lab hours. In addition, doing the hardest task first thing in the morning contributes to the sense of accomplishment during the rest of the day. This positive feeling spills over into our work and life and has a very positive effect on our overall attitude.\n\n## Rule 1: Create regular time blocks for writing as appointments in your calendar and keep these appointments.\n\n# 2. Start with an outline\n\nNow that you have scheduled time, you need to decide how to start writing. The best strategy is to start with an outline. This will not be an outline that you are used to, with Roman numerals for each section and neat parallel listing of topic sentences and supporting points. This outline will be similar to a template for your paper. Initially, the outline will form a structure for your paper; it will help generate ideas and formulate hypotheses. Following the advice of George M. Whitesides, \". . . start with a blank piece of paper, and write down, in any order, all important ideas that occur to you concerning the paper\" \\[3\\]. Use <a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a> as a starting point for your outline. Include your visuals (figures, tables, formulas, equations, and algorithms), and list your findings. These will constitute the first level of your outline, which will eventually expand as you elaborate.\n\nThe next stage is to add context and structure. Here you will group all your ideas into sections: Introduction, Methods, Results, and Discussion\/Conclusion (<a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a>). This step will help add coherence to your work and sift your ideas.\n\nNow that you have expanded your outline, you are ready for the next step: discussing the ideas for your paper with your colleagues and mentor. Many universities have a writing center where graduate students can schedule individual consultations and receive assistance with their paper drafts. Getting feedback during early stages of your draft can save a lot of time. Talking through ideas allows people to conceptualize and organize thoughts to find their direction without wasting time on unnecessary writing. Outlining is the most effective way of communicating your ideas and exchanging thoughts. Moreover, it is also the best stage to decide to which publication you will submit the paper. Many people come up with three choices and discuss them with their mentors and colleagues. Having a list of journal priorities can help you quickly resubmit your paper if your paper is rejected.\n\n## Rule 2: Create a detailed outline and discuss it with your mentor and peers.\n\n# 3. Continue with drafts\n\nAfter you get enough feedback and decide on the journal you will submit to, the process of real writing begins. Copy your outline into a separate file and expand on each of the points, adding data and elaborating on the details. When you create the first draft, do not succumb to the temptation of editing. Do not slow down to choose a better word or better phrase; do not halt to improve your sentence structure. Pour your ideas into the paper and leave revision and editing for later. As Paul Silvia explains, \"Revising while you generate text is like drinking decaffeinated coffee in the early morning: noble idea, wrong time\" \\[2\\].\n\nMany students complain that they are not productive writers because they experience writer's block. Staring at an empty screen is frustrating, but your screen is not really empty: You have a template of your article, and all you need to do is fill in the blanks. Indeed, writer's block is a logical fallacy for a scientist \u2015 it is just an excuse to procrastinate. When scientists start writing a research paper, they already have their files with data, lab notes with materials and experimental designs, some visuals, and tables with results. All they need to do is scrutinize these pieces and put them together into a comprehensive paper.\n\n# 3.1. Starting with Materials and Methods\n\nIf you still struggle with starting a paper, then write the Materials and Methods section first. Since you have all your notes, it should not be problematic for you to describe the experimental design and procedures. Your most important goal in this section is to be as explicit as possible by providing enough detail and references. In the end, the purpose of this section is to allow other researchers to evaluate and repeat your work. So do not run into the same problems as the writers of the sentences in (1):\n\n> 1a. Bacteria were pelleted by centrifugation.\n>\n> 1b. To isolate T cells, lymph nodes were collected.\n\nAs you can see, crucial pieces of information are missing: the speed of centrifuging your bacteria, the time, and the temperature in (1a); the source of lymph nodes for collection in (b). The sentences can be improved when information is added, as in (2a) and (2b), respectfully:\n\n> 2a. Bacteria were pelleted by centrifugation at 3000g for 15 min at 25\u00b0C.\n>\n> 2b. To isolate T cells, mediastinal and mesenteric lymph nodes from Balb\/c mice were collected at day 7 after immunization with ovabumin.\n\nIf your method has previously been published and is well-known, then you should provide only the literature reference, as in (3a). If your method is unpublished, then you need to make sure you provide all essential details, as in (3b).\n\n> 3a. Stem cells were isolated, according to Johnson \\[23\\].\n>\n> 3b. Stem cells were isolated using biotinylated carbon nanotubes coated with anti-CD34 antibodies.\n\nFurthermore, cohesion and fluency are crucial in this section. One of the malpractices resulting in disrupted fluency is switching from passive voice to active and vice versa within the same paragraph, as shown in (4). This switching misleads and distracts the reader.\n\n> 4\\. Behavioral computer-based experiments of Study 1 were programmed by using E-Prime. We took ratings of enjoyment, mood, and arousal as the patients listened to preferred pleasant music and unpreferred music by using Visual Analogue Scales (SI Methods). The preferred and unpreferred status of the music was operationalized along a continuum of pleasantness \\[4\\].\n\nThe problem with (4) is that the reader has to switch from the point of view of the experiment (passive voice) to the point of view of the experimenter (active voice). This switch causes confusion about the performer of the actions in the first and the third sentences. To improve the coherence and fluency of the paragraph above, you should be consistent in choosing the point of view: first person \"we\" or passive voice \\[5\\]. Let's consider two revised examples in (5).\n\n> 5a. We programmed behavioral computer-based experiments of Study 1 by using E-Prime. We took ratings of enjoyment, mood, and arousal by using Visual Analogue Scales (SI Methods) as the patients listened to preferred pleasant music and unpreferred music. We operationalized the preferred and unpreferred status of the music along a continuum of pleasantness.\n>\n> 5b. Behavioral computer-based experiments of Study 1 were programmed by using E-Prime. Ratings of enjoyment, mood, and arousal were taken as the patients listened to preferred pleasant music and unpreferred music by using Visual Analogue Scales (SI Methods). The preferred and unpreferred status of the music was operationalized along a continuum of pleasantness.\n\nIf you choose the point of view of the experimenter, then you may end up with repetitive \"we did this\" sentences. For many readers, paragraphs with sentences all beginning with \"we\" may also sound disruptive. So if you choose active sentences, you need to keep the number of \"we\" subjects to a minimum and vary the beginnings of the sentences \\[6\\].\n\nInterestingly, recent studies have reported that the Materials and Methods section is the only section in research papers in which passive voice predominantly overrides the use of the active voice \\[5,7,8,9\\]. For example, Mart\u00ednez shows a significant drop in active voice use in the Methods sections based on the corpus of 1 million words of experimental full text research articles in the biological sciences \\[7\\]. According to the author, the active voice patterned with \"we\" is used only as a tool to reveal personal responsibility for the procedural decisions in designing and performing experimental work. This means that while all other sections of the research paper use active voice, passive voice is still the most predominant in Materials and Methods sections.\n\nWriting Materials and Methods sections is a meticulous and time consuming task requiring extreme accuracy and clarity. This is why when you complete your draft, you should ask for as much feedback from your colleagues as possible. Numerous readers of this section will help you identify the missing links and improve the technical style of this section.\n\n## Rule 3: Be meticulous and accurate in describing the Materials and Methods. Do not change the point of view within one paragraph.\n\n# 3.2. Writing Results Section\n\nFor many authors, writing the Results section is more intimidating than writing the Materials and Methods section . If people are interested in your paper, they are interested in your results. That is why it is vital to use all your writing skills to objectively present your key findings in an orderly and logical sequence using illustrative materials and text.\n\nYour Results should be organized into different segments or subsections where each one presents the purpose of the experiment, your experimental approach, data including text and visuals (tables, figures, schematics, algorithms, and formulas), and data commentary. For most journals, your data commentary will include a meaningful summary of the data presented in the visuals and an explanation of the most significant findings. This data presentation should not repeat the data in the visuals, but rather highlight the most important points. In the \"standard\" research paper approach, your Results section should exclude data interpretation, leaving it for the Discussion section. However, interpretations gradually and secretly creep into research papers: \"Reducing the data, generalizing from the data, and highlighting scientific cases are all highly interpretive processes. It should be clear by now that we do not let the data speak for themselves in research reports; in summarizing our results, we interpret them for the reader\" \\[10\\]. As a result, many journals including the *Journal of Experimental Medicine* and the *Journal of Clinical Investigation* use joint Results\/Discussion sections, where results are immediately followed by interpretations.\n\nAnother important aspect of this section is to create a comprehensive and supported argument or a well-researched case. This means that you should be selective in presenting data and choose only those experimental details that are essential for your reader to understand your findings. You might have conducted an experiment 20 times and collected numerous records, but this does not mean that you should present all those records in your paper. You need to distinguish your results from your data and be able to discard excessive experimental details that could distract and confuse the reader. However, creating a picture or an argument should not be confused with data manipulation or falsification, which is a willful distortion of data and results. If some of your findings contradict your ideas, you have to mention this and find a plausible explanation for the contradiction.\n\nIn addition, your text should not include irrelevant and peripheral information, including overview sentences, as in (6).\n\n> 6\\. To show our results, we first introduce all components of experimental system and then describe the outcome of infections.\n\nIndeed, wordiness convolutes your sentences and conceals your ideas from readers. One common source of wordiness is unnecessary intensifiers. Adverbial intensifiers such as \"clearly,\" \"essential,\" \"quite,\" \"basically,\" \"rather,\" \"fairly,\" \"really,\" and \"virtually\" not only add verbosity to your sentences, but also lower your results' credibility. They appeal to the reader's emotions but lower objectivity, as in the common examples in (7):\n\n> 7a. Table 3 clearly shows that \u2026\n>\n> 7b. It is obvious from figure 4 that \u2026\n\nAnother source of wordiness is nominalizations, i.e., nouns derived from verbs and adjectives paired with weak verbs including \"be,\" \"have,\" \"do,\" \"make,\" \"cause,\" \"provide,\" and \"get\" and constructions such as \"there is\/are.\"\n\n> 8a. We tested the hypothesis that there is a disruption of membrane asymmetry.\n>\n> 8b. In this paper we provide an argument that stem cells repopulate injured organs.\n\nIn the sentences above, the abstract nominalizations \"disruption\" and \"argument\" do not contribute to the clarity of the sentences, but rather clutter them with useless vocabulary that distracts from the meaning. To improve your sentences, avoid unnecessary nominalizations and change passive verbs and constructions into active and direct sentences.\n\n> 9a. We tested the hypothesis that the membrane asymmetry is disrupted.\n>\n> 9b. In this paper we argue that stem cells repopulate injured organs.\n\nYour Results section is the heart of your paper, representing a year or more of your daily research. So lead your reader through your story by writing direct, concise, and clear sentences.\n\n## Rule 4: Be clear, concise, and objective in describing your Results.\n\n# 3.3. now it is time for your Introduction\n\nNow that you are almost half through drafting your research paper, it is time to update your outline. While describing your Methods and Results, many of you diverged from the original outline and re-focused your ideas. So before you move on to create your Introduction, re-read your Methods and Results sections and change your outline to match your research focus. The updated outline will help you review the general picture of your paper, the topic, the main idea, and the purpose, which are all important for writing your introduction.\n\nThe best way to structure your introduction is to follow the three-move approach shown in <a href=\"#T3\" data-ref-type=\"table\">Table 3<\/a>.\n\nThe moves and information from your outline can help to create your Introduction efficiently and without missing steps. These moves are traffic signs that lead the reader through the road of your ideas. Each move plays an important role in your paper and should be presented with deep thought and care. When you establish the territory, you place your research in context and highlight the importance of your research topic. By finding the niche, you outline the scope of your research problem and enter the scientific dialogue. The final move, \"occupying the niche,\" is where you explain your research in a nutshell and highlight your paper's significance. The three moves allow your readers to evaluate their interest in your paper and play a significant role in the paper review process, determining your paper reviewers.\n\nSome academic writers assume that the reader \"should follow the paper\" to find the answers about your methodology and your findings. As a result, many novice writers do not present their experimental approach and the major findings, wrongly believing that the reader will locate the necessary information later while reading the subsequent sections \\[5\\]. However, this \"suspense\" approach is not appropriate for scientific writing. To interest the reader, scientific authors should be direct and straightforward and present informative one-sentence summaries of the results and the approach.\n\nAnother problem is that writers understate the significance of the Introduction. Many new researchers mistakenly think that all their readers understand the importance of the research question and omit this part. However, this assumption is faulty because the purpose of the section is not to evaluate the importance of the research question in general. The goal is to present the importance of your research contribution and your findings. Therefore, you should be explicit and clear in describing the benefit of the paper.\n\nThe Introduction should not be long. Indeed, for most journals, this is a very brief section of about 250 to 600 words, but it might be the most difficult section due to its importance.\n\n## Rule 5: Interest your reader in the Introduction section by signalling all its elements and stating the novelty of the work.\n\n# 3.4. Discussion of the results\n\nFor many scientists, writing a Discussion section is as scary as starting a paper. Most of the fear comes from the variation in the section. Since every paper has its unique results and findings, the Discussion section differs in its length, shape, and structure. However, some general principles of writing this section still exist. Knowing these rules, or \"moves,\" can change your attitude about this section and help you create a comprehensive interpretation of your results.\n\nThe purpose of the Discussion section is to place your findings in the research context and \"to explain the meaning of the findings and why they are important, without appearing arrogant, condescending, or patronizing\" \\[11\\]. The structure of the first two moves is almost a mirror reflection of the one in the Introduction. In the Introduction, you zoom in from general to specific and from the background to your research question; in the Discussion section, you zoom out from the summary of your findings to the research context, as shown in <a href=\"#T4\" data-ref-type=\"table\">Table 4<\/a>.\n\nThe biggest challenge for many writers is the opening paragraph of the Discussion section. Following the moves in <a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>, the best choice is to start with the study's major findings that provide the answer to the research question in your Introduction. The most common starting phrases are \"Our findings demonstrate . . .,\" or \"In this study, we have shown that . . .,\" or \"Our results suggest . . .\" In some cases, however, reminding the reader about the research question or even providing a brief context and then stating the answer would make more sense. This is important in those cases where the researcher presents a number of findings or where more than one research question was presented. Your summary of the study's major findings should be followed by your presentation of the importance of these findings. One of the most frequent mistakes of the novice writer is to assume the importance of his findings. Even if the importance is clear to you, it may not be obvious to your reader. Digesting the findings and their importance to your reader is as crucial as stating your research question.\n\nAnother useful strategy is to be proactive in the first move by predicting and commenting on the alternative explanations of the results. Addressing potential doubts will save you from painful comments about the wrong interpretation of your results and will present you as a thoughtful and considerate researcher. Moreover, the evaluation of the alternative explanations might help you create a logical step to the next move of the discussion section: the research context.\n\nThe goal of the research context move is to show how your findings fit into the general picture of the current research and how you contribute to the existing knowledge on the topic. This is also the place to discuss any discrepancies and unexpected findings that may otherwise distort the general picture of your paper. Moreover, outlining the scope of your research by showing the limitations, weaknesses, and assumptions is essential and adds modesty to your image as a scientist. However, make sure that you do not end your paper with the problems that override your findings. Try to suggest feasible explanations and solutions.\n\nIf your submission does not require a separate Conclusion section, then adding another paragraph about the \"take-home message\" is a must. This should be a general statement reiterating your answer to the research question and adding its scientific implications, practical application, or advice.\n\nJust as in all other sections of your paper, the clear and precise language and concise comprehensive sentences are vital. However, in addition to that, your writing should convey confidence and authority. The easiest way to illustrate your tone is to use the active voice and the first person pronouns. Accompanied by clarity and succinctness, these tools are the best to convince your readers of your point and your ideas.\n\n## Rule 6: Present the principles, relationships, and generalizations in a concise and convincing tone.\n\n# 4. Choosing the best working revision strategies\n\nNow that you have created the first draft, your attitude toward your writing should have improved. Moreover, you should feel more confident that you are able to accomplish your project and submit your paper within a reasonable timeframe. You also have worked out your writing schedule and followed it precisely. Do not stop \u2015 you are only at the midpoint from your destination. Just as the best and most precious diamond is no more than an unattractive stone recognized only by trained professionals, your ideas and your results may go unnoticed if they are not polished and brushed. Despite your attempts to present your ideas in a logical and comprehensive way, first drafts are frequently a mess. Use the advice of Paul Silvia: \"Your first drafts should sound like they were hastily translated from Icelandic by a non-native speaker\" \\[2\\]. The degree of your success will depend on how you are able to revise and edit your paper.\n\nThe revision can be done at the macrostructure and the microstructure levels \\[13\\]. The macrostructure revision includes the revision of the organization, content, and flow. The microstructure level includes individual words, sentence structure, grammar, punctuation, and spelling.\n\nThe best way to approach the macrostructure revision is through the outline of the ideas in your paper. The last time you updated your outline was before writing the Introduction and the Discussion. Now that you have the beginning and the conclusion, you can take a bird's-eye view of the whole paper. The outline will allow you to see if the ideas of your paper are coherently structured, if your results are logically built, and if the discussion is linked to the research question in the Introduction. You will be able to see if something is missing in any of the sections or if you need to rearrange your information to make your point.\n\nThe next step is to revise each of the sections starting from the beginning. Ideally, you should limit yourself to working on small sections of about five pages at a time \\[14\\]. After these short sections, your eyes get used to your writing and your efficiency in spotting problems decreases. When reading for content and organization, you should control your urge to edit your paper for sentence structure and grammar and focus only on the flow of your ideas and logic of your presentation. Experienced researchers tend to make almost three times the number of changes to meaning than novice writers \\[15,16\\]. Revising is a difficult but useful skill, which academic writers obtain with years of practice.\n\nIn contrast to the macrostructure revision, which is a linear process and is done usually through a detailed outline and by sections, microstructure revision is a non-linear process. While the goal of the macrostructure revision is to analyze your ideas and their logic, the goal of the microstructure editing is to scrutinize the form of your ideas: your paragraphs, sentences, and words. You do not need and are not recommended to follow the order of the paper to perform this type of revision. You can start from the end or from different sections. You can even revise by reading sentences backward, sentence by sentence and word by word.\n\nOne of the microstructure revision strategies frequently used during writing center consultations is to read the paper aloud \\[17\\]. You may read aloud to yourself, to a tape recorder, or to a colleague or friend. When reading and listening to your paper, you are more likely to notice the places where the fluency is disrupted and where you stumble because of a very long and unclear sentence or a wrong connector.\n\nAnother revision strategy is to learn your common errors and to do a targeted search for them \\[13\\]. All writers have a set of problems that are specific to them, i.e., their writing idiosyncrasies. Remembering these problems is as important for an academic writer as remembering your friends' birthdays. Create a list of these idiosyncrasies and run a search for these problems using your word processor. If your problem is demonstrative pronouns without summary words, then search for \"this\/these\/those\" in your text and check if you used the word appropriately. If you have a problem with intensifiers, then search for \"really\" or \"very\" and delete them from the text. The same targeted search can be done to eliminate wordiness. Searching for \"there is\/are\" or \"and\" can help you avoid the bulky sentences.\n\nThe final strategy is working with a hard copy and a pencil. Print a double space copy with font size 14 and re-read your paper in several steps. Try reading your paper line by line with the rest of the text covered with a piece of paper. When you are forced to see only a small portion of your writing, you are less likely to get distracted and are more likely to notice problems. You will end up spotting more unnecessary words, wrongly worded phrases, or unparallel constructions.\n\nAfter you apply all these strategies, you are ready to share your writing with your friends, colleagues, and a writing advisor in the writing center. Get as much feedback as you can, especially from non-specialists in your field. Patiently listen to what others say to you \u2015 you are not expected to defend your writing or explain what you wanted to say. You may decide what you want to change and how after you receive the feedback and sort it in your head. Even though some researchers make the revision an endless process and can hardly stop after a 14th draft; having from five to seven drafts of your paper is a norm in the sciences. If you can't stop revising, then set a deadline for yourself and stick to it. Deadlines always help.\n\n## Rule 7: Revise your paper at the macrostructure and the microstructure level using different strategies and techniques. Receive feedback and revise again.\n\n# 5. It is time to submit\n\nIt is late at night again. You are still in your lab finishing revisions and getting ready to submit your paper. You feel happy \u2015 you have finally finished a year's worth of work. You will submit your paper tomorrow, and regardless of the outcome, you know that you can do it. If one journal does not take your paper, you will take advantage of the feedback and resubmit again. You will have a publication, and this is the most important achievement.\n\nWhat is even more important is that you have your scheduled writing time that you are going to keep for your future publications, for reading and taking notes, for writing grants, and for reviewing papers. You are not going to lose stamina this time, and you will become a productive scientist. But for now, let's celebrate the end of the paper.\n\n###### Outline \u2014 Level 1\n\n|                                            |\n|--------------------------------------------|\n| 1\\. What is the topic of my paper?         |\n|                                            |\n| 2\\. Why is this topic important?           |\n|                                            |\n| 3\\. How could I formulate my hypothesis?   |\n|                                            |\n| 4\\. What are my results (include visuals)? |\n|                                            |\n| 5\\. What is my major finding?              |\n\n###### Outline \u2014 Level 2\n\n|                                                          |\n|----------------------------------------------------------|\n| **Introduction**                                         |\n|                                                          |\n| 1\\. Why is your research important?                      |\n|                                                          |\n| 2\\. What is known about the topic?                       |\n|                                                          |\n| 3\\. What are your hypotheses?                            |\n|                                                          |\n| 4\\. What are your objectives?                            |\n|                                                          |\n| **Materials and Methods**                                |\n|                                                          |\n| 1\\. What materials did you use?                          |\n|                                                          |\n| 2\\. Who were the subjects of your study?                 |\n|                                                          |\n| 3\\. What was the design of your research?                |\n|                                                          |\n| 4\\. What procedure did you follow?                       |\n|                                                          |\n| **Results**                                              |\n|                                                          |\n| 1\\. What are your most significant results?              |\n|                                                          |\n| 2\\. What are your supporting results?                    |\n|                                                          |\n| **Discussion and Conclusions**                           |\n|                                                          |\n| 1\\. What are the studies major findings?                 |\n| 2\\. What is the significance\/implication of the results? |\n\n###### Moves in Research Paper Introductions\n\n|  |\n|----|\n| **Move 1. Establish a research territory** |\n|  |\n| a\\. Show that the general research area is important, central, interesting, and problematic in some way; |\n|  |\n| **Move 2. Find a niche** |\n|  |\n| a\\. Indicate a gap in the previous research, or extend previous knowledge in some way. |\n|  |\n| **Move 3. Occupy the niche** |\n|  |\n| a\\. Outline purposes or state the nature of the present research; |\n|  |\n| b\\. List research questions or hypotheses; |\n|  |\n| c\\. Announce principle findings; |\n|  |\n| d\\. State the value of the present research; |\n|  |\n| e\\. Indicate the structure of the research paper. |\n\nAdapted from Swales and Feak \\[11\\].\n\n###### Moves in Research Paper Discussions.\n\n|  |\n|----|\n| **Move 1. The study's major findings** |\n|  |\n| a\\. State the study's major findings. |\n|  |\n| b\\. Explain the meaning and importance of your finding. |\n|  |\n| c\\. Consider alternative explanations of the findings. |\n|  |\n| **Move 2. Research Context** |\n|  |\n| a\\. Compare and contrast your findings with those of other published results. |\n|  |\n| b\\. Explain any discrepancies and unexpected findings. |\n|  |\n| c\\. State the limitations, weaknesses, and assumptions of your study. |\n|  |\n| **Move 3. Closing the paper** |\n|  |\n| a\\. Summarize the answers to the research questions. |\n| b\\. Indicate the importance of the work by stating applications, recommendations, and implications. |\n\nAdapted from Swales and Feak and Hess \\[11,12\\].","meta":{"dup_signals":{"dup_doc_count":202,"dup_dump_count":30,"dup_details":{"curated_sources":2,"2023-40":9,"2021-39":1,"2021-25":1,"2021-04":1,"2020-45":2,"2020-29":1,"2020-24":14,"2020-16":14,"2020-10":4,"2020-05":5,"2019-51":1,"2019-47":4,"2019-43":13,"2019-39":2,"2019-35":12,"2019-30":19,"2019-26":20,"2019-22":22,"2019-18":13,"2019-13":1,"2019-09":1,"2018-51":9,"2018-47":6,"2018-43":5,"2018-39":3,"2018-34":5,"2018-30":3,"2018-26":5,"2018-17":3,"2018-05":1}},"file":"PMC3178846"},"subset":"pubmed_central"}
{"text":"abstract: A report on the Jackson Laboratory 'Advances in nanostructural genomics II' meeting, Bar Harbor, USA, 3-6 October 2002.\nauthor: Joan Politz; Roel van Driel; Markus Sauer; Ana Pombo\ndate: 2003\ninstitute: 1University of Massachusetts Medical School, Department of Biochemistry and Molecular Pharmacology, 377 Plantation Street, Worcester, MA 01605, USA; 2Swammerdam Institute for Life Sciences, University of Amsterdam, PO Box 94062,1090 GB Amsterdam, The Netherlands; 3Physikalisch-Chemisches Institut, Universit\u00e4t Heidelberg, Im Neuenheimer Feld 253, 69120 Heidelberg, Germany; 4MRC - Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital Campus, Du Cane Road, London W12 ONN, UK; Corrspondence: Ana Pombo. E-mail: firstname.lastname@example.com\ntitle: From linear genome sequence to three-dimensional organization of the cell nucleus\n\nIn the modern era of biochemical science, the isolation, identification and characterization of biologically relevant molecules have become routine. The challenge remains, however, to discover how these molecules are assembled within cells to form the dynamic network of molecular machines that supports living organisms. Because molecular machines are only several tens of nanometers in size (for example, a ribosome is about 30 nm wide), new optical methods with both increased sensitivity and higher resolution are required to define the spatial relationships and dynamic interplay between interacting biomolecules. Furthermore, techniques that provide dynamic, three-dimensional images of individual molecules *in vivo* will be necessary if we are to study the detailed organization of 'nanomachines' within the cell. The 'Advances in nanostructural genomics II' meeting at The Jackson Laboratory (Bar Harbor, ME, USA) brought together workers from the diverse areas of research that address these challenges. The topics covered ranged from genome sequence analysis and gene expression and organization of the cell nucleus to high-resolution imaging methods, nanolithography and mathematical modeling.\n\nOne of us (R.v.D.) opened the meeting by summarizing the current state of research on gene expression, chromatin structure and nuclear organization, and highlighting the areas that would benefit from a multidisciplinary approach. These include studies of chromatin structure *in vivo*, the function of subnuclear structures, the relationship between gene clustering and functional nuclear domains, and how the mobility of nuclear components relates to their activities. A newly assembled magneto-microscope was also described; it was developed by a consortium of groups (called 'Cellular Submarine') who came together after the 'Genomics meets nanoscience' meeting last year (Politz and Pombo, *Genome Biol* 2002,**3:**reports4007.1-4007.3). It can guide micro-magnetic beads that have factors or enzymes bound through the nucleus, like an 'intracellular submarine', to measure and\/or interfere with nuclear function at the nanoscale level.\n\n# Higher-order chromatin organization\n\nThe availability of the nearly complete human and mouse genome sequences has increased the need for robust tools for sequence annotation. Carol Bult (The Jackson Laboratory, Bar Harbor, USA) and Ross Hardison (Pennsylvania State University, University Park, USA) described the development of annotation methods, such as those available at the websites of the Penn State Bioinformatics Group <http:\/\/bio.cse.psu.edu\/> and of the UCSC Genome Bioinformatics Group <http:\/\/www.genome.ucsc.edu>; they pointed out that currently no single search program can consistently identify all coding sequences within a region, and that the detection of functional non-coding regions is even more difficult. As Tim O'Brien (also at The Jackson Laboratory) explained, more detailed information can currently be obtained from specific genomic regions when computational and genetic methods are combined. As a model for functional genomic analyses he used the complex, 40 Mb large piebald region encoding coat color on mouse chromosome 14. Five coding genes per megabase were found in this region, together with a large number of non-coding sequences, some of which are essential. Although further studies are needed to determine which of these are associated with biological function, initial studies have shown that experimentally defined auto-regulatory elements and putative transcription-factor-binding sites are contained within these conserved blocks of sequence. Giacomo Cavalli (National Centre for Scientific Research (CNRS), Institute of Human Genetics, Montpellier, France) and Doug Engel (University of Michigan, Ann Arbor, USA) showed that long-range interactions *in trans* between different sequences, such as locus control regions and Polycomb repressor elements, affected gene activity in *Drosophila* and mouse. Cavalli demonstrated that these sequences physically interact within the three-dimensional space of the *Drosophila* nucleus, and Engel echoed the idea that our definition of a gene must change, if we wish it to include all sequences that control a particular gene's transcription.\n\nLong-range interactions were further discussed by Job Dekker (Harvard University, Cambridge, USA) who presented a novel high-throughput method, chromosome conformation capture or '3Cs', used to study the conformations of chromosomal sequences *in vivo*. In brief, sequences that interact *in vivo* are fixed within whole nuclei, chromatin is digested, and crosslinked fragments are ligated at low concentration to promote intra-molecular ligation. Ligated DNA fragments were quantified by PCR using oligonucleotides spanning a genomic area of interest. Dekker used this method to determine the frequencies of physical interactions between pairs of genomic loci on chromosome III of *Saccharomyces cerevisiae* and found the chromosome to be quite flexible with a circular configuration of only 300 nm in diameter; the most active domains occurred in more extended conformations. Siegfried Janz (National Cancer Institute, National Institutes of Health (NIH), Bethesda, USA) and Michael Difilippantonio (Center for Cancer Research, NIH, Bethesda, USA) discussed interchromosomal interactions that result in oncogenic chromosome translocations and gene amplifications. They used the murine t(12;15) translocation as a model for human Burkitt's lymphoma, in which interchromosomal DNA rearrangements lead to aberrant expression of the oncogene c-*myc* and to increased propensity for chromosome segregation errors and tumorigenesis. Difilippantonio reported that the joining of non-homologous broken chromosome ends may trigger the aberrant DNA rearrangement, as non-homologous end joining is involved in repairing programmed double-strand breaks in DNA that occur naturally during lymphocyte development, for example, when V, D and J gene segments in the immunoglobulin loci are rearranged to create the variable antigen receptor region (VDJ recombination), or when the gene segments that define different antibody classes are recombined (isotype switching). Janz also studied the fine structure of the t(12;15) translocation but used mice that have intact non-homologous end-joining pathways by using c-*myc* knock-ins, which he argued can mimic translocations between the c-*myc* and the immunoglobulin H (IgH) gene more accurately than previous mouse models. He found that DNA rearrangements were most frequently caused by aberrant isotype switching, and rarely by VDJ recombination.\n\nUnderstanding the complexities of chromatin and its interactions with chromatin-modifying factors were highlighted in a thought-provoking talk by Chris Woodcock (University of Massachusetts, Amherst, USA). If we are to fully understand the nucleus we will need to describe its biochemical and biophysical parameters. We have begun to understand the substrates (chromatin being perhaps the main one), the catalysts (enzymes that act on the substrates), and the kinetics of interaction, but it remains a daunting challenge to understand the complexity of the kinetic parameters of a long polymer with local modifications that is acted upon only locally by specific catalysts. Woodcock highlighted that a wide range of techniques will be necessary (with examples from his own work on the yeast SWI\/SNF nucleosome remodeling complex) if we are to define chromatin structure at four levels - at the primary, nucleosome level; the secondary level of the 30 nm DNA fibre; the tertiary folding beyond the 30 nm fibre; and the quaternary structure of the whole chromosome within the nucleus. Astrid Visser (Scripps Research Institute, La Jolla, USA) discussed the need for combined efforts to sequence the histone modification 'code', which posits that different combinations of histone modifications signal different chromatin states, to give insights into higher-order chromatin structure. This could include capture of specific chromatin conformations, followed by linearization of the chromatin and reading of the histone code. Chromatin configurations could also be captured either after DNA stretching and visualization by light or electron microscopy, or possibly with technology that might allow sequencing the histone code in the future.\n\n# Improvements in nanotechnology\n\nAlthough research on nanostructures is one of the most rapidly growing fields in modern science, the lack of higher-resolution substrate patterning methods has prevented direct examination of these structures at the level of resolution needed to study macromolecular activity within the cell. Joachim Spatz (University of Heidelberg, Germany) demonstrated that non-conventional methods of patterning surfaces onto glass coverslips allow the preparation of 5 nm gold dots that are separated by distances of 30-100 nm - a scale at which molecular interactions within and between cells can be monitored.\n\nRapid advances in microfluidics and microinstrumentation are also taking place. Gabriel Lopez (University of New Mexico, Albuquerque, USA) described the development of high-throughput microfluidic devices that allow cells that are cultured in 96-well plates to be washed completely between medium changes, by using flow, rather than diffusion, as the mixing impetus. Scott Collins (University of Maine, Orono, USA) discussed refined instrumentation for single-cell experiments, such as microperfusion chambers that can be used to mimic arteries in arteriosclerosis studies and optical traps that can be used to manipulate individual cells. He also discussed a nano-sequencing technique in which DNA is electrophoresed through a nanopore and the sequence determined by measuring the current modulation caused by different bases as they move through the pore. Currently, however, only purines and pyrimidines can be differentiated. Watt Webb (Cornell University, Ithaca, USA) demonstrated that the activity of single polymerase molecules that are immobilized inside a zero-mode waveguide (subwavelength holes in a metal film) can be followed by timing the fluorescence bursts that are emitted from labeled nucleotide analogs that enter the active site of the enzyme and are incorporated into the growing DNA strand. This technique is also being developed into a new DNA sequencing method.\n\nMagnetic tweezers are a promising tool in cell biology; they allow objects inside cells to be moved while the forces exerted by biological systems in the pico- to nano-Newton range are measured. In contrast to optical tweezers, they do not use laser light that is potentially harmful, particularly when objects are below the micrometer size range. Gunther Reiss (University of Bielefeld, Germany) described a magnetic trap with which the two-dimensional and three-dimensional position of magnetic nanoparticles can be controlled with high sensitivity and precision.\n\nHorst Weller (University of Hamburg, Germany) and Avi Ulmann (Brooklyn Polytechnic Institute, New York, USA) discussed how nanoparticles can be used for biolabeling and new surface chemistry techniques, for example, biotin-conjugated particles can be used to visualize streptavidin that is used in immunolabeling procedures or that is attached to a glass coverslip. Advances in the chemical synthesis of highly luminescent semiconductor nanocrystals, such as gold nanocrystals coated with silica, often referred to as quantum dots, allow the development of alternative fluorescent or luminescent labels that can be used instead of standard organic dyes, such as fluorescein isothiocyanate (FITC).\n\n# Improvements in microscopy\n\nConventional fluorescence microscopy can be used to determine the positions of objects only when they are separated by distances greater than a few hundred nanometers - a restriction imposed by the diffraction limit of light. In contrast, fluorescence resonance energy transfer (FRET) techniques can be used to measure distances within one to ten nanometers. To visualize objects whose distance lies between these limits, alternative techniques are being developed. One of us (M.S.) discussed the use of dyes, such as Cy5 and JF9, that have different fluorescence life times and of time-resolved fluorescence spectroscopy to distinguish and measure distances between two dye molecules that are separated by less than 30 nm. With fluorescence correlation spectroscopy (FCS) several properties of fluorescent labels can be measured and the mobility of single labeled molecules in live cells can be monitored. Samuel T. Hess (National Institute of Child Health and Human Development, NIH, Bethesda, USA) and Thomas Weidemann (German Cancer Research Center, Heidelberg, Germany) used this technique to study the properties of proteins tagged with green fluorescence protein (GFP) and their intracellular mobility in living cells. Hess discussed different properties of fluorescent proteins and their application as biological markers. For example, enhanced GFP (eGFP) flickers between dark and bright states at low pH, such that it may be used as a pH sensor. Weidemann used FCS to determine the concentration of nucleosomes labeled with H2B-eYFP in the HeLa cell nucleus and in mitotic chromosomes.\n\nOther new microscopy techniques, such as laser holography and X-ray microscopy, are being used to study the three-dimensional organization of the nucleus. J\u00fcrgen Kreuzer (Dalhousie University, Halifax, Canada) showed that laser holography allows the acquisition of detailed images of living organisms without prior labeling. The image not only looks 'real' but appears to float in space and to move when the viewer moves, as with a real object. So far, Kreuzer has imaged heart cells and diatoms, and has tracked swimming algae using this technique. G\u00fcnther Schmahl (University of G\u00f6ttingen, Germany) and Gerd Schneider (Lawrence Berkeley National Laboratory, Berkeley, USA) discussed the application of X-ray microscopy to studying the nuclear distribution of the *Drosophila* dosage compensation protein MSL-1, which elevates the transcriptional activity of the X chromosome in males. Structural preservation is excellent under cryogenic conditions and the natural contrast between macromolecules and water in the cell allows the visualization of fine structural details. The distribution of immuno-gold stained MSL-1 protein was resolved to a level of about 60 nm; for example, the protein was observed to localize close to, but not within, the nucleolus. Michael Grunze (University of Heidelberg, Germany) discussed developments in the use of newly available synchrotron radiation sources of high brilliance to yield X rays and other types of radiation for electron- and soft X-ray-based microscopy of biological systems. With such a source of radiation, separate imaging of different chemical elements may become possible and we may see the development of novel types of microscopic imaging with spatial resolution at the atomic level.\n\n# Nuclear biology\n\nKevin Sullivan (Scripps Research Institute) summarized the current understanding of the DNA sequence and protein content of kinetochores, which form at the centromeres of mitotic chromosomes. Although centromeres are rich in satellite DNA, the sequence itself does not specify the function of centromeres, which is instead inherited epigenetically. CENP-A, a histone-like protein that binds alphoid satellite DNA, is made during G2 phase of the cell cycle and incorporated into the inner kinetochore plate until mitosis. Using stretched DNA fibers, it was found that centromeres contain a mixture of CENP-A and histone 4. The Aurora B kinase, which phosphorylates CENP-A and H3, assembles at pericentric heterochromatin in a CENP-A-dependent process; Aurora moves to other chromosomal regions only after phosphorylation is complete at this site. This mechanism ensures an epigenetic mark of centromeres. Jim McNally (National Cancer Institute) addressed the relationship between transcriptional activation and chromatin decondensation. Several genetically engineered systems that consist of clusters of tandemly repeated genes display such relationship, but this behavior could be specific to artificially amplified gene clusters. McNally therefore looked for naturally occurring clusters of genes that are transcriptionally coactivated, for example, clusters that are controlled by extracellular signals. Such a cluster was found on mouse chromosome 22 in a microarray gene-expression analysis, and McNally showed that this naturally occurring cluster shows a decondensation after transcriptional activation that is very similar to that of the artificial gene clusters.\n\nActive RNA polymerase I transcription sites are spatially organized into superstructures within the nucleolus. Making use of quantitative high-resolution light and electron microscopy, another of us (A.P.) reported that polymerases II and III also seem to form clusters of approximately eight enzymes in the nucleoplasm; such 'factories' are about 50 nm in diameter and each is surrounded by a cloud of nascent RNA. Antibodies against phosphorylated (active) polymerase II provided evidence that the factories contain different forms of polymerase.\n\nThe main cytological features of a eukaryotic nucleus are the nucleolus, chromosome territories, and the ill-defined interchromatin space. Thoru Pederson (University of Massachusetts Medical School, Worcester, USA) reviewed our current understanding of the interchromatin space, focusing mainly on the diffusion properties of RNA and other particles. He argued that interchromatin domains are active regions that are defined by and simultaneously interact with genes from different chromosomes. One of us (J.P.) showed that both the RNA and proteins that make up the signal recognition particle (SRP), which is required for targeting membrane and secretory proteins to membranes, are present within nucleoli, suggesting that the assembly of SRP might occur in this compartment. SRP RNA was found concentrated in subnucleolar domains that do not overlap with sites of ribosome assembly, suggesting that the nucleolus contains previously unrecognized domains devoted to novel functions.\n\nChristoph Cremer (University of Heidelberg) first described that small, gene-dense chromosomal territories are located near the center of the nucleus, whereas small, gene-poor chromosomal territories are located near the periphery. He also found that larger chromosomes tend to be located closer to the periphery of the nucleus. He explained that current modeling techniques cannot account for all of these observations. In an overview of new and forthcoming approaches in the nuclear structure field, he then discussed new microscopic technologies that are being developed in his group that will allow structural analysis to a precision level of 10 nm. Cremer stressed that beyond the development of new microscopy methods, quantitative modeling of biological structures and processes must be refined and developed, and better methods for labeling biomolecules must be discovered if we are to visualize specific proteins and RNA and DNA sequences *in vivo* with minimal disturbance of biological structures. So far, we have only had a glimpse at cellular structures. But with the rapid development of methods that allow us to study nanostructures, we may soon get a clearer picture.","meta":{"dup_signals":{"dup_doc_count":118,"dup_dump_count":42,"dup_details":{"curated_sources":4,"2022-05":1,"2021-39":1,"2020-40":1,"2020-29":1,"2019-22":1,"2018-51":1,"2018-30":2,"2018-13":1,"2018-09":1,"2017-47":1,"2017-34":1,"2017-26":1,"2017-17":1,"2017-09":8,"2016-44":1,"2016-40":1,"2016-36":10,"2016-30":10,"2016-22":1,"2016-18":1,"2016-07":1,"2015-48":3,"2015-40":1,"2015-35":2,"2015-32":3,"2015-27":3,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":6,"2014-42":8,"2014-41":3,"2014-35":4,"2014-23":4,"2014-15":4,"2022-33":1,"2015-18":3,"2015-11":3,"2015-06":3,"2014-10":3,"2013-48":3,"2013-20":1}},"file":"PMC153456"},"subset":"pubmed_central"}
{"text":"abstract: In their recent study, Sohn and colleagues identify multiple plasma proteins in the synovial fluid of patients with osteoarthritis (OA) and demonstrate the capacity of several of the proteins to activate macrophages via the innate immune receptor Toll-like receptor-4 (TLR-4). The authors speculate that the plasma proteins transit into the synovial compartment at sites of tissue damage where the endothelial barrier may be compromised, thus bringing them into contact with the articular surface and cells within the synovium. These results demonstrate a novel mechanism by which synovial inflammation can be initiated in patients with OA and how this process may contribute to the pathogenesis of OA joint pathology.\nauthor: Steven R Goldring; Carla R Scanzello\ndate: 2012\ninstitute: 1Hospital for Special Surgery, Weill Cornell Medical Center, New York, NY, USA; 2Section of Rheumatology, Departments of Immunology\/Microbiology and Biochemistry, Rush University Medical Center, Chicago, IL 60612, USA\nreferences:\ntitle: Plasma proteins take their toll on the joint in osteoarthritis\n\nSee related research by Sohn *et al.*, <http:\/\/arthritis-research.com\/content\/14\/1\/R7M>\n\nThe view of osteoarthritis (OA) as strictly a non-inflammatory joint disease has evolved as imaging and histopathological studies have demonstrated that synovial inflammation is present in many patients, and importantly, is associated with increased pain, poorer joint function, and accelerated deterioration of joint structures. In the current article by Sohn and colleagues \\[1\\], the authors advance the understanding of how inflammation may be initiated by demonstrating that certain plasma proteins present in OA synovial fluids (SFs) can activate macrophages via the innate immune receptor Toll-like receptor (TLR)-4.\n\nThe authors began with a proteomic analysis, utilizing two-dimensional PAGE followed by liquid chromatography-mass spectrometry, to survey proteins in SF from five OA patients. The analysis identified 108 proteins, many of which previously were shown to be increased in OA SF compared to normal, including proteins related to inflammation and immunity and products of extracellular matrix degradation and remodeling \\[2\\]. Of these, 64 proteins were novel and included histonerelated and macrophage-associated proteins, proinflammatory receptors and proteins related to the proinflammatory transcription factor nuclear factor kappa B. As the approach was not optimal for detection of small molecules such as cytokines, they went on to perform multiplex bead-based immunoassays to measure cytokines and chemokines involved in inflammatory processes and compared the levels in normal sera, OA and rheumatoid arthritis sera and OA and rheumatoid arthritis SF. In general, the levels in OA sera samples were only slightly higher than the normal levels and those in the rheumatoid arthritis sera were markedly higher. Some novel mediators were identified by this comparison, including T cell attracting chemokine (CTAK) and IL-9, which were significantly higher in the OA compared to the normal sera, supporting a role for T cells in OA inflammation. Among the OA samples, multiple proinflammatory cytokines and chemokines were much higher in the SF compared to the sera, consistent with overproduction of these cytokines in the joints.\n\nInterestingly, they found that over one- third of proteins identified in the proteomic screen of OA SF were plasma proteins. This is not unexpected, since SF is partially a plasma filtrate, but many of the plasma proteins were increased in OA compared to normal SF. Together with evidence that another plasma protein, fibrinogen, can incite inflammation by stimulating the innate receptor TLR-4, they tested whether other plasma proteins identified in the screen could do the same and demonstrated that three (\u03b11m, \u03b12m, and Gc-globulin) induced tumor necrosis factor-\u03b1, IL- 1\u03b2, IL-6, and vascular endothelial growth factor release from cultured macrophages. Very recently, other members of this group demonstrated that the complement membrane-attack complex plays a role in the development of inflammation and joint degeneration in an animal model of OA \\[3\\]. As plasma can be a source of many proteins, including complement components, the authors speculated that these proteins transit into the synovial compartment at sites of tissue damage where the endothelial barrier may be compromised, thus bringing them into contact with the articular surface and cells such as macrophages and synovial fibroblasts.\n\nThe authors demonstrated that the effect of these plasma proteins is mediated through TLR-4, as macrophages from mice lacking TLR-4 were unresponsive to the protein treatments. These results are significant for several reasons. First, the authors present data linking SF proteins present in OA joints, including plasma proteins, to activation of inflammatory processes. Second, TLR activation has previously been demonstrated to impact cartilage breakdown \\[4,5\\]. As macrophages are important mediators of synovial inflammation in OA, the present results extend the potential impact of this pathway to synovitis in OA. Finally, reports of TLR activation by non-microbial products often have been called into question, as low-level endotoxin contamination can often lead to false positive results. This issue was addressed using two different approaches. First, they demonstrated that treatment of the cultures with polymyxin B, which binds lipopolysaccharide by charge interactions and prevents it from associating with TLR-4, reverses the lipopolysaccharide response, but does not significantly affect the response to \u03b11m, \u03b12m, and Gc-globulin. Secondly, they demonstrated that proteinase K treatment fully or partially reversed the macrophage response to the three plasma proteins, while not affecting lipopolysaccharide.\n\nIn conclusion, the article by Sohn and colleagues contributes to our understanding of how TLRs may be activated locally in the setting of OA, by pointing to potential stimuli beyond matrix-derived and stress-related molecules derived from joint tissues. The observation that plasma proteins present in the OA joint can stimulate macrophages provides important rationale for further study of this pathway in OA models, but also may implicate this pathway in a variety of other local joint conditions that may produce alterations in synovial diffusion properties.\n\n# Abbreviations\n\nIL: interleukin; OA: osteoarthritis; SF: synovial fluid; TLR: Toll-like receptor.\n\n# Competing interests\n\nThe authors declare that they have no competing interests.\n\n## Acknowledgements\n\nCRS is supported by 1K08 AR057859, Mentored Clinician Scientist Career Development Award from the National Institute of Arthritis, Musculoskeletal and Skin Diseases.","meta":{"dup_signals":{"dup_doc_count":129,"dup_dump_count":32,"dup_details":{"curated_sources":1,"2021-43":1,"2021-31":1,"2021-17":1,"2021-04":1,"2020-45":1,"2020-34":1,"2020-05":1,"2019-51":1,"2019-39":1,"2017-09":11,"2016-44":1,"2016-40":1,"2016-36":8,"2016-30":10,"2016-22":1,"2016-18":1,"2016-07":10,"2015-48":4,"2015-40":4,"2015-35":4,"2015-32":4,"2015-27":4,"2015-22":4,"2015-14":4,"2014-52":4,"2014-49":9,"2014-42":11,"2014-41":7,"2014-35":5,"2014-23":7,"2014-15":4,"2023-40":1}},"file":"PMC3446406"},"subset":"pubmed_central"}
{"text":"author: Duncan T Odom\ndate: 2013\ninstitute: 1University of Cambridge, Cancer Research UK - Cambridge Institute, Cambridge, CB2 0RE, UK\ntitle: How to evaluate a graduate studentship, or choosing the right doctoral advisor\n\nA potential graduate student may spend more time in deciding what trendy digital device to buy than the environment and conditions of a studentship that guide their future career - I know this is true, because I got a really good Walkman, back in the day. This brief essay summarizes the core advice that I give folks who are about to take the first steps on the (potentially perilous) path towards an academic or industrial research career in biological sciences.\n\n# The golden rules\n\nThis article is written for someone who is passionate about science, and who knows that having a doctorate is the first magical step toward a principal investigator position in a research institute or a university. You have made all the right moves so far in your undergraduate studies; so you have (a) solid to excellent grades, and (b) done substantial amounts of research with a faculty member who can vouch for your competence (this means by the way, ideally more than a year). Now, famous people in storied places as diverse as MIT, Oxford and Singapore have invited you to interview for PhD studentships, which means you have been favorably evaluated by these programs. Congratulations!\n\nBut...how do *you* evaluate *them*: that is, the group leaders whose studentships are offered to you? In other words, how do you maximize the chances of surviving the next five (all too often brutal) years of your professional life?\n\nMy advice is based on my own experiences as a graduate student in chemistry (at Caltech, 5 years), a postdoctoral fellow in genomics (at the Whitehead and MIT, 6 years), and a faculty member in comparative and functional genomics (at University of Cambridge, 6 years). I currently run a medium-sized research group with a steady state of two to four postdocs, two to three graduate students, and about four staff scientists of various flavors. Here, I emphasize points relevant to biological sciences, but some of the ideas are applicable more widely.\n\nTo begin, let's ask a quick, basic, rarely-asked question. What do you need to accomplish as a graduate student in order to get a great postdoctoral position, from which you could launch a high-caliber faculty job? Although many accomplishments might be desirable, the most basic is this:\n\nYou must obtain your doctoral degree in a reasonable amount of time (think: ideally less than five years) with (1) an intact, enthusiastic spirit of curiosity, unbroken by terrible experiences, and (2) at least one solid, first-author publication that has been peer-reviewed.\n\nThis clear, unambiguous goal for survival should really help guide your choice of graduate advisor with open and critical eyes. Working in a Nobel-winning laboratory and producing publications in the highest impact journals might be ideal, but might not be a realistic (or even desirable) strategy for surviving your first years as a young scientist. Since the biggest variable you can control for is the scientific identity and character of your PhD advisor, you should evaluate this *before* you join a laboratory.\n\n# Established laboratories\n\nThe easiest situation to evaluate is the established laboratory, where you should start by asking hard-nosed questions about their prior record, both in general, and specifically with graduate students' training.\n\n## History with students\n\nHow many people have accepted PhD studentships in the past?\n\nDid they all publish first-author papers somewhere?\n\nIf not, who didn't publish, and what fraction of accepted students does that represent?\n\nHow many students have left the group, successfully and otherwise?\n\nWhere did they go?\n\nWhy?\n\nAll of these questions assume that your experience will mirror that of a typical person in that lab.\n\n## The lab's scientific leadership\n\nHow is the advisor as a leader and scientific role model?\n\nAn interview is your chance to ask pointed questions, probably in private, with as many folks as possible. Consider soliciting gossip, either from your social network or from other labs that neighbor your prospective host lab. This advice may be particularly uncomfortable to follow, since we all want to assume the best of everyone.\n\nBut think about it this way: if you were about to adventure-drive across the Sahara desert, would you just buy any random vehicle on the word of the owner, or would you have an independent car mechanic in to evaluate its mechanical condition, and thus to maximize your survival? The first four to five years as a scientist in large part shapes one's future identity irrevocably. Again, the single biggest influence will be your advisor, so choose wisely and be as informed as possible.\n\nTo state the obvious, certain character traits are undesirable in a mentor (well, at least when present in excess). These include, but are not limited to: conflict avoidance, being a bully, having an unstable temper, overt narcissism, passive-aggressive patterns, un-professionalism (such as outright intoxication at an institute beer hour or multiple paper retractions), crippling insecurity, excessive perfectionism (never submitting papers, for example), a damaging history of playing favorites.\n\nUse your common sense, and always remember that you are trying to avoid only serious, Titanic-sinking phenotypes. You should not be demanding perfection - as everyone is human and we all have limitations and make mistakes - just look for survivability.\n\nHow much does your candidate lab's group leader travel?\n\nAsk the laboratory if the group leader's travel to conferences and talks interferes with the lab's smooth function and publication rate. When my travel gets heavy, papers and grants turn around more slowly - and so my lab suffers. During trips, work time is more fragmented and less gets done, even when I set aside time. And, even in this wired world, I am simply much more effective in my own office, where my folks can just drop in.\n\n## Lab dynamics\n\nHow big is the prospective lab?\n\nHaving now run a group for a few years, I know from hard experience that I can only manage about five to six researchers, who are themselves actively running research projects. My realization of this limitation has meant that my lab naturally equilibrates at about ten people, counting the four staff scientists who do not normally run larger projects. If my lab got bigger than it currently is, then I would be forced to neglect people's hard-fought discoveries. My caveat here is that some faculty are much better at managing folks than I am, which leads to the next point.\n\nAre the lab's publications spread (somewhat) evenly across all the lab members, or do publications concentrate for excessively long stretches with one or two people as first authors?\n\nFor your own sake, you should be ruthless in your evaluations, but, to be fair, do consider lab size carefully. If someone has five graduate students, and they publish three solid papers a year with different lead authors, that sounds like a potentially promising environment (averaging one paper per person, every year and a half or so). Instead, though, what if there are a total of fifteen postdocs and grad students in a single lab, and two solid papers a year get published? That situation would mean one paper published per person, every seven and a half years or so. Not quite so good.\n\n# Fresh-faced group leaders\n\nWith a newly minted junior group leader, you probably have less actual history to go on. However, you can certainly ask them questions about what kind of lab they *intend* to build.\n\nHow many people do they want\/intend to have in the lab in five years? In what roles? Why?\n\nMost senior postdocs do not think strongly about these issues, as most of their efforts are spent in trying to get a faculty position. However, the starting group leaders who have thought these issues through would be expected to be more prepared to train promising graduate students like you. There are powerful advantages of starting work for a very junior group, not least that your interests and theirs are exactly aligned in ways that senior group leaders cannot be. In particular, untenured group leaders will be wanting your first stories published at least as much as you do, possibly more.\n\nAn aside: I believe that the first four or five people who join a nascent laboratory have the best chances of hitting a scientific gold mine. It does, however, require courage to take a chance on an unknown group leader.\n\n# Medics\n\nWith a medically trained group leader, you have a huge advantage in easier access to patient materials, which facilitates looking at questions directly relating to human disease biology. But there are certain drawbacks.\n\nWhat is the allocation of the clinician-scientist's time between clinic and lab?\n\nMany clinician scientists stay engaged with patient care, which is often an excellent strategy, but the cost of this dedication can at times be how much attention they can give their laboratory. Furthermore, many areas of translational medicine are scientifically over-subscribed and extraordinarily competitive. Remember that as long as you, as a grad student, get your solid publication or two, you are fine. However, if the clinician-scientist group leader is aiming for a big splash in a top-tier journal, then lower-impact stories that make up the bedrock of a successful graduate studentship might get neglected along the way.\n\n# And finally\n\nGood luck!","meta":{"dup_signals":{"dup_doc_count":106,"dup_dump_count":53,"dup_details":{"curated_sources":2,"2023-14":1,"2022-27":1,"2021-43":1,"2021-31":1,"2021-04":1,"2020-50":1,"2020-45":1,"2020-40":1,"2020-34":1,"2019-51":1,"2019-47":1,"2019-39":1,"2019-30":1,"2019-22":1,"2019-13":1,"2019-04":2,"2018-47":1,"2018-43":2,"2018-30":2,"2018-17":1,"2018-09":2,"2018-05":1,"2017-47":1,"2017-39":1,"2017-34":1,"2017-22":1,"2017-17":1,"2017-09":8,"2016-44":1,"2016-40":1,"2016-36":8,"2016-30":8,"2016-22":1,"2016-18":1,"2016-07":3,"2015-48":2,"2015-40":1,"2015-35":2,"2015-32":2,"2015-27":2,"2015-22":2,"2015-14":2,"2014-52":2,"2014-49":4,"2014-42":5,"2014-41":4,"2014-35":4,"2014-23":3,"2023-40":1,"2015-18":2,"2015-11":1,"2015-06":2,"2024-22":1}},"file":"PMC3663100"},"subset":"pubmed_central"}
{"text":"abstract: **Objectives** To investigate the association between opium use and subsequent risk of death.\n  .\n  **Design** Prospective cohort study.\n  .\n  **Setting** The Golestan Cohort Study in north-eastern Iran collected detailed validated data on opium use and other exposures at baseline. Participants were enrolled between January 2004 and June 2008 and were followed to May 2011, with a follow-up success rate of over 99%.\n  .\n  **Participants** 50\u2009045 participants aged 40-75 at baseline.\n  .\n  **Main outcomes** Mortality, all cause and major subcategories.\n  .\n  **Results** 17% (n=8487) of the participants reported opium use, with a mean duration of 12.7 years. During the follow-up period 2145 deaths were reported. The adjusted hazard ratio for all cause mortality associated with ever use of opium was 1.86 (95% confidence interval 1.68 to 2.06). Opium consumption was significantly associated with increased risks of deaths from several causes including circulatory diseases (hazard ratio 1.81) and cancer (1.61). The strongest associations were seen with deaths from asthma, tuberculosis, and chronic obstructive pulmonary disease (11.0, 6.22, and 5.44, respectively). After exclusion of people who self prescribed opium after the onset of major chronic illnesses, the associations remained strong with a dose-response relation.\n  .\n  **Conclusion** Opium users have an increased risk of death from multiple causes compared with non-users. Increased risks were also seen in people who used low amounts of opium for a long period and those who had no major illness before use.\nauthor: Hooman Khademi; Reza Malekzadeh; Akram Pourshams; Elham Jafari; Rasool Salahi; Shahryar Semnani; Behrooz Abaie; Farhad Islami; Siavosh Nasseri-Moghaddam; Arash Etemadi; Graham Byrnes; Christian C Abnet; Sanford M Dawsey; Nicholas E Day; Paul D Pharoah; Paolo Boffetta; Paul Brennan; Farin KamangarCorrespondence to: R Malekzadeh email@example.com, P Brennan <firstname.lastname@example.com>\ndate: 2012\nreferences:\ntitle: Opium use and mortality in Golestan Cohort Study: prospective cohort study of 50\u2009000 adults in Iran\n\n# Extra material supplied by the author\n\nAppendix: Supplementary tables A-E\n\nClick here for additional data file.\n\n# Introduction\n\nUse of opium (lachryma papaveris) for medicinal or recreational purposes has a long history in many parts of the world1 and continues to be common. In 2008 an estimated 13-22 million people worldwide used opium or its derivatives as illicit drugs: 6.5-12.5 million in Asia; 3.3-3.8 million in Europe; 2.3-2.4 million in the Americas; and the rest in Africa and Oceania.2\n\nA small number of retrospective case-control studies or case series have described the possible role of opium in oesophageal cancer,3 4 bladder cancer,5 6 7 coronary heart disease,8 9 10 11 and a few other select health conditions.12 13 14 There is little information from well designed prospective epidemiological studies on the effect of the use of opium on overall or cause specific mortality, partly because of relatively low opium use in most established cohort studies and the difficulty of obtaining validated data. There is also scant information on the effect of different types of opium with respect to health outcomes.\n\nThe Golestan Cohort Study (GCS), a prospective study established in north eastern Iran,15 where opium consumption is exceptionally common,16 has collected detailed validated data on opium use from over 50\u2009000 people.17 This study has collected data on typical amounts used, duration of use, and the four major types of opiates used locally\u2014that is, teriak, shireh, sukhteh, and heroin. The first two can be smoked or ingested. Sukhteh can only be ingested, and heroin can be injected, sniffed, or smoked.4 Using data on over 2000 deaths identified in the initial four years of follow-up, we report the effects of opium use, including by subtypes,4 18 on overall and cause specific mortality.\n\n# Methods\n\n## Study population\n\nThe Golestan Cohort Study was launched in January 2004 in north eastern Iran after completion of a pilot study in 2003.16 In brief, a total of 50\u2009045 people aged 40-75 were recruited from January 2004 to June 2008. The cohort study was designed to investigate the causes of oesophageal squamous cell carcinoma in Golestan province, where the risk of this cancer is extremely high. Study participants were enrolled from those who lived in Gonbad City and 326 villages in Golestan province, north eastern Iran. Further details have been discussed elsewhere.15\n\n## Assessment of exposure\n\nTrained interviewers used a general questionnaire and a food frequency questionnaire to collect data. The general questionnaire included detailed queries on opium use (see below), tobacco use, and alcohol drinking and questions on demographic characteristics, residential history, occupation, education, and medical history. Height, weight, and waist and hip circumferences were also measured. Blood pressure for each participant was measured twice from both arms, with 10 minute intervals, while the person was seated and relaxed. Nutritional data were collected with a food frequency questionnaire that was specifically designed and validated for this area.19 Biospecimens included 10 ml of blood, 4.5 ml of midstream urine, and hair and nail samples from each individual.\n\nDetailed data on opium use were collected as opium use is common in Golestan and could be a risk factor for oesophageal cancer. Data were obtained on age of start and end of opium use, typical amount of use in nokhod (the local unit for opium use, equivalent to about 0.2 g), frequency of use (how many days a week if weekly or more), and routes of administration for the different types of opiates commonly used\u2014namely, teriak, sukhteh, shireh, and heroin. If participants used multiple types of opium, or if they used opium intermittently, data were recorded separately for each type and period of use.\n\nTeriak, or raw opium, is directly obtained from the seed capsule of the opium poppy, *Papaver somniferum*.20 The ripening poppy capsules are incised to extract a milky juice, which turns into a dark sticky or crumbly mass, known locally as teriak. Teriak can be taken orally or smoked after direct heating with burning charcoal in specialised devices such as an opium pipe. Sukhteh is the opium dross remaining after the opium is smoked and is taken orally. This form of opium, which is generally cheaper than teriak, has been shown to have high mutagenic activity.18 Shireh is a refined product of opium that is mainly obtained from sukhteh, with or without adding teriak, boiled in water and filtered several times to remove the insoluble materials.18 Shireh can be ingested or smoked, by indirect heating, in a special type of opium pipe. As indirect heating is used, the vapour of opiate is inhaled, not its smoke. In this region users who smoke opium generally do not mix it with any other substances such as tobacco.\n\n## Ascertainment of cause of death\n\nAs nearly all study participants have telephones, they are actively followed by annual telephone calls. Detailed questions are asked regarding their health status and any admission to hospital or outpatient procedures. All data are recorded on hard copies as well as on a computerised database. If a study participant is inaccessible through telephone calls, after seven attempts during a two week period, researchers contacted friends or local health workers. Using this method, we successfully obtained follow-up information for over 99% of all participants.15 21 On learning of a reported death through the phone calls or through the local health workers or monthly provincial reports of death registration, a general practitioner who is a member of the follow-up team visits the home of the dead person and completes a validated verbal autopsy questionnaire21 by interviewing the closest relative of the dead person. At the same time other members of the team gather all available and relevant medical documents including medical charts, radiographs, pathology reports, hospital discharge documents, etc, from all hospitals or pathology centres, either within the province or neighbouring provinces.\n\nFor 65% of all deaths, in addition to the verbal autopsy, extensive medical documents\u2014including physician notes, test results, findings on electrocardiography and radiology, endoscopy results, and pathology reports\u2014were retrieved to determine cause of death.21 For the remaining deaths, cause was determined only by verbal autopsy. Our previous studies show high accuracy (all measures of accuracy above 81%) and reliability (\u03ba \\>0.75), particularly for major groups of cause of death, for this verbal autopsy.21 Two separate internists independently reviewed all collected documents, including the verbal autopsy information and medical charts, and the cause of death was documented with ICD-10 codes (international classification of diseases, 10th revision). If the results were concordant, a diagnosis was made. In case of discordance a third, more experienced internist reviewed all documents and the two initial diagnoses and made the final diagnosis. If a final diagnosis could not be made for any reason, the cause of death was coded as \"unknown.\"\n\nThough cause of death should ideally be assessed by someone blinded to the exposure data, the physician who completed the verbal autopsy by interviewing the next of kin of the dead person also collected some information on medical history and habits (such as smoking and alcohol and opium use). Nevertheless, total deaths, the main outcome of the study, could not be biased by knowledge of the exposure. Furthermore, as the internists who determined the cause of death had no prior belief regarding the association between opium use and any specific cause of death, except perhaps for oesophageal cancer, we believe such information should have minimal impact on classification of cause of death.\n\nThe major categories of death evaluated in this analysis were the most prevalent causes of death among the participants, including circulatory, cancer, respiratory, digestive, infectious, external (mainly from motor vehicle crashes or other unintentional injury and suicide), unknown, or other.\n\n## Statistical analysis\n\nAfter examining the proportional hazard assumption using Schoenfeld residuals, we fitted a Cox proportional hazards model, with age as the timescale, to estimate unadjusted and adjusted hazard ratios and 95% confidence intervals for total and cause specific mortality in relation to the exposures of interest, including any opium use, type of opium used, duration of use, cumulative use, and the method of opium consumption. Follow-up continued until loss to follow-up, death, or 20 May 2011, whichever came first. While opium use is a potentially important factor in all cause mortality, age is still the most important risk factor for death and is biologically more important than the time of recruitment into the cohort. Hence we chose age as the Cox regression time variable, implying that all risk comparisons are made between individuals of the same age. As deaths before recruitment were not observed, participants were left censored at the age of enrolment. We also used accumulated exposure at the time of enrolment as a fixed variable, rather than allowing it to increment with time in the study as might seem more natural. This was done because we had no data on continued exposure after enrolment; we do not know how the risk from accumulated exposure changes after cessation of consumption; and the time within the study is typically short compared with the accumulated years of exposure. We adjusted for potential confounders\u2014including sex, place of residence (urban or rural), marital status, highest educational level, ethnicity (Turkmen or others), and cigarette smoking\u2014by including indicator variables in the Cox regression models. Further inclusion of alcohol consumption, body mass index (BMI, either categorical or continuous), height (as a measure of health during early adulthood), and consumption of fruit and vegetables did not materially change the hazard ratios. As adjustment for a composite wealth score (including information on ownership of car or motorbike, black and white TV, colour TV, refrigerator, freezer, vacuum cleaner, and washing machine, as well as having bath in the home) that has been previously used to estimate socioeconomic status in Golestan Province22 did not change the results, we have presented the main results without adjustment for these variables. Interactions were examined by including appropriate interaction terms in the Cox regression models.\n\nFor the purpose of this analysis, we defined opium users as those who consumed opium at least once a week for at least six months. Opium users were further classified as former users and current users. Opium types analysed in this study were teriak, sukhteh, shireh, and heroin. Among current users, duration of use, which was calculated by summing the years using any type of opium, was categorised into fifths. We then calculated cumulative use by multiplying duration of use by amount used. If study participants used opium intermittently during several periods of their life, we obtained overall cumulative use by summing cumulative use during each period of use. The methods of opium consumption were classified into three categories: smoking, ingestion, or both. As the number of heroin users was small, we did not consider injection as one of the categories of use.\n\nPeople of this area may use opium to alleviate pain from chronic illnesses, which could lead to associations from reverse causality. To assess this possibility, we used data collected at enrolment to exclude from analysis any participants who started opium use after experiencing ischaemic heart disease, cerebrovascular event, hypertension, or diabetes mellitus. To further study reverse causality, we also conducted a sensitivity analysis by excluding deaths that occurred in the first 6, 12, 18, and 24 months of follow-up.\n\nWe calculated both age and age-sex standardised mortality rates based on World Population 2000. We also calculated the fraction of deaths attributed to opium use in this population using the hazard ratios and prevalence of opium use. All statistical analyses were conducted with Stata statistical software, version 11 (StataCorp, College Station, TX).\n\n# Results\n\nData from all 50\u2009045 cohort participants were used for this study, with a total of 234\u2009928 person years of follow-up (median 4.7 years per participant). The mean age of participants at baseline was 52.1 (SD 8.9) years. Most participants were women (58%), 74% were Turkmen, 80% lived in rural areas, 88% were married, 83% were non-smokers, and 70% had no formal education.\n\nOf the 17% (n=8487) of cohort members who reported ever using opium, the mean age at enrolment was 53.4 (SD 9.2), the mean age at which they started using opium was 39.7 (SD 9.2), and the mean reported duration of use was 12.7 (SD 11.1) years. The median daily amount of opium used was 0.6 g (25th-75th centile 0.2-1.2g). Opium users were more likely than the rest of the cohort to be men, rural dwellers, and cigarette smokers. The proportion of Turkmen people, married individuals, and those with no formal education was similar to the entire cohort.\n\nDuring the follow-up period until 20 May 2011, 293 (0.6%) of the study participants, including 226 (0.5%) of those who had never used opium and 67 (0.8%) of the opium users, were lost to follow-up, and 2145 deaths were reported, resulting in a crude death rate of 913 per 100\u2009000 person years. As shown in table 1<a href=\"#tbl1\" data-ref-type=\"table\"><\/a>, an overall increased risk of death was observed for being male, being illiterate, not being married (single, widowed, divorced), and medical history of myocardial infarction, cerebrovascular events, hypertension, and diabetes, whereas ethnicity and place of residence were not associated with an increased risk.\n\n\u2002Demographic and anthropometric characteristics, medical history, and habits in relation to death in participants of Golestan Cohort Study\n\n| Variables | No of participants (person years) | No of deaths (crude rate\\*) | SMR\u2020 | Relative rate |\n|----|----|----|----|----|\n| Sex: |  |  |  |  |\n| \u2003Female | 28 811 (136 279) | 893 (655) | 727 | Reference |\n| \u2003Male | 21 234 (98 649) | 1252 (1269) | 1207 | 1.66 |\n| Ethnicity: |  |  |  |  |\n| \u2003Non-Turkmen | 12 792 (57 038) | 520 (912) | 972 | Reference |\n| \u2003Turkmen | 37 253 (177 890) | 1625 (914) | 959 | 0.99 |\n| Education (highest level): |  |  |  |  |\n| \u2003No formal | 35 118 (163 647) | 1708 (1044) | 1050 | Reference |\n| \u2003Up to eight years | 10 708 (49 753) | 316 (635) | 704 | 0.67 |\n| \u2003High school | 3155 (15 883) | 89 (560) | 460 | 0.44 |\n| \u2003University | 1064 (5644) | 32 (567) | 505 | 0.48 |\n| Marital status: |  |  |  |  |\n| \u2003Married | 43 890 (205 921) | 1732 (841) | 924 | Reference |\n| \u2003Single | 302 (1449) | 22 (1519) | 1738 | 1.88 |\n| \u2003Widow\/er | 5603 (26 422) | 376 (1423) | 1703 | 1.84 |\n| \u2003Divorced\/other | 135 (635) | 10 (1575) | 1700 | 1.84 |\n| Place of residence: |  |  |  |  |\n| \u2003Rural | 40 012 (179 745) | 1625 (904) | 979 | Reference |\n| \u2003Urban | 10 033 (55 183) | 520 (942) | 942 | 0.96 |\n| History of ischaemic heart disease: |  |  |  |  |\n| \u2003No | 46 994 (221 407) | 1797 (812) | 886 | Reference |\n| \u2003Yes | 3051 (13 521) | 348 (2574) | 2032 | 2.29 |\n| History of cerebrovascular event: |  |  |  |  |\n| \u2003No | 49 616 (233 040) | 2078 (892) | 948 | Reference |\n| \u2003Yes | 429 (1,888) | 67 (3548) | 2646 | 2.79 |\n| Hypertension: |  |  |  |  |\n| \u2003No | 40 170 (189 871) | 1388 (731) | 827 | Reference |\n| \u2003Yes | 9875 (45 057) | 757 (1680) | 1627 | 1.97 |\n| History of diabetes mellitus: |  |  |  |  |\n| \u2003No | 46 591 (219 541) | 1836 (836) | 897 | Reference |\n| \u2003Yes | 3454 (15 387) | 309 (2008) | 1997 | 2.23 |\n| Cigarette smoking: |  |  |  |  |\n| \u2003Never smoker | 41 385 (194 823) | 1537 (789) | 878 | Reference |\n| \u2003Ever smoker | 8660 (40 105) | 608 (1516) | 1429 | 1.63 |\n| Opium use: |  |  |  |  |\n| \u2003Never user | 41 558 (196 825) | 1440 (732) | 818 | Reference |\n| \u2003Ever user | 8487 (38 103) | 705 (1850) | 1650 | 2.02 |\n\n\\*Crude rates calculated in 10^5^ person years.\n\n\u2020Age and sex standardised mortality rate based on 2000 world standard population.\n\nIn all, 8660 (17%) participants reported ever smoking cigarettes, with an adjusted hazard ratio of 1.53 (95% confidence interval of 1.37 to 1.70) for all causes of death. The hazard ratio for death associated with opium use was 1.98 (1.80 to 2.17), before adjustment for cigarette smoking and 1.86 (1.68 to 2.06) after adjustment for cigarette smoking.\n\nTable 2<a href=\"#tbl2\" data-ref-type=\"table\"><\/a> compares hazard ratios for overall mortality among opium users stratified by sex, ethnicity, education, marital status, place of residence, history of chronic diseases, BMI, and categories of cigarette smoking, with tests for interaction across each group. With the exception of people with history of cerebrovascular events, a relatively small group, opium use was associated with an increased risk of mortality among all strata. Significantly higher increased risks of mortality were observed among women than men (P\\<0.001), urban than rural dwellers (P=0.008), obese than non-obese people (P=0.020), and those who had never smoked than those who did (P=0.001).\n\n\u2002Hazard ratios for association between opium use and mortality by subgroups in participants in Golestan Cohort Study\n\n| Variable | No (%) who never used opium | No (%) who ever used opium | Cox hazard ratio\\* (95% CI) |  | P for interaction |\n|----|----|----|----|----|----|\n|  |  |  | Unadjusted | Adjusted\u2020 |  |\n| All cohort | 41 558 | 8487 | 2.26 (2.06 to 2.47) | 1.86 (1.68 to 2.06) | \u2014 |\n| Sex: |  |  |  |  |  |\n| \u2003Women | 26 456 (63.7) | 2355 (27.7) | 2.54 (2.16 to 2.98) | 2.43 (2.05 to 2.88) | \\<0.001 |\n| \u2003Men | 15 102 (36.3) | 6132 (72.3) | 1.82 (1.62 to 2.03) | 1.63 (1.44 to 1.84) |  |\n| Ethnicity: |  |  |  |  |  |\n| \u2003Turkmen | 30 701 (73.9) | 6552 (77.2) | 2.24 (2.02 to 2.48) | 1.82 (1.62 to 2.05) | 0.578 |\n| \u2003Non-Turkmen | 10 857 (26.1) | 1935 (22.8) | 2.28 (1.89 to 2.76) | 1.99 (1.62 to 2.45) |  |\n| Education: |  |  |  |  |  |\n| \u2003Illiterate | 29 490 (71.0) | 5628 (66.3) | 2.24 (2.02 to 2.48) | 1.89 (1.68 to 2.11) | 0.558 |\n| \u2003Up to 8 years | 8480 (20.4) | 2228 (26.3) | 2.23 (1.77 to 2.82) | 1.76 (1.35 to 2.29) |  |\n| \u2003High school | 2601 (6.30) | 554 (6.50) | 2.68 (1.71 to 4.18) | 1.61 (0.98 to 2.67) |  |\n| \u2003University | 987 (2.30) | 77 (0.90) | 3.90 (1.55 to 9.81) | 3.65 (1.28 to 10.4) |  |\n| Marital status: |  |  |  |  |  |\n| \u2003Married | 36 345 (87.7) | 7545 (89.0) | 2.22 (2.00 to 2.45) | 1.80 (1.61 to 2.02) | 0.629 |\n| \u2003Single | 240 (0.60) | 62 (0.70) | 3.22 (1.32 to 7.81) | 2.36 (0.81 to 6.90) |  |\n| \u2003Widow\/er | 4760 (11.5) | 843 (9.90) | 2.40 (1.92 to 2.99) | 2.16 (1.69 to 2.75) |  |\n| \u2003Divorced\/other | 108 (0.20) | 27 (0.40) | 2.22 (0.60 to 8.26) | 2.10 (0.41 to 10.9) |  |\n| Place of residence: |  |  |  |  |  |\n| \u2003Rural | 32 527 (78.3) | 7485 (88.2) | 2.12 (1.91 to 2.35) | 1.77 (1.58 to 1.99) | 0.008 |\n| \u2003Urban | 9031 (21.7) | 1002 (11.8) | 2.97 (2.43 to 3.61) | 2.32 (1.85 to 2.92) |  |\n| History of ischaemic heart disease: |  |  |  |  |  |\n| \u2003No | 39 210 (94.3) | 7784 (91.7) | 2.30 (2.09 to 2.54) | 1.87 (1.67 to 2.09) | 0.053 |\n| \u2003Yes | 2348 (5.70) | 703 (8.30) | 1.82 (1.46 to 2.28) | 1.58 (1.23 to 2.04) |  |\n| History of cerebrovascular event: |  |  |  |  |  |\n| \u2003No | 41 237 (99.2) | 8379 (98.7) | 2.28 (2.08 to 2.50) | 1.88 (1.69 to 2.08) | 0.164 |\n| \u2003Yes | 321 (0.80) | 108 (1.30) | 1.34 (0.78 to 2.33) | 1.12 (0.59 to 2.13) |  |\n| Hypertension: |  |  |  |  |  |\n| \u2003No | 33 148 (79.8) | 7022 (82.7) | 2.30 (2.06 to 2.57) | 1.81 (1.59 to 2.05) | 0.685 |\n| \u2003Yes | 8410 (20.2) | 1465 (17.3) | 2.39 (2.04 to 2.79) | 1.94 (1.63 to 2.32) |  |\n| History of diabetes mellitus: |  |  |  |  |  |\n| \u2003No | 38 647 (93.0) | 7944 (93.6) | 2.33 (2.12 to 2.57) | 1.87 (1.67 to 2.08) | 0.320 |\n| \u2003Yes | 2911 (7.00) | 543 (6.40) | 2.00 (1.55 to 2.57) | 1.67 (1.26 to 2.22) |  |\n| Body mass index (BMI): |  |  |  |  |  |\n| \u2003Underweight | 1328 (3.20) | 1080 (12.7) | 1.76 (1.32 to 2.35) | 1.54 (1.12 to 2.10) | 0.020 |\n| \u2003Normal weight | 13 585 (32.7) | 4344 (51.2) | 1.99 (1.74 to 2.28) | 1.77 (1.53 to 2.06) |  |\n| \u2003Overweight | 14 884 (35.8) | 2088 (24.6) | 2.30 (1.92 to 2.75) | 1.83 (1.50 to 2.23) |  |\n| \u2003Obese | 11 753 (28.3) | 971 (11.5) | 2.93 (2.30 to 3.72) | 2.25 (1.72 to 2.93) |  |\n| Cigarette smoking: |  |  |  |  |  |\n| \u2003Never smoked | 37 373 (89.9) | 4012 (47.3) | 2.25 (1.99 to 2.53) | 2.07 (1.83 to 2.35) | 0.001 |\n| \u2003Ever smoked | 4185 (10.1) | 4475 (52.7) | 1.53 (1.30 to 1.81) | 1.50 (1.27 to 1.77) |  |\n\n\\*Hazard ratio of dying in opium users in each stratum.\n\n\u2021Adjusted for variables including sex, ethnicity, education level, marital status, residential place, and cigarette smoking, except if each of them is considered as stratifying variable.\n\nTable 3<a href=\"#tbl3\" data-ref-type=\"table\"><\/a> shows the hazard ratios (95% confidence intervals) for major causes of death in relation to opium use. Opium use was associated with an increased risk of death in all major categories, except the category classified as \"external,\" which mainly included unintentional injuries and trauma. The strongest associations were found for infections (adjusted hazard ratio 5.47), respiratory diseases (3.78), and digestive diseases (3.12). Consistent with the overall association, the hazard ratios were higher in women than in men for most causes of deaths. Among deaths from circulatory causes, opium use was associated with an increased risk of ischaemic heart disease and cerebrovascular events (table 3<a href=\"#tbl3\" data-ref-type=\"table\"><\/a>). Among deaths from cancer, opium use was associated with an increased risk of three common cancers that have been shown to be related to tobacco smoking\u2014that is, oesophageal, gastric, and lung cancers\u2014though this was significant only for lung cancer. Additional examination of the association between opium use and deaths from respiratory conditions showed that the adjusted hazard ratios were 11.0 (95% confidence interval 3.97 to 30.6) for asthma, 6.22 (2.36 to 16.4) for tuberculosis, and 5.44 (2.03 to 14.5) for chronic obstructive pulmonary disease.\n\n\u2002Hazard ratios for major causes of death in relation to opium use in Golestan Cohort Study\n\n| Cause of death | Total (n=50 045) | No (%) of opium users (n=8487, 17%) | Cox hazard ratio\\* (95% CI) |  |  |\n|----|----|----|----|----|----|\n|  |  |  | Total | Men | Women |\n| Any | 2145 | 705 (33) | 1.86 (1.68 to 2.06) | 1.63 (1.44 to 1.84) | 2.43 (2.05 to 2.88) |\n| Circulatory: |  |  |  |  |  |\n| \u2003All | 1073 | 335 (31) | 1.81 (1.56 to 2.09) | 1.53 (1.28 to 1.82) | 2.52 (1.98 to 3.20) |\n| \u2003Ischaemic heart disease | 624 | 208 (33) | 1.90 (1.57 to 2.29) | 1.58 (1.26 to 1.98) | 2.90 (2.10 to 3.99) |\n| \u2003Cerebrovascular event | 352 | 98 (28) | 1.68 (1.29 to 2.18) | 1.49 (1.07 to 2.08) | 1.97 (1.30 to 2.97) |\n| \u2003Other circulatory | 97 | 29 (30) | 1.73 (1.06 to 2.82) | 1.30 (0.71 to 2.36) | 2.98 (1.35 to 6.58) |\n| Cancer: |  |  |  |  |  |\n| \u2003All | 449 | 137 (31) | 1.61 (1.28 to 2.03) | 1.70 (1.30 to 2.24) | 1.42 (0.92 to 2.20) |\n| \u2003Oesophagus | 100 | 30 (30) | 1.51 (0.93 to 2.43) | 1.12 (0.62 to 2.04) | 2.40 (1.13 to 5.10) |\n| \u2003Stomach | 75 | 25 (33) | 1.32 (0.77 to 2.26) | 1.33 (0.74 to 2.40) | 1.23 (0.33 to 4.65) |\n| \u2003Lung | 37 | 18 (49) | 2.27 (1.07 to 4.80) | 2.65 (1.13 to 6.21) | 1.19 (0.15 to 9.72) |\n| \u2003Other cancer | 237 | 64 (27) | 1.67 (1.21 to 2.31) | 2.09 (1.40 to 3.13) | 1.10 (0.59 to 2.07) |\n| Respiratory: |  |  |  |  |  |\n| \u2003All | 95 | 52 (55) | 3.78 (2.36 to 6.04) | 2.64 (1.50 to 4.73) | 6.64 (3.30 to 13.3) |\n| \u2003Asthma | 23 | 17 (74) | 11.0 (3.97 to 30.6) | 21.5 (2.60 to 177) | 8.04 (2.26 to 28.6) |\n| \u2003COPD | 24 | 17 (71) | 5.44 (2.03 to 14.5) | 3.18 (1.11 to 9.14) | 34.1 (3.29 to 353) |\n| \u2003Other respiratory | 48 | 18 (38) | 1.87 (0.94 to 3.71) | 1.15 (0.49 to 2.70) | 4.08 (1.51 to 11.0) |\n| Digestive: |  |  |  |  |  |\n| \u2003All | 69 | 32 (46) | 3.12 (1.82 to 5.37) | 3.05 (1.60 to 5.79) | 3.32 (1.24 to 8.84) |\n| \u2003Liver cirrhosis | 36 | 14 (39) | 2.28 (1.05 to 4.97) | 2.90 (1.14 to 7.34) | 1.17 (0.21 to 6.40) |\n| \u2003Other digestive | 33 | 18 (55) | 4.35 (2.01 to 9.39) | 3.25 (1.34 to 7.90) | 8.89 (2.20 to 35.9) |\n| Infectious: |  |  |  |  |  |\n| \u2003All | 51 | 30 (59) | 5.47 (2.87 to 10.4) | 3.44 (1.54 to 7.69) | 8.85 (3.51 to 22.3) |\n| \u2003Tuberculosis | 24 | 16 (67) | 6.22 (2.36 to 16.4) | 4.12 (1.33 to 12.7) | 10.6 (2.33 to 48.7) |\n| \u2003Other infectious | 27 | 14 (52) | 5.07 (2.11 to 12.2) | 2.90 (0.91 to 9.23) | 8.11 (2.48 to 26.5) |\n| External | 135 | 30 (22) | 0.86 (0.54 to 1.35) | 0.73 (0.44 to 1.21) | 1.45 (0.58 to 3.64) |\n| Other | 150 | 43 (29) | 1.66 (1.11 to 2.48) | 1.21 (0.73 to 2.02) | 2.78 (1.52 to 5.06) |\n| Unknown | 123 | 46 (37) | 2.42 (1.59 to 3.67) | 2.66 (1.55 to 4.55) | 2.03 (1.02 to 4.03) |\n\nCOPD=chronic obstructive pulmonary disease.\n\n\\*Cox hazard ratios for opium use, adjusted for sex (except when stratified by sex), ethnicity, education level, marital status, residential place, and cigarette smoking\n\nAmongst 8487 opium users, most used teriak only (7308), shireh only (721), or a combination of the two. Other forms of opium use were far less common. Increased mortality was seen for each subtype of opium (see table A in appendix), with the strongest risk with heroin, albeit with wide confidence intervals. Use of shireh (adjusted hazard ratio 2.19) was associated with a slightly higher hazard of death than teriak (1.83), but the difference was not significant.\n\nTo assess the dose-response relation, we classified opium users as former users and current users. Current opium users had a higher hazard of death than former users (table 4<a href=\"#tbl4\" data-ref-type=\"table\"><\/a>). Among current users, longer duration of use was not associated with a higher mortality hazard (P=0.497 for trend). When we excluded 542 people who started opium use after receiving a diagnosis of a major illness (6.4% of all opium users), however, we found a dose-response association with duration of opium use (P=0.001 for trend). This pattern was also seen for several major causes of death. Assessment of the dose-response relation with cumulative use yielded similar results (see table B in appendix). Further sensitivity analysis with exclusion of deaths that occurred in the first 6, 12, 18, and 24 months of follow up did not have a material effect on the point estimates (see table C in appendix). Further adjustment did not change the results meaningfully (see table D in appendix), reducing the possibility of residual and unmeasured confounding. When we created a \"healthy group\" by excluding participants with any history of hypertension, ischemic heart disease, diabetes mellitus, or cerebrovascular events and underweight participants and those who had ever smoked cigarettes, the adjusted Cox hazard ratio for opium use in relation to overall mortality was 1.90 (95% confidence interval of 1.55 to 2.33).\n\n\u2002Cox hazard ratios\\* (95% confidence intervals) by duration of opium use in relation to all mortality and cause specific mortality overall and after exclusion of participants who started using opium after diagnosis of serious disease (ischaemic heart disease, cerebrovascular event, hypertension, or diabetes mellitus) in Golestan Cohort Study\n\n|  | Never users | Former users | Current users | Fifth of duration (years) of opium use in current opium users |  |  |  |  | P for trend\u2020 |\n|----|----|----|----|----|----|----|----|----|----|\n|  |  |  |  | 1 (\u22643 years) | 2 (4-7 years) | 3 (8-12 years) | 4 (13-20 years) | 5 (\u226521 years) |  |\n| **All causes** |  |  |  |  |  |  |  |  |  |\n| No of deaths | 1440 | 63 | 642 | 131 | 107 | 99 | 122 | 183 | \u2014 |\n| Overall | Reference | 1.29 (1.00 to 1.67) | 1.94 (1.75 to 2.16) | 2.14 (1.79 to 2.57) | 1.85 (1.51 to 2.26) | 1.97 (1.59 to 2.42) | 1.81 (1.49 to 2.19) | 1.94 (1.64 to 2.29) | 0.497 |\n| Before diagnosis\u2021 | Reference | 1.27 (0.98 to 1.65) | 1.70 (1.52 to 1.90) | 1.62 (1.31 to 1.99) | 1.39 (1.09 to 1.78) | 2.00 (1.62 to 2.48) | 1.79 (1.46 to 2.18) | 1.90 (1.60 to 2.25) | 0.001 |\n| **Circulatory causes** |  |  |  |  |  |  |  |  |  |\n| No of deaths | 738 | 38 | 297 | 63 | 53 | 50 | 59 | 72 | \u2014 |\n| Overall | Reference | 1.57 (1.12 to 2.20) | 1.84 (1.58 to 2.14) | 2.09 (1.61 to 2.72) | 1.87 (1.41 to 2.49) | 2.05 (1.52 to 2.75) | 1.79 (1.36 to 2.35) | 1.55 (1.20 to 2.01) | 0.490 |\n| Before diagnosis\u2021 | Reference | 1.40 (0.98 to 1.99) | 1.53 (1.30 to 1.80) | 1.21 (0.86 to 1.69) | 1.36 (0.95 to 1.94) | 1.99 (1.47 to 2.70) | 1.76 (1.32 to 2.34) | 1.48 (1.13 to 1.93) | 0.039 |\n| **Cancer causes** |  |  |  |  |  |  |  |  |  |\n| No of deaths | 312 | 8 | 129 | 13 | 22 | 17 | 30 | 47 | \u2014 |\n| Overall | Reference | 0.77 (0.38 to 1.57) | 1.73 (1.37 to 2.18) | 0.96 (0.55 to 1.68) | 1.71 (1.10 to 2.66) | 1.52 (0.92 to 2.51) | 2.01 (1.36 to 2.98) | 2.26 (1.61 to 3.17) | 0.003 |\n| Before diagnosis\u2021 | Reference | 0.81 (0.39 to 1.64) | 1.79 (1.41 to 2.26) | 1.57 (1.00 to 2.47) | 1.19 (0.68 to 2.10) | 1.70 (1.05 to 2.78) | 1.87 (1.23 to 2.86) | 2.32 (1.65 to 3.26) | 0.016 |\n| **Respiratory causes** |  |  |  |  |  |  |  |  |  |\n| No of deaths | 43 | 4 | 48 | 9 | 5 | 6 | 8 | 20 | \u2014 |\n| Overall | Reference | 2.02 (0.70 to 5.84) | 4.05 (2.52 to 6.52) | 4.25 (2.02 to 8.93) | 2.64 (1.02 to 6.83) | 3.52 (1.45 to 8.56) | 3.36 (1.52 to 7.46) | 5.75 (3.11 to 10.6) | 0.117 |\n| Before diagnosis\u2021 | Reference | 2.01 (0.69 to 5.82) | 3.85 (2.36 to 6.29) | 3.75 (1.71 to 8.23) | 2.42 (0.84 to 6.92) | 2.98 (1.14 to 7.77) | 3.56 (1.60 to 7.92) | 5.51 (2.94 to 10.3) | 0.078 |\n| **Digestive causes** |  |  |  |  |  |  |  |  |  |\n| No of deaths | 37 | 4 | 28 | 5 | 6 | 8 | 5 | 4 | \u2014 |\n| All | Reference | 3.01 (1.02 to 8.85) | 3.14 (1.80 to 5.47) | 2.98 (1.14 to 7.75) | 3.93 (1.61 to 9.60) | 5.60 (2.49 to 12.6) | 2.60 (0.98 to 6.88) | 1.58 (0.53 to 4.67) | 0.168 |\n| Before diagnosis\u2021 | Reference | 3.06 (1.04 to 9.02) | 3.16 (1.78 to 5.60) | 3.01 (1.15 to 7.88) | 3.15 (1.09 to 9.12) | 6.22 (2.76 to 14.0) | 2.87 (1.08 to 7.63) | 1.66 (0.56 to 4.93) | 0.219 |\n| **Infectious causes** |  |  |  |  |  |  |  |  |  |\n| No of deaths | 21 | 1 | 29 | 11 | 2 | 4 | 5 | 7 | \u2014 |\n| Overall | Reference | 1.37 (0.18 to 10.5) | 6.05 (3.17 to 11.6) | 12.3 (5.69 to 26.7) | 2.23 (0.50 to 9.80) | 5.54 (1.81 to 16.9) | 5.07 (1.81 to 14.2) | 4.70 (1.80 to 12.3) | 0.270 |\n| Before diagnosis\u2021 | Reference | 1.25 (0.16 to 9.70) | 5.23 (2.66 to 10.3) | 8.62 (3.63 to 20.5) | 2.54 (0.57 to 11.2) | 7.06 (2.52 to 19.8) | 4.22 (1.37 to 13.0) | 3.80 (1.38 to 10.5) | 0.424 |\n\n\\*Adjusted for sex, ethnicity, education level, marital status, residential place, and cigarette smoking.\n\n\u2020P for trend calculated by assigning consecutive integers to fifths of duration of opium use in current opium users.\n\n\u2021Excludes those who started opium use after diagnosis with serious disease.\n\nTable E in the appendix shows the results by broad categories of opium use\u2014that is, ingestion and smoking. After exclusion of rare methods of opium use\u2014for instance, the four and six participants who exclusively used heroin and sukhteh, respectively\u2014of the remaining individuals, 5804 only smoked opium, 2176 exclusively ingested opium, and 497 reported both smoking and ingestion. Both methods were associated with increased risk of mortality: adjusted hazard ratio 2.08 for ingestion; 1.68 for smoking; and 2.34 for both. There was a similar pattern for most major causes of death, both before and after adjustment for either duration of opium use or its cumulative consumption.\n\nThe figure<a href=\"#fig1\" data-ref-type=\"fig\"><\/a> presents Cox hazard ratios in former users by years after cessation of opium use until they were enrolled into the study. The longer duration of cessation was associated with a decreasing hazard ratio (P=0.003 for trend).\n\nBased on the overall adjusted hazard ratio for opium use of 1.86 (1.68 to 2.06), and assuming this represents a causal association, we calculate the fraction of deaths attributable to opium in this population as 14.9% (12.3% to 17.5%).\n\n# Discussion\n\n## Major findings\n\nOpium use is common in this population in Iran and is associated with an estimated 86% increased risk of death overall; in women the increase was 143%. This increased risk was present for several major causes of death, including death from circulatory disorders and cancer, for different subtypes of opium (teriak, shireh, heroin, or combinations), for various routes of use (smoking and ingestion), and in several study subgroups (sex, ethnicity, residence, and others).\n\n## Evidence for causality\n\nThis large prospective study looked at opium use in relation to overall mortality and cause specific mortality. Although causal inferences from this study could be premature, the findings are consistent with those from retrospective case-control studies that have shown increased risk of oesophageal cancer,3 4 bladder cancer,5 6 7 coronary heart disease,8 10 11 and other diseases12 13 14 in relation to opium use. Also, several of our findings point toward a causal role for opium in increasing mortality. A nearly twofold risk of mortality in opium users is substantial and is stronger than the estimated risk posed by tobacco use in this population. After adjustment for several factors, including tobacco use, the associations diminished only slightly but were still strong and significant.\n\nPeople in this region might use opium to alleviate pain from chronic conditions, which could result in associations from reverse causality. To avoid misleading conclusions, we carried out a sensitivity analysis by excluding participants who started using self prescribed opium after the onset of four major chronic diseases. This resulted in some attenuation of risk that was restricted to recent users, as expected, but little change in risks for longer term users of eight years or more. Further sensitivity analysis excluding deaths occurring in the first 24 months of follow-up did not make a material difference either. Finally, longer opium cessation was associated with lower risk of death.\n\nOpium alkaloid (such as morphine) and non-alkaloid (such as meconic acid) constituents, and other chemicals added during processing or generated during smoking, have a wide range of effects and can potentially explain the diverse increased risk of morbidity and mortality with opium use. Activation of \u00b5-opioid receptors in pre-B\u00f6tzinger complex, located in the ventrolateral medulla, can reduce breathing frequency, tidal volume, chest wall compliance, and respiratory response to increased arterial carbon dioxide concentration.23 Opium use can also cause upper airway constriction through inhibition of acetylcholine release23 and increased bronchial tone through provocation of histamine release from mast cells.11 24 Smoking opium can also cause histological damage to the bronchi and lungs.20 Prolonged administration of exogenous opioids can have inhibitory effects on both humoral and cell mediated immune response.25 All of these mechanisms could underlie the strong associations seen with deaths from asthma, tuberculosis, and chronic obstructive pulmonary disease. Smoking opium can expose the body to carcinogenic polycyclic aromatic hydrocarbons, heterocyclic aromatic hydrocarbons, and primary aromatic amines.26 There is a growing body of evidence indicating that opiates could act as cancer promoters through impairing immune function, activating angiogenesis and tumour neovascularisation,25 increasing the concentrations of N-nitrosamines through modifying their pharmacokinetics,27 increasing the release of nitric oxide,28 or inhibiting production and release of hydrogen peroxide.29 Although opium use might have cardioprotective effects in short term use,30 it can have deleterious effects in the long term through causing hypotension, bradycardia, and respiratory depression that could decrease myocardial oxygenation and hence increase the infarct size and mortality.31 Morphine, like other opiate alkaloids, is metabolised principally in the liver via conjugation with glucuronic acid.32 Similar to other substances metabolised by the liver, morphine can result in hepatotoxicity.33 Extensive animal research has shown that morphine and morphine-like agents can increase liver enzyme activity and lead to perivenular and focal hepatocellular necrosis.34 35 36 37 Chronic administration of morphine in rats is thought to increase inflammatory infiltration and focal parenchymal necrosis in the liver, in addition to an increase in total lipid content of hepatocytes, all leading to lipid fibrosis and hepatic damage.38\n\nOpium users in our cohort reported relatively low amounts of use (median 0.6 g\/day), considerably lower than ranges of 1-24 g reported elsewhere.20 Although study participants might under-report the amount used, average daily use is likely to be low in this group as these were people who were able and willing to participate in the study, had low rates of loss to follow-up (\\<1%), and whose education, ethnic background, and marital status was similar to non-users. Therefore, the opium users perhaps had a lifestyle that was not substantially different from others. These findings are in favour of a causal connection and suggest that even relatively small amounts of opium use over a long period can have a substantial effect on mortality.\n\nBoth opium ingestion and opium smoking were associated with an increased risk of death. Compared with ingestion, smoking exposes individuals to more polycyclic and heterocyclic aromatic hydrocarbons and primary aromatic amines26 but also exposes users to nearly 10 times less morphine. Almost 50% of opium that is smoked adheres to the opium pipe and of the other half that is vaporised in the pipe, a large proportion is lost in the smoke that escapes into the surrounding air.20\n\nWe found that opium increased the risk of death in nearly all subgroups of the study participants, including men and women, Turkmen and non-Turkmen people, people who lived in urban and rural areas, people with and without major chronic diseases, and smokers and non-smokers. The strength of association, however, differed in some subgroup analyses. Most importantly, the hazards ratios were higher in women than in men and in non-smokers than tobacco smokers.\n\nOpium use is probably accurately reported in this population. A previous pilot study nested in this population showed that there was a strong correspondence between self reported opium use and opium metabolites in urine, with a sensitivity and specificity of 93% and 89%, respectively.17 Unlike alcohol, opium is not considered to be strictly religiously forbidden in this area, and recreational low dose consumption is culturally acceptable. Although there is evidence that the participants' report of any opium use was valid, we do not know whether their reported amount of use was also correct. In our analyses, cumulative use of opium, estimated by multiplying duration and amount of use, did not result in stronger associations than duration of use alone. Therefore, responses to amount of use could be subject to misclassification.\n\nWe used several methods to control for the effect of known and unknown confounders. Statistical adjustment for important potential confounders such as age, sex, place of residence, levels of education, marital status, BMI, height, intake of fruit and vegetables, a composite wealth score, alcohol consumption, and cigarette smoking status had little effect on the risk estimates. Compared with those who never used opium, stratified analyses showed a consistently significant increased risk of mortality among people who had ever used opium in almost all strata, including in those who had ever or never smoked cigarettes. Dose-response analyses showed a significant increasing trend of overall and cause specific risks of mortality for increasing doses of opium use. Finally, sensitivity analyses, through both classic and customised methods in our study, consistently showed the same results for the effect of opium use on risks of mortality. Although there could still be some unmeasured confounders or residual confounding from modelling or measurement errors, we believe their presence and effect would not be considerable because of extensive adjustment for several known confounders.39 40 Confounding factors should be strongly associated with both the exposure and outcomes to have a material effect,41 and we have adequately adjusted for age, smoking, and other confounders strongly related to both opium use and death; other unknown factors are unlikely to be significantly associated with both opium use and death. Whereas large scale randomised controlled clinical trials are the best design to eliminate or limit residual confounding, conducting randomised studies of opium and mortality is not ethically feasible; therefore prospective observational studies remain the design of choice in this setting.\n\nAssuming a causal relation, the impact of opium use on mortality is substantial. About 20 million people in the world use opium or its derivatives, and these individuals are at a substantially increased risk of death.\n\n## Strengths and limitations of the study\n\nThe strengths of this study include its prospective design, large sample size, availability of data to adjust for confounders, prior validation of exposure and outcome measurement, and minimal loss to follow-up. This study also has limitations, which include its observational design and therefore the potential for residual confounding and reverse causality. There is also a possibility of outcome misclassification as the cause of death was determined solely based on verbal autopsy in 35% of the cases. However, we used several methods to increase the validity of our outcome determination and to reduce the possibility of confounding and reverse causality.\n\n## Conclusions and implications\n\nIn summary, in this large scale prospective study we found strong increased risks of death from multiple causes in opium users compared with non-users, even among those who used low doses of opium. These results are consistent with results of previous case-control studies in humans and animal studies suggesting deleterious health consequences of exogenous opioids such as morphine. Further epidemiological studies on opium use in relation to morality are needed. Also, follow-up studies of patients taking opioid analgesics long term for treatment of pain could shed further light on this issue.\n\n### What is already known on this topic\n\n1.  A small number of retrospective case-control studies or case series have described the possible role of opium in causing oesophageal cancer, bladder cancer, coronary heart disease, and a few other select health conditions\n\n2.  There is little information from well designed prospective epidemiological studies on the effect of opium use, particularly low doses over a long period, on overall or cause specific mortality\n\n### What this study adds\n\n1.  Long term opium use, even in relatively low doses, is associated with an estimated 86% increased risk of death overall\n\n2.  Users have an increased risk for several major causes of death, including circulatory disorders and cancer\n\nCite this as: *BMJ* 2012;344:e2502","meta":{"dup_signals":{"dup_doc_count":175,"dup_dump_count":52,"dup_details":{"curated_sources":2,"2023-06":1,"2022-27":1,"2021-43":1,"2021-21":1,"2020-40":2,"2020-34":1,"2019-30":1,"2019-26":1,"2018-22":2,"2018-17":3,"2018-13":3,"2018-09":2,"2018-05":2,"2017-51":2,"2017-47":2,"2017-43":4,"2017-39":2,"2017-34":4,"2017-30":2,"2017-26":3,"2017-22":3,"2017-17":3,"2017-09":1,"2017-04":1,"2016-50":1,"2016-44":2,"2016-40":2,"2016-36":4,"2016-30":4,"2016-26":5,"2016-22":5,"2016-18":4,"2016-07":5,"2015-48":5,"2015-40":3,"2015-35":4,"2015-32":4,"2015-27":5,"2015-22":5,"2015-14":4,"2014-52":5,"2014-49":5,"2014-42":11,"2014-41":6,"2014-35":5,"2014-23":10,"2023-50":1,"2017-13":5,"2015-18":5,"2015-11":4,"2015-06":5,"2024-26":1}},"file":"PMC3328545"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  This study examines whether young never smokers in Scotland, UK, who have tried an e-cigarette are more likely than those who have not, to try a cigarette during the following year.\n  .\n  # Methods\n  .\n  Prospective cohort survey conducted in four high schools in Scotland, UK during February\/March 2015 (n=3807) with follow-up 1\u2009year later. All pupils (age 11\u201318) were surveyed. Response rates were high in both years (87% in 2015) and 2680\/3807 (70.4%) of the original cohort completed the follow-up survey. Analysis was restricted to baseline 'never smokers' (n=3001\/3807), 2125 of whom were available to follow-up (70.8%).\n  .\n  # Results\n  .\n  At baseline, 183 of 2125 (8.6%) never smokers had tried an e-cigarette and 1942 had not. Of the young people who had not tried an e-cigarette at baseline, 249 (12.8%) went on to try smoking a cigarette by follow-up. This compares with 74 (40.4%) of those who had tried an e-cigarette at baseline. This effect remained significant in a logistic regression model adjusted for smoking susceptibility, having friends who smoke, family members' smoking status, age, sex, family affluence score, ethnic group and school (adjusted OR 2.42 (95% CI 1.63 to 3.60)). There was a significant interaction between e-cigarette use and smoking susceptibility and between e-cigarette use and smoking within the friendship group.\n  .\n  # Conclusions\n  .\n  Young never smokers are more likely to experiment with cigarettes if they have tried an e-cigarette. Causality cannot be inferred, but continued close monitoring of e-cigarette use in young people is warranted.\nauthor: Catherine Best; Farhana Haseen; Dorothy Currie; Gozde Ozakinci; Anne Marie MacKintosh; Martine Stead; Douglas Eadie; Andy MacGregor; Jamie Pearce; Amanda Amos; John Frank; Sally HawCorrespondence to Dr Catherine Best, Faculty of Health Sciences and Sport, University of Stirling, Stirling, FK9 4LA, UK; <email@example.com>\ndate: 2018-07\ninstitute: 1 Faculty of Health Sciences and Sport, University of Stirling, Stirling, UK; 2 Child and Adolescent Health Research Unit, School of Medicine, University of St Andrews, St Andrews, UK; 3 School of Medicine, University of St Andrews, St Andrews, UK; 4 Institute for Social Marketing, Faculty of Health Sciences and Sport, University of Stirling, Stirling, UK; 5 ScotCen Social Research, Edinburgh, UK; 6 Centre for Research on Environment Society and Health, School of GeoSciences, University of Edinburgh, Edinburgh, UK; 7 The Usher Institute of Population Health Sciences and Informatics, College of Medicine and Veterinary Medicine, University of Edinburgh, Edinburgh, UK\nreferences:\ntitle: Relationship between trying an electronic cigarette and subsequent cigarette experimentation in Scottish adolescents: a cohort study\n\n# Introduction\n\nIn the UK and many other countries, e-cigarette use among young people is largely confined to those who have already tried tobacco and is mostly experimental in nature.1 2 That is, most young people who have never tried tobacco smoking, hereon referred to as never-smokers, do not engage in regular e-cigarette use that is sustained over\u00a0time. Nevertheless, there remains concern that trying an e-cigarette could ease the pathway to experimentation with tobacco smoking for young never-smokers.\n\nEight longitudinal studies, all conducted in the USA with follow-up after 63 4 and\/or 12 months,5\u201310 have explored the relationship between e-cigarette use and smoking initiation in young never-smokers. They found that young people who had ever used an e-cigarette at baseline were more likely to have tried a cigarette by follow-up.\n\nMost of the evidence from prospective cohort studies of young never smokers, e-cigarette use and smoking initiation has come from the USA. It is important this evidence can be compared with studies from different countries because varied national contexts, such as different tobacco control regulations, historical and cultural factors around tobacco use, availability and supply of products, ethnic composition of the population and investment in advertising of products, make it difficult to generalise findings across national boundaries. For example, in Poland 27.4% of adolescents report using an e-cigarette in the past month.11 Poland is a major European tobacco and e-cigarette producer. Recently smoking rates have increased among Polish female adolescents although they are stable in men12\u201314 and by late adolescence most Polish e-cigarette users are dual users (tobacco and e-cigarette use). A recent study found 21.8% of students (16\u201318\u2009years) were dual users and this was not associated with reduced cigarette consumption compared with tobacco-only users.15 The case of Poland highlights the potential role of national factors such as tobacco production and industry involvement in affecting levels of use in young people.\n\nIn Scotland, the prevalence of cigarette smoking among young people has steadily fallen over the last two decades. In 2015, only 2% of 13\u2009year olds and 7% of 15\u2009year olds were regular smokers.16 However, current smoking among young people aged 16\u201324\u2009years in Scotland is significantly higher at 21%.17 This disparity suggests that smoking initiation may now be delayed until early adulthood. Therefore early risk factors for later smoking initiation require further investigation.\n\nPrevious cross-sectional research has shown a positive association between e-cigarette use and weakened intentions not to smoke in children aged 10\u201311 years in Wales.1 Recently the ever use of e-cigarettes among young non-smokers has increased in Scotland with 10% of non-smoking 15\u2009year olds having tried them in 2013 and 24% in 2015.16 Levels of regular e-cigarette use among young people in Wales have also increased with 2.7% of young people aged 11\u201318\u2009years reporting using them at least once a week in 2015.18 These increases were preceded by a marked growth in the retail availability of e-cigarettes with the proportion of retailers with displays of e-cigarettes doubling between 2013 and 2014.19 When this study was conducted, within store advertising and promotion of e-cigarettes was not regulated and there was no age restriction on the legal purchase of e-cigarettes in the UK. This study is one of the first to examine e-cigarette use and cigarette experimentation in a UK longitudinal sample.\n\n# Methods\n\nThe data presented here are drawn from the Determining the Impact of Smoking Point-of-Sale Legislation Among Youth\u00a0(DISPLAY) study.20 The DISPLAY study is a 5-year multimodal study designed to measure the impact of UK legislation to ban point-of-sale displays of tobacco products on the smoking attitudes and behaviours of young people. One element of the DISPLAY study is an annual school survey conducted in four Scottish secondary schools located in communities that differ in terms of their socioeconomic and urban\u2013rural profiles. The data presented here are from the 2015 and 2016 surveys which included all pupils (aged 11\u201318) in the four schools. All four schools had pupils across the age range 11\u201318 years and a breakdown of participant numbers by school and by year group is given in <a href=\"#T1\" data-ref-type=\"table\">table 1<\/a>. The survey was administered by class teachers under exam conditions and took on average 40\u2009min to complete. Pupils who were absent on the day of the survey were given opportunity during the following 2\u2009weeks to complete the survey.\n\nNumber of 'never smoking' respondents by school and year group\n\n<table>\n<thead>\n<tr>\n<th style=\"text-align: left;\"><\/th>\n<th style=\"text-align: left;\">Year 1<\/th>\n<th style=\"text-align: left;\">Year 2<\/th>\n<th style=\"text-align: left;\">Year 3<\/th>\n<th style=\"text-align: left;\">Year 4<\/th>\n<th style=\"text-align: left;\">Year 5<\/th>\n<th style=\"text-align: left;\">Year 6<\/th>\n<th style=\"text-align: left;\">Total<\/th>\n<\/tr>\n<\/thead>\n<tbody>\n<tr>\n<td style=\"text-align: left;\">School 1<br \/>\nAccessible small town\/medium\u2013low deprivation<\/td>\n<td style=\"text-align: left;\">207<br \/>\n24.1%<\/td>\n<td style=\"text-align: left;\">184<br \/>\n21.5%<\/td>\n<td style=\"text-align: left;\">193<br \/>\n22.5%<\/td>\n<td style=\"text-align: left;\">129<br \/>\n15.0%<\/td>\n<td style=\"text-align: left;\">93<br \/>\n10.8%<\/td>\n<td style=\"text-align: left;\">52<br \/>\n6.1%<\/td>\n<td style=\"text-align: left;\">858<br \/>\n100%<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">School 2Urban\/medium\u2013low deprivation<\/td>\n<td style=\"text-align: left;\">147<br \/>\n19.9%<\/td>\n<td style=\"text-align: left;\">175<br \/>\n23.7%<\/td>\n<td style=\"text-align: left;\">136<br \/>\n18.4%<\/td>\n<td style=\"text-align: left;\">134<br \/>\n18.2%<\/td>\n<td style=\"text-align: left;\">85<br \/>\n11.5%<\/td>\n<td style=\"text-align: left;\">61<br \/>\n8.3%<\/td>\n<td style=\"text-align: left;\">738<br \/>\n100%<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">School 3<br \/>\nOther urban\/high deprivation<\/td>\n<td style=\"text-align: left;\">177<br \/>\n26.3%<\/td>\n<td style=\"text-align: left;\">160<br \/>\n23.8%<\/td>\n<td style=\"text-align: left;\">106<br \/>\n15.8%<\/td>\n<td style=\"text-align: left;\">125<br \/>\n18.6%<\/td>\n<td style=\"text-align: left;\">62<br \/>\n9.2%<\/td>\n<td style=\"text-align: left;\">42<br \/>\n6.3%<\/td>\n<td style=\"text-align: left;\">672<br \/>\n100%<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">School 4<br \/>\nUrban\/high deprivation<\/td>\n<td style=\"text-align: left;\">151<br \/>\n20.6%<\/td>\n<td style=\"text-align: left;\">197<br \/>\n26.9%<\/td>\n<td style=\"text-align: left;\">122<br \/>\n16.6%<\/td>\n<td style=\"text-align: left;\">126<br \/>\n17.2%<\/td>\n<td style=\"text-align: left;\">94<br \/>\n12.8%<\/td>\n<td style=\"text-align: left;\">43<br \/>\n5.9%<\/td>\n<td style=\"text-align: left;\">733<br \/>\n100%<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Total<\/td>\n<td style=\"text-align: left;\">682<\/td>\n<td style=\"text-align: left;\">716<\/td>\n<td style=\"text-align: left;\">557<\/td>\n<td style=\"text-align: left;\">513<\/td>\n<td style=\"text-align: left;\">333<\/td>\n<td style=\"text-align: left;\">197<\/td>\n<td style=\"text-align: left;\">3001<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Mean age (SD)<\/td>\n<td style=\"text-align: left;\">12.5 (0.34)<\/td>\n<td style=\"text-align: left;\">13.5<br \/>\n(0.34)<\/td>\n<td style=\"text-align: left;\">14.6<br \/>\n(0.34)<\/td>\n<td style=\"text-align: left;\">15.6<br \/>\n(0.35)<\/td>\n<td style=\"text-align: left;\">16.6<br \/>\n(0.36)<\/td>\n<td style=\"text-align: left;\">17.6<br \/>\n(0.32)<\/td>\n<td style=\"text-align: left;\">14.4<br \/>\n(1.58)<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\nEthical approval was obtained from the University of St Andrews, University Teaching and Research Ethics Committee (UTREC). Parental opt-out consent was obtained prior to pupils completing the survey. Pupils also provided active consent by completing the survey.\n\n## Derivation of variables\n\n### Smoking status\n\nRespondents were asked \"Have you ever smoked cigarettes or hand-rolled cigarettes (roll-ups), even if it is just one or two puffs?\" to which they could respond 'yes' or 'no'. Young people who responded 'no' were deemed to be never-smokers at that point.\n\n### E-cigarette use\n\nRespondents were asked whether or not they had heard of e-cigarettes. Pupils who answered that they had not heard of e-cigarettes were routed past further questions on e-cigarettes. Pupils that had heard of e-cigarettes were then asked \"Which ONE of the following is closest to describing your experience of e-cigarettes\/vapourisers\/shisha pens?\" with response options of 'I have never used them', 'I have tried them once or twice', 'I use them sometimes (more than once a month)' or 'I use them often (more than once a week)'. Young people who responded that they had never heard of e-cigarettes were coded as having 'never used them'.\n\nFor the logistic regression analysis, due to low frequencies in the categories reflecting regular use, participants were divided into those who had never tried e-cigarettes versus those who had tried e-cigarettes.\n\n### Susceptibility to smoking\n\nSusceptibility to smoking was assessed through two questions \"If one of your friends offered you a cigarette or hand-rolled cigarettes (roll-ups), would you smoke it?\" and \"Do you think you will smoke a cigarette or hand-rolled cigarettes (roll-ups) at any time during the next year?\". The response option for these questions was 'definitely yes', 'probably yes', 'probably not' and 'definitely not'. If respondents answered anything other than 'definitely not' to either of these questions then they were coded as being susceptible to smoking. These measures of smoking susceptibility have been used in related studies6 and are based on validated measures.21\n\n### Number of friends and family who smoke\n\nRespondents were asked \"How many of your friends smoke cigarettes or hand-rolled cigarettes (roll-ups)?\" and could respond 'most of them', 'about half of them', 'some of them', 'none of them' or 'don't know'. 'Don't know' responses were coded as missing and then a binary variable was generated distinguishing those who responded 'none of them' versus any other response.\n\nRespondents were asked \"which if any of the following people smoke cigarettes or hand rolled cigarettes (roll-ups)?\". Options included their mother or female carer, father or male carer, brother (eldest if more than one) and sister (eldest if more than one). A binary variable was created splitting participants who had responded that any of these family members smoked versus those that reported no smokers in their immediate family.\n\n### Demographic variables\n\nRespondents were asked their gender, ethnic group and date of birth. Individual family material well-being was assessed through the Family Affluence Scale\u00a0(FAS).22 The FAS consists of four questions (own bedroom, number of family cars, number of computers and number of family holidays abroad per year). The\u00a0FAS raw scores were transformed though categorical principal component analysis into single-dimensional scores that were then divided into tertiles of high, medium and low FAS.\n\n## Analysis\n\nAnalysis was conducted in Stata\u00a0V.14 (StataCorp).\n\nNever smokers were divided into those who had tried an e-cigarette at baseline and those who had not and these groups were compared in terms of the proportion of participants that reported having experimented with cigarettes by follow-up. Tobacco experimentation in this study was defined as any cigarette use, even just one or two puffs.\n\nMultivariate logistic regression was used to control for potential confounding factors\u2014sex, age, ethnicity, family affluence, smoking within the family, smoking by friends and susceptibility to smoking. The model was built in three blocks, first with only e-cigarette use and smoking-related variables as independent variables and in the second block demographic variables were added and an indicator for school was included in the model. Including school as a covariate makes explicit the effect of school as school-level smoking norms are an important influence on smoking behaviour.23 In the third block interactions between e-cigarette use, smoking susceptibility and smoking within friendship group were included. The risk ratio\u00a0(RR) for the unadjusted model was obtained from a binomial log-linear regression and for the adjusted models a Poisson regression model with a robust variance estimator.24\n\nTo test the effect of missing data on the parameter estimates, we used multiple imputation by chained equations (Stata\u00a0V.14: mi impute chained). Further information on the imputation procedure is given in the\u00a0online <a href=\"#SP1\" data-ref-type=\"supplementary-material\">supplementary materials<\/a>.\n\n10.1136\/tobaccocontrol-2017-053691.supp1\n\n# Results\n\n## Sample characteristics\n\nIn 2015, there were 3001 never smokers in our sample, of these 9.4% had tried an e-cigarette. Twenty-six per\u00a0cent were coded as susceptible to smoking, 32.8% had a family member who smoked and 23.8% reported having at least one friend who smoked.\n\nOur final sample included 2125 young people for whom we had data on e-cigarette use and smoking status at baseline and follow-up. Of these, 183 (8.6%) had tried an e-cigarette at baseline and 1942 (91.4%) had not. <a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a> shows the year group distribution of the sample by school.\n\n## Relationship between baseline e-cigarette use and smoking status at follow-up in baseline never smokers\n\nOf the young people who had tried an e-cigarette at baseline (n=183), 74 (40.4%) went on to initiate smoking cigarettes by follow-up. This compares with 249 (12.8%) of those who reported never having used an e-cigarette at baseline (n=1942) and went on to initiate smoking cigarettes by follow-up. <a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a> shows the bivariate relationship between e-cigarette use in 2015 and smoking status in 2016.\n\nBaseline e-cigarette use in 2015 and follow-up smoking status in 2016\n\n|  |  | Have you ever smoked cigarettes or roll-ups, even if it is just one or two puffs? (2016) |  | Total |\n|----|----|----|----|----|\n|  |  | No | Yes |  |\n| E-cigarette use (2015) | I have never used an e-cigarette | 1693 | 249 | 1942 |\n|  |  | 87.2% | 12.8% | 100% |\n|  | I have only used them once or twice | 104 | 65 | 169 |\n|  |  | 61.5% | 38.5% | 100% |\n|  | I use them sometimes (monthly) | 3 | 5 | 8 |\n|  |  | 37.5% | 62.5% | 100% |\n|  | I use them often (weekly) | 2 | 4 | 6 |\n|  |  | 33.3% | 66.7% | 100% |\n| Total |  | 1802 | 323 | 2125 |\n|  |  | 84.9% | 15.2% | 100% |\n\n## Logistic regression on 'experimented with cigarettes by follow-up'\n\nBaseline e-cigarette use is a significant predictor of experimentation with cigarettes. In an unadjusted model, the OR for ever-smoking at follow-up in ever e-cigarette users versus never e-cigarette users was 4.62 (95% CI 3.34 to 6.38), giving a RR of 3.15 (95% CI 2.55 to 3.89). <a href=\"#T3\" data-ref-type=\"table\">Table 3<\/a> below shows the ORs, p values and 95% CIs for the OR for each of the models. All the models below were adjusted for sex, age centred on the mean (ie, individual age minus the mean age of the sample)\u00a0FAS, ethnic group and school.\n\nMultivariate logistic regressions on 'ever smoked a cigarette' in 2016\n\n| Variable | Model 1\u2014adjusted main effects model (n=1806) |  |  | Model 2\u2014adjusted model including interactions (n=1806) |  |  | Model 3\u2014imputed model with interactions (n=2520) |  |  |\n|----|----|----|----|----|----|----|----|----|----|\n|  | OR | CI | p | OR | CI | p | OR | CI | p |\n| E-cigarette ever use 2015 | 2.42 | 1.63 to 3.60 | \\<0.001 | 5.97 | 3.12 to 11.40 | \\<0.001 | 6.64 | 3.60 to 12.26 | \\<0.001 |\n| Susceptibility to smoking 2015 | 3.65 | 2.70 to 4.94 | \\<0.001 | 4.13 | 2.98 to 5.72 | \\<0.001 | 5.19 | 3.74 to 7.21 | \\<0.001 |\n| Any family member smokes 2015 | 1.89 | 1.40 to 2.56 | \\<0.001 | 1.93 | 1.43 to 2.61 | \\<0.001 | 1.83 | 1\\. 37 to 2.44 | \\<0.001 |\n| 'At least some' friends smoke\u00a02015 | 1.33 | 0.95 to 1.85 | 0.094 | 1.56 | 1.09 to 2.25 | 0.016 | 1.51 | 1.07 to 2.14 | 0.020 |\n| Interaction between e-cigarette\u00a0and susceptibility |  |  |  | 0.42 | 0.19 to 0.94 | 0.036 | 0.42 | 0.20 to 0.88 | 0.021 |\n| Interaction between e-cigarette and friends smoking |  |  |  | 0.49 | 0.23 to 1.07 | 0.072 | 0.52 | 0.25 to 1.09 | 0.082 |\n\nModel 1 RR for e-cigarette use is 1.72 (95% CI 1.31 to 2.26), model 2 RR for e-cigarette use is 4.09 (95% CI 2.57 to 6.52), RR for e-cigarette\\*susceptibility interaction is 0.43 (95% CI 0.25 to 0.72), RR for e-cigarette\\*friend smokes interaction 0.62 (95% CI 0.39 to 0.99), model 3 RR for e-cigarette use is 4.22 (95% CI 2.83 to 6.36), RR for e-cigarette\\*susceptibility interaction is 0.41 (95% CI 0.26 to 0.64) and RR for e-cigarette\\*friend smokes interaction 0.65 (95% CI 0.44 to 0.97).\n\n<a href=\"#F1\" data-ref-type=\"fig\">Figure 1<\/a> shows that the impact of having tried an e-cigarette at baseline on probability of tobacco experimentation at follow-up is much greater for young people who were non-susceptible to smoking at baseline. The contrast of predicted probabilities is significant (\u03c7^2^=53.93,\u2009p\\<0.001).\n\n<a href=\"#F2\" data-ref-type=\"fig\">Figure 2<\/a> shows that the impact of having tried an e-cigarette at baseline on probability of tobacco experimentation at follow-up is much greater for young people who have no friends who smoke. The contrast of predicted probabilities is significant (\u03c7^2^=4.91, p=0.042).\n\nFurther information on characteristics of missing cases is given in online\u00a0<a href=\"#SP1\" data-ref-type=\"supplementary-material\">supplementary materials<\/a>. To test the effect of missing data on our parameter estimates we used multiple imputation by chained equations. Model 3 shows the estimates from an imputed model (m=100). The model estimates are stable under complete case analysis and imputation.\n\n# Discussion\n\nThis study found that young 'never-smokers' who had tried an e-cigarette were more likely to try a cigarette during the following year than young never-smokers who had not tried an e-cigarette. This is consistent with the results of all previous published longitudinal studies of the relationship between e-cigarettes and tobacco experimentation in young people from the USA,3\u20139 providing further confirmation in a non-US context.\n\nIt is possible that the relationship between e-cigarettes and tobacco experimentation may not be causal if young never-smokers who try an e-cigarette would have gone on to initiate smoking anyway due to being already favourably disposed towards tobacco use. In other words, it is possible that e-cigarette use and tobacco experimentation have common liability25 and the former is incidental to tobacco experimentation. To address this possibility, we controlled for factors associated with transition to smoking such as smoking susceptibility26 and smoking among friends and family27 in the analysis. However, even when these items were included in the model e-cigarette use remained a significant predictor of cigarette experimentation. Importantly, there was also an interaction between smoking susceptibility and e-cigarette use and between e-cigarette use and having friends who smoked. These data indicate that e-cigarette use had a greater effect on the odds of cigarette experimentation in young people not traditionally thought to be high risk,\u00a0that is, those with a firm intention not to smoke and\/or those with no smokers in their friendship group.\n\nThere is some evidence from other studies that young people who try e-cigarettes before tobacco have different characteristics to those who go straight to smoking. Wills and colleagues28 found that those who used an e-cigarette first were less rebellious and more likely to receive social support from their parents. Miech and colleagues10 found that young never-smokers who had tried e-cigarettes were more likely to move away from the perception that cigarettes were a 'great risk' over the following year. Wills and colleagues29 also found that young never smokers who used e-cigarettes were also more likely to increase their positive smoking expectancies (such as beliefs that smoking would make them more confident, help them relax and reduce boredom) and were more likely to become friends with smokers and subsequently try smoking. However, with only 1-year follow-up these studies were not able to determine whether changes in expectancies or affiliations preceded smoking. Further research on this topic is required over longer follow-up periods.\n\nSchneider and Diehl have outlined a 'catalyst model' of e-cigarette influence on smoking uptake in adolescence.30 This is intended as an alternative to 'gateway theory'31 32 as an explanation of the relationship between e-cigarette and tobacco use. They break the process down into two stages: factors influencing transition from 'no use' to 'e-cigarette use' and then the factors influencing the second stage of transition from 'e-cigarette use' to 'tobacco use'. The first-stage mechanisms include easing the process of initial trial, for example, with sweet flavours. The second-stage mechanisms include increased accessibility and learning of smoking rituals. Thus, there are a number of paths within the catalyst model whereby e-cigarette use, even single trial, might facilitate smoking uptake. There are also pathways by which e-cigarettes could mitigate against a transition to regular smoking. For those young people who are curious to try the performative aspects of smoking (the hand to mouth action and inhalation process), the act of trying e-cigarettes may result in lower motivation to try tobacco smoking.\n\nLevy and colleagues have modelled the public health impacts of e-cigarettes and estimate that under a range of conditions, e-cigarettes may have a positive net impact on public health at a population level because of the greater benefits conferred on smokers relative to the potential harm to young people.33 Further studies could usefully examine e-cigarette use, smoking and smoking-related attitudes over longer time periods to determine the conditions under which e-cigarettes enhance adult quit rates without facilitating uptake in young people.\n\nThe importance of research findings about the relationship between e-cigarette use and smoking initiation has been debated on the basis that most e-cigarette use among young people is occasional and therefore unlikely to be directly harmful or be sufficient to influence other behaviours. However, some argue that the influence of e-cigarette experimentation may be psychosocial rather than chemical; it has been suggested that e-cigarettes '(convey) to young apprehensive would-be smokers that nicotine is a benign drug and potentially weaken the established message that smoking kills'.34 E-cigarette advertising has emphasised the commonalities between the products with the message that e-cigarettes can give the psychological and social benefits of smoking without the health or social costs.35 There are some signs that these messages confuse young people about the harms of smoking. For example, a recent study found that after viewing an e-cigarette advert young people were more likely to rate occasional cigarette smoking as less harmful.36\n\nAt the time this research was conducted there were no legal restrictions on sales or advertisement of e-cigarettes. However, in the UK e-cigarettes are now banned from sale to people under 1837 38 and advertising on television, print media and radio is prohibited under the Tobacco Products Directive and associated UK regulations,39 40 although at present point-of-sale marketing is still permitted. It will be important to ascertain if this legislation is sufficient to prevent or reduce the numbers of young people trying e-cigarettes.\n\n## Strengths and limitations\n\nThe strengths of this study are its prospective design, large sample and high response and follow-up rates. Importantly, the multiple imputation models indicate that model estimates are not biased by missing data. However, there are a number of limitations. First, most of the young people whom we categorised as having initiated smoking may have only taken one or two puffs of a cigarette during the follow-up period. Therefore, we do not know whether any of these young people will transition to regular smoking. Transition from never-smoker to smoker is often conceptualised as a multistep pathway.41\u201343 Recent research suggests that any experimentation with cigarettes is a strong predictor of transition to regular smoking, with experimentation at baseline identifying two-thirds of regular smokers at 2-year follow-up with a false positive rate of only 8%.44\n\nSecond, participants were drawn from only four schools in Scotland and therefore may not be representative of the Scottish school population. However, comparison of the demographic characteristics of our sample with a nationally representative one does not indicate any significant deviation.45 Third, the study is based on self-reports and we do not yet know the reliability of young people's self-reported use of e-cigarettes.\n\nThe age range of the sample (11\u201318 years) is broader than in some other research in this area. Therefore, we split our sample in half by age and repeated the analysis on the split samples. The results we obtained were the same and are presented in the online\u00a0<a href=\"#SP1\" data-ref-type=\"supplementary-material\">supplementary materials<\/a>.\n\nFinally, although we have used validated measures of smoking susceptibility, they were developed more than 20 years ago and there may be other aspects of common liability to tobacco and e-cigarette use that are not assessed by existing measures of susceptibility.\n\n# Conclusions\n\nThis UK longitudinal study found that young never-smokers who try e-cigarettes are at elevated risk of initiating smoking compared with young never-smokers who do not try e-cigarettes. Further research with longer follow-up is required to discover how many of the full sample of young people, if any, transition to regular smoking and to explore the longitudinal relationship between use of e-cigarettes and changes in attitudes to smoking. Careful and regular monitoring of smoking rates and e-cigarette use among young people is necessary over the coming years. This needs to be set within the context of the rapidly changing landscape of tobacco and nicotine product availability, recent changes in the regulation of advertising and strategies used by industry, particularly the tobacco industry, to promote these products.\n\n###### What this paper adds\n\n-   Eight prospective studies in the USA have reported a temporal relationship between trying an e-cigarette and subsequent experimentation with cigarettes.\n\n-   Consistent with the US studies, this study indicates a positive relationship between e-cigarette use in never smokers and their subsequent first experimentation with cigarettes by follow-up 1\u2009year later.\n\n-   This UK study found that e-cigarette use had a greater impact on the odds of cigarette experimentation in young never smokers not traditionally thought to be high risk, that is, those with a firm intention not to smoke and\/or no smokers in their friendship group.\n\n# References","meta":{"dup_signals":{"dup_doc_count":105,"dup_dump_count":46,"dup_details":{"curated_sources":1,"2023-50":4,"2023-40":2,"2023-23":4,"2023-14":5,"2023-06":3,"2022-33":3,"2022-27":4,"2022-21":1,"2022-05":4,"2021-49":1,"2021-43":3,"2021-39":3,"2021-31":4,"2021-25":1,"2021-21":2,"2021-17":3,"2021-04":2,"2020-45":2,"2020-40":2,"2020-34":4,"2020-29":2,"2020-24":3,"2020-05":2,"2019-51":1,"2019-47":1,"2019-43":2,"2019-35":1,"2019-30":1,"2019-22":1,"2019-18":2,"2019-13":1,"2019-09":1,"2018-51":3,"2018-47":3,"2018-43":1,"2018-39":3,"2018-34":2,"2018-30":3,"2018-26":2,"2018-22":1,"2018-17":1,"2018-13":2,"2018-09":2,"2018-05":1,"2017-51":3,"2017-47":2}},"file":"PMC6047138"},"subset":"pubmed_central"}
{"text":"abstract: Cenozoic temperature, sea level and CO~2~ covariations provide insights into climate sensitivity to external forcings and sea-level sensitivity to climate change. Climate sensitivity depends on the initial climate state, but potentially can be accurately inferred from precise palaeoclimate data. Pleistocene climate oscillations yield a fast-feedback climate sensitivity of 3\u00b11^\u00b0^C for a 4\u2009W\u2009m^\u22122^ CO~2~ forcing if Holocene warming relative to the Last Glacial Maximum (LGM) is used as calibration, but the error (uncertainty) is substantial and partly subjective because of poorly defined LGM global temperature and possible human influences in the Holocene. Glacial-to-interglacial climate change leading to the prior (Eemian) interglacial is less ambiguous and implies a sensitivity in the upper part of the above range, i.e. 3\u20134^\u00b0^C for a 4\u2009W\u2009m^\u22122^ CO~2~ forcing. Slow feedbacks, especially change of ice sheet size and atmospheric CO~2~, amplify the total Earth system sensitivity by an amount that depends on the time scale considered. Ice sheet response time is poorly defined, but we show that the slow response and hysteresis in prevailing ice sheet models are exaggerated. We use a global model, simplified to essential processes, to investigate state dependence of climate sensitivity, finding an increased sensitivity towards warmer climates, as low cloud cover is diminished and increased water vapour elevates the tropopause. Burning all fossil fuels, we conclude, would make most of the planet uninhabitable by humans, thus calling into question strategies that emphasize adaptation to climate change.\nauthor: James Hansen; Makiko Sato; Gary Russell; Pushker Kharechae-mail: <firstname.lastname@example.com>[^1]\ndate: 2013-10-28\ninstitute: 1The Earth Institute, Columbia University, New York, NY 10027, USA; 2NASA Goddard Institute for Space Studies, New York, NY 10027, USA\nreferences:\ntitle: Climate sensitivity, sea level and atmospheric carbon dioxide\n\n# Introduction\n\nHumanity is now the dominant force driving changes in the Earth's atmospheric composition and climate\u00a0\\[1\\]. The largest climate forcing today, i.e. the greatest imposed perturbation of the planet's energy balance\u00a0\\[1,2\\], is the human-made increase in atmospheric greenhouse gases (GHGs), especially CO~2~ from the burning of fossil fuels.\n\nEarth's response to climate forcings is slowed by the inertia of the global ocean and the great ice sheets on Greenland and Antarctica, which require centuries, millennia or longer to approach their full response to a climate forcing. This long response time makes the task of avoiding dangerous human alteration of climate particularly difficult, because the human-made climate forcing is being imposed rapidly, with most of the current forcing having been added in just the past several decades. Thus, observed climate changes are only a partial response to the current climate forcing, with further response still 'in the pipeline'\u00a0\\[3\\].\n\nClimate models, numerical climate simulations, provide one way to estimate the climate response to forcings, but it is difficult to include realistically all real-world processes. Earth's palaeoclimate history allows empirical assessment of climate sensitivity, but the data have large uncertainties. These approaches are usually not fully independent, and the most realistic eventual assessments will be ones combining their greatest strengths.\n\nWe use the rich climate history of the Cenozoic era in the oxygen isotope record of ocean sediments to explore the relation of climate change with sea level and atmospheric CO~2~, inferring climate sensitivity empirically. We use isotope data from Zachos *et al.*\u00a0\\[4\\], which are improved over data used in our earlier study\u00a0\\[5\\], and we improve our prescription for separating the effects of deep ocean temperature and ice volume in the oxygen isotope record as well as our prescription for relating deep ocean temperature to surface air temperature. Finally, we use an efficient climate model to expand our estimated climate sensitivities beyond the Cenozoic climate range to snowball Earth and runaway greenhouse conditions.\n\n# Overview of Cenozoic climate and our analysis approach\n\nThe Cenozoic era, the past 65.5 million years (Myr), provides a valuable perspective on climate \\[5,6\\] and sea-level change\u00a0\\[7\\], and Cenozoic data help clarify our analysis approach. The principal dataset we use is the temporal variation of the oxygen isotope ratio (*\u03b4*^18^O relative to *\u03b4*^16^O; <a href=\"#RSTA20120294F1\" data-ref-type=\"fig\">figure\u00a01<\/a>*a* right-hand scale) in the shells of deep-ocean-dwelling microscopic shelled animals (foraminifera) in a near-global compilation of ocean sediment cores\u00a0\\[4\\]. *\u03b4*^18^O yields an estimate of the deep ocean temperature (<a href=\"#RSTA20120294F1\" data-ref-type=\"fig\">figure\u00a01<\/a>*b*), as discussed in \u00a73. Note that coarse temporal resolution of *\u03b4*^18^O data in the intervals 7\u201317, 35\u201342 and 44\u201365\u2009Myr reduces the apparent amplitude of glacial\u2013interglacial climate fluctuations (see electronic supplementary material, figure S1). We use additional proxy measures of climate change to supplement the *\u03b4*^18^O data in our quantitative analyses.\n\nCarbon dioxide is involved in climate change throughout the Cenozoic era, both as a climate forcing and as a climate feedback. Long-term Cenozoic temperature trends, the warming up to about 50\u2009Myr before present (BP) and subsequent long-term cooling, are likely to be, at least in large part, a result of the changing natural source of atmospheric CO~2~, which is volcanic emissions that occur mainly at continental margins due to plate tectonics (popularly 'continental drift'); tectonic activity also affects the weathering sink for CO~2~ by exposing fresh rock. The CO~2~ tectonic source grew from 60 to 50\u2009Myr\u2009BP as India subducted carbonate-rich ocean crust while moving through the present Indian Ocean prior to its collision with Asia about 50\u2009Myr\u2009BP\u00a0\\[8\\], causing atmospheric CO~2~ to reach levels of the order of 1000\u2009ppm at 50\u2009Myr\u2009BP\u00a0\\[9\\]. Since then, atmospheric CO~2~ declined as the Indian and Atlantic Oceans have been major depocentres for carbonate and organic sediments while subduction of carbonate-rich crust has been limited mainly to small regions near Indonesia and Central America\u00a0\\[10\\], thus allowing CO~2~ to decline to levels as low as 170\u2009ppm during recent glacial periods\u00a0\\[11\\]. A climate forcing due to a CO~2~ change from 1000 to 170\u2009ppm is more than 10\u2009W\u2009m^\u22122^, which compares with forcings of the order of 1\u2009W\u2009m^\u22122^ for competing climate forcings during the Cenozoic era\u00a0\\[5\\], specifically long-term change of solar irradiance and change of planetary albedo (reflectance) owing to the overall minor displacement of continents in that era.\n\nSuperimposed on the long-term trends are occasional global warming spikes, 'hyperthermals', most prominently the Palaeocene\u2013Eocene Thermal Maximum (PETM) at approximately 56\u2009Myr\u2009BP\u00a0\\[12\\] and the Mid-Eocene Climatic Optimum at approximately 42\u2009Myr\u2009BP\u00a0\\[13\\], coincident with large temporary increases of atmospheric CO~2~. The most studied hyperthermal, the PETM, caused global warming of at least 5^\u00b0^C coincident with injection of a likely 4000\u20137000\u2009Gt of isotopically light carbon into the atmosphere and ocean\u00a0\\[14\\]. The size of the carbon injection is estimated from changes in the stable carbon isotope ratio ^13^C\/^12^C in sediments and from ocean acidification implied by changes in the ocean depth below which carbonate dissolution occurred.\n\nThe potential carbon source for hyperthermal warming that received most initial attention was methane hydrates on continental shelves, which could be destabilized by sea floor warming\u00a0\\[15\\]. Alternative sources include release of carbon from Antarctic permafrost and peat\u00a0\\[16\\]. Regardless of the carbon source(s), it has been shown that the hyperthermals were astronomically paced, spurred by coincident maxima in the Earth's orbit eccentricity and spin axis tilt\u00a0\\[17\\], which increased high-latitude insolation and warming. The PETM was followed by successively weaker astronomically paced hyperthermals, suggesting that the carbon source(s) partially recharged in the interim\u00a0\\[18\\]. A high temporal resolution sediment core from the New Jersey continental shelf\u00a0\\[19\\] reveals that PETM warming in at least that region began about 3000 years prior to a massive release of isotopically light carbon. This lag and climate simulations\u00a0\\[20\\] that produce large warming at intermediate ocean depths in response to initial surface warming are consistent with the concept of a methane hydrate role in hyperthermal events.\n\nThe hyperthermals confirm understanding about the long recovery time of the Earth's carbon cycle\u00a0\\[21\\] and reveal the potential for threshold or 'tipping point' behaviour with large amplifying climate feedback in response to warming\u00a0\\[22\\]. One implication is that if humans burn most of the fossil fuels, thus injecting into the atmosphere an amount of CO~2~ at least comparable to that injected during the PETM, the CO~2~ would stay in the surface carbon reservoirs (atmosphere, ocean, soil, biosphere) for tens of thousands of years, long enough for the atmosphere, ocean and ice sheets to fully respond to the changed atmospheric composition. In addition, there is the potential that global warming from fossil fuel CO~2~ could spur release of CH~4~ and CO~2~ from methane hydrates or permafrost. Carbon release during the hyperthermals required several thousand years, but that long injection time may have been a function of the pace of the astronomical forcing, which is much slower than the pace of fossil fuel burning.\n\nThe Cenozoic record also reveals the amplification of climate change that occurs with growth or decay of ice sheets, as is apparent at about 34\u2009Myr\u2009BP when the Earth became cool enough for large-scale glaciation of Antarctica and in the most recent 3\u20135\u2009Myr with the growth of Northern Hemisphere ice sheets. Global climate fluctuated in the 20\u2009Myr following Antarctic glaciation with warmth during the Mid-Miocene Climatic Optimum (MMCO, 15\u2009Myr\u2009BP) possibly comparable to that at 34\u2009Myr\u2009BP, as, for example, Germany became warm enough to harbour snakes and crocodiles that require an annual temperature of about 20^\u00b0^C or higher and a winter temperature more than 10^\u00b0^C\u00a0\\[23\\]. Antarctic vegetation in the MMCO implies a summer temperature of approximately 11^\u00b0^C warmer than today\u00a0\\[24\\] and annual sea surface temperatures ranging from 0^\u00b0^C to 11.5^\u00b0^C\u00a0\\[25\\].\n\nSuperimposed on the long-term trends, in addition to occasional hyperthermals, are continual high-frequency temperature oscillations, which are apparent in <a href=\"#RSTA20120294F1\" data-ref-type=\"fig\">figure\u00a01<\/a> after 34\u2009Myr\u2009BP, when the Earth became cold enough for a large ice sheet to form on Antarctica, and are still more prominent during ice sheet growth in the Northern Hemisphere. These climate oscillations have dominant periodicities, ranging from about 20 to 400\u2009kyr, that coincide with variations in the Earth's orbital elements\u00a0\\[26\\], specifically the tilt of the Earth's spin axis, the eccentricity of the orbit and the time of year when the Earth is closest to the Sun. The slowly changing orbit and tilt of the spin axis affect the seasonal distribution of insolation\u00a0\\[27\\], and thus the growth and decay of ice sheets, as proposed by Milankovitch\u00a0\\[28\\]. Atmospheric CO~2~, CH~4~ and N~2~O have varied almost synchronously with global temperature during the past 800\u2009000 years for which precise data are available from ice cores, the GHGs providing an amplifying feedback that magnifies the climate change instigated by orbit perturbations\u00a0\\[29\u201331\\].\n\nOcean and atmosphere dynamical effects have been suggested as possible causes of some climate change within the Cenozoic era; for example, topographical effects of mountain building\u00a0\\[32\\], closing of the Panama Seaway\u00a0\\[33\\] or opening of the Drake Passage\u00a0\\[34\\]. Climate modelling studies with orographic changes confirm significant effects on monsoons and on Eurasian temperature\u00a0\\[35\\]. Modelling studies indicate that closing of the Panama Seaway results in a more intense Atlantic thermohaline circulation, but only small effects on Northern Hemisphere ice sheets\u00a0\\[36\\]. Opening of the Drake Passage surely affected ocean circulation around Antarctica, but efforts to find a significant effect on global temperature have relied on speculation about possible effects on atmospheric CO~2~\u00a0\\[37\\]. Overall, there is no strong evidence that dynamical effects are a major direct contributor to Cenozoic global temperature change.\n\nWe hypothesize that the global climate variations of the Cenozoic (<a href=\"#RSTA20120294F1\" data-ref-type=\"fig\">figure\u00a01<\/a>) can be understood and analysed via slow temporal changes in Earth's energy balance, which is a function of solar irradiance, atmospheric composition (specifically long-lived GHGs) and planetary surface albedo. Using measured amounts of GHGs during the past 800\u2009000 years of glacial\u2013interglacial climate oscillations and surface albedo inferred from sea-level data, we show that a single empirical 'fast-feedback' climate sensitivity can account well for the global temperature change over that range of climate states. It is certain that over a large climate range climate sensitivity must become a strong function of the climate state, and thus we use a simplified climate model to investigate the dependence of climate sensitivity on the climate state. Finally, we use our estimated state-dependent climate sensitivity to infer Cenozoic CO~2~ change and compare this with proxy CO~2~ data, focusing on the Eocene climatic optimum, the Oligocene glaciation, the Miocene optimum and the Pliocene.\n\n# Deep ocean temperature and sea level in the Cenozoic era\n\nThe *\u03b4*^18^O stable isotope ratio was the first palaeothermometer, proposed by Urey\u00a0\\[38\\] and developed especially by Emiliani\u00a0\\[39\\]. There are now several alternative proxy measures of ancient climate change, but the *\u03b4*^18^O data (<a href=\"#RSTA20120294F1\" data-ref-type=\"fig\">figure\u00a01<\/a>*a*) of Zachos *et al.*\u00a0\\[4\\], a conglomerate of the global ocean sediment cores, is well suited for our purpose as it covers the Cenozoic era with good temporal resolution. There are large, even dominant, non-climatic causes of *\u03b4*^18^O changes over hundreds of millions of years\u00a0\\[40\\], but non-climatic change may be small in the past few hundred million years\u00a0\\[41\\] and is generally neglected in Cenozoic climate studies. The principal difficulty in using the *\u03b4*^18^O record to estimate global deep ocean temperature, in the absence of non-climatic change, is that *\u03b4*^18^O is affected by the global ice mass as well as the deep ocean temperature.\n\nWe make a simple estimate of global sea-level change for the Cenozoic era using the near-global *\u03b4*^18^O compilation of Zachos *et al.*\u00a0\\[4\\]. More elaborate and accurate approaches, including use of models, will surely be devised, but comparison of our result with other approaches is instructive regarding basic issues such as the vulnerability of today's ice sheets to near-term global warming and the magnitude of hysteresis effects in ice sheet growth and decay.\n\nDuring the Early Cenozoic, between 65.5 and 35\u2009Myr\u2009BP, the Earth was so warm that there was little ice on the planet and the deep ocean temperature is approximated by\u00a0\\[6\\]\n\n![](rsta20120294-e1.jpg)\n\nHansen *et al.*\u00a0\\[5\\] made the approximation that, as the Earth became colder and continental ice sheets grew, further increase in *\u03b4*^18^O was due, in equal parts, to deep ocean temperature change and ice mass change,\n\n![](rsta20120294-e2.jpg)\n\nEqual division of the *\u03b4*^18^O change into temperature change and ice volume change was suggested by comparing *\u03b4*^18^O at the endpoints of the climate change from the nearly ice-free planet at 35\u2009Myr\u2009BP (when *\u03b4*^18^O approx. 1.75) with the Last Glacial Maximum (LGM), which peaked approximately 20\u2009kyr\u2009BP. The change of *\u03b4*^18^O between these two extreme climate states (approx.\u00a03) is twice the change of *\u03b4*^18^O due to temperature change alone (approx.\u00a01.5), with the temperature change based on the linear relation (??eq3.1) and estimates of *T*~do~\u223c5^\u00b0^C at 35\u2009Myr\u2009BP (<a href=\"#RSTA20120294F1\" data-ref-type=\"fig\">figure\u00a01<\/a>) and approximately \u22121^\u00b0^C at the LGM\u00a0\\[42\\].\n\nThis approximation can easily be made more realistic. Although ice volume and deep ocean temperature changes contributed comparable amounts to *\u03b4*^18^O change on average over the full range from 35\u2009Myr to 20\u2009kyr\u2009BP, the temperature change portion of the *\u03b4*^18^O change must decrease as the deep ocean temperature approaches the freezing point\u00a0\\[43\\]. The rapid increase in *\u03b4*^18^O in the past few million years was associated with the appearance of Northern Hemisphere ice sheets, symbolized by the dark blue bar in <a href=\"#RSTA20120294F1\" data-ref-type=\"fig\">figure\u00a01<\/a>*a*.\n\nThe sea-level change between the LGM and Holocene was approximately 120\u2009m\u00a0\\[44,45\\]. Thus, two-thirds of the 180\u2009m sea-level change between the ice-free planet and the LGM occurred with formation of Northern Hemisphere ice (and probably some increased volume of Antarctic ice). Thus, rather than taking the 180\u2009m sea-level change between the nearly ice-free planet of 34\u2009Myr\u2009BP and the LGM as being linear over the entire range (with 90\u2009m for *\u03b4*^18^O\\<3.25 and 90\u2009m for *\u03b4*^18^O\\>3.25), it is more realistic to assign 60\u2009m of sea-level change to *\u03b4*^18^O 1.75\u20133.25 and 120\u2009m to *\u03b4*^18^O\\>3.25. The total deep ocean temperature change of 6^\u00b0^C for the change of *\u03b4*^18^O from 1.75 to 4.75 is then divided two-thirds (4^\u00b0^C) for the *\u03b4*^18^O range 1.75\u20133.25 and 2^\u00b0^C for the *\u03b4*^18^O range 3.25\u20134.75. Algebraically,\n\n![](rsta20120294-e3.jpg)\n\n![](rsta20120294-e4.jpg)\n\n![](rsta20120294-e5.jpg)\n\nand\n\n![](rsta20120294-e6.jpg)\n\nwhere SL is the sea level and its zero point is the Late Holocene level. The coefficients in equations\u00a0(<a href=\"#RSTA20120294M3x3\" data-ref-type=\"disp-formula\">3.4<\/a>) and\u00a0(<a href=\"#RSTA20120294M3x6\" data-ref-type=\"disp-formula\">3.6<\/a>) account for the fact that the mean LGM value of *\u03b4*^18^O is approximately 4.9. The resulting deep ocean temperature is shown in <a href=\"#RSTA20120294F1\" data-ref-type=\"fig\">figure\u00a01<\/a>*b* for the full Cenozoic era.\n\nSea level from equations\u00a0(<a href=\"#RSTA20120294M3x3\" data-ref-type=\"disp-formula\">3.3<\/a>) and\u00a0(<a href=\"#RSTA20120294M3x4\" data-ref-type=\"disp-formula\">3.4<\/a>) is shown by the blue curves in <a href=\"#RSTA20120294F2\" data-ref-type=\"fig\">figure\u00a02<\/a>, including comparison (<a href=\"#RSTA20120294F2\" data-ref-type=\"fig\">figure\u00a02<\/a>*c*) with the Late Pleistocene sea-level record of Rohling *et al.*\u00a0\\[47\\], which is based on analysis of Red Sea sediments, and comparison (<a href=\"#RSTA20120294F2\" data-ref-type=\"fig\">figure\u00a02<\/a>*b*) with the sea-level chronology of de Boer\u00a0*et al.*\u00a0\\[46\\], which is based on ice sheet modelling with the *\u03b4*^18^O data of Zachos *et al.*\u00a0\\[4\\] as a principal input driving the ice sheet model. Comparison of our result with that of de Boer *et al.*\u00a0\\[46\\] for the other periods of <a href=\"#RSTA20120294F2\" data-ref-type=\"fig\">figure\u00a02<\/a> is included in the electronic supplementary material, where we also make available our numerical data. Deep ocean temperature from equations\u00a0(<a href=\"#RSTA20120294M3x5\" data-ref-type=\"disp-formula\">3.5<\/a>) and\u00a0(<a href=\"#RSTA20120294M3x6\" data-ref-type=\"disp-formula\">3.6<\/a>) is shown for the Pliocene and Pleistocene in <a href=\"#RSTA20120294F3\" data-ref-type=\"fig\">figure\u00a03<\/a> and for the entire Cenozoic era in\u00a0<a href=\"#RSTA20120294F1\" data-ref-type=\"fig\">figure\u00a01<\/a>.\n\nDifferences between our inferred sea-level chronology and that from the ice sheet model\u00a0\\[46\\] are relevant to the assessment of the potential danger to humanity from future sea-level rise. Our estimated sea levels have reached +5 to 10\u2009m above the present sea level during recent interglacial periods that were barely warmer than the Holocene, whereas the ice sheet model yields maxima at most approximately 1\u2009m above the current sea level. We find the Pliocene sea level varying between about +20\u2009m and \u221250\u2009m, with the Early Pliocene averaging about +15\u2009m; the ice sheet model has a less variable sea level with the Early Pliocene averaging about +8\u2009m. A 15\u2009m sea-level rise implies that the East Antarctic ice sheet as well as West Antarctica and Greenland ice were unstable at a global temperature no higher than those projected to occur this century\u00a0\\[1,48\\].\n\nHow can we interpret these differences, and what is the merit of our simple *\u03b4*^18^O scaling? Ice sheet models constrained by multiple observations may eventually provide our best estimate of sea-level change, but as yet models are primitive. Hansen\u00a0\\[49,50\\] argues that real ice sheets are more responsive to climate change than is found in most ice sheet models. Our simple scaling approximation implicitly assumes that ice sheets are sufficiently responsive to climate change that hysteresis is not a dominant effect; in other words, ice volume on millennial time scales is a function of temperature and does not depend much on whether the Earth is in a warming or cooling phase. Thus, our simple transparent calculation may provide a useful comparison with geological data for sea-level change and with results of ice sheet models.\n\nWe cannot *a priori* define accurately the error in our sea-level estimates, but we can compare with geological data in specific cases as a check on reasonableness. Our results (<a href=\"#RSTA20120294F2\" data-ref-type=\"fig\">figure\u00a02<\/a>) yield two instances in the past million years when sea levels have reached heights well above the current sea level: +9.8\u2009m in the Eemian (approx. 120\u2009kyr\u2009BP, also known as Marine Isotope Stage 5e or MIS-5e) and +7.1\u2009m in the Holsteinian (approx. 400\u2009kyr\u2009BP, also known as MIS-11). Indeed, these are the two interglacial periods in the Late Pleistocene that traditional geological methods identify as probably having a sea level exceeding that in the Holocene. Geological evidence, mainly coral reefs on tectonically stable coasts, was described in the review of Overpeck *et al.*\u00a0\\[51\\] as favouring an Eemian maximum of +4 to more than 6\u2009m. Rohling *et al.*\u00a0\\[52\\] cite many studies concluding that the mean sea level was 4\u20136\u2009m above the current sea level during the warmest portion of the Eemian, 123\u2013119\u2009kyr\u2009BP; note that several of these studies suggest Eemian sea-level fluctuations up to +10\u2009m, and provide the first continuous sea-level data supporting rapid Eemian sea-level fluctuations. Kopp *et al.*\u00a0\\[53\\] made a statistical analysis of data from a large number of sites, concluding that there was a 95% probability that the Eemian sea level reached at least +6.6\u2009m with a 67% probability that it exceeded 8\u2009m.\n\nThe Holsteinian sea level is more difficult to reconstruct from geological data because of its age, and there has been a long-standing controversy concerning a substantial body of geological shoreline evidence for a +20\u2009m Late Holsteinian sea level that Hearty and co-workers have found on numerous sites\u00a0\\[54,55\\] (numerous pros and cons are contained in the references provided in our present paragraph). Rohling *et al.*\u00a0\\[56\\] note that their temporally continuous Red Sea record 'strongly supports the MIS-11 sea level review of Bowen\u00a0\\[57\\], which also places MIS-11 sea level within uncertainties at the present-day level'. This issue is important because both ice core data\u00a0\\[29\\] and ocean sediment core data (see below) indicate that the Holsteinian period was only moderately warmer than the Holocene with similar Earth orbital parameters. We suggest that the resolution of this issue is consistent with our estimate of the approximately +7\u2009m Holsteinian global sea level, and is provided by Raymo & Mitrovica\u00a0\\[58\\], who pointed out the need to make a glacial isostatic adjustment (GIA) correction for post-glacial crustal subsidence at the places where Hearty and others deduced local sea-level change. The uncertainties in GIA modelling led Raymo & Mitrovica\u00a0\\[58\\] to conclude that the peak Holsteinian global sea level was in the range of +6 to 13\u2009m relative to the present. Thus, it seems to us, there is a reasonable resolution of the long-standing Holsteinian controversy, with substantial implications for humanity, as discussed in later sections.\n\nWe now address differences between our sea-level estimates and those from ice sheet models. We refer to both the one-dimensional ice sheet modelling of de Boer *et al.*\u00a0\\[46\\], which was used to calculate sea level for the entire Cenozoic era, and the three-dimensional ice sheet model of Bintanja *et al.*\u00a0\\[59\\], which was used for simulations of the past million years. The differences most relevant to humanity occur in the interglacial periods slightly warmer than the Holocene, including the Eemian and Hosteinian, as well as the Pliocene, which may have been as warm as projected for later this century. Both the three-dimensional model of Bintanja *et al.*\u00a0\\[59\\] and the one-dimensional model of de Boer *et al.*\u00a0\\[46\\] yield maximum Eemian and Hosteinian sea levels of approximately 1\u2009m relative to the Holocene. de Boer *et al.*\u00a0\\[46\\] obtain approximately +8\u2009m for the Early Pliocene, which compares with our approximately +15\u2009m.\n\nThese differences reveal that the modelled ice sheets are less susceptible to change in response to global temperature variation than our *\u03b4*^18^O analysis. Yet the ice sheet models do a good job of reproducing the sea-level change for climates colder than the Holocene, as shown in <a href=\"#RSTA20120294F2\" data-ref-type=\"fig\">figure\u00a02<\/a> and electronic supplementary material, figure S2. One possibility is that the ice sheet models are too lethargic for climates warmer than the Holocene. Hansen & Sato\u00a0\\[60\\] point out the sudden change in the responsiveness of the ice sheet model of Bintanja *et al.*\u00a0\\[59\\] when the sea level reaches today's level (figs\u00a03 and\u00a04 of Hansen & Sato\u00a0\\[60\\]) and they note that the empirical sea-level data provide no evidence of such a sudden change. The explanation conceivably lies in the fact that the models have many parameters and their operation includes use of 'targets'\u00a0\\[46\\] that affect the model results, because these choices might yield different results for warmer climates than the results for colder climates. Because of the potential that model development choices might be influenced by expectations of a 'correct' result, it is useful to have estimates independent of the models based on alternative assumptions.\n\nNote that our approach also involves 'targets' based on expected behaviour, albeit simple transparent ones. Our two-legged linear approximation of the sea level (equations\u00a0(<a href=\"#RSTA20120294M3x3\" data-ref-type=\"disp-formula\">3.3<\/a>) and\u00a0(<a href=\"#RSTA20120294M3x4\" data-ref-type=\"disp-formula\">3.4<\/a>)) assumes that the sea level in the LGM was 120\u2009m lower than today and that the sea level was 60\u2009m higher than today 35\u2009Myr\u2009BP. This latter assumption may need to be adjusted if glaciers and ice caps in the Eocene had a volume of tens of metres of sea level. However, Miller *et al.*\u00a0\\[61\\] conclude that there was a sea level fall of approximately 55\u2009m at the Eocene\u2013Oligocene transition, consistent with our assumption that Eocene ice probably did not contain more than approximately 10\u2009m of sea level.\n\nReal-world data for the Earth's sea-level history ultimately must provide assessment of sea-level sensitivity to climate change. A recent comprehensive review\u00a0\\[7\\] reveals that there are still wide uncertainties about the Earth's sea-level history that are especially large for time scales of tens of millions of years or longer, which is long enough for substantial changes in the shape and volume of ocean basins. Gasson *et al.*\u00a0\\[7\\] plot regional (New Jersey) sea level (their fig.\u00a014) against the deep ocean temperature inferred from the magnesium\/calcium ratio (Mg\/Ca) of deep ocean foraminifera\u00a0\\[62\\], finding evidence for a nonlinear sea-level response to temperature roughly consistent with the modelling of de Boer *et al.*\u00a0\\[46\\]. Sea-level change is limited for Mg\/Ca temperatures up to about 5^\u00b0^C above current values, whereupon a rather abrupt sea-level rise of several tens of metres occurs, presumably representing the loss of Antarctic ice. However, the uncertainty in the reconstructed sea level is tens of metres and the uncertainty in the Mg\/Ca temperature is sufficient to encompass the result from our *\u03b4*^18^O prescription, which has comparable contributions of ice volume change and deep ocean temperature change at the Late Eocene glaciation of Antarctica.\n\nFurthermore, the potential sea-level rise of most practical importance is the first 15\u2009m above the Holocene level. It is such 'moderate' sea-level change for which we particularly question the projections implied by current ice sheet models. Empirical assessment depends upon real-world sea-level data in periods warmer than the Holocene. There is strong evidence, discussed above, that the sea level was several metres higher in recent warm interglacial periods, consistent with our data interpretation. The Pliocene provides data extension to still warmer climates. Our interpretation of *\u03b4*^18^O data suggests that Early Pliocene sea-level change (due to ice volume change) reached about +15\u2009m, and it also indicates sea-level fluctuations as large as 20\u201340\u2009m. Sea-level data for Mid-Pliocene warm periods, of comparable warmth to average Early Pliocene conditions (<a href=\"#RSTA20120294F3\" data-ref-type=\"fig\">figure\u00a03<\/a>), suggest sea heights as great as +15\u201325\u2009m\u00a0\\[63,64\\]. Miller *et al.*\u00a0\\[61\\] find a Pliocene sea-level maximum of 22\u00b110\u2009m (95% confidence). GIA creates uncertainty in sea-level reconstructions based on shoreline geological data\u00a0\\[65\\], which could be reduced via appropriately distributed field studies. Dwyer & Chandler\u00a0\\[64\\] separate Pliocene ice volume and temperature in deep ocean *\u03b4*^18^O via ostracode Mg\/Ca temperatures, finding sea-level maxima and oscillations comparable to our results. Altogether, the empirical data provide strong evidence against the lethargy and strong hysteresis effects of at least some ice sheet models.\n\n# Surface air temperature change\n\nThe temperature of most interest to humanity is the surface air temperature. A record of past global surface temperature is required for empirical inference of global climate sensitivity. Given that climate sensitivity can depend on the initial climate state and on the magnitude and sign of the climate forcing, a continuous record of global temperature over a wide range of climate states would be especially useful. Because of the singularly rich climate story in Cenozoic deep ocean *\u03b4*^18^O (<a href=\"#RSTA20120294F1\" data-ref-type=\"fig\">figure\u00a01<\/a>), unrivalled in detail and self-consistency by alternative climate proxies, we use deep ocean *\u03b4*^18^O to provide the fine structure of Cenozoic temperature change. We use surface temperature proxies from the LGM, the Pliocene and the Eocene to calibrate and check the relation between deep ocean and surface temperature change.\n\nThe temperature signal in deep ocean *\u03b4*^18^O refers to the sea surface where cold dense water formed and sank to the ocean bottom, the principal location of deep water formation being the Southern Ocean. Empirical data and climate models concur that surface temperature change is generally amplified at high latitudes, which tends to make temperature change at the site of deep water formation an overestimate of global temperature change. Empirical data and climate models also concur that surface temperature change is amplified over land areas, which tends to make temperature change at the site of deep water an underestimate of the global temperature. Hansen *et al.*\u00a0\\[5\\] and Hansen & Sato\u00a0\\[60\\] noted that these two factors were substantially offsetting, and thus they made the assumption that benthic foraminifera provide a good approximation of global mean temperature change for most of the Cenozoic era.\n\nHowever, this approximation breaks down in the Late Cenozoic for two reasons. First, the deep ocean and high-latitude surface ocean where deep water forms are approaching the freezing point in the Late Cenozoic. As the Earth's surface cools further, cold conditions spread to lower latitudes but polar surface water and the deep ocean cannot become much colder, and thus the benthic foraminifera record a temperature change smaller than the global average surface temperature change\u00a0\\[43\\]. Second, the last 5.33\u2009Myr of the Cenozoic, the Pliocene and Pleistocene, was the time that global cooling reached a degree such that large ice sheets could form in the Northern Hemisphere. When a climate forcing, or a slow climate feedback such as ice sheet formation, occurs in one hemisphere, the temperature change is much larger in the hemisphere with the forcing (cf. examples in Hansen *et al.*\u00a0\\[66\\]). Thus, cooling during the last 5.33\u2009Myr in the Southern Ocean site of deep water formation was smaller than the global average cooling.\n\nWe especially want our global surface temperature reconstruction to be accurate for the Pliocene and Pleistocene because the global temperature changes that are expected by the end of this century, if humanity continues to rapidly change atmospheric composition, are of a magnitude comparable to climate change in those epochs\u00a0\\[1,48\\]. Fortunately, sufficient information is available on surface temperature change in the Pliocene and Pleistocene to allow us to scale the deep ocean temperature change by appropriate factors, thus retaining the temporal variations in the *\u03b4*^18^O while also having a realistic magnitude for the total temperature change over these epochs.\n\nPliocene temperature is known quite well because of a long-term effort to reconstruct the climate conditions during the Mid-Pliocene warm period (3.29\u20132.97\u2009Myr\u2009BP) and a coordinated effort to numerically simulate the climate by many modelling groups\u00a0(\\[67\\] and papers referenced therein). The reconstructed Pliocene climate used data for the warmest conditions found in the Mid-Pliocene period, which would be similar to average conditions in the Early Pliocene (<a href=\"#RSTA20120294F3\" data-ref-type=\"fig\">figure\u00a03<\/a>). These boundary conditions were used by eight modelling groups to simulate Pliocene climate with atmospheric general circulation models. Although atmosphere\u2013ocean models have difficulty replicating Pliocene climate, atmospheric models forced by specified surface boundary conditions are expected to be capable of calculating global surface temperature with reasonable accuracy. The eight global models yield Pliocene global warming of 3\u00b11^\u00b0^C relative to the Holocene\u00a0\\[68\\]. This Pliocene warming is an amplification by a factor of 2.5 of the deep ocean temperature change.\n\nSimilarly, for the reasons given above, the deep ocean temperature change of 2.25^\u00b0^C between the Holocene and the LGM is surely an underestimate of the surface air temperature change. Unfortunately, there is a wide range of estimates for LGM cooling, approximately 3\u20136^\u00b0^C, as discussed in \u00a76. Thus, we take 4.5^\u00b0^C as our best estimate for LGM cooling, implying an amplification of surface temperature change by a factor of two relative to deep ocean temperature change for this climate interval.\n\nWe obtain an absolute temperature scale using the Jones *et al.*\u00a0\\[69\\] estimate of 14^\u00b0^C as the global mean surface temperature for 1961\u20131990, which corresponds to approximately 13.9^\u00b0^C for the 1951\u20131980 base period that we normally use\u00a0\\[70\\] and approximately 14.4^\u00b0^C for the first decade of the twenty-first century. We attach the instrumental temperature record to the palaeo data by assuming that the first decade of the twenty-first century exceeds the Holocene mean by 0.25\u00b10.25^\u00b0^C. Global temperature probably declined over the past several millennia\u00a0\\[71\\], but we suggest that warming of the past century has brought global temperature to a level that now slightly exceeds the Holocene mean, judging from sea-level trends and ice sheet mass loss. Sea level is now rising 3.1\u2009mm per year or 3.1\u2009m per millennium\u00a0\\[72\\], an order of magnitude faster than the rate during the past several thousand years, and Greenland and Antarctica are losing mass at accelerating rates\u00a0\\[73,74\\]. Our assumption that global temperature passed the Holocene mean a few decades ago is consistent with the rapid change of ice sheet mass balance in the past few decades\u00a0\\[75\\]. The above concatenation of instrumental and palaeo records yields a Holocene mean of 14.15^\u00b0^C and Holocene maximum (from five-point smoothed *\u03b4*^18^O) of 14.3^\u00b0^C at 8.6\u2009kyr\u2009BP.\n\nGiven a Holocene temperature of 14.15^\u00b0^C and LGM cooling of 4.5^\u00b0^C, the Early Pliocene mean temperature 3^\u00b0^C warmer than the Holocene leads to the following prescription:\n\n![](rsta20120294-e7.jpg)\n\nand\n\n![](rsta20120294-e8.jpg)\n\nThis prescription yields a maximum Eemian temperature of 15.56^\u00b0^C, which is approximately 1.4^\u00b0^C warmer than the Holocene mean and approximately 1.8^\u00b0^C warmer than the 1880\u20131920 mean. Clark & Huybers\u00a0\\[76\\] fit a polynomial to proxy temperatures for the Eemian, finding warming as much as +5^\u00b0^C at high northern latitudes but global warming of +1.7^\u00b0^C 'relative to the present interglacial before industrialization'. Other analyses of Eemian data find global sea surface temperature warmer than the Late Holocene by 0.7\u00b10.6^\u00b0^C\u00a0\\[77\\] and all-surface warming of 2^\u00b0^C\u00a0\\[78\\], all in reasonable accord with our prescription.\n\nOur first estimate of global temperature for the remainder of the Cenozoic assumes that *\u0394T*~s~=*\u0394T*~do~ prior to 5.33\u2009Myr\u2009BP, i.e. prior to the Plio-Pleistocene, which yields a peak *T*~s~ of approximately 28^\u00b0^C at 50\u2009Myr\u2009BP (<a href=\"#RSTA20120294F4\" data-ref-type=\"fig\">figure\u00a04<\/a>). This is at the low end of the range of current multi-proxy measures of sea surface temperature for the Early Eocene Climatic Optimum (EECO)\u00a0\\[79\u201381\\]. Climate models are marginally able to reproduce this level of Eocene warmth, but the models require extraordinarily high CO~2~ levels, for example 2240\u20134480\u2009ppm\u00a0\\[82\\] and 2500\u20136500\u2009ppm\u00a0\\[83\\], and the quasi-agreement between data and models requires an assumption that some of the proxy temperatures are biased towards summer values. Moreover, taking the proxy sea surface temperature data for the peak Eocene period (55\u201348\u2009Myr\u2009BP) at face value yields a global temperature of 33\u201334^\u00b0^C (fig.\u00a03 of Bijl *et al.*\u00a0\\[84\\]), which would require an even larger CO~2~ amount with the same climate models. Thus, below we also consider the implications for climate sensitivity of an assumption that *\u0394T*~s~=1.5\u00d7*\u0394T*~do~ prior to 5.33\u2009Myr\u2009BP, which yields *T*~s~ approximately 33^\u00b0^C at 50\u2009Myr\u2009BP (see electronic supplementary material, figure S3).\n\n# Climate sensitivity\n\nClimate sensitivity (*S*) is the equilibrium global surface temperature change (*\u0394T*~eq~) in response to a specified unit forcing after the planet has come back to energy balance,\n\n![](rsta20120294-e9.jpg)\n\ni.e. climate sensitivity is the eventual (equilibrium) global temperature change per unit forcing. Climate sensitivity depends upon climate feedbacks, the many physical processes that come into play as climate changes in response to a forcing. Positive (amplifying) feedbacks increase the climate response, whereas negative (diminishing) feedbacks reduce the response.\n\nWe usually discuss climate sensitivity in terms of a global mean temperature response to a 4\u2009W\u2009m^\u22122^ CO~2~ forcing. One merit of this standard forcing is that its magnitude is similar to an anticipated near-term human-made climate forcing, thus avoiding the need to continually scale the unit sensitivity to achieve an applicable magnitude. A second merit is that the efficacy of forcings varies from one forcing mechanism to another\u00a0\\[66\\]; so it is useful to use the forcing mechanism of greatest interest. Finally, the 4\u2009W\u2009m^\u22122^ CO~2~ forcing avoids the uncertainty in the exact magnitude of a doubled CO~2~ forcing\u00a0\\[1,48\\] estimate of 3.7\u2009W\u2009m^\u22122^ for doubled CO~2~, whereas Hansen *et al.*\u00a0\\[66\\] obtain 4.1\u2009W\u2009m^\u22122^, as well as problems associated with the fact that a doubled CO~2~ forcing varies as the CO~2~ amount changes (the assumption that each CO~2~ doubling has the same forcing is meant to approximate the effect of CO~2~ absorption line saturation, but actually the forcing per doubling increases as CO~2~ increases\u00a0\\[66,85\\]).\n\nClimate feedbacks are the core of the climate problem. Climate feedbacks can be confusing, because in climate analyses what is sometimes a climate forcing is at other times a climate feedback. A CO~2~ decrease from, say, approximately 1000\u2009ppm in the Early Cenozoic to 170\u2013300\u2009ppm in the Pleistocene, caused by shifting plate tectonics, is a climate forcing, a perturbation of the Earth's energy balance that alters the temperature. Glacial\u2013interglacial oscillations of the CO~2~ amount and ice sheet size are both slow climate feedbacks, because glacial\u2013interglacial climate oscillations largely are instigated by insolation changes as the Earth's orbit and tilt of its spin axis change, with the climate change then amplified by a nearly coincident change of the CO~2~ amount and the surface albedo. However, for the sake of analysis, we can also choose and compare periods that are in quasi-equilibrium, periods during which there was little change of the ice sheet size or the GHG amount. For example, we can compare conditions averaged over several millennia in the LGM with mean Holocene conditions. The Earth's average energy imbalance within each of these periods had to be a small fraction of 1\u2009W\u2009m^\u22122^. Such a planetary energy imbalance is very small compared with the boundary condition 'forcings', such as changed GHG amount and changed surface albedo that maintain the glacial-to-interglacial climate change.\n\n## Fast-feedback sensitivity: Last Glacial Maximum\u2013Holocene\n\nThe average fast-feedback climate sensitivity over the LGM\u2013Holocene range of climate states can be assessed by comparing estimated global temperature change and climate forcing change between those two climate states\u00a0\\[3,86\\]. The appropriate climate forcings are the changes in long-lived GHGs and surface properties on the planet. Fast feedbacks include water vapour, clouds, aerosols and sea ice changes.\n\nThis fast-feedback sensitivity is relevant to estimating the climate impact of human-made climate forcings, because the size of ice sheets is not expected to change significantly in decades or even in a century and GHGs can be specified as a forcing. GHGs change in response to climate change, but it is common to include these feedbacks as part of the climate forcing by using observed GHG changes for the past and calculated GHGs for the future, with calculated amounts based on carbon cycle and atmospheric chemistry models.\n\nClimate forcings due to past changes in GHGs and surface albedo can be computed for the past 800\u2009000 years using data from polar ice cores and ocean sediment cores. We use CO~2~\u00a0\\[87\\] and CH~4~\u00a0\\[88\\] data from Antarctic ice cores (<a href=\"#RSTA20120294F5\" data-ref-type=\"fig\">figure\u00a05<\/a>*a*) to calculate an effective GHG forcing as follows:\n\n![](rsta20120294-e10.jpg)\n\nwhere Fa is the adjusted forcing, i.e. the planetary energy imbalance due to the GHG change after the stratospheric temperature has time to adjust to the gas change. Fe, the effective forcing, accounts for variable efficacies of different climate forcings\u00a0\\[66\\]. Formulae for Fa of each gas are given by Hansen *et al.*\u00a0\\[89\\]. The factor 1.4 converts the adjusted forcing of CH~4~ to its effective forcing, Fe, which is greater than Fa mainly because of the effect of CH~4~ on the tropospheric ozone and the stratospheric water vapour\u00a0\\[66\\]. The factor 1.12 approximates the forcing by N~2~O changes, which are not as well preserved in the ice cores but have a strong positive correlation with CO~2~ and CH~4~ changes\u00a0\\[90\\]. The factor 1.12 is smaller than the 1.15 used by Hansen *et al.*\u00a0\\[91\\], and is consistent with estimates of the N~2~O forcing in the current Goddard Institute for Space Studies (GISS) radiation code and that of the Intergovernmental Panel on Climate Change (IPCC)\u00a0\\[1,48\\]. Our LGM\u2013Holocene GHG forcing (<a href=\"#RSTA20120294F5\" data-ref-type=\"fig\">figure\u00a05<\/a>*c*) is approximately 3\u2009m^\u22122^, moderately larger than the 2.8\u2009W\u2009m^\u22122^ estimated by IPCC\u00a0\\[1,48\\] because of our larger effective CH~4~ forcing.\n\nClimate forcing due to surface albedo change is a function mainly of the sea level, which implicitly defines ice sheet size. Albedo change due to LGM\u2013Holocene vegetation change, much of which is inherent with ice sheet area change, and albedo change due to coastline movement are lumped together with ice sheet area change in calculating the surface albedo climate forcing. An ice sheet forcing does not depend sensitively on the ice sheet shape or on how many ice sheets the ice volume is divided among and is nearly linear in sea-level change (see electronic supplementary material, figure S4, and\u00a0\\[5\\]). For the sake of simplicity, we use the linear relation in Hansen *et al.*\u00a0\\[5\\] and electronic supplementary material, figure S4; thus, 5\u2009W\u2009m^\u22122^ between the LGM and ice-free conditions and 3.4\u2009W\u2009m^\u22122^ between the LGM and Holocene. This scale factor was based on simulations with an early climate model\u00a0\\[3,92\\]; comparable forcings are found in other models (e.g. see discussion in\u00a0\\[93\\]), but results depend on cloud representations, assumed ice albedo and other factors; so the uncertainty is difficult to quantify. We subjectively estimate an uncertainty of approximately 20%.\n\nGlobal temperature change obtained by multiplying the sum of the two climate forcings in <a href=\"#RSTA20120294F5\" data-ref-type=\"fig\">figure\u00a05<\/a>*c* by a sensitivity of 3\/4^\u00b0^C per W\u2009m^\u22122^ yields a remarkably good fit to 'observations' (<a href=\"#RSTA20120294F6\" data-ref-type=\"fig\">figure\u00a06<\/a>), where the observed temperature is 2\u00d7*\u0394T*~do~, with 2 being the scale factor required to yield the estimated 4.5^\u00b0^C LGM\u2013Holocene surface temperature change. The close match is partly a result of the fact that sea-level and temperature data are derived from the same deep ocean record, but use of other sea-level reconstructions still yields a good fit between the calculated and observed temperature\u00a0\\[5\\]. However, exactly the same match as in <a href=\"#RSTA20120294F6\" data-ref-type=\"fig\">figure\u00a06<\/a> is achieved with a fast-feedback sensitivity of 1^\u00b0^C per W\u2009m^\u22122^ if the LGM cooling is 6^\u00b0^C or with a sensitivity of 0.5^\u00b0^C per W\u2009m^\u22122^ if the LGM cooling is 3^\u00b0^C.\n\nAccurate data defining LGM\u2013Holocene warming would aid empirical evaluation of fast-feedback climate sensitivity. Remarkably, the range of recent estimates of LGM\u2013Holocene warming, from approximately 3^\u00b0^C\u00a0\\[94\\] to approximately 6^\u00b0^C\u00a0\\[95\\], is about the same as at the time of the CLIMAP\u00a0\\[96\\] project. Given today's much improved analytic capabilities, a new project to define LGM climate conditions, analogous to the Pliocene Research, Interpretation and Synoptic Mapping (PRISM) Pliocene data reconstruction\u00a0\\[97,98\\] and Pliocene Model Intercomparison Project (PlioMIP) model intercomparisons\u00a0\\[67,68\\], could be beneficial. In \u00a77*b*, we suggest that a study of Eemian glacial\u2013interglacial climate change could be even more definitive. Combined LGM, Eemian and Pliocene studies would address an issue raised at a recent workshop\u00a0\\[99\\]: the need to evaluate how climate sensitivity varies as a function of the initial climate state. The calculations below were initiated after the workshop as another way to address that question.\n\n## Fast-feedback sensitivity: state dependence\n\nClimate sensitivity must be a strong function of the climate state. Simple climate models show that, when the Earth becomes cold enough for the ice cover to approach the tropics, the amplifying albedo feedback causes rapid ice growth to the Equator: 'snowball Earth' conditions\u00a0\\[100\\]. Real-world complexity, including ocean dynamics, can mute this sharp bifurcation to a temporarily stable state\u00a0\\[101\\], but snowball events have occurred several times in the Earth's history when the younger Sun was dimmer than today\u00a0\\[102\\]. The Earth escaped snowball conditions owing to limited weathering in that state, which allowed volcanic CO~2~ to accumulate in the atmosphere until there was enough CO~2~ for the high sensitivity to cause rapid deglaciation\u00a0\\[103\\].\n\nClimate sensitivity at the other extreme, as the Earth becomes hotter, is also driven mainly by an H~2~O feedback. As climate forcing and temperature increase, the amount of water vapour in the air increases and clouds may change. Increased water vapour makes the atmosphere more opaque in the infrared region that radiates the Earth's heat to space, causing the radiation to emerge from higher colder layers, thus reducing the energy emitted to space. This amplifying feedback has been known for centuries and was described remarkably well by Tyndall\u00a0\\[104\\]. Ingersoll\u00a0\\[105\\] discussed the role of water vapours in the 'runaway greenhouse effect' that caused the surface of Venus to eventually become so hot that carbon was 'baked' from the planet's crust, creating a hothouse climate with almost 100 bars of CO~2~ in the air and a surface temperature of about 450^\u00b0^C, a stable state from which there is no escape. Arrival at this terminal state required passing through a 'moist greenhouse' state in which surface water evaporates, water vapour becomes a major constituent of the atmosphere and H~2~O is dissociated in the upper atmosphere with the hydrogen slowly escaping to space\u00a0\\[106\\]. That Venus had a primordial ocean, with most of the water subsequently lost to space, is confirmed by the present enrichment of deuterium over ordinary hydrogen by a factor of 100\u00a0\\[107\\], the heavier deuterium being less efficient in escaping gravity to space.\n\nThe physics that must be included to investigate the moist greenhouse is principally: (i) accurate radiation incorporating the spectral variation of gaseous absorption in both the solar radiation and thermal emission spectral regions, (ii) atmospheric dynamics and convection with no specifications favouring artificial atmospheric boundaries, such as between a troposphere and stratosphere, (iii) realistic water vapour physics, including its effect on atmospheric mass and surface pressure, and (iv) cloud properties that respond realistically to climate change. Conventional global climate models are inappropriate, as they contain too much other detail in the form of parametrizations or approximations that break down as climate conditions become extreme.\n\nWe use the simplified atmosphere\u2013ocean model of Russell *et al.*\u00a0\\[108\\], which solves the same fundamental equations (conservation of energy, momentum, mass and water substance, and the ideal gas law) as in more elaborate global models. Principal changes in the physics in the current version of the model are use of a step-mountain C-grid atmospheric vertical coordinate\u00a0\\[109\\], addition of a drag in the grid-scale momentum equation in both atmosphere and ocean based on subgrid topography variations, and inclusion of realistic ocean tides based on exact positioning of the Moon and Sun. Radiation is the *k*-distribution method of Lacis & Oinas\u00a0\\[110\\] with 25 *k*-values; the sensitivity of this specific radiation code is documented in detail by Hansen *et al.*\u00a0\\[111\\]. Atmosphere and ocean dynamics are calculated on 3^\u00b0^\u00d74^\u00b0^ Arakawa C-grids. There are 24 atmospheric layers. In our present simulations, the ocean's depth is reduced to 100\u2009m with five layers so as to achieve a rapid equilibrium response to forcings; this depth limitation reduces poleward ocean transport by more than half. Moist convection is based on a test of moist static stability as in Hansen *et al.*\u00a0\\[92\\]. Two cloud types occur: moist convective clouds, when the atmosphere is moist statically unstable, and large-scale super-saturation, with cloud optical properties based on the amount of moisture removed to eliminate super-saturation, with scaling coefficients chosen to optimize the control run's fit with global observations\u00a0\\[108,112\\]. To avoid long response times in extreme climates, today's ice sheets are assigned surface properties of the tundra, thus allowing them to have a high albedo snow cover in cold climates but darker vegetation in warm climates. The model, the present experiments and more extensive experiments will be described in a forthcoming paper\u00a0\\[112\\].\n\nThe equilibrium response of the control run (1950 atmospheric composition, CO~2~ approx. 310\u2009ppm) and runs with successive CO~2~ doublings and halvings reveals that snowball Earth instability occurs just beyond three CO~2~ halvings. Given that a CO~2~ doubling or halving is equivalent to a 2% change in solar irradiance\u00a0\\[66\\] and the estimate that solar irradiance was approximately 6% lower 600\u2009Ma at the most recent snowball Earth occurrence\u00a0\\[113\\], <a href=\"#RSTA20120294F7\" data-ref-type=\"fig\">figure\u00a07<\/a> implies that about 300\u2009ppm CO~2~ or less was sufficiently small to initiate glaciation at that time.\n\nClimate sensitivity reaches large values at 8\u201332\u00d7CO~2~ (approx. 2500\u201310\u2009000\u2009ppm; <a href=\"#RSTA20120294F7\" data-ref-type=\"fig\">figure\u00a07<\/a>*b*). High sensitivity is caused by increasing water vapour as the tropopause rises and diminishing low cloud cover, but the sensitivity decreases for still larger CO~2~ as cloud optical thickness and planetary albedo increase, as shown by Russell *et al.*\u00a0\\[112\\]. The high sensitivity for CO~2~ less than 4\u00d7CO~2~ is due partly to the nature of the experiments (Greenland and Antarctic ice sheets being replaced by the tundra). High albedo snow cover on these continents largely disappears between 1\u00d7CO~2~ and 4\u00d7CO~2~, thus elevating the calculated fast-feedback sensitivity from approximately 4^\u00b0^C to approximately 5^\u00b0^C. In the real world, we would expect the Greenland and Antarctic ice sheets to be nearly eliminated and replaced by partially vegetated surfaces already at 2\u00d7CO~2~ (620\u2009ppm) equilibrium climate. In other words, if the Greenland\/Antarctic surface albedo change were identified as a slow feedback, rather than as a fast-feedback snow effect as it is in <a href=\"#RSTA20120294F7\" data-ref-type=\"fig\">figure\u00a07<\/a>, the fast-feedback sensitivity at 1\u20134\u00d7CO~2~ would be approximately 4^\u00b0^C. Thus, the sensitivity approximately 8^\u00b0^C per CO~2~ doubling in the range of 8\u201332\u00d7CO~2~ is a very large increase over sensitivity at smaller CO~2~ amounts.\n\nHow confident are we in the modelled fast-feedback sensitivity (<a href=\"#RSTA20120294F7\" data-ref-type=\"fig\">figure\u00a07<\/a>*b*)? We suspect that the modelled water vapour feedback may be moderately exaggerated, because the water vapour amount in the control run exceeds observed amounts. In addition, the area of sea ice in the control run exceeds observations, which may increase the modelled sensitivity in the 1\u20134\u00d7CO~2~ range. On the other hand, we probably underestimate the sensitivity at very high CO~2~ amounts, because our model (such as most climate models) does not change the total atmospheric mass as the CO~2~ amount varies. Mass change due to conceivable fossil fuel loading (up to say 16\u00d7CO~2~) is unlikely to have much effect, but sensitivity is probably underestimated at high CO~2~ amounts owing to self-broadening of CO~2~ absorption lines. The increased atmospheric mass is also likely to alter the cloud feedback, which otherwise is a strongly diminishing feedback at very large CO~2~ amounts. Atmospheric mass will be important after the Earth has lost its ocean and carbon is baked into the atmosphere. These issues are being examined by Russell *et al.*\u00a0\\[112\\].\n\nEarth today, with approximately 1.26 times 1950 CO~2~, is far removed from the snowball state. Because of the increase in solar irradiance over the past 600\u2009Myr and volcanic emissions, no feasible CO~2~ amount could take the Earth back to snowball conditions. Similarly, a Venus-like baked-crust CO~2~ hothouse is far distant because it cannot occur until the ocean escapes to space. We calculate an escape time of the order of 10^8^\u201310^9^ years even with the increased stratospheric water vapour and temperature at 16\u00d7CO~2~. Given the transient nature of a fossil fuel CO~2~ injection, the continuing forcing required to achieve a terminal Venus-like baked-crust CO~2~ hothouse must wait until the Sun's brightness has increased on the billion year time scale. However, the planet could become uninhabitable long before that.\n\nThe practical concern for humanity is the high climate sensitivity and the eventual climate response that may be reached if all fossil fuels are burned. Estimates of the carbon content of all fossil fuel reservoirs including unconventional fossil fuels such as tar sands, tar shale and various gas reservoirs that can be tapped with developing technology\u00a0\\[114\\] imply that CO~2~ conceivably could reach a level as high as 16 times the 1950 atmospheric amount. In that event, <a href=\"#RSTA20120294F7\" data-ref-type=\"fig\">figure\u00a07<\/a> suggests a global mean warming approaching 25^\u00b0^C, with much larger warming at high latitudes (see electronic supplementary material, figure S6). The result would be a planet on which humans could work and survive outdoors in the summer only in mountainous regions\u00a0\\[115,116\\]\u2014and there they would need to contend with the fact that a moist stratosphere would have destroyed the ozone layer\u00a0\\[117\\].\n\n# Earth system sensitivity\n\nGHG and surface albedo changes, which we treated as specified climate forcings in evaluating fast-feedback climate sensitivity, are actually slow climate feedbacks during orbit-instigated Pleistocene glacial\u2013interglacial climate swings. Given that GHG and albedo feedbacks are both strong amplifying feedbacks, indeed accounting by themselves for most of the global Pleistocene climate variation, it is apparent that today's climate sensitivity on millennial time scales must be substantially larger than the fast-feedback sensitivity.\n\nClimate sensitivity including slow feedbacks is described as 'Earth system sensitivity'\u00a0\\[118\u2013120\\]. There are alternative choices for the feedbacks included in Earth system sensitivity. Hansen & Sato\u00a0\\[60\\] suggest adding slow feedbacks one by one, creating a series of increasingly comprehensive Earth system climate sensitivities; specifically, they successively move climate-driven changes in surface albedo, non-CO~2~ GHGs and CO~2~ into the feedback category, at which point the Earth system sensitivity is relevant to an external forcing such as changing solar irradiance or human-made forcings. At each level, in this series, the sensitivity is state dependent.\n\nOur principal aim here is to use Cenozoic climate change to infer information on the all-important fast-feedback climate sensitivity, including its state dependence, via analysis of Earth system sensitivity. CO~2~ is clearly the dominant forcing of the long-term Cenozoic cooling, in view of the abundant evidence that CO~2~ reached levels of the order of 1000\u2009ppm in the Early Cenozoic\u00a0\\[9\\], as discussed in the Overview above. Thus, our approach is to examine Earth system sensitivity to CO~2~ change by calculating the CO~2~ history required to produce our reconstructed Cenozoic temperature history for alternative state-independent and state-dependent climate sensitivities. By comparing the resulting CO~2~ histories with CO~2~ proxy data, we thus assess the most realistic range for climate sensitivity.\n\nTwo principal uncertainties in this analysis are (i) global temperature at the EECO approximately 50\u2009Myr\u2009BP and (ii) CO~2~ amount at that time. We use EECO approximately 28^\u00b0^C (<a href=\"#RSTA20120294F4\" data-ref-type=\"fig\">figure\u00a04<\/a>) as our standard case, but we repeat the analysis with EECO approximately 33^\u00b0^C (see electronic supplementary material, figure S3), thus allowing inference of how the conclusions change if knowledge of Eocene temperature changes.\n\nSimilarly, our graphs allow the inferred climate sensitivity to be adjusted if improved knowledge of CO~2~ 50\u2009Myr\u2009BP indicates a value significantly different from approximately 1000\u2009ppm.\n\nTo clarify our calculations, let us first assume that fast-feedback climate sensitivity is a constant (state-independent) 3^\u00b0^C for doubled CO~2~ (0.75^\u00b0^C per W\u2009m^\u22122^). It is then trivial to convert our global temperature for the Cenozoic (<a href=\"#RSTA20120294F4\" data-ref-type=\"fig\">figure\u00a04<\/a>*a*) to the total climate forcing throughout the Cenozoic, which is shown in the electronic supplementary material, figure S4*a*, as are results of subsequent steps. Next, we subtract the solar forcing, a linear increase of 1\u2009W\u2009m^\u22122^ over the Cenozoic era due to the Sun's 0.4% irradiance increase\u00a0\\[121\\], and the surface albedo forcing due to changing ice sheet size, which we take as linear at 5\u2009W\u2009m^\u22122^ for the 180\u2009m sea-level change from 35\u2009Myr\u2009BP to the LGM. These top-of-the-atmosphere and surface forcings are moderate in size, compared with the total forcing over the Cenozoic, and partially offsetting, as shown in the electronic supplementary material, figure S4*b*. The residual forcing, which has a maximum of approximately 17\u2009W\u2009m^\u22122^ just prior to 50\u2009Myr\u2009BP, is the atmospheric forcing due to GHGs. Non-CO~2~ GHGs contribute 25% of the total GHG forcing in the period of ice core measurements. Atmospheric chemistry simulations\u00a0\\[122\\] reveal continued growth of non-CO~2~ gases (N~2~O, CH~4~ and tropospheric O~3~) in warmer climates, at only a slightly lower rate (1.7\u20132.3\u2009W\u2009m^\u22122^ for 4\u00d7CO~2~, which itself is approx. 8\u2009W\u2009m^\u22122^). Thus, we take the CO~2~ forcing as 75% of the GHG forcing throughout the Cenozoic in our standard case, but we also consider the extreme case in which non-CO~2~ gases are fixed and thus contribute no climate forcing.\n\nA CO~2~ forcing is readily converted to the CO~2~ amount; we use the equation in table\u00a01 of Hansen *et al.*\u00a0\\[89\\]. The resulting Cenozoic CO~2~ history required to yield the global surface temperature of <a href=\"#RSTA20120294F4\" data-ref-type=\"fig\">figure\u00a04<\/a>*a* is shown in <a href=\"#RSTA20120294F8\" data-ref-type=\"fig\">figure\u00a08<\/a>*a* for state-independent climate sensitivity with non-CO~2~ GHGs providing 25% of the GHG climate forcing. The peak CO~2~ in this case is approximately 2000\u2009ppm. If non-CO~2~ GHGs provide less than 25% of the total GHG forcing, then the inferred CO~2~ amount would be even greater. Results for alternative sensitivities, as in <a href=\"#RSTA20120294F8\" data-ref-type=\"fig\">figure\u00a08<\/a>*b*, are calculated for a temporal resolution of 0.5\u2009Myr to smooth out glacial\u2013interglacial CO~2~ oscillations, as our interest here is in CO~2~ as a climate forcing.\n\nWe focus on the CO~2~ amount 50\u2009Myr\u2009BP averaged over a few million years in assessing the realism of our inferred CO~2~ histories, because CO~2~ variations in the Cenozoic remain very uncertain despite the success of Beerling & Royer\u00a0\\[9\\] in eliminating the most extreme outliers. Beerling & Royer\u00a0\\[9\\] find a best-fit CO~2~ at 50\u2009Myr\u2009BP of about 1000\u2009ppm\u2014see their <a href=\"#RSTA20120294F1\" data-ref-type=\"fig\">figure\u00a01<\/a>, which also indicates that CO~2~ at 50\u2009Myr\u2009BP was almost certainly in the range of 750\u20131500\u2009ppm, even though it is impossible to provide a rigorous confidence interval.\n\nWe conclude that the average fast-feedback climate sensitivity during the Cenozoic is larger than the canonical 3^\u00b0^C for 2\u00d7CO~2~ (0.75^\u00b0^C per W\u2009m^\u22122^) that has long been the central estimate for current climate. An average 4^\u00b0^C for 2\u00d7CO~2~ (1^\u00b0^C per W\u2009m^\u22122^) provides a good fit to the target 1000\u2009ppm CO~2~, but the sensitivity must be still higher if non-CO~2~ GHG forcings amplify the CO~2~ by less than one-third, i.e. provide less than 25% of the total GHG forcing.\n\n## State-dependent climate sensitivity\n\nMore realistic assessment should account for the state dependence of climate sensitivity. Thus, we make the same calculations for the state-dependent climate sensitivity of the Russell climate model, i.e. we use the fast-feedback climate sensitivity of <a href=\"#RSTA20120294F7\" data-ref-type=\"fig\">figure\u00a07<\/a>*b*. In addition, for the purpose of assessing how the results depend upon climate sensitivity, we consider a second case in which we reduce the Russell sensitivity of <a href=\"#RSTA20120294F7\" data-ref-type=\"fig\">figure\u00a07<\/a>*b* by the factor two-thirds.\n\nThe estimated 1000\u2009ppm of CO~2~ at 50\u2009Myr\u2009BP falls between the Russell sensitivity and two-thirds of the Russell sensitivity, though closer to the full Russell sensitivity. If the non-CO~2~ GHG forcing is less than one-third of the CO~2~ forcing, the result is even closer to the full Russell sensitivity. With these comparisons at 50\u2009Myr\u2009BP in mind, we can use <a href=\"#RSTA20120294F9\" data-ref-type=\"fig\">figure\u00a09<\/a> to infer the likely CO~2~ amount at other times. The End-Eocene transition began at about 500\u2009ppm and fell to about 400\u2009ppm. The Mid-Miocene warmth, which peaked at about 15\u2009Myr\u2009BP, required a CO~2~ increase of only a few tens of ppm with the Russell sensitivity, but closer to 100\u2009ppm if the true sensitivity is only two-thirds of the Russell sensitivity. The higher (full Russell) sensitivity requires much less CO~2~ change to produce the Mid-Miocene warming for two reasons: (i) the greater temperature change for a specified forcing and (ii) the smaller CO~2~ change required to yield a given forcing from the lesser CO~2~ level of the higher sensitivity case. The average CO~2~ amount in the Early Pliocene is about 300\u2009ppm for the Russell sensitivity, but could reach a few tens of ppm higher if the true sensitivity is closer to two-thirds of the Russell sensitivity.\n\n## Comparison with van de Wal *et al.* model\n\nvan de Wal *et al.*\u00a0\\[123\\] used the same Zachos *et al.*\u00a0\\[4\\] *\u03b4*^18^O data to drive an inverse model calculation, including an ice sheet model to separate ice volume and temperature, thus inferring CO~2~ over the past 20\u2009Myr. They find an MMCO CO~2~ approximately 450\u2009ppm, which falls between the Russell and two-thirds Russell sensitivities (<a href=\"#RSTA20120294F9\" data-ref-type=\"fig\">figure\u00a09<\/a>). The van de Wal *et al.*\u00a0\\[123\\] model has a 30^\u00b0^C change in Northern Hemisphere temperature (their model is hemispheric) between the MMCO and average Pleistocene conditions driven by a CO~2~ decline from approximately 450\u2009ppm to approximately 250\u2009ppm, which is a forcing of approximately 3.5\u2009W\u2009m^\u22122^. Thus, the implied (Northern Hemisphere) Earth system sensitivity is an implausible approximately 35^\u00b0^C for a 4\u2009W\u2009m^\u22122^ CO~2~ forcing. The large temperature change may be required to produce substantial sea-level change in their ice sheet model, which we suggested above is unrealistically unresponsive to climate change. However, they assign most of the temperature change to slow feedbacks, thus inferring a fast-feedback sensitivity of only about 3^\u00b0^C per CO~2~ doubling.\n\n## Inferences from the Palaeocene\u2013Eocene Thermal Maximum and Early Cenozoic climate\n\nFinally, we use the largest and best documented of the hyperthermals, the PETM, to test the reasonableness of the Russell state-dependent climate sensitivity. Global warming in the PETM is reasonably well defined at 5\u20136^\u00b0^C and the plausible range for carbon mass input is approximately 4000\u20137000\u2009Pg C\u00a0\\[14\\]. Given that the PETM carbon injection occurred over a period of a few millennia, carbon cycle models suggest that about one-third of the carbon would be airborne as CO~2~ following complete injection\u00a0\\[21\\]. With a conversion factor of 1\u2009ppm CO~2~\u223c2.12\u2009Gt C, the 4000\u20137000\u2009Gt C source thus yields approximately 630\u20131100\u2009ppm CO~2~. We can use <a href=\"#RSTA20120294F10\" data-ref-type=\"fig\">figure\u00a010<\/a>, obtained via the same calculations as described above, to see how much CO~2~ is required to yield a 5^\u00b0^C warming. The Russell sensitivity requires approximately 800\u2009ppm CO~2~ for a 5^\u00b0^C warming, whereas two-thirds of the Russell sensitivity requires approximately 2100\u2009ppm CO~2~. Given the uncertainty in the airborne fraction of CO~2~ and possible non-CO~2~ gases, we cannot rule out the two-thirds Russell sensitivity, but the full Russell sensitivity fits plausible PETM carbon sources much better, especially if the PETM warming is actually somewhat more than 5^\u00b0^C (see <a href=\"#RSTA20120294F10\" data-ref-type=\"fig\">figure\u00a010<\/a> for quantitative implications).\n\nThis analysis is for Earth system sensitivity with CO~2~ as the forcing, as is appropriate for the PETM because any carbon injected as CH~4~ would be rapidly oxidized to CO~2~. Feedbacks in the PETM do not include large ice sheets, but non-CO~2~ GHGs are an unmeasured feedback. If a warming climate increases the amount of N~2~O and CH~4~ in the air, the required carbon source for a given global warming is reduced, because the amount of carbon in airborne CH~4~ is negligible. Any non-CO~2~ GHG feedback increases the CO~2~-forced Earth system sensitivity, potentially by a large amount (<a href=\"#RSTA20120294F10\" data-ref-type=\"fig\">figure\u00a010<\/a>). The CO~2~-forced Earth sensitivity is the most relevant climate sensitivity, not only for the PETM but for human-made forcings. Although present enhanced amounts of airborne CH~4~ and N~2~O are mostly a climate forcing, i.e. their increases above the pre-industrial level are mainly a consequence of human-made sources, they also include a GHG feedback. Climate sensitivity including this GHG feedback is the most relevant sensitivity for humanity as the CO~2~ forcing continues to grow.\n\nIf the EECO global temperature exceeded 28^\u00b0^C, as suggested by multi-proxy data taken at face value (see above), climate sensitivity implied by the EECO warmth and the PETM warming is close to the full Russell climate sensitivity (see electronic supplementary material, figures S7\u2013S9). We conclude that the existing data favour a climate sensitivity of at least two-thirds of the Russell sensitivity, and probably closer to the full Russell sensitivity. That lower limit is just over 3^\u00b0^C for 2\u00d7CO~2~ for the range of climate states of immediate relevance to humanity (<a href=\"#RSTA20120294F7\" data-ref-type=\"fig\">figure\u00a07<\/a>*b*).\n\n# Summary discussion\n\nCovariation of climate, sea level and atmospheric CO~2~ through the Cenozoic era is a rich source of information that can advise us about the sensitivity of climate and ice sheets to forcings, including human-made forcings. Our approach is to estimate Cenozoic sea level and temperature from empirical data, with transparent assumptions and minimal modelling. Our data are available in the electronic supplementary material, allowing comparison with other data and model results.\n\n## Sea-level sensitivity\n\nHansen\u00a0\\[49,50\\] argues that real ice sheets are more responsive to warming than in most ice sheet models, which suggests that large ice sheets are relatively stable. The model of Pollard & DeConto\u00a0\\[124\\], for example, requires three to four times the pre-industrial CO~2~ amount to melt the Antarctic ice sheet. This stability is, in part, a result of hysteresis: as the Earth warms, the ice sheet size as a function of temperature does not return on the same path that it followed as temperature fell and the ice sheet grew. We do not question the reality of mechanisms that cause ice sheet hysteresis, but we suspect they are exaggerated in models. Thus, as an extreme alternative that can be compared with ice sheet models and real-world data, we assume that hysteresis effects are negligible in our approximation for sea level as a function of temperature.\n\nIce sheets in question are those on Greenland and Antarctica, ice sheets that could shrink with future warming. Despite the stability of those ice sheets in the Holocene, there is evidence that sea level was much more variable during the Eemian, when we estimate the peak global temperature was only +1.0^\u00b0^C warmer than in the first decade of the twenty-first century. Rohling *et al.*\u00a0\\[52\\] estimate an average rate of Eemian sea-level change of 1.4\u2009m per century, and several studies noted above suggest that the Eemian sea level reached heights of +4\u20136\u2009m or more relative to today.\n\nThe MMCO provides one test of hysteresis. Our sea-level approximation (<a href=\"#RSTA20120294F2\" data-ref-type=\"fig\">figure\u00a02<\/a>) suggests that the Antarctic ice sheet nearly disappeared at that time. John *et al.*\u00a0\\[125\\] provide support for that interpretation, as well as evidence of numerous rises and falls of sea level by 20\u201330\u2009m during the Miocene. These variations are even larger than those we find (<a href=\"#RSTA20120294F2\" data-ref-type=\"fig\">figure\u00a02<\/a>), but the resolution of the *\u03b4*^18^O data we use is not adequate to provide the full amplitude of variations during that period (electronic supplementary material, figure S1).\n\nThe Mid-Pliocene is a more important test of ice sheet variability. We find sea-level fluctuations of at least 20\u201340\u2009m, much greater than in ice sheet models (<a href=\"#RSTA20120294F2\" data-ref-type=\"fig\">figure\u00a02<\/a>), with global temperature variations of only a few degrees. Independent analyses designed to separate ice volume and temperature change, such as Dwyer & Chandler\u00a0\\[64\\], find sea-level maxima and variability comparable to our estimates. Altogether, the empirical data support a high sensitivity of the sea level to global temperature change, and they provide strong evidence against the seeming lethargy and large hysteresis effects that occur in at least some ice sheet models.\n\n## Fast-feedback climate sensitivity\n\nEstimates of climate sensitivity cover a wide range that has existed for decades\u00a0\\[1,48,99\\]. That range measures our ignorance; it does not mean that climate response from a specified state is stochastic with such inherent uncertainty. God (Nature) plays dice, but not for such large amounts. Indeed, one implication of the tight fit of calculated and measured temperature change of the past 800\u2009000 years (<a href=\"#RSTA20120294F6\" data-ref-type=\"fig\">figure\u00a06<\/a>) is that there is a single well-defined, but unknown, fast-feedback global climate sensitivity for that range of climate, despite large regional climate variations and ocean dynamical effects\u00a0\\[31\\].\n\nImproved empirical data can define climate sensitivity much more precisely, provided that climate-induced aerosol changes are included in the category of fast feedbacks (human-made aerosol changes are a climate forcing). Empirical assessment of fast-feedback climate sensitivity is obtained by comparing two quasi-equilibrium climate states for which boundary condition climate forcings (which may be slow feedbacks) are known. Aerosol changes between those climate states are appropriately included as a fast feedback, not only because aerosols respond rapidly to changing climate but also because there are multiple aerosol compositions, they have complex radiative properties and they affect clouds in several ways, thus making accurate knowledge of their glacial\u2013interglacial changes inaccessible.\n\nThe temporal variation of the GHG plus surface albedo climate forcing closely mimics the temporal variation of either the deep ocean temperature (<a href=\"#RSTA20120294F6\" data-ref-type=\"fig\">figure\u00a06<\/a>) or Antarctic temperature\u00a0\\[5,31\\] for the entire 800\u2009000 years of polar ice core data. However, the temperature change must be converted to the global mean to allow inference of climate sensitivity. The required scale factor is commonly extracted from an estimated LGM\u2013Holocene global temperature change, which, however, is very uncertain, with estimates ranging from approximately 3^\u00b0^C to approximately 6^\u00b0^C. Thus, for example, the climate sensitivity (1.7\u20132.6^\u00b0^C for 2\u00d7CO~2~) estimated by Schmittner *et al.*\u00a0\\[94\\] is due largely to their assumed approximately 3^\u00b0^C cooling in the LGM, and in lesser part to the fact that they defined some aerosol changes (dust) to be a climate forcing.\n\nClimate sensitivity extracted from Pleistocene climate change is thus inherently partly subjective as it depends on how much weight is given to mutually inconsistent estimates of glacial-to-interglacial global temperature change. Our initial assessment is a fast-feedback sensitivity of 3\u00b11^\u00b0^C for 2\u00d7CO~2~, corresponding to an LGM cooling of 4.5^\u00b0^C, similar to the 2.2\u20134.8^\u00b0^C estimate of PALAEOSENS\u00a0\\[99\\]. This sensitivity is higher than estimated by Schmittner *et al.*\u00a0\\[94\\], partly because they included natural aerosol changes as a forcing. In addition, we note that their proxies for LGM sea surface cooling exclude planktic foraminifera data, which suggest larger cooling\u00a0\\[126\\], and, as noted by Schneider von Deimling *et al.*\u00a0\\[95\\], regions that are not sampled tend to be ones where the largest cooling is expected. It should be possible to gain consensus on a narrower range for climate sensitivity via a community project for the LGM analogous to PRISM Pliocene data reconstruction\u00a0\\[97,98\\] and PlioMIP model intercomparisons\u00a0\\[67,68\\].\n\nHowever, we suggest that an even more fruitful approach would be a focused effort to define the glacial-to-interglacial climate change of the Eemian period (MIS-5e). The Eemian avoids the possibility of significant human-made effects, which may be a factor in the Holocene. Ruddiman\u00a0\\[127\\] suggests that deforestation and agricultural activities affected CO~2~ and CH~4~ in the Holocene, and Hansen *et al.*\u00a0\\[91\\] argue that human-made aerosols were probably important. Given the level of Eemian warmth, approximately +1.8^\u00b0^C relative to 1880\u20131920, with a climate forcing similar to that for LGM\u2013Holocene (<a href=\"#RSTA20120294F5\" data-ref-type=\"fig\">figure\u00a05<\/a>), we conclude that this relatively clean empirical assessment yields a fast-feedback climate sensitivity in the upper part of the range suggested by the LGM\u2013Holocene climate change, i.e. a sensitivity of 3\u20134^\u00b0^C for 2\u00d7CO~2~. Detailed study is especially warranted because Eemian warmth is anticipated to recur in the near term.\n\n## Earth system sensitivity\n\nWe have shown that global temperature change over the Cenozoic era is consistent with CO~2~ change being the climate forcing that drove the long-term climate change. Proxy CO~2~ measurements are so variable and uncertain that we only rely on the conclusion that the CO~2~ amount was of the order of 1000\u2009ppm during peak Early Eocene warmth. That conclusion, in conjunction with a climate model incorporating only the most fundamental processes, constrains average fast-feedback climate sensitivity to be in the upper part of the sensitivity range that is normally quoted\u00a0\\[1,48,99\\], i.e. the sensitivity is greater than 3^\u00b0^C for 2\u00d7CO~2~. Strictly this Cenozoic evaluation refers to the average fast-feedback sensitivity for the range of climates from ice ages to peak Cenozoic warmth and to the situation at the time of the PETM. However, it would be difficult to achieve that high average sensitivity if the current fast-feedback sensitivity were not at least in the upper half of the range of 3\u00b11^\u00b0^C for 2\u00d7CO~2~.\n\nThis climate sensitivity evaluation has implications for the atmospheric CO~2~ amount throughout the Cenozoic era, which can be checked as improved proxy CO~2~ measurements become available. The CO~2~ amount was only approximately 450\u2013500\u2009ppm 34\u2009Myr\u2009BP when large-scale glaciation first occurred on Antarctica. Perhaps more important, the amount of CO~2~ required to melt most of Antarctica in the MMCO was only approximately 450\u2013500\u2009ppm, conceivably only about 400\u2009ppm. These CO~2~ amounts are smaller than suggested by ice sheet\/climate models, providing further indication that the ice sheet models are excessively lethargic, i.e. resistant to climate change. The CO~2~ amount in the earliest Pliocene, averaged over astronomical cycles, was apparently only about 300\u2009ppm, and decreased further during the Pliocene.\n\n## Runaway greenhouse\n\nOur climate simulations, using a simplified three-dimensional climate model to solve the fundamental equations for conservation of water, atmospheric mass, energy, momentum and the ideal gas law, but stripped to basic radiative, convective and dynamical processes, finds upturns in climate sensitivity at the same forcings as found with a more complex global climate model\u00a0\\[66\\]. At forcings beyond these points the complex model 'crashed', as have other climate models (discussed by Lunt *et al.*\u00a0\\[83\\]). The upturn at the 10\u201320\u2009W\u2009m^\u22122^ negative forcing has a simple physical explanation: it is the snowball Earth instability. Model crashes for large positive forcings are sometimes described as a runaway greenhouse, but they probably are caused by one of the many parametrizations in complex global models going outside its range of validity, not by a runaway greenhouse effect.\n\nThe runaway greenhouse effect has several meanings ranging from, at the low end, global warming sufficient to induce out-of-control amplifying feedbacks, such as ice sheet disintegration and melting of methane hydrates, to, at the high end, a Venus-like hothouse with crustal carbon baked into the atmosphere and a surface temperature of several hundred degrees, a climate state from which there is no escape. Between these extremes is the moist greenhouse, which occurs if the climate forcing is large enough to make H~2~O a major atmospheric constituent\u00a0\\[106\\]. In principle, an extreme moist greenhouse might cause an instability with water vapour preventing radiation to space of all absorbed solar energy, resulting in very high surface temperature and evaporation of the ocean\u00a0\\[105\\]. However, the availability of non-radiative means for vertical transport of energy, including small-scale convection and large-scale atmospheric motions, must be accounted for, as is done in our atmospheric general circulation model. Our simulations indicate that no plausible human-made GHG forcing can cause an instability and runaway greenhouse effect as defined by Ingersoll\u00a0\\[105\\], in agreement with the theoretical analyses of Goldblatt & Watson\u00a0\\[128\\].\n\nOn the other hand, conceivable levels of human-made climate forcing could yield the low-end runaway greenhouse. A forcing of 12\u201316\u2009W\u2009m^\u22122^, which would require CO~2~ to increase by a factor of 8\u201316 times, if the forcing were due only to CO~2~ change, would raise the global mean temperature by 16\u201324^\u00b0^C with much larger polar warming. Surely that would melt all the ice on the planet, and probably thaw methane hydrates and scorch carbon from global peat deposits and tropical forests. This forcing would not produce the extreme Venus-like baked-crust greenhouse state, which cannot be reached until the ocean is lost to space. A warming of 16\u201324^\u00b0^C produces a moderately moist greenhouse, with water vapour increasing to about 1% of the atmosphere's mass, thus increasing the rate of hydrogen escape to space. However, if the forcing is by fossil fuel CO~2~, the weathering process would remove the excess atmospheric CO~2~ on a time scale of 10^4^\u201310^5^ years, well before the ocean is significantly depleted. Baked-crust hothouse conditions on the Earth require a large long-term forcing that is unlikely to occur until the sun brightens by a few tens of per cent, which will take a few billion years\u00a0\\[121\\].\n\n## Global habitability\n\nBurning all fossil fuels would produce a different, practically uninhabitable, planet. Let us first consider a 12\u2009W\u2009m^\u22122^ greenhouse forcing, which we simulated with 8\u00d7CO~2~. If non-CO~2~ GHGs such as N~2~O and CH~4~ increase with global warming at the same rate as in the palaeoclimate record and atmospheric chemistry simulations\u00a0\\[122\\], these other gases provide approximately 25% of the greenhouse forcing. The remaining 9\u2009W\u2009m^\u22122^ forcing requires approximately 4.8\u00d7CO~2~, corresponding to fossil fuel emissions as much as approximately 10,000\u2009Gt C for a conservative assumption of a CO~2~ airborne fraction averaging one-third over the 1000 years following a peak emission\u00a0\\[21,129\\].\n\nOur calculated global warming in this case is 16^\u00b0^C, with warming at the poles approximately 30^\u00b0^C. Calculated warming over land areas averages approximately 20^\u00b0^C. Such temperatures would eliminate grain production in almost all agricultural regions in the world\u00a0\\[130\\]. Increased stratospheric water vapour would diminish the stratospheric ozone layer\u00a0\\[131\\].\n\nMore ominously, global warming of that magnitude would make most of the planet uninhabitable by humans\u00a0\\[132,133\\]. The human body generates about 100\u2009W of metabolic heat that must be carried away to maintain a core body temperature near 37^\u00b0^C, which implies that sustained wet bulb temperatures above 35^\u00b0^C can result in lethal hyperthermia\u00a0\\[132,134\\]. Today, the summer temperature varies widely over the Earth's surface, but wet bulb temperature is more narrowly confined by the effect of humidity, with the most common value of approximately 26\u201327^\u00b0^C and the highest approximately of 31^\u00b0^C. A warming of 10\u201312^\u00b0^C would put most of today's world population in regions with wet a bulb temperature above 35^\u00b0^C\u00a0\\[132\\]. Given the 20^\u00b0^C warming we find with 4.8\u00d7CO~2~, it is clear that such a climate forcing would produce intolerable climatic conditions even if the true climate sensitivity is significantly less than the Russell sensitivity, or, if the Russell sensitivity is accurate, the CO~2~ amount required to produce intolerable conditions for humans is less than 4.8\u00d7CO~2~. Note also that increased heat stress due to warming of the past few decades is already enough to affect health and workplace productivity at low latitudes, where the impact falls most heavily on low- and middle-income countries\u00a0\\[135\\].\n\nThe Earth was 10\u201312^\u00b0^C warmer than today in the Early Eocene and at the peak of the PETM (<a href=\"#RSTA20120294F4\" data-ref-type=\"fig\">figure\u00a04<\/a>). How did mammals survive that warmth? Some mammals have higher internal temperatures than humans and there is evidence of evolution of surface-area-to-mass ratio to aid heat dissipation, for example transient dwarfing of mammals\u00a0\\[136\\] and even soil fauna\u00a0\\[137\\] during the PETM warming. However, human-made warming will occur in a few centuries, as opposed to several millennia in the PETM, thus providing little opportunity for evolutionary dwarfism to alleviate impacts of global warming. We conclude that the large climate change from burning all fossil fuels would threaten the biological health and survival of humanity, making policies that rely substantially on adaptation inadequate.\n\nLet us now verify that our assumed fossil fuel climate forcing of 9\u2009W\u2009m^\u22122^ is feasible. If we assume that fossil fuel emissions increase by 3% per year, typical of the past decade and of the entire period since 1950, cumulative fossil fuel emissions will reach 10\u2009000\u2009Gt C in 118 years. However, with such large rapidly growing emissions the assumed 33% CO~2~ airborne fraction is surely too small. The airborne fraction, observed to have been 55% since 1950\u00a0\\[1\\], should increase because of well-known nonlinearity in ocean chemistry and saturation of carbon sinks, implying that the airborne fraction probably will be closer to two-thirds rather than one-third, at least for a century or more. Thus, the fossil fuel source required to yield a 9\u2009W\u2009m^\u22122^ forcing may be closer to 5000\u2009Gt C, rather than 10\u2009000\u2009Gt C.\n\nAre there sufficient fossil fuel reserves to yield 5000\u201310\u2009000\u2009Gt\u2009C? Recent updates of potential reserves\u00a0\\[114\\], including unconventional fossil fuels (such as tar sands, tar shale and hydrofracking-derived shale gas) in addition to conventional oil, gas and coal, suggest that 5\u00d7CO~2~ (1400\u2009ppm) is indeed feasible. For instance, using the emission factor for coal from IPCC\u00a0\\[48\\], coal resources given by the Global Energy Assessment\u00a0\\[114\\] amount to 7300\u201311\u2009000\u2009Gt\u2009C. Similarly, using emission factors from IPCC\u00a0\\[48\\], total recoverable fossil energy reserves and resources estimated by GEA\u00a0\\[114\\] are approximately 15\u2009000\u2009Gt C. This does not include large 'additional occurrences' listed in ch. 7\u2009of GEA\u00a0\\[114\\]. Thus, for a multi-centennial CO~2~ airborne fraction between one-third and two-thirds, as discussed above, there are more than enough available fossil fuels to cause a forcing of 9\u2009W\u2009m^\u22122^ sustained for centuries.\n\nMost of the remaining fossil fuel carbon is in coal and unconventional oil and gas. Thus, it seems, humanity stands at a fork in the road. As conventional oil and gas are depleted, will we move to carbon-free energy and efficiency\u2014or to unconventional fossil fuels and coal? If fossil fuels were made to pay their costs to society, costs of pollution and climate change, carbon-free alternatives might supplant fossil fuels over a period of decades. However, if governments force the public to bear the external costs and even subsidize fossil fuels, carbon emissions are likely to continue to grow, with deleterious consequences for young people and future generations.\n\nIt seems implausible that humanity will not alter its energy course as consequences of burning all fossil fuels become clearer. Yet strong evidence about the dangers of human-made climate change have so far had little effect. Whether governments continue to be so foolhardy as to allow or encourage development of all fossil fuels may determine the fate of humanity.\n\n## Acknowledgements\n\nWe thank James Zachos for the deep ocean oxygen isotope data; Chris Brierly, Mark Chandler, Bas de Boer, Alexey Fedorov, Chris Hatfield, Dorothy Peteet, David Rind, Robert Rohde and Cynthia Rosenzweig for helpful information; Andy Ridgwell for useful editorial suggestions and patience; Eelco Rohling for ably organizing the palaeoclimate workshop that spurred the writing of this paper; Gerry Lenfest (Lenfest Foundation), ClimateWorks, Lee Wasserman (Rockefeller Family Foundation), Stephen Toben (Flora Family Foundation) and NASA program managers Jack Kaye and David Considine for research support.\n\n# References\n\n[^1]: One contribution of 11 to a Discussion Meeting Issue '[Warm climates of the past\u2014a lesson for the future?](http:\/\/rsta.royalsocietypublishing.org\/content\/371\/2001.toc)'.","meta":{"dup_signals":{"dup_doc_count":107,"dup_dump_count":41,"dup_details":{"curated_sources":2,"2018-47":2,"2018-39":4,"2018-34":1,"2018-30":1,"2018-26":1,"2018-22":1,"2018-17":1,"2018-13":1,"2018-09":2,"2018-05":1,"2017-47":3,"2017-43":1,"2017-39":3,"2017-34":1,"2017-30":3,"2017-26":3,"2017-22":2,"2017-17":3,"2017-09":3,"2017-04":4,"2016-50":4,"2016-44":5,"2016-40":4,"2016-36":3,"2016-30":4,"2016-26":4,"2016-22":4,"2016-18":2,"2016-07":3,"2015-48":3,"2015-40":2,"2015-35":3,"2015-32":3,"2015-27":3,"2015-22":3,"2015-14":3,"2014-52":2,"2017-13":2,"2015-18":3,"2015-11":2,"2015-06":2}},"file":"PMC3785813"},"subset":"pubmed_central"}
{"text":"abstract: We are now well entering the exciting era of stem cells. Potential stem cell therapy holds great promise for the treatment of many diseases such as stroke, traumatic brain injury, Alzheimer's disease, Parkinson's disease, amyotrophic lateral-sclerosis, myocardial infarction, muscular dystrophy, diabetes, and etc. It is generally believed that transplantation of specific stem cells into the injured tissue to replace the lost cells is an effective way to repair the tissue. In fact, organ transplantation has been successfully practiced in clinics for liver or kidney failure. However, the severe shortage of donor organs has been a major obstacle for the expansion of organ transplantation programs. Toward that direction, generation of transplantable organs using stem cells is a desirable approach for organ replacement and would be of great interest for both basic and clinical scientists. Here we review recent progress in the field of organ generation using various methods including single adult tissue stem cells, a blastocyst complementation system, tissue decellularization\/recellularization and a combination of stem cells and tissue engineering.\nauthor: Yunying Liu; Ru Yang; Zuping He; Wei-Qiang Gao\ndate: 2013-01-22\nreferences:\ntitle: Generation of functional organs from stem cells\n\n# Introduction\n\nStem cells are undifferentiated cells found in the body which have the ability to continuously divide, self-renew themselves and differentiate into various kinds of cells. With the capability of self-renewal, pluripotency and differentiation, stem cells have been believed to be useful for treatment of a wide variety of diseases in the future, including stroke, traumatic brain injury, Alzheimer's disease, Parkinson's disease, spinal cord injury, baldness, blindness, deafness, wound healing, amyotrophic lateral-sclerosis, myocardial infarction, muscular dystrophy, osteoarthritis rheumatoid arthritis, Crohn's disease, and diabetes. Amongst the applications, a number of adult stem cell therapies have already been practiced clinically. As an example, hematopoietic stem cell transplantation has been successfully applied to treat leukemia.\n\nIn addition to cell replacement therapy using stem cells, organ transplantation has been successfully practiced in clinics for organ failure of the liver or kidney. However, the severe shortage of donor organs has become the main obstacle to expand the organ transplant program. Generation of biological or semi-biological organs could be an alternative approach to solve the problem of the donor organ shortage. Notably, researchers have been hunting for ways to establish a whole organ using stem cells.\n\nIn recent years encouraging approaches for functional organ generation have emerged. The present manuscript provides an overview of organ generation using a single adult tissue stem cell, a blastocyst complementation system coupled with a specific stem cell niche, a method of decellularization and recellularization of bio-scaffold, and a combinatorial approach of tissue engineering and stem cells.\n\n## Generation of a functional organ from a single adult tissue stem cell\n\nTo demonstrate whether there are true stem cells in a given tissue, one needs to show that a single stem cell purified from the tissue has the capability of generating the entire organ. Up to present, it has been elegantly shown by independent groups that the mammary gland and the prostate can be generated in vivo from a single adult tissue stem cell \\[1\u20133\\].\n\nTwo laboratories have reported independently that single stem cells isolated from adult mouse mammary glands are able to produce secretory mammary glands when they are transplanted in the fat-pad in mice \\[1, 2\\]. It is long believed that there are stem cells in the mammary glands because this organ has the capability of undergoing an extensive growth at puberty and a second phase of expansion and retraction during pregnancy under the regulation of estrogen \\[4\\]. However, due to the lack of defined markers, there has been no reliable method to isolate mammary stem cells. This hypothesis was not proven until the work by the two groups. Based on some previous work by Clark and colleague \\[5\\], Shackleton et al. isolated putative mouse mammary stem cells using specific cell-surface markers (Lin^-^, CD29^hi^, CD24^+^) by FACS. They demonstrated these Lin^-^CD29^hi^CD24^+^ mammary cells have *in vitro* sphere formation capability and the ability to repopulate all mammary epithelial cells after transplantation into the fat-pad. Importantly, the investigators employed a lineage-tracer in the stem cells so to follow their ultimate phenotypes *in vivo*. They showed elegantly that a single cell within the Lin^-^CD29^hi^CD24^+^ population of the mouse mammary gland, marked with a LacZ report transgene, can reconstitute a completely functional mammary gland *in vivo*. Notably, the transplanted cell contributes to both the luminal and myoepithelial lineages and generates milk-producing lobuloalveolar units during pregnancy. This is the first report to demonstrate that a single adult tissue stem cell has multi-lineage differentiation capacity to produce a functional organ in an *in vivo* setting.\n\nSimilarly, using a colony-formation *in vivo* assay and an *in vivo* renal capsule transplantation approach, Gao and his colleagues have also reported that a single stem cell isolated from the adult mouse prostate epithelium has the capacity to generate a functional prostate \\[3\\]. Actually, prostate stem cells were postulated to exist because of its regenerative feature following androgen deprivation and replacement more than twenty years ago \\[6\\]. Because cell death mainly occurs in the luminal cell compartment after castration and cell proliferation mainly happens in the basal compartment following androgen replacement, stem cells are generally believed to reside in basal cell compartment and are able to repopulate the entire prostate epithelial cells.\n\nFirst, this group identified a new marker for prostate stem cells, CD117, based on the following 6 features: 1) It is enriched in proximal region of the prostate; 2) It is mainly expressed in basal cell population; 3) It is upregulated after castration and returned to normal levels following androgen replacement; 4) Only CD117^+^ cells, but not the CD117^-^ cells, can form colony structures with lumen *in vitro*; 5) It can generate prostate epithelial glandular structures *in vivo*; 6) CD117+ cells from 2^nd^ and 3^rd^ generation grafts also have the self-renewal capability.\n\nTo demonstrate whether a single stem cell has the ability to generate a prostate structure, the investigators first further enriched the stem cell population by sorting the cells expressing multiple stem cell markers, Sca-1^+^CD133^+^CD44^+^CD117^+^, into individual wells of a 96-well plate, verified them under a microscope, and transplanted them in a combination with rat embryonic urogenital sinus mesenchymal cells (rUGM) under the renal capsule of athymic nu\/nu mouse hosts. Three months later, they removed the kidneys and analyzed the fate of the grafted cells. Of the 97 single-cell transplants, there were about 1\/7 Sca-1^+^CD133^+^CD44^+^CD117^+^ grafts that demonstrate a branching pattern with epithelial tubules composed of prostatic basal, luminal and neuroendocrine lineages by the histological and immunocytochemical examination.\n\nThe generation of mammary gland and prostate acini from single stem cell implants in mice is a major breakthrough. These findings raise the possibility that people who have lost their mammary glands or prostates due to cancer could grow new ones. On the other hand, it is believed that in some tissues, there are cancer stem cells that are tumor-initiating cells. Cancer stem cells might be derived from normal stem cells in which specific tumor suppressor genes are mutated or lost \\[7\\]. Therefore, although the prostate glands were grown in mice but the research may aid the identification of markers for cancer stem cells, which may help diagnosis and more efficient treatment of prostate cancer in humans. These studies on reconstruction of mammary and prostate glands using single adult stem cells have important ramifications not only for tissue repair\/regeneration, but also for identification of mammary and prostate cancer stem cells. In other words, we envision that the potential use of single stem cells in clinic will ultimately change the treatment paradigm for human disorders more than mammary gland injury and prostatic disease.\n\n## Generation of organs using a blastocyst complementation system\n\nIn addition to single stem cells prepared from specific adult tissue, embryonic stem cells (ESCs) have been shown to be able to produce specific organs by using a strategy of injection of ESCs from one species into the blastocyst of another species. The development of a specific organ can be precluded by genetic manipulation in the recipient species but still providing a niche for organ development. The pluripotent stem cell-derived cells from the donor species would then developmentally compensate for the defect and produce the missing organ.\n\nThe blastocyst complementation system was first reported by Chen et al. by implanting normal mouse embryonic stem cells (mESCs) into the blastocysts derived from Rag2^\u2212\/\u2212^ mice to generate T and B lymphocyte lineages \\[8\\]. In recent years, this system was applied to generate pluripotent stem cell (PSC)\u2013derived donor organs *in vivo* due to the fact that complex cellular interactions among and within tissues that are required for organogenesis are difficult to recapitulate *in vitro*. For this purpose, PSCs were injected into blastocysts obtained from mutant mice in which the development of a certain organ was precluded by genetic manipulation just to supply as a niche for organ development. The PSC-derived cells would form the missing organ as a result of developmental compensation for the defect.\n\nKobayashi and colleagues had showed proof-of-principle findings of pancreas generation by injection of PSCs into the blastocysts of pancreas-deficient *Pdx1* ^\u2212\/\u2212^ mouse (pancreatogenesis-disabled) \\[9\\]. More specifically, they injected induced pluripotent stem cells (iPS) taken from rats into the blastocysts of mice that were unable to grow their own pancreas and were unable to produce insulin. The rat stem cells grew in the special mouse environment in the absent of mouse pancreas. When the mice matured to adulthood, they developed a rat pancreas from the injected rat stem cells. The origin and function of pancreatic cells were analyzed in the PSC-derived rat pancreas in adult mice. Glucose load was tested and the mice showed no signs of diabetes. These findings clearly indicate that functional rat pancreas are successfully generated in *Pdx1* ^\u2212\/\u2212^ mice using the inter-species blastocyst complementation.\n\nThe same technique has been also used to generate kidney using *Sall1* ^\u2212\/\u2212^ mouse blastocysts \\[10\\]. Rat ESC or PSC are injected into *Sall1* ^\u2212\/\u2212^ mouse blastocysts and the entire rat kidneys are formed by the injected mouse ESC- or PSC-derived cells. These ESC\/PSC-derived kidneys connect with ureters and function well. The bladders are full of urine.\n\nGeneration of pancreas and kidney via blastocyst complementation is an innovative approach. It demonstrates that this technique can be used to generate complex organ using donor PSCs. With an empty developmental niche, a solid organ will be produced developmentally from PSCs-derived cellular progeny in the vacant space.\n\nThe current organ shortage means that patients must endure a long time on waiting lists for transplantation. This blastocyst complementation system may therefore provide a novel approach for organ supply. The ultimate goal of blastocyst complementation system is to generate specific human organs from various pluripotent stem cells. However, such inter-species technology using human stem cells involves ethical issues which has to meet the scientific and ethical guidelines proposed by the International Society for Stem Cell Research (ISSCR) and requires approval from related ethical committees.\n\n## Engineering a whole organ via decellularization of matrix bioscaffolds and recellularization with stem cells\n\nOrgan formation requires not only stem cells but also the surrounding stem cell niche or microenvironment and extracellular matrix participation. In fact, three-dimensional tissue scaffold has been demonstrated to be very critical for organ regeneration.\n\nDecellularization and recellularization of matrix bioscaffolds have been shown to be a promising approach for whole-organ tissue engineering in recent years \\[11\\]. Donor organs such as the heart \\[12\\], liver \\[13\\], lung \\[14\\], kidney \\[15\\] and bladder \\[16\\] can be decellularized to an acellular biologic scaffold material and then be recellularized with functional parenchymal cells and\/or selected progenitor cell populations. Encouraging results have emerged in animal model studies.\n\nTaylor and colleagues have generated a bioartificial heart which has a heart structure with appropriate cell composition and cardiac pump function using this attractive technique in 2008 \\[12\\]. They perfused heart with 1% SDS, a strong ionic detergent, to produce acellular scaffolds, preserved extracellular matrix and original microvascular network, and then repopulated these heart-like constructs with neonatal cardiomyocytes and aortic endothelial cells. When proper physiological stimuli were given, the recellularized organ in the culture was able to further mature and display beating behaviors. Rhythmic contractions were observed by day 4, pump function generated by day 8 and cardiac architecture was formed, as confirmed by histological analysis. The bioartificial heart pumped with an amazing function, which was equivalent to about 2% heart power of an adult or 25% heart function of a 16-week fetus.\n\nUygun and colleagues modified Taylor's perfusion decellularization technique to achieve transplantable liver grafts in 2010 \\[13\\]. The ischemic livers were perfused through the portal vein with SDS. The intact lobular structure and vascular network were retained in the decellularized liver bioscaffold. The reseeded adult hepatocytes and nonparenchymal cells were able to repopulate to create a recellularized whole liver graft. The graft was transplanted as auxiliary heterotopic graft with portal vein arterializations and animal was shown to be viable for 8 hours after transplantation. The hepatocytes in the recellularized liver graft were metabolically active with functions of urea and albumin secretion. Similarly, Niklason and colleagues regenerated lung grafts using the decellularization and recellularization strategy in 2010 \\[14\\]. They removed cellular components from lung matrix, preserved hierarchical branching structures of the airways and vasculature bed in the extracellular scaffold, and reseeded pulmonary and vascular endothelial cells in the acellular lung matrix. The engineered lungs presented pneumonocyte viability and function after implantation, as evidenced by oxygen exchange.\n\nThese exciting studies on complex organs, such as the heart, liver and lung as described above, provide insights into the power of the methodology. The technology has multiple advantages. First, a bioartificial organ could theoretically solve the problem of lifelong immunosuppression after organ transplantation. Second, the preserved extracellular matrix and three-dimensional scaffold provide important signals for engraftment of repopulated cells, survival of engrafted cells, and function of newborn organ. Third, the vascular bed in the decellularized bioscaffold allows oxygen and nutrient rapid to be delivered after recellularization and reconnection to the circulation.\n\nThe most important concern in the technology is the cell type and the source of cells used for filling the decellularized organ matrix. Different types of cells are needed to reconstruct the parenchyma, vasculature, and support structures underneath. An ideal strategy is that a stem or progenitor cell can self-renew and differentiate into heterogeneous types of cells as needed to form a functional organ. Currently, cell sources are broadly categorized. Stem or progenitor cells used for recellularization include embryonic stem cells (ESCs), fetal cells, adult-derived stem or progenitor cells and adult tissue-derived inducible pluripotent stem cells (iPSCs). Non-stem or progenitor cells used for repopulation are usually parenchymal cells (e.g. cardiomyocytes, hepatocytes, pneumonocytes), vascular cells (e.g. endothelial cells) and supportive cells (e.g. fibroblasts). Whether autologous or allogeneic cells are utilized will depend on cell numbers required and if the need is immediate.\n\nIn short, decellularization and recellularization technique is a potential breakthrough in whole-organ tissue engineering. With further development, organs (e.g. heart, liver, lung, kidney, pancreas) can be stripped of their cells and regrow to pump blood, to generate albumin and urea, to exchange oxygen to produce urine and to secret insulin\/glucagon with replaced cardiocytes, hepatocytes, alveolar type 2 cells, renal medulla cells and islet \u03b1, \u03b2 cells.\n\n## Generation of organs by a combination of tissue-engineering and seeded stem cells\n\nTissue-engineering techniques have been used to combine specific stem cells with biocompatible and biodegradable polymer scaffolds to produce a cell polymer implant. The field of tissue engineering has been developed to solve the problem of donor-tissue scarcity either as whole organs or as reconstructive grafts, such as cartilage frameworks for total ear reconstruction \\[17, 18\\]. Cao and colleagues were the first to apply polyglycolic acid-polylactic acid to form a polymer template in the shape of a human auricle and seeded chondrocytes into the construct. New tissue-engineered cartilage grew for 12 weeks after implantation under the dorsal skin of mice. The polyglycolic acid-polylactic acid construct degraded gradually following transplantation, but the cartilage was formed and confirmed by morphological and histological analysis \\[17\\]. The technique is particularly useful for plastic and reconstructive surgery.\n\nBut this system, for quite a long time, seems mainly focused on the exterior instead of the interior of the function of the organ because of the key challenge to integrate fully functioning vascular architectures into the engineered construct. Therefore, the generation of functional human vascular network by Takebe and colleagues provides an innovative strategy to regenerate larger, well-vascularized organs including the liver and seems to solve the major obstacles in regenerating thick, complex tissues. They co-cultured human umbilical vein endothelial cells and mesenchymal stem cells in a matrix made of collagen and fibronectin. The human umbilical vein endothelial cells were labeled with GFP and the human mesenchymal stem cells were with kusabira orange. Vessel-like constructs were formed and detected under a fluorescence microscope *in vitro* seven days after cultivation. More importantly, when the vascular network constructs were implanted into an immunodeficient mouse, they were able to infuse the structures with rhodamine dextran to confirm that the structures can act as functional vessels. Study in this direction will facilitate future reconstitution of vascularized human organs \\[19\\].\n\n# Conclusions\n\nIn summary, as described above, there are four major ways of generation of functional organs using stem cells (Figure <a href=\"#Fig1\" data-ref-type=\"fig\">1<\/a>). Each of them has its own uniqueness. Specifically, using adult stem cells such as adult tissue epithelial stem cells, there will be a very low risk of tumorigenesis, more selective differentiation capability and better integration into the related tissue mass and acquisition of relevant organ functions. However, the number of the adult tissue stem cells that can be isolated is a limiting factor. Although using blastocyst complementation system is an attractive approach, particularly using a human-porcine inter-species strategy, injection of human stem cells into an organ-deficient porcine embryo is currently forbidden for ethical reasons. In fact, blastocyst complementation across species of large livestock animals such as porcine and canine has not been experimentally proven, more studies are needed in this direction. While the decellularization and recellularization research on various types of organs including the heart, liver, lung, kidney, bladder, etc. has yielded exciting results, but it is not practical to get the donor organs for such approach from the human beings. In addition, the acquisition of the organ functions from these studies is still limited. Tissue engineering using various biomaterial scaffolds holds hopes for particular medical needs such as plastic and reconstructive surgery, however the scaffolds using particular biomaterials are still primitive and require further development and research. Finally, the stem cell types that can be considered to be used for generation of functional organs may include ESC, iPSC and adult tissue cells. Each of these cell types has different advantages and disadvantages, in terms of proliferation, differentiation and tumorigenicity risk, which have been well-discussed in the literature.\n\nAlthough organogenesis is a complex process, the organ generation systems described above using stem cells or a combination of stem cells and tissue engineering may be applied, or at least raise the hope, to treat organ failure in humans in the near future. Besides adult tissue stem cells and embryonic stem cells, recent burgeoning and promising development of technology of induced pluripotent stem cells opens a new avenue for potential cell replacement and organ generation. Relevant to generation of functional organs, it is worth-mentioning that functional hepatocyte-like cells can be generated from induced pluripotent stem cells and the liver can be partially reconstituted in Fah\u2212\/\u2212 mice \\[20\\].\n\nTaken together, with the above initial steps and further improvement, stem cell therapy may one day not only repair tissue damage but also generate new tissues for tissue\/organ transplantation. Even though it is still at an infancy stage, these studies may hold promise for generation of specific functional organs for organ transplantation, to help solve the clinical problem of donor shortage.\n\n## Acknowledgements\n\nThe study is supported by funds from the Chinese Ministry of Science and Technology (2012CB966800 and 2013CB945600 to WQG and RY and 2012CB967900), the National Natural Science Foundation of China (81130038 to WQG, 31171422 and 31230048 to ZH), Science and Technology Commission of Shanghai Municipality (Pujiang program to WQG, 11PJ1406400 to ZH), Shanghai Education Committee Key Disciplines and Specialties Foundation (J50208 to WQG), Shanghai Health Bureau Key Disciplines and Specialties Foundation (to WQG), KC Wong foundation (to WQG) and the China Postdoctoral Science Foundation (2012M510835 to YYL).\n\n# References","meta":{"dup_signals":{"dup_doc_count":104,"dup_dump_count":64,"dup_details":{"curated_sources":4,"2023-14":1,"2022-40":1,"2022-27":1,"2022-05":1,"2021-43":1,"2021-21":1,"2021-10":1,"2021-04":1,"2020-50":1,"2020-40":1,"2020-29":2,"2020-24":1,"2020-16":1,"2020-05":1,"2019-47":1,"2019-39":1,"2019-35":1,"2019-30":2,"2019-22":1,"2019-13":1,"2019-09":1,"2018-51":1,"2018-43":1,"2018-39":1,"2018-30":1,"2018-22":1,"2018-09":1,"2017-47":2,"2017-34":1,"2017-26":1,"2017-17":1,"2017-09":2,"2017-04":1,"2016-50":1,"2016-44":1,"2016-40":1,"2016-36":1,"2016-30":1,"2016-22":1,"2016-18":1,"2015-48":2,"2015-40":2,"2015-35":2,"2015-32":2,"2015-27":2,"2015-22":2,"2015-14":2,"2014-52":2,"2014-49":3,"2014-42":6,"2014-41":3,"2014-35":4,"2014-23":3,"2014-15":5,"2023-40":1,"2024-10":1,"2017-13":2,"2015-18":2,"2015-11":2,"2015-06":2,"2014-10":2,"2013-48":2,"2013-20":1,"2024-26":1}},"file":"PMC4230490"},"subset":"pubmed_central"}
{"text":"abstract: # OBJECTIVE\n  .\n  Time-related biases in observational studies of drug effects have been described extensively in different therapeutic areas but less so in diabetes. Immortal time bias, time-window bias, and time-lag bias all tend to greatly exaggerate the benefits observed with a drug.\n  .\n  # RESEARCH DESIGN AND METHODS\n  .\n  These time-related biases are described and shown to be prominent in observational studies that have associated metformin with impressive reductions in the incidence of and mortality from cancer. As a consequence, metformin received much attention as a potential anticancer agent; these observational studies sparked the conduction of randomized, controlled trials of metformin as cancer treatment. However, the spectacular effects reported in these studies are compatible with time-related biases.\n  .\n  # RESULTS\n  .\n  We found that 13 observational studies suffered from immortal time bias; 9 studies had not considered time-window bias, whereas other studies did not consider inherent time-lagging issues when comparing the first-line treatment metformin with second- or third-line treatments. These studies, subject to time-related biases that are avoidable with proper study design and data analysis, led to illusory extraordinarily significant effects, with reductions in cancer risk with metformin ranging from 20 to 94%. Three studies that avoided these biases reported no effect of metformin use on cancer incidence.\n  .\n  # CONCLUSIONS\n  .\n  Although observational studies are important to better understand the effects of drugs, their proper design and analysis is essential to avoid major time-related biases. With respect to metformin, the scientific evidence of its potential beneficial effects on cancer would need to be reassessed critically before embarking on further long and expensive trials.\nauthor: Samy Suissa; Laurent AzoulayCorresponding author: Samy Suissa, <email@example.com>.\ndate: 2012-12\nreferences:\nsubtitle: : Time-related biases in observational studies\ntitle: Metformin and the Risk of Cancer\n\nTime-related biases are common in observational studies of drug effects and have been described extensively in several therapeutic areas. However, these biases have not been described in detail in the field of diabetes. Time-related biases include immortal time bias, a bias introduced with time-fixed cohort analyses that misclassify unexposed time as exposed; time-window bias, a bias introduced because of differential exposure opportunity time windows between subjects; and time-lag bias, a bias introduced by comparing treatments given at different stages of the disease (1,2). These biases are known to exaggerate downward the effect of a drug, thus making a drug seem to be protective when in fact it may have no effect. In this review, we show that several of the observational studies investigating the association between metformin and cancer incidence and mortality are affected by these time-related biases.\n\nMetformin is a drug of choice for the management of type 2 diabetes mellitus (3,4). It reduces insulin resistance, improves glycemic control, and can be combined safely with other antidiabetic drugs (5). In 2005, an observational study using data from Tayside, Scotland, reported a significant 23% reduction in the incidence of any cancer with metformin use, thus putting forward the hypothesis that metformin could lower the risk of cancer onset in patients with diabetes (6). This study generated great interest in metformin as an agent in the prevention and treatment of cancer, and many preclinical studies showed that metformin can inhibit the growth of cancer cells in vitro and in vivo (7,8).\n\nIn parallel, a series of observational studies conducted in various databases generally reported similar beneficial results with metformin, thus \"confirming\" the findings of the 2005 study. Two meta-analyses including some of these observational studies reported a highly significant reduction in cancer incidence or mortality associated with metformin use (risk reductions ranged between 31 and 34%) (9,10). This convergence of evidence from multiple preclinical and epidemiological studies formed the impetus to recommend the conduct of randomized, controlled trials (RCTs) of metformin in the prevention and treatment of cancer (11\u201314). However, as discussed later, several of these observational studies had important time-related biases that likely exaggerated the potential antitumor effects of metformin.\n\n# STUDIES OF CANCER PREVENTION\n\nThe majority of studies published to date have reported the role of metformin in preventing the occurrence of cancer in general and of some cancers in particular. To describe the different variations of this bias, we use only some of these studies as a model; the list of studies that incurred the same biases is provided in <a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a> (15\u201337).\n\nTime-related biases in observational studies investigating the effects of metformin on cancer incidence and as a cancer treatment\n\n![](2665tbl1)\n\n## Immortal time bias\n\nThe first published epidemiological study to verify this hypothesis involved a cohort of patients of 10,309 new users of oral hypoglycemic agents (OHAs) identified from the Saskatchewan Health databases (15). Patients entered the cohort at the time of their first OHA prescription during 1991\u20131996. They were followed through 1999 for death due to cancer, which occurred in 407 patients. Exposure was classified as sulfonylureas only or metformin, with the latter including users of only metformin and those who also used sulfonylureas at some point in time during follow-up. A Cox proportional hazards model was used to estimate the hazard ratio (HR), adjusted for age, sex, insulin use, and a chronic disease score. The adjusted HR of cancer mortality for sulfonylurea versus metformin was 1.3 (95% CI 1.1\u20131.6), which, when reversed, gives the HR for metformin relative to sulfonylurea as 0.8 (0.6\u20130.9), suggesting a protective effect of metformin.\n\nImmortal time bias is introduced in this study from its definition of exposure and related analysis. The 3,340 subjects who entered the cohort while using sulfonylureas and remained on these throughout the entire follow-up and the 1,229 who used only metformin from day 1 throughout follow-up pose no problem to the definition of exposure. The bias is introduced by classifying some of the remaining 5,740 who used sulfonylureas and metformin at various times during follow-up as \"metformin users.\" Although those whose cohort entry prescription was metformin are classified correctly in the metformin-exposed group, those whose cohort entry prescription was a sulfonylurea are not (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<\/a>). Indeed, the time between the sulfonylurea prescription at entry into the cohort and the first metformin prescription during follow-up is called \"immortal\" (thick gray line in <a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<\/a>) because the patient must be alive to have received their first metformin prescription (1). Moreover, this immortal person-time should be classified as sulfonylurea-exposed until the start of metformin, at which point the remaining person-time can be classified as metformin-exposed. In this study, the authors misclassified this immortal person-time as metformin-exposed, which will lead to immortal time bias.\n\nBecause the data necessary to assess the magnitude of the bias are not provided in the article, for the sake of illustration we assume that the mean delay between cohort entry and the first metformin prescription was 1 year among the 5,740 combined users. This would result in 5,740 immortal person-years of sulfonylurea exposure misclassified as metformin exposure. The reported rates of cancer death for metformin (245\/39,026 = 6.3 per 1,000 person-years) and for sulfonylureas (162\/16,700 = 9.7 per 1,000 person-years), which were based on misclassified denominators, produced a crude rate ratio of 0.65. By properly reclassifying exposure (per our assumption), the rates would become 245\/(39,026\u20135,740) = 7.4 per 1,000 person-years for metformin and 162\/(16,700+5,740) = 7.2 per 1,000 person-years for sulfonylureas, resulting in a corrected crude rate ratio of 1.0. There are more sophisticated ways to estimate the rate ratio while properly accounting for such time-varying exposures, such as Cox proportional hazards models with time-dependent factors. An alternative approach simply is to use an intent-to-treat approach (as is done in RCTs) that classifies exposure according to the first OHA prescription type, irrespective of switching to or adding the other agent, with, of course, the limitations of interpretation inherent to this approach.\n\nIn a subsequent publication reanalyzing the same data, the authors recognized the presence of immortal time bias, claiming \"we extended our analyses by classifying time-varying exposure of metformin, sulfonylureas and insulin therapy\" (16). The reported an adjusted HR of 0.80 (95% CI 0.65\u20130.98; *P* = 0.03) for metformin use relative to sulfonylurea monotherapy and the corresponding rates of cancer mortality (6.3 vs. 9.7 per 1,000 person-years) remained identical to those reported previously, which were subject to immortal time bias. This perfect equality of rates is numerically impossible and thus casts doubt on whether the time-varying statistical model was applied correctly to metformin exposure to properly reclassify the misclassified exposure in this second publication.\n\nImmortal time bias also was evident in a study using Taiwanese National Health Insurance data in 2000 (18). It included 12,005 patients with type 2 diabetes who received at least two prescriptions of metformin during 2000\u20132007; these patients were compared with 4,597 patients who did not use metformin but had other OHAs and 8,643 who did not use any OHA during this period. Cancer events occurring 1 year after entry into the cohort until the end of 2007 were identified. Using the Cox proportional hazards model to adjust for covariates, the use of metformin was associated with lower incidence of cancer with HRs of any cancer of 0.12 (95% CI 0.08\u20130.19), of colorectal cancer (0.36 \\[0.13\u20130.98\\]), liver cancer (0.06 \\[0.02\u20130.16\\]), and pancreatic cancer (0.15 \\[0.03\u20130.79\\]).\n\nImmortal time bias is introduced in this study from the definition of metformin exposure. Indeed, the 12,005 subjects defined as exposed to metformin during 2000\u20132007 had to have at least two prescriptions for metformin during this period. The bias is introduced because the time between the first and second metformin prescriptions during follow-up is immortal, that is, the patient must be cancer-free to have received the second metformin prescription, which could have been dispensed much later. Moreover, this person-time should be classified as unexposed until the second metformin prescription, at which point the remaining person-time should be classified as metformin-exposed. Thus, the analysis misclassified all this person-time as metformin exposure, which led to immortal time bias. In contrast, the comparison group of patients who did not use any OHA did not have such a condition of a second prescription.\n\nAnother example of this bias is the study using the Hong Kong Diabetes Registry to form a cohort of 2,658 patients diagnosed with type 2 diabetes who did not use metformin when enrolled in the study between 1996 and 2005 or in the 2.5 years before enrollment, with follow-up until 2005 (22). During a median 5.5 years of follow-up, 129 patients developed cancer. A Cox proportional hazards model was used to estimate the HR of cancer, which was adjusted for several covariates. The use of metformin during follow-up was associated with significant reductions in cancer incidence, with HRs of 0.51 (95% CI 0.31\u20130.82) among patients with HDL cholesterol \u22651.0 mmol\/L and 0.30 (0.13\u20130.70) among patients with HDL cholesterol \\<1.0 mmol\/L.\n\nImmortal time bias also is present in this study because none of the patients were using metformin at the time of entry into the cohort or during the 2.5 years before but were classified in the analysis as metformin users if they received a prescription for metformin during the follow-up. The bias then is introduced by classifying the time between entry into the cohort and the first metformin prescription as metformin-exposed. This person-time is immortal and should have been classified as unexposed until the start of metformin, at which point the remaining person-time should be classified as metformin-exposed. Misclassifying all this person-time as metformin-exposed leads to immortal time bias.\n\nThis bias also is present in a recent study investigating the association between metformin and the incidence of lung cancer (23). In that study, the authors used administrative databases from the Taiwanese National Health Insurance program to extract a cohort of 19,624 patients who were treated with antidiabetic agents between 2000 and 2005. In Cox proportional hazards models, the use of metformin was associated with a 45% risk reduction of lung cancer (HR 0.55 \\[95% CI 0.37\u20130.82\\]). Similar results were observed for thiazolidinediones and \u03b1-glucosidase inhibitors (0.55 \\[0.32\u20130.94\\] and 0.61 \\[0.38\u20130.98\\], respectively), whereas null results were observed for insulin and sulfonylureas (1.00 \\[0.68\u20131.45\\] and 1.27 \\[0.75\u20132.15\\], respectively). As with the studies described earlier, immortal time bias was introduced by misclassifying unexposed person-time as exposed person-time. Overall, the 19,624 patients generated 108,213 person-years of follow-up. Based on the data presented in the study, the 16,616 patients who ever used metformin generated 93,987 person-years of exposure, an average of 5.7 years per patient. However, the latter included immortal person-time (i.e., time between entry into the cohort and the first metformin prescription, during which no event could have occurred). Assuming an average 1-year delay between entry into the cohort and the first metformin prescription, a total of 16,616 immortal person-years would have been misclassified as exposed. As shown in <a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a>, by correctly reclassifying this person-time, the rate of the unexposed group would become 33\/(14,266 + 16,616) = 10.7 per 10,000 person-years instead of 23.2 per 10,000 person-years, whereas the rate in the metformin-exposed group would become 96\/(93,987 \u2013 16,616) = 12.4 per 10,000 person-years instead of 10.2 per 10,000 person-years, resulting in a corrected crude rate ratio of 1.16. Based on similar calculations, a delay of only 9.6 months between entry into the cohort and the first metformin prescription would have been necessary to bring the crude rate ratio to the null value. Thus, it is important to recognize that in the context of a highly prevalent exposure such as metformin, even small misclassifications of just a few months can result in greatly biased rate ratios.\n\nComparison between biased time-fixed and corrected time-dependent data analyses for the cohort study of metformin and lung cancer incidence (23)\n\n![](2665tbl2)\n\nAs summarized in <a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>, immortal time bias affected nine observational studies evaluating the association between metformin and cancer incidence or mortality. Because of important misclassifications of exposure, the risk reductions observed in these studies were greatly exaggerated in favor of metformin. Furthermore, the variations in the reported risk reductions, ranging between 20 and 94% (<a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>), are a direct reflection of the different magnitude of misclassification of immortal time in the studies, leading to bias of different magnitudes.\n\n## Time-window bias\n\nA study design that has been used extensively is the nested case-control analysis conducted, in this first study, within a cohort of 22,621 women newly treated with OHAs between 1994 and 2005 who were identified using the U.K. General Practice Research Database (32). During follow-up, 309 women developed breast cancer and were matched to 1,153 controls selected from the cohort on age, general practice, and index date. Overall, the use of metformin was not associated with a decreased risk of breast cancer (odds ratio \\[OR\\] 1.03 \\[95% CI 0.76\u20131.39\\]). However, for patients who were prescribed at least 40 prescriptions, metformin was associated with a significant risk reduction (0.44 \\[0.24\u20130.82\\]).\n\nIn this study, the authors used risk-set sampling, making metformin exposure time-dependent and thus avoiding immortal time bias. However, the cases and controls were not matched on duration of follow-up or, more specifically, on duration of exposure opportunity time, which can lead to time-window bias (2). Matching on duration of follow-up ensures the same opportunity time of being exposed for cases and matched controls. In this study, the null finding for overall use of metformin likely indicates that cases and matched controls had, on average, similar follow-up times. However, it is possible that in certain subgroups, such as in those who received at least 40 prescriptions, the follow-up may have been different between cases and controls. As such, it is possible that controls had a longer follow-up period than cases and hence a greater opportunity to receive additional prescriptions. The opposite could have occurred as well, and therefore the direction of this bias highly depends on the distribution of follow-up among the randomly selected controls. Indeed, in a subsequent study using similar methods to investigate the effects of metformin on the incidence of colorectal cancer, the same authors reported an increased risk among patients who received at least 50 prescriptions (OR 1.43 \\[95% CI 1.08\u20131.90\\]) (36). This time-window bias also likely was present in other studies by the same authors who used this same approach to investigate the effect of metformin use on the risk of ovarian and pancreatic cancers (34,35).\n\nAnother example of time-window bias is a study investigating the association between antidiabetic drugs and the risk of hepatocellular carcinoma (31). This was a single-center, hospital-based, case-control study from Texas, involving 420 cases of hepatocellular carcinoma and 1,104 hospital controls identified between 2000 and 2008. The use of metformin and thiazolidinediones was associated with a decreased risk of hepatocellular carcinoma (OR 0.3 \\[95% CI 0.2\u20130.6\\] and 0.3 \\[0.1\u20130.7\\], respectively). It is interesting that cases were more likely than controls to be controlled with diet (13.1 vs. 2.3%, respectively). A priori, diet-controlled diabetes would have been expected to be associated with the lowest risk of cancer because it is used early in the natural history of the disease primarily in patients with a new diagnosed of type 2 diabetes (based on the assumption that disease severity is associated with an increased risk of cancer). However, it is surprising that diet-controlled diabetes was associated strongly with an increased risk of hepatocellular carcinoma (7.8 \\[1.5\u201340.0\\]). Whereas duration of diabetes was generally similar between the cases and controls, this finding reveals that duration of *treated* diabetes was largely differential between cases and controls. Indeed, the much lower prevalence of diet-controlled diabetes among the controls suggests that these patients had a longer treatment history than the cases. As such, on the basis of their longer treatment history, controls had a greater opportunity to use metformin and other antidiabetic drugs. This differential treatment time window between cases and controls likely led to the apparent increased risk of hepatocellular carcinoma with diet-controlled diabetes and consequently to an apparent decreased risk with metformin and thiazolidinediones.\n\nAs discussed earlier and summarized in <a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>, time-window bias can lead to spurious associations even when proper time-dependent analyses are performed. In some studies, there was an attempt to either adjust or match on duration of diabetes (28,31). Although controlling for this important variable certainly minimizes confounding by disease severity, it does not correct for the differential lengths of the treatment time windows in the cases and controls. Furthermore, it is difficult to predict the direction of the time-window bias because it depends on the distribution of treatment duration in the randomly selected controls compared with the cases. It is fortunate that this bias can easily be avoided at the design stage by properly accounting for duration of treatment in the cases and controls (2).\n\n## Bias from time lag and latency\n\nSome studies that have used cohorts of patients treated with antidiabetic drugs have compared the exposure time of metformin use with exposure time of other antidiabetic treatments, including sulfonylureas and insulin. Several methodological aspects have to be considered when a first-line therapy such as metformin is compared with second- or third-line therapies (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<\/a>). First, patients are unlikely to be at the same stage of diabetes, which can induce confounding by disease duration: longer duration of diabetes may be associated with higher cancer incidence, independent of age. In such a case, the proper comparison would require matching on disease duration so that the cohort would be formed as in <a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<\/a> (*bottom*) rather than the time-lagged comparison suggested by <a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<\/a> (*top*). Second, if the first treatment used is associated with an increased incidence of cancer, albeit after a long period of exposure, it is likely that the cancer will occur during exposure to the second-line agent, in which case attribution of the cancer to either treatment is challenging\u2014thus the importance of considering latency time windows.\n\nA study that was affected by confounding by disease stage is one that used The Health Information Network, a U.K. general practice database (17). In this study, the authors identified 62,809 users of OHAs or insulin between 2000 and 2007. The study population was then subdivided into four cohorts: cohort 1 included new users of metformin monotherapy; cohort 2 comprised new users of sulfonylurea monotherapy; cohort 3 included those who switched from monotherapy with metformin or sulfonylurea to a combination of both drugs; and cohort 4 included those who switched from OHAs to insulin. Incidence rates of cancer (specifically breast, pancreas, colorectal, and prostate cancers) were calculated within each of the aforementioned cohorts, and Cox proportional hazards models were used to compare cohorts 2, 3, and 4 with cohort 1 (metformin monotherapy). After a mean follow-up of 2 years, the HRs (95% CIs) were 1.4 (1.2\u20131.5) for sulfonylurea monotherapy, 1.1 (1.0\u20131.2) for the metformin-sulfonylurea combination, and 1.4 (1.3\u20131.6) for insulin. Among patients treated with insulin, the concomitant use of metformin was associated with a statistically significant risk reduction (0.54 \\[0.43\u20130.66\\]). However, this strong inverse association was likely due to immortal time bias because exposure to metformin was assessed at any time during follow-up and thus included person-time during which patients were not yet exposed to this therapy (<a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>). Furthermore, as noted by others, the survival curves of the different cohorts presented by the authors diverged as early as during the first year of treatment, thus arguing against a causal effect, which is biologically inconsistent with the long latencies of cancers (38). Aside from reverse causality and confounding, which are certainly important considerations, such early divergence in the survival curves can also be explained by two other factors. First, comparing the different cohorts, which consisted of second- and third-line treatments, with metformin monotherapy, a first-line treatment, inherently introduces biases related to disease duration and progression that may not be dealt with adequately during analysis. Second, the consideration of cancers diagnosed early after entry into the cohort is problematic, especially for patients with a history of OHA use (i.e., cohorts 3 and 4), because it ignores the fact that previous exposure might have affected future cancer risk. Indeed, patients may remain at risk for an extended period of time long after discontinuation of treatment. For example, former smokers with a history of smoking one pack per day for a period of 1 year have a nearly threefold increased risk of lung cancer after 30 years of cessation (39). One approach to minimize this bias is to apply time-lag periods to exclude cancers diagnosed within a specified time period after entry into the cohort. This method works reasonably well, especially if there is clustering of cancers early during the treatment, as was observed in this study (17).\n\nA study that introduced a variation of bias due to time-lagging of comparison cohorts was that based on the Diabetes Audit and Research in Tayside Study cohort of patients diagnosed with type 2 diabetes in Tayside, Scotland (37). All 4,085 eligible new users of metformin between 1994 and 2003 were matched individually with 4,085 nonusers on year of diabetes diagnosis; each pair was assigned the date of entry into the cohort as the date of the first metformin prescription of the user. Of the subjects who were cancer free at cohort entry, 771 developed cancer during follow-up. Cancer was diagnosed in 7.3% of the metformin users compared with 11.6% of nonusers. A Cox proportional hazards model was used to estimate the HR of cancer, adjusted for several covariates. The adjusted HR of cancer associated with metformin use was 0.63 (95% CI 0.53\u20130.75), suggesting a strong protective effect of metformin.\n\nAlthough this study avoided biases associated with prevalent exposures by using a new-user cohort of patients treated with metformin, this approach was not used for the comparison group, which was not based on a specific comparison drug. Because the comparison metformin nonuser does not have a prescription date to define time zero, it was assigned the date of the first metformin prescription of its matched user, a date called the index date. In choosing among potential nonusers for a given user, a variation of bias from time lags was introduced. Indeed, if the potential matched nonuser had a diagnosis of cancer or had died prior to the index date, it was discarded. However, it \"was potentially available for a different metformin user\u2026this process was repeated until suitable comparators were identified.\"\n\nNonusers should be alive and cancer free at entry into the cohort just like the matched users, but the net effect of selecting the nonusers with replacement is twofold. First, the final nonuser group had zero subjects excluded because of cancer before the index date, in contrast with the 239 excluded from the metformin group. Second, the nonusers who should have been excluded (and not reused) because of a previous cancer were included as nonusers matched to other users. As a result, cancers that should have been an exclusion criterion were transferred to the follow-up period after the index date and counted as outcome events, thus artificially increasing the rate of cancer in the nonuser group. This phenomenon is noticeable in the Kaplan-Meier curves that suggest a clustering of cancers in the first 2 years of follow-up of the nonuser group. Thus, by forming the nonuser group, the authors artificially induced a cohort that was immortal and cancer free before the index date but had more deaths and cancers after entry into the cohort. As a result of this transfer, the nonuser group had 474 cancers diagnosed during follow-up compared with 297 in the metformin group. If the exclusion of 239 cancers before the first metformin prescription is applied to the nonuser group, the 474 would be reduced to 177.\n\n# STUDIES OF CANCER TREATMENT\n\nA number of studies investigated the effects of metformin among patients with cancer. Several of these studies reported significant risk reductions for all-cause mortality, cancer-specific mortality, and cancer progression, ranging between 34 and 62% (24\u201327). All of the studies reporting these important risk reductions suffered from immortal time bias (summarized in <a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>). We describe below selected studies to illustrate this bias in the setting of cancer treatment.\n\nIn the first study, the authors reviewed the records of 233 patients diagnosed with prostate cancer between 1999 and 2008 at a medical institution in Texas (24). They evaluated whether antidiabetic treatments were associated with all-cause mortality in this patient population. Cox proportional hazards models were constructed, with ever-use of insulins and OHAs entered as independent variables, and adjusted for potential confounders. Their results indicate that metformin (HR 0.55 \\[95% CI 0.32\u20130.96\\]) and thiazolidinediones (0.45 \\[0.21\u20130.97\\]) were associated with a significant decrease in all-cause mortality. No statistically significant associations were observed with the other antidiabetic agents, although few patients were exposed in this small dataset. Misclassifying as exposed the time between cohort entry and first prescription of metformin or a thiazolidinedione introduced immortal time bias, thus greatly exaggerating the benefits of these drugs on all-cause mortality. That exposure was defined as any time during the 10-year follow-up period contributed to compound the effect of this misclassification. As explained earlier, the appropriate analysis would have treated exposures to the different antidiabetic agents as time-dependent variables.\n\nThe authors of another study identified 595 patients who had been diagnosed with both colorectal cancer and diabetes mellitus and compared the 258 taking metformin any time during follow-up with the 337 nonusers (25). After a median follow-up of 41 months, the adjusted HR of all-cause mortality with metformin use was 0.66 (95% CI 0.45\u20130.98), whereas for colorectal cancer, mortality was also 0.66 (0.48\u20130.92). Here again, these mortality reductions are the result of immortal time bias caused by misclassifying as exposed to metformin the time between cohort entry and first prescription, when in fact it is unexposed.\n\nImmortal time bias also was present in a cohort study of 1,983 consecutive patients with HER2+ breast cancer treated between 1 January 1998 and 30 September 2010 (26). All-cause and cancer-specific deaths were identified. Among the patients with diabetes, the HR of all-cause death associated with metformin was 0.52 (95% CI 0.28\u20130.97), whereas for breast cancer\u2013specific mortality it was 0.47 (0.24\u20130.90). These mortality reductions again are the result of immortal time bias caused by misclassifying as exposed the time between cancer diagnosis and first prescription of metformin given during follow-up, when in fact it is unexposed to metformin.\n\n# CONCLUSIONS\n\nWe have shown that a large number of observational studies reporting significant reductions in the incidence of or mortality from cancer associated with metformin use are afflicted with time-related biases such as immortal time bias, time-window bias, and time-lag bias. By not classifying metformin exposure during follow-up or measuring metformin exposure over different time intervals properly, the resulting analyses produced apparent reductions in risk that were created artificially by the misclassification of metformin exposure. These biases, regrettably common in pharmacoepidemiology, have been described extensively in different therapeutic areas (1,2,40\u201342) but do not seem to have sufficiently penetrated the fields of diabetes and cancer. Although two of the studies recognized the possibility of immortal time bias, the technique of data analysis required to address it was clearly not appropriate (16,18).\n\nOther studies have been conducted with varying results; some have found no association between metformin use and cancer incidence or prognosis (43\u201348), others have found lower rates of cancer with metformin (49\u201357), and one study found an increased risk (58). Although these studies were designed and analyzed with no obvious time-related biases, it is of course possible that other epidemiological biases such as selection, information, or confounding could have affected them. This article focused specifically on time-related biases, where metformin was associated with strong risk reductions, ranging from relative risks of 0.80 (20% risk reduction) to 0.06 (94% risk reduction), likely due to flaws in their designs and methods of analysis.\n\nThree recent studies that specifically used the time-dependent techniques needed to avoid both immortal time and time-window biases found no association between metformin use and cancer incidence (59\u201361). The first study used the U.K. General Practice Research Database and found a rate ratio of prostate cancer incidence of 1.23 (95% CI 0.99\u20131.52) with metformin use. With more than 36 prescriptions the rate ratio was significantly elevated at 1.40 (1.03\u20131.89). The second study used the Kaiser Permanente database and found no effect of metformin on the incidence of the 10 different cancers studied, with HRs ranging between 0.8 (0.6\u20131.1) for melanoma to 1.3 (1.0\u20131.6) for kidney\/renal and pelvic cancers. Finally, no effect of metformin was found on lung cancer incidence (61).\n\nIt is also important to recognize that most observational studies focused on the incidence of cancer as it relates to the effects of metformin on carcinogenesis, which is distinct from its effects on proliferation of established tumors. Nevertheless, large RCTs using patients with established tumors currently are being planned or already are in progress. One such study is the MA.32 being conducted by the National Cancer Institute of Canada, a multicenter phase III RCT that includes almost 3,600 women with early breast cancer and is comparing metformin to placebo as adjuvant therapy on the primary end point of invasive, disease-free survival over a 5-year follow-up (12,13). The authors claim that \"we believe the science underlying such a trial is strong, the novelty of the intervention is high, and the potential for benefit is large\" (12). Although it is possible that the in vitro science is strong and metformin may lead to positive results, we have shown that the pharmacoepidemiological evidence from observational studies is weak at best.\n\nIn conclusion, time-related biases are common in the field of diabetes and can be avoided using appropriate study designs and methods of analysis. In the case of the association between metformin and cancer, we believe that methodologically inaccurate studies, even if their results are replicated in different settings, should not be the driving force behind long and expensive trials. At this time, it may be important to question and reassess the scientific evidence of the potential beneficial effects of metformin on cancer. Although the in vitro science seems to be strong, certainly all observational studies identified as potentially biased would need to be redone properly to correct for the bias. Moreover, new observational studies using more rigorous designs and analyses that avoid these biases should urgently be undertaken before more randomized trials are initiated in vain.\n\n## Acknowledgments\n\nS.S. is the recipient of the James McGill Chair, and L.A. is the recipient of a Chercheur-Boursier Award from the Fonds de la recherche en sant\u00e9 du Qu\u00e9bec.\n\nNo potential conflicts of interest relevant to this article were reported.\n\n# References","meta":{"dup_signals":{"dup_doc_count":109,"dup_dump_count":66,"dup_details":{"curated_sources":2,"2022-40":1,"2022-21":1,"2022-05":1,"2021-49":1,"2021-43":2,"2021-39":1,"2021-31":3,"2021-25":2,"2021-21":1,"2020-50":1,"2020-45":3,"2020-40":3,"2020-16":3,"2020-10":2,"2019-51":2,"2019-43":2,"2019-35":2,"2019-30":1,"2019-26":2,"2019-09":1,"2019-04":1,"2018-47":2,"2018-43":1,"2018-39":1,"2018-30":2,"2018-17":1,"2018-09":2,"2017-47":2,"2017-39":3,"2017-30":2,"2017-26":1,"2017-22":2,"2017-17":1,"2017-09":3,"2017-04":2,"2016-50":2,"2016-44":2,"2016-40":2,"2016-36":2,"2016-30":2,"2016-26":1,"2016-22":2,"2016-18":1,"2016-07":1,"2015-48":1,"2015-40":1,"2015-35":1,"2015-32":1,"2015-27":1,"2015-22":1,"2015-14":1,"2014-52":1,"2014-49":2,"2014-42":4,"2014-41":2,"2014-35":2,"2014-23":1,"2014-15":1,"2023-50":1,"2024-10":2,"2017-13":3,"2015-18":1,"2015-11":1,"2015-06":1,"2013-48":1,"2024-30":1}},"file":"PMC3507580"},"subset":"pubmed_central"}
{"text":"abstract: # Introduction\n  .\n  Angiotensin-converting enzyme (ACE) 2, a homolog of ACE, converts angiotensin (Ang) II into Ang(1-7), and the vasoprotective effects of Ang(1-7) have been documented. We explored the hypothesis that serum autoantibodies to ACE2 predispose patients with connective tissue diseases to constrictive vasculopathy, pulmonary arterial hypertension (PAH), or persistent digital ischemia.\n  .\n  # Methods\n  .\n  Serum was examined from 42 patients with systemic lupus erythematosus (SLE), scleroderma, or mixed connective tissue disease. Eighteen vasculopathy patients with PAH (five cases) and\/or persistent digital ischemia (16 cases) were compared with 24 patients without these vasculopathies (control patients) for serum reactivity to purified recombinant human ACE2, using an ELISA.\n  .\n  # Results\n  .\n  The sera from 17 of the 18 (94%) vasculopathy patients had ELISA scores above the baseline level determined using control sera from 28 healthy subjects, and the mean ELISA score in the vasculopathy patients was significantly higher than that in the control patients (*P* \\< 0.0005). The relative activity of serum ACE2, which was defined using a reference serum, correlated inversely with the ELISA scores for serum anti-ACE2 antibodies in the 18 vasculopathy patients (*R*^2^ = 0.6872). The IgG fraction from vasculopathy patients, but not from healthy subjects, inhibited ACE2 activities *in vitro*. Consistent with this, immunosuppressive therapy given to one SLE patient with digital necrosis markedly decreased the anti-ACE2 antibody titer and restored serum ACE2 activity.\n  .\n  # Conclusions\n  .\n  Autoantibodies to ACE2 may be associated with constrictive vasculopathies.\nauthor: Yuko Takahashi; Shiori Haga; Yukihito Ishizaka; Akio Mimori\ndate: 2010\ninstitute: 1Division of Rheumatic Diseases, Research Institute, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan; 2Department of Intractable Diseases, Research Institute, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan\nreferences:\ntitle: Autoantibodies to angiotensin-converting enzyme 2 in patients with connective tissue diseases\n\nSee related editorial by Chappell, <http:\/\/arthritis-research.com\/content\/12\/3\/128>\n\n# Introduction\n\nAngiotensin-converting enzyme (ACE) 2, a homolog of ACE, is a carboxypeptidase that degrades angiotensin (Ang) II to Ang(1-7) \\[1\\]. Ang(1-7) has vasodilating, antiproliferative, and antithrombotic properties that antagonize the action of Ang II and play vasoprotective roles \\[2-4\\]. Recent studies have demonstrated the therapeutic effects of ACE2 activation by a synthetic molecule \\[5\\] or of *ACE2* gene transfer \\[6\\] in experimental pulmonary hypertension models.\n\nPulmonary arterial hypertension (PAH), a vasculopathy of unknown etiology, is a serious complication of connective tissue disease (CTD) \\[7\\]. One clinical study found reduced metabolism of ACE synthetic substrate in the pulmonary vascular bed of PAH-CTD patients, but not in primary PAH patients \\[8\\]. Persistent digital ischemia, which manifests as skin ulcers or necrotic lesions, is another intractable vasculopathy of CTD, and is strongly associated with Raynaud's phenomenon. A correlation between Raynaud's phenomenon and elevated systolic pulmonary arterial pressure has been reported in patients with systemic lupus erythematosus (SLE) \\[9\\]. PAH or persistent digital ischemia is less frequent than Raynaud's phenomenon, and these three vascular abnormalities are involved in CTD patients across different disease entities, including SLE, systemic sclerosis (SSc), and mixed connective tissue disease (MCTD).\n\nOur preliminary examination suggested the presence of novel autoantibodies to ACE2 in the sera of two patients: a patient with SLE suffering from severe digital necrosis, and a patient with SSc accompanied by lethal PAH. Furthermore, the sera of the two patients lacked ACE2 activity. These findings prompted us to conduct the present study in order to explore the hypothesis that serum autoantibodies to ACE2 predispose patients with CTD to constrictive vasculopathies; that is, PAH and persistent digital ischemia.\n\n# Materials and methods\n\n## Study design\n\nAs many patients as possible among those with CTD and PAH or persistent digital ischemia (vasculopathy patients) in our hospital at time of the study were enrolled. Sera from these patients were studied in comparison with those from CTD patients without vasculopathy or from healthy subjects. The ethics committee of our hospital approved this study, and written informed consent was obtained from all patients and control subjects.\n\n## Serum sampling\n\nFresh serum was obtained from all of the patients and normal subjects for the present study. Each serum sample was aliquoted to avoid repeated thawing and was stocked at -20\u00b0C until assayed.\n\n## Diagnosis of connective tissue disease\n\nForty-two patients with SLE, SSc, or MCTD were studied. SLE was diagnosed according to the classification criteria of the American College of Rheumatology \\[10\\]. Patients with SSc met the classification criteria for the diffuse (n = 3) or limited (n = 6) form of SSc, as described in the literature \\[11\\]. Patients with MCTD met the criteria for MCTD from Kasukawa and colleagues \\[12\\] and the original definition of MCTD by Sharp and colleagues \\[13\\].\n\n## Diagnosis of pulmonary arterial hypertension\n\nPAH had been diagnosed in five patients with SSc, based on dyspnea on exertion, elevated plasma brain natriuretic peptide levels \\>100 pg\/ml, right ventricular outflow and peak tricuspid regurgitant pressure gradient exceeding 30 mmHg on echocardiography, exclusion of pulmonary thromboembolism by high-resolution computed tomography or pulmonary scintigraphy, and no deteriorated lung fibrosis that could cause pulmonary hypertension.\n\n## Diagnosis of persistent digital ischemia\n\nEach of the 16 patients in this category had persistent cyanotic lesions on the digits, present for more than 2 years at the time of the present study, and a history of or existing digital ulcers or necrosis. The radial artery pulse in each patient was normal, and atherosclerotic ischemia was excluded clinically. Three of the 16 patients also had PAH.\n\n## Control patients\n\nSerum samples from 24 CTD patients who had neither PAH nor persistent digital ischemia were collected randomly (that is, independent of the activity of their CTD) on the ward or in the outpatient clinic. These samples served as disease controls.\n\n## Normal control subjects\n\nSerum samples were obtained from 28 healthy volunteers (23 females and five males) who were free from any active or chronic diseases, including hypertension. Before bleeding, each subject was confirmed to have normal blood pressure in the arms. The mean age of the 28 subjects was 32.0 \u00b1 9.5 years.\n\n## Detection of anti-ACE2 antibodies in patient serum\n\nWe established an ELISA using purified recombinant human ACE2. A plasmid DNA-encoding human ACE2 cDNA, which was kindly gifted from Dr Hyeryun Choe (Harvard Medical School, Cambridge, MA, USA), was introduced into 293 free-style cells, according to the manufacturer's protocol (Invitrogen, Carlsbad, CA, USA). Culture supernatant was harvested on day 2 after transfection, dialyzed against 25 mM Tris-Cl (pH 8.5), and applied onto a DEAE column. ACE2 was eluted with 250 mM NaCl, and was dialyzed against for further use. About 12.5 \u03bcg\/ml recombinant human ACE2 were first coated overnight onto a 96-well plate with bicarbonate buffer (pH 9.6) at 4\u00b0C. The wells were then treated with a blocking buffer composed of 5% BSA in PBS and washed with buffer composed of 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween 20. The sera of the patients and normal healthy volunteers were added to the plate and incubated for 1 hour at room temperature. Bound ACE2 antibodies were detected using horseradish peroxidase-conjugated anti-human IgG antibody. The optical density at 450 nm was measured after a 30-minute incubation with SureBlue TMB microwell peroxidase substrate (Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD, USA). All samples were analyzed in triplicate, and the values were normalized with the positive control.\n\n## Measurement of serum ACE2 activity\n\nZinc-dependent ACE2 enzymatic activity was measured according to the methods described previously \\[14,15\\]. Briefly, 20 \u03bcM (7-methoxycoumarin-4-yl) acetyl-APK-(2,4-dinitrophenyl)-OH (amino acids depicted by single letters) (AnaSpec Inc., Fremont, CA, USA) - a fluorogenic substrate \\[14\\] - was incubated with 1 \u03bcl test serum. Fluorescence was monitored using Safire2 (TECAN, M\u00e4nnedorf, Switzerland) at the excitation and emission wavelengths of 320 nm and 450 nm, respectively.\n\nPreliminary experiments revealed that the relative fluorescence unit (RFU) value was not increased when the substrate was simply incubated without the enzyme (data not shown). Among 20 healthy humans, two samples showed significantly high autofluorescence - in these cases, the RFU values decreased during the incubation even with the substrate. We excluded these samples because it was not possible to evaluate the ACE2 enzyme activity.\n\nThe RFU values obtained by the APK substrate were almost completely inhibited by an ACE2 inhibitor, DX600 (Phoenix Pharmaceuticals Inc., Burlingame, CA, USA) (Additional file <a href=\"#S1\" data-ref-type=\"supplementary-material\">1<\/a>). Additionally, the RFU values were statistically significant at both 60 and 70 minutes of the incubation (Additional file <a href=\"#S2\" data-ref-type=\"supplementary-material\">2<\/a>). Based on these observations, we used the RFU values at 70 minutes without subtracting the residual RFU counts resistant to DX600 \\[15\\]. The relative ACE2 activity (%) in each sample was further calculated based on the level of ACE2 activity in a mixture of serum samples from 28 healthy subjects (a reference serum). The assay was performed in triplicate and the mean values of three independent measurements of the ACE2 ELISA and ACE2 activity assay were used for the statistical analysis by Student's *t* test.\n\n## Detection of serum ACE2 protein\n\nAn anti-human ACE2 goat polyclonal antibody (R&D Systems, Minneapolis, MN, USA) was used for immunoprecipitation. Serum was first treated for 15 minutes with 100 mM MgCl~2~, 70 units DNase I (Takara Bio Inc., Shiga, Japan) and 500 \u03bcg\/ml Ribonuclease A (Sigma, St Louis, MO, USA) at room temperature, and was then incubated with anti-human ACE2 antibody at 4\u00b0C for 2 hours. After incubation, the immune complexes were recovered using Protein G Sepharose\u2122 4 Fast Flow (GE Healthcare, Amersham, Buckinghamshire, UK) and subjected to SDS-PAGE, followed by western blot analysis. To detect ACE2 proteins, an anti-human ACE2 mouse monoclonal antibody was used (R&D Systems).\n\n## Purification of IgG from serum\n\nFive microliters of serum were incubated with Protein G Sepharose\u2122 4 Fast Flow in 20 mM sodium phosphate buffer (pH 7.0) for 2 hours at 4\u00b0C. The immune complexes were then washed and the IgG-bound beads eluted with 0.1 M glycine-HCl (pH 2.7). The recovered IgG was immediately neutralized with 1 M Tris-HCl (pH 8.0). IgG was detected by SDS-PAGE followed by Coomassie Brilliant Blue staining. IgG fractions were prepared from three vasculopathy patients and three healthy subjects. The effect of each IgG on the activity of recombinant human ACE2 (standard rACE2; R&D Systems) was examined as described.\n\n# Results\n\n## Disease status of patients at the time of the present study\n\nThe demographics of patients with CTD and PAH or persistent digital ischemia (vasculopathy patients, n = 18) and of the control CTD patients (n = 24) are presented in Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>. The control patients had neither manifestations nor a history of these vasculopathies.\n\nPatient demographics\n\n| Parameter | Vasculopathy patients | Control patients |\n|----|----|----|\n| Number of patients | 18 (17 females, 1 male) | 24 (21 females, 3 males) |\n| Age (years) | 50.5 \u00b1 14.3 | 46.8 \u00b1 17.8 |\n| Disease entities | SLE (4), SSc (6), MCTD (8) | SLE (21), SSc (2), MCTD (1) |\n| Constrictive vasculopathies | PAH (5); all patients had SSc^a^; persistent digital ischemia (16) | None |\n\nData presented as n or mean \u00b1 standard deviation. PAH, pulmonary arterial hypertension; MCTD, mixed connective tissue disease; SLE, systemic lupus erythematosus; SSc, systemic sclerosis. ^a^Three patients also had digital ulcers; of these, one patient had a history of scleroderma renal crisis.\n\n### Vasculopathy patients\n\nOf the 18 vasculopathy patients, three (Patients 1, 5, and 12) were hospitalized for treatment of an exacerbation of their CTD. Patient 1, with SLE, had fever, pancytopenia, and progressing digital necrosis of the fingers and toes. Patient 5, with the diffuse form of SSc, was referred to our hospital and admitted because of heart failure caused by severe PAH of recent origin. Despite high-dose steroid therapy for PAH and conventional therapy for heart failure, she died of sudden cardiac arrest. Patient 12, with SLE, had fever, pancytopenia, diffuse erythema, and painful digital ischemia accompanied by chilblain lupus.\n\nThe remaining 15 outpatients (all had persistent digital ischemia and four also had PAH) were being treated with prostacyclins or calcium antagonists without apparent improvement, and all of the patients with digital ischemia had suffered from digital ulcers or digital necrosis within 2 years of the present study. In the five patients with PAH, the mean \u00b1 standard deviation peak tricuspid regurgitant pressure gradient was 51 \u00b1 13 mmHg (range, 39 to 70 mmHg; median, 50 mmHg) on echocardiography within 6 months of the present study, and the mean plasma brain natriuretic peptide level was 639 \u00b1 623 pg\/ml (range, 107 to 1,312 pg\/ml; median, 568 pg\/ml) at the time of this study. Systemic hypertension was observed in four SLE patients and in one SSc patient. Three of the patients had been treated with a calcium antagonist or ACE inhibitor for years, and hypertension developed in the remaining two patients (Patients 1 and 12) during steroid therapy at the time of study.\n\n### Control connective tissue disease patients\n\nOf these 24 patients, 11 were hospitalized: nine patients for exacerbated SLE (six with lupus nephritis, two with neuropsychiatric lupus, and one with thrombocytopenia), one SLE patient for aseptic bone necrosis, and one SSc patient for accidental cholecystitis. The remaining 13 outpatients had stable disease on a maintenance dose of steroid therapy. Recent Raynaud's phenomena were observed in two SLE patients, two SSc patients, and one MCTD patient. Systemic hypertension was observed in one SLE patient, who had been treated with an Ang II receptor blocker.\n\n## ELISA for serum anti-ACE2 antibodies\n\nAll of the patients had received a maintenance dose of steroid therapy by the time of blood sampling. Serum samples were obtained from inpatients just before starting additional immunosuppressive therapy for a disease flare-up, and from outpatients. Sera from 17 of the 18 (94%) vasculopathy patients showed reactivity to ACE2 with ELISA values above the baseline level value, which was determined as the mean ELISA score in the 28 normal control subjects. The mean ELISA score was significantly higher in the vasculopathy patients than in the control patients (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>). These results suggest that anti-ACE2 antibodies in the serum are associated with constrictive vasculopathies, PAH, or persistent digital ischemia.\n\n## Inhibition of ACE2 activity by IgG purified from patient serum\n\nIgG fractions from the sera of three healthy subjects and three vasculopathy patients (Patients 1, 5, and 6) were prepared (Figure <a href=\"#F2\" data-ref-type=\"fig\">2a<\/a>). The activity of standard rACE2 was measured in 5 \u03bcg aliquots. The IgG fractions from the vasculopathy patients, but not from the healthy subjects, suppressed the rACE2 enzyme activity significantly compared with that in the absence of IgG (Figure <a href=\"#F2\" data-ref-type=\"fig\">2b<\/a>).\n\n## Serum ACE2 activity in vasculopathy patients, control patients, and healthy subjects\n\nFirst, the RFU value of the ACE2 enzyme activity, which was blocked by DX600 (Additional file <a href=\"#S1\" data-ref-type=\"supplementary-material\">1<\/a>), was determined in 18 patients with vasculopathy, in 16 control patients without vasculopathy, and in 26 healthy subjects. The relative rate of serum ACE2 enzyme activity was then compared with a reference mixture composed of sera from healthy subjects. Serum ACE2 activity could not be measured in one control patient and two healthy subjects due to high fluorescence at the start of incubation, and sera from the remaining seven control patients were not available for these experiments. The mean \u00b1 standard deviation relative ACE2 activity was significantly lower in the vasculopathy patients (64.8 \u00b1 16.4, n = 18) compared with that in the healthy subjects (86.6 \u00b1 32.2, n = 26, *P* = 0.0055) and control patients (165.1 \u00b1 99.1, n = 16, *P* = 0.0010).\n\nTo clarify the correlation between ACE2 activity and the presence of autoantibodies to ACE2, the ELISA scores for serum anti-ACE2 antibodies and serum ACE2 activity were plotted (Figure <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>). Our data indicate that the titers of anti-ACE2 antibodies were inversely proportional to ACE2 peptidase activity in the vasculopathy patients (Figure <a href=\"#F3\" data-ref-type=\"fig\">3c<\/a>). In contrast, no correlation was observed in the healthy subjects or control patients, suggesting that the low serum ACE2 activities in most of the vasculopathy patients were due to the presence of anti-ACE2 antibodies. To assess ACE2 expression in the vasculopathy patients, we tested for ACE2 by immunoprecipitation followed by immunoblotting. As shown in Figure <a href=\"#F3\" data-ref-type=\"fig\">3d<\/a>, the ACE2 levels in Patients 1 and 5, who were deficient in ACE2 activity (Figure <a href=\"#F3\" data-ref-type=\"fig\">3c<\/a>), were comparable with those in three healthy subjects, clearly indicating that the decreased ACE2 activity in the vasculopathy patients was due to a functional deficiency. The relative ACE2 protein levels in the patients are shown in Additional file <a href=\"#S3\" data-ref-type=\"supplementary-material\">3<\/a>. The signal intensities of ACE2 protein and the IgG heavy chain shown in Figure <a href=\"#F3\" data-ref-type=\"fig\">3d<\/a> were measured and normalized.\n\n## Recovery of serum ACE2 activity after therapy in one patient\n\nThe exacerbated SLE and progressive necrosis of the fingers and toes in Patient 1, a 25-year-old female, were treated with intravenous methylprednisolone at a dose of 500 mg\/day for 3 days, followed by 30 mg\/day oral prednisolone combined with three sessions of double-filtration plasmapheresis and conventional therapy, including prostanoids, stellate ganglion anesthesia, and local care. Her cutaneous lesions healed completely before discharge, without amputation of any digits. Serum tests 2 months after the start of therapeutic intervention showed that the ELISA score for anti-ACE2 antibodies was reduced markedly (*P* \\< 0.05) (Figure <a href=\"#F4\" data-ref-type=\"fig\">4a<\/a>) and the ACE2 activity had recovered significantly (*P* \\< 0.05) (Figure <a href=\"#F4\" data-ref-type=\"fig\">4b<\/a>), compared with the values before therapy. An ELISA for anti-ACE2 antibodies in the waste double-filtration plasmapheresis fluid was negative, and thus we could not evaluate whether double-filtration plasmapheresis had removed these antibodies from the patient's blood. The anti-ACE2 antibodies were probably decreased as a result of immunosuppressive therapy using high-dose pulse steroids.\n\nThe other two vasculopathy inpatients (Patients 5 and 12) also received immunosuppressive therapy during the present study and had high ELISA scores for anti-ACE2 antibodies before therapy. The serum of Patient 5 was not studied after therapy, because she died suddenly. For Patient 12, the ELISA scores before therapy and after 2 months of therapy with 30 mg\/day prednisolone were 0.68 \u00b1 0.02 and 0.36 \u00b1 0.01, respectively. These results were not significantly different, and the titer tended to decrease after therapy. The relative rates (%) of serum ACE2 activity in this patient before and after therapy were 52.2 \u00b1 19.1 and 63.1 \u00b1 4.8 (*P* = NS), based on the level of activity detected in the reference sample, respectively.\n\n# Discussion\n\nPersistent digital ischemia and clinically evident PAH are intractable and relatively infrequent complications of SLE, SSc, and MCTD - although latent forms of vasculopathies, including sporadic abnormalities such as Raynaud's phenomena, may be more prevalent in CTD patients. Immunosuppressive therapy can improve PAH in some patients with SLE, SSc, or MCTD \\[16\\], and in most SLE patients \\[17\\] via an unknown therapeutic mechanism. The treatment of digital ischemia remains an unsolved clinical problem for physicians. Our study suggests, for the first time, that autoantibodies to ACE2 are present in the serum of patients with persistent constrictive vasculopathies. Furthermore, the elevated ELISA scores for anti-ACE2 antibodies correlated with reduced ACE2 activity. The serum autoantibodies to ACE2 may therefore inhibit ACE2 activity, and the inhibition may be reversible after immunosuppressive therapy, based on the data from Patient 1. There were small numbers of patients with systemic hypertension in both the vasculopathy and control patient groups. We could not determine whether their hypertension was essential hypertension, or was caused by steroid therapy or ACE2 inhibition.\n\nThe inhibition of ACE2 by autoantibodies may result in reduced physiological levels of the vasoprotective agent Ang(1-7) in the local vascular milieu, which may induce vasculopathies in patients with underlying disease such as CTD. Vasodilating drugs - including prostanoids, endothelin-receptor antagonists, and phosphodiesterase type-5 inhibitors - have been used widely to treat PAH and have been partially effective for some PAH patients \\[18\\]. No effective drugs have been found for persistent digital ischemia \\[19\\]. Our study suggests that ACE2 activation or administration in experimental PAH \\[5,6\\] may be applicable as a therapy for PAH or persistent digital ischemia in patients with CTD.\n\n# Conclusions\n\nAnti-ACE2 autoantibodies may be associated with constrictive vasculopathy in patients with connective tissue disease.\n\n# Abbreviations\n\nACE: angiotensin-converting enzyme; Ang: angiotensin; BSA: bovine serum albumin; CTD: connective tissue disease; ELISA: enzyme-linked immunosorbent assay; MCTD: mixed connective tissue disease; PAH: pulmonary arterial hypertension; PBS: phosphate-buffered saline; rACE2: recombinant human angiotensin-converting enzyme 2; RFU: relative fluorescence unit; SLE: systemic lupus erythematosus; SSc: systemic sclerosis.\n\n# Competing interests\n\nThe authors declare that they have no competing interests.\n\n# Authors' contributions\n\nYT and SH equally contributed to this study. YT performed the ELISA, clinical practice, and wrote the paper. SH prepared a recombinant ACE2 protein, carried out the assays for ACE2 activities, and wrote the methods. YI designed the experiments. AM designed the research and completed the paper.\n\n# Supplementary Material\n\n###### Additional file 1\n\n**Inhibition of the enzyme activity by an ACE2 inhibitor**. By preincubation with DX600 for 30 minutes, the enzyme activity was almost completely blocked (*P* \\< 0.01). H, healthy volunteer; P, patients with vasculopathy; CP, control patients without vasculopathy. The activity was also blocked by the addition of ethylenediamine tetraacetic acid (EDTA) (data not shown), indicating that the activity would depend on the presence of zinc ion.\n\nClick here for file\n\n###### Additional file 2\n\n**Optimization of the measurement analysis of ACE2 activity**. ACE2 activity was measured chronologically with the fluorogenic substrate and plotted for 90 min. The relative fluorescence unit (RFU) values increased within 70 minutes and then declined. Each plot is a representative result of three independent experiments. In each experiment, a rACE2 (a standard) and a reference serum (a mixture of sera from 28 healthy subjects) were assayed simultaneously with sample sera from healthy subjects, control patients, or vasculopathy patients. The difference of the ACE2 enzyme activity between healthy subjects and vasculopathy patients was statistically significant (*P* \\< 0.01) both at 60 and 70 minutes of the incubation.\n\nClick here for file\n\n###### Additional file 3\n\n**Relative intensity of the ACE2 protein levels in patients**. The signal intensities of ACE2 protein and the IgG heavy chain shown in Figure <a href=\"#F3\" data-ref-type=\"fig\">3d<\/a> were measured and normalized. Each relative intensity was standardized with that of sample H1.\n\nClick here for file\n\n## Acknowledgements\n\nThe authors thank Dr Hyeryun Choe (Harvard Medical School, Cambridge, MA, USA) for a plasmid DNA encoding human ACE2 cDNA. The present work was supported by Grants-in-Aid for Research on Intractable Diseases from the Ministry of Health, Labor, and Welfare of Japan.","meta":{"dup_signals":{"dup_doc_count":138,"dup_dump_count":35,"dup_details":{"curated_sources":4,"2022-49":1,"2022-21":1,"2021-39":1,"2021-17":1,"2020-34":1,"2020-24":1,"2017-39":1,"2017-17":1,"2017-09":14,"2016-44":1,"2016-40":1,"2016-36":18,"2016-30":16,"2015-48":3,"2015-40":2,"2015-35":3,"2015-32":3,"2015-27":3,"2015-22":4,"2015-14":3,"2014-52":3,"2014-49":3,"2014-42":11,"2014-41":4,"2014-35":5,"2014-23":6,"2014-15":4,"2023-14":1,"2015-18":3,"2015-11":3,"2015-06":3,"2014-10":3,"2013-48":2,"2013-20":3,"2024-26":1}},"file":"PMC2911869"},"subset":"pubmed_central"}
{"text":"author: Hans Erik B\u00f8tker; Niels M\u00f8llerCorresponding author: Niels M\u00f8ller, <email@example.com>.\ndate: 2013-08\nreferences:\ntitle: ON NO\u2014The Continuing Story of Nitric Oxide, Diabetes, and Cardiovascular Disease\n\nNitric oxide (NO) is a simple chemical compound\u20141 nitrogen and 1 oxygen atom coupled together\u2014with complex biological actions (1,2). A singularly prominent feature of NO is its ability to cause vasodilation, a quality that is used pharmacologically when treating ischemic heart disease with NO precursors such as nitroglycerin.\n\nIn 1980, Furchgott and Zawadzki (3) showed that vascular relaxation induced by acetylcholine was dependent on the presence of endothelium and provided evidence for the release of a volatile humoral factor. This substance, later called endothelium-derived relaxing factor and now recognized as NO (4), is a significant component of the insulin-signaling cascade (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<\/a>), executing microvascular vasodilation stimulated by local NO production from the vascular endothelium (5). Vasodilation has the potential to decrease systemic blood pressure and increase local tissue blood flow in tissues such as muscle. The combination of decreased blood pressure and increased tissue blood flow, together with specific beneficial endothelial effects, may serve to prevent hypertension, cardiovascular disease, and insulin resistance (5,6). However, these effects remain exquisitely sensitive to impaired insulin signaling within the endogenous cardiovascular NO system and appear to be compromised in the presence of insulin resistance (7). So there is evidence that impaired NO-dependent vasodilation causes hypertension and insulin resistance and vice versa: that insulin resistance, such as that observed in diabetes, the metabolic syndrome, and hypertension, impairs NO-dependent vasodilation. The existence of this vicious cycle is supported by epidemiological data from the Framingham Heart Study and other studies showing that hypertension, diabetes, and cardiovascular disease cosegregate (8,9).\n\nIn the vasculature, NO is synthesized from the guanidine group of the amino acid arginine under critical control of the enzyme endothelial NO synthase (eNOS). eNOS activity is stimulated by insulin (and estrogen), exercise, and caloric restriction (10,11), whereas gluco\/lipotoxicity, inflammation, and proinflammatory cytokines, together with specific arginine metabolites, ADMA (asymmetric dimethylarginine), LMMA (<span class=\"smallcaps\">l<\/span>-monomethylarginine), and SDMA (symmetric dimethylarginine), inhibit eNOS (10,12,13). It is important to emphasize that insulin has dual actions (11): it acts after binding to its receptor and activates phosphatidylinositol 3-kinase (leading to eNOS stimulation, NO production, and vasodilation) and mitogen-activated protein kinase (leading to endothelin-1 stimulation and vasoconstriction\/cell adhesion), as illustrated in <a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<\/a>. Given the complex nature of the regulation of vascular tone and endothelial function, it is of major importance to define and describe the underlying mechanisms leading to vascular dysfunction and insulin resistance. From a clinical point of view, there is a specific need for experimental human studies assessing NO synthesis and clearance rates and whether abnormalities of NO function relate to altered kinetics and impaired insulin stimulation.\n\nIn this issue of *Diabetes*, Tessari et al. (14) report basal and insulin-stimulated synthesis rates for mononitrogen oxides (NO and NO~2~ \u2013 NOx) in 26 subjects covering a wide range of age, BMI, cholesterol, glucose tolerance, and renal function. In these experiments, the investigators use a precursor product, isotope dilution technique. Because arginine is the precursor for NO (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<\/a>), arginine labeled with nitrogen-15, a stable isotope, is infused, and the percentage of labeled arginine (precursor) and its product (NO) is measured by mass spectrometry. Once the whole system is at steady state, synthesis\/appearance rates for arginine and NO can be calculated depending on the \"intensity\" of labeling. Compared with normal subjects, the new data showed that NO synthesis was lower in the elderly and in people with type 2 diabetes and generally increased after insulin stimulation. Regression analysis using data for all subjects showed that NOx synthesis was inversely correlated with arginine metabolites (ADMA, SDMA) and age, but not with insulin sensitivity. The authors conclude that whole-body NOx production is decreased in aging and type 2 diabetes and that arginine metabolites, not insulin resistance, appear to be negative regulators of in vivo NOx production.\n\nThese are timely and pertinent data from a well-conducted human study, and the findings not only expand our understanding of the field but also suggest that insulin resistance and NO dysfunction may not be as intimately linked as hitherto believed. A particular strength of the study is that it combines a complicated clinical setup in a relative large number of subjects with a state-of-the-art kinetic tracer technique.\n\nA recent study, using a saliva oral nitrate test and an intravenous glucose tolerance test, reported a correlation between insulin sensitivity and NO synthesis (15), whereas the current study, using steady state isotope dilution and clamp techniques, fails to make this connection. Insulin sensitivity is expressed by a glucose clamp\u2013derived M (or glucose infusion rate) value, which predominantly reflects glucose uptake in muscle (16). NO synthesis is measured systemically, leaving the contributing tissues unidentified. Hence, the new results do not necessarily reflect conditions in muscle. Furthermore, the design of the study is quite complex, with many different subgroups and mixed pathologic features. It is therefore possible that the heterogeneity of the study sample may have underpowered the study. Whether clearance\/disappearance is altered in insulin-resistant states is not known, and it is possible that NO-dependent alterations in blood flow might contribute to a feedback loop in which NO removal from its target tissues increases with increasing vasodilation and blood flow.\n\nThe clinical implication of the current study relates to the effect of insulin resistance on endothelial dysfunction. Although Tessari et al. (14) did not measure endothelial function or any balance between insulin-regulated NO-dependent vasodilator actions versus endothelin-1\u2013dependent vasoconstrictor actions, the data support the idea that insulin resistance may only partly explain the impairment of endothelial function with increasing age, obesity, and type 2 diabetes. Several studies using multivariate analyses that adjust for other potential modulators of endothelial function have shown that insulin resistance may not be an independent predictor of endothelial function (17\u201319). On the other hand, the UK Prospective Diabetes Study clearly showed that metformin intervention, aimed at improving insulin sensitivity and endothelium-dependent vasodilation, led to a significant reduction in cardiovascular events in patients with insulin resistance (20). The extent to which other interventions that improve insulin sensitivity, such as caloric restriction, physical activity, and pharmacological agents, act via NO to improve cardiovascular outcomes are issues to be addressed by future studies.\n\n## ACKNOWLEDGMENTS\n\nNo potential conflicts of interest relevant to this article were reported.\n\n# REFERENCES","meta":{"dup_signals":{"dup_doc_count":128,"dup_dump_count":45,"dup_details":{"curated_sources":2,"2022-05":1,"2021-43":1,"2018-22":3,"2018-17":1,"2018-13":2,"2018-05":2,"2017-51":2,"2017-47":1,"2017-43":6,"2017-39":1,"2017-34":3,"2017-30":5,"2017-26":4,"2017-22":2,"2017-17":7,"2017-09":5,"2017-04":10,"2016-50":4,"2016-44":3,"2016-40":4,"2016-36":4,"2016-30":4,"2016-26":3,"2016-22":3,"2016-18":1,"2016-07":2,"2015-48":3,"2015-40":2,"2015-35":1,"2015-32":2,"2015-27":1,"2015-22":2,"2015-14":2,"2014-52":3,"2014-49":1,"2014-42":5,"2014-41":4,"2014-35":1,"2014-23":2,"2023-06":1,"2017-13":4,"2015-18":3,"2015-11":2,"2015-06":2,"2024-10":1}},"file":"PMC3717860"},"subset":"pubmed_central"}
{"text":"author: Russell L. Blaylock\\***Corresponding author:** Russell L. Blaylock, Theoretical Neuroscience Research, LLC, Ridgeland, Mississippi, United States. <email@example.com>\ndate: 2022\ninstitute: Retired Neurosurgeon, Theoretical Neuroscience Research, LLC, Ridgeland, Mississippi, United States.\nreferences:\ntitle: COVID UPDATE: What is the truth?\n\n![](SNI-13-167-inline001.jpg)\n\nThe COVID-19 pandemic is one of the most manipulated infectious disease events in history, characterized by official lies in an unending stream lead by government bureaucracies, medical associations, medical boards, the media, and international agencies.\\[3,6,57\\] We have witnessed a long list of unprecedented intrusions into medical practice, including attacks on medical experts, destruction of medical careers among doctors refusing to participate in killing their patients and a massive regimentation of health care, led by non-qualified individuals with enormous wealth, power and influence.\n\nFor the first time in American history a president, governors, mayors, hospital administrators and federal bureaucrats are determining medical treatments based not on accurate scientifically based or even experience based information, but rather to force the acceptance of special forms of care and \"prevention\"\u2014including remdesivir, use of respirators and ultimately a series of essentially untested messenger RNA vaccines. For the first time in history medical treatment, protocols are not being formulated based on the experience of the physicians treating the largest number of patients successfully, but rather individuals and bureaucracies that have never treated a single patient\u2014including Anthony Fauci, Bill Gates, EcoHealth Alliance, the CDC, WHO, state public health officers and hospital administrators.\\[23,38\\]\n\nThe media (TV, newspapers, magazines, etc), medical societies, state medical boards and the owners of social media have appointed themselves to be the sole source of information concerning this so-called \"pandemic\". Websites have been removed, highly credentialed and experienced clinical doctors and scientific experts in the field of infectious diseases have been demonized, careers have been destroyed and all dissenting information has been labeled \"misinformation\" and \"dangerous lies\", even when sourced from top experts in the fields of virology, infectious diseases, pulmonary critical care, and epidemiology. These blackouts of truth occur even when this information is backed by extensive scientific citations from some of the most qualified medical specialists in the world.\\[23\\] Incredibly, even individuals, such as Dr. Michael Yeadon, a retired ex-Chief Scientist, and vice-president for the science division of Pfizer Pharmaceutical company in the UK, who charged the company with making an extremely dangerous vaccine, is ignored and demonized. Further, he, along with other highly qualified scientists have stated that no one should take this vaccine.\n\nDr. Peter McCullough, one of the most cited experts in his field, who has successfully treated over 2000 COVID patients by using a protocol of early treatment (which the so-called experts completely ignored), has been the victim of a particularly vicious assault by those benefiting financially from the vaccines. He has published his results in peer reviewed journals, reporting an 80% reduction in hospitalizations and a 75% reduction in deaths by using early treatment.\\[44\\] Despite this, he is under an unrelenting series of attacks by the information controllers, none of which have treated a single patient.\n\nNeither Anthony Fauci, the CDC, WHO nor any medical governmental establishment has ever offered any early treatment other than Tylenol, hydration and call an ambulance once you have difficulty breathing. This is unprecedented in the entire history of medical care as early treatment of infections is critical to saving lives and preventing severe complications. Not only have these medical organizations and federal lapdogs not even suggested early treatment, they attacked anyone who attempted to initiate such treatment with all the weapons at their disposal\u2014loss of license, removal of hospital privileges, shaming, destruction of reputations and even arrest.\\[2\\]\n\nA good example of this outrage against freedom of speech and providing informed consent information is the recent suspension by the medical board in Maine of Dr. Meryl Nass' medical license and the ordering of her to undergo a psychiatric evaluation for prescribing Ivermectin and sharing her expertise in this field.\\[9,65\\] I know Dr, Nass personally and can vouch for her integrity, brilliance and dedication to truth. Her scientific credentials are impeccable. This behavior by a medical licensing board is reminiscent of the methodology of the Soviet KGB during the period when dissidents were incarcerated in psychiatric gulags to silence their dissent.\n\n# OTHER UNPRECEDENTED ATTACKS\n\nAnother unprecedented tactic is to remove dissenting doctors from their positions as journal editors, reviewers and retracting of their scientific papers from journals, even after these papers have been in print. Until this pandemic event, I have never seen so many journal papers being retracted\u2014 the vast majority promoting alternatives to official dogma, especially if the papers question vaccine safety. Normally a submitted paper or study is reviewed by experts in the field, called peer review. These reviews can be quite intense and nit picking in detail, insisting that all errors within the paper be corrected before publication. So, unless fraud or some other major hidden problem is discovered after the paper is in print, the paper remains in the scientific literature.\n\nWe are now witnessing a growing number of excellent scientific papers, written by top experts in the field, being retracted from major medical and scientific journals weeks, months and even years after publication. A careful review indicates that in far too many instances the authors dared question accepted dogma by the controllers of scientific publications\u2014especially concerning the safety, alternative treatments or efficacy of vaccines.\\[12,63\\] These journals rely on extensive adverting by pharmaceutical companies for their revenue. Several instances have occurred where powerful pharmaceutical companies exerted their influence on owners of these journals to remove articles that in any way question these companies' products.\\[13,34,35\\]\n\nWorse still is the actual designing of medical articles for promoting drugs and pharmaceutical products that involve fake studies, so-called ghostwritten articles.\\[49,64\\] Richard Horton is quoted by the Guardian as saying \"journals have devolved into information laundering operations for the pharmaceutical industry.\"\\[13,63\\] Proven fraudulent \"ghostwritten\" articles sponsored by pharmaceutical giants have appeared regularly in top clinical journals, such as JAMA, and New England Journal of Medicine\u2014never to be removed despite proven scientific abuse and manipulation of data.\\[49,63\\]\n\nGhostwritten articles involve using planning companies whose job it is to design articles containing manipulated data to support a pharmaceutical product and then have these articles accepted by high-impact clinical journals, that is, the journals most likely to affect clinical decision making of doctors. Further, they supply doctors in clinical practice with free reprints of these manipulated articles. The Guardian found 250 companies engaged in this ghostwriting business. The final step in designing these articles for publication in the most prestigious journals is to recruit well recognized medical experts from prestigious institutions, to add their name to these articles. These recruited medical authors are either paid upon agreeing to add their name to these pre- written articles or they do so for the prestige of having their name on an article in a prestigious medical journal.\\[11\\]\n\nOf vital importance is the observation by experts in the field of medical publishing that nothing has been done to stop this abuse. Medical ethicists have lamented that because of this widespread practice \"you can't trust anything.\" While some journals insist on disclosure information, most doctors reading these articles ignore this information or excuse it and several journals make disclosure more difficult by requiring the reader to find the disclosure statements at another location. Many journals do not police such statements and omissions by authors are common and without punishment.\n\nAs concerns the information made available to the public, virtually all the media is under the control of these pharmaceutical giants or others who are benefitting from this \"pandemic\". Their stories are all the same, both in content and even wording. Orchestrated coverups occur daily and massive data exposing the lies being generated by these information controllers are hidden from the public. All data coming over the national media (TV, newspaper and magazines), as well as the local news you watch every day, comes only from \"official\" sources\u2014most of which are lies, distortions or completely manufactured out of whole cloth\u2014all aimed to deceive the public.\n\nTelevision media receives the majority of its advertising budget from the international pharmaceutical companies\u2014this creates an irresistible influence to report all concocted studies supporting their vaccines and other so-called treatments.\\[14\\] In 2020 alone the pharmaceutical industries spent 6.56 billion dollars on such advertising.\\[13,14\\] Pharma TV advertising amounted to 4.58 billion, an incredible 75% of their budget. That buys a lot of influence and control over the media. World famous experts within all fields of infectious diseases are excluded from media exposure and from social media should they in any way deviate against the concocted lies and distortions by the makers of these vaccines. In addition, these pharmaceutical companies spend tens of millions on social media advertising, with Pfizer leading the pack with \\$55 million in 2020.\\[14\\]\n\nWhile these attacks on free speech are terrifying enough, even worse is the virtually universal control hospital administrators have exercised over the details of medical care in hospitals. These hirelings are now instructing doctors which treatment protocols they will adhere to and which treatments they will not use, no matter how harmful the \"approved\" treatments are or how beneficial the \"unapproved\" treatments are.\\[33,57\\]\n\nNever in the history of American medicine have hospital administrators dictated to its physicians how they will practice medicine and what medications they can use. The CDC has no authority to dictate to hospitals or doctors concerning medical treatments. Yet, most physicians complied without the slightest resistance.\n\nThe federal Care Act encouraged this human disaster by offering all US hospitals up to 39,000 dollars for each ICU patient they put on respirators, despite the fact that early on it was obvious that the respirators were a major cause of death among these unsuspecting, trusting patients. In addition, the hospitals received 12,000 dollars for each patient that was admitted to the ICU\u2014explaining, in my opinion and others, why all federal medical bureaucracies (CDC, FDA, NIAID, NIH, etc) did all in their power to prevent life- saving early treatments.\\[46\\] Letting patients deteriorate to the point they needed hospitalization, meant big money for all hospitals. A growing number of hospitals are in danger of bankruptcy, and many have closed their doors, even before this \"pandemic\".\\[50\\] Most of these hospitals are now owned by national or international corporations, including teaching hospitals.\\[10\\]\n\nIt is also interesting to note that with the arrival of this \"pandemic\" we have witnessed a surge in hospital corporate chains buying up a number of these financially at-risk hospitals.\\[1,54\\] It has been noted that billions in Federal Covid aid is being used by these hospital giants to acquire these financially endangered hospitals, further increasing the power of corporate medicine over physician independence. Physicians expelled from their hospitals are finding it difficult to find other hospitals staffs to join since they too may be owned by the same corporate giant. As a result, vaccine mandate policies include far larger numbers of hospital employees. For example, Mayo Clinic fired 700 employees for exercising their right to refuse a dangerous, essentially untested experimental vaccine.\\[51,57\\] Mayo Clinic did this despite the fact that many of these employees worked during the worst of the epidemic and are being fired when the Omicron variant is the dominant strain of the virus, has the pathogenicity of a common cold for most and the vaccines are ineffective in preventing the infection.\n\nIn addition, it has been proven that the vaccinated asymptomatic person has a nasopharyngeal titer of the virus as high as an infected unvaccinated person. If the purpose of the vaccine mandate is to prevent viral spread among the hospital staff and patients, then it is the vaccinated who present the greatest risk of transmission, not the unvaccinated. The difference is that a sick unvaccinated person would not go to work, the asymptomatic vaccinated spreader will.\n\nWhat we do know is that major medical centers, such as Mayo Clinic, receive tens of millions of dollars in NIH grants each year as well as monies from the pharmaceutical makers of these experimental \"vaccines\". In my view, that is the real consideration driving these policies. If this could be proven in a court of law the administrators making these mandates should be prosecuted to the fullest extent of the law and sued by all injured parties.\n\nThe hospital bankruptcy problem has grown increasingly acute due to hospitals vaccine mandates and resulting large number of hospitals staff, especially nurses, refusing to be forcibly vaccinated.\\[17,51\\] This is all unprecedented in the history of medical care. Doctors within hospitals are responsible for the treatment of their individual patients and work directly with these patients and their families to initiate these treatments. Outside organizations, such as the CDC, have no authority to intervene in these treatments and to do so exposes the patients to grave errors by an organization that has never treated a single COVID-19 patient.\n\nWhen this pandemic started, hospitals were ordered by the CDC to follow a treatment protocol that resulted in the deaths of hundreds of thousands of patients, most of whom would have recovered had proper treatments been allowed.\\[43,44\\] The majority of these deaths could have been prevented had doctors been allowed to use early treatment with such products as Ivermectin, hydroxy-chloroquine and a number of other safe drugs and natural compounds. It has been estimated, based on results by physicians treating the most covid patients successfully, that of the 800,000 people that we are told died from Covid, 640,000 could have not only been saved, but could have, in many cases, returned to their pre-infection health status had mandated early treatment with these proven methods been used. This neglect of early treatment constitutes mass murder. That means 160,000 would have actually died, far less than the number dying at the hands of bureaucracies, medical associations and medical boards that refused to stand up for their patients. According to studies of early treatment of thousands of patients by brave, caring doctors, seventy-five to eighty percent of the deaths could have been prevented.\\[43,44\\]\n\nIncredibly, these knowledgeable doctors were prevented from saving these Covid-19 infected people. It should be an embarrassment to the medical profession that so many doctors mindlessly followed the deadly protocols established by the controllers of medicine.\n\nOne must also keep in mind that this event never satisfied the criteria for a pandemic. The World Health Organization changed the criteria to make this a pandemic. To qualify for a pandemic status the virus must have a high mortality rate for the vast majority of people, which it didn't (with a 99.98% survival rate), and it must have no known existing treatments\u2014which this virus had\u2014in fact, a growing number of very successful treatments.\n\nThe draconian measures established to contain this contrived \"pandemic\" have never been shown to be successful, such as masking the public, lockdowns, and social distancing. A number of carefully done studies during previous flu seasons demonstrated that masks, of any kind, had never prevented the spread of the virus among the public.\\[60\\]\n\nIn fact, some very good studies suggested that the masks actually spread the virus by giving people a false sense of security and other factors, such as the observation that people were constantly breaking sterile technique by touching their mask, improper removal and by leakage of infectious aerosols around the edges of the mask. In addition masks were being disposed of in parking lots, walking trails, laid on tabletops in restaurants and placed in pockets and purses.\n\nWithin a few minutes of putting on the mask, a number of pathogenic bacteria can be cultured from the masks, putting the immune suppressed person at a high risk of bacterial pneumonia and children at a higher risk of meningitis.\\[16\\] A study by researchers at the University of Florida cultured over 11 pathogenic bacteria from the inside of the mask worn by children in schools.\\[40\\]\n\nIt was also known that children were at essentially no risk of either getting sick from the virus or transmitting it.\n\nIn addition, it was also known that wearing a mask for over 4 hours (as occurs in all schools) results in significant hypoxia (low blood oxygen levels) and hypercapnia (high CO2 levels), which have a number of deleterious effects on health, including impairing the development of the child's brain.\\[4,72,52\\]\n\nWe have known that brain development continues long after the grade school years. A recent study found that children born during the \"pandemic\" have significantly lower IQs\u2014yet school boards, school principals and other educational bureaucrats are obviously unconcerned.\\[18\\]\n\n# TOOLS OF THE INDOCTRINATION TRADE\n\nThe designers of this pandemic anticipated a pushback by the public and that major embarrassing questions would be asked. To prevent this, the controllers fed the media a number of tactics, one of the most commonly used was and is the \"fact check\" scam. With each confrontation with carefully documented evidence, the media \"fact checkers\" countered with the charge of \"misinformation\", and an unfounded \"conspiracy theory\" charge that was, in their lexicon, \"debunked\". Never were we told who the fact checkers were or the source of their \"debunking\" information\u2014we were just to believe the \"fact checkers\". A recent court case established under oath that facebook \"fact checkers\" used their own staff opinion and not real experts to check \"facts\".\\[59\\] When sources are in fact revealed they are invariably the corrupt CDC, WHO or Anthony Fauci or just their opinion. Here is a list of things that were labeled as \"myths\" and \"misinformation\" that were later proven to be true.\n\n-   The asymptomatic vaccinated are spreading the virus equally as with unvaccinated symptomatic infected.\n\n-   The vaccines cannot protect adequately against new variants, such as Delta and Omicron.\n\n-   Natural immunity is far superior to vaccine immunity and is most likely lifelong.\n\n-   Vaccine immunity not only wanes after several months, but all immune cells are impaired for prolonged periods, putting the vaccinated at a high risk of all infections and cancer.\n\n-   COVID vaccines can cause a significant incidence of blood clots and other serious side effects\n\n-   The vaccine proponents will demand numerous boosters as each variant appears on the scene.\n\n-   Fauci will insist on the covid vaccine for small children and even babies.\n\n-   Vaccine passports will be required to enter a business, fly in a plane, and use public transportation\n\n-   There will be internment camps for the unvaccinated (as in Australia, Austria and Canada)\n\n-   The unvaccinated will be denied employment.\n\n-   There are secret agreements between the government, elitist institutions, and vaccine makers\n\n-   Many hospitals were either empty or had low occupancy during the pandemic.\n\n-   The spike protein from the vaccine enters the nucleus of the cell, altering cell DNA repair function.\n\n-   Hundreds of thousands have been killed by the vaccines and many times more have been permanently damaged.\n\n-   Early treatment could have saved the lives of most of the 700,000 who died.\n\n-   Vaccine-induced myocarditis (which was denied initially) is a significant problem and clears over a short period.\n\n-   Special deadly lots (batches) of these vaccines are mixed with the mass of other Covid-19 vaccines\n\nSeveral of these claims by those opposing these vaccines now appear on the CDC website\u2014most still identified as \"myths\". Today, extensive evidence has confirmed that each of these so-called \"myths\" were in fact true. Many are even admitted by the \"saint of vaccines\", Anthony Fauci. For example, we were told, even by our cognitively impaired President, that once the vaccine was released all the vaccinated people could take off their masks. Oops! We were told shortly afterward\u2014 the vaccinated have high concentrations (titers) of the virus in their noses and mouths (nasopharynx) and can transmit the virus to others in which they come into contact\u2014especially their own family members. On go the masks once again\u2014 in fact double masking is recommended. The vaccinated are now known to be the main superspreaders of the virus and hospitals are filled with the sick vaccinated and people suffering from serious vaccine complications.\\[27,42,45\\]\n\nAnother tactic by the vaccine proponents is to demonize those who reject being vaccinated for a variety of reasons. The media refers to these critically thinking individuals as \"anti-vaxxers\", \"vaccine deniers\", \"Vaccine resisters\", \"murders\", \"enemies of the greater good\" and as being the ones prolonging the pandemic. I have been appalled by the vicious, often heartless attacks by some of the people on social media when a parent or loved one relates a story of the terrible suffering and eventual death, they or their loved one suffered as a result of the vaccines. Some psychopaths tweet that they are glad that the loved one died or that the dead vaccinated person was an enemy of good for telling of the event and should be banned. This is hard to conceptualize. This level of cruelty is terrifying, and signifies the collapse of a moral, decent, and compassionate society.\n\nIt is bad enough for the public to sink this low, but the media, political leaders, hospital administrators, medical associations and medical licensing boards are acting in a similar morally dysfunctional and cruel way.\n\n# LOGIC, REASONING, AND SCIENTIFIC EVIDENCE HAS DISAPPEARED IN THIS EVENT\n\nHas scientific evidence, carefully done studies, clinical experience and medical logic had any effect on stopping these ineffective and dangerous vaccines? Absolutely not! The draconian efforts to vaccinate everyone on the planet continues (except the elite, postal workers, members of Congress and other insiders).\\[31,62\\]\n\nIn the case of all other drugs and previous conventional vaccines under review by the FDA, the otherwise unexplained deaths of 50 or less individuals would result in a halt in further distribution of the product, as happened on 1976 with the swine flu vaccine. With over 18,000 deaths being reported by the VAERS system for the period December 14, 2020 and December 31^st^, 2021 as well as 139,126 serious injuries (including deaths) for the same period there is still no interest in stopping this deadly vaccine program.\\[61\\] Worse, there is no serious investigation by any government agency to determine why these people are dying and being seriously and permanently injured by these vaccines.\\[15,67\\] What we do see is a continuous series of coverups and evasions by the vaccine makers and their promoters.\n\nThe war against effective cheap and very safe repurposed drugs and natural compounds, that have proven beyond all doubt to have saved millions of lives all over the world, has not only continued but has stepped up in intensity.\\[32,34,43\\]\n\nDoctors are told they cannot provide these life-saving compounds for their patients and if they do, they will be removed from the hospital, have their medical license removed or be punished in many other ways. A great many pharmacies have refused to fill prescriptions for lvermectin or hydroxy- chloroquine, despite the fact that millions of people have taken these drugs safely for over 60 years in the case of hydroxy chloroquine and decades for Ivermectin.\\[33,36\\] This refusal to fill prescriptions is unprecedented and has been engineered by those wanting to prevent alternative methods of treatment, all based on protecting vaccine expansion to all. Several companies that make hydroxy chloroquine agreed to empty their stocks of the drug by donating them to the Strategic National Stockpile, making this drug far more difficult to get.\\[33\\] Why would the government do that when over 30 well-done studies have shown that this drug reduced deaths anywhere from 66% to 92% in other countries, such as India, Egypt, Argentina, France, Nigeria, Spain, Peru, Mexico, and others?\\[23\\]\n\nThe critics of these two life-saving drugs are most often funded by Bill Gates and Anthony Fauci, both of which are making millions from these vaccines.\\[48,15\\]\n\nTo further stop the use of these drugs, the pharmaceutical industry and Bill Gates\/Anthony Fauci funded fake research to make the case that hydroxy chloroquine was a dangerous drug and could damage the heart.\\[34\\] To make this fraudulent case the researchers administered the sickest of covid patients a near lethal dose of the drug, in a dose far higher than used on any covid patient by Dr. Kory, McCullough and other \"real\", and compassionate doctors, physicians who were actually treating covid patients.\\[23\\]\n\nThe controlled, lap-dog media, of course, hammered the public with stories of the deadly effect of hydroxy- chloroquine, all with a terrified look of fake panic. All these stories of ivermectin dangers were shown to be untrue and some of the stories were incredibly preposterous.\\[37,43\\]\n\nThe attack on Ivermectin was even more vicious than against hydroxy-chloroquine. All of this, and a great deal more is meticulously chronicled in Robert Kennedy, Jr's excellent new book\u2014*The Real Anthony Fauci. Bill Gates, Big Pharma, and the Global War on Democracy and Public Health*.\\[32\\] If you are truly concerned with the truth and with all that has occurred since this atrocity started, you must not only read, but study this book carefully. It is fully referenced and covers all topics in great detail. This is a designed human tragedy of Biblical proportions by some of the most vile, heartless, psychopaths in history.\n\nMillions have been deliberately killed and crippled, not only by this engineered virus, but by the vaccine itself and by the draconian measures used by these governments to \"control the pandemic spread\". We must not ignore the \"deaths by despair\" caused by these draconian measures, which can exceed hundreds of thousands. Millions have starved in third world countries as a result. In the United States alone, of the 800,000 who died, claimed by the medical bureaucracies, well over 600,000 of these deaths were the result of the purposeful neglect of early treatment, blocking the use of highly effective and safe repurposed drugs, such as hydroxy-chloroquine and Ivermectin, and the forced use of deadly treatments such as remdesivir and use of ventilators. This does not count the deaths of despair and neglected medical care caused by the lockdown and hospital measures forced on healthcare systems.\n\nTo compound all this, because of vaccine mandates among all hospital personnel, thousands of nurses and other hospital workers have resigned or been fired.\\[17,30,51\\] This has resulted in critical shortages of these vital healthcare workers and dangerous reductions of ICU beds in many hospitals. In addition, as occurred in the Lewis County Healthcare System, a specialty-hospital system in Lowville, N.Y., closed its maternity unit following the resignation of 30 hospital staff over the state's disastrous vaccine mandate orders. The irony in all these cases of resignations is that the administrators unhesitatingly accepted these mass staffing losses despite rantings about suffering from short staffing during a \"crisis\". This is especially puzzling when we learned that the vaccines did not prevent viral transmission and the present predominant variant is of extremely low pathogenicity.\n\n# DANGERS OF THE VACCINES ARE INCREASINGLY REVEALED BY SCIENCE\n\nWhile most researchers, virologists, infectious disease researchers and epidemiologists have been intimidated into silence, a growing number of high integrity individuals with tremendous expertise have come forward to tell the truth\u2014that is, that <u>these vaccines are deadly<\/u>.\n\nMost new vaccines must go through extensive safety testing for years before they are approved. New technologies, such as the mRNA and DNA vaccines, require a minimum of 10 years of careful testing and extensive follow-up. These new so-called vaccines were \"tested\" for only 2 months and then the results of these safety test were and continue to be kept secret. Testimony before Senator Ron Johnson by several who participated in the 2 months study indicates that virtually no follow-up of the participants of the pre-release study was ever done.\\[67\\] Complains of complications were ignored and despite promises by Pfizer that all medical expenses caused by the \"vaccines\" would be paid by Pfizer, these individuals stated that none were paid.\\[66\\] Some medical expenses exceed 100,000 dollars.\n\nAs an example of the deception by Pfizer, and the other makers of mRNA vaccines, is the case of 12-year-old Maddie de Garay, who participated in the Pfizer vaccine pre-release safety study. At Sen. Johnson's presentation with the families of the vaccine injured, her mother told of her child's recurrent seizures, that she is now confined to a wheelchair, must be tube fed and suffers permanent brain damage. On the Pfizer safety evaluation submitted to the FDA her only side effect is listed as having a \"stomachache\". Each person submitted similar horrifying stories.\n\nThe Japanese resorted to a FOIA (Freedom of Information Act) lawsuit to force Pfizer to release its secret biodistribution study. The reason Pfizer wanted it kept secret is that it demonstrated that Pfizer lied to the public and the regulatory agencies about the fate of the injected vaccine contents (the mRNA enclosed nano-lipid carrier). They claimed that it remained at the site of the injection (the shoulder), when in fact their own study found that it rapidly spread throughout the entire body by the bloodstream within 48 hours.\n\nThe study also found that these deadly nano-lipid carriers collected in very high concentrations in several organs, including the reproductive organs of males and females, the heart, the liver, the bone marrow, and the spleen (a major immune organ). The highest concentration was in the ovaries and the bone marrow. These nano-lipid carriers also were deposited in the brain.\n\nDr. Ryan Cole, a pathologist from Idaho reported a dramatic spike in highly aggressive cancers among vaccinated individuals, (not reported in the Media). He found a frighteningly high incidence of highly aggressive cancers in vaccinated individuals, especially highly invasive melanomas in young people and uterine cancers in women.\\[26\\] Other reports of activation of previously controlled cancers are also appearing among vaccinated cancer patients.\\[47\\] Thus far, no studies have been done to confirm these reports, but it is unlikely such studies will be done, at least studies funded by grants from the NIH.\n\nThe high concentration of spike proteins found in the ovaries in the biodistribution study could very well impair fertility in young women, alter menstruation, and could put them at an increased risk of ovarian cancer. The high concentration in the bone marrow, could also put the vaccinated at a high risk of leukemia and lymphoma. The leukemia risk is very worrisome now that they have started vaccinating children as young as 5 years of age. No long-term studies have been conducted by any of these makers of Covid-19 vaccines, especially as regards the risk of cancer induction. Chronic inflammation is intimately linked to cancer induction, growth and invasion and vaccines stimulate inflammation.\n\nCancer patients are being told they should get vaccinated with these deadly vaccines. This, in my opinion, is insane. Newer studies have shown that this type of vaccine inserts the spike protein within the nucleus of the immune cells (and most likely many cell types) and once there, inhibits two very important DNA repair enzymes, BRCA1 and 53BP1, whose duty it is to repair damage to the cell's DNA.\\[29\\] Unrepaired DNA damage plays a major role in cancer.\n\nThere is a hereditary disease called xeroderma pigmentosum in which the DNA repair enzymes are defective. These ill-fated individuals develop multiple skin cancers and a very high incidence of organ cancer as a result. Here we have a vaccine that does the same thing, but to a less extensive degree.\n\nOne of the defective repair enzymes caused by these vaccines is called BRCA1, which is associated with a significantly higher incidence of breast cancer in women and prostate cancer in men.\n\nIt should be noted that no studies were ever done on several critical aspects of this type of vaccine.\n\n-   They have never been tested for long term effects\n\n-   They have never been tested for induction of autoimmunity\n\n-   They have never been properly tested for safety during any stage of pregnancy\n\n-   No follow-up studies have been done on the babies of vaccinated women\n\n-   There are no long-term studies on the children of vaccinated pregnant women after their birth (Especially as neurodevelopmental milestone occur).\n\n-   It has never been tested for effects on a long list of medical conditions:\n\n    -   Diabetes\n\n    -   Heart disease\n\n    -   Atherosclerosis\n\n    -   Neurodegenerative diseases\n\n    -   Neuropsychiatric effects\n\n    -   Induction of autism spectrum disorders and schizophrenia\n\n    -   Long term immune function\n\n    -   Vertical transmission of defects and disorders\n\n    -   Cancer\n\n    -   Autoimmune disorders\n\nPrevious experience with the flu vaccines clearly demonstrates that the safety studies done by researchers and clinical doctors with ties to pharmaceutical companies were essentially all either poorly done or purposefully designed to falsely show safety and coverup side effects and complications. This was dramatically demonstrated with the previously mentioned phony studies designed to indicate that hydroxy Chloroquine and Ivermectin were ineffective and too dangerous to use.\\[34,36,37\\] These fake studies resulted in millions of deaths and severe health disasters worldwide. As stated, 80% of all deaths were unnecessary and could have been prevented with inexpensive, safe repurposed medications with a very long safety history among millions who have taken them for decades or even a lifetime.\\[43,44\\]\n\nIt is beyond ironic that those claiming that they are responsible for protecting our health approved a poorly tested set of vaccines that has resulted in more deaths in less than a year of use than all the other vaccines combined given over the past 30 years. Their excuse when confronted was\u2014\"we had to overlook some safety measures because this was a deadly pandemic\".\\[28,46\\]\n\nIn 1986 President Reagan signed the National Childhood Vaccine Injury Act, which gave blanket protection to pharmaceutical makers of vaccines against injury litigation by families of vaccine injured individuals. The Supreme Court, in a 57-page opinion, ruled in favor of the vaccine companies, effectively allowing vaccine makers to manufacture and distribute dangerous, often ineffective vaccines to the population without fear of legal consequences. The court did insist on a vaccine injury compensation system which has paid out only a very small number of rewards to a large number of severely injured individuals. It is known that it is very difficult to receive these awards. According to the Health Resources and Services Administration, since 1988 the Vaccine Injury Compensation Program (VICP) has agreed to pay 3,597 awards among 19,098 vaccine injured individuals applying amounting to a total sum of \\$3.8 billion. This was prior to the introduction of the Covid-19 vaccines, in which the deaths alone exceed all deaths related to all the vaccines combined over a thirty-year period.\n\nIn 2018 President Trump signed into law the \"right-to-try\" law which allowed the use of experimental drugs and all unconventional treatments to be used in cases of extreme medical conditions. As we have seen with the refusal of many hospitals and even blanket refusal by states to allow Ivermectin, hydroxy-chloroquine or any other unapproved \"official\" methods to treat even terminal Covid-19 cases, these nefarious individuals have ignored this law.\n\nStrangely, they did not use this same logic or the law when it came to Ivermectin and Hydroxy Chloroquine, both of which had undergone extensive safety testing by over 30 clinical studies of a high quality and given glowing reports on both efficacy and safety in numerous countries. In addition, we had a record of use for up to 60 years by millions of people, using these drugs worldwide, with an excellent safety record. It was obvious that a group of very powerful people in conjunction with pharmaceutical conglomerates didn't want the pandemic to end and wanted vaccines as the only treatment option. Kennedy's book makes this case using extensive evidence and citations.\\[14,32\\]\n\nDr. James Thorpe, an expert in maternal-fetal medicine, demonstrates that these covoid-19 vaccines given during pregnancy have resulted in a 50-fold higher incidence of miscarriage than reported with all other vaccines combined.\\[28\\] When we examine his graph on fetal malformations there was a 144-fold higher incidence of fetal malformation with the Covid-19 vaccines given during pregnancy as compared to all other vaccines combined. Yet, the American Academy of Obstetrics and Gynecology and the American College of Obstetrics and Gynecology endorse the safety of these vaccines for all stages of pregnancy and among women breast feeding their babies.\n\nIt is noteworthy that these medical specialty groups have received significant funding from Pfizer pharmaceutical company. The American College of Obstetrics and Gynecology, just in the 4^th^ quarter of 2010, received a total of \\$11,000 from Pfizer Pharmaceutical company alone.\\[70\\] Funding from NIH grants are much higher.\\[20\\] The best way to lose these grants is to criticize the source of the funds, their products or pet programs. Peter Duesberg, because of his daring to question Fauci's pet theory of AIDS caused by HIV virus, was no longer awarded any of the 30 grant applications he submitted after going public. Prior to this episode, as the leading authority on retroviruses in the world, he had never been turned down for an NIH grant.\\[39\\] This is how the \"corrupted\" system works, even though much of the grant money comes from our taxes.\n\n# HOT LOTS\u2014DEADLY BATCHES OF THE VACCINES\n\nA new study has now surfaced, the results of which are terrifying.\\[25\\] A researcher at Kingston University in London, has completed an extensive analysis of the VAERs data (a subdepartment of the CDC which collects voluntary vaccine complication data), in which he grouped reported deaths following the vaccines according to the manufacturer's lot numbers of the vaccines. Vaccines are manufactured in large batches called lots. What he discovered was that the vaccines are divided into over 20,000 lots and that one out of every 200 of these batches (lots) is demonstrably deadly to anyone who receives a vaccine from that lot, which includes thousands of vaccine doses.\n\nHe examined all manufactured vaccines\u2014Pfizer, Moderna, Johnson and Johnson (Janssen), etc. He found that among every 200 batches of the vaccine from Pfizer and other makers, one batch of the 200 was found to be over 50x more deadly than vaccines batches from other lots. The other vaccine lots (batches) were also causing deaths and disabilities, but nowhere near to this extent. These deadly batches should have appeared randomly among all \"vaccines\" if it was an unintentional event. However, he found that 5% of the vaccines were responsible for 90% of the serious adverse events, including deaths. The incidence of deaths and serious complications among these \"hot lots\" varied from over 1000% to several thousand percent higher than comparable safer lots. If you think this was by accident\u2014think again. This is not the first time \"hot lots\" were, in my opinion, purposefully manufactured and sent across the nation\u2014usually vaccines designed for children. In one such scandal, \"hot lots\" of a vaccine ended up all in one state and the damage immediately became evident. What was the manufacture's response? It wasn't to remove the deadly batches of the vaccine. He ordered his company to scatter the hot lots across the nation so that authorities would not see the obvious deadly effect.\n\nAll lots of a vaccine are numbered\u2014for example Modera labels them with such codes as 013M20A. It was noted that the batch numbers ended in either 20A or 21A. Batches ending in 20A were much more toxic than the ones ending in 21A. The batches ending in 20A had about 1700 adverse events, versus a few hundred to twenty or thirty events for the 21A batches. This example explains why some people had few or no adverse events after taking the vaccine while others are either killed or severely and permanently harmed. To see the researcher's explanation, go to <https:\/\/www.bitchute.com\/video\/6xIYPZBkydsu\/> In my opinion these examples strongly suggest an intentional alteration of the production of the \"vaccine\" to include deadly batches.\n\nI have met and worked with a number of people concerned with vaccine safety and I can tell you they are not the evil anti-vaxxers you are told they are. They are highly principled, moral, compassionate people, many of which are top researchers and people who have studied the issue extensively. Robert Kennedy, Jr, Barbara Lou Fisher, Dr. Meryl Nass, Professor Christopher Shaw, Megan Redshaw, Dr. Sherri Tenpenny, Dr. Joseph Mercola, Neil Z. Miller, Dr. Lucija Tomjinovic, Dr. Stephanie Seneff, Dr. Steve Kirsch and Dr. Peter McCullough just to name a few. These people have nothing to gain and a lot to lose. They are attacked viciously by the media, government agencies, and elite billionaires who think they should control the world and everyone in it.\n\n# WHY DID FAUCI WANT NO AUTOPSIES OF THOSE WHO DIED AFTER VACCINATION?\n\nThere are many things about this \"pandemic\" that are unprecedented in medical history. One of the most startling is that at the height of the pandemic so few autopsies, especially total autopsies, were being done. A mysterious virus was rapidly spreading around the world, a selected group of people with weakened immune systems were getting seriously ill and many were dying and the one way we could rapidly gain the most knowledge about this virus\u2014an autopsy, was being discouraged.\n\nGuerriero noted that by the end of April, 2020 approximately 150,000 people had died, yet there were only 16 autopsies performed and reported in the medical literature.\\[24\\] Among these, only seven were complete autopsies, the remaining 9 being partial or by needle biopsy or incisional biopsy. Only after 170,000 deaths by Covid-19 and four months into the pandemic were the first series of autopsies actually done, that is, more than ten. And only after 280,000 deaths and another month, were the first large series of autopsies performed, some 80 in number.\\[22\\] Sperhake, in a call for autopsies to be done without question, noted that the first full autopsy reported in the literature along with photomicrographs appeared in a medico-legal journal from China in February 2020.\\[41,68\\] Sperhake expressed confusion as to why there was a reluctance to perform autopsies during the crisis, but he knew it was not coming from the pathologists. The medical literature was littered with appeals by pathologist for more autopsies to be performed.\\[58\\] Sperhake further noted that the Robert Koch Institute (The German health monitoring system) at least initially advised against doing autopsies. He also knew that at the time 200 participating autopsy institutions in the United States had done at least 225 autopsies among 14 states.\n\nSome have claimed that this dearth of autopsies was based on the government's fear of infection among the pathologists, but a study of 225 autopsies on Covid-19 cases demonstrated only one case of infection among the pathologist and this was concluded to have been an infection contracted elsewhere.\\[19\\] Guerriero ends his article calling for more autopsies with this observation: \"Shoulder to shoulder, clinical and forensic pathologists overcame the obstructions of autopsy studies in Covid-19 victims and hereby generated valuable knowledge on the pathophysiology of the interaction between the SARS-CoV-2 and the human body, thus contributing to our understanding of the disease.\"\\[24\\]\n\nSuspicion concerning the worldwide reluctance of nations to allow full post mortem studies of Covid-19 victims may be based on the idea that it was more than by chance. There are at least two possibilities that stand out. First, those leading the progression of this \"non-pandemic\" event into a perceived worldwide \"deadly pandemic\", were hiding an important secret that autopsies could document. Namely, just how many of the deaths were actually caused by the virus? To implement draconian measures, such as mandated mask wearing, lockdowns, destruction of businesses, and eventually mandated forced vaccination, they needed very large numbers of covid-19 infected dead. Fear would be the driving force for all these destructive pandemic control programs.\n\nElder et al in his study classified the autopsy findings into four groups.\\[22\\]\n\n1.  Certain Covid-19 death\n\n2.  Probably Covid-19 death\n\n3.  Possible Covid-19 death\n\n4.  Not associated with Covid-19, despite the positive test.\n\nWhat possibly concerned or even terrified the engineers of this pandemic was that autopsies just might, and did, show that a number of these so-called Covid-19 deaths in truth died of their comorbid diseases. In the vast majority of autopsy studies reported, pathologists noted multiple comorbid conditions, most of which at the extremes of life could alone be fatal. Previously it was known that common cold viruses had an 8% mortality in nursing homes.\n\nIn addition, valuable evidence could be obtained from the autopsies that would improve clinical treatments and could possibly demonstrate the deadly effect of the CDC mandated protocols all hospitals were required to follow, such as the use of respirators and the deadly, kidney-destroying drug remdesivir. The autopsies also demonstrated accumulating medical errors and poor-quality care, as the shielding of doctors in intensive care units from the eyes of family members inevitably leads to poorer quality care as reported by several nurses working in these areas.\\[53-55\\]\n\nAs bad as all this was, the very same thing is being done in the case of Covid vaccine deaths\u2014very few complete autopsies have been done to understand why these people died, that is, until recently. Two highly qualified researchers, Dr. Sucharit Bhakdi a microbiologist and highly qualified expert in infectious disease and Dr. Arne Burkhardt, a pathologist who is a widely published authority having been a professor of pathology at several prestigious institutions, recently performed autopsies on 15 people having died after vaccination. What they found explains why so many are dying and experiencing organ damage and deadly blood clots.\\[5\\]\n\nThey determined that 14 of the fifteen people died as a result of the vaccines and not of other causes. Dr. Burkhardt, the pathologist, observed widespread evidence of an immune attack on the autopsied individuals' organs and tissues\u2014 especially their heart. This evidence included extensive invasion of small blood vessels with massive numbers of lymphocytes, which cause extensive cell destruction when unleashed. Other organs, such as the lungs and liver, were observed to have extensive damage as well. These findings indicate the vaccines were causing the body to attack itself with deadly consequences. One can easily see why Anthony Fauci, as well as public health officers and all who are heavily promoting these vaccines, publicly discouraged autopsies on the vaccinated who subsequently died. One can also see that in the case of vaccines, that were essentially untested prior to being approved for the general public, at least the regulatory agencies should have been required to carefully monitor and analyze all serious complications, and certainly deaths, linked to these vaccines. The best way to do that is with complete autopsies.\n\nWhile we learned important information from these autopsies what is really needed are special studies of the tissues of those who have died after vaccination for the presence of spike protein infiltration throughout the organs and tissues. This would be critical information, as such infiltration would result in severe damage to all tissues and organs involved\u2014especially the heart, the brain, and the immune system. Animal studies have demonstrated this. In these vaccinated individuals the source of these spike proteins would be the injected nanolipid carriers of the spike protein producing mRNA. It is obvious that the government health authorities and pharmaceutical manufacturers of these \"vaccines\" do not want these critical studies done as the public would be outraged and demand an end to the vaccination program and prosecution of the involved individuals who covered this up.\n\n# CONCLUSIONS\n\nWe are all living through one of the most drastic changes in our culture, economic system, as well as political system in our nation's history as well as the rest of the world. We have been told that we will never return to \"normal\" and that a great reset has been designed to create a \"new world order\". This has all been outlined by Klaus Schwab, head of the World Economic Forum, in his book on the \"Great Reset\".\\[66\\] This book gives a great deal of insight as to the thinking of the utopians who are proud to claim this pandemic \"crisis\" as their way to usher in a new world. This new world order has been on the drawing boards of the elite manipulators for over a century.\\[73,74\\] In this paper I have concentrated on the devastating effects this has had on the medical care system in the United States, but also includes much of the Western world. In past papers I have discussed the slow erosion of traditional medical care in the United States and how this system has become increasingly bureaucratized and regimented.\\[7,8\\] This process was rapidly accelerating, but the appearance of this, in my opinion, manufactured \"pandemic\" has transformed our health care system over night.\n\nAs you have seen, an unprecedented series of events have taken place within this system. Hospital administrators, for example, assumed the position of medical dictators, ordering doctors to follow protocols derived not from those having extensive experience in treating this virus, but rather from a medical bureaucracy that has never treated a single COVID-19 patient. The mandated use of respirators on ICU Covid-19 patients, for example, was imposed in all medical systems and dissenting physicians were rapidly removed from their positions as caregivers, despite their demonstration of markedly improved treatment methods. Further, doctors were told to use the drug remdesivir despite its proven toxicity, lack of effectiveness and high complication rate. They were told to use drugs that impaired respiration and mask every patient, despite the patient's impaired breathing. In each case, those who refused to abuse their patients were removed from the hospital and even faced a loss of license\u2014or worse.\n\nFor the first time in modern medical history, early medical treatment of these infected patients was ignored nationwide. Studies have shown that early medical treatment was saving 80% of higher number of these infected people when initiated by independent doctors.\\[43,44\\] Early treatment could have saved over 640,000 lives over the course of this \"pandemic\". Despite the demonstration of the power of these early treatments, the forces controlling medical care continued this destructive policy.\n\nFamilies were not allowed to see their loved ones, forcing these very sick individuals in the hospitals to face their deaths alone. To add insult to injury, funerals were limited to a few grieving family members, who were not allowed to even sit together. All the while large stores, such as Walmart and Cosco were allowed to operate with minimal restrictions. Nursing home patients were also not allowed to have family visitations, again being forced to die a lonely death. All the while, in a number of states, the most transparent being in New York state, infected elderly were purposefully transferred from hospitals into nursing homes, resulting in a very high death rates of these nursing home residents. At the beginning of this \"pandemic\" over 50% of all death were occurring in nursing homes.\n\nThroughout this \"pandemic\" we have been fed an unending series of lies, distortions and disinformation by the media, the public health officials, medical bureaucracies (CDC, FDA and WHO) and medical associations. Physicians, scientists, and experts in infectious treatments who formed associations designed to develop more effective and safer treatments, were regularly demonized, harassed, shamed, humiliated, and experience a loss of licensure, loss of hospital privileges and, in at least one case, ordered to have a psychiatric examination.\\[2,65,71\\]\n\nAnthony Fauci was given essentially absolute control of all forms of medical care during this event, including insisting that drugs he profited from be used by all treating physicians. He ordered the use of masks, despite at first laughing at the use of masks to filter a virus. Governors, mayors, and many businesses followed his orders without question.\n\nThe draconian measures being used, masking, lockdowns, testing of the uninfected, use of the inaccurate PCR test, social distancing, and contact tracing had been shown previously to be of little or no use during previous pandemics, yet all attempts to reject these methods were to no avail. Some states ignored these draconian orders and had either the same or fewer cases, as well as deaths, as the states with the most strictly enforced measures. Again, no amount of evidence or obvious demonstration along these lines had any effect on ending these socially destructive measures. Even when entire countries, such as Sweden, which avoided all these measures, demonstrated equal rates of infections and hospitalization as nations with the strictest, very draconian measures, no policy change by the controlling institutions occurred. No amount of evidence changed anything.\n\nExperts in the psychology of destructive events, such as economic collapses, major disasters and previous pandemics demonstrated that draconian measures come with an enormous cost in the form of \"deaths of despair\" and in a dramatic increase in serious psychological disorders. The effects of these pandemic measures on children's neurodevelopment is catastrophic and to a large extent irreversible.\n\nOver time tens of thousands could die as a result of this damage. Even when these predictions began to appear, the controllers of this \"pandemic\" continued full steam ahead. Drastic increases in suicides, a rise in obesity, a rise in drug and alcohol use, a worsening of many health measures and a terrifying rise in psychiatric disorders, especially depression and anxiety, were ignored by the officials controlling this event.\n\nWe eventually learned that many of the deaths were a result of medical neglect. Individuals with chronic medical conditions, diabetes, cancer, cardiovascular disease, and neurological diseases were no longer being followed properly in their clinics and doctor's offices. Non-emergency surgeries were put on hold. Many of these patients chose to die at home rather than risk going to the hospitals and many considered hospitals \"death houses\".\n\nRecords of deaths have shown that there was a rise in deaths among those aged 75 and older, mostly explained by Covid-19 infections, but for those between the ages of 65 to 74, deaths had been increasing well before the pandemic onset.\\[69\\] Between ages of 18 and aged 65 years, records demonstrate a shocking hike in non-Covid-19 deaths. Some of these deaths were explained by a dramatic increase in drug-related deaths, some 20,000 more than 2019. Alcohol related deaths also increased substantially, and homicides increased almost 30% in the 18 to 65-year group.\n\nThe head of the insurance company OneAmerica stated that their data indicated that the death rate for individuals aged 18 to 64 had increased 40% over the pre-pandemic period.\\[21\\] Scott Davidson, the company's CEO, stated that this represented the highest death rate in the history of insurance records, which does extensive data collections on death rates each year. Davidson also noted that this high of a death rate increase has never been seen in the history of death data collection. Previous catastrophes of monumental extent increased death rates no more than 10 percent, 40% is unprecedented.\n\nDr. Lindsay Weaver, Indiana's chief medical officer, stated that hospitalizations in Indiana are higher than at any point in the past five years. This is of critical importance since the vaccines were supposed to significantly reduce deaths, but the opposite has happened. Hospitals are being flooded with vaccine complications and people in critical condition from medical neglect caused by the lockdowns and other pandemic measures.\\[46,56\\]\n\nA dramatic number of these people are now dying, with the spike occurring after the vaccines were introduced. The lies flowing from those who have appointed themselves as medical dictators are endless. First, we were told that the lockdown would last only two weeks, they lasted over a year. Then we were told that masks were ineffective and did not need to be worn. Quickly that was reversed. Then we were told the cloth mask was very effective, now it's not and everyone should be wearing an N95 mask and before that that they should double mask. We were told there was a severe shortage of respirators, then we discover they are sitting unused in warehouses and in city dumps, still in their packing crates. We were informed that the hospitals were filled mostly with the unvaccinated and later found the exact opposite was true the world over. We were told that the vaccine was 95% effective, only to learn that in fact the vaccines cause a progressive erosion of innate immunity.\n\nUpon release of the vaccines, women were told the vaccines were safe during all states of pregnancy, only to find out no studies had been done on safety during pregnancy during the \"safety tests\" prior to release of the vaccine. We were told that careful testing on volunteers before the EUA approval for public use demonstrated extreme safety of the vaccines, only to learn that these unfortunate subjects were not followed, medical complications caused by the vaccines were not paid for and the media covered this all up.\\[67\\] We also learned that the pharmaceutical makers of the vaccines were told by the FDA that further animal testing was unnecessary (the general public would be the Guinea pigs.) Incredibly, we were told that the Pfizer's new mRNA vaccines had been approved by the FDA, which was a cleaver deception, in that another vaccine had approval (comirnaty) and not the one being used, the BioNTech vaccine. The approved comirnaty vaccine was not available in the United States. The national media told the public that the Pfizer vaccine had been approved and was no longer classed as experimental, a blatant lie. These deadly lies continue. It is time to stop this insanity and bring these people to justice.\n\n## Disclaimer\n\nThe views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the Journal or its management.\n\n# REFERENCES","meta":{"dup_signals":{"dup_doc_count":156,"dup_dump_count":15,"dup_details":{"curated_sources":2,"2023-50":18,"2023-40":14,"2023-23":5,"2023-14":18,"2023-06":13,"2022-49":18,"2022-40":8,"2022-33":8,"2022-27":23,"2022-21":3,"2024-30":4,"2024-26":4,"2024-22":4,"2024-18":9,"2024-10":5}},"file":"PMC9062939"},"subset":"pubmed_central"}
{"text":"date: 2020\ntitle: GLOBAL PARTNERSHIP TO MAKE AVAILABLE 120 MILLION AFFORDABLE, QUALITY COVID-19 RAPID TESTS FOR LOW- AND MIDDLE-INCOME COUNTRIES\n\n**28 September 2020** - A set of agreements to make available, for low and middle-income countries, affordable, high-quality COVID-19 antigen rapid tests were today announced by the Access to COVID-19 Tools (ACT) Accelerator. Organizations involved in the milestone agreement include the Africa Centres for Disease Control and Prevention (Africa CDC), the Bill & Melinda Gates Foundation, the Clinton Health Access Initiative (CHAI), the Foundation for Innovative New Diagnostics (FIND), the Global Fund, Unitaid, and the World Health Organization (WHO).\n\nAs part of this comprehensive, end-to-end effort, the Bill & Melinda Gates Foundation has executed separate volume guarantee agreements with rapid diagnostic test (RDT) producers Abbott and SD Biosensor. These two arrangements will make available to LMICs 120 million antigen rapid diagnostic tests (Ag RDTs) \u2013 priced at a maximum of US\\$5 per unit \u2013 over a period of six months. These tests provide results in 15\u201330 minutes, rather than hours or days, and will enable expansion of testing, particularly in countries that do not have extensive laboratory facilities or trained health workers to implement molecular (polymerase-chain reaction or PCR) tests.\n\nThe tests developed by Abbott and SD Biosensor are highly portable, reliable, and easy to administer, making testing possible in near-person, decentralized healthcare settings. Both companies' tests are faster and cheaper than laboratory-based tests, enabling countries to increase the pace of testing, tracing and treating people for COVID-19 at the point of care particularly in areas with under-resourced health systems. A number of other Ag RDTs are at various stages of development and assessment.\n\nTo scale up the Ag RDTs, the Global Fund today announced that it has made available an initial US\\$50 million from its COVID-19 Response Mechanism to enable countries to purchase at least 10 million of the new rapid tests for LMICs at the guaranteed price, with the first orders expected to be placed this week through the Global Fund's pooled procurement mechanism.\n\nFIND and WHO are working together to accelerate appropriate use by supporting implementation research that will optimize Ag RDT use in multiple LMICs, in line with WHO guidance. This includes provision of catalytic volumes of tests to understand how Ag RDTs can best fit into health systems.\n\nUnitaid and Africa CDC will combine resources to initiate the roll out of these tests in up to 20 countries in Africa starting in October 2020. This multi-million-dollar intervention, currently undergoing final sign-off by their Boards, is designed to engage multiple partners active in the COVID-19 response in these countries, such as CHAI, African Society for Laboratory Medicine (ASLM) and local organizations. This will bolster efforts by the African Union's Partnership to Accelerate COVID-19 Testing (PACT) initiative, launched in August 2020 to mobilize experts, community workers, supplies and other resources to minimize the impact of the pandemic on the African continent by testing, tracing, and treating COVID-19 cases in a timely manner.\n\nTesting is a critical cornerstone of the COVID-19 response, enabling countries to trace and contain the virus now, and to prepare for the roll-out of vaccines once available. Effective testing strategies rely on a portfolio of test types that can be used in different settings and situations. While molecular tests started to be rolled out within a month of the virus being sequenced, these tests are mainly laboratory based, relying on infrastructure and trained personnel to conduct them. Rapid tests to detect the presence of the virus at the point of care, which are faster and cheaper, are a vital addition to the testing arsenal needed to contain and fight COVID-19.\n\nWHO guidance published on 11 September 2020 highlights the value of these tests in areas where community transmission is widespread and where nucleic acid amplification-based diagnostic (NAAT) testing is either unavailable or where test results are significantly delayed. As well as supporting test-trace-isolate strategies, the tests can help identify or confirm new outbreaks, support outbreak investigations through screening; monitor disease trends; and potentially test asymptomatic contacts.\n\nThe ACT-Accelerator Diagnostics Pillar is co-convened by FIND and the Global Fund, working closely with WHO and over 30 global health expert partners to accelerate innovation and overcome the technical, financial, and political obstacles to achieving equitable access to effective and timely testing. Such unprecedented global collaboration has enabled development and deployment of the first WHO EUL-approved Ag RDT within eight months of the first identification of the virus. In comparison, it took nearly five years to develop the first RDT for HIV. Several more antigen RDTs for COVID-19 are currently under WHO EUL review. Overall, the ACT-Accelerator Diagnostic Pillar aims to facilitate the supply of 500 million tests to LMICs within 12 months.\n\nThese agreements are critical to fulfil the key objective of the ACT-Accelerator: to ensure all countries, regardless of income, have fair access to new tests and tools to fight COVID-19. The exceptional speed with which the Ag RDT access package has been created demonstrates the breadth of the impact of the ACT-Accelerator initiative, and this and future achievements in testing will complement similar milestones anticipated to emerge from the Vaccines and Therapeutics Pillars.\n\n**Dr Tedros Adhanom Ghebreyesus**, Director General of WHO, said: \"High-quality rapid tests show us where the virus is hiding, which is key to quickly tracing and isolating contacts and breaking the chains of transmission. The tests are a critical tool for governments as they look to reopen economies and ultimately save both lives and livelihoods.\"\n\n**Mark Suzman**, Chief Executive Officer of the Gates Foundation, said: \"Testing is an essential tool in the fight against COVID-19. We are delighted to join a partnership that will help ensure that the latest, high-quality diagnostics do not just go to the highest bidder but will be available at an affordable price to the world's lower income countries. In addition, all of the actions announced today point to the growing success of the ACT-Accelerator in catalyzing global cooperation for a fair and effective response to this global crisis.\"\n\n**Dr Iain Barton**, Chief Executive Officer of CHAI, said: \"These agreements will help ensure that millions of people in low- and middle-income countries have access to high-quality rapid testing in villages and towns as well as cities. This has the potential to revolutionize government's ability to respond to the pandemic, enabling quick diagnosis and response to contain localized virus outbreaks before they spread.\"\n\n**Andrea F. Wainer**, Executive Vice President of Abbott's rapid and molecular diagnostics businesses, said: \"Abbott is pleased to bring our Panbio COVID-19 rapid antigen test and Sympheos digital solution to people and health authorities in low- and middle-income countries through this innovative partnership. We have long been committed to making sure our life-changing technologies are affordable and accessible, and for decades have been supporting many of these countries with our rapid tests for malaria, HIV, hepatitis, and other deadly infectious diseases.\"\n\n**Hyo-Keun Lee**, Chief Executive Officer of SD Biosensor, said: \"We, SD Biosensor, are pleased to supply our STANDARD Q COVID-19 rapid antigen tests for people who really need fast and accurate COVID-19 diagnosis. Through this partnership, we will keep striving do our best to provide the best quality of COVID-19 antigen rapid kits for fighting COVID-19.\"\n\n**Dr John Nkengasong**, Director of the Africa CDC, said: \"Antigen tests are an important complement to PCR testing, and are crucial to expand testing capacity throughout Africa. The beauty of antigen testing is that it is fast and gives quick results. It will allow healthcare workers to quickly isolate cases and treat them while tracing their contacts to cut the transmission chain.\"\n\n**Dr Philippe Duneton**, Unitaid's Executive Director a.i., said: \"Access to these point-of-care rapid tests with be a game changer in the fight against COVID-19. We are working to support countries to rapidly deploy and use these new tests in the best possible way. Today's news shows what the ACT-A partners working together can deliver in our efforts against the COVID-19 pandemic.\"\n\n**Dr Carolyn Gomes, Special Advisor for the Board, ProActividad, Jamaica, and Alternate Board Member (Developing Country NGOs), The Global Fund** \"Ensuring equitable access to rapid diagnostic tests is essential for controlling COVID-19 in all countries and to opening up economies across the world. Ensuring an affordable price is a major step forward. Tests that can be used at the point of care by front-line workers will greatly facilitate community access to testing. To ensure equity in access for those who need it most, there will need to be much greater support of the ACT-Accelerator and the Diagnostics Pillar in particular. Much more money is needed to meet the needs of the most vulnerable.\"\n\n**Peter Sands**, Executive Director of the Global Fund, said: \"This is the ACT-Accelerator in action. It is proof that by working together at a massive global scale, we can develop and deploy a vital new tool to help contain and fight the pandemic. This is not just a new test \u2013 it's the money and the deployment plan to get it to where it's needed, fast. This is the power of global collaboration.\"\n\n**Dr Catharina Boehme**, Chief Executive Officer of FIND, said: \"With this Ag RDT package, the ACT-Accelerator partners have secured much-needed tools for LMICs to dramatically increase COVID-19 testing. With the financial support of several countries, we have made great progress, but to ensure we reach all those who need testing and bring the prices down, we urgently need substantial funding from public, philanthropic, and multilateral sources.\"\n\n**About the ACT-Accelerator**\n\nThe Access to COVID-19 Tools (ACT) Accelerator, is a new, ground-breaking global collaboration to accelerate the development production, and equitable access to COVID-19 tests, treatments, and vaccines. It was set up in response to a call from G20 leaders in March 2020 and launched by WHO, the European Commission, France and the Bill & Melinda Gates Foundation in April 2020. The ACT-Accelerator is not a decision-making body or a new organization, but works to speed up collaborative efforts among existing organizations to end the pandemic. It is a framework for collaboration that has been designed to bring key players around the table with the goal of ending the pandemic as quickly as possible through the accelerated development, equitable allocation, and scaled up delivery of tests, treatments and vaccines, thereby protecting health systems and restoring societies and economies in the near term. It draws on the experience of leading global health organizations which are tackling the world's toughest health challenges, and who, by working together, are able to unlock new and more ambitious results against COVID-19. Its members share a commitment to ensure all people have access to all the tools needed to defeat COVID-19 and to work with unprecedented levels of partnership to achieve it. The ACT-Accelerator has four areas of work: diagnostics, therapeutics, vaccines and the health system connector. Cross-cutting all of these is the workstream on Access & Allocation.\n\nThe Diagnostics Pillar of the ACT-Accelerator is focused on ensuring that everyone who needs a test can get one. Workstreams span research and development, market readiness, procurement, and country preparedness. Achievements to date include laboratory trainings in partnership with Africa CDC in early February, and a suite of online courses deployed within weeks. Nearly 20 million tests have been procured with the Diagnostics Consortium, ensuring diagnostic access for LMICs and readiness for test-and-treat implementation in these countries. Independent evaluations of antibody tests are also being conducted, as high-quality antibody tests are essential to understand population immunity for future vaccine roll out..\n\nAvailable from: <https:\/\/www.who.int\/news-room\/detail\/28-09-2020-global-partnership-to-make-available-120-million-affordable-quality-covid-19-rapid-tests-for-low--and-middle-income-countries>","meta":{"dup_signals":{"dup_doc_count":157,"dup_dump_count":27,"dup_details":{"curated_sources":2,"2023-50":2,"2023-40":5,"2023-23":2,"2023-14":2,"2023-06":5,"2022-49":5,"2022-40":4,"2022-33":3,"2022-27":6,"2022-21":1,"2022-05":9,"2021-49":3,"2021-43":7,"2021-39":8,"2021-31":8,"2021-25":3,"2021-21":9,"2021-17":4,"2021-10":6,"2021-04":13,"2020-50":5,"2020-45":33,"2024-26":4,"2024-22":3,"2024-18":1,"2024-10":3,"2024-30":1}},"file":"PMC7841515"},"subset":"pubmed_central"}
{"text":"abstract: Although published material exists about the skills required for a *successful* bioinformatics career, strangely enough no work to date has addressed the matter of how to excel at *not* being a bioinformatician. A set of basic guidelines and a code of conduct is hereby presented to re-address that imbalance for fellow-practitioners whose aim is to *not* to succeed in their chosen bioinformatics field. By scrupulously following these guidelines one can be sure to regress at a highly satisfactory rate.\nauthor: Manuel Corpas; Segun Fatumo; Reinhard Schneider\ndate: 2012\ninstitute: 1Independent, Cambridge, UK; 2Covenant University Nigeria, Department of Computer and Information Sciences, KM 10 Idiroko road, Ota, Nigeria; 3Data Integration and Knowledge Management, European Molecular Biology Laboratory (EMBL), Meyerhofstr. 1, 69117, Heidelberg, Germany\nreferences:\ntitle: How Not to Be a Bioinformatician\n\n# Introduction\n\nThe advent of fast 3D gaming PCs, the Internet and massive sequencing efforts have attracted hackers and failed wet-lab biologists to the bioinformatics field. To make matters worse, the looming prospect of massive lay-offs in the financial sector or the new hoped-for thrill to be working on things that matter \\[1\\] following a mid-life crisis, have all contributed to the resurgence of a new breed of a mutant, super-resistant bioinformatician species.\n\nThe resulting ecosystem of practitioners is an eclectic mixture of individuals, including a) those whose graphics card for video games would preferably be hammered out with algorithmic computations for the elucidation of the meaning of life, b) those submerged in the muddy waters of the ever increasing \u2013omics subfields, and c) those fulfilling their role as annotation monkeys. As if that wasn't enough, a myriad of new packages, formats and databases keep on mushrooming daily throughout the biomedical arena, surreptitiously embracing complexity just for the sake of it. Bioinformatics has evolved into the cheap form of Biology during cycles of funding shortages. Its tentacles now spread into pretty much every branch of life sciences. So much so that the ways of defining it are nearly as numerous as there are individuals in the field. After quite some time of wrestling with the idea of what Bioinformatics is, we decided that it would probably make more sense to define how *not* to become one of the troupe. So what follows is a compendium of 10 \"sarcastic\" guidelines that illustrate how a few months on the computer can save a few hours in the library (or on Google) \\[2\\].\n\n1\\. **Stay low level at every level**. Develop your code by anecdote: avoid planning phases, requirement analysis exercises or any structure to your code. Stay away from object oriented programming. Build up your own little myriad of helper scripts. Do not document either inside or outside your code. Your coding style should only be understood by you. Make sure your software does not scale. Refuse to model or abstract and always choose the quick and dirty fix.\n\n2\\. **Be open source without being open.** Error messages should never be provided. If error messages are provided, they should be utterly cryptic so as to convey as little information as possible to the end user \\[3\\]. If you create the application, make it difficult to build. Have plenty of hidden dependencies and bizarre variables. Don't bother to debug or provide backwards compatibility. Ensure that your code is not portable, it only works in outdated operating systems and assume only you will use your application. Take for granted that everyone will be able to understand it.\n\n3\\. **Make tools that make no sense to biologists.** The less they resemble any intelligible biological question the better. If you provide a help document, bombard scientists with abbreviations and provide as much unnecessary technical information as possible. The typical biologist hates mathematics, so use mathematical formulas extensively throughout the documentation. Integrate your workflows with as many irrelevant services as possible, so you'll have greater the chances of a potential dead link.\n\n4\\. **Do not provide a graphical user interface: command line is always more effective.** Force your end-users to use the command line. It helps if the parameter name does not relate to the intended action. For example, never use \u2013o for specifying an output file; a \"k\" or \"B\" creates a more memorable impression. If you provide a graphical user interface, a) make sure there is no logic behind it, b) it is not intuitive to the user and c) support as few formats as you can, preferably html or text only. Forget HTTP-XML or SOAP. To make sure that the user experience is a nightmare, here are some guiding principles: 1) Provide thousands of menu options and pop up windows that make little or no sense. 2) Ask the user to make impossible decisions. 3) Change your interface\/format whenever you feel like it, especially if many people and applications depend on it.\n\n5\\. **Make sure the output of your application is unreadable, unparseable and does not comply to any known standards.** Just use plain ASCII text, or better still, provide your own obfuscated format. Do not use ontologies, XML, or any other inter-exchangeable standard. If you use XML, make sure that your data file is impossible to validate and that it does not follow any XML schema. You can also invent a new name for your gene if a) it doesn't match any available identifier from reference databases and b) you don't tell anyone about it.\n\n6\\. **Be unreachable and isolated.** Configure your contact email to either bounce back or permanently set it to vacation. Miss key meetings or seminars where other colleagues may be presenting their seminal results and never, ever make any attempt at remembering their names or where they work. Reinvent the wheel. Do not keep up with the literature on current methods of research if you possibly can.\n\n7\\. **Never maintain your databases, web services or any information that you may provide at any time.** Provide unstable data, unstable models and unstable services. Your ultimate goal in data curation should be to propagate as many errors as possible from one database to another, while still making sure that they sound realistic. Your curated data should only partially reflect the science of the papers you don't read. When curating your data, make as many new categories as exceptions you find to your classifications. Forget about the final scientific question you are trying to answer and stay well away from convention.\n\n8\\. **Blindly believe in the predictions given, P-values or statistics.** Select instances for your training set that you know will give you the answer you want. Produce arbitrary cut-offs on rank-ordered result lists. Absolute truth above, absolute falsehood below \\[2\\]. Always work with default parameters and never explore other options algorithms might provide. If you get a list of hits, only look at the first one. Do not believe in the mantra \"rubbish in, rubbish out\"; just make sure that your rubbish data doesn't smell.\n\n9\\. **Do not ever share your results and do not reuse.** Never discuss your results before your submission has been accepted in a lost conference proceeding. Consider that the work others are doing is probably a waste of time. Ignore whatever new algorithms and methods your colleagues have developed in the last two decades.\n\n10\\. **Make your algorithm or analysis method irreproducible.** The less testing you carry out in your experiments, the more revolutionary results you should expect to get. When testing your algorithm, compare it against methods developed before the past decade: your performance levels will look much better. Include irrelevant variables in your equations and make them unnecessarily complex, so your reviewers will be very impressed by the complexity of the astonishing predictions you get.\n\n# Conclusions\n\nHere we have highlighted a series of disastrous practices in the bioinformatics field. We hope that readers will not only find them stimulating but useful. Embrace them fully and we guarantee their efficacy.\n\n# Competing interests\n\nThe authors declare that they have no competing interest.\n\n# Authors' contributions\n\nMC, SF and RS conceived and wrote the paper. All authors have read and approved the final manuscript.\n\n## Acknowledgements\n\nThe authors would like to thank Nils Gehlenborg for helpful comments.","meta":{"dup_signals":{"dup_doc_count":108,"dup_dump_count":71,"dup_details":{"curated_sources":2,"2023-14":1,"2022-33":1,"2022-21":1,"2021-43":1,"2021-21":1,"2021-17":1,"2021-04":1,"2020-45":1,"2020-40":1,"2020-29":2,"2020-24":2,"2020-16":1,"2020-10":2,"2020-05":1,"2019-51":1,"2019-47":1,"2019-43":1,"2019-35":1,"2019-30":1,"2019-22":1,"2019-13":2,"2019-04":1,"2018-51":2,"2018-47":1,"2018-43":1,"2018-34":1,"2018-26":1,"2018-17":2,"2018-13":1,"2018-09":2,"2018-05":2,"2017-51":2,"2017-47":1,"2017-43":1,"2017-39":2,"2017-34":3,"2017-30":2,"2017-26":2,"2017-22":2,"2017-17":2,"2017-09":2,"2017-04":2,"2016-50":2,"2016-44":3,"2016-40":3,"2016-36":3,"2016-30":3,"2016-26":1,"2016-22":1,"2016-18":1,"2015-48":2,"2015-40":1,"2015-35":1,"2015-32":1,"2015-27":1,"2015-22":1,"2015-14":1,"2014-52":1,"2014-49":2,"2014-42":4,"2014-41":1,"2014-35":2,"2014-23":2,"2014-15":2,"2023-23":1,"2015-11":1,"2015-06":1,"2014-10":1,"2013-48":1,"2013-20":1,"2015-18":1}},"file":"PMC3430599"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  Although nursing education aims to equip nursing students to provide care to dying patients and their families, nurses often feel ill-prepared to cope with the emotional labour involved in end-of-life care.\n  .\n  # Objectives\n  .\n  The aim of the study was to explore and describe nursing students' experiences of end-of-life care through experiential learning within a constructivist educational model.\n  .\n  # Method\n  .\n  A qualitative, descriptive design was used. As part of introducing experiential learning, innovative educational practices were initiated during a second year level undergraduate nursing module on end-of-life care. Qualitative data on second-year nursing students' experiences were collected through written reflections and analysed using open coding.\n  .\n  # Results\n  .\n  The themes that emerged revealed participants' sensory and emotional experiences during the learning opportunities. Participants reflected on what they learnt and clarified their values related to death and dying. They indicated how they would apply the new meanings constructed in clinical practice.\n  .\n  # Conclusion\n  .\n  A constructivist educational model of experiential learning holds potential to enhance value clarification and nursing students' sensory and emotional awareness of death and dying. Experiential learning is recommended to develop nursing students' competency in providing end-of-life care.\nauthor: Anna E. van der Wath; Pieter H. du Toit**Correspondence to:** Anna van der Wath, **Email:**<email@example.com>**Postal address:** Private Bag x 323, Arcadia 0007, South Africa[^1]\ndate: 2015-11-05\nreferences:\ntitle: Learning end-of-life care within a constructivist model: Undergraduate nursing students**'** experiences\n\n# Introduction\n\nLearning how to face death and dying is inevitable for nurses who are often the caregivers at the end-of-life (Todaro-Franceschi & Lobelo 2014:171). Mastering end-of-life care competence often confronts nursing students with their own mortality and their personal beliefs about death and dying (Peters *et al.* 2013:14).\n\nTo facilitate learning of end-of-life-care is also a challenging task for nurse educators (Dobbins 2011:159). Death and dying is a sensitive topic and educators may feel 'cautious and over-conscious' regarding the emotional side of end-of-life care (Venkatasalu, Jackson & Walsh 2014:A34). Chan and Tin (2012:900) believe that to prepare nurses for the demands of end-of-life care requires more than the traditional teaching methods that focus on transferring knowledge and skills. They believe that 'self-competence', referring to personal resources, existential coping, and emotional coping, is a necessary competency to deal with death, dying and bereavement (Chan & Tin 2012:899). Experiential learning, which includes self-reflection to integrate and apply knowledge, skills and self-competence, is recommended as s strategy to facilitate self-competence in end-of-life care (Chan & Tin 2012:909\u2013910). Value clarification, a process of self-reflection to identify and consider personal beliefs about death and the worth of life, may also help to prepare nurses for ethical decision-making during end-of-life care (Tarzian & Schwarz 2010:35\u201336).\n\n# Background\n\nThere is a clear call for a paradigm shift in nursing education. Instead of lecture-based teaching where students are passive recipients, models and methods enhancing active student participation are proposed (Brandon & All 2010:89; Freed & McLaughlin 2013:174; Rieger & Chernomas 2013:53; Stanley & Dougherty 2010:378). Brandon and All (2010:89) proposed a constructivist paradigm to enhance nursing student-centred interactive learning. An example of this is a reflection-centred curriculum designed to enhance the intra- and interpersonal development of nursing students (Horton-Deutsch, McNelis & O' Haver Day 2012:341).\n\nNursing care requires nurses to be emotionally competent and able to deal with others' emotions, whilst regulating their own (Vilelas & Diogo 2014:147\u2013148). In end-of-life care, nursing education should make provision for nursing students to experience and manage their own emotional reactions towards death and dying. Gillan, Van der Riet and Jeong (2014:341) recommended that educators consider students' personal and professional experiences with death and dying and to provide different learning opportunities to meet all students' needs during end-of-life care education.\n\n## Literature review\n\nIn a study by Todaro-Franceschi (2011:318) professional nurses indicated that their training did not equip them with the competence to care for the dying and their families (Todaro-Franceschi 2011:318). Twenty five per cent of oncology nurses felt they were inadequately prepared to care for dying patients (White & Coyne 2011:714). Professional nurses in New Zealand reported that their early experiences with death and dying, for which they felt unprepared, left a lasting personal and professional impression. They experienced helplessness, distress and guilt when a patient died (Kent, Anderson & Owens 2012:1255). Fear of death and a negative attitude towards end-of-life care is more evident in younger nurses (Peters *et al.* 2013:14). When feeling unprepared to deal with death and dying, nursing students may avoid emotional involvement with dying patients (Mutto *et al.* 2012:97). Nurses who experience challenges in caring for dying patients may experience poor professional quality of life and increased risk for compassion fatigue and burnout (Todaro-Franceschi & Lobelo 2014:170).\n\nA literature review by Gillan *et al.* (2014:341) found that nursing students feel unprepared to deal with death and dying and schools of nursing are not adequately preparing nursing students for end-of-life care, leading to inadequate care once they are employed. The findings highlighted the lack of end-of-life content in textbooks and lack of content on end-of-life care in undergraduate nursing curricula. Venkatasalu *et al.* (2014:A34) described this phenomenon as 'hidden death' which relates to the 'lack of explicit curricular objectives around end-of-life care topics' that 'results in limited exposure to death and dying topics in pre-registration programmes'. They also point out a lack of support structures to help educators and students deal with the emotional issues surrounding end-of-life education (Venkatasalu *et al.* 2014:A34).\n\nMany examples of experiential opportunities to facilitate learning for end-of-life care are available in scholarly literature. Dobbins (2011:161) mentioned 'cinemeducation', the use of movies or movie clips, as a method to incorporate an educational experience that serves as an opportunity for critical self-reflection. Self-awareness and self-reflection during facilitating learning are also reported to have helped students to gain experiential knowledge to provide end-of-life care (Liu *et al.* 2011:861). Real-life cases and open-group discussions have been reported to facilitate emotional intelligence, the management of emotional labour and exploration of personal emotions (Bailey & Hewison 2014:3555). Storyboarding, a process that engages the creative right brain and the critical left brain, has helped students to identify cultural aspects and feelings related to the dying patient (Lillyman, Gutteridge & Berridge 2011:179). Two experiential educational strategies, high fidelity and gaming simulation, were also used by Kopp and Hanson (2012:e99\u2013e101) in end-of-life care education to help students develop awareness of the issues surrounding terminal illness and the importance of having empathy with these patients. In this study, students verbalised increased awareness of the losses patients suffer, whilst considering how they would respond in a similar situation. Another example is how a workshop enhancing students' reflections on life and death helped them to gain experiential knowledge on end-of-life care, cherish life and explore their feelings related to facing death (Liu *et al.* 2011:860).\n\nExperiential learning is described as a philosophy and a teaching method that focuses on the relationship between students, their concrete experiences and their reflective processes about the experience (Fenwick 2001:16). As described by Kolb 1984:\n\n> To learn is not the special province of a single specialized realm of human functioning such as cognition or perception. It involves the integrated functioning of the total organism-thinking, feeling, perceiving and behaving. (p. 31)\n\nExperiential learning stems from Sch\u00f6n's 'epistemology of practice' (1983:49, 66\u201368), Mezirow's transformative learning theory (Mezirow 1991:7\u20138, 14, 77), the phenomenological perspectives of Boud, Cohen and Walker (1993:8\u201316) and Kolb's cycle of experiential learning (1984:33).\n\nThe constructivist view underpins experiential learning (Fenwick 2001:9). Constructivism assumes that learning takes place as students create new meanings which are integrated with existing constructs (Kanselaar 2002:1). Stemming from the theories of Vygotsky (1896\u20131934), socio-constructivists add to constructivist assumptions by viewing learning as a social process that occur through engagement in social interactions (Vygotsky 1978:38). In a constructivist view of experiential learning, students are believed to interpret their worlds actively and construct knowledge through processes of reflection (Fenwick 2001:10, 14). A constructivist model of experiential learning was developed by Kolb (1984:33). The student learns through cycles of concrete experience (a simulated or real-life experience), reflective observation (questioning the observations, awareness and meaning of the experience), abstract conceptualisation (reflecting on principles and new understandings) and active experimentation (reflecting on future application by revising and reshaping the learning) (Fenwick 2001:10\u201311; Kolb 1984:30\u201333).\n\n## Problem statement\n\nEnd-of-life care is considered to be a complex area of practice in intensive care units, and nurses expressed the need for emotional support (Ranse, Yates & Coyer 2012:14). Research indicated that undergraduate nursing students often feel unprepared to deal with dying patients and their families (Gillan *et al.* 2014:331).\n\nTeaching and learning that focus on knowledge and skills development only is not considered the most effective way to equip nursing students to cope with the emotional and existential challenges of end-of-life care (Chan & Tin 2012:900). Insufficient focus on emotional aspects may result in students feeling ill-prepared to care for dying patients and their families (Bailey & Hewison 2014:3555\u20133560). When students are involved in purposefully designed educational strategies that focus on knowledge, skills and attitudes necessary to provide compassionate end-of-life care, they will be better prepared to deal with the bio-psychosocial and spiritual needs of dying patients (Dobbins 2011:165). Nurse educators need to find effective strategies of facilitating learning to prepare nursing students for care of dying patients (Dobbins 2011:159). Nursing education that focuses on self-awareness and own beliefs and emotions regarding death and dying seem to have a positive effect on students' attitudes towards end-of-life care (Bailey & Hewison 2014:3555\u20133560).\n\nGillan *et al.* (2014:341) identified a lack of qualitative research on end-of-life teaching and the need to consider students' experiences with death and dying during the facilitation of learning. Venkatasalu *et al.* (2014:A34) are of the opinion that further research to evaluate end-of-life care strategies of facilitating learning and how these strategies relate to clinical care outcomes, is needed.\n\n# Aim of the study\n\nThe aim of the study was to explore and describe nursing students' experiences regarding end-of-life care through experiential learning within a constructivist model.\n\n# Definition of key concepts\n\n*Constructivism* assumes that there are multiple representations of reality. Knowledge is constructed through collaborative participation in authentic tasks in real-life environments and reflection on these experiences (Jonassen 1999:217\u2013219). In this article 'constructivism' refers to an experiential learning model where students are believed to construct knowledge through collaborative participation and processes of reflection.\n\n*Experience* was described by Dewey, an influential educational theorist of the twentieth century, as a process of interaction 'going on between an individual and objects and other persons. An experience is always what it is because of a transaction taking place between an individual and what, at the time, constitutes his environment \u2026' (Dewey 1938:43). In this article 'experience' refers to the interaction taking place in the environment where nursing students learn to provide end-of-life nursing care.\n\n*End-of-life-care nursing education* aims to increase nursing students' understanding of patient care for dying patients and the related ethical-legal, cultural and spiritual issues. It also cultivates a respectful attitude towards death (Liu *et al.* 2011:857). In this article 'end-of-life care nursing education' refers to a module in the second year where undergraduate nursing students learn to provide care to dying patients and their families.\n\n*Undergraduate nursing students* in this article refer to second-year nursing students at a higher education institution in Gauteng Province enrolled for a degree in nursing and registered as learners at the South African Nursing Council (2005:27).\n\n# Contribution to field\n\nThe study findings may contribute to nursing education, practice and research. The article illustrates the use of a constructivist model of experiential learning (Kolb 1984:33) in end-of-life care nursing education for undergraduate nursing students. The findings may help to understand the ways in which the learning experiences contribute to sensory, cognitive and emotional awareness; value clarification and nursing students' perceived competencies regarding end-of-life care. The innovative educational practices introduced in the article may help to ensure nurses feel more prepared to provide end-of-life care to patients and their families. Finally, this research may contribute to the research-based body of knowledge on end-of-life nursing care.\n\n# Research design and methods\n\n## Design\n\nA qualitative design (Polit & Beck 2012:14) was used to explore and describe nursing students' experiences of end-of-life care through experiential learning within a constructivist educational model.\n\nA psychiatric nursing module for second-year students, dealing with end-of-life care, was used to integrate innovative educational practices such as co-operative learning and reflective processes. Opportunities were created to engage students in experiential learning. Students were divided into co-operative learning groups. Each group was assigned the learning task of presenting their experiences on a theme related to death and dying based on an applicable film, an article, a story, a poem or a case study of their choice. Students used a variety of methods (demonstrations, acts, posters, video clips and mind maps) to present the themes during four learning opportunities as scheduled on the timetable. Each presentation was followed by an open class discussion where applicable theoretical models and practical interventions were introduced, for example, 'breaking the bad news'.\n\n## Participants\n\nSixty four students registered for the second year of a four-year undergraduate nursing science programme in a higher education institution constituted the population for the study. All the students participated in the experiential learning opportunities and were invited to write reflective narratives on the experience of learning of end-of-life care. Sixty two students voluntarily handed in written reflections.\n\n## Data collection method\n\nQualitative data were collected through the use of students' written reflections which served as a narrative account of their experiences (Mendieta 2013:140). To facilitate deep self-reflection, Sch\u00f6n's model of reflection (1983:26, 61\u201362) was used as a guideline. The model explicates reflection-in-action as a way of attending to experiences and coming in touch with feelings to create new understanding. Reflection-on-action is done after the experience to explore and think about actions. Students were requested to write a reflection during each learning opportunity, using a layout based on the acronym, LEARN (College of Nurses of Ontario 1991, in Brown, Matthew-Maich & Royle 2001:133):\n\n-   Look back on the situation.\n\n-   Examine the detail: What happened? How were you involved? How did you feel? What were you thinking? Who else was involved? What did others say?\n\n-   Analyse: What influenced the experience? What role did the values of different people play? What have you learned? Will it influence your practice?\n\n-   Revise: What would you do differently next time? Are there any learning issues? How can you use what has been learned? What needs to change?\n\n-   New perspective: Recommendations for learning in similar future situations.\n\n## Data analysis\n\nQualitative data analysis of written reflections was done according to the steps as described by Tesch (cited in Creswell 2009:85). The researcher read through the data to obtain a general sense of the total experience and reflected on the meanings students attached to the learning opportunities. Text sections were segmented and themes labelled with appropriate terms. The themes were described as derived from the data and interrelationships between the themes noted. Guided by the principle of data saturation, written reflections were analysed until no new themes emerged (Polit & Beck 2012:742).\n\n## Context of the study\n\nThe research setting constitutes a four-year undergraduate nursing science programme in a higher education institution in Gauteng Province, South Africa.\n\nThis programme includes a module on end-of-life care in the second year.\n\n# Results\n\nThe themes 'I saw', 'I heard' and 'I did' that emerged from participants' reflective narratives illustrate their sensory experiences during the learning opportunities. The feelings participants experienced during the presentations are reflected in the theme, 'I felt'. The theme 'I learnt' indicate what and how participants learnt. The theme 'I clarified my values' reveals participants' reflections on their values related to death and dying. Participants applied what they have learnt to practice: what 'I will do' differently next time when encountering death and dying in clinical practice. The results are discussed according to these themes, supported by excerpts from different students' verbatim narratives.\n\n## 'I saw'\n\nDifferent participants specifically mentioned that the visual material enhanced their learning as in the following examples:\n\n> 'We looked at various videos and we watched various physical performances by our classmates. In these, various principles of how to deal with grief and how grief present itself, was presented.' (Participant 15, male)\n>\n> 'The experience was influenced by the use of video clips, visual learning made it easy for me to relate the topic with the actual reality.' (Participant 22, female)\n>\n> 'I found the videos are an effective way of showing us how children grieve in a visual way \u2013 a way that can easily be remembered.' (Participant 30, female)\n>\n> 'Different groups drew pictures and made posters to help us visualise the process.' (Participant 46, female)\n\nParticipants who observed other students demonstrating some of the skills indicated that it also enhanced learning:\n\n> 'I think the acting out part was also helpful because most of the time what you have seen is not easily forgotten.' (Participant 12, female)\n>\n> 'Making the students demonstrate the management allows other learners to learn in a more realistic way.' (Participant 35, male)\n\nParticipants observed each other's emotional expressions during the learning opportunities and sensed the emotional atmosphere elicited by the presentations:\n\n> '\u2026 expression of sadness could be seen through their faces, especially the group members.' (Participant 5, male)\n>\n> 'Students that were sitting next to me were smiling because of the wonderful lessons that we got.' (Participant 32, female)\n>\n> 'The rest of the class also became quiet and depressed.' (Participant 43, female)\n\n## 'I heard'\n\nSome participants (fewer than in the 'I saw' group) mentioned that hearing \u2013 hearing music, poetry and others' experiences \u2013 enhanced their learning experience:\n\n> 'The issue of calm music is also recommended to facilitate the audience's emotions.' (Participant 12, female)\n>\n> 'I appreciated the music to explain the class content. I feel music is a great indirect way of explaining a subject.' (Participant 24, female)\n>\n> 'In poems and scenarios read we witnessed different maladaptive behaviours that people manifest.' (Participant 41, female)\n>\n> 'I was listening.' (Participant 52, female)\n>\n> 'Others express a feeling of sadness, by shouting out or verbalising words like hah.' (Participant 60, female)\n\n## 'I did'\n\nParticipants commented on and appreciated their own and others' participation and involvement in the co-operative learning opportunities:\n\n> 'We spoke about principles in management of grief \u2026 about different ways in which children may respond \u2026 different cultural needs \u2026 I was involved in the presentation.' (Participant 8, female)\n>\n> 'I was involved by giving the feedback on each topic discussed; this made me feel more involved.' (Participant 13, female)\n>\n> 'Class interaction is very crucial.' (Participant 28, female)\n>\n> 'I read out the case study and applied the learning content \u2026 I felt good because I at least contributed something in the class.' (Participant 36, female)\n>\n> 'My other group members were involved.' (Participant 42, female)\n>\n> 'Others said they have also been in similar situations.' (Participant 55, female)\n>\n> 'We got to participate, do some presentations and acting. This helped me to concentrate throughout the lecture and to be willing to know more.' (Participant 58, female)\n>\n> 'At first I was shy until I got involved and gain my confidence.' (Participant 60, female)\n\n## 'I felt'\n\nMost participants mentioned their emotional experiences during the learning opportunity as significant. The learning material triggered intrapersonal experiences of loss and bereavement:\n\n> 'During the presentations I felt very interested in what was said, but some of the clips got me emotional. Because grief is a difficult subject to understand as people experience it so differently, I felt a level of sadness.' (Participant 4, female)\n>\n> '\u2026 they really dragged my emotions into reality.' (Participant 13, female)\n>\n> 'Well during the presentation I got emotional \u2026 I have been faced with the loss of two grandparents \u2026 so I could relate somehow.' (Participant 22, female)\n>\n> 'A young girl lost her close friend in a movie, it made me feel so sad and I started imagining myself losing the one I love then I cried. Others said it's good to cry.' (Participant 28, female)\n>\n> 'I was thinking how I reacted or it just reminded me of the loved one that I have lost few years back \u2026 my friends and I and the whole class was involved in the class discussion.' (Participant 39, female)\n>\n> 'I also related to the grief because I also experienced the same stages of grief.' (Participant 53, female)\n>\n> 'It brings back old memories of what happened to me \u2026 I almost cried during class discussion.' (Participant 58, female)\n\nSome participants felt guilty for getting emotionally involved:\n\n> 'And next time I will try not to identify myself with the clips we view in class.' (Participant 10, female)\n>\n> 'Next time I will make sure that I try to be as objective as possible and that I do not let my emotions overwhelm me.' (Participant 24, female)\n>\n> 'Next time I will listen effectively, I won't let my past interfere with my attention \u2026 I want to learn about death without being sad.' (Participant 36, female)\n\n## 'I learnt'\n\nParticipants expressed their appreciation for the way in which learning was facilitated:\n\n> 'The effective manner in which learning was enhanced leaves me with no idea of how I could change anything.' (Participant 15, male)\n>\n> 'I have experienced that if the lessons are taught with the use of presentations, some acting and some pictures it gives a lot of understanding and it is not easy to forget.' (Participant 23, female)\n>\n> 'I have learnt a world's worth of information on reflection and feel that I have grown in leaps and bounds as a person. Thank you!' (Participant 49, female)\n\nParticipants also experienced the joy of learning:\n\n> 'I enjoyed the acts the students put on because they made it funny and I think it will be easy for me to remember.' (Participant 12, female)\n>\n> 'Thank God for school! I really learnt a lot.' (Participant 16, female)\n>\n> 'Videos, case studies and presentations made the learning fun and understandable.' (Participant 37, female)\n>\n> 'The topic was very emotional and sensitive; however some plays made us laugh and move away from the sorrow.' (Participant 50, female)\n\nNew meaning was constructed:\n\n> '\u2026 as the class went on and I learnt more skills; I saw it is possible to be there for someone who is grieving.' (Participant 11, female)\n>\n> 'I now better understand why I need to go through the stages \\[*of grief*\\] to process all the emotions.' (Participant 32, female)\n>\n> 'I never understood why children act differently \u2026 what I've learned I'll be able to use throughout my life.' (Participant 37, female)\n>\n> 'I never considered how much different these processes are for children than for adults.' (Participant 54, female)\n>\n> 'Death can have a calming or beautiful effect when done in a gracious and gentle manner.' (Participant 56, female)\n\n## 'I clarified my values'\n\nParticipants shared the existential intelligence they obtained through the learning opportunities:\n\n> 'What I should do differently is to talk about death. I was afraid of talking about it previously. I have realised that if we are afraid of death we are afraid of life itself.' (Participant 14, female)\n>\n> 'Dying is part of life. We all get born and live our lives and eventually will die.' (Participant 47, female)\n\nThey also obtained interpersonal intelligence on how to appreciate and respect others' values with regards to death and dying:\n\n> 'I will be more understanding and empathic \u2026 I will have more respect for people and how they grief.' (Participant 11, female)\n>\n> 'I will not bring my own frame of reference into other people's situations especially when they experience loss.' (Participant 36, female)\n>\n> 'The clip that was shown was very beautiful and showed the respect that was needed to take care of someone that died.' (Participant 48, female)\n\n## 'I will do'\n\nMost participants were able to relate the learning to their clinical practice and how it will affect the care they provide. They verbalised feeling more competent to deal with challenging clinical situations:\n\n> '\u2026 we should actually give time to people that are grieving and be there for them \u2026 this will influence the way I view other people's culture \u2026 I won't judge.' (Participant 2, female)\n>\n> 'I learnt a lot about dying, how to break the bad news, what to say and not to say.' (Participant 7, female)\n>\n> 'I now, however feel I can provide the support I need to give to a grieving patient.' (Participant 15, male)\n>\n> 'I will comfort the grieving family members and not judge them.' (Participant 22, female)\n>\n> '\u2026 principles that can help each of us to be caring and helpful.' (Participant24, female)\n>\n> 'Compassion is important.' (Participant 31, female)\n>\n> '\u2026 it is important to positively regard other people's feelings of loss as important.' (Participant 47, female)\n>\n> 'I can use the lessons to different culture groups, in the children's wards because I now have a knowledge of the process of grieving.' (Participant 48, female)\n>\n> 'Don't ever deny people the opportunity to cry and go through grief \u2026 don't always feel the necessity to say something. If you have nothing to say just reflect.' (Participant 55, female)\n\n# Ethical considerations\n\nPermission to conduct the research was obtained from the Research Ethics Committee (Information removed to ensure blind peer review). The principles of the Belmont Report (Polit & Beck 2012:152\u2013253) assisted in avoiding exploitation of participants and ensured that their rights were protected.\n\n## Potential benefits and hazards\n\nStudents benefited directly from the research, and the findings may contribute to educational nursing practice. Students were invited to discuss any emotional discomfort and personal experiences they experienced during participation in private with the researcher, a psychiatric nursing specialist, or with the student advisor who is available to all students at the faculty. Two students made use of the opportunity and shared the way they benefited from the learning opportunity as it facilitated their own grieving. They did not require referral for emotional support.\n\n## Recruitment procedures\n\nStudents were invited and given freedom to choose to either participate in the study or discontinue at any given time without the risk of incurring adverse consequences. Students had an equal chance to be included in and benefit from the research.\n\n## Informed consent\n\nParticipants were provided with a participant information leaflet to explain the nature of and likely consequences of the research and signed informed consent prior to participation.\n\n## Data protection\n\nReflective reports were assigned a code number to ensure confidentiality. Reflective reports will be stored for 15 years in a safe place at the Nursing Science Department.\n\n# Trustworthiness\n\nLincoln and Guba's model (cited in Polit & Beck 2012:589) for assessing the trustworthiness of data was applied. Credibility was obtained through spending sufficient time with students to generate an in-depth understanding of their experiences of the learning material and opportunities. The researcher reflected on any personal values that could have affected data collection and analysis by keeping a reflective journal to stay in contact with her own experiences. To obtain transferability and dependability, sufficient descriptive information of the methodology and setting were provided to allow for comparison should readers be interested to apply the findings to similar situations. Confirmability was obtained through keeping the raw data as supportive evidence of the findings. Authenticity, the extent to which the research fairly and faithfully shows a range of realities, was obtained by supporting the findings with students' narratives.\n\n# Discussion\n\n## Outline of the results\n\nVideo clips, music, demonstrations, posters, case studies, poems and pictures were used during the learning opportunities. The participants reflected on their sensory (visual, auditory and kinaesthetic) and emotional experiences. They experienced co-operative learning in groups (Johnson, Johnson & Stanne 2000:2) and reflected on their own learning. The quotes reflect the meaning participants constructed about death and dying. Some participants clarified the values they attached to death and dying. They reflected on how they would apply the new meaning they constructed in clinical practice, whilst some of them perceived themselves as more competent to deal with death and dying.\n\nParticipants in this research identified certain competencies they acquired during the learning opportunities. Chan and Tin (2012:899) identified similar competencies that health care professionals require when they are working with death, dying and bereavement. The competencies included knowledge (cognitive), practical (clinical) experience and self-competence which requires personal resources, emotional and existential (value-driven) coping. The same authors (2012:909) similarly used creative and interactive means such as movies, videos, songs and group discussions, to facilitate helping professionals' preparation for what they called 'death work'.\n\nSensory awareness enhances learning. The senses offer a means of contact and communication with the external world, but the raw sense data need to be constructed through perception (Beard & Wilson 2013:166\u2013167). Whilst experiential learning is about observing, reflecting and doing, the three dominant thinking patterns \u2013 visual, auditory and kinaesthetic (Beard & Wilson 2013:177, 179) \u2013 are evident in students' reflections on the learning opportunities they were exposed to. The concept of 'embodied pedagogy' is described by Nguyen and Larson (2015:n.p.) as learning that takes place through a 'bodily and spatial awareness of sensation and movement' and mind-body interaction in a 'socio-cultural context'. The authors asked the question of how to engage the body, the reflective mind and the social being of the student in learning to construct knowledge and meaning. The findings in this article illustrate how participants used their bodily senses to engage in learning and construct new meaning.\n\nConfrontation with death and dying, be it a direct or indirect experience, elicits emotions. The participants acknowledged their feelings of sadness during the experiential learning. The encounter with dying in clinical practice may trigger more than sadness. For example, medical and nursing students in a study in Argentina described feelings of helplessness, vulnerability and sympathy for patients (Mutto *et al.* 2012:95). Students given the opportunity to experience and reflect on their emotional responses in a safe and supportive environment begin to learn how to manage their emotions related to death and dying. Identification of emotional labour is the first step towards emotional intelligence which has a positive effect on nurses' attitudes towards end-of-life care (Bailey & Hewison 2014:3561\u20133562). A literature review revealed that nurses' unresolved personal experiences with death can play a role in the way they approach death and dying (Wilson & Kirshbaum 2011:559).\n\nNursing curricula that incorporated competencies associated with emotional intelligence positively related to students' well-being and effective stress-management skills. Emotional intelligence focuses on self-awareness and identifying and monitoring own and others' emotions to guide thoughts and behaviour (Por *et al.* 2011:856, 859, 860). Nurses' empathetic capacity may also be enhanced through emotional intelligence (Williams & Stickley 2010:754).\n\nSome students who participated in the study reported in this article felt guilty about getting emotionally involved; this might be a product of socialisation or the expectation that nurses must suppress their emotions. In the class discussion the students had the opportunity to talk about emotional expression: If to be encouraged in patients, why do nurses have to feel guilty when they experience sadness and pain? A study by Jack and Wibberley (2014:905\u2013906) describe the complex emotional needs of nursing students who felt the need to change on an emotional level, but struggled to find ways to deal with the emotional demands of nursing. Although they wished to engage in discourse to find meaning in the emotional experiences they encountered, they felt lost and unable to share their feelings. The authors emphasise the need to find ways to facilitate student nurses' emotional coping.\n\nParticipants in the study also experienced positive emotions as they expressed their appreciation for the learning opportunities. A storyboarding technique to address end-of-life experiences in practice yielded positive experiences as students appreciated the opportunity to discuss their feelings in a supportive group (Lillyman *et al.* 2011:183). In a study by Liu *et al.* (2011:856), experiential learning was also found to increase students' motivation to learn.\n\nSome students who participated in this study expressed existential awareness as they realised the different values people assign to dying and clarified their own values related to death and dying. They reflected on dying as part of life, similar to nursing students exposed to end-of-life curriculum content in an USA study (Dobbins 2011:164) who 'demonstrated an increased acceptance of death as an inevitable reality of life'. Students valued discussions on the meaning of suffering and death which is sometimes covered in modules such as philosophy and ethics (Mutto *et al.* 2012:95). Nurses need to be aware of how their personal beliefs and cultural considerations may affect their responses towards a dying patient (Peters *et al.* 2013:20) and their willingness to learn about end-of-life-care (Todaro-Franceschi 2011:318).\n\nThe results suggested nursing students' perceived clinical competency. The use of cinemeducation as a strategy of facilitating learning yielded similar results as students experienced a decrease in fear of death and increased comfort to care for dying patients (Dobbins 2011:164). Film can serve as learning support to enhance reflection and the understanding of experiences of health and suffering in the caring sciences. A film that has the potential to transform theory into a life world experience transcends the learning situation (H\u00f6rberg & Ozolins 2012:1).\n\n## Practical implications\n\nThe results indicate that students who actively participate in learning opportunities experience sensory and emotional awareness. Nurse educators need to plan learning opportunities that will facilitate learning about end-of-life- care to raise students' emotional awareness and provide opportunities for students to clarify personal values about death and dying. The learning opportunities need to be followed by open discussions focusing on emotional intelligence: ways to express and manage the complicated emotions nurses may experience during the provision of end-of-life care once in practice.\n\n# Limitations of the study\n\nIn this study only qualitative data were collected. The data provided in-depth insight into the students' learning experiences, but quantitative data, for example student questionnaires to rate the facilitation of learning, could have provided the researcher with an opportunity to identify areas of progress and areas to improve on.\n\nThe findings did not illuminate real-life experiences. It would have been helpful to assess how the experiential learning opportunities affected students' real-life encounters with death and dying in clinical practice.\n\n# Recommendations\n\nA variety of innovative educational methods can be utilised to facilitate experiential learning to teach end-of-life care. Nurse educators need to use creative experiential learning opportunities to ensure students participate and use their senses during learning end-of-life care. The learning opportunities should also be designed to facilitate emotional awareness and invite students to clarify their values in relation to death and dying.\n\nIt is essential for nurse educators to obtain feedback (both qualitative and quantitative) on students' learning experiences. These findings need to be published so that other educators can benefit from the process. Research in different areas of nursing education and the use of different educational methods will enhance the quality of nursing education. Action research is proposed as an appropriate research design to accomplish exactly this.\n\n# Conclusion\n\nExperiential learning opportunities opened doors for students and the nurse educator in question responsible for facilitating learning. Participants' creative presentations facilitated their own and other participants' learning. The reflections illustrated participants' constructivist learning experiences that enhanced the construction of meaning. Participants experienced heightened emotional awareness as they reflected on their own encounters with death and dying. Participants were challenged to clarify their values, but realised their obligation to respect others' values. They perceived themselves as more competent to care for dying patients and bereaved families with compassion and empathy.\n\n## Acknowledgements\n\nThe authors wish to acknowledge the contributions of the undergraduate nursing students who freely shared their experiences. To observe their openness and willingness to learn is an uplifting experience.\n\n## Competing interests\n\nThe authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article.\n\n## Authors**'** contributions\n\nA.E.v.d.W. (University of Pretoria) was responsible for the title, methodology, analysis and interpretation of data, revision of the manuscript and approval of the final version. P.H.d.T. (University of Pretoria) contributed in a supervisory capacity and assisted with the revision of the manuscript and approval of the final version.\n\n# References\n\n[^1]: **How to cite this article:** Van der Wath, A.E. & Du Toit, P.H., 2015, 'Learning end-of-life care within a constructivist model: Undergraduate nursing students' experiences', *Curationis* 38(2), Art. \\#1537, 9 pages. <http:\/\/dx.doi.org\/10.4102\/curationis.v38i2.1537>","meta":{"dup_signals":{"dup_doc_count":106,"dup_dump_count":30,"dup_details":{"curated_sources":2,"2019-09":1,"2018-51":1,"2018-43":1,"2018-34":1,"2018-26":2,"2018-22":3,"2018-17":2,"2018-13":2,"2018-09":2,"2018-05":3,"2017-51":1,"2017-47":3,"2017-43":3,"2017-39":3,"2017-34":3,"2017-30":3,"2017-26":3,"2017-22":4,"2017-17":4,"2017-09":5,"2017-04":7,"2016-50":5,"2016-44":6,"2016-40":6,"2016-36":6,"2016-30":4,"2016-26":7,"2016-22":6,"2019-30":1,"2017-13":6}},"file":"PMC6091757"},"subset":"pubmed_central"}
{"text":"author: Tracy G. Anthony; Christopher D. Morrison; Thomas W. GettysCorresponding author: Thomas W. Gettys, <firstname.lastname@example.com>.\ndate: 2013-08\nreferences:\ntitle: Remodeling of Lipid Metabolism by Dietary Restriction of Essential Amino Acids\n\nAll forms of life share the ability to sense environmental change and respond in real time by engaging adaptive homeostatic programs. In higher organisms, the composition of consumed meals presents a complex, constantly changing nutritional environment, and we are only beginning to understand the sensing and signaling systems that detect and coordinate the transcriptional, physiological, and behavioral responses to meal components. Although it has long been axiomatic that dietary composition plays a deterministic role in overall health, the growing prevalence of obesity and metabolic syndrome has prompted a refocus of attention on the significance of dietary macronutrients to health outcomes. Findings from earlier investigations of potential roles for dietary carbohydrates and fat in disease progression or remediation (1\u20134) are now being reframed by experimental evidence linking dilution of dietary protein to compensatory increases in ingestive behavior (5,6). Recent studies also illustrate that modifying dietary essential amino acid (EAA) composition has profound effects on lipid metabolism and energy balance (7\u201310). A consensus is emerging to support the view that protein intake is monitored through nutrient-sensing mechanisms that detect changes in EAAs and provide regulatory input to both energy balance and the integration of carbohydrate and lipid metabolism (5,6,11,12). We will examine the transcriptional and metabolic responses to EAA deprivation or restriction to make the broader case that EAA sensing and signaling play an essential role in coordinating metabolic responses to dietary composition, particularly the content and composition of dietary protein.\n\n# Nutrient sensing and protein nutrition\n\n## Protein leverage hypothesis.\n\nSimpson and Raubenheimer (11) used an integrative modeling approach to develop a geometric framework for evaluating the impact of dietary macronutrients on response variables such as nutrient selection, body composition, longevity, and reproduction. The geometric framework model of nutrition was originally developed and tested in experiments in which life span or fecundity were measured in three different insect species given ad libitum access to 1 of 28 different diets varying in their protein-to-carbohydrate ratio. The authors summarized the responses to all 28 diets by plotting the protein consumed per day on the *x* axis, the carbohydrate consumed per day on the *y* axis, and the biological response (e.g., longevity, fecundity, etc.) to each nutritional combination as a heat map in the *z* plane (13,14). The heat maps identified the specific ratios of protein to carbohydrate that optimized each response, and in all three species, the ratio producing optimal life span differed from the ratio producing greatest fecundity (15). The effectiveness of this approach in describing biological responses to nutritional complexity led to its application in examining how varying dietary macronutrient composition affects ingestive behavior, and how animals prioritize macronutrient intake when given a choice (11,14,16,17). It was found that lowering the percentage of protein in the diet causes a concomitant increase in energy intake to maintain constant protein intake. This leveraging of carbohydrate and fat intake that occurs with dilution of dietary protein represents the conceptual basis for the protein leverage hypothesis (16). A key prediction of the hypothesis is that the overconsumption of total energy that occurs with low-protein diets has the potential to predispose to the development of obesity and metabolic disease (12). However, if the protein-leveraged overconsumption of energy were accompanied by a commensurate increase in energy expenditure (EE), the target protein intake could be achieved without effect on overall energy balance. Alternatively, the hypothesis predicts that high protein diets would be more satiating and reduce energy intake. This element of the prediction has experimental support from work with humans (12,18,19). Collectively, the emerging evidence is consistent with the conclusion that both quantitative and compositional measures of protein intake are being sensed and factor into the regulation of ingestive behavior. It is far less clear how protein nutrition is actually monitored on an ongoing basis, what indices of protein nutrition and amino acid metabolism are being sensed, and the extent to which leveraged changes in overall energy intake are coordinated with changes in EE.\n\n## Metabolic responses to protein restriction.\n\nThe protein leverage hypothesis infers the superimposition of a protein intake target to the regulation of ingestive behavior that is functionally linked to the animal's protein requirements for maintenance and growth. Dietary protein and amino acid requirements are normally highest in young, growing animals and decline with age during the approach to maturity. The orexigenic responses to variations in dietary protein have been studied extensively, and several overall conclusions are supported by this body of work (6,20,21). First, mild to moderate protein restriction produces an increase in consumption of a protein-diluted diet, and the magnitude of the response is dependent upon the ongoing protein requirements when it is imposed. For example, the response to moderate restriction is much more robust in young growing animals (e.g., higher protein requirements) as compared with a mature animal when protein needs are lower (22). Second, protein restriction beyond a certain critical level causes food aversion in both young and mature animals (23\u201325). Considered together, these responses imply the operation of one or more sensing systems that have detection limits and function within certain concentration ranges for both the orexigenic and anorexigenic responses to protein restriction. And in the case of the orexigenic component, the amplitude of the response is incrementally modulated within a defined range of protein restriction that is a function of the individual amino acid requirements when the restriction is imposed (22).\n\nLow-protein diets, particularly when given to young animals, also influence the expenditure component of energy balance. In 32-day-old rats, restricting dietary protein to 8% increases EE, circulating thyroid hormones, and produces changes in brown adipose tissue mass and function (24,26,27). Although the increase in EE functions to counterbalance the increase in energy intake, in many cases protein restriction still leads to increases in body fat and carcass energy stores (24,25,28). In contrast, protein restriction initiated after physical maturity is perceived as a less severe restriction (22) because of the lower protein requirements and greater protein mass available for mobilization and redeployment. However, it is exactly under these physiological circumstances that even a modest leveraging of additional energy intake by lower dietary protein could favor a systematic increase in adiposity. A significant unanswered question is how the determinants of protein nutrition are interpreted and translated relative to the physiological state when protein restriction is imposed.\n\n# Amino acid sensing and protein nutrition\n\n## EAAs as markers of protein nutrition.\n\nA strong case can be made that the systems that monitor dietary protein provide regulatory input to the control of energy balance and metabolism, but the associated sensing and signaling mechanisms that coordinate the elements of the response are poorly understood. Given that a subgroup of the amino acids (e.g., EAAs) that make up proteins cannot be synthesized endogenously, the ability to detect and respond to dietary EAA deficiencies is clearly an indispensable survival mechanism. Molecular and cellular mechanisms of EAA sensing have been identified and well described (29\u201336). Although their existence does not preclude important roles for additional sensing mechanisms, the well-documented in vivo responses to perturbation of dietary EAA composition make a compelling case that EAAs play a dominant role as mediators of the effects of dietary protein on metabolism and energy balance (7\u201310,37\u201339). Thus, the focus of this perspective is on EAAs, with special emphasis on the EAA-sensing and -signaling mechanisms that have been characterized using dietary manipulation of EAAs as models. A feature common to these dietary models is that they produce significant changes in tissue-specific lipid metabolism, the integration of substrate utilization among tissues, and not surprisingly, overall energy balance.\n\n## Mechanisms of EAA sensing and signaling.\n\nRestricting the availability of EAAs effectively limits charging of tRNA with its cognate amino acid and triggers a cascade of signaling events and transcriptional responses collectively referred to as the amino acid response (AAR) (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<\/a>). The transcriptional responses linked to the AAR are modulated in timing and magnitude by the perceived intensity of the nutritional stress (40\u201345). Evaluation of the in vivo responses to limitation of dietary EAAs makes a compelling case that the sequence of signaling and gene expression events occurs as part of an adaptive program to reestablish homeostasis. Among the well-documented responses of particular interest are the coordinated, tissue-specific effects on genes involved in lipid metabolism (7\u20139,39,46\u201348). This illustrates that the adaptive response to the perception of protein deprivation includes an additional integrative program involving the remodeling of lipid metabolism.\n\n## In vitro responses to EAA deprivation on lipogenic genes.\n\nThe transcriptional effects of EAA deprivation on lipogenic genes were initially identified in human HepG2 cells, where media lacking any single EAA rapidly decreases transcriptional initiation and expression of fatty acid synthase (FAS) (49). FAS is also partially reduced in cells deprived of cysteine or glutamate, but not with any other non-EAAs (49). Since this initial report, transcriptional profiling with HepG2, HepG2\/C3a, and mouse embryonic fibroblasts has been used to explore the overall systems biology of the AAR (35,50\u201352). Though it is beyond the scope of the current perspective to explore the profiles in detail, a common feature of the response in each cell line is a negative effect on genes associated with lipid metabolism (35,51,53). Loss of function approaches show that downregulation of many lipogenic genes is dependent on general control nonderepressible 2 (GCN2) (35). However, the findings from this and other studies (39) make a strong case for the involvement of additional GCN2-independent signaling mechanisms in mediating the in vitro responses to EAA deprivation. As will become apparent in subsequent sections, a similar conclusion is supported from in vivo studies using GCN2 loss-of-function approaches. It should also be noted that in vitro models of EAA deprivation evaluate an extreme cellular situation incompatible with life and in the absence of interorgan amino acid metabolism. The transcriptional responses of peripheral tissues to in vivo restriction of dietary protein or single EAAs reflect the cumulative and highly integrated inputs following both central and peripheral detection of the restriction. In a similar sense, net changes in adiposity also reflect how the perceived changes in dietary protein or specific EAAs affect the intake and expenditure components of energy balance. A deeper understanding of how gene expression patterns in individual tissues relate to substrate utilization and EE will help determine how dietary restriction of single EAAs may benefit overall metabolism.\n\n# In vivo responses to dietary limitations of EAAs\n\n## Dietary models of EAA deprivation versus restriction.\n\nThe conserved nature and ubiquitous expression of molecules associated with EAA sensing and signaling underscores the high priority placed on defending against EAA deficiency in the organism, but the broader and more difficult task is to understand how EAA sensing systems are anatomically organized to produce the highly integrated physiological and behavioral responses that occur when an EAA deficiency is detected. The primary experimental strategy used to study EAA deficiency involves formulation of diets with individual amino acids as the source of protein, and removing or limiting the amount of specific EAAs in the diet. The two most common models involve *1*) deprivation of an EAA or *2*) restriction of a specific EAA to low but not devoid levels in the diet. Much of the early work on EAA deprivation examined the acute behavioral responses to diets devoid of threonine (34,54\u201356), whereas later work used diets devoid of leucine to examine the transcriptional, translational, and associated signaling events regulating protein balance, insulin sensitivity, and lipid metabolism (30,57\u201359). The second approach involves dietary formulations in which an EAA is reduced but not absent (e.g., EAA restricted). This model has received far less attention than EAA deprivation, but that changed after an initial report showed that restricting dietary methionine from normal levels (e.g., \u223c0.8 g\/100 g diet) to 0.17% (e.g., 0.17 g \/100 g diet) produced a remarkable improvement in the metabolic phenotype of rats while increasing their life span by \u223c25% (60). In contrast, dietary methionine deprivation, like leucine deprivation, produces a rapid loss of weight, adiposity, and deterioration in health of the animal. Thus, the relatively small difference in methionine produced by dietary methionine deprivation (0%) versus restriction (0.17%) has a profound difference on the physiological responses to the respective diets. Essentially, all work to date on dietary restriction of an EAA has focused on methionine, but it will be important in future studies to restrict other EAAs and determine whether the beneficial responses to methionine restriction (MR) are specific to methionine or can be reproduced in whole or part by restricting other EAAs.\n\nThe mechanistic basis for the fundamental difference between the responses to MR versus methionine deprivation represents a critical gap in our understanding of the underlying sensing systems that detect and respond to changes in dietary EAAs. In the years since the initial report of the beneficial responses to restricting dietary methionine to 0.17% (60), significant effort has been devoted to understanding the mechanistic basis for the effects of dietary MR on energy balance, lipid metabolism, and insulin sensitivity. In the subsequent sections, the similarities and differences between leucine deprivation and MR will be presented to illustrate potential future lines of inquiry.\n\n## Physiological and transcriptional responses to dietary leucine deprivation.\n\nMetabolic phenotyping of mice subjected to short-term (e.g., 7 or 17 days) dietary leucine, valine, or isoleucine deprivation shows a strong anorexigenic response (30,39) in conjunction with a significant increase in EE (9,10). Studies of EAA deprivation beyond 3 weeks are typically not permitted or possible because of extensive weight loss and increased mortality, so most work on leucine deprivation examines responses after 7 days on the diet. Mice presented with diets lacking leucine decrease intake by 20\u201330% in the first 4 h after introduction of the imbalanced diet (34,36) and maintain the lowered rate of consumption after 7 days and 17 days (30,39). The significant decrease in energy intake is accompanied by increased sympathetic outflow to adipose tissues and a concomitant increase in lipid mobilization, oxidation, and uncoupled respiration (9,10). The combination of decreased energy intake and increased EE has a profound effect on energy balance such that after 7 days body weight is reduced by \u223c15\u201320% (30) and adipose tissue mass is two- to threefold lower (10,39). After 17 days of leucine deprivation, body weight is 30% lower and virtually no dissectable fat mass remains (39). The effects of 7 days of leucine deprivation on energy balance are accompanied by a significant reduction in fasting insulin (30,39), which is reflective of increases in tissue-specific and overall insulin sensitivity (58). The inclusion of a pair-fed control group supports the conclusion that the beneficial effects of the diet on insulin sensitivity are independent of the effects of the diet on energy balance (58). The authors propose a mechanism for the diet-induced increase in hepatic insulin sensitivity involving increased AMP-activated protein kinase activation and a GCN2-dependent decrease in mammalian target of rapamycin (mTOR)\/S6 kinase 1 (S6K1) signaling (30,58). However, previous studies show that the ability of leucine deprivation to decrease fasting insulin is not compromised in GCN2-null mice (30,39).\n\nThe transcriptional effects of leucine deprivation include targeted effects on genes involved in lipid metabolism in both liver and white adipose tissue (WAT). In liver, 7 days on the diet produces significant decreases in genes associated with fatty acid and triglyceride synthesis but not fatty acid transport or oxidation (39). In WAT, leucine deprivation increases expression of genes involved in fatty acid oxidation and thermogenesis while simultaneously repressing lipogenic genes (9). In a subsequent study using mice lacking \u03b2-adrenergic receptors, the ability of the diet to increase uncoupling protein 1, activate thermogenesis, increase EE, and reduce adiposity is compromised (10), suggesting that the sympathetic nervous system (SNS) is an essential mediator of the effect of leucine deprivation on fat deposition. As expected, the diet-induced effects on genes associated with oxidative and thermogenic responses in WAT and brown adipose tissue are blocked in \u03b2-less mice, but it remains unclear whether the previously observed downregulation of lipogenic genes in both liver and WAT requires intact signaling through \u03b2-adrenergic receptors (10).\n\n## Physiological and transcriptional responses to dietary MR.\n\nDietary MR produces a highly integrated series of behavioral, physiological, and biochemical responses that improve biomarkers of metabolic health and increase longevity in rodents (60\u201362) and flies (63). The metabolic responses are rapid in onset but persist indefinitely and include hyperphagia, increased EE, reduced fat deposition, reduced circulating lipids and leptin, and increased plasma adiponectin (7,8,64). The diet-induced reduction in fat deposition is particularly interesting because it occurs despite a paradoxical increase in food intake (60,64,65). Subsequent studies establish that dietary MR produces a simultaneous increase in EE that compensates for the increase in energy intake (7,8). More importantly, the diet enhances metabolic flexibility (7,8) and produces a significant increase in overall insulin sensitivity that develops quickly after introduction of the diet and persists after long-term MR (64). In most studies, MR is initiated after weaning in young growing animals, but the beneficial metabolic responses are also observed when MR is initiated after physical maturity (7). In this case, initiation of dietary MR at 6 months of age prevents any further increase in adiposity over the following 6 months by producing a coordinated increase in energy intake and expenditure (7). Dietary MR also increases metabolic flexibility, and after 3 and 6 months on the diet, fasting insulin, plasma triglycerides, and liver triglycerides are two- to threefold lower than that in rats on the control diet (7; T.W. Gettys, unpublished observation). It is unclear whether the reductions in hepatic and circulating neutral lipids are products of dietary effects on energy balance, direct effects of dietary MR in the liver, or some combination of both mechanisms.\n\nMost of the early work on MR was conducted with rats, but the responses to dietary MR in mice are comparable in almost every respect (8). A summary of the metabolic profile of mice after short-term (8 week) consumption of dietary MR from our work illustrates that the offsetting effects on energy intake and expenditure effectively limit fat deposition while producing significant changes in endocrine function of WAT (<a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>). WAT is also a transcriptional target of dietary MR, with the diet increasing expression of genes involved in both lipid oxidation and synthesis (8) (<a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>). The upregulation of FAS and stearoyl-CoA desaturase-1 (SCD-1) in WAT is complemented by downregulation of these lipogenic genes in the liver, potentially compromising its capacity to synthesize and export triglyceride. This prediction is supported by data from both preclinical studies (<a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>) and in patients with metabolic syndrome, in which dietary MR for 16 weeks produced a significant reduction in hepatic lipid content (66). These data illustrate the complex but highly beneficial effects that accrue from consumption of a diet in which methionine is limited but not absent. Although the combined effects of dietary MR on energy homeostasis, WAT endocrine function, and remodeling of tissue-specific lipid metabolism contribute to the observed increase in metabolic flexibility, the relative importance of the component responses to the improvement in overall insulin sensitivity remains to be established.\n\nResponses of male C57BL\/6J mice to dietary MR for 8 weeks\n\n![](2635tbl1)\n\n## Role of GCN2 in in vivo responses to EAA deprivation.\n\nDiets deficient in an EAA activate GCN2 in the anterior piriform cortex and produce an aversive feeding response within 20\u201340 min (34,36). Loss of function studies using either global or neuron-specific GCN2-null mice establish that the acute aversive response to EAA deprivation is mediated by GCN2 (34,36). EAA deprivation also produces a chronic anorexigenic response that remains evident after 7 and 17 days (30,39) on the diet. However, both wild-type and GCN2-null mice reduce consumption of a diet lacking leucine by 20\u201330% over the 1\u20132 weeks (30,39) following its introduction, indicating that GCN2 is not required for the chronic anorexigenic response to leucine deprivation.\n\nThe hypothalamus is also an important target and mediator of the effects of EAA deprivation on energy intake because it both receives axonal projections from the anterior piriform cortex (67,68) and responds directly to depletion and repletion of EAAs (69). Recent studies have shown that EAA deprivation produces rapid changes in anorexigenic neuropeptides and signaling in hypothalamic feeding centers (70\u201372) and that the hypothalamus is responsive to repletion of the limiting EAA or altered neuropeptide (71). The mediobasal hypothalamus is thought to play a role in responding to changes in protein quality and quantity through leucine-dependent activation of mammalian target of rapamycin complex 1 (73) and provide corresponding input to the control of energy intake (74). An integrated picture of the anatomical organization and temporal development of hypothalamic responses to EAA deprivation has yet to emerge but will clearly be essential to understanding the full range of adaptive responses.\n\nThe coordinated downregulation of the lipogenic gene program in both liver and WAT by leucine deprivation is mediated in part by a decrease in expression of sterol regulatory element binding protein-1c\u00a0(SREBP-1c) in both tissues (39,75). In wild-type mice, these changes are associated with a 40% decrease in serum triglyceride but no change in liver triglyceride (39). The diet-induced decrease in hepatic SREBP-1c and associated lipogenic genes (e.g., FAS, SCD-1, acetyl-CoA carboxylase-1, ATP-citrate lyase) is absent in GCN2-null mice, but the decrease in serum triglycerides is unaltered, as is the decrease in fasting insulin (39). However, compared with wild-type mice, hepatic triglycerides are increased twofold by leucine deprivation in GCN2-null mice (39). Although the question has not been addressed experimentally, these findings suggest the interesting possibility that leucine deprivation limits lipid export into VLDLs secreted from the liver, and the additional absence of GCN2 may further compromise lipoprotein signal peptide abundance, apoliprotein B synthesis, and VLDL secretion. Considered together, these findings make a compelling case that the overall effects of leucine deprivation are mediated through both GCN2-dependent and -independent mechanisms. Studies evaluating the role of GCN2 as a mediator of the responses to dietary MR are ongoing and suggest that the orexigenic response to MR does not require GCN2 (T.G. Anthony, T.W. Gettys, unpublished data).\n\n## Fundamental differences between dietary MR and EAA deprivation.\n\nIn contrast to the acute and chronic anorectic responses to EAA deprivation, dietary MR produces a hyperphagic response within 6\u20137 days after introduction of the diet, and the 20\u201325% increase in consumption of the diet continues indefinitely (7,64). A compilation of the short-term responses to dietary MR and leucine deprivation are summarized in <a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<\/a>, with differences highlighted in yellow. The acute responses to the diets also share several similarities, including comparable transcriptional effects on lipogenic genes in the liver, increased oxidative genes in WAT, increased EE, and enhanced insulin sensitivity (7\u201310,30,39,48,58,76,77). A key similarity is that both diets increase SNS stimulation of adipose tissue, which induces oxidative and thermogenic gene programs, resulting in increased EE. Recent studies provide evidence that leucine deprivation activates the SNS by increasing expression of corticotropin-releasing hormone in the hypothalamic paraventricular nucleus (10) through a mechanism involving inhibition of S6K1 (76). The central sensing and signaling mechanisms of dietary MR have received far less attention to date, so it is unknown whether dietary MR acts through a similar mechanism to activate the SNS. In contrast to their similar effects on the SNS, the two diets have opposite effects on ingestive behavior, and not surprisingly, different effects on body weight and adiposity (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<\/a>). Whereas the effect of MR is to slow the deposition of adipose tissue relative to body weight, leucine deprivation causes mobilization and loss of adipose tissue at a rate exceeding the loss of body weight (39). A third major difference between the responses in adipose tissue is that leucine deprivation produces a significant downregulation of SREBP-1c and the primary genes involved in de novo lipogenesis and triglyceride synthesis (9). In contrast, dietary MR produces a significant and persistent increase in lipogenic gene expression in WAT depots (47), which suggests retention of the capacity to conduct de novo lipogenesis in the fed state to compensate for the loss of lipogenic potential in the liver (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<\/a>). Recently we assessed the physiological significance of this by measuring ^2^H-enrichment in palmitate 12 h after injecting control and MR mice with ^2^H~2~O (T.W. Gettys, unpublished data). As expected, the calculated rates of de novo lipogenesis in WAT from control mice were quite low but were increased almost fourfold by MR. These findings are consistent with the exaggerated increases in respiratory quotient and EE observed in rats on the MR diet in the fed state (7). The associated increase in metabolic flexibility comes at the expense of metabolically inefficient conversion of glucose to lipid (78), but this may be a key component of the mechanism through which dietary MR produces a disproportionate increase in EE in the fed state (7). Conversely, MR reduced hepatic de novo lipogenesis by fivefold compared with controls, consistent with a remodeling of the integration of lipid metabolism between liver and adipose tissue. Collectively, the tissue-specific changes in de novo lipogenesis are fully consistent with the MR-induced changes in lipogenic gene expression among the tissues, as well as the relative retention of adipose tissue mass after chronic consumption of the diet. Lastly, the fundamental differences in the adaptive responses between diets should be viewed in the context that dietary MR extends life span, producing a beneficial metabolic phenotype in the process; in contrast EAA deprivation cannot sustain life beyond a few weeks.\n\n# Perspectives and future directions\n\nA comparison of the responses to leucine deprivation and MR has been provided to illustrate their tissue-specific and physiological responses and to better understand the sensing and signaling mechanisms being engaged. A conceptual model of the anatomical organization of the communication networks that are either known or proposed to be involved in coordinating the complex physiological responses to EAA sensing are presented in <a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<\/a>. The illustration is equally applicable to EAA deprivation or restriction, and can also be used to guide a comparison of the temporal relationships among responses to leucine deprivation, MR, or other dietary modifications of EAAs (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<\/a>). Irrespective of dietary model, it is difficult to distinguish the direct effects of an EAA limitation in a particular tissue from responses that are modulated by detection of the deficiency in another anatomical site that may then provide secondary signaling or endocrine input to the initial site. Thus, in addition to their spatial organization, the individual components of the response to an EAA deficiency are also temporally organized, developing in a reproducible progression after introduction of the diet. Our model proposes that there are at least four potential sites of sensing amino acid depletion or restriction: *1*) Direct sensing of luminal or absorbed EAAs by the gut; *2*) sensing of EAAs in the portal circulation and\/or liver, perhaps through the afferent vagus; *3*) direct sensing of EAAs in the general circulation by tissues; and *4*) sensing of EAAs in various regions of the brain. The model is also presented as a way to illustrate gaps in our understanding of how these and as yet unknown sensing components function together to mediate the integrated physiological responses to changes in dietary EAA content. Several areas needing further exploration include the following: *1*) To what extent does the gut participate in sensing dietary changes in protein or amino acid content and does such a sensing system use endocrine signals (e.g., GLP-1, cholecystokinin, ghrelin, peptide YY) or vagal afferents to communicate this information to the brainstem and other central sites for integration? *2*) How are the central amino acid sensing systems organized to provide integrated regulation of the components of energy balance and communication to peripheral tissues? *3*) How are other AAR signaling mechanisms besides GCN2 (e.g., mTOR, S6K1, leucyl-tRNA synthetases, mitogen-activated protein kinases) involved in detecting changes in dietary amino acids? *4*) What components of the adaptive response to EAA deprivation or restriction mediate the tissue-specific and overall enhancement of insulin sensitivity? *5*) Are the beneficial metabolic responses to MR unique to methionine or will restriction of other EAAs reproduce the phenotype? The overall metabolic phenotype produced by EAA deprivation versus restriction is the product of a series of responses that are anatomically and temporally organized and in many cases, interdependent. Therefore, a significant ongoing challenge within the field will be to develop experimental approaches that distinguish between the direct, tissue-specific responses to EAA deprivation versus restriction and the responses perceived in one anatomical site and modulated in another.\n\nThe comparison of the physiological responses to dietary MR and leucine deprivation suggest that the EAAs are in a sense functioning as ligands with responses determined by the degree of the restriction. It follows from this observation that an important future objective will be to examine systematically the physiological responses to incremental restriction of methionine, leucine, and other EAAs. In addition to identifying a range of restrictions that are most beneficial, this approach could also provide important mechanistic insights regarding EAA-specific sensing mechanisms and temporal interdependence among components of the overall phenotype. Returning to the concept of the geometric framework, Piper et al. (14) proposed to evaluate biological responses (e.g., longevity, fecundity, etc.) to nutritional complexity by representing variations in dietary composition in the *x*-*y* plane and plotting individual responses to the nutritional matrix in the *z* plane. We suggest that this may be a particularly productive approach to examine the physiological responses to variations in the dietary content of specific amino acids as part of a comprehensive strategy to assess the impact of EAA nutrition in model organisms. In our work on MR, we have identified a range of dietary methionine concentrations that produces profound improvements in biomarkers of metabolic health. The important next steps are to refine our understanding of the degree of MR linked to each component and extend this work to the other EAAs and their collective roles in the mechanistic basis of nutrient sensing in protein nutrition. Another important unexplored area involves the effects of EAA restriction on protein turnover within narrow ranges of EAA restriction such as those identified in studies of MR. Understanding the signals that trigger remodeling of protein metabolism alongside that of lipid and energy metabolism will provide an integrative view of the benefits of EAA restriction.\n\nLastly, the translational potential of the concepts developed in preclinical studies of dietary MR were recently evaluated in a human cohort meeting the criteria for metabolic syndrome (66). Dietary MR was accomplished using the semisynthetic medical food, Hominex-2 (Abbott Nutrition, Columbus, OH) in a short-term study (16 weeks) to evaluate the metabolic consequences of limiting dietary methionine from 35 mg\/kg body weigh\/day to 2 mg\/kg body weight\/day. The experimental diet (Hominex-2) is a commercial food designed to provide nutritional support for patients with pyridoxine-unresponsive homocystinuria or hypermethioninemia. However, it is comprised in part of elemental amino acids, and their associated low palatability resulted in high withdrawal rates and raised questions about compliance and achieving the desired degree of MR. Notwithstanding these experimental limitations, we were able to conclude that dietary MR has metabolic responses that are beneficial in subjects with metabolic disease. An important remaining challenge will be to develop palatable solutions that will allow the full potential of dietary MR to be evaluated in a variety of translational contexts.\n\n## ACKNOWLEDGMENTS\n\nThis work was supported in part by the National Institutes of Health (\\[NIH\\] RO1 HD-070487 to T.G.A.; RO1 DK-081563 to C.D.M.; RO1 DK-096311 to T.W.G.), the American Diabetes Association (1-12-BS-58 to T.W.G.), and a subcontract from the Mouse Metabolic Phenotyping Center Consortium (NIH U24 DK-076169 to T.W.G.). This work made use of the Genomics and Cell Biology and Bioimaging core facilities supported by NIH P20-GM-103528 (T.W.G.) and NIH P30 DK-072476.\n\nNo potential conflicts of interest relevant to this article were reported.\n\nT.G.A. and C.D.M. contributed to the writing and editing of the manuscript. T.W.G. contributed to the writing and editing of the manuscript and analyzed the data used to produce illustrations.\n\nThe authors acknowledge Drs. Jean-Marc Schwarz of the University of California, San Francisco Diabetes Center and Jennifer Rood of the Pennington Stable Isotope core facility for conducting the de novo lipogenesis analysis.\n\n# REFERENCES","meta":{"dup_signals":{"dup_doc_count":120,"dup_dump_count":47,"dup_details":{"curated_sources":2,"2021-04":1,"2020-50":1,"2020-24":1,"2019-39":1,"2019-35":1,"2018-26":2,"2018-17":3,"2018-09":3,"2017-51":1,"2017-47":2,"2017-43":3,"2017-39":3,"2017-34":1,"2017-30":6,"2017-26":2,"2017-22":2,"2017-17":6,"2017-09":4,"2017-04":7,"2016-50":3,"2016-44":4,"2016-40":3,"2016-36":3,"2016-30":3,"2016-26":3,"2016-22":3,"2016-18":2,"2016-07":3,"2015-48":3,"2015-40":1,"2015-35":3,"2015-32":3,"2015-27":1,"2015-22":2,"2015-14":2,"2014-52":2,"2014-49":3,"2014-42":4,"2014-41":3,"2014-35":2,"2022-05":1,"2024-22":1,"2017-13":4,"2015-18":3,"2015-11":1,"2015-06":1,"2024-26":1}},"file":"PMC3717867"},"subset":"pubmed_central"}
{"text":"abstract: In this article, the 2010 European League against Rheumatism (EULAR) recommendations for the management of rheumatoid arthritis (RA) with synthetic and biological disease-modifying antirheumatic drugs (sDMARDs and bDMARDs, respectively) have been updated. The 2013 update has been developed by an international task force, which based its decisions mostly on evidence from three systematic literature reviews (one each on sDMARDs, including glucocorticoids, bDMARDs and safety aspects of DMARD therapy); treatment strategies were also covered by the searches. The evidence presented was discussed and summarised by the experts in the course of a consensus finding and voting process. Levels of evidence and grades of recommendations were derived and levels of agreement (strengths of recommendations) were determined. Fourteen recommendations were developed (instead of 15 in 2010). Some of the 2010 recommendations were deleted, and others were amended or split. The recommendations cover general aspects, such as attainment of remission or low disease activity using a treat-to-target approach, and the need for shared decision-making between rheumatologists and patients. The more specific items relate to starting DMARD therapy using a conventional sDMARD (csDMARD) strategy in combination with glucocorticoids, followed by the addition of a bDMARD or another csDMARD strategy (after stratification by presence or absence of adverse risk factors) if the treatment target is not reached within 6\u2005months (or improvement not seen at 3\u2005months). Tumour necrosis factor inhibitors (adalimumab, certolizumab pegol, etanercept, golimumab, infliximab, biosimilars), abatacept, tocilizumab and, under certain circumstances, rituximab are essentially considered to have similar efficacy and safety. If the first bDMARD strategy fails, any other bDMARD may be used. The recommendations also address tofacitinib as a targeted sDMARD (tsDMARD), which is recommended, where licensed, after use of at least one bDMARD. Biosimilars are also addressed. These recommendations are intended to inform rheumatologists, patients, national rheumatology societies and other stakeholders about EULAR's most recent consensus on the management of RA with sDMARDs, glucocorticoids and bDMARDs. They are based on evidence and expert opinion and intended to improve outcome in patients with RA.\nauthor: Josef S Smolen; Robert Landew\u00e9; Ferdinand C Breedveld; Maya Buch; Gerd Burmester; Maxime Dougados; Paul Emery; C\u00e9cile Gaujoux-Viala; Laure Gossec; Jackie Nam; Sofia Ramiro; Kevin Winthrop; Maarten de Wit; Daniel Aletaha; Neil Betteridge; Johannes W J Bijlsma; Maarten Boers; Frank Buttgereit; Bernard Combe; Maurizio Cutolo; Nemanja Damjanov; Johanna M W Hazes; Marios Kouloumas; Tore K Kvien; Xavier Mariette; Karel Pavelka; Piet L C M van Riel; Andrea Rubbert-Roth; Marieke Scholte-Voshaar; David L Scott; Tuulikki Sokka-Isler; John B Wong; D\u00e9sir\u00e9e van der Heijde[^1]Correspondence to Professor Josef S Smolen, Division of Rheumatology, Department of Medicine 3, Medical University of Vienna, Waehringer Guertel 18-20, Vienna A-1090, Austria; <email@example.com>[^2]\ndate: 2014-03\ninstitute: 1Division of Rheumatology, Department of Medicine 3, Medical University of Vienna, Vienna, Austria; 22nd Department of Medicine, Hietzing Hospital Vienna, Vienna, Austria; 3Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; 4Atrium Medical Center, Heerlen, The Netherlands; 5Department of Rheumatology, Leiden University Medical Center, Leiden, The Netherlands; 6Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Chapel Allerton Hospital, Leeds, UK; 7NIHR Leeds Musculoskeletal Biomedical Research Unit, Leeds Teaching Hospitals NHS Trust, Leeds, UK; 8Department of Rheumatology and Clinical Immunology, Charit\u00e9-University Medicine, Free University and Humboldt University, Berlin, Germany; 9Clinical Immunology Free University and Humboldt University, Berlin, Germany; 10Department of Rheumatology B, Cochin Hospital, Ren\u00e9 Descartes University, Paris, France; 11Department of Rheumatology, N\u00eemes University Hospital, Montpellier I University, Nimes, France; 12Rheumatology Department, Paris 06 UPMC University, AP-HP, Pite-Salpetriere Hospital, Paris, France; 13Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; 14Hospital Garcia de Orta, Almada, Portugal; 15Oregon Health and Science University, Portland, Oregon, USA; 16EULAR Standing Committee of People with Arthritis\/Rheumatism in Europe (PARE), Zurich, Switzerland; 17Department of Rheumatology and Clinical Immunology, University Medical Center Utrecht, Utrecht, The Netherlands; 18VU University Medical Center, Amsterdam, The Netherlands; 19Service d'Immuno-Rhumatologie, Montpellier University, Lapeyronie Hospital, Montpellier, France; 20Academic Clinical Unit of Rheumatology, Department of Internal Medicine, University of Genova, Genova, Italy; 212nd Hospital Department, Institute of Rheumatology, University of Belgrade Medical School, Belgrade, Serbia; 22Department of Rheumatology, Erasmus MC, University Medical Center, Dr Molewaterplein, Rotterdam, The Netherlands; 23Department of Rheumatology, Diakonhjemmet Hospital, Oslo, Norway; 24Hopitaux Universitaires Paris Sud, AP-HP, and Universit\u00e9 Paris-Sud, Le Kremlin Bicetre, France; 25Institute of Rheumatology and Clinic of Rheumatology, Charles University, Prague, Czech Republic; 26Department of Rheumatology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; 27Department of Internal Medicine, University of Cologne, Cologne, Germany; 28King's College School of Medicine, Weston Education Centre, London, UK; 29Jyv\u00e4skyl\u00e4 Central Hospital, Jyv\u00e4skyl\u00e4, Finland; 30Medcare Oy, \u00c4\u00e4nekoski, Finland; 31Division of Clinical Decision Making, Informatics and Telemedicine, Tufts University School of Medicine, Boston, Massachusetts, USA\nreferences:\ntitle: EULAR recommendations for the management of rheumatoid arthritis with synthetic and biological disease-modifying antirheumatic drugs: 2013 update\n\nThe management of rheumatoid arthritis (RA) rests primarily on the use of disease-modifying antirheumatic drugs (DMARDs). These agents are commonly characterised by their capacity to reduce or reverse signs and symptoms, disability, impairment of quality of life, inability to work, and progression of joint damage and thus to interfere with the entire disease process.1 DMARDs form two major classes: synthetic chemical compounds (sDMARDs) and biological agents (bDMARDs). In this respect, a new nomenclature for DMARDs was recently proposed which we will adhere to in this report.2 Consequently, the term conventional sDMARDs (csDMARDs) will be used to include chemical agents such as methotrexate (MTX), sulfasalazine and leflunomide, whereas tofacitinib, a new sDMARD specifically designed to target janus kinases (JAKs), will be designated as a targeted sDMARD (tsDMARD). The five available tumour necrosis factor (TNF) inhibitors (adalimumab, certolizumab pegol, etanercept, golimumab and infliximab), the T cell costimulation inhibitor, abatacept, the anti-B cell agent, rituximab, and the interleukin (IL)-6 receptor (IL-6R)-blocking monoclonal antibody, tocilizumab, as well as the IL-1 inhibitor, anakinra, will be subsumed as biological originator (bo) DMARDs, while biosimilars (bs), such as bs-infliximab, recently approved by the European Medicines Agency (EMA), will be named bsDMARDs.2\n\nWith abundant therapeutic options available and insufficient information on differential efficacy and safety, making treatment decisions in clinical practice remains challenging. To this end, the European League Against Rheumatism (EULAR) has recently developed recommendations for the management of RA with these drugs.3 These recommendations were based on five systematic literature reviews (SLRs)4\u20138 and focused on indications for the use of, and suggestions for, differential and strategic employment of csDMARDs and bDMARDs based on treatment targets, disease risk assessment, safety aspects and contraindications. While some of the individual recommendations have elicited extensive discussions, all of them were based on the evidence available at that point in time4\u20138 and on the results of the discussions and votes by the expert committee. Moreover, the EULAR recommendations have been used and adopted widely, as suggested by their application as a template for many national and regional recommendations after their publication.9\u201312 However, as with most recommendations and especially in a rapidly evolving field such as RA, it was anticipated that the 2010 recommendations would need updating within a few years.3 Indeed, more experience and additional evidence on agents approved at that time, as well as data on new compounds, have become available over the last 3\u20134\u2005years, motivating us to update the recommendations as described here.\n\n# Methods\n\nWith the approval of the EULAR Executive Committee, the convenor (JSS) and epidemiologist (RL) who led the 2010 activity formed a Steering Group and a Task Force with the aim of updating the 2010 EULAR recommendations for the management of RA.\n\n## Task force\n\nComprised of 33 members from 11 European countries and the USA, this EULAR Task Force included four patient representatives, 24 rheumatologists, an infectious disease specialist, a health economist and three fellows; care was taken to have a good representation of clinicians and experts experienced in RA clinical trials and their analysis from all European regions.\n\nInitially, a Steering Group prioritised research questions and search terms for the three SLRs. These searches expanded and updated the available published information on efficacy of csDMARDs (as monotherapy or combination therapy, with and without glucocorticoids), efficacy of bDMARDs (as monotherapy or combined with csDMARDs) and safety aspects of csDMARDs and bDMARDs; treatment strategies were contained in the present SLRs rather than being separate as in 2010.7 Although the SLRs informing the 2010 EULAR recommendations also included a search on economic evaluations,8 the Steering Group felt that re-evaluation was not necessary because the approval status and price of new agents such as bsDMARDs was unknown.\n\nSubsequently, with the help of their mentors, the three fellows performed the respective SLRs using established databases, including registry data for safety outcomes, and abstracts, especially from recent meetings (American College of Rheumatology 2012, EULAR 2012 and 2013). Details on and results of the SLRs are reported separately.13\u201315 Levels of evidence and grades of recommendation were determined according to the standards of the Oxford Centre for Evidence-Based Medicine.16\n\n## Consensus finding\n\nAt a subsequent meeting, these data were presented first to the Steering Group consisting of nine rheumatologists, an infectious disease specialist and a patient representative, who drafted a preliminary set of new recommendations based on their discussions. The search results as well as the drafted proposal for the recommendations were subsequently presented to the whole Task Force and discussed in detail in four break-out groups focusing on (i) csDMARDs and tsDMARDs, (ii) glucocorticoids, (iii) bDMARDs and (iv) safety aspects. After these deliberations, each subgroup reported their respective results and made new proposals for the recommendations to the entire group. After discussion, the Task Force then amended them as deemed appropriate to achieve final consensus, ultimately voting on each individual recommendation. When an initial majority of 70% in favour of\u2014or against\u2014a recommendation or formulation was not achieved, the contents or wordings were amended until a majority of the Task Force members approved the individual item. The results of the final ballot are presented for each of the recommendations as a percentage of voting members. An ultimate round of wording refinements was carried out via electronic communication but with no changes of the meaning permitted. This was accompanied by anonymous voting on the strength of recommendation (level of agreement) for each item on a 0\u201310 scale (0, no agreement at all; 10, full agreement).\n\nA few principal considerations had already been developed by the Steering Group before the SLRs and were subsequently approved by the whole Task Force: (i) all of the 2010 recommendations should be reconsidered on the basis of new available supportive or contradicting evidence and voted upon; (ii) any of the previous recommendations could be kept as had been formulated, could undergo textual amendments, could be totally abandoned or could be shifted from a prominent place in the table listing the individual recommendations to the text accompanying them; (iii) although not yet approved and used in clinical practice outside the USA at the start of the current activity, it was deemed important to at least discuss and possibly formulate a recommendation on the application of tofacitinib based on the evidence from the literature; (iv) while not yet approved or used in practice in Europe or North America, it was also deemed important to at least discuss and potentially express a view on the place of biosimilars in the therapeutic arena based on available evidence.\n\nIn line with these a priori considerations and the potential need to provide some totally new recommendations, each of the three overarching principles and 15 recommendations of the consensus published in 2010 underwent thorough re-evaluation for their validity based on information that had become available from trials and registries during the years since the last SLR and consensus finding; where no new evidence had been found, the evidence from the 2010 searches was applied.\n\n# Results\n\n## General aspects\n\nAs with the 2010 recommendations, this 2013 update reflects the balance of efficacy and safety, but does not deal with the toxicity of DMARDs in detail; this can be derived from the results of the safety SLR; all three SLRs provide an important adjunct to these recommendations, since they establish the evidence base. Thus, in line with the 2010 recommendation, the 2013 update primarily considers agents with toxicity that appears to be manageable, assuming that prescribers are either aware of the respective risks or will adhere to the information provided in the package inserts. However, where toxicity appears to be a major issue, a general warning is included in the respective recommendation.\n\n## Overarching principles\n\nIn line with the 2010 recommendations, the Task Force felt again that some of the principles of treating RA are of such a generic nature that they should be separated from individual recommendations on individual therapeutic approaches or compounds. However, the sequence of these principles was changed and the wording refined (<a href=\"#ANNRHEUMDIS2013204573TB1\" data-ref-type=\"table\">table 1<\/a>).\n\n1.  *Treatment of RA patients should aim at the best care and must be based on a shared decision between the patient and the rheumatologist*. This principle was originally ranked as B,3 but the Task Force decided that decision-sharing by patient and rheumatologist is of such overwhelming importance that it should spearhead the recommendations. Shared decision-making includes the need to inform the patient of the risks of RA and the benefits of reaching the targeted disease activity states as well as the pros and cons of respective therapies. It also means two-way communication and joint or shared decision-making on the therapeutic target and management plan as well as support for the patient to develop personal preferences. The term 'best care' inherently refers to the recommendations provided here.\n\n2.  *Rheumatologists are the specialists who should primarily care for RA patients*. Shifting this item from rank A to B was not at all meant to diminish the role of the rheumatologist in the care of patients with RA. Indeed, the wording of this principle remained unchanged and the rheumatologist is already mentioned in item A and should constitute the main counselling anchor for patients with RA. Further, the evidence for provision of better care by rheumatologists in comparison with other physicians (see item A: 'best care') has been briefly reviewed in the 2010 recommendations and further corroborated since then.17 18 The term 'primarily' constitutes a short cut with several thoughts behind it that go even beyond the considerations expressed in 2010: first, it reflects the necessity to involve other physicians experienced in the care of RA patients, including experience in novel therapies and their potential complications, where there is a lack of trained rheumatologists; second, it is consistent with multiprofessional care and thus with current trends in some countries for an increasing role of non-physician health professionals who are well trained in the care of patients with RA, such as rheumatology nurses,19 as long as the responsibility in general is in the hands of the rheumatologist; and third, the term 'primarily' should also remind the rheumatologist that multidisciplinary care may sometimes be needed, especially when dealing with comorbidities, such as cardiovascular disease,20 or complications of applied therapies, such as serious infections.\n\n3.  *RA incurs high individual, societal and medical costs, all of which should be considered in its management by the treating rheumatologist*. Slightly reworded, the meaning of this principle has not changed from last time. It consists of two parts. The first part relates to the costs incurred by RA for the individual patient\/family and society and mentions the costs of modern therapies. It has been well established that RA incurs a substantial socioeconomic burden,21 and this has recently been supported by the Global Burden of Disease studies.22 23 In this context, the cost-effectiveness of treating RA has been repeatedly addressed, and the impact of modern therapies on late, costly consequences of RA, such as joint replacement surgery, is noteworthy.24 25 While rheumatologists cannot generally be held responsible for costs of treatment when attempting to provide best care in line with overarching principle A, this item does reiterate the responsibility of the rheumatologist to consider economic implications when selecting between treatment strategies or modalities with similar efficacy and safety in the short or intermediate term. Comparative meta-analyses and head-to-head studies help us to judge similarities or differences between therapies,26\u201330 although\u2014as will be discussed below\u2014the qualities of the studies may differ, which should be thoroughly weighed in therapeutic decision-making. Cost considerations by rheumatologists will become more important once biosimilar biological agents become available.31 In parallel, payers (governments or social security agencies) ought to take the overall individual and societal implications of RA into account when making decisions on medical costs.\n\n2013 Update of the EULAR recommendations (the table of 2010 recommendations can be seen in the online supplement or the original publication)\n\n|  |  |\n|:---|:---|\n|  | ***Overarching principles*** |\n| A. | Treatment of RA patients should aim at the best care and must be based on a shared decision between the patient and the rheumatologist |\n| B. | Rheumatologists are the specialists who should primarily care for RA patients |\n| C. | RA incurs high individual, societal and medical costs, all of which should be considered in its management by the treating rheumatologist |\n|  | ***Recommendations*** |\n| 1\\. | Therapy with DMARDs should be started as soon as the diagnosis of RA is made |\n| 2\\. | Treatment should be aimed at reaching a target of remission or low disease activity in every patient |\n| 3\\. | Monitoring should be frequent in active disease (every 1\u20133\u2005months); if there is no improvement by at most 3\u2005months after the start of treatment or the target has not been reached by 6\u2005months, therapy should be adjusted |\n| 4\\. | MTX should be part of the first treatment strategy in patients with active RA |\n| 5\\. | In cases of MTX contraindications (or early intolerance), sulfasalazine or leflunomide should be considered as part of the (first) treatment strategy |\n| 6\\. | In DMARD-na\u00efve patients, irrespective of the addition of glucocorticoids, csDMARD monotherapy or combination therapy of csDMARDs should be used |\n| 7\\. | Low-dose glucocorticoids should be considered as part of the initial treatment strategy (in combination with one or more csDMARDs) for up to 6\u2005months, but should be tapered as rapidly as clinically feasible |\n| 8\\. | If the treatment target is not achieved with the first DMARD strategy, in the absence of poor prognostic factors, change to another csDMARD strategy should be considered; when poor prognostic factors are present, addition of a bDMARD should be considered |\n| 9\\. | In patients responding insufficiently to MTX and\/or other csDMARD strategies, with or without glucocorticoids, bDMARDs (TNF inhibitors\\*, abatacept or tocilizumab, and, under certain circumstances, rituximab\u2020) should be commenced with MTX |\n| 10\\. | If a first bDMARD has failed, patients should be treated with another bDMARD; if a first TNF inhibitor therapy has failed, patients may receive another TNF inhibitor\\* or a biological agent with another mode of action |\n| 11\\. | Tofacitinib may be considered after biological treatment has failed |\n| 12\\. | If a patient is in persistent remission after having tapered glucocorticoids, one can consider tapering\u2021 bDMARDs\u00a7, especially if this treatment is combined with a csDMARD |\n| 13\\. | In cases of sustained long-term remission, cautious reduction of the csDMARD dose could be considered, as a shared decision between patient and physician |\n| 14\\. | When therapy needs to be adjusted, factors apart from disease activity, such as progression of structural damage, comorbidities and safety issues, should be taken into account |\n\n\\*TNF inhibitors: adalimumab, certolizumab pegol, etanercept, golimumab, infliximab, biosimilars (as approved according to a thorough approval process, such as by EMA and\/or FDA).\n\n\u2020The 'certain circumstances', which include history of lymphoma or a demyelinating disease, are detailed in the accompanying text.\n\n\u2021Tapering is seen as either dose reduction or prolongation of intervals between applications.\n\n\u00a7Most data are available for TNF inhibitors, but it is assumed that dose reduction or interval expansion is also pertinent to biological agents with another mode of action.\n\nDMARD, disease-modifying antirheumatic drug; EMA, European Medical Agency; EULAR, European League against Rheumatism; FDA, Food and Drug Administration; MTX, methotrexate; RA, rheumatoid arthritis; TNF, tumour necrosis factor.\n\n## Recommendations\n\nThe discussion process of the Task Force led to 14 (rather than the previous 15) recommendations. This reduction is due to the elimination of three of the 2010 items (Nos 10, 11 and 14) and the addition of two new recommendations. The decision to delete old item 10 (mentioning the potential use of 'azathioprine, cyclosporine A \\[or\u2026cyclophosphamide\\]') was taken unanimously; the decision to remove old item 11 ('Intensive medication strategies should be considered in every patient, although patients with poor prognostic factors have more to gain') was likewise taken unanimously because those treatment strategies are now well established, and several of the revised recommendations inherently incorporate a strategic approach to treating RA intensively. Finally, a 94% majority vote supported deleting previous recommendation 14 ('DMARD-na\u00efve patients with poor prognostic markers might be considered for combination therapy of MTX plus a biological'); for more details see explanations on new No 9. However, it was simultaneously decided to mention these therapeutic considerations in the text accompanying pertinent recommendations.\n\nThe 14 recommendations arising from the current activity are presented in <a href=\"#ANNRHEUMDIS2013204573TB1\" data-ref-type=\"table\">table 1<\/a> and discussed in detail below. With the exception of the first two items, which are the mainstay of the therapeutic approach to RA, they are not primarily weighted by an order of importance, but rather follow a logical sequence and procedural hierarchy. They are summarised in abbreviated form in the algorithm presented in <a href=\"#ANNRHEUMDIS2013204573F1\" data-ref-type=\"fig\">figure 1<\/a>. <a href=\"#ANNRHEUMDIS2013204573TB2\" data-ref-type=\"table\">Table\u00a02<\/a> displays the levels of evidence and grades of recommendation based on the Oxford Levels of Evidence assessment, as well as the primary voting results at the Task Force meeting and level of agreement\/strength of recommendation voting by the Task Force.\n\n1.  *Therapy with DMARDs should be started as soon as the diagnosis of RA is made.* This recommendation is almost the same as in 2010; the term 'synthetic' before DMARDs was omitted to emphasise the generic nature of this recommendation, focusing particularly on the importance of diagnosing RA early and treating it appropriately as soon as such a diagnosis is presumed. To this end, the 2010 American College of Rheumatology (ACR)\u2013EULAR classification criteria (which had only been in development when the 2010 EULAR RA management recommendations were discussed and are now well established)32 should be used to support diagnosis and facilitate early introduction of effective therapy in RA. Although diagnosis relies on the individual rheumatologist's judgement about the disease in a particular patient at a particular point in time, whereas classification relates to the group level and is important primarily for clinical studies, the new classification establishes general criteria for early diagnosis. In the course of its discussions, the Task Force reiterated both the importance of the presence of clinical synovitis in at least one joint (in line with the 2010 classification criteria) and the essential importance of starting DMARD therapy as soon as possible.\n\n2.  *Treatment should be aimed at reaching a target of remission or low disease activity in every patient.* The definition of the treatment target was deemed of such fundamental importance that the Task Force decided that aspects of patient follow-up should not dilute it. Therefore the former recommendation 2 is now split into two recommendations, items 2 and 3. When the 2010 EULAR recommendations were set forth to target remission,3 33 the ACR\u2013EULAR remission definition was still in development; in the meantime, more stringent criteria have been published34 by ACR and EULAR and should be applied in the context of these recommendations for the actual definition of remission as the optimal treatment target. Remission as defined by the Disease Activity Score based on 28 joint counts (DAS28\\<2.6) is not regarded as sufficiently stringent to define remission.34 The proportion of patients reaching remission by the ACR\u2013EULAR criteria in clinical trials and practice is sufficiently large to warrant their preferential and widespread use in daily care of RA patients.35\u201338 A large array of data has confirmed the value of reaching stringent remission not only with regard to signs and symptoms of RA, but also with regard to achieving maximal functional improvement and halting progression of structural damage39\u201344; thus good outcomes in terms of physical function and structural changes are implicitly included in targeting good clinical outcome. Moreover, the Task Force agreed with the 2010 recommendations and similar recommendations by another expert committee,27 namely that low disease activity defined by composite measures45 is a good alternative goal for many patients who cannot attain remission even today, especially those with long-standing disease who actually constitute the majority of patients in clinical care. Indeed, although somewhat worse than remission, low disease activity conveys much better functional and structural outcomes than moderate or high disease activity.38 40 46 Because a significant proportion of patients in clinical practice still do not attain a state of remission,37 47 48 implementation of this combined therapeutic target appears to be particularly relevant and significant. Also, once any patient has reached a low disease activity that is close to remission, the individual disease activity variables have to be considered in detail before major therapeutic changes are made.\n\n3.  *Monitoring should be frequent in active disease (every 1\u20133\u2005months); if there is no improvement by at most 3\u2005months after treatment start or the target has not been reached by 6\u2005months, therapy should be adjusted.* In contrast with the second half of prior recommendation 2, which also dealt with follow-up and treatment adjustments that could be interpreted or used differently than intended, statement 3 of the updated recommendations is very specific and clarifies any potential incongruity. First, monitoring should be performed as frequently as disease activity necessitates, namely more frequently (such as every 1\u20133\u2005months) with active disease and less frequently (such as every 6\u201312\u2005months) once the treatment target has been stabilised. EULAR also advocates the use of composite measures of disease activity, which include formal joint counts and the application of the ACR\u2013EULAR criteria for remission.34 45 Further, this item clearly specifies that the treatment target (remission or at least low disease activity, see item 2) should be attained within 6\u2005months and not necessarily within 3\u2005months; the 3-month time point relates solely to assessing improvement, meaning reduction of disease activity from a high to at least a moderate state by composite measures.45 If there is no improvement in disease activity (such as persistence of high disease activity as assessed by composite scores) after 3\u2005months and provided that therapy has already been adjusted to maximise treatment effect, the ongoing therapeutic regimen is usually unlikely to lead to the treatment goal in many additional patients even by 1\u2005year and should be modified.49 Maximisation of treatment effects includes reaching an optimal MTX dose within a few weeks and maintaining the maximal dose (25\u201330\u2005mg weekly) for at least 8\u2005weeks.50 If improvement is achieved at 3\u2005months,51 52 it must be borne in mind that maximal efficacy will not be seen before 6\u2005months in many patients with most treatment strategies. This is true for all types of therapies, including most biological agents. A similar approach should be made if the treatment target (remission or low disease activity) is not attained at 6\u2005months. Of note, individual patients may be well on their way to reaching the targets of low disease activity or remission at 6\u2005months and might just take slightly more time to attain this desired state. Therefore the change in disease activity from treatment start to the 6-month time point will have to be taken into account when making final treatment decisions in the individual patient.\n\n4.  *MTX should be part of the first treatment strategy in patients with active RA.* This statement (previously No 3) remains unchanged. The Task Force felt reassured by the respective SLR13 that MTX is a highly effective agent both as monotherapy and in combination with glucocorticoids, other csDMARDs and bDMARDs, and thus continues to serve as an anchor drug in RA.53 As monotherapy with or without glucocorticoids, it is effective in DMARD-na\u00efve patients and leads to low disease activity states or 70% improvement rates according to the criteria of the ACR (which correspond to nearly a state of low disease activity)54 in about 25\u201350% of patients with early RA within 6\u201312\u2005months.35 55\u201360 Generally, this statement combines three aspects: first, by using the term 'part of the first treatment strategy', it implies that MTX, although effective as monotherapy, may be combined with other agents, such as glucocorticoids but also other csDMARDs (see above and below); second, by stating 'active disease' (suggested definitions: Clinical Disease Activity Index (CDAI)\\>10, DAS28\\>3.2 or Simplified Disease Activity Index (SDAI)\\>11),45 it implies that some patients with low disease activity (defined as CDAI\u226410, DAS28\\<3.2, SDAI\u226411) may not need MTX and can do well on alternative csDMARD therapies; the third aspect relates to patients previously treated with other csDMARDs who should receive MTX at a sufficient dose and for sufficient time before progressing to potentially more intensive therapies. Important aspects include dose optimisation,50 optimal use of folic acid,61 and recognition that the maximum effect of MTX is attained only after 4\u20136\u2005months55\u201359; in this respect, the optimal dose (25\u201330\u2005mg a week with folate substitution, or somewhat less in the case of dose-limiting side effects62), should be maintained for at least 8\u2005weeks as an important aspect on the way to ultimate treatment success.50 For patients with contraindications to MTX, other drugs should be used (see next recommendation).\n\n5.  *In cases of MTX contraindications (or early intolerance), leflunomide or sulfasalazine should be considered as part of the (first) treatment strategy.* While MTX is usually quite well tolerated, especially with folate supplementation,61 63 safety issues do exist62 64 and contraindications to MTX include hepatic or renal disease; a further safety aspect of concern may be MTX-induced lung disease. Sulfasalazine and leflunomide were also included as alternatives for MTX in 2010 in statement 4, so this item has only been shifted. Both sulfasalazine and leflunomide have shown clinical, functional and structural efficacy,65\u201369 and, although MTX doses in respective comparative trials may not have been optimal, both have shown efficacies similar to MTX. No new studies have disproved this conclusion, and both drugs have been used effectively in combination with biological agents.70 71 Optimal therapeutic dosing of sulfasalazine is 3\u20134\u2005g\/day as enteric coated tablets72\u201376; the usual leflunomide dose is 20\u2005mg\/day. As with all other agents mentioned, safety risks and contraindications should be considered, and\u2014aside from its higher cost\u2014some issues stated in relation to MTX above may also pertain to leflunomide. Of note, sulfasalazine is considered to be safe during pregnancy.77 Prior recommendation No 4 also mentioned injectable gold salts as an alternative to MTX. While the Task Force does not at all withdraw its evidence-based opinion on the efficacy of parenteral gold salts (which is similar to that of MTX in clinical, functional and structural terms) set forth in 2010, gold salts are used rarely and, indeed, are unavailable in many countries. Although some members of the Task Force expressed safety concerns, these were not found to be substantiated in the previous SLR4; however, no study has evaluated intramuscular gold since the last SLR was performed. Therefore it was decided to remove gold salts from its relatively prominent place in the table, while acknowledging that its efficacy remains established by high-quality evidence.78 Further, antimalarials, such as hydroxychloroquine and chloroquine, are used in RA, especially in combination therapy, but also as monotherapy in patients with very mild disease.79 Interestingly, beyond their mild DMARD activity, antimalarials exhibit a variety of positive metabolic effects and are also considered to be safe during pregnancy.80 81 Because they may not retard progression of joint damage to the same extent as other agents,65 82 they have not been mentioned more prominently in this statement, although patients with low disease activity have a low propensity for joint destruction. Finally, compared with the previous statement on these drugs, the term 'early' has now been added to 'intolerance' to indicate the Task Force's view that early intolerance to MTX (within 6\u2005weeks) should be viewed as a contraindication and not as a failure of the first treatment strategy. Of note, the Task Force decided unanimously to delete recommendation 10, which also dealt with potential alternative therapies for desperate cases ('In the case of refractory severe RA or contraindications to biological agents or the previously mentioned sDMARDs, the following sDMARDs might be also considered, as monotherapy or in combination with some of the above: azathioprine, cyclosporine A \\[or exceptionally cyclophosphamide\\]') from the table of recommendations. Given the many currently available effective csDMARDs and bDMARDs and the view that the benefit\/risk ratio of the mentioned drugs was not convincingly favourable, especially in relation to other therapies, their use in a first treatment strategy should be restricted to rare, exceptional situations (for details see 2010 recommendations).3\n\n6.  *In DMARD-na\u00efve patients, irrespective of the addition of glucocorticoids, csDMARD monotherapy or combination therapy of csDMARDs should be used.* In the previous set of recommendations, item 5 read: 'In DMARD-na\u00efve patients, irrespective of the addition of glucocorticoids, sDMARD monotherapy rather than combination therapy of sDMARDs may be applied.' This wording expressed a preference for monotherapy based on the respective SLRs,64 83 which had revealed no superiority of combination therapy using csDMARDs when excluding the concomitant use of glucocorticoids. However, by saying 'may', that statement did not generally oppose the use of csDMARD combination therapy; this was also reflected in the respective figure depicting the proposed algorithm. Since then, several additional studies suggest that csDMARD combination may be superior to MTX monotherapy, and some even found efficacy to be similar to that of bDMARDs.84\u201388 Nevertheless, although these trials yielded similar results strengthening their interpretation, controversy persists because of methodological limitations of these studies,13 which were also clearly stated in some of the reports themselves. Moreover, additional recent data suggest that sequential monotherapy is as effective as combination therapy in clinical, functional and structural outcomes89 90 and that stepping up from MTX monotherapy to a biological agent has significant superiority over a combination of csDMARDs.89 Nonetheless, the Task Force agreed unanimously that the use of csDMARD combination therapy should be mentioned as an appropriate alternative strategy alongside the use of csDMARD monotherapy, with or without glucocorticoids. The Committee thus felt that both monotherapy and combination therapy of csDMARDs are effective and that patient preferences and expectations of adverse events should be considered when discussing treatment options with them. In general, combination therapy with csDMARDs should include MTX, since other combinations have not been sufficiently studied. Finally, the Task Force recognised the limitations of meta-analyses in the light of new studies84 86 contradicting a meta-analysis that had suggested similar structural efficacy for csDMARD combinations and bDMARD treatment.91\n\n7.  *Low-dose glucocorticoids should be considered as part of the initial treatment strategy (in combination with one or more csDMARDs) for up to 6\u2005months, but should be tapered as rapidly as clinically feasible.* As before, the Task Force heavily debated the role of glucocorticoids (previously recommendation 6). Indeed, this item was reworded (previously: 'Glucocorticoids added at low to moderately high doses to sDMARD monotherapy \\[or combinations of sDMARDs\\] provide benefit as initial short term treatment, but should be tapered as rapidly as clinically feasible.'). Rather than just making the general statement that glucocorticoids may 'provide benefit', the Task Force now recommends that they should be considered as part of the initial therapeutic approach. This change is based on the respective SLR13 which includes additional information accrued over the last few years.85 92 Low dose refers primarily to a dose of 7.5\u2005mg prednisone or equivalent per day or less.93 Mentioning glucocorticoids in a separate recommendation results from their proven capacity to increase clinical, functional and structural efficacy when combined with csDMARDs,92 94\u201396 and this combination has similar efficacy when compared with TNF inhibitors plus MTX60 97; thus glucocorticoids, both in initially high and rapidly tapered regimens (eg, COBRA) and at lower doses extended over a year or two, may increase DMARD activity and are even effective in this regard as monotherapy.98 99 However, glucocorticoid monotherapy is not specifically recommended by the Task Force and should only be used in exceptional cases when all other DMARDs have contraindications. A separate EULAR committee has concluded that the literature on safety of long-term glucocorticoid therapy at low doses still has important gaps, but in general does not support the notion of unacceptable safety issues100; subsequently, that committee formulated management guidelines that also address preventive measures against glucocorticoid-induced adverse events.101 The current SLRs13 15 are not in disagreement with any of the above findings. Nevertheless, the adverse event profile and comorbidity implications of glucocorticoids (and thus their benefit\/risk profile) elicited a fierce debate within the Task Force. A compromise (based on expert opinion) to be more specific with respect to the time frame of their application by stating 'up to 6\u2005months' rather than just 'short term' ultimately led to a majority vote; however, only 73% of the members approved this item (the lowest majority level of all recommendations), reflecting divergent opinions, with both proponents of a stronger and a weaker recommendation voting against. However, the level of agreement (strength of recommendation) was quite high (mean of 8.9) upon final anonymous grading. Thus, the Task Force suggests using them only as bridging therapy and limiting their use to a maximum of 6\u2005months, ideally tapering them at earlier time points. However, neither chronic use of glucocorticoids in established RA nor intra-articular glucocorticoid applications were discussed. Of note, it was also decided to change the algorithm in <a href=\"#ANNRHEUMDIS2013204573F1\" data-ref-type=\"fig\">figure 1<\/a> from the 2010 version by downsizing the '\u2212' compared with the '+' in the '\u00b1' symbol to reflect the increasing agreement of the Task Force that glucocorticoids should be combined with MTX or other csDMARD regimens.\n\n8.  *If the treatment target is not achieved with the first DMARD strategy, in the absence of poor prognostic factors, change to another csDMARD strategy should be considered; when poor prognostic factors are present, addition of a bDMARD should be considered.* Slightly reworded compared with 2010, this statement reiterates the unanimous view of the Task Force that risk stratification is an important aspect in the therapeutic approach to RA. These risks have been well defined over the years and include a high disease activity state, autoantibody positivity (rheumatoid factor and\/or antibodies to citrullinated proteins) and the early presence of joint damage.102 103 In patients with a low risk of poor RA outcome, another csDMARD strategy (plus glucocorticoids) would be preferred, while in patients with a high risk, the addition of a bDMARD would be preferred. It should be noted that the Task Force changed the sequence compared with the 2010 recommendation, since it assumed that many patients may not be at high risk after a first DMARD strategy, especially in terms of a reduced disease activity and maybe even lower autoantibody levels, and that a rapid change of the csDMARD regimen within 6\u2005months, in line with recommendation 3, may convey further efficacy for a significant proportion of patients. 'Change' rather than the previously used 'switch' is semantically more in line with potentially adding drugs, especially in patients initially treated with MTX monotherapy, and inherently also comprises switching. 'Another conventional DMARD strategy' has to be seen in relation to the first DMARD strategy; if the first DMARD was MTX monotherapy, then a switch to, or the addition of, other csDMARDs would be the appropriate choice; if the first DMARD strategy was combination therapy of MTX, sulfasalazine and hydroxychloroquine, then the next csDMARD strategy to choose in patients at low risk of poor outcome may be leflunomide, all of this under the proviso that optimal doses of the csDMARDs have already been used before (see above). The term 'considered' was used in both instances to reflect the Committee's preferences, but inherently acknowledges and implies that treatment decisions have to be made individually and that using a bDMARD after a first csDMARD also in a patient with lower risk of a poor outcome may be appropriate, just as using another csDMARD strategy in a patient at high risk of a poor outcome may be appropriate, as long as the target-oriented strategy to attain remission or low disease activity within 6\u2005months remains paramount; the latter approach, indeed, may be a common or enforced approach in many healthcare systems. Studies suggesting that step-up csDMARD combination therapy was as effective as a step-up combination of a biological agent with MTX87 88 104 were seen to be in conflict with results from other studies showing better efficacy of addition of a bDMARD.89 Obviously, and for reasons of clarity, when speaking of csDMARDs, the Task Force had only the hitherto employed csDMARDs in mind and not any potential new targeted synthetic (ts) DMARD, such as a kinase inhibitor.\n\n9.  *In patients responding insufficiently to MTX and\/or other csDMARD strategies, with or without glucocorticoids, bDMARDs (TNF inhibitors, abatacept or tocilizumab, and, under certain circumstances, rituximab) should be commenced with MTX.* This point was approved as worded by 90% of the participants. First, the Task Force reiterated here that bDMARDs should primarily be started when patients did not achieve the therapeutic target after treatment with csDMARDs for 6\u2005months (or had no improvement at 3\u2005months). Second, it explicitly defined the agents it meant when mentioning 'biological DMARDs'. In the 2010 recommendations, the Committee had added 'current practice would be to start a TNF inhibitor', and explained this expert opinion with the long-term use of TNF blockers and the availability of registry data when compared with abatacept and tocilizumab; this was simply an expression of a preference based on their larger and longer evidence base and was not intended to preclude use of other biological agents after csDMARD failures. Also, at that time, their application in patients with an inadequate response to csDMARDs was not yet approved for tocilizumab in the USA and for abatacept in Europe. Meanwhile, the approval status has changed for both drugs, the clinical experience with these agents has now grown for several years, and initial registry data do not seem to reveal differences in their safety profiles from the clinical trial data or when compared with TNF inhibitors.105\u2013109 Moreover, a direct comparison of abatacept and adalimumab in patients with active disease despite MTX revealed very similar efficacy and overall safety.110 Therefore the Task Force decided by a 90% majority vote that no preference of one over another biological agent should be expressed in the 2013 update of the recommendations. However, the Task Force recognised that there was still more experience with TNF inhibitors than with other bDMARDs, and that more safety data from registries would be desirable for the newer bDMARDs. Notably, IL-1 inhibitors have not shown strong efficacy when compared with other bDMARDs in meta-analyses, so anakinra is not specifically mentioned in the abbreviated recommendation; nevertheless, some patients may respond to this bDMARD. Thirdly, the Task Force intentionally added 'under certain circumstances rituximab'; while rituximab is approved for use after patients have responded insufficiently to TNF blockers, the Committee acknowledged that trial data in patients who were na\u00efve for csDMARDs and those who had an inadequate response to csDMARDs have been published111 112 (level 1 evidence) and that, in the presence of certain contraindications for other agents\u2014such as a recent history of lymphoma, latent tuberculosis (TB) with contraindications to the use of chemoprophylaxis, living in a TB-endemic region, or a previous history of demyelinating disease\u2014rituximab may be considered as a first-line biological agent. Some rheumatologists also prioritise this drug in patients with a recent history of any malignancy, because there are no indications that rituximab use is associated with the occurrence of cancers113 114; furthermore, rituximab is the least expensive biological agent at present. Fourth, when speaking of TNF inhibitors, the Task Force listed the presently approved agents, adalimumab, certolizumab pegol, etanercept, golimumab and infliximab, but also decided to mention biosimilars under the proviso that they become approved in the USA and\/or Europe; current data suggest that at least one biosimilar, CT-P13, has a similar efficacy and safety profile to the original antibody, infliximab, in RA and axial spondyloarthritis.115 116 Fifth, the Task Force felt that all bDMARDs should be used preferentially in combination with MTX or other csDMARDs. For neither TNF inhibitors nor rituximab or abatacept has monotherapy been consistently found to be superior to MTX alone, whereas combination therapy has; a dose of 10\u2005mg MTX or more a week appears to be effective and appropriate for use with adalimumab and infliximab63 117 and, until proven otherwise, also with all other TNF inhibitors. Only tocilizumab has been repeatedly demonstrated to be superior as a monotherapy over MTX or other csDMARDs, although the Japanese study had an oplen label design and the MTX dose was low.118 119 In 2010, the Committee explicitly mentioned that a three-arm trial in early RA is needed to gain full insight, and, most recently, these data became available, albeit only in abstract form,120 revealing that only in combination with MTX tocilizumab (8\u2005mg\/kg) showed consistent significant superiority over MTX with regard to clinical, functional and structural outcomes. The other arms, tocilizumab monotherapy (8\u2005mg\/kg) and tocilizumab (4\u2005mg\/kg) in combination with MTX, showed superiority mainly in reaching the primary clinical end point (DAS28\u2013erythrocyte sedimentation rate \\<2.6, a measure confounded by placing a high weight on acute phase reactant levels121), with only numeric differences in most of the clinical and functional secondary end points, most of which did not reach statistical significance. On the other hand, a head-to-head trial in patients with established RA who stopped MTX therapy revealed tocilizumab monotherapy to be superior to adalimumab monotherapy in most (though not all) endpoints.36 Thus, if biological monotherapy must be initiated, tocilizumab has some supportive evidence, but taken together, the data strongly support the use of all biological agents in combination with MTX. While clinical response is usually maintained even on withdrawal of MTX in patients receiving established therapy with MTX plus tocilizumab or a TNF inhibitor (and therefore presumably also other bDMARDs),122\u2013124 there is rarely a reason to withdraw MTX, since, with established therapy, it is usually well tolerated. Also monotherapy is not a major recommendation by the Task Force, which clearly preferred maintenance of combination therapy. Finally, it is important to note that the Task Force agreed to abandon former recommendation No 14: 'DMARD-na\u00efve patients with poor prognostic markers might be considered for combination therapy of MTX plus a biological'. In 2010 it was already stated that early use of a biological agent should only be considered in exceptional patients; however, as it stood, this statement could have been misinterpreted as advocating use of biological agents even before an initial csDMARD strategy had failed. With the current decision, the use of bDMARDs before trying a csDMARD approach is even more strongly discouraged than signified by the 2010 recommendation. The majority of the current Committee members felt that using a treat-to-target strategy that gave patients the initial opportunity to respond to treatment in line with items 4, 5 and 7 still provides the option of adding a biological agent within 6\u2005months\u2014and thus quite early in the disease course or therapeutic chronology\u2014if the treatment target was not reached. This approach was supported by several recent clinical trials.29 88 125 126 Although bDMARDs in comparison with csDMARDs in early disease confer a significant structural benefit, this benefit was deemed to be neither sufficiently large nor sufficiently frequent, provided that a treat-to-target strategy with treatment adaptations within 6\u2005months was adhered to.125\u2013127 This decision was probably the most heavily debated one: an initial ballot revealed a 94% majority for deleting the old recommendation No 14; subsequently, after thorough discussions, a re-voting process was solicited, but only 33% of the members voted for re-inclusion of old recommendation No 14. In this context it should be clarified that the Task Force was fully aware of the data in patients with early RA who have significantly higher response rates with bDMARDs combined with MTX than MTX alone; however, embedded within this responder population are good responders to MTX monotherapy who experience about 25% (or more) ACR70 improvement (corresponding to a low disease activity state including remission) across studies without any additional treatment adaptations being allowed over a 1-year trial period; in clinical practice, many of these patients would have been overtreated if they had received biological agents instead, placing them at a potentially increased risk of serious adverse events and incurring high costs. Also, it must be borne in mind that the BeSt study showed that csDMARDs plus glucocorticoids led to very similar initial clinical, functional and structural results as with bDMARDs,97 and the IMPROVED trial showed a very high rate of good outcomes using MTX plus low-dose glucocorticoids within a few months.60 Nevertheless, there may still be an exceptional patient in whom the use of a bDMARD in combination with MTX as the first DMARD strategy is considered appropriate. One can imagine patients whose professional or family life entirely depends on a rapid complete resolution of the joint symptoms and where glucocorticoids as adjunctive therapy with csDMARDs have failed or cannot be used because of contraindications. In such cases, the exceptional use of a biological agent is not precluded now that we have dismissed this recommendation from its former place in the table, but the prominence that could be misinterpreted has now been removed. Importantly, however, there are studies suggesting that early induction therapy with anti-TNF plus MTX may result in good outcomes even after withdrawal of the biological agent.128 129 Nevertheless, another study, although partly confirming these results, resulted in a relatively high relapse rate on withdrawal of the TNF inhibitor.130 Thus, more data are needed to support induction therapy, but further evidence confirming the maintenance of remission after withdrawal of the biological agent may lead to a paradigm change in our approach to treating RA (see item 12).\n\n10. *If a first bDMARD has failed, patients should be treated with another bDMARD; if a first TNF inhibitor therapy has failed, patients may receive another TNF inhibitor or a biological agent with another mode of action.* A consequence of item 9, recommendation 10 simply states that once the treatment target has not been reached with an initial biological therapy, other bDMARDs should be used; no preference is stated. The second part of this recommendation focusing on patients who have initially received a TNF inhibitor may seem somewhat redundant. However, it has two purposes: (i) to express the conclusion of the Task Force that current evidence does not suggest any one agent to be better than another TNF inhibitor when active disease prevails despite initial treatment with a TNF blocker; (ii) over the next few years, new biological agents targeting the IL-6 receptor (sarilumab) or IL-6 (clazakizumab, sirukumab) may become available131\u2013133; without specific note on the options after failure of an initial TNF inhibitor therapy, one could infer that potentially approved new IL-6 inhibitors might be used after failure of tocilizumab, but in contrast with TNF inhibition, the efficacy of such an approach is currently unknown for IL-6 inhibition (or costimulation blockers or rituximab). Of note, with biosimilars approaching, it is self-evident that an infliximab biosimilar cannot be regarded as 'another TNF inhibitor' in patients with an insufficient response to infliximab. This recommendation was voted for by 97% of the members.\n\n11. *Tofacitinib may be considered after biological treatment has failed.* Tofacitinib, a JAK inhibitor, was approved for the treatment of RA in the USA, Japan and Russia at the time of the Task Force's meeting on 9 April 2013. For reasons stated above, an a priori decision had been made to address tofacitinib in the recommendations based on evidence of efficacy and safety available from the literature and accrued in the course of the respective SLR.13 Tofacitinib is not a bDMARD, but a synthetic chemical compound. It is a targeted molecule interfering with specific signal-transduction pathways and thus could not be subsumed within the term 'conventional synthetic DMARDs'. Therefore the Task Force decided to address its use in a separate recommendation as a tsDMARD, rather than as part of csDMARDs or bDMARDs.2 The evidence from published papers and abstracts has convinced the Committee that tofacitinib is sufficiently efficacious in improving clinical, functional and structural outcomes to be considered a DMARD.134\u2013136 The fact that the 5\u2005mg dose approved in the USA and Japan just misses statistical significance for inhibition of joint damage progression compared with placebo at 12\u2005months (p=0.06)137 did not preclude the Task Force from recognising its structural efficacy, given significant radiological differences at this dose in another trial.138 However, little is currently known about its long-term safety. Data from clinical trials reveal a numerical increase in serious infection rates compared with controls; herpes zoster infections in particular appear to be more common than seen with TNF inhibitors;134 139 several cases of TB and non-TB opportunistic infections have been reported; lymphocytopenia and anaemia also occur, and haemoglobin levels appear to increase less upon clinical improvement than seen with csDMARDs and bDMARDs. In light of the many available csDMARDs and bDMARDs that have long-standing clinical experience data, the Task Force felt that tofacitinib should primarily be used when bDMARDs have been insufficiently effective, even though it is already approved in the USA, Japan, Russia and meanwhile Switzerland for use after failure of csDMARDs. Indeed, the discussion initially focused on whether tofacitinib should only be recommended for use only after failure with two bDMARDs with different modes of action, but ultimately it was decided to just reflect this discussion item in the accompanying text and not in the recommendation. More clinical experience and safety data from registries, with a particular focus on serious infections, herpes zoster and malignancies, will be needed before the actual place of tofacitinib in the treatment sequence can be clarified, and at present the Committee did not feel that tofacitinib was safer or more efficacious than rituximab, which, according to currently existing labelling, should be used after TNF inhibitor failure. While the Task Force mainly focused on efficacy and safety, it also considered economic aspects, as supported by GRADE140 and as occurred in the 2010 recommendations when all recommendations were supported by cost-effectiveness data, with the exception of starting biological agents before sDMARDs.8 Given constrained healthcare budgets, the inefficient use of healthcare resources (ie, funding those interventions that are not cost-effective) often results in either a lost opportunity to improve the health of other individuals with cost-effective interventions or increased costs through taxes or insurance premiums. Hence the Committee noticed that the annual cost of tofacitinib in the USA and Switzerland is currently about US\\$25\u2005000 and CHF25\u2005000, respectively, placing it at a similar level to biological agents.141 142 Notably, the first biosimilar has been approved in Europe in the meantime143 144 and is expected to be priced at lower levels than the currently available bDMARDs.31 Therefore, although the Task Force appreciates that tofacitinib is an oral first-in-class drug with a different mechanism of action and is aware of the approval situation in the USA, Japan and other countries, it did not believe it was yet possible to conclude that tofacitinib has a similar safety profile to tocilizumab or other biological agents for which far more person-years of exposure have been accumulated and reported to date. Thus, additional long-term safety data and clinical experience will be needed to determine an overall benefit\/harm ratio. Also, a proper cost-effectiveness analysis would be desirable. Accordingly, the Committee preferred not to recommend tofacitinib after MTX failure as it did for other biological agents. Among the Task Force members, 90% voted in favour of this recommendation as phrased here. Of particular importance, at the time of the Task Force's meeting on 9 April 2013, it was unknown when the EMA would release its decision on the approval of tofacitinib. Thus, the discussions, formulation of recommendation, explanatory stipulations and on-site voting of the Committee occurred before EMA published its first and second negative decision on tofacitinib (with resubmission being planned by the company).145\u2013147 Anonymous voting on the level of agreement (strength of recommendation), however, occurred electronically after the first EMA decision became known and was the lowest of all items (7.6 on a scale of 0\u201310), which may have been influenced by this information.\n\n12. *If a patient is in persistent remission after having tapered glucocorticoids, one can consider tapering bDMARDs, especially if this treatment is combined with a csDMARD.* In contrast with 2010\u2014when a similar recommendation was stated\u2014more evidence is now available and there was unanimous approval within the Task Force. In established RA, the available data suggest that most patients flare upon withdrawal of a TNF inhibitor,148\u2013151 and more profound and persistent responses increase the likelihood of maintenance of a good outcome with csDMARDs even after withdrawal of the bDMARD.150 In the PRESERVE trial, the time frame was at least 4\u2005months.152 However, for early RA, the data are somewhat contradictory. While the primary target in early RA clearly should be stringent remission,33 34 most data on withdrawal of bDMARDs come from patients who are in sustained low disease activity. The OPTIMA trial showed that a 6-month induction regimen with adalimumab plus MTX soon after diagnosis may be sufficient to allow most patients to maintain low disease activity or remission after open label and even after double-blind withdrawal of the TNF inhibitor128 153 154; however, while similar findings on withdrawal of a TNF blocker were obtained in an open label fashion in the HIT HARD study,129 somewhat contradicting data were seen in the PRIZE trial, where dose reduction but not withdrawal of the biological agent was accompanied by maintenance of good outcome.130 Thus, only if further and more broadly confirmed can short-term inclusion of a biological agent in a first DMARD strategy become a true option (see discussion to recommendation No 9). On the other hand, reduction of the TNF inhibitor dose after attainment of DAS28\\<2.6 in early RA allows excellent outcomes to be maintained,130 as also seen in established RA.152 155 While most studies on dose reduction or withdrawal have been performed with TNF blockers, some data on other bDMARDs are emerging with similar overall results,156\u2013158 but clearly more information is needed in this regard. Importantly, before bDMARDs are tapered, glucocorticoids should have been withdrawn in line with point 7. Also of note, reinstitution of bDMARDs appears to allow the good outcome to be recaptured.150 156 159\n\n13. *In cases of sustained long-term remission, cautious reduction of the csDMARD dose could be considered, as a shared decision between patient and physician.* Except for minimal rewording (the term 'titration' is replaced by 'reduction'), this item is identical with point 13 of the 2010 recommendations; it refers solely to those patients in whom glucocorticoids, if used, have already been stopped and\/or who have attained and maintained the targeted therapeutic state on csDMARDs or those in whom bDMARDs have been successfully withdrawn (see above). This recommendation received 100% of the votes. As stated then, it must be borne in mind that stopping csDMARDs in patients with established RA in remission is followed by flares in about 70% of patients, twice as frequently as maintaining therapy irrespective of regimen.160 161 Therefore the focus of this item is on csDMARD reduction rather than cessation. On the other hand, drug-free remission may be an option in patients in whom therapy was initiated very early and who therefore also had achieved remission early in their disease course.162 Of note, in light of the availability of the ACR\u2013EULAR remission definitions, the term 'persistent remission' in this recommendation primarily pertains to these definitions; however, formal DMARD withdrawal studies using them have not yet been performed.\n\n14. *When therapy needs to be adjuusted, factors apart from disease activity, such as progression of structural damage, comorbidities and safety issues, should be taken into account.* Again, this point is essentially identical with the last recommendation of 2010 and was unanimously approved at the Task Force meeting. It is intended to raise awareness that reaching the outcome of low disease activity or remission is not an absolute prerequisite and that it is equally important to account for comorbidities and other contraindications when targeting a good outcome. Conversely, high disease activity is typically associated with comorbidities,163 164 so effective therapeutic intervention may also prevent comorbidity.165\u2013167 Finally, some patients with low disease activity may still develop seriously progressive radiographic joint damage, so after potential lag periods have been accounted for to recognise progression,168 such patients may then need intensification of therapy.\n\nLevels of evidence (LoE), grades of recommendations (GoR), strength of recommendation (SoR; = level of agreement), and % of votes for the respective items as worded\n\n<table>\n<thead>\n<tr>\n<th style=\"text-align: left;\"><\/th>\n<th style=\"text-align: left;\">LoE<\/th>\n<th style=\"text-align: left;\">GoR<\/th>\n<th style=\"text-align: left;\">SoR<\/th>\n<th style=\"text-align: left;\">%<\/th>\n<\/tr>\n<\/thead>\n<tbody>\n<tr>\n<td style=\"text-align: left;\">A.<\/td>\n<td style=\"text-align: left;\">na<\/td>\n<td style=\"text-align: left;\">na<\/td>\n<td style=\"text-align: left;\">9.8\u00b10.9<\/td>\n<td>100<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">B.<\/td>\n<td style=\"text-align: left;\">na<\/td>\n<td style=\"text-align: left;\">na<\/td>\n<td style=\"text-align: left;\">9.8\u00b10.5<\/td>\n<td>100<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">C.<\/td>\n<td style=\"text-align: left;\">na<\/td>\n<td style=\"text-align: left;\">na<\/td>\n<td style=\"text-align: left;\">9.6\u00b10.6<\/td>\n<td>100<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\"><\/td>\n<td style=\"text-align: left;\"><\/td>\n<td style=\"text-align: left;\"><\/td>\n<td style=\"text-align: left;\"><\/td>\n<td><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">1.<\/td>\n<td style=\"text-align: left;\">1a<\/td>\n<td style=\"text-align: left;\">A<\/td>\n<td style=\"text-align: left;\">9.8\u00b10.5<\/td>\n<td>97<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">2.<\/td>\n<td style=\"text-align: left;\">1a<\/td>\n<td style=\"text-align: left;\">A<\/td>\n<td style=\"text-align: left;\">9.6\u00b10.7<\/td>\n<td>100<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">3.<\/td>\n<td style=\"text-align: left;\">2b<\/td>\n<td style=\"text-align: left;\">B<\/td>\n<td style=\"text-align: left;\">9.5\u00b11.0<\/td>\n<td>100<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">4.<\/td>\n<td style=\"text-align: left;\">1a<\/td>\n<td style=\"text-align: left;\">A<\/td>\n<td style=\"text-align: left;\">9.6\u00b10.9<\/td>\n<td>100<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">5.<\/td>\n<td style=\"text-align: left;\">1a<\/td>\n<td style=\"text-align: left;\">A<\/td>\n<td style=\"text-align: left;\">9.0\u00b11.7<\/td>\n<td>87<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">6.<\/td>\n<td style=\"text-align: left;\">1a<\/td>\n<td style=\"text-align: left;\">A<\/td>\n<td style=\"text-align: left;\">9.5\u00b10.8<\/td>\n<td>100<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">7.<\/td>\n<td style=\"text-align: left;\">1a\u2212<\/td>\n<td style=\"text-align: left;\">A<\/td>\n<td style=\"text-align: left;\">8.9\u00b11.2<\/td>\n<td>73<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">8.<\/td>\n<td style=\"text-align: left;\">5<\/td>\n<td style=\"text-align: left;\">D<\/td>\n<td style=\"text-align: left;\">8.9\u00b11.3<\/td>\n<td>100<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">9.<\/td>\n<td style=\"text-align: left;\">1b<\/td>\n<td style=\"text-align: left;\">A<\/td>\n<td style=\"text-align: left;\">9.2\u00b11.2<\/td>\n<td>90<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">10.<\/td>\n<td style=\"text-align: left;\">1a<\/td>\n<td style=\"text-align: left;\">A<\/td>\n<td style=\"text-align: left;\">9.4\u00b10.8<\/td>\n<td>97<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">11.<\/td>\n<td style=\"text-align: left;\">1b*<br \/>\n5\u2020<\/td>\n<td style=\"text-align: left;\">A*<br \/>\nD\u2020<\/td>\n<td style=\"text-align: left;\">7.6\u00b11.8<\/td>\n<td>90<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">12.<\/td>\n<td style=\"text-align: left;\">2b<\/td>\n<td style=\"text-align: left;\">B<\/td>\n<td style=\"text-align: left;\">8.7\u00b11.8<\/td>\n<td>100<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">13.<\/td>\n<td style=\"text-align: left;\">4<\/td>\n<td style=\"text-align: left;\">C<\/td>\n<td style=\"text-align: left;\">8.9\u00b11.0<\/td>\n<td>100<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">14<\/td>\n<td style=\"text-align: left;\">3b<\/td>\n<td style=\"text-align: left;\">C<\/td>\n<td style=\"text-align: left;\">9.7\u00b10.7<\/td>\n<td>100<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\nLoE and GoR are based on the recommendations of the Oxford Centre for Evidence-Based Medicine.\n\n\\*The general statement is evidence based.\n\n\u2020The place in the treatment algorithm is based on expert consensus opinion.\n\nna, not applicable.\n\n# Discussion\n\nThe 2013 update of the EULAR RA management recommendations comprises three overarching principles and 14 recommendations. The overarching principles bring the patient into even closer focus than in 2010 by moving shared decision-making to become principle A.\n\nIn recommendation 2 the Task Force reconfirmed that the therapeutic target was low disease activity (especially in patients with established RA) or remission (especially in patients with early, newly diagnosed RA), although the target may have to be modified in accordance with comorbidities and safety considerations (item 14); importantly, since publication of the 2010 recommendations, ACR and EULAR have provided a new index- and Boolean-based definition of remission, for both clinical trials and practice, and these criteria should be used accordingly.34\n\nCompared with the 2010 recommendations, the current update continues to advocate the efficacy of csDMARDs as monotherapy or combination therapy as the initial DMARD treatment strategy, ideally combined with glucocorticoids, which\u2014as now proposed\u2014should primarily be of low dose (\u226410\u2005mg per day) and applied for only a limited time (6\u2005months; item 7). Intra-articular application of glucocorticoids was not part of the search activities, but is evidently an important aspect in the treatment of RA, especially when there is residual joint activity in patients who do well on DMARD therapy otherwise.169 170\n\nThe Task Force now also regards all currently approved bDMARDs as being similarly effective (with the exception of anakinra) and generally safe for use as an initial biological therapy after csDMARD failure. While the 2010 Task Force had recognised a shortage of longer-term and, especially, registry data on non-TNF inhibitor biological agents, these have now become available to a sufficient extent to warrant this change. However, there is still a need for more observational\/registry data for abatacept, rituximab and tocilizumab; there are still many fewer 'real world' safety data available for these three compounds than for the TNF inhibitors.\n\nFurther, in the 2013 update, the preference to use a bDMARD in combination with csDMARDs, particularly MTX, rather than as monotherapy is reiterated. Moreover, although the 2010 Task Force made clear statements disfavouring biological therapy as part of the initial DMARD strategy, the 2013 Task Force wished to enforce this point and avoid any misunderstanding by abandoning any statement in this respect within the abbreviated recommendation statements. The Task Force had sufficient evidence and was in full agreement that disease-modifying therapy should start with csDMARDs, ideally combined with glucocorticoids, and that, in a treat-to-target approach, patients who do not attain the therapeutic target by 6\u2005months and have poor prognostic markers would benefit to a similar extent from the addition of biological agents as if they had received them from the beginning. This approach prevents overtreatment of a significant proportion of patients who can achieve low disease activity or remission with an initial csDMARD plus glucocorticoid strategy. There was also agreement that the current lack of support for initiating the therapeutic cascade with bDMARDs might fade if the promise of persistent good outcomes following withdrawal of biological agents after a respective induction therapy in early RA is maintained.129 154\n\nThe Task Force now also briefly addressed the use of bsDMARDs. Currently, data are available for one biosimilar infliximab poduct115 which shows similar efficacy and safety profiles to the original biological agent and was placed alongside the other TNF inhibitors in the therapeutic cascade; it is assumed that the price of this biosimilar will be significantly lower. Finally, the Task Force also dealt with the available data for tofacitinib.13 The abundance of efficacy data available has convinced the Task Force that tofacitinib at the twice daily 5\u2005mg dose has clinical, functional and structural efficacy resembling that seen with bDMARDs; this may not be surprising given its pharmacological profile, namely inhibition of the JAK pathway, which is involved, among others, in IL-6 signalling. However, as detailed in the Results section, some safety aspects of tofacitinib are of concern and precluded recommendation of its use before the failure of at least one and preferably two biological agents, since many bDMARDs are currently available on the market and familiar to rheumatologists. In the absence of a cost-effectiveness analysis, the Task Force was also concerned about the remarkably high price in the USA (and meanwhile also in Switzerland).141 142 While the Task Force's major focus was and should be efficacy and safety of available therapies, it did not ignore its own overarching principle C, which includes cost considerations of medications in general and in its own therapeutic recommendations, as evidenced by inclusion of a health economist in the Task Force. Thus, the current recommendation for the first tsDMARD considers its entire net profile (risk\/benefit\/costs); this aspect was also addressed for other therapies, especially boDMARDs, in 2010.3 8 However, it is evident that economic approaches will differ between countries depending on their healthcare systems. This recommendation was voted on while the Task Force had knowledge about the US label and literature on tofacitinib, but before the negative opinion of EMA became known.\n\nFinally, tapering bDMARDs was addressed, as new data had become available (item 12) that suggest that, once low disease activity and, especially, remission is sustained, a dose reduction of bDMARDs will allow maintenance of the good outcome.\n\nThe Task Force did not address issues of immunogenicity reported for some but not other bDMARDs, since the SLRs performed did not reveal any significant differences in efficacy among the different bDMARDs and also since agents that induce drug antibodies were not shown to convey worse outcome than agents that do not.171 In addition, although there are data available that suggest that baseline TNF levels may predict response to TNF inhibitor therapy,150 the Task Force did not focus on predictors of response and regarded this aspect as part of the research agenda.\n\nThus, looking at the major changes in comparison with the 2010 recommendations,3 this update placed combination therapy of csDMARDs at the same level as MTX monotherapy as a first-line DMARD strategy (in addition to its potential use as a second csDMARD strategy after insufficient efficacy of MTX in patients without adverse prognostic markers), all preferably in combination with glucocorticoids. For the use of biological agents as a second-line DMARD strategy in patients with adverse prognostic signs, a preference for TNF inhibitors is no longer maintained, and the use of bDMARDs in combination with csDMARDs is generally advocated. Further, biosimilars and tofacitinib are addressed. Compared with the 2012 update of the ACR management recommendations,172 the EULAR update is of a more general nature and avoids discussing individual case scenarios, focuses less on safety aspects (which are covered in the respective SLR and are widely available in the respective package inserts), addresses glucocorticoids, disregards minocycline, does not advocate the use of biological agents as monotherapy or part of the initial treatment strategy, places tocilizumab at the same level as other biological agents, and also discusses tofacitinib and biosimilars.\n\nOne of the most important aspects in the context of developing recommendations or guidelines is their implementation173 and actual application. Implementation is a multistep procedure, which benefits from the adoption of international recommendations by local societies, as was the case for the 2010 EULAR recommendations.9\u201312 Nevertheless, adoption of therapeutic targets and means to reach these targets in clinical practice have been shown to be far from ideal,174 and, in a very recent analysis of the implementation of guidelines and recommendations across Europe, there was some room for further improvement.175 Thus, it will clearly be a challenge for EULAR to ensure and find ways to monitor whether these updated recommendations are at least considered widely in clinical care.\n\nThe 2013 update of the EULAR recommendations was developed by a Task Force consisting of 33 members from 11 European countries and the USA; among them were four patients, an infectious disease specialist and a health economist. While it may be seen to be a limitation that the rheumatologists of the Task Force came only from Europe and none was from the USA, Japan or other countries, it is important to state that most of these recommendations are based on a large body of evidence and only a few reflect elements of expert opinion (see also <a href=\"#ANNRHEUMDIS2013204573TB2\" data-ref-type=\"table\">table 2<\/a>). Even if these recommendations are regarded to reflect primarily a European view, they can be used as a template for slightly amended versions by other national or international rheumatological societies outside Europe, as has, indeed, been the case with the 2010 recommendations.9 10 The recommendations are intended to assist and inform rheumatologists, patients, hospital managers, representatives of social security agencies, regulatory authorities and government officials. Although some of the medications discussed are not yet licensed at all or in all countries, they are expected to receive this status in due course, and sufficient literature was available to address them accordingly. Importantly, most of the recommendations have a very high level of evidence, received a large majority vote, and have a high strength of recommendation. Nevertheless, some items were developed just by expert opinion or comprise a mixture of high evidence level and expert opinion, and this drove the research agenda presented in <a href=\"#bx1\" data-ref-type=\"boxed-text\">box 1<\/a>. It is rewarding to see that some of the items of the research agenda presented in 2010 (eg, items 2, 4, 6)3 have already been partly or fully addressed and this, indeed, informed the current update.\n\n###### Research agenda\n\n1.  After insufficient response to MTX, is step-up therapy using a combination of csDMARDs as efficacious as step-up therapy using a bDMARD? Such trials should be thoroughly performed by defining an appropriate end point, adhering to the a priori primary end point, and recruiting\/evaluating sufficient numbers of patients in accordance with the original power calculation.\n\n2.  Can triple therapy with MTX, sulfasalazine and hydroxychloroquine be regarded as a treatment with 'three different DMARDs' or is it just a 'single DMARD strategy'?\n\n3.  What is the most successful tapering strategy of glucocorticoids after bridging or longer-term therapy?\n\n4.  What is the balance of benefit\/harm of long-term (\\>6\u2005months) treatment with glucocorticoids at doses up to 10\u2005mg\/day in established RA?\n\n5.  How long can low-dose glucocorticoids be applied with benefit and without causing harm?\n\n6.  How do biological agents plus MTX compare with MTX plus low-dose glucocorticoids in patients with early RA?\n\n7.  Is induction therapy with bDMARDs plus MTX as a first treatment strategy followed by withdrawal of the biological agent after 6\u201312\u2005months as promising an option for abatacept and tocilizumab as it appears to be for TNF inhibitors, and can therefore an induction regimen with bDMARDs plus MTX become a new therapeutic paradigm?\n\n8.  With respect to the efficacy and safety of tofacitinib, can biological agents be safely used after tofacitinib (with or without a washout period) and can tofacitinib be safely and effectively used after abatacept, rituximab and tocilizumab?\n\n9.  How comparable are the different biological agents to each other and to tofacitinib?\n\n10. Are there, aside from rituximab, differences in responsiveness to bDMARDs between seropositive and seronegative patients?\n\n11. Is there a difference between reducing dose and increasing interval when tapering biological agents after the targeted state has been reached?\n\n12. Is it correct that, when patients have not reached the target on MTX, those with risk factors for bad outcome benefit more from the addition of a biological agent than from switching to or addition of csDMARDs?\n\n13. Is it correct that, when patients have not reached the target on MTX, those with no risk of bad outcome benefit equally from switching to or addition of csDMARDs as they would from addition of a biological agent?\n\n14. Can we find common or specific predictors of response to the different biological agents, csDMARDs and tsDMARDs?\n\n15. What are the risk factors that define patients who benefit from a more intensive initial treatment modality?\n\n16. Which factors predict who will be able to successfully withdraw bDMARDs and who not?\n\n17. How big is the difference in clinical, functional and structural efficacy when treatment strategies aiming to achieve remission are compared with those aiming to achieve low disease activity?\n\n18. How can immunogenicity of bDMARDs explain the similarity of clinical trial data observed with both immunogenic and non-immunogenic compounds?\n\n19. How good is patient adherence to biological agents and can lack of adherence be related to loss of efficacy?\n\n20. Is measurement of serum drug and\/or drug antibody levels useful in clinical practice?\n\n21. Which degree of improvement is needed at 3 months to ensure reaching the treatment target at 6 months and beyond?\n\n22. How long should we aim to use concomitant GC therapy in RA?\n\n23. To understand more in detail how the molecular mechanisms of genomic and non-genomic GC actions (and their dose dependency!) mediate the clinically wanted benefits but also the known adverse effects.\n\n24. To improve treatment with conventional GCs (eg, in respect of timing and circadian rhythms) and develop innovative GC or novel GC receptor ligands.\n\n25. To evaluate further possibilities to reduce the (subjective) adverse events of MTX, the anchor drug in treating RA.\n\n26. Long-term safety data in real life (registries) are needed for non-TNF inhibitor biological agents and tofacitinib.\n\n27. Is tocilizumab monotherapy as efficacious and safe as other bDMARDs plus MTX?\n\n28. Can bDMARDs and\/or sDMARDs be safely withdrawn in patients with established disease who have long-standing (\\>6\u2005months) remission according to the ACR\u2013EULAR definition?\n\nACR, American College of Rheumatology; bDMARD, biological DMARD; csDMARD, conventional synthetic DMARD; DMARD, disease-modifying antirheumatic drug; EULAR, European League against Rheumatism; GC, ; MTX, methotrexate; RA, rheumatoid arthritis; TNF, tumour necrosis factor; tsDMARD, targeted synthetic DMARD.\n\nThe 2013 update of the EULAR recommendations provides the current state of thinking in the field of RA management from a mainly European perspective. The updated recommendations comprise the synthesis of available information based primarily on efficacy and safety of the agents addressed, with inclusion of some health economic considerations. They should enable optimal outcomes in our patients. However, a significant proportion of patients may still not reach the desired therapeutic target. Therefore new therapies are still needed and, indeed, are on the horizon. Also, some items will need to be further developed in the context of future research activities. Consequently, we will carefully follow the developments in the field and anticipate that yet another update may be needed in 2\u20133\u2005years. Until then, we hope that the current recommendations will find their way into clinical practice either directly or through national societies that may wish to use them as a framework for development of local guidance documents.\n\n# Supplementary Material\n\n###### Web table\n\nWe would like to thank the European League Against Rheumatism (EULAR) for providing the funds to perform this task.\n\n# References\n\n[^1]: **Handling editor** Francis Berenbaum\n\n[^2]: JSS and RL serve as joint first authors","meta":{"dup_signals":{"dup_doc_count":267,"dup_dump_count":62,"dup_details":{"curated_sources":2,"2023-50":4,"2023-40":8,"2023-23":7,"2023-14":3,"2023-06":5,"2022-49":3,"2022-40":7,"2022-33":8,"2022-27":10,"2022-21":9,"2022-05":3,"2021-49":4,"2021-43":11,"2021-39":7,"2021-31":9,"2021-25":5,"2021-21":3,"2021-17":3,"2021-10":7,"2021-04":7,"2020-50":7,"2020-45":8,"2020-40":10,"2020-34":2,"2020-29":2,"2020-24":1,"2020-16":6,"2020-10":2,"2020-05":4,"2019-51":5,"2019-47":8,"2019-39":3,"2019-35":2,"2019-30":5,"2019-26":3,"2019-18":3,"2019-13":2,"2019-09":1,"2019-04":2,"2018-51":1,"2018-47":5,"2018-39":1,"2018-30":2,"2018-26":1,"2018-22":2,"2018-17":2,"2018-13":3,"2018-05":5,"2017-51":2,"2017-47":3,"2017-43":1,"2017-39":3,"2017-34":4,"2017-26":4,"2017-22":2,"2017-17":1,"2017-09":1,"2024-30":3,"2024-26":6,"2024-22":9,"2024-18":2,"2017-13":3}},"file":"PMC3933074"},"subset":"pubmed_central"}
{"text":"abstract: We present a different strategy for synthesizing the Au-\u03b3-Fe~2~O~3~ bifunctional nanoparticle by using a larger (50\u2009nm) Au nanoparticle as the core surrounded by smaller (10\u2009nm) \u03b3-Fe~2~O~3~ nanoparticles. The synthesis of the composite nanoparticles is quite facile based on a simple redox process whereby Fe^2+^ is used to reduce Au^3+^. The morphology and composition of the product is measured by transmission electron microscopy, X-ray powder diffraction and UV\u2013vis spectroscopy. We demonstrate the utility of these as-prepared Au-\u03b3-Fe~2~O~3~ nanoparticles by showing they can be used to separate proteins in solution. For example, bovine serum is efficiently removed from an aqueous solution with the simple addition of the NPs and application of a small magnet. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis is performed to evaluate the fidelity and efficiency of the protein separation procedure.\nauthor: Yu Song; Ling Tao; Xiangchun Shen\ndate: 2012\ninstitute: 1Department of Pharmacy, Third Affiliated Hospital of Southern Medical University, Guangzhou 510630, China; 2Research Division of Pharmacology, Guiyang Medical College, Guiyang 550004, China\nreferences:\ntitle: Synthesis of new type of Au-magnetic nanocomposite and application for protein separation thereof\n\n# Background\n\nNanoparticles (NPs) containing two completely different elemental compositions (i.e., bifunctional nanomaterials) enable a single particle to have physical properties vastly superior to those made solely from the individual elements. Due to their increased versatility, such bifunctional nanomaterials have enhanced potential for the development of new applications in many different areas, especially in biotechnology. For example, a single composite nanoparticle derived from gold (Au) and iron oxide nanoparticle subunits is quite versatile, having excellent surface chemistry, superior optical characteristics of gold and superparamagnetic properties of iron oxide \\[1-7\\].\n\nCommonly, such Au-maghemite (\u03b3-Fe~2~O~3~) bifunctional nanoparticles have a \u03b3-Fe~2~O~3~ core, either a solid Au shell or smaller Au nanoparticles surrounding the core \\[8,9\\]. We present a different strategy for synthesizing the Au-\u03b3-Fe~2~O~3~ bifunctional nanoparticle by using a larger (50\u2009nm) Au nanoparticle as the core surrounded by smaller (10\u2009nm) \u03b3-Fe~2~O~3~ nanoparticles. The synthesis of the composite nanoparticles is quite facile based on an easy redox process whereby Fe^2+^ was used to reduce Au^3+^. One advantage of this composition is that the size of the bifunctional nanoparticle is easily tuned by changing the size of the Au nanoparticle core while still maintaining a strong magnetic response since a significant amount of magnetic material composes the single particle.\n\nWe demonstrate the utility of these as-prepared Au-\u03b3-Fe~2~O~3~ nanoparticles by showing they can be used to separate proteins in solution. For example, bovine serum is efficiently removed from an aqueous solution with the simple addition of the NPs and application of an external magnetic field. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is performed to evaluate the fidelity and efficiency of the protein separation procedure.\n\n# Methods\n\nAll chemicals were purchased from Sigma-Aldrich Corporation (MO, USA) and used as received without further purification. Deionized water was used throughout. The TEM images were taken using a JEOL 2000EX transmission electron microscope (JEOL Ltd., Tokyo, Japan) at an accelerating voltage of 200 kv. The UV\u2013vis spectra were taken by Lambda 950 UV\u2013vis spectrometer (PerkinElmer, MA, USA).\n\nIn this method, magnetite nanoparticles were prepared first in water solvent by the chemical precipitation method. Then, gold precursor was added in the solution and reduced by Fe^2+^ which was oxidized to Fe^3+^. Magnetite change to maghemite and attach on gold nanoparticles (as shown in Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>). In a typical process, a mixture of 0.1\u2009mmol of FeCl~2~\u00b73H~2~O, 0.2\u2009mmol of FeCl~3~\u00b76H~2~O and 0.1\u2009mmol D-lysine in 50\u2009ml deionized water was stirred and bubbled with N~2~ for 30\u2009min, and then, 0.6\u2009ml of 5\u2009N ammonium was added in the mixture under N~2~ protection. The color of the mixture changed to black immediately while the mixture was continually stirred for another 30\u2009min and added with 2\u2009ml of 0.05\u2009M HAuCl~4~ aqueous solution drop by drop into the black mixture. The color changed to purple black slowly while on continuous stirring for 60\u2009min. Separated by external magnate, the liquid was almost colorless, and the paste was purple black which was washed by deionized water and separated by external magnate three times. The final product, which was purple black solution, was redispersed in deionized water for further measurement. Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a> shows the procedure of formation of maghemite-gold bifunctional nanomaterials. Inset photo is the final product in water, which shows the purple-black solution.\n\nProtein separation is one of the basic applications of this kind of bifunctional nanomaterials. To use these materials for protein separation, the sample was washed several times to make sure there is no free gold nanoparticle in the solution, and bovine serum solution was added in the sample. Then, by using external magnetic separation, the sample was divided to liquid and paste, which was redispersed in water, for SDS-PAGE electrophoresis gel stained by Coomassie blue. Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a> shows the mechanism for protein separation by the bifunctional nanocomposite.\n\n# Results and discussion\n\nTo compare with the bifunctional nanocomposite, gold (50\u2009nm) and magnetite (10\u2009nm) nanoparticles were synthesized and characterized separately. From TEM images and AFM image (Figure <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>), the morphology of Fe~3~O~4~ (magnetite) and Au nanoparticle is shown clearly. After the addition of gold precursor to the magnetite solution, the structure of 50-nm gold nanoparticle surrounded by 10-nm maghemite nanoparticles was formed (Figure <a href=\"#F4\" data-ref-type=\"fig\">4<\/a>). Due to this kind of structure, the UV peak for gold shifted to 565\u2009nm.\n\nThe composition of the synthesized materials was identified by XRD, as shown in Figure <a href=\"#F5\" data-ref-type=\"fig\">5<\/a>. The left pattern is the data for magnetite (Fe~3~O~4~) (black line in left figure), which is before the addition of Au precursor, and the right pattern is the data for Au-\u03b3-Fe~2~O~3~ composite (black line in right figure). The results match the data in the 2003 JCPDS-International Centre for Diffraction Data for magnetite (89\u20130951) (red line in left figure), maghemite (89\u20135894) (blue line in right figure) and gold (89\u20133697) (red line in right figure). These results can provide two facts: first is after magnetic separation; the gold is still in the sample. Second is that magnetite was reduced to maghemite.\n\nTo test these materials for protein separation, bovine serum solution was added in the sample. Then, by using external magnetic separation, the sample was divided to liquid and paste which was redispersed in water for SDS-PAGE electrophoresis gel.\n\nThe Bradford protein assay protocol (Coomassie Blue G-250) was used as an instant method to examine for the existence of protein in those two parts of sample \\[10\\]. If a certain part contains proteins, it will turn the originally brown Coomassie Blue G-250 solution into a blue color, while the part without protein in it will leaves Coomassie Blue G-250 brown. As shown in the above photos of Figure <a href=\"#F6\" data-ref-type=\"fig\">6<\/a>, sample 1 is the original sample solution which was before the magnetic separation. It contained protein since the color of Coomassie Blue solution was turned into blue. Sample 2 is the supernatant part after magnetic separation. It contained no protein since the color of Coomassie Blue solution remained brown. Sample 3 is the solid part after magnetic separation which was redissolved in buffer. It contains proteins since it turned the solution into blue.\n\nSodium dodecyl sulfate-\u2009polyacrylamide gel electrophoresis was then applied according to the literature \\[11\\]. As shown in Figure <a href=\"#F6\" data-ref-type=\"fig\">6<\/a>, compared to the marker in lane 0, the original mixture solution (as shown in lane 1) contained proteins of various molecular sizes. After magnetic separation, those proteins were removed from the solution (lane 2). For the solid sample after separation, the proteins were obtained again (lane 3), so the protein was effectively separated and collected.\n\n# Conclusions\n\nWe reported the synthesis of gold-maghemite nanoparticles and their use in separating proteins. The as-prepared nanocomposites combined the merits of both gold and magnetic nanoparticles, and were produced by a very easy method. Furthermore, this experiment has also suggested a new way to synthesize various bifunctional or multifunctional composite nanomaterials through simple redox process.\n\n# Competing interests\n\nThe authors declare that they have no competing interests.\n\n# Authors' contributions\n\nYS carried out the synthesis, TEM and XRD measurements. LT carried out Bradford protein assay and SDS\u2009\u2212\u2009PAGE, and participated in the design of the study. XS conceived of the study, participated in its design and coordination, and drafted the manuscript. All authors read and approved the final manuscript.\n\n# Authors' information\n\nYS is a an MD and a pharmacist-in-charge. YS's research areas are pharmaceutics and Chinese medicine pharmacology, and is affiliated to Department of Pharmacy, Third Affiliated Hospital of Southern Medical University, Guangzhou, 510630, China. LT is an MD whose research is on pharmaceutics and Chinese medicine pharmacology, and is affiliated to the Research Division of Pharmacology, Guiyang Medical College, Guiyang, 550004, China. XS is also an MD and PI whose research areas are on nanotechnology, pharmaceutics and Chinese medicine pharmacology. XS is affiliated to the Research Division of Pharmacology, Guiyang Medical College, Guiyang, 550004, China.\n\n## Acknowledgments\n\nThis research is funded by the National Natural Science Foundation of China (grant no.: 30701024), Guizhou Province Special Assistant to Funding High-Level Talent (TZJF-2006-13), Guizhou International Technology Cooperation Fund (grant no.: G \\[2009\\] 700115); and the Provincial Key Technologies R&D Program of Guizhou (grant no.: S \\[2011\\] 3010).","meta":{"dup_signals":{"dup_doc_count":111,"dup_dump_count":24,"dup_details":{"curated_sources":2,"2022-40":1,"2020-29":1,"2019-51":1,"2015-48":4,"2015-40":4,"2015-35":5,"2015-32":2,"2015-27":5,"2015-22":5,"2015-14":5,"2014-52":4,"2014-49":8,"2014-42":12,"2014-41":6,"2014-35":4,"2014-23":5,"2014-15":7,"2023-40":1,"2015-18":5,"2015-11":5,"2015-06":4,"2014-10":5,"2013-48":5,"2013-20":5}},"file":"PMC3423036"},"subset":"pubmed_central"}
{"text":"date: 2019\ntitle: WHO RELEASES FIRST GUIDELINE ON DIGITAL HEALTH INTERVENTIONS\n\n**17 April 2019** - WHO today released new recommendations on 10 ways that countries can use digital health technology, accessible via mobile phones, tablets and computers, to improve people's health and essential services.\n\n\"Harnessing the power of digital technologies is essential for achieving universal health coverage,\" says WHO Director-General Dr Tedros Adhanom Ghebreyesus. \"Ultimately, digital technologies are not ends in themselves; they are vital tools to promote health, keep the world safe, and serve the vulnerable.\"\n\nOver the past two years, WHO systematically reviewed evidence on digital technologies and consulted with experts from around the world to produce recommendations on some key ways such tools may be used for maximum impact on health systems and people's health.\n\nOne digital intervention already having positive effects in some areas is sending reminders to pregnant women to attend antenatal care appointments and having children return for vaccinations. Other digital approaches reviewed include decision-support tools to guide health workers as they provide care; and enabling individuals and health workers to communicate and consult on health issues from across different locations.\n\n\"The use of digital technologies offers new opportunities to improve people's health,\" says Dr Soumya Swaminathan, Chief Scientist at WHO. \"But the evidence also highlights challenges in the impact of some interventions.\"\n\nShe adds: \"If digital technologies are to be sustained and integrated into health systems, they must be able to demonstrate long-term improvements over the traditional ways of delivering health services.\"\n\nFor example, the guideline points to the potential to improve stock management. Digital technologies enable health workers to communicate more efficiently on the status of commodity stocks and gaps. However, notification alone is not enough to improve commodity management; health systems also must respond and take action in a timely manner for replenishing needed commodities.\n\n\"Digital interventions, depend heavily on the context and ensuring appropriate design,\" warns Dr Garrett Mehl, WHO scientist in digital innovations and research. \"This includes structural issues in the settings where they are being used, available infrastructure, the health needs they are trying to address, and the ease of use of the technology itself.\"\n\n**Digital health interventions are not sufficient on their own**\n\nThe guideline demonstrates that health systems need to respond to the increased visibility and availability of information. People also must be assured that their own data is safe and that they are not being put at risk because they have accessed information on sensitive health topics, such as sexual and reproductive health issues.\n\nHealth workers need adequate training to boost their motivation to transition to this new way of working and need to use the technology easily. The guideline stresses the importance of providing supportive environments for training, dealing with unstable infrastructure, as well as policies to protect privacy of individuals, and governance and coordination to ensure these tools are not fragmented across the health system.\n\nThe guideline encourages policy-makers and implementers to review and adapt to these conditions if they want digital tools to drive tangible changes and provides guidance on taking privacy considerations on access to patient data.\n\n\"Digital health is not a silver bullet,\" says Bernardo Mariano, WHO's Chief Information Officer. \"WHO is working to make sure it's used as effectively as possible. This means ensuring that it adds value to the health workers and individuals using these technologies, takes into account the infrastructural limitations, and that there is proper coordination.\"\n\nThe guideline also makes recommendations about telemedicine, which allows people living in remote locations to obtain health services by using mobile phones, web portals, or other digital tools. WHO points out that this is a valuable complement to face-to-face-interactions, but it cannot replace them entirely. It is also important that consultations are conducted by qualified health workers and that the privacy of individuals' health information is maintained.\n\nThe guideline emphasizes the importance of reaching vulnerable populations, and ensuring that digital health does not endanger them in any way.\n\n**WHO's work on digital health**\n\nThis guideline represents the first of many explorations into the use of digital technologies and has only covered a fraction of the many aspects of digital health.\n\nIn 2018, governments unanimously adopted a World Health Assembly resolution calling on WHO to develop a global strategy on digital health to support national efforts to achieve universal health coverage. That strategy is scheduled to be considered at the World Health Assembly in 2020.\n\nAlthough WHO is expanding its focus on digital health, the Organization has been working in this area for years, for example, through the development of the eHealth Strategy Toolkit in 2012, published in collaboration with International Telecommunications Union (ITU).\n\nTo support governments in monitoring and coordination of digital investments in their country, WHO has developed the Digital Health Atlas, an online global repository where implementers can register their digital health activities. WHO has also established innovative partnerships with the ITU, such as the BeHe@lthy, BeMobile initiative for the prevention and control of noncommunicable diseases, as well as efforts for building digital health capacity through the WHO Regional Office for Africa.\n\nOver the years, WHO has released a number of resources to strengthen digital health research and implementation, including the mHealth Assessment and Planning for Scale (MAPS) toolkit, a handbook for Monitoring and Evaluation of Digital Health, and mechanisms to harness digital health to end TB.\n\nOn 6 March 2019, Dr Tedros announced the creation of the Department of Digital Health to enhance WHO's role in assessing digital technologies and support Member States in prioritizing, integrating and regulating them.\n\nAvailable from: <https:\/\/www.who.int\/news-room\/detail\/17-04-2019-who-releases-first-guideline-on-digital-health-interventions>","meta":{"dup_signals":{"dup_doc_count":157,"dup_dump_count":41,"dup_details":{"curated_sources":2,"2023-50":4,"2023-40":3,"2023-23":2,"2023-14":3,"2023-06":3,"2022-49":2,"2022-40":4,"2022-33":1,"2022-27":3,"2022-21":3,"2022-05":3,"2021-43":4,"2021-39":3,"2021-31":4,"2021-25":2,"2021-21":1,"2021-17":6,"2021-10":1,"2021-04":2,"2020-50":4,"2020-45":5,"2020-40":3,"2020-34":3,"2020-29":2,"2020-24":7,"2020-16":6,"2020-10":3,"2020-05":4,"2019-47":3,"2019-43":7,"2019-39":6,"2019-35":8,"2019-30":7,"2019-26":3,"2019-22":17,"2019-18":2,"2024-26":5,"2024-22":1,"2024-18":2,"2024-10":2,"2024-30":1}},"file":"PMC6535172"},"subset":"pubmed_central"}
{"text":"abstract: In undifferentiated Drosophila cells, differentiation-associated genes have monovalent, not bivalent histone modifications, in contrast to differentiation-associated genes in stem cells.\nauthor: Qiang Gan; Dustin E Schones; Suk Ho Eun; Gang Wei; Kairong Cui; Keji Zhao; Xin Chen\ndate: 2010\ninstitute: 1Department of Biology, The Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA; 2Laboratory of Molecular Immunology, National Heart, Lung and Blood Institute, National Institutes of Health, 10 Center Drive, Building 10, Bethesda, MD 20892, USA\nreferences:\ntitle: Monovalent and unpoised status of most genes in undifferentiated cell-enriched *Drosophila* testis\n\n# Background\n\nExtensive studies indicate that embryonic stem cells (ESCs), lineage-committed adult stem cells and early progenitor cells maintain their identities by a unique transcriptional network and chromatin structure (reviewed by \\[1,2\\]). In particular, the bivalent domains harboring both the active H3K4me3 and repressive H3K27me3 marks label developmental regulators \\[3\\]. The H3K4me3 and H3K27me3 marks are placed by the Trithorax group (TrxG) complex \\[4-6\\] and the Polycomb group (PcG) complex \\[7,8\\], respectively. Increasing evidence indicates that the PcG and the TrxG complexes play critical roles in the choice between the proliferating progenitor cell state and terminal differentiation program \\[4,9\\]. It has been reported that bivalent genes in ESCs or early progenitor cells are bound by PcG proteins and are maintained at a 'poised' status by recruitment of RNA Polymerase II (Pol II), in preparation for lineage-specific expression upon differentiation \\[10-12\\]. In various stem cell lineages, reversal of repression by the PcG silencing machinery may act as the first step toward robust activation of terminal differentiation genes \\[13,14\\].\n\nThe *Drosophila* male germline stem cell (GSC) lineage is a naturally existing adult stem cell system and has emerged as an excellent system for studying the molecular mechanisms that control stem cell maintenance versus differentiation \\[15\\]. Each GSC divides asymmetrically to self-renew and give rise to a gonialblast, the daughter cell that first undergoes a transit-amplifying spermatogonial stage before converting to differentiating spermatocytes \\[16\\]. The maintenance of GSCs and spermatogonia in an undifferentiated and proliferative state, as well as the subsequent reversal of these controls to allow terminal differentiation, are both critical to continuous production of gametes throughout lifetime. Despite extensive genetic studies on maintenance of GSCs, it is unclear how epigenetic mechanisms may establish and maintain a unique chromatin structure for their undifferentiated status; and how mis-regulation of such a structure may lead to mis-determination of their fate \\[14\\].\n\nPrevious studies in this system have shown that PcG transcriptional silencing proteins repress the genes required for terminal differentiation in undifferentiated germ cells. Developmental programs reverse Polycomb silencing and activate the expression of differentiation genes in spermatocytes \\[17\\]. This work uncovered an intriguing parallel between *Drosophila* GSC and ESC lineages, because PcG proteins play an extensive role in keeping developmental regulators at a silent status in murine and human ESCs \\[10,11\\]. To investigate whether other features in mammalian ESCs apply to *Drosophila*, we studied the chromatin structure in the undifferentiated-cell-enriched *Drosophila* testis. Our results revealed two distinct features in this tissue: a monovalent chromatin signature and lack of paused RNA Pol II at the differentiation genes. Both features are different from what have been reported for ESCs and other mammalian adult stem cells, suggesting a potential novel mechanism of regulating the germ cell differentiation program in *Drosophila* testis.\n\n# Results and discussion\n\n## Summary of the ChIP-seq results in undifferentiated-cell-enriched *Drosophila* testis\n\nSince it is unfeasible to obtain a sufficient number of naturally existing GSCs for epigenomic mapping using current chromatin immunoprecipitation (ChIP) techniques, we took advantage of the *bag of marbles* (*bam*) mutant strain. Mutations in the *bam* gene inhibit the transition from spermatogonial progenitor stage to differentiating spermatocytes, which results in an accumulation of undifferentiated cells, including GSCs, transit-amplifying spermatogonial cells, as well as somatic cells \\[18,19\\]. Although the *bam* testes are not a pure source of GSCs, they are enriched with undifferentiated germ cells and have been used for transcription profiling by microarray \\[20\\] or RNA-seq \\[21\\] to search for undifferentiated-cell-enriched genes. These analyses further confirmed the undifferentiated status of cells in *bam* testes, in which the expression of meiotic and terminal differentiation genes was extremely low or undetectable.\n\nTo investigate the epigenetic mechanisms that regulate the male germline cellular differentiation program, we used *bam* testes for chromatin landscape mapping using ChIP followed by high-throughput sequencing (ChIP-seq). The ChIP-seq technique has been demonstrated to be a highly sensitive method to detect binding sites of chromatin-associated proteins at a genome-wide coverage (reviewed by \\[22,23\\]). More importantly, this technique is compatible with small amounts of starting material, such as the hand-dissected fly testes in our experiments. To compare our data with the previous results using mammalian cells, we used the same set of antibodies, including antibodies against RNA Pol II, H3K4me3, H3K36me3, H3K27me3 and unmodified histone H3, to perform ChIP-seq experiments using *bam* testes (Materials and methods; Additional file <a href=\"#S1\" data-ref-type=\"supplementary-material\">1<\/a>). The anti-Pol II antibody (4H8) used in our assays recognizes both the initiating and elongating forms of Pol II according to published work \\[12,24\\]. Sequencing depth analysis indicated that we have reached or almost reached the plateau of peak detection for each of these histone modifications as well as Pol II (Materials and methods; Additional file <a href=\"#S2\" data-ref-type=\"supplementary-material\">2<\/a>a-d).\n\nTo validate our ChIP-seq data, we compared the relationship between gene expression level and enrichment of RNA Pol II as well as distinct histone modifications. The *Drosophila* genome is highly compact, including many overlapping genes and genes with transcriptional start sites (TSSs) within a short distance of one another, complicating interpretations of ChIP-seq results. To avoid this, we classified 9,459 annotated genes that are applicable for ChIP-seq analysis into four groups (Materials and methods) according to their expression levels determined by RNA-seq in RPKM values \\[21,25,26\\] (Figure <a href=\"#F1\" data-ref-type=\"fig\">1a<\/a>; RPKM is sequencing reads per kilobase of exon per million mapped reads \\[26\\]). Our data demonstrated that both RNA Pol II and H3K4me3 were highly elevated near the TSSs of annotated genes. They also positively correlated with gene expression level (Figure <a href=\"#F1\" data-ref-type=\"fig\">1b, c<\/a>). And H3K36me3 was enriched downstream of the TSSs and also positively correlated with expression level (Figure <a href=\"#F1\" data-ref-type=\"fig\">1d<\/a>). In contrast, H3K27me3 was negatively correlated with gene expression, as expected for a repressive mark (Figure <a href=\"#F1\" data-ref-type=\"fig\">1e<\/a>). These results are consistent with observations in human T cells \\[27\\] and mammalian ESCs \\[28\\].\n\n## Monovalent chromatin signature is prevalent in undifferentiated-cell-enriched *bam* testis\n\nThe coexistence of active H3K4me3 and repressive H3K27me3 marks is detected in a large number of developmental regulator genes in mammalian ESCs \\[3,29,30\\] and adult stem\/progenitor cells \\[31,32\\]. These 'bivalent' modifications are proposed to poise genes for rapid induction during cellular differentiation to become different cell lineages. However, the mechanisms that contribute to the bivalency of genes may be diverse under different circumstances, and still remain unsolved at the structural level \\[33\\].\n\nTo test whether bivalency also applies to the undifferentiated cells of *Drosophila* testis, we first examined several critical genes that regulate cellular differentiation in the GSC lineage. The *Enhancer of Zeste* (*E(z)*) gene encodes a PcG complex component that is highly expressed in undifferentiated cells, but abruptly down-regulated upon differentiation initiation in spermatocytes. The co-existence of H3K27me3 and H3K4me3 was not observed at the *E(z)* gene locus near the TSS. Instead, we found only the active H3K4me3 mark at the *E(z)* gene locus, enriched near the TSS (Materials and methods; Figure <a href=\"#F2\" data-ref-type=\"fig\">2a, b<\/a>). The *spermatocyte arrest* (*sa*) gene, which encodes a testis-specific homolog of TBP associated factor (tTAF), is turned on in spermatocytes. The *tTAF* gene acts to antagonize PcG repression and regulate a cell-type-specific transcription program for terminal differentiation \\[17,34\\] (Figure <a href=\"#F2\" data-ref-type=\"fig\">2b<\/a>). In contrast to the *E(z)* gene, we found that the chromatin of the *sa* gene locus was only associated with the repressive H3K27me3 mark near its TSS in *bam* testis (Figure <a href=\"#F2\" data-ref-type=\"fig\">2c<\/a>). More examples of the histone modification patterns of differentially expressed genes are shown in Additional file <a href=\"#S3\" data-ref-type=\"supplementary-material\">3<\/a>. These data suggest that developmental regulator genes in the germline lineage may be associated with a 'monovalent' modification (either H3K4me3 or H3K27me3 mark) in undifferentiated cells of *bam* testis.\n\nTo test the prevalence of monovalency at a genome-wide scale, we analyzed all 9,459 annotated genes that are applicable for ChIP-seq analysis (Materials and methods). We identified 3,360 genes that were enriched with a significant level of active H3K4me3 and 1,631 genes enriched significantly with repressive H3K27me3 (*P* \\< 0.05; Materials and methods). Surprisingly, only 91 genes were associated with both H3K4me3 and H3K27me3 marks (Figure <a href=\"#F2\" data-ref-type=\"fig\">2d<\/a>).\n\nAlthough we could validate several bivalent genes using independent ChIP followed by real-time PCR analysis (Additional file <a href=\"#S4\" data-ref-type=\"supplementary-material\">4<\/a>), we found that, different from the silent status of bivalent genes in ESCs \\[3\\], 56% of the bivalent genes in *bam* testis were actively expressed (Additional file <a href=\"#S5\" data-ref-type=\"supplementary-material\">5<\/a>; the RPKM cutoff for expressed genes is based on Additional file <a href=\"#S6\" data-ref-type=\"supplementary-material\">6<\/a>), indicating that the bivalency may be a result of temporally and\/or spatially regulated gene expression, as has been reported in *Xenopus* embryos \\[35,36\\]. One particular example is the *benign gonial cell neoplasm* (*bgcn*) gene (Additional file <a href=\"#S4\" data-ref-type=\"supplementary-material\">4<\/a>b), whose product acts with the Bam protein in the transition of germ cells from the proliferative to the differentiating stage \\[18\\]. We reasoned that the bivalency at the *bgcn* gene may have resulted from mixed cell types in *bam* testes. The active status of *bgcn* in spermatogonial cells is likely associated with the enriched active H3K4me3 mark, whereas its repressive status in GSCs and somatic cells may contribute to the detection of the repressive H3K27me3 mark. Consistent with this, we found that none of the 91 putative bivalent genes we identified in *bam* testis retained their bivalency in the cultured *Drosophila* cells, including S2, BG3 and D23 cell lines \\[37\\] (Additional files <a href=\"#S5\" data-ref-type=\"supplementary-material\">5<\/a> and <a href=\"#S7\" data-ref-type=\"supplementary-material\">7<\/a>; Materials and methods). Although the culture cells were not synchronized, they were more pure than tissues with mixed types and staged cells. In addition, we performed Gene Ontology analysis to search for significantly enriched gene categories in both molecular function and biological process terms (Materials and methods). Surprisingly the only category that is significantly enriched (*P* \\< 0.01) for these 91 putative bivalent genes is the 'multicellular organismal process' category, further suggesting that the apparent bivalency may come from different cells and is not specifically involved in cellular differentiation during spermatogenesis.\n\nTo test the prevalence of monovalency for all differentiation genes in testis, we compared the expression profile of fully differentiated wild-type testis with undifferentiated *bam* testis using the RNA-seq data \\[21\\]. We found 1,894 genes that were silent in the undifferentiated-cell-enriched *bam* testis (RPKM \\< 0.5) but were turned on in the differentiated cells from wild-type testis (RPKM \u2265 1). We defined these genes as differentiation genes (the RPKM cutoffs are based on Additional file <a href=\"#S6\" data-ref-type=\"supplementary-material\">6<\/a>). Among these 1,894 differentiation genes, 1,304 are applicable for ChIP-seq analysis (Materials and methods). Examination of the histone modifications on these 1,304 genes revealed that only 18 genes were associated with both active H3K4me3 and repressive H3K27me3 in *bam* testis (Figure <a href=\"#F2\" data-ref-type=\"fig\">2d<\/a>; Additional file <a href=\"#S5\" data-ref-type=\"supplementary-material\">5<\/a>), whereas 448 were associated with only H3K27me3, 48 were associated with only H4K4me3 and 790 were not associated with either of these modifications. We also checked all genes that are up-regulated at least two-fold in wild-type testis, regardless of their expression level in *bam* testis (RPKM in wild-type testis\/*bam* testis \u2265 2). We found 3,377 genes that fall into this category, including all differentiation genes. Among these 3,377 up-regulated genes, 2,188 are applicable for ChIP-seq analysis. We analyzed the H3K27me3 and H3K4me3 enrichment of these genes and found only 25 genes enriched with both histone marks (Figure <a href=\"#F2\" data-ref-type=\"fig\">2d<\/a>). These results indicate that most differentiation genes in undifferentiated-cell-enriched testis are marked by either a monovalent chromatin signature or no modification (H3K4me3 or H3K27me3), which is very different from the modification patterns in mammalian ESCs \\[3\\] or progenitor cells \\[32\\]. Consistent with our findings, previous studies demonstrated the paucity of bivalent domains in fly embryos, which contain progenitor cells mainly for somatic tissues \\[38\\]. In summary, our results reveal that monovalent modification is a prevalent chromatin signature of differentiation genes in undifferentiated-cell-enriched *Drosophila* testis.\n\n## Most genes are unpoised in the undifferentiated-cell-enriched *bam* testis\n\nFor the purpose of discussion here, we use 'poised' to describe the prepared status of a gene for transcription \\[3,24,39-42\\], which is associated with the promoter proximal binding of RNA Pol II at a 'paused' status \\[43-49\\] and\/or with active histone modification marks \\[12,32\\]. Previous studies in mammalian ESCs \\[12\\] and *Drosophila* embryos \\[39\\] have suggested that many differentiation genes are unexpressed yet have paused RNA Pol II associated with their promoters, in order to stay at a poised state ready for robust transcription upon developmental stimuli. For example, more than 13% of the ectodermal differentiation genes were repressed but have paused RNA Pol II in the *Toll*^*10b*^ mutant *Drosophila* embryos, in which all cells are transformed to the mesodermal fate \\[39\\]. Here we examined whether paused Pol II is also prevalent in the undifferentiated-cell-enriched male gonads. Surprisingly, we found only 63 (4.8%) of the 1,304 differentiation genes were bound by Pol II (*P* \\< 0.05) in undifferentiated-cell-enriched *bam* testis (Figure <a href=\"#F3\" data-ref-type=\"fig\">3a<\/a>). These data indicate that most genes that are silent in undifferentiated cells (RPKM in *bam* testis \\<0.5) but turned on upon differentiation (RPKM in wild-type testis \u2265 1) remained at an unpoised status in undifferentiated cells.\n\nOn the other hand, analyses of the expression levels of all genes with significant Pol II binding (*P* \\< 0.05) demonstrated that approximately 95% of genes (3,796 genes) that are enriched with Pol II (4,008 genes) were actively expressed (RPKM in *bam* testis \u2265 1; Figure <a href=\"#F3\" data-ref-type=\"fig\">3b<\/a>). Only 5% of Pol II-enriched genes (212 genes) were expressed at very low to undetectable levels (RPKM \\<1), which may be candidate poised genes in this cell lineage but only comprised 2% of all genes analyzed (9,459 genes). Interestingly, we found that a subset of these potentially poised genes (43%; 92 genes in Additional file <a href=\"#S4\" data-ref-type=\"supplementary-material\">4<\/a>b) are up-regulated upon differentiation (RPKM in wild-type testis\/*bam* testis \u2265 2), with the significantly (*P* \\< 0.01) enriched ontology category 'transcription regulator activity'. In contrast, 75 genes (35%) remain silent throughout spermatogenesis (RPKM \\<0.5 in both wild-type and *bam* testis). Consistent with their low or undetectable expression (RPKM \\<1) in *bam* testis, we found that only 28 out of the 212 (13.2%) potentially poised genes are enriched with H3K36me3, a transcription elongation epigenetic mark \\[27\\]. Furthermore, we compared these 92 putative poised genes with the 91 potential bivalent genes in *bam* testis. Surprisingly, only two genes (*CG8517* and *CG13611*) overlapped. These data demonstrate that different from what has been reported in mammalian ESC lineages \\[10-12\\], most differentiation genes stay at an unpoised status before exposure to the developmental signals in the undifferentiated-cell-enriched *Drosophila bam* testis.\n\nIn addition to RNA Pol II, H3K4me3 is another well-established mark for active chromatin status, and is associated with many unexpressed but poised genes in ESCs \\[12\\]. In our analysis, we found 95.4% of all H3K4me3-enriched genes (3,360 genes) are actively expressed (3,204 genes in Additional file <a href=\"#S8\" data-ref-type=\"supplementary-material\">8<\/a>). This result further confirmed that most genes in undifferentiated-cell-enriched *Drosophila* testis are either actively expressed or silent, without an intermediate poised status.\n\n## Direct comparison of RNA-seq and microarray data reveals genes with low expression levels may contribute to the paused Pol II phenomenon\n\nOne explanation for the difference in our results from previous publications is that previous analysis of gene expression mainly relied on microarray studies, which may not be sensitive enough to distinguish genes with low expression levels from genes that are absolutely silent \\[50\\]. Indeed, when we compared the gene expression profiles in *bam* testis determined by microarray versus RNA-seq techniques, we found that 670 genes considered as silent based on microarray analysis (three 'absent' calls in all three biological replicates) were actually expressed according to their RPKM values in the RNA-seq data set (RPKM \u2265 1; Figure <a href=\"#F3\" data-ref-type=\"fig\">3c<\/a>). Approximately 24.8% of these genes (107 of the 432 genes that are applicable for ChIP-seq analysis among all 670 genes) had significant enrichment of Pol II (*P* \\< 0.05) at their promoter regions. Therefore, our results suggest that some of the 'poised' genes may actually be expressed but below the detection threshold in microarray analysis.\n\nSince the Pol II antibody we used in our ChIP-seq experiment recognizes both the initiating and elongating (Ser5 phosphorylated) forms \\[12,24\\], we used the Pol II ChIP-seq data to calculate the stalling index for each gene (\\[39\\] and Materials and methods). Using this assay, we identified 695 genes that have paused Pol II at their promoter regions in *bam* testis (Additional file <a href=\"#S9\" data-ref-type=\"supplementary-material\">9<\/a>a; Materials and methods). Ontology analysis revealed significant functional categories (*P* \\< 0.01) for these genes (Additional file <a href=\"#S9\" data-ref-type=\"supplementary-material\">9<\/a>b). However, approximately 93.0% (647 out of 695) of these genes were expressed (RPKM \u2265 1), albeit at relatively low levels (Figure <a href=\"#F3\" data-ref-type=\"fig\">3d<\/a>).\n\n## Do different techniques contribute to the different features of the chromatin landscape?\n\nThere are two major differences between our work and published studies in *Drosophila* or mammalian cells: first, we used direct sequencing-based techniques (ChIP-seq and RNA-seq) instead of hybridization-based techniques (ChIP-chip and microarray); and second, we used a tissue that is enriched with undifferentiated germ cells instead of embryos \\[39\\], embryo-derived cell lines \\[51\\], or mammalian cells \\[12\\], which mainly comprise cells with somatic fate. In order to make a more direct comparison to address whether the difference is due to distinct techniques or different cell types, we performed ChIP-seq and RNA-seq analyses using the same cell type - *Drosophila* S2 cells - that was used in a previous study \\[51\\].\n\nWe analyzed the chromatin status in *Drosophila* S2 cells using antibodies against RNA Pol II, H3K4me3, H3K36me3 and H3K27me3 (Additional file <a href=\"#S10\" data-ref-type=\"supplementary-material\">10<\/a>; Materials and methods). Sequencing depth analysis indicated that most of these assays almost reached (Additional file <a href=\"#S2\" data-ref-type=\"supplementary-material\">2<\/a>e,g) or reached (Additional file <a href=\"#S2\" data-ref-type=\"supplementary-material\">2<\/a>f,h) the detection saturation plateau. For libraries that are not completely saturated, the strongest peaks enriched with the corresponding modified histones or Pol II should be detected first and are therefore identified by our assays. Through analyzing the ChIP-seq data, we identified 3,742 genes with enriched active H3K4me3 and 2,095 genes with enriched repressive H3K27me3 in S2 cells (*P* \\< 0.05; Materials and methods). Among them, we found only 27 genes enriched in both H3K4me3 and H3K27me3 (Figure <a href=\"#F4\" data-ref-type=\"fig\">4a<\/a>; Additional file <a href=\"#S7\" data-ref-type=\"supplementary-material\">7<\/a>), indicating that bivalency is also not a prevalent chromatin signature for genes in S2 cells. To compare the ChIP-seq data with the gene expression level, we profiled the S2 cell transcriptome using the RNA-seq method (Materials and methods). We found that 25 out of the 27 (93%) bivalent genes in non-synchronized S2 cells are actively expressed (RPKM \u2265 1), indicating that the bivalency may be a result of temporally regulated gene expression during cell cycle. Our S2 cell data are consistent with a previous report using fly embryos \\[38\\], where the authors identified 4,893 H3K4me3- and 2,480 H3K27me3-enriched regions, with only 161 overlapping regions. The even fewer bivalent genes in S2 cells (27 genes) than those in *bam* testis (91 genes) may reflect a higher homogeneity of cultured cells than of dissected tissues. We also identified 3,956 genes that have significant binding of RNA Pol II at their promoter region (*P* \\< 0.05), and 5,281 genes that are unbound by Pol II (Figure <a href=\"#F4\" data-ref-type=\"fig\">4b<\/a>). We found that among all the Pol II-enriched genes, 93% are expressed genes (RPKM \u2265 1; Figure <a href=\"#F4\" data-ref-type=\"fig\">4b<\/a>). In contrast, only 260 genes that were expressed at very low to undetectable levels (RPKM \\<1) have enriched Pol II binding at their promoter region; these genes comprise approximately 6.6% of the entire Pol II-enriched genes and 2.7% of the total genes analyzed (9,459 genes). Consistently, more than 99.8% of H3K4me3-enriched genes are actively expressed (3,720 genes out of 3,728 genes with significant enrichment of H3K4me3 have RPKM values \u2265 1; Additional file <a href=\"#S11\" data-ref-type=\"supplementary-material\">11<\/a>). When we compared the gene expression profiles in S2 cells determined by microarray versus RNA-seq techniques, we found that 770 genes considered as silent based on microarray analysis (two 'absent' calls in both biological replicates) were actually expressed according to their RPKM values (RPKM \u2265 1; Figure <a href=\"#F4\" data-ref-type=\"fig\">4c<\/a>) in the RNA-seq data set.\n\nWe next applied the ChIP-seq data using antibodies against RNA Pol II to compute the stalling index of individual genes. Through this assay, we identified 1,821 genes with stalled Pol II in S2 cells (Additional file <a href=\"#S9\" data-ref-type=\"supplementary-material\">9<\/a>a; Materials and methods and \\[39\\]). We compared these 1,821 genes with the genes with promoter-proximal enrichment of polymerase, which were identified in the previous study using S2 cell \\[51\\]. In fact, approximately 86.2% genes with promoter-proximal enrichment of polymerase overlapped with the genes with stalled Pol II identified in our analysis (Additional file <a href=\"#S9\" data-ref-type=\"supplementary-material\">9<\/a>a), indicating that the criteria we used to define genes with stalled Pol II is consistent with previous studies. Ontology assays further confirmed that the significant categories (*P* \\< 0.01) of genes identified using either method are similar (Additional file <a href=\"#S9\" data-ref-type=\"supplementary-material\">9<\/a>c,d). Noticeably, compared to genes with active Pol II, the genes with stalled Pol II have lower expression levels (Figure <a href=\"#F4\" data-ref-type=\"fig\">4d<\/a>), although most (88.5%) of them are actively expressed (RPKM \u2265 1). These results suggest that Pol II binding correlates well with active gene expression status. More sensitive techniques, such as RNA-seq, can accurately detect transcript level for comparison with chromatin structure, in order to understand how epigenetic mechanisms regulate gene expression. To further confirm that our results are not algorithm-specific, we re-analyzed the data with an independent method, the SICER software \\[52\\] (Materials and methods). This software uses different algorithms to identify Pol II or modified histone-enriched genomic regions, regardless of their associations with genes. The SICER software has been used to identify bivalent domains in multipotent hematopoietic stem cell lineages in mammals \\[31,32\\]. Using SICER, we confirmed that most H3K4me3- and H3K27me3-enriched regions are not overlapping (data not shown) and therefore are monovalent in *Drosophila bam* testis and cultured S2 cells. Using SICER, we also confirmed that most genes associated with Pol II-enriched regions in these two samples are actively expressed (data not shown).\n\n## Does cell type specificity dictate chromatin architecture?\n\nOne possible reason for the different chromatin features identified in our data versus previously published mammalian studies is cell type differences. The *bam* testis we used for ChIP-seq analysis is mainly enriched with transit-amplifying spermatogonial cells, and the chromatin differences we found could come from the distinction between transit-amplifying cells and *bona fide* stem cells. Another possibility for such differences between undifferentiated germ cell-enriched testis samples and ESCs is that germ cells are part of a unilineage stem cell system, but ESCs are pluripotent. Thus, in the germline lineage, differentiation genes have a unidirectional switch from silent to activated status during spermatogenesis, whereas developmental genes in ESC lineages have multi-directional switches during lineage-specific differentiation. The bivalent signature of ESC genes may enable more dynamic and refined activation upon perceiving additional signals (Figure <a href=\"#F5\" data-ref-type=\"fig\">5a<\/a>). Indeed, a permanganate footprint assay using early staged *Drosophila* embryos, which are pluripotent, showed evidence of paused Pol II at several genes. In contrast, there was no such evidence when using S2 cells derived from older embryos, at which stage the pluripotency may be lost \\[39\\]. Alternatively, these differences may simply reflect distinctive features between somatic cells and germ cells. Germ cells give rise to gametes, which are the most 'immortal' cell type due to their ability to produce the next generation of an entire organism upon fertilization \\[53\\]. This requirement to reset totipotency may distinguish germ cells from somatic cells with regard to their chromatin features (Figure <a href=\"#F5\" data-ref-type=\"fig\">5a<\/a>).\n\n## Does species specificity dictate chromatin architecture?\n\nThe chromatin structure we observed in undifferentiated germ cell-enriched *bam* testis may also apply to *Drosophila* S2 cells, raising another possibility that species difference should be considered. It has been demonstrated that bivalent genes in mammalian ESCs are also enriched with PcG activities \\[10-12\\]. These PcG components have the capability to recruit DNA methyltransferases to methylate DNA and silence gene expression in a more permanent manner \\[54-56\\]. In contrast, the active H3K4me3 mark placed by the TrxG complex \\[4-6\\] may keep chromatin in a relatively open state by recruiting RNA Pol II and initiating low level transcription to prevent DNA methylation (Figure <a href=\"#F5\" data-ref-type=\"fig\">5b<\/a>). Indeed, many bivalent domains in ESCs reside at the highly conserved non-coding elements, which are enriched with CpG islands and potentiated for DNA methylation \\[3\\]. In contrast to mammalian systems, the DNA methylase activity is almost negligible in adult flies \\[57-60\\]. Therefore, differentiation genes in *Drosophila* are not subject to a 'stable' repression caused by DNA methylation as in the mammalian systems, which may promote more dynamic gene regulation while rendering bivalency and paused RNA Pol II status dispensable in *Drosophila*. Noticeably, recent work in *Xenopus* embryos also demonstrated that active H3K4me3 and repressive H3K27me3 regulate distinct groups of genes in a spatially controlled manner. It will be interesting to study monovalency versus bivalency during animal development, as well as evolution.\n\n# Conclusions\n\nRecent data have suggested two non-mutually exclusive mechanisms of gene expression regulation, paused Pol II and bivalent modifications, in mammalian ESCs and other stem or progenitor cells. In these primitive cell types, developmental regulators are characterized by bivalent domains harboring both the H3K4me3 and H3K27me3 marks \\[3\\]. These genes may also be bound by paused Pol II, which may poise them for rapid induction during differentiation or in response to developmental and environmental stimuli \\[2,3,12\\]. Here our analysis of undifferentiated-cell-enriched *Drosophila bam* testis, by combining highly sensitive ChIP-seq and RNA-seq methods, indicates that most differentiation genes are not enriched with the bivalent signature. In addition, these genes are unpoised with no significant binding of paused Pol II. These data suggest that transcription of differentiation genes in this system is mainly controlled at the Pol II recruitment step but not at the elongation step. These chromatin signatures we identified could reflect cell type specificity, species specificity, or both.\n\n# Materials and methods\n\n## Fly strains and husbandry\n\nFlies were raised using standard medium at 25\u00b0C unless stated otherwise. The *bam*^1^\/TM3 stock was obtained from Bloomington *Drosophila* Stock Center. The *bam*^*114-97*^\/TM6B stock was a gift from Dr Margaret Fuller. The *bam*^*1*^*\/bam*^*114-97*^ testis was used for immunostaining with antibodies against a germ cell-specific mark Vasa \\[61\\] and a somatic cell-specific mark Traffic jam (Tj) \\[62\\] to demonstrate that most of the cells in this tissue are germ cells (Additional file <a href=\"#S12\" data-ref-type=\"supplementary-material\">12<\/a>).\n\n## Culturing S2 cells\n\nThe *Drosophila* S2 cells (ATCC, CRL-1963, Lot#5054622, Manassas, VA, USA) were cultured following the manufacturer's protocol. Briefly, one vial of S2 cell stock was taken from liquid nitrogen and thawed quickly at room temperature. Once the cells were completely thawed, they were transferred to a 25 cm^2^ flask (Corning, CLS430639-20EA, Lowell, MA, USA) containing 5 ml of room temperature complete Schneider's *Drosophila* medium (GIBCO, \\#11720-034, Carlsbad, CA, USA; contains 10% heat-inactivated fetal bovine serum (GIBCO \\#16140-071)). After incubating at 25\u00b0C for 30 minutes, the S2 cells were centrifuged at 1,000 rpm, the medium was removed and the cells were transferred into a 25 cm^2^ flask (Corning, CLS430639-20EA) containing 5 ml of room temperature complete Schneider's *Drosophila* medium. The S2 cells were harvested at the exponentially growing stage after incubating at 25\u00b0C for about 90 hours.\n\n## Microarray experiments and data analysis\n\nTotal RNA from approximately 200 pairs of *bam* (*bam*^*1*^\/*bam*^*114-97*^) fly testes was extracted using TRIzol (Invitrogen, \\#15596-018, Carlsbad, CA, USA) following the manufacturer's instructions and the genomic DNA was degraded using 2 Units of DNase I (Fermentas, \\#EN0521, Glen Burnie, MD, USA) at 37\u00b0C for 20 minutes. Total RNA from *Drosophila* S2 cells was extracted using the Qiagen RNeasy Mini Kit (catalogue number 74014, Valencia, CA, USA), and the genomic DNA was degraded with RNase-Free DNase (catalogue number 79254). RNA integrity was checked by gel electrophoresis (1% agarose). Approximately 4 \u03bcg of total RNA from each biological replicate were used to generate labeling probes to hybridize with the Affymetix GeneChip *Drosophila* Genome 2.0 Array according to the Affymetrix protocol. Three biological replicates were performed for *bam* testes and two biological replicates for S2 cells.\n\nMicroarray hybridization was processed at the Genomics Core Facility at the National Heart, Lung and Blood Institute and the raw data were exported from the Affymetrix Microarray Suite (MAS). The CEL files were used for signal normalization with RMA as part of the limma package from the Bioconductor R packages \\[63\\]. The 'Present (P)', 'Absent (A)' and 'Marginal (M)' calls were retrieved with the code of 'eset' in the Affy package.\n\n## RNA-seq\n\nExtraction of RNA using *bam* testes or S2 cells was performed using similar methods as described for microarray experiments. For the total RNA from *bam* testes (8.5 \u03bcg) and S2 cells (approximately 20 \u03bcg), we performed two rounds of mRNA isolation using Dynabeads mRNA purification kit (Invitrogen, \\#610-06), according to the manufacturer's instructions. The final mRNAs were eluted in 13.5 \u03bcl 10 mM Tris-HCl (pH 7.5) and immediately used to generate the first strand cDNA, using 4 \u03bcl random hexamers (ABI, \\#N8080127, Foster City, CA, USA) and SuperScript II Reverse Transcription Kit (Invitrogen, \\#18064-014) in a 30 \u03bcl final volume, following the manufacturer's instructions. The second strand cDNA was generated with the following recipe: 10 \u03bcl 5\u00d7 second strand buffer (500 mM Tris-HCl pH7.8, 50 mM MgCl~2~, 10 mM DTT), 30 nmol dNTPs (Invitrogen, \\#18427-013), 2 Units of RNase H (Invitrogen, \\#18021-014) and 50 Units of DNA Pol I (Invitrogen, \\#18010-025). The entire reaction mix was incubated at 16\u00b0C for 2.5 hours. The double-stranded DNA (dsDNA) was purified with a QIAquick PCR purification kit (Qiagen, \\#28106) and the concentration was quantified using a Qubit fluorometer (Invitrogen).\n\nTo generate sequencing libraries, about 300 ng dsDNA from each sample was fragmented by sonication using Bioruptor (Diagenode, UCD-200-TM-EX, Sparta, NJ, USA) using medium power output for 30 minutes in ice water. The resulting DNA fragments were analyzed by agarose gel to verify they were within the approximately 100 to 300 bp size range. Sequencing libraries were prepared as follows: end-repair (DNA end-repair kit from Epicenter, \\#ER0720, Madison, WI, USA); A-tailing (300 ng dsDNA, 5 \u03bcl Thermo buffer, 10 nmol dATP, 15 Units of Taq polymerase, at 70\u00b0C for 30 minutes); Solexa adaptor ligation (300 ng dsDNA, 4 \u03bcl DNA Ligase buffer, 1 \u03bcl Solexa adaptor mix, 3 ul DNA Ligase, at 70\u00b0C overnight); PCR (98\u00b0C 10 s, 65\u00b0C 30 s, 72\u00b0C 30 s for 16 cycles; then additional 72\u00b0C for 5 minutes) amplification with adaptor primers and size selection (200 to 400 bp). Then the library dsDNA for S2 cells was used on an Illumina Genome Analyzer II at a concentration of 10 ng per lane.\n\nWe obtained 20,041,035 and 9,780,523 total reads from an Illumina Genome Analyzer II for *bam* testis and S2 cell samples, respectively. And 10,163,916 (*bam* testis) and 6,263,318 (S2 cell) unique and non-redundant reads were used for downstream data analysis.\n\nThe Gene Expression Omnibus accession number for the raw and analyzed RNA-seq data is \\[GEO:GSE19325\\].\n\n## Comparison of microarray results with RNA-seq data\n\nTo compare the RPKM value from RNA-seq data with microarray results, we first retrieved 12,728 *Drosophila* genes from the Ensembl database, which also have probe(s) in the Affymetix GeneChip *Drosophila* Genome 2.0 Array. Genes with multiple probes were filtered out if different probes gave inconsistent 'Present (P)' or 'Absent (A)' calls. We then analyzed the RPKM distribution for genes with all 'Absent' calls or all 'Present' calls in microarray datasets, for both *bam* testis and S2 cell samples.\n\nThe histograms were generated using the 'hist' function in the R programming environment (R version 2.5.0 \\[64\\]). To calculate the log~2~ RPKM values of individual genes, all their original RPKM values were added a pseudo-count of 1.\n\n## Chromatin immunoprecipitation\n\nFor each modified histone and Pol II ChIP experiment, we dissected approximately 200 pairs of *bam* testes in cold phosphate-buffered saline (PBS) and grouped them in 200 \u03bcl PBS that contained protease inhibitor (Roche complete mini, \\#11836153001, Nutley, NJ, USA) and 0.5 mM phenylmethanesulfonyl fluoride (PMSF; MP Biomedicals, \\#195381, Solon, OH, USA). Approximately 1,000 cells could be extracted from one *bam* testis. We then added 5.5 \u03bcl 37% fresh formaldehyde (Supelco, \\#47083-U, Bellefonte, PA, USA) and incubated at 37\u00b0C for 15 minutes. The testes were washed twice with 450 \u03bcl cold 1\u00d7 PBS (with inhibitors and PMSF). Then 200 \u03bcl lysis buffer (50 mM Tris-HCl, pH7.6, 1 mM CaCl~2~, 0.2% Triton X-100, 5 mM butyrate, 1\u00d7 protease inhibitor cocktail, and 0.5 mM fresh PMSF) was added and the tissues were homogenized thoroughly followed by incubation at room temperature for 10 minutes. Chromatin was sheared into approximately 200-bp fragments by sonication using Microtip (Misonix, Inc., Microson XL-2000, Farmingdale, NY, USA) with the following procedure: 4 s at power 20, rest for 50 s, 4 to 5 times, followed by spinning at 14k rpm for 10 minutes at 4\u00b0C. The chromatin was diluted 10\u00d7 with RIPA buffer (10 mM Tris, pH7.6, 1 mM EDTA, 0.1% SDS, 0.1% Na-Deoxycholate, 1% Triton X-100, with protease inhibitors and PMSF) and 50 \u03bcl of this dilution was reverse cross-linked with 0.25 M NaCl for 2 hours at 65\u00b0C and used as input for real-time PCR analysis.\n\nWe washed 40 \u03bcl of Dynabeads Protein A (Invitrogen, \\#100.01D) with 600 \u03bcl 1\u00d7 PBS. We then added 100 \u03bcl 1\u00d7 PBS with 4 \u03bcg antibody and incubated the antibody-Protein A beads mixture at room temperature for 40 minutes with occasional tapping. After the unbound antibody was removed using the manipulator (Invitrogen, DYNAL MPC-S), 1 ml of the chromatin extract was added to the beads and the mixture was rotated at 4\u00b0C overnight. The beads were then washed twice with 1 ml RIPA buffer, twice with 1 ml RIPA buffer containing 0.3 M NaCl, once with LiCl wash buffer (0.25 M LiCl, 0.5% NP40, and 0.5% sodium deoxycholate), once with 1 ml TE (10 mM Tris-HCl, pH 8.0 and 1 mM EDTA) containing 0.2% Triton X-100, and once with 1 ml TE. The beads were then suspended in 100 \u03bcl 1\u00d7 TE containing 3 \u03bcl 10% SDS and 5 \u03bcl 20 mg\/ml proteinase K, followed by incubation at 65\u00b0C overnight. After the supernatant was collected, the beads were washed once more with 100 \u03bcl TE with 0.5 M NaCl. The supernatant from this wash was combined with the previous supernatant. The combined samples were treated by Phenol\/Chloroform extraction, salt\/EtOH precipitation, and dissolved in 50 \u03bcl 1\u00d7 TE. The products were either used for real-time PCR analyses or processed for Solexa sequencing according to the established protocol. Antibodies used include those against H3K4me3 (Abcam, \\#ab8580, Cambridge, MA, USA), H3K27me3 (Millipore, \\#07-449, Billerica, MA, USA), H3K36me3 (Abcam, \\#ab9050), H3 (Abcam, \\#ab1791) and RNA Pol II (Abcam, ab5408).\n\n## ChIP experiment using S2 cells\n\nExponentially growing S2 cells were harvested and dissolved in digestion buffer (50 mM Tris-HCl, pH7.6, 1 mM CCl~2~, 0.2% Triton X-100, 5 mM butyrate, 1\u00d7 protease inhibitor cocktail and 0.5 mM PMSF). Chromatin was prepared and ChIP-seq experiments were performed as described previously \\[27\\] with antibodies against Pol II (Abcam, \\#ab5408), H3K4me3 (Abcam, \\#ab8580), H3K27me3 (Millipore\/Upstate, \\#07-449), and H3K36me3 (Abcam, \\#ab9050).\n\n## Solexa pipeline analysis\n\nThe 25-bp sequencing reads were obtained from the Illumina Genome Analyzer pipeline. All reads were aligned to the *Drosophila* genome (dm3) using the ELAND (Efficient Local Alignment of Nucleotide Data) software, allowing up to two mismatches with the reference sequence. Only uniquely mapped reads were retained. For multiple identical reads, at most three copies were retained to reduce the possibility of biases from PCR amplification. The output of the Genome Analyzer pipeline was converted to browser extensible data (BED) files. The wig files used for visualization on the UCSC browser were generated from the uniquely mapped reads using a 4-bp window and 160 bp as the DNA fragment size, as previously described \\[27\\]. The size of the DNA fragment was determined by the distance from the 5' to the 3' peak of the mapped reads, as shown in Additional file <a href=\"#S13\" data-ref-type=\"supplementary-material\">13<\/a>.\n\nThe Gene Expression Omnibus accession number for the raw and analyzed ChIP-seq data is \\[GEO:GSE19325\\].\n\n## Defining genomic regions for analyzing modified histone and Pol II enrichment\n\nTo determine the regions used for modified histone and Pol II occupancy of the annotated transcripts in Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>, we first plotted each histone modification and Pol II using the entire annotated transcripts that are applicable for ChIP-seq analysis. Based on the overall enrichment plot, we used the region from -250 to +250 bp (the TSS was defined as 0) to calculate the Pol II enrichment of individual transcripts. A 0 to +500-bp window was used for H3K4me3 and H3K27me3, and a +500 to 1,500-bp window was used for H3K36me3 enrichment calculations. For genes with a transcript size longer than 1 kb, a +500 to +1,000-bp window was defined as the gene body region to calculate the stalling index.\n\n## *Drosophila* genes used for ChIP-seq analysis\n\n*Drosophila* genes used for ChIP-seq analysis were derived from the UCSC database (April 2006\/BDGP R5\/dm3), which contained 14,058 genes and 21,243 transcripts. The coordinates for these transcripts were downloaded (August 2009) from the UCSC table browser \\[65\\]. Compared to human and mouse genomes, the *Drosophila* genome is more gene-dense, including many overlapping genes and short distances between TSSs, which may lead to incorrect conclusions from ChIP-seq analysis. To avoid this, we chose transcripts for analysis using the following three steps. First, exclude transcripts shorter than 500 bp (transcript size is defined as the distance between the annotated transcription start and end sites) because these short transcripts will affect the promoter analysis for H3K4me3 and H3K27me3. A total of 740 transcripts (736 genes) were excluded using this criterion. Second, exclude overlapping transcripts from different genes (Additional file <a href=\"#S14\" data-ref-type=\"supplementary-material\">14<\/a>) and transcripts that have short distances between their TSSs; 6,497 transcripts (4,535 genes) overlap with at least one other transcript, but 3,832 transcripts (2,555 genes) among them were useful for promoter analysis (-250 to +250 bp, or 0 to +500 bp), because the overlapping regions do not affect promoter analysis of these genes. A total of 3,558 non-overlapping transcripts (2,729 genes with opposite transcription direction) have a distance between TSSs shorter than 400 bp, which will affect the Pol II promoter analysis (-250 to +250 bp), and were thus excluded. Third, after removal of certain transcripts using the above two criteria, 14,849 transcripts (9,459 genes) were retained for ChIP-seq promoter analysis. In total, 7,799 genes were applicable for stalling index analysis, and 6,400 genes for H3K36me3 enrichment analysis. Among these 9,459 genes, 2,612 have multiple transcripts, but only one transcript for each was used for ChIP-seq analysis.\n\n## Choosing the dominant transcript for multi-isoform genes\n\nFor genes with multiple isoforms, we used the most abundant one for the comparison between ChIP-seq and RNA-seq data.\n\n### *bam* Pol II versus testis RPKM, and S2 Pol II versus cell RPKM\n\n1, The dominant transcript was determined as that with the highest number of Pol II ChIP-seq reads in the -250 to +250-bp window with respect to the TSS region. If multiple transcripts have the same number of reads in this region, criterion 2 is used. 2, For transcripts that are longer than 1 kb, the dominant transcript was determined as that with the highest number of ChIP-seq reads in the gene body (+500 to +1,000-bp with respect to the TSS). For those transcripts that are shorter than 1 kb, the dominant transcript was determined as that with the highest number of ChIP-seq reads for H3K4me3 in the region 0 to +500 bp with respect to the TSS. If multiple transcripts have the same numbers of reads, criterion 3 is used. 3, The dominant transcript was determined as that with the longest transcript. If all isoforms have the same length, one was chosen randomly.\n\n### *bam* H3K4me3 versus H3K27me3, bam H3K4me3 versus testis RPKM, S2 cell H3K4me3 versus H3K27me3, and S2 H3K4me3 versus RPKM\n\n1, The dominant transcript was determined as that with the highest number of H3K4me3 ChIP-seq reads in the region 0 to +500 bp with respect to the TSS. If multiple transcripts have the same number of reads, criterion 2 is used. 2, For transcripts that are longer than 1.5 kb, the dominant transcript was determined as that with the highest number of ChIP-seq reads for H3K36me3. For transcripts that are shorter than 1.5 kb, and thus are not applicable to calculate H3K36me3 reads, the dominant transcript was determined as that with the highest number of ChIP-seq reads for Pol II in the region -250 to +250 bp with respect to the TSS. If multiple transcripts have the same number of reads, criterion 3 is used. 3, The dominant transcript was determined as that with the longest transcript. If all isoforms have the same length, one was chosen randomly.\n\n### *bam* H3K36me3 versus testis RPKM, and S2 cell H3K36me3 versus RPKM\n\n1, For the 6,400 genes that have at least one transcript longer than 1.5 kb, the dominant transcript was determined as that with the highest number of H3K36me3 ChIP-seq reads in the region +500 to +1,500 bp with respect to the TSS. If multiple transcripts have the same number of reads, criterion 2 is used. 2, The dominant transcript was determined as that with the highest number of ChIP-seq reads for H3K4me3 in the region 0 to +500 bp with respect to the TSS. If multiple transcripts have the same number of reads, criterion 3 is used. 3, The dominant transcript was determined as that with the longest transcript. If all isoforms have the same length, one was chosen randomly.\n\n## Comparison of ChIP-seq results with the RNA-seq data\n\nTo generate the plots in Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>, we retrieved annotated *Drosophila* genes from the Ensembl database for ChIP-seq analysis. We classified them into silent and expressed genes according to RPKM value (genes with RPKM = 0 were classified as the silent group, and genes with RPKM \u2265 1 were classified as the expressed group). The expressed group was further classified into low, moderate and high groups based on RPKM values. The coordinates of these transcripts were downloaded from the UCSC table browser \\[65\\]. The read density was calculated in a 5-bp window across the genome.\n\n## Comparison of our ChIP-seq data with the published ChIP-chip data\n\nWe compared our 91 bivalent genes in *bam* testis with the ChIP-chip data using BG3 and D23 cells \\[37\\]. We first downloaded both the H3K4me3 and H3K27me3 sgr files from the NCBI website. We retained the probes whose ChIP\/input hybridization intensity ratios are \u2265 2, to be consistent with the authors' definition of enriched regions. We then mapped the probes to the promoter regions (0 to +500 bp) of our 91 bivalent genes. For BG3 cells, we found 6 out of the 91 *bam* testis bivalent genes contained an enriched H3K4me3 signal; and 4 of these 91 bivalent genes contained an enriched H3K27me3 signal. However, none of these 91 bivalent genes is enriched with both H3K4me3 and H3K27me3 in BG3 cells. Similarly, for D23 cells, 5 and 11 out of these 91 *bam* testis bivalent genes contained enriched H3K4me3 and H3K27me3 signals, respectively. Again, none of these 91 bivalent genes is enriched with both H3K4me3 and H3K27me3 in D23 cells.\n\nWe also checked the ChIP-on-chip data using *Drosophila* embryos \\[38\\] and found 4,893 H3K4me3 and 2,480 H3K27me3 enriched regions. However, only 161 of them overlap with each other. We then compared the 91 bivalent genes we identified in *bam* testis with these 161 bivalent regions in embryos. From this comparison, we found only two genes (*CG4637* and *CG9610*) for which the promoter region (0 to +500 bp) overlaps two bivalent regions in embryos (chr3R: 18965718-18967662 and chr3R: 4158335-4159501).\n\n## Determination of the *P*-value of enrichment of ChIP-seq reads within a 500-bp window\n\nSince we used a 500-bp window to detect enrichment of Pol II and histone modifications (for example, active H3K4me3 and repressive H3K27me3), a sequencing read count threshold was chosen according to the Poisson distribution, which distributes the total and unique reads randomly across the *Drosophila* genome that can be mapped. For every 500-bp window with read number ranging from 1 to 99, the *P*-value was calculated. The threshold was chosen as the minimal number of reads that reached a significant enrichment compared to the random distribution (that is, *P* \\< 0.05). For example, for the total 1,342,075 reads of anti-Pol II ChIP-seq in *bam* testis, a 500-bp window containing 11 read counts has a *P*-value of 0.03, which passes the threshold *P*-value of \u2264 0.05. We then set the threshold of significant enrichment within a 500-bp window in this data set to be 11 read counts.\n\n## Analysis of the RNA Pol II stalling index\n\nTo analyze the RNA Pol II stalling index, we modified a method that was adapted from published work \\[39,66\\]. Basically, the stalling index reflects differential Pol II binding at the promoter region versus the gene body region. Therefore, we defined the -250 to +250-bp region around the TSS as the promoter region (with respect to the TSS), and the +500 to +1,000-bp region (with respect to the TSS) as the gene body region. To calculate the stalling index, we first counted the total Pol II reads at the promoter region and at the gene body region. A stalling index was defined as the ratio of total reads in the promoter region divided by the total reads in the gene body region. Based on the stalling index, we classified genes into active, stalled or no Pol II categories based on the following criteria: active Pol II genes, stalling index \u2264 3 and significant Pol II enrichment (*P* \\< 0.05); stalled Pol II genes, stalling index \u2265 5 and significant Pol II enrichment (*P* \\< 0.05); no Pol II genes, no significant Pol II enrichment (*P* \\> 0.05).\n\n## Scatter plot analysis\n\nThe scatter plots delineate comparisons of different chromatin modifications in *bam*. All plots were generated in the R programming environment (R version 2.5.0 \\[64\\]). Transformation to the log value was used to compare chromatin modifications with RPKM values.\n\n## Box plot analysis\n\nThe distribution of gene expression level was analyzed using box plots in the R programming environment (R version 2.5.0 \\[64\\]). The box represents the 25th and 75th percentiles, with the 50th percentile as a black bar. The whiskers refer to outliers that are at least 1.5\u00d7 the interquartile range from the box. The y-axis represents the RPKM value.\n\n## Identification of Pol II or modified histone-enriched regions using SICER\n\nTo identify the significantly enriched regions, we used SICER software \\[52\\] with the following parameters: window size = 200 bp, gap size = 0 bp and E-value = 100 for Pol II and H3K4me3; window size = 400 bp, gap size = 0 bp and E-value = 100 for H3K27me3 and H3K36me3. The reason we do not allow any gap is due to the density of genes in the *Drosophila* genome.\n\n## Sequencing depth analysis\n\nTo analyze the sequencing depth of ChIP-seq, we first shuffled the reads and their corresponding genomic loci, then extracted subsamples (2.5%, 5%, 7.5%, and so on until 100% of the total unique reads), and then identified the enriched regions in each subsample using SICER software as described previously. The E-value was increased from the first subsample (E-value = 3) to the last subsample (E-value = 120) by an increment of 3. We then plotted the correlation between subsamples and the enriched Pol II or modified histones in the corresponding regions, as shown in Additional file <a href=\"#S2\" data-ref-type=\"supplementary-material\">2<\/a>.\n\n## Gene Ontology assay\n\nThe gene function ontology analyses were performed using the DAVID 2008 informatics tools \\[67\\], based on the Gene Ontology Consortium \\[68\\]. All Ensembl annotated genes were used as a background comparison. Two particular Gene Ontology annotations (molecular function and biological process) were analyzed with a cutoff P value of \\< 0.01.\n\n# Abbreviations\n\n*bam*: *bag-of-marbles*; *bgcn*: *benign gonial cell neoplasm*; ChIP: chromatin immunoprecipitation; ChIP-seq: chromatin immunoprecipitation followed by high-throughput sequencing; dsDNA: double-stranded DNA; ESC: embryonic stem cell; *E(z)*: *Enhancer of Zeste*; GSC: germline stem cell; PBS: phosphate-buffered saline; PcG: Polycomb group; PMSF: phenylmethanesulfonyl fluoride; Pol II: RNA Polymerase II; RPKM: sequencing reads per kilobase of exon per million mapped reads; *sa*: *spermatocyte arrest*; TrxG: Trithorax group; TSS: transcriptional start site.\n\n# Authors' contributions\n\nQG, SE, GW, KC and XC performed the experiments; QG and DS analyzed the data; QG, DS, KZ and XC wrote the paper.\n\n# Supplementary Material\n\n###### Additional file 1\n\nSummary of unique and non-redundant reads in each sample.\n\nClick here for file\n\n###### Additional file 2\n\n**Sequencing depth of ChIP-seq using various antibodies in both *bam* testis and S2 cell samples**. Sequencing depth is analyzed by calculating the enriched regions for each subsample of the corresponding ChIP-seq experiment. The blue line represents the trend line. The x-axis of the plot indicates the percentage of subsample reads compared to the total unique reads, whereas the y-axis indicates the identified islands.\n\nClick here for file\n\n###### Additional file 3\n\n**More examples of the monovalent genes in *bam* testis**. **(a)** Transcription levels of two representative H3K4me3-monovalent genes, *Decondensation factor 31* (*Df31*) and *Rpd3*, and two representative H3K27me3-monovalent genes, *don juan* (*dj*) and *fuzzy onion* (*fzo*), in *bam* and wild-typetestis, respectively. **(b-e)** UCSC genome browser screenshot showing the active H3K4me3 monovalency at (b) *Df31* and (c) *Rpd3* genes, as well as the repressive H3K27me3 monovalency at (d) *dj* and (e) *fzo* genes, respectively. These examples are chosen based on their expression profiles in \\[21\\].\n\nClick here for file\n\n###### Additional file 4\n\n**Validation of bivalent genes using ChIP followed by real-time PCR analyses on five gene loci**. **(a)** *Rpl7A* gene is used as a positive control for the active H3K4me3 enrichment and *don juan* (*dj*) is used as a positive control for the repressive H3K27me3 mark. All five bivalent genes have H3K4me3 and H3K27me3 at levels between the two controls. The y-axis denotes the ChIP-ed DNA\/input DNA percentage. Primer pairs used for PCR validation are shown as black boxes in each panel of (b-f). Primer sequence information is in Materials and methods. **(b-f)** UCSC genome browser screenshots showing H3K4me3 and H3K27me3 enrichment at each of the following genes' loci: (b) *bgcn*; (c) *Neurochondrin*; (d) *CG14834*; (e) *NPC2*; and (f) *retn*.\n\nClick here for file\n\n###### Additional file 5\n\n**Bivalent genes in undifferentiated cells in *bam* testis**. In total, 91 bivalent genes were identified in undifferentiated cells in *bam* testis, including 18 differentiation genes (RPKM in *bam* \\< 0.5, in wild-type \u2265 1), which are highlighted in red.\n\nClick here for file\n\n###### Additional file 6\n\n**Determination of the cutoff RPKM value for expressed genes by comparing microarray and RNA-seq data**. The histogram shows the distribution of RPKMs for genes that are unambiguously called 'Present' in all three replicates from the gene expression microarray data. The RPKM values are adjusted by adding a pseudo-count of 1 prior to the logarithm. Threshold RPKM values of 0.5 and 1 are shown as dashed lines in blue and green, respectively. Approximately 99% of genes that are considered unambiguously 'Present' (3P calls) in the microarray data have an RPKM \u2265 1. Therefore, we assigned RPKM \u2265 1 as the cutoff for expressed genes. Conversely, only 0.4% of these genes have an RPKM \\< 0.5, and we assigned RPKM \\< 0.5 as the cutoff for absent or silent genes.\n\nClick here for file\n\n###### Additional file 7\n\n**Bivalent genes in *Drosophila* S2 cells**. In total, 27 bivalent genes were identified in *Drosophila* S2 cells.\n\nClick here for file\n\n###### Additional file 8\n\n**Most genes enriched with H3K4me3 are actively expressed in *bam* testis**. Scatter plot for H3K4me3 enrichment and RPKM values for all annotated genes. All labels are the same as in Figure <a href=\"#F2\" data-ref-type=\"fig\">2d<\/a>, except RPKM = 1 is used as a cutoff for expressed genes.\n\nClick here for file\n\n###### Additional file 9\n\n**Comparison of our ChIP-seq data with published ChIP-chip data. (a)** Flow chart to compare our ChIP-seq data in *bam* testis and S2 cells with published ChIP-chip data in S2 cells \\[51\\] and embryos \\[39\\]. **(b-d)** Ontology analysis of (b) the 695 genes with stalled Pol II in *bam* testis, (c) the 1,821 genes with stalled Pol II in S2 cells, and (d) the 1,014 genes with promoter-proximal enrichment of Pol II \\[51\\].\n\nClick here for file\n\n###### Additional file 10\n\n**Summary of the ChIP-seq results using *Drosophila* S2 cells**. **(a)** The four groups of genes were classified according to their RPKM value based on the RNA-seq results. The numbers in brackets denote genes used for H3K36me3 (K36) analysis. \\*See Materials and methods for gene selection criteria. **(b-e)** Antibodies used for ChIP-seq are: (b) anti-RNA Pol II (Pol II); (c) anti-H3K4me3 (K4); (d) anti-H3K36me3 (K36); and (e) anti-H3K27me3 (K27). Enrichment of each histone modification and RNA Pol II is plotted over a -5 to +5-kb region with respect to the TSSs of the genes.\n\nClick here for file\n\n###### Additional file 11\n\n**Most genes enriched with H3K4me3 are actively expressed in S2 cells**. Scatter plot of H3K4me3 enrichment and RPKM values for all annotated genes. All labels are the same as in Figure <a href=\"#F2\" data-ref-type=\"fig\">2d<\/a>, except RPKM = 1 was used as a cutoff for expressed genes.\n\nClick here for file\n\n###### Additional file 12\n\nImmunostaining of the *bam* testis with antibodies against the germ cell marker Vasa and the somatic marker Traffic jam (Tj).\n\nClick here for file\n\n###### Additional file 13\n\n**Determination of the fragment size for ChIP-seq analysis, based on the distance between the 5' and 3' sequencing read peaks**. All expressed genes (RPKM \u2265 1) were used for the modification level plots over a -2 to +2-kb window with respect to the TSS. **(a-e)** Antibodies used for ChIP-seq in *bam* testis were: (a) anti-RNA Pol II; (b) anti-H3K4me3; (c) anti-H3K36me3; (d) anti-H3K27me3; and (e) anti-H3.\n\nClick here for file\n\n###### Additional file 14\n\n**Cartoons showing overlapping transcripts from different genes that were retained for data analysis**. The transcripts labeled by blue letters were retained for data analysis, in each situation shown for overlapping genes.\n\nClick here for file\n\n## Acknowledgements\n\nWe thank Caitlin Choi and Ankit Vartak for technical assistance with testis dissection. We also thank Chen lab members for their critical reading and insightful suggestions. The gene expression profiling using microarrays was performed by the Genomics Core Facility of the National Heart, Lung and Blood Institute. This work is supported in part by research grant no. 05-FY09-88 from the March of Dimes Foundation, the R00HD055052 NIH Pathway to Independence Award from NICHD, the 49th Mallinckrodt Scholar Award from the Edward Mallinckrodt, Jr Foundation and the Johns Hopkins University start-up funding (XC), and support of Division of Intramural Research, the National Heart, Lung and Blood Institute, NIH (KZ).","meta":{"dup_signals":{"dup_doc_count":106,"dup_dump_count":30,"dup_details":{"curated_sources":3,"2022-21":1,"2021-49":1,"2019-43":1,"2017-47":1,"2017-17":1,"2016-44":1,"2016-36":1,"2015-48":4,"2015-40":3,"2015-35":4,"2015-32":5,"2015-27":4,"2015-22":4,"2015-14":3,"2014-52":4,"2014-49":7,"2014-42":9,"2014-41":6,"2014-35":5,"2014-23":6,"2014-15":5,"2023-23":1,"2024-10":1,"2015-18":4,"2015-11":4,"2015-06":3,"2014-10":5,"2013-48":4,"2013-20":4,"2024-26":1}},"file":"PMC2884545"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  Breast cancer may be classified into luminal A, luminal B, HER2+\/ER-, basal-like and normal-like subtypes based on gene expression profiling or immunohistochemical (IHC) characteristics. The aim of our study is to show the molecular profile characteristic of breast cancer in the North African population of Morocco. This work showed preliminary results and correlations with clinicopathological and histological parameters. Three hundred and ninety primary breast carcinomas tumor tissues were immunostained for ER, PR, HER2, CK5\/6, CK8\/18 and Ki67 using paraffin tissue.\n  .\n  # Methods\n  .\n  We reviewed 390 cases of breast cancer diagnosed on January 2008 to December 2011 at the Department of pathology, Hassan II teaching hospital, Fez, Morocco. Age, size tumor, metastatic profile, node involvement profile, histological type and immunohistochemical profile were studied.\n  .\n  # Results\n  .\n  The average age was 46 years; our patients were diagnosed late with a high average tumor size. Luminal B subtype was more prevalent (41.8%), followed by luminal A (30.5%), basal-like (13, 6%), Her2-overexpressing (9, 2%), and unclassified subtype (4.9%).\n  .\n  # Conclusion\n  .\n  This study showed that molecular classification and biological profile may be different according to geographical distribution, to encourage further studies to know the genomic profile of tumors and the environment.\n  .\n  # Virtual slide\n  .\n  <http:\/\/www.diagnosticpathology.diagnomx.eu\/vs\/1675272504826544>\nauthor: Hinde El Fatemi; Sanae Chahbouni; Sofia Jayi; Kaoutar Moumna; My Abdelilah Melhouf; Abdelaziz Bannani; Omar Mesbahi; Afaf Amarti\ndate: 2012\ninstitute: 1Department of Pathology, Hassan II teaching hospital, Fez, Morocco; 2Department of Gynecology, Hassan II teaching hospital, Fez, Morocco; 3Department of Oncology, Hassan II teaching hospital, Fez, Morocco\nreferences:\ntitle: Luminal B tumors are the most frequent molecular subtype in breast cancer of North African women: an immunohistochemical profile study from Morocco\n\n# Background\n\nBreast cancer is a heterogeneous disease such that they may have different prognoses and respond to therapy differently despite similarities in histological types, grade and stage. Based on the presence or absence of expression of the estrogen receptor (ER), breast cancer is divided in two groups: ER+ and ER-. Genetic expression profile has identified two subtypes of the ER+ tumors: luminal A and luminal B. ER- tumors also include two subtypes, the HER2+ and the basal type. These subtypes differ in their biology and both demonstrate short disease-free periods after treatment and poorer outcome. In Morocco, it's the first cancer in women and is currently a major public health problem. The molecular classification in breast carcinomas is now based upon gene expression analysis using DNA microarrays and allows to identify at least five groups: luminal A, luminal B, HER2-overexpressing, basal-like and normal breast-like \\[1-3\\]. However, large-scale subtyping using gene expression profiling from formalin-fixed, paraffin-embedded samples is not currently feasible and remains very expensive. Therefore, immunohistochemical markers have been used as surrogates tools for DNA microarray in subtyping breast cancer \\[4,5\\]. Several studies used routinely panels of immunohistochemical markers to classify breast cancers into subtypes similar to those previously defined using gene expression analyses \\[6\\]. The recent study was realized by Prat et al. \\[4\\]. They defined several immunohistochemical subtypes: (luminal A, Luminal B, HER2- enriched, basal-like) and a normal breast-like group that show significant differences in incidence, survival and response to therapy. Luminal A (ER positive (ER+) and\/or PR positive (PR+), Her2 negative (Her2-)) with ki67\\<14%, luminal B (ER + and\/or PR+ with ki67\\>14%, Her2 positive or negative (Her2+\/-), Her2+\/ER \u2212 subtype (Her2+, ER\u2212, PR\u2212) and basal-like (ER\u2212, PR\u2212, Her2\u2212, Cytokeratin 5\/6 positive (CK5\/6+) and\/or Her1+ (EGFR)). Tumors which were negative at immunohistochemical staining for all markers (ER, PR, Her2, Her1, and CK5\/6) were considered unclassified subtype \\[4\\]. According to this classification, we performed immunohistochemical staining for ER, PR, Her2, Her1, CK8\/18, basal CK5\/6 and KI67 in paraffin sections from blocks of breast cancer. The aim of the present study was to estimate the prevalence of breast cancer subtypes in patients of the north east region of Morocco, and to correlate between clinical and pathological characteristics.\n\n# Methods\n\nPatients this study was approved by Ethical Committee of Hassan II teaching hospital Center. A total of three hundred and ninety patients diagnosed with invasive breast carcinoma were examineted. Clinical information was retrieved from the medical records. Breast cancers were classified according to the World Health Organization (WHO, 2003) \\[7\\] while histological grading and staging were performed according to Modified Bloom-Richardson classification \\[8\\] and American Joint Committee on Cancer (AJCC) \\[9\\], respectively.\n\n## Immunohistochemical study\n\nTumors sections were deparaffinized and rehydrated. Peroxide blocking was done with 0.4% H2O2. Antigen retrieval was achieved by heat retrieval using a pressure cooker. After washing, the slides were treated with protein blocking agent (UltraTech HRP, Immunotech) then incubated with the following primary antibodies: anti-human ER (ER1D5, Immunotech), PR (PR10A9, Immunotech), CK5\/6 (D5\/16B4, Cell Marque), CK8\/18 (RTU-5D3, Novocastra), Ki67 (SP6 Cell Marque). After rinsing with PBS, the slides were incubated with a secondary biotinylated antibody (Immunotech). The slides were then rinsed with PBS. Sections were then incubated with streptavidin-peroxidase reagent. Staining for the slides was developed with Amino-Ethyl-Carbazole (Ultra Tech AEC, Immunotech) and then the slides were counterstained with hematoxylin, hydrated, and mounted. For Her2, immunohistochemical was carried out using with HercepTest (A0485, Dako) according to the commercial instructions for use.\n\nImmunohistochemical surrogate biomarkers of molecular classification Immunohistochemical subtypes were defined as follows: Luminal A (ER + and\/or PR+, Her2-, KI67\\<14%), luminal B (ER + and\/or PR+, Her2+\/-, Ki67\\>14%), basal-like (ER-, PR-, Her2-, and CK5\/6+, CK14+), Her2+\/ER-, and unclassified subtype (negative for all markers) (Table\u2009<a href=\"#T1\" data-ref-type=\"table\">1<\/a>). CK8\/18 expression was used for confirmation the luminal subtypes.\n\nImmunohistochemical characterization of molecular subtypes of breast cancer\n\n| **Molecular subtype** | **Immunohistochemical characterization**     |\n|:----------------------|:---------------------------------------------|\n| Luminal A             | ER (+) et Her-2 (-) et CK8\/18 (+), Ki67\\<14% |\n| Luminal B             | ER (+) et Her-2 (+)                          |\n|                       | ou ER (+), Her-2 (-) et Ki67\\>14%            |\n| Her-2                 | ER (-) et Her-2 (+)                          |\n| Basal                 | ER (-) et Her-2 (-) et CK5\/6 (+)             |\n\nPositive controls were included in each staining run and consisted of breast cancers known to express each of the antigens of interest. Cases were considered positive for ER and PR according to standardized guidelines using a cut-off of \u22651% stained tumour nuclei. Fluorescence in situ hybridization (FISH) study\n\nAll Her2 score 2+ cases were analyzed by FISH. They were performed using the PathVysion HER2 DNA Probe (Abbott Vysis Inc., Downers Grove, IL) according to the manufacturer's protocol. The probe cocktail included the LSI HER-2\/neu probe and the CEP17 probe. Fluorescence signals were analyzed and digitalized using the CytoVisionTM image analysis system (Applied Imaging International Ltd., Newcastle-Upon-Tyne, UK). Between 60 and 100 nuclei were scored for each case. Signal ratios (HER2: CEP17) \u2265 2, 2 were classified as amplified. In the absence of positive FISH data, tumors scored 2+ by IHC were considered as negative for HER-2. Her2 was scored based on a 0 to 3 scale according to the criteria set by ASCO (American Society of Clinical Oncology\/College of American Pathologists) \\[10\\]. Scores 0 and 1+ were considered as negative; score 2+ was considered borderline; and score 3+ was considered as strongly positive. FISH was performed on the borderline cases (score 2+).\n\n## KI67\n\nIn breast cancer, immunohistochemical assessment of the proportion of cells staining for the nuclear antigen Ki67 has become the most widely used method for comparing proliferation between tumor samples. Potential uses include prognosis, prediction of relative responsiveness or resistance to chemotherapy or endocrine therapy, estimation of residual risk in patients on standard therapy and as a dynamic biomarker of treatment efficacy in samples taken before, during, and after neoadjuvant therapy, particularly neoadjuvant endocrine therapy. Cases were considered positive for ki67 according to standardized guidelines using a cut-off of \u226514% stained tumour nuclei with moderate intensity (Figure\u2009<a href=\"#F1\" data-ref-type=\"fig\">1<\/a>).\n\n## Statistical analysis\n\nStatistical analysis was performed in the Department of Epidemiology, of the Faculty of Medicine and Pharmacy of Fez and was carried out using Epi-Info (3.3.2).\n\n# Results and discussion\n\nThe study was achieved on 390 patients diagnosed with infiltrating breast cancer and managed at the Department of pathology in Hassan II University Hospital in Fez. The patient's average age at diagnosis was 46 years (ranging from 22 to 92 years). The tumor clinical stage on first diagnosis, according to American Joint Committee on Cancer Staging Systems, showed that 47 women (14.5%) are at stage I, 108 (33%) are at stage II, 114 (35%) are at stage III and 57 (17.5%) at stage IV. After histological analysis, the tumor average size was 3.7 \u00b1 2.6 cm (ranging from 0.2 to 16 cm). Most of these tumors (87, 2%) were diagnosed as invasive carcinoma (IC NOS) while 5, 4% were invasive lobular carcinomas (ILC), 2% were metaplastic carcinoma (MC), and few patients had cancers of rare histology (5, 4%), which were summarized as \"other types\" in our study. The histological grade distribution for luminal B were grade II (59.9%), grade III (30.6%) but few patients only were grade I (9,6%) with p\\<0,0001. Vascular emboli were detected in 71.2% of patient's luminal B. The status of lymph nodes was determined for 326 patients among which 68, 8% had positive lymph nodes and 12.5% had distant metastasis.\n\nThe immunohistochemical study showed that 67, 1% patients were ER positives, 63, 7% were PR positive, and 24, 7% were Her2 positive. Therefore, Luminal B subtype was more prevalent (41.8%), followed by luminal A (30.5%), basal-like (13, 6%), Her2-overexpressing (9, 2%), and unclassified subtype (4.9%). The luminal B subtypes present a higher median tumor size (T2 and T3) as like basal-like and Her2-overexpressing. IHC subtypes were significantly different by histological grade (p\\< 0.00001). The luminal B also represented a higher percentage of cases with histological grade II and III (90, 5%) like, basal-like and Her2-overexpressing subtypes (90, 2% and 96% respectively) and a very low percentage of tumors with histological grade I (9,6%, 9,8% and 4%, respectively). The Luminal B subtype had 71, 2% vascular emboli, approach Her2-overexpressing subtypes (75,6%). In this study, the luminal B tumors had average percentage of lymph node metastasis (68, 8%) and distant metastasis (12, 5%). Table\u2009<a href=\"#T2\" data-ref-type=\"table\">2<\/a> Twenty-two patients (12.2%) died because of cancer-related events during the follow-up. Among these patients, 25% belong to basal-like, 22% belong to unclassified subtype group. 20% to Her2-overexpressing group, 17% to luminal B and only 5% in luminal A.\n\nPrevalence of intrinsic subtypes and clinico-pathological characteristics\n\n| **subtype** | **luminal A** | **luminal B** | **Her-2** | **Basallike** | **Unclassified** |\n|:---|:--:|:--:|:--:|:--:|:--:|\n| **Histological type %** | \u00a0 | \u00a0 | \u00a0 | \u00a0 | \u00a0 |\n| \u2003**IDC Nos** | **30,5** | **41,1** | **10,9** | **13,9** | **3,6** |\n| \u2003**ILC** | **47,6** | **42,9** | **4,8** | **0,0** | **4,8** |\n| **Other types** | **25** | **8,3** | **16,7** | **50** | **0,0** |\n| **P\\<0,00001** | \u00a0 | \u00a0 | \u00a0 | \u00a0 | \u00a0 |\n| **Histological grade%** | \u00a0 | \u00a0 | \u00a0 | \u00a0 | \u00a0 |\n| \u2003\u2009**I**\u2003 | **28,3** | **9,6** | **9,8** | **4** | **15,8** |\n| \u2003\u2009**II**\u2009 | **59,2** | **59,9** | **56,1** | **32** | **26,3** |\n| \u2003\u2009**III**\u2003\u2009 | **12,5** | **30,6** | **34,1** | **64** | **57,9** |\n| **P\\<0,0001** | \u00a0 | \u00a0 | \u00a0 | \u00a0 | \u00a0 |\n| **Vascular emboli %**\u2003 | \u00a0 | \u00a0 | \u00a0 | \u00a0 | \u00a0 |\n| **Positive** | **60,8** | **71,2** | **75,6** | **64,4** | **63,2** |\n| **negative**\u2002 | **39,2** | **28,8** | **24,4** | **35,3** | **36,8** |\n| **p=0,29** | \u00a0 | \u00a0 | \u00a0 | \u00a0 | \u00a0 |\n| **Intraductal carcinoma %** | \u00a0 | \u00a0 | \u00a0 | \u00a0 | \u00a0 |\n| \u2003\u2009**Yes** | **55** | **39,5** | **41,5** | **25,5** | **78,9** |\n| \u2003\u2009**No** | **45** | **60,5** | **58,5** | **74,5** | **21,1** |\n| \u2003\u2009**P=0,0013** | \u00a0 | \u00a0 | \u00a0 | \u00a0 | \u00a0 |\n| **Tumour size (cm)** | \u00a0 | \u00a0 | \u00a0 | \u00a0 | \u00a0 |\n| \u2003\u2009**T1** | **42,9** | **31,8** | **9,1** | **12,5** | **25** |\n| \u2003\u2009**T2** | **34,5** | **44,76** | **45,5** | **46,9** | **25** |\n| \u2003\u2009**T3** | **14,3** | **18,8** | **27,3** | **15,6** | **12,5** |\n| \u2003\u2009**T4** | **8,4** | **4,7** | **18,2** | **25** | **37,5** |\n| **P=0,0003** | \u00a0 | \u00a0 | \u00a0 | \u00a0 | \u00a0 |\n| **Necrosis %** | \u00a0 | \u00a0 | \u00a0 | \u00a0 | \u00a0 |\n| **Present** | **8,5** | **17,8** | **22,2** | **22,6** | **10,5** |\n| **Absent** | **91,5** | **82,8** | **77,8** | **77,4** | **89,5** |\n| **P=0,0698** | \u00a0 | \u00a0 | \u00a0 | \u00a0 | \u00a0 |\n| **Positive Lymph nodes %** | **52,4** | **68,8** | **86,7** | **81,3** | **-** |\n| **P=0,0066** | \u00a0 | \u00a0 | \u00a0 | \u00a0 | \u00a0 |\n| **Distant metastasis %** | \u00a0 | \u00a0 | \u00a0 | \u00a0 | \u00a0 |\n| **Yes** | **4,5** | **12,5** | **17,9** | **26,7** | **28,6** |\n| **No** | **95,5** | **87,5** | **82,1** | **73,3** | **71,4** |\n| **P=0,0030** | \u00a0 | \u00a0 | \u00a0 | \u00a0 | \u00a0 |\n\nMolecular studies of breast cancer revealed biological heterogeneity of the disease and opened new perspectives for personalized therapy. While multiple gene expression-based systems have been developed, current clinical practice is largely based upon conventional clinical and pathologic criteria. Digital image analysis (DA) with multivariate statistics of the data opens new opportunities in this field \\[11\\].\n\nRecently, gene expression studies, using microarray technology, confirmed that the heterogeneity of clinical response could be correlated with different molecular profiles of breast cancers \\[1\\]. Arvydas and al reported a series of tissue microarrays of 109 patients with breast ductal carcinoma, were stained for a set of 10 IHC markers (ER, PR, HER2, Ki67, AR, BCL2, HIF-1\u03b1, SATB1, p53, and p16) \\[12\\]. This study demonstrates that factor analysis of multiple IHC biomarkers measured by automated DA is an efficient exploratory tool clarifying complex interdependencies in the breast ductal carcinoma IHC profiles. Major factor of the aggressive disease behaviour (i-Grade) is characterized by opposite loadings of ER\/PR\/AR\/BCL2 and Ki67\/HIF-1\u03b1. The i-Grade factor scores represent integral quantitative characteristics that reveal bimodal distribution and are strongly associated with the histological grade and relevant intrinsic subtypes. In HR-positive tumours, the aggressiveness of the tumour is best reflected by the combination of Ki67 and ER, rather than Ki67 and BCL2. Inverse relation between HER2 and PR expression in the HR-positive tumours which, along with the inverse relation between Ki67 and ER, may shed the light into the differential information conveyed by the ER and PR expression. Remarkably, SATB1 along with HIF-1\u03b1 reflected the second major factor of variation in patients with breast cancer; furthermore, in the HR-positive group they were inversely associated with the HR and BCL2 expression and represented the major factor contributing to the variation in the IHC data set. However, this factor was not associated with the clinicopathologic categories studied. Biological meaning of this variation remains unclear: HIF-1\u03b1 and SATB1 may convey important biological messages other than the aggressiveness of the disease reflected by Ki67 expression and histological grade. Meanwhile, the authors, support the notion that the suggested prognostic significance of SATB1 may be related to its inverse relation to the ER expression. Finally, this analysis confirms high expression levels of p16 in Triple-negative tumours \\[12\\]. In our study, based on recent updated IHC subtype definition by Prat et al. \\[4\\], we estimated the prevalence of breast cancer subtypes in patients from the north east Moroccan region and established the correlations between clinico-pathological characteristics. The patients recruited in our university hospital were younger than in western series; the average age at diagnosis was 46.8 years. In terms of clinical staging, only 14.5% patients were diagnosed at stage I, while 33% were at stage II, 35% at stage III and 17.5% at stage IV. On the other hand, after histological analysis the average tumor size was 3.7 cm and 75% of cases measured more than 2 cm. A majority of tumors were stage II or III. Our data showed that more than 50% of patients of all subtypes presented positive lymph nodes and more than 12% of cases had distant metastasis on first diagnosis. This could be due to late consultation during the progression of the disease in our region as well as to lack of the Medicare coverage, lack of screening mammography program and women's awareness trainings particularly in rural area. The predominant histology type in this study was invasive cancer (87.4%), similar to most breast cancer studies worldwide. Overexpression of the protein and\/or amplification of the HER2 gene have been reported in approximately 20 to 30% of breast cancers, similar to what was found in our patients (24,7%). Her2+ tumors are associated with either poor prognosis or with response to trastuzumab \\[13\\]. Our results showed a distribution of breast cancer subtypes non similar to what was reported by other immunohistochemical studies \\[4\\]. In this study, the prevalence of luminal B (41, 8%) and is the more frequent subtype. Considering the fact that all patients came from a university hospital the possible effects of a selection bias should be regarded. Cancer registry available includes only data from the university hospital and not all North African population of Morocco. Triple-negative subtype represented approximately 13, 6% of our series and 78% of them were basal-like tumors. These results were similar to what was found in other studies \\[4,14\\]. The basal-like group was defined by immunohistochemistry, as being negative for ER, PR, and Her2 and positive for Ck5\/6, CK14 and\/or Her1 (EGFR). It is important to study whether EGFR is overexpressed in patients with breast cancer since these patients can be given specific EGFR molecule tyrosine kinase inhibitors such as gefitinib and lapatinib \\[15,16\\]. There are only a few reports regarding the overexpression of EGFR, with these studies indicating 8-36% of breast cancers over express this protein. However, systematic studies appraising EGFR gene amplification and mutations in the same set of cases among Chinese female patients with breast cancer are absent \\[17-19\\]. EGFR gene mutations are infrequent in breast cancers. This suggested that EGFR mutation analysis is not useful as a screening test for sensitivity to anti-EGFR therapy for breast cancers \\[20\\]. In literature, the luminal B tumors are associated with poor recurrence-free and disease-specific survivals in all adjuvant systemic treatment categories including hormone therapy; the identification of specific signaling pathways driving luminal B biology is paramount to improve treatment. Sircoulomb et al. and Holland et al. have independently identified the ZNF703 gene, located in chromosomal region 8p12, as preferentially amplified in luminal B tumours \\[21\\].\n\nThe natural history of breast cancer involves progression through defined pathological and clinical stages, starting with ductal hyperproliferation, with subsequent evolution into in situ and invasive carcinomas, and finally into metastatic disease. The majority of invasive breast cancer develop over long periods of time from certain pre-existing benign lesions. The best characterized premalignant lesions recognized today are referred to as atypical ductal hyperplasia (ADH), atypical lobular hyperplasia (ALH), ductal carcinoma in situ (DCIS) and lobular carcinoma in situ (LCIS). Douglas and al, \\[22\\] reported that Lobular neoplasia (LN) and invasive lobular carcinoma (ILC) are detected together frequently in the same specimen and location of the tumor--in up to 90% of cases of ILC \\[23\\]. Invasive carcinomas were present with LN in 45.6% of cases, with similar rates of association with invasive carcinoma and ILC (47.2%). Bratthauer and Tavassoli stratified the LNs as \"lobular intraepithelial neoplasias\" (LIN) and evaluated the frequency of association between LIN subtypes (1, 2, and 3) and invasive carcinoma. The percentage of LIN 1 (equivalent to ALH) that was associated with invasive carcinoma was 14%, and 89% of these tumors were IDCs. In the patients with LIN 3 (equivalent to LCIS), the frequency of association with IDC and ILC was 23% and 86%, respectively. The authors concluded that the advance from LIN 1 to LIN 3 was linked to a 64% increase in the frequency of invasive carcinoma and a greater than 70% rise in the likelihood of ILC \\[24\\]. Apoptosis is a frequent phenomenon in breast cancer and it can be detected by light microscopy in conventional histopathological sections or by special staining techniques. Balance between expression status of anti-apoptotic and pro-apoptotic proteins determines cells to be alive or not. The key event of apoptosis occurrence is cascade activation of caspases, and inhibitor of apoptosis proteins (IAPs) play a important role in caspase inhibition. XIAP is the most potent caspase inhibitor and Smac is one of the antagonists of XIAP. Down regulation of XIAP expression or applying exogenous Smac mimics can sensitize tumor cells, especially for breast cancer cells, to chemotherapeutics and promote apoptosis \\[25,26\\]. In immunohistochemistry, XIAP and Smac were positive in cytoplasm of tumor cells with strong or moderate intensity, respectively. The positive ratio of XIAP (84.3%, 86\/102) was more higher than that of Smac (33.3%, 34\/102), and immunoscore of XIAP was higher than Smac in IDC too (*P* \\< 0.0001). It was noteworthy that 44 IDC samples were nuclear positive for XIAP, but none was for Smac. And cytoplasm positive status of XIAP nuclear positive group was stronger than the negative group (*P* = 0.030, 0.047). Otherwise, Smac immunoscore was prevalent in HER2 positive group than negative group (*P* \\< 0.0001). Remaining data revealed that the expression status of XIAP\/Smac was not correlated with patient age, tumor size, lymph node status, histologic grading, expression of ER and PR. XIAP was a potent protein for apoptosis inhibition and Smac was an important negative regulator of the former. Disturbed balance of expression between XIAP and Smac probably contributed to carcinogenesis and XIAP positive nuclear labeling was a sign of unfavourable prognosis in breast invasive ductal carcinoma \\[27\\]. Others studies have demonstrated that claudin-6 functions as a cancer suppressor in human MCF-7 breast cancer cells. The growth inhibitory effect could be attributed to inhibition of cell proliferation and induction of apoptosis. Claudins (CLDNs) constitute a family of integral membrane proteins and have been identified as prominent structural components of tight junction ( TJ ) strands \\[28,29\\]. TJs are junctional complexes which mediate cell-to-cell adhesion in epithelial and endothelial cellular sheets \\[30\\], and which affect cell polarity and tight junction formation \\[28\\]. Guo and al reported that the apoptosis signal-regulating kinase 1 (ASK1) expression is low in breast cancer, and the levels of ASK1 mRNA and protein expression are correlated with that of claudin-6. They have identified a novel mechanism responsible for the pro-apoptosis function of claudin-6, and ASK1 may become a target for breast cancer treatments. The integrins, a family of transmembrane glycoproteins, play a major role in invasive and metastatic processes. Integrins are involved in cell adhesion in both cell-extracellular matrix and cell-cell interactions, and particularly, beta 1 integrin is involved in proliferation and differentiation of cells in the development of epithelial tissues. The putative role of beta 1 integrin expression on survival and metastasis in patients with breast invasive ductal carcinoma (IDC) was studied by Santos and al \\[31\\]. His study showed that beta1 integrin can be a marker of poor prognosis in breast cancer. Beta 1 integrin was overexpressed in 32.8% of IDC and was related with HER-2 and VEGF expression, and it had a significant relationship with metastasis and death , survival analysis showed that the overexpression of this protein is very significant in specific survival (number of months between diagnosis and death caused by the disease). Immunohistochemistrey is important to determinate the tumors of unknown origin (5-15%) and specially breast cancer metastasis (1.5%). Mammaglobin (MAG) antibody in the immunohistochemical panel for the detection of tumors of unknown origin contributed to the detection of metastasis of breast cancer. The diagnostic strategy with the highest positive predictive value (88%) included hormone receptors and mammaglobin in serial manner \\[32\\].\n\n# Conclusions\n\nWe have shown that simple IHC-based classification of breast tumors can be helpful. Since the predictive power of IHC criteria appears to be similar to that of gene expression analysis, this information can be used to improve therapeutic decisions, mainly for luminal B, Her2- overexpressing and basal-like subtypes. The luminal B subtype was associated with a poor prognosis and unfavorable clinico-pathological characteristics. In addition, findings concerning tumors stages are alarming and highlight the importance of early screening and the urgent need to improve women's awareness of breast cancer in our region. Our results should be confirmed by large studies to be conducted in other institutions and hospitals including patients coming from different regions of Morocco.\n\n# Abbreviations\n\nHER2: Epithelial human receptor 2; ER: Estrogen receptor; PR: Progesterone receptor; IHC: Immunohistochemistry; CK: Cytokeratin; WHO: World Health Organization; AJCC: American Joint Committee on Cancer; FISH: Fluorescence in situ hybridization; IC NOS: Invasive carcinoma not otherwise specified; ILC: Invasive lobular carcinoma; MC: Metaplastic carcinoma; DA: Digital image analysis; EGFR: Epidermal growth factor receptor; ADH: Atypical ductal hyperplasia; ALH: Atypical lobular hyperplasia; DCIS: Ductal carcinoma in situ; LCIS: Lobular carcinoma in situ; LN: Lobular neoplasia; LIN: Lobular intraepithelial neoplasia; ASK1: Apoptosis signal-regulating kinase 1.\n\n# Competing interests\n\nThe authors declare that they have no competing interests.\n\n# Authors' contributions\n\nAll authors analyzed, interpreted and approved the final manuscript.\n\n# Funding\n\nThis study received no specific grant from any funding agency in the public, commercial or not-for-profit sectors","meta":{"dup_signals":{"dup_doc_count":131,"dup_dump_count":20,"dup_details":{"curated_sources":2,"2015-48":6,"2015-40":3,"2015-35":6,"2015-32":5,"2015-27":4,"2015-22":6,"2015-14":4,"2014-52":6,"2014-49":5,"2014-42":15,"2014-41":9,"2014-35":8,"2014-23":8,"2014-15":8,"2015-18":5,"2015-11":6,"2015-06":6,"2014-10":8,"2013-48":5,"2013-20":6}},"file":"PMC3538531"},"subset":"pubmed_central"}
{"text":"abstract: This paper presents a low-cost inkjet dosing system capable of continuous, two-dimensional spatiotemporal regulation of gene expression via delivery of diffusible regulators to a custom-mounted gel culture of *E. coli*. A consumer-grade, inkjet printer was adapted for chemical printing; *E. coli* cultures were grown on 750 \u00b5m thick agar embedded in micro-wells machined into commercial compact discs. Spatio-temporal regulation of the *lac* operon was demonstrated via the printing of patterns of lactose and glucose directly into the cultures; X-Gal blue patterns were used for visual feedback. We demonstrate how the bistable nature of the *lac* operon's feedback, when perturbed by patterning lactose (inducer) and glucose (inhibitor), can lead to coordination of cell expression patterns across a field in ways that mimic motifs seen in developmental biology. Examples of this include sharp boundaries and the generation of traveling waves of mRNA expression. To our knowledge, this is the first demonstration of reaction-diffusion effects in the well-studied *lac* operon. A finite element reaction-diffusion model of the *lac* operon is also presented which predicts pattern formation with good fidelity.\nauthor: Daniel J. Cohen; Roberto C. Morfino; Michel M. Maharbiz\\* E-mail: <email@example.com>[^1]\ndate: 2009\ninstitute: 1 Department of Bioengineering, University of California, Berkeley, California, United States of America; 2 Department of Electrical Engineering, \u00c9cole Polytechnique F\u00e9d\u00e9rale de Lausanne, Lausanne, Switzerland; 3 Department of Electrical Engineering and Computer Science, University of California, Berkeley, California, United States of America; Baylor College of Medicine, United States of America\nreferences:\ntitle: A Modified Consumer Inkjet for Spatiotemporal Control of Gene Expression\n\n# Introduction\n\nThe development of methods that introduce spatiotemporal perturbations into developing, multi-cellular systems via soluble molecules has a long history \\[1\\]\u2013\\[4\\] and a rich, recent body of literature. Specifically, advances in microfluidics \\[5\\]\u2013\\[10\\] and biochemistry \\[11\\]\u2013\\[14\\] are beginning to open the door to direct modulation of developmental pattern formation at the spatial and temporal scale of the cell's control circuitry. Such devices can provide spatially rich, real-time input-output (I\/O) signals to bias toward developing cells into specific phenotypes. In the context of synthetic biology, such interfaces would add a degree of control over the pattern formation dynamics in multi-cellular structures that are expressing genetic circuits intended to coordinate activity through soluble molecules \\[15\\]. Initial efforts in building synthetic multicellular constructs have already begun \\[15\\], \\[16\\] and, as these mature, a robust chemical interface will be invaluable in addressing and biasing the development of patterns. In the context of control theory, such devices would allow an exploration of equilibria, stability criteria, and the non-linear dynamics of regulatory circuits. In the context of regenerative medicine and tissue engineering, these devices could potentially provide active, spatiotemporal control of morphogenesis \\[8\\], \\[17\\], \\[18\\].\n\nWhile it is clear that these applications call for systems capable of high-resolution dosing of multiple chemicals onto ensembles of cells, it is less clear how best to achieve this in a way that is low-cost, versatile, and open-source. Although a number of microfluidics-based attempts have been published \\[5\\], \\[9\\], \\[19\\], \\[20\\], all have limitations in resolution, complexity of fabrication, or ease of use. As an alternative to microfluidics, we considered inkjet technology. To date, inkjets have been incorporated into a variety of biological techniques including: direct cell printing for patterning \\[21\\], \\[22\\] and tissue engineering \\[23\\]\u2013\\[27\\], assorted cell factor printing to regulate cell positioning and behavior \\[27\\]\u2013\\[30\\], and DNA micro-array fabrication \\[31\\]. This list demonstrates the versatility of the platform, although inkjets have yet to be used for active regulation of cellular behavior. Commercialized research-grade inkjet systems such as the Fujifilm Dimatix system exist but cost orders of magnitude more than consumer-grade printers and usually only print one ink at a time.\n\nThis paper presents the adaptation of a commercial, low-cost, piezoelectric inkjet printer and commercial compact discs (CDs) for use as a chemical interface system designed to actively regulate cellular development (<a href=\"#pone-0007086-g001\" data-ref-type=\"fig\">Figure 1<\/a>). The printer is capable of addressing up to six different soluble chemicals and subsequently delivering precise doses of these chemicals to cell cultures at 226 dots\/mm. The platform can be integrated with inline microscopy to acquire data at specific time points post-dosing. Additionally, no custom software is required for our approach, making the whole system simple and user-friendly. While the CD platform is compatible with the rich toolset of polymer microfluidics \\[32\\], \\[33\\] and could be adapted into a more sophisticated device in future work, its use here was solely as a convenient, readily modifiable substrate that was compatible with the printer. In this study we used the inkjet to control the spatiotemporal reaction-diffusion dynamics of gene expression in the *lac* regulatory system by printing specific patterns of lactose and glucose onto a field of *E. coli*.\n\nThe choice of *lac* was deliberate, in that it is commonly used in synthetic biology and has a number of interesting control features, including well-characterized feedback and multiple stable states \\[34\\]\u2013\\[36\\]. We were surprised by the observation that the bistable nature of the *lac* operon's feedback system, when perturbed by patterns of lactose (inducer) and glucose (inhibitor), can lead to coordination of cell expression patterns across a field in ways that mimic motifs seen in developmental biology. Examples of this behavior include sharp gene expression boundaries and the generation of traveling waves of mRNA expression from single 'trigger' patterns. In this context, lactose and glucose are analogous to morphogens in a developing system, with the *lac* operon acting as a general template for exploring how spatially-graded perturbations generate rich behavior in bistable circuits. This is especially interesting given that *lac*, while employing positive and negative feedback and diffusible molecules (i.e. glucose and lactose), is not usually considered a reaction-diffusion system capable of generating pattern. By contrast, there is a vast literature on Turing-type and other reaction-diffusion systems and their applications to biological pattern formation \\[37\\].\n\n# Methods\n\n## Theory and Model\n\nThe *lac* operon is one of the best studied regulatory pathways in microbes \\[34\\]\u2013\\[36\\], \\[38\\]. In *E. coli*, kinetic data for the entire *lac* operon is available and robust models have been developed \\[34\\], \\[35\\]. Moreover, the dynamics of the system are well understood, and are known to exhibit multiple stable points \\[34\\]\u2013\\[36\\]. In the canonical *lac* system (<a href=\"#pone-0007086-g002\" data-ref-type=\"fig\">Figure 2<\/a>), extra-cellular lactose is taken up by lactose permease, where it is converted to allolactose by \u03b2-galactosidase. Allolactose upregulates the production of both \u03b2-galactosidase and lactose permease by inhibiting the repressor of the *lac* promoter. This lactose-based, positive feedback loop has been shown to be bistable \\[34\\]; below a certain lactose threshold, little lactose is converted while, above this threshold, the system jumps to a much higher consumption rate. Glucose acts to inhibit the conversion of lactose by lowering the transcription rate of the *lac* operon via cAMP and the catabolite repressor protein (CRP). This acts as negative feedback for the conversion of lactose. Both lactose and glucose are soluble in the extra-cellular space. We chose to model (<a href=\"#pone-0007086-g002\" data-ref-type=\"fig\">Figure 2<\/a>) the *lac* system as a set of coupled differential equations, following models developed by \\[34\\], \\[35\\] (see <a href=\"#pone.0007086.s001\" data-ref-type=\"supplementary-material\"> <em>Appendix S1<\/em> <\/a>). The well-known X-Gal assay \\[39\\] introduces the soluble X-Gal compound, which is cleaved by \u03b2-galactosidase into 5-bromo-4-chloro-3-hydroxyindole, in turn oxidizing to 5,5\u2032-dibromo-4,4\u2032-dichloro-indigo, finally resulting in an insoluble blue product. This allows for \u03b2-galactosidase activity to be assayed.\n\n## Experimental overview\n\nAll experiments involved a series of four steps, detailed below. First, bacteria were cultured in advance of an inkjet print run. Once at the proper optical density (OD~600~), they were spread over an agar substrate containing X-Gal and incubated until printing time. During the incubation period, the printer was sterilized and loaded with lactose and glucose. At the appropriate time, the bacteria were removed from the incubator, loaded onto the substrate CD, printed upon, and returned to the incubator. Additional information is provided in <a href=\"#pone.0007086.s001\" data-ref-type=\"supplementary-material\"> <em>Appendix S1<\/em> <\/a>.\n\n## Cell culture\n\n*E. coli* strain MG1655Z1 was used in this study \\[40\\]; the strain contained a low-copy number plasmid (pNS2-\u03c3VL) with three reporters, one of which is GFP driven by the *lac* promoter. The initial culture, suspended in LB Agar (Sigma LB at 20 g\/L and Sigma Bacteriological Agar at 14 g\/L), was prepared for freezing with the addition of 50% glycerol. Colony plates were created by taking a sterile loop and transferring a sample from the freezer stock to 5 mL of LB Broth (Sigma, 20 g\/L) containing *Kanamycin* (50 \u00b5g\/mL). This culture was incubated at 37\u00b0C for 12 hours and then spread, via sterile probe, onto the surface of an LB Agar plate containing *Kanamycin* (50 \u00b5g\/mL). The colony plate was incubated at 37\u00b0C for a further 12 hours, before being transferred to a refrigerator maintained at 4\u00b0C.\n\n## Print medium preparation\n\nLB Agar was poured to a depth of 750 microns in Petri dishes and chilled overnight at 4\u00b0C. Prior to plating the cells, the plates were removed from the refrigerator, and 200 \u00b5L of X-Gal solution (Sigma, 20 mg\/ml in DMSO) was bead-spread onto the surface of each plate. Ten minutes were allowed for the X-Gal to penetrate the agar and for the DMSO to evaporate. Finally, 150 \u00b5L of an overnight *E. coli* suspension (diluted to OD~600~ 0.8) was bead-spread onto the surface and allowed to sit for 10 minutes, after which the plates were wrapped with Parafilm and placed in a 37\u00b0C incubator for 3.5 hours, at which point they had just entered the exponential phase \\[41\\].\n\n## Inkjet modification and printing\n\nAs detailed descriptions of the modifications made to the Epson R280 are included in the <a href=\"#pone.0007086.s001\" data-ref-type=\"supplementary-material\"> <em>Appendix S1<\/em> <\/a> and <a href=\"#pone.0007086.s002\" data-ref-type=\"supplementary-material\"> <em>Movie S1<\/em> <\/a>, an abbreviated summary is presented here. The necessary modifications require commonly available tools and several hours to complete, and the procedure should be adaptable to a number of different printer types.\n\nWe used an Epson R280 inkjet for several reasons. Epson printers use piezoelectric print-heads, as opposed to thermal jet heads. While both types of print-head would probably suffice for our experiments, the mechanical nature of piezoelectric heads means that they can safely print a greater array of chemicals, and they do not impose temperature fluctuations on the printed fluid. Additionally, the R280 has the ability to print on rigid substrates (compact discs), which is not a common feature. Finally, the whole system is low cost (\u223c\u2592100) and widely available.\n\nThere are three fundamental challenges related to manipulating a printer: loading customized inks, uniquely specifying which inks are used during a print job, and interfacing with the biological substrate. Using an Epson R280 printer, we were able to load our lactose and glucose inks by interrupting the ink-charging process and manually injecting, by syringe pump, our solutions into specific color reservoirs (300 mM lactose, 500 mM glucose). This technique requires no manipulation of the ink cartridges themselves; we inject ink downstream of the cartridge, meaning that the printer functions completely normally but prints the injected solutions rather than ink. This is simpler, less prone to damage of the printer, and does not require the use of third-party hardware. Having primed the printer, the final step was to prepare it to accept a cell-bearing substrate.\n\nWe took advantage of the R280's ability to print directly onto the surface of compact discs and milled 800-micron deep wells directly into the surface of CDs. The size, geometry, and position of these wells were selected so as not to interfere with any of the printer's mechanisms (feed rollers or carriage drive system). By using a CD template in Adobe Photoshop, it was possible to create any planar pattern, uniquely specify the inks to be used, and print directly into the wells.\n\n## Printing of chemicals onto cultured E. coli\n\nWe used sterile shim-stock to cut out individual pieces of cell-bearing agar and transfer them to the appropriate wells on the surface of the CD. The CD was then loaded into the printer, and the print job sent. No run lasted longer than two minutes, and at no point did the cells come into contact with any components of the printer, which had previously been sterilized with 70% ethanol. Post-printing, the agar slices were transferred to hydrated Petri-dishes, placed in the incubator, and observed over a period of 15 hours.\n\n## Microscopy and image analysis\n\nStatic images were taken at 15 hours post-printing and were captured using a fluorescent backlight and an 8 mega-pixel digital camera (Canon A590). Time-lapse data was collected using an Intel QX3 microscope positioned within the incubator. All images were subsequently grey-scaled, and intensity profiles were calculated using ImageJ. The resulting intensity profiles were normalized so that higher intensity values imply a greater transmittance of light. All simulation data was obtained according to the parameter set specified in <a href=\"#pone.0007086.s001\" data-ref-type=\"supplementary-material\"> <em>Appendix S1<\/em> <\/a>.\n\n# Results and Discussion\n\nThere were three key goals for this work. First, we aimed to demonstrate the feasibility of using a commercial inkjet printer as a micro-dosing chemical interface for cellular systems. Second, we wished to determine whether inclusion of diffusion terms into a partial differential equation (PDE) model of the *lac* operon would predict the gene expression patterns generated by the printer. Lastly, we hoped to explore the types of morphogenetic-like behaviors that could be induced solely through direct, chemical manipulation of the *lac* operon.\n\n## The bistability of the lac operon generates sharp gene expression boundaries\n\nWe first characterized the resolution and pattern-formation capability of the printer system. As we were printing into hydrated agar (which would allow for diffusion of any dosed molecule), we could not rely on the resolution specifications of the print head. Concentrated lactose (300 mM) was printed in parallel bars of varying widths onto samples and the resulting X-Gal pattern was recorded (<a href=\"#pone-0007086-g003\" data-ref-type=\"fig\">Figure 3<\/a>). By fitting this data (in addition to the transient data presented below) to our finite element reaction-diffusion model, the effective diffusion rates were calculated. The fact that competition between diffusion and reaction rates biases the bistable response of the *lac* operon is evident in <a href=\"#pone-0007086-g003\" data-ref-type=\"fig\">Figure 3<\/a>. Below a certain width of printed inducer, diffusion reduces the peak concentration, and the lac operon never switches to its ON state (note how the fourth bar shows a marked, non-linear decrease in induction). For 300 mM lactose and our agar formulation, features smaller than 700 \u00b5m tended not to visibly induce. Thus, by varying the diffusion constant of the medium and the concentration of dosed inducer, the exact minimum width of an induced feature can be precisely controlled. A demonstration of this involves using half-toning to produce size-graded, two-dimensional features across a field of cells (<a href=\"#pone-0007086-g004\" data-ref-type=\"fig\">Figure 4<\/a>). Here, we see blurring between closely spaced, large features, but more well defined smaller features. The implication is that, by taking the transport characteristics of a system into account, we can modulate how features interact with each other.\n\nBearing this in mind, <a href=\"#pone-0007086-g005\" data-ref-type=\"fig\">Figure 5<\/a> shows a cross-hatched pattern used to test the uniformity of response (and resolution) across a large field of cells. This also demonstrates how the inkjet, in conjunction with a bistable circuit, can be readily used to produce sharp boundaries enclosing non-induced material even in the presence of an inter-diffusion zone. By taking advantage of bistability in the presence of weak gradients, we can achieve fairly sharp boundaries (see below), a motif observed in embryonic developmental programs \\[42\\]. Given a well-tuned simulation tool, it is possible to design and print almost any induction pattern within the resolution constraint given above.\n\n## Inline microscopy can be used to acquire time-lapse pattern formation data\n\n<a href=\"#pone-0007086-g006\" data-ref-type=\"fig\">Figure 6<\/a> shows the development of X-Gal pattern over time subsequent to lactose induction. Time-lapse microscopy was performed within an incubator, with images being taken every 20 minutes for a period of 3 hours (see <a href=\"#s2\" data-ref-type=\"sec\">Methods<\/a>). This data was used to fit the finite element model diffusion rates. Typically, induction becomes visible by eye after 45 minutes, and will then plateau at around 1.5 hours.\n\n## Inkjet printing allows for the printing of 2-D spatial chemical gradients\n\n<a href=\"#pone-0007086-g007\" data-ref-type=\"fig\">Figure 7<\/a> shows results for a piece-wise continuous lactose gradient across a field of cells. Working from a grayscale image generated on a commercial drawing program (CorelDraw 11.0), the first bar contains 0.24 M lactose, and each subsequent bar is 20% less concentrated than the previous bar. Such a pattern is not easily attainable without a patterning device, such as the inkjet, and demonstrates the ability to produce customized, finely controlled patterns, in turn allowing fine control of cellular behavior.\n\n## Patterns of multiple chemicals can be printed\n\nWe took advantage of the R280's ability to print multiple types of ink by creating patterns composed of both lactose and glucose. Specifically, we first printed a large, uniform field of lactose (300 mM) over an entire sample, immediately followed by a narrow bar of glucose (550 mM) printed on top of the lactose. Glucose is an exceptionally strong transcriptional inhibitor for the *lac* promoter, and this effect is demonstrated both by the complete lack of induction underneath the glucose bar, and the graded level of induction propagating out from that region (<a href=\"#pone-0007086-g008\" data-ref-type=\"fig\">Figure 8<\/a>).\n\n## Subtle reaction-diffusion dynamics arise from patterned activator-inhibitor dosing to a bistable system\n\nIn addition to predicting general features of X-Gal production patterns, our model predicts two pattern formation phenomena that arise as a result of spatial heterogeneity in the initial conditions (<a href=\"#pone-0007086-g009\" data-ref-type=\"fig\">Figure 9<\/a>).\n\n1.  a)Size invariance in a printed field (<a href=\"#pone-0007086-g009\" data-ref-type=\"fig\">Figure 9a<\/a>). The multi-stable nature of the lac operon \\[34\\]\u2013\\[36\\] allows for counter-gradients of glucose and lactose to generate an ON\/OFF boundary (as in <a href=\"#pone-0007086-g008\" data-ref-type=\"fig\">Figure 8<\/a>) at the same location relative to the size of the field (<a href=\"#pone-0007086-g009\" data-ref-type=\"fig\">Figure 9a<\/a>). This is a classical motif in developmental biology known as the French Flag problem \\[43\\]; it is interesting to observe that, even in a regulatory system not intended for pattern formation, such behavior arises simply as a consequence of coupling bistable gene regulation with weak (linear) gradients.\n\n2.  b)Traveling pulses of gene expression emerge from a single initial printed pattern (<a href=\"#pone-0007086-g009\" data-ref-type=\"fig\">Figure 9b<\/a>). The coupling of the *lac* operon's reactions and the diffusion of lactose and glucose lead to interesting dynamical behavior at boundaries between glucose and lactose patterns (<a href=\"#pone-0007086-g009\" data-ref-type=\"fig\">Figure 9b<\/a>). Regions dosed with lactose above the threshold for induction will immediately begin to uptake (and metabolize lactose); conversely, areas dosed with high amounts of glucose will not uptake lactose until all of the glucose is taken up. This creates a reservoir of unused lactose in the glucose-dosed regions which begins to diffuse into the lactose-dosed regions where it is taken up. This phenomenon leads to the darker regions seen at the boundaries (and predicted by simulation) in <a href=\"#pone-0007086-g008\" data-ref-type=\"fig\">Figure 8<\/a>. More interestingly, as both reaction and diffusion of glucose near the boundary deplete glucose, inhibition for lactose uptake progressively weakens and a traveling pulse of *lac* mRNA transcription arises, originating from the lactose region and extending into the glucose-rich areas.\n\n## Conclusions\n\nThis work proposed and developed a low-cost, simple interface for regulating spatiotemporal gene expression through the use of a consumer-level inkjet printer. The system was successfully demonstrated using lactose and glucose to manipulate the *lac* operon in *E. coli*. By printing a particular chemical dosing pattern in both space and time, the printer is able to regulate the micro-chemical environments within a field of cells.\n\nWe note two fundamental biological observations from our work. First, even genetic regulatory systems not used for cell signaling (*lac*, in this case), can be adapted to mimic developmental, morphogenetic regulatory systems. As long as spatiotemporal control of chemical dosing can be achieved, many such systems might be manipulated to perform in a manner that mimics more traditional developmental pathways. Second, the ability to disturb chemical boundary conditions in space and time (especially if used in conjunction with inline imaging) may reveal new and rich dynamics even in classical gene circuits (like the canonical *lac* operon). Moreover, apart from the ability to print simple line and dot-based patterns, the printer allows for graded dosing profiles to be created. Owing to the non-linearity of reaction-diffusion systems, there are important dynamics that likely cannot be seen without graded initial conditions. Ultimately, as the system improves, the loop can be closed with real-time monitoring of genetic activity coupled to a controller that can actively correct or redirect activity by regulating the temporal aspect of the dose profiles. This system can be informed by using computational simulations as a design tool to obtain specific patterns (in much the way that Computer Aided Design systems work). Our system and techniques are simple, rapid, and inexpensive, making them an attractive option for others interested in exploring the effects of spatiotemporal dosing.\n\n# Supporting Information\n\nExplanation of simulation methodology and procedure for modifying the inkjet.\n\n(1.36 MB DOC)\n\nClick here for additional data file.\n\nDemonstration of flushing the print-head. Video details the general method for cleaning and priming the print head of the Epson R280.\n\n(10.47 MB MOV)\n\nClick here for additional data file.\n\nThe authors thank Will Holtz (UC Berkeley) and Professor Jay Keasling (UC Berkeley) for helpful biological advice, and the Maharbiz Research Group (UC Berkeley) for support and suggestions.\n\n# References\n\n[^1]: Conceived and designed the experiments: DJC MMM. Performed the experiments: DJC RCM. Analyzed the data: DJC RCM MMM. Contributed reagents\/materials\/analysis tools: DJC MMM. Wrote the paper: DJC MMM.","meta":{"dup_signals":{"dup_doc_count":106,"dup_dump_count":43,"dup_details":{"curated_sources":2,"2021-43":1,"2021-39":1,"2020-10":1,"2019-47":1,"2018-30":1,"2018-17":1,"2018-05":1,"2017-51":3,"2017-47":4,"2017-43":2,"2017-39":1,"2017-34":4,"2017-30":4,"2017-26":2,"2017-22":3,"2017-17":4,"2017-09":1,"2016-36":1,"2016-30":1,"2016-26":1,"2016-22":2,"2016-18":2,"2016-07":2,"2015-48":3,"2015-40":2,"2015-35":4,"2015-32":4,"2015-27":5,"2015-22":4,"2015-14":1,"2014-52":2,"2014-49":3,"2014-42":3,"2014-41":4,"2014-35":5,"2014-23":6,"2014-15":2,"2023-14":1,"2024-18":2,"2015-18":3,"2015-11":3,"2014-10":1,"2013-48":2}},"file":"PMC2739290"},"subset":"pubmed_central"}
{"text":"abstract: Algorithms and information, fundamental to technological and biological organization, are also an essential aspect of many elementary physical phenomena, such as molecular self-assembly. Here we report the molecular realization, using two-dimensional self-assembly of DNA tiles, of a cellular automaton whose update rule computes the binary function XOR and thus fabricates a fractal pattern\u2014a Sierpinski triangle\u2014as it grows. To achieve this, abstract tiles were translated into DNA tiles based on double-crossover motifs. Serving as input for the computation, long single-stranded DNA molecules were used to nucleate growth of tiles into algorithmic crystals. For both of two independent molecular realizations, atomic force microscopy revealed recognizable Sierpinski triangles containing 100\u2013200 correct tiles. Error rates during assembly appear to range from 1% to 10%. Although imperfect, the growth of Sierpinski triangles demonstrates all the necessary mechanisms for the molecular implementation of arbitrary cellular automata. This shows that engineered DNA self-assembly can be treated as a Turing-universal biomolecular system, capable of implementing any desired algorithm for computation or construction tasks.\nauthor: Paul W. K Rothemund; Nick Papadakis; Erik Winfree\ndate: 2004-12\ninstitute: **1**Computation and Neural Systems, California Institute of TechnologyPasadena, CaliforniaUnited States of America; **2**Computer Science, California Institute of TechnologyPasadena, CaliforniaUnited States of America\nreferences:\ntitle: Algorithmic Self-Assembly of DNA Sierpinski Triangles\n\n# Introduction\n\nHow is complex organization produced and maintained by physical processes? One may look to biology, where we find the most sophisticated organization of matter, often spanning more than 24 orders of magnitude from component molecules (0.1 attograms) to entire organism (100 kilograms). This organization is information-based: DNA sequences refined by evolution encode both the components and the processes that guide their development into an organism\u2014the developmental program. For a language to describe this carefully orchestrated organization, it is tempting to turn to computer science, where the concepts of programming languages, data structures, and algorithms are used to specify complex organization of information and behavior. Indeed, the importance of universal computation for autonomous fabrication tasks was recognized in von Neumann's seminal work on self-reproducing automata, where he postulated a *universal constructor* that, by reading an input tape specifying an algorithm for what to build, could carry out the commands necessary to construct an arbitrary object (von Neumann 1966). If algorithmic concepts can be successfully adapted to the molecular context, the algorithm would join energy and entropy as essential concepts for understanding how physical processes create order. Unfortunately, the study of molecular algorithms has been hampered by the lack of suitable physical systems on which to hone such a theory: nature provides us with elementary chemical reactions too simple to program, full-blown life too complex to use as a model system, and few systems in between. This gap may be explored by synthesizing programmable biochemical systems in vitro, where we can implement and study a variety of molecular algorithms ranging gradually from simple to complex.\n\nBiomolecular self-assembly is particularly attractive for the exploration of molecular algorithms that control nanofabrication tasks. Attesting to its power, self-assembly is used pervasively in biology to create such structures as virus capsids, microtubules, and flagella. In each case, the binding interactions between a small number of protein species is sufficient to dictate the form of the final structure, often via a complex sequence of cooperative assembly steps. This can be viewed loosely as a form of programmable nanofabrication, where the program is the set of molecular species involved. For synthetic approaches, Seeman (1982, 2003) has demonstrated that DNA provides an alternative to protein that can be readily programmed by Watson\u2013Crick complementarity. A seminal paper by Adleman (1994) used one-dimensional (1D) DNA self-assembly to operate as a finite-state machine, establishing the first experimental connection between DNA self-assembly and computation. This work inspired a theoretical proposal (Winfree 1996) that builds on Wang's (1961, 1962) embedding of computation in geometrical tilings to show that two-dimensional (2D) self-assembly of DNA can perform Turing-universal computation\u2014which implies that *any* algorithm can in principle be embedded in, and guide, a potentially aperiodic crystallization process. In this \"algorithmic self-assembly\" paradigm, a set of molecular Wang tiles is viewed as the program for a particular computation or molecular fabrication task (Reif 1999; Rothemund and Winfree 2000; Adleman et al. 2001). (This framework differs from previous approaches relating tiling theory to crystalline ground-states \\[Radin 1985\\] in that kinetic phenomena are essential here.) Whereas 1D algorithmic self-assembly offers limited computational power (Winfree et al. 1998b) and has been experimentally demonstrated (Adleman 1994; Mao et al. 2000), 2D algorithmic self-assembly offers not only new capabilities for computation and construction, but also presents a new range of physical phenomena and experimental challenges as well.\n\nA natural Turing-universal model of computation that can be implemented by 2D algorithmic self-assembly is the class of 1D cellular automata. A simple but interesting choice for the local cellular automaton rule is the exclusive\u2013or (XOR) function: at each time step, each cell is computed as the XOR of its two neighbors. Beginning with a row of all '0's punctuated by a single central '1,' snapshots of the cellular automaton's state at successive time steps may be stacked one on top of the other to produce a space\u2013time history identical to Pascal's triangle (Bondarenko 1993) modulo 2 (<a href=\"#pbio-0020424-g001\" data-ref-type=\"fig\">Figure 1<\/a>A, left), which is a discrete form of Sierpinski's fractal triangle (<a href=\"#pbio-0020424-g001\" data-ref-type=\"fig\">Figure 1<\/a>A, right). To represent this cellular automaton as a tiling, each local context present in the space\u2013time history must have a corresponding Wang tile whose shape represents the input and output occurring at that location (<a href=\"#pbio-0020424-g001\" data-ref-type=\"fig\">Figure 1<\/a>B). Thus, we need four tiles, one for each entry of the truth table for XOR, and a linear input row representing the initial state of the cellular automaton (<a href=\"#pbio-0020424-g001\" data-ref-type=\"fig\">Figure 1<\/a>C). Given these tiles and the input row there is a unique way to tile the upper half-plane without mismatches or missing tiles\u2014the Sierpinski Tiling\u2014which reproduces the cellular automaton's space\u2013time history (<a href=\"#pbio-0020424-g001\" data-ref-type=\"fig\">Figure 1<\/a>D).\n\nWhereas execution of a cellular automaton occurs perfectly and synchronously, molecular self-assembly is asynchronous and may have many types of errors. To be successful, an implementation of cellular automata by molecular tiling must address four challenges: (1) The abstract tiles must be translated into molecules (molecular tiles) that readily form 2D crystals. (2) Molecular tiles must be programmed with specific binding domains that match the logic of the chosen abstract tiles. (3) The binding of molecular tiles must be sufficiently cooperative to enforce the correct order of assembly and prevent errors. (4) Assembly of molecular tiles must occur on a specified nucleating structure, and spurious nucleation must be suppressed. These properties are necessary and sufficient for implementing not only the XOR cellular automaton, but also any other 1D cellular automaton. All four have been shown individually: several types of DNA Wang tiles have been designed and shown to grow into micron-scale 2D periodic crystals (Winfree et al. 1998a; Mao et al. 1999; LaBean et al. 2000b); the interactions between these tiles can be programmed by sequence-specific hybridization (Winfree et al. 1998a; Mao et al. 2000); cooperative binding of multiple domains ensures specificity\u2014the right tile attaches in the right place (Winfree et al. 1998b; Mao et al. 2000); and input to the self-assembly process can be provided by a single-stranded template (LaBean et al. 2000a; Yan et al. 2003a). Here we demonstrate, via self-assembly of Sierpinski triangles, that all four challenges can be simultaneously overcome, thus establishing all the mechanisms necessary to implement arbitrary cellular automata. The Sierpinski tiling, then, gives rise to a new type of aperiodic crystal\u2014an algorithmic crystal.\n\n# Results\n\n## Modeling Tile-Based Self-Assembly\n\nPreventing the types of errors mentioned above may seem impossible. For example, if a single binding domain is strong enough to hold a tile in place (red arrows in <a href=\"#pbio-0020424-g001\" data-ref-type=\"fig\">Figure 1<\/a>C), then one would expect roughly 33% of tiles to mismatch with tiles in the layer below. Simulations of self-assembly shed light on how to avoid such dire circumstances. We use two levels of abstraction that isolate and address critical issues for the design and analysis of our algorithmic self-assembly experiments. How crystal morphology and patterning can be programmed by tile design in an inherently asynchronous assembly process is addressed by the abstract Tile Assembly Model (aTAM) (Winfree 1996, 1998a). To explore how physical parameters, such as tile concentration and temperature, affect crystal growth and influence error rates, we use the kinetic Tile Assembly Model (kTAM) based on reversible tile association and dissociation rates (Winfree 1998a).\n\nControl over the order of assembly is obtained by exploiting the cooperativity of binding. The aTAM models cooperativity via a threshold, \u03c4, representing the number of bonds that must be made for an association event to be thermodynamically stable: a tile may be added to a crystal if at least \u03c4 binding domains match the existing crystal. Black arrows in <a href=\"#pbio-0020424-g001\" data-ref-type=\"fig\">Figure 1<\/a>C indicate four potential association events that could occur at \u03c4 = 2; red arrows indicate two additional association events that would be permitted at \u03c4 = 1, but not at \u03c4 = 2. Simulation of cellular automata is designed to work at \u03c4\u2009= 2. Isolated tiles cannot associate at \u03c4 = 2 and so growth and computation must begin with a preformed nucleating structure (<a href=\"#pbio-0020424-g001\" data-ref-type=\"fig\">Figure 1<\/a>C, blue) that represents the input to the computation. Importantly, at \u03c4 = 2 no tile may be added until *both* preceding tiles are already present, guaranteeing a deterministic outcome despite the asynchronous order of events. Thus, assembly from an input row containing a solitary '1' domain produces the Sierpinski triangle pattern (<a href=\"#pbio-0020424-g001\" data-ref-type=\"fig\">Figures 1<\/a>D and <a href=\"#pbio-0020424-g002\" data-ref-type=\"fig\">2<\/a>A) regardless of the order in which permitted associations occur. If a small number of additional \u03c4 = 1 associations are permitted to occur, then mismatches between neighboring tiles (mismatch errors) may result. In this case, or if there are several '1's in the input row, the resulting pattern can appear to be qualitatively different: owing to propagation of information and the linearity of XOR, the resulting pattern is the superposition of Sierpinski triangles initiated at input '1's and at mismatch error sites (see <a href=\"#pbio-0020424-g001\" data-ref-type=\"fig\">Figure 1<\/a>E).\n\nThe rate at which such errors occur can be understood using the kTAM. In this model, all tiles (regardless of how well they match) may associate at a given site at a rate, *r~f~* , proportional to their concentration: *r~f~* = *k*\\[*tile*\\] = *ke* ^\u2212*G~mc~*^ , where *k* is a forward rate constant and *G~mc~* \\> 0 is the nondimensionalized entropy lost due to association\u2014thus it represents the monomer concentration. Dissociation rates depend on how many binding domains match correctly: a tile with *b* correctly matching binding domains has a dissociation rate given by *r~r,b~* =*ke* ^\u2212*bG~se~*^ , where *G~se~* \\> 0 is the nondimensionalized free energy for a single binding domain\u2014thus it represents the sticky end strength. *G~se~* decreases with increased temperature. Thus, if *G~se~\\<* *G~mc~* \\< 2*G~se~*, a reaction wherein the tile matches at a single domain would have *r~f~* \\< *r~r,~* ~1~ and thus would be thermodynamically unfavorable, while a reaction wherein the tile matches at two domains would have *r~f~* \\> *r~r,~* ~2~ and thus would be thermodynamically favorable. This model is a reasonable first-order approximation for the tile-based self-assembly of single crystals. For *G~mc~* \u2248 2*G~se~*, as *G~mc~* and *G~se~* become arbitrarily large, the \u03c4 = 2 aTAM is approximated arbitrarily well, and error rates go to zero\u2014concomitantly, assembly speed goes to zero (Winfree 1998a). For ranges of *G~mc~* and *G~se~* compatible with current experimental conditions, assuming thermodynamic and kinetic parameters extrapolated from the literature of DNA duplex hybridization (Bloomfield et al. 2000), this model (<a href=\"#sg001\" data-ref-type=\"supplementary-material\">Figure S1<\/a>) predicts mismatch error rates between 0.1% and 1.0% (<a href=\"#pbio-0020424-g002\" data-ref-type=\"fig\">Figure 2<\/a>B).\n\nThe effects of non-idealities can also be explored in this model. For example, <a href=\"#pbio-0020424-g002\" data-ref-type=\"fig\">Figure 2<\/a>C shows growth when the concentrations of the T-00 and T-11 tiles are twice that of the T-01 and T-10 tiles, and the nucleating structure grows slowly from special nucleating tiles rather than being preformed. Under this condition there is a preferential association of '0's on the facets of the growing crystal, causing characteristic errors that terminate Sierpinski triangles at corners and result in large all-zero patches (<a href=\"#pbio-0020424-g003\" data-ref-type=\"fig\">Figures 3<\/a>A and <a href=\"#sg002\" data-ref-type=\"supplementary-material\">S2<\/a>). The mechanism responsible for these errors appears to be preferential nucleation of T-00 tiles on all-zero facets, due to their higher concentration (<a href=\"#sg003\" data-ref-type=\"supplementary-material\">Figure S3<\/a>). If nucleation occurs on an all-zero facet both above and below a '1' tile, correct growth from the '1' will be sandwiched between '0's and therefore further errors will be forced (<a href=\"#pbio-0020424-g003\" data-ref-type=\"fig\">Figure 3<\/a>B). The further errors could be either (1) termination of the Sierpinski triangle by addition of a mismatched '0' tile at the corner site, or (2) sideways propagation creating a new small triangle by addition of a mismatched '1' tile on the facet below the corner site (arrow in <a href=\"#pbio-0020424-g003\" data-ref-type=\"fig\">Figure 3<\/a>A). Thus, slight quantitative variations in the model parameters can lead to striking qualitative differences in the observed error morphologies, which are effectively never seen under growth conditions with equimolar tile concentrations or with preformed borders (see <a href=\"#sg002\" data-ref-type=\"supplementary-material\">Figure S2<\/a>).\n\nThe kTAM also provides insights into a second kind of error, the spontaneous 2D nucleation of untemplated crystals in the absence of the nucleating structure. For *G~mc~* \u2248 2*G~se~*, which corresponds to the melting temperature of the crystals, such untemplated nucleation is inhibited by a kinetic barrier\u2014the existence of a critical nucleus size (Davey and Garside 2000) that decreases with increasing supersaturation. The growth rate of untemplated crystals also increases with supersaturation since their growth occurs by spontaneous 1D nucleation of a new layer of tiles on any of four facets. Via any single binding domain, there are always two tiles that can bind, so such nucleation must effectively invent a new bit of information. This bit may be propagated quickly forward or sideways (wherein tiles attach by one input and one output domain) to complete the layer without error according to the logic of XOR. Consequently, such crystals have none of the qualitative appearance of Sierpinski triangles even though they may contain no mismatched tiles (see <a href=\"#pbio-0020424-g002\" data-ref-type=\"fig\">Figure 2<\/a>D). If *G~se~* is increased, corresponding to lowering the temperature, nucleation occurs more rapidly but errors are more frequent. Backward growth, in which tiles attach to a crystal by both of their output domains, is especially error-prone since every one of these associations involves the invention of information (<a href=\"#pbio-0020424-g003\" data-ref-type=\"fig\">Figure 3<\/a>C). Whenever two backward-growing domains meet and disagree on the information that they have invented, growth can only proceed via an error. Under fast growth conditions, significantly below the melting temperature as in <a href=\"#pbio-0020424-g002\" data-ref-type=\"fig\">Figure 2<\/a>E, this gives rise to higher error rates in the reverse growth direction. Near the melting temperature, however, this effect is insignificant. The most noticeable effect for untemplated crystals is due to the non-ideality discussed above (doubling the relative concentration of T-00 and T-11 tiles): the statistical preference for all-zero patches actually *increases* the frequency and size of perfect Sierpinski patterns (see <a href=\"#pbio-0020424-g002\" data-ref-type=\"fig\">Figure 2<\/a>F).\n\nThese simulation studies suggest that all three difficulties (asynchronous association of tiles, mismatch errors, and untemplated nucleation) in principle can be controlled by slowing down the growth processes, making experimental investigations the appropriate next step.\n\n## Design and Preparation of DNA Tiles\n\nAbstract Wang tiles are implemented as DNA tiles according to the scheme described earlier (Winfree et al. 1998a): each molecular Wang tile is a DNA double-crossover molecule (Fu and Seeman 1993) with four sticky-ends (5-base single-stranded overhangs) that serve as the programmable binding domains. We rendered the four Sierpinski rule tiles using two types of double-crossover molecule, known as DAE-E and DAO-E molecules (Winfree 1996), resulting in two independent molecular implementations (<a href=\"#pbio-0020424-g004\" data-ref-type=\"fig\">Figure 4<\/a>, sequences are as given in <a href=\"#sg004\" data-ref-type=\"supplementary-material\">Figures S4\u2013S7<\/a>). The DAE-E Sierpinski tile set (<a href=\"#pbio-0020424-g004\" data-ref-type=\"fig\">Figure 4<\/a>A) consists of four molecular tiles, each composed of five strands whose sequences were designed to minimize the potential for forming alternative structures (Seeman 1990), as confirmed by non-denaturing gel electrophoresis (<a href=\"#sg008\" data-ref-type=\"supplementary-material\">Figure S8<\/a>).\n\nSince untemplated crystals were not expected to produce recognizable Sierpinski triangles, it was necessary to create a proper nucleating structure to provide the initial input for the algorithmic self-assembly. Previous work using DNA tiles to self-assemble an initial boundary had proven to be difficult (Schulman et al. 2004), so in this work we took an alternative approach of using assembly PCR (Stemmer et al. 1995) to create a long single-stranded molecule which could serve as a scaffold (LaBean et al. 2000a; Yan et al. 2003a) for the assembly of a row of input tiles (<a href=\"#pbio-0020424-g004\" data-ref-type=\"fig\">Figures 4<\/a>A and <a href=\"#sg009\" data-ref-type=\"supplementary-material\">S9\u2013S11<\/a>). Because this nucleating strand serves as the bottom of these tiles, only four strands are needed to assemble the input tiles, and an additional capping strand is used to form a double-helix between input tiles on the nucleating strand. By doping the assembly PCR mix with a small fraction of the strands coding for an input tile outputting a single '1,' we ensure that each nucleating structure contains a few randomly located sites from which a Sierpinski triangle should grow.\n\nThe DAO-E Sierpinski tile set (<a href=\"#pbio-0020424-g004\" data-ref-type=\"fig\">Figure 4<\/a>B) consists of six molecular tiles, due to peculiarities of the DAO-E motif. First, consideration of the 5\u2032 and 3\u2032 orientation of strands\u2014particularly the fact that the sticky ends at the top and bottom of a DAO-E tile have opposite polarity\u2014demands that each tile binds only to \"upside-down\" neighbors, resulting in layers of tiles with alternating orientation, which we refer to here as R-type and S-type tiles. Furthermore, the sugar\u2013phosphate backbone of the DAO-E tiles has a dyad symmetry axis, implying that the R-01 and S-01 tiles each can play the roles of both the T-01 and T-10 tiles. Likewise, the R-00, R-11, S-00, and S-11 tiles can each bind in two orientations in a site where both inputs match.\n\nIn order for the nucleating structure for the DAO-E lattice to assemble onto a long PCR-generated nucleating strand, the tiles on the input row must be of the DAE-O variant. Further, we simplified the construction so that all nucleating strands contain the same repetitive sequence, but the input tile strands are doped with a fraction of strands containing a '1' sticky-end, and again the nucleating structure contains a few randomly located sites from which a Sierpinski triangle should grow.\n\n## Self-Assembly of DNA Sierpinski Triangles\n\nIn principle, two approaches can be taken for initiating algorithmic self-assembly of DNA tiles. In the preformed tile approach, each tile is prepared separately by mixing a stoichiometric amount of each component strand in the hybridization buffer and then annealing from 90 \u00b0C to room temperature over the course of several hours. The nucleating structure is similarly prepared by annealing the nucleating strand with input tile and capping strands. Then the rule tiles and nucleating structure are mixed together at a temperature appropriate for crystal growth. In the bulk annealing approach, the nucleating strand, the capping and input tile strands, and the strands for all rule tiles are initially mixed together and then annealed. Since, at the concentrations we use, the tiles themselves have melting temperatures between roughly 60 \u00b0C and 70 \u00b0C while the crystals have a melting temperature within a few degrees of 40 \u00b0C (<a href=\"#sg012\" data-ref-type=\"supplementary-material\">Figure S12<\/a>), during annealing the tiles themselves will first form, and only later will the fully formed tiles assemble into crystals, presumably growing from the nucleating structure prior to overcoming the barrier to spontaneous nucleation. Both approaches work, but because of the convenience of the bulk annealing approach, all samples reported here were prepared using that method, with a final concentration of 0.2 \u03bcM each tile. After self-assembly in solution, samples are deposited onto mica and imaged by tapping mode atomic force microscopy (AFM).\n\nResults for the DAE-E tile set are shown in <a href=\"#pbio-0020424-g005\" data-ref-type=\"fig\">Figures 5<\/a> and <a href=\"#sg013\" data-ref-type=\"supplementary-material\">S13\u2013S15<\/a>. The majority of DNA crystals we observed were similar to those in <a href=\"#pbio-0020424-g005\" data-ref-type=\"fig\">Figure 5<\/a>A: in addition to many small and indistinctly formed fragments, larger crystals are typically thin and long (up to several microns) with '1' tiles clearly visible. Crystals consisting exclusively of VE-00 tiles (upper arrow in <a href=\"#pbio-0020424-g005\" data-ref-type=\"fig\">Figure 5<\/a>A) were particularly common; further investigation revealed that some (perhaps all) of these crystals formed as DNA tubes, and subsequently broke open and lay flat on the mica (see <a href=\"#sg015\" data-ref-type=\"supplementary-material\">Figure S15<\/a>; Rothemund et al. 2004). A '011011'-striped pattern (lower arrow in <a href=\"#pbio-0020424-g005\" data-ref-type=\"fig\">Figure 5<\/a>A) was also quite common; it can be constructed from the RE-01, SE-10, and UE-11 tiles. Growth may have been biased to form '011011' patterns by the depletion of VE-00 tiles, a stoichiometric disproportionation of tiles, due to growth of tubes early during annealing. Crystals that clearly grew from the nucleating structure were also apparent; <a href=\"#pbio-0020424-g005\" data-ref-type=\"fig\">Figure 5<\/a>B\u2013<a href=\"#pbio-0020424-g005\" data-ref-type=\"fig\">5<\/a>E show examples with particularly few errors. In several of these crystals, individual tiles could be identified and a compatible arrangement of abstract tiles (and thus errors) could be determined. Large error-free domains containing more than eight rows of perfect Sierpinski triangle were observed. In these examples, the mismatch error rate was about 2% over a wider selection of fragments, the error rate varied between 1% and 10%. We partly attribute this variation to changes in the physical conditions during annealing that result in a disproportionation of tiles. In addition to errors due to incorporation of the wrong tile, we also observed missing tiles and lattice dislocations. Frequently, as in <a href=\"#pbio-0020424-g005\" data-ref-type=\"fig\">Figure 5<\/a>E, the identity of obscured or missing tiles was deduced from the neighboring tiles by assuming correct information propagation (the imperfection often being caused by sample preparation or by interaction with the AFM tip rather than by errors during assembly).\n\nShown in <a href=\"#pbio-0020424-g006\" data-ref-type=\"fig\">Figures 6<\/a> and <a href=\"#sg016\" data-ref-type=\"supplementary-material\">S16\u2013S18<\/a>, the DAO-E tiles also succeeded in producing recognizable Sierpinski triangles. However, the DAO-E tiles self-assembled into considerably larger sheets than the DAE-E tiles, presumably because of the DAO-E tiles' symmetries that result in cancellation of strain and thus encourage flat sheets. Templated crystals were observed that had grown more than 70 rows (<a href=\"#pbio-0020424-g006\" data-ref-type=\"fig\">Figure 6<\/a>A). Because the R-11 tile does not appear in an error-free templated crystal, in some experiments (<a href=\"#pbio-0020424-g006\" data-ref-type=\"fig\">Figure 6<\/a>A) we did not include this tile; however, we observed no qualitative difference between samples prepared with and without R-11. In these crystals we almost always observed subregions with a characteristic pattern of errors that coincidentally resulted in termination of Sierpinski triangles at their corners and tops, creating large patches of zeros. Even untemplated crystals (<a href=\"#pbio-0020424-g006\" data-ref-type=\"fig\">Figure 6<\/a>B) contained recognizable subregions of the Sierpinski pattern. These features may be explained as follows: although the DAO-E tiles were mixed with equal quantities, the R-00, S-00, R-11, and S-11 tiles can bind to any permitted site in two orientations, thus making the experimental conditions analogous to the simulations of <a href=\"#pbio-0020424-g002\" data-ref-type=\"fig\">Figure 2<\/a>C\u2013<a href=\"#pbio-0020424-g002\" data-ref-type=\"fig\">2<\/a>F wherein the concentration of the corresponding tiles is doubled; slow growth of the input row in the simulations may correspond to slow straightening out of the nucleating strand, which is initially a random coil (see <a href=\"#sg017\" data-ref-type=\"supplementary-material\">Figure S17<\/a>). Large crystals often have strikingly different tile distributions and error rates, as can be seen in the amalgamation of several large crystals shown in <a href=\"#pbio-0020424-g006\" data-ref-type=\"fig\">Figure 6<\/a>C and <a href=\"#sv001\" data-ref-type=\"supplementary-material\">Video S1<\/a>. Again, this may be attributed to the disproportionation of tiles during annealing, or to sideways growth as the nucleating structure straightens out. <a href=\"#pbio-0020424-g006\" data-ref-type=\"fig\">Figure 6<\/a>D\u2013<a href=\"#pbio-0020424-g006\" data-ref-type=\"fig\">6<\/a>E shows particularly clear examples of Sierpinski triangles, averaged from several scans of the same crystal. Attempts to optimize the reaction conditions to produce Sierpinski triangles with lower error rates did not yield dramatic improvements (<a href=\"#sg018\" data-ref-type=\"supplementary-material\">Figure S18<\/a>).\n\n# Discussion\n\nThe self-assembly of DNA Sierpinski triangles demonstrates all four features necessary for Turing-universal computation by crystallization: formation of extended crystals, programmable interactions between DNA tiles determined by sticky-end sequences, selective associations of tiles enforced by the cooperative binding of more than one sticky end, and controlled nucleation of growth initiated by a template containing input information. This tiling approach could be used to implement other cellular automaton rules. Given a set *S* of possible states for the memory cells and an update function *f : S \u00d7 S \u2192 S*, one can create a set of \\|*S*\\|^2^ tiles according to the scheme of <a href=\"#pbio-0020424-g001\" data-ref-type=\"fig\">Figure 1<\/a>B, one tile for each possible input pair. The need for binding specificity limits the number of sticky-end sequences (and hence \\|*S*\\|) to about 20 for the DAO-E and DAE-E tile designs used here, but this is already sufficient to implement several known universal Turing machines and cellular automata (Lindgren and Nordahl 1990; Rogozhin 1996). A larger set of sticky-end sequences could be achieved by redesigning the DNA tile molecules to use longer sticky-ends, provided that the melting temperatures of tiles and crystals remain well separated. Thus, DNA crystallization is programmable and Turing-universal. Furthermore, for fabrication purposes, computation by self-assembly could be used to control the direction and extent of growth, thus allowing arbitrary shapes to be created efficiently (Soloveichik and Winfree 2005)\u2014demonstrating that algorithmic self-assembly is not limited to the simulation of cellular automata or Turing machines.\n\nThe main obstacle currently limiting attempts to compute or fabricate using algorithmic self-assembly is the presence of several types of errors. We observed lattice dislocations, a structural error; untemplated tubes and untemplated crystals, an error in the control of nucleation; and mismatched tiles, an error in the growth process. Accurate quantitative models of algorithmic self-assembly will be valuable for developing methods to control and reduce such errors. The kTAM simulations described here, while qualitatively insightful, predict mismatch error rates an order of magnitude smaller than those observed\u2014motivating experimental measurements of errors and refinement of the model. Although it may be possible to reduce the error rates by carefully controlling the assembly conditions, a more promising route is the creation of fault-tolerant tile sets that perform the same logic (Winfree and Bekbolatov 2004; Chen and Goel 2005; Reif et al. 2005; Schulman and Winfree 2005). For the same assembly conditions, and thus roughly the same growth rate, the kTAM predicts that these tile sets can reduce the mismatch error rates by many orders of magnitude\u2014a conclusion likely to hold in spite of inaccuracies in the model.\n\nSelf-assembly has been touted as a possible successor to photolithography, a basis for nanotechnology and a route to complexity in chemistry (Whitesides et al. 1991). Algorithmic self-assembly\u2014whether using DNA tiles as demonstrated here or using appropriately designed small molecules, proteins, or even macroscopic tiles (Bowden et al. 1997; Rothemund 2000)\u2014extends the range of structures accessible by bottom-up fabrication techniques. For example, an abstract tile set that enumerates binary numbers\u2014a binary counter\u2014uses just four tiles, yet it can be used to define the size of self-assembled structures (Rothemund and Winfree 2000), thus addressing the synthetic chemistry challenge of creating monodisperse particles with programmable size. Furthermore, attachment of suitable logic gates to '0' and '1' tiles would yield a demultiplexer for a RAM circuit. This and other interesting digital circuits (Cook et al. 2004) might be created by using algorithmic crystals as templates for further chemical processing (Braun et al. 1998; Yan et al. 2003b).\n\nThe Turing-universality of self-assembly allows theoretical insights from computer science to be applied to self-assembly. For example, many questions phrased using the aTAM\u2014such as \"Will a certain tile type, say tile type \\#5, ever be incorporated into the assembly?\" or \"Will the final assembled shape have 4-fold symmetry?\"\u2014are formally undecidable as a consequence of the undecidability of the halting problem (Turing 1936; Adleman et al. 2002). This suggests that there exists no generally applicable method for predicting the behavior or properties of crystals. A concrete instance of this dilemma is whether quasicrystals' 5-fold symmetry and aperiodicity could arise from self-assembly. Crystallographers have argued that, if so, definitions of order based on X-ray diffraction must be modified to include the new structures (Senechal 1995). The growth of Sierpinski triangles, demonstrated here, shows unequivocally that self-assembly can create aperiodic structures based on local rules. Furthermore, traditional methods of measuring order, such as X-ray diffraction, will not recognize order that exists in certain algorithmic crystals. For example, an algorithmic crystal simulating a pseudo-random number generator (Wolfram 1986; Jen 1990; Knuth 1997) would appear disordered, yet each molecule would be precisely and deterministically positioned. Thus, the growth of algorithmic crystals motivates the use of algorithmic definitions of order (Kolmogorov 1965; Levin 1984; Bennett 1995) that generalize crystallography (Mackay 1975).\n\nFinally, we ask whether the study of algorithmic self-assembly might further our understanding of biological self-assembly. Algorithmic crystals composed of simple sugar-based tiles have appeared in science fiction as a form of life (Egan 1995); indeed, the simplicity and versatility of crystalline self-assembly suggests that templating, as a basis for simple organisms (Penrose and Penrose 1957; Cairns-Smith 1971), may be more natural than previously supposed. However, examination of self-assembly in modern organisms reveals many mechanisms beyond those considered here, including conformational changes, dissipative mechanisms such as ATP hydrolysis, and interactions with genetic regulatory networks\u2014themselves biochemical information processors. The development of a theory of molecular algorithms that encompasses these additional mechanisms, if successful, will deepen our understanding of the complex processes found in nature, their fundamental limits, and their remarkable potential.\n\n# Materials and Methods\n\n### kTAM simulations\n\nSimulations described in this paper were performed with the xgrow program, written by Erik Winfree and available, along with tile sets used here, from <http:\/\/www.dna.caltech.edu\/SupplementaryMaterial>.\n\nThe xgrow program simulates the kTAM for a set of square Wang tiles (see <a href=\"#sg001\" data-ref-type=\"supplementary-material\">Figure S1<\/a>), beginning with a single seed tile. The tile set used here consists of the four Sierpinski rule tiles T-00, T-11, T-01, and T-10, augmented by three border tiles B-0, B-1, and B-B, the latter being used as the seed tile. To simulate the presence of a nucleating structure, the binding domain that joins border tiles is considered to be twice as strong as the other bonds\u2014that is, it counts as two bonds in the sum *b* that determines off-rates *r~r,b~* =*ke* ^\u2212*bG~se~*^ . The border row grows\u2014simulating the long nucleating structure becoming straight\u2014by association of border tiles at the rate *r~f~* = *k*\\[*tile*\\] = *kS~i~e* ^\u2212*G~mc~*^ , where *S~i~* is the stoichiometry of border tile *i* relative to the concentration of the four Sierpinski rule tiles. Since we have no knowledge of how quickly DNA nucleating structures straighten in our experiments, we considered two cases: (1) A rigid or quickly straightening nucleating structure was simulated by setting *S~i~* = 4, so that near the crystal melting temperature where *G~mc~* \u2248 2*G~se~*, the border growth is strongly favorable. This was used for <a href=\"#pbio-0020424-g002\" data-ref-type=\"fig\">Figure 2<\/a>B, where the seed tile stoichiometry was also set to zero, so that exactly one seed tile would be incorporated into the nucleating structure. (2) A floppy and slowly straightening nucleating structure was simulated by setting *S~i~* = 0.25 for the border tiles; in this case, near the melting temperature border growth requires stabilization by growth of rule tiles, resulting in faceted crystals. In combination with doubled concentrations of T-00 and T-11 (*S~i~* = 2), this case was used for <a href=\"#pbio-0020424-g002\" data-ref-type=\"fig\">Figure 2<\/a>C, where additionally the seed tile stoichiometry was set to 0.01 so that roughly 1% of border tiles output a '1,' in rough agreement with the observed fraction of '1's within the DNA nucleating structures in our DAO-E experiments.\n\nThe strong effect of these variations may be seen in <a href=\"#sg002\" data-ref-type=\"supplementary-material\">Figure S2<\/a>. Slow border growth significantly increases the mismatch error rate, resulting in the information contained in the border being lost in a few layers. The primary effect of doubled T-00 and T-11 concentrations is to increase the predominance of all-zero patches in the resulting crystal; not only are all-zero patches typically larger, but all-zero information in the border is propagated more reliably. Additionally, under these conditions spontaneous nucleation almost exclusively involves an initial all-zero nucleus.\n\nSimulations confirm the preferential nucleation of T-00 tiles on all-zero facets when T-00 and T-11 concentrations are doubled (<a href=\"#sg003\" data-ref-type=\"supplementary-material\">Figure S3<\/a>). In contrast, preferential nucleation on facets is *not* seen for the T-11 tile, despite its increased concentration. This is because, regardless of what information is presented on the facet, there is no way to create a layer containing more than 50% T-11 tiles and no mismatches; T-01 or T-10 tiles must intervene. Thus the nucleation rate is substantially reduced, relative to T-00 nucleation on all-zero facets. This can be assessed in simulations by measuring the probability, *p(L),* that a T-00 tile will be found after *L* layers of growth from a facet. Simulations with parameters similar to <a href=\"#pbio-0020424-g002\" data-ref-type=\"fig\">Figure 2<\/a>C (doubled T-00 and T-11 concentrations) show that *p(L)*\u2009\u2248\u20090.66*e^\u2212L\/27^* + 0.34 for all-zero facets, indicating strongly preferential nucleation, but for all other facets *p(L)* relaxes quickly to the asymptotic distribution. Simulations with parameters similar to <a href=\"#pbio-0020424-g002\" data-ref-type=\"fig\">Figure 2<\/a>B (normal T-00 and T-11 concentrations) show no preferential nucleation, as *p(L)* relaxes to the asymptotic distribution immediately for every facet type investigated.\n\n### DNA sequence design\n\nDesign of DNA Wang tiles occurs in three steps. First, the tile and lattice geometry must be determined. Here, the sizes (number of base-pairs) of each double-helical domain and sticky end, and other structural adornments such as contrast hairpins, are decided. These decisions impact the stability of each tile molecule, as the natural geometry of the DNA double-helix (10.5 bp for a full turn of B-form DNA) (Wang 1979; Rhodes and Klug 1980) constrains, for example, the separation between crossover points to be an integral number of half-turns. For the double-crossover motif used here (Fu and Seeman 1993), the acronym DAE-E refers to some of these choices at the structural level: double-crossover; antiparallel orientation of non-crossover strands at each junction; even number of half-turns (21 bp) between crossover points within each molecules; and even number of half-turns (21 bp) between nearest crossover points in two molecules joined by sticky ends. DAO-E refers to a similar set of choices, except that an odd number of half-turns (16 bp) separates the crossover points within each molecule. Where hairpin sequences were inserted for AFM contrast, we included two unpaired Ts at the bulged three-arm junctions, which has been shown to encourage stacking in the original helix domain (Ouporov and Leontis 1995) without significantly affecting the rigidity of the molecule (Li et al. 1996).\n\nAt the second level, specific sequences must be chosen. The issue here is that we wish to prevent undesired associations between strands that might inhibit formation of the correct molecular structure. We used the heuristic principle of sequence symmetry minimization (Seeman 1982, 1990) to minimize the length and number of unintentional Watson\u2013Crick complementary subsequences among all strands in each system (DAO-E and DAE-E). Violations that occurred within a single strand were weighed more heavily than violations between two strands; similarly, violations between strands in the same tile were weighed more heavily than violations between strands in different molecules. A simple adaptive walk algorithm was found to be effective in minimizing the violations and arriving at acceptable sequences. Sticky-end sequences were chosen with particular care to minimize the possibility of erroneous hybridization.\n\nThe third level of design concerns variations. We conceptualize DNA Wang tiles as consisting of three modules: the sticky ends, the core helical regions, and adornments such as the hairpin structures that provide contrast for AFM imaging. A given double-crossover core can be given different sticky ends (reprogrammed) by replacing just one or two strands, thus allowing reuse of core designs to implement different tile sets. In our experience, the structural and thermodynamic stability of a given core is not significantly affected by changes in the sticky-end sequences. Similarly, using additional strands, a given core can be used with or without the hairpin adornments, which can be inserted at various locations. Although the hairpin adornments can affect the integrity of a DNA tile (e.g., strand dimers or other high molecular weight species), we have seldom found the undesired products to exceed 20% of the material.\n\nThe core sequences for R-00 and S-00 are identical to the A and B tiles from a previous study (Winfree et al. 1998a). We usually give tiles names that indicate their core, sticky ends, and adornments. However, in the main text of this paper we have dispensed with the indication of these variations for clarity. For example, R-01 would more properly be called R-01n-23JC; S-01 called S-01-23JC; RE-01 called RE-01-15J; and SE-10 called SE-10-15J to specify which component strands have hairpins, and where those hairpins are. (The shorter names properly refer to unadorned tiles.)\n\n### DNA tile preparation and gel electrophoresis\n\nAll oligonucleotides were synthesized by standard methods (Integrated DNA Technologies), PAGE purified, and quantitated by UV absorption at 260 nm in H~2~O (purified by a Milli-Q system, Millipore, Bedford, Massachusetts, United States) based on extinction coefficients estimated using a nearest-neighbor model (Bloomfield et al. 2000). DNA tiles were prepared by mixing stoichiometric quantities of each component strand in a TAE\/Mg^2+^ buffer, as described in Winfree et al. (1998a). Proper formation of each of the ten DAE-E and DAO-E tile cores was confirmed in non-denaturing PAGE (10%\u201315% 19:1 bis:acrylamide, 3\u20135 h at 15 V\/cm and 4C, 2 pmol complex\/lane, Sybr Gold \\[Molecular Probes, Eugene, Oregon, United States\\] stained for 20\u201330 min, excited at 488 nm, imaged with 530 bandpass filter on a Bio-Rad \\[Hercules, California, United States\\] Molecular Imager FX Pro Plus) by observing a single major band (see <a href=\"#sg008\" data-ref-type=\"supplementary-material\">Figure S8<\/a>; typically between 5% and 20% of the total material appears in bands identified as partial products, such as incomplete tiles with missing strands). We redesigned the core sequences for one tile (R01) that initially did not form clean gel bands; the new tile (R01n) was used exclusively in this study. Most notably in DAE-E tiles, some lanes containing subsets of a tile's component strands showed ill-formed or heavy species such as dimers, but these difficulties were not pronounced in lanes containing the complete tile. Formation gels also allow us to estimate the relative accuracy of our concentration measurements: mismatches in stoichiometry would result in excess single-stranded or partial complexes. Concentrations appear to be accurate to **\u00b1**10%. This suggests that purification of tile complexes could result in cleaner self-assembly reactions and lower error rates.\n\n### Synthesis of the nucleating strand\n\nThe single-stranded nucleating strands were synthesized using a procedure based on Stemmer's assembly PCR (Stemmer et al. 1995). In assembly PCR, a long, repetitive, double-stranded product is generated by performing PCR on a set of splints, primer-like short (typically 40 nt) oligos that are subsequences of the desired repeat sequence as shown in <a href=\"#sg009\" data-ref-type=\"supplementary-material\">Figure S9<\/a>. To generate the single-stranded product needed for subsequent assembly of tiles on the nucleating strand, asymmetric PCR with primers for just one of the two complementary product strands could be used, in principle. In practice, we have found that such reactions result in more double-stranded product and little or no single-stranded product\u2014probably because the repetitive nature of the assembly PCR product means that every 3\u2032 end, including those of the undesired strand, may act as a primer. Thus we designed the long covalent strand of our nucleating structures to contain exclusively As, Cs, and Ts and generated single-stranded nucleating strands from the output of an assembly PCR by performing synthesis using a reaction mixture containing just dATP, dCTP, and dTTP. Although predominantly single-stranded, the output of this reaction has both single- and double-stranded material in it. We do not purify the single strands and thus double-stranded material persists in our experiments (<a href=\"#sg017\" data-ref-type=\"supplementary-material\">Figure S17<\/a>). The splint strands for generating the DAE-E nucleating strands and the DAO-E nucleating strands are given in <a href=\"#sg010\" data-ref-type=\"supplementary-material\">Figure S10<\/a>. Note that in order to have 20 base overlaps, some splint strands complement the same central three base sections of complementary splints.\n\nAssembly reactions for DAO-E and DAE-E nucleating strands were designed using slightly different principles. The improved design used for DAO-E nucleating strands is simpler: A single periodic sequence is generated. The fraction of '1' sites is determined by the stoichiometry of input tile strands used in subsequent self-assembly reactions\u2014strands A4SV and A4-S00 both assemble in the input tile in the same place, but one carries a '1' sticky end while the other carries a '0' sticky end. The approach used for DAE-E nucleating strands is more complex but more powerful for generating non-trivial input patterns. By having multiple splints that can overlap a given sequence, the assembly can be directed to non-deterministically choose one of several ways to extend a sequence. Thus, assembly PCR can be used to generate any regular language (Winfree 1998b). In this work, we used a combination of splint strands that generates substrings of the language (NRE NUE**^+^**)**\\***. The fraction of NRE subsequences is controlled by the amount of SplintNREUE2 and SplintNUERE2, which mediate the transitions into and out of the NRE sequence. (Here, we used these splints at one-fifth the concentration of other splints.) The NRE input tile outputs '0' and '1,' while the NUE input tile outputs '0' and '0'. To generate a different language, or a different distribution of sequences in the same language, a new assembly PCR must be run. (The simpler design approach used for DAO-E could also be used for DAE-E nucleating structures.)\n\nFor both methods, the PCR protocol has four stages, the first three for assembly PCR and the last to generate single-stranded material. PCR was performed in a Stratagene (La Jolla, California, United States) MX 4000 real-time PCR instrument using a Perkin-Elmer (Torrance, California, United States) GeneAMP XL kit that uses rTth polymerase. In stage 1, a 20 \u03bcl reaction mixture containing 1 pmol total of splints (of which there are *N* types) is prepared without polymerase (Mix A, per 20 \u03bcl: 1 \u03bcl of 1 \u03bcM mixed splints, 1\/*N* \u03bcM each; 1.6 \u03bcl of 10 mM dNTPs, 2.5 mM each; 1 \u03bcl of 25 mM magnesium acetate, 6 \u03bcl of 3.3X GeneAMP XL PCR buffer; 10 \u03bcl water). To avoid mispriming events, the splints are annealed in the reaction mixture at 37 \u00b0C for 5 min. The polymerase (0.4 \u03bcl) is added and the reaction is subjected to an initial 72 \u00b0C extension step, followed by 40 cycles (94 \u00b0C for 15 s, 40 \u00b0C for 30 s, 72 \u00b0C for 10 s + 1 s\/cycle; about 2 h). In stage 2, 40 \u03bcl of new PCR Mix B (Mix A minus the splints, plus 0.4 \u03bcl of polymerase, water adjusted to 20 \u03bcl), is added to the first reaction volume and the reaction cycled for an additional 25 cycles (94 \u00b0C for 15 s, 40 \u00b0C for 30 s, 72 \u00b0C for 45 s + 1 s\/cycle; about 1.5 h). In stage 3, the 60 \u03bcl reaction volume is split into three 20 \u03bcl volumes, an additional 40 \u03bcl of Mix B is added to each and an additional 20 cycles (94 \u00b0C for 15 s, 40 \u00b0C for 30 s, 72 \u00b0C for 70 s + 1 s\/cycle; about 1.3 h) are performed. At this point, long double-stranded product should be formed. (We have observed that such products remain in the well of an agarose gel long after a 20 kb marker has entered the gel.) Also the dNTPs in the mixture are presumably nearly exhausted\u2014specifically there is little dGTP left. (Any remaining dGTP will be used up early in stage 4.) In stage 4, to create single-stranded nucleating strands, 5 \u03bcl of the stage 3 product are mixed with 55 \u03bcl of fresh PCR mixture (Mix B with 1.6 \u03bcl of a mixture containing 2.5 mM each dATP, dCTP, and dTTP, rather than all four dNTPs) for additional 60 cycles of the stage 3 program (94 \u00b0C for 15 s, 40 \u00b0C for 30 s, 72 \u00b0C for 70 s + 1 s\/cycle). While addition of asymmetric primers at this stage might yield more single-stranded product, a satisfactory yield of single-stranded product results without doing so. After the final PCR, the reaction mixture is extracted with phenol:chloroform:isoamyl alcohol (Sigma, St. Louis, Missouri, United States), ethanol precipitated, and resuspended in purified water; the yield was estimated by UV absorbance. Typically, three 60 \u03bcl tubes of stage 4 product were pooled in a single recovery step and DNA was resuspended in 200 \u03bcl of water. Absorbance measurements of freshly resuspended material appeared unstable, perhaps because clumps of nucleating material scatter light. Long single-stranded DNAs may be prone to hydrolysis in water or strand-breakage upon freeze\u2013thaw. However, after storage in water at 4 \u00b0C for a year, the nucleating structure still works well (as in <a href=\"#sg018\" data-ref-type=\"supplementary-material\">Figure S18<\/a>).\n\nTo check whether the output of stage 4 is suitable as a nucleating strand for self-assembly, one can estimate the binding capacity of the nucleating strand material. <a href=\"#sg011\" data-ref-type=\"supplementary-material\">Figure S11<\/a> shows such a gel (non-denaturing PAGE, 5% 19:1 bis:acrylamide, 1 h, 150 V) for the DAO-E nucleating strand, examining how much of the fluorescently labeled Cy3-cpBr1 can be bound. We observe several things. First, stage 3 double-stranded material does not bind Cy3-cpBr1 well, as expected. Second, stage 4 material does bind Cy3-cpBr1 well, quantitatively absorbing the full amount (1 \u03bcl) added. Third, stage 4 material cannot absorb 2 \u03bcl of Cy3-cpBr1, giving us an estimate of the binding capacity of the nucleating strands. This is important for determining how much of the input tile strands must be added to ensure that a tile assembles onto nearly every site on the nucleating strand. Fourth, the presence of Sybr Green I during PCR does not appear to affect the quality of double or single-stranded material generated.\n\n### UV melts of tiles and crystals\n\nMelting temperatures for tiles and crystals were estimated based on UV^260^ melts of S-00 and R-00-23J and a mixture of both tiles (<a href=\"#sg012\" data-ref-type=\"supplementary-material\">Figure S12<\/a>). These tiles, also used in Winfree et al. (1998a), are identical to the R-00 and S-00 tiles for the DAO-E Sierpinski system, but with hairpins added to the R-00 tile. Individual tiles were at 0.4 \u03bcM of each component strand in TAE\/Mg^2+^. The mixture of R-00-23J and S-00, which forms crystals when annealed slowly, contained each strand at 0.2\u03bcM. Melts were performed on an Aviv model 14NT-UV-VIS spectrophotometer (Aviv Instruments\/Protein Solutions, Lakewood, NJ, United States), and began with preannealed samples at 15 \u00b0C, increasing to 80 \u00b0C over the course of several hours. Single-tile melts were superimposable with the reanneal from 80 \u00b0C back to 15 \u00b0C, indicating that equilibrium values were measured. Raw absorbance values were normalized. Whereas S-00 has a sharp melting transition (also seen for most other tiles lacking hairpins) near 65 \u00b0C, the R-00-23J tile has a somewhat more gradual transition, which we attribute to the presence of the hairpin. Above 40 \u00b0C, the absorbance of the mixture equals the average absorbance of the individual tiles, indicating that crystals have completely melted by that point. Prior to the crystal melting transition between 36 \u00b0C and 40 \u00b0C, there is significant noise in the measurement, presumably due to light scattering.\n\nWe have not performed UV^260^ melts of all tiles; however, several other DAO-E and DAE-E tiles have similar transitions between 50 \u00b0C and 70 \u00b0C. Therefore we assume that the templated and untemplated Sierpinski crystals also melt at approximately 40 \u00b0C and that at that temperature, the DNA tiles are reasonably well formed.\n\n### Self-assembly reactions\n\nSelf-assembly was performed by bulk annealing of all relevant rule tile, input tile, capping, and nucleating strands in a (typically) 50 \u03bcl volume of 1\u00d7 TAE\/Mg^2+^ buffer (40 mM Tris\u2013acetate \\[pH 8.0\\], 2 mM EDTA, 12.5 mM Mg^2+^), annealing from 90 \u00b0C to 20 \u00b0C at a rate of 1 \u00b0C\/min (taking about 1 h). Longer annealing schedules (e.g., 1 \u00b0C\/min from 90 \u00b0C to 50 \u00b0C followed by 1 \u00b0C\/30 min in the critical region from 50 \u00b0C to 20 \u00b0C, a total of about 15 h) did not seem to decrease the error rate or the number of untemplated tubes or crystals.\n\nDAO-E reactions contained nucleating strands sufficient to bind 0.004 \u03bcM of input tile (as estimated from binding capacity gels), 0.2 \u03bcM of each capping and input tile strand (A1S, A2, A3-nick, A4-S00, cpBr1, and 1\/100 as much A4SV), and 0.2 \u03bcM of each rule tile strand (for each of the five or six tiles used). An excess of input tile strands was used to ensure complete coverage of the nucleating strand. The excess partial input tiles appeared not to significantly interfere with the self-assembly of algorithmic crystals.\n\nDAE-E reactions contained nucleating strands sufficient to bind 0.002\u20130.008 \u03bcM of input tile (as inferred from the estimated yield of the PCR), 0.2 \u03bcM of each capping and input tile strand (NRE1 to NRE4, NUE1 to NUE4, CapNRERE and CapNUERE), and 0.2 \u03bcM of each tile strand (for each of the four tiles used). Again, an excess of input tile strands was used to ensure complete coverage of the nucleating strand.\n\n### AFM imaging\n\nAFM imaging was performed in tapping mode under TAE\/Mg^2+^ buffer on a Digital Instruments Nanoscope III (Veeco Metrology, Woodbury, New York, United States) equipped with a nano-Analytics Q-control III (Asylum Research, Santa Barbara, California, United States) and a vertical engage J-scanner, using the roughly 9.4 kHz resonance of the narrow 100 \u03bcM, 0.38 N\/m force constant cantilever of an NP-S oxide-sharpened silicon nitride tip (Veeco Metrology). After self-assembly is complete, samples were prepared for AFM imaging by deposition of 5 \u03bcl onto a freshly cleaved mica surface (Ted Pella) attached by hot melt glue to a 15\u2009mm\u2009metal puck; an additional 30 \u03bcl of buffer was added to both sample and cantilever (mounted in the standard tapping mode fluid cell) before the sample and fluid cell were positioned in the AFM head. The tapping amplitude setpoint, after engage, was typically 0.2\u20130.4 V, the drive amplitude was typically 100\u2013150 mV, scan rates ranged from 2 to 5 Hz. Individual tiles are most clearly resolved for low amplitude setpoint and high drive amplitude values. However, under such conditions, the greatest damage is done to the sample and the hairpin labels are less distinct, sometimes disappearing entirely. Thus, to prevent damage to samples, amplitude setpoint was maximized and\/or drive amplitude minimized subject to the constraint that tiles and their hairpin labels be visible.\n\nAfter acquisition, most images were flattened by subtracting a low-order polynomial from each scan line, or by adjusting each scan line to match intensity histograms. For some images (see <a href=\"#pbio-0020424-g006\" data-ref-type=\"fig\">Figures 6<\/a>D\u2013<a href=\"#pbio-0020424-g006\" data-ref-type=\"fig\">6<\/a>E and <a href=\"#sg018\" data-ref-type=\"supplementary-material\">S18<\/a>, bottom), multiple scans were aligned using hand-picked fiducial marks and averaged in Matlab (The Mathworks, Inc., Natick, Massachusetts, United States).\n\n# Supporting Information\n\n###### Representations and Tile Sets Used in Simulations\n\n(57 KB PDF).\n\nClick here for additional data file.\n\n###### Behavior of Simulated Crystal Growth\n\n(160 KB PDF).\n\nClick here for additional data file.\n\n###### Simulations of Growth on Large Facets\n\n(126 KB PDF).\n\nClick here for additional data file.\n\n###### DAE-E Strand Sequences\n\n(16 KB PDF).\n\nClick here for additional data file.\n\n###### DAE-E Tile Diagrams\n\n(21 KB PDF).\n\nClick here for additional data file.\n\n###### DAO-E Strand Sequences\n\n(16 KB PDF).\n\nClick here for additional data file.\n\n###### DAO-E Tile Diagrams\n\n(22 KB PDF).\n\nClick here for additional data file.\n\n###### Formation Gels for Representative DAO-E and DAE-E Tiles\n\n(244 KB PDF).\n\nClick here for additional data file.\n\n###### Using Assembly PCR to Generate Long, Repetitive, Single-Stranded DNA\n\n(22 KB PDF).\n\nClick here for additional data file.\n\n###### Assembly PCR Scheme for DAE-E and DAO-E Nucleating Strands\n\n(25 KB PDF).\n\nClick here for additional data file.\n\n###### Binding Capacity Gel for Determining Nucleating Strand Stoichiometry\n\n(53 KB PDF).\n\nClick here for additional data file.\n\n###### Melts of R-00-23J and S-00 and Their Mixture\n\n(33 KB PDF).\n\nClick here for additional data file.\n\n###### AFM Images Showing the Context and Distribution of DAE-E Crystals\n\n(234 KB PDF).\n\nClick here for additional data file.\n\n###### AFM Images Showing the Context and Distribution of DAE-E Crystals\n\n(203 KB PDF).\n\nClick here for additional data file.\n\n###### AFM Images of DAE-E Crystals and Tubes\n\n(226 KB PDF).\n\nClick here for additional data file.\n\n###### AFM Images Showing the Context and Distribution of DAO-E Crystals\n\n(256 KB PDF).\n\nClick here for additional data file.\n\n###### AFM Images of Boundary Assemblies and Untemplated DAO-E Crystals\n\n(234 KB PDF).\n\nClick here for additional data file.\n\n###### AFM Images of DAO-E Crystals Grown under Constant-Temperature, Near-Constant Concentration Conditions\n\n(146 KB PDF).\n\nClick here for additional data file.\n\n###### This file contains <a href=\"#sg001\" data-ref-type=\"supplementary-material\">Figures S1\u2013S18<\/a> and their captions in a single file for convenient printing\n\n(1.7 MB PDF).\n\nClick here for additional data file.\n\n###### Composite of 64 AFM Images Taken Sequentially at Scales from 24 \u03bcm to 24 nm\n\nEach frame is an average of three raw images. At the center is an amalgamation of many individual algorithmic crystals, each with its own characteristic pattern of tiles (e.g., mostly zero, bearing small triangles, or apparently random). While no large undamaged Sierpinski triangles were seen in this series of images, in some frames it is possible to see both double-helices within the tiles, as well as the major and minor grooves within the helices.\n\n(17.8 MB MPG).\n\nClick here for additional data file.\n\nFor discussions, insights, and advice, we thank John Hopfield, Ned Seeman, Len Adleman, Matt Cook, Hui Wang, Rebecca Schulman, Shaun Lee, Rizal Hariadi, and Jason Rolfe. Experiments described in <a href=\"#sg018\" data-ref-type=\"supplementary-material\">Figure S18<\/a> were performed by Jason Rolfe. We thank the Caltech Molecular Materials Research Center for use of their AFM scanners. PWKR was supported by a Beckman Fellowship. This work was supported in part by the National Science Foundation PECASE EIA-0093486, DARPA BioComputation F30602-01-2-0561, NASA NRA2-37143, and GenTel Corporation.\n\n# References\n\n## Abbreviations\n\nAFM\n\n:   atomic force microscopy\n\naTAM\n\n:   abstract Tile Assembly Model\n\n1D\n\n:   one-dimensional\n\n2D\n\n:   two-dimensional\n\nDNA\n\n:   deoxyribonucleic acid\n\nDAE-E\n\n:   double-crossover\n\nDAO-E\n\n:   double-crossover\n\nkTAM\n\n:   kinetic Tile Assembly Model\n\nPCR\n\n:   polymerase chain reaction\n\nRAM\n\n:   random-access memory\n\nXOR\n\n:   exclusive or","meta":{"dup_signals":{"dup_doc_count":126,"dup_dump_count":51,"dup_details":{"curated_sources":2,"2023-14":1,"2022-33":1,"2021-49":1,"2021-17":1,"2021-10":1,"2019-04":1,"2018-51":1,"2018-47":1,"2018-30":2,"2018-26":1,"2018-05":2,"2017-51":1,"2017-47":1,"2017-43":1,"2017-39":1,"2017-30":2,"2017-22":2,"2017-17":1,"2017-09":8,"2017-04":1,"2016-50":1,"2016-44":1,"2016-40":1,"2016-36":2,"2016-30":4,"2016-26":1,"2016-22":1,"2016-18":1,"2016-07":1,"2015-48":4,"2015-40":4,"2015-35":7,"2015-32":4,"2015-27":4,"2015-22":2,"2015-14":4,"2014-52":3,"2014-49":4,"2014-42":7,"2014-41":7,"2014-35":5,"2014-23":7,"2014-15":3,"2023-40":1,"2024-22":1,"2017-13":1,"2015-18":3,"2015-11":5,"2014-10":2,"2013-48":1,"2024-26":1}},"file":"PMC534809"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  Back problems are often recurring or chronic. It is therefore not surprising that chiropractors wish to prevent their return or reduce their impact. This is often attempted with a long-term treatment strategy, commonly called maintenance care. However, some aspects of maintenance care are considered controversial. It is therefore relevant to investigate the scientific evidence forming the basis for its use.\n  .\n  # Objectives\n  .\n  A review of the literature was performed in order to obtain answers to the following questions: What is the exact definition of maintenance care, what are its indications for use, and how is it practised? How common is it that chiropractors support the concept of maintenance care, and how well accepted is it by patients? How frequently is maintenance care used, and what factors are associated with its use? Is maintenance care a clinically valid method of approach, and is it cost-effective for the patient?\n  .\n  # Results\n  .\n  Thirteen original studies were found, in which maintenance care was investigated. The relative paucity of studies, the obvious bias in many of these, the lack of exhaustive information, and the diversity of findings made it impossible to answer any of the questions.\n  .\n  # Conclusion\n  .\n  There is no evidence-based definition of maintenance care and the indications for and nature of its use remains to be clearly stated. It is likely that many chiropractors believe in the usefulness of maintenance care but it seems to be less well accepted by their patients. The prevalence with which maintenance care is used has not been established. Efficacy and cost-effectiveness of maintenance care for various types of conditions are unknown. Therefore, our conclusion is identical to that of a similar review published in 1996, namely that maintenance care is not well researched and that it needs to be investigated from several angles before the method is subjected to a multi-centre trial.\nauthor: Charlotte Leboeuf-Yde; Lise Hestb\u00e6k\ndate: 2008\ninstitute: 1Nordic Institute of Chiropractic and Clinical Biomechanics, part of Clinical Locomotion Science, University of Southern Denmark, Forskerparken 10, DK-5230 Odense M, Denmark\nreferences:\ntitle: Maintenance care in chiropractic \u2013 what do we know?\n\n# Background\n\nChiropractors all over the world are consulted for spinal pain and dysfunction. Because many spinal pain complaints are chronic or recurrent in nature \\[1,2\\], it is understandable that, once improvement has been achieved, chiropractors attempt to prevent new events or maintain patients at their optimal level. This is usually done by scheduling additional visits over a prolonged period of time but at longer time intervals than during the acute event. Among chiropractors, this approach is named \"maintenance care\", whereas in public health terms it is described as secondary or tertiary prevention. Secondary prevention is aimed at preventing new events, whereas tertiary prevention means that improved patients with incurable conditions are maintained at the best possible level.\n\nAlthough it appears perfectly logical to use maintenance care in chronic and recurrent conditions, when informally discussing this phenomenon with chiropractors, we have often detected either a disinclination to discuss, or an ardour of arguments, often resulting in an embarrassing change of subject. In other words, maintenance care appears to be, for some, a politically incorrect topic.\n\nThis might be because the indications for treatment in asymptomatic patients depend solely on tests and observations, such as palpation findings, none of which has been shown to be clearly valid \\[3\\]. When treating an acute problem, however, this lack of valid examination tests is of little or no concern, as the patient's reaction to the treatment will provide feedback on the construct validity of the various treatment procedures. Therefore, there appears to be disagreement among chiropractors as to whether chiropractic treatment is mainly effective in the acute phase or whether it is possible also to prevent the underlying disorder, regardless of whether the patient is symptomatic at the time of examination and treatment.\n\nJamison has discussed the preventive aspect of maintenance care, when encompassing other than the musculoskeletal conditions. She points out that some chiropractors believe \"that subluxations can cause, and spinal adjustments correct, diverse problems ranging from pain to more subtle endocrine, visceral and autonomic dysfunctions\" and warns that this scientifically untested theory has considerable ill effects in the scientific and medical communities \\[4\\]. In general, if chiropractors believe that \"spinal health\" equals good health, it is understandable that they would try to convince patients to have regular preventive chiropractic treatments. Jamison discusses this in a second paper, where she also mentions the negative repercussions of such practice \\[5\\]. It could also be that the overzealous use of maintenance care has resulted in problems with various reimbursement systems, as Mitchell warned already in 1980 \\[6\\]. Some individuals' short-term financial gains could be seen as having negative long-term repercussions for the whole profession.\n\nThe concept of maintenance care, therefore, seems to be associated with the very core of disagreement between chiropractors and their styles of practice; those who treat mainly musculoskeletal conditions and those who attempt to treat also other conditions. In addition, it may divide those who believe that their examination method is objective and valid and those who depend (also) on patients' signs and symptoms for their diagnosis and treatment.\n\nNevertheless, maintenance care seems to be commonly employed, and if it is a useful model of preventive treatment, it should be recognized as such; but if it is ineffective, it should not be part of the chiropractic patient management strategy. Maintenance care therefore, merits being taken seriously and to be subjected to scientific scrutiny.\n\nIn 1993, the Mercy Guidelines \\[7\\] attempted to perform a literature review on this subject but ended up making its recommendations largely on clinical experience \"of nearly 100 years\". The report suggested that the use of chiropractic adjustments in a regiment of preventive\/maintenance care has merit. There are no statements in the guideline in relation to indications, type of treatment, duration and frequency of treatment, nor on effectiveness. It is merely written that maintenance care is \"discretionary and elective on the part of the patient\" and that when recommended, \"it is necessary for the practitioner to clearly identify the type and nature of this care and to give proper patient disclosure\".\n\nAker and Martel, three years later, performed a narrative review and concluded on the basis of the sparse literature that \"there is no scientific evidence to support the claim that maintenance care improves health status\" and went on to recommend a series of research actions to be taken \\[8\\]. Our continued monitoring of the literature revealed several additional studies since the time of their publication.\n\n# Objectives\n\nTherefore, a new literature review of this subject appeared timely, with the intent of:\n\n1\\. Defining maintenance care and the indications for and nature of its use.\n\n2\\. Describing to what degree chiropractors believe in maintenance care and to what degree it is accepted by patients.\n\n3\\. Establishing the prevalence with which chiropractors use maintenance care, and factors associated with its use.\n\n4\\. Determining its efficacy and cost-effectiveness for various types of conditions.\n\nBecause of the few articles that could be traced in relation to the number of questions, only a narrative review could be undertaken.\n\n## Search strategy and inclusion criteria\n\nA librarian-assisted electronic literature search was attempted using the Medline and Mantis databases, with no limitations for language or time period. The search terms were \"chiropractic\", \"maintenance care\", and \"prevention\", both as Mesh-term and free text. However, fewer articles than those already known by the authors appeared, and therefore a manual search was undertaken as well. The index lists of chiropractic journals were examined, as well as reference lists of articles on the topic, which resulted in the 13 articles used in this review. In addition, guidelines and chiropractic textbooks were consulted, but not exhaustively, in order to bring forth some background information on the topic.\n\nThe only inclusion criteria for this review were that the texts were research articles published in journals, that they dealt with chiropractors or chiropractic patients, and that they discussed the issue of maintenance care, also described as secondary prevention, tertiary prevention, or regular, long-term chiropractic care. Articles exclusively dealing with primary prevention were not eligible for the review.\n\n# Results\n\n## General description of the studies\n\nWe found 13 original articles, in which the issue of maintenance care was investigated. Eleven could be described as surveys and have been briefly described in Table 1 \\[Additional file <a href=\"#S1\" data-ref-type=\"supplementary-material\">1<\/a>\\]. Eight were cross-sectional \\[4,5,9-14\\], one was a file inspection study \\[15\\], one was a prospective study \\[16\\], and one was a one-year multi-centre prospective outcome study, in which the prevalence of maintenance care was retrospectively investigated at the one-year follow-up \\[17\\]. The two non-survey articles consisted of a case-report \\[18\\] and a preliminary randomized controlled clinical trial, investigating the efficacy of maintenance care in patients with chronic LBP \\[19\\]. The first of the studies was published in 1976 with data collected in 1973\u20134 \\[15\\] and the last 30 years later \\[19\\].\n\nFive of the surveys were from Australia, three from the USA, two from UK, and one from Norway (Table 1 \\[Additional file <a href=\"#S1\" data-ref-type=\"supplementary-material\">1<\/a>\\]). The clinical trial would have been carried out in Canada \\[19\\], whereas it is unclear in which country the case-report was produced \\[18\\]. The sample sizes ranged from a case-report of 1 patient \\[18\\] to 2056 case files \\[15\\].\n\nFive of the 11 surveys had low response rates: 20% \\[5\\], 22% \\[4\\], 35% \\[13\\], 44% \\[11\\], 51% \\[10\\], and in one survey the response rate does not appear to have been reported \\[12\\]. In the file inspection study, the proportion of participants was higher (71%) than the percentage of practitioners whose files were inspected (35%) \\[15\\]. Two of the surveys with higher response rates included specific study samples that nevertheless probably did not represent the general chiropractic profession; one of recently graduated Australian chiropractors \\[9\\] and one in which Australian chiropractors were included on the basis of whether they practised in a chiropractor-dense area or not \\[16\\].\n\nIn summary, there were only few studies, covering a wide time-period, unevenly distributed across the world, and the study participants were often unlikely to be representative of their target populations.\n\n## Definitions of maintenance care\n\nThe definitions of maintenance care that we found were not based on scientific evidence of the clinical validity of maintenance care but perhaps more on opinion and consensus.\n\nBecause there is a conviction among some chiropractors that spinal adjustments\/manipulations have a preventive effect not only on musculoskeletal problems but also on the general state of health, it is not surprising that one \"officially recognized and approved\" definition of maintenance care that we found was rather vague. This would allow the chiropractor to apply it according to his\/her own scope of practice. The definition is \"Appropriate treatment directed toward maintaining optimal body function. This is treatment of the symptomatic patient who has reached pre-clinical status or maximum medical improvement, where condition is resolved or stable\" \\[20\\].\n\nAnother definition found in the literature is \"...a regimen designed to provide for the patient's continued well-being or for maintaining the optimum state of health while minimizing recurrences of the clinical status\" in brief also \"continuing care\" \\[6\\]. These definitions resemble that used by Breen, when reporting his study, in 1976: \"... treatment, either scheduled or elective, which occurred after optimum recorded benefit was reached, provided there was no evidence of relapse.\" \\[15\\]. But on the other hand, according to a recent British study of chiropractors, osteopaths and physiotherapists, at least 10% of each profession reported that they sometimes continue to treat patients with low back pain who show almost no improvement \\[14\\]. Therefore, it appears that maintenance care is also offered to patients who have *not* improved. Boline and Sawyer \\[10\\] report on \"regular chiropractic care\", which we interpreted as meaning maintenance care.\n\nOther authors, who studied this subject, did not define maintenance care at all \\[9,13,16,17\\] and in one study such non-definition was stated to be purposeful, in order to prevent bias of the investigation process \\[11\\].\n\n## Indications for maintenance care\n\nThere was only sparse information in the literature on the indications for maintenance care and there was no information on specific indications for particular conditions.\n\nThe chiropractors in a North-American study by Rupert \\[11\\] generally agreed that the purposes of maintenance care were to minimize recurrence or exacerbation, maintain or optimize state of health, prevent conditions from developing, provide palliative care for \"incurable\" conditions, and determine and treat subluxations (all statements with over 80% agreement). Fifty-six percent meant that the purpose of maintenance care was to prevent subluxations, and this was confirmed in a similar study of Australian chiropractors \\[13\\]. Most of these statements reflect secondary or tertiary preventive approach. Nevertheless, some could also be interpreted as referring to a primary preventive approach.\n\nInterestingly, the concept of \"prevention and health promotion\" was used in a North American study of maintenance care in relation to chiropractic patients aged at least 65 \\[12\\]. Boline and Sawyer explored similar concepts, also in a study from North America, in which they investigated attitudes among chiropractors in relation to counselling of patients on a healthy lifestyle, i.e. including elements of primary prevention, as well as regular chiropractic care. \\[10\\]. This shows that they mean that chiropractors should participate in health promotion and prevention of disease, outside the realm of the purely musculoskeletal.\n\nAccording to a prospective multicenter study of 115\/205 Norwegian chiropractors and 832 patients with persistent low back pain, patients, who after one year reported definite improvement, were treated only a few times and those with poorer outcome had a larger number of consultations over a period of one year. This might indicate that tertiary prevention is offered to and accepted by some of those patients with persistent low back pain, who fail to recover, but that secondary prevention in patients with more satisfactory outcome is less common \\[17\\]. This information is the only indication of how maintenance care is actually employed in clinical practice.\n\n## The nature of the use of maintenance care\n\nAccording to the Mercy Guidelines \\[7\\], spinal adjustments are central in maintenance care. However, the research literature is vague on the contents of a maintenance care consultation and the frequency of treatments required for different types of patients.\n\nRupert \\[11\\] asked his North-American study subjects to describe the therapeutic components of maintenance care and concluded that they were adjustments\/spinal manipulation, exercises, proper eating habits, patient education, and vitamin supplementation. This finding was confirmed in a similar a study of Australian chiropractors \\[13\\] and in another study of Rupert et al in North American patients, aged 65 or older \\[12\\].\n\nWenban described the outcome in relation to the various outcomes measures used when treating one female patient with a complaint of uncomplicated chronic low back pain \\[18\\]. His indications for treatment were \"tenderness of the patient's vertebral spinous processes, S2 spinous process, and the superior aspect of the posterior superior iliac spines\". According to the author, this indicated a \"subluxation\", and providing that there were no other \"more serious indicators\" (not further specified), treatment was provided. The treatment consisted of adjustments only, using a combination of diversified and sacro-occipital techniques (a reference is provided to two textbooks\\].\n\nWenban also described the frequency of visits, after a 12 weeks intensive care period, as 2 times per week for 6 weeks, 1 time per week for 2 weeks, and 1 time per 2 weeks for the reminder of the study period of 5.5 months. In addition, Jamison offers some information on this subject. According to the participants in one of her studies: \"Maintenance adjustments should be offered on a basis of once a month to once every three to four months\" \\[4\\]. According to one of Rupert's studies, North American patients who agree to receive maintenance care, average 14 visits per year \\[11\\], and in another study, elderly maintenance care patients (65+ years of age) average 17 visits per year \\[12\\]. This amounts to a little more than one visit per month.\n\nThere were no studies of different types of strategies in relation to different types of patients or conditions.\n\n## Beliefs among chiropractors and acceptance among patients\n\nBoline and Sawyer in a 1987-survery of North American chiropractors noted that 98% of their participants believed that \"regular chiropractic care would be important for the 'average' person\" \\[10\\]. Jamison found that 93% of Australian chiropractors considered that at least some patients would require maintenance care (and that 41% thought that all patients would) \\[4\\]. In another survey, she found that 92% believed that spinal adjustments promote health in asymptomatic patients \\[5\\].\n\nHowever, the last two studies dealt primarily with the concept of spinal adjustments and the prevention of endocrine, visceral and autonomic dysfunction, which may have incited chiropractors of specific opinions to participate and, indeed, the very low response rates (22% and 20%, respectively) indicate that this may have been the case.\n\nThese beliefs are underpinned by the finding in the study by Rupert \\[11\\], in which 40% of the chiropractic respondents believe that there is adequate research to support the concept of maintenance care. The Australian respondents were less na\u00efve, with only 22% supporting this statement \\[13\\].\n\nWe found no information on the patients' perspective of maintenance care. However, in one study it was stated that 79% of patients are recommended for maintenance care and that 34% of those \"elect to receive these services\" \\[11\\]. It is not clear, whether these estimates are based on an objective count of patient files or on the participating chiropractors' opinions. However, the figure of 34% does not appear unreasonable, given the personal experience of many chiropractors that only some patients are willing and able to continue treatment past the acute event.\n\nEven among patients with persistent low back pain, maintenance care might not be attractive. Of the 832 participants in a large Norwegian multi-centre prospective outcome study, all with persistent low back pain at base line, only 14% were reported by their chiropractors to have received some type of maintenance care during the subsequent year \\[17\\].\n\nIn summary, it is possible that most chiropractors believe in maintenance care but data from unbiased samples are missing. The opinion of patients is unknown, although it might be less positive, as a relatively low percentage of patients seem to accept to continue treatment past the initial treatment program.\n\n## Prevalence of use and factors associated with its use\n\nAlthough the concept of maintenance care seems to be firmly ensconced in the chiropractic profession, the frequency of its use has not been clearly described.\n\nBreen reported in 1976 that 36% of 2987 case files belonged to patients who received maintenance care \\[15\\]. These patients were obtained from a sample belonging to 24 British chiropractors, i.e. 35% of the practitioners at that time. They had been selected to give a proportional representation of practitioners in the British hospital regions, and a 20% randomly selected sample of up to 1000 case files was taken from each practice. There were proportionally fewer files from newly established practices. Most of these chiropractors were educated in North America, practising at a time when there were relatively few manipulating practitioners but also at a time when chiropractic was relatively unknown. It was noted that most patients consulted for \"rheumatic conditions and in particular low back pain\" whereas non-musculoskeletal problems were very rare. This appears to be the first investigative study on this subject, and it probably provides a fair picture of the use of maintenance care in the UK at that time.\n\nOthers have reported the proportion of patients who receive maintenance care in the literature. Webb and Leboeuf in 1987 found that 44% of newly graduated Australian chiropractors estimated that at least 34% of their patients were on maintenance care \\[9\\].\n\nA similar estimate was obtained in another Australian study published two years later \\[16\\]. In both these two studies, only 6% of the respondents reported that more than 2\/3 of their patients received maintenance care. However, although the response rates were better in these, two studies (65% and 82%, respectively); their estimates were based on the practitioners' opinion rather than exact counts of patient files.\n\nObviously, the use of maintenance care will affect the clinic income. According to Rupert's participants, 23% of practices' incomes was generated from maintenance care \\[11\\]. That the use of maintenance care can affect the patient turnover was shown in the Leboeuf et al study from Australia \\[16\\].\n\nWe were unable to find any information on which factors play a role, when a chiropractor decides to offer a maintenance care program to a patient, and no information seems to be available on what considerations patients take into account when deciding to accept such a program.\n\n## Efficacy and cost-effectiveness for various types of conditions\n\nIt remains also to study the efficacy and cost-effectiveness of maintenance care for various types of conditions.\n\nTo our knowledge, it has only been attempted to test the efficacy of maintenance care in one well-designed pilot study, in which 29 patients with chronic low back pain were randomly allocated to either a non-maintenance care group or a maintenance care group (for a 9-month treatment after one initial month of treatment). At follow-up, there was no difference in pain but the group that received maintenance care had lower disability scores than the control group \\[19\\]. A full-scale trial is presently underway on patients with neck pain (personal communication \u2013 M. Descarreaux).\n\n# Discussion\n\nThis literature review reveals that more than 30 years of ad hoc research into maintenance care does not provide much information. In fact, the relative paucity of studies, the obvious biases in many of these, the lack of exhaustive information, and the diversity of findings made it impossible to answer any of our questions.\n\nIt is not clear whether chiropractors use maintenance care mainly as a secondary or tertiary preventive measure, and if so how frequently and for which conditions. It is possible that there are considerable differences between countries and perhaps during certain periods. The Australian literature in the 1990s dealt with some fundamental concepts, such as \"the healthy spine \u2013 freedom from disease\" concept, which the UK study 20 years earlier did not concern itself with. In between, Mitchell in 1980 \\[6\\] transmitted a warning on over-servicing, which is the negative side of maintenance care if used unwisely. None of the studies looked at this very difficult balance.\n\nSeveral studies indicated that almost all chiropractors believe in the value of maintenance care, but newer studies on unbiased study samples are needed to confirm these findings. In addition, it is possible that differences exist between different groups of chiropractors. Would chiropractors who graduated from a university-based chiropractic institution have the same beliefs as those coming from chiropractic colleges of a more traditional type? Further, the density of chiropractors in a region might have an effect on this aspect.\n\nPatients' opinions and expectations of maintenance care and their satisfaction with maintenance care appear not to have been studied. This is, nevertheless, an important subject. Unless expectations and results match, patients are likely to become unsatisfied.\n\nThe treatment program, including contents and time schedule, requires further study. Only one report deals explicitly with the timing of treatments. Wenban \\[18\\] describes a pre hoc determined and rigid plan for a large number of treatment visits in a young patient with \"uncomplicated\" low back pain for more than three months. We know, from personal experience that also other, more flexible, treatment plans are used, but how common are these respective approaches?\n\nDespite the bulk of the literature being of poor quality, there seems to be light at the end of the tunnel. It has been shown that it is feasible to conduct a randomized controlled clinical trial of maintenance care, and a full-scale study is presently underway. However, several aspects other than efficacy need to be investigated. For example, it would be relevant to test the cost-effectiveness for patients who choose to be treated regularly vs. those who receive treatment only when symptoms arise. This should be investigated for different types of conditions and using different treatment programs. In other words, the aim should be to be able to say, not only, if maintenance care is a clinically valid treatment approach but, also, for whom it should be used, how and when.\n\nHowever, before testing the clinical validity of maintenance care, it would be necessary to find out what it is, how it is practised and on which indications, to make sure that such a trial does reflect the situation in real life.\n\n# Conclusion\n\n\u2022 There is no evidence-based definition of maintenance care and the indications for and nature of its use remains to be clearly stated.\n\n\u2022 It is likely that many chiropractors believe in the usefulness of maintenance care but it seems to be less well accepted by their patients.\n\n\u2022 The prevalence with which maintenance care is used has not been established.\n\n\u2022 Efficacy and cost-effectiveness of maintenance care for various types of conditions are unknown.\n\nTherefore, the recommendations given by Aker and Martel \\[8\\] more than a decade ago are still relevant, namely that \"before a large-scale, multicentered clinical trial can be pursued, a series of preliminary studies need to be conducted to delineate the parameters of care to be used in the clinical trial, the outcome measures to be used, and the feasibility of conducting such a complicated and resource-intensive study.\"\n\n# Competing interests\n\nThe authors declare that they have no competing interests.\n\n# Authors' contributions\n\nCY did the literature search. Both authors read the articles and abstracted the data. CY made the first draft of the manuscript. LH revised it critically for intellectual content. Both authors read and approved the final manuscript.\n\n# Supplementary Material\n\n###### Additional file 1\n\nTable 1. Description of 11 surveys in a review on the use of maintenance care among chiropractors. The table provides an overview of the reviewed articles, including author, year of publication, year and country of study, population, response rate, sampling method, data collection and objectives as they relate to the present review.\n\nClick here for file\n\n### Acknowledgements\n\nThe manuscript was preared within the scope of the authors' positions at The Nordic Institute of Chiropractic and Clinical Biomechanics. The only role of the Institute was to pay the authors' salary. Otherwise, the Institute had no role in the study design; in the collection, analyis an interpretation of data; in the writing of the manuscript; or in the decision to submit the manuscript for publication.","meta":{"dup_signals":{"dup_doc_count":101,"dup_dump_count":59,"dup_details":{"curated_sources":2,"2022-27":1,"2021-39":1,"2021-04":2,"2020-40":2,"2020-29":1,"2019-51":1,"2019-43":1,"2019-30":2,"2019-22":1,"2019-09":1,"2018-51":1,"2018-43":1,"2018-34":1,"2018-22":1,"2018-13":1,"2018-09":1,"2018-05":2,"2017-51":2,"2017-47":2,"2017-43":1,"2017-39":2,"2017-34":2,"2017-30":2,"2017-26":3,"2017-22":3,"2017-17":3,"2017-09":2,"2017-04":2,"2016-50":2,"2016-44":2,"2016-40":3,"2016-36":2,"2016-30":1,"2016-22":1,"2016-18":1,"2015-48":1,"2015-35":1,"2015-32":2,"2015-27":2,"2015-22":2,"2015-14":1,"2014-52":2,"2014-49":1,"2014-42":5,"2014-41":2,"2014-35":2,"2014-23":2,"2014-15":3,"2023-40":1,"2024-26":1,"2024-22":1,"2024-10":1,"2017-13":2,"2015-18":2,"2015-11":1,"2015-06":2,"2014-10":2,"2013-48":2,"2024-30":1}},"file":"PMC2396648"},"subset":"pubmed_central"}
{"text":"date: 2013-03-15\ntitle: Findings of research misconduct.\n\nFindings of Research Misconduct\n\n**Notice Number:**\u00a0 NOT-OD-13-049**  \n  \n  \n**\n\n\u00a0 NOT-OD-13-049**  \n  \n  \n**\n\nKey Dates\n\n**Release Date:** \u00a0\u00a0March 13, 2013 \u00a0\n\nIssued by\n\nDepartment of Health and Human Services\u00a0\n\nPurpose\n\nSUMMARY: Notice is hereby given that the Office of Research Integrity (ORI) has taken final action in the following case:\n\nAdam C. Savine, Washington University in St. Louis: Based on the report from Washington University in St. Louis (WUSTL) and Respondent's admission, ORI found that Mr. Adam C. Savine, former doctoral student, Department of Psychology, WUSTL, engaged in research misconduct in research supported by National Institute of Mental Health (NIMH), National Institutes of Health (NIH), grant R56 MH066078, National Institute on Drug Abuse (NIDA), NIH, grants F31 DA032152 and R21 DA027821, and National Institute on Aging (NIA), NIH, grant T32 AG00030.\n\nORI found that the Respondent engaged in research misconduct by falsifying data that were included in the following three publications and six conference abstracts:\n\nPublications\n\n1\\. Savine, A.C., & Braver, T.S. \\`\\`Local and global effects of motivation on cognitive control.' Cogn Affect Behav Neurosci. 12(4):692-718, 2012 Dec. (hereafter referred to as Cogn Affect Behav Neurosci. 2012).\n\n2\\. Savine, A.C., McDaniel, M.A., Shelton, J.T., Scullin, M.K. \\`\\`A characterization of individual differences in prospective memory monitoring using the Complex Ongoing Serial Task.' J Exp Psychol Gen. 141(2):337-62, 2012 May (hereafter referred to as J Exp Psychol Gen. 2012).\n\n3\\. Savine, A.C., & Braver, T.S. \\`\\`Motivated cognitive control: Reward incentives modulate preparatory neural activity during task- switching.' J Neurosci. 30(31):10294-305, 2010 Aug 4 (hereafter referred to as J Neurosci. 2010).\n\nConference Abstracts\n\n1\\. Savine, A.C., & Braver, T.S. (November 2010) \\`\\`The contextual and local effects of motivation on cognitive control.' Psychonomics Society, St. Louis, MO.\n\n2\\. Savine, A.C., & Braver, T.S. (November 2010) \\`\\`A model-based characterization of the individual differences in prospective memory monitoring.' Psychonomics Society, St. Louis, MO.\n\n3\\. Savine, A.C., & Braver, T.S. (November 2010) \\`\\`Motivated cognitive control: Reward incentives modulate preparatory neural activity during task-switching.' Society for Neuroscience, San Diego, CA.\n\n4\\. Savine, A.C., & Braver, T.S. (June 2010) \\`\\`Motivated cognitive control: Reward incentives modulate preparatory neural activity during task-switching.' Motivation and Cognitive Control Conference, Oxford, England.\n\n5\\. Savine, A.C., & Braver, T.S. (January 2010) \\`\\`Neural correlates of the motivation\/cognitive control interaction: Activation dynamics and Performance prediction during task-switching.' Genetic and Experiential Influences on Executive Function, Boulder, CO.\n\n6\\. Savine, A.C., & Braver, T.S. (June 2009) \\`\\`Incentive Induced Changes in Neural Patterns During Task-Switching.' Organization for Human Brain Mapping, San Francisco, CA.\n\nAs a result of the Respondent's admission, the senior authors will request that the published papers be retracted or corrected.\n\nORI finds that Respondent falsified data and related text in Cogn Affect Behav Neurosci. 2012, J Exp Psychol Gen. 2012, J Neurosci. 2010, and in six (6) meeting abstracts, by altering the experimental data to improve the statistical results. Specifically, Respondent:\n\n1\\. Falsified data in Cogn Affect Behav Neurosci. 2012 to show an unambiguous dissociation between local and global motivational effects. Specifically, Respondent exaggerated (1) the effect of incentive context on response times and error rates in Table 1 and Figures 1 and 3 for experiment 1 and (2) the effect of incentive cue timing on response times and error rates in Table 2 and in Figures 6, 9, and S2 for experiment 2.\n\n2\\. Falsified data in J Exp Psychol Gen. 2012 to show that prospective memory is influenced by three dissociable underlying monitoring patterns (attentional focus, secondary memory retrieval, information thresholding), which are stable within individuals over time and are influenced by personality and cognitive differences. Specifically, Respondent modified the data to support the three category model and to show (1) that individuals fitting into each of the three categories exhibited differential patterns of prospective memory performance and ongoing task performance in Tables 1-3; Figures 5-8, and (2) that certain cognitive and personality differences were predictive of distinct monitoring approaches within the three categories in Figure 9.\n\n3\\. Falsified data in J Neurosci. 2010 and mislabeled brain images to show that motivational incentives enhance task-switching performance and are associated with activation of reward-related brain regions, behavioral performance, and trial outcomes. Specifically, Respondent modified the data so that he could show a stronger relationship between brain activity and behavior in Table 2 and Figure 4 and used brain images that fit the data rather than the images that corresponded to the actual Talairach coordinates in Figure 3.\n\nMr. Savine has entered into a Voluntary Settlement Agreement and has voluntarily agreed for a period of three (3) years, beginning on February 22, 2013:\n\n\\(1\\) To have his research supervised; Respondent agreed that prior to the submission of an application for U.S. Public Health Service (PHS) support for a research project on which his participation is proposed and prior to his participation in any capacity on PHS-supported research, Respondent shall ensure that a plan for supervision of his duties is submitted to ORI for approval; the supervision plan must be designed to ensure the scientific integrity of his research contribution; he agreed that he shall not participate in any PHS-supported research until such a supervision plan is submitted to and approved by ORI; Respondent agreed to maintain responsibility for compliance with the agreed upon supervision plan;\n\n\\(2\\) That any institution employing him shall submit, in conjunction with each application for PHS funds, or report, manuscript, or abstract involving PHS-supported research in which Respondent is involved, a certification to ORI that the data provided by Respondent are based on actual experiments or are otherwise legitimately derived and that the data, procedures, and methodology are accurately reported in the application, report, manuscript, or abstract;\n\n\\(3\\) To exclude himself voluntarily from serving in any advisory capacity to PHS including, but not limited to, service on any PHS advisory committee, board, and\/or peer review committee, or as a consultant; and\n\n\\(4\\) That the senior authors will request that the following papers be retracted or corrected: Cogn Affect Behav Neurosci. 2012, J Exp Psychol Gen. 2012, and J Neurosci. 2010.\n\nInquiries\n\nPlease direct all inquiries to:**  \n  \n** David E. Wright, Ph.D.**  \n** Director**  \n** Office of Research Integrity**  \n** 1101 Wootton Parkway, Suite 750**  \n** Rockville, MD 20852","meta":{"dup_signals":{"dup_doc_count":128,"dup_dump_count":35,"dup_details":{"curated_sources":2,"2017-30":1,"2017-22":1,"2017-17":1,"2017-09":2,"2017-04":1,"2016-50":1,"2016-44":1,"2016-40":1,"2016-36":1,"2016-26":1,"2016-22":1,"2016-18":1,"2016-07":6,"2015-48":6,"2015-40":5,"2015-35":7,"2015-32":5,"2015-27":5,"2015-22":5,"2015-14":3,"2014-52":6,"2014-49":8,"2014-42":7,"2014-41":8,"2014-35":7,"2014-23":6,"2014-15":6,"2018-05":1,"2015-18":3,"2015-11":5,"2015-06":3,"2014-10":4,"2013-48":3,"2013-20":3,"2024-18":1}},"file":"PMC4259747"},"subset":"pubmed_central"}
{"text":"author: John M Barry\ndate: 2004\ninstitute: 1Distinguished Visiting Scholar, the Center for Bioenvironmental Research of Tulane and Xavier Universities, New Orleans, Louisiana, USA\nreferences:\ntitle: The site of origin of the 1918 influenza pandemic and its public health implications\n\nThe 1918\u20131919 influenza pandemic killed more people than any other outbreak of disease in human history. The lowest estimate of the death toll is 21 million, while recent scholarship estimates from 50 to 100 million dead. World population was then only 28% what is today, and most deaths occurred in a sixteen week period, from mid-September to mid-December of 1918.\n\nIt has never been clear, however, where this pandemic began. Since influenza is an endemic disease, not simply an epidemic one, it is impossible to answer this question with absolute certainty. Nonetheless, in seven years of work on a history of the pandemic, this author conducted an extensive survey of contemporary medical and lay literature searching for epidemiological evidence \u2013 the only evidence available. That review suggests that the most likely site of origin was Haskell County, Kansas, an isolated and sparsely populated county in the southwest corner of the state, in January 1918 \\[1\\]. If this hypothesis is correct, it has public policy implications.\n\nBut before presenting the evidence for Haskell County it is useful to review other hypotheses of the site of origin. Some medical historians and epidemiologists have theorized that the 1918 pandemic began in Asia, citing a lethal outbreak of pulmonary disease in China as the forerunner of the pandemic. Others have speculated the virus was spread by Chinese or Vietnamese laborers either crossing the United States or working in France.\n\nMore recently, British scientist J.S. Oxford has hypothesized that the 1918 pandemic originated in a British Army post in France, where a disease British physicians called \"purulent bronchitis\" erupted in 1916. Autopsy reports of soldiers killed by this outbreak \u2013 today we would classify the cause of death as ARDS \u2013 bear a striking resemblance to those killed by influenza in 1918 \\[2\\].\n\nBut these alternative hypotheses have problems. After the 1918\u20131919 pandemic, many investigators searched for the source of the disease. The American Medical Association sponsored what is generally considered the best of several comprehensive international studies of the pandemic conducted by Dr. Edwin Jordan, editor of ***The Journal of Infectious Disease***. He spent years reviewing evidence from all over the world; the AMA published his work in 1927.\n\nSince several influenza pandemics in preceding centuries were already well-known and had come from the orient, Jordan first considered Asia as the source. But he found no evidence. Influenza did surface in early 1918 in China, but the outbreaks were minor, did not spread, and contemporary Chinese scientists, trained by Rockefeller Institute for Medical Research (now Rockefeller University) investigators, stated they believed these outbreaks were endemic disease unrelated to the pandemic \\[3\\]. Jordan also looked at the lethal pulmonary disease cited by some historians as influenza, but this was diagnosed by contemporary scientists as pneumonic plague. By 1918 the plague bacillus could be easily and conclusively identified in the laboratory \\[3\\]. So after tracing all known outbreaks of respiratory disease in China, Jordan concluded that none of them \"could be reasonably regarded as the true forerunner\" of the pandemic \\[3\\].\n\nJordan also considered Oxford's theory that the \"purulent bronchitis\" in British Army camps in 1916 and 1917 was the source. He rejected it for several reasons. The disease had flared up, true, but had not spread rapidly or widely outside the affected bases; instead, it seemed to disappear \\[3\\]. As we now know a mutation in an existing influenza virus can account for a virulent flare-up. In the summer of 2002, for example, an influenza epidemic erupted in parts of Madagascar with an extremely high mortality and morbidity; in some towns it sickened an outright majority \u2013 in one instance sixty-seven percent \u2013 of the population. But the virus causing this epidemic was an H3N2 virus that normally caused mild disease. In fact, the epidemic affected only thirteen of 111 health districts in Madagascar before fading away \\[4\\]. Something similar may have happened in the British base.\n\nJordan considered other possible origins of the pandemic in early 1918 in France and India. He concluded that it was highly unlikely that the pandemic began in any of them \\[3\\].\n\nThat left the United States. Jordan looked at a series of spring outbreaks there. The evidence seemed far stronger. One could see influenza jumping from Army camp to camp, then into cities, and traveling with troops to Europe. His conclusion: the United States was the site of origin.\n\nA later equally comprehensive, multi-volume British study of the pandemic agreed with Jordan. It too found no evidence for the influenza's origin in the Orient, it too rejected the 1916 outbreak among British troops, and it too concluded, \"The disease was probably carried from the United States to Europe \\[5\\].\"\n\nAustralian Nobel laureate MacFarlane Burnet spent most of his scientific career working on influenza and studied the pandemic closely. He too concluded that the evidence was \"strongly suggestive\" that the disease started in the United States and spread with \"the arrival of American troops in France \\[6\\].\"\n\nBefore dismissing the conclusions of these contemporary investigators who lived through and studied the pandemic, one must remember how good many of them were. They were very good indeed.\n\nThe Rockefeller Institute, whose investigators were intimately involved in the problem, alone included extraordinary people. By 1912 its head Simon Flexner \u2013 his brother wrote the \"Flexner report\" that revolutionized American medical education \u2013 used immune serum to bring the mortality rate for meningococcal meningitis down from over 80% to 18%; by contrast, in the 1990s at Massachusetts General Hospital a study found a 25% mortality rate for bacterial meningitis. Peyton Rous won the Nobel Prize in 1966 for work he did at the institute in 1911; he was that far ahead of the scientific consensus. By 1918 Oswald Avery and others at Rockefeller Institute had already produced both an effective curative serum and a vaccine for the most common pneumococcal pneumonias. At least partly because of the pandemic, Avery would spend the rest of his career studying pneumonia. That work led directly to his discovery of the \"transforming principle\" \u2013 his discovery that DNA carries the genetic code.\n\nThe observations of investigators of this quality cannot be dismissed lightly. Jordan was of this quality.\n\nMore evidence against Oxford's hypothesis comes from Dr. Jeffrey Taubenberger, well-known for his work extracting samples of the 1918 virus from preserved tissue and sequencing its genome. He initially believed, based on statistical analysis of the rate of mutation of the virus that it existed for two or three years prior to the pandemic. But further work convinced him that the virus emerged only a few months prior to the pandemic (personal communication with the author from J Taubenberger, June 5^th^ 2003).\n\nSo if the contemporary observers were correct, if American troops carried the virus to Europe, where in the United States did it begin?\n\nBoth contemporary epidemiological studies and lay histories of the pandemic have identified the first known outbreak of epidemic influenza as occurring at Camp Funston, now Ft. Riley, in Kansas. But there was one place where a previously unknown \u2013 and remarkable \u2013 epidemic of influenza occurred.\n\nHaskell County, Kansas, lay three hundred miles to the west of Funston. There the smell of manure meant civilization. People raised grains, poultry, cattle, and hogs. Sod-houses were so common that even one of the county's few post offices was located in a dug-out sod home. In 1918 the population was just 1,720, spread over 578 square miles. But primitive and raw as life could be there, science had penetrated the county in the form of Dr. Loring Miner. Enamored of ancient Greece \u2013 he periodically reread the classics in Greek \u2013 he epitomized William Welch's comment that \"the results \\[of medical education\\] were better than the system.\" His son was also a doctor, trained in fully scientific ways, serving in the Navy in Boston.\n\nIn late January and early February 1918 Miner was suddenly faced with an epidemic of influenza, but an influenza unlike any he had ever seen before. Soon dozens of his patients \u2013 the strongest, the healthiest, the most robust people in the county \u2013 were being struck down as suddenly as if they had been shot. Then one patient progressed to pneumonia. Then another. And they began to die. The local paper ***Santa Fe Monitor***, apparently worried about hurting morale in wartime, initially said little about the deaths but on inside pages in February reported, \"Mrs. Eva Van Alstine is sick with pneumonia. Her little son Roy is now able to get up... Ralph Lindeman is still quite sick... Goldie Wolgehagen is working at the Beeman store during her sister Eva's sickness... Homer Moody has been reported quite sick... Mertin, the young son of Ernest Elliot, is sick with pneumonia... Pete Hesser's children are recovering nicely... Ralph McConnell has been quite sick this week (Santa Fe Monitor, February 14^th^, 1918).\"\n\nThe epidemic got worse. Then, as abruptly as it came, it disappeared. Men and women returned to work. Children returned to school. And the war regained its hold on people's thoughts.\n\nThe disease did not, however, slip from Miner's thoughts. Influenza was neither a reportable disease, nor a disease that any state or federal public health agency tracked. Yet Miner considered this incarnation of the disease so dangerous that he warned national public health officials about it. ***Public Health Reports*** (now ***Morbidity and Mortality Weekly Report***), a weekly journal produced by the U.S. Public Health Service to alert health officials to outbreaks of communicable diseases throughout the world, published his warning. In the first six months of 1918, this would be the only reference in that journal to influenza anywhere in the world.\n\nHistorians and epidemiologists have previously ignored Haskell most likely because his report was not published until April and it referred to deaths on March 30, after influenza outbreaks elsewhere. In actuality, by then the county was free of influenza. Haskell County, Kansas, is the first recorded instance anywhere in the world of an outbreak of influenza so unusual that a physician warned public health officials. It remains the first recorded instance suggesting that a new virus was adapting, violently, to man.\n\nIf the virus did not originate in Haskell, there is no good explanation for how it arrived there. There were no other known outbreaks anywhere in the United States from which someone could have carried the disease to Haskell, and no suggestions of influenza outbreaks in either newspapers or reflected in vital statistics anywhere else in the region. And unlike the 1916 outbreak in France, one can trace with perfect definiteness the route of the virus from Haskell to the outside world.\n\nAll Army personnel from the county reported to Funston for training. Friends and family visited them at Funston. Soldiers came home on leave, then returned to Funston. ***The Monitor*** reported in late February, \"Most everybody over the country is having lagrippe or pneumonia (Santa Fe Monitor, February 21^st^ 1918).\" It also noted, \"Dean Nilson surprised his friends by arriving at home from Camp Funston on a five days furlough. Dean looks like soldier life agrees with him.\" He soon returned to the camp. Ernest Elliot left to visit his brother at Funston as his child fell ill. On February 28, John Bottom left for Funston. \"We predict John will make an ideal soldier,\" said the paper (Santa Fe Monitor February 28^th^, 1918).\n\nThese men, and probably others unnamed by the paper, were exposed to influenza and would have arrived in Funston between February 26 and March 2. On March 4 the first soldier at the camp reported ill with influenza at sick call. The camp held an average of 56,222 troops. Within three weeks more than eleven hundred others were sick enough to require hospitalization, and thousands more \u2013 the precise number was not recorded \u2013 needed treatment at infirmaries scattered around the base.\n\nWhether or not the Haskell virus did spread across the world, the timing of the Funston explosion strongly suggests that the influenza outbreak there did come from Haskell. Meanwhile Funston fed a constant stream of men to other American locations and to Europe, men whose business was killing. They would be more proficient at it than they knew.\n\nSoldiers moved uninterrupted between Funston and the outside world, especially to other Army bases and France. On March 18, Camps Forrest and Greenleaf in Georgia saw their first cases of influenza and by the end of April twenty-four of the thirty-six main Army camps suffered an influenza epidemic \\[3\\]. Thirty of the fifty largest cities in the country also had an April spike in excess mortality from influenza and pneumonia \\[7\\]. Although this spring wave was generally mild \u2013 the killing second wave struck in the fall \u2013 there were still some disturbing findings. A subsequent Army study said, \"At this time the fulminating pneumonia, with wet hemorrhagic lungs, fatal in from 24 to 48 hours, was first observed \\[8\\].\" (Pathology reports suggest what we now call ARDS.) The first recorded autopsy in Chicago of an influenza victim was conducted in early April. The pathologist noted, \"The lungs were full of hemorrhages.\" He found this unusual enough to ask the then-editor of ***The Journal of Infectious Diseases*** \"to look over it as a new disease\" \\[3\\].\n\nBy then, influenza was erupting in France, first at Brest, the single largest port of disembarkation for American troops. By then, as MacFarlane Burnet later said, \"It is convenient to follow the story of influenza at this period mainly in regard to the army experiences in America and Europe \\[6\\].\"\n\nThe fact that the 1918 pandemic likely began in the United States matters because it tells investigators where to look for a new virus. They must look everywhere.\n\nIn recent years the World Health Organization and local public health authorities have intervened several times when new influenza viruses have infected man. These interventions have prevented the viruses from adapting to man and igniting a new pandemic. But only 83 countries in the world \u2013 less than half \u2013 participate in WHO's surveillance system (WHO's flunet website <http:\/\/rhone.b3e.jussieu.fr\/flunet\/www\/docs.html>). While some monitoring occurs even in those countries not formally affiliated with WHO's surveillance system, it is hardly adequate. If the virus did cross into man in a sparsely populated region of Kansas, and not in a densely populated region of Asia, then such an animal-to-man cross-over can happen anywhere. And unless WHO gets more resources and political leaders move aggressively on the diplomatic front, then a new pandemic really is all too inevitable.","meta":{"dup_signals":{"dup_doc_count":101,"dup_dump_count":70,"dup_details":{"curated_sources":2,"2023-40":2,"2023-14":1,"2022-49":1,"2022-40":1,"2022-21":1,"2021-43":2,"2021-31":1,"2021-17":2,"2021-04":1,"2020-45":1,"2020-34":1,"2020-24":1,"2020-16":1,"2020-05":1,"2019-39":1,"2019-30":1,"2019-26":1,"2019-22":1,"2019-13":1,"2019-09":2,"2019-04":1,"2018-47":2,"2018-39":1,"2018-30":2,"2018-26":1,"2018-13":1,"2018-09":1,"2018-05":1,"2017-51":1,"2017-47":1,"2017-43":1,"2017-39":2,"2017-34":2,"2017-30":2,"2017-26":2,"2017-22":2,"2017-17":1,"2017-09":2,"2017-04":2,"2016-50":2,"2016-44":2,"2016-40":2,"2016-36":2,"2016-30":3,"2016-26":1,"2016-22":1,"2016-18":1,"2016-07":1,"2015-48":1,"2015-40":1,"2015-35":1,"2015-32":2,"2015-27":1,"2015-22":1,"2014-52":1,"2014-49":1,"2014-42":3,"2014-41":1,"2014-35":3,"2014-23":2,"2014-15":2,"2024-26":1,"2024-22":2,"2017-13":2,"2015-18":1,"2015-11":1,"2015-06":1,"2014-10":2,"2013-20":1,"2024-30":1}},"file":"PMC340389"},"subset":"pubmed_central"}
{"text":"abstract: A report on the 2006 Keystone Conference on Signaling Networks, Vancouver, Canada, 30 January-4 February 2006.\nauthor: Nevan J Krogan; Timothy R Hughes\ndate: 2006\ninstitute: 1Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94143, USA; 2Banting and Best Department of Medical Research, University of Toronto, Toronto, ON, M5S 3E1, Canada\ntitle: Signals and systems\n\nThe boundaries between traditional notions of cellular signaling and more genomic and systematic approaches to biology are becoming increasingly blurred, and the recent Keystone conference on signaling networks reflected an expanded view of signaling. It is becoming increasingly accepted that genes, proteins, cells, and organisms function as components of larger systems, rather than independent activities contributing to a single defined outcome, and many presentations at the conference reflected this. If there was a single theme, it was the heavy reliance on technical approaches in functional genomics, proteomics, and computational biology, such that conceptual and technical discussions often dominated the resulting biology.\n\n# RNA interference screening\n\nThe impact of technology on the study of signaling networks was most evident in the widespread application of RNA interference (RNAi) screens. Screens to study signaling networks in multicellular organisms using RNAi technology are being performed with success, although several hurdles clearly remain. One of these is the apparently high rate of false positives. In his keynote lecture, Norbert Perrimon (Harvard Medical School, Boston, USA) predicted an uncomfortably high rate of false positives due to off-target effects, at least in *Drosophila*. Consistent with this, Phil Beachy (Johns Hopkins University School of Medicine, Baltimore, USA) described an ongoing RNAi screen in *Drosophila* looking for proteins involved in the Wingless signaling pathway. Following efforts to study a previously uncharacterized gene identified by this screen, it was noted that there were 16 bases in the interfering RNA that were identical to *armadillo*, a known gene in the pathway, suggesting that it is an unanticipated off-target that could completely explain the phenotype conferred by the interfering RNA. Perrimon proposed that incorporating other types of data, such as protein-protein interaction information, can help decipher which hits are physiologically relevant. Rene Bernards (Netherlands Cancer Institute, Amsterdam, The Netherlands) commented that reporting lists of unconfirmed hits from genome-wide RNAi screens in humans might be detrimental to future work by creating distractions from more productive lines of research, and that scientists (and journals) perhaps need to be more mindful of this.\n\nDespite these limitations, work presented on *Caenorhabditis elegans* showed unquestionably that useful biological information could be extracted from RNAi screens. Gary Ruvkun (Massachusetts General Hospital, Boston, USA) and colleagues have been involved in a variety of screens looking for factors involved in lifespan, molting, defects in fat storage, and the RNAi pathway itself. Ruvkun described the complete list of hits from the molting screen as a \"rogues' gallery\" of phenotypes and molecular functions. One could argue that this might be evidence not so much of a high false-positive rate as of the dependence of a system on many components and processes; a corollary of this is that perturbation of an individual gene can have a myriad of physiological effects. Andrew Fraser (Wellcome Trust Sanger Institute, Cambridge, UK) presented work that supports this view. His group has been performing synthetic interaction screens in *C. elegans* in which previously characterized worm mutants defective in the epidermal growth factor (EGF)-Ras-Raf-MAP kinase pathway are treated with libraries encoding double-stranded RNAs, in order to identify synthetic genetic interactions. A significant outcome of this work was that RNAi of genes in certain functional categories results in synthetic effects with a high proportion of all mutants tested, suggesting that some cellular functions buffer or canalize many physiological traits, rather than perform a single physiological function.\n\nDavid Sabatini (Massachusetts Institute of Technology, Cambridge, USA) presented a thorough overview of technical aspects of the development and use of genome-scale lentiviral RNAi libraries for human and mouse. A consortium of groups in the public and private sectors is contributing to the creation of the libraries, which are sold by both Sigma (St. Louis, USA) and Open Biosystems (Huntsville, USA) and are available as glycerol stocks, plasmids and viruses. Sabatini commented that while this effort is still incomplete, some functional categories (for example, transcription factors) are fairly comprehensively covered. The efficacy of the system has been validated by targeting tyrosine kinases, and Sabatini reported that 90% of these genes have at least one target that results in a significant knockdown of expression. Sabatini also discussed issues of high-content screening, such as image archiving (a screen can generate 1 Tb of images), image analysis, and defining a 'hit'. He described Cell Profiler <http:\/\/jura.wi.mit.edu\/cellprofiler\/>, a free, open-source software package for analysis of thousands of cell images, which among other statistics can output cell counts, DNA content and mitotic index.\n\nSabatini gave his view on how to define true positives: as a rule of thumb, he suggested requiring two independent hairpins that work (the consortium library contains several for each gene), dose dependence, and complementation if possible. Finally, he noted that as antibodies are not available for the majority of human proteins, it is currently difficult to ascertain whether protein levels are impacted by RNAi. On a similar theme, Sumit Chanda (Genomic Institute of the Novartis Research Foundation, San Diego, USA) pointed out that 50% of work published in humans corresponds to less than 10% of the genome, with few (or no) publications for most known and predicted genes, which provides a justification for comprehensive screening efforts.\n\n# Chemical screening\n\nChemicals have long been used to perturb specific biological activities in experiments, and they have a particular appeal because of their potential application as drugs. Justin Lamb (Broad Institute, Cambridge, USA) described an ongoing effort to construct a transcriptional connectivity map for biomedical discovery, which relates microarray gene-expression profiles that are due to drug effects, disease, and gene perturbations. An initial goal is to find off-target uses for existing drugs; Lamb's group is currently analyzing 1,500 drugs approved by the US Food and Drug Administration (FDA), 500 non-drug bioactive compounds used as research tools, and the effects of the knockdown of 500 potential drug targets using lentivirally delivered short hairpin RNAs (shRNAs). A distinguishing feature of the connectivity map is the use of the Kalmogorov-Smirnov statistic to score a relationship between two experiments, even in the absence of an overall correlation. Lamb presented evidence from these analyses to indicate that connections can be drawn between drugs and diseases even if the experiments are performed in different cell types, or possibly even different organisms, which is significant because large-scale RNAi screens are typically performed in well behaved cultured cells.\n\nAn alternative assay for chemical activities was presented by Soren Jensby Nielsen (BioImage, Soeborg, Denmark), who described screening compounds for their impact on protein translocation. He reported the effects of around 50 compounds on the localization of a dozen different green fluorescent protein (GFP) fusion proteins. A surprising number of effects were observed, and although effects appear to be protein-specific (that is, they do not correlate with growth inhibition or other general effects), Nielsen stated that this screening approach has not yet identified any primary targets, indicating that the observed mislocalizations are probably downstream effects of perturbing the primary target. Nevertheless, this is an intriguing approach to screening, especially as it requires only a relatively basic setup compared with high-content assays.\n\n# Protein-interaction networks\n\nGiulio Superti-Furga (CeMM Center for Molecular Medicine, Vienna, Austria) and one of us (N.K.) both described efforts to purify protein complexes for the entire proteome of *Saccharomyces cerevisiae*. The two groups used a similar strategy involving the immunoprecipitation of affinity-tagged proteins followed by mass spectrometry. Although proteomic studies have been previously published in budding yeast, the current studies attempt to tag and purify approximately 10 times as many unique proteins than any previous study. Both studies have just been published and comparison of the datasets with each other and with others previously generated should allow the compilation of the most comprehensive protein-interaction map ever generated for an organism.\n\nA next goal will be to define similar physical interaction maps in higher organisms. Marc Vidal (Dana-Farber Cancer Institute, Boston, USA) presented work on uncovering interacting proteins in human, focusing on two-hybrid analyses that are then confirmed by pulldowns. Like Perrimon, Vidal was enthusiastic about weighting the significance of interactions on the basis of multiple independent data types, most notably phenotypic profiles.\n\n# Pathways and networks\n\nThe term signaling pathway implies a cause-and-effect relationship among a series of steps. Two talks concerned the development of computational methods for the inference of cause and effect, or for determining the importance of the nodes in a network, from quantitative genetic and phenotypic analysis. Eric Schadt (Rosetta Inpharmatics, Seattle, USA) pointed out that in genetic crosses information can only flow from genotype to phenotype. In contrast, gene-expression patterns can be either a cause or an effect of phenotypes. He gave a conceptual overview of the statistical approaches his group is using to distinguish whether gene-expression phenotypes are upstream or downstream of other phenotypes in large mouse crosses. In summary, one can ask which network structure is most consistent with the observed correlations, with quantitative trait loci used as an anchor. Alon Kaufman (Hebrew University, Jerusalem, Israel) presented algorithms for calculating the contribution of genes to a function that has been quantified via set of multiple mutants in factors that influence that function. These algorithms, known as the 'multi-perturbation shapely value analysis' and the 'functional influence network' algorithms, can recreate some features of known networks and should aid in predicting aspects of unknown pathways.\n\nAnalysis of the consequences of multiple perturbations has historically been a primary approach to dissecting genetic and signaling networks. On the other hand, these computational analyses require a collection of quantitative genetic data. Two talks described methods for generating such data on a large scale in budding yeast. Jef Boeke (Johns Hopkins University, Baltimore, USA) described a comprehensive genetic analysis of approximately 70 genes involved in DNA integrity. Analysis of the genetic patterns enables the establishment of 'congruence groups', functionally related clusters that share the same pattern of synthetic sick or lethal (SSL) interactions, but may not participate in synthetic sick or lethal relationships among themselves. One of us (N.K.) described the generation of an epistatic miniarray profile (a quantitative analysis of double-mutant growth phenotypes among a logically-selected subset of genes) of approximately 850 genes involved in most aspects of chromosome function. This analysis detected not only synthetic sick or lethal interactions, but also cases in which double deletions grow better than would be expected from the growth of each single mutant when crossed to most other mutants on the array. The genes involved in these latter interactions often displayed similar sets of genetic profiles and correlated well with physical interactions. Will RNAi enable the generation of such networks in higher organisms? Perhaps to some degree; however, in the question-and-answer period, Fraser suggested that his group had encountered a high false-negative rate in RNAi x RNAi 'crosses' in *C. elegans*, relative to RNAi x germline mutation, presumably because most RNAi knockdowns are incomplete.\n\nBiological networks tend to have distinctive mathematical properties, most notably a scale-free degree distribution, the cause of which has been debated extensively. Ravi Iyengar (Mount Sinai Medical Center, New York, USA) and Albert-Laszlo Barabasi (University of Notre Dame, South Bend, USA) both presented arguments and modeling results supporting the notion that the origin of scale-free network structures is likely to be a consequence of selection pressure following gene duplication. Briefly, the idea is that newly duplicated genes have increased chances of survival if they develop a function that is tied to that of a gene that is already functionally coupled to many other genes, creating a 'rich get richer' scenario. The results of simulations of this paradigm appear to match experimentally derived network structures.\n\nIn contrast to all the network technology, a biological highlight was the description by Cori Bargmann (Rockefeller University, New York, USA) of her lab's demonstration that *C. elegans* can learn to smell which bacteria it should and should not eat, using a network of only a few hundred neurons. Overall, the conference successfully integrated a broad range of biological topics, including novel technologies and computational strategies that biologists can apply to obtain meaningful systems-level biological information on signaling networks.","meta":{"dup_signals":{"dup_doc_count":119,"dup_dump_count":45,"dup_details":{"curated_sources":4,"2023-14":1,"2022-21":1,"2021-43":1,"2020-34":1,"2020-24":1,"2019-47":1,"2019-35":1,"2019-26":1,"2019-18":1,"2019-04":1,"2018-47":1,"2018-05":1,"2017-39":1,"2017-30":1,"2017-22":1,"2017-17":1,"2017-09":7,"2016-44":1,"2016-40":1,"2016-36":8,"2016-30":7,"2016-18":1,"2016-07":6,"2015-48":3,"2015-40":2,"2015-35":2,"2015-32":3,"2015-27":3,"2015-22":3,"2015-14":3,"2014-52":2,"2014-49":4,"2014-42":7,"2014-41":5,"2014-35":3,"2014-23":5,"2014-15":3,"2023-40":1,"2015-18":3,"2015-11":3,"2015-06":3,"2014-10":3,"2013-48":3,"2013-20":3,"2017-13":1}},"file":"PMC1557984"},"subset":"pubmed_central"}
{"text":"abstract: We assess five years of usage of the major genome-wide collections of mutants from *Saccharomyces cerevisiae*.\nauthor: Bart Scherens; Andre Goffeau\ndate: 2004\ninstitute: 1Institut de Recherches Microbiologiques J.M. Wiame, Campus CERIA, Av. E. Gryson 1, 1070 Bruxelles, Belgium; 2Institut des Sciences de la Vie, Universit\u00e9 Catholique de Louvain, Croix du Sud 2-20, 1348 Louvain-la-Neuve, Belgium\nreferences:\ntitle: The uses of genome-wide yeast mutant collections\n\nIn April 1996, the completely annotated genome sequence of the yeast *Saccharomyces cerevisiae* was made publicly available \\[1,2\\], the first eukaryotic genome sequence to be completed. Eight years later, thanks to the united efforts of the large yeast research community and to the unique genetic and physiological properties of yeast, this humble servant of mankind provides by far the best annotated eukaryotic genome \\[3\\]. The completeness of the yeast genome sequence has allowed the development of many novel tools for analyzing all molecular components of the cell and their interactions. These tools include three high-throughput collections of mutants that were first produced in 1999 and that have been analyzed in the five years since then. Here, we review the uses of these collections and their contribution to the identification of the components of basic physiological and developmental pathways of *S. cerevisiae*.\n\n# The yeast deletion mutant collection\n\nA set of over 20,000 knockout strains was created by a consortium of European and North American laboratories \\[4,5\\]. The collection currently contains homozygous and heterozygous diploid strains corresponding to deletions of each of 5,916 genes (including 1,159 essential genes) and one haploid strain of each mating type for every non-essential gene (4,757 genes). Each knockout strain is marked by two unique 20-nucleotide 'bar codes', allowing quantitative and qualitative identification by DNA microarray hybridization of each strain in the pools used to assess the strains under different growth conditions (see Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>). The original article \\[4\\] describing this collection has been cited more then 560 times in the five years since its publication, according to the ISI Web of Science \\[6\\]. The complete collection of strains can be obtained at low cost from Euroscarf \\[7\\], ATCC \\[8\\] and Invitrogen \\[9\\].\n\nThe deletion collection has been used in dozens of novel exhaustive screens for phenotypes that occur under a variety of physiological conditions; these include growth in minimal medium, in high salt and low salt, in galactose or sorbitol, at pH 8, after heat or cold shock, under stress by hydrogen peroxide (all in \\[10\\]); growth on non-fermentable carbon substrates \\[11\\], in saline conditions \\[12\\] or after treatment by ionizing radiation or DNA-damaging agents \\[13-17\\]; and the collection has also been screened for defects in meiosis, sporulation and germination \\[18,19\\]. This approach has uncovered numerous new putative components of well-known pathways; for instance, the number of genes known to have sporulation or germination phenotypes when deleted has been doubled by these analyses \\[18\\].\n\nMore sophisticated screens, for example for suppressors of the accumulation of mutations \\[20\\], have been developed more recently, as well as screens involving transformation of the deletion strains in order to identify genes needed for non-homologous DNA end-joining \\[21\\]. Novel protocols requiring individual transformations of each mutant have allowed the identification of host factors that influence the fate of the Ty family of long-terminal-repeat retrotransposable elements \\[22\\] and of genes involved in the unfolded protein response induced by heterologous introduction of mutant human Huntingtin protein or fragments of \u03b1-synuclein, both of which form disease-associated aggregates \\[23\\]. Similarly, proteins that interfere with the assembly of endoplasmic-reticulum structures termed karmellae, which are induced by elevated levels of HMG-CoA reductase under specific growth and genetic conditions, have been identified using the collection \\[24\\]. Several morphological screens have been developed, for example for defects in the selection of the bipolar bud site \\[25\\], in cell-size distribution \\[26,27\\], in cell morphology \\[28\\] and in meiotic chromosomal segregation \\[29\\]. Another approach is the screening of individual colonies. For instance, mis-sorting and secretion of vacuolar carboxypeptidase Y were detected by colony immunoblotting \\[30\\]. A second example of colony screening is the transformation of each single-deletion strain to express viral replicase proteins and an RNA replication template in which the capsid gene was replaced by a luciferase reporter gene, which was used to monitor viral expression in yeast colonies \\[31\\]. Finally, to identify the genes affecting glycogen storage, the deletion mutant colonies were blotted and stained by iodine vapor; the intensity of coloration allowed assessment of glycogen accumulation \\[32\\].\n\n## Use of the yeast deletion collection in screens for synthetic lethal mutants and to study drug targets\n\nSynthetic lethality is the phenomenon that occurs when two mutations that are each viable are combined and the double mutant is lethal. A method has been developed for the systematic construction of double mutants, which is called synthetic genetic array analysis (SGA) \\[33,34\\]. Haploid strains with mutations in non-essential genes were crossed to an array of the whole haploid deletion collection; the resulting diploid cells were made to sporulate and the lethal combinations identified, indicating the existence of essential interactions between gene products. In a first SGA screen using eight query genes, 291 interactions among 204 genes were identified \\[33\\]. Three years later \\[34\\], the search was expanded to 132 query genes, and 4,000 interactions were identified among 1,000 genes with roles in cytoskeletal organization, cell-wall biosynthesis, microtubule-based chromosome segregation and DNA metabolism.\n\nA more recent development of this approach is the use of DNA-DNA hybridization protocols to assess lethality, termed 'synthetic lethality analysis by microarray' (SLAM). In this method, a pool of haploid deletion strains is transformed with a cassette that replaces the gene of interest with either a deletion construct or the wild-type form. Transformants are pooled and genomic DNA is isolated; the barcodes are amplified by PCR and labeled with either Cy3 (green) or Cy5 (red) fluorescent dyes; and hybridization to an array containing all the deletion tags allows identification of the synthetic-lethal combinations (which are missing). The SLAM method has been validated by identifying members of the DNA helicase interaction network \\[35\\]. Synthetic genetic arrays have also been used for high-resolution genetic mapping of suppressor mutations \\[36\\]; this method, termed SGA mapping (SGAM), is in principle also applicable to the analysis of multigenic traits.\n\nThe yeast deletion collection has also been used to identify members of the pathways modified by more than 25 different chemical ligands. This approach has identified the L-carnitine transporter Agp2p as a novel transporter of bleomycin in yeast, implicating membrane transport as a key determinant of resistance to this widely used anticancer agent \\[37\\]. In another study, the transcription factor Rpn4p was shown to compensate for proteasome inhibition by PS-341, a drug that is being studied as a treatment for cancer \\[38\\]. Other ligands tested include the phosphatidyl kinase inhibitor wortmannin \\[39\\]; anticancer agents \\[10,37,40,41\\]; antifungal agents including nystatin and calcofluor \\[10,41\\]; antibiotics including hygromycin B \\[42\\] and rapamycin \\[43\\]; statins (a class of drugs that lower cholesterol), the smooth muscle relaxant alverine citrate, the local anaesthetic dyclonine, yeast K1 killer toxin \\[44\\], 6-azauracil (a drug that depresses cellular nucleoside triphosphate levels \\[45\\]) and even the detergent sodium dodecyl sulfate \\[44\\].\n\n## Major scientific contributions arising from the use of the yeast deletion collection\n\nIn a total of 33 publications that have used the deletion collection, in which over 100 different conditions were explored, more then 5,000 genes were attributed a phenotype (see Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a> for an overview). Many of these genes were not previously known to contribute to the pathways scrutinized, even though in many cases - such as in studies of sporulation, the cell cycle, radiation damage and growth in defined media - there had previously been apparently exhaustive classical mutagenesis or DNA microarray screens. Altogether, these analyses have shown 71 genes previously thought to be non-essential to have a nonviable phenotype \\[46\\]; and the systematic deletion approach has been claimed to be five-fold more sensitive then a microarray gene-expression analysis for finding genes involved in pathways of interest \\[11\\].\n\nSome new phenotypes have emerged from these analyses, although many known phenotypes have not yet been explored \\[47\\]. As an example of recent novel information provided by the systematic-deletion approach, over 100 new genes resulting in a defective bipolar budding pattern \\[25\\] have been identified. Other examples are the identification of a new putative bleomycin transporter \\[37\\]; the *NEJ1* gene involved in non-homologous DNA end-joining and claimed to be one of the guardians of the cancer cells \\[21\\]; and 52 genes, the deletion of which causes lethality in yeast transfected with a fragment of heterologous human Huntingtin \\[23\\]. The latter set, which includes genes involved in lipid metabolism, vesicle transport and of unknown function, may help to define the still poorly defined pathway termed the unfolded protein response \\[23\\].\n\n## Uncertainties in the results obtained from systematic deletion mutants\n\nThe yeast deletion collection allows powerful novel screens and has revealed many novel components of complex metabolic and developmental pathways. The data produced should be examined with great care, however, as there are several problems with the use of the collection (for further details, see two reviews by Elisabeth Winzeler \\[46,48\\]). A first issue is that the collection is incomplete, as a few hundred genes were missed in the first annotation of the genome that was used as the basis of the deletions (see updates of yeast genome data in *Saccharomyces* genome database (SGD) \\[49\\]). Moreover, relative amounts of some deleted strains may have been distributed unequally during the initial creation of the pools and further amplified \\[4\\]. Secondly, there can be problems with mutations or variations elsewhere in the genome that affect the phenotype. For instance, because the transformation procedure that was used to create the deletion library is a mutagenic event, mutations in sites some distance away from the intended deletion might be present in some strains \\[50\\]. Also, up to 8% of the deleted strains are known to retain a wild-type copy of the targeted gene, presumably because of aneuploidy or some duplication event \\[50\\]. Even in diploid strains, mutations that apparently have no phenotype may in fact modify the phenotype of other mutations in subtle ways (a phenomenon termed haploinsufficiency) \\[51\\].\n\nWhen deletions are moved into a different genetic background, their phenotypes may change; for instance, up to 18% of synthetic-lethal interactions were not confirmed in different backgrounds \\[33,46\\]. Another source of discrepancy might be genetic variation among the individual isolates of the strains that are amplified under given selective conditions. For instance, the phenotype for a given mutant strain can be masked by extracellular complementation by a protein product of another strain in the pool.\n\nOther factors are related to the use of the barcodes. Their presence may modify the phenotype or, conversely, the stress created by the knockout procedure may mutagenize the barcode. About 17% of the 4,600 homozygous strains in the collection have a hybridization signal similar to that of the background. Moreover, trivial technical factors may affect the hybridization data, for example when the wrong piece of DNA is deposited on the microarray or when the tag modifies the secondary structures and thus causes poor hybridization. Also, some published datasets have too few repetitions of experiments to be statistically valid.\n\nThese and other problems concerning the specificity and sensitivity of the screen used must be taken into account when interpreting data obtained using the yeast deletion collection. As a gross estimate, over one third of the data may consist of false positives or false negatives. As a golden rule, therefore, the data from a primary screen of the collection should always be confirmed by independent approaches.\n\n# Collections of strains mutated using transposons\n\nTransposons are mobile genetic elements that can be used to disrupt genes in a non-targeted fashion. Insertional mutagenesis using transposons can disrupt non-coding as well as coding regions and can lead to partial loss or gain of function; it is thus complementary to deletion of genes \\[52\\]. The 'mini-Mu' transposon modified for insertional mutagenesis in yeast was pioneered by Daignan-Fornier and Bolotin-Fukuhara \\[53\\]. Ross-Macdonald *et al*. \\[52\\] used a version of the same transposon including a reporter gene (*lacZ*), an epitope tag (encoding hemagglutinin) and selectable markers for yeast and bacteria to perform random transposon mutagenesis. The approach uses a minitransposon that was introduced into the yeast genome by a two step method: a yeast genomic DNA library was mutagenized in *Escherichia coli* using the minitransposon; the library plasmids were then cut to excise the yeast genomic DNA with the transposon insertion, which was transformed into diploid yeast, replacing one chromosomal copy by homologous recombination \\[52\\]. This transposon-mutagenesis system can be used for the analysis of gene expression, detection of subcellular localization and identification of phenotypes caused by the insertion, and the transposon can be deleted using the Cre-LoxP recombinase system, removing the selectable marker. The insertion point in the genome can be determined for each mutant by sequencing outwards from the transposon sequence into the surrounding yeast sequences. The article describing the first such screen \\[52\\] has been cited 209 times in the five years since its publication \\[6\\]. The collection of transposon-inserted strains created in this work \\[52\\] is available free of charge from Michael Snyder's laboratory \\[54\\].\n\nA database called 'transposon-insertion phenotypes, localization and expression in *Saccharomyces cerevisiae*' (TRIPLES) \\[55,56\\] contains the results obtained from exploiting the mutants obtained by the transposon-insertion approach. A total of 240,768 yeast insertional mutants were screened for transposon-encoded \u03b2-galactosidase activity, and a collection of 28,428 yeast strains was generated that produce \u03b2-galactosidase under conditions of vegetative growth and\/or sporulation. The insertion point of 23,191 of these strains has been sequenced, identifying 3,750 different yeast genes, and the subcellular localization of 2,744 different yeast proteins has been documented (A. Kumar, personal communication).\n\nThe transposon-insertion collection \\[52,54\\] has been used to identify genes involved in sporulation and vegetative growth. Analysis of the collection has also identified 137 new genes that were not originally annotated by the yeast genome sequencing consortium \\[57\\]. Ferreira *et al*. \\[58\\] have used the collection - which was produced in the S288C strain used to sequence the yeast genome \\[52\\] - to mutagenize the W303-1A strain (which is known to be more responsive to gene inactivation than the S288C). Ferreira *et al*. \\[58\\] identified genes required for growth in high-salt medium (61 insertions in 31 genes) and survival of hypo-osmotic shock and growth at 15\u00b0C (31 insertion in 10 genes). A similar approach was also used by Takahashi *et al*. \\[59\\] to screen in other strains for ethanol tolerance and modification of the cell wall, and by Suzuki *et al*. \\[60\\] to screen for the effects of insertions on the response to nitrogen starvation, on invasive or filamentous growth and on sporulation.\n\nOther approaches for insertional mutagenesis have also been applied to yeast. Firstly, instead of bacterial mini-Mu transposons, T-DNA from *Agrobacterium tumefaciens* has been used to mutagenize yeast on a genome-wide scale \\[61\\]. A second approach is the use of non-homologous insertion of DNA cassettes not derived from mobile elements. This process, which exploits the cellular machinery that normally repairs DNA double-strand breaks by non-homologous end joining, is increased by gamma irradiation \\[62\\]. Thirdly, Blanc and Adams \\[63\\] used mutations resulting from insertion of the Ty1 transposon to identify yeast mutations that generate evolutionarily significant phenotypes by causing small but positive increments of fitness. Finally, an application of transposon mutagenesis called direct allele replacement technology (DART) allows rapid transfer of any insertion allele into any strain \\[64\\]. A transposon library consisting of a collection of plasmids containing yeast genomic DNA with transposon insertions is sequenced to identify the exact insertion point in the yeast genomic DNA \\[64\\]. After excision from the plasmid, the yeast genomic DNA containing the transposon is used to transform a yeast strain of choice by homologous recombination. The procedure was validated by identification of 29 insertions into 17 genes involved in apical growth \\[64\\].\n\nInsertional mutagenesis using transposons has several potential advantages over targeted deletion: for instance, insertion occurs in the non-coding as well as in the coding segments, so regulatory regions and other non-genie regions can be disrupted. Moreover, depending on the site of insertion, transposon mutagenesis may lead to partial loss of function or gain of function and hence to the identification of novel functions that would not be found from studies of complete knockouts of genes \\[52\\]. Conditional alleles may be generated, as well as mutants in promoter or terminal regions. Also, apart from phenotype analysis of the mutation, the level of expression of the targeted gene can be measured *in vivo* and the subcellular localization of its product can be determined. A disadvantage, however, is that transposon insertions are not random, and this method may therefore never cover all the genes in the genome. Also, several of the problems that were mentioned above for the analysis of the yeast deletion collection apply equally to the transposon mutant collections, including the observation that phenotypes are often very strongly background-dependent.\n\n# Other genome-wide mutant collections and their uses\n\nSeveral databases have been developed to catalog the subcellular localizations of yeast proteins as identified by fluorescence microscopy. The yeast protein localization database \\[65,66\\] describes the results obtained using a library of yeast genes fused to a green fluorescent protein (GFP) reporter. The TRIPLES database \\[55,56\\] includes the use of the transposon-insertion libraries to determine protein localization for 5,504 insertions. The yeast GFP fusion localization database \\[67,68\\] presents the localization of 4,156 proteins into 22 distinct subcellular locations, as determined using a library of GFP-tagged proteins compared with reference strains expressing proteins of known localization tagged with red fluorescent protein (the strains used are available from Invitrogen \\[9\\]).\n\nIn a recent study \\[69\\], an exhaustive global analysis of protein expression in yeast was reported. Each open reading frame was marked by an insertion cassette consisting of a modified version of the tandem affinity purification (TAP) tag, a yeast selectable marker to drive homologous recombination, and regions homologous to yeast genes. The level of expression of each protein was determined by very sensitive western-blotting analysis that could detect less than 50 protein molecules per cell. The level of expression of 4,251 gene products was identified in exponential growth conditions \\[69\\].\n\nLike the sequencing of the yeast genome, the construction of the single and double deletion libraries and that of the major transposon insertion library has required a considerable investment by the international yeast research community. The three original papers \\[4,33,52\\] presenting the libraries have a total of 83 authors, funded by public agencies from the USA, from the European Union and from Canada. Was this effort worthwhile?\n\nOver the last five years, the three seminal papers have been cited more than 950 times \\[6\\]. We have found over 50 experimental papers reporting on different phenotypic screens carried out with these genome-wide libraries. More than 100 experimental conditions have been tested, representing near a million individual mutant screens. We estimate that more than 5,000 novel phenotypic traits have been assigned to yeast genes of known or unknown molecular functions. This undoubtedly represents a considerable amount of progress towards the ultimate goal of a full description of the functions and interactions of all the molecular components of a basic eukaryotic cell. Several recent improvements of the use of the original genome-wide mutant libraries have been reported, such as the DART \\[64\\] and SLAM \\[35\\] approaches described above. New genome-wide libraries using more sensitive protein tagging are being developed \\[69\\] for global measurements of protein levels or subcellular expression. In all this work, yeast continues to be a very convenient test-bed for the development of novel high-throughput tools that rely on the availability of the complete genome sequence.\n\nBut a word of caution is appropriate. As was the case for other high-throughput tools that were also pioneered using yeast, such as two-hybrid protein-interaction screening, DNA-hybridization microarrays and proteomic analyses, the information provided by large screens of genome-wide libraries contains an appreciable number of false-positive and false-negative data points. It is therefore essential to confirm each result by several independent approaches. Ultimately, we will have to return to 'reductionist' biochemical approaches to demonstrate fully the molecular function suggested by a large primary screen.\n\nFinally, there is an urgent need to build databases to collect and organize all the data obtained from all the large screening approaches. The SGD \\[49\\] and the Munich information center for protein sequences (MIPS) database \\[70\\] are making good progress in this respect. The authors of these databases should, however, be encouraged to further develop procedures that take into account the difficult assessment of the uncertainties associated with much of the data. Here again, yeast is expected to become a pioneer.\n\n## Figures and Tables\n\nScreens performed so far on the yeast deletion strain collection and transposon-insertion collection\n\n| Condition, treatment or phenotype | Number of genes responding to the treatments\\* | References |\n|----|----|----|\n| **Phenotype screens of the deletion collection** |  |  |\n| Fitness after growth on minimal medium, high salt, low salt, galactose, sorbitol, pH 8, heat shock, cold shock, or hydrogen peroxide | ? | \\[10\\] |\n| Growth on non-fermentable carbon substrates | 466 | \\[11\\] |\n| Response to DNA-damaging agents, ionizing radiation, UV radiation, cisplatin, or hydrogen peroxide | \\>170 | \\[13-16\\] |\n| Chemical DNA damage (methane sulfonate sensitivity) | 103 | \\[17\\] |\n| Enhanced sporulation proficiency | 102 | \\[18,19\\] |\n| Sporulation defects | 261 | \\[18,19\\] |\n| Post-germination defects | 158 | \\[18,19\\] |\n| Genes essential for sporulation | 334 | \\[18,19\\] |\n| Saline response | About 500 | \\[12\\] |\n| Suppression of the accumulation of mutations | 33 | \\[20\\] |\n| Non-homologous DNA end-joining | About 30 | \\[21\\] |\n| Ty1 retrotransposition cycle | 101 | \\[22\\] |\n| Response to the introduction of human Huntingtin or \u03b1-synuclein fragments (unfolded protein response) | 52\/86 | \\[23\\] |\n| Karmellae assembly | 120 | \\[24\\] |\n| Bipolar bud-site selection | 127 | \\[25\\] |\n| Cell-size distribution | 500 | \\[26,27\\] |\n| Altered cell morphology | ? | \\[28\\] |\n| Meiotic chromosomal segregation | ? | \\[29\\] |\n| Vacuolar protein sorting | 146 | \\[30\\] |\n| Host genes affecting replication of a positive-strand RNA virus | 100 | \\[31\\] |\n| Glycogen storage (low, high) | 324, 242 | \\[32\\] |\n|  |  |  |\n| **Sensitivity of the deletion collection to drugs or chemicals** |  |  |\n| Rapamycin | 106 | \\[43\\] |\n| PS-341 | ? | \\[38\\] |\n| Bleomycin (hypersensitivity, resistance) | 231, 5 | \\[37,40\\] |\n| Amoxillin, penicillin, rifampin, vancomycin, (oxy)tetracycline, or gentamicin | 23 | \\[42\\] |\n| Anticancer, antifungal agents, statins, alverine citrate, dyclonine, or nystatin | About 10 | \\[10,41\\] |\n| K1 killer toxin, hygromycin B, calcofluor or sodium dodecyl sulfate | 268 | \\[44\\] |\n| 6-Azauracil | 42 | \\[45\\] |\n| Wortmannin (resistance, sensitivity) | 591, 476 | \\[39\\] |\n|  |  |  |\n| **Phenotypes of transposon-insertion libraries** |  |  |\n| Phenotypes under 20 conditions, expression levels and subcellular localization (TRIPLES database) | ? | \\[55\\] |\n| High salt | 31 | \\[58\\] |\n| Survival of hypo-osmotic shock and growth at 15\u00b0C | 10 | \\[58\\] |\n| Ethanol tolerance and cell-wall modifications | 5 | \\[59\\] |\n| Nitrogen starvation, invasive or filamentous growth and sporulation | About 105 | \\[60\\] |\n\n\\*The number of open reading frames responding to the relevant screen is reported whenever this information could be deduced from the relevant article; ? indicates that the information could not be deduced.","meta":{"dup_signals":{"dup_doc_count":120,"dup_dump_count":46,"dup_details":{"curated_sources":2,"2022-05":1,"2021-39":1,"2021-31":1,"2021-25":1,"2019-26":1,"2019-22":1,"2019-13":1,"2019-04":1,"2018-47":2,"2018-39":2,"2018-30":1,"2018-22":1,"2018-13":1,"2017-47":1,"2017-34":1,"2017-26":1,"2017-22":1,"2017-09":9,"2016-44":1,"2016-40":1,"2016-36":10,"2016-30":8,"2016-22":1,"2016-18":1,"2015-48":3,"2015-40":2,"2015-35":3,"2015-32":3,"2015-27":3,"2015-22":3,"2015-14":2,"2014-52":2,"2014-49":6,"2014-42":7,"2014-41":5,"2014-35":3,"2014-23":4,"2014-15":4,"2022-33":1,"2015-18":3,"2015-11":3,"2015-06":3,"2014-10":3,"2013-48":3,"2013-20":1,"2024-26":1}},"file":"PMC463272"},"subset":"pubmed_central"}
{"text":"abstract: This blinded field safety study was conducted in Senegal to assess safety and immunogenicity of administration of the registered dose of Rift Valley fever virus (RVFV) Clone 13 vaccine (Onderstepoort Biological Products) to sheep and goats of West African breeds under natural conditions. A total of 267 small ruminants (220 sheep, 47 goats) were included; half received RVFV Clone 13 vaccine at the recommended dose and half received the diluent (as placebo) only. The study was performed on three commercial farms in the northern and eastern region of Senegal in accordance with veterinary good clinical practices. The animals were observed daily for 3 days after vaccination, and then weekly for 1 year. In both sheep and goats vaccinated against RVFV seroconversion rates above 70% were recorded. No seroconversion related to RVFV was observed in placebo-treated animals. No statistically significant differences were determined between placebo and vaccinated groups for mean rectal temperatures for the first 3 days after administration (*p* \\> 0.05). No abnormal clinical signs related to treatment were noted, and only one slight injection site reaction was observed in one vaccinated animal for 2\u00a0days after vaccination. Out of 176 births assessed over 1 year (93 from the vaccinated group, 83 from the placebo group), 9 were abnormal in the placebo group and 3 in the vaccinated group (*p* \\> 0.05). The frequency of adverse events was similar in the placebo and vaccinated groups. RVFV Clone 13 vaccine administered according to the manufacturer's instructions was safe and well tolerated in West African breeds of sheep and goats, including animals of approximately 6 months of age and pregnant females, under field conditions in Senegal. Antibody levels persisted up to 1 year after vaccination.\nauthor: Modou M. Lo; Victor Mbao; Pascale Sierra; Yaya Thiongane; Mariame Diop; Meritxell Donadeu; Baptiste Dungu**Correspondence to:** Victor Mbao **Email:**<email@example.com>**Postal address:** Private Bag A 130, Lilongwe 03, Malawi[^1]\ndate: 2015-05-29\nreferences:\ntitle: Safety and immunogenicity of Onderstepoort Biological Products' Rift Valley fever Clone 13 vaccine in sheep and goats under field conditions in Senegal\n\n# Introduction\n\nRift Valley fever (RVF) is an acute mosquito-borne disease affecting mainly ruminant animals and humans (Swanepoel & Coetzer 2004). It causes abortions and high mortality in young animals (Bird & Nichol 2012; Ikegami & Makino 2011). In humans it causes a severe influenza-like disease, with occasionally more serious central nervous system complications and death (Swanepoel & Coetzer 2004). RVF is caused by a single-stranded ribonucleic acid virus of the *Phlebovirus* genus in the family Bunyaviridae (Pepin *et al.* 2010). Major epidemics have occurred in many African countries, including Senegal, where the virus is believed to be endemic (Chevalier, Thiongane & Lancelot 2009).\n\nThe control of RVF has essentially relied on vaccination, using the live attenuated Smithburn vaccine or the inactivated vaccine (Smithburn 1949; Swanepoel & Coetzer 2004). Whilst these currently available RVF vaccines have been used extensively for the control of the disease, they have several shortcomings, such as poor immunogenicity, or toxicity when used in pregnant animals (Coetzer 1982; Dungu, Donadeu & Bouloy 2013). This has encouraged many research groups to develop new vaccine candidates that may address a large number of the current challenges and be suitable for use in currently RVF-free regions as well as being included in different contingency and emergency- preparedness strategies.\n\nA number of research groups have been working on an alternative vaccine, the RVFV Clone 13 vaccine, which has been developed, evaluated (Dungu *et al.* 2010; Muller *et al.* 1995) and registered for use in cattle, sheep and goats in South Africa. The vaccine is based on a natural RVF virus mutant that carries a large in-frame deletion in the NSs gene. Evidence so far indicates that the vaccine is highly immunogenic and does not cause abortion or foetal teratogenicity in ewes and goats vaccinated during pregnancy. The risk of reversion to virulence is believed to be negligible given the substantial genetic deletion present in the vaccine virus (Muller *et al.* 1995).\n\nDespite the occurrence of a number of RVF outbreaks in West Africa, more specifically Senegal and Mauritania (Caminade *et al.* 2014; Ndione *et al.* 2008), vaccination has never been used for the control of the disease in this region. This is mainly due to safety concerns associated with the RVF Smithburn vaccine and challenges with vaccine availability. The RVFV Clone 13 was considered to be a safer alternative for use in the region. In order to provide confidence to livestock owners and veterinary authorities, this study was designed and was aimed at evaluating the safety and efficacy (through the evaluation of immunogenicity) of Onderstepoort Biological Products' (OBP) Clone 13 RVFV vaccine, (Onderstepoort, Republic of South Africa) in Senegalese sheep and goat breeds under local conditions, and assessing the possible spread of virus from vaccinated to unvaccinated animals.\n\nThe OBP RVFV Clone 13 vaccine was tested in Senegal under field conditions, as it may potentially represent a safer and more effective option to protect livestock in West Africa than RVF vaccines currently being used elsewhere.\n\nThis trial was designed to demonstrate the safety and immunogenicity of OBP RVFV Clone 13 vaccine in sheep and goats when administered according to the manufacturer's recommendations, and to assess spread of virus from vaccinated to unvaccinated animals in local Senegalese goats and sheep under field conditions. The trial was conducted as a controlled, randomised and blinded study following the principles of good clinical practice as detailed by the International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products guideline number nine.\n\n# Research method and design\n\n## Setting\n\nThe study was conducted from August 2011 to October 2012 on three commercial farms in the northern (Mpal, Thille Boubacar) and eastern (Diawara) regions of Senegal, using animals bred locally and kept under local farm conditions. The locations were favoured because of active surveillance implemented in these areas for more than 15 years that demonstrated endemicity of RVF (Thiongane *et al.* 1994).\n\n## Animals\n\nA total of 267 sheep (Touabir and Warale breeds) and goats (Sahelian breed) were included, as indicated in <a href=\"#T0001\" data-ref-type=\"table\">Table 1<\/a>. They included 220 sheep (191 females and 29 males), and 47 goats (41 females and 6 males).\n\nExperimental animals.\n\n| Categories        | Diawara |         | Mpal  |         | Thille |         | Total   |\n|-------------------|---------|---------|-------|---------|--------|---------|---------|\n|                   | Males   | Females | Males | Females | Males  | Females |         |\n| Goats, placebo    | 0       | 1       | 0     | 0       | 4      | 18      | 23      |\n| Goats, vaccinated | 0       | 2       | 0     | 0       | 2      | 20      | 24      |\n| Sheep, placebo    | 5       | 40      | 1     | 26      | 9      | 29      | 110     |\n| Sheep, vaccinated | 4       | 40      | 0     | 28      | 10     | 28      | 110     |\n| **Subtotal**      | **9**   | **83**  | **1** | **54**  | **25** | **95**  | **267** |\n\nAge determination was based on incisors: the presence of lactating (milk) teeth (LT) only or the number of adult teeth (AT). LT: less than 14 months old \u2013 68 females and 31 males; 2 AT: between 14 and approximately 20 months old (29 females and 3 males); 4 AT: between 20 and 28 months old (27 females, 0 males); 6 AT: between 28 and 36 months old (36 females, 0 males) and 8 AT: more than 36 months old (72 females, 1 male).\n\nAt the time of vaccination 123 animals were pregnant (99 sheep and 24 goats), and 53 animals (46 sheep and 7 goats) became pregnant during the study, as shown in <a href=\"#T0002\" data-ref-type=\"table\">Table 2<\/a>.\n\nNumbers of sheep and goats that gave birth in relation to time of vaccination using Onderstepoort Biological Products Rift Valley fever virus Clone 13 vaccine.\n\n| Pregnancy status at vaccination | Goats |  | Sheep |  |\n|----|----|----|----|----|\n|  | Vaccinated | Placebo | Vaccinated | Placebo |\n| Vaccinated during first trimester of gestation | 3 | 3 | 18 | 21 |\n| Vaccinated during second trimester of gestation | 5 | 5 | 16 | 13 |\n| Vaccinated during third trimester of gestation | 5 | 3 | 18 | 13 |\n| Pregnant within 2 months of vaccination | 1 | 3 | 11 | 8 |\n| Pregnant 2 months or more after vaccination | 1 | 2 | 15 | 12 |\n| **Total** | **15** | **16** | **78** | **67** |\n\nAll animals were identified by ear tag. Appropriate treatments were given to animals in the 2 weeks preceding vaccination or placebo injection, including antiparasitic therapy with ivermectin and clorsulon.\n\nAll animals were seronegative to RVF at the start of the study, except for one goat and one sheep (both were positive at day 0 by enzyme-linked immunosorbent assay \\[ELISA\\] but not the viral neutralisation test \\[VNT\\]).\n\n## Vaccination\n\nThe RVFV Clone 13 vaccine manufactured by OBP was used. The batch number was 13, with an expiry date of 01 January 2012. The placebo was the diluent of the same vaccine, batch number 8593, expiry date 01 April 2014. Storage conditions of the vaccine and diluent were appropriately monitored and complied with the manufacturer's instructions.\n\nEach animal received either vaccine or placebo, at a dose of 1 mL subcutaneously. Vaccines were injected with sterile needles and syringes that were changed for each animal.\n\n## Study personnel\n\nAll personnel were well experienced in conducting vaccine trials, and were formally trained to good clinical practice guidelines by an independent study monitor. The independent study monitor attended the crucial study time-points, including pre-study, vaccination, observation period and end of study, and maintained regular contact with Institut S\u00e9n\u00e9galais de Recherches Agricoles's (ISRA's) team throughout the observation period. Animals were examined at least weekly by the local veterinary representative and every 2 months by the investigator from ISRA Dakar.\n\n## Serology and virology\n\nAll serology (VNT and ELISAs for IgG and IgM) and virology (virus isolation using Vero cells and conventional polymerase chain reaction) analyses were conducted at Laboratoire National de l'Elevage et de Recherches V\u00e9t\u00e9rinaires (LNERV) at ISRA, Dakar.\n\nSera were taken from all animals before the study commenced and then from a selection of approximately 20 animals per group per site every 2 months. The samples were analysed by virus neutralisation (Davies, Jacobsen & Sylla 1988), RVF inhibition ELISA to detect IgM (National Institute for Communicable Diseases, Sandringham, South Africa; Paweska *et al.* 2005) for all samples up to day 135 and RVF recIgG ELISA to detect IgG (National Institute for Communicable Diseases, Sandringham, South Africa; Paweska, Jansen van Vuren & Swanepoel 2007) for all samples between days 195 and 365. The ELISAs used were commercial kits manufactured by Biological Diagnostic Supplies Limited, Ayrshire, Scotland.\n\n## Study design\n\nThis study was a randomised, blinded and placebo-controlled trial conducted under field conditions. Vaccine safety was assessed by monitoring local (injection site) and general clinical reactions, including rectal temperature, once daily for the first 3 days then at least once weekly for 1 year following vaccination or placebo injection. All abnormal health observations, medical treatments and births or abortions were recorded for the year of observation.\n\nBlood samples were taken on the day of vaccination and afterwards every 2 months (+\/\u2212 10 days) until day 365 to assess RVF antibody levels prior to vaccination and every 2 months afterwards.\n\nRandomisation schedules were prepared for each species (sheep or goat) and each study site; half of the animals received vaccine and the other half received placebo based on order of catch.\n\nTo maintain blinding the person who administered treatments was never present at clinical examinations and the randomisation lists and treatment record forms were kept in a secure location which was not accessible to personnel conducting the health and safety observations.\n\n## Statistical methods\n\nMean rectal temperatures were calculated daily for each treatment group and compared using two-factor analysis of variance (time and treatment). The frequencies of adverse events and abnormal births were compared between groups using the Chi-square test.\n\n# Results\n\n## Rectal temperatures\n\nMean and standard deviations for rectal temperatures from day 0 (prior to treatment) to day 3 (approximately 72 hours after treatment) are shown in <a href=\"#T0003\" data-ref-type=\"table\">Table 3<\/a> and <a href=\"#F0001\" data-ref-type=\"fig\">Figure 1<\/a>.\n\nMean and standard deviations for rectal temperature in \u00b0C from day 0 to day 3.\n\n| Treatment            | Mean RT and SD | Study day |       |       |       |\n|----------------------|----------------|-----------|-------|-------|-------|\n|                      |                | Day 0     | Day 1 | Day 2 | Day 3 |\n| Placebo              | Mean RT\u2020       | 38.62     | 38.14 | 38.11 | 38.79 |\n|                      | SD\u2020 RT         | 0.82      | 4.76  | 4.75  | 0.51  |\n| RVF Clone 13 vaccine | Mean RT        | 38.62     | 37.52 | 38.03 | 38.41 |\n|                      | SD RT          | 0.81      | 6.63  | 4.74  | 3.49  |\n\nRVF, Rift Valley fever; RT, rectal temperature; SD, standard deviation.\n\n\u2020, rectal temperature in \u00b0C.\n\nNo statistically significant difference was noted between treatment groups for mean rectal temperature between days 0 and 3 (*p* = 0.27 or *p* \\> 0.05) and also no significant time x treatment interaction (*p* = 0.79 or *p* \\> 0.05) was recorded.\n\n## Local tolerance: Injection site reactions\n\nOf 267 animals that received either the placebo or the RVFV Clone 13 vaccine, only one sheep was observed to have slight swelling at the injection site on days 1 and 2. The signs disappeared after day 2 and no other abnormal signs were observed. None of the other animals presented any injection site reaction.\n\n## General health observations\n\nAll animals were individually observed for 3 days after vaccination. During this period only one goat was recorded to have abnormal general health, diagnosed as foot rot, which was not considered to be related to the study.\n\nIn the year following vaccination or placebo injection all animals were observed weekly for abnormal health. Only those animals with health abnormalities were then individually examined. In total 45 health observations were recorded by the veterinarian (veterinary reports) in the placebo group and 50 in the vaccine group, as well as 12 potential adverse events (6 in each group). The frequency of veterinary reports was not significantly different between treatment groups (Chi-square test, *p* \\> 0.05). No abnormal health observations were related to the trial.\n\n## Births and abortions\n\nA total of 176 births were observed during the 1-year study period, 31 in does and 145 in ewes. Ninety-three births occurred in the vaccinated group and 83 in the placebo group, and of these 12 were considered abnormal (3 in the vaccine group and 9 in the placebo group). Two abnormal births were noted less than a week after the start of the trial, one in each treatment group. All other abnormal births occurred at least one month (one animal) but generally more than 2 months after injection. The offspring from the ewe that aborted 5 days after vaccination was tested for RVF by polymerase chain reaction and isolation in Vero cells, and the results were negative. The abortion was considered not to be related to vaccination. The frequency of abnormal births was not significantly different between the vaccinated group and the placebo group (Chi-square test with Yate's correction, *p* \\> 0.05).\n\n## Seroconversion in the placebo group\n\nNo seroconversion to RVFV was observed in any animal from the placebo group, which supports the concept of no passage of the RVFV vaccine strain from the vaccinated animals to the non-vaccinated animals.\n\nNatural infection did not occur, as demonstrated by lack of clinical signs and or seroconversion of the non-vaccinated animals, as well as lack of clinical signs in of neighbouring herds.\n\n## Seroconversion after vaccination\n\nOn day 0 one goat and one sheep from the vaccinated group were positive on ELISA to IgM (RVF inhibition ELISA), and no animal was positive by VNT. In the placebo group the frequency of animals which were seropositive, both by ELISA (testing for IgM until day 135, and for IgG afterwards) and VNT stayed low throughout the observation period (as reflected in <a href=\"#T0004\" data-ref-type=\"table\">Tables 4<\/a> and <a href=\"#T0005\" data-ref-type=\"table\">5<\/a>). Amongst the animals that regularly had blood sampled, only one tested positive by VNT on consecutive samplings on days 195, 260 and 315, but remained negative on ELISA, except on day 195. This animal was then sold. Two other animals had antibody titres on VNT of 160 at one or more time-points, but became seronegative again at the following time-point. These animals were not positive on ELISA at these time-points. One animal was seropositive on day 365 only, on testing both by VNT and by ELISA.\n\nEnzyme-linked immunosorbent assay and viral neutralisation test results in goats.\n\n| Categories | Day 0 | Day 60 | Day 120 | Day 195 | Day 260 | Day 315 | Day 365 |\n|----|----|----|----|----|----|----|----|\n| Number of vaccinated goats sampled | 24 | 11 | 11 | 11 | 6 | 8 | 7 |\n| Goats positive ELISA | 1 | 5 | 4 | 7 | 4 | 7 | 6 |\n| % goats positive on ELISA | 4.17 | 45.45 | 36.36 | 63.64 | 66.67 | 87.50 | 85.71 |\n| Goats positive on VNT 160 | 0 | 4 | 4 | 6 | 4 | 7 | 5 |\n| Goats doubtful on VNT 80 | 0 | 2 | 4 | 2 | 1 | 1 | 2 |\n| % Goats \u2265 80 VNT | 0.00 | 54.55 | 72.73 | 72.73 | 83.33 | 100.00 | 100.00 |\n| **Number placebo goats sampled** | 22 | 6 | 4 | 5 | 5 | 6 | 7 |\n| Goats positive ELISA | 0 | 0 | 0 | 0 | 0 | 0 | 1 |\n| % goats positive ELISA | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 14.29 |\n| Goats positive VNT 160 | 0 | 0 | 0 | 0 | 0 | 0 | 1 |\n| Goats doubtful VNT 80 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |\n| % Goats \u2265 80 VNT | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 14.29 |\n\nELISA, enzyme-linked immunosorbent assay; VNT, viral neutralisation test.\n\nEnzyme-linked immunosorbent assay and viral neutralisation test results in sheep.\n\n| Categories | Day 0 | Day 60 | Day 120 | Day 195 | Day 260 | Day 315 | Day 365 |\n|----|----|----|----|----|----|----|----|\n| **Number vaccinated sheep sampled** | 110 | 64 | 61 | 58 | 58 | 58 | 55 |\n| Sheep positive ELISA | 1 | 31 | 17 | 25 | 6 | 27 | 22 |\n| % sheep positive ELISA | 0.91 | 48.44 | 27.87 | 43.10 | 10.34 | 46.55 | 40.00 |\n| Sheep positive VNT 160 | 0 | 35 | 23 | 31 | 38 | 26 | 30 |\n| Sheep doubtful VNT 80 | 0 | 12 | 10 | 10 | 3 | 6 | 9 |\n| % sheep \u2265 80 VNT | 0.00 | 73.44 | 54.10 | 70.69 | 70.69 | 55.17 | 70.91 |\n| **Number placebo sheep sampled** | 109 | 62 | 48 | 58 | 52 | 44 | 55 |\n| Sheep positive ELISA | 0 | 1 | 1 | 5 | 1 | 0 | 0 |\n| % sheep positive ELISA | 0.00 | 1.61 | 2.08 | 8.62 | 1.92 | 0.00 | 0.00 |\n| Sheep positive VNT 160 | 0 | 1 | 1 | 1 | 0 | 0 | 0 |\n| Sheep doubtful VNT 80 | 0 | 0 | 0 | 1 | 2 | 0 | 1 |\n| % sheep \u2265 80 VNT | 0.00 | 1.61 | 2.08 | 3.45 | 3.85 | 0.00 | 1.82 |\n\nELISA, enzyme-linked immunosorbent assay; VNT, viral neutralisation test.\n\nIn the vaccine group seroconversion was observed by ELISA and VNT in more than 70% of the animals, starting on day 60. The seropositivity rates remained high throughout the observation period, including on the last study day, when more than 70% of the sheep and goats had neutralising antibody titres above the positivity threshold. Neutralising antibody titre results were generally more consistent over time for the same animal. Many animals in the vaccinated group regularly had an ELISA titre very close to but lower than the test threshold and were therefore not counted as positive. However, they definitely presented a significant rise in antibody titres over time.\n\nThe percentage of ELISA-positive animals at day 60 was as expected, based on the authors' previous experience.\n\n# Ethical considerations\n\nThe study protocols were reviewed and approved by the scientific committees of the Global Alliance for Livestock Veterinary Medicines and the ISRA of Dakar. The study did not involve killing, administration of pain-causing substances or invasive procedures in animals. Only the registered vaccine was used and animals were routinely bled according to a standard operating procedure. Permission to conduct the study was granted by the Ministry of Livestock in Senegal.\n\n# Discussion\n\nVaccination is the most effective tool to control RVF, accompanied by other measures such as effective surveillance, a good diagnostic strategy and reliable emergency-preparedness (Dungu *et al.* 2013). Preventing RVFV infection of livestock by vaccination is a key element in breaking the chain of human epidemics, and could lead to control of this significant public health threat (Bird & Nichol 2012). A human RVF vaccine is currently not commercially available.\n\nThe vaccine used in this trial, OBP RVFV Clone 13, has been registered and used in South Africa. It is based on an avirulent RVF virus isolated from a non-fatal case of RVF in the Central African Republic that had been passaged in mice and Vero cells and then plaque purified in order to study the homogeneity of virus subpopulations (Muller *et al.* 1995). A Clone designated 13 did not react with specific monoclonal antibodies against NSs, and when further investigated was found to be avirulent in mice, yet immunogenic (Muller *et al.* 1995; Vialat *et al.* 2000). This vaccine has been evaluated for safety and efficacy in sheep and cattle (Dungu *et al.* 2010; Von Teichman *et al.* 2011).\n\nUnlike the Smithburn and MP-12 strains of RVFV vaccines, RVFV Clone 13 vaccine does not appear to induce abortions in pregnant ewes, making it one of the most promising live attenuated vaccine candidates thus far (Boshra *et al.* 2011). Results of experiments in sheep demonstrated that RVFV Clone 13 vaccine also has a better safety profile than other RVFV mutants, such as MP-12 and Smithburn (Dungu *et al.* 2010). The safety and efficacy of RVFV Clone 13 vaccine in cattle and sheep under the recommended conditions of use are also supported by the administration of more than 10 million doses during the 2009\u20132010 RVF outbreak in South Africa, where the vaccine played a key role in the control of the disease (Dungu *et al.* 2013). This vaccine has the advantage over inactivated vaccines as it is more cost-effective, requiring only one vaccination instead of two administrations, and can be produced in larger batches than the inactivated vaccine (Dungu *et al.* 2010).\n\nThe RVFV Clone 13 vaccine has not been used in West Africa, despite the presence of RVF in the region (Chevalier et\u00a0 al. 2009; Clements *et al.* 2007). Prior to the field evaluation described here, this vaccine was tested in West African goats and sheep under laboratory conditions at ISRA Dakar (data not shown) as well as in field conditions in Kenya. In both studies the vaccine proved to be safe and induced a high level of seroconversion (B. Dungu, pers. comm., January 2012).\n\nThis is the first report on safety and immunogenicity data for the OBP RVFV Clone 13 vaccine in goats and sheep under field conditions in Senegal. The results are very encouraging, as the vaccine was confirmed to be safe in local breeds of both species in West Africa. Efficacy, evaluated as seroconversion after vaccination, was observed in more than 70% of the animals, which should ensure protective immunity at herd level. In experimental studies very good neutralising antibody responses measured by the World Organisation for Animal Health-recommended VNT were recorded in all RVFV OBP Clone 13 vaccinated sheep from day 7, and in some sheep up to day 60 post-vaccination (Dungu *et al.* 2010). In addition, in this field trial no signs of toxicity resulting from vaccination were detected when it was used in pregnant animals.\n\nIn conclusion, a total of 267 sheep and goats of local breeds were included in this field study to evaluate the safety and immunogenicity of the administration of the registered dose of RVFV Clone 13 vaccine. The animals were kept under local conditions and observed for 1 year after vaccination or placebo injection. More than 70% of the sheep and goats vaccinated with the OBP RVFV Clone 13 vaccine showed long-term seroconversion that persisted for the 1 year during which the trial was conducted. No signs of local or general intolerance and no signs of toxicity to vaccination of pregnant ewes and goats were noted. Vaccination with RVFV Clone 13 vaccine was safe and well tolerated in sheep and goats of Senegalese breeds, including pregnant females, under field conditions. These results suggest that vaccination of small ruminants with this vaccine could be effective in controlling RVF.\n\n## Acknowledgements\n\nThe authors are grateful to all the technicians at LNERV, ISRA, Dakar as well as Ismaila Seck, field veterinary at Mpal, Maniang Diokh field veterinary at Thille Boubacar and Serigne Mbaye field veterinary at Diawara for their valuable contribution to the study. This work was funded by the Bill & Melinda Gates Foundation & the UK Government's Department for International Development (DFID).\n\n### Competing interests\n\nThe authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article.\n\n### Authors' contributions\n\nP.S. (independent veterinary consultant) worked on the experimental design, data analysis and statistics, M.M.L. (ISRA) worked on the experimental design and reviewed the manuscript, Y.T. (ISRA) carried out data analysis, M.D. (ISRA) performed the ELISA for IgM and IgG, V.M. (Global Alliance for Livestock Veterinary Medicines) and M.D. (Global Alliance for Livestock Veterinary Medicines) conceived the study and edited the manuscript and B.D. (Global Alliance for Livestock Veterinary Medicines) was involved with conception of the study and manuscript preparation.\n\n# References\n\n[^1]: **How to cite this article:** Lo, M.M., Mbao, V., Sierra, P., Thiongane, Y., Diop, M., Donadeu, M. *et al*., 2015, 'Safety and immunogenicity of Onderstepoort Biological Products' Rift Valley fever Clone 13 vaccine in sheep and goats under field conditions in Senegal', *Onderstepoort Journal of Veterinary Research* 82(1), Art. \\#857, 6 pages. <http:\/\/dx.doi.org\/10.4102\/ojvr.v82i1.857>","meta":{"dup_signals":{"dup_doc_count":111,"dup_dump_count":31,"dup_details":{"curated_sources":2,"2019-30":1,"2019-09":1,"2018-51":1,"2018-43":1,"2018-34":1,"2018-26":1,"2018-22":1,"2018-17":6,"2018-13":1,"2018-09":2,"2018-05":4,"2017-51":1,"2017-47":4,"2017-43":3,"2017-39":4,"2017-34":3,"2017-30":4,"2017-26":4,"2017-22":2,"2017-17":5,"2017-09":6,"2017-04":5,"2016-50":6,"2016-44":5,"2016-40":6,"2016-36":7,"2016-30":5,"2016-26":7,"2016-22":6,"2021-31":1,"2017-13":5}},"file":"PMC6238691"},"subset":"pubmed_central"}
{"text":"abstract: The number of published systematic reviews of studies of healthcare interventions has increased rapidly and these are used extensively for clinical and policy decisions. Systematic reviews are subject to a range of biases and increasingly include non-randomised studies of interventions. It is important that users can distinguish high quality reviews. Many instruments have been designed to evaluate different aspects of reviews, but there are few comprehensive critical appraisal instruments. AMSTAR was developed to evaluate systematic reviews of randomised trials. In this paper, we report on the updating of AMSTAR and its adaptation to enable more detailed assessment of systematic reviews that include randomised or non-randomised studies of healthcare interventions, or both. With moves to base more decisions on real world observational evidence we believe that AMSTAR 2 will assist decision makers in the identification of high quality systematic reviews, including those based on non-randomised studies of healthcare interventions*.*\nauthor: Beverley J Shea; Barnaby C Reeves; George Wells; Micere Thuku; Candyce Hamel; Julian Moran; David Moher; Peter Tugwell; Vivian Welch; Elizabeth Kristjansson; David A HenryCorrespondence to: B J Shea <firstname.lastname@example.com>\ndate: 2017\nreferences:\ntitle: AMSTAR 2: a critical appraisal tool for systematic reviews that include randomised or non-randomised studies of healthcare interventions, or both\n\n# Summary points\n\n1.  Systematic reviews of studies of healthcare interventions effects often include non-randomised studies\n\n2.  AMSTAR is a popular instrument for critically appraising systematic reviews of randomised controlled clinical trials\n\n3.  AMSTAR underwent further development to enable appraisal of systematic reviews of randomised and non-randomised studies of healthcare interventions\n\n4.  The revised instrument (AMSTAR 2) retains 10 of the original domains, has 16 items in total (compared with 11 in the original), has simpler response categories than the original AMSTAR, includes a more comprehensive user guide, and has an overall rating based on weaknesses in critical domains\n\n5.  AMSTAR 2 is not intended to generate an overall score\n\n6.  With moves to base more decisions on real world observational evidence, AMSTAR 2 should assist in the identification of high quality systematic reviews\n\nWith the rapid increase in biomedical publishing, keeping up with primary research has become almost impossible for healthcare practitioners and policy makers.1 Consequently, healthcare decision makers rely on systematic reviews as one of the key tools for achieving evidence based healthcare.2 Systematic reviews provide an opportunity to base decisions on accurate, succinct, credible, and comprehensive summaries of the best available evidence on a topic.2\n\nUncritically accepting the results of a single systematic review has risks. One of us (DM) led efforts to improve standards for reporting of systematic reviews, which led to the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) statement.3 The reporting guide for systematic reviews of observational (non-randomised) studies is MOOSE (Meta-analysis of Observational Studies in Epidemiology).4 The quality of reporting of a systematic review may, however, more accurately reflect authors' ability to write in a comprehensible manner rather than the way they conducted their review. This underscores the need for guidelines that evaluate the way in which reviews are planned and conducted.5 6\n\nThe Cochrane Collaboration Handbook provides a comprehensive guide for review authors, but it does not provide a concise critical appraisal instrument for completed reviews.5 Several instruments have been designed to evaluate individual studies that are being included in systematic reviews or how certain steps (eg, meta-analysis, testing for publication bias) should be conducted.7 8 9 10 11 12 13 14 15 But relatively few instruments assess all important steps in the conduct of a review.16 17 18 19 20 21\n\nAMSTAR (A MeaSurement Tool to Assess systematic Reviews), published in 2007, is one of the most widely used instruments.22 23 24 AMSTAR was designed by us and our colleagues as a practical critical appraisal tool for use by health professionals and policy makers who do not necessarily have advanced training in epidemiology, to enable them to carry out rapid and reproducible assessments of the quality of conduct of systematic reviews of randomised controlled trials of interventions. Since publication, several critiques of the instrument have been published.25 26 27 28 29 30 31 These critiques plus feedback received at workshops and developments in the science of systematic reviews pointed to a need to revise and update the original AMSTAR instrument.\n\n# Inclusion of non-randomised studies in systematic reviews\n\nAlmost half of published systematic reviews now include non-randomised studies of intervention effects.4 32 33 34 There are many concerns about the conduct and reporting of systematic reviews of non-randomised studies.32 35 36 To summarise, non-randomised studies of healthcare interventions (an important focus of this revision of AMSTAR) are subject to a range of biases that are either not present or are less noticeable in randomised controlled trials, thus requiring different risk of bias assessments. Observational studies are increasingly conducted within large population databases, sometimes with hundreds of thousands or even millions of recipients of healthcare interventions. These generate precise estimates of intervention effects, which may be inaccurate because of residual biases. If these estimates are combined with those from the (generally smaller) randomised controlled trials, the meta-estimates will be weighted towards the observational study estimates. The original AMSTAR instrument did not include an assessment of the risk of bias in non-randomised studies included in a review, which is a key issue given the diversity of designs that such studies may use and the biases that may affect them.\n\n# Development of AMSTAR 2\n\nThe development and validation of the original AMSTAR instrument (published in 2007) has been described in detail elsewhere.22 23 24 Briefly, the original list of items was created from the results of a scoping review of the then available rating instruments. This review identified many over-lapping appraisal items, mainly from two extensively cited reports.16 17 The lists of items from these reports were combined and reduced by factor analysis. After pilot testing, items were reworded as needed and the reliability and usability of the tool was assessed. A modified version was validated externally and performed well against the global judgments of a panel of content experts.23 The publications describing the original AMSTAR instrument were widely cited and the instrument has been used and critiqued extensively.22 23 24 25 26 27 28 29 30 31\n\nWe convened an expert group, comprising authors of the original instrument, members with expertise in the conduct of non-randomised studies, development of appraisal instruments, biostatistics, and study designs. The expert group met for a day in Ottawa, Canada and members were presented with the results of updated literature reviews on relevant critical appraisal instruments, the results of surveys of AMSTAR users, recorded experience of participants in AMSTAR workshops at Cochrane Colloquiums in 2015 and 2016, feedback from the AMSTAR website ([www.amstar.ca](http:\/\/www.amstar.ca)), and published critiques of the original instrument.16 17 18 19 20 21 22 23 24 25 26 The perspective adopted by the expert group was to increase the value of AMSTAR as a broad critical appraisal instrument designed primarily for systematic reviews of studies of healthcare interventions. The expert group considered that revisions should address all aspects of the conduct of a systematic review, and the challenges of including non-randomised studies. They also thought the revised instrument should function as a teaching aid and as a concise checklist for those conducting reviews. The revisions were not intended to deal with the special requirements of diagnostic test reviews, individual patient data meta-analyses or network meta-analyses, scoping reviews, or realist reviews.37 38 39 40 41\n\nWe used a nominal group technique to propose and then prioritise specific changes to the instrument and to agree on the draft wording of items. Based on their experience of the instrument and the presentations made at the meeting, participants were asked to record their ideas independently and privately. The ideas were then enunciated in a round-robin format. One idea was collected from everyone, in turn, and presented to the group by the facilitator. This process was continued until all ideas had been listed. Individuals then privately recorded their judgments and rankings. These were aggregated statistically to derive the group judgments. The following changes were agreed on (these are not listed in order of priority as all were considered important enough to mandate modifications to the instrument):\n\n-   Simplify the response categories\n\n-   Align the definition of research questions with the PICO (population, intervention, control group, outcome) framework\n\n-   Seek justification for the review authors' selection of different study designs (randomised and non-randomised) for inclusion in systematic reviews\n\n-   Seek more details on reasons for exclusion of studies from the review\n\n-   Determine whether the review authors had made a sufficiently detailed assessment of risk of bias for the included studies (whether randomised or non-randomised)\n\n-   Determine whether risk of bias with included studies was considered adequately during statistical pooling of results (if this was performed)\n\n-   Determine whether risk of bias with included studies was considered adequately when interpreting and discussing the review findings.\n\nA description was formulated for each of the draft items. A small subgroup refined the wording of the items and assembled the draft instrument for testing. Initial pilot testing was performed by group members. Draft versions were presented at workshops held at the Cochrane Colloquiums in 2015 and 2016, where feedback directed further modifications and redrafting of the instrument. The version of the instrument presented here was subject to inter-rater reliability and usability testing.\n\n# Comparison with the original instrument\n\nThe supplementary figure provides details of the new instrument (AMSTAR 2). Ten domains were retained from the original tool, with changes to the wording of items based on feedback about the original instrument and experience of testing drafts of the new instrument. Two domains were given more detailed coverage in AMSTAR 2 than in the original instrument: duplicate study selection and data extraction now have their own items (they were combined in the original tool). The possible influence of funding sources is now considered separately for individual studies included in the review and for the review itself. Previously they were combined in one item. We added more detailed and separate considerations of risk of bias for randomised and non-randomised studies. Both sub-items are based on content from the Cochrane risk of bias instruments for randomised and non-randomised (ROBINS-I) studies.42 43 One domain was removed\u2014consideration of grey literature, previously a separate item, is now handled in the item on literature searching.\n\nIn total, four domains were added. Two of these came directly from the ROBINS-I tool\u2014namely, elaboration of the PICO and the way in which risk of bias was handled during evidence synthesis.43 One of the other new domains\u2014discussion of possible causes and significance of heterogeneity\u2014is an elaboration of content in the original AMSTAR tool. Another new domain\u2014justification of selection of study designs\u2014was part of the adaptation of AMSTAR to deal with non-randomised designs.\n\nThe domain specific questions in AMSTAR 2 are framed so that a \"Yes\" answer denotes a positive result. We removed the \"not applicable\" and \"cannot answer\" options in the original AMSTAR instrument because we believe that all domains are relevant to contemporary systematic reviews of healthcare interventions. If no information is provided to rate an item, the review authors should not be given the benefit of doubt and the item should be rated as a \"No.\" We have provided a \"partial Yes\" response in some instances where we considered it worthwhile to identify partial adherence to the standard.\n\n# Rationale for selection of items\n\nHere we summarise our thinking behind the items in AMSTAR 2, which are numbered as in the instrument (see supplementary figure). Supplementary appendix 1 provides a more complete user's guide.\n\n*1. Did the research questions and inclusion criteria for the review include the components of PICO?*\n\nIt is common practice to use the PICO description (population, intervention, control group, and outcome) as a convenient and easily memorised framework for a study question. Sometimes a timeframe should be added if this is critical in determining the likelihood of a study capturing relevant clinical outcomes (eg, an effect of the intervention is only expected after several years).\n\n2\\. *Did the report of the review contain an explicit statement that the review methods were established prior to the conduct of the review and did the report justify any significant* *deviations from the protocol?*\n\nSystematic reviews are a form of observational research, and the methods for the review should be agreed on before the review commences. Adherence to a well developed protocol reduces the risk of bias in the review. Authors should show that they worked with a written protocol with independent verification.\n\n3\\. *Did the review authors explain their selection of the study designs for inclusion in the review?*\n\nFor some questions, for instance the effects of policy changes, or for ethical reasons, non-randomised studies may be the only studies addressing the review question. With an expansion of AMSTAR 2 to appraise reviews that include randomised controlled trials or non-randomised studies, or both, it is important that authors justify the inclusion of different study designs in systematic reviews. The authors should indicate that they followed a strategy. When both randomised and non-randomised studies address the same question about the effects of an intervention, we believe that authors should consider whether a review that is restricted to randomised controlled trials will give an incomplete summary of the important effects of a treatment.\n\n*4. Did the review authors use a comprehensive literature search strategy?*\n\nThe importance of adequate literature searching in systematic reviews is well established.5 This item was carried over with minimal changes to the wording from the original instrument. We have made the response options clearer in AMSTAR 2 and provide more detailed guidance on completion of the item, particularly in relation to the identification of non-randomised studies (see supplementary appendix 1).\n\n*5. Did the review authors perform study selection in duplicate?*\n\nBest practice requires two review authors to determine eligibility of studies for inclusion in systematic reviews.5 This involves checking the characteristics of a study against the elements of the research question. In the original AMSTAR, this item covered determining both study eligibility and data extraction. The expert group believed that they were sufficiently distinct processes to merit separate items in AMSTAR 2.\n\n*6. Did the review authors perform data extraction in duplicate?*\n\nThe expert group recognised that data extraction might be more complex for non-randomised studies of healthcare interventions as it usually involves extraction of measures of treatment effects and other associations that have been adjusted for potential confounding, rather than raw outcome data from treated and control groups. A study report may present multiple treatment effects; judgment is therefore needed to select the one that conforms best to the PICO question and is at lowest risk from confounding.\n\n*7. Did the review authors provide a list of excluded studies and justify the exclusions?*\n\nIn the revised instrument we consider excluded and included studies separately. Excluded studies should be accounted for fully by review authors, otherwise there is a risk that they remain invisible and the impact of their exclusion from the review is unknown.\n\n*8. Did the review authors describe the included studies in adequate detail?*\n\nThe revised instrument requires review authors to provide detail about research designs, study populations, interventions, comparators, and outcomes. The detail should be sufficient for appraisers to make a judgment about the extent to which the studies were appropriately chosen (in relation to the PICO) and whether the study populations and interventions were relevant to their questions. This information is needed to determine the extent to which the results of different studies should be combined, help explain heterogeneity, and assist those applying the results.\n\n*9. Did the review authors use a satisfactory technique for assessing the risk of bias (RoB) in individual studies that were included in the review?*\n\nBiases can be introduced at several stages in the design, planning, conduct, and analysis of a study. This item replaces a less detailed item on \"scientific quality.\" The item specifies domains of bias for randomised and non-randomised studies that should have been considered by reviewers, based on the relevant Cochrane instruments.42 43 In AMSTAR 2 we ask whether the review authors made an adequate assessment of study level efforts to avoid, control, or adjust for baseline confounding, selection biases, bias in measurement of exposures and outcomes, and selective reporting of analyses or outcomes, or both. The guidance document (see supplementary appendix 1) and the ROBINS-I report provide more detail.43 We decided not to include assessment of time varying confounding, performance biases, and biases due to missing data, although they are currently included in ROBINS-I.43 This was because of the complex nature of techniques used to adjust for these potential sources of bias and the frequent lack of data (in contemporary primary studies) to enable assessment of these items. Version 2.0 of the Cochrane risk of bias instrument for randomised controlled trials is now available in draft form, and AMSTAR 2 will be aligned with this in the future.44\n\n*10. Did the review authors report on the sources of funding for the studies included in the review?*\n\nWe added a consideration of funding sources in the light of evidence from several sources that the results of industry funded studies sometimes favoured sponsored products, and that industry funded studies were less likely to be published than those that were independently funded.45 46 47 Such influences may not be detected as flaws in design or methods (item 9).\n\n*11. If meta-analysis was performed, did the review authors use appropriate methods for statistical combination of results?*\n\nThis is a modified version of an item in the original instrument and is judged separately for randomised and non-randomised studies. Review authors should have stated explicitly in the review protocol the principles on which they based their decision to perform meta-analysis of data from the included studies. This includes the extent to which the studies are compatible (in terms of patients, controls, and interventions) and the value of a single pooled effect (for instance from several compatible but underpowered studies). Where reviewers consider it appropriate to conduct a meta-analysis, the inclusion of non-randomised studies increases the complexity of the analyses and may increase heterogeneity (see supplementary appendix 1).\n\n*12. If meta-analysis was performed, did the review authors assess the potential impact of RoB in individual studies on the results of the meta-analysis or other evidence synthesis?*\n\nThis is a new item that requires reviewers to examine how results vary with inclusion or exclusion of primary studies judged to be at high risk of bias. In cases where review authors have chosen to include only high quality randomised controlled trials there may be little discussion of the potential impact of bias on the results. But where they have included randomised controlled trials of variable quality or non-randomised studies they should assess the impact of study level risk of bias on the results of the review.48\n\n*13. Did the review authors account for RoB in primary studies when interpreting\/discussing the results of the review?*\n\nThis is a modification of an item from the original instrument. With a greater emphasis on assessing risk of bias, the expectation is that reviewers will make explicit reference to the potential impacts of risk of bias when interpreting and discussing the results of their review and in drawing conclusions or making recommendations.\n\n*14. Did the review authors provide a satisfactory explanation for, and discussion of, any heterogeneity observed in the results of the review?*\n\nThis item is carried over with modified wording from the original instrument. It is important that reviewers investigate possible causes of heterogeneity, including variation in those elements included in the PICO framework (see item 1) and those arising from design and methodological considerations (see item 9). With the inclusion of non-randomised studies, variations in design and analysis may contribute to heterogeneity.\n\n*15. If they performed quantitative synthesis did the review authors carry out an adequate investigation of publication bias (small study bias) and discuss its likely impact on the results of the review?*\n\nThis item is carried over from the original instrument but with modified wording. Publication bias is an important problem but it can be difficult for authors to resolve completely. Typically, statistical tests (several are available) or graphical displays are used and if the results are positive they indicate the presence of publication bias. Negative test results are not a guarantee of the absence of publication bias as they are insensitive. A minimum of 10 studies are required to show funnel plot asymmetry.5 The underlying tendency to selectively publish small positive studies may be compounded by the effects of lower methodological quality of small studies, a greater tendency to selectively report results, and increased clinical heterogeneity when conducted in patient subgroups.49\n\n*16. Did the review authors report any potential sources of conflict of interest, including any funding they received for conducting the review?*\n\nThis item is carried over with modified wording from the original instrument and is now separate from consideration of funding of the primary studies included in the review (item 10). As with primary studies, review authors should report their funding sources.50 51\n\n## Identification of critical domains\n\nAll steps in the conduct of a systematic review and meta-analysis are important, but we believe that seven domains can critically affect the validity of a review and its conclusions (box 1). Two of these concern risk of bias, whether it has been assessed adequately and how it can influence the results of a review. The prominence we give to risk of bias is because AMSTAR 2 is going to be used to appraise many systematic reviews that include non-randomised studies.\n\n### Box 1 AMSTAR 2 critical domains\n\n-   Protocol registered before commencement of the review (item 2)\n\n-   Adequacy of the literature search (item 4)\n\n-   Justification for excluding individual studies (item 7)\n\n-   Risk of bias from individual studies being included in the review (item 9)\n\n-   Appropriateness of meta-analytical methods (item 11)\n\n-   Consideration of risk of bias when interpreting the results of the review (item 13)\n\n-   Assessment of presence and likely impact of publication bias (item 15)\n\nWe recognise that the items listed in box 1 will not always be regarded as critical; for example, risk of bias related items may be considered less important when a review is confined to high quality randomised controlled trials. Other circumstances where the critical nature of items may be questioned are when a review team are using meta-analysis to summarise a known literature base (eg, the output from one or more established clinical trial collaborative groups). In this circumstance the adequacy of the literature search (item 4), listing of excluded studies (item 7), and possibility of publication bias (item 15) may not be considered critical. If a meta-analysis was not performed, the item covering the appropriateness of the meta-analytical methods (item 11) will not apply. However, it is important in this circumstance that appraisers are alert to the possible impact of risk of bias when review authors select individual studies to highlight in a narrative summary.\n\nFlaws in the items that we have identified as critical may not be fatal if further information (eg, directly from the review authors) indicates that the original response option was wrong. This may provide reassurance about the review findings or enable an amendment of the review through additional analyses. We emphasise that our listing is a suggestion and appraisers may add or substitute other critical domains. For example, the failure to include non-randomised studies (item 3) in a review of adverse outcomes of treatment may be a critical flaw, as would the inability to explain large variations in treatment effects across a body of studies (item 14).\n\n## Applying AMSTAR 2 to systematic reviews\n\nIf one or more systematic reviews will be the basis of important practice and policy decisions we recommend that the appraisal team agree on how the AMSTAR 2 items should be applied. This includes the practice or policy context and the questions that should be addressed, based on the relevant PICO components. For example, available systematic reviews may have included studies with different comparators or different follow-up times, and their relevance to the policy relevant questions needs to be established. The likely sources of bias should also be agreed on. For instance, in observational studies of intervention effects, confounding by indication (or disease severity) may be problematic when interventions are reserved for certain subgroups of patients.52 It is good practice to recruit new users of a technology or drug into studies to avoid prevalence bias.53 If the start of one intervention tends to be delayed the choice of comparator may introduce immortal time bias.54 Measurement errors can misclassify exposure and outcomes and may be unbalanced across comparison groups. Selective reporting among multiple analyses and outcomes may give an inaccurate measure of intervention effects.\n\nSupplementary appendix 1 provides guidance on sections of AMSTAR 2. Some of the judgments (particularly whether review authors have adequately assessed risk of bias with individual non-randomised studies) are complex, and advice on both methodology and content may be needed. Content knowledge is sometimes necessary to determine if the review authors have made an adequate assessment of the relevant PICO elements (item 1), and to identify potential confounders.\n\nWe strongly recommend that individual item ratings are not combined to create an overall score.55 56 Rather, users should consider the potential impact of an inadequate rating for each item.\n\nIn box 2 we propose a scheme for interpreting weaknesses detected in critical and non-critical items. This is advisory and appraisers should decide which items are most important for the reviews under consideration.\n\n### Box 2 Rating overall confidence in the results of the review\n\n1.  **High**\n\n2.  *No or one non-critical weakness*: the systematic review provides an accurate and comprehensive summary of the results of the available studies that address the question of interest\n\n3.  **Moderate**\n\n4.  *More than one non-critical weakness*\\*: the systematic review has more than one weakness but no critical flaws. It may provide an accurate summary of the results of the available studies that were included in the review\n\n5.  **Low**\n\n6.  *One critical flaw with or without non-critical weaknesses*: the review has a critical flaw and may not provide an accurate and comprehensive summary of the available studies that address the question of interest\n\n7.  **Critically low**\n\n8.  *More than one critical flaw with or without non-critical weaknesses*: the review has more than one critical flaw and should not be relied on to provide an accurate and comprehensive summary of the available studies\n\n## Inter-rater reliability of AMSTAR 2\n\nWe measured inter-rater agreement with three pairs of raters and three sets of systematic reviews (see supplementary appendix 2). The first pair of raters was involved in the development of AMSTAR 2 (coauthors MT and CH). They individually appraised 20 systematic reviews derived from a rapid search (conducted in 2015 on the terms \"systematic review\" and \"meta-analysis\" in the title) using Google Scholar. From the first 200 we selected 20 systematic reviews of any healthcare intervention. The other two pairs of raters were experienced in the appraisal of systematic reviews and were not involved in the development of AMSTAR or AMSTAR 2. They applied AMSTAR 2 during their routine work, performing appraisals of systematic reviews of two topics: interventions to reduce medication errors (14 reviews) and non-pharmacological therapies for Parkinson's disease (20 reviews) (see references in supplementary appendix 2). In both cases systematic reviews had been identified through comprehensive literature searches (details available on request). All raters had access to the user guide (see supplementary appendix 1), applied the instrument individually, and did not try to achieve consensus. In total, six raters applied the instrument to 54 systematic reviews, of which 20 included only randomised controlled trials, 18 included only non-randomised studies of interventions, and 16 included a mixture of both designs.\n\nSupplementary appendix 2 provides summaries of the \u03ba scores for agreement between the three pairs of raters across the three sets of reviews. The values varied substantially across items and between pairs of raters. Most values were in an acceptable range, with 46 of the 50 \u03ba scores falling in the range of moderate or better agreement and 39 displaying good or better agreement. There were no large differences between raters, and those who had been involved in the development of AMSTAR 2 did not have higher levels of agreement than the rater who was not involved. Items 9, 12, and 13 are concerned with measurement of risk of bias and how this is handled during discussion of the meta-analysis and interpretation of the results. The ranges of \u03ba scores for these items were similar to those seen with other items in the instrument (see supplementary appendix 2). For items 9 and 11 the \u03ba values for risk of bias judgments for randomised controlled trials were similar to those for non-randomised studies.\n\n## Usability of AMSTAR 2\n\nThe completion times for the 20 reviews used by reviewers 1 and 2 ranged from 15-32 minutes. These estimates do not include the time taken to read the reviews. This is almost twice the time taken to complete the original AMSTAR instrument (range 10-15 minutes), when it was applied to systematic reviews that were limited to randomised controlled trials.57 The comments from the reviewers included: that the removal of the \"can't answer\" and \"not applicable\" response options in the original instrument forced them to make judgments; that it takes longer to evaluate the non-randomised and mixed study reviews, but this requires the reviewer to confront important methodological issues; that it was common for review authors to mention the presence or absence of publication bias, but not provide any evidence; and that review authors would disclose their potential competing interests but not how they managed them.\n\n# Discussion\n\nAMSTAR 2 is a major revision of the original AMSTAR instrument, which was designed to appraise systematic reviews that included randomised controlled trials.22 23 24 The main modifications include simplified response categories; a more detailed consideration of risk of bias with included studies, and how this was handled by review authors in summarising and interpreting the results of their reviews; better alignment with the PICO framework for research questions; a more detailed justification of selection of study designs for inclusion in a review; and more information on studies that were excluded from reviews. In addition, we recommend defining critical domains before starting an appraisal of a systematic review. Identification of weaknesses in these domains should undermine confidence in the results of a systematic review.\n\nWe stress that responses to AMSTAR 2 items should not be used to derive an overall score.55 56 The original AMSTAR instrument was often used for this purpose and this was facilitated by the website ([www.amstar.ca](http:\/\/www.amstar.ca)). We accept that an overall score may disguise critical weaknesses that should diminish confidence in the results of a systematic review and we recommend that users adopt the rating process based on identification of critical domains (see box 2), or some variation based on these principles.56\n\nWe envisage that AMTAR 2, like its predecessor, may have a role as a convenient teaching aid and as a brief checklist for those conducting systematic reviews. However, we stress that the instrument does not explain in detail the logic and methods of conducting systematic reviews, and those looking for comprehensive advice should consult the Cochrane Handbook.5\n\nThe consideration of risk of bias in individual studies is equally important for randomised and non-randomised studies of healthcare interventions but is generally better understood with the former. Large non-randomised studies, often conducted in large administrative databases, are increasingly being used to assess the real world impact of a wide range of healthcare technologies and practices. Although such studies often use sophisticated methods, residual confounding or failure to deal with other sources of bias may lead to inaccurate estimates of effect. Inclusion of large observational studies in meta-analyses may generate precise but biased estimates of intervention effects.32\n\nThe items in AMSTAR 2 that deal with risk of bias identify domains specified in the Cochrane risk of bias instruments for randomised and non-randomised studies.42 43 These represent a consensus, in each case developed with input from more than 30 experts in methodology. However, AMSTAR 2 does not currently specify which risk of bias instruments review authors should have used to assess non-randomised studies included in a systematic review. The ROBINS-I instrument, which is the most comprehensive tool for non-randomised studies evaluating the effects of healthcare interventions, was released in 2016 and it is unrealistic to expect authors of reviews started before its release to have used it.43 Presently, AMSTAR 2 leaves it to the review authors and those appraising the review to satisfy themselves that the risk of bias instrument used by review authors has sufficient discriminatory ability for the specified risk of bias domains. A review by Sanderson and colleagues identified 86 tools for assessing quality of observational studies, without a clear preference among them.58 The authors pointed to the need to agree on critical elements for assessing susceptibility to bias in observational epidemiology. In part this review led to the development of ROBINS-I.43 Popular appraisal instruments for individual studies, such as the Newcastle Ottawa Scale and the Scottish Intercollegiate Guidelines Network (SIGN) checklist may not focus on validity alone.59 60 The Newcastle Ottawa Scale appears to lack sensitivity and is sometimes used to generate an overall score, something that is not recommended because it may disguise critical weaknesses in a review.56 61\n\nAMSTAR 2, as a critical appraisal instrument for systematic reviews, joins several published instruments designed for this purpose.3 4 16 17 19 20 25 62 Two prominent examples are concerned with guidelines for reporting systematic reviews, rather than their conduct.3 4 Two highly cited instruments were the basis for the development of the original AMSTAR tool.16 17 22 Two published instruments are direct derivatives of the original AMSTAR.19 25 Another publication includes a checklist used to appraise systematic reviews that are being included in an umbrella review.20 Overlap between the content of this checklist and the original AMSTAR is considerable.22\n\nAMSTAR 2 provides a broad assessment of quality, including flaws that may have arisen through poor conduct of the review (with uncertain impact on findings). In this respect it differs from another instrument, the Risk Of Bias In Systematic reviews (ROBIS).62 ROBIS is a sophisticated three phase instrument that focuses specifically on the risk of bias introduced by the conduct of the review. It covers most types of research question, including diagnosis, prognosis, and aetiology. In contrast, AMSTAR 2 is intended to be used for reviews of healthcare interventions. Inevitably there is overlap in the items considered by ROBIS and AMSTAR 2; indeed, two investigators (BCR, BJS) were involved in the development of both.\n\nIn developing AMSTAR 2 we sought to maintain its familiar and popular stepwise checklist approach and augmented this by the addition and modification of items. AMSTAR 2 will be familiar to users of the original instrument, although more demanding to use for reasons discussed previously. Because AMSTAR 2 is structured around the key sequential steps in the conduct of a systematic review, it may be used as a brief teaching aid or as a checklist by those conducting systematic reviews.\n\nUnlike the original instrument, AMSTAR 2 identifies critical weaknesses (see box 1) that should reduce confidence in the findings of a review, and it asks users to prespecify how this list will vary for the review topic. We understand that there will be debate about membership of this list and propose that users may wish to prespecify a different set of critical items for a specific PICO research question or setting.\n\nWe did not perform an extensive validation of the revised AMSTAR 2 tool. In its development, 10 domains were retained from the original validated tool, albeit with some wording changes based on feedback and extensive experience of using it. Two domains were given more detailed coverage: duplicate study selection and data extraction now have their own items (they were combined in the original tool); we have added more detailed, and separate, considerations of risk of bias for randomised and non-randomised studies. The sub-items were derived from widely used Cochrane instruments. One domain was removed; consideration of grey literature, previously a separate item, is now handled in the item on literature searching. In total, four domains were added. Two of these come directly from the ROBINS-I tool\u2014namely, elaboration of PICO in the review and the way in which risk of bias was handled during evidence synthesis.43 One of the other new domains, discussion of possible causes and importance of heterogeneity, is elaboration of content in the original AMSTAR tool.22 The final domain, justification of selection of study designs, is justified by adapting AMSTAR to deal with non-randomised designs. We do not think this needs validation because we believe it is obvious that authors of systematic reviews should justify why they have included study designs that are more susceptible to bias.\n\nThe levels of agreement achieved by the three pairs of raters varied across items, but they were moderate to substantial for most items. Notably, the agreement between two raters involved in the development of AMSTAR 2 was no higher than that achieved by experienced raters who had not been involved its development. We did not expect perfect agreement, and differences between raters reflect the demanding nature of some item level judgments and should prompt group discussion of their causes and importance, and, if needed, consultation with experts in subject matter and methods.\n\nIn developing AMSTAR 2 we relied heavily on the consensus of the expert panel, but we also received extensive feedback from users of the original instrument in the form of direct communications, website comments, and evaluations made at teaching workshops at Cochrane Colloquiums. In the later phases of development of AMSTAR 2 we had access to, and discussed, recently published critiques of AMSTAR.25 26 27 28 29 30 31\n\nOur experience of releasing and using the original AMSTAR instrument is that judgments need to be made and users may sometimes decide to make modifications to the instrument.25 26 30 We encourage investigators to provide feedback, and, if they adapt the instrument for particular settings, to report their experience at [www.amstar.ca](http:\/\/www.amstar.ca).\n\nWe thank for their assistance in the development of the AMSTAR critical appraisal instruments: Lex Bouter, Maarten Boers, Alonso Carrasco-Labra, Jeremy Grimshaw, Ranjeeta Mallick, Jordi Pardo-Pardo, and Larissa Shamseer, and for conducting the reliability studies reported in this manuscript: Brian Hutton, Pauline Barbeau, Fatemeh Yazdi, Vesa Basha, and Roxanne Ward.\n\nExtra material supplied by the authors\n\n**Supplementary figure:** the AMSTAR 2 instrument\n\n**Supplementary appendix 1:** AMSTAR 2 guidance document\n\n**Supplementary appendix 2:** Inter-rater reliability of AMSTAR 2 items and references for studies included in the analyses","meta":{"dup_signals":{"dup_doc_count":127,"dup_dump_count":49,"dup_details":{"curated_sources":2,"2023-50":6,"2023-40":6,"2023-23":2,"2023-06":5,"2022-49":2,"2022-40":5,"2022-33":5,"2022-27":3,"2022-21":6,"2022-05":4,"2021-49":3,"2021-43":5,"2021-39":4,"2021-31":3,"2021-25":4,"2021-21":5,"2021-17":4,"2021-10":1,"2021-04":1,"2020-50":3,"2020-45":3,"2020-29":2,"2020-24":2,"2020-16":1,"2020-10":2,"2019-51":1,"2019-47":1,"2019-43":1,"2019-39":2,"2019-35":1,"2019-30":1,"2019-26":3,"2019-22":1,"2019-13":1,"2019-09":1,"2019-04":1,"2018-51":1,"2018-47":2,"2018-43":2,"2018-34":3,"2018-26":2,"2018-17":2,"2018-05":1,"2017-51":1,"2017-43":2,"2024-26":3,"2024-22":2,"2024-18":2,"2024-30":1}},"file":"PMC5833365"},"subset":"pubmed_central"}
{"text":"abstract: Interest in how the gut microbiome can influence the metabolic state of the host has recently heightened. One postulated link is bacterial fermentation of \"indigestible\" prebiotics to short-chain fatty acids (SCFAs), which in turn modulate the release of gut hormones controlling insulin release and appetite. We show here that SCFAs trigger secretion of the incretin hormone glucagon-like peptide (GLP)-1 from mixed colonic cultures in vitro. Quantitative PCR revealed enriched expression of the SCFA receptors *ffar2* (*grp43)* and *ffar3 (gpr41)* in GLP-1\u2013secreting <span class=\"smallcaps\">L<\/span> cells, and consistent with the reported coupling of GPR43 to Gq signaling pathways, SCFAs raised cytosolic Ca^2+^ in <span class=\"smallcaps\">L<\/span> cells in primary culture. Mice lacking *ffar2* or *ffar3* exhibited reduced SCFA-triggered GLP-1 secretion in vitro and in vivo and a parallel impairment of glucose tolerance. These results highlight SCFAs and their receptors as potential targets for the treatment of diabetes.\nauthor: Gwen Tolhurst; Helen Heffron; Yu Shan Lam; Helen E. Parker; Abdella M. Habib; Eleftheria Diakogiannaki; Jennifer Cameron; Johannes Grosse; Frank Reimann; Fiona M. GribbleCorresponding authors: Fiona M. Gribble, <email@example.com>, and Frank Reimann, <email@example.com>.\ndate: 2012-02\nreferences:\ntitle: Short-Chain Fatty Acids Stimulate Glucagon-Like Peptide-1 Secretion via the G-Protein\u2013Coupled Receptor FFAR2\n\nTargeting the release of anorectic and antidiabetic gut peptides is the focus of many ongoing drug development programs, as evidence is accumulating that enhanced secretion of glucagon-like peptide (GLP)-1 and peptide YY (PYY) from intestinal L cells may translate into beneficial effects in subjects with diabetes and obesity (1). Already, the diabetes field has witnessed the impact of therapeutic GLP-1 mimetics and dipeptidyl peptidase (DPP)4 inhibitors, which, respectively, mimic endogenous active GLP-1 and slow its enzymatic degradation in the circulation (2). The injectable GLP-1 mimetics, in particular, are associated not only with improved blood glucose control and reduced incidence of hypoglycemia but also with significant weight reduction (3). The observed correlation between elevated postprandial GLP-1 levels and improved glucose homeostasis in patients after bariatric surgery indicates that the body has excess capacity in the GLP-1 axis that can be recruited in obese diabetic patients, with downstream beneficial effects on food intake and diabetes control (4). Identifying and validating pharmaceutical strategies to enhance GLP-1 secretion are central to many ongoing L cell\u2013targeting research programs.\n\nL cells are a component of the enteroendocrine system, diffusely located along the length of the intestinal epithelium (5). They make contact with the gut lumen via apical processes and are believed to respond directly to luminal signals. Among the best characterized triggers of GLP-1 secretion are sugars, amino acids, and long-chain fatty acids, which stimulate L cells by a variety of pathways including transporter-associated uptake, metabolism, and G-protein\u2013coupled receptor activation (5,6). L cells are, however, found in highest density in the colonic epithelium, where these nutrients are unlikely to reach significant concentrations (7). Short-chain fatty acids (SCFAs), derived from bacterial fermentation of macrofibrous material reaching the distal gut, by contrast are known to reach high concentrations under physiological conditions in the colons of healthy subjects. Intraluminal concentrations beyond 100 mmol\/L, comprising \\~60% acetate (C2), 25% propionate (C3), and 15% butyrate (C4), have been reported (8). Plasma levels of SCFA are also dominated by acetate but are generally below \u223c200 \u03bcmol\/L unless elevated by ethanol metabolism (9,10). Nondigestible and fermentable dietary fiber, as well as SCFAs themselves, has been shown to increase GLP-1 secretion in humans (11,12) and rodents (13,14), and enhanced PYY release has been proposed as a link between luminal SCFAs and altered gut motility (15,16).\n\nSCFAs act as a local nutrient source but can also trigger cell-specific signaling cascades by activation of the G-protein\u2013coupled free fatty acid receptor (FFAR)2 (GPR43) and FFAR3 (GPR41) (17,18). These two receptors share \\~40% amino acid sequence similarity and are conserved across several mammalian species (17\u201319). Both receptors respond to SCFAs containing two to five carbons, although a preference of FFAR2 for C2 and C3 fatty acids and of FFAR3 for C3\u2013C5 carbon chain lengths has been reported (17,18,20). The receptors differ in their intracellular signaling capabilities, with FFAR2 reportedly coupling to either G~q~ or G~i\/o~ and FFAR3 exclusively activating G~i\/o~ pathways (17,18,20). The finding that both receptors are located in colonic L cells by immunostaining (21\u201323) suggests that SCFAs may use this pathway to modulate L-cell function. Experimental data in support of this idea are, however, still lacking.\n\nThe aim of the current study was to establish and explore the link between SCFAs and GLP-1 secretion, making use of our transgenic mouse model (GLU-Venus) in which L cells are identifiable by their expression of a yellow fluorescent protein derivative, Venus (24).\n\n# RESEARCH DESIGN AND METHODS\n\nAll animal procedures were approved by the local ethics review committees and conformed to Home Office regulations. Experiments were performed using mice on a C57B\/6 background, except in the case of the *ffar2*^\u2212\/\u2212^ and *ffar3*^\u2212\/\u2212^ mice and littermate controls, which were on a 129\/SvEv background.\n\n## Generation of knockout mice.\n\nFor generation of the targeting vectors, homology arms (*ffar3*, 1,250 base pairs \\[bp\\] 5\u2032 and 3,914 bp 3\u2032; *ffar2*, 1,761 bp 5\u2032 and 4,199 bp 3\u2032) were PCR amplified from genomic DNA and cloned into a vector containing internal ribosomal entry site *\u03b2-gal* reporter gene, neomycin selection marker, and two thymidine kinase negative selection markers ([Supplementary Figs. 1 and 2](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db11-1019\/-\/DC1)). The linearized vector was electroporated into embryonic stem cells (129\/SvEv), and homologous recombination was detected by screening PCR and Southern blot. Chimeras were generated by blastocyst injection, bred with 129\/SvEv animals, and maintained inbred in this background.\n\n## Glucose-stimulated GLP-1 secretion in vivo.\n\nExperiments were performed on 3- to 4-month-old *ffar2*^\u2212\/\u2212^ and *ffar3*^\u2212\/\u2212^ mice or wild-type littermate control 129\/SvEv mice. No significant differences in body weight were observed between the wild-type and knockout groups. Mice were fasted for 4 h and dosed per os with 20 mg\/kg DPP4 inhibitor (cat. no. KR-62436; Sigma). Thirty minutes later, mice were dosed by gavage with 1.5 g\/kg glucose solution (Fisher Scientific). For initial GLP-1 measurement, 150 \u03bcL blood was collected from awake mice via tail bleed into EDTA-coated capillary tubes (Bilbate) before glucose dosing. Thirty minutes after glucose dosing, mice were killed by CO~2~ inhalation and blood was collected via cardiac puncture into tubes containing aprotinin (0.6 trypsin inhibiting units\/mL). Blood samples were centrifuged immediately, and plasma was frozen on dry ice before assay in duplicate for active GLP-1 (MesoScale, Gaithersburg, Maryland).\n\n## Oral glucose tolerance test.\n\nThree- to four-month-old *ffar2*^\u2212\/\u2212^, *ffar3*^\u2212\/\u2212^, and wild-type littermate control 129\/SvEv mice were fasted for 14 h and then dosed by gavage with 1.5 g\/kg glucose. Blood glucose was measured using a handheld glucometer (OneTouch Ultra) via tail bleed. Samples for insulin measurements were taken via tail bleed from awake mice into heparinized capillary tubes (Bilbate) and assayed by ELISA (Crystalchem Ultra sensitive mouse insulin ELISA). Mice undergoing the procedure were of similar body weight: *ffar2^\u2212\/\u2212^*, 22.5 \u00b1 0.4 g (*n* = 11), wild type, 21.5 \u00b1 0.5 g (*n* = 8); *ffar3^\u2212\/\u2212^*, 22.7 \u00b1 1.5 g (*n* = 6), and wild type, 20.8 \u00b1 0.6 g (*n* = 7).\n\n## Insulin tolerance test.\n\nThree- to four-month-old *ffar2^\u2212\/\u2212^*, *ffar3^\u2212\/\u2212^*, and wild-type littermate control 129\/SvEv mice were fasted for 4 h and then dosed with 0.75 units\/kg insulin i.p. (Actrapid insulin, supplied by our Named Veterinary Surgeon). Blood glucose was measured using a handheld glucometer (OneTouch Ultra) via tail bleed from awake mice.\n\n## Colonic tissue and cell preparation.\n\nMice aged 6\u201326 weeks were killed by cervical dislocation, and colons were collected in ice-cold Leibovitz-15 medium. Cultures for secretion and calcium imaging experiments were prepared as previously described (24). Colonic tissue used for RNA and protein extraction was washed in PBS and placed in RNA later or a protein lysis buffer, respectively, and frozen until processed.\n\nGLUTag cells were cultured in Dulbecco's modified Eagle's medium (5.5 mmol\/L glucose) supplemented with 10% FBS, 2 mmol\/L <span class=\"smallcaps\">L<\/span>-glutamine, 100 units\/mL penicillin, and 0.1 mg\/mL streptomycin.\n\n## RNA extraction and quantitative PCR.\n\nTotal RNA from cells sorted with fluorescence-activated cell sorter (FACS) prepared from GLU-Venus transgenic mice (24) was isolated using a microscale RNA isolation kit (Ambion). Total RNA from GLUTag cells and murine colonic tissue was prepared following the Tri-Reagent protocol (Sigma). All samples were reverse transcribed according to standard protocols. Quantitative RT-PCR was performed with a 7900 HT Fast Real-Time PCR system (Applied Biosystems) using Taqman probes for \u03b2-actin*, gcg, pyy*, *ffar2*, and *ffar3* from Applied Biosystems. Expression was compared with that of \u03b2-actin measured in parallel on the same sample, giving a \u0394Ct for \u03b2-actin minus the test gene. If the test gene was undetectable, it was assigned a Ct value of 40. Means \u00b1 SE were calculated and statistics were performed for the \u0394Ct data and only converted to relative expression levels (2^\u0394Ct^) for presentation in the Figures.\n\n## Colonic protein analysis.\n\nTissue was mechanically homogenized in lysis buffer. Active GLP-1 was assessed by ELISA (Millipore, Watford, U.K.) and expressed relative to total protein content, measured using a Bradford assay (Sigma).\n\n## Secretion from primary mixed colonic culture.\n\nSecretion studies were performed 24\u201336 h after culture preparation. Where applicable, cultures were preincubated with 0.2 \u03bcg\/mL pertussis toxin for 18 h. Cultures were washed with standard saline and incubated with test substances for 2 h at 37\u00b0C. Secreted and cellular GLP-1 was extracted as previously described (24), and active GLP-1 was quantified by ELISA (Millipore). GLP-1 secretion was expressed as a fraction of the total hormone (secreted plus extracted) and normalized to basal secretion measured in the same set of experiments.\n\n## Ca^2+^ imaging.\n\nExperiments were performed 4\u20137 days postplating, using colonic tissue from GLU-Venus transgenic mice with <span class=\"smallcaps\">L<\/span> cell\u2013specific Venus expression (24). Cells were loaded with 7 \u03bcmol\/L Fura2-AM (Invitrogen, Paisley, U.K.), 0.01% pluronic F127, and 300 \u03bcmol\/L eserine in standard saline solution for 30 min at 37\u00b0C. Single-cell imaging was performed using an inverted fluorescence microscope (Olympus IX71; Southall, U.K.) with a 40\u00d7 oil-immersion objective. Fura2 was excited at 340\/6 and 380\/4 nm and Venus at 475\/10 nm, using a 75-W xenon arc lamp and a monochromator (Cairn Research, Faversham, U.K.) controlled by MetaFluor software (Molecular Devices, Wokingham, U.K.). Emission was recorded with an Orca ER CCD camera (Hamamatsu, Welwyn Garden City, U.K.) using a dichroic mirror and a 510-nm long-pass filter. Test substances were added to the bath solution and perfused at \u223c1 mL\/min. Fura2 fluorescence measurements were taken every 2 s, background corrected, and expressed as the 340:380 nm ratio. Average fluorescence ratios from individual cells were determined over 20-s periods before addition and during perfusion of a test agent. Peak responses to a test agent were expressed as the mean ratio of the test agent divided by the averaged ratios measured prior to drug application.\n\n## Solutions.\n\nStandard in vitro saline solution contained (in millimoles per liter) 4.5 KCl, 138 NaCl, 4.2 NaHCO~3~, 1.2 NaH~2~PO~4~, 2.6 CaCl~2~, 1.2 MgCl~2~, 10 glucose, and 10 HEPES, pH 7.4 (NaOH). Essentially fatty acid\u2013free BSA (0.1%) was added to solutions used for static secretion experiments. SCFAs were dissolved directly in saline solution and pH corrected if necessary. All other drugs for in vitro experiments were prepared as 1,000\u00d7 stocks. The protein and secretion lysis buffer contained 50 mmol\/L Tris-HCl, 150 mmol\/L NaCl, 1% IGEPAL-CA 630, 0.5% deoxycholic acid, and one tablet of complete EDTA-free protease inhibitor cocktail (Roche). All reagents were supplied by Sigma (Poole, U.K.) unless otherwise indicated. The synthetic GPR43 agonist (S)-2-(4-chlorophenyl)-*N*-(5-fluorothiazol-2-yl)-3-methylbutanamide (CFMB) (25) was synthesized, its identity was confirmed by nuclear magnetic resonance analysis, and it was dissolved in DMSO as a 1,000\u00d7 stock.\n\n## Data analysis.\n\nComparisons between conditions were made using Student *t* test (Microsoft Excel) or by one- or two-way ANOVA with post hoc Bonferroni correction or Dunnett test (Prism5; GraphPad), as indicated, with a threshold for significance of *P* \\< 0.05. All data are expressed as means \u00b1 SEM.\n\n# RESULTS\n\n## SCFAs enhance GLP-1 release.\n\nIn primary murine colonic cultures, acetate and propionate (1 mmol\/L) significantly stimulated GLP-1 secretion over a 2-h incubation period (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>A<\/em><\/a>). Secretion was further enhanced in the presence of 100 \u03bcmol\/L phosphodiesterase inhibitor isobutyl methyl xanthine (IBMX), and under these conditions a significant stimulation by 1 mmol\/L butyrate was also observed. To mimic the higher SCFA content of the colonic lumen, we also examined the effect of a 140 mmol\/L mixture containing 80 mmol\/L acetate, 20 mmol\/L butyrate, and 40 mmol\/L propionate, which was added to the standard bath solution and compared with 140 mmol\/L additional NaCl as an osmotic control. The effect of the high-SCFA mixture was similar to that triggered by the 1 mmol\/L concentrations, enhancing GLP-1 secretion \u223c1.3-fold compared with the osmotic control (*n* = 5\u20136, *P* \\< 0.01) (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>B<\/em><\/a>). By contrast, GLUTag cells were not found to secrete GLP-1 in response to 1 or 10 mmol\/L SCFA (data not shown).\n\n## SCFA-induced calcium responses in primary mouse colonic L cells.\n\nL cells in mixed cultures were identified by their expression of Venus (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<em>B<\/em><\/a>), and the calcium concentration, \\[Ca^2+^\\]~i~, was monitored in both L cells and their nonfluorescent neighbors after loading of the monolayer with Fura2-AM (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<em>A<\/em><\/a>). \\[Ca^2+^\\]~i~ is represented in the figures by the 340:380 fluorescence ratio. Propionate (1 mmol\/L) triggered a transient Ca^2+^ response (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<em>A<\/em>, <em>C<\/em>, and <em>D<\/em><\/a>), with a mean 1.5 \u00b1 0.1-fold increase (*n* = 17, *P* \\< 0.01) above baseline. Acetate (1 mmol\/L) triggered a comparable \\[Ca^2+^\\]~i~ response of 1.3 \u00b1 0.1-fold (*n* = 6, *P* \\< 0.05) (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<em>D<\/em><\/a>). The SCFA-induced Ca^2+^ mobilization appeared specific to L cells, as intracellular \\[Ca^2+^\\]~i~ in the non\u2013<span class=\"smallcaps\">L-<\/span>cell population was unaffected by acetate and only marginally elevated by propionate (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<em>D<\/em><\/a>).\n\n## *ffar2* and *ffar3* expression is enriched in primary L cells.\n\nExpression of *ffar2* and *ffar3* was investigated by quantitative RT-PCR using mRNA from FACS-sorted intestinal L cells from transgenic GLU-Venus mice (24). Nonfluorescent cells collected in parallel were used to represent the mixed non\u2013<span class=\"smallcaps\">L-<\/span>cell population. As shown in <a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<\/a>, *ffar2* and *ffar3* were expressed in small intestinal and colonic L cells and significantly enriched in <span class=\"smallcaps\">L-<\/span>cell compared with non\u2013<span class=\"smallcaps\">L-<\/span>cell populations (*P* \\< 0.05). GLUTag cells expressed *ffar3* but had barely detectable levels of *ffar2*.\n\n## Acute SCFA-induced GLP-1 secretion is not mediated via a G~i~-signaling pathway.\n\nThe findings that SCFAs trigger Ca^2+^ elevation in L cells and enhance GLP-1 secretion are consistent with the involvement of a G~q~-mediated pathway, potentially, therefore, implicating FFAR2. Hence, we tested whether a synthetic phenylacetamide agonist for FFAR2, CFMB (25), could also mobilize Ca^2+^ and found that 30 \u03bcmol\/L CFMB indeed elevated intracellular Ca^2+^ in L cells (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<em>D<\/em><\/a>). A smaller but significant increase was also observed in the non\u2013<span class=\"smallcaps\">L-<\/span>cell population. To evaluate any opposing contribution from G~i~ signaling, we examined GLP-1 release in the presence of the G~i~ inhibitor pertussis toxin. Whereas SCFAs were stimulatory in the presence of IBMX (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>B<\/em><\/a>), somatostatin (100 nmol\/L), used as a positive control for G~i~-coupled pathways, abolished GLP-1 secretion under these conditions (*n* = 3, *P* \\< 0.001) (<a href=\"#F4\" data-ref-type=\"fig\">Fig. 4<em>A<\/em><\/a>), an effect that was partially reversed by preincubation with 0.2 \u03bcg\/mL pertussis toxin (*n* = 3, *P* \\< 0.001). By contrast, pertussis toxin did not have a significant effect on the response to 1 mmol\/L propionate (<a href=\"#F4\" data-ref-type=\"fig\">Fig. 4<em>B<\/em><\/a>), suggesting that there is little signaling by SCFA through G~i~-coupled pathways during 2-h secretion studies in vitro.\n\n## FFAR2 mediates SCFA-induced GLP-1 release.\n\nTo evaluate the relative contributions of FFAR2 and FFAR3 to SCFA-triggered secretion, we examined the effects of propionate and acetate on primary colonic cultures from mice with knockout of either *ffar2* (*ffar2*^\u2212\/\u2212^) or *ffar3* (*ffar3*^\u2212\/\u2212^). While GLP-1 secretion was triggered by 1 mmol\/L propionate and acetate in cultures from wild-type littermate controls (<a href=\"#F5\" data-ref-type=\"fig\">Fig. 5<em>A<\/em><\/a>), the response to propionate was reduced by 70% (*P* \\< 0.001) and the response to acetate was abolished (*P* \\< 0.001) in the *ffar2* knockout tissue. Knockout of *ffar3* also impaired secretory responses to both acetate and propionate (*P* \\< 0.01) (<a href=\"#F5\" data-ref-type=\"fig\">Fig. 5<em>A<\/em><\/a>), although to a lesser extent than *ffar2* knockout. Similar, but more pronounced, effects were observed in the presence of 100 \u03bcmol\/L IBMX (<a href=\"#F5\" data-ref-type=\"fig\">Fig. 5<em>A<\/em><\/a>). The higher concentration of mixed SCFA (140 mmol\/L, as described above) also failed to enhance GLP-1 secretion significantly in both the *ffar2* and *ffar3* knockout mouse models (<a href=\"#F5\" data-ref-type=\"fig\">Fig. 5<em>B<\/em><\/a>).\n\nAnalysis of mRNA expression in whole colonic tissue extracts from wild-type and knockout mice revealed that, as expected, *ffar2*^\u2212\/\u2212^ mice lacked *ffar2* mRNA and *ffar3*^\u2212\/\u2212^ mice lacked *ffar3* (<a href=\"#F5\" data-ref-type=\"fig\">Fig. 5<em>C<\/em> and <em>D<\/em><\/a>). *ffar3* expression was also significantly decreased in *ffar2*^\u2212\/\u2212^ mice, but, although we also observed a trend toward a reduced *ffar2* expression in *ffar3*^\u2212\/\u2212^ mice, this did not reach statistical significance.\n\n## *ffar2* knockout decreases GLP-1 content.\n\nTo examine whether the receptors have additional effects on L cells operating over a longer time scale, we examined whether colonic tissue from mice lacking *ffar2* or *ffar3* had altered levels of mRNA for glucagon (*gcg*) (which includes the coding sequence for GLP-1), *pyy*, or active GLP-1 peptide. Knockout of *ffar3* did not have a significant effect on colonic *gcg* mRNA or active GLP-1. There was a trend toward reduced colonic *gcg* and *pyy* mRNA expression in the *ffar2* receptor knockout model that did not reach statistical significance (<a href=\"#F5\" data-ref-type=\"fig\">Fig. 5<em>E<\/em> and <em>F<\/em><\/a>). *ffar2*-deficient mice, however, had significantly reduced colonic GLP-1 protein content (*n* = 6, *P* \\< 0.05) (<a href=\"#F5\" data-ref-type=\"fig\">Fig. 5<em>G<\/em><\/a>).\n\n## FFAR2 and FFAR3 affect GLP-1 release in vivo.\n\nTo evaluate whether the reduced in vitro GLP-1 secretory capacity of *ffar2*- and *ffar3*-deficient mice translates into impaired hormone secretion in vivo, we examined plasma GLP-1\u2013level responses to oral glucose administration. Basal levels of active GLP-1 were reduced by \u223c40% in *ffar2*-deficient mice (*P* \\< 0.01) (<a href=\"#F6\" data-ref-type=\"fig\">Fig. 6<em>A<\/em><\/a>) and were also lower, although not significantly, in *ffar3*-deficient mice compared with those in wild-type littermate controls. Following an oral glucose load, more pronounced impairments of plasma GLP-1 responses were observed in both *ffar2* and *ffar3* knockout models (*n* = 5, *P* \\< 0.05) (<a href=\"#F6\" data-ref-type=\"fig\">Fig. 6<em>A<\/em><\/a>). Coinciding with the reduced circulating GLP-1 concentrations, *ffar2*- and *ffar3*-deficient mice also exhibited impaired glucose tolerance when tested with gavage administration of 1.5 g\/kg glucose (<a href=\"#F6\" data-ref-type=\"fig\">Fig. 6<em>B<\/em> and <em>C<\/em><\/a>). This correlated with reduced plasma insulin levels, although again this reached significance in mice lacking *ffar2* but not *ffar3* when compared with their respective littermate controls (<a href=\"#F6\" data-ref-type=\"fig\">Fig. 6<em>D<\/em> and <em>E<\/em><\/a>). To test for possible differences in insulin sensitivity, *ffar2*^\u2212\/\u2212^ and *ffar3*^\u2212\/\u2212^ mice and littermate controls were injected with 0.75 units\/kg insulin after a 4-h fast. No significant differences were found between the different genotypes (<a href=\"#F6\" data-ref-type=\"fig\">Fig. 6<em>F<\/em> and <em>G<\/em><\/a>).\n\n# DISCUSSION\n\nOur results demonstrate a direct link between SCFA activation of FFAR2, elevation of intracellular Ca^2+^ in L cells, and enhanced GLP-1 secretion from primary colonic cultures. A stimulatory role for FFAR2 in vivo is supported by the finding that knockout of *ffar2* lowers both basal and glucose-stimulated GLP-1 concentrations.\n\nBy quantitative PCR, we could demonstrate that expression of mRNA for *ffar2* and *ffar3* is enriched in L cells, consistent with the detection of these receptors in PYY- and GLP-1\u2013positive cells in rat and human colon by immunohistochemistry (21\u201323). Interestingly, *ffar2* expression was very low in the GLP-1\u2013secreting cell line GLUTag, perhaps explaining the poor responsiveness of GLUTag cells to SCFAs (data not shown) and emphasizing the importance of studying primary L cells in parallel with cell line models.\n\nFFAR2 reportedly couples to G~q~- or G~i\/o~-signaling pathways and FFAR3 exclusively to G~i~. The finding that GLP-1 secretion is enhanced, rather than inhibited, by SCFAs suggests that G~q~-coupled pathways predominate over any G~i~ activation, and indeed, we did not observe any evidence that G~i~ coupling blunts SCFA-triggered GLP-1 secretion either in secretion studies with added pertussis toxin or after knockout of *ffar3*. Somatostatin, by contrast, inhibited IBMX-triggered GLP-1 release in a pertussis toxin\u2013sensitive manner, indicating that downstream G~i~-coupled pathways are globally functional in L cells.\n\nThe idea that FFAR2 is coupled to G~q~-signaling pathways in L cells is suggested by the Ca^2+^ imaging data, which showed that acetate and propionate triggered intracellular Ca^2+^ responses in the <span class=\"smallcaps\">L-<\/span>cell population. In support of this idea, knockout of *ffar2* abolished SCFA-triggered GLP-1 secretion in vitro. The explanation for the blunted GLP-1 secretory response to SCFA in cultures from *ffar3*^\u2212\/\u2212^ mice is less clear, as FFAR3 has not been reported to couple to stimulatory G~q~- or G~s~-signaling pathways. One contributing factor could be the reduced *ffar2* expression that was observed in *ffar3*^\u2212\/\u2212^ mice, although this did not reach statistical significance in our samples. Reduced expression of *ffar2* has, however, previously been reported in adipose tissue of *ffar3* knockout mice (26). Alternatively, there may be a previously unidentified component to FFAR3 signaling in L cells that also contributes to SCFA-triggered GLP-1 secretion. This is not, however, supported by the observation that stimulation of GLP-1 release was not observed in GLUTag cells, which express *ffar3* but very little *ffar2*. A dominant role of FFAR2 over FFAR3 in SCFA-triggered <span class=\"smallcaps\">L-<\/span>cell activation is further suggested by the acute Ca^2+^-elevations seen in primary L cells in response to the FFAR2-specific agonist CFMB, which reportedly does not have activity against FFAR3 (25).\n\nIt is not known whether FFAR2 and FFAR3 reside on the apical or basolateral membrane of L cells or whether they primarily detect luminal or plasma SCFA. SCFA levels in the colonic lumen are in the area of 100 mmol\/L. Although this is considerably above the half maximal effective concentrations (EC~50~s) of FFAR2 and FFAR3 for SCFA (0.5\u20131 mmol\/L) (17,18,20), SCFA concentrations may be lower in the immediate vicinity of the L cells as a result of the diffusional barrier provided by the mucous layer and active uptake by neighboring enterocytes. Circulating plasma SCFA concentrations are in the range of 100\u2013200 \u03bcmol\/L (10), which is closer to the working range of the receptors, and while most studies have examined how luminally applied SCFAs affect GLP-1 release (14,27,28) it has also been shown that systemically infused acetate can enhance GLP-1 secretion (28). As luminal SCFA concentrations are not predicted to change markedly in response to acute food ingestion, it is possible that SCFAs produced by colonic fermentation provide a chronic stimulatory tone on L cells via apical or basolaterally located SCFA receptors. This could account for the circulating levels of active GLP-1 that are detectable even in the fasting state and amplify the responses to ingested nutrients, mediated, for example, via neurohormonal signals triggered by nutrient arrival higher up the gastrointestinal tract.\n\nConsistent with the impaired responsiveness of *ffar2*^\u2212\/\u2212^ and *ffar3*^\u2212\/\u2212^ colonic cultures to SCFAs in vitro, we also observed lower GLP-1 levels in the knockout mice in vivo. As *ffar2*^\u2212\/\u2212^ mice exhibited both reduced colonic GLP-1 content and impaired SCFA-triggered secretion in vitro, we cannot conclude from our data which of these components underlies the reduced basal and glucose-triggered GLP-1 secretory responses in vivo*.* In *ffar3*^\u2212\/\u2212^ mice, however, the colonic GLP-1 content was not reduced, suggesting that the lower plasma GLP-1 levels reflect the impaired SCFA-triggered secretory response.\n\nIn addition to having low circulating GLP-1 levels, *ffar2*^\u2212\/\u2212^ and *ffar3*^\u2212\/\u2212^ mice exhibited impaired glucose tolerance. The reduced GLP-1 concentrations are likely to contribute to the impaired glucose homeostasis but may not be the only cause because *ffar2* and *ffar3* are not exclusively expressed in intestinal cells. In fact, they are better characterized as mediators of SCFA effects on immune cells and adipocytes (17,18,20), where they have been implicated in modulating neutrophil activation (18,20), reducing inflammation by inhibition of cytokine and chemokine expression, enhancing leptin production (29), stimulating adipogenesis, and inhibiting lipolysis (30,31). They are also expressed in pancreatic \u03b2 and \u03b1 cells (G. Tolhurst, H. Parker, A. Habib, F. Reimann, and F. Gribble, unpublished observations). Global knockout of these receptors may therefore affect adipocyte function, inflammation, or pancreatic \u03b2-cell function, which would themselves impact on glucose tolerance. The fact that we observed reduced insulin levels in the *ffar2*^\u2212\/\u2212^ mice during an oral glucose tolerance test but did not observe differences in insulin tolerance indicates that the observed impaired glucose tolerance in our model reflects an impairment of insulin secretion, possibly in part due to an impaired incretin axis. Another *ffar2*^\u2212\/\u2212^ mouse model was recently reported to exhibit increased food intake but had lower body weight and insulin levels compared with wild-type controls after a prolonged period on a high-fat diet (32). No explanation was found for the increased food intake, but our data suggest that it may be related to reduced secretion of <span class=\"smallcaps\">L-<\/span>cell peptides like GLP-1 and PYY. Unlike the findings of our study, those of Bjursell et al. (32) did not show a significant difference in glucose tolerance between chow-fed *ffar2*^\u2212\/\u2212^ and control mice. The apparent differences between the two knockout models may reflect the fact that the mice were maintained on different genetic backgrounds (C57B\/6JOlaHsd vs. 129\/SvEv) or that they were studied at different ages.\n\nInterest in the gut microbiome has heightened recently, with the recognition that our complement of colonic bacteria may not simply reflect factors such as diet and illness but can also in return influence food intake and metabolism. Probiotics, prebiotics, and high-fiber diets are promoted for their potential beneficial effects on obesity, diabetes, inflammation, and immunity, as well as local effects on the health and integrity of the gut. One potential link between colonic microflora and systemic activity are SCFAs, which are produced by bacterial fermentation of fiber and which not only act as a local nutrient source but also trigger the release of anorectic hormones like GLP-1 and PYY. Our data suggest that SCFAs produced by bacterial fermentation in the gut can directly influence L cells to enhance the release of peptides such as GLP-1 and PYY. Whether this interrelationship is altered by metabolic conditions such as diabetes and obesity remains to be determined. FFAR2 should perhaps be added to the list of target receptors that may be exploitable for the pharmacological stimulation of the enteroendocrine system.\n\n## ACKNOWLEDGMENTS\n\nThis work was funded by Wellcome Trust grants to F.M.G. and F.R. (WT088357 and WT084210, respectively) and the European Union Seventh Framework Programme (FP7\/2007-2013, grant agreement no. 266408).\n\nThe in vivo experiments on the knockout and control mice were performed at Takeda Cambridge Ltd, and the knockout mice used were originally created for Takeda Cambridge Ltd. The ffar2 selective agonist CFMB used in the study was synthesized for Takeda Cambridge Ltd. No other potential conflicts of interest relevant to this article were reported.\n\nG.T., H.H., Y.S.L., H.E.P., A.M.H., E.D., and J.C. researched data. J.G. designed the study. F.R. and F.M.G. designed the study and wrote the manuscript.\n\nThe authors thank Keith Burling and the Medical Research Council Centre for Obesity and Related Disorders (Cambridge, U.K.) for performing plasma GLP-1 assays. Daniel Drucker (Samuel Lunenfeld Research Institute, Toronto, Canada) kindly provided the GLUTag cell line.\n\n# REFERENCES","meta":{"dup_signals":{"dup_doc_count":111,"dup_dump_count":62,"dup_details":{"curated_sources":2,"2022-05":1,"2021-39":1,"2021-31":1,"2021-21":1,"2021-17":1,"2021-10":5,"2021-04":1,"2020-34":1,"2020-29":3,"2020-24":1,"2020-16":1,"2019-43":1,"2019-26":1,"2019-22":1,"2019-13":1,"2019-04":1,"2018-43":1,"2018-22":2,"2018-17":1,"2018-13":1,"2018-05":2,"2017-51":1,"2017-47":1,"2017-43":3,"2017-39":2,"2017-34":1,"2017-30":5,"2017-26":2,"2017-22":1,"2017-17":5,"2017-09":3,"2017-04":5,"2016-50":2,"2016-44":3,"2016-40":2,"2016-36":2,"2016-30":2,"2016-26":2,"2016-22":2,"2016-18":1,"2016-07":2,"2015-48":2,"2015-40":2,"2015-35":2,"2015-32":1,"2015-27":1,"2015-22":2,"2015-14":1,"2014-52":2,"2014-49":1,"2014-42":1,"2014-41":1,"2014-35":2,"2014-23":1,"2014-15":2,"2023-40":1,"2017-13":3,"2015-18":2,"2015-11":2,"2015-06":1,"2014-10":2,"2024-30":1}},"file":"PMC3266401"},"subset":"pubmed_central"}
{"text":"author: Fumihiko UranoCorresponding author: Fumihiko Urano, <email@example.com>.\ndate: 2014-03\nreferences:\ntitle: Wolfram Syndrome iPS Cells: The First Human Cell Model of Endoplasmic Reticulum Disease\n\nWolfram syndrome is a rare autosomal recessive genetic disorder with clinical signs apparent in early childhood. This condition is characterized by childhood-onset diabetes, optic nerve atrophy, deafness, diabetes insipidus, and neurodegeneration, and it results in death in middle adulthood (1\u20133). Genetic and experimental evidence strongly suggest that endoplasmic reticulum (ER) dysfunction is a critical pathogenic component of Wolfram syndrome (4,5). However, there is a lack of complete understanding of the pathways and biomarkers involved in the disease process due to the limitations of animal models that do not accurately reflect human patients. As a result, despite the underlying importance of ER dysfunction in Wolfram syndrome, there are currently no therapies that target the ER, a deficiency that points to the urgent need to develop a human cell model of this condition. In this issue, Shang et al. (6) report that this has been successfully accomplished.\n\nER is a membrane network within the cytoplasm of cells that is involved in protein synthesis, calcium storage, redox regulation, steroid synthesis, and cell death. Recent clinical and genetic evidence indicate that acquired or inherited ER dysfunction can cause rare genetic diseases such as Wolfram syndrome, as well as many common diseases, including type 1 and type 2 diabetes, atherosclerosis, and neurodegenerative diseases (7\u201311). Thus, ER is an emerging target for both rare and common chronic conditions. However, it has been challenging to study the efficacy of pharmacological agents that could potentially reverse ER dysfunction due to the lack of a human cell model of ER disorders.\n\nIn Wolfram syndrome, pancreatic \u03b2-cells and neuronal cells are selectively destroyed as a consequence of mutations in the *WFS1* gene. This gene encodes a transmembrane protein localized to the ER, suggesting that ER dysfunction is a major pathogenic component of Wolfram syndrome. In animal and cell models of Wolfram syndrome, WFS1 mutations lead to elevated ER stress levels, pancreatic \u03b2-cell dysfunction, and initiation of ER stress\u2013associated cell death (5,12,13). Shang et al. showed that Wolfram syndrome patient induced pluripotent stem (iPS) cell\u2013derived \u03b2-cells also showed increased levels of ER stress molecules, as well as decreased insulin content. Upon exposure to experimental ER stress, Wolfram syndrome patient iPS cell\u2013derived \u03b2-cells showed impaired insulin processing and failed to increase insulin secretion in response to glucose and other secretagogues. These observations were all consistent with previous findings and could validate the roles of WFS1 in insulin production, secretion, and protection against ER stress (14). Moreover, Shang et al. showed that 4-phenyl butyric acid (4PBA), a chemical protein folding and trafficking chaperone, could restore normal insulin synthesis and the ability to upregulate insulin secretion in Wolfram syndrome patient iPS cell\u2013derived \u03b2-cells, a finding that suggested 4PBA is a candidate drug for treating patients with Wolfram syndrome.\n\nWe can expect Wolfram syndrome patient iPS cell lines and Wolfram iPS cell\u2013derived \u03b2-cells to be cornerstones for developing novel therapeutic modalities for Wolfram syndrome and other diseases involving ER dysfunction. We can use these cells to screen and identify drugs for treating patients with Wolfram syndrome and other ER-associated diseases. Chemical chaperones such as 4PBA and tauroursodeoxycholic acid (TUDCA) are good candidates. It has been shown that these compounds can mitigate ER stress in peripheral tissues, such as liver, in mouse models of diabetes (15). Glucagon-like peptide 1 agonists and rapamycin are encouraging compounds because they have been shown to prevent ER stress\u2013mediated \u03b2-cell death in mouse and cell models (16,17) (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<\/a>). Clinical trials evaluating the efficacy of these drugs should be seriously considered after we carefully test these compounds in \u03b2-cells and neurons derived from Wolfram iPS cells. It has been shown that WFS1 polymorphisms and mutations are associated with type 2 diabetes and adult-onset diabetes, suggesting that drugs effective for Wolfram syndrome may also be useful for the treatment of more prevalent forms of diabetes, type 1 and type 2 diabetes (18,19). Thus, the potential public health implications of the new findings are considerable.\n\nIn the future, we can use these cells to regenerate damaged tissues including pancreatic \u03b2-cells, retinal ganglion cells, and neurons in patients with Wolfram syndrome (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<\/a>). Rapid progress in genetic editing technologies and regenerative medicine will make it possible to correct WFS1 mutations in patient-specific iPS cell lines and regenerate patients' damaged cells. Shang et al. (6) have taken us a step closer toward these goals. As a physician and scientist who is working on Wolfram syndrome, a devastating form of juvenile diabetes, I truly appreciate their important accomplishment. We should make the best use of these cells to develop treatments for Wolfram syndrome\u2014efforts that may lead to breakthroughs in diabetes treatment.\n\n## Article Information\n\n**Acknowledgments.** The author thanks all of the participants in the Wolfram Syndrome International Registry (<http:\/\/wolframsyndrome.dom.wustl.edu\/>) and the Washington University Wolfram Clinic for their time and effort. The author also thanks the Washington University Wolfram Study Group members and the study staff for advice and support.\n\nThe author dedicates this article to M. Alan Permutt, MD, whose untimely death from cancer was a great loss for us all.\n\n**Funding.** This work was supported by grants from National Institutes of Health (DK-067493, DK-016746, DK-020579, and UL1-TR-000448), JDRF (47-2012-760 and 17-2013-512), American Diabetes Association (1-12-CT-61), Samuel E. Schechter Professorship, the Ellie White Foundation for Rare Genetic Disorders, and the Jack and J.T. Snow Scientific Research Foundation to F.U.\n\n**Duality of Interest.** No potential conflicts of interest relevant to this article were reported.\n\n# References","meta":{"dup_signals":{"dup_doc_count":175,"dup_dump_count":54,"dup_details":{"curated_sources":2,"2022-40":2,"2022-21":1,"2021-17":1,"2019-51":2,"2019-43":1,"2019-18":1,"2019-13":1,"2019-09":1,"2019-04":1,"2018-47":1,"2018-39":1,"2018-30":1,"2018-26":2,"2018-22":1,"2018-17":2,"2018-13":2,"2018-09":2,"2017-51":5,"2017-43":10,"2017-39":1,"2017-34":3,"2017-30":6,"2017-26":5,"2017-17":10,"2017-09":6,"2017-04":10,"2016-50":5,"2016-44":5,"2016-40":5,"2016-36":5,"2016-30":5,"2016-26":3,"2016-22":3,"2016-18":3,"2016-07":3,"2015-48":4,"2015-40":3,"2015-35":4,"2015-32":3,"2015-27":2,"2015-22":1,"2015-14":3,"2014-52":4,"2014-49":4,"2014-42":5,"2014-41":3,"2014-35":3,"2014-23":1,"2023-40":1,"2017-13":5,"2015-18":2,"2015-11":4,"2015-06":4,"2024-18":1}},"file":"PMC3931391"},"subset":"pubmed_central"}
{"text":"abstract: In March, 2012 we held the first Mideast conference on \"Depression and Anxiety Spectrum disorders: from basic science to the clinic and back\", at the University of Amman, Jordan. This event brought together both clinical and basic scientists with expertise in depression and anxiety spectrum disorders. The meeting took place in a large lecture hall at the University of Jordan Medical School. The audience included faculty, residents, and students. The Dean of the Medical School opened the meeting, welcoming the guest speakers and participants.\nauthor: Suzanne N Haber; Ziad Safadi; Mohammad R Milad\ndate: 2013\ninstitute: 1Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA\nreferences:\ntitle: Meeting report: \"Depression and Anxiety Spectrum disorders: from basic science to the clinic and back\"\n\nAnxiety disorders are generally characterized by irrational and excessive fear along with chronically elevated levels of anxiety. The trigger for the inappropriate fear and anxiety may be clear and well-defined in some of the disorders, such as specific phobias, posttraumatic stress disorder, or social anxiety disorder, or may be unspecific or not defined in other disorders such as panic disorder and generalized anxiety disorder. Depression is commonly characterized by lost of interest in engaging in daily activities, feelings of hopelessness, lack of energy and concentration, and feelings of incompetence. Depression and anxiety share common clinical features. Between 1\/3 and 2\/3 of patients who suffer from major depression experience some form of anxiety as part of their illness \\[1\\]. Moreover, the prevalence of major depression in patients with anxiety disorders is significantly higher than in the general population. Co-morbidity between anxiety and depression is common \\[2,3\\]. This relationship extends to family studies. Twin studies suggest shared genetic vulnerability to depression and anxiety. Effective treatments for depression and anxiety also notably overlap: medications acting on monoaminergic systems, and psychotherapies based on fear extinction and other methods geared toward reducing avoidance and increasing goal-directed, rewarding activities. Importantly, there is also a common neural network associated with these diseases. This network is central to the pharmacological and behavioral approaches for treatment and the hypotheses that drive the research of the underlying pathophysiology. The network includes the prefrontal cortex, specifically the orbital and anterior cingulate cortices, the ventral striatum, amygdala, and the brainstem catacholaminergic systems, particularly dopamine and serotonin \\[4\\].\n\nAlthough there are no 'hard' data available for most Middle East countries \\[5\\], the population afflicted with these diseases is growing according to our colleagues practicing in there. Moreover, these diseases are massively under diagnosed and under treated. This meeting provided a unique opportunity to discuss the basic and clinical science that focus on the biological underpinnings of these mental health disorders.\n\nThe goal of the meeting was three-fold: first, to bring together experts on the pathophysiology and treatment of depression and anxiety spectrum disorders in a culturally diverse environment, where such disorders are prevalent, but exposure to cutting-edge therapies and research is limited; second, to explore the directions in which science is driving this field by presenting data on experimental and therapeutic approaches to understanding and treating these syndromes; and finally, to address the possibilities for future training and research in the region. The last goal was particularly important, as this meeting provided a forum to explore needs of the region and possible future collaborations.\n\nThere were two general topics, with sub-topics within each. The first general topic, \"Biological basis of mood and anxiety disorders: A clinical and basic science overview\", focused on an overview of where the field is now, including a brief history. This session started with 2 general overviews, one on clinical aspects of mood and anxiety disorders (Dr. Greenberg) and a general discussion of the reward circuit that underlies these disorders (Dr. Haber). These general introductions were followed by talks focused specifically on state of the art clinical and basic science approaches. Dr. Eskandar gave an overview of neurosurgical and physiological studies in both animal and human studies; Dr. Akil gave an overview of the underlying biology associated with stress and depression; and Dr. Dougherty discussed using neuroimaging approaches to understanding these diseases. The second topic, \"Biological basis of mood and anxiety disorders: New advances\", focused on ongoing research. Dr. Moghaddam discussed reward and anxiety processing in adolescents, their vulnerability to disease, and goals for prevention; Dr. Aston-Jones talked about ongoing studies focused on the locus coeruleus; Dr. Greenberg discussed the clinical and basic science perspective of obsessive-compulsive disorder while Dr. Grace focused on animal models of the disease. Finally, the last afternoon of the meeting was dedicated to trauma and fear, with Dr. Pitman discussing stress, anxiety, and trauma, Dr. Milad focusing in fear and fear extinction, and Dr. Quirk discussing the neurobiology of fear learning. In addition there were some specialized talks, giving a perspective from the regions. These included a presentation from Dr. Jadiry on the prevalence of anxiety and mood disorders. Dr. Sarhan summarized cognitive behavioral therapy of Islamic religious OCD, and Dr. BaniMustafa on depression in primary care patients. We summarize the talks below.\n\nThe meeting was very well received and prompted numerous thoughtful questions and discussions. Students made comments such as \"This meeting has completely changed my thinking and now I want to do research in neuroscience.\" Faculty comments were equally positive; \"The meeting was a success by all standards this is not personal view alone but the feedback I got from meeting people who attended.\" Several also indicated how important it was to have this meeting. \"The meeting about anxiety and depression was a great opportunity for clinicians and neuroscientists to meet to discuss the latest research in the field, which is not given the attention it deserves in undergraduate and postgraduate medical education taking in consideration that more than half of those who suffer from anxiety and depression go unrecognized and never receive treatment.\" This impact on medical and graduate students is perhaps the most important legacy of the symposium.\n\nTopic 1: Biological basis of mood and anxiety disorders: A clinical and basic science overview.\n\n## 1A. Clinical overview and the reward circuit\n\nDr. Benjamin D. Greenberg from the Departments of Psychiatry and Human Behavior in the Alpert Medical School of Brown University gave the first talk, entitled \"*Overview of mood & anxiety disorders\".* Dr. Greenberg emphasized several main points: that patients with the same categorical diagnosis as defined in the DSM system are in fact heterogeneous; that longitudinal stability of most diagnoses is poor; and that co-occurrence (\"comorbidity\") of diagnoses tends to be the rule. For example, one- to two-thirds of individuals with an anxiety disorder will have a lifetime mood disorder; conversely, two-thirds of individuals who develop major depression or dysthymia will also have an anxiety disorder during their lifetimes.\n\nIn this context, he noted that the development of medication treatments in psychiatry was initially focused on how medications affected core clinical features of affected individuals as much as (or more than) than on treating illnesses as sepble entities. Such core features include an impaired ability to respond to reward after motivated behaviors, or positive emotional and social experiences (anhedonia). He proposed that an essential step towards making it possible for clinicians to target specific treatments to those individuals most likely to benefit from them, was to seek to understand behavioral and brain mechanisms underlying how effective behavioral and medication treatments may change such clinical features. The same approach should allow refinement of existing therapies, and development of new ones. Moreover, progress in this domain should enhance public understanding of psychiatric illnesses as disorders of the brain and behavior, which, in time, should reduce stigma and encourage treatment seeking, across cultures.\n\nDr. Suzanne Haber Professor at the Department of Pharmacology and physiology at the University of Rochester Medical School gave a presentation entitled, \"*Understanding the Reward Circuit: Implications for Depression and Anxiety Spectrum Disorders*\". The presentation began with a brief overview of the background and the history of research of the reward system, followed by describing the reward network. She noted that while many brain regions respond to reward, central to the network are structures embedded within the cortico-basal ganglia system and include, the orbital, and anterior cingulate cortices, the ventral striatum, the ventral tegmental, midbrain dopamine neurons. In addition, other structures including the dorsal prefrontal cortex, amygdala, hippocampus, thalamus, lateral habenular n., and specific brainstem structures, such as the pedunculopontine n., and the raphe n., are key components in regulating the reward circuit. Connectivity between these areas forms a complex neural network. This network mediates different aspects of reward processing, including value, magnitude, and anticipation. Dr. Haber discussed how delineating these neurocircuits are key elements for understanding anatomical substrates of psychiatric diseases.\n\nClassically, the cortico-basal ganglia system has been functionally divided into septe and segregated circuits. However, while the topographic organization of cortico-striatal projections are well documented, there is increasing evidence that there are also regions of interface between terminals from different cortical areas, suggesting functional integration to link motivation to behavioral output, critical for developing appropriate action plans. Dr. Haber demonstrated two ways in which cortico-basal ganglia circuits can integrate this information across functional circuits, through cortico-striatal projections, and through striatal-midbrain-striatal connections.\n\nProjections from the cortical regions associated with motivation terminate in specific striatal regions, but also converge with terminals from associative areas. This complex interweaving and convergence provides an anatomical substrate for modulation between these motivation and cognition. This creates activation sites that may be particularly important for incentive-based learning. In addition, there is an interface between different functional striatal regions via the midbrain DA cells. Dr. Haber demonstrated how this interface is organized in an ascending spiral interconnecting different functional regions of the striatum. This anatomical arrangement creates a hierarchy of information flow, and provides an anatomical basis for the limbic\/cognitive\/motor interface via the midbrain dopamine neurons.\n\nDr. Haber ended her talk by describing translational studies, linking animal anatomical studies with neuroimaging. Diffusion tensor imaging (DTI) is increasingly used to develop models of prefrontal connections, to examine chances in white matter (WM) integrity associated with disease, and to target surgical interventions, including deep brain stimulation for psychiatric disease. Dr, Haber described how her lab uses nonhuman primate tracing studies coupled with 3-D reconstructions of WM bundles to delineate the rules frontal cortex axons follow to reach their targets. She then demonstrated that human prefrontal WM pathways follow these organizational rules. Taken together, these data provide the foundation for dividing white matter bundles into functional components and for predicting what is likely to be carried at different points through each bundle. Understanding this organization is key for determining specific connections that are likely to be captured at different neurosurgical targets.\n\n## 1B. What we can learn from clinical and basic science\n\nDr. Emad Eskandar, MD, from the Department of Neurosurgery Massachusetts General Hospital presented two talks at the symposium. The first talk was entitled \"Physiological Mechanisms of Basal Ganglia Disorders\". In this presentation, Dr. Eskandar presented data on the role of the basal ganglia and movement psychiatric disorders. First, Dr Eskandar gave a description of the relevant anatomy and physiology, followed by a reviewed surgical treatment options such as deep brain stimulation. This portion of talk discussed the rational for treatment and the key technical aspects of the surgery. Finally, Dr. Eskandar reviewed a number of prospective randomized clinical trials illustrating the benefits and limitations of the surgical treatments. Later in the symposium, Dr. Eskandar gave a talk entitled \"Insights from Human and Primate Neurophysiology\" which focused on the important role played by basic physiologic research in translating scientific findings into useful treatments for human disorders.\n\nDr. Huda Akil Professor of Neuroscience & Psychiatry and Director of the Molecular and Behavioral Neuroscience Institute, at the University of Michigan. She began her talk entitled \"The Neurobiology of Stress and Depression: Animal Models and Human Studies\" by noting that her late father, Dr. Fakher Akil (Akel) was one of the founders of the University of Jordan almost 50 years ago, and a member of the faculty of its Psychology Department when it was first established. She shared photographs of Dr. Akil with His Majesty the late King Hussein and other founding members at the dedication of the University of Jordan, and she noted that this gave this meeting a very special meaning for her.\n\nDr. Akil emphasized the burden of depression around the world, and the underlying complexity of the illness, as it has many genetic and environmental causes, converging to disturb the functioning of brain circuits that control our responses to stress and our short term and long-term moods. But currently available treatments for depression only work for a subset of patients, and at least a third of depressed individuals do not respond to any of the current treatments and become increasingly more depressed, some of them ending their lives tragically. So, how can science discover the biological causes of depression and identify new molecules that may become targets for new types of antidepressant drugs?\n\nDr. Akil described how her laboratory has created animal models of increased vulnerability to depression. One model uses a genetic approach in mice, and shows that increasing the activity of a gene that responds to stress only in the brain, and only during early development, is sufficient to produce an anxious and emotionally labile animal that serves as an excellent model for bipolar (manic-depressive) disorder. Thus, the stress the glucocorticoid receptor gene, GR, may be a good target for treatment in some patients.\n\nAnother approach was to create models of \"temperament\" by developing genetically bred lines of rats that are either prone to anxiety and depression-like behaviors or resistant to stress. These animals show characteristics of \"internalizing disorders\" versus \"externalizing disorders\". Studies of developmental, molecular and neural bases of these differences have already yielded many surprising discoveries.\n\nDr. Akil then spoke about a complementary approach, where she and her collaborators begin their search in the brains of patients who had died when severely depressed compared to non-depressed controls. Using broad genetic and gene expression strategies, they identify molecules in specific brain regions that differentiate the severely depressed brains. Then, they return to the animal models to examine the function of these molecules. Using this strategy, they discovered that a family of growth factors, the FGF system, is severely disrupted in major depression. Studies in the animal models show that FGF2, which is low in depressed human brains, serves as a natural anti-depressant and anti-anxiety agent that is present in all brains, but differences in its activity can modify mood. Thus, animals that are prone to anxiety and depression, like depressed humans, have low levels of FGF2. Social stress also lowers the levels of FGF2. Importantly, replacing FGF2 in the brains of anxiety-prone animals reverses these tendencies, acts as an antidepressant and increases their activity and exploration. Thus FGF family has become a new target for studies of depression and as a potential target for developing new types of antidepressants.\n\nIn sum, combining basic science research and human clinical studies, and using genetic, molecular, anatomical and behavioral approaches are yielding exciting new insights into the causes of depression and hold promise for developing new treatment strategies for these stress-related brain disorders.\n\nDr. Darin D. Dougherty from the Department of Psychiatry at Massachusetts General Hospital and Harvard Medical School gave a presentation entitled, *\"Neuroimaging, what we know and what we need to know\"*. The presentation began with a brief overview of studies utilizing modern neuroimaging technology that have helped to elucidate regarding the pathophysiology of major depressive disorder (MDD) and obsessive-compulsive disorder (OCD). In brief, when compared to healthy volunteers, MDD appears to be associated with decreased resting activity in more dorsal neocortical brain regions associated with cognitive processes and increased resting activity in more ventral limbic and plimbic brain regions associated with the neurovegetative symptoms common to MDD. In addition, during neuroimaging studies that use a variety of activation pdigms in subjects with MDD and healthy volunteers, these dorsal regions are hyporeactive during cognitive tasks and these ventral regions are hyperreactive during affective tasks in subjects with MDD. Lastly, most of these abnormalities are attenuated following successful treatment (with either pharmacotherapy or psychotherapy) of the major depressive episode. In contrast, when compared to healthy volunteers, subjects with OCD exhibit greater resting activity in a specific cortico-thalamo-striato-cortical pathway that includes the orbitofrontal cortex (OFC), thalamus, and head of the caudate nucleus and these abnormalities can be amplified during pdigms that temporarily worsen OCD symptomatology. As is the case with MDD, these abnormalities are attenuated following successful treatment (with either pharmacotherapy or psychotherapy) of OCD.\n\nAfter describing the rudimentary pathophysiology of both MDD and OCD, Dr. Dougherty described initial attempts to understand the effects of deep brain stimulation (DBS) at the ventral capsule\/ventral striatum (VC\/VS) target on these circuits using functional neuroimaging techniques. Because of the presence of metal in the DBS leads, most studies to date have used positron emission tomography (PET). The use of ^15^O isotopes (either in CO~2~ in room air or in water) allows for the study of acute changes in blood flow. Interestingly, studies using PET and ^15^O in subjects with VC\/VS DBS for OCD have found acute blood flow changes in all nodes (OFC, thalamus, caudate) of the very circuit implicated in the pathophysiology of OCD. Initial data from studies using ^18^FDG and PET in subjects with VC\/VS DBS for OCD to identify chronic (e.g., following 3-6 months of DBS) changes are suggesting that the same brain regions that exhibit these acute changes in blood flow continue to demonstrate changes from baseline following chronic treatment with VC\/VS DBS. Compble studies in subjects receiving VC\/VS DBS for MDD are ongoing. Dr. Dougherty concluded with a review of potential future directions, including using baseline neuroimaging data to identify individuals who are more likely to respond following DBS and to possibly tailor electrode targeting.\n\nTopic 2: Biological basis of mood and anxiety disorders: New advances.\n\n## 2A. Depression and Stress\n\nDr. Bita Moghaddam from the department of neuroscience at University of Pittsburgh gave a talk entitled \" Neuronal mechanisms for behavioral and psychiatric vulnerability in adolescents\". Dr. Moghaddam discussed how adolescents often respond differently than adults to the same salient motivating contexts, like peer interactions and stressors. Adolescence to early adulthood is also a period of psychiatric vulnerability and the typical time of symptom onset for several psychopathologies, including depression, addiction and schizophrenia. Thus, while the great majority of animal and human research related to these illnesses has focused on adults, research findings on the adolescent to early adulthood developmental age range may be more relevant to understanding the biological mechanisms that may contribute to the disease process and prevention. Adolescence encompasses puberty, a time of reproductive development, but also characterized by a myriad of non-reproductive socio-behavioral changes. Concomitant with these behavioral changes, adolescents undergo significant neurodevelopmental changes, including axonal growth, myelination and synaptic pruning, and changes in modulatory regulation of prefrontal cortex activity. Despite its importance, this stage of development remains understudied at mechanistic levels that are relevant to motivated behavior. This has been in part due to technical limitations associated with adolescent human and animal models. We believe that age-related changes in the ways salient stimuli are processed in key brain regions could underlie the unique predilections and vulnerabilities of adolescence. Because motivated behavior is the central issue, it is critical that age-related comparisons of brain activity be undertaken within motivational contexts. Dr. Moghaddam presented single neuron and large-scale electrophysiological data showing that the pattern of activation of cortico-striatal circuits, and the inhibitory-excitatory balance in these circuits, is different during motivated behavior in adolescent rats. These data indicate that there is reduced efficiency in the processing of cortical neural activity and related behaviors in adolescents. Furthermore, during this developmental period, regions critically involved in learning and habit formation are highly responsive to reward. This work highlights neuronal processing differences in adolescence that suggest possible mechanisms contributing to the vulnerability that this period presents for psychiatric illnesses.\n\nDr. Gary Aston-Jones, from the Medical University of South Carolina gave a talk entitled \"The cortex in context: Locus coeruleus, optimal performance, and maximal utility\" Optimal performance on a specific task requires selective attention to filter out extraneous stimuli and behaviors. It is also important, however, to disengage from the current task and find a new behavioral goal if motivation or the stimulus context changes. This tension between selective attention and the search for a new task in an altered context has been characterized as the exploitation-exploration tradeoff, and is critical for adaptive behavior. LC neurons exhibit two patterns or modes of activity in monkeys performing cognitive tasks: a *phasic mode* in which these cells are transiently activated selectively following decisions that were made, and a *tonic mode* when these cells have elevated tonic levels of discharge but no phasic responses. We hypothesize that the phasic mode of LC activity promotes selective behavioral responding to task-defined stimuli (selective attention), whereas the tonic mode produces a broader sampling of stimuli in the environment (search for new task, high behavioral flexibility). Both of these modes serve to increase the utility of behavior: The phasic mode increases utility on a short time-scale by facilitating selective responding in the task at hand, whereas the tonic mode increases long time-scale utility by promoting the identification of a new task that is adaptive in a changed context. In view of this functional framework, impairments of LC activity and ability to fluidly and adaptively change activity modes can produce deficits involved in several mental disorders, including attention deficit-hyperactivity disorder (persistent tonic mode), autism (persistent phasic mode), or sleep disorders and depression (persistently low activity).\n\n## 2B. OCD and Anxiety\n\nDr. Benjamin D. Greenberg from the Departments of Psychiatry and Human Behavior in the Alpert Medical School of Brown University gave second talk entitled OCD*, clinical and basic science perspectives\".* He discussed one specific diagnosis, obsessive-compulsive disorder (OCD). He presented data on the population prevalence, course, clinical features (including subtypes based on differing symptom profiles) and the full range of current treatment approaches from behavior therapy to neurosurgery. The presentation of the range of clinical features was intended to give clinicians and others in the audience assistance in diagnosing OCD, the prevalence of which has historically been under-appreciated throughout the world. While current epidemiological information suggests that OCD, which typically has its onset in childhood or adolescence, affects about one percent of adults in any given year, many affected individuals fail to seek treatment, or may do so only after a long delay. As for other psychiatric illnesses, accurate diagnosis is often best accomplished via obtaining information from multiple sources, including potentially family members.\n\nThen, after describing conventional behavioral and medication treatments, Dr. Greenberg turned to the subset of OCD sufferers with disabling, \"intractable\" illness, in whom surgical approaches might be considered. Determining if the strict selection criteria for, e.g., deep brain stimulation (DBS) are met requires exhaustive presurgical evaluations in multiple domains of symptoms, comorbidities, cognition and daily functioning. In the main stimulation approach, DBS leads implanted in the ventral anterior limb of the internal capsule and adjacent ventral striatum (VC\/VS) are intended to modulate the same prefrontal cortex-basal ganglia-thalamic circuitry targeted by lesion procedures. Related approaches include targeting VS subterritories (nucleus accumbens or ventral caudate) using DBS.\n\nOutcomes data are mainly open-label, though small studies have used sham-controlled DBS. In one case a sham gamma knife lesion was compared to actual ablations in the VC\/VS, in the first controlled trial of a lesion procedure for any psychiatric illness. Across procedures, meaningful benefit appeared in 30-60% when assessed after varying delays. The longstanding clinical impression that exposure-based behavioral therapies (exposure and response prevention, or ERP) can become more effective after vs. before neurosurgery has continued. This has led to explicit incorporation of ERP early in the post-surgery course in at least one study.\n\nMean severity decreases from \"very severe\" to \"moderate\" illness were usually but not always plleled by functional gains. Given that the chronically severe symptoms in these patients are profoundly disruptive to the life course, capturing possible changes in functioning might require a year-long period of observation, even if symptom lessening appears earlier. The prognostic significance of differences in OCD clinical phenomenology to surgical outcomes is beginning to be addressed. Ego-dystonic OCD symptoms primarily based in harm avoidance might improve most, although symptoms of \"incompleteness\" (motivated by an intense desire to achieve a \"just right\" feeling) have lessened in some patients as well.\n\nTranslational research mapping ventral prefrontal-basal circuitry in primates has delineated potential pathways of DBS effect. Stimulation-related changes in behavioral phenotypes related to fear conditioning and extinction are the focus of ongoing collaborative work, in concert with our controlled multicenter trial of VC\/VS DBS for intractable OCD. For OCD sufferers as a whole, the major promise of this line of research is that better delineation of the neurocircuit and behavioral mechanisms necessary to therapeutic benefit will have application using less invasive treatment strategies.\n\nDr. Anthony A. Grace from the Departments of Neuroscience, Psychiatry and Psychology at the University of Pittsburgh presented a talk entitled \"Animal Models of OCD: Frontal Cortical Disruption and Modulation by DBS.\" In this talk Dr. Grace covered what we have found about the mechanisms of deep brain stimulation (DBS) action and how it can relate to therapy in humans. An issue in understanding DBS is a better understanding of its mechanism of action. DBS has been carried out in the treatment of neurological disorders by stimulating regions that previous studies have targeted for lesions to achieve a therapeutic action. This gave rise to the \"functional lesion\" hypothesis of DBS action. However, the stimulation frequency typically used for DBs is 130 Hz, which for neurophysiologists typically is associated with long-term potentiation rather than inhibition. Therefore, we examined the effects of DBS using the ventral striatal circuit that is typically used for the treatment of Obsessive Compulsive Disorder (OCD) in humans.\n\nOCD is typically associated with hyperactivity in frontal cortical structures, notably the orbitofrontal cortex. We found that DBS of the ventral striatum for 90 minutes caused a decrease in the firing rate of orbitofrontal neurons, along with firing through antidromic activation of a small percentage of the pyramidal output neurons in this area. In contrast, when we presented single stimuli, we found that there was a classical NMDA-dependent long-term potentiation of the evoked field potential, which is a recording of the total electrical signal originating in the orbitofrontal cortex caused by the stimulation. Moreover, interneurons in the cortex were found to be more responsive to stimulation. This led to the conclusion that DBS caused antidromic activation of a small number of pyramidal neurons and, via local axon collaterals, long-term potentiation of the excitation of interneurons. Therefore, the DBS was not causing a \"functional lesion,\" but instead caused activation of interneurons to increase inhibition within the orbitofrontal cortex. This is consistent with the concept that the overactivity in the orbitofrontal cortex may have been due to deficits in interneuron inhibition.\n\nBesides inhibiting pyramidal neurons, interneurons have another important function within the cortex and other structures - they control the generation of rhythmic activity within these structures. Rhythmic activity is believed to represent the functional state of a region, with beta and gamma oscillations representing neuronal activation that correlates with increases in metabolism seen with functional imaging. In awake animals, the effects of DBS are transient in nature, wearing off minutes after turning off the stimulation; this suggests that the brain is organized in a manner to \"push back\" from the effects of DBS. However, when the stimulation is done for at least 5 days, there are significant changes in these frequencies. First, the short-term increases in power seem to give way to changes in coherence, or the organization of activity between regions. In particular, the orbitofrontal cortex showed increased coherence between itself and other regions, suggesting that the orbitofrontal cortex was now more responsive to inputs. This is consistent with the functional pathology of OCD - i.e., the orbitofrontal cortex is responsible for devaluing a stimulus; something that OCD patients fail to do. Therefore, when an OCD patient finds that a door is locked, instead of devaluing its significance in the face of data, their orbitofrontal cortex keeps \"reminding\" them that the door may indeed be unlocked. By restoring the ability of the orbitofrontal cortex to respond to this information, DBS may enable the patient to \"let go\" of inappropriate stimuli in the face of evidence to the contrary.\n\n## 2C. Trauma and fear\n\nDr. Roger Pitman a Professor of Psychiatry at Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA delivered a lecture entitled, \"Stress, Anxiety, and Trauma.\" Because of the breadth of this topic, Dr. Pitman focused on his group's research into biological aspects of posttraumatic stress disorder (PTSD). He noted that although PTSD is still largely regarded as a psychological phenomenon, ultimately the impact of an environmental event, such as a psychological trauma, must be understood at organic, cellular, and molecular levels. Dr. Pitman reviewed research in the psychophysiology laboratory beginning in the 1980's indicating that trauma victims diagnosed with PTSD showed larger heart rate, skin conductance, and facial electromyogram responses during personal, script-driven imagery of their traumatic events. When the script-driven imagery method was taken into the positron emission tomography (PET) scanner, results indicated that PTSD subjects also showed greater concurrent activation of amygdalocentric anterior plimbic brain areas thought to underlie emotional experience. However, one brain region, the ventromedial prefrontal cortex (vmPFC), showed pdoxically reduced activation in PTSD subjects during traumatic imagery. Further work with functional magnetic resonance imaging (fMRI) revealed an inverse association between vmPFC and amygdala activation, suggesting that vmPFC activation acts to inhibit amygdala and thereby confers resilience.\n\nPitman also presented results of a 15-year ongoing study of identical twins discordant for combat exposure in Vietnam, which has addressed the origin of biological abnormalities in PTSD. If his combat-unexposed co-twin shares an abnormality in a PTSD veteran, it is unlikely to have been acquired as a result of combat trauma but rather likely represents a familial vulnerability factor. If it is not shared, it is likely to have been acquired. This research has suggested that greater heart rate response to loud (startling) tones, lower electroencephalographic P300 event-related potential (ERP) response, increased P2 intensity dependence, lower vmPFC volume according to voxel-based morphometry, and impaired retention of extinction of a de novo conditioned fear response are acquired PTSD biomarkers, whereas more neurological soft signs (suggestive of subtle central nervous system impairment), diminished volume of the hippocampus, and both hypermetabolism and hyperreactivity of dorsal anterior cingulate cortex (a brain region found to promote the fear response) are familial vulnerability factors for PTSD.\n\nDr. Mohammed R Milad from the Department of Psychiatry at the Department of Psychiatry and Massachusetts General Hospital presented a talk entitled \"Neurobiology of learning not to fear: implications to PTSD and beyond\". In his presentation, Dr. Milad presented data on the neural correlates of fear extinction using a translational and multi-modal approach. After a brief definition and description of the classical fear conditioning pdigms used to study fear extinction, Dr. Milad presented data describing the basic mechanisms of how fear extinction learning is formed and stored with a network of brain regions including the ventromedial prefrontal cortex (vmPFC), the amygdala and the hippocampus. Dr. Milad presented data regarding the apparently apposing effects of two prefrontal regions on fear expression: the activation of the dorsal anterior cingulate cortex (dACC) seems to enhance the expression of conditioned fear, and the vmPFC seems to quell the expression of fear. The function of these brain regions within the context of fear learning and extinction seems to be impaired in patients diagnosed with PTSD, and that such functional impairment seem to be associated with exaggerated fear expression in PTSD patients.\n\nIn the second part of his talk, Dr. Milad described the potential role of gonadal hormones, such as estrogen, on fear extinction and how these hormones may contribute to sex differences in fear learning and extinction and may also contribute to the differences in the epidemiology of PTSD between men and women. In brief, Dr. Milad presented data showing that an increased estrogen level in both female rats and in women is associated with enhanced consolidation of extinction memory. The elevated levels of estrogen seem to interact with the functional activation of the fear extinction network, specifically, the higher the estrogen levels, the more activation the vmPFC exhibits during extinction recall in women. The importance of assessing hormonal status in women during treatment for PTSD and other anxiety disorders was highlighted in the presentation.\n\nDr. Gregory Quirk from the University of Puerto Rico presented a talk entitled \"Neurobiological Basis of Conditioned Fear and Extinction\" in which he talked about the neural mechanism of fear conditioning, using rats as a model organism. Fear conditioning has become a popular model of emotional learning, in which neurons in the amygdala, hippocampus, and prefrontal cortex acquire information about stimuli (such as tones) that predict danger (such as shocks). Disruption of such circuits leads to excessive fear seen in anxiety disorders such as PTSD and OCD. Quirk showed that expression of fear in the amygdala is under bidirectional control by the prelimbic and infralimbic subdivisions of prefrontal cortex. Modulation of activity in these circuits can increase fear either by facilitating expression or by impairing extinction of fear. In addition to modulating fear (such as freezing), these areas also modulate avoidance behaviors, in which rats escape a shock by moving to a safe place. Deep brain stimulation (DBS) of the ventral striatum has been shown to help some patients suffering from OCD, and Quirk showed rodent data suggesting that DSB may act by augmenting plasticity in extinction circuits located in amygdala and prefrontal areas. In collaboration with Dr. Haber, Quirk is characterizing the striatal-cortical circuits by which DBS enhances extinction of fear.\n\nTopic 3: Anxiety and depression: perspective from the region.\n\nDr. Abdul-Monaf Al-Jadiry, Professor and chair of Psychiatry, Department of Medicine, Medical Faculty, The University of Jordan, Amman, Jordan gave a presentation entitled, \"Prevalence of Anxiety and Mood Disorders and the link to Trauma: An Iraqi Perspective\". The presentation initiated with a general conclusion, based on literature review, about the impact of trauma on mental health in general, and the prevalence of anxiety and mood disorders in particular. Dr Al-Jadiry presented data from Iraqi Mental Health Survey (IMHS) to demonstrate the link between the two. IMHS is the first nationwide mental health epidemiological survey in Iraq and the second in the Middle East after Lebanon. The IMHS was undertaken in the years 2006-207, amidst circumstances of extreme instability and minimal security following the second gulf war. IMHS used World Mental Health Survey (WMHS) methodology and diagnostic instrument to provide data on the prevalence of mental health disorders, their impact and treatment, and their relationship to violence in the population. Trained interviewers used the \"Composite International Diagnostic Interview (CIDI), Version 3\" as a diagnostic tool for the assessment of mental disorders. The IMHS for the first time provides evidence-based data about the prevalence of mental health problems and mental health of Iraqi people. The survey provides an initial indicator of lifetime prevalence, and 12 months and 30 days prevalence rates alongside the experience of trauma. The importance of the Iraqi example springs from the fact that the state of Iraqis has been unique, as they have suffered more than any other people from decades of war trauma, sanctions and violence, which are still going on.\n\nThe IMHS demonstrated that 11.58% of population suffered from any anxiety disorder, including stress disorder, lifetime, and 7.82% from any affective disorder lifetime, with a significant difference between women and men, age groups, and several other socio-demographic variables. Moreover, the overall lifetime exposure to traumatic events, according to the survey was 60.02%. In the lifetime prevalence rate of these disorders cases had significant higher exposure to trauma. The most striking finding of the survey is the very high exposure of the population to various types of trauma and the relative low prevalence rates of anxiety and mood disorders though in line with prevalence rates reported in Lebanon and Afghanistan. Dr. Al-Jadiry attributed this discrepancy to the biological nature of people vulnerability for anxiety and mood disorders in addition to inherent protective sociocultural characteristics of Iraqis; these include: development of resilience, high community support, rituals to canalize distress and bereavement in a socially acceptable manner, and viewing losses as divine states. Moreover, the effect of stigma of mental illness is to make some patients hide their symptoms. Dr Al\u2013Jadiry concluded, despite massive exposure to traumatic events, the prevalence of anxiety and mood disorders amongst Iraqis is relatively lower than expected, though in accordance with the literature from post conflict zones.\n\nDr. Walid Sarhan a consultant Psychiatrist from Amman Jordan gave a talk entitled \"Cognitive behavioral therapy of Islamic religious OCD\". The talk started by brief background of Islamic religion with emphases on how its comprehensive application to people's daily lives and its rules to be followed in praying, washing, cleaning may relate to OCD symptoms expressed in some of the patients in the region. In managing those patients, Dr. Sarhan discussed how therapists may need to be aware of the compatibility of the cognitive behavioral therapy with the Islamic religion. The clinical experience of dealing with cleaning, abolition, praying, divorce and doubts in beliefs were discussed in light of the literature, while at the same time addressing the common clinical challenges presented by Arab Muslim patients suffering from OCD. Dr. Sarhan discussed how religious individuals with authority to declare legal rulings or decrees (Fatwa) on some aspects of the Islamic religion may also influence patient's symptoms. The clinician's knowledge of some of these rulings or \"fatwas\" may be necessary to understanding how some aspects of the OCD symptoms (especially those related to cleansing) came to exist.\n\nIn obsessions related to one's faith, Dr. Sarhan suggested that clinician's knowledge of the Islamic religion might be used as an additional tool to help aid the treatment; a clinician could make use of the teachings of the prophet to his followers as the key element in cognitive therapy.\n\nDr. Radwan A. BaniMustafa from the University of Jordan Medical School & Hospital gave a talk entitled \"Depression in Primary patients with chronic medical diseases\" he described a study which included a group of 407 primary care patients with chronic medical diseases. These patients were seen in the primary care clinics in Jordan University hospital in 2011. Prevalence of depression in this sample was 45.5%. Depression was significantly associated with young age, low level of education, chronic headache and chronic musculoskeletal diseases. In addition, it was associated but not significantly so with having more than three medical problems, chronic cardiopulmonary disorders, chronic Skin and thyroid disorders, female sex and single status.\n\n## Acknowledgements\n\nFunding for this meeting was made possible by NIMH (PI: Haber) and the International Brain Organization (IBRO). The meeting was hosted and supported by the University of Jordan, School of Medicine and the University of Rochester.","meta":{"dup_signals":{"dup_doc_count":103,"dup_dump_count":58,"dup_details":{"curated_sources":3,"2023-23":1,"2022-33":1,"2022-05":1,"2021-43":1,"2021-31":1,"2021-17":1,"2021-04":1,"2020-40":1,"2020-29":1,"2020-16":1,"2020-05":1,"2019-51":1,"2019-43":1,"2019-35":1,"2019-26":1,"2019-18":1,"2019-09":1,"2018-51":1,"2018-43":1,"2018-34":1,"2018-26":1,"2018-13":1,"2017-47":1,"2017-34":1,"2017-26":1,"2017-22":1,"2017-17":1,"2017-09":9,"2017-04":1,"2016-50":1,"2016-44":1,"2016-40":1,"2016-36":1,"2016-30":1,"2016-22":1,"2016-18":1,"2015-48":2,"2015-40":2,"2015-35":3,"2015-32":2,"2015-27":2,"2015-22":3,"2015-14":2,"2014-52":2,"2014-49":3,"2014-42":6,"2014-41":4,"2014-35":4,"2014-23":4,"2014-15":4,"2023-50":1,"2017-13":1,"2015-18":2,"2015-11":2,"2015-06":2,"2014-10":2,"2013-48":2,"2024-22":1}},"file":"PMC3621545"},"subset":"pubmed_central"}
{"text":"abstract: There are over 165 studies that have focused on Thimerosal, an organic-mercury (Hg) based compound, used as a preservative in many childhood vaccines, and found it to be harmful. Of these, 16 were conducted to specifically examine the effects of Thimerosal on human infants or children with reported outcomes of death; acrodynia; poisoning; allergic reaction; malformations; auto-immune reaction; Well's syndrome; developmental delay; and neurodevelopmental disorders, including tics, speech delay, language delay, attention deficit disorder, and autism. In contrast, the United States Centers for Disease Control and Prevention states that Thimerosal is safe and there is \"no relationship between \\[T\\]himerosal\\[-\\]containing vaccines and autism rates in children.\" This is puzzling because, in a study conducted directly by CDC epidemiologists, a 7.6-fold increased risk of autism from exposure to Thimerosal during infancy was found. The CDC's current stance that Thimerosal is safe and that there is no relationship between Thimerosal and autism is based on six specific published epidemiological studies coauthored and sponsored by the CDC. The purpose of this review is to examine these six publications and analyze possible reasons why their published outcomes are so different from the results of investigations by multiple independent research groups over the past 75+ years.\nauthor: Brian Hooker; Janet Kern; David Geier; Boyd Haley; Lisa Sykes; Paul King; Mark Geier\\*Brian Hooker: <email@example.com>[^1]\ndate: 2014\ninstitute: ^1^Simpson University, 2211 College View Drive, Redding, CA 96001, USA; ^2^Institute of Chronic Illness, Inc., 14 Redgate Court, Silver Spring, MD 20905, USA; ^3^University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75235, USA; ^4^University of Kentucky, Lexington, KY 40506, USA; ^5^CoMeD, Inc., Silver Spring, MD, USA\nreferences:\ntitle: Methodological Issues and Evidence of Malfeasance in Research Purporting to Show Thimerosal in Vaccines Is Safe\n\n# 1. Introduction\n\nThimerosal is an organic-mercury (Hg) based compound, used as a preservative in many childhood vaccines, in the past and present. To date, there have been over 165 studies that focused on Thimerosal and found it to be harmful \\[1, 2\\]. (A comprehensive list of these studies is shown at <http:\/\/mercury-freedrugs.org\/docs\/20140329_Kern_JK_ExcelFile_TM_sHarm_ReferenceList_v33.xlsx>.) Of these studies, 16 were conducted to specifically examine the effects of Thimerosal on human infants and\/or children \\[3\u201318\\]. Within these studies, which focused on human infants and\/or children, the reported outcomes following Thimerosal exposure were (1) death \\[3\\]; (2) acrodynia \\[4\\]; (3) poisoning \\[5\\]; (4) allergic reaction \\[6\\]; (5) malformations \\[7\\]; (6) autoimmune reaction \\[8\\]; (7) Well's syndrome \\[9\\]; (8) developmental delay \\[10\u201313\\]; and (9) neurodevelopmental disorders, including tics, speech delay, language delay, attention deficit disorder, and autism \\[10, 11, 14\u201318\\].\n\nHowever, the United States (US) Centers for Disease Control and Prevention (CDC) still insists that there is \"no relationship between \\[T\\]himerosal\\[-\\]containing vaccines and autism rates in children\" \\[19\\]. This is a puzzling conclusion because, in a study conducted directly by the CDC, epidemiologists assessed the risk for neurologic and renal impairment associated with past exposure to Thimerosal-containing vaccine (TCV) using automated data from the Vaccine Safety Datalink (VSD) and found a 7.6-fold increased risk of autism from exposure to Thimerosal during infancy \\[20\\]. The database for that study was \"from four health maintenance organizations \\[HMOs\\] in Washington, Oregon, and California, containing immunization, medical visit, and demographic data on over 400,000 infants born between 1991 and 1997.\" In that initial study, Verstraeten et al. \\[20\\] \"categorized the cumulative ethyl-Hg exposure from \\[T\\]himerosal\\[-\\]containing vaccines after one month of life and assessed the subsequent risk of degenerative and developmental neurologic disorders and renal disorders before the age of six.\" They \"applied proportional hazard models adjusting for HMO, year of birth, and gender, and excluded premature babies.\" The reported results showed that \"the relative risk (RR) of developing a neurologic development disorder was 1.8 (95% confidence intervals \\[CI\\] 1.1\u20132.8) when comparing the highest exposure group at 1 month of age (cumulative dose \\> 25\u2009*\u03bc*g) to the unexposed group.\" Similarly, they \"also found an elevated risk for the following disorders: autism (RR 7.6, 95% CI = 1.8\u201331.5), nonorganic sleep disorders (RR 5.0, 95% CI = 1.6\u201315.9), and speech disorders (RR 2.1, 95% CI = 1.1\u20134.0)\" in the highest exposure group.\n\nConsidering the many peer-reviewed published research studies that have shown harm from Thimerosal, including studies in which Thimerosal exposure is associated with the subsequent diagnosis of neurodevelopmental disorders (16 studies) such as autism, and the just-described evidence from the CDCs own research, which found evidence of a relationship between the level of Thimerosal exposure and the risk of a subsequent autism diagnosis, how does the CDC conclude that there is no evidence of that relationship? The foundation for the CDC's current stance apparently is based primarily on six specific published epidemiological studies that the CDC has completed, funded, and\/or cosponsored, starting in the late 1990s. These studies include (1) the Madsen et al. \\[21\\] ecological study of autism incidence versus Thimerosal exposure in Denmark, (2) the Stehr-Green et al. \\[22\\] ecological study of autism incidence versus Thimerosal exposure in Denmark, Sweden, and California, (3) the Hviid et al. \\[23\\] study of autism incidence versus Thimerosal exposure in Denmark (also ecological), (4) the Andrews et al. \\[24\\] cohort study of autism incidence and Thimerosal exposure in the United Kingdom, (5) the published Verstraeten et al. \\[25\\] CDC cohort study of autism incidence and Thimerosal exposure in the United States, and (6) the more recent Price et al. \\[26\\] case-control study of autism incidence and Thimerosal exposure in the United States. Although the CDC cites several other publications to purport the safety of Thimerosal, only these six specifically consider its putative relationship to autism.\n\nThe purpose of this review is to examine these six publications \\[21\u201326\\] which were \"overseen\" by the CDC and which claim that prenatal and early childhood vaccine-derived Thimerosal exposures are not related to the risk of a subsequent diagnosis of autism or autism spectrum disorder (ASD). This review analyzes possible reasons why their published outcomes are so different from the results of investigations by multiple independent research groups over the past 75+ years. The review begins with an examination of the Madsen et al. \\[21\\] study.\n\n# 2. The Madsen et al. 2003 Study\n\nThe CDC-sponsored Madsen et al. \\[21\\] study examined whether discontinuing the use of TCVs in Denmark led to a decrease in the incidence of autism. Data were obtained from the Danish Psychiatric Central Research Register, which contains all psychiatric admissions since 1971 and all outpatient contacts in psychiatric departments in Denmark since 1995. The study authors examined the data from 1971 to 2000 and reported that rate of autism increased with the removal of Thimerosal from vaccines (starting in 1992, the year that Thimerosal-containing early childhood vaccines were phased out).\n\nAlthough there are several concerns about the methodology used, the most serious concern involves diagnosis. As described in the paper, estimates of total autism cases in Denmark were only based on diagnoses occurring during inpatient visits from 1971 to 1994 and then during both inpatient and outpatient visits from 1995 to the last year of the study in 2000. Thus, the inclusion criteria are greatly expanded two years after the phaseout of Thimerosal from infant vaccines in Denmark, creating an \"artificial increase\" in autism prevalence. The authors conceded that \"the proportion of outpatient to inpatient activities was about 4 to 6 times as many outpatients as inpatients with variations across time and age bands.\" However, in an earlier publication by Madsen et al. \\[27\\], the same authors had stated regarding this same data, \"in our cohort, 93.1% of the children were treated only as outpatients\u2026\" Unlike the statement in the Madsen et al. \\[21\\] study, the 2002 paper indicates that the ratio between outpatients and inpatients in the 1971\u20132000 dataset was 13.5\u2009:\u20091, which would account for an even greater increase in cases diagnosed starting in 1995 (i.e., after the probable completion of the phaseout of TCVs that started in 1992).\n\nIn addition, the authors stated that the Danish registry which was used to count cases did not include a large Copenhagen clinic before 1993. This clinic accounted for as many as 20% of the autism cases nationwide, which would again artificially inflate the autism incidence observed in Denmark after the phaseout of TCVs was initiated in 1992. The authors do not mention this change in inclusion criteria (i.e., the addition of a new clinic in the registry) neither do they attempt to adjust their analysis in accordance with the anomaly. It was revealed, instead, in a similar paper by Stehr-Green et al. \\[22\\] where the authors state regarding the Denmark registry of autistic patients, \"Prior to 1992, the data in the national register did not include cases diagnosed in one large clinic in Copenhagen (which accounts for approximately 20% of cases occurring nationwide).\"\n\nAlso, the diagnosis criteria for \"autism\" changed within the course of the study. From 1971 to 1993, the ICD-8 standards for diagnosis (psychosis protoinfantilis 299.00 or psychosis infantilis 299.01) were used to measure autism incidence. However, from 1994 to 2000, the ICD-10 standard (infantile autism, F84.1) was used. Although the authors did not address the impact of the change in diagnostic criteria, this could result in as much as a 25-fold increase in cases as the instantaneous change in autism prevalence in Denmark, due to this change, went from a low of 1.2\/10,000 to a high of 30.8\/10,000 \\[28\\].\n\nAnother disconcerting methodological issue was that the 2001 data, which showed a strong downward trend in autism rates in at least two of the three age groups (continuing from 1999 through 2001), was not included in the final publication. This was apparent because when the paper was initially submitted for publication, it included the 2001 data. After the paper was rejected for publication by the Journal of the American Medical Association (JAMA) and the Lancet, it was submitted to the journal Pediatrics again including the 2001 data. As stated by one of the peer-reviewers of the Pediatrics submission, \"The drop of incidence shown for the most recent years is perhaps the most dramatic feature of the figure, and is seen in the oldest age group as well as the youngest. The authors do not discuss whether incomplete ascertainment in the youngest children or delay in recording of data in the most recent years might play a role in this decline, or the possibility that this decrease might have come about through elimination of \\[T\\]himerosal\" (January 23, 2003, communication between Dr. Poul Thorsen, Aarhus University, and Dr. Coleen Boyle, CDC scientist). In response to this criticism, the authors removed the 2001 incidence numbers. The authors' decision to withhold these data resembles scientific malfeasance, especially when coupled with the previously discussed problematic methods for counting autism cases. If the scientists believed that downward trend between 1999 and 2001 was caused by some phenomenon unrelated to the phaseout of the TCVs, these scientists should have included those data and then explained the trend within the discussion of the data.\n\nIf the 2001 data had been included in the final publication, the results would have been consistent with a more recent CDC study \\[29\\] where a decreasing trend of autism prevalence in Denmark after the removal of Thimerosal in 1992 was reported. Instead of large increases in autism prevalence after 1992, the recent Danish study revealed that the autism spectrum disorder prevalence in Denmark fell steadily from a high of 1.5% in 1994-95 (when children receiving Thimerosal-free formulations were too young to receive an autism diagnosis and, because of the known offset in diagnosis, most of those being diagnosed had been born 4 to 8 years earlier \\[from 1985 to 1990\\]) to a low of 1.0% in 2002\u20132004 (more than 10 years after the phasein of the use of Thimerosal-free vaccine formulations was started in 1992).\n\n# 3. The Stehr-Green et al. 2003 Study\n\nThe CDC's Stehr-Green et al. \\[22\\] study compared the prevalence\/incidence of autism in California, Sweden, and Denmark with average exposures to TCVs. Graph-based ecologic analyses were used to examine population data from the state of California (national immunization coverage surveys and counts of children diagnosed with autism-like disorders seeking special education services in California); Sweden (national inpatient data on autism cases, national vaccination coverage levels, and information on use of all vaccines and vaccine-specific amounts of Thimerosal); and Denmark (national registry of inpatient\/outpatient-diagnosed autism cases, national vaccination coverage levels, and information on use of all vaccines and vaccine-specific amounts of Thimerosal).\n\nThe study followed and appeared to be conducted in response to California study data \\[30\\], which was presented to the Institute of Medicine's Immunization Safety Review Committee. The California data showed that increased uptake of Thimerosal-containing vaccines in California during the 1990s correlated with a corresponding increase in autism diagnoses. In the Stehr-Green et al. \\[22\\] study, the researchers stated that the reliability of the autism prevalence data, citing that the California data included autism spectrum disorder diagnoses such as pervasive development disorder (PDD), could account for the increase. However, in a published response to this paper, Blaxill and Stehr-Green \\[31\\] stated that the California prevalence rates were reported based solely on autism cases.\n\nIn the Stehr-Green paper, the Sweden autism prevalence data showed an increase in autism rates from 5- 6 cases per 100,000 in 1980\u201382 to a peak of 9.2 cases per 100,000 in 1993. In Sweden, TCVs were phased out starting in 1987. Denmark's autism prevalence data was identical to that reported in the Madsen et al. \\[21\\] study critiqued previously. For Denmark, the authors reported an astounding 20-fold increase in autism prevalence between 1990 and 1999, despite the phaseout of TCVs that started in 1992.\n\nIn addition, the data from Sweden were based on inpatient (hospital) visits only. This limitation (counting a small fraction of the total number of cases) likely accounted for the erratic swings in the annual numbers of autism cases reported in that country. Also, the Thimerosal exposure level based on the Swedish vaccination schedule during this time period was much less (a nominal maximum of 75\u2009*\u03bc*g of Hg by two years of age) than that possible in California (and the United States as a whole) where developing children nominally received up to 237.5\u2009*\u03bc*g of Hg by 18 months of age through the standard immunization schedule. In conclusion, the Stehr-Green et al. study was problematic in its attempt to combine ecological data from three different countries that, relative to each other, demonstrated different vaccination policies and widely different Thimerosal exposure levels.\n\n# 4. The Hviid et al. (2003) Study\n\nThe Hviid et al. \\[23\\] population-based cohort study, widely cited by the CDC, compared rates of autism prevalence among individuals who received Thimerosal-free vaccines to those receiving TCVs. The authors report that there was no evidence of increased autism prevalence with Thimerosal exposure.\n\nThe study authors stated that the mean age of autism diagnosis within their population was 4.7 years with a standard deviation of 1.7 years. However, cases and controls as young as 1 year of age were included within the analysis. Accordingly, controls that were less than the mean age of diagnosis minus two standard deviations (1.3 years) from that age had a 97.5% probability of actually being individuals who will later develop autism and are therefore possibly misclassified. Similarly, in this study, the mean age for an ASD diagnosis was 6.0 years with a standard deviation of 1.9 years. Thus, the study methodology is questionable because it appears to have underascertained the number of cases diagnosed with autism and ASD.\n\nIn addition, rather than counting persons within the cohort, the authors counted \"person-years of follow up.\" With this technique, each age group (one-year-olds, two-year-olds, etc.) was considered equally, despite the fact that younger age groups were much less likely to receive an autism diagnosis. This again contributed to the undercounting of the cases with a diagnosis of autism and ASD and biased the study towards the null hypothesis (that there is no statistically significant Thimerosal exposure effect on the outcomes observed).\n\n# 5. The Andrews et al. (2004) Study\n\nThe Andrews et al. \\[24\\] study was a retrospective cohort study completed using records from a database in the United Kingdom, where autism prevalence rates were compared for children receiving Thimerosal-containing DTaP and DT vaccines. In the Andrews et al. \\[24\\] study, Cox's proportional-hazards ratios were used to evaluate periods of followup in the cohort examined by the investigators using the records in the general practitioner research database (GPRD), a database that was known to have a significant level of errors. These investigators reported that increased organic-Hg exposure from TCVs was associated with a significantly reduced risk for diagnosed general developmental disorders and for unspecified developmental delay (although there was a significantly higher risk for diagnosed tics).\n\nConsidering that there are several studies conducted by independent investigators that have found that exposure to Thimerosal is a risk factor for neurodevelopmental delay and disorders \\[10, 11, 16\\], the reduced rate of neurodevelopmental delay and disorders with Thimerosal exposure found in the Andrews et al. \\[24\\] study suggests possible methodological issues.\n\nThis result may have occurred, in part, because other studies examined cohorts with significantly different childhood vaccine schedules and with different diagnostic criteria for outcomes. This difference may also exist because these other studies that found Thimerosal to be a risk factor for neurodevelopmental delay and disorders employed different epidemiological methods, especially with respect to the issue of follow-up period for individuals in the cohorts examined. The method used to measure follow-up period for individuals is a critical issue in all studies examining the relationship between exposures and the subsequent risk of a neurodevelopmental disorder diagnosis, especially in those instances where the postexposure periods for all of the participants in the study are essentially the same. This is because the risk of an individual being diagnosed with a neurodevelopmental disorder is not uniform throughout his\/her lifetime. As observed in the present study, the initial mean age for any neurodevelopmental disorder diagnosis was 2.62 years old, and the standard deviation of mean age of the initial diagnosis of neurodevelopmental disorder was 1.58 years old. These findings are highly problematic because (1) any follow-up method that fails to consider the lag time between birth and age of initial neurodevelopmental disorder diagnosis will likely not be able to observe the true relationship between exposure and the subsequent risk of a neurodevelopmental disorder diagnosis and (2) statistically, the mean and standard deviation age of diagnosis as reported lead to the nonsensical result that a significant portion (2.5%) of the children in this study were diagosed with a neurodevelopmental disorder more than six months before they were born (i.e., the mean age minus two standard deviations, 2.62 \u2212 \\[2 \u00d7 1.58\\] = \u22120.54 years of age).\n\nAnother issue with this study is that the authors used a nontransparent, multivariate regression technique to analyze vaccine uptake and autism prevalence data. The study included one dependent variable (autism) and multiple independent variables, including two independent variables (Thimerosal exposure levels and year of birth) that were \"correlated\" with each other, since Thimerosal exposures increased with time. Thus, the researchers did not report a statistical analysis of the effect of Thimerosal exposure on autism incidence, despite the fact that the authors stated that no such effect was observed. Moreover, the methods used in this study can create a problem in regression known as \"multicolinearity.\" In this case, since the time variable and the vaccine exposure variable are correlated, they actually compete to explain the outcome effect. Inclusion of the time variable reduces the significance of the exposure variable. Yet, the authors did not explain why they included a time variable that competes with the exposure variable. Unfortunately, the authors of this study never released the raw data so that a valid single-variable analysis could be conducted to ascertain the probability of an association between Thimerosal exposure and the risk of autism.\n\nIt is also important to note that the UK Thimerosal exposure (a maximum of 75\u2009*\u03bc*g of Hg by 4 months of age) was not comparable to that in the United States (a maximum of 75\u2009*\u03bc*g of Hg by 2 months of age and 187.5\u2009*\u03bc*g of Hg by 6 months of age). Thus, this study should not be extrapolated to the probability of an autism-Thimerosal association based on the US vaccination schedule.\n\n# 6. The Verstraeten et al. (2003) Study\n\nThe CDC's published Verstraeten et al. \\[25\\] study consists of a cohort analysis of a subset of records from the medical records databases for several of the HMOs whose records were maintained in a central data repository, the Vaccine Safety Datalink (VSD). This study was conducted in at least five separate phases. In the final phase (i.e., the results reported in the publication), the authors stated that there was no relationship between Thimerosal exposure in vaccines and autism incidence. However, no data are reported in the published study to support this conclusion.\n\nResults from the first phase of the study released in an internal presentation abstract by Verstraeten et al. \\[20\\] (mentioned earlier) using records from four (4) HMOs showed that infants who were exposed to greater than 25\u2009*\u03bc*g of Hg in vaccines and immunoglobulins at the age of one month were 7.6 times more likely to have an autism diagnosis than those not exposed to any vaccine-derived organic Hg. Within the same abstract, Verstraeten reports that the risk for any neurodevelopmental disorder was 1.8, the risk for speech disorder was 2.1, and the risk for nonorganic sleep disorder was 5.0. All relative risks were statistically significant.\n\nIn the second phase of the study, a different approach was taken: exposure was compared at 3 months of age, rather than one month. Results of this phase showed that children exposed to the maximum amount of organic Hg in infant vaccines (62.5\u2009*\u03bc*g) were 2.48 times more likely to have autism diagnosis compared to those exposed to less than 37.5\u2009*\u03bc*g of Hg in vaccines. These results were also statistically significant. No assessment against a \"no exposure\" control was apparently completed in this study phase.\n\nIn the third phase of the study, in which more data stratification methods and different inclusion\/exclusion criteria were applied to the analysis, the relative risk of autism for children at three months of Thimerosal exposure dropped to 1.69. At this point, evidence in an email from Verstraeten, the lead investigator, written to a colleague outside of the CDC (obtained by the authors via the US Freedom of Information Act of 1950 as amended), suggests that Verstraeten could have been receiving pressure within the CDC to apply unsound statistical methods to deny a causal relationship between Thimerosal and autism. In this email, Verstraeten states (<a href=\"#fig1\" data-ref-type=\"fig\">Figure 1<\/a>), \"I do not wish to be the advocate of the anti-vaccine lobby and sound like being convinced that thimerosal is or was harmful, but at least I feel we should use sound scientific argumentation and not let our standards be dictated by our desire to disprove an unpleasant theory.\"\n\nThe fourth and fifth phase of the study used records from only two of the original HMOs and incorporated a third HMO, Harvard Pilgrim, into the analysis. Some critics of the study questioned the use of Harvard Pilgrim, as this HMO appeared to be riddled with uncertain record keeping practices, and the state of Massachusetts had been forced to take it over after it declared bankruptcy. In addition, the HMO used different diagnostic codes than the other two HMOs used in phases 2 and 3. Other criticisms include that the study used younger children, from 0 to 3 years of age, even though the average age for an autism diagnosis at the time was 4.4 years. Since half of the children receiving an autism diagnosis would be over 4.4 years of age, far greater than the maximum age in the study at 3 years, this analysis excluded more than 50% of all autism cases from this HMO. Also, the cohort from this HMO contained 7 times fewer individuals than the main cohort from the previous study (i.e., HMO B), and there was no apparent attempt to assess the power of this HMO to show any statistically significant effect.\n\nAlso of note is the lack of variability within strata among the different HMOs in the Verstraeten et al. \\[25\\] study. By design, a cohort study seeking to assess the effect of some treatment on a subsequent outcome should be designed to maximize the range of the independent \"treatment\" variable (Thimerosal exposure in this instance) in order to determine if there is indeed an \"effect\" in the dependent postexposure outcome variable (neurological disorders in this study). However, the authors knowingly stratified the analysis based on the participants' gender, year of birth, month of birth, and clinic most often visited. This effectively reduced the variability of Thimerosal exposure within the strata to the point that it reduced the capability of the final analysis to find any but the \"strongest\" Thimerosal exposure-related outcome effects. The problems with such \"overmatching\" practices have been discussed in detail in peer-reviewed scientific literature and will be treated in greater detail in the forthcoming review of the CDC's Price et al. \\[26\\] paper.\n\nAnother methodological concern about the Verstraeten et al. \\[25\\] study is related to the issue of the minimum follow-up period required for individuals in the cohorts examined to ensure that all the cases in the cohort will have been identified with a high degree of certainty. This issue has been mentioned as a problem in the previous studies. As mentioned earlier, the method used to determine the minimum follow-up period for individuals is a critical issue in all studies examining the relationship between exposures and the subsequent risk of a neurodevelopmental disorder diagnosis, especially in those instances where the exposures to all participants in the study are the same or essentially the same. This is the case because the risk of an individual being diagnosed with a neurodevelopmental disorder is not uniform throughout his\/her lifetime. Any follow-up method that fails to consider the lag time between birth and age of initial neurodevelopmental disorder diagnosis will likely not be able to observe the true relationship between exposure and the subsequent risk of a neurodevelopmental disorder diagnosis. Verstraeten et al. \\[25\\] included children in the control group who were too young (down to \"0\" years of age) to receive a neurodevelopmental disorder diagnosis.\n\nWithin this study, Verstraeten et al. \\[25\\] still found significantly increased risk ratios for tics and language delay. However, the authors stated that, because these results were not consistent between the HMOs tested, these significantly increased risk ratios could not be used to make a determination of the potential adverse consequences of organic-Hg exposure from TCVs.\n\n# 7. The Price et al. 2010 Study\n\nIn 2010, the CDC published another epidemiology study on Thimerosal and autism \\[26\\]. This case-control study was conducted using the records from three managed care organizations (MCOs) consisting of 256 children with an ASD diagnosis and 752 controls that were matched by birth year, gender, and MCO to the children with an ASD diagnosis. Exposure to Thimerosal in vaccines and immunoglobulin preparations was determined from electronic immunization registries, medical charts, and parent interviews. Conditional logistic regression was used to assess associations between ASD, autistic disorder (AD), and ASD with regression and exposure to ethyl-Hg during prenatal, birth-to-1-month, birth-to-7-month, and birth-to-20-month periods. Their published finding was that prenatal and infant Thimerosal exposure from TCVs and Thimerosal-containing immunoglobulin posed no statistically significant risk of autism.\n\nAs mentioned earlier, in case-control studies, the main methodological concern is the phenomenon called \"overmatching.\" This concern for overmatching in the Price et al. \\[26\\] study was voiced previously by DeSoto and Hitlan \\[32\\]. In their comprehensive analysis of overmatching errors specific to the Price paper, DeSoto and Hitlan \\[32\\] stated that \"Matching cannot\u2014or should not\u2014be done in a way that artificially increases the chance that within\\[-\\] strata exposure is the same; this happens when a matching variable is a significant predictor of exposure and is called overmatching.\"\n\nCases were matched with controls of the same age and sex, within the same HMO and essentially the same vaccination schedule, using the same vaccine manufacturers. DeSoto and Hitlan then state further, regarding the lack of variability of Thimerosal exposure in the Price study, \"Across the different years, the average cumulative exposure varies from 42.3\u2009*\u03bc*g to 125.46\u2009*\u03bc*g; while within the birth year stratas (sic), the mean exposures do not vary by more than 15 micrograms.\" In other words, the maximum level of variation in Thimerosal exposure in the cases and controls being compared was 15\u2009*\u03bc*g of Hg, as compared to the \"83\"\u2009*\u03bc*g of Hg range for the average cumulative exposures in the cohort studies. Moreover, this range is much less than the range of Thimerosal exposures that could have been used to determine risk including (a) 0 to 50\u2009*\u03bc*g of Hg for one-month exposures, (b) 0 to 190\u2009*\u03bc*g of Hg for seven-month exposures, and (c) 0 to 300\u2009*\u03bc*g of Hg for 20-month exposures. Finally, regarding the Price study, DeSoto and Hitlan \\[32\\] concluded, \"this paper is flawed. Unfortunately, there is not an analytic fix for overmatching: it is \\[a\\] design flaw.\"\n\nPrenatal Thimerosal exposure for the children within the study arose from the Thimerosal-preserved inactivated-influenza vaccine given during pregnancy and the Rho immunoglobulin administered to pregnant women to prevent Rh-factor incompatibility injury to the developing child. Unlike postnatal exposure from TCVs in the recommended childhood vaccination schedule, prenatal exposures would not be overmatched in a study design that stratified the participants based on their birth year or HMO. Evidence from the background CDC report regarding the Price study showed a significant risk of regressive autism due to prenatal Thimerosal exposure levels, at exposure levels as low as 16\u2009*\u03bc*g of Hg \\[33\\]. However, the risk of regressive autism due to prenatal Thimerosal exposure reported in that paper was 1.86 and yielded a *P* value of 0.072 which was deemed as insignificant based on the authors' \"cut-off\" value of *P* \\< 0.05. However, *P* values between 0.05 and 0.10 are \"marginally significant\" and should merit further study. In addition, upon further analysis, it was found that the 2009 background report \\[33\\] to the Price et al. \\[26\\] study showed that the prenatal Thimerosal exposure model was run in six different ways and that the most reliable methods (those that factored out the postnatal Thimerosal exposure effects) found highly statistically significant relative risks of up to 8.73 (*P* = 0.009) for regressive ASD due to prenatal Thimerosal exposures from Thimerosal-containing influenza vaccines and Rho immunoglobulin products relative to no such prenatal Thimerosal exposures. Curiously, these more compelling results were not reported in the paper. Withholding these data from the publication and, instead, reporting a significantly lower value could appear to constitute scientific malfeasance on the part of the authors of this study.\n\n# 8. Conclusion\n\nAs seen in this review, the studies upon which the CDC relies and over which it exerted some level of control report that there is no increased risk of autism from exposure to organic Hg in vaccines, and some of these studies even reported that exposure to Thimerosal appeared to decrease the risk of autism. These six studies are in sharp contrast to research conducted by independent researchers over the past 75+ years that have consistently found Thimerosal to be harmful. As mentioned in the Introduction section, many studies conducted by independent investigators have found Thimerosal to be associated with neurodevelopmental disorders. Several studies, for example, including three of the six studies covered in this review, have found Thimerosal to be a risk factor for tics \\[10, 17, 24, 25, 34, 35\\]. In addition, Thimerosal has been found to be a risk factor in speech delay, language delay, attention deficit disorder, and autism \\[10, 11, 15\u201317, 24, 25, 34\\].\n\nConsidering that there are many studies conducted by independent researchers which show a relationship between Thimerosal and neurodevelopmental disorders, the results of the six studies examined in this review, particularly those showing the protective effects of Thimerosal, should bring into question the validity of the methodology used in the studies. A list of the most common methodological issues with these six studies is shown in <a href=\"#tab1\" data-ref-type=\"table\">Table 1<\/a>. Importantly, other than the Hviid et al. \\[23\\] study, five of the publications examined in this review were directly commissioned by the CDC, raising the possible issue of conflict of interests or research bias, since vaccine promotion is a central mission of the CDC. Conceivably, if serious neurological disorders are found to be related to Thimerosal in vaccines, such findings could possibly be viewed as damaging to the vaccine program.\n\n## Acknowledgment\n\nFunding was provided by the nonprofit Institute of Chronic Illnesses, Inc. and CoMeD, Inc.\n\n## Conflict of Interests\n\nAll of the investigators on the present study have been involved in vaccine\/biologic litigation.\n\nMethodological issues most common in each of the six reviewed studies.\n\n<table>\n<thead>\n<tr>\n<th style=\"text-align: left;\">Study reviewed<\/th>\n<th style=\"text-align: left;\">Methodological issues<\/th>\n<\/tr>\n<\/thead>\n<tbody>\n<tr>\n<td style=\"text-align: left;\">Madsen et al. [<span class=\"citation\" data-cites=\"B21\">21<\/span>]<\/td>\n<td style=\"text-align: left;\">(i) Changing entrance criteria in ecological studies.<br \/>\n(ii) Withholding important results from the final publication.<br \/>\n(iii) Conclusions not generalizable to the US vaccination schedule due to widely different vaccination schedules and different levels of Thimerosal dosing in other countries.<\/td>\n<\/tr>\n<tr>\n<td colspan=\"2\" style=\"text-align: left;\"><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Stehr-Green et al. [<span class=\"citation\" data-cites=\"B22\">22<\/span>]<\/td>\n<td style=\"text-align: left;\">(i) Changing entrance criteria in ecological studies.<br \/>\n(ii) Withholding important results from the final publication.<br \/>\n(iii) Conclusions not generalizable to the US vaccination schedule due to widely different vaccination schedules and different levels of Thimerosal dosing in other countries.<\/td>\n<\/tr>\n<tr>\n<td colspan=\"2\" style=\"text-align: left;\"><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Hviid et al. [<span class=\"citation\" data-cites=\"B23\">23<\/span>]<\/td>\n<td style=\"text-align: left;\">(i) Accounting for \"person-years\" regarding exposure rather than actual exposure levels.<br \/>\n(ii) Conclusions not generalizable to the US vaccination schedule due to widely different vaccination schedules and different levels of Thimerosal dosing in other countries.<\/td>\n<\/tr>\n<tr>\n<td colspan=\"2\" style=\"text-align: left;\"><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Andrews et al. [<span class=\"citation\" data-cites=\"B24\">24<\/span>]<\/td>\n<td style=\"text-align: left;\">(i) Accounting for \"person-years\" regarding exposure rather than actual exposure levels.<br \/>\n(ii) Conclusions not generalizable to the US vaccination schedule due to widely different vaccination schedules and different levels of Thimerosal dosing in other countries.<\/td>\n<\/tr>\n<tr>\n<td colspan=\"2\" style=\"text-align: left;\"><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Verstraeten et al. [<span class=\"citation\" data-cites=\"B25\">25<\/span>]<\/td>\n<td style=\"text-align: left;\">(i) Cohort of children too young for followup for an autism diagnosis.<br \/>\n(ii) \"Overmatching\" phenomena due to too closely matched cases and controls.<br \/>\n(iii) Withholding important results from the final publication.<\/td>\n<\/tr>\n<tr>\n<td colspan=\"2\" style=\"text-align: left;\"><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Price et al. [<span class=\"citation\" data-cites=\"B26\">26<\/span>]<\/td>\n<td style=\"text-align: left;\">(i) \"Overmatching\" phenomena due to too closely matched cases and controls.<br \/>\n(ii) Withholding important results from the final publication.<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n[^1]: Academic Editor: Jyutika Mehta","meta":{"dup_signals":{"dup_doc_count":128,"dup_dump_count":67,"dup_details":{"curated_sources":3,"2022-49":3,"2022-33":3,"2022-27":1,"2022-21":1,"2022-05":3,"2021-43":1,"2021-39":2,"2021-17":2,"2021-10":2,"2021-04":1,"2020-50":1,"2020-45":1,"2020-34":1,"2020-29":2,"2020-24":3,"2020-16":5,"2020-10":3,"2020-05":3,"2019-51":3,"2019-47":1,"2019-43":2,"2019-39":3,"2019-35":4,"2019-26":4,"2019-18":1,"2019-09":4,"2018-51":2,"2018-47":2,"2018-43":3,"2018-39":4,"2018-34":2,"2018-30":3,"2018-26":2,"2018-22":1,"2018-17":4,"2018-09":2,"2018-05":2,"2017-51":2,"2017-47":1,"2017-43":2,"2017-39":1,"2017-34":2,"2017-30":2,"2017-26":1,"2017-22":2,"2016-50":1,"2016-40":1,"2016-30":1,"2016-26":1,"2016-22":1,"2016-18":1,"2016-07":1,"2015-48":1,"2015-40":1,"2015-35":1,"2015-32":1,"2015-27":1,"2015-22":1,"2014-52":1,"2014-49":1,"2014-42":2,"2014-41":1,"2014-35":2,"2014-23":2,"2023-06":1,"2017-13":1,"2024-18":1}},"file":"PMC4065774"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  The National Health Policy 2000 - 2009 and Health sector strategic plans I & II emphasized that Primary Health Care (PHC) would be the main strategy for national development and would be operationalized through provision of the minimum health care package. Commitment was to spend an increasing proportion of the health budget for the provision of the basic minimum package of health services which was interpreted to mean increasing spending at health centre level. This analysis was undertaken to gain a better understanding of changes in the way recurrent funding is allocated in the health sector in Uganda and to what extent it has been in line with agreed policy priorities.\n  .\n  # Methods\n  .\n  Government recurrent wage and non-wage expenditures - based on annual releases by the Uganda Ministry of Finance, Planning and Economic Development were compiled for the period 1997\/1998 to financial year 2007\/2008. Additional data was obtained from a series of Ministry of Health annual health sector reports as well as other reports. Data was verified by key government officials in Ministry of Finance, Planning and Economic Development and Ministry of Health. Analysis of expenditures was done at sector level, by the different levels in the health care system and the different levels of care.\n  .\n  # Results\n  .\n  There was a pronounced increase in the amount of funds released for recurrent expenditure over the review period fueled mainly by increases in the wage component. PHC services showed the greatest increase, increasing more than 70 times in ten years. At hospital level, expenditures remained fairly constant for the last 10 years with a slight reduction in the wage component.\n  .\n  # Conclusion\n  .\n  The policy aspiration of increasing spending on PHC was attained but key aspects that would facilitate its realization were not addressed. At any given level of funding for the health sector, there is need to work out an optimal balance in investment in the different inputs to ensure efficiency in health spending. Equally important is the balance in investment between hospitals and health centers. There is a need to look comprehensively at what it takes to provide PHC services and invest accordingly.\nauthor: Frederick Mugisha; Juliet Nabyonga-Orem\ndate: 2010\ninstitute: 1Economic Policy Research Centre, Makerere University Kampala; 2Health Systems Unit, World Health Organization, Uganda Country Office\nreferences:\ntitle: To what extent does recurrent government health expenditure in Uganda reflect its policy priorities?\n\n# Background\n\nThe National Health policy for Uganda (NHP) 2000 - 2009 \\[1\\] emphasized that Primary Health Care (PHC) would be the main philosophy and strategy for national development and would be made operational through provision of the minimum health care package. A minimum health care package, with interventions addressing the biggest burden of diseases affecting majority of the population, would form the primary focus of the health care delivery system. The welfare of the poor was to be given special consideration. Even though there was a reduction in poverty levels - from 38% in 2002\/03 to 31% in 2005\/2006 -, the population in poverty was still considered significant and more pronounced in rural areas \\[2,3\\].\n\nThe NHP\\[1\\] committed to allocating and spending an increasing proportion of the health budget for the provision of the basic minimum package of health services. This was interpreted during implementation to mean increasing spending at health centre level, where majority of the population, especially the rural and poor, lived and sought public health services (district health services). It is noteworthy that health centers' main focus is primary health care while hospitals' main focus is tertiary care. However, the policy provides for hospitals to offer primary health services in the absence of health centers. Spending at the central level and on referral and tertiary hospitals was to be held constant in real terms. The Health sector strategic plan (HSSP) I; 2000\/01 - 2004\/05 similarly stated ensuring effectiveness, efficiency, and equity in allocation and utilization of resources and expenditure on most relevant and cost effective priority health interventions, with a clear bias on protecting the poor and most vulnerable \\[4\\]. Review of the HSSP I noted that some progress had been made. There was an increase in funding to PHC services in absolute and relative terms targeted at peripheral health units \\[5\\].\n\nHSSP II 2005\/06 - 2009\/10 committed to allocating resources in the health sector in line with efficiency and equity principles\\[6\\]. Preferential allocation of resources to cost effective activities and increasing consumption of services were measures envisaged to address allocative and technical efficiency issues respectively. Targeting increasing allocations to health care inputs with a large impact on quality of services especially drugs and other health supplies, and increasing the proportion of resources allocated to the district health services where the majority of population, especially the poor, live were among the strategies to address equity concerns.\n\nUganda has a decentralized system of health service delivery with roles and responsibilities for the centre and districts clearly stated. The central level is responsible for setting policies, standards and guidelines, resource mobilization, capacity building, coordination of service delivery, monitoring and supervision. The decentralized levels (districts) are responsible for service delivery.\n\nThe objective of this paper is to provide a better understanding of changes in the way recurrent funding (wage and non-wage) is allocated in the health sector in Uganda and to what extent it has been in line with agreed policy priorities.\n\nUnderstanding changes in the way funding is allocated provides government and development partners an opportunity to examine not only the importance attached to its priorities but also because it provides a tool for monitoring the benefits expected from increased spending. In general expenditure on health is one of the determinants of health status. For example, in Lesotho, public expenditure on health was one of the important determinants of life expectancy at birth, infant mortality and under-five mortality \\[7\\]. Similar results were found in Pacific Island Countries while examining the relationship between per capita public expenditure and three measures of health outcomes (infant and under-five mortality rates and crude death rates)\\[8\\]. The results showed strong evidence that per capita health expenditure is an important factor in determining health outcomes. More important is the fact that when this expenditure is targeted, it is expected to yield better outcomes than when it is not. For example, Vietnam introduced a Health Care Fund for the Poor to increase access to health care and reduce the financial burden of health expenditure faced by the poor and ethnic minorities. The results suggest that despite numerous administrative challenges, the fund helped increase utilization and reduced out-of-pocket expenditure for the program's target population\\[9\\].\n\n# Methods\n\n## Data\n\nQuantitative research methods were used. The main dataset used in this paper is the government recurrent wage and non-wage expenditures - based on outturns by the Uganda Ministry of Finance, Planning and Economic Development (MoFPED). Expenditure outturns are actual releases of funds from central government to various spending centers. Possible alternative expenditure data is that based on actual accountability for expenses made at the end of each financial year. This data is unavailable. Accountability for expenditure is meant for audit purposes and therefore is not consistently captured.\n\nThe expenditure data based on central government outturns was compiled from approved estimates of revenue and expenditure reports that MoFPED publishes annually \\[10\\]. The data was complied for ten financial years - from the financial year 1997\/1998 to financial year 2007\/2008. This data does not include funds that are recurrent in nature not expended through the common government basket. Additional data was obtained from a series of annual health sector reports \\[11-13\\] as well as other reports.\n\nData was verified by key government officials in MoFPED and Ministry of Health (MoH). The key aspect required in the verification exercise was to confirm whether the data was correct and provide input in explaining the observed patterns. The Ministry of Finance, Planning and Economic Development being the source of the data provided all the necessary data reports. Officials from the planning department of the Ministry of Health responsible for finance and budget - given the subject matter - were asked to review the data and also comment on the emerging trends. The authors shared with them the data and the paper. They made few collections on the figures and shared with the authors their own compilation - which were reconciled.\n\n## Analysis\n\nIn order to understand the pattern of and further examine the recurrent government expenditure for PHC, the recurrent budget was categorized into two service care levels - the hospital services and the health center services. The Uganda health service structure is organized broadly in this manner. The primary health care services include services offered at Health Center I or Village Health team, Health Center II, Health Center III, and Health Center IV. Hospital services are provided at a General Hospital, Regional Referral Hospital and the National Referral Hospital.\n\nThe structure of the health sector outturns as presented in the estimates of revenue and expenditure is shown in Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>. Hospital expenditure includes that of Mulago Hospital Complex, Butabika Hospital, Regional Referral Hospitals, General Hospitals and NGO hospitals. NGO subvention is spent at NGO Hospital and Health Centre levels. The percent of NGO expenditure on hospitals was used in computing the actual expenses for hospital services. The part of the district NGO hospitals or primary health care that is spent on hospital care was varied across the years (see Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>).\n\nExample of the Health Sector Budget structure\n\n| FY 2007\/08 Out-turn |  |  |  |  |  |\n|----|----|----|----|----|----|\n| **SECTOR\/VOTE HEALTH** |  | **Wage** | **Non-Wage Recurrent** | **Domestic Dev** | **Donor Project** |\n|  |  |  |  |  |  |\n| 14 | Health | 2.86 | 42.78 | 19.62 |  |\n| 107 | Uganda Aids Commission (Statutory) | 0.69 | 0.63 | 1.51 |  |\n| 134 | Health Service Commission | 0.45 | 1.25 | 0.04 |  |\n| 151 | Uganda Blood Transfusion Service (UBTS) | 0.84 | 0.85 |  |  |\n| 161 | Mulago Hospital Complex | 12.42 | 17.16 | 1.32 |  |\n| 162 | Butabika Hospital | 1.58 | 1.8 | 6.8 |  |\n| 163-173 | Regional Referral Hospitals | 16.93 | 8.82 |  |  |\n| 501-850 | NGO subvention |  | 15.93 |  |  |\n| 501-850 | District Primary Health Care | 78.9 | 19.85 | 6.31 |  |\n| 501-850 | General Hospitals |  | 9.61 |  |  |\n|  |  |  |  |  |  |\n|  | SUB-TOTAL HEALTH | 114.66 | 118.7 | 35.6 |  |\n\nSource: MFPED, Estimates of revenue and expenditure (recurrent and development) FY 2007\/08. 2008, Ministry of Finance, Planning and Economic Development: Kampala, Uganda.\n\nThe total expenditure on PHC is shown as \"District Primary Health Care\" plus the amount expended on NGO Health Centers. Expenditure by the MoH headquarters and district health offices is included as a separate category. The MoH headquarters and district health offices do conduct management functions on behalf of the Health Centers and Hospitals.\n\nExpenditure on Uganda Aids Commission, Health Service Commission and Uganda Blood Transfusion Service is considered to be other expenditure. Other expenditure items that have been phased out over the years include the Health Training Schools and the lunch allowances. The training schools were transferred to be under the Ministry of Education while the lunch allowance was consolidated in the wage component.\n\n# Results\n\n## Overall Recurrent Expenditure\n\nFigure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a> shows recurrent (wage and non-wage) expenditure for the health sector over a period of ten financial years.\n\nThree messages are noticeable from the results. First, during the year 2000-01, there was a pronounced increase in the amount of funds released for recurrent expenditure. It increased by about UGX 60 Billion or 76 percent between 2000\/01 - 2001\/02. This coincided with Government of Uganda policy to focus on PHC as a strategy.\n\nSecond, prior to 2005-06 the non-wage component of recurrent expenditure was higher than the wage. However after this financial year, the wage component caught-up with the non-wage component.\n\nThird, we notice that the wage component of the recurrent expenditure increased consistently each year during the period under review while the non-wage did not show significant increase. This suggests that increases in the wage component contributed most to the increase in recurrent expenditure.\n\n## Recurrent Expenditure Across Levels\n\nFigure <a href=\"#F3\" data-ref-type=\"fig\">3<\/a> shows the recurrent expenditure across different levels in the health system.\n\nPHC services showed the greatest increase over the ten year period. Between 1997 and 2000, primary health care services attracted less recurrent expenditure than hospital services or the ministerial and district headquarters. In the financial year 2000-01, a policy directive was made to focus spending on PHC as a strategy to bring services closer to the population. Indeed, following this pronouncement, in the financial year 2001-02, expenditure on PHC shot up beyond that of hospital services. The rate of increase after this time continued to be higher than that of hospital services. Recurrent expenditure at district and ministry headquarters, and other health institutions remained rather constant over this period. In the rest of the paper, no further analysis of ministerial and district headquarters is done. Further analysis was done for hospital services and primary health care services.\n\n## Recurrent Expenditure On Phc Services\n\nIn line with the policy decision, recurrent expenditure on PHC services has gone up from 1.5 billion in 1997-98 to 106 billion in 2007-08 in nominal terms, increasing more than 70 times in ten years (see Figure <a href=\"#F4\" data-ref-type=\"fig\">4<\/a>). We notice that the increase was mainly driven by wage component of the recurrent expenditure. In fact the non-wage component actually started to decline in 2003\/2004 and continued to decline.\n\nConsidering the wage and non-wage components as percentages of the total, Figure <a href=\"#F5\" data-ref-type=\"fig\">5<\/a> shows that the percentage of non-wage has continued to decline. This is in contrast to the wage, which moved from a negligible percentage to about 80 percent in 2007-08. This result creates an impression that expansion of health services was pushed by the wage component with increasingly less resources to deliver the services.\n\n## Recurrent Expenditure On Hospital Services\n\nRecurrent expenditure for hospital services went up from 27 billion in 1997\/1997 to 76 billion in 2007\/2008 in nominal terms, increasing almost 3 times in ten years (Figure <a href=\"#F6\" data-ref-type=\"fig\">6<\/a>). Unlike with PHC services, non-wage expenditure was consistently higher than the wage component. The expenditure pattern at hospital level differs from that seen at PHC level.\n\nFigure <a href=\"#F7\" data-ref-type=\"fig\">7<\/a> shows the two components of recurrent expenditure - wage and non-wage - in terms of percentage. The results show that, unlike the expenditure on PHC services that on hospital services is consistent. The percent expenditure on wage is consistently lower than that of non-wage for all the years.\n\n# Discussion\n\nWe begin the discussion with a comment on the scope of the paper. The paper does examine recurrent government recurrent expenditure. It therefore leaves out government development expenditure. It also leaves out expenditure that is not done through the government medium expenditure framework (MTEF), that is, the off budget expenditure. This off budget expenditure is done mainly by development partners and non-government organizations. The proportion of the government recurrent expenditure to the total expenditure of the sector is difficult to estimate. No data is collected either for the non-government organizations or for the development partners. However, analysis of recurrent expenditure in itself is a an important aspect of understanding government priorities.\n\nSimultaneous changes as part of the implementation of the policy are noteworthy. A one off capital investment for Primary Health Care was made four years into the implementation of the policy. Capital expenditure increased from Ushs 10.6 billion in the 2001-2002 financial year to Ushs 75.8 billion shillings in the next financial year (2002-2003) - Based on analysis of the mid-term expenditure framework. This investment enabled further expansion of health centers with an additional 400 HCIIs. In addition, the recruitment of health workers was re-instituted in 2001. By mid 2002, more than 85% (2,538) of health workers under the Poverty action funds targeting PHC services had been recruited\\[5\\].\n\nThe sector wide analysis has shown an increase in recurrent expenditure largely accounted for by increase in the wage component while the non wage recurrent remained fairly constant. The noted increase in the wage bill may be explained by several reasons, increase in salaries, adjustment for inflation and recruitment of more health workers. We note that increase in public servants wages have been very modest, for example, between 1999 and 2002 wages grew by 4.8% per annum\\[14\\] while between 2002 - 2007, average monthly earnings only increased by 13%\\[15\\]. Inflation was maintained below 8% between 2000 and 2008\\[16\\]. Percentage of approved posts filled by trained staff in the health sector improved from 33% in 1999\/00 to 69% in 2004\/05\\[11\\] and given this, increase in number of health workers accounts for the increasing wage bill.\n\nThe availability of human resource without adequate inputs affects quality of care and leads to further loses, having to pay salaries for health workers who are not providing services. Underfunding other key inputs has affected service delivery, for example, medicines stock outs is a long standing problem. The percentage of health facilities registering stock outs in essential medicines has consistently been over 60% for the last 10 years \\[11-13\\]. Per capita expenditure on essential medicines and health supplies has remained below US\\$2 per capita for the last six years \\[11-13,17-19\\]. Functionality of available equipment ranges from as low as 33% at the general hospital level to 52% at the Health Centre four (HC IV) level\\[19\\]. Available investments cannot be put to optimal use.\n\nLooking at recurrent expenditure by level, the policy decision of capping of expenditures at tertiary level hospitals, which were viewed to serve a small urban population was achieved. Similarly, the high and increasing recurrent expenditure at the district level shows that the policy decision to target peripheral units serving mainly the poor \\[17\\]was also met. Eighty percent (80%) of the population live in rural areas and poverty is more prevalent in rural areas compared to urban areas However, expenditure at the national level health institutions remained the lowest and constant over the review period. These institutions offer support services and their proper functioning is crucial for realization of PHC provision. As a result of underinvestment in these institutions, key aspects of PHC have not been effectively provided. Inadequate blood transfusion services for example have affected delivery of emergency obstetric care services. The mid-term review report noted the markedly inadequate and poorly furnished blood transfusion structure and lack of enough blood supply\\[18\\]. Out of the seven recommended regional blood banks, only five are in place with no expansion made for the last three years\\[20\\]. Operations of the Health service commission, charged with the responsibility of establishing an efficient health workforce continue to be constrained\\[18\\]. Occasionally, funding for the wage bill has been returned to the treasury because of failure to recruit health workers.\n\nThe patterns of expenditure within hospitals and health centers have been analyzed. We have noted that between 2000\/01 and 2001\/02, at the hospital level, there was a reduction in the wage and an increase in the non wage recurrent while at the health centre level, there was a reduction in the non wage recurrent and an increase in the wage. This has affected availability of essential supplies, for example, percentage of health centre IIs experiencing stock outs of essential medicines has been close to 80% for the last 3 years compared to the national average of close 70%. Only 52% of HC II were able to provide antenatal care compared to hospitals at 95% \\[21\\]. Percentage of HCIIs, offering child immunization with all equipment available, was only 55% compared to hospitals at 90%. Preferential investment in wage at the expense of the non wage recurrent at HC level has affected service delivery at this level with resultant congestion at hospitals of cases that could be treated at lower levels. Similarly, evidence suggests that human resource utilization is suboptimal when investment in other areas like of the capital (i.e. medical equipment, etc.) is inadequate. They recommended that reorientation of the resource allocation towards the capital investments would save more lives \\[22\\].\n\n# Conclusion\n\nIn this analysis we have shown that the policy aspiration of increasing spending on PHC was attained but key aspects that would facilitate its realization were not addressed. Support services like blood transfusion and the human resource commission which facilitates recruitment of health workers were not supported. At any given level of funding for the health sector, there is need to work out an optimal balance in investment in the different inputs to ensure efficiency in health spending. Much as improving availability of health workers is important, they must have necessary inputs to provide services. Equally important is the balance in investment between hospitals and health centres. There is need to look comprehensively at what it takes to provide PHC services and invest accordingly.\n\n# Competing interests\n\nThe authors declare that they have no competing interests.\n\n# Authors' contributions\n\nBoth authors contributed substantially in the conception and design of the paper, acquisition of data, analysis and interpretation of data. Both authors drafted the manuscript, revised it critically for important intellectual content and gave final approval of the version to be published.\n\n## Acknowledgements\n\nWe acknowledge the contribution of government of Uganda for compiling the data on an annual basis. The Global Development Network is also acknowledged for facilitating the exercise. Economic Policy Research Centre and World Health Organization Uganda Office provided technical guidance.","meta":{"dup_signals":{"dup_doc_count":115,"dup_dump_count":34,"dup_details":{"curated_sources":4,"2018-05":1,"2017-39":1,"2017-30":1,"2017-22":1,"2017-17":1,"2017-09":6,"2017-04":1,"2016-50":1,"2016-44":1,"2016-36":1,"2016-22":1,"2016-18":1,"2015-48":17,"2015-40":9,"2015-35":3,"2015-32":3,"2015-27":3,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":4,"2014-42":9,"2014-41":3,"2014-35":5,"2014-23":5,"2014-15":3,"2022-27":1,"2015-18":3,"2015-11":3,"2015-06":3,"2014-10":4,"2013-48":3,"2013-20":3,"2024-18":1}},"file":"PMC2984410"},"subset":"pubmed_central"}
{"text":"abstract: Science and religion; never the twain shall meet.\nauthor: Gregory A Petsko\ndate: 2009\ninstitute: 1Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02454-9110, USA\ntitle: Render unto Darwin\n\nYou probably haven't encountered a website for something called BioLogos. If you have, you will undoubtedly already have formed a strong opinion about it - it's that kind of site. If you haven't, you really ought to check it out <http:\/\/www.biologos.org>. It's the website for something called the BioLogos Foundation. According to its mission statement, \"The BioLogos Foundation promotes the search for truth in both the natural and spiritual realms seeking harmony between these different perspectives.\" The foundation was established by Francis Collins with a grant from the John Templeton Foundation, a much older organization with a similar mission. And that, apart from its intrinsic interest, is why you should check it out, and why I'm wagering you will have strong opinions when you do. Francis Collins is the scientist who headed the publicly funded Human Genome Sequencing Project in the 1990s. Until last August he was the head of the National Human Genome Institute at the National Institutes of Health (NIH), which continues that work and funds much of the genome biology in the United States. And he just might be the next director of the NIH, the largest scientific research funding organization in the world.\n\nIn a public statement, Collins said that he established the BioLogos Foundation \"to address the escalating culture war between science and faith in the United States. On one end of the spectrum, 'new atheists' argue that science removes the need for God. On the other end, religious fundamentalists argue that the Bible requires us to reject much of modern science. Many people - including scientists and believers in God - do not find these extreme options attractive. BioLogos represents the harmony of science and faith. It addresses the central themes of science and religion and emphasizes the compatibility of Christian faith with scientific discoveries about the origins of the universe and life. To communicate this message to the general public and add to the ongoing dialog, The BioLogos Foundation created BioLogos.org.\"\n\nLet's dissect this statement, because if ever there was a statement that needed dissecting, this is one. I completely agree with Collins that there is a culture war between science and faith in the United States. But I do not agree that the war is due primarily to the clash between the extremists on both sides. Take the \"new atheists\", for example. There are many atheists in the United States, and some of them are scientists. But only a handful would take the extreme - and, to my mind, incorrect - position that science disproves the existence of God. The British scientist Richard Dawkins might, but he doesn't speak for the majority of scientists I know, and his eloquent but strident voice has only served to inflame the opposition by preaching to the converted. There are many more agnostics, who simply believe that there is no compelling evidence to believe in any deity.\n\nNow let's look at that opposition, the \"religious fundamentalists\" who argue that \"the Bible requires us to reject much of modern science\". There are a lot more of those, especially in the United States, but - and this is a crucial distinction, as we shall see - they are almost entirely evangelical Christians, not \"religious fundamentalists\" in general. Evangelical Christians often take the Bible literally, and a literal reading of the Bible is certainly incompatible with many of the findings of science.\n\nOne of the missions of the BioLogos website is to advance the idea of theistic evolution, a concept discussed in depth in Collins' book *The Language of God*, which is also promoted on biologos.org. Essentially, theistic evolution means that evolution is the way God created life. I was first clued into this website by Jonathan Eisen, an evolutionary biologist and open access publishing maven at the University of California, Davis. Eisen, whose blog *The Tree of Life* <http:\/\/phylogenomics.blogspot.com\/> is a delightful and thoughtful commentary on the worlds of both genomics and scientific publishing, wrote about BioLogos on 5 May. So that you will know where he's coming from, here's his opening statement: \"I am all for trying to have discussions about science and religion. But I do not think the two topics are really compatible in the sense of merging them together. Science (and medicine) should be about, well, science. And religion can be about whatever it wants to be. And when we can get religious and scientific leaders together to talk about the implications of each area on the other and on the world, fine too. But merging the two together into one hybrid such as Christian Science and Creation Science? Not for me.\"\n\nHe goes on to make a pointed criticism of the underlying logic - or lack thereof, in his view - behind BioLogos. \"The details of Collins' attempt to merge science and religion into a version of theistic evolution are really unclean. Basically, he is trying to argue that on the one hand science and religion are completely separate activities (I support this) but at the same time argues that God can intervene in the setting up of natural laws and in providing some guidance here and there in order to, for example, produce human beings in his image. The website repeats some things from Collins' book that are equally illogical - such as saying that altruism can be explained by science (and even specifically saying that science is the way to explain the natural world) but then turning around and saying that science cannot explain extreme forms of altruism (and therefore implying that actually, the natural world cannot be explained by science). Which is it? Is science for the natural world or not?\" Eisen is right that this, and some of BioLogos' other talking points, smack of setting up a straw man.\n\nBut in the end, BioLogos aims to show that the findings of science are not inconsistent with the existence of God. And not just any God. BioLogos is all about the Christian God. It even says so: \"The creation story of BioLogos is compatible with many faith traditions, and there is no way to give a scientific proof for one monotheistic faith over another. Therefore, this response will simply show the compatibility of Christianity with BioLogos.\" And again, more forcefully, in their mission statement: \"the website is a reliable source of scholarly thought on contemporary issues in science and faith that highlights the compatibility of modern science with traditional Christian beliefs.\"\n\nHere's another example: \"For believers, these \\[scientific\\] discoveries must ultimately be compatible with the truth that is revealed in the Bible, and it is the conviction of BioLogos that this compatibility is not only desirable but also possible. The limitation is that our access to all forms of truth, including scientific and religious, is at best partial.\" The statement that it is Biblical truth that science must be compatible with (and there are other comments that make it clear BioLogos means the Christian Bible, especially the New Testament) marks a clear attempt to link science with one brand of religion.\n\nThe creators of BioLogos have every right to make the foundation and website about whatever they want. And I suppose you could argue that, as I see it, because it is evangelical Christians that are the chief opponents of modern science, especially evolution, it is sensible for scientists to promote the compatibility of science with Christian beliefs. But I don't agree. I think it's a huge mistake.\n\nGK Chesterton, a devout Roman Catholic, has his priest-detective Father Brown say, in the superb short story *The Sign of the Broken Sword*, \"When will people understand that it is useless for a man to read his Bible unless he also reads everybody else's Bible? A printer reads the Bible for misprints. A Mormon reads his Bible and finds polygamy; a Christian Scientist reads his and finds we have no arms and legs.\" I've always liked this quotation, and not just because I agree that one huge problem with putting your faith in the literal reading of a book is that you can find justification in that book for almost any form of behavior, from altruism to genocide to slavery. I like it for a reason that Chesterton probably never intended: it reminds me that there are many more religions than Christianity, and many more people of faith than monotheists. I think if you are going to understand people of faith and try to see how we as scientists can find common ground with them, it is discriminatory - and possibly something worse - to focus on Christians or even monotheists (which, in the modern world, pretty much consists of Muslims, Christians and Jews - Zoroastrians being in short supply nowadays - and I'm pretty sure that the BioLogos folks would not include Islam in their mission, given that faith's denial of the divinity of Christ). If you are going to read the Christian Bible you should also read the Jewish Bible. And the Book of Mormon. And the Koran. And the Bhagavad Gita. If you really care about making contact with people of faith you should not exclude most of them just because they worship different gods from yours.\n\nIn some temples in India, during services the priest will read from the Hebrew or Christian Bible and the Muslim Koran as well as the Hindu Gita, moving from one to the other as though it did not matter what precise words were being spoken as long as there was something greater than the individual self that was being worshipped. How can we as scientists find common ground with people of faith unless we recognize the commonalities they share with each other? At its best, all religion is about a love for the natural world, a desire to help other people, and a sense that life is well lived only when it is not lived selfishly and pettily - values that typically underlie most scientific research. At its worst, religion is about unquestionable certainty, authoritarianism, exclusion and discrimination - things that have no place in science either. Scientists can make common cause with people of faith through the values we share, but must reject the extremist, intolerant views that poison both spheres.\n\nAnd in the end, that's my big problem with BioLogos - at its heart, it strikes me as implicitly exclusionary (plus I agree with Eisen that its logic is shaky). I would have much preferred a clearcut effort to emphasize the non-connectedness of science and faith: that science is about evidence and testable hypotheses, whereas religion is about believing in things for which there is no evidence whatsoever, and cannot be. That would place them in separate realms, but with common ground as I defined it above. The moment you start trying to say that data from science is compatible with the Christian religion in particular, you imply that, for example, polytheistic religions are wrong, and maybe not just as a matter of faith, but as a matter of science.\n\nNothing is more dangerous than such absolutism. It sets one type of religion as being true and therefore can be used to support the branding of all the others as false. However well-intentioned, BioLogos isn't likely to bring peace to the war between science and religion if it is oriented so strongly towards one religion.\n\nI would have loved to see the resources that the Templeton and BioLogos Foundations spent on biologos.com - both financial and in terms of human effort - devoted to clarifying and promoting the distinctions between science and religion and to a search for a common ground that does not exclude anyone of faith. That's something I could support (and, I bet, something that Jonathan Eisen and possibly Charles Darwin could support, too). But the idea that science provides information that cannot be explained by science alone - and therefore that science 'needs' the Christian God for a complete description of the universe - strikes me as the wrong thing to do. Render unto Darwin the things that are Darwin's, and unto God the things that are God's. But for God's sake (or should that be Darwin's?), don't mix them together.","meta":{"dup_signals":{"dup_doc_count":109,"dup_dump_count":53,"dup_details":{"curated_sources":2,"2023-23":1,"2022-33":1,"2021-39":1,"2021-21":1,"2020-34":1,"2020-24":1,"2019-47":2,"2019-30":1,"2019-22":1,"2019-09":1,"2019-04":1,"2018-51":1,"2018-47":1,"2018-43":1,"2018-39":1,"2018-34":1,"2018-30":1,"2018-26":1,"2018-13":1,"2018-05":1,"2017-39":2,"2017-22":1,"2017-09":9,"2017-04":1,"2016-44":1,"2016-40":1,"2016-36":9,"2016-30":7,"2016-22":1,"2016-18":1,"2016-07":6,"2015-48":2,"2015-40":1,"2015-35":2,"2015-32":2,"2015-27":2,"2015-22":2,"2015-14":2,"2014-52":2,"2014-49":2,"2014-42":3,"2014-41":4,"2014-35":3,"2014-23":4,"2014-15":2,"2023-50":1,"2015-18":2,"2015-11":2,"2015-06":2,"2014-10":2,"2013-48":2,"2013-20":2,"2024-26":1}},"file":"PMC2718506"},"subset":"pubmed_central"}
{"text":"abstract: The momentum towards open-access publishing has continued to build in the months since *Journal of Biology* was launched.\nauthor: Theodora Bloom\ndate: 2003\ninstitute: 1Editor, *Journal of Biology*\ntitle: Editorial\n\nOne of the unusual features of *Journal of Biology* is its commitment to publish each research article online as it is ready, and to assemble a print issue around as few or as many research articles as are available at one time. This print issue includes two research articles along with associated commentary, and this reflects a change in the pace at which suitable articles are being submitted to the journal - although we continue to decline tens of articles for every one that is accepted for publication, as befits a journal that aims to join the top rank.\n\nLike all peer-reviewed research journals, this one can only select for publication from among the articles submitted to it. Given our aspiration to cover the full spectrum of biology, it is gratifying to note that the research articles published so far span a broad range of biological subjects. The first articles described genome-wide characteristics of gene expression (<http:\/\/jbiol.com\/content\/1\/1\/5>) and a chemical approach to studying cell signaling during development and disease (<http:\/\/jbiol.com\/content\/1\/2\/10>). Now, the articles in this issue describe the application of a new combination of biophysicial technologies to single-molecule measurements (<http:\/\/jbiol.com\/content\/2\/1\/6>) and new insights into the coordination of cell growth with cell division in mammalian cells (<http:\/\/jbiol.com\/content\/2\/1\/7>). In the forthcoming issues, which are now being prepared, readers can look forward to further diversity, and we hope that potential authors will continue to be reassured that we do intend to cover their favorite subject area.\n\nA notable attribute of the second research article in this print issue is that the senior author is the journal's Editor-in-Chief, Martin Raff. There will be some readers who question the propriety of the Editor-in-Chief publishing in 'his own journal' - but in a classic double bind, if he chose instead to publish in another high-profile journal there would be those who doubted his commitment to *Journal of Biology*. We can only reassure the sceptics that the peer-review process was, as usual, stringent and anonymous, and that the journal's usual standards and practices were applied especially scrupulously to this article. We hope that others, whether formally associated with this journal or not, will follow the Editor-in-Chief's lead and submit their best work to this journal and so help promote open access as the preferred method of publishing in biology.\n\nThe momentum towards open-access publishing has continued to build in the months since *Journal of Biology* was launched. The high-profile group of scientists behind the Public Library of Science (<http:\/\/www.plos.org\/>) plan to launch their own open-access journals later this year. In addition, announcements of support for the open-access publishing model have continued to pile up from around the world, from both funding agencies (see, for example, those listed at <http:\/\/www.biomedcentral.com\/info\/about\/apcfaq>) and individual institutions (see <http:\/\/www.biomedcentral.com\/inst\/>). Ever since the first circulation in 1999 of an idea from Harold Varmus for an NIH-funded public repository of open-access research articles, it has been clear that every author who chooses to publish their research in an open-access journal rather than one that charges a subscription fee is benefiting the community of interested researchers both immediately and in perpetuity. We urge you to bear these issues in mind when considering where to publish your next important piece of research.\n\n**Editor's note:** The authors of the second research article in this print issue (<http:\/\/jbiol.com\/content\/2\/1\/7>) have both had close associations with *Journal of Biology*, and Martin Raff continues to do so. Neither author was involved in the refereeing of this article, in the decision to publish it, or in the choice of accompanying commentary.","meta":{"dup_signals":{"dup_doc_count":127,"dup_dump_count":41,"dup_details":{"curated_sources":2,"2019-09":1,"2018-51":1,"2018-43":1,"2018-30":1,"2018-13":1,"2017-47":1,"2017-34":1,"2017-26":1,"2017-22":1,"2017-17":1,"2017-09":10,"2016-50":1,"2016-44":1,"2016-40":1,"2016-36":10,"2016-30":8,"2016-22":1,"2016-18":1,"2016-07":9,"2015-48":3,"2015-40":3,"2015-35":2,"2015-32":3,"2015-27":3,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":3,"2014-42":8,"2014-41":4,"2014-35":5,"2014-23":4,"2014-15":6,"2019-22":1,"2015-18":3,"2015-11":3,"2015-06":3,"2014-10":3,"2013-48":3,"2013-20":3,"2024-18":1}},"file":"PMC156592"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  Homosexual patients are affected by social factors in their environment, and as a result may not have easy access to existing health care services. Prejudice against homosexuality and homosexual patients remains a barrier to them seeking appropriate health care. The concern is that lesbians and gays might delay or avoid seeking health care when they need it because of past discrimination or perceived homophobia within the health care thereby putting their health at risk.\n  .\n  # Aim of the study\n  .\n  The aim of the study was to explore and describe the experiences of homosexual patients utilising primary health care (PHC) services in Umlazi in the province of KwaZulu-Natal (KZN).\n  .\n  # Method\n  .\n  A qualitative, exploratory, descriptive study was conducted which was contextual in nature. Semi-structured interviews were conducted with 12 participants. The findings of this study were analysed using content analysis.\n  .\n  # Results\n  .\n  Two major themes emerged from the data analysis, namely, prejudice against homosexual patients by health care providers and other patients at the primary health care facilities, and, homophobic behaviour from primary health care personnel.\n  .\n  # Conclusion\n  .\n  Participants experienced prejudice and homophobic behaviour in the course of utilising PHC clinics in Umlazi, which created a barrier to their utilisation of health services located there. Nursing education institutions, in collaboration with the National Department of Health, should introduce homosexuality and anti-homophobia education programmes during the pre-service and in-service education period. Such programmes will help to familiarise health care providers with the health care needs of homosexual patients and may decrease homophobic attitudes.\nauthor: Nokulunga H. Cele; Maureen N. Sibiya; Dudu G. Sokhela**Correspondence to:** Maureen Sibiya, **Email:**<email@example.com>**Postal address:** PO Box 1334, Durban 4000, South Africa[^1]\ndate: 2015-09-28\nreferences:\ntitle: Experiences of homosexual patients' access to primary health care services in Umlazi, KwaZulu-Natal\n\n# Introduction\n\nAccess to health care is a fundamental principle of health care systems around the world and is a fundamental human right of all citizens irrespective of race, class, religion, gender or sexual orientation. Access to health care can be defined as the ability of an individual to identify their health needs and to seek health care services and obtain the relevant care for their health needs (Levesque, Harris & Russell 2013:1). The Constitution of the Republic of South Africa, Section 27(1)(a), states that all citizens have the right to access health care services (Republic of South Africa 1996:6\u20138). The priority for public health is to eliminate health disparities in all spheres together with adverse health outcomes by improving access and utilisation of health care services (Graves 2009:46). Despite the recognition and increased awareness of homosexuality and the health needs of homosexual patients, negative attitudes persist and affect the way in which they can access health care services (Hayman *et al.* 2013:121).\n\nA 2009 survey conducted amongst South African students in the Western Cape regarding their attitudes and beliefs towards homosexuality, found that 44% of them disapproved of the idea of homosexuality being acceptable in South Africa, and 41% believed that granting equal rights to homosexual individuals by the constitution was correct and acceptable (Mwaba 2009:803). The results of this survey indicate the general attitude some members of the community have towards homosexual patients and homosexuality. Homophobia, which is 'the irrational fear and hatred of people who are attracted to the same sex' (M\u00fcller 2013:2), is most threatening for lesbians. Many South African lesbians are living in fear of being 'found out' and becoming victims of discrimination and possible physical harm, including 'corrective rape' (Ochse 2011:3\u20134). Such sexual violence, driven by homophobia, places lesbians at high risk of HIV infection (M\u00fcller 2013:2).\n\nMen who have sex with men (MSM) reported having a fear of being judged by health care providers and getting treated badly as a result of disclosure (Rispel *et al.* 2011:47). Other studies (Kennedy *et al.* 2013:3; Rosenstreich, Comfort & Martin 2011:2) assert that homosexual patients experience stigmatisation when accessing health services. O' Byrne and Watts (2014:21) point out that many homosexual patients avoid doing an HIV test because of hesitance and fear of stigmatisation. Some people in society believe that homosexual patients have violated cultural gender norms, and that stigma also adds barriers to access health care settings (Rounds, McGrath & Walsh 2013:99).\n\nMany studies (for example Banwari *et al.* 2015; O' Byrne & Watts 2014; Ochse 2011; Rispel *et al.* 2011) reveal that lesbians (females) and gays (males), commonly known as homosexuals, experience challenges regarding access to health care services, and this has become a serious concern. The concern is that lesbians and gays might delay or avoid seeking health care when they need it because of past discrimination or perceived homophobia within the health care, thereby putting their health at risk (M\u00fcller 2013:2). For example, Bjorkman and Malterud (2009:239) found that lesbians were not following the sequence of screening programmes for females, such as pap smears. They emphasised that lesbians might suffer from health problems to a greater extent than heterosexuals or the general female population as a result of marginalisation.\n\nRispel *et al.* (2011:138) conducted a survey in South Africa regarding perceptions, preferences and utilisation of health care services by homosexual patients. They found that negative attitudes from health care providers discouraged them from disclosing their sexual orientation, often resulting in inadequate treatment and unmet health care needs.\n\nHomosexual patients are affected by a range of social and structural factors around their environment, and as a result have unique health needs that might not be met by existing health care services because of ignorance by health care providers but also fear of disclosure by homosexual patients (Mitra & Globerman 2014).\n\nIn their study, Rounds *et al.* (2013:99) found that homosexual patients experience verbal abuse as well as many other forms of discrimination and a general lack of satisfactory care, including from health care professionals refusing to provide them with treatment. Eaton *et al.* (2015) report on a study in which homosexual men state that the health services offered to them were substandard. Rispel *et al.* (2011:147\u2013149), in their study of South African homosexual men, found that some participants perceived the private health care sector to be more understanding than the public health sector.\n\n# Profile of the researcher\n\nThe principal investigator was employed as a Clinical Nurse Specialist in one of the PHC (primary health care) facilities at Umlazi township in KZN at the time of conducting this study. She noticed that very few homosexual patients visited the clinic and that the majority of them only reported to the clinic during late stages of their illnesses or when they had complications.\n\n# Problem statement\n\nThe rights of all South African citizens are enshrined in the Constitution of the Republic of South Africa which ensures non-discrimination by sexual orientation (Republic of South Africa 1996:13), yet it seems that homosexual patients are still discriminated against when seeking health care. They are not yet free in South African society as a result of the challenges that they daily encounter such as social stigma and homophobic attacks (Ochse 2011:4), and are often fearful and hesitant to access health care services owing to the possibility of discrimination by health care providers.\n\n# Aim of the study\n\nThe aim of the study was to explore and describe the experiences of homosexual patients utilising PHC services in Umlazi in KZN.\n\n# Research method design\n\n## Design\n\nA qualitative research study that was exploratory, descriptive and contextual in nature was conducted. A qualitative study has a subjective approach and is used to define life experiences and give them meaning (Grove, Burns & Gray 2013:23). The study provides an overview of the real life situation of homosexual patients, particularly lesbians, regarding access to health care services at the PHC level in Umlazi.\n\nThe exploratory design explores the dimensions of a phenomenon in order to better understand the context in which an intervention unfolds. The exploratory design sheds light on the various ways in which a phenomenon is manifested and on underlying processes (Polit & Beck 2012:640). The researcher used an exploratory design to better understand the accessibility of PHC services for homosexual patients.\n\nA descriptive research design provides an accurate account of the characteristics of a particular group in real-life situations for the purposes of describing what exists, determining the frequency with which something occurs, and categorising information (Grove *et al.* 2013:26). The study was contextual in nature as the validity of the findings is only claimed in a specific context. This design was used to obtain more information about characteristics of homosexual patients regarding their experiences of utilising primary health care services in Umlazi. The purpose of a descriptive study is to provide a picture of what is happening in real life situations (Grove *et al.* 2013:215).\n\n## Sampling and sampling technique\n\nPurposive sampling was used to select the study participants. In qualitative research, purposive sampling is used to recruit participants for whom the research topic is relevant and the goal is to gather information about their experiences (Knudsen *et al.* 2012:85). Purposive sampling was used because it enabled the researcher to select informative cases that could provide rich and substantial information based on the personal experiences of homosexual patients regarding accessing and using PHC services in the Umlazi area. Inclusion criteria were 18 years of age or older, self-identified as either lesbian or gay, experience of PHC services in Umlazi within the past year, and able to conduct a conversation in either English or isiZulu.\n\nPurposive sampling was modified by time-space probability based sampling. This technique is used when there is a relatively small number of people who usually gather at a specific place and time and who experience stigma and discrimination. These samples are often called hard-to-reach populations and include homosexual patients (Berry *et al.* 2013:39; Semaan 2010:61). Potential participants were approached at a gay club with the permission of the club owners. The recruitment flyers were posted at the clubhouse one week before the researcher began data collection.\n\nAccording to Polit and Beck (2012:521), qualitative research has no direct fixed sample size; it is driven by the information needs of the study. The sample size was guided by the principle of data saturation. This point is reached when no new information is obtained from participants during the later stages of data collection, when subsequent interviews produce repetitions of information, indicating the completeness of the data gathered. In this study, data saturation was reached after interviewing 10 participants, but 2 more were interviewed to confirm that no new information could be elicited.\n\n## Data collection\n\nData collection took place from September to November 2014. The researcher approached the club-goers whom she did not know personally and introduced herself and screened them based on the inclusion criteria. If the participant met the study's inclusion criteria, the researcher explained the purpose and the importance of the study. Participants were interviewed in a quiet locked room in the club to allow for privacy and minimise disruptions during interviews. Potential participants were given an information letter to read and were encouraged to ask questions to clarify matters. Consent forms were signed by those who agreed to participate. Semi-structured interviews were conducted using an interview guide. The following four questions were asked:\n\n-   Describe what made you visit the PHC clinic during the past year.\n\n-   Describe your experience at the PHC clinic starting from the time you entered the clinic, enquiries, waiting for help (in the queue), the service point (the health provider's room), waiting for medicine and receiving the medicine, leaving the clinic.\n\n-   How were you treated? Was that the way you would like to be treated? If yes, why? If not, why?\n\n-   Describe how accessible PHC services are to you.\n\nAll interviews were conducted by the researcher in English or isiZulu according to the language preference of the participant and lasted 30 to 45 minutes. All interviews were recorded using a digital recorder supplemented by field notes.\n\n## Pre-testing of the interview guide\n\nThe interview guide was pre-tested to assess whether the research questions were realistic and understood by the pre-test participants. Pre-test participants are patients who meet the inclusion criteria but their data are not included in the actual study (Brink, Van der Walt & Van Rensburg 2012:174). The interview questions were pre-tested on two consenting participants at the clinic where the researcher worked. The data collected were not included in the final data analysis. No amendments were made to the interview questions.\n\n## Data analysis\n\nAccording to Polit and Beck (2012:556), data analysis involves organising the data that the researcher has collected, providing structure and eliciting meaning from it. Data were analysed using content analysis. Content analysis is used to analyse and interpret data by classifying the words in the text into categories in order to give it meaning. Repeated ideas or patterns of thought are grouped by organising data into categories and concepts that the researcher has developed (Grove *et al.* 2013:281).\n\n## Ethical considerations\n\nAll consenting participants were given written and verbal information about the process of the study. They signed consent forms after reading and understanding the information letter. Privacy was maintained by not disclosing participants' information to any third party. Justice was maintained because the participants were informed that they could decline participation at any stage of the research, or withdraw during the interview, and could do so without incurring any negative consequences. The right to confidentiality was ensured throughout the research process; names would be known only to the researcher and codes were used to identify data associated with them. The master list of participants and their codes was kept under lock and key. No family member, health worker or any other person had \u2013 or will have \u2013 access to the raw data to prevent breach of confidentiality. The Durban University of Technology Institutional Research Ethics Committee provided ethical clearance for the research proposal (IREC number 61\/14).\n\n## Trustworthiness\n\nGuba's model of trustworthiness was applied to ensure the trustworthiness of this study in regard to credibility, dependability, confirmability and transferability (Brink *et al.* 2012:172). Credibility was maintained through using the same interview guide for all participants. The researcher visited the club over a period of three months, meeting with different participants. Member checks were conducted whereby the researcher returned to give feedback to participants and checked their reactions to themes emerging from the data. In order to ensure dependability the researcher developed an audio trail by keeping all the original audio records of interviews and discussions on a disc. To ensure confirmability, the researcher interpreted and analysed the data through identifying themes and subthemes which were supported by direct quotations from the participants in order to eliminate subjectivity and bias. A thorough description of the research report was provided to ensure transferability so that other researchers could evaluate and test the applicability of the data in other contexts.\n\n# Results\n\nTwo major themes emerged from the data analysis:\n\n**Theme 1:** Prejudice against homosexual patients by health care providers and other patients at PHC facilities.\n\n**Theme 2:** Homophobic behaviour from PHC personnel.\n\nSeveral sub-themes emerged from the interview in line with the two major themes. The themes and sub-themes are presented in <a href=\"#T0001\" data-ref-type=\"table\">Table 1<\/a>.\n\nThemes and sub-themes.\n\n<table>\n<thead>\n<tr>\n<th style=\"text-align: left;\">Themes<\/th>\n<th style=\"text-align: left;\">Sub-themes<\/th>\n<\/tr>\n<\/thead>\n<tbody>\n<tr>\n<td style=\"text-align: left;\">Prejudice against homosexual patients by health care providers and other patients at primary health care facilities.<\/td>\n<td style=\"text-align: left;\"><ul>\n<li><p>Stigma from health care providers.<\/p><\/li>\n<li><p>Rejection by other patients.<\/p><\/li>\n<li><p>Homosexual patients' satisfaction and dissatisfaction with primary health care services.<\/p><\/li>\n<\/ul><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Homophobic behaviour from primary health care personnel.<\/td>\n<td style=\"text-align: left;\"><ul>\n<li><p>Lack of understanding of homosexuality.<\/p><\/li>\n<li><p>Attitudes of homosexual patients towards health care providers.<\/p><\/li>\n<li><p>Influence of religious and cultural beliefs of health care providers on their perceptions of homosexual patients.<\/p><\/li>\n<li><p>Heteronormativity amongst health care providers.<\/p><\/li>\n<li><p>Attitudes of health care providers towards homosexual patients.<\/p><\/li>\n<li><p>Inappropriate curiosity of health care providers regarding homosexuality.<\/p><\/li>\n<li><p>Personal involvement of health care providers with homosexual patients.<\/p><\/li>\n<\/ul><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n## Prejudice against homosexual patients by health care providers and other patients at primary health care facilities\n\nParticipants cited experiences of prejudice and stigmatisation as the major reasons for not receiving fair treatment from the health care providers after gaining entry to such services. Discrimination was noted by participants when they received treatment which was different from that of heterosexuals. Their perception of being stigmatised was when they were labelled on the basis of their appearance or dress code. This prejudice was experienced as coming from other patients as well as from health care providers. The sub-themes that emerged were stigma from health care providers, rejection by other patients, lack of equality for homosexual patients in PHC services, and their satisfaction and dissatisfaction with PHC services.\n\n# Stigma from health care providers\n\nParticipants reported that they felt stigmatised by health care providers. They reported that they were being judged because of their personality and because of the way they dressed, talked or walked. Their perceptions of being stigmatised are evident in the following quotations:\n\n> 'Are you here for family planning? The nurse asked me while she was holding and reading my card. As I was getting ready for the injection she continued to say 'you are also wearing men's underwear?' I was there for a different injection but a nurse kept on asking about family planning, saying lesbians do not do prevention \u2026' (P8)\n>\n> '\u2026 \\[*F*\\]rom the nurses, umm, you get lots of questions? In the consultation room, the first question that the nurse asked me was what type of a person I was. I ignored the question and told her that I was sick \u2026' (P1)\n>\n> '\u2026 \\[*A*\\]t the PHC facility people have their own prejudices and there is a lot of stigmatisation. If you are a homosexual you go there looking for help for your sickness but they always include things that are unnecessary. Despite telling the nurse that you are a male and gay, they always make you to explain yourself and emphasise that you are different. They repeatedly ask if you are male or female.' (P11)\n\n# Rejection by other patients\n\nParticipants reported that they felt rejected by other patients in the PHC facility's waiting area. Some patients in the waiting area made comments which scared participants, causing some of them to leave the PHC facility before they were attended to by the health care practitioner. Rejection by other patients is revealed in the following quotations:\n\n> '\u2026 \\[*W*\\]hen I went to the PHC facility, at the gate, I enquired from the security guard, an old man, who looked at me, clicked his tongue and swore at me. But I could not care less because I am used to things like that. When I got inside the PHC facility patients laughed at me because I was wearing tight pants and they were looking at my private parts, but still I did not care. I put my earphones on, ignored them and sat in the queue \u2026' (P7)\n>\n> 'When I was still sitting in the queue there were women behind me who were laughing, but I did not know what they were laughing at, but I noticed when the woman next to me also started laughing. When I listened carefully they were debating about homosexuals. Someone said it was a fashion. The other woman added; if I can see a homosexual here I can put a tyre around her neck and burn her. I could not stay long, I decided to go back home because these people made me uncomfortable and I felt that they could harm me. I decided that when I have money I will go to the private doctor.' (P2)\n>\n> '\u2026 I felt very disturbed and unhappy because people were sitting away from me because I am gay.' (P5)\n\n# Homosexual patients' satisfaction and dissatisfaction with primary health care services\n\nOnly one participant reported satisfaction with the treatment received from the health care provider who had good communication skills and treating all patients the same. This was expressed in the following excerpt:\n\n> '\u2026 I don't want to lie; I was so satisfied with the treatment that I got from the health care provider who was helping me. That nurse did not treat me differently from heterosexuals. She did not have that in her mind that I am a homosexual client.' (P6)\n\nThe majority of participants reported dissatisfaction with the treatment received from health care providers. This was mainly because of the experience of participants regarding health care providers' attitudes toward homosexual patients and of being treated differently to the way heterosexuals were being treated. This is expressed in the following quotations:\n\n> \u2026 \\[*I*\\]t is so difficult to use PHC facility. I was so dissatisfied with the treatment that I received and please do not ask how it ended. I just left the clinic without receiving any help.' (P4)\n>\n> '\u2026 I was uncomfortable, as a result I could not disclose my real problem because I had an anal problem, I decided to go to a private doctor.' (P7)\n>\n> 'Our clinics are not in a good standard they don't accommodate all of us as we having different sexual orientations \u2026' (P8)\n>\n> 'It was almost over in 15 minutes, a nurse busy asking me questions about being gay, and not helping me and I was no longer there I wanted help and go home but I felt very disappointed about everything.' (P5)\n\n# Homophobic behaviour from primary health care personnel\n\nParticipants reported that they experienced a range of homophobic behaviours from health care providers and in the waiting area from other patients. The following seven sub-themes emerged from this: lack of understanding of homosexual patients; attitudes of homosexual patients towards health care providers; influence of religious and cultural beliefs of health care providers on their perceptions of homosexual patients; heteronormativity amongst health care providers; attitudes of health care providers towards homosexual patients; inappropriate curiosity of health care providers regarding homosexuality; personal involvement of health care providers with homosexual patients.\n\n# Lack of understanding of homosexuality\n\nThe findings of the study revealed that health care providers and members of the public who were patients in the PHC lacked an understanding of homosexuality. This was evident from the questions they asked and the disbelief that often accompanied the answers. Participants were not believed nor taken seriously when they reported that they had been infected with sexually transmitted infections, as noted in the following excerpts:\n\n> '\u2026 \\[*W*\\]hen I got into the consultation room a nurse asked what I was suffering from and I told her that I had a problem in my private part and I think it is a sexually transmitted infection. She asked me how come I have a sexually transmitted infection while I am in a relationship with a female. The nurse asked me to explain how I have sexual intercourse. I told her that I could not explain something like that, and I asked her to help me with treatment as I wanted to leave the room.' (P9)\n>\n> '\u2026 \\[*T*\\]he nurse asked me about my HIV status as I was having some sort of sexually transmitted infection and I told her that I was HIV negative. She asked if I was using condoms and I told her that I was not, and I told her I did not even know homosexuals' condoms, I had never seen them before.' (P5).\n\n# Attitudes of homosexuals towards health care providers\n\nSome participants who went to the PHC facility had a positive attitude towards health care providers or health facilities because of previous positive experience. One participant expressed her experience of a positive attitude regarding the PHC provider by saying:\n\n> 'The nurse who attended to me did not treat me differently. I did not feel discriminated.' (P6)\n\nOther participants reported that their own attitudes were negative towards the PHC providers prior to them reaching the PHC facility, because of preconceived ideas about health care providers which might or might not have been factual. This caused problems for the participants as they became agitated without provocation from the health care providers. The participants who had experienced negative attitudes from health care providers had this to say:\n\n> '\u2026 \\[*W*\\]hat I don't like most with the clinic is the attitude of nurses towards homosexuals. I always see it \u2026' (P6)\n>\n> 'However, I was lazy to go there because of what my friend told me, the questions he went through. Before I got helped I was asked many questions too and criticised too much.' (P11)\n>\n> '\u2026 Umm \u2026 It is not easy to use the clinic; you end up getting hurt.' (P7)\n\n# Influence of religious and cultural beliefs of health care providers on their perceptions of homosexuals\n\nThe religious beliefs of health care providers were perceived as problematic by participants. The participants reported that health care providers tended to want to convert or impose their religious beliefs on them and expected them to behave and lead their lives according to those beliefs. An example of such a view is expressed in the following excerpt:\n\n> '\u2026 \\[*I*\\]t wasn't good news to a nurse to discover that I am a lesbian. The nurse started preaching saying she feels sorry for my mother, I need to go to church and pray because what I am doing is against God's will \u2026' (P12)\n\nSome patients believed that homosexuality could be 'corrected or healed' using cultural methods they were familiar with:\n\n> 'The conversation continued until a person next to me said, being homosexual is not right, because it is Satanism and it is kind of a fashion and someone said homosexuals need slaughtering of goats because this is against culture.' (P6)\n\n# Heteronormativity amongst health care providers\n\nWhen health care providers managed patients, they never seemed to give a thought to the fact that there could be homosexual patients seeking medical assistance. They tended to treat everyone as if they were heterosexual. Participants witnessed heteronormativity in the following instances:\n\n> '\u2026 I told the nurse that I wanted to do an HIV test. The first question was; why do you want to do HIV test? I told her that I want to know my status; she then asked if I use condoms. Hey! I was taken aback for a moment, seriously I was confused by the question \u2026 I answered no, now a nurse gave me health education on using condoms and asked if am not aware of the importance of using condoms, I told her I knew. She did not ask me about the type of partner I have or the reason for not using condoms. Later on, she asked me if my partner has tested, and I told her that she's outside. She then asked me to call him so that she will give us both health education about the use of condoms. When my partner got inside the nurse just changed her facial expression \\[*frowning*\\] she was surprised and looked very shocked to see that it was a female. And she said: 'this is the reason you are not using condoms', I said 'yes'.' (P3)\n>\n> '\u2026 \\[*T*\\]he nurse asked the usual questions as to a female, she asked about pregnancy, my last menstrual periods, and use of condoms? When I said no to using condoms, she immediately educated me on the importance of using them. I explained my sexuality to her and she was taken aback and was speechless for some time. It wasn't good news to a nurse to discover that I'm a lesbian. Nurse started preaching me saying she feel ashamed for my mother \u2026' (P12)\n\n# Attitudes of health care providers towards homosexuals\n\nParticipants reported negative attitudes from health care providers. They verbalised moments where their sexual orientation seemed to upset the health care providers. The negative attitudes of health care providers towards homosexual patients are portrayed in the following excerpts:\n\n> '\u2026 \\[*W*\\]hen I entered to a consultation room the first thing a nurse said to me was 'what's going on here?', and I looked back because I thought maybe there was something happening behind me, and a nurse said no I am asking you 'what type of a person are you really?' (P8)\n>\n> '\u2026 \\[*W*\\]hen I got into the consultation room I explained to a nurse that I needed contraceptives, a doctor had referred me because of my abnormal menstrual periods, a nurse refused to give me contraceptives, no matter how hard I tried to explain that I am not sleeping with males I need it for abnormal periods. A nurse said I am a lesbian and I don't need contraceptives and she did not give them to me.' (P10)\n\n# Inappropriate curiosity of health care providers regarding homosexuality\n\nThe majority of participants reported that they had been asked about how they engaged in sexual intercourse by health care providers. The participants perceived this to be inappropriate curiosity on the part of the health care providers, and they felt that this question was irrelevant to the history taking or the diagnosis and treatment as noted in the following excerpts:\n\n> '\u2026 \\[*I*\\]f things were done my way, I wish lesbians and gays had a dedicated clinic in order not to get tortured by nurses with lots of questions, that are private like asking how do we engage in sexual intercourse with the person of the same sex?' (P12)\n>\n> '\u2026 \\[*T*\\]he question that followed was; how do you engage in sexual intercourse as you're both females?' (P8)\n>\n> '\u2026 \\[*T*\\]he nurse asked me to explain how I have sexual intercourse. I told her that I could not explain something like that, and I asked her to please help me with treatment as I wanted to leave the room.' (P9)\n\n# Personal involvement of health care providers with homosexuals\n\nSometimes health care providers were somewhat personally affected by homosexuality either having a homosexual child or the health care provider admitting to having feelings for homosexual patients. Some participants felt offended whilst others felt that this helped the health care provider understand homosexuality better. An example of a participant who felt offended is provided below:\n\n> '\u2026 \\[*W*\\]hen I got inside the consulting room the male nurse asked if he should write female or male, I told him that I am a gay male, I am not a female. After that he told me that he was married but wished to have relations with a gay person like me. I became so angry because I thought that he was provoking me. He then told me that we will book a place where we can sleep. I was so annoyed and uncomfortable as he was continuing with the conversation \u2026' (P7)\n\n# Discussion\n\n## Prejudice against homosexual patients by health care providers and other patients at primary health care facilities\n\nParticipants identified prejudice against them as an obstacle to their utilisation of health care services. This is consistent with findings of other studies (for example Kennedy *et al.* 2013; M\u00fcller 2013; Rounds *et al.* 2013). Rosenstreich *et al.* (2011:302) state that throughout the world health care providers and communities still discriminate against and marginalise homosexual patients. Some health care providers lack the tolerance required to work with them because of their personal affiliations which interfere with their professionalism (R\u00f6ndahl 2009:146). Many health care providers found it difficult to render care to them as they cannot tolerate their sexuality and end up failing to give quality nursing care equal to that given to heterosexuals (R\u00f6ndahl 2009:147). Participants in this study reported that they were not treated in the same way as heterosexuals. Only one participant reported the experience of being treated fairly. Two studies report that patients verbalised satisfaction with health care services when their health needs were met (McNair, Szelacha & Hughes 2011; Tjepkema 2008). These authors further assert that satisfaction is reported to be higher when health care providers show acceptance and respect towards patients' homosexuality and display positive attitudes towards them. Rounds *et al.* (2013:99) reported that homosexual patients received substandard care from health care providers.\n\nParticipants verbalised that some members of the public who were patients showed signs of prejudice by rejecting homosexual patients in the waiting areas at the clinics where all patients queue whilst waiting for assistance from the health care providers. Some reported leaving the PHC facilities before consultation with the health care providers because of this. According to Ara\u00fajo *et al.* (2009:667), homosexual patients usually experience situations of prejudice in the community. The burden of stigma might be more severe for patients who look like homosexual patients compared to those who are not easily identifiable. These authors further state that homosexual patients might be humiliated and mocked by some members of society even in the health care settings because of their homosexuality.\n\n## Homophobic behaviour from primary health care personnel\n\nThe participants in the current study reported experiencing homophobic behaviour from PHC personnel during their utilisation of health care services. According to Akhan and Barlas (2013:435) homophobia lies behind the judgements, negative attitudes and unfair treatment of homosexual patients in health care facilities. Berry *et al.* (2013:40) state that members of the public are generally not educated about, and lack an understanding, of homosexual people and once they discover that someone is homosexual, they change their attitudes and start looking down upon them.\n\nParticipants reported that there were moments where the health care providers preached to them instead of providing the services that they required. R\u00f6ndahl (2009:149) argues that the religious health care providers can be problematic in the nursing context, especially when nursing homosexual patients. They became more concerned about influencing homosexual patients to become heterosexuals instead of treating patients holistically. This attitude can also be present in married health care providers who associate homosexuality as being against God's will for what a family should be and therefore it needs to be changed (Akhan & Barlas 2013:435). Some participants in this study experienced criticism from members of the public who were also patients in the PHC, and who believed that homosexuality was against the cultural background of African people. According to Akhan and Barlas (2013:435), these homophobic attitudes are influenced by the social arrangements and the patriarchal structures of countries. An example of patriarchal heteronormative behaviour was experienced by participants in the current study when it was assumed that they were heterosexual because health care providers did not ask about their sexual behaviour. Lesbians are becoming more visible in society but health care providers still tend to treat all females as if they are heterosexuals (Hayman *et al.* 2013:121).\n\nOne participant in the current study verbalised that she had a positive meeting with the health care provider who showed good communication skills and acknowledgement of homosexuality. According to R\u00f6ndahl (2009:146), the insecurities of homosexual patients sometimes affect their interaction with health care providers as not all health care providers display negative attitudes towards them.\n\nIn this study some health care providers indicated inappropriate curiosity when they constantly enquired about how homosexuals engage in sexual relations. Hayman *et al.* (2013:120) report cases of health care providers asking about how conception takes place, laughing and joking about pregnancies amongst homosexual women. However, health care providers sometimes ask homosexual patients about their sexuality and sexual activities in order to understand their personal issues. Akhan and Barlas (2013:435) suggest that when health care providers meet homosexual patients they should seize the opportunity to educate themselves and their families about their lifestyle.\n\n# Limitations of the study\n\nResearching homosexual patients in South Africa is challenging because they are hard to access as many have not 'come out' about their homosexuality, especially homosexual males; hence, only those who visited a particular gay club in Umlazi were interviewed. The research was conducted on those residing in Umlazi only. The experiences of health care providers were not studied, only that of homosexual patients. Participants were not asked about their experiences of accessing and utilising private health care services, as this aspect fell beyond the scope of the current study.\n\n# Recommendations\n\nThe following recommendations emerged:\n\n-   Nursing education institutions, in collaboration with the National Department of Health, should introduce homosexuality and anti-homophobia education programmes during the pre-service and in-service education period. Such programmes will help to familiarise health care providers with the health care needs of homosexual patients and may decrease homophobic attitudes.\n\n-   Guidelines on history taking from homosexual patients should be compiled. This will improve their ability to offer relevant health information and management.\n\n-   Health care providers should not show patients that they are surprised about their sexual orientation. They need to set a relaxed tone and be aware of their negative body language.\n\n-   Campaigns to educate the public about homosexuality could create a more conducive environment for homosexual patients so that they can access health facilities without fear of prejudice and discrimination by members of the public who are also patients at the PHCs. Such education sessions could be provided in PHC clinic waiting areas and during HIV and\/or AIDS health education sessions.\n\n-   The possibility should be investigated of offering PHC services at gay clubs, for instance one evening per week, by health care providers who are willing to treat homosexual patients with respect and without prejudice.\n\n-   Future studies should endeavour to identify the attitudes of health care providers working at PHC clinics towards homosexual patients, as well as their knowledge of and perceptions of them.\n\n# Conclusion\n\nThe findings indicate that some participants encountered challenges to accessing and using PHC services in the Umlazi area. The major patient-related challenge was that some participants feared discrimination at the PHC clinics and consequently they postponed or abandoned their PHC clinic visits. The major service-related challenges involved the non-professional attitudes of some health care providers who discriminated against some participants' sexual orientations, asked inappropriate questions and failed to provide adequate services. Some of the other patients in the waiting areas also showed disrespect towards participants causing some of them to fear for their safety to the extent that they left the clinic without consulting any health care professional.\n\nHealth care providers should receive pre-service and in-service education about the necessity of treating homosexual patients professionally and respectfully. Members of the public should also be educated about homosexuality and about avoiding discriminating against homosexual patients. Such education sessions could be provided in PHC clinic waiting areas, at women's and men's gatherings and during HIV and\/or AIDS health education sessions. Implementation of some of these recommendations could help to facilitate homosexual patients access to and utilisation of PHC services in the Umlazi area of the KwaZulu-Natal province.\n\n## Acknowledgements\n\n### Competing interests\n\nAuthors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article.\n\n### Authors' contributions\n\nN.H.C. was the Master's student and principal investigator, M.N.S. the primary supervisor providing guidance to the student in writing the manuscript. D.G.S. was the co-supervisor for N.H.C.\n\n# References\n\n[^1]: **How to cite this article:** Cele, N.H., Sibiya, M.N. & Sokhela, D.G., 2015, 'Experiences of homosexual patients' access to primary health care services in Umlazi, KwaZulu-Natal', *Curationis* 38(2), Art. \\#1522, 9 pages. <http:\/\/dx.doi.org\/10.4102\/curationis.v38i2.1522>","meta":{"dup_signals":{"dup_doc_count":105,"dup_dump_count":30,"dup_details":{"curated_sources":2,"2019-09":1,"2018-51":1,"2018-43":1,"2018-34":1,"2018-26":2,"2018-22":4,"2018-17":2,"2018-13":3,"2018-09":3,"2018-05":1,"2017-51":2,"2017-47":3,"2017-43":3,"2017-39":3,"2017-34":3,"2017-30":4,"2017-26":3,"2017-22":5,"2017-17":2,"2017-09":5,"2017-04":6,"2016-50":5,"2016-44":5,"2016-40":6,"2016-36":7,"2016-30":3,"2016-26":6,"2016-22":6,"2019-26":1,"2017-13":6}},"file":"PMC6092696"},"subset":"pubmed_central"}
{"text":"abstract: A report on the joint Cold Spring Harbor Laboratory\/Wellcome Trust Conference 'Genome Informatics', 15-19 September 2010, Hinxton, Cambridge, UK.\nauthor: Zemin Ning; Stephen B Montgomery\ndate: 2010\ninstitute: 1Sequencing Informatics, The Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK; 2Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva 1211, Switzerland\ntitle: Out of the sequencer and into the wiki as we face new challenges in genome informatics\n\n# Meeting report\n\nNext generation sequencing (NGS) analysis, open-source software, cloud computing and wiki-style genomics were among the hot topics and discussions at the recent Genome Informatics meeting at the Wellcome Trust Genome Campus, Cambridge, UK. Here we summarize some highlights of the meeting.\n\n# Accuracy of polymorphism detection\n\nComparison of related genomes can generate a wealth of knowledge about genome evolution and function. Recent advances in NGS technologies have greatly increased the scale and scope with which we can interrogate novel genomes and uncover genetic variation. However, for variation detection and statistical analysis, there are false positive errors for various reasons, notably incompleteness of reference genomes, read mapping errors or limitations, and sequencing-induced features. Benjamin Dickins (Penn State University, University Park, USA) discussed an approach to estimate polymorphism accuracy from NGS data by deeply sequencing a small plasmid genome and comparing it with Sanger sequencing.\n\nElliott Margulies (National Human Genome Research Institute, Bethesda, USA) gave an enticing presentation on this topic entitled 'Analysis of identical twins' genomes reveals sources of false-positive variation detection'. With 55\u00d7 and 50\u00d7 depth of read coverage from each twin's sample, they initially identified 83,538 discordant genotype calls across 97.6% of the human reference genome. Through inspection of a random set of discordantly genotyped positions, he revealed that a majority occurred in regions with poorly aligned reads. Margulies noted that he would be highly suspicious of genotype calls in regions with high coverage but with low mapping scores. When these events were filtered out, the number dropped to 13,140, a reduction of 84%. By then further introducing other filtering mechanisms, such as incorrect alignments of short reads across indels, Q20 (99% confidence) evidence in the other twin and 10% allele frequency, Margulies' final number of discordant genotype calls was only in the range of 500 to 1,000.\n\nMargulies' suspicion was certainly shared by Richard Durbin (Wellcome Trust Sanger Institute, Cambridge, UK), who told the audience, \"If someone tells me that his accuracy on variant detection is 99%, I should be cautious\". Durbin described that in the pilot phase of the 1000 Genomes Project, the sequencing strategy with low coverage (2\u00d7 to 4\u00d7) worked well, and it can efficiently find shared sequence variation with good accuracy. The consortium has now started sequencing 2,500 people in five different ethnic groups. Furthermore, Durbin highlighted the recently launched UK 10 K project, which, in collaboration with clinical investigators, will involve sequencing 4,000 cohort samples with rich phenotypes. With massive sample collections under way, further advances in informatics pipelines to reduce errors in variation detection will be indispensable.\n\n# Genome assembly and read alignment\n\nNGS data provide challenging but exciting prospects for *de novo* assembly. There were several presentations that focused on development and evaluation of genome assemblers using such data. David Jaffe (Broad Institute, Cambridge, USA) outlined a practical and general laboratory\/computational method for generating high-quality *de novo* genome assemblies. Three types of data were produced with the Illumina platform: 180 bp short-insert reads; 3,000 bp mate-pair reads; and 40,000 bp fosmid ends. The short-insert data were mainly intended to ensure contig base accuracy, whereas the longer jumping fragments provide potential for long-range connectivity. Jaffe generated data for mouse C57BL\/6 and human Yoruban NA18507 and then compared the short-read assembler, ALLPATHS, with the reference assembly that used the capillary long-read assembler, Arachne. From the comparison of the results, the quality of short-read assemblies was found to be close to that of capillary assemblies, in terms of both base accuracy and contig connectivity.\n\nTranscriptome data also offer great value and unique challenges in helping to identify and assemble gene sets for multiple species. Daniel Zerbino (University of California, Santa Cruz, USA) presented a new methodology, Oasis, which is built in with the popular short-read assembler Velvet. This new program takes in a preliminary assembly produced by Velvet and exploits the read sequence and pairing information to produce transcript isoforms. When possible, it also detects and reports standard alternative splicing events. It is specifically designed to get around the issues of unequal expression levels and alternative splicing breakpoints.\n\nShort-read alignment saw further new developments at the meeting. New tools include: SMALT by Hannes Ponstingl (Wellcome Trust Sanger Institute, Cambridge, UK); NextGenMap by Fritz Sedlazeck (Max F Perutz Laboratories, Vienna, Austria); and YASRAT by Masahiro Kasahara (The University of Tokyo, Kashiwa, Japan). One distinguishing feature in these new alignment tools is that they all try to address the cases in which there is a higher level (over 2%) of base-pair errors in the raw reads.\n\n# Databases and data visualization\n\nAs sequence availability has increased, data access, representation, analysis and visualizations pose significant challenges in the community. Enis Afgan (Emory University, Atlanta, USA) has developed a solution that allows experimentalists to perform large-scale analysis using cloud-computing resources (<http:\/\/usegalaxy.org\/cloud>). Because the solution is built on the open-source Galaxy framework, analyses using this service are accessible, transparent and reproducible. Furthermore, popular tools and workflows for sequence analysis from various types of experiment are already built in and ready to run. Marc Fiume (University of Toronto, Canada) presented the Sequence Annotation Visualization and ANalysis Tool (Savant), a fast and interactive genome browser that can display sequence, read alignment, single nucleotide polymorphism and other genomic datasets stored in standard file formats. Harminder Sehra (European Bioinformatics Institute, Hinxton, UK) described how the well established UniProt Knowledgebase (UniProKB) has developed from manual to automatic annotation. Marcin Piechota (Institute of Pharmacology, Krakow, Poland) described <http:\/\/genes2mind.org>, an online resource for the genomic profiling of psychoactive drugs. The database contains comparisons of the effects of various classes of psychoactive drugs on transcriptional alternations of about 20,000 genes in the mouse brain. And Maximillian Hauessler (University of Manchester, UK) presented text2genome, a method for annotating the genome using sequences that are reported in scientific publications.\n\nAlthough NGS has required the development and improvement of many novel visualization tools, Martin Krzywinski (British Columbia Cancer Agency, Vancouver, Canada), the author of the Circos tool, took us back to basics by redefining network diagrams in his compelling talk in which he introduced a technique for turning network diagrams from incomprehensible 'hairballs' into information-rich schematics.\n\n# New insights from sequencing repetitive elements\n\nRepetitive element sequencing is revealing some interesting new biology. Karen Hayden (Duke University, Durham, USA) demonstrated unique patterns of repeats in human centromeres, which could facilitate completion and understanding of specific biology in these hard-to-assemble regions. Kateryna Makova (Penn State University) highlighted the lifecycle of microsatellites and their potential functional impact near genes. And Mark Batzer (Louisiana State University, Baton Rouge, USA) showed from 37 orangutans clear differences in retrotransposon polymorphism, which could support the separation of the Sumatran from the Bornean orangutan.\n\nRepeats are not always a good thing. There were also some presentations of genome analysis on large plant genomes. The highly repetitive wheat genome, with a size of 17 GB, poses significant challenges using the current sequencing platforms, in physical mapping as well as *de novo* assembly. Frederic Choulet (Institut National de la Recherche Agronomique, Clermont-Ferrand, France) described the effort to sequence and analyze wheat chromosome 3B. Contiguous bacterial artificial chromosome pools were sequenced by combining Roche 454 and Illumina platform sequencing. Shiran Pasternak (Cold Spring Harbor Laboratory, Cold Spring Harbor, USA) described the current progress on assembling the wheat D-genome, and Rachel Brenchley (University of Liverpool, UK) highlighted the advantages of comparative genome analysis in distinguishing genic loci from the abundance of repetitive content in the wheat genome.\n\n# RNA-seq and epigenomics\n\nThere was one dedicated session on RNA sequencing (RNA-Seq) and its application to genome annotation, in which various issues were discussed. Specifically, identifying the underlying biases in the RNA-Seq presents a significant analytical challenge. Projects such as the RNA-Seq Genome Annotation Assessment Project (RGASP) are examples of successful collaborations that are pushing the development of algorithms that can infer accurate transcript quantification. RGASP, led by Jen Harrow (Wellcome Trust Sanger Institute, Cambridge, UK), and Felix Scheslinger (Cold Spring Harbor Laboratory) showed the utility of spike-ins in RNA-Seq experiments to achieve adequate normalization. Many talks also highlighted how RNA-Seq was used for novel interrogating of the impact of genetic variation, such as that of Emilie Graison (University of Lausanne, Switzerland) who demonstrated how RNA-Seq can be used to uncover interesting connections between copy number variants and transcript diversity.\n\nChromatin immunoprecipitation sequencing (ChIP-Seq) provides an extraordinary window into the dynamics of protein-DNA binding. Xin Feng (Stony Brook University, New York, USA) presented PeakRanger, a new algorithm to call peaks from ChIP-Seq data and explore complex peak structures in regions of interest. It works by identifying broad regions of enriched binding and integrates topological clues to detect narrow peaks.\n\n# Wikis can change the way we do science\n\nThe growth of sequencing data is far outpacing the rate of curation. Previously successful models are not meeting the demands of genome biology and, in many historical cases, the majority of annotations were from a small number of contributors. A view that was heard in the conference was that it is necessary to incorporate the community to a greater extent into the annotation process using tools such as wikis. Although lack of recognition and credit may prevent scientists from actively participating, this may be only part of the story. Reasons for not giving back to the community need to be investigated. In his keynote speech, Alex Bateman (Wellcome Trust Sanger Institute, Hinxton, UK) described new approaches to engage the community of RNA biologists to improve the annotation of the Rfam RNA database as well as more generally contribute to the annotation of RNA in Wikipedia. The journal *RNA Biology*, in collaboration with Rfam, has pioneered a new model of scientific publication where scientists are required to write a Wikipedia article to go alongside their manuscript paper describing new families of non-coding RNAs. At the same time, the Wikipedia article will also be under a full peer review process. Complementing this approach, Daniel Renfro (Texas A&M University, College Station, USA) presented community-focused annotation resources using the Mediawiki software. Currently this includes two model bacterial species: *Escherichia coli* (EcoliWiki) and *Bacillus subtilis* (SubtilisWiki).\n\nIn recent years, we have seen significant changes in the bioinformatics community: from massive data flow to complicated pipelines; from big computational farms to cloud computing; from a single genome or chromosome to many thousand genomes; from different sequencing platforms to various data types and error models. The themes reviewed here reflect the fact that the field of genome informatics is constantly under change, responding with new solutions, new algorithms and new technologies. The future presents us with a new challenge in how we transform the scale of data into collective knowledge, and it is likely that, as open-source software is accelerating bioinformatics and cloud computing is helping us to scale up, the growing online collaboration offers a chance to tackle this.\n\n## Acknowledgements\n\nSBM is funded by the Louis-Jeantet Foundation.","meta":{"dup_signals":{"dup_doc_count":115,"dup_dump_count":41,"dup_details":{"curated_sources":2,"2019-09":1,"2018-51":1,"2018-43":1,"2018-34":1,"2018-30":1,"2018-13":1,"2017-47":1,"2017-34":1,"2017-26":1,"2017-17":2,"2017-09":7,"2017-04":1,"2016-50":1,"2016-44":1,"2016-40":1,"2016-36":10,"2016-30":6,"2016-22":1,"2016-18":1,"2015-48":3,"2015-40":1,"2015-35":4,"2015-32":3,"2015-27":2,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":4,"2014-42":10,"2014-41":4,"2014-35":3,"2014-23":5,"2014-15":4,"2019-22":1,"2015-18":3,"2015-11":3,"2015-06":3,"2014-10":3,"2013-48":3,"2013-20":4,"2024-18":1}},"file":"PMC3218654"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  An imbalance between pro-oxidants and antioxidant systems has been suggested to be implicated in the physiopathology of acute myocardial infarction (AMI). We aimed to evaluate the antioxidant capacity in Tunisian patients and to assess the possible relationship between erythrocyte catalase enzyme activity and hyperhomocysteinaemia.\n  .\n  # Methods\n  .\n  108 patients with AMI and 81 healthy subjects were enrolled in this study. Catalase erythrocyte enzyme activity was determined spectrophotometrically whereas \"total antioxidant status\" (TAS) concentration was measured by a commercially available method. Serum total homocysteine (tHcy) level was determined by a fluorescence polarization immunoassay (FPIA). Lipid peroxidation was measured with a fluorimetric method as \"thiobarbituric acid reactive substances\" (TBARS).\n  .\n  # Results\n  .\n  Compared with healthy subjects, patients with AMI had significantly lower catalase activity (*P\\<0.001*), TAS concentrations (*P\\<0.001*), and significantly higher serum tHcy (*P\\<0.001*) and TBARS levels (*P\\<0.001*). Erythrocyte catalase enzyme activity was negatively correlated with serum tHcy and TBARS while serum tHcy and TBARS were in positive correlation. Furthermore, the unbalance between pro-oxidants and antioxidants seems to be more aggravated in patients with Q wave AMI compared to patients with non-Q wave AMI.\n  .\n  # Conclusion\n  .\n  Our results suggest the involvement of hyperhomocysteinaemia in the drop of erythrocyte catalase activity related to myocardial ischemia reperfusion. Hyperhomocysteinaemia may increase the myocardial wall dysfunction under ischemia reperfusion by excessive production of reactive oxygen species which is made evident by increased lipid peroxidation.\n  .\n  # Virtual slides\n  .\n  The virtual slide(s) for this article can be found here: <http:\/\/www.diagnosticpathology.diagnomx.eu\/vs\/1623509866881834>\nauthor: Yosri Noichri; Abdelkader Chalghoum; Latifa Chkioua; Bruno Baudin; Samia Ernez; Salima Ferchichi; Abdelh\u00e9di Miled\ndate: 2013\ninstitute: 1Biochemistry Laboratory CHU Farhat HACHED, Street Doctor Moreau, 4000 Sousse, Tunisia; 2Department of Biochemistry, Hospital Saint-Antoine, 184 Street Faubourg Saint-Antoine, 75571 Paris Cedex 12, France; 3Department of Cardiology, CHU Farhat Hached, Street Doctor Moreau, 4000 Sousse, Tunisia\nreferences:\ntitle: Low erythrocyte catalase enzyme activity is correlated with high serum total homocysteine levels in tunisian patients with acute myocardial infarction\n\n# Background\n\nMyocardial infarction is a major cause of morbidity and mortality worldwide. Recently, an increase in the incidence of coronary heart disease (CHD) has been recorded in Tunisian cardiovascular disease register. Among diabetes, hypertension, abdominal obesity and smoking, a family lipoprotein disorder such as a high level of serum apolipoprotein B (Apo B) or\/and a lower level of serum apolipoprotein A-1 accounted for almost all the population attributable risk of AMI \\[1,2\\]. Similarly, hyperhomocysteinaemia related to nutritional or genetic factors, such as methylenetetrahydrofolate reductase, endothelial nitric oxide synthase genes or with low paraoxonase activity led to increased risk of CHD severity. It can induce sustained injury of arterial endothelial cells and proliferation of arterial smooth muscle cells \\[3\\].\n\nAtherosclerotic plaques are the major cause of Myocardial infarction. Other unusual causes of MI can be related to myocardial necrosis, or Thiamine deficiency causing alterations in heart metabolism \\[4,5\\]. Coronary artery occlusion can result in a reduction in myocardial blood flow \\[6\\]. In fact, myocardial ischemia occurs when myocardial oxygen demand exceeds the oxygen supply. Reperfusion of the ischemic myocardium can restore, afterwards, the blood flow but sudden massive increase in oxygen supply can lead to additional myocardial cell dysfunction and cell necrosis. Excessive production of Reactive Oxygen Species (ROS) has been most importantly proposed to mediate ischemia reperfusion injury. These species are toxic and may cause potential biological damage to all cellular components \\[7\\]. This occurs when there is an overproduction of ROS on one side and a deficit or inadequate availability of antioxidant systems on the other \\[6\\]. ROS generation may induce irreversible myocardial cell necrosis or apoptosis through triggering DNA fragmentation and caspase activation and causing myocardial injury \\[8,9\\]. Exposure to ROS from a variety of sources led to develop a series of defense mechanisms to neutralize these species and so protect cells against their toxic effects. This is achieved mainly by enzymatic antioxidants such as catalase, or by non-enzymatic antioxidants such as vitamins. Catalase is believed to play a major role in the first line of enzymatic antioxidant defense. It is a tetrameric enzyme, consisting of four identical subunits that contain a single ferriprotoporphoryn group per subunit. Catalase reacts efficiently in peroxisomes with hydrogen peroxide (H202) to form water and molecular oxygen \\[10,11\\]. Hyperhomocysteinaemia and other cardiovascular risk factors have been suggested to be implicated in the imbalance aggravation between pro-oxidants and antioxidants linked to its pro-oxidant properties or in the impairment of antioxidant systems. The aim of our study is to evaluate the total antioxidant capacity and erythrocyte catalase activity in patients hospitalized for AMI. We aimed to access the possible relationships between erythrocyte catalase activity, hyperhomocysteinaemia and the severity of myocardial dysfunction under ischemia reperfusion.\n\n# Materials and methods\n\n## Study population\n\nThe study consisted of 108 patients with a mean age of 63 *\u00b1* 12 years admitted to the department of cardiology in Farhad Hached hospital in Tunisia. The diagnostic of AMI was established according to universal clinical criteria: chest pain which lasted for up 3 hours, ECG changes and serum troponin elevation. The control group consisted of 81 healthy volunteers with no history of coronary artery disease, diabetes, hypertension or inflammatory disease. Their mean of age was 59 *\u00b1* 9 years.\n\nThe electrocardiographic changes were recorded and assessed by 2 cardiologists who were unaware that the patients are included in our study.\n\nCoronary risk factors have been referred in our study population according to universal definitions. Dyslipidemia was defined when total cholesterol concentration was \u2265 5,68 mmol\/l, or triglyceride concentration \u2265 2,28 mmol\/l. Hyperhomocysteinaemia was defined by a raised blood total homocysteine, exceeding 15 \u03bcmol\/l.\n\nInformed written consent was obtained from each patient and healthy subject according to the guidelines of our ethics' committee.\n\n## Blood collection and biochemical methods\n\nVenous blood was collected after overnight fasting and within 24 hours after admission for chest pain. Serum was separated by centrifugation at 1500 g for 10 min, and then stored at \u221280\u00b0C until the day of analysis. Serum was used for the estimation of lipid peroxidation, total homocysteine (tHcy), apolipoproteins (Apo A-1 and Apo B) levels and total antioxidant status (TAS) concentration.\n\nTotal homocysteine in serum was measured by the \"Abbott homocysteine assay\", a fully automated fluorescence polarization immunoassay (FPIA) from Abbott diagnostics. Serum apolipoproteins (Apo A-1 and Apo B) were measured by immunonephelometry (Dade Behring, Marburg, Germany). Plasma TAS was measured with commercial kit (Randox, Antrim, UK) according to the method of Miller et al. \\[12\\]. It can evaluate the total capacity of all antioxidants found in serum to neutralize the oxidative action of free radicals. Lipid peroxidation level was estimated by measurement of thiobarbituric acid reactive substances (TBARS) in serum according to the fluorimetric method of Yagi \\[13\\]. The pink chromogen produced by the reaction of thiobarbituric acid with lipid peroxidation products such as malondialdehyde (MDA) was estimated using 1,1,3,3-tetraethoxypropane as standard MDA. Serum TBARS levels were measured at 515 nm excitation and 553 nm emission.\n\nCatalase activity was determined in washed red cells prepared immediately after sampling from whole blood anticoagulant with heparin. Then, cell lysates were stored frozen. Catalase activity was measured according to the spectrophotometric method of Goth \\[14\\]. This assay is based on the ability of hydrogen peroxide to form a stable stained complex with ammonium molybdate measured at 405 nm.\n\n## Statistical analysis\n\nDatabase management and statistical analyses were carried out using SPSS (Statistical Package for the Sociological Sciences), version 17.0. Results are presented as means \u00b1 SD, or percentages. Means were compared using Student for independent samples. The relations between variables were assessed with Pearson's correlation analysis. The significance threshold was set at 5% (p\\<0.05).\n\n# Results\n\nClinical characteristics of patients with AMI and control groups are illustrated in Table\u00a0<a href=\"#T1\" data-ref-type=\"table\">1<\/a>. No difference was found between the 2 groups for the mean of age and sex. Among cardiovascular factors risk, diabetes, hypertension and dyslipidemia are greatly mentioned in patients compared with healthy subjects. Apolipoproteins disorder was observed in patients. There was a significant increase in serum Apo B level (*\\<0.001)* and a decreased serum Apo A-1 level (*\\<0.001)* compared with serum apolipoprotein levels in healthy subjects. In other hand, tHcy and TBARS, as a marker of lipid peroxidation, tended to be increased in patients whereas serum TAS concentrations and erythrocyte catalase activity were found lower than in controls.\n\nClinical and biochemical features of patients with acute myocardial infarction and controls\n\n| \u00a0 | ***Patients (n= 108)*** | ***Controls (n =81)*** | ***P*** |\n|:---|:--:|:--:|:--:|\n| *Age (years)* | 63 *\u00b1* 12 | 59 *\u00b1 9* | *NS* |\n| *Men (%)* | 70 | 38 | \u00a0 |\n| *BMI (kg\/m*^*2*^*)* | 27 *\u00b1 6* | *23 \u00b1 3* | *NS* |\n| *Diabetes mellitus (%)* | 61 | 0 | \u00a0 |\n| *Dyslipidemia (%)* | 45 | 0 | \u00a0 |\n| *Hypertension (%)* | 70 | 0 | \u00a0 |\n| *Smokers (%)* | 50 | 6 | \u00a0 |\n| *Alcohol (%)* | 25 | 0 | \u00a0 |\n| *Apo A1 (g\/L)* | 1.26 \u00b1 0.19 | 1.5 \u00b1 0.17 | *\\<0.001* |\n| *Apo B (g\/L)* | 1.29 \u00b1 0.36 | 0.92 \u00b1 0.22 | *\\<0.001* |\n| *TAS (mmol\/L)* | 1.47 \u00b1 0.38 | 1.71 \u00b1 0.22 | *\\<0.001* |\n| *Catalase (kU\/g Hb)* | 283 \u00b1 141 | 652 \u00b1 104 | *\\<0.001* |\n| *Homocysteine (\u03bcmol\/L)* | 26.9 \u00b1 15.47 | 14.75 \u00b1 2.69 | *\\<0.001* |\n| *TBARS (\u03bcmol\/L)* | 1.31 \u00b1 0.36 | 0.71 \u00b1 0.12 | *\\<0.001* |\n| *Troponine TnIc (\u03bcg\/L)* | *5,04* \u00b1 2,90 | 0 | \u00a0 |\n| \u2003\u2009Q wave AMI | 9,13 \u00b1 2,18 | \u00a0 | \u00a0 |\n| \u2003\u2009Non Q wave AMI | 4,09 \u00b1 2,03 | \u00a0 | \u00a0 |\n| *Treatment* | \u00a0 | \u00a0 | \u00a0 |\n| *Statins* (%) | 38 | 0 | \u00a0 |\n| *Beta-Blockers* (%) | 40 | 0 | \u00a0 |\n| *Ca-Blockers* (%) | 28 | 0 | \u00a0 |\n| *Diuretics* (%) | 37 | 0 | \u00a0 |\n\nValues are expressed as mean \u00b1 *SD* or percentage (%).\n\nNS: not significant, BMI: body mass index, TAS: total antioxidant status.\n\nWe showed in Table\u00a0<a href=\"#T2\" data-ref-type=\"table\">2<\/a> that erythrocyte catalase activity tended to decrease with smoking and\/or arterial hypertension as cardiovascular risk factors. Serum tHcy level was significantly higher in hypertensive patients compared to the normotensive patients.\n\nErythrocyte catalase activity and serum total homocysteine in patients with different cardiovascular risk factors\n\n| ***Risk factor*** | \u00a0 | ***Catalase (kU\/g Hb)*** | ***P*** | ***Homocysteine (\u03bcmol\/L)*** | ***P*** |\n|:---|:---|:---|:---|:---|:---|\n| *Sex* | Men | 294 \u00b1 129 | NS | 25.8 \u00b1 11.6 | NS |\n|  | *Women* | 271 \u00b1 152 |  | 28 \u00b1 18.4 |  |\n| *Diabetes* | *Yes* | 290 \u00b1 152 | NS | 26.3 \u00b1 15.9 | NS |\n|  | *No* | 268 \u00b1 121 |  | 27.8 \u00b1 14.9 |  |\n| *Dyslipidemia* | *Yes* | 282 \u00b1 151 | NS | 26.5 \u00b1 12.3 | NS |\n|  | *No* | 279 \u00b1 124 |  | 27.6 \u00b1 19.6 |  |\n| Obesity | *Yes* | 298 \u00b1 131 | NS | 24.7 \u00b1 13.2 | NS |\n|  | *No* | 268 \u00b1 150 |  | 29.1 \u00b1 17.2 |  |\n| *Hypertension* | *Yes* | *211* \u00b1 90 | \\< 0.001 | 30 \u00b1 17 | \\< 0.001 |\n|  | *No* | *412* \u00b1 95 |  | 20.3 \u00b1 8.2 |  |\n| *Smoking* | *Yes* | *224* \u00b1 105 | \\< 0.001 | 28.4 \u00b1 13.1 | NS |\n|  | *No* | *377* \u00b1 142 |  | 24.5 \u00b1 18.7 |  |\n| *Alcohol* | *Yes* | *275* \u00b1 148 | NS | 24.5 \u00b1 18.7 | NS |\n| \u00a0 | *No* | *387* \u00b1 135 | \u00a0 | 28.6 \u00b1 11 | \u00a0 |\n\nValues are expressed as mean \u00b1 SD.\n\nNS: not significant.\n\n## Erythrocyte catalase activity and serum tHcy level according to the severity of the myocardial dysfunction\n\nThirty one patients had a distinct increase of Q wave in the electrocardiogram established at the diagnostic. Patients with Q wave AMI had a significantly lower erythrocyte catalase activity, higher serum tHcy and TBARS levels compared to patients with non Q wave AMI (Figure\u00a0<a href=\"#F1\" data-ref-type=\"fig\">1<\/a>).\n\n## Results of Pearson correlation\n\nWe studied the Pearson correlation between serum tHcy, erythrocyte catalase activity and serum TBARS in patients. We found that erythrocyte catalase activity is negatively correlated with both serum tHcy and TBARS (r=\u22120.38, p\\<0.001; r=\u22120.41, p\\<0.001) (Figures\u00a0<a href=\"#F2\" data-ref-type=\"fig\">2<\/a> and <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>). Furthermore, serum tHcy was found positively correlated with serum TBARS level (r=0.4; p\\<0.001) (Figure\u00a0<a href=\"#F4\" data-ref-type=\"fig\">4<\/a>).\n\n# Discussion\n\nMyocardial infarction is a complex and multifactor disease in which the cellular and molecular mechanisms contributing to myocardial injury need to be more defined. In our data, investigation about oxidative stress parameters shows an increased serum TBARS level, as a lipid peroxidation marker, and a drop in the total antioxidant capacity in patients with AMI compared to healthy subjects. According to our observation, Pasupathi et al. have considered that the increased serum TBARS is a consequence to excessive ROS generation. The authors reported it to the raised xanthine oxidase activity under ischemia reperfusion \\[15\\]. Other enzymatic and cellular sources are suggested to be implicated in ROS generation. Some of these are related to the uncoupling of mitochondrial electron transport in consequence to the lack of oxygen supply under ischemia, or to the impairment of NO synthase activity \\[7\\].\n\nIn our study, we observed a decrease in erythrocyte catalase activity in patients compared to controls. Accumulation of hydrogen peroxide under ischemia reperfusion can also inhibit antioxidant enzymes (high substrate inhibition) and alter its enzymatic conformation. According to Senthil et al., the decreased activity of catalase in patients with CHD could be due to the inactivation of this enzyme by cross linking or to the impairment of NO synthase. Nitrite oxide (NO) can bind reversibly to ferric iron, inhibiting afterwards catalase activity \\[16,17\\]. Erythrocyte catalase activity tended to decrease in patients with hypertension and smoking as the major cardiovascular risk factors. Cigarette smoke is an abundant source of free radicals. It contains more than 10^15^ free radicals including superoxide anion and NO. Chemical oxidants in cigarette smoke can cause the oxidation of DNA encoding for antioxidant proteins \\[18\\]. On the other hand, essential Hypertension is subjected to increased oxidative stress. It may damage the endothelium and impair endothelium-dependent vascular relaxation. ROS can act on angiotensin converting enzyme to increase its catalytic activity resulting in the increase in angiotensin II production, which, in turn, is a major endogenous inducer of NADPH oxidase. Oxidative stress, endothelial dysfunction and inflammation represent a key triad for the development and progression of Coronary heart disease \\[19,20\\].\n\nThe imbalance between pro-oxidants and antioxidants occurred under ischemia reperfusion can be aggravated by hyperhomocysteinaemia. The moderate hyperhomocysteinaemia revealed in patients can be reported to a hereditary defect of any of the Hcy metabolic enzymes (cystathionine \u03b2-synthase, methylene tetrahydrofolate reductase) or to the depletion in folic acid and vitamin B6 or B12 \\[3\\]. Increased serum tHcy may cause endothelial dysfunction by promoting free radicals. Homocysteine is readily oxidized. Its auto-oxidation is catalyzed by transition metal ions, such as copper leading to homocystine, homocysteine mixed disulfides and homocysteine thiolactone formation \\[21,22\\]. Homocysteine reduces the transition metal ion (M^n+^) to generate a thioyl radical (Hcy\u00b0). It is thought to react with homocysteine to generate a free radical intermediate that reduces oxygen to superoxide anion (O~2~\u00b0) and then to peroxide hydrogen generation \\[3,23\\]. In our data, we have shown a negative correlation between hyperhomocysteinaemia and erythrocyte catalase activity in patients with AMI. Homocysteine has the ability to bind proteins and to form disulphide bridges with cysteine residues within proteins. Milton N et al. suggest that modification of cysteine residues by Homocysteine may alter the enzymatic activity of catalase \\[24\\]. The excessive generation of ROS under ischemia reperfusion can affect the red cell metabolism and the possible hemolysis when more than 98% in blood catalase is located in erythrocytes \\[25\\]. Now this is in line with other works suggesting that Hcy can affect the antioxidant enzyme expression. Nanako et al. reported that homocysteine reduced the expression of superoxide dismutase (SOD) mRNA in cultured rat smooth muscle cells \\[26\\]. Other studies showed a positive correlation between plasma Hcy and genomic damage related to DNA hypomethylation which let us to suggest that Hcy can exert genotoxic effects on DNA genes encoding for antioxidant proteins \\[27\\].\n\nReactive oxygen species generated by Hcy auto-oxidation are involved through Fenton type reaction in lipid peroxidation. The serum tHcy and TBARS levels were found higher in patients' with electrocardiogram presenting Q wave AMI compared to patients with non Q wave MI. Myocardial Infarction with Q wave can be used as a predictor of morbidity and mortality patterns after Myocardial events. Desmarais PL and al showed that individuals who had non-Q wave MI had better survival rates for the first 3 years after myocardial rehabilitation than did those who had Q wave MI \\[28\\]. Excessive lipid peroxidation can be directly involved in the myocardial necrosis manifesting as a myocardial wall dysfunction. Polyunsaturated fatty acids (PUFAs) such as arachidonic, linolenic and linoleic acids present the major targets for free radical attack. It has also been suggested that lipid peroxidation might proceed not only in plasma membranes but also in the nuclear membranes close to chromosomes, due to the loss of membrane integrity in cell membranes consisting of phospholipids. Lipid peroxidation products, such as 4-hydroxynonenal (HNE) and Malondialdehyde (MDA) are toxic. HNE leads to a decrease in protein thiols, disturbance of calcium homeostasis, inhibition of DNA, RNA and protein synthesis, inhibition of respiration and glycolysis \\[29\\]. Furthermore, HNE leads to mammalian cell death. It increases the expression of p53 family members as well as an increase of expression of the Bax pro-apoptotic gene \\[30\\]. MDA is able to interact with nucleic acid bases to form several different adducts and to exacerbate DNA oxidative damage, including genes encoding for antioxidant proteins such as catalase, glutathione peroxidase and SOD.\n\n# Conclusion\n\nMolecular mechanisms of myocardial necrosis under ischemia reperfusion need to be more defined. We have shown that hyperhomocysteinaemia, as a cardiovascular risk factor, may be involved in the drop of the enzymatic antioxidant activity. Hyperhomocysteinaemia can be involved in the excessive lipid peroxidation due to its pro-oxidant proprieties, aggravating the magnitude of the oxidative stress and the severity of the myocardial wall dysfunction. Further insights into the molecular mechanisms of the metabolic basis of hyperhomocysteinaemia will prove invaluable in the treatment and the prevention of cardiovascular diseases related to atherosclerosis.\n\n# Abbreviations\n\nAMI: Acute myocardial infarction; TAS: Total antioxidant status; tHcy: Total homocysteine; TBARS: Thiobarbituric acid reactive substances; ROS: Reactive oxygen species; ECG: Electrocardiogram; NO: Nitric oxide; SOD: Superoxide dismutase\n\n# Competing interests\n\nThe authors declare that they have no competing interests.\n\n# Authors' contributions\n\nAll authors have done all the work in the laboratory. All authors have done the analysis of the results. All authors have given final approval of the version to be published. All authors read and approved the final manuscript.\n\n## Acknowledgments\n\nThe authors thank the staff of the Biochemistry Laboratory and the Cardiology Department of CHU Farhat Hached of Sousse and Mrs Imen ANNABI in French Institute (Tunis) for their valuable contribution in this work.","meta":{"dup_signals":{"dup_doc_count":116,"dup_dump_count":28,"dup_details":{"curated_sources":2,"2020-34":1,"2017-17":1,"2017-09":19,"2016-36":1,"2016-30":1,"2016-22":1,"2016-18":1,"2015-48":4,"2015-40":2,"2015-35":4,"2015-32":4,"2015-27":4,"2015-22":4,"2015-14":3,"2014-52":5,"2014-49":4,"2014-42":11,"2014-41":5,"2014-35":5,"2014-23":7,"2014-15":5,"2023-40":1,"2015-18":4,"2015-11":4,"2015-06":4,"2014-10":4,"2013-48":4,"2024-26":1}},"file":"PMC3651277"},"subset":"pubmed_central"}
{"text":"abstract: Greg's two dogs are impressed that only a few genes control the different lengths, growth patterns and curl of their coats.\nauthor: Gregory A Petsko\ndate: 2009\ninstitute: 1Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02454-9110, USA\ntitle: The dog days of autumn\n\n*In the world which we know, among the different and primitive geniuses that preside over the evolution of the several species, there exists not one, excepting that of the dog, that ever gave a thought to the presence of man*. Maurice Maeterlinck\n\n*Greg Petsko is tied up with teaching, so, by popular demand - actually, he wonders why there never seems to be a demand for more from* him- *his column will be guest-written this month by his two dogs, the mixed poodle\/spaniel Clifford and the chocolate Labrador retriever Mink (Figure* <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>). *They are not strangers to these pages, having written before, to much acclaim. Precisely how they manage to type their text is unclear*.\n\n**Mink**: Did you see the paper in the issue of *Science* for 2 October 2009 (**326:**150-153) by Cadieu and coworkers? It's entitled *Coat variation in the domestic dog is governed by variants in three genes*.\n\n**Clifford**: What's a domestic dog?\n\n**Mink**: I'm not sure. I think maybe it's the opposite of a foreign dog.\n\n**Clifford**: Are you a foreign dog? After all, you're from Labrador.\n\n**Mink**: No, I'm from New England. My ancestors were from Labrador. And yours were from France and England.\n\n**Clifford**: Does that mean I'm not a domestic dog? I don't want to be a foreign dog! I don't speak French!\n\n**Mink**: Calm down. We're both domestic dogs, I'm sure. But we're getting off the subject here. Did you see that paper?\n\n**Clifford**: No, I didn't. Was it written in French?\n\n**Mink**: Will you forget about French! It was written in scientific English, which means it's not easy for a little puppy to understand, but I'll explain it to you. It's about the genes that control different coats in dogs.\n\n**Clifford**: You mean like how your coat is dark brown and mine is like wheat?\n\n**Mink**: No, the genes that govern coat color have been known for quite a while. This paper is about the genes that control coat length, growth pattern, and curl. For example, I have a fur coat that's all one color, and it only grows to a certain length and then it stops. I shed in winter -\n\n**Clifford**: I'll say you do! I've never seen so much brown fur flying around! Why, the carpet in the family room is covered with little mounds of -\n\n**Mink**: Yes, yes, I know. I can't help it. But as I was saying, I have solid brown, straight fur while you have patchy off-white and beige curly hair. Your coat would just keep growing forever and curl into huge mats if you didn't get taken to the groomer for -\n\n**Clifford**: I hate the groomer! Hate them!\n\n**Mink**: Can we stay focused here? I know you hate the groomer. You make that perfectly plain every time Greg tries to take you there. I haven't seen such a performance of suffering since we watched that television broadcast of *King Lear* with Greg last spring.\n\n**Clifford (sotto voce)**: Hate them!\n\n**Mink**: OK, we've established that. But the point I'm trying to make is: look how different our coats are.\n\n**Clifford**: Mine's better. Except for having to go to the groomer. I hate -\n\n**Mink (quickly)**: I'm glad you like your coat. I think mine is perfectly fine, too. And I don't have to go to the groomer. So there.\n\n**Clifford (sullenly)**: What was your point about the paper?\n\n**Mink**: Oh, yes. The paper. Well, our coats are so completely different, you would think that there would be many genes that were involved in determining those different properties. But the authors of this paper found that's simply not the case. They carried out what are called genome-wide association studies (which is basically just looking for variations in gene sequence that correlate with changes in some property) of more than 1,000 dogs from 80 domestic breeds to identify genes associated with canine fur phenotypes. They were able to take advantage of both inter- and intrabreed variability.\n\n**Clifford**: What does that mean?\n\n**Mink**: I think it means that, although dogs' coats vary a lot from breed to breed, like with you and me, they also vary a bit within breeds. Not all poodles have the same kinds of coat, as any groomer can tell you.\n\n**Clifford**: I hate the groomer!\n\n**Mink**: Right. Nothing more about groomers, I promise. Anyway, it's an advantage when you have small variations within a breed, because you can use that to find the small number of genes that most likely account for those variations (they stand out against a background that doesn't vary so much since all the dogs are from the same breed), and then you can pay particular attention to those genes when you look for what controls the much larger variations between breeds. That makes genome-wide association studies in dogs much easier and more rewarding than genome-wide association studies in people, where it's harder to find candidate genes, so you have to look at thousands of individuals and it's very expensive.\n\n**Clifford (proudly)**: Dogs are better than people.\n\n**Mink**: Of course we are. But as I was saying, Greg has talked about this before. He is convinced that, for association studies in people, it would be smart to use the relatively common mutations that give rise to autosomal recessive diseases and examine the carriers for association with other diseases. For example, people with Gaucher Disease are much more likely to get multiple myeloma, so an obvious thing to do would be to see if Gaucher carriers are overrepresented among myeloma patients. Greg thinks that's what the human genome people ought to be doing if they want to make rapid progress on diseases, because the carrier mutations are known to affect the functions of those proteins, so they're much more likely to do something than the common variants that the gene association studies mostly look at. Greg says those people are barking up the wrong tree.\n\n**Clifford**: Barking up the wrong tree? Why would anybody bark up the wrong tree?\n\n**Mink**: I have no idea.\n\n**Clifford**: Can we get back to talking about dogs?\n\n**Mink**: Sorry. As I was saying, with dogs you can get a good idea what genes to look at as well, from variations within a breed. That's how the people in this paper started their project. The team of scientists, which was headed by Elaine Ostrander of the National Institutes of Health -\n\n**Clifford**: I've heard of her! She's a genome biologist. We like her. She works on genes responsible for cancer susceptibility in people and dogs. Cancer is the number one killer of dogs. We hate cancer! We hate it almost as much as we hate the gr -\n\n**Mink (even more quickly)**: Yes, she is a great benefactor of the canine race. You may remember that, about two years ago, she headed the team that studied height variation in dogs (*Science*, **316:**112-115, 2007). Dogs have the greatest variation in height of any mammalian species. She discovered that the default for dogs is to be tall, like me, but that a mutation in a single gene, insulin-like growth factor 1, could account for the fact that many dogs are quite small, like Chihuahuas, fox terriers, and, well, like you.\n\n**Clifford**: I'm not small! I just have short legs for my body height.\n\n**Mink**: Whatever. The point is, it was a big surprise that one gene could account for such big differences.\n\n**Clifford**: How did they find that gene? I forget.\n\n**Mink**: Exactly the same way they found the genes in this study. They first looked at variation in height within a breed where it varies a lot: Portuguese water dogs. That allowed them to home in on the likely gene. Then they checked it across breeds.\n\n**Clifford**: President Barack Obama has a Portuguese water dog named Bo, doesn't he? I wonder why he didn't pick a poodle\/spaniel mix.\n\n**Mink**: Or a chocolate Lab. Well, nobody's perfect. Anyway, that discovery sort of made sense because insulin-like growth factor is one of the genes that controls cell growth and lifespan.\n\n**Clifford (musing)**: I'd like to meet Bo. Do you think President Obama would let him play with us?\n\n**Mink**: Can we stay on the topic here? This column'll be over soon.\n\n**Clifford**: OK. Did they use Portuguese water dogs in this new study about coat variation too?\n\n**Mink**: As a matter of fact, they did. One of their same-breed groups comprised 76 Portuguese water dogs, because it's a breed that varies a lot in hair curl. They looked at three phenotypes, actually: hair curl, hair length, and the presence or absence of what they call 'furnishings' - you know, that little moustache and bushy eyebrows you have.\n\n**Clifford (proudly)**: I am well furnished.\n\n**Mink**: Of course you are. Well, after they looked at a few same-breed groups, they then examined genetic variation across 903 dogs from 80 different breeds. They found that distinct mutations in just three genes, *RSPO2*, *FGF5*, and *KRT71*, together account for most coat phenotypes in pure-bred dogs in the United States.\n\n**Clifford**: You mean my coat is controlled by just three genes?\n\n**Mink**: Maybe not. They only looked at purebreds, and you're a mixture of two breeds.\n\n**Clifford**: Are you insulting my mother? I'm just as pure as -\n\n**Mink**: No, not at all. It's just that, er, uh, more sophisticated dogs like you are too complex for simple genetic analysis.\n\n**Clifford**: That's me, all right. I'm complicated.\n\n**Mink**: You can say that again. Anyway, *RSPO2* largely controls furnishing, which is interesting, because the gene codes for a protein called R-spondin-2, which is a signaling regulator that synergizes with the Wnt pathway to activate \u03b2-catenin, and Wnt signaling is required for the establishment of hair follicles in mammals. The mutation doesn't seem to change the protein sequence; it probably affects the mRNA level. You know, this same pathway is involved in the development of hair-follicle tumors, or pilomatricomas, which occur most frequently in breeds that have furnishings. Recent studies have shown that a mutation in the *EDAR* gene, also involved in the Wnt pathway, is responsible for a coarse East-Asian hair type found in humans, and as you know, that hair type has some similarity to canine wirehair.\n\n**Clifford**: Do you think this pathway controls Greg's hair?\n\n**Mink**: He's a middle-aged man. What hair?\n\n**Clifford**: How about the other two phenotypes?\n\n**Mink**: Curl seems to be determined by the *KRT71* gene, which codes for one of the forms of keratin, the major protein component of hair.\n\n**Clifford**: That makes sense. Does the mutation change the protein sequence?\n\n**Mink**: Yes, it does. It replaces one amino acid, an arginine, with a tryptophan. But why that leads to curly hair is not obvious. The third gene, *FGF5*, is involved in hair length.\n\n**Clifford**: What does that protein do?\n\n**Mink**: It makes one of the fibroblast growth factors. Makes sense, right?\n\n**Clifford**: It does. Amazing. And if a dog has all three genes mutated...\n\n**Mink**: He's a wire-hair.\n\n**Clifford**: Like our friend Max in the park. Cool. But why is this important - besides the fact that it refers to dogs, of course?\n\n**Mink**: Isn't that enough? Well, I guess one other reason is that it explains how so many different sizes, shapes and appearances of dog could have arisen in only about 15,000 years of accidental and deliberate breeding. If combinations of only a few genes can have a big effect on morphology and so forth, it won't take that many generations to produce a large number of possibilities. In fact, it's thought that most of the breeds we see today originated since about 1800, so it really can happen fast. Dog evolution is much faster than evolution of other mammals in the wild.\n\n**Clifford**: That's because we're a superior species.\n\n**Mink**: Obviously. After all, who lies around all day and gets fed, while the other species works to support us?\n\n**Clifford**: Isn't evolution wonderful?\n\n**Mink**: It is, but in our case, I prefer the term intelligent design.","meta":{"dup_signals":{"dup_doc_count":113,"dup_dump_count":51,"dup_details":{"curated_sources":2,"2022-21":1,"2020-34":1,"2020-05":1,"2019-51":1,"2019-43":1,"2019-26":1,"2019-13":1,"2019-04":1,"2018-47":1,"2018-39":1,"2018-30":2,"2018-13":1,"2017-47":2,"2017-34":1,"2017-30":1,"2017-26":2,"2017-22":1,"2017-17":2,"2017-09":9,"2017-04":1,"2016-50":1,"2016-44":1,"2016-40":2,"2016-36":10,"2016-30":9,"2016-18":1,"2016-07":4,"2015-48":2,"2015-40":2,"2015-35":2,"2015-32":2,"2015-27":1,"2015-22":2,"2015-14":1,"2014-52":2,"2014-49":5,"2014-42":7,"2014-41":2,"2014-35":2,"2014-23":2,"2014-15":3,"2023-14":1,"2024-10":1,"2017-13":1,"2015-18":2,"2015-11":2,"2015-06":2,"2014-10":2,"2013-48":2,"2013-20":2,"2024-30":1}},"file":"PMC2784313"},"subset":"pubmed_central"}
{"text":"abstract: Sodium glucose cotransporter 2 (SGLT2) inhibition is a novel and promising treatment for diabetes under late-stage clinical development. It generally is accepted that SGLT2 mediates 90% of renal glucose reabsorption. However, SGLT2 inhibitors in clinical development inhibit only 30\u201350% of the filtered glucose load. Why are they unable to inhibit 90% of glucose reabsorption in humans? We will try to provide an explanation to this puzzle in this perspective analysis of the unique pharmacokinetic and pharmacodynamic profiles of SGLT2 inhibitors in clinical trials and examine possible mechanisms and molecular properties that may be responsible.\nauthor: Jiwen (Jim) Liu; TaeWeon Lee; Ralph A. DeFronzoCorresponding authors: Jiwen (Jim) Liu, <email@example.com>, and Ralph A. DeFronzo, <firstname.lastname@example.com>.\ndate: 2012-09\nreferences:\ntitle: Why Do SGLT2 Inhibitors Inhibit Only 30\u201350% of Renal Glucose Reabsorption in Humans?\n\nType 2 diabetes is a serious global health issue that has reached epidemic proportions in both developed and developing countries over the last two decades (1). With currently available medicines, many diabetic patients fail to achieve optimal glycemic control (HbA~1c~ \\<6.5\u20137.0%). With the exception of the glucagon-like peptide 1 analogs and the thiazolidinediones (2), other antidiabetic medications lose their effectiveness to control hyperglycemia over time, partially due to the progressive decline of \u03b2-cell function (2\u20134). As a consequence, many patients receive multiple antidiabetic medicines and eventually require insulin therapy, which often fails to achieve the desired glycemic goal and is associated with weight gain and hypoglycemia (5,6). Failure to achieve glycemic targets is the primary factor responsible for the microvascular complications (retinopathy, neuropathy, nephropathy) and, to a lesser extent, macrovascular complications (2,7). In addition, the majority of diabetic patients are overweight or obese, and many of the current therapies are associated with weight gain, which causes insulin resistance and deterioration in glycemic control (2).\n\nGiven the difficulty in achieving optimal glycemic control (8,9) for many diabetic patients using current therapies, there is an unmet medical need for new antidiabetic agents. Although it has been known for 50 years (10,11) that renal glucose reabsorption is increased in type 2 diabetic patients, only recently have the clinical therapeutic implications of this observation been recognized (2,12). Inhibition of renal tubular glucose reabsorption, leading to a reduction in blood glucose concentration through enhanced urinary glucose excretion, provides a novel insulin-independent therapy (2,12) that in animal models of diabetes has been shown to reverse glucotoxicity and improve insulin sensitivity and \u03b2-cell function (13,14). The majority (\u223c80\u201390%) of filtered plasma glucose is reabsorbed in the early proximal tubule by the high-capacity, low-affinity sodium glucose cotransporter (SGLT) 2 (15,16). The remaining 10\u201320% of filtered glucose is reabsorbed by the high-affinity, low-capacity SGLT1 transporter in the more distal portion of the proximal tubule. After glucose is actively reabsorbed by SGLT2 and SGLT1 into the proximal tubular cells, it is diffused out of the cells from the basolateral side into blood through facilitative GLUT 2 and 1 (15). Because the majority of glucose reabsorption occurs via the SGLT2 transporter, pharmaceutical companies have focused on the development of SGLT2 inhibitors, and multiple SGLT2 inhibitors currently are in human phase II and III clinical trials (17). This class of antidiabetic medication effectively lowers blood glucose levels and offers additional benefits, including weight loss, low propensity for causing hypoglycemia, and reduction in blood pressure. The SGLT2 inhibitors are effective as monotherapy and in combination with existing therapies (2,12,14,15,17), including insulin (18). Because of their unique mechanism of action (12,15), which is independent of the severity of insulin resistance and \u03b2-cell failure, type 2 diabetic individuals with recent-onset diabetes (\\<1 year) respond equally well as type 2 diabetic patients with long-standing diabetes (\\>10 years) (19).\n\nDapagliflozin is the most advanced SGLT2 inhibitor in clinical trials (12,17,20). In addition, multiple other SGLT2 inhibitors are in phase II to III trials (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<\/a>) (17,21). However, none of these SGLT2 inhibitors are able to inhibit \\>30\u201350% of the filtered glucose load, despite in vitro studies indicate that 100% inhibition of the SGLT2 transporter should be achieved at the drug concentrations in humans (22,23). In this perspective, we shall examine potential explanations for this apparent paradox. Resolution of the paradox has important clinical implications with regard to the efficacy of this class of drugs and the development of more efficacious SGLT2 inhibitors.\n\n# PUZZLE ABOUT SGLT2 INHIBITORS\n\nIn healthy nondiabetic humans, \u223c160\u2013180 g of plasma glucose is filtered daily (glomerular filtration rate \\[GFR\\] = 180 L\/day \u00d7 plasma glucose = 900\u20131000 mg\/L), and essentially all of the filtered glucose is reabsorbed in the proximal tubule of the kidneys. It is generally believed that SGLT2 reabsorbs 80\u201390% of the filtered glucose load (15,16). However, SGLT2 inhibitors in clinical development induce a maximum of 50\u201380 g of urinary glucose excretion (UGE) per day (i.e., only 30\u201350% of the filtered glucose load) in healthy volunteers. Some SGLT2 inhibitors cause a maximum daily UGE at a low dose and cannot augment UGE even with a \\>10-fold increase in dose (22,23). For example, dapagliflozin produces a maximum UGE of \u223c60 g\/day at a dose of 20 mg\/day in healthy human volunteers, and UGE remains at 60 g\/day when the dose is increased to 500 mg\/day (23). Why can these inhibitors not block 90% of the filtered glucose load in humans?\n\nA number of explanations have been proposed to explain this paradox (<a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>, explanations 1\u20135), but they are insufficient to account for many of the data and observations. In this perspective, we will analyze these explanations and attempt to provide a more satisfactory solution to this puzzle. We will start by examining the pharmacokinetic and pharmacodynamic (PK\/PD) characteristics of SGLT2 inhibitors, which are varied and very unique.\n\nPotential explanations for why SGLT2 inhibitors cannot inhibit \\>30\u201350% of the filtered glucose load\n\n![](2199tbl1)\n\n# PK\/PD CHARACTERISTICS OF SGLT2 INHIBITORS\n\nFirst, in healthy human volunteers, there are large differences in the dose-dependent PD responses between empagliflozin (BI 10773) and ipragliflozin (ASP-1941) (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<\/a>), despite sharing many similarities (22,24). Both SGLT2 inhibitors have similar chemical structures, human PK profiles, and in vitro potencies. The in vitro SGLT2 half-maximal inhibitory concentration (IC~50~) of empagliflozin and ipragliflozin is 3.1 and 7.4 nmol\/L, respectively. The half-lives (*t*~1\/2~) of empagliflozin and ipragliflozin in the blood are 8.6\u201313 and 10\u201314 h, respectively, and the time to maximum plasma concentration (*t*~max~) is 1.5\u20132.5 and 1.0\u20132.3 h, respectively. The area under the curve (AUC) of empagliflozin at the 2.5-mg dose is 396 nmol\u00b7h\/L and that of ipragliflozin at the 5-mg dose is 810 nmol\u00b7h\/L. However, UGE over 24 h of empagliflozin at 2.5-mg dose is 30.6 g, whereas that of ipragliflozin at 5-mg dose is only 3.1 g. Differences in plasma protein binding (PPB) can result in different PD responses, but it is likely (although not measured) that the PPB of the two compounds is similar, because the time course PK profiles of the two compounds (as characterized by their *t*~1\/2~, *t*~max~, and AUC) are almost identical. A difference in PPB would be expected to change the time course PK profile, because this would change the clearance and volume of distribution of the drugs and thus the *t*~1\/2~. Further, there are no reports in the literature that describe a major difference in PPB of the SGLT2 inhibitors in current clinical development.\n\nSecond, the unbound plasma concentrations of dapagliflozin do not seem to account for the drug's effect on UGE. In phase I single and multiple ascending-dose studies of dapagliflozin, a near-maximal UGE was maintained in healthy volunteers for 24 h following a dose of 20 mg, whereas the plasma concentration decreased to a range of 10\u201320 nmol\/L at 24 h from a maximum concentration (*C*~max~) of 600\u2013700 nmol\/L (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<\/a>) (21). Taking into account PPB, this corresponds to an unbound dapagliflozin plasma concentration of \\<2 nmol\/L 24 h after the dose. This concentration is in the vicinity of the drug's in vitro SGLT2 IC~50~ (1.1 nmol\/L), yet dapagliflozin is still capable of generating a near maximum UGE response. To our knowledge, there are no published data that suggest that in vitro potency overestimates the in vivo drug concentration needed to inhibit SGLT2. To the contrary, much higher concentrations of T-1095A (an *O*-glucoside inhibitor) were required to inhibit glucose uptake into brush border membrane vesicles prepared from animal kidneys than into cells overexpressing SGLT2 (25). It is possible the SGLT2 expression levels in the renal proximal tubule are higher than those on SGLT2-transfected cells. This possibility is supported by studies that have measured the binding of tritium-labeled dapagliflozin to the same amount of protein from mouse kidneys or transfected cells (Amgen, unpublished data). It was found that SGLT2 expression (calculated from the amount of radioactivity bound) in the mouse kidney tissue was several fold higher than in the proteins of transfected cells ([Supplementary Fig. 1](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-0052\/-\/DC1); T.L., unpublished observations). Because proximal tubular proteins only comprise a small portion of the kidney tissue, the SGLT2 expression levels in the proximal tubule could be substantially higher than those of SGLT2-transfected cells. Considering dapagliflozin's high selectivity and specificity for SGLT2, nonspecific binding is unlikely to be a significant contributing factor.\n\nThird, there are disconnects between the UGE effect of empagliflozin and its plasma concentration. After administration of single doses of empagliflozin to healthy humans, a rapid onset of UGE response occurred and was maintained long after the plasma concentrations diminished to very low levels (<a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<\/a>) (22,26). The empagliflozin plasma concentrations peaked \u223c2 h postdose, whereas the maximal UGE rate occurred at about 7 h across a range of doses (10 to 800 mg). Further, the UGE rates did not decline nearly as rapidly as the decrease in plasma concentrations. Following the 100- and 400-mg doses of empagliflozin, the UGE rates at 60 h postdose dropped about threefold from the peak values, whereas the plasma concentrations at the same doses dropped \\>50-fold from their peak levels during the same period of time.\n\nThe unbound plasma drug concentration usually correlates well with PD response. In the case of SGLT2 inhibitors, it also appears reasonable to assume that the unbound plasma concentration reflects the concentration in the location of the SGLT2 transporters. Unbound SGLT2 inhibitors are filtered through glomeruli and travel a short distance to reach the SGLT2 transporters, which are located in the early segment of the renal proximal tubule. However, the three observations above indicate that the UGE responses induced by the SGLT2 inhibitors do not correlate with the drug's unbound plasma concentrations. Why? In the following discussion, we provide a possible answer to this question, followed by an explanation for the puzzle described in the title.\n\n# EXPLANATION OF THE PARADOX\n\nAs described above, SGLT2 inhibitors, such as dapagliflozin and empagliflozin, demonstrate unique PK\/PD relationships. Robust UGE was maintained long after the plasma drug concentrations diminished (22,23) (<a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<\/a>). If SGLT2 inhibitors are actively secreted (in addition to filtered) into the renal proximal tubule, the renal secretion can deliver higher drug concentrations to the area of the SGLT2 transporter and thus contribute to the robust UGE effect, especially when the plasma concentrations are low. Another possible contributing factor that may explain the prolonged UGE effect is a slow off rate of SGLT2 inhibitors. Compounds with a slow off rate can maintain their PD effect even when their plasma concentrations are low. Empagliflozin has a slow off rate with a dissociation *t*~1\/2~ of 1 h both in the absence and presence of 20 mmol\/L glucose (27). If SGLT2 inhibitors also block the transporter from inside the cells, their inhibition effect can last longer than indicated by their dissociation *t*~1\/2~ (28).\n\nTo the best of our knowledge, renal secretion has not been considered as a factor for the PK\/PD relationship in the literature. There are several potential reasons for the lack of consideration of renal secretion. First, the reported renal clearance of SGLT2 inhibitors is low across species and insignificant compared with their hepatic clearance (29,30). Dapagliflozin has a low renal clearance of 5.6 mL\/min in humans, half of its GFR\\*fu (10.8 mL\/min), in which fu is fraction unbound (29). When renal clearance is insignificant and less than the product of GFR\\*fu, renal secretion is not considered to be a significant factor for drug\u2013drug interaction (DDI), so it is not critical to study renal secretion and the transporters involved for compounds with low renal clearances (31). Second, even if renal transporters are studied, the ones most often examined for DDI are limited, such as P-glycoprotein, breast cancer\u2013resistant protein, organic anion transporters, and organic cation transporters (OCTs) (31). There are many more transporters (peptide transporters 1 and 2, multidrug resistance\u2013associated protein 2 and 4, OCTN1, and OCTN2, etc.) involved in renal secretion, but often they are not studied for DDI (31).\n\nLow renal clearance does not mean lack of renal secretion, because significant renal secretion can be masked by renal reabsorption, if compounds have good passive permeability (32\u201334). SGLT2 inhibitors generally have good permeability, as indicated by high oral bioavailability and permeability measurement (29). Although there is no direct evidence in the literature to support renal secretion of SGLT2 inhibitors, indirect evidence supports such a mechanism. It is interesting to note that following oral administration of a 1 mg\/kg dose of TS-071 (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<\/a>), rats exhibited kidney\/plasma ratios of 35 at 4 h postdose, despite the fact that TS-071 primarily is excreted by hepatic metabolism (35). After 24 h, TS-071 was no longer detected in rat kidneys. Renal secretion and slow off rate from targets in the kidney can make compounds preferentially distributed into the kidney.\n\nNeutral drugs that have high lipophilicity (indicating good permeability), like dapagliflozin, tend to have a low renal clearance (33). Even though the renal clearance of dapagliflozin is low, many drugs that have similar PPB (indicated by GFR\\*fu) and similar lipophilicity (indicated by cLogD or cLogP) to those of dapagliflozin actually have a much lower renal clearance than dapagliflozin (34). The reason why dapagliflozin does not have a lower renal clearance may be due to its renal secretion.\n\nThe sustained robust UGE and different timing of peak UGE and *C*~max~ of empagliflozin also suggest renal secretion (<a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<\/a>) (22,26). If glomerular filtration were the only way for SGLT2 inhibitors to reach the site of action, one would expect the timing of peak UGE to match the *t*~max~ of the plasma concentration and UGE to decrease in concert with the reduction of the plasma concentration. The onset of UGE response is rapid after SGLT2 inhibition. Although slow off rate of SGLT2 inhibitors can contribute to the slow decrease of UGE response, it cannot explain the vastly slower decline of UGE response compared with the plasma concentration.\n\nThe human metabolite profile of dapagliflozin suggests that active metabolites do not significantly contribute to the PD response, because dapagliflozin is primarily eliminated as a pharmacologically inactive glucuronide metabolite (29). The possibility of superpotent minor metabolites contributing to the PD effects cannot be ruled out, but there is no evidence to support this.\n\nIf SGLT2 inhibitors are secreted by the renal tubule, this could provide a solution to the puzzle as to why SGLT2 inhibitors are unable to inhibit 80\u201390% of the filtered glucose load in humans. First, even though renal secretion may deliver sufficient drug to achieve robust UGE for a longer duration, it can saturate at high doses and thus limit SGLT2 inhibitors from maximally inhibiting renal glucose reabsorption (<a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>, explanation 6). Second, location of renal secretion relative to that of the SGLT2 transporters can affect the efficiency of SGLT2 inhibition. Downstream renal secretion of drug cannot affect glucose reabsorption, if the SGLT2 transporters are located upstream (<a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>, explanation 7). Third, full inhibition of the SGLT2 transporter may require significantly higher concentrations of the inhibitors than suggested by the in vitro SGLT2 potency, because the expression levels of SGLT2 in the renal proximal tubule may be much higher than those on the transfected cells used in the in vitro assays (<a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>, explanation 8). Fourth, SGLT2 inhibitors delivered through glomerular filtration may have an impact on UGE during the initial few hours after drug administration, but exert limited effect at later times even at high doses, because the plasma concentration declines rapidly after the *C*~max~ is reached. Taken collectively, it is possible that glomerular filtration cannot deliver impactful levels of the SGLT2 inhibitors over a long duration even when high doses of the drugs are given. Rather, renal tubular secretion and slow off rate of the inhibitors may be responsible for the sustained drug levels in the proximal tubule, which in turn results in a sustained UGE response. Full inhibition of SGLT2 may be limited by the capacity of renal secretion and\/or the proximity of the site of renal secretion and the SGLT2 transporters.\n\nIt also is possible that excessive reabsorption of SGLT2 inhibitors in the proximal tubule contributes to the inability of SGLT2 inhibitors to block \\>30\u201350% of glucose reabsorption (<a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>, explanation 9). Excessive active reabsorption through transporters could result in significantly lower drug concentrations in the proximal tubule than the plasma unbound concentration and thus limit the inhibition. But the lower tubular concentration is not consistent with the prolonged robust UGE effect, unless inhibition of the SGLT2 transporter also can take place intracellularly. Active reabsorption can accumulate SGLT2 inhibitors inside the tubular cells and result in the prolonged UGE effect (especially in combination with slow off rate), if the inhibitors also block from inside the cells. In this case, the capacity of the active reabsorption and\/or its location relative to the SGLT2 transporters dictates the extent of SGLT2 inhibition. However, there is no evidence in the literature regarding possible intracellular inhibition of the SGLT2 transporter by the SGLT2 inhibitors in clinical trials. Passive reabsorption does not have the same effect as active reabsorption, because it is driven by the drug concentration gradient from the renal tubule to the bloodstream. Therefore, it only makes the drug tubular concentration approach the plasma unbound concentration.\n\nRenal secretion (and\/or reabsorption) also can explain several other important observations. First, as discussed above, empagliflozin and ipragliflozin have very different dose-dependent UGE responses, even though they share many similar properties, such as potency and PK properties. It is possible that differences in renal secretion (and\/or reabsorption) and dissociation *t*~1\/2~ are responsible for the different UGE responses. Second, in contrast to humans, SGLT2 inhibitors can block \\>70% of the glucose reabsorption in Sprague-Dawley rats. In these rats, about 3.6 g\/day\/200 g body weight of glucose is filtered through glomeruli (GFR = 1.4 mL\/min\/100 g body weight = 4 L\/day\/200 g body weight \u00d7 0.9 g\/L = plasma glucose level) (36). The maximum UGE\/day\/200 g body weight induced by SGLT2 inhibitors in Sprague-Dawley rats is 2.6 g for dapagliflozin and PF-04971729 (37,38), 2.9 g for EGT1442 (39), and 3.7 g for canagliflozin (40). Potency and PK cannot explain this species difference. This difference may be explained by a greater renal secretory (and\/or reabsorption) capacity and\/or more upstream renal secretion (and\/or reabsorption) in rodents compared with humans.\n\nThere are other potential explanations (<a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>, explanations 1\u20135) for this puzzle, but they are unlikely to explain the paradox. The first explanation in <a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a> is that increased levels of glucose in the proximal tubule, as a result of SGLT2 inhibition, compete with the drug and thus prevent the full inhibition of SGLT2. However, this is not consistent with the observation that a \\>20-fold increase in the dose of dapagliflozin from 20 to 500 mg\/day only prolongs the PD effect and does not further increase the level of inhibition (\u223c40% inhibition of the glucose reabsorption) (23). The second explanation in <a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a> is that GLUT other than SGLT2 may be responsible for a much greater fraction of the glucose reabsorption. However, this cannot explain why some patients with severe SGLT2 mutations excrete \\>150 g\/day of glucose into the urine, unless the SGLT2 mutation also affects other glucose transporters (i.e., SGLT5 or SGLT1, for which genes are not located on the same chromosome as SGLT2). In addition, LX-4211, a SGLT2\/SGLT1 dual inhibitor in phase II clinical trials, does not generate a larger glucosuric effect than selective SGLT2 inhibitors (41). SGLT5 has been suggested as a transporter that may be responsible for a greater fraction of glucose reabsorption, because of its almost exclusive expression in the renal tubule and its unidentified function. However, recent studies have demonstrated that SGLT5 is a mannose fructose transporter (27). Further, remogliflozin is a SGLT2\/SGLT5 dual inhibitor with IC~50~ for the two transporters of 12 and 480 nmol\/L, respectively (42). In healthy humans, remogliflozin etabonate (prodrug of remogliflozin) could not induce more than \u223c40 g\/day of UGE, even when its dose was increased from 150 to 1000 mg (43). Based upon these observations, the role of SGLT5 in renal glucose reabsorption appears to be insignificant. Furthermore, knockout of SGLT2 in the mouse results in the excretion of 70\u201380% of the filtered glucose load (16), although one could argue that other GLUTs play a more dominant role in man. Last and most important, SGLT2 antisense oligonucleotides, which knock down SGLT2 mRNA expression up to 80%, have a much greater glucosuric effect than the orally administered SGLT2 inhibitors in multiple species, including rats and dogs as well as monkeys (44,45).\n\nExplanations 3\u20135 in <a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a> also are unlikely. Based upon animal studies that have localized the SGLT2 transporter to the brush border membrane of the proximal tubule (16), inability of the filtered SGLT2 inhibitor to interact with the SGLT2 transporters because of their anatomical location seems unlikely (<a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>, explanation 3). The high PPB of some SGLT2 inhibitors should not limit the extent of inhibition if the dose is increased (<a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>, explanation 4). High PPB limits the percentage of unbound drug and thus limits the amount of drug filtered through the glomeruli. However, the amount of filtered (unbound) drug can be increased, if the dose is increased. For example, unbound *C*~max~ of dapagliflozin increases from \u223c60 nmol\/L to \\>1 \u03bcmol\/L (calculated using 91% PPB and its PK in healthy humans) (23,29) when its dose is increased from 20 to 500 mg. The similar magnitude of glucosuria on days 1\u20133 versus day 14 after administration of dapagliflozin strongly argues against upregulation of other SGLTs or other GLUTs distal to the SGLT2 transporter (<a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>, explanation 5) (46). Moreover, phlorizin-treated diabetic rats do not upregulate GLUT2 (47). Although SGLT2 inhibition does not appear to cause upregulation of SGLTs or other GLUTs in humans, there is evidence for increased GLUT2 expression in diabetic rodent models (17,19). However, in healthy humans in whom upregulation of GLUTs and\/or SGLTs does not occur, SGLT2 inhibitors still cannot block \\>30\u201350% of renal glucose reabsorption. In addition, if there were significant contribution to glucose reabsorption from GLUTs or SGLTs other than SGLT2, it would not explain the unique PD effects of SGLT2 inhibitors, such as prolonged maximum UGE response at diminished plasma levels and different timing between the maximum PD response of empagliflozin and its maximum plasma concentration.\n\n# CONCLUSIONS\n\nThe SGLT2 inhibitors in human clinical trials have good efficacy, but they do not inhibit \\>30\u201350% of the filtered glucose load. Based upon their PK\/PD relationship, we postulate that their mechanism of action is related to secretion and\/or active reabsorption in the proximal tubule and slow off rate from the SGLT2 target. Renal micropuncture studies with radiolabeled SGLT2 inhibitors and comparison of drug radioactivity in the glomerular filtrate and proximal tubule will help to define the relative contributions of tubular secretion and\/or reabsorption versus glomerular filtration in establishing the drug concentration at the site of the SGLT2 transporters in the proximal tubule. A better understanding of the renal handling of the SGLT2 inhibitors will help to develop more effective medications that are capable of inhibiting a greater percentage of filtered glucose load and causing a greater reduction in HbA~1c~.\n\n## ACKNOWLEDGMENTS\n\nR.A.D. received grants from Amylin and Takeda; served on the advisory boards of Amylin, Takeda, BMS, Novo Nordisk, Jansen, and Boehringer-Ingelheim; and served as a member of the Speakers Bureau at Novo Nordisk. No other potential conflicts of interest relevant to this article were reported.\n\nJ.L. and R.A.D. wrote, reviewed, and edited the manuscript. T.L. researched data and reviewed and edited the manuscript. J.L. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.\n\nThe authors thank Dr. Ernest M. Wright of David Geffen School of Medicine at University of California Los Angeles and Dr. Jeff Jasper of Cytokinetics for editorial comments.\n\n# REFERENCES","meta":{"dup_signals":{"dup_doc_count":151,"dup_dump_count":58,"dup_details":{"curated_sources":2,"2021-49":1,"2021-43":1,"2021-39":1,"2021-31":1,"2021-25":1,"2021-21":2,"2020-10":1,"2020-05":3,"2019-51":1,"2019-47":1,"2019-35":1,"2019-30":1,"2018-39":1,"2018-30":1,"2018-22":2,"2018-17":1,"2018-13":3,"2018-09":1,"2018-05":1,"2017-51":2,"2017-47":2,"2017-43":7,"2017-39":3,"2017-34":1,"2017-30":8,"2017-26":2,"2017-22":4,"2017-17":11,"2017-09":5,"2017-04":10,"2016-50":3,"2016-44":4,"2016-40":3,"2016-36":3,"2016-30":4,"2016-26":3,"2016-22":3,"2016-18":3,"2016-07":2,"2015-48":3,"2015-40":3,"2015-35":3,"2015-32":2,"2015-27":2,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":2,"2014-42":2,"2014-35":3,"2014-23":3,"2022-05":1,"2017-13":2,"2015-18":2,"2015-11":1,"2015-06":1,"2014-10":1,"2024-30":1}},"file":"PMC3425428"},"subset":"pubmed_central"}
{"text":"abstract: The Canadian Society for Exercise Physiology, in partnership with the Public Health Agency of Canada, has initiated a review of their physical activity guidelines to promote healthy active living for Canadian children, youth, adults and older adults; previous guidelines were released in 2002, 2002, 1998 and 1999 respectively. Several background papers from this project were published recently and provide foundation evidence upon which to base new guidelines. Furthermore, comprehensive systematic reviews were completed to ensure a rigorous evaluation of evidence informing the revision of physical activity guidelines for asymptomatic populations. The overall guideline development process is being guided and assessed by the AGREE II instrument. A meeting of experts was convened to present the evidence complied to inform the guideline revisions. An independent expert panel was assembled to review the background materials and systematic reviews; listen to the presentations and discussions at the expert meeting; ask for clarification; and produce the present paper representing their interpretation of the evidence including grading of the evidence and their identification of needs for future research. The paper includes also their recommendations for evidence-informed physical activity guidelines.\nauthor: Antero Kes\u00e4niemi; Chris J Riddoch; Bruce Reeder; Steven N Blair; Thorkild IA S\u00f8rensen\ndate: 2010\ninstitute: 1Institute of Clinical Medicine, Department of Internal Medicine and Biocenter Oulu, University of Oulu, PO Box 5000, FIN-90014, Finland; 2School for Health, University of Bath, 6.4 Norwood House, Bath BA2 7AY, UK; 3School of Public Health, Department of Community Health and Epidemiology, University of Saskatchewan, 107 Wiggins Road, Saskatoon, Saskatchewan S7N 5E5, Canada; 4Department of Exercise Science, Public Health Research Building, University of South Carolina, 225, 921 Assembly Street, Columbia, South Carolina, 29208, USA; 5Institute of Preventive Medicine, Copenhagen University Hospital, Centre for Health and Society, \u00d8ster S\u00f8gade 18, 1st fl, DK-1357 Copenhagen K, Denmark\nreferences:\ntitle: Advancing the future of physical activity guidelines in Canada: an independent expert panel interpretation of the evidence\n\n# Review\n\nThere is extensive research supporting the importance of regular physical activity (PA) in reducing all-cause mortality and improving several health outcomes. Canada has played an important role in the development of PA guidelines and guides for individuals of all ages. In 1998, the Canadian Society for Exercise Physiology (CSEP), in partnership with Health Canada (now the Public Health Agency of Canada) and others, released *Canada's Physical Activity Guide to Healthy Active Living* for adults between the ages 20 and 55 years \\[1\\]. This was followed by *Canada's Physical Activity Guide to Healthy Active Living for Older Adults* in 1999 and *Canada's Physical Activity Guides for Children and Youth* in 2002 \\[2,3\\]. Research in the PA sciences has advanced remarkably since these publications. Therefore, there has been a need to review the current research evidence to determine whether updated guidelines are required to help Canadians improve their health through regular PA.\n\nIn 2007 CSEP, with support from the Public Health Agency of Canada, completed an extensive review of PA measurement and guidelines in Canada, with 14 background papers published as a combined supplement of the *Canadian Journal of Public Health* and *Applied Physiology, Nutrition, and Metabolism* \\[4\\]. Further to this, on January 14-16, 2009, CSEP and the Public Health Agency of Canada organized an international consensus conference to discuss the current research evidence in asymptomatic individuals (i.e., with no pre-existing conditions health conditions) and to determine possible modifications to the existing guidelines. At the conference, experts presented evidence from detailed systematic reviews in which they updated findings on PA and health outcomes, including dose-response relationships and possible adverse effects of PA \\[5-9\\]. Detailed evidence of the health benefits of PA in school-aged children and youth \\[5\\], adults \\[6\\], older adults \\[7\\], Aboriginal populations, individuals with a disability, pre-school children, and pregnant women were presented. A rigorous, evidence-based approach was used to develop the levels of evidence for the relationship between PA and health outcomes using the procedures adopted by the Canadian Obesity Guidelines \\[10\\].\n\nAn independent expert panel (the Consensus Panel) comprised of five individuals (two with subject-specific knowledge in PA and health (CR, SB) and three with expertise from the associated disciplines of public health (BR), general and clinical epidemiology (TS), and medicine (AK)) was tasked with interpreting and rating the evidence compiled and presented for the first three categories mentioned (school-aged children and youth, adults, and older adults). The Consensus Panel reviewed and evaluated the evidence presented, assigned it to an evidence category (according to the classification systems shown in Tables <a href=\"#T1\" data-ref-type=\"table\">1<\/a> and <a href=\"#T2\" data-ref-type=\"table\">2<\/a>), and identified needs for future research. For the determination of the dose-response relationship between PA and health outcomes, the authors identified studies that evaluated the relationship between at least three different levels of PA. The Consensus Panel agreed with this approach for the determination of the shape of the curve between PA and health outcomes, but also considers the information based on only two levels of PA informative.\n\nCriteria for assigning a level of evidence to recommendations\n\n| Level of Evidence | Criteria |\n|----|----|\n| Level 1 | Randomized control trials without important limitations |\n| Level 2 | Randomized control trials with important limitations |\n|  | Observational studies (non-randomized clinical trials or cohort studies) with overwhelming evidence |\n| Level 3 | Other observational studies (prospective cohort studies, case-control studies, case series) |\n| Level 4 | Inadequate or no data in population of interest |\n|  | Anecdotal evidence or clinical experience |\n\nCriteria for assigning a grade to recommendations\n\n<table>\n<thead>\n<tr>\n<th style=\"text-align: left;\">Grade of Evidence<\/th>\n<th style=\"text-align: left;\">Criteria<\/th>\n<\/tr>\n<\/thead>\n<tbody>\n<tr>\n<td style=\"text-align: left;\">Grade A<\/td>\n<td style=\"text-align: left;\">Strong recommendation (action can apply to most individuals in most circumstances)<br \/>\nBenefits clearly outweigh risks (or vice-versa)<br \/>\nEvidence is at Level 1, 2, or 3<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Grade B<\/td>\n<td style=\"text-align: left;\">Weak recommendation (action may differ depending on individual's characteristics or other circumstances)<\/td>\n<\/tr>\n<tr>\n<td><\/td>\n<td style=\"text-align: left;\">Unclear if benefits outweigh risks<br \/>\nEvidence is at Level 1, 2, or 3<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Grade C<\/td>\n<td style=\"text-align: left;\">Consensus recommendations (alternative actions may be equally reasonable)<\/td>\n<\/tr>\n<tr>\n<td><\/td>\n<td style=\"text-align: left;\">Unclear if benefits outweigh risks<br \/>\nEvidence is at Level 3 or 4<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\nIn preparing the current document, the Consensus Panel observed that some important information was not available in the systematic reviews presented. Therefore, the Consensus Panel has used additional sources of information to cover these areas. Accordingly, the Consensus Panel's consensus statement, presented here, is based on (1) the background articles published in the 2007 supplement \\[4\\], (2) the systematic reviews presented at the consensus conference \\[5-9\\], and (3) additional information available in the literature (4) discussion and debate at the international consensus conference. This consensus statement provides information and recommendations for school-aged children and youth, adults, and older adults, and a discussion of the adverse effects of PA and needs for future research. Short summaries of the evidence are provided for each set of recommendations, with complete details available in the source documents \\[4-9\\]; the purpose of this paper is to provide a short summary, independent interpretation and list of recommendations based on the detailed work previously done. All of the recommendations presented herein represent the unanimous views of the panel. Because of the heterogeneity of data across studies, we did not directly link the evidence to individual studies, and refer the reader to each of the systematic reviews. Details on the history of this project and an assessment of the quality and rigor of processes informing these recommendations are provided in the paper by Tremblay et al \\[11\\].\n\n# School-aged children and youth, aged 5-19 years\n\n## Introduction\n\nThe systematic review \\[5\\] examined evidence with respect to seven key health indicators known to be associated with either PA or fitness: blood cholesterol, blood pressure, overweight\/obesity, bone mineral density, metabolic syndrome, depression, and injuries. After removing duplicates, a total of 437 articles were identified for cholesterol, 1,151 for depression, 2,505 for injuries, 1,181 for bone mineral density, 1,677 for blood pressure, 5,824 for obesity, and 1,677 for the metabolic syndrome, for a total of 14,452. Many of these articles were retrieved for two or more health outcomes, and after removing these duplicates there was a total of 11,088 unique citations. After review of the titles and abstracts, full-text copies of 454 citations were obtained and reviewed. Of these, 86 passed the eligibility criteria, reported dichotomous outcomes, and were included in the systematic review. Several of the papers included had results for two or more of the health outcomes.\n\nEvidence was reviewed with respect to the following questions:\n\n1\\. How much (volume) PA is needed for minimal and optimal health benefits?\n\n2\\. What types of PA are needed to produce health benefits?\n\n3\\. What is the appropriate PA intensity?\n\n4\\. Does the effect of PA on health in school-age children and youth vary by sex and\/or age?\n\nThe current recommendations within Canada's PA Guide for Children and Youth are as follows \\[3\\]:\n\n1\\. Increase the time currently spent on PA, starting with 30 minutes\/day more, and progress over approximately 5 months to 90 minutes\/day more.\n\n2\\. Build up PA throughout the day in periods of at least 5-10 minutes.\n\n3\\. The 90-minute increase in PA should include at least 1 hour of moderate activity (e.g., brisk walking, skating, bicycle riding) and 30 minutes of vigorous activity (e.g., running, basketball, soccer).\n\n4\\. Combine three types of physical activities--endurance, flexibility, and strength activities--to achieve the best results.\n\n5\\. Reduce non-active time spent watching television and video, playing computer games, and surfing the internet. Start with 30 minutes\/day less of such activities and progress over the course of approximately 5 months to 90 minutes\/day less.\n\nA brief summary of the current evidence of the associations between PA\/fitness and health outcomes for children and youth is presented below.\n\n## Associations with health outcomes\n\n### Cholesterol and blood lipids\n\nA total of nine articles examining blood lipids and lipoproteins met the inclusion criteria, one of which was observational in nature and eight of which were experimental. Interventions ranged from 6 to 24 weeks in duration and included from 1 to 4 hours\/week of prescribed exercise (mean: 9-34 minutes\/day). Most of the exercise programs were aerobic in nature and included various forms of moderate- to vigorous-intensity PA.\n\nThe one observational (cross-sectional) study indicated that unfit girls (bottom quintile of fitness) were 1.89 times (95% confidence interval \\[CI\\] 1.12-3.17) more likely to have hypercholesterolemia than moderately and highly fit girls. Unfit boys were 3.68 times (95% CI 2.55-5.31) more likely to have hypercholesterolemia than moderately and highly fit boys.\n\nThree of the eight experimental studies reported that exercise training had significant beneficial effects on at least one lipid\/lipoprotein variable. These studies tended to prescribe aerobic exercise. Other studies, which included interventions based on resistance training and circuit training, reported small and\/or insignificant findings for the variables examined. The interventions were predominantly found to be favorable in \"high-risk\" participants (e.g. those with obesity, high cholesterol).\n\nThe nature of the dose-response relationship between PA and blood lipids remains unclear. The effects of age and sex have not been adequately studied.\n\n### Blood pressure\n\nThree observational studies (two cross-sectional studies and one prospective cohort study) and eight experimental studies were included. Interventions ranged from 4 to 25 weeks in duration. The experimental studies were limited to children and youth with high blood pressure or obesity. With one exception, interventions included between 60 and 180 minutes\/week of prescribed exercise (mean: 9-30 minutes\/day). Most of the exercise programs were aerobic in nature and included various forms of moderate- to vigorous-intensity PA.\n\nWithin the observational studies, the relationship between PA or fitness and hypertension was weak, and in one case was not significant. Results from the intervention studies were positive, with the majority reporting modest but significant reductions (approximately 5-7%) in systolic blood pressure in response to exercise training. A few of the studies also reported modest but significant changes (approximately 4-6%) in diastolic blood pressure in response to exercise. Unlike the aerobic-based exercise programs, the two studies that employed resistance training did not report significant reductions in blood pressure.\n\nDose-response associations and the effects of age and sex have not been adequately studied.\n\n### Metabolic syndrome\n\nThe existence of true \"metabolic syndrome\" in children is debated. Nevertheless, elements of adult metabolic syndrome are known to cluster in children, as they do in adults. In children, a variety of definitions are used to describe the clustering of risk factors that comprise the metabolic syndrome in adults. In this review, obesity and cholesterol levels--two constituent components of the metabolic syndrome--are discussed as separate health outcomes. This section includes studies that have addressed clustering of any constituent components and those measuring the effect of PA on insulin levels and\/or insulin resistance--a main element of the metabolic syndrome.\n\nNine observational studies (eight cross-sectional, one prospective) were included, including some with large and diverse samples of participants. There were nine experimental studies (six randomized controlled trials, three quasi-experimental), all but one of which was conducted in overweight\/obese children. Interventions ranged from 6 to 40 weeks in duration and included anywhere from 80 to 200 minutes\/week of prescribed exercise. Most of the exercise programs were aerobic in nature and included various forms of moderate- to vigorous-intensity PA.\n\nIn three cross-sectional studies using self-reported PA, associations between PA and the metabolic syndrome were either weak or modest in strength, and all were non-significant. By comparison, the two studies that used accelerometers to measure PA and the four studies that used direct measures of cardiorespiratory fitness all reported strong and significant associations with the metabolic syndrome. In the experimental studies, half of the studies reported significant improvements in metabolic syndrome indicators with increased PA. Both aerobic and resistance training programs had positive effects. Programs with longer durations (\\>20 weeks) tended to report stronger effects.\n\nIn the observational studies that used more precise measures of PA and\/or fitness, clear dose-response relationships with metabolic syndrome indicators were observed. However, the shape of the curve (e.g., linear or curvilinear) was unclear. Comparison of risk estimates in boys and girls suggests that PA\/fitness may be more preventive of the metabolic syndrome in boys.\n\nThe effects of PA dose, intensity, age and sex have not been adequately studied.\n\n### Overweight and Obesity\n\nThirty-one observational studies (25 cross-sectional, three prospective cohort, two case-control, one mixed) and 25 intervention studies (13 randomized controlled trials, 12 quasi-experimental) were included in this review. The studies ranged in length from 1 month to 2 years, with most being 4-6 months in duration. The amounts of exercise prescribed typically ranged from 2 to 3.5 hours\/week (mean: 17-30 minutes\/day). Approximately half of the studies were limited to overweight\/obese participants. Observational studies using self-report measures of PA tended to report weak to moderate inverse associations of PA with overweight\/obesity, with many risk estimates being non-significant. Eight studies using either objective measures of PA (one pedometer, three accelerometer) or cardiorespiratory fitness reported significant associations that were modest to strong in magnitude. Studies that were limited to more vigorous forms of exercise tended to produce stronger effect estimates. The majority of studies involving exercise interventions that were aerobic in nature observed significant changes in measures of total fat and abdominal fat in response to training. Changes in body-mass index and weight were far less consistent in these studies. Most studies that employed other training modalities (four resistance training, two circuit training, one Pilates) did not observe significant improvements in measures of total or abdominal fat in response to training.\n\nThere is good evidence of an inverse dose-response association between PA and being overweight or obese, but the shape of the curve is unclear because both linear and curvilinear patterns were observed. Visual inspection of risk estimates suggests that PA and fitness are more strongly associated with weight in boys than in girls.\n\n### Bone mineral density\n\nNo observational studies in the literature search met the systematic review criteria of presenting the effects of PA on bone mineral density as a dichotomous outcome. Eleven experimental studies were included. The programs typically consisted of moderate- to high-intensity anaerobic, high-impact activities such as jumping. These programs were performed from 3 to 60 minutes in length on at least 2 or 3 days of the week, and lasted from a few months to 2 years in duration.\n\nAs little as 10 minutes of moderate- to high-impact activities performed on as little as 2 or 3 days of the week was found to have a positive impact on bone mineral density when combined with more general weight-bearing aerobic activities that are also beneficial for cardiovascular risk factors and obesity prevention (e.g., jogging, play).\n\nDose-response relationships and age and gender differences have not been adequately studied.\n\n### Depression\/depressive symptoms\n\nThree observational (all cross-sectional) and three experimental studies (two randomized controlled trials) were included. All of the experimental studies prescribed aerobic exercise. Programs for children with depressive symptoms were approximately 8-12 weeks in length and prescribed modest amounts of exercise (60-90 minutes\/week).\n\nThe observational studies reported small and insignificant or modest relationships between PA and depression. Two of the experimental studies observed significant improvements in depressive symptoms. One of the studies included both high-intensity and moderate-intensity PA programs, and only the high-intensity program resulted in significant improvements in depression scores in comparison to the control group, which performed flexibility exercises.\n\nDose-response associations and age and gender differences have not been adequately studied.\n\n### Injuries\n\nThree cross-sectional studies were included. These studies focused on participants who had been injured or groups composed entirely of athletes (e.g., football players, ballet dancers), and addressed medically treated injuries, the severity of which was poorly reported.\n\nAll three of the studies reported higher rates of injury in physically activity children and youth. There was clear evidence of a dose-response relationship between PA participation and the likelihood of injury. That is, as the PA level increased, the likelihood of injury increased in a graded fashion. More vigorous activity seemed to be related to high rates of injury.\n\nAge and gender differences have not been adequately studied.\n\n## Recommendations\n\nBased on the 2007 supplement \\[4\\], the evidence presented at the conference, the systematic review \\[5\\] and other materials, the Consensus Panel makes three recommendations for school-age children and youth. Following these recommendations should stimulate sound growth and development and confer protection against known risk factors for adult chronic disease.\n\n### Recommendation 1\n\nChildren and youth aged 5-19 years of age should accumulate at least 1 hour and up to several hours of at least moderate-intensity PA on a daily basis to achieve most of the health benefits associated with PA (evidence: level 3, grade A). Some health benefits can be achieved through 30 minutes\/day of moderate-intensity PA, and this should be used as a \"stepping stone\" for currently sedentary children (evidence: level 2, grade A).\n\n#### Interpretation of evidence and justification\n\nWith the exception of injuries, the collective evidence from observational studies for several health outcomes suggests that the majority of health benefits are achieved at the higher end of the PA spectrum. Normal lifestyle-embedded PA needs to be added to the purposeful PA most often used for the intervention studies. This justifies the optimal level of PA as \u2265 1 hour\/day, despite the lack of experimental evidence or randomized trials. Experimental studies for several health outcomes suggest that participating in as little as 2 or 3 hours\/week of at least moderate-intensity PA is associated with health benefits. This is supported by evidence from observational studies that have demonstrated dose-response relationships between PA and health outcomes, with differences in health risk between the least active (or fit) and the second least active groups. Thus, it would seem appropriate to set a minimal \"stepping stone\" level of 30 minutes\/day of moderate-intensity activity, which will confer some health benefits.\n\n### Recommendation 2\n\nVigorous-intensity activities should be incorporated or added when possible, including activities that strengthen muscle and bone (evidence: level 3, grade B).\n\n#### Interpretation of evidence and justification\n\nAlthough few studies have systematically evaluated the differential effects of various intensities of PA, the available information suggests that vigorous-intensity activities may provide additional health benefits beyond moderate-intensity activities. Furthermore, many of the experimental studies that observed significant changes in health variables prescribed exercise that would fall within the vigorous-intensity range (\u2265 7 METS) or in the upper end of the moderate-intensity range (5-7 METS).\n\n### Recommendation 3\n\nAerobic activities should make up the majority of the daily PA. Muscle- and bone-strengthening activities should be incorporated on at least 3 days of the week (evidence: level 2, grade A).\n\n#### Interpretation of evidence and justification\n\nMany of the health outcomes examined, particularly obesity and measures of cardiometabolic health, responded almost exclusively to aerobic-type activities. However, bone health was more favorably effected by modest amounts of resistance training and other high-impact activities (e.g., jumping) that were performed on at least 2-3 days of the week. It should be noted that the evidence supports the applicability of these recommendations through an age range of 5-19 years.\n\n# Summary\n\nThere is a level of consistency for the beneficial effects of PA across health outcomes. Some more recent, large, epidemiological studies that have used more robust measurement methods have suggested the strongest associations. The main recommendations (recommendations 1 and 2) reflect the linear or curvilinear dose-response association observable for some of the main health outcomes. In general, the three recommendations are justified by the level and strength of the evidence. Although not reviewed here, it is likely that reducing sedentary behaviour is important for health. The Canadian Paediatric Society recommends a maximum of 2 hours\/day of television-viewing time \\[12\\]. We endorse that recommendation.\n\n# Adults aged 19-65 years\n\n## Introduction\n\nTo assess the physical activity guidelines for asymptomatic adults a systematic, evidence-based approach was taken by the Consensus Panel. The evidence considered included the 2007 supplement \\[4\\], the systematic review presented at the consensus conference\\[6\\], and reviews conducted by scientists in the US \\[13\\] and UK \\[14\\]. The systematic review critically evaluated scientific literature published in English from January 1966 to March 2008 that examined the relationship between PA and all-cause mortality or PA and the incidence of one or more of seven chronic diseases (cardiovascular disease \\[excluding stroke\\], stroke, hypertension, colon cancer, breast cancer, type 2 diabetes, and osteoporosis) in adults aged 19-65 years. Any measure of PA (e.g., self-report, pedometer, accelerometer) or fitness (e.g., maximal aerobic power (VO2 max) was eligible for inclusion; however, only studies that presented at least three different levels of PA were included.\n\nIn adults, PA has been promoted for health benefits since the release of the *Canada's Physical Activity Guide to Healthy Active Living* in 1998 \\[1\\]. With the present review, the Consensus Panel examined evidence for the following:\n\n1\\. The relationship between PA and all-cause mortality.\n\n2\\. The relationship between PA and the incidence of seven chronic conditions: cardiovascular disease (excluding stroke), stroke, hypertension, colon cancer, breast cancer, type 2 diabetes, and osteoporosis. These conditions were identified *a priori* as diseases of major public health importance for which an evidence base exists.\n\n3\\. The dose-response relationship between PA and these health outcomes.\n\nThis review focuses on the primary prevention of the seven conditions in asymptomatic adults, not on the role of PA in secondary prevention or treatment. Canada's current recommendations for PA for adults aged 20-55 years are as follows \\[1\\]:\n\n1\\. Accumulate 20-60 minutes of PA every day in periods of at least 10 minutes each. The time needed depends on effort: light effort 60 minutes, moderate effort 30-60 minutes, and vigorous effort 20-30 minutes.\n\n2\\. Perform three types of activity to keep your body healthy: endurance activities (4-7 days a week), flexibility activities (4-7 days a week), and strength activities (2-4 days a week).\n\nA brief summary of the current evidence of the associations between PA\/fitness and health outcomes for adults is presented below.\n\n## Associations with health outcomes\n\nA total of 261 articles met the eligibility criteria. These examined the relationships between PA\/fitness and premature all-cause mortality (*N* = 72), cardiovascular disease (*N* = 51), stroke (*N* = 25), hypertension (*N* = 13), colon cancer (*N* = 34), breast cancer (*N* = 43), type 2 diabetes (*N* = 20), and osteoporosis (*N* = 2).\n\n### All-cause mortality\n\nA total of 2,040 relevant citations were identified, with 72 articles eligible for inclusion. These studies followed over 1,600,000 participants for an average of 11.7 years, and identified over 111,000 deaths (all-cause). The vast majority of studies used a prospective cohort design.\n\nA strong and consistent inverse relationship between PA and all-cause mortality was observed in both men and women, with a mean of 31% lower risk in the most active compared to the least active group. In studies that used objective measures of aerobic fitness, the risk reduction averaged 50%. The current Canadian guidelines (gross energy expenditure of approximately 4.2 MJ\/week, 1,000 kcal\/week) are estimated to be associated with at least a 20% lower risk for premature all-cause mortality \\[15-17\\]. An inverse curvilinear dose-response relationship is seen with both PA and fitness (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>).\n\n### Cardiovascular disease\n\nThe associations of PA with stroke and other cardiovascular disease were examined separately. For cardiovascular disease, a total of 9,408 citations were identified and 51 articles deemed eligible for inclusion in the systematic review. These studies followed 658,747 participants for an average of 14.6 years and observed 30,292 incident cases of disease. A consistent inverse relationship between PA and cardiovascular disease was seen, with a mean of 33% lower risk in the most active compared to the least active groups. This risk reduction was observed in both men and women and in Caucasian as well as non-Caucasian populations. In studies that used objective measures of aerobic fitness, the risk reduction averaged 50%.\n\n### Stroke\n\nA total of 1,104 citations that examined the relationship between PA and stroke were identified and 25 articles were deemed eligible for inclusion in the systematic review. These prospective cohort studies followed 479,336 participants for an average of 13.2 years and observed 12,361 cases of stroke. As with other cardiovascular disease, an inverse relationship was noted between PA and stroke, with a mean of 31% lower risk in the most active compared to the least active group. Greater risk reductions were observed in studies that measured fitness than in those that measured PA. The shape of the dose-response relationship between PA and stroke varied between studies, and it is uncertain if the inverse relationship seen with total and ischemic stroke also applies to hemorrhagic stroke.\n\n### Hypertension\n\nA total of 6,287 citations were identified that examined the relationship between PA and hypertension and 13 studies were included in the systematic review. These primarily prospective cohort studies followed 113,524 participants for an average of 8.7 years and observed 11,695 cases of hypertension. An inverse relationship was seen between PA and the incidence of hypertension, with the most active individuals experiencing a mean of 32% lower risk than the least active. The shape of the dose-response relationship, however, was unclear. Some evidence from the review and randomized controlled trials suggests that moderate to vigorous activity is required to produce an observable reduction in the incidence of hypertension. Furthermore, a body of evidence indicates that moderate-intensity resistance training can reduce blood pressure.\n\n### Colon cancer\n\nA total of 252 citations were identified that examined the relationship between PA and the incidence of colon cancer and 34 articles were included in the systematic review. These case-control, prospective cohort, and cross-sectional studies used diverse methods to measure both occupational and leisure-time PA. The studies involved a total of 1,322,900 participants and 14,625 cases of colon cancer. The length of follow-up in the 11 prospective cohort studies averaged 10.7 years. Although considerable variability in study findings is seen, those in the most active groups had, on average, a 30% lower risk of developing colon cancer than the least active. The shape of the dose-response relationship is unclear.\n\n### Breast cancer\n\nA total of 571 citations were identified that examined the relationship between PA and the incidence of breast cancer and 43 articles were included in the systematic review. These prospective cohort and case-control studies followed 1,860,660 participants and observed 80,138 cases of breast cancer. Follow-up for the prospective cohort studies averaged 10.5 years. An inverse relationship was seen between PA and the incidence of breast cancer, with the most active individuals experiencing, on average, a 20% lower risk than the least active. The majority of evidence suggests that 30-60 minutes\/day of moderate- to vigorous-intensity activity is required to reduce the incidence of breast cancer, and that the relationship is stronger in post-than in pre-menopausal women.\n\n### Type 2 diabetes\n\nA total of 3,655 citations were identified that examined the relationship between PA and the incidence of type 2 diabetes and 20 articles were included in the systematic review. These prospective cohort studies followed 624,952 participants for an average of 9.3 years and observed 19,325 cases of diabetes. A consistent inverse relationship was observed between levels of PA or fitness and type 2 diabetes. When comparing the most active\/fit to the least active\/fit group, an average of 42% risk reduction is seen. An inverse curvilinear dose-response relationship exists. Although it is difficult to separate exercise volume from intensity in the evidence available, small changes in PA levels yield substantial reductions in the risk of developing diabetes. Randomized controlled trials of individuals at high risk for the development of diabetes suggest that 150 minutes\/week of moderately vigorous activity results in a 58% reduction in the risk of developing diabetes.\n\n### Osteoporosis\/bone mineral density\n\nNumerous exercise intervention trials and systematic reviews have demonstrated that aerobic and resistance activities enhance bone mineral density across the lifespan and reduce the incidence of fractures. However, little research has examined the relationship between PA and the incidence of osteoporosis. Although 3,655 studies were identified in the systematic review only 2 were included. These studies revealed an inverse relationship between PA level and the prevalence of osteoporosis.\n\n## Recommendations\n\nBased on the 2007 supplement \\[4\\], the evidence presented at the conference, the systematic review \\[6\\] and other materials \\[13,14\\], the Consensus Panel makes three recommendations for adults aged 19-65 years.\n\n### Recommendation 1\n\nAdults aged 19-65 years should accumulate 150 minutes\/week of moderate-intensity PA or 90 minutes\/week of vigorous-intensity PA in periods of at least 10 minutes each. Greater amounts of activity and more vigorous activity provide additional benefits (evidence: level 2, grade A).\n\n#### Interpretation of evidence and justification\n\nThe evidence indicates that 150-180 minutes\/week of moderate or 90 minutes\/week of vigorous PA is associated with a 30% reduction in the risk of all-cause mortality and reductions in the incidence of cardiovascular disease, stroke, hypertension, colon and breast cancer, and type 2 diabetes. The inverse dose-response relationship seen with these conditions demonstrates that additional health benefits are observed with greater amounts of or more vigorous PA. The greatest benefit is seen when the PA is distributed throughout the week and in periods of at least 10 minutes' duration. This volume of exercise may also prevent weight gain in some individuals; however, greater volumes may be required for others.\n\n### Recommendation 2\n\nEngage in resistance activities on 2-4 days\/week (evidence: level 2, grade A).\n\n#### Interpretation of evidence and justification\n\nRecent reviews \\[4,13-15\\] have demonstrated that musculoskeletal fitness improves blood pressure, bone mineral density, mobility and functional independence, and overall quality of life, and reduces premature mortality and the risk of falls.\n\n### Recommendation 3\n\nEngage in flexibility activities on 4-7 days\/week (evidence: level 3, grade A).\n\n#### Interpretation of evidence and justification\n\nThe inclusion of flexibility activities as part of an adult's PA routine may enhance mobility and functional independence, and decrease the risk of falls.\n\n# Older adults aged \u2265 65 years\n\n## Introduction\n\nThe Consensus Panel reviewed several sources of evidence regarding PA and health in older adults (\u2265 65 years). These included the 2007 supplement \\[4,18\\], systematic reviews presented at the consensus conference \\[7\\], and reviews conducted by scientists in the US \\[13\\] and UK \\[14\\]. Collectively, these sources provide an extensive and thorough review of the literature on PA and health.\n\nCanada's current recommendations for PA for adults aged \u2265 64 years are as follows \\[2\\]:\n\n1\\. Increase endurance activities--4-7 days a week.\n\n2\\. Increase flexibility activities--daily.\n\n3\\. Increase strength and balance activities--2-4 days a week.\n\nA brief summary of the current evidence of the associations between PA\/fitness and health outcomes for older adults is presented below.\n\n## Associations with health outcomes\n\n### Morbidity from chronic disease and all-cause mortality\n\nExtensive reviews of studies on PA\/fitness and various health outcomes have been published \\[4,13-15\\]. These reviews reported strong, consistent, and biologically plausible evidence for the benefits of regular PA on numerous health outcomes. There is a dose-response gradient for morbidity and mortality across levels of PA. An example for cardiorespiratory fitness and all-cause mortality is shown in Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>\\[19\\]. With the least fit quintile as the reference group, individuals in the second quintile of fitness had approximately a 50% lower mortality risk. The risk of death continued to decline across other fitness groups, with the most fit 20% having approximately a 79% lower risk compared with the referent group. Numerous other studies \\[6,7,13-18\\] have shown similar dose-response relationships between PA or fitness for a variety of health outcomes, although the gradient tends to be steeper in fitness studies than in PA studies (see Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>). This is perhaps due to greater misclassification of the exposure to PA in the latter studies.\n\n### Functional independence and disability\n\nLow levels of PA in older adults are likely to lead to a decline in cardiorespiratory fitness and muscular strength that may reach levels that are incompatible with some aspects of functional independence. Thus, it is important to maintain cardiorespiratory fitness and muscular strength to prevent crossing \"functional thresholds\" \\[7\\].\n\nA systematic review that evaluated the relationship of the PA of healthy, community-dwelling older (\\>65 years) adults with outcomes related to functional limitations, disability, or loss of independence was presented at the consensus conference \\[7\\]. The review included epidemiological investigations and exercise-training intervention studies. A total of 66 articles on the relationship between PA and functional independence met the inclusion criteria for the review, and 34 articles included cognitive function as an outcome. The review did not include persons who, at baseline, had clinical conditions, functional limitations, disability, or frailty or who were \\>85 years of age. PA was assessed by self-report in most studies. Most studies presented data in two PA groups, but a few included three or four levels of PA. Two studies included cardiorespiratory fitness as the exposure. A few studies assessed muscular strength. Functional outcomes included self-reported functional ability or disability, self-reported PA limitations, and performance measures. Outcomes were typically reported in dichotomous categories. The review also included 35 prospective studies, with sample sizes ranging from 200-10,000 individuals. Follow-up ranged from 2.5 to 30 years. Physically active individuals had an approximately 50% lower risk of disability in activities of daily living and instrumental activities of daily living, with only two negative studies. Studies with more than two categories of PA tended to show an inverse dose-response trend.\n\nPhysically active individuals also tended to have a slower rate of functional decline, with those who were active at approximately 50% lower risk compared to the inactive. There was consistency across studies and across a wide range of outcome measures. The benefits were observed in both short-term and long-term follow-up. There was some evidence of a dose-response relationship.\n\nFourteen exercise-training studies were reviewed, and they were consistent in showing improved functional performance in the exercise groups. A majority of the studies used moderate-intensity aerobic PA as the intervention, typically walking on at least 3 days\/week. Some studies also combined aerobic exercise with resistance training, and reported greater functional improvements with combined aerobic and resistance programs versus a resistance or aerobic programs alone.\n\n### Cognitive function\n\nThe search found a total of 861 citations of which 32 met the inclusion criteria; an additional 2 papers were found from checking reference lists and the authors files, therefore 34 studies were included in the systematic review \\[7\\]. Cognitive measures varied widely across studies, and included reaction time, motor function, memory, executive function, and visual attention. Seventy-one percent (24\/34) of the studies showed a positive relationship between PA and some aspect of cognitive function. Prospective studies indicated a reduced risk of dementia and Alzheimer's disease in physically active individuals. The majority (7\/12) of the exercise-training studies showed a positive effect on at least one cognitive outcome.\n\n## Recommendations\n\nBased on evidence from the sources described the Consensus Panel makes three recommendations for PA for older adults. These recommendations apply to apparently healthy individuals and not to populations with clinical disease. Following the recommendations should reduce the risk of chronic disease, premature mortality, loss of function and disability, and cognitive decline.\n\n### Recommendation 1\n\nOlder adults should participate in moderate-intensity aerobic activity for a total of 150 minutes\/week, or in vigorous-intensity activity for a total of 90 minutes\/week. Moderate- and vigorous-intensity activities are defined as approximately 50% and 60-70% of maximal aerobic capacity, respectively. For many older adults, walking at 3.0-3.5 mph is a good example of moderate-intensity activity. The dose of PA described here is *in addition* to the routine and light-intensity activities of daily living, and can be expected to reduce the risk of several chronic diseases and premature death by 20-30%. Regular PA is also important for maintaining a healthy body weight. The PA recommendation presented here will be sufficient for many people to prevent weight gain, while others may need to engage in more PA to achieve this benefit. Obviously, caloric intake also must be considered as a tool for managing body weight.\n\nHigher doses of PA, and more vigorous-intensity activity provide additional health benefits. The weekly dose of PA may be accumulated in sessions of at least 10 minutes of moderate-intensity activity (evidence: level 2, grade A).\n\n#### Interpretation of evidence and justification\n\nA previous publication by Paterson et al. \\[18\\] and the systematic review presented at the consensus conference \\[7\\] provide strong evidence of the benefits of aerobic PA for older adults. The evidence comes from large cohort studies and controlled exercise trials. The evidence is highly consistent, with few negative studies. Regular aerobic PA has an inverse dose-response relationship with major chronic diseases (coronary heart disease, type 2 diabetes, depression, some cancers, dementia, disability, and loss of function). The recommended dose of aerobic PA reduces the risk of these conditions and functional limitations by 30-50%, and higher doses of PA provide further benefits.\n\n### Recommendation 2\n\nIn addition to the aerobic PA recommendation, older adults should also engage in resistance exercises on 2 days\/week. Resistance exercise should involve the major muscle groups of the body, and should consist of 8-12 repetitions at \\>60% of 1 repetition maximum (RM). Daily activities that involve lifting, carrying, and pushing tasks should be maintained because they can also benefit muscular and bone health (evidence: level 2, grade A).\n\n#### Interpretation of evidence and justification\n\nA previous publications by Paterson et al. \\[18\\] and a systematic review presented at the consensus conference \\[7\\] provide evidence for the benefits of resistance exercise in older adults. The major benefit of resistance training is preservation of muscle mass and prevention of age-related sarcopenia. The evidence supports the potential benefits of resistance training in preventing chronic diseases such as the metabolic syndrome and in delaying mortality. Greater strength--and particularly muscle power--results in maintaining function and potentially preventing disability.\n\n### Recommendation 3\n\nGood balance helps prevent falls, and older adults should participate in activities that improve and maintain balance. Such activities include dancing, walking on uneven surfaces such as a field or in a forest, and various exercises such as standing on one leg. Stretching exercises should be done regularly to maintain good flexibility. Inflexibility can interfere with routine daily tasks and in participation in leisuretime PA (evidence: level 2, grade A).\n\n#### Interpretation of evidence and justification\n\nThere is growing evidence that balance activities result in a lower risk of falling, a major health concern for more frail older adults. Balance training, along with activities to strengthen the muscles of the legs, is the best strategy to reduce falls and complications from falls. Stretching exercises will help maintain joint flexibility, which can help maintain function.\n\n# Adverse effects\n\nThere are extensive health benefits of regular PA, as discussed in detail in the three main sections of this paper. However it is important to be cognizant of the potential adverse events of PA. Fortunately, the risk of adverse events is low, especially for those doing moderate amounts and intensities of PA. The *2008 Physical Activity Guidelines for Americans* report that musculoskeletal injuries are the most common adverse event, but that the injury rate is approximately one event for every 1,000 hours of walking for exercise \\[13\\]. It is a bit higher, at four events per 1,000 hours, for running. Serious adverse events, such as cardiac arrest, are more likely during exercise than at rest, but are still relatively rare. Although serious adverse events are higher during the exercise period, the overall risk for regular exercisers is still substantially lower over the entire day than it is for non-exercisers. Thus, the benefits of regular PA outweigh the risks \\[13\\].\n\nIndividuals need to be aware of the potential risks of physical activity and should consult their primary-care provider if they have questions. Individuals should also be aware of the early warning signs of potential serious adverse events, such as chest pain or discomfort with exercise, dizziness, abdominal pain, or other major symptoms. In such cases, a medical care authority should be consulted. In addition, the risks of exercising with or shortly after a febrile infection should be understood and prevented.\n\n# Future research\n\nThe preparatory work for the revision of the Canadian guidelines has led to the identification of numerous questions for which adequate answers are not available in the literature and that warrant further research. Many of the questions raised here are of potentially great interest to extending our understanding of the relationship between PA and health. This section has restricted proposals for future research to questions that were felt to provide results that will improve the basis for future revisions of the PA guidelines.\n\n## General methodological issues\n\nBecause guidelines for PA aim at improving PA in segments of the population where the it is inadequate, there is clearly a need for more studies that provide solid evidence for how to make changes in the best way, and evidence for the health effects of these changes. Many of the studies that have formed the evidence for these guidelines did not directly address these fundamental questions and, moreover, were of rather poor or suboptimal quality for a variety of reasons. Thus, there is a need for more high-quality, large-scale, randomized intervention trials testing methods of increasing PA and testing the health effects of increasing PA where applicable.\n\nFor research questions that realistically cannot be addressed in randomized trials, such as the longterm effects of behavioural changes at the population level on morbidity and mortality, there is a similarly strong need for high-quality, large-scale, long-lasting, observational prospective cohort studies with repeated assessment of PA, allowing the identification of groups who have changed PA, confounders, and health outcomes. In both randomized trials and observational cohort studies, there is a need to apply far more precise and accurate measurement tools, especially for PA, but also for several health outcomes and potential confounders. Also, reporting of randomized clinical trials should be in accordance with the CONSORT guidelines or of non-randomized trials using the TREND guidelines \\[20\\].\n\n## Population segments\n\nConsidering the heterogeneity of the population in Canada, investigations of corresponding differences between groups within the population are needed with regard to the distribution of PA, conditions for improving PA, and the health effects of given amounts and intensities PA, all of which may justify different guidelines for various population groups. Such groups could be defined, for example, by sex, age range, ethnicity, socioeconomic status, family setting, disability, puberty, or pregnancy. With advances in genomic research, there is a prospect of defining groups by genetic profile. Groups may also be defined by characteristics that themselves can be manipulated or modified, such as other lifestyle factors. The best possible epidemiological-statistical tools (e.g. interaction analysis) should be applied to investigate whether it is really necessary to make distinctions between groups.\n\nIt must be taken into account that the more groups into which the population is divided, the more difficult it is to validly demonstrate differential effects. It is a major challenge to identify the optimal cost-benefit balance between interventions customized to particular groups--or even tailored to particular individuals--versus fewer and more broadly applicable interventions. A need for different interventions may apply to individuals who already suffer from various diseases, but PA in such groups should be separated into PA aimed at general preventive effects among these individuals and PA aimed at specific therapeutic effects for these individuals as patients (clinical guidelines).\n\n## Health outcomes\n\nPA is related to numerous health outcomes, many of which constitute the core of the evidence for the PA guidelines. However, there are also many other outcomes of potential relevance for which the evidence is too weak to provide a compelling argument for recommendations. These include, for example, mental health and cognitive ability, especially in children, adolescents, and the elderly; common musculoskeletal disorders that also create great demands on the health care sector, such as osteoarthritis and osteoporosis with related fractures; and chronic medical disorders such as chronic heart failure and chronic obstructive pulmonary disease. Obviously, the guidelines that are applicable to a single individual should not differ for different health outcomes. The task is to integrate the evidence for different health outcomes and thereby allow recommendations that are generally applicable irrespective of differences in relationships to different health outcomes.\n\nResearch that translates PA-related health outcomes to quality-of-life measures is needed with regard to immediate, short-term and long-term effects. This applies to all age groups and draws attention to definitions of good and poor health, which may need to be specific for different age groups, especially for young children and the oldest adults. The consistent finding of an inverse association between allcause mortality and PA should be extended to quality-adjusted life years or disability-free life years.\n\n## PA assessment\n\nCharacterization of PA requires measurements of frequency, duration, intensity, volume (intensity \u00d7 duration), and mode, all that vary over time intervals from seconds to years. Most studies so far have been based on self-reported (for children, parent-reported) PA, often very crudely categorized data, and this information about PA obviously suffers from several types of severe measurement error. This leads to misclassifications of individuals' PA and hence to biases in the assessment of the relationship between PA and health outcomes, between putative determinants of PA and PA, and between interventions aimed at changing PA and achieved changes in PA. It is a great challenge to obtain evidence based on better measurement technology. The current options are various types of devices that continuously monitor body movements (e.g., accelerometers); however, although these do provide a more objective measure of PA, they still do not cover all aspects of PA. Cardiorespiratory fitness has been used as a proxy for the cumulative effects of PA in the preceding period, but is also dependent on other factors (e.g., genetic constitution). Furthermore, there is a need to study the impact of a sedentary lifestyle as a separate entity from typically assessed PA, based on the suspicion that there are both determinants of and effects of a sedentary lifestyle that are not simply equivalent to \"low PA\" \\[21,22\\]. NEAT (non-exercise activity thermogenesis) may play an important role in mediating, moderating or determining the relationship between PA or sedentary behaviour and health outcomes and deserves further investigation \\[23\\].\n\n## Effect measurement and dose-response\n\nThe magnitude of effects and the dose-response relationship, both for given levels of PA and for changes in PA, needs more investigation based on better measurement technology. When assessing effects in general and their dependence on the starting level of risk, it is of particular importance to pay attention to the measures of effects (relative versus absolute effect, where the former is heavily dependent on the reference level of risk that defines the denominator of the measure). Assessment of dose-response functions requires specification of the definition of \"dose\" (better characterization of PA) and of \"response,\" including the time relationship (e.g., immediate, delayed, or cumulative investigations of the dose-response relationships should also address various compositions of the \"dose,\" for example whether very-low-intensity PA for longer periods can a substitute high-intensity PA for short periods. Statistically convincing distinctions between different shapes of dose-response curves, including determination of threshold effects, will require very large sample sizes.\n\n## Mediators\n\nIn the context of analytical epidemiological research (in both observational and interventional studies) addressing cause-effect relationships, a mediator is defined as a modifiable factor or variable that is influenced by the cause in question and thereby induces the effect that through the mediator is attributable to the cause. Obviously, causes may induce effects both through and beyond mediation of a particular mediator. From a biological or psychological point of view, the mediation process can be considered as the \"mechanism\" or \"pathway\" by which the PA is linked upstream to determinants of PA and downstream to health effects. Understanding the mediation process may enhance the opportunities for interventions to improve PA, and for increased PA to improve health, not least because different types of PA may be related to different mediators \\[24,25\\]. The current evidence on mediation is rather limited, and there is a great need for more research, both regarding the mediation of interventions aimed at changing PA (particularly psycho-behavioural factors, e.g., self-regulation and self-efficacy) and the mediation of changes in PA on health outcomes (e.g., via biological coronary heart disease risk factors, cardiorespiratory fitness, energy expenditure). Demonstration of the associations between a putative mediator and the cause, and between this mediator and the effect, may not suffice as evidence for mediation.\n\n## Hazards and adverse effects\n\nAlthough the evidence so far indicates that there are few hazards or adverse effects of increasing PA or keeping a high level of PA, the possibility of adverse effects should be kept in mind in conducting future studies; for the sake of transparency, developing clinical practice guidelines and when building evidence for increasing PA.\n\n## Special biases\n\nIn addition to the usual type of biases that affect randomized trials and cohort studies, some special biases need consideration--namely, those based on continuously operating behaviours, such as PA, as determinants of concurrent or later chronic disease health outcomes, such as obesity and coronary heart disease, respectively. Reverse causation implies that the disease process is already ongoing and, although not yet clinically diagnosed, may lead to changes in PA or associated confounders. For a condition such as obesity, there is the additional problem that this condition tends to fluctuate (statistically with the effect of regression toward the mean) and behavioural lifestyle factors (e.g., PA) may change in response to this fluctuation \\[26,27\\]. Therefore, an apparent association may be created between these lifestyle factors and subsequent changes in the opposite direction.\n\n## Systematic review process\n\nThe systematic review process is of fundamental importance to the synthesis of evidence forming the basis for guidelines \\[11,28\\]. Where the outcome of a review can be supported by a meta-analysis providing pertinent quantitative effect estimates, this should be done. Adherence to internationally well-established standards for systematic reviews and meta-analyses, for example as developed and implemented by the Cochrane Collaboration, will increase the value and trustworthiness of the output of the process. Particular attention needs to be paid to the correspondence between the aim of the review and the criteria for literature selection in order to avoid missing potentially informative, high-quality studies. Where available, previously performed systematic and high quality reviews should be taken as the starting point so that the review process can take advantage of what has already been done by others, while of course maintaining a critical analytical approach \\[29\\].\n\n## Scoring systems\n\nThe application and interpretation of scoring systems for the quality and message of the evidence to the output should be based on scales that are adapted to the type of evidence obtainable. The current scoring systems are typically based on an assessment of evidence that can be generated in large-scale therapeutic randomized trials (e.g., drug trials). However, it is not possible to use such trial designs when addressing questions about the long-term effects on mortality of behavioural preventive measures such as PA. Future research should explore how existing medical-oriented evidence assessment procedures require modification to accommodate population and lifestyle behavioural modifications. One example of such modifications is that assessment of the intervention by comparative trial design may have to be based on randomisation of a few clusters in which the intervention is set up by facilitating or encouraging physical activity in various ways in institutions or regions. Another possibly necessary modification is that the evaluation of the outcome rather than being based on changes in risk of clinically relevant endpoints, namely morbidity and mortality, may have to be based on what else would be considered as surrogate endpoints such as well being, improved fitness and strength, improved risk factor profile, e.g. improved blood pressure, blood lipids, glucose tolerance.\n\n## Harmonization of physical activity guidelines\n\nGuidelines for PA are being prepared by governmental and non-governmental organizations in many countries (e.g., US, UK) and by several international institutions (e.g., the World Health Organization). Future revisions of guidelines in all countries should take advantage--alongside the systematic reviews being conducted conditional on previous reviews--of comparisons between different guidelines in order to identify the reasons for differences (e.g., evidence, cultural, autonomy) and use this information to contribute to the improvement of future guidelines. In the process of such harmonization it would also be of great value to make the rationale and criteria applied for choosing particular thresholds for duration and intensity of recommended PA of particular types explicit, given that the available evidence seldom leads to unambiguous thresholds and that feasibility in the target populations necessarily must be considered as well.\n\n## Healthy lifestyle approach\n\nLifestyle risk factors, such as PA, diet, smoking, alcohol intake and sleep, are clustered and possibly interact, both in terms of their determinants (lifestyle habits may substitute for each other) and effects on health. There is an obvious need for a healthy lifestyle approach in researching these factors and thereby forming the basis for more comprehensive guidelines that may be adequate for large segments of the population \\[30,31\\].\n\n# Conclusions\n\nPublic health challenges exacerbated by low levels of physical activity and high levels of sedentary behaviour make the careful preparation and dissemination of physical activity guidelines particularly important. Research evidence completed since the release of Canada's Physical Activity Guidelines \\[1-3\\] suggests the guidelines were appropriate but may now need some minor revisions to remain evidence-informed. Almost all people without existing disease can benefit from an increase in physical activity, including predominantly aerobic activity, but also activities to improve strength\/resistance\/power and flexibility. Adverse effects of an increase in physical activity are relatively rare and usually minor, leading to a net health promoting effect. Further research, with better measurement methodology and improved study quality, is required to better describe and detail the relationships among PA, sedentary behaviour and health outcomes to continue to inform physical activity guidelines for populations and various population sub-groups.\n\n# Competing interests\n\nTIAS is collaborating with various industries on obesity research, see here <http:\/\/www.ipm.regionh.dk\/person\/tias\/Disclosures.html>. The other authors declare that they have no competing interests.\n\n# Authors' contributions\n\nAll authors had an equal role in the formulation and drafting of this manuscript. All authors have read and approved the final manuscript.\n\n## Acknowledgements\n\nThe authors, Antero Kes\u00e4niemi, Chris J. Riddoch, Bruce Reeder, Steven N. Blair, and Thorkild I.A. S\u00f8rensen, comprised the Consensus Panel for the Canadian Society for Exercise Physiology.\n\nThe Consensus Panel would like to acknowledge the assistance of Ashlee McGuire and Mark Tremblay in the preparation of this manuscript. The Consensus Panel would also like to thank the authors of the systematic reviews and Mary Duggan of the Canadian Society for Exercise Physiology for making the work of the Panel as efficient as possible. Production of this paper has been made possible through a financial contribution from the Public Health Agency of Canada. The views expressed herein do not necessarily represent the views of the Public Health Agency of Canada.","meta":{"dup_signals":{"dup_doc_count":101,"dup_dump_count":48,"dup_details":{"curated_sources":8,"2021-39":1,"2020-05":1,"2019-47":1,"2019-35":1,"2018-51":1,"2018-43":1,"2018-34":1,"2018-30":1,"2018-22":1,"2018-09":1,"2018-05":1,"2017-39":1,"2017-30":2,"2017-22":1,"2017-17":1,"2017-09":13,"2017-04":1,"2016-50":1,"2016-44":1,"2016-40":1,"2016-36":1,"2016-30":1,"2016-22":1,"2016-18":1,"2015-48":2,"2015-40":2,"2015-35":2,"2015-32":2,"2015-27":2,"2015-22":2,"2015-14":2,"2014-52":2,"2014-49":3,"2014-42":6,"2014-41":3,"2014-35":4,"2014-23":4,"2014-15":4,"2022-21":1,"2024-10":1,"2017-13":1,"2015-18":2,"2015-11":2,"2015-06":2,"2014-10":2,"2013-48":2,"2013-20":2,"2024-22":1}},"file":"PMC2876061"},"subset":"pubmed_central"}
{"text":"author: Sanjay Rajagopalan; Robert D. BrookCorresponding author: Sanjay Rajagopalan, <firstname.lastname@example.com>.\ndate: 2012-12\nreferences:\nsubtitle: : Mechanistic Insights\ntitle: Air Pollution and Type 2 Diabetes\n\n# Epidemic of Cardiometabolic Disease in Developing Nations: A Threat to Global Prosperity\n\nAccording to the International Diabetes Federation in the year 2011, diabetes mellitus (DM) affects at least 366 million people worldwide, and that number is expected to reach 566 million by the year 2030. Over 99% of all diabetes cases represent type 2 DM with most of these projected to occur in low- to middle-income countries. Technology innovations, globalization with its free movement of food and services, seismic shifts in agrarian practices, and nutritional transition to freely available high-caloric diets have irrevocably altered energy expenditures during work and leisure. These and other factors are helping to foster the continued epidemiological transition occurring across the globe. Scientific effort over the last few decades has focused primarily on components of urbanization such as inactivity and dietary factors. More recent observations have provided additional links between exposure to environmental factors in air\/water and propensity to chronic diseases (1). This issue is of importance given the extraordinary confluence of high levels of airborne and water pollutants in urbanized environments. Multiple studies in China, India, and other rapidly urbanizing economies demonstrate a steep gradient in urban\u2013rural prevalence.\n\nThis review will summarize recent evidence on how outdoor air pollution may represent an underappreciated yet critical linkage between urbanization and the emergence of cardiometabolic diseases, with a focus on type 2 DM. We define cardiometabolic disease as the confluence of cardiovascular disease and type 2 DM in recognition of the fact that the milieu of diabetes fundamentally alters the pathophysiology of coronary, cerebrovascular, and peripheral arterial disease. Thus, alteration in susceptibility to DM automatically increases the likelihood of cardiovascular disease. Indoor air pollution is not discussed owing to the paucity of data. It should be noted that our current understanding of air pollution\u2013mediated cardiometabolic disease is derived from outdoor air pollution studies, with there being no good reasons to believe that the dose-response relationship to indoor air pollution will be any different. An understanding of potentially reversible environmental factors responsible for this rapid burgeoning of cardiometabolic disorders among developing nations is crucial in order to devise a societal response that is proportionate and adequate (2). In this review, the association between air pollution and type 2 DM is discussed unless this distinction cannot be made in the cited study (typically health registry data sets).\n\n# Exposure to Environmental Toxins and Metabolic Disease\n\nEpidemiologic studies that have attempted to investigate environmental factors that accentuate risk for development of cardiometabolic disorders have uncovered a number of factors other than traditional suspects related to diet and exercise. These variables include factors such as stress (mental and emotional), cultural and socioeconomic variables, chronic low-grade infection, and environmental pollutants (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<\/a>). In many instances, these factors are strongly correlated, rendering isolation of cause and effect difficult.\n\nThe plausibility that environmental exposures are linked to metabolic disease is exemplified by persistent organic pollutants, toxins that have consistently shown to associate with insulin resistance (IR) and type 2 DM. Prospective cohort studies of subjects exposed to 2,3,7,8-tetrachlorodibenzo-*p*-dioxin or other persistent organic pollutants in occupational and other settings have reported increased risk of DM and IR (1,3). Air pollution in Asia, Latin America, and Africa is a significant public health burden, especially given the often extraordinarily high concentrations of pollutants (e.g., particulate matter), high population density, and pervasive nature of air pollution. <a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a> lists the top countries for particulate matter (PM) air pollution in the world, all of which have rapidly urbanized populations based on a World Health Organization (WHO) database that reviewed pollution data in \\>1,100 cities in 91 countries. The mean annual average for the top 10 countries is roughly fivefold higher than the U.S. National Ambient Air Quality Standard of 15 \u03bcg\/m^3^ for PM \\<2.5 \u03bcg mass (PM~2.5~) and the WHO standards of 10 \u03bcg\/m^3^. Given the worldwide burden of air-pollution effects and their continuous and omnipresent nature, even small adverse health associations for individuals represent an enormous public health issue that deserves broad changes in public health policy (4).\n\nAnnual PM data from 47 countries with the highest reported PM~10~ levels\n\n![](3037tbl1)\n\n# Epidemiologic Evidence Linking Air Pollution and Type 2 Diabetes\n\nThere are now at least six published epidemiologic studies showing some degree of association between PM- or traffic-related air pollutants and DM. At least two other studies have demonstrated a relationship between ambient levels of air pollutants and markers of insulin sensitivity in humans. The main characteristics and principal findings of these studies are summarized in <a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a>. In most of these studies, no distinction was made between type 2 DM and type 1, and hence, the overall prevalence of DM is described. Because the vast majority of patients with diabetes are type 2, the associations likely describe an effect of type 2 DM.\n\nEpidemiological associations among air pollutants, diabetes, and insulin resistance\n\n![](3037tbl2)\n\nThough not all findings from every study were positive, taken together, the majority of observations support an association between air pollution, in particular traffic-related sources, and DM. Nonetheless, not all aspects of this relationship have been consistently reported nor are they fully elucidated at this time. The varying associations noted between studies may relate to numerous differences. These include the population characteristics, risk factors, individual susceptibilities, robustness of the cohort data, prevalence of type 2 DM, technical aspects of the exposure assessment methodologies, pollution types\/sources, and the degree and duration of air-pollution exposures. The sex-specific differences seen in some of these studies may relate to true differences in biologic susceptibility, a finding mirrored by observations in the Women's Health Study that also demonstrated a greater susceptibility of obese women to air pollution\u2013mediated cardiovascular events (5). In contrast, it is also possible that the sex predilection may relate to exposure assessment error, particularly in males, who tend to be more mobile compared with females. Additional considerations include cosegregation of factors such as low socioeconomic status, stress, and poorly characterized pollutants often pervasive in the urban environment.\n\n## Type 2 DM and metabolic syndrome as a susceptibility factor for air-pollution effects.\n\nDiabetic patients have previously been shown to be more susceptible to air pollution\u2013induced cardiovascular morbidity and mortality (6,7). A few studies have examined the underlying mechanisms. In a study in Boston-area residents, 6-day moving averages of PM~2.5~ and black carbon (BC) were associated with decreased vascular reactivity among patients with diabetes (8). Effects were stronger in type 2 than type 1 DM. In another study in Boston involving patients participating in unrelated clinical trials who provided blood samples, BC concentrations from a regional monitoring station were significantly associated with increased levels of inflammatory markers (9). Although many of the estimates were imprecise owing to limited sample size, the overall trend of the point estimates was positive, consistent with epidemiological and experimental data. These data are supported by prospective panel studies in a small population of type 2 DM patients. Flow-mediated dilatation decreased with PM~2.5~ during the first 24 h. These PM~2.5~-associated decrements in endothelial function were greater among participants with high hemoglobin A~1c~, low adiponectin, and elevated myeloperoxidase levels on the examination day (10). Thus, alterations in vascular tone and inflammation may represent potential mechanisms that may explain susceptibility.\n\n# Experimental Evidence of Mechanisms of Diabetes Association With Air Pollution\n\n## PM~2.5~ as a mediator of endothelial dysfunction and IR.\n\nAir-pollution exposure alters endothelial function in both animals and humans (11,12). Alterations in endothelial function often precede changes in IR and have been implicated in reduced peripheral glucose uptake (13). In the first experimental investigation directly linking inhalational exposure of PM~2.5~ with DM, exposure in conjunction with high-fat diet feeding, increased fasting, postprandial glucose, insulin, and Homeostasis Model Assessment-IR (HOMA-IR) measures. The changes in IR measures seen with PM~2.5~ were incremental to that of high-fat diet alone over a period of 24 weeks (14). The mean concentration of PM~2.5~ was 60 \u00b1 5 \u00b5g\/m^3^ (\u223c10-fold concentration from ambient levels). Tumor necrosis factor (TNF)-\u03b1, interleukin-6 (IL-6), resistin, and leptin levels were elevated following PM~2.5~ exposure, in keeping with a proinflammatory insulin-resistant state. PM exposure also resulted in elevations in prothrombotic adipokines such as plasminogen activator inhibitor 1 and increased circulating adhesion molecules such as intracellular adhesion molecule-1 and E-selectin. The latter are important in promoting leukocyte adherence in postcapillary venular endothelium (15). PM exposure was associated with impairment in phosphatidylinositol 3-kinase\u2013Akt\u2013endothelial nitric oxide synthase signaling in the aorta and decreased tyrosine phosphorylation of IRS-1 in the liver (15), providing evidence for abnormal insulin signaling in the vasculature.\n\nIn subsequent experiments, the effect of PM~2.5~ exposure early in life with and without concomitant exposure to a high-fat diet was evaluated. C57BL\/6 mice fed a normal diet but exposed for 10 weeks exhibited metabolic abnormalities including an increase in HOMA-IR and postprandial glucose that approached those seen with high-fat chow diet\u2013fed mice exposed to filtered air (16). In another study, intratracheal exposure of PM~2.5~ potentiated IR at the end of 3 weeks in high-fat\u2013fed male Sprague-Dawley rats (17). Taken together, these experiments suggest an important interaction of PM~2.5~ exposure with high-fat diet, and they raise the possibility that early life may represent a vulnerable period of enhanced susceptibility to PM~2.5~ exposure effects. There are currently no studies on the effect of air-pollution exposure on \u03b2-cell function.\n\n## Inflammation including visceral adipose tissue effects of air pollution.\n\nThere is evidence that exposure to ambient PM can be associated with elevated systemic proinflammatory biomarkers. Several reports have detailed association between day-to-day variation in acute-phase proteins, such as C-reactive protein (CRP), IL-6, fibrinogen, or white blood cell counts and circulating soluble adhesion molecules as reviewed previously (4). In an analysis of 1,003 myocardial infarction survivors, ambient particle number concentration and PM with diameter \\<10 \u03bcm (PM~10~) were associated with increased IL-6 and fibrinogen (18). Pollutants associated with primary combustion (e.g., elemental and BC, primary organic carbon) and ultrafine particles rather than PM~2.5~ appeared to be strongly associated with adverse responses. A positive association between white blood cell count and estimated long-term 1-year exposure to PM~10~ was reported in the Third National Health and Nutrition Examination Survey (19). Among 4,814 adults in Germany, small increases in annual mean PM~2.5~ (3.9 \u03bcg\/m^3^) were associated with increases in high-sensitivity CRP by 24% and fibrinogen by 4% among men. It is important to note that several studies, including some with improved exposure assessment (20) and large population cohorts (21,22), have not found a relationship between particulate exposure and inflammation. It is thus conceivable that variations in the particulate chemistry and duration\/intensity of exposure as well as susceptibility factors may be at play. Subjects with underlying risk factors such as type 2 DM and metabolic syndrome may exhibit stronger associations (19,23,24). As pointed out earlier, type 2 DM may represent a unique susceptibility factor that may potentiate inflammation (8,9).\n\nIn experimental animal models, PM~2.5~ exposure results in an increase in adipose tissue macrophages with a shift to a proinflammatory phenotype characterized by an increase in TNF-\u03b1 and IL-6 and a decrease in IL-10 gene expression (15). The finding of increased innate immune cells in visceral adipose tissue (VAT) is a pathophysiologic hallmark of type 2 DM. To test the mechanism by which increased proinflammatory monocytes permeate VAT, a transgenic model of yellow fluorescent protein (YFP) expression, driven by a monocyte-lineage promoter (and therefore restricted to monocytes, c-fms^YFP^), was employed to follow the migration of cells into the VAT compartment. The animals were initially rendered insulin-resistant with a high-fat diet and then subject to air-pollution exposure. Intravital microscopic studies were conducted to detect leukocyte\u2013endothelial interactions. Pollution exposure resulted in a doubling in the number of endothelial adherent YFP^+^ cells in mesenteric fat with a sixfold increase in monocytes within adipose (15). Thus, PM mediated adhesion and migration of YFP^+^ cells into visceral fat depots. These changes in adipose occurred with concomitant low-grade inflammation in the lung (25). In subsequent studies, PM~2.5~ exposure alone (normal chow diet) resulted in a heightened chemotactic ability of adipose tissue from PM~2.5~-exposed mice (16). In keeping with the well-known link between air-pollution exposure with oxidant stress, PM~2.5~ exposure was associated with oxidative stress in VAT and increased phosphorylation of a key cytosolic subunit of NADPH oxidase, and p47. p47^phox\u2212\/\u2212^ mice were protected from the effects of PM~2.5~ exposure and did not exhibit impairment in IR, vascular function, and visceral inflammation in response to PM~2.5~ (16). Thus, data from experimental animal models suggest that air pollution exposure may direct an innate immune response in VAT.\n\n## Hepatic IR and endoplasmic reticulum stress.\n\nDefective insulin signaling in tissues such as the liver are fundamental to the pathogenesis of IR\/DM. Increased serine phosphorylation of IRS-1 and decreased tyrosine phosphorylation results in defective phosphatidylinositol 3-kinase\u2013Akt signaling and suppression of insulin-stimulated GLUT4 translocation. PM~2.5~ exposure decreases phosphorylation of Akt in the liver and skeletal muscle compared with filtered air\u2013exposed control, and these changes were accompanied by hepatic lipid deposition and decreased gluconeogenesis (26,27). Endoplasmic reticulum (ER) stress, also called unfolded protein response (UPR), is an evolutionarily conserved pathway designed to alleviate protein misfolding in response to diverse pathophysiologic stressors (28). In vitro-exposure studies have demonstrated that PM~2.5~ is capable of inducing ER stress and the UPR in vitro and may be a mechanism by which PM~2.5~ exerts toxicity. PM exposure results in significant increase in the UPR-associated proteins activating transcription factor (ATF) 4, heat shock proteins 70 and 90, and binding immunoglobulin protein (BiP). In response to inhalational PM~2.5~, glucose regulatory peptide 94 and BiP increase in lungs and liver compared with minimal induction in aorta and spleen, indicating activation of the ATF6 pathway in these organs (14). ATF6 is one of three key main sensors of ER stress (the others being: inositol requiring 1a and double-stranded RNA-activated protein kinase-like ER kinase \\[PERK\\]). Phosphorylated PERK and eukaryotic translation initiation factor 2a were increased in the liver along with induction of C\/EBP homologous transcription factor CHOP\/GADD153 (14). The latter correlated with apoptosis in the lung and liver. The UPR is known to intersect with a variety of inflammatory and stress-signaling systems including the nuclear factor (NF)-\u03baB and c-Jun *N*-terminal kinase pathways as well oxidative stress responses, all of which may influence lipid and glucose metabolism. In these studies, a critical role for oxidant stress mediated via NADPH oxidase in activation of the ER stress response was also demonstrated (14). In a subsequent article, Zheng et al. (27) have demonstrated that PM~2.5~ exposure causes a nonalcoholic steatohepatitis-like phenotype and reduction of hepatic glycogen storage in animals. PM~2.5~ exposure lead to activation of the inflammatory pathway through c-Jun *N*-terminal kinase and downregulation of the insulin receptor substrate 1 (IRS1)-mediated signaling and peroxisome proliferator-activated receptor \u03b32 expression in the liver. These changes were associated with abnormalities in IR and glucose homeostasis (27).\n\n## Mitochondrial dysfunction and brown adipose tissue alterations.\n\nRecent data in adult humans suggest an important link between brown adipose tissue (BAT)\u2013mediated thermogenesis and obesity. Defective fatty acid metabolism through \u03b2-oxidation in mitochondria lead to accumulation of intracellular metabolites, including fatty-acyl CoA, diacylglycerol, and ceramide in both skeletal muscle and liver contributing to IR\/DM (29). Prior studies have shown that both cigarette smoke and hypercholesterolemia greatly increase mitochondrial damage (30). With long-term exposure to PM~2.5~ (10 months), visible decreases in interscapular BAT and mitochondrial size were noted (25). These changes were accompanied by an increase in excess oxidative and nitrosative stress in BAT, coordinated with phase II antioxidant gene induction, including NF-E2\u2013related factor 2, NAD(P)H quinone oxidoreductase 1, and glutamate-cysteine ligase modifier subunit. BAT expression of Ucp1 and peroxisome proliferator\u2013activated receptor \u03b3 coactivator 1\u03b1 were decreased with PM~2.5~ exposure, whereas Prdm16, Pgc-1\u03b1, and Ppar\u03b32 were significantly decreased in the white adipose tissue, suggestive of downregulation of pathways that modulate insulin sensitivity in adipose (31). Similar results were also seen with a different model of IR (31).\n\n# Integrated Model of Mechanisms Involved in Air Pollution\u2013Mediated Diabetes\n\n## How may signals perceived in the lung lead to metabolic abnormalities?\n\nThe notion that alteration in lung function may lead to metabolic dysfunction is not new (32). Chronic inflammation is a sine qua non for type 2 DM and obesity (metaflammation), is well known to occur with air-pollution exposure, and may represent a potential link between air-pollution exposure and metabolic dysfunction. Type 2 DM in humans and animal models is associated with increased levels of inflammatory cytokines and recruitment and\/or activation of innate immune cells in depots such as visceral adipose. Animal models of type 2 DM\/obesity have provided strong evidence that diet-induced oxidative stress\/inflammation plays a critical role in the pathogenesis of type 2 DM (33). In addition to classical inflammatory pathways, air pollutant\u2013mediated alterations in autonomic balance may further exacerbate systemic insulin resistance via overactivity of the sympathetic nervous system. Numerous pulmonary receptors such as transient receptor potential ankyrin 1 can be stimulated by pollutant inhalation and prompt sympathetic activation through centrally mediated pathways (34). The robust literature linking PM exposure with impaired heart rate variability supports such a mechanism (4). Unpublished observations from one of our studies demonstrate that elevations in ambient levels of fine PM in the Detroit area were associated with worsening of insulin sensitivity among healthy adults over a 5-day period of exposure (S.R., R.D.B.). Reductions in heart rate variability were linked to both exposures and the worsened insulin sensitivity, suggesting that (at least relatively acutely) autonomic pathways might be contributing to PM-induced alterations in metabolic insulin sensitivity. Other potential pathways that have not as of yet been explored may also be involved. Plausible mechanisms that could be impacted by air pollutants include activation of hypothalamic\u2013pituitary\u2013adrenal axis, impaired insulin-sensitive tissue perfusion due to endothelial dysfunction or vasoconstriction, dysfunctional activity of high-density lipoprotein particles, and\/or epigenetic alterations of critical modulators of cellular insulin signaling (e.g., PGC-1\u03b1).\n\n## Toll-like receptors\/nucleotide-binding oligomerization domain-like receptors as sensors for environmental signals: links between immunity and type 2 DM.\n\nToll-like receptors (TLRs) and nucleotide-binding oligomerization domain-like receptors (NLRs) are widely expressed on immune cells, with a substantive body of evidence linking these pathways in experimental and human obesity\/DM (35,36). In contrast to dietary signals, the alveolar macrophage and bronchial epithelial cells are principal initial cellular sensors of PM and express TLRs and NLRs. Biologic components intrinsic to PM such as lipopolysaccharide (LPS) and peptides and gaseous copollutants such as ozone can directly activate TLRs (37,38). The NLR Nalp3 has been shown to sense a diversity of particulate components and induce production of IL-1\u03b2 (39). Although the level of LPS is lower in PM~2.5~ versus PM~10~, there are data linking levels of such components with IR. In a recent prospective study by Sun et al. (40) in an urban population in Shanghai, an important predictor for the development of type 2 DM on multivariate analysis after adjustment of most risk factors including CRP was LPS binding protein. LPS binding protein is a better surrogate for LPS in plasma, and emerging studies suggest that this may serve as a surrogate for inflammatory disorders resulting from activation of the innate immune system (38,41). Endogenous danger-associated molecular patterns (DAMPs) that are released in response to PM may represent additional mechanisms for TLR\/NLR activation that may potentiate already overactive pathways in obesity\/IR. In a recent study, a key role for lipotoxicity-associated ceramide accumulation in the pathogenesis of type 2 DM via activation of Nlp3 was demonstrated (36). A number of DAMPs released in response to PM and\/or gaseous components have been demonstrated, including oxidized phospholipid components and hyaluronan fragments (26,37). Oxidation products of palmitoyl-arachidonyl phosphocholine, an abundant phospholipid in lung lavage fluid, has been implicated in a diverse variety of lung-injury signals to activate TLR4 (42). Release of oxidized palmitoyl-arachidonyl phosphocholine may facilitate innate immune activation in the lung and function as a mechanism to release chemokines such as CC chemokine ligand (CCL)-2 that may then secondarily mediate efflux of inflammatory monocytes (43). Ozone exposure in animal models may mediate degradation of hyaluronan, which can then activate TLR4 via myeloid differentiating factor 88 pathways (37). Thus, oxidized phospholipids, hyaluronan fragments, and possibly ceramide as a consequence of diet and\/or air-pollution coexposure may represent mechanisms that couple IR\/type 2 DM development with air pollution.\n\nHow may innate immune signals in the lung secondarily lead to systemic inflammation susceptibility to IR\/type 2 DM? In this regard, a number of key pathways have been posited: *1*) direct inflammatory\/oxidative stress of cells such as alveolar macrophages particularly under conditions of overload typified by continual exposure to PM may release innate immune cytokines such as IL-1, TNF-\u03b1, IL-6, and chemokine triggers from the lung, including CCL-2 and CCL-5, that may mediate a cellular response from the bone marrow\/spleen (26,44). Oxidant stress may be critical in activation of these pathways (26), with prior studies demonstrating a role for NADPH oxidases in TLR4- and Nalp3-mediated inflammation (39,42,45). *2*) Uptake by macrophages of particulates may also lead to presentation via dendritic cells to T cells in secondary lymphoid organs resulting in adaptive immune responses (43). *3*) Direct penetration of leechable components such as reactive oxygen species\/transition metals and stable organic compounds (e.g., quinines, semiquinones, and aldehydes) into the systemic vasculature has also been hypothesized and may lead to vascular inflammation and potentially IR (46,47). *4*) Finally, emerging data support a role for central mechanisms for inflammation via afferent pathways linking the lung with the brain (34). <a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<\/a> provides a hypothetical framework for these interactions and illustrates how inhalational stimuli may interact with overnutrition to entrain a state of chronic oxidative stress and inflammation.\n\n## Homeostatic mechanisms to prevent exuberant lung inflammation.\n\nTeleologically, it is thought that pattern recognition receptors were meant to represent a crude but critical early-warning system to rapidly sense changes in lung microenvironment but also, equally importantly, to dissipate early to prevent unfettered inflammation. Thus, the notion that continual activation of these receptors may occur in a feed-forward manner and in concert with other stimuli without dissipation may be somewhat simplistic. However, it is also true that as humans, we did not evolve to be continually exposed to dietary and inhalational stimuli over the years, and such chronic exposure in vivo may have very different effects that we insufficiently understand. Alveolar macrophages continually exposed to PM may result in particle overload resulting in a state of perpetual low-grade inflammation. Additional counterregulatory mechanisms that prevent excess TLR activation including proteins and phospholipids in the bronchoalveolar fluid may also be rendered dysfunctional with chronic PM exposure and participate in inflammation. The protein component of bronchoalveolar lavage includes the collectins such as surfactant A and surfactant D, both of which inhibit TLR4 and TLR2 signaling and upregulate anti-inflammatory functions including efferocytosis and dissipation of oxidant stress. Surfactants may play a dual role in the presence of DAMPs and facilitate inflammation (48). Oxidative modification of surfactants and phospholipid components has been reported that may lead to a facilitatory role in air pollution\u2013mediated effects (43,49,50).\n\nEmerging data from prospective human studies suggest a relationship between surfactant proteins (SPs) and propensity to inflammation and atherosclerosis. In one study, serum SP-D concentration was significantly decreased in subjects with obesity and type 2 DM (*P* = 0.005) and negatively associated with fasting\/postload glucose, HbA1C, plasma triglycerides, insulin sensitivity, and inflammation (TNF-R levels) (51). Smoking subjects, in contrast, showed significantly higher serum SP-D concentration than nonsmokers. In the Vancouver Angiography cohort, plasma SP-D levels correlated with cardiovascular mortality, and values \\>176 ng\/mL associated with a fourfold excess risk. Addition to SP-D levels to traditional risk factors improved c-statistic (from 0.76 to 0.78) and net reclassification across all levels of risk (52). In the Dallas Heart Study, increasing levels of SP-B was associated with other traditional cardiac risk factors and higher levels of inflammatory biomarkers. In multivariable analyses after adjusting for risk factors, SP-B remained associated with aortic plaque in smokers (odds ratio 1.87, fourth versus first quartiles; *P* \\< 0.0001) (53).\n\nHow does one reconcile increases in plasma SP levels in population studies to increase susceptibility? Increased levels of surfactants in plasma seen in smoking and lung inflammation have been hypothesized to indicate translocation from the lung to the circulation with lung damage. Surfactants A and D are assembled as large multimeric units composed of lectin-containing globular domains and a collagenous domain. In the presence of DAMPs, they may exert proinflammatory effects by binding to CD47 (thrombospondin receptor) (48). It is also highly possible that increased levels may indicate oxidatively modified forms of surfactant that are not functional. Surfactants are often assembled as multimers and are well-known to undergo oxidative modification to oligomeric forms. Current assays for SPs do not distinguish between these various forms.\n\n# Future Directions\n\nA growing body of evidence has implicated inflammatory responses to diet and environmental factors as a key mechanism that help explain the emerging epidemic in diabetes and cardiovascular disease. Both genetic and environmental factors undoubtedly play a role, although the role of the physical and social environment in determining susceptibility appears to be critical. Nontraditional factors such as air pollution that are pervasive in the urban environment may provide low-level synergism with other dominant factors in accelerating propensity for type 2 DM. Emerging data from both experimental and epidemiologic studies are beginning to provide insights into this association. There are a number of areas that would benefit from further studies and enable additional insights into the mechanisms by which environmental signals modulate susceptibility to metabolic disease. The effects of air-pollution exposure on \u03b2-cell function, counterregulatory hormones such as glucagon, and effects on insulinotropic mechanisms deserve further study. The effects of air pollution on hypothalamic mechanisms of appetite and satiety are areas of emerging interest, as it is entirely possible that air pollutants may modulate inflammation in key brain homeostatic centers. In addition, the effects on central autonomic control of peripheral inflammation may represent additional pathways by which environmental triggers may play an important role in determining peripheral inflammation. The societal costs of this link, if indeed true, are staggering given the ubiquitous nature of air pollution and the economic costs of obesity\/DM-related complications. Given the already established nature of the links between air pollution and cardiovascular disease and regulations already in place, at least in countries like the U.S. and Europe, these additional links, if they can be established in additional large cohorts, would provide persuasive rationale for limiting exposure to air pollution.\n\n## ACKNOWLEDGMENTS\n\nThis work was supported by National Institute of Environmental Health Sciences (NIEHS) grants R01-ES-017290, R01-ES-015146, and R01-ES-019616 (to S.R.) and the Environmental Protection Agency\u2013supported Great Lakes Center for Environmental Research (to S.R. and R.D.B.).\n\nNo potential conflicts of interest relevant to this article were reported.\n\nS.R. and R.D.B. researched the data and wrote and reviewed the manuscript.\n\nThe authors thank Geoffrey Gatts, Wexner Medical Center at The Ohio State University, for excellent editorial assistance and data collection.\n\n# REFERENCES","meta":{"dup_signals":{"dup_doc_count":191,"dup_dump_count":56,"dup_details":{"curated_sources":2,"2021-43":1,"2021-25":1,"2020-24":1,"2019-43":2,"2019-30":1,"2018-51":2,"2018-39":3,"2018-26":2,"2018-22":2,"2018-17":3,"2018-13":3,"2018-09":1,"2018-05":3,"2017-51":3,"2017-47":3,"2017-43":7,"2017-39":2,"2017-34":4,"2017-30":4,"2017-26":4,"2017-22":5,"2017-17":7,"2017-09":7,"2017-04":9,"2016-50":6,"2016-44":7,"2016-40":7,"2016-36":7,"2016-30":5,"2016-26":5,"2016-22":7,"2016-18":2,"2016-07":3,"2015-48":3,"2015-40":3,"2015-35":1,"2015-32":2,"2015-27":5,"2015-22":5,"2015-14":1,"2014-52":4,"2014-49":1,"2014-42":4,"2014-41":2,"2014-35":5,"2014-23":2,"2014-15":1,"2022-05":1,"2024-22":1,"2017-13":6,"2015-18":4,"2015-11":3,"2015-06":3,"2014-10":1,"2013-48":1,"2024-26":1}},"file":"PMC3501850"},"subset":"pubmed_central"}
{"text":"author: Corresponding author: David M. Nathan, <firstname.lastname@example.com>.[^1]\ndate: 2009-07\nreferences:\ntitle: International Expert Committee Report on the Role of the A1C Assay in the Diagnosis of Diabetes\n\n*An International Expert Committee with members appointed by the American Diabetes Association, the European Association for the Study of Diabetes, and the International Diabetes Federation was convened in 2008 to consider the current and future means of diagnosing diabetes in nonpregnant individuals. The report of the International Expert Committee represents the consensus view of its members and not necessarily the view of the organizations that appointed them. The International Expert Committee hopes that its report will serve as a stimulus to the international community and professional organizations to consider the use of the A1C assay for the diagnosis of diabetes.*\n\nDiabetes is a disease characterized by abnormal metabolism, most notably hyperglycemia, and an associated heightened risk for relatively specific long-term complications affecting the eyes, kidney, and nervous system. Although diabetes also substantially increases the risk for cardiovascular disease, cardiovascular disease is not specific to diabetes and the risk for cardiovascular disease has not been incorporated into previous definitions or classifications of diabetes or of subdiabetic hyperglycemia.\n\n# Background\n\n## Diagnosing diabetes based on the distribution of glucose levels\n\nHistorically, the measurement of glucose has been the means of diagnosing diabetes. Type 1 diabetes has a sufficiently characteristic clinical onset, with relatively acute, extreme elevations in glucose concentrations accompanied by symptoms, such that specific blood glucose cut points are not required for diagnosis in most clinical settings. On the other hand, type 2 diabetes has a more gradual onset, with slowly rising glucose levels over time, and its diagnosis has required specified glucose values to distinguish pathologic glucose concentrations from the distribution of glucose concentrations in the nondiabetic population. Virtually every scheme for the classification and diagnosis of diabetes in modern times has relied on the measurement of plasma (or blood or serum) glucose concentrations in timed samples, such as fasting glucose; in casual samples independent of prandial status; or after a standardized metabolic stress test, such as the 75-g oral glucose tolerance test (OGTT).\n\nEarly attempts to standardize the definition of diabetes relied on the OGTT, but the performance and interpretation of the test were inconsistent and the number of subjects studied to define abnormal values was very small (1\u20136). Studies in the high-risk Pima Indian population that demonstrated a bimodal distribution of glucose levels following the OGTT (7,8) helped establish the 2-h value as the diagnostic value of choice, even though most populations had a unimodal distribution of glucose levels (9). Of note, a bimodal distribution was also seen in the fasting glucose samples in the Pimas and other high-risk populations (10,11). However, a discrete fasting plasma glucose (FPG) or 2-h plasma glucose (2HPG) level that separated the bimodal distributions in the Pimas was difficult to identify, with potential FPG and 2HPG cut points ranging from 120 to 160 mg\/dl (6.7\u20138.9 mmol\/l) and from 200 to 250 mg\/dl (11.1\u201313.9 mmol\/l), respectively.\n\nIn 1979, the National Diabetes Data Group (NDDG) provided the diagnostic criteria that would serve as the blueprint for nearly two decades (12). The NDDG relied on distributions of glucose levels, rather than on the relationship of glucose levels with complications, to diagnose diabetes despite emerging evidence that the microvascular complications of diabetes were associated with a higher range of fasting and OGTT glucose values (11,13\u201315). The diagnostic glucose values chosen were based on their association with decompensation to \"overt\" or symptomatic diabetes.\n\nWhen selecting the threshold glucose values, the NDDG acknowledged that \"there is no clear division between diabetics and nondiabetics in the FPG concentration or their response to an oral glucose load,\" and consequently, \"an arbitrary decision has been made as to what level justifies the diagnosis of diabetes.\" The diagnosis of diabetes was made when *1*) classic symptoms were present; *2*) the venous FPG was \u2265140 mg\/dl (\u22657.8 mmol\/l); or *3*) after a 75-g glucose load, the venous 2HPG and levels from an earlier sample before 2 h were \u2265200 mg\/dl (\u226511.1 mmol\/l). An intermediate group was classified as having \"impaired glucose tolerance\" (IGT) with FPG \\<140 mg\/dl (7.8 mmol\/l) and a 2HPG value between 140 and 200 mg\/dl (7.8\u201311.1 mmol\/l). IGT was identified on the basis of its relatively higher risk of progression to diabetes compared with that of \"normal\" glucose tolerance, low frequency of \"diabetic symptoms,\" high probability of reverting to normal glucose tolerance or continuing to have IGT, and rarity of \"clinically significant\" microvascular disease. The NDDG recommendations were also promulgated by the contemporaneous report of the World Health Organization (WHO) (16).\n\n## Diagnosing diabetes based on the relationship between glucose levels and long-term complications\n\nIn 1997, the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus (17) reexamined the basis for diagnosing diabetes. This committee made two seminal contributions: First, they refocused attention on the relationship between glucose levels and the presence of long-term complications as the basis for the diagnosis of diabetes. Second, they summarized data negating the widespread hypothesis that the 2HPG was the gold-standard test for diagnosing diabetes. The committee examined data from three cross-sectional epidemiological studies that included an Egyptian population (*n* = 1,018), Pima Indians (*n* = 960), and the U.S. National Health and Nutrition Examination Survey (NHANES) population (*n* = 2,821). Each assessed retinopathy with fundus photography or direct ophthalmoscopy and measured glycemia as FPG, 2HPG, and A1C. These studies demonstrated glycemic levels below which there was little prevalent retinopathy and above which the prevalence of retinopathy increased in an apparently linear fashion (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<\/a>). When the prevalence of retinopathy was expressed by deciles of glycemia for each of the three measures, the deciles at which retinopathy began to increase were the same for each measure within each population. Moreover, the glycemic values above which retinopathy increased were similar among the populations. These data showed a clear relationship between glycemia and the risk for retinopathy that would supplant the previous notion of risk for progression to overt, symptomatic diabetes as the basis for diagnosing diabetes.\n\nIn comparing the relationship between FPG and 2HPG values and retinopathy, it was apparent that the previous FPG cut point of \u2265140 mg\/dl (7.8 mmol\/l) was substantially above the glucose level at which the prevalence of retinopathy began to increase. As a result, the committee recommended that the FPG cut point be lowered to \u2265126 mg\/dl (7.0 mmol\/l) so that this cut point would represent a degree of hyperglycemia that was \"similar\" to the 2HPG value and diagnosis with either measure would result in a similar prevalence of diabetes in the population. The 1997 committee report acknowledged that even at the lower FPG cut point, the FPG and OGTT (2HPG) were not perfectly concordant. An individual could have diabetes using one test but not the other. This discrepancy has been confirmed in numerous subsequent reports and may be due, in part, to the fact that although both tests are measures of glycemia, they reflect different physiological measures of acute glucose metabolism (18). The debate regarding the relative roles of FPG and 2HPG in the diagnosis of diabetes in the nonpregnant adult has continued (19\u201321).\n\nThe 1997 report also recommended that the FPG level, rather than the 2HPG, be the preferred test to diagnose diabetes because it was more convenient for patients and less costly and time consuming and the repeat-test reproducibility was superior (17). In addition, the committee introduced the term \"impaired fasting glucose\" (IFG) to differentiate the metabolic state between a normal state (FPG \\<110 mg\/dl or \\<6.1 mmol\/l) and diabetes (\u2265126 mg\/dl or \u22657.0 mmol\/l) when the FPG test was used. If an OGTT was performed, the intermediate glycemic state continued to be called IGT, with the 2HPG (between 140 and 200 mg\/dl \\[7.8 and 11.1 mmol\/l\\]) the same as that as in the NDDG report. A WHO consultation (22) adopted most of the above recommendations except they concluded that, whenever feasible, individuals with IFG should be given an OGTT to exclude the presence of diabetes that would otherwise be missed and that the OGTT should remain the \"gold standard.\" A 2003 follow-up report from the expert committee refined the fasting glucose value range for IFG from \u2265110 but \\<126 mg\/dl to \u2265100 but \\<126 mg\/dl (\u22656.1 but \\<7.0 mmol\/l to \u22655.6 but \\<7.0 mmol\/l) to make it more comparable with the IGT value (21). The WHO did not change its previous recommendations (23).\n\n# Can the A1C test be used to diagnose diabetes?\n\nIf chronic hyperglycemia sufficient to cause diabetes-specific complications is the hallmark of diabetes, common sense would dictate that laboratory measures that capture long-term glycemic exposure should provide a better marker for the presence and severity of the disease than single measures of glucose concentration. Observational studies that have assessed glycemia with measures that capture longer-term exposure (i.e., A1C) or with single or longitudinal measurements of glucose levels have consistently demonstrated a strong correlation between retinopathy and A1C (24\u201326) but a less consistent relationship with fasting glucose levels (27). In one study that measured both FPG and A1C, there was a stronger correlation between A1C and retinopathy than between fasting glucose levels and retinopathy (25). The correlation between A1C levels and complications has also been shown in the setting of controlled clinical trials in type 1 (28) and type 2 (29) diabetes, and these findings have been used to establish the widely accepted A1C treatment goals for diabetes care (30).\n\nAll of these observations suggest that a reliable measure of chronic glycemic levels such as A1C, which captures the degree of glucose exposure over time (31,32) and which is related more intimately to the risk of complications than single or episodic measures of glucose levels, may serve as a better biochemical marker of diabetes and should be considered a diagnostic tool. Although the 1997 expert committee report considered this option, it recommended against using A1C values for diagnosis in part because of the lack of assay standardization (17). The 2003 follow-up report noted that, while the National Glycohemoglobin Standardization Program (33) had succeeded in standardizing the vast majority of assays used in the U.S., the use of A1C for diagnosis still had \"disadvantages,\" and it reaffirmed the previous recommendation that A1C not be used to diagnose diabetes (21).\n\nAn updated examination of the laboratory measurements of glucose and A1C by the current International Expert Committee indicates that with advances in instrumentation and standardization, the accuracy and precision of A1C assays at least match those of glucose assays. The measurement of glucose itself is less accurate and precise than most clinicians realize (34). A recent analysis of the performance of a variety of clinical laboratory instruments and methods that measure glucose revealed that 41% of instruments have a significant bias from the reference method that would result in potential misclassification of \\>12% of patients (35). There are also potential preanalytic errors owing to sample handling and the well-recognized lability of glucose in the collection tube at room temperature (36,37). Even when whole blood samples are collected in sodium fluoride to inhibit in vitro glycolysis, storage at room temperature for as little as 1 to 4 h before analysis may result in decreases in glucose levels by 3\u201310 mg\/dl in nondiabetic individuals (36\u201339).\n\nBy contrast, A1C values are relatively stable after collection (40), and the recent introduction of a new reference method to calibrate all A1C assay instruments should further improve A1C assay standardization in most of the world (41\u201343). In addition, between- and within-subject coefficients of variation have been shown to be substantially lower for A1C than for glucose measurements (44). The variability of A1C values is also considerably less than that of FPG levels, with day-to-day within-person variance of \\<2% for A1C but 12\u201315% for FPG (45\u201347). The convenience for the patient and ease of sample collection for A1C testing (which can be obtained at any time, requires no patient preparation, and is relatively stable at room temperature) compared with that of FPG testing (which requires a timed sample after at least an 8-h fast and which is unstable at room temperature) support using the A1C assay to diagnose diabetes.\n\nIn summary, compared with the measurement of glucose, the A1C assay is at least as good at defining the level of hyperglycemia at which retinopathy prevalence increases; has appreciably superior technical attributes, including less preanalytic instability and less biologic variability; and is more clinically convenient. A1C is a more stable biological index than FPG, as would be expected with a measure of chronic glycemia levels compared with glucose concentrations that are known to fluctuate within and between days (<a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>).\n\nAdvantages of A1C testing compared with FPG or 2HPG for the diagnosis of diabetes\n\n<table>\n<tbody>\n<tr>\n<td style=\"text-align: left;\"><ul>\n<li><p>Standardized and aligned to the DCCT\/UKPDS; measurement of glucose is less well standardized<\/p><\/li>\n<li><p>Better index of overall glycemic exposure and risk for long-term complications<\/p><\/li>\n<li><p>Substantially less biologic variability<\/p><\/li>\n<li><p>Substantially less preanalytic instability<\/p><\/li>\n<li><p>No need for fasting or timed samples<\/p><\/li>\n<li><p>Relatively unaffected by acute (e.g., stress or illness related) perturbations in glucose levels<\/p><\/li>\n<li><p>Currently used to guide management and adjust therapy<\/p><\/li>\n<\/ul><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n# What is the most appropriate A1C cut point for the diagnosis of diabetes?\n\nAs shown in the 1997 committee report, the prevalence of retinopathy increases substantially at A1C values starting between 6.0 and 7.0% (17) (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<\/a>). A recent analysis derived from DETECT-2 (48) and including the 3 that were included in the 1997 report examined the association between A1C and retinopathy, objectively assessed and graded by fundus photography (S. Colagiuri, personal communication). This analysis included \u223c28,000 subjects from nine countries and showed that the glycemic level at which the prevalence of \"any\" retinopathy begins to rise above background levels (any retinopathy includes minor changes that can be due to other conditions, such as hypertension), and for the more diabetes-specific \"moderate\" retinopathy, was 6.5% when the data were examined in 0.5% increments (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<\/a>). Among the \\>20,000 subjects who had A1C values \\<6.5%, \"moderate\" retinopathy was virtually nonexistent. The receiver operating characteristic curve analysis of the same data indicated that the optimal cut point for detecting at least moderate retinopathy was an A1C of 6.5%.\n\nIn summary, the large volume of data from diverse populations has now established an A1C level associated with an increase in the prevalence of moderate retinopathy and provides strong justification for assigning an A1C cut point of \u22656.5% for the diagnosis of diabetes. A recently published population-based study of 3,190 adults of Malay ethnicity independently concluded that A1C levels \"in the range 6.6 to 7% were optimal for detecting microvascular complications\" (26).\n\nAny suggestion that the relationship between chronic glycemic levels and the long-term complications of diabetes may be better expressed as a continuum, rather than as a strictly dichotomous relationship, is belied by the retinopathy findings presented herein. There is a low prevalence of \"any\" retinopathy at A1C levels \\<6.5% that may reflect a continuum of risk; alternatively, retinopathy related to conditions other than diabetes (e.g., hypertension) or inaccurate assessment of long-term glycemic levels with a single A1C measurement may contribute to this observation. However, the substantial increase in the prevalence of moderate retinopathy at A1C levels \u22656.5% supports a threshold level of glycemia that results in retinopathy most characteristic of diabetes.\n\nThis cut point should not be construed as an absolute dividing line between normal glycemia and diabetes; however, the A1C level of 6.5% is sufficiently sensitive and specific to identify individuals who are at risk for developing retinopathy and who should be diagnosed as diabetic. The A1C level is at least as predictive as the current FPG and 2HPG values. In selecting a diagnostic A1C level \u22656.5%, the International Expert Committee balanced the stigma and costs of mistakenly identifying individuals as diabetic against the minimal clinical consequences of delaying the diagnosis in someone with an A1C level \\<6.5%. The committee agreed to emphasize specificity rather than sensitivity. This decision was aided by the parallel decision to recommend effective prevention strategies for the highest at-risk group with an A1C between 6.0 and 6.5%. (See below.)\n\n# Limitations of A1C as the recommended means of diagnosing diabetes\n\nThe A1C assay is the test of choice for the chronic management of diabetes and is now being recommended for its diagnosis; however, there are parts of the world where the costs of providing the assay preclude its routine use. In such circumstances, clinicians should continue to use the previously recommended approaches to diagnose diabetes based on glucose measurements. The International Expert Committee encourages clinicians worldwide to move as quickly as possible to A1C testing using standardized methods and instrumentation. However, the decision to change to A1C assays as the means of diagnosing diabetes should take into account the performance of local A1C assays and the local prevalence of conditions that may interfere with the assay. (See below.)\n\nAlthough the discussion above argues for using the A1C assay for the diagnosis of diabetes in nonpregnant individuals, there are patient conditions that either will require a specific A1C assay method or will preclude A1C testing. First, some hemoglobin traits, such as HbS, HbC, HbF, and HbE, interfere with some A1C assay methods (49). Currently, many assay methods can correct for the presence of the most common hemoglobin traits ([www.ngsp.org](http:\/\/www.ngsp.org)), and affinity assays that are unaffected by hemoglobin traits may be used (49). Second, any condition that changes red cell turnover, such as hemolytic anemia, chronic malaria, major blood loss, or blood transfusions, will lead to spurious A1C results. Clinicians must be aware of these conditions, particularly in populations in which they are more prevalent. As in the setting where A1C assays are unavailable, the traditional diagnostic tests (e.g., FPG, 2HPG) must be used in individuals in whom interpreting the A1C is problematic. Third, A1C levels appear to increase with age (50), but the extent of the change, whether it relates to factors other than glucose metabolism, and the effect of the age-related increases on the development of complications are not sufficiently clear to adopt age-specific values in a diagnostic scheme. Similarly, racial disparities in A1C, based on putative differences in the relationship between glucose levels and A1C, have been suggested (51); however, here too, their etiology and significance are unclear, and it is premature to establish race-specific diagnostic values. Finally, there are rare clinical settings, such as rapidly evolving type 1 diabetes, where the A1C level will not have had time to \"catch up\" with the acute elevations in glucose levels; however, in these very rare cases, diabetes should be diagnosable with typical symptoms and casual glucose levels \\>200 mg\/dl (11.1 mmol\/l) despite a nondiagnostic A1C level.\n\nNotwithstanding the above limitations of A1C testing, the assay has numerous important advantages compared with the currently used laboratory measurements of glucose (<a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>). The prevalence of diabetes in some populations may not be the same when diagnosis is based on A1C compared with diagnosis with glucose measurements, and one method may identify different individuals than the other. Because the measurements of glucose levels and A1C reflect different aspects of glucose metabolism, this is to be expected. However, establishing identical prevalences should not be the goal in defining a new means of diagnosing diabetes. The ultimate goal is to identify individuals at risk for diabetes complications so that they can be treated. The A1C diagnostic level of 6.5% accomplishes this goal.\n\n# Can A1C measurements define a specific subdiabetic \"high-risk\" state?\n\nThe 2003 International Expert Committee report reduced the lower bound of IFG from 110 mg\/dl (6.1 mmol\/l) to 100 mg\/dl (5.6 mmol\/l) on the grounds that the lower level optimized the sensitivity and specificity for predicting future diabetes and also increased the proportion of those with IGT who could be identified with an FPG test (21). While previous studies have shown a powerful effect of IFG and\/or IGT on the subsequent development of diabetes diagnosed with glucose values (52\u201354), recent reports have demonstrated a graded risk of diabetes development at glycemic levels well within what was previously considered \"normal,\" i.e., FPG \\<100 mg\/dl (5.6 mmol\/l) and A1C \\<6.0% (55,56). In addition, metabolic derangements related to diabetes have been documented at similarly low glycemic levels, increasing in severity with higher glucose values within the nondiabetic range (57,58).\n\nAs with measures of glucose, a continuum of risk for the development of diabetes based on A1C levels has been demonstrated (59\u201361). Thus, while there appears to be an approximate glycemic threshold above which the risk for retinopathy escalates, there does not appear to be a specific level at which risk for diabetes clearly begins. A continuum of risk for the development of diabetes across a wide range of subdiabetic A1C levels may make the classification of individuals into categories similar to IFG and IGT equally problematic for A1C, as it implies that we actually know where risk begins or becomes clinically important. The continuum of risk in the subdiabetic glycemic range argues for the elimination of dichotomous subdiabetic classifications, such as \"pre-diabetes,\" IFG, and IGT. However, as A1C levels approach the diagnostic level for diabetes, the risk of developing diabetes becomes greatest (59,60,62).\n\n# Should A1C testing be used to identify individuals at high risk for diabetes?\n\nThe screening tests to identify individuals at elevated risk for diabetes are the same as the diagnostic tests; therefore, the technical advantages of A1C testing compared with glucose testing apply to the detection of individuals at high risk as they do to the diagnosis of diabetes. Therapeutic decisions should be based on how close A1C levels are to the diagnosis of diabetes. In the absence of a specific identifiable lower threshold defining when prevention efforts should be implemented, and with potentially limited resources taken into consideration, individuals whose A1C values are close to the 6.5% A1C threshold of diabetes (i.e., \u22656.0%) should receive demonstrably effective interventions (63,64). By identifying this very high-risk subdiabetic group, the International Expert Committee is implying not that populations at lower A1C levels are not at risk but, rather, that they are at lower risk. All individuals at risk for diabetes should receive counseling to maintain normal weight, lose weight if necessary, and become more physically active.\n\nOther risk factors for diabetes development in addition to A1C have been identified, including elevated levels of triglycerides, blood pressure, BMI, and family history of diabetes (59,60), and these should be taken into account in determining when to initiate interventions in individuals with A1C \\<6.0%. The use of well-validated risk assessment tools may be valuable in that regard. At the population level, the A1C value at which prevention services are provided will depend on the resources available, the size of the target population, and the nature of the intervention.\n\n# What are the practical issues related to A1C testing?\n\nA1C tests to diagnose diabetes should be performed using clinical laboratory equipment. Point-of-care instruments have not yet been shown to be sufficiently accurate or precise for diagnosing diabetes. Although this International Expert Committee has concluded that the attributes of the A1C assay with regard to diagnosing diabetes and detecting individuals at high risk support its use over the FPG or 2HPG tests, the superiority of A1C testing does not invalidate the diagnostic criteria based on glucose testing. In circumstances when A1C testing cannot be performed, the diagnostic glucose tests are acceptable alternatives.\n\nWhichever of the three different tests now available to diagnose diabetes (A1C, FPG, and 2HPG) is used, both initial and confirmatory testing should be performed with the same test. As the three tests are not completely concordant, using different tests could easily lead to confusion. The only exception to the need to confirm the diagnosis of diabetes with the same test would be the presence of clinical symptoms characteristic of diabetes and glucose levels \\>200 mg\/dl (\\>11.1 mmol\/l). Confirmatory testing is also not required to establish risk status in individuals identified as in the highest-risk group for diabetes (A1C of 6.0 to \\<6.5%).\n\nMost cases of type 1 diabetes, particularly in children and adolescents, are diagnosed by the classical symptoms of polyuria, polydipsia, polyphagia, unexplained weight loss, and a casual glucose \\>200 mg\/dl. If diabetes is suspected in the absence of those conditions, A1C testing is warranted.\n\n# Recommendations and conclusions\n\nBased on the above discussion, the International Expert Committee has concluded that the best current evidence supports the following recommendations, summarized in <a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a>.\n\nRecommendation of the International Expert Committee\n\n<table>\n<tbody>\n<tr>\n<td style=\"text-align: left;\">For the diagnosis of diabetes:<br \/>\n&#10;<ul>\n<li><p>The A1C assay is an accurate, precise measure of chronic glycemic levels and correlates well with the risk of diabetes complications.<\/p><\/li>\n<li><p>The A1C assay has several advantages over laboratory measures of glucose.<\/p><\/li>\n<li><p>Diabetes should be diagnosed when A1C is \u22656.5%. Diagnosis should be confirmed with a repeat A1C test. Confirmation is not required in symptomatic subjects with plasma glucose levels &gt;200 mg\/dl (&gt;11.1 mmol\/l).<\/p><\/li>\n<li><p>If A1C testing is not possible, previously recommended diagnostic methods (e.g., FPG or 2HPG, with confirmation) are acceptable.<\/p><\/li>\n<li><p>A1C testing is indicated in children in whom diabetes is suspected but the classic symptoms and a casual plasma glucose &gt;200 mg\/dl (&gt;11.1 mmol\/l) are not found.<\/p><\/li>\n<\/ul><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">For the identification of those at high risk for diabetes:\n<ul>\n<li><p>The risk for diabetes based on levels of glycemia is a continuum; therefore, there is no lower glycemic threshold at which risk clearly begins.<\/p><\/li>\n<li><p>The categorical clinical states pre-diabetes, IFG, and IGT fail to capture the continuum of risk and will be phased out of use as A1C measurements replace glucose measurements.<\/p><\/li>\n<li><p>As for the diagnosis of diabetes, the A1C assay has several advantages over laboratory measures of glucose in identifying individuals at high risk for developing diabetes.<\/p><\/li>\n<li><p>Those with A1C levels below the threshold for diabetes but \u22656.0% should receive demonstrably effective preventive interventions. Those with A1C below this range may still be at risk and, depending on the presence of other diabetes risk factors, may also benefit from prevention efforts.<\/p><\/li>\n<li><p>The A1C level at which population-based prevention services begin should be based on the nature of the intervention, the resources available, and the size of the affected population.<\/p><\/li>\n<\/ul><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n## For the diagnosis of diabetes\n\n-   There is no single assay related to hyperglycemia that can be considered the gold standard, as it relates to the risk for microvascular or macrovascular complications.\n\n-   A measure that captures chronic glucose exposure is more likely to be informative regarding the presence of diabetes than is a single measure of glucose.\n\n-   The A1C assay provides a reliable measure of chronic glycemia and correlates well with the risk of long-term diabetes complications.\n\n-   The A1C assay (standardized and aligned with the Diabetes Control and Complications Trial\/UK Prospective Diabetes Study assay) has several technical, including preanalytic and analytic, advantages over the currently used laboratory measurements of glucose.\n\n-   For the reasons above, the A1C assay may be a better means of diagnosing diabetes than measures of glucose levels.\n\n-   The diagnosis of diabetes is made if the A1C level is \u22656.5%. Diagnosis should be confirmed with a repeat A1C test unless clinical symptoms and glucose levels \\>200 mg\/dl (\\>11.1 mmol\/l) are present.\n\n-   If A1C testing is not possible owing to patient factors that preclude its interpretation (e.g., hemoglobinopathy or abnormal erythrocyte turnover) or to unavailability of the assay, previously recommended diagnostic measures (e.g., FPG and 2HPG) and criteria should be used. Mixing different methods to diagnose diabetes should be avoided.\n\n-   In children and adolescents, A1C testing is indicated when diabetes is suspected in the absence of the classical symptoms or a plasma glucose concentration \\>200 mg\/dl (\\>11.1 mmol\/l).\n\n-   The diagnosis of diabetes during pregnancy, when changes in red cell turnover make the A1C assay problematic, will continue to require glucose measurements.\n\n## For the identification of individuals at high risk for diabetes\n\n-   Individuals with an A1C level \u22656% but \\<6.5% are likely at the highest risk for progression to diabetes, but this range should not be considered an absolute threshold at which preventative measures are initiated.\n\n-   The classification of subdiabetic hyperglycemia as pre-diabetes is problematic because it suggests that all individuals so classified will develop diabetes and that individuals who do not meet these glycemia-driven criteria (regardless of other risk factor values) are unlikely to develop diabetes\u2014neither of which is the case. Moreover, the categorical classification of individuals as high risk (e.g., IFG or IGT) or low risk, based on any measure of glycemia, is less than ideal because the risk for progression to diabetes appears to be a continuum. The glucose-related terms describing subdiabetic hyperglycemia will be phased out of use as clinical diagnostic states as A1C measurements replace glucose measurements for the diagnosis of diabetes.\n\n-   When assessing risk, implementing prevention strategies, or initiating a population-based prevention program, other diabetes risk factors should be taken into account. In addition, the A1C level at which to begin preventative measures should reflect the resources available, the size of the population affected, and the anticipated degree of success of the intervention. Further analyses of cost-benefit should guide the selection of high-risk groups targeted for intervention within specific populations.\n\n# Supplementary Material\n\n###### Accompanying Editorial\n\n## Acknowledgments\n\nNo potential conflicts of interest relevant to this article were reported.\n\n### APPENDIX\n\nInternational Expert Committee members: David M. Nathan, MD (Chair); Beverly Balkau, PhD; Enzo Bonora, MD, PhD; Knut Borch-Johnsen, MD, DMSc; John B. Buse, MD, PhD; Stephen Colagiuri, MD; Mayer B. Davidson, MD; Ralph DeFronzo, MD; Saul Genuth, MD; Rury R. Holman, FRCP; Linong Ji, MD; Sue Kirkman, MD; William C. Knowler, MD, Dr PH; Desmond Schatz, MD; Jonathan Shaw, MD; Eugene Sobngwi, MD; Michael Steffes, MD, PhD; Olga Vaccaro, MD; Nick Wareham, MD; Bernard Zinman, MD; and Richard Kahn, PhD.\n\n# References\n\n[^1]: \\*A list of members of the International Expert Committee can be found in the <span class=\"smallcaps\">appendix<\/span>.","meta":{"dup_signals":{"dup_doc_count":204,"dup_dump_count":74,"dup_details":{"curated_sources":2,"2023-50":2,"2023-40":1,"2023-23":2,"2023-14":2,"2022-40":1,"2022-21":1,"2022-05":2,"2021-49":9,"2021-43":10,"2021-39":7,"2021-31":7,"2021-25":5,"2021-21":5,"2021-17":4,"2021-10":4,"2021-04":3,"2020-50":6,"2020-45":10,"2020-40":4,"2020-34":6,"2020-29":3,"2020-24":6,"2020-16":4,"2020-10":5,"2020-05":3,"2019-51":4,"2019-47":8,"2019-43":11,"2019-39":2,"2019-35":1,"2019-30":2,"2019-26":2,"2019-22":4,"2019-18":1,"2019-13":1,"2019-09":1,"2019-04":2,"2018-51":5,"2018-47":2,"2018-43":1,"2018-39":4,"2018-34":2,"2018-30":1,"2018-26":1,"2018-22":2,"2018-09":1,"2018-05":1,"2017-47":1,"2017-43":1,"2017-39":2,"2017-30":1,"2017-26":1,"2017-22":2,"2017-09":1,"2016-44":2,"2016-40":1,"2016-36":1,"2016-22":1,"2016-18":1,"2016-07":1,"2015-48":1,"2015-40":1,"2015-27":1,"2015-22":1,"2014-42":1,"2014-41":2,"2014-35":1,"2014-23":1,"2015-18":1,"2015-11":1,"2015-06":1,"2014-10":1,"2013-48":1,"2024-26":1}},"file":"PMC2699715"},"subset":"pubmed_central"}
{"text":"abstract: A multi-parametric genetic screening approach sheds light on integrated control of the endocytic pathway in mammalian cells.\nauthor: Peter Hein; Mark von Zastrow\ndate: 2010\ninstitute: 1Department of Psychiatry, Department of Cellular and Molecular Pharmacology and Program in Cell Biology, University of California, N212 Genentech Hall, 600 16th Street, San Francisco, CA 94158-2140, USA\nreferences:\ntitle: Membrane traffic in the post-genomic era\n\n# Research highlight\n\nA defining feature of eukaryotic cells is the presence of an elaborate network of internal membrane compartments that communicate between themselves and with the cell surface via specific membrane fission and fusion reactions \\[1,2\\]. Such 'membrane trafficking' processes can be viewed as a network of intracellular transport pathways, whose operation is critical to normal physiology and disturbed in disease. A major goal in the field of cell biology, therefore, is to elucidate the mechanistic basis of these fundamental membrane trafficking processes and how they are regulated. Historically, genetic approaches have been instrumental in this effort, particularly forward genetic screens in model eukaryotes, such as budding yeast, by the traditional route of mutagenesis, phenotype selection, and subsequent identification of the affected gene. Such screens have led to the identification of a variety of essential proteins mediating membrane traffic in the biosynthetic pathway of yeast, many of which have orthologs in mammals \\[3,4\\].\n\nA long-standing barrier to more comprehensive analysis of membrane-trafficking processes in mammalian cells has been the relative intractability of these cells to forward genetic analysis. The main barrier is that mammalian cell culture lines, unlike yeast, cannot be maintained in a haploid state. Therefore, traditional genetic methods based on mutations in the genome, because they typically disrupt only a single copy of a particular gene, rarely produce a screenable phenotype. This barrier is beginning to break down, however, based on the development of alternative methods. The sequencing and annotation of animal genomes, combined with the use of RNA interference (RNAi) to knock down specific gene expression, are ushering in a new era of forward genetic analysis that extends to mammalian cells \\[5\\]. A recent study published in *Nature* from Marino Zerial's group in Dresden (Collinet *et al.*) \\[6\\], illustrates how such approaches are beginning to be applied to study the integrated function of the endocytic pathway in human cells.\n\nMajor gaps exist in our understanding of membrane traffic in mammalian cells compared with that in yeast. Such gaps quickly become evident when one begins to consider how membrane trafficking is integrated with other essential cellular processes. Endocytic membrane traffic is essential not only for 'classical' functions such as nutrient uptake from the extracellular milieu, but also plays critical roles in a wide range of superficially unrelated processes. One of the best recognized of these relationships is with cellular signal transduction. Multicellular life is dependent on a diversity of receptor-mediated signaling mechanisms, and animals have greatly expanded the representation of signaling receptors in their genome compared with yeast. Membrane trafficking of many signaling receptors in the endocytic pathway is essential for the proper organization and regulation of downstream information transfer. Such effects are not only critical for organized cell-cell communication under normal physiological conditions, but disturbances in the endocytic trafficking of receptors play a causative or supporting role in disease states such as cancer. There is also compelling and accumulating evidence for regulation in the converse direction - of the membrane machinery by signaling - at multiple stages of both the membrane-biosynthetic and endocytic pathways \\[7,8\\].\n\n# Analysis of endocytic pathways in mammalian cells\n\nThe main new advance introduced in the study of Collinet *et al.* is automated phenotyping of the endocytic pathway, using quantitative fluorescence microscopy. The investigators applied this method to carry out unbiased analysis of the phenotypes produced by knocking down gene expression using RNAi. Using the HeLa human cell line, Collinet *et al.* monitored two receptor-mediated endocytic processes - the uptake of the iron-transport protein transferrin bound to its receptor and the uptake of epidermal growth factor (EGF) bound to its receptor (a receptor tyrosine kinase), which are important to cellular nutrition and cellular signaling, respectively (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>, which also illustrates the core membrane-trafficking pathways in the cell). Endocytosis of these two receptor-ligand complexes is thought to utilize much of the same 'core' endocytic machinery, yet each pathway differs significantly in its regulation, and in the specificity with which internalized ligands are trafficked to different internal membrane compartments (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>). These two processes are also a good choice from the experimental perspective, because fluorochrome-conjugated ligands enable the visualization of these processes by fluorescence microscopy.\n\nFifty-eight different parameters describing, for example, vesicle amount, size and intracellular distribution, were extracted using a computer-controlled algorithm from automatic confocal images of HeLa cells. The investigators screened multiple libraries of synthetic small interfering RNAs (siRNAs) and an endoribonuclease-prepared siRNA (esiRNA) library, each covering every human gene several-fold. Cluster analysis of these 58 parameters led to 10 parameter groups describing distinct classes of endocytosis phenotypes. This approach resulted in 161,492 knockdowns and around 2.5 \u00d7 10^6^ cofocal microscope images, requiring 4.5 \u00d7 10^6^ computing hours on a 2,584-core computer cluster to analyze. This is, first, an approach that excludes the subjective bias of a human observer. Second, the multi-parametric description of phenotypes potentially allows the detection of effects on endocytosis that would be missed by more conventional approaches, which are typically limited to relatively severe (or lethal) phenotypes. Taking advantage of their multi-parametric analysis, combined with deep coverage of the expressed genome, the authors have developed an impressively rich database of the effects of genetic disruption on the endocytic pathway in a human cell line.\n\nWhat emerges from this analysis is both exciting and cautionary. On the exciting side, the authors identified a remarkably large number of genes - more than 4,000 - whose knockdown reliably affected some parameter of the endocytic analysis. On the cautionary side, this is a remarkably high hit rate - around 15% of the coding genome. The authors emphasize that their goal was not to identify particular genes that directly mediate a particular trafficking step or pathway but, instead, to develop a larger genetic profile that would enable appreciation of integrated 'design principles' of the endocytic pathway in mammalian cells. From this perspective, the list of implicated genes supports the existence of exquisitely close relationships, both direct and indirect, between the endocytic pathway and diverse cellular processes.\n\nReturning to the question of how membrane trafficking is related to signal transduction, Collinet *et al.* identified a particularly large number of genes that encode signaling receptors and mediator proteins. For example, the primary hit list includes a large number of seven-pass transmembrane receptors, including 'orphan' receptors whose physiological significance is currently not established. The potential of this forward genetic screening approach to reveal new links in the signaling-endocytosis nexus is indeed very exciting. On the cautionary side, some of the identified hits (such as several neuropeptide receptors) are thought not to be expressed at significant levels in HeLa cells. Thus, despite the careful attention paid to verifying hits with multiple siRNA targets, the possibility that the current list still includes a number of false positives must be kept in mind. All in all, the recent work by Collinet *et al*. represents a bold and interesting effort, with great potential but also significant challenges.\n\nIn future studies we can anticipate integration of the strategy used by Collinet *et al*. with proteomics and protein biochemical methods, which will help distinguish direct from indirect genetic effects and provide insight into biochemical mechanisms. Further advances in automation and computational power may allow practical genetic analysis of endocytic effects produced by changes in the cellular environment or the activation of particular signaling pathways. We can also look forward to extension of the genetic approach to paired or combinatorial knockdowns, which may help organize the large number of hits identified into coherent genetic pathways. Such analysis could also provide crucial insight to the significance of hits representing the remarkably large number of human disease-linked genes identified, most of which have not been implicated previously in endocytosis. The paper by Collinet *et al*. indeed makes a bold step into the future, and provides an intriguing preview of a new era in cell biological research.","meta":{"dup_signals":{"dup_doc_count":105,"dup_dump_count":48,"dup_details":{"curated_sources":3,"2021-39":1,"2021-21":1,"2021-04":1,"2020-50":1,"2020-45":1,"2020-40":1,"2019-51":1,"2019-47":1,"2019-39":1,"2019-26":1,"2019-13":1,"2019-04":2,"2018-47":2,"2018-34":1,"2018-22":1,"2018-05":1,"2017-39":1,"2017-22":1,"2017-09":10,"2016-44":1,"2016-40":1,"2016-36":10,"2016-30":7,"2016-22":1,"2016-18":1,"2015-48":2,"2015-40":1,"2015-35":2,"2015-32":2,"2015-27":2,"2015-22":2,"2015-14":2,"2014-52":2,"2014-49":3,"2014-42":5,"2014-41":3,"2014-35":2,"2014-23":3,"2014-15":4,"2023-14":1,"2024-10":1,"2015-18":2,"2015-11":2,"2015-06":2,"2014-10":2,"2013-48":3,"2013-20":2,"2024-26":1}},"file":"PMC2898079"},"subset":"pubmed_central"}
{"text":"abstract: Dogs were present in pre-Columbian America, presumably brought by early human migrants from Asia. Studies of free-ranging village\/street dogs have indicated almost total replacement of these original dogs by European dogs, but the extent to which Arctic, North and South American breeds are descendants of the original population remains to be assessed. Using a comprehensive phylogeographic analysis, we traced the origin of the mitochondrial DNA lineages for Inuit, Eskimo and Greenland dogs, Alaskan Malamute, Chihuahua, xoloitzcuintli and perro s\u00edn pelo del Peru, by comparing to extensive samples of East Asian (*n* = 984) and European dogs (*n* = 639), and previously published pre-Columbian sequences. Evidence for a pre-Columbian origin was found for all these breeds, except Alaskan Malamute for which results were ambigous. No European influence was indicated for the Arctic breeds Inuit, Eskimo and Greenland dog, and North\/South American breeds had at most 30% European female lineages, suggesting marginal replacement by European dogs. Genetic continuity through time was shown by the sharing of a unique haplotype between the Mexican breed Chihuahua and ancient Mexican samples. We also analysed free-ranging dogs, confirming limited pre-Columbian ancestry overall, but also identifying pockets of remaining populations with high proportion of indigenous ancestry, and we provide the first DNA-based evidence that the Carolina dog, a free-ranging population in the USA, may have an ancient Asian origin.\nauthor: Barbara van Asch; Ai-bing Zhang; Mattias C. R. Oskarsson; Cornelya F. C. Kl\u00fctsch; Ant\u00f3nio Amorim; Peter Savolainene-mail: <firstname.lastname@example.com>[^1]\ndate: 2013-09-07\ninstitute: 1IPATIMUP, Rua Dr Roberto Frias s \/ n, 4200-465 Porto, Portugal; 2Faculdade de Ci\u00eancias da Universidade do Porto, Rua do Campo Alegre s\/n, 4169-007 Porto, Portugal; 3Science for Life Laboratory, School of Biotechnology, Division of Gene Technology, KTH-Royal Institute of Technology, SE-171 65 Solna, Sweden; 4Bethany College, Lindsborg, KS 67456, USA; 5College of Life Sciences, Capital Normal University, Beijing 100048, People's Republic of China\nreferences:\ntitle: Pre-Columbian origins of Native American dog breeds, with only limited replacement by European dogs, confirmed by mtDNA analysis\n\n# Introduction\n\nThe dog is now well established as the most ancient domestic animal and is unique as the only domesticate present in human societies on every continent in ancient times \\[1\\]. Previous studies of mitochondrial (mt) DNA have indicated southern East Asia as the geographical centre for the origin of dogs \\[2\u20135\\]. This has been largely supported by analyses of Y-chromosomal DNA \\[5,6\\], while a recent single nucleotide polymorphism (SNP)-based study argued that Middle Eastern wolves primarily contributed to the extant dog gene pool \\[7\\]. Regardless of the exact geographical origin of domestic dogs, it seems clear that American dogs originate from the Old World since analysis of ancient samples from several pre-Columbian archaeological sites were shown to have the same mtDNA haplogroups as Old World dogs \\[8\\].\n\nA remaining question is if today's American dogs trace their origin to the dogs originally introduced from Asia via the Bering Straits in pre-Columbian time, or if this ancestry has been diluted or even completely erased by European dogs brought across the Atlantic following the arrival of Europeans in America. Several studies of mtDNA have attempted to elucidate this question, but did not include comprehensive samples across American breeds and\/or of reference samples from the Old World, necessary for a comprehensive analysis \\[8\u201311\\]. Importantly, with the detailed knowledge of the worldwide dog mtDNA diversity presented recently \\[4\\], an opportunity has opened up to identify whether mtDNA haplotypes carried by American dogs originate from East Asia in ancient times or from Europe in the post-Columbian era.\n\nArchaeological data and historic records have provided abundant evidence that dogs in pre-Columbian times were part of native cultures in the American continent long before the dawn of transoceanic travel in the fifteenth century \\[12\\]. Dogs were used for a large number of different purposes, for example, for hunting, sledging, freighting, protection and company, for religious and medicinal purposes and as a food resource. The earliest archaeological evidence for presence of dogs in the Americas has been dated to 10 000\u20138500 years ago, the dog thus being the sole domesticate in America during several thousand years \\[13,14\\]. According to current hypotheses, the Americas started to be colonized by south Siberian peoples at least 15 000 years ago immediately after deglaciation of the Pacific coastal corridor \\[15\\]. Thus, pre-Columbian dogs must have been brought along by Paleo-Indians of Asian origin in their expansions throughout the American continent, although not necessarily in connection with the first waves of humans.\n\nA small number of extant breeds in the American continent, for example, the Mexican Chihuahua, the xoloitzcuintli (Mexican hairless dog) and the Peruvian perro s\u00edn pelo (Peruvian hairless dog) have been claimed to descend from pre-Columbian populations. Archaeological findings in South America are abundant in evidence of dogs increasingly entwined in human societies \\[16\\]. In the Arctic region, the presence of well-adapted dogs and their use by Native American peoples prior to 1492 is also well recorded \\[17\\]. Many of those dogs, such as the hare Indian dog and the Tahltan bear dog have disappeared as the aboriginal hunting methods declined. Modern Arctic breeds thought to be indigenous include the Inuit sled dog, the Canadian Eskimo dog and the Greenland dog (all three thought to share the same origin) and the Alaskan Malamute. Some modern American breeds such as the dogo Argentino, the fox Paulistinha, the fila brasileiro and the cimarr\u00f3n Uruguayo have historical origins in European dogs and are not putatively indigenous. Similarly, the Alaskan husky and the American Eskimo dog have a known origin from Siberian spitzes and European dogs.\n\nBesides breed dogs, free-ranging dogs are abundantly found across the American continent. These dogs commonly have heterogeneous morphologies suggesting an origin predominantly from a mix of European breeds. However, in remote areas of southeastern USA (South Carolina and Georgia) there is a group of free-ranging dogs (called the Carolina dog) which morphologically resemble the Australian dingo and South Asian pariah dogs. Based on this resemblance to 'primitive' dogs, an origin from indigenous pre-Columbian dogs rather than from run-away breed dogs of European origin has been suggested \\[18\\].\n\nThe strong impact of the introduction of domestic species by European settlers and explorers, and its deleterious effects on Native American cultures, is indisputable. The replacement or dilution of Native American dog populations with European dogs is thought to have been severe, with consequent loss of historic Native breeds \\[19\\]. However, the extent of this replacement or admixture remains largely undisclosed. Did the ancient migrants leave descendants in the modern American gene pool or were they completely erased by European dogs brought across the Atlantic in the post-Columbian era, and are extant dog populations direct descendents of the ancient populations in the same geographical region?\n\nA very recent study of mtDNA in pre-Columbian dogs from Alaska and Greenland \\[9\\] showed that the ancient population and the modern Inuit sled dog population in Greenland carry predominantly the unique haplotype A31, strongly indicating local ancestry for this group of Arctic dogs. By contrast, a study of mtDNA among village dogs and street dogs from across America indicated that the maternal lineages of the indigenous American dog population have been almost completely replaced with European dogs \\[10\\]. However, this study did not include American breed dogs. Furthermore, the results were based on comparisons with a restricted source of information, that is, with the haplotypes found among 19 ancient American dog samples analysed in one previous study \\[8\\].\n\nAnother approach, possibly allowing for a more comprehensive picture of the geographical ancestry of mtDNA lineages among American dogs, is to compare extant American dogs with European and East Asian dogs, assuming that today's populations are good approximations for the ancient ones. The possibility of tracing the geographical origin of mtDNA lineages has opened up with the comprehensive picture of the worldwide dog mtDNA diversity presented recently \\[4\\]. This study showed distinct differences between the European and East Asian mtDNA gene pools, offering the possibility to trace whether mtDNA haplotypes among American dogs may have originated from Europe or East Asia. All populations across the Old World share a common gene pool of three major phylogenetic groups called clades A, B and C. In East Asia, the full diversity of these genetic groups is represented, but European dogs (together with all other populations west of the Himalayas and the Urals) have a limited diversity. Out of totally 206 mtDNA haplotypes identified globally \\[3\\], there are 15 haplotypes which are represented in virtually every dog population across the Old World, called universal types (UTs). In Europe, 76.7% of the dogs carry one of these 15 haplotypes and totally 92.6% carry a haplotype which is a UT or differs by a single substitution from one of the UTs. In addition, haplogroup D (which is absent in East Asia) has been found in Europe, in 5.9% of the dogs. Thus, totally 98.5% of European dogs carry an mtDNA which is a UT, differs by one substitution from a UT or belongs to haplogroup D. Consequently, if an American dog carries a haplotype which does not belong to this group of haplotypes, it is unlikely that its mtDNA lineage derives from Europe. In East Asia and Siberia, only 54.9% and 50% of the dogs, respectively, carry a UT; instead, there is a large number of unique haplotypes distinct from those in Europe. Therefore, it may be assumed that approximately 50% of the dogs that entered America via Bering Strait in pre-Columbian time carried a haplotype which was not a UT or belonged to haplogroup D and is therefore potentially informative for excluding a European origin.\n\nImportantly, the high frequency of the 15 UTs and haplotypes differing by a single substitution from the UTs is a universal phenomenon west of the Himalayas, with for example 94.5% of dogs in southwest Asia and 93.1% in Africa, carrying such haplotypes \\[3,4\\]. Therefore, the high proportion of these haplotypes in today's European population most probably reflects the genetic make-up across western Eurasia in ancient times, and thus also in Europe at the time of European colonization of America. The assumption that today's European population can be used to represent the situation 500 years ago therefore seems justified, offering a firm basis for the analyses in this study.\n\nIn this study, the origins of American dogs were addressed by comparison of mtDNA sequences from American, European and East Asian dogs. This dataset includes new samples for several American breeds and free-ranging dog populations and an extensive collection of European dogs. Together with previous data for extant dogs across the world \\[4\\] and sequences derived from ancient American samples \\[8\\], we obtained a comprehensive picture of the mtDNA gene pools in America, Europe and East Asia. Based on this, we identified the sharing of haplotypes and haplogroups among these regions and between ancient and modern samples. Through this analysis, we traced the maternal ancestry of the modern New World dog populations, assessing for the first time the extent to which American dog breeds descend from the original pre-Columbian population, and reanalysing the ancestry of free-ranging populations in greater phylogeographical detail.\n\n# Material and methods\n\n## Samples, PCR amplification and sequencing\n\nA sample of American dogs, of supposedly indigenous breeds as well as street dogs from both North and South America, was analysed by comparison with a comprehensive sample of European and East Asian dogs (see the electronic supplementary material, table S1). The Old World samples (total, *n* = 1872) were from Europe (*n* = 639), East Asia excluding Siberia (*n* = 889), Siberia (*n* = 95), Africa (*n* = 57), southwest Asia (*n* = 133) and India (*n* = 59). The New World samples (total, *n* = 347) included the Arctic breeds Alaskan Malamute (*n* = 9), Canadian Eskimo dog (*n* = 9), Greenland dog (*n* = 11) and Inuit sled dog (*n* = 18), the North American breeds Chihuahua (*n* = 14) and xoloitzcuintli (*n* = 43), and the South American breed perro s\u00edn pelo (Peru, *n* = 53). The American breeds were sampled from pure-bred dogs avoiding known common female ancestry among individuals. Sampling was obtained from European as well as American lineages of the breeds, and for the perro s\u00edn pelo, 19 samples were obtained directly from the Peruvian stock. Two American breeds of known Old World origin were also studied: fox Paulistinha (Brazil, *n* = 16) and dogo Argentino (Argentina, *n* = 40). In addition, free-ranging dogs were studied: from North America (USA), the pariah-like Carolina dog \\[20\\] (*n* = 19; captive dogs descending from free-ranging dogs); from South America, dogs collected at rural locations (total, *n* = 108); Argentina (Cordova and Buenos Aires regions, *n* = 54; Brazil (Rio de Janeiro and Rio Grande do Norte regions), *n* = 19; Colombia (Bucaramanga region), *n* = 35).\n\nThe samples were collected in the form of buccal cells or total blood. Buccal cells were collected by swabbing the inside of the cheek using sponge swabs, and applied to Whatman FTA cards (Whatman International Ltd., Maidstone, UK), according to the manufacturer's instructions. Total blood was also sampled on Whatman FTA cards. DNA extractions, PCR amplifications and sequence analysis were performed as previously described \\[2\\]. A 582 base-pair fragment of the mt control region (CR) was surveyed using previously published and newly sequenced samples (see the electronic supplementary material, table S1). All novel haplotypes identified in this study have been deposited in GenBank under accession numbers [HQ452424](HQ452424)\u2013[HQ452430](HQ452430) and [HQ452435](HQ452435)\u2013[HQ452438](HQ452438). In a previous study \\[4\\], complete mt genomes of 169 individuals representing almost all parts of the CR networks resulted in the identification of subclades within the original clades A, B and C, which are informative since some of them have not been found in European dogs (<a href=\"#RSPB20131142F1\" data-ref-type=\"fig\">figure 1<\/a>). Individuals sequenced only for the CR can be assigned to subclades by mutations within the CR which are diagnostic for each subclade \\[4\\].\n\nWe also compared our dataset with published data from ancient American dog samples \\[8\\] and modern American non-breed dogs \\[10\\]. Since the sequences of these two datasets overlapped only partly with our sequences, comparisons were performed separately from the main analysis. Thus, our modern sequences were compared with 19 ancient DNA sequences obtained from dog remains from South America (Bolivia, Peru and Mexico) and Alaska (Fairbanks area) \\[8\\]. These sequences overlap by 244 bp with our modern sequence data. We also compared our samples with sequences from Castroviejo-Fisher *et al*. \\[10\\] which consisted of mtDNA sequences from '400 dogs from rural and isolated areas as well as street dogs' from all across America. These sequence overlap by 342 bp with our sequences.\n\n## Phylogenetic analysis\n\nSequences were aligned with the MUSCLE tool implemented in G<span class=\"smallcaps\">eneious<\/span> v. 5.5.3 P<span class=\"smallcaps\">ro<\/span> \\[21\\]. Comparisons of sequences and identification of haplotypes were performed with D<span class=\"smallcaps\">na<\/span>S<span class=\"smallcaps\">p<\/span> v. 5 \\[22\\]. Minimum-spanning networks and trees were constructed manually based on the minimum distances between haplotypes calculated with A<span class=\"smallcaps\">rlequin<\/span> v. 3.5 software \\[23\\] ignoring indels. Unique and shared haplotypes among dog populations or breeds from different geographical regions were identified using the program UPST.EXE (available by request to <firstname.lastname@example.com>).\n\n# Results\n\nAmong the American samples, all haplotypes belonged to the universally occurring clades A, B and C, indicating an Old World origin for all maternal lineages. Among the Old World samples, 38 haplotypes were unique to Europe, 121 unique to East Asia (excluding Siberia) and 12 unique to Siberia (<a href=\"#RSPB20131142F1\" data-ref-type=\"fig\">figure 1<\/a>). This dataset included 302 new European samples relative to a previous study \\[4\\], but the large difference between European and East Asian samples observed previously did not alter. Thus, the diversity among European dogs was very limited, with practically all haplotypes centred around the 15 UTs (<a href=\"#RSPB20131142F1\" data-ref-type=\"fig\">figure 1<\/a>). By contrast, approximately 50% of East Asian dogs carried haplotypes on large distance from those found in Europe, many of which belonging to sub-haplogroups not detected in Europe, implying the possibility of tracing the geographical origins of a large proportion of the American maternal lineages.\n\n## Breed dogs\n\n### Arctic America\n\nInuit sled dogs, Canadian Eskimo dogs and Greenland dogs had similar mtDNA gene pools, in that the most frequent haplotype for all three breeds was A31, unique to this group of breeds (<a href=\"#RSPB20131142TB1\" data-ref-type=\"table\">table 1<\/a> and <a href=\"#RSPB20131142F1\" data-ref-type=\"fig\">figure 1<\/a>). The Inuit sled dog and the Greenland dog also shared a haplotype unique to the two breeds (A124). In addition, the Inuit sled dog and the Eskimo dog had one unique haplotype each (A181 and A63, respectively). Three of these four private haplotypes differ by at least two substitutions from any of the European haplotypes. In total, 30\/38 individuals in this group of dogs had haplotypes unique to the group, and the remaining eight dogs had universally occurring haplotypes, present in both Europe and East Asia. Importantly, a very recent article \\[9\\] showed that pre-Columbian samples from Greenland had a high frequency of A31, strongly suggesting ancestry from the pre-Columbian population for this group of dogs.\n\nmtDNA control region haplotypes found among American breed dogs and Carolina dog, and their geographical distribution in both the Old World and the New World. Subclade refers to the 10 subclades of clades A, B and C; only haplotypes belonging to subclades other than the four universal subclades a1, b1, c1 and c2 are indicated. UT, universal types of DNA haplotype found worldwide \\[4\\]; PT, private types found only in a specific breed, population and\/or region.\n\n| breed (code) | *n* | haplotype (subclade) | frequency (*n*) | geographical distribution |\n|:---|:---|:---|:---|:---|\n| Inuit sled dog (ISD) | 18 | A181 | 6% (1) | PT |\n|  |  | A124 | 6% (1) | PT in Arctic America (ISD, GD) |\n|  |  | A31 | 78% (14) | PT in Arctic America (ED, GD, ISD) |\n|  |  | A20 | 11% (2) | UT |\n| Eskimo dog (ED) | 9 | A31 | 67% (6) | PT in Arctic America (ED, GD, ISD) |\n|  |  | A63 | 11% (1) | PT |\n|  |  | A18 | 22% (2) | UT |\n| Greenland dog (GD) | 11 | A31 | 55% (6) | PT in Arctic America (ED, GD, ISD) |\n|  |  | A124 | 9% (1) | PT in Arctic America (ISD, GD) |\n|  |  | A11; A17; A20 | 36% (4) | UT |\n| Alaskan Malamute (AM) | 9 | A29 (a2) | 78% (7) | Siberia, Japan, China, Indonesia |\n|  |  | A11; A17 | 22% (2) | UT |\n| Chihuahua (CH) | 14 | A185 (a4) | 36% (5) | PT |\n|  |  | C16 | 7% (1) | South America (CH and PSP), Siberia |\n|  |  | A11; A17; B1 | 57% (8) | UT |\n| xoloitzcuintle (Xo) | 43 | A24 | 2% (1) | Europe, Colombia |\n|  |  | B12 | 2% (1) | Europe, Japan |\n|  |  | A2; A16; A17; B1; C1; C2; C3 | 95% (41) | UT |\n| Carolina dog (CD) | 19 | A184 (a5) | 37% (7) | PT |\n|  |  | A39 (a2) | 5% (1) | Japan, China |\n|  |  | A16; A18; A19; B1 | 58% (11) | UT |\n| perro sin pelo Del Peru (PSP) | 53 | C16 | 62% (33) | Siberia, South America (CH, PSP) |\n|  |  | A1 | 2% (1) | Europe , China, Korea, Brazil |\n|  |  | B8 | 2% (1) | Europe |\n|  |  | C8 | 2% (1) | Europe |\n|  |  | A2; A11; A17; A18; A19; B1 | 32% (17) | UT |\n\nAmong the Alaskan Malamute, 7\/9 individuals had haplotype A29, which belongs to subclade a2 (not represented in Europe) and otherwise found only in East Asia (including Siberia) and island Southeast Asia. The two other individuals had universally occurring haplotypes. Haplotype A29 was also found in pre-Columbian samples from Alaska \\[8,9\\]. This suggests ancestry of modern Alaskan Malamute from the pre-European Alaskan population (<a href=\"#RSPB20131142F2\" data-ref-type=\"fig\">figure 2<\/a>) but, since A29 is also commonly found among Siberian husky with which Alaskan huskys were interbred in the early 1900s, the evidence for this is not clear cut. The Alaskan Malamute and the Eskimo\/Greenland\/Inuit group had almost totally different mtDNA gene pools, only 2\/9 and 2\/38 individuals, respectively, sharing haplotypes between the groups.\n\n### North America\n\nFor the Mexican breed Chihuahua, the most frequent haplotype (carried by 5\/14 individuals) was A185, which was unique to this breed among the modern dogs. Importantly, this haplotype was also found in one pre-Columbian sample from Mexico (<a href=\"#RSPB20131142F2\" data-ref-type=\"fig\">figure 2<\/a>), suggesting direct ancestry of Chihuahua from ancient Mexican dogs. One individual had haplotype C16, the most common type among the South American breed perro s\u00edn pelo del Peru. C16 was also reported for xolo at a frequency of 16% in a previous study \\[24\\]. Among the Old World samples, C16 was found only among Siberian dogs. The remaining Chihuahua had universally occurring haplotypes. The Mexican 'naked breed' xoloitzcuintle (xolo) had only haplotypes occurring universally and two haplotypes found in Europe. However, as noted above, C16 was found in a previous study of xolo, at a frequency of 16% \\[24\\]. Notably, 79% of the xolo carried haplotypes shared with perro s\u00edn pelo del Peru, the South American 'naked breed'.\n\n### South America\n\nFor the Peruvian 'naked breed' perro s\u00edn pelo del Peru, the majority of dogs (33\/53) had haplotype C16, absent among the European samples and shared only with the other 'naked breed' xolo, and with Chihuahua and Siberian dogs. Two individuals had haplotypes otherwise unique to Europe, and the remaining individuals had universally occurring haplotypes. Eighty-one per cent of the individuals shared a haplotype with xolo.\n\nIn conclusion, all investigated breeds of presumed indigenous origin across America carried haplotypes indicating pre-European origin, except for Alaskan Malamute for which the results were ambigous. All these breeds except xolo had a high frequency of haplotypes absent in Europe: 79% of the Arctic Inuit, Eskimo and Greenland dogs, and a total of 35% of dogs belonging to the Meso\/South American breeds Chihuahua, xolo and perro s\u00edn pelo del Peru. Only half of the lineages introduced to America from Asia are expected to differ from European sequences, since the frequency of UTs is approximately 50% in Siberia and East Asia. Therefore, even if 100% of the lineages would have an ancient origin from Asia, only 50% of the individuals would be expected to carry haplotypes absent in Europe. Hence, our data indicates that the Arctic breeds Inuit, Eskimo and Greenland dog (having a frequency above 50% of haplotypes absent in Europe) have remained practically uninfluenced by European lineages, and that among the Meso\/South American breeds (35% of the dogs carrying haplotypes distinct from European haplotypes), 70% of the maternal lineages originate from the pre-Columbian population. Thus, replacement of indigenous American breeds by European dogs seems to have been relatively limited.\n\nWe also analysed dogs belonging to the fox Paulistinha and dogo Argentino breeds. These breeds originate from dogs of known European ancestry. In accordance, these dogs carried only haplotypes that are frequently found in Europe, five universal haplotypes and one other haplotype frequent in Europe (see the electronic supplementary material, table S2).\n\n## Comparison of pre-Columbian sequences with a comprehensive modern sample\n\nWe compared our dataset with ancient American mtDNA sequences from Mexico, Peru, Bolivia and Alaska reported in an earlier study (\\[8\\]; <a href=\"#RSPB20131142F2\" data-ref-type=\"fig\">figure 2<\/a>). This analysis is the first comparison of these ancient American sequences with a comprehensive collection of sequences from modern East Asian dogs and American breed dogs, instead of predominantly European breeds as in earlier studies. This allowed us to investigate possible geographical continuity over time among American breed dogs and to explore the possibility of an East Asian origin of the ancient American dog mtDNA lineages.\n\nWe found two cases where ancient and modern samples from the same geographical region had the same non-universal haplotype, suggesting continuity since pre-European time for these populations. In one case, haplotype A185 was found exclusively in ancient Mexican samples (D25) and in modern samples of the Mexican Chihuahua, strongly indicating direct ancestry of Chihuahua from Mexican pre-Columbian dogs. Furthermore, a haplotype corresponding to A29 (D18), otherwise found only in Siberia, East Asia and Oceania, was identified in ancient Alaskan samples as well as in the modern Arctic breed Alaskan Malamute but, as related above, the presence of A29 also among Siberian husky offers an alternative to genetic continuity in Alaska. Notably, a very recent study of pre-Columbian dogs from Alaska and Greenland \\[9\\] showed that, in Greenland, both the ancient population and the modern Inuit sled dog population carry predominantly the unique haplotype A31, strongly indicating ancient local ancestry for this group of Arctic dogs.\n\nImportantly, Leonard *et al*. reported one phylogenetic group of haplotypes (containing haplotypes D27, D28, D29, D30, D31 and D33) to be unique for the ancient South\/Meso American samples (<a href=\"#RSPB20131142F2\" data-ref-type=\"fig\">figure 2<\/a>). However, we found the central haplotype D28 in East Asia (in Korea, haplotype A161), thus placing the origins of these sequences in East Asia. Eight of the 19 ancient haplotypes correspond to haplotypes found in the modern samples, all of which, except the American specific A185, found in East Asia. The remaining haplotypes were unique to the ancient samples, but differ by one or two substitutions from extant haplotypes found in East Asia, except one ancient haplotype (D40). D40 is separated by at least four substitutions from any other dog haplotype and seemingly represents a separate phylogenetic clade (<a href=\"#RSPB20131142F2\" data-ref-type=\"fig\">figure 2<\/a>). Therefore, D40 is possibly the result of a crossbreeding between a dog and a female wolf in America.\n\nIn conclusion, we find a strong indication that the Mexican breed Chihuahua has a direct ancestry from Mexican pre-Columbian dogs, and a similar link was recently shown for Inuit dogs and pre-Columbian samples in Greenland \\[9\\]. Importantly, except D40, all ancient American samples can be linked to haplotypes found among East Asian dogs.\n\n## Free-ranging dogs\n\nAmong the 'free-ranging' Carolina dogs of southern USA, the most frequent haplotype was A184, which was unique to these dogs (it is a novel halpotype not previously reported in GenBank) and belongs to the East Asian-specific phylogenetic subclade a5 (<a href=\"#RSPB20131142F1\" data-ref-type=\"fig\">figure 1<\/a>). One individual had haplotype A39, otherwise found only among Chinese non-breed dogs and the Japanese breed shiba inu. The remaining dogs had universal haplotypes. This gives a strong support to the hypothesis that the Carolina dog has indeed originated from pre-Columbian dogs.\n\nBy contrast, South American free-ranging dogs from Argentina, Brazil and Colombia had mainly universally occurring haplotypes (83.3% carrying UTs) and several European specific haplotypes (see the electronic supplementary material, table S2). There were also three unique haplotypes (A189, A190, B45), a single step from haplotypes found also in Europe, and three haplotypes (A38, B2 and B5) absent from Europe but present in East and West Asian breeds (e.g. chow chow, Afghan hound and akita) and rural non-breed dogs. Thus, South American free-ranging dogs in our sample seem to originate mainly from European dogs, although traces of native dogs cannot be totally excluded.\n\nThis is in good agreement with a previous study by Castroviejo-Fisher *et al*. \\[10\\] of 400 dogs from rural and isolated areas, as well as street dogs from across America. This study reported that a very low proportion of the mtDNA lineages among these modern American non-breed dogs (less than 10%) derive from the pre-European dog population, based on a comparison with the 19 ancient American dog samples reported by Leonard *et al.* \\[8\\]. This indicated that almost all indigenous dogs were replaced by European dogs. However, comparing the dataset in Castroviejo-Fisher *et al*. with our comprehensive sample of modern dogs, we found that these dogs carry several potentially indigenous mtDNA haplotypes which are unique to America or otherwise found exclusively in East Asia (see the electronic supplementary material, figure S1 and table S3). These haplotypes were carried by 7.8% of all dogs, which indicates that approximately 15% of the lineages have pre-European ancestry, considering that at least 50% of indigenous American dogs are expected to carry non-informative haplotypes (UTs) also found in Europe. More importantly, in a detailed analysis of the different subpopulations, we also noted that the putatively indigenous haplotypes were not evenly distributed. Thus, 9.8% of the Mexican dogs and 14.8% of the Bolivian dogs carry these haplotypes (indicating 20\u201330% Asian ancestry), but only 2.3% of the dogs from Argentina. This agrees with our findings that a large proportion of Carolina dogs carry seemingly indigenous American haplotypes, whereas our sample of free-ranging Argentinan dogs has almost exclusively European haplotypes. Therefore, it seems that while free-ranging American dogs generally derive from European dogs, there are pockets of rural populations, for example, the Carolina dog and populations in Mexico and Bolivia, with a high proportion of pre-Columbian ancestry. Importantly, comparing with a dataset from Europe \\[25\\], only two of 94 dogs of European origin (2.1%) carried a unique haplotype, confirming that the majority of the unique American haplotypes have an indigenous origin and are not merely European haplotypes not yet sampled.\n\nCastroviejo-Fisher *et al.* \\[10\\] also reported that a large proportion of haplotypes found in the dataset (23 out of 40) were unique to the American dogs compared with dogs of the Old world. However, the comparison was performed against a limited dataset of non-American dogs \\[26\\], and comparing instead to our more comprehensive dataset of dogs from across the Old World, we identified the majority of these 23 haplotypes. For the 342 bp region overlapping with our dataset, there were 34 haplotypes in the dataset of Castroviejo-Fisher *et al*. Of these, 27 were shared with dogs in Eurasia, leaving only seven haplotypes unique to America.\n\n# Discussion\n\nThis study provides clear evidence for the ancient Asian ancestry of extant American dog breeds. All breeds of presumed indigenous American origin carried haplotypes absent in a comprehensive sample of European dogs, and our data indicates that only 30% or less of the female lineages in indigenous American breeds have a European origin. Importantly, the Mexican breed Chihuahua shared a haplotype uniquely with Mexican pre-Columbian samples, showing genetic continuity over time and geographical region and corroborating the Mexican origins of the Chihuahua. The data also once more confirmed that American dogs have a common origin with Old World dogs, since no distinct haplogroups unique to American dogs were found and all haplotypes fell into the previously described universal phylogenetic clades A, B and C. However, we note that one pre-European Alaskan dog reported by Leonard *et al*. \\[8\\] had a haplotype (D40) separated by four substitutions from the closest modern dog haplotype, which we believe may derive from a dog\u2013wolf hybridization.\n\nAn important finding is that not only American breed dogs but also some populations of free-ranging dogs seem to stem from indigenous American dogs. Thus, we here give genetic evidence that feral dogs from the USA, the so-called Carolina dog, may have an indigenous American origin and are not just 'run-away' dogs of European descent. The dingo-like appearance of the Carolina dogs may therefore be a remnant of their ancient past. The most frequent haplotype (A184) is unique to Carolina dogs and belongs to an East Asian-specific phylogenetic subclade, offering a clear indication of an East Asian origin for these dogs. Also haplotype A39, otherwise found only among East Asian dogs including the shiba inu, may have an ancient East Asian origin, but the possibility remains that this haplotype derives from abandoned shiba. The South American free-ranging dogs carried mainly universal or typically European haplotypes, but a few individuals had haplotypes carried by East Asian dogs and absent among European breeds. However, all these haplotypes are also carried by East Asian breeds present in the Western world dog population and could have resulted from integration of abandoned Asian breed dogs. We conclude that the South American free-ranging dogs mainly originate from dogs brought by the Europeans, although traces of native dog cannot be totally excluded.\n\nIn general agreement with our data, the study by Castroviejo-Fisher *et al.* \\[10\\] reported that at most 10% of the mtDNA lineages among American rural and street dogs have indigenous American origin. However, in a more detailed phylogeographical analysis of these samples, we note that in the subsamples from Mexico and Bolivia approximately 25% of the lineages seem to retain mtDNA haplotypes with an Asian origin. Therefore, pockets of remaining populations with high proportion of indigenous pre-European origin seem to persist among American non-breed dogs, as well as among the indigenous American breeds.\n\nWe conclude that the ancestry of the American dog population is complex. There are several different types of populations of American dogs of possibly pre-Columbian origin, and these populations retain different proportions of pre-Columbian ancestry. The dog breeds of supposedly indigenous American origin carry predominantly mtDNA haplotypes of pre-Columbian origin. By contrast, free-ranging dogs, including dogs from rural areas as well as street dogs, generally show no or very little pre-Columbian ancestry, but also among these dogs there remain populations with considerable proportions of indigenous American ancestry.\n\nThe studied Arctic American dogs seem to consist of two separate populations. The Alaskan Malamute is thought to descend from dogs bred by the Mahlemut people of the upper-western Alaska. A majority of these dogs carried haplotype A29, which seemingly presents both spacial and temporal clues to the origins of the breed. A29 (absent in Europe) is found among East Asian dogs and, significantly, among Siberian husky, a sled dog originally bred by the Chukchi people on the northeastern tip of Siberia. This indicates genetic links between East Asia, Siberia and Arctic America. A29 was also found in ancient samples from Alaska, indicating the presence of the Alaskan Malamute ancestors in the pre-Columbian era. However, a complicating factor is that Alaskan Malamutes were interbred with Siberian husky in the gold rush era; analyses of additional markers will be necessary to establish how the modern Alaskan Malamutes relates to the dogs of the Mahlemut people. The other modern Arctic breeds (Inuit, Eskimo and Greenland dogs) seem to constitute a separate genetic group, as only 2\/38 individuals shared haplotypes with Alaskan Malamutes. The three breeds share a common gene pool including a unique haplotype (A31) found in the majority of individuals. These three breeds, in accordance with the mtDNA data, are often considered a single breed and are thought to descend from sled dogs bred by Thule people, the ancestors of the modern Inuits. This culture developed in coastal Alaska by 1000 years ago and rapidly reached Greenland a few centuries later, having presented the first evidence of consistent use of sled dogs in the Arctic \\[27\\]. Importantly, a very recent study of pre-Columbian arctic dogs \\[9\\] showed that the related ancient dog population in Greenland carried almost exclusively A31. Thus, the ancient and modern populations share the unique haplotype A31 at high frequency, confirming the ancestry of modern Inuit, Eskimo and Greenland dogs from the local ancient population. The almost total lack of shared haplotypes between these breeds and the Alaskan Malamute possibly reflects sequential arrival to America of the related human populations \\[15\\].\n\nOverlap was also minimal between the Arctic breeds and other dogs across America. By contrast, North American (excluding the Arctic) and South American breeds showed genetic links. Chihuahua shared one haplotype (C16) exclusively with perro s\u00edn pelo and Siberian dogs. This haplotype was absent from our xolo sample but was found in this breed in a previous study \\[24\\]. Furthermore, around 80% of the individuals of the Mexican 'naked breed' xolo and the Peruvian 'naked dogs' shared haplotypes, indicating a geographical link connected with the shared morphology, in agreement with all naked breeds carrying the same causative mutation \\[28\\]. It is notable that genetic data for both dogs and humans indicate an initial migration forming the North and South American populations, followed by subsequent waves into the Arctics \\[15\\].\n\nComparison of the modern data with ancient sequences suggested genetic continuity from pre-Columbian into modern times in Mexico and, possibly, Alaska, adding to the evidence for genetic continuity in Greenland presented very recently by Brown *et al.* \\[9\\]. Modern Chihuahua and ancient Mexico had the same unique haplotype (A185 and D25, respectively), and ancient Alaska and modern Alaskan Malamute shared a non-European haplotype (D18 and A29, respectively). Interestingly, A185 and A29 have been reported also for modern Puerto Rican 'street dogs' \\[29\\], but the possible link to ancient American samples was not noted in that study. This suggests that A185 and A29 may have been widespread across ancient America but, since the two haplotypes are found among modern Chihuahua and Siberian husky, there remains the possibility that A185 and A29 in Puerto Rico originate from abandoned dogs of these breeds. An important finding when comparing the new modern sequences presented in this study with the ancient DNA samples was that the central haplotype (D28) in a formerly 'ancient America-specific' clade \\[8,10\\] was found among East Asian dogs (haplotype A161). This implies that all ancient American sequences except one (D40; possibly the result of dog\u2013wolf crossbreeding) can now be linked to haplotypes present in East Asia or Siberia. We also note a large proportion of unique haplotypes among the ancient samples. However, the ancient sequences harbour an unusual amount of transversions, suggesting that these may be single base substitution artefacts, a known problem in the sequencing of ancient samples. For example, the five unique haplotypes surrounding D28 (D27, D29, D30, D31 and D33) are defined by three transversions (in previously non-variable positions) and two transitions, compared with six transversions and 50 transitions found among the 1555 modern samples in clade A. It is therefore possible that several of the haplotypes surrounding D28 are in reality D28 (modern A161), and thus identical to modern East Asian samples.\n\nIn the modern sample, we identified nine haplotypes, five in the Arctic and four in North and South America, which were distinct from haplotypes found among European dogs. Only 50% of the pre-European dogs are expected to have carried haplotypes absent in Europe, implying that several more of the American lineages probably have an Asian origin. There were also at least four additional haplotypes found in the ancient American samples. Thus, it seems that the American dog population was not formed through a severe genetic bottleneck, but that several lineages were brought to America from Asia in the pre-Columbian era, probably in several waves of migration. The analyses in this study are based on mtDNA, which is a single genetic marker inherited maternally, putting obvious limitations to the inferences. To assess fully the pre- and post-Columbian contribution of genetic material to the American breeds, as well as the possibility of hybridization between American dogs and wolves, studies based on additional markers, suggestively the Y-chromosome as well as autosomal markers will be necessary. Possibly, improved phylogeographic fine mapping of both American humans and dogs may show whether the genetic diversity of domestic dogs mirrors that of humans and provide clues for understanding the colonization of the New World.\n\n## Acknowledgements\n\nWe thank numerous donors of samples, especially Vet. Dr Liliana Arbulu Perez Vargas, Sue Hamilton, Marina Kuzina, Gunvor Bergstr\u00f6m, Sonja Kolijn, Martin Whittle, Carlos Vullo, Eliseu Fagundes de Carvalho, Pedro Balonas, Adriana Castillo, Eduardo Arroyo and Jane Gunnell for organizing of sampling.\n\n## Funding statement\n\nThis study was supported by grants from the Swedish Research Council, OE and Edla Johanssons Scientific Foundation, the Wenner-Gren Foundations and by a research grant to B.v.A. (SFRH\/BDE\/15581\/2006) from Funda\u00e7\u00e3o para a Ci\u00eancia e a Tecnologia. P.S. is a Royal Swedish Academy of Sciences Research Fellow supported by a grant from the Knut and Alice Wallenberg Foundation and was during the work with this study, a Pearson Distinguished Professor at Bethany College supported by a grant from Gerald Pearson. IPATIMUP is an Associate Laboratory of the Portuguese Ministry of Ministry of Science, Technology and Higher Education and is partially supported by FCT, the Portuguese Foundation for Science and Technology.\n\n# References\n\n[^1]: These authors contributed equally to this study.","meta":{"dup_signals":{"dup_doc_count":104,"dup_dump_count":40,"dup_details":{"curated_sources":2,"2018-43":1,"2018-39":1,"2018-34":1,"2018-30":1,"2018-26":1,"2018-22":2,"2018-13":1,"2018-09":1,"2018-05":2,"2017-47":2,"2017-39":3,"2017-30":2,"2017-26":1,"2017-22":2,"2017-17":1,"2017-09":4,"2017-04":3,"2016-50":2,"2016-44":4,"2016-40":3,"2016-36":3,"2016-30":4,"2016-26":2,"2016-22":3,"2016-18":3,"2016-07":4,"2015-48":4,"2015-40":3,"2015-35":4,"2015-32":4,"2015-27":4,"2015-22":4,"2015-14":3,"2014-52":4,"2014-49":3,"2018-47":1,"2017-13":2,"2015-18":4,"2015-11":1,"2015-06":4}},"file":"PMC3730590"},"subset":"pubmed_central"}
{"text":"abstract: # Introduction\n  .\n  The finding of antinuclear antibody (ANA) positivity in a healthy individual is usually of unknown significance and in most cases is benign. However, a subset of such individuals is at risk for development of autoimmune disease. We examined demographic and immunological features that are associated with ANA positivity in clinically healthy persons to develop insights into when this marker carries risk of progression to lupus.\n  .\n  # Methods\n  .\n  Biological samples from healthy individuals and patients with systemic lupus erythematosus (SLE) were obtained from the Dallas Regional Autoimmune Disease Registry (DRADR). Measurements carried out on serum samples included ANA, extractable nuclear antibodies (ENA) and autoantibody profiling using an array with more than 100 specificities. Whole blood RNA samples from a subset of individuals were used to analyze gene expression on the Illumina platform. Data were analyzed for associations of high ANA levels with demographic features, the presence of other autoantibodies and with gene expression profiles.\n  .\n  # Results\n  .\n  Overall, ANA levels are significantly higher in females than in males and this association holds in patients with the autoimmune diseases lupus and rheumatoid arthritis (RA) as well as in healthy controls (HC). Age was not significantly associated with ANA levels and the elevated ANA values could not be explained by higher IgG levels. Another autoantibody, anti- cyclic citrullinated peptide (CCP), did not show gender dimorphism in rheumatoid arthritis (RA) or healthy individuals. The autoantigen array showed significant elevations of other autoantibodies in high ANA HCs. Some of these autoantibodies were directed to antigens in skin and others were related to autoimmune conditions of kidney, thyroid or joints. Gene expression analyses showed a greater prevalence of significantly upregulated genes in HCs with negative ANA values than in those with significant ANA positivity. Genes upregulated in high ANA HCs included a celiac disease autoantigen and some components of the Type I interferon (IFN) gene signature.\n  .\n  # Conclusions\n  .\n  Risks for ANA positivity include female gender and organ-specific autoimmunity. Upregulation of skin-specific autoantibodies may indicate that early events in the break of tolerance take place in cutaneous structures. Some of these changes may be mediated by Type I IFN. Blood profiling for expressed autoantibodies and genes has the potential to identify individuals at risk for development of autoimmune diseases including lupus.\nauthor: Quan-Zhen Li; David R Karp; Jiexia Quan; Valerie K Branch; Jinchun Zhou; Yun Lian; Benjamin F Chong; Edward K Wakeland; Nancy J Olsen\ndate: 2011\ninstitute: 1Department of Immunology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas TX 75390-9093, USA; 2Department of Medicine, The Division of Rheumatic Diseases, The Simmons Arthritis Center, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas TX 75390-8884, USA; 3Department of Dermatology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas TX 75390-9069, USA; 4Current address: Division of Rheumatology, Penn State Hershey Medical Center, 500 University Drive, Hershey PA 17033, USA\nreferences:\ntitle: Risk factors for ANA positivity in healthy persons\n\n# Introduction\n\nAntinuclear antibodies (ANAs) are measurable in approximately 25% of the population, and the prevalence of significantly elevated levels may be 2.5% \\[1\\]. Findings from numerous studies show remarkable consistency across ethnically and racially diverse study populations despite the use of many different methods for ANA measurement. The persistence of this type of autoreactivity in the human population suggests that antinuclear antibodies may be an important component of the normal immune response. Most individuals with a positive ANA do not have an autoimmune disease and most also are unlikely to develop one. This is consistent with the fact that the prevalence of all autoimmune disorders is 5 to 7% \\[2\\]. Furthermore, the disease that is most closely linked to ANA positivity, systemic lupus erythematosus (SLE), is relatively rare, affecting no more than 1 to 1.5 per 1,000 persons (0.1 to 0.15%) in the United States \\[3\\]. Nevertheless, since ANA positivity is for all practical purposes a requirement for SLE diagnosis, it must also be assumed that individuals who are in preclinical disease stages are represented in the ANA positive healthy population. Although many consultations for ANA positivity seen in rheumatology practice are not associated with any identifiable pathology, it is also true that if early detection of SLE is to become feasible, focus on the ANA positive population will be necessary.\n\nWe have considered the possibility that other blood markers could be used to differentiate benign ANA positivity from that which carries a high risk of autoimmune disease. These markers may include other autoantibodies, since it is well-known that autoantibody positivity increases in quantity and complexity in years preceding a diagnosis of SLE \\[4\\]. Gene dysregulation in peripheral blood cells has been closely associated with SLE diagnosis and disease status, so changes in gene expression may also signal a condition with enhanced risk.\n\nTo address these questions, we studied healthy individuals and patients with autoimmune diseases who had been enrolled in the Dallas Regional Autoimmune Disease Registry (DRADR). A subgroup of healthy controls that were found to have high ANA levels was examined in greater detail using autoantigen and gene expression arrays. The findings suggest the feasibility of identifying risk markers for development of SLE in the setting of ANA positivity, using both demographic features and profiling of autoantibodies and expressed genes in peripheral blood.\n\n# Materials and methods\n\n## Patients and healthy controls\n\nSamples for study were obtained from the Dallas Regional Autoimmune Disease Registry (DRADR). Individuals are classified according to clinical diagnosis or healthy condition at the time of enrollment into the registry. Disease features and the presence of SLE criteria were determined by patient interview and medical record review \\[5\\]. Blood samples were obtained for banking of serum aliquots and whole blood samples were stored in PaxGene tubes for later isolation of RNA. All samples were maintained at -80\u00b0C until use. The overall study group included 1,159 individuals from DRADR: 401 healthy controls (HC) who were negative for current or past autoimmune disease, 116 first-degree relatives (FDR), 294 patients with SLE, 151 patients with less than 4 SLE criteria and considered as having incomplete lupus (ILE), 154 with rheumatoid arthritis (RA) and 43 with other miscellaneous conditions including scleroderma, Sjogren's syndrome, ankylosing spondylitis and vasculitis. More detailed analyses were carried out on a subset of HC individuals with high ANA values (*n* = 18) and these were compared to gender- and age-matched HC with negative ANA values (*n* = 16) and to SLE patients with high ANA levels of \\>100 E.U. (*n* = 14). In one experiment, plasma samples from the Dallas Heart Study population, which has been described previously \\[1\\], were employed. All subjects gave written informed consent for entry into the Dallas Regional Autoimmune Disease Registry. Research carried out under the auspices of this registry has been approved by the UT Southwestern Institutional Review Board.\n\n## Antibody assays\n\nSerum ANA levels were measured by ELISA (Inova, San Diego, CA, USA) using the manufacturer's suggested cut-off of \\>20 units to define positive results. Other ELISA kits were used to measure CCP antibodies (Inova), thyroglobulin autoantibodies (Genway Biotech Inc., San Diego, CA, USA) and total IgG (Bethyl Laboratories Inc., Montgomery, TX, USA). The extractable nuclear antibody (ENA) panel for eight additional specificities was a Luminex-based multiplex assay, and positive results were as defined by the manufacturer (Inova). Serum reactivity to a panel of approximately 101 autoantigens and 6 controls was measured on a slide-based array that has been described previously (Additional file <a href=\"#S1\" data-ref-type=\"supplementary-material\">1<\/a>) \\[6\\]. Serum samples (1 \u03bcL, diluted 1:100) were added to each array in duplicate and autoantibodies were detected with Cy3-labelled anti-human IgG and Cy-5 labeled anti-human IgM simultaneously. Images were generated for analysis and mean fluorescence intensities (MFI) were determined as previously described. Heat maps were generated using Cluster and Treeview software (Michael Eisen, Berkeley CA, USA) \\[7\\]. On the heat map, intensities higher than the row mean are colored red, those below the mean are green and cells with signals close to the mean are black. Gray was used to denote missing data.\n\n## Gene expression analysis\n\nTotal RNA was prepared from 2.5 to 5.0 ml of blood collected in Paxgene tubes that had been stored at -80\u00b0C. Purity and concentration of the isolated RNA was determined using Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) and Nanodrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). We used 250 ng of total RNA to generate biotinylated cRNA using a TotalPrep RNA Amplification kit (Ambion, Austin, TX, USA). The samples were applied to Ilumina HumanWG-6 v3.0 Expression Bead Chips (Illlumina Inc., San Diego, CA, USA) following the manufacturer's directions. The resulting array data were analyzed using Ilumina GenomeStudio software (version 3) and statistical analyses were carried out using Partek Genomic Suite (version 6; Partek Inc., Gladstone, MI, USA). Heat maps were generated using the Cluster and Treeview programs \\[7\\].\n\n## Statistical analyses\n\nData are presented as mean values and standard errors of the mean. Continuous variables in experiments with three or more groups of samples were analyzed using a one-way ANOVA with Tukey's multiple comparison test for post test analysis of pairs of samples or the Kruskal-Wallis test for data that did not fit a normal distribution. Analyses of experimental data with two comparison groups used an unpaired 2-tailed Student's *t*-test, with Welch's correction for groups with unequal variances or Mann-Whitney U for data that did not fit a normal distribution. Pearson's correlation coefficient was used to compare continuous variables. Discontinuous variables were compared using Fisher's Exact Test. Graph pad PRISM software (version 5.0a; GraphPad Software, La Jolla, CA, USA) was used for data analysis and graphics. *P*-values \\<0.05 were considered significant.\n\n# Results\n\n## ANA and autoantibodies\n\nUsing the defined cut-off of \\>20 ELISA units (EU), 615 individuals out of the 1,159 tested were ANA positive. For the subset of 401 HC, the average ANA was 19.5 EU and 101 individuals were in the positive range. This rate of 25% HC positivity is very close to what we have reported previously \\[1\\]. Healthy FDRs had a slightly higher overall average ANA (24.4 EU) and a prevalence of ANA positivity of 34%. These values were not significantly different than in the HCs (*P* \\>0.07 for both). For the overall group, ANA values were significantly higher in females than in males (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>) and no males had values \\>240 EU while 3.2% of females had values in this range (*P* = 0.0030). When the analysis was limited to HC, females again showed significantly higher ANA levels than males (21.4 vs. 15.6 EU; *P* = 0.033) and ANA positivity was also more prevalent in HC females (29%) than in HC males (17%; *P* = 0.014). This corresponds to a relative risk of ANA positivity in healthy females vs. males of 1.21 (95% confidence interval = 1.059 to 1.390). The highest ANA levels were seen almost exclusively in non-Hispanic females (data not shown), although the overall mean values for Hispanic and non-Hispanic females were not significantly different (*P* = 0.7). African-American (AA) HC (*n* = 32) had a higher mean ANA value than non-AAs (27.47 \u00b1 5.8 EU vs. 18.1 \u00b1 5.8 EU), but the difference did not reach statistical significance (*P* = 0.064). Within each gender, AA individuals also had higher values than non-AA individuals (females: 30.38 \u00b1 8.12 EU vs. 20.59 \u00b1 1.80 EU; males 21.09 \u00b1 5.55 EU vs. 15.17 \u00b1 1.31 EU); these differences were also not significant (*P* \\>0.1 for each). However, the results are highly suggestive of higher ANA values in AA HC, and the differences might achieve significance in a larger sample size. Patients with SLE did not show gender differences in overall ANA values, although the very highest ANA values were again seen exclusively in females (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>).\n\nWe compared these findings to another autoimmune disease, RA, which is associated with the CCP autoantibody. While RA patients showed significantly higher ANA levels in females than in males (Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>), antibodies to CCP did not show gender differences in either the control population represented by the Dallas Heart Study or in RA patients from DRADR (Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>). These findings suggest that female gender is a general risk factor for ANA but is not necessarily a correlate with other autoimmune disease-specific antibodies.\n\nAge was not related to ANA positivity in HCs; high values were scattered throughout the age spectrum (R^2^ = 0.01). The high ANA levels in HC also were not explained by overall increases in total IgG as the two measures were not significantly correlated (R^2^ = 0.11; *P* = 0.2).\n\nTo further understand high ANA levels in HC, a subgroup analysis was done using HC subjects with ANA values greater than 40 EU as the index group. This value is approximately one standard deviation greater than the overall mean ANA for HC. A total of 18 of the 401 HC, or about 4%, fit this definition of high ANA. Two comparator groups, HC with negative ANA (ANA values \\<12 EU) and SLE patients with high ANA (\\>100 EU) were generally matched for demographic features including gender, race and ethnicity (Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>). Autoantibodies on the ENA panel were generally not elevated in the high ANA HC group. Only 3 of the 18 individuals showed positive ENA results, and in all three the lone positive specificity was anti-chromatin.\n\nDemographic features of study groups\n\n| Group | Age (yrs) | ANA (EU) | Female (%) | AA (%) | Hispanic (%) |\n|----|----|----|----|----|----|\n| HC High ANA (*n* = 18) | 33.8 \u00b1 3.4\\* | 78.4 \u00b1 10.0\\* | *83* | *22* | 6 |\n| HC Low ANA (*n* = 16) | 43.1 \u00b1 3.7 | 7.0 \u00b1 0.6 | *75* | *25* | 0 |\n| SLE (*n* = 14) | 38.0 \u00b1 4.0 | 182.4 \u00b1 15.4 | *93* | *43* | 7 |\n| *P*\\*\\* | 0.2 | 0.001^\\#^ | 0.4 | 0.4 | 0.6 |\n\n\\*Values represent mean and standard error of the mean. \\*\\**P-*values were calculated by one-way ANOVA or Fisher's exact test. ^\\#^ Mean ANA in SLE was significantly higher than in the High ANA HC group (*P* \\<0.05). ANA, antinuclear antibodies; SLE, systemic lupus erythematosus.\n\nThe three study groups were then compared using the autoantigen array for both IgG and IgM autoantibodies (Figures <a href=\"#F3\" data-ref-type=\"fig\">3<\/a> and <a href=\"#F4\" data-ref-type=\"fig\">4<\/a>). Two major IgM clusters showed a tendency for the SLE patients to cluster together, but this was not statistically significant (*P* = 0.08); the high ANA HC individuals had a similar tendency to be in the non-SLE cluster (*P* = 0.06; Figure <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>). For IgG, two primary IgG clusters were identified, and all of the SLE patients were in one of these clusters (*P* = 0.009; Figure <a href=\"#F4\" data-ref-type=\"fig\">4<\/a>).\n\nAntibodies detected on the arrays were then examined for specificities appearing in HC that were lupus-like. This was defined as overall mean values for a given specificity showing no difference between the high ANA HC and SLE groups (*P* \\>0.1) while at the same time having lower values in the low ANA HC group compared to SLE (*P* \\<0.1). The antibodies in these groups were examined to choose those with stepwise increases in the groups (low ANA HC \\<High ANA HC \\<SLE) and to exclude any with distributions that were highly skewed by one or two individuals. This analysis yielded 14 IgGs; 9 of these with the most significant *P*-values (Kruskal-Wallis test) are shown (Figure <a href=\"#F5\" data-ref-type=\"fig\">5<\/a>). Four of the nine specificities are directed against autoantigens found in skin tissues (DSG4, MMP1, recombinant human collagen, integrin\u03b16\u03b24) \\[8-10\\]. Others are associated with autoimmune kidney disease (GBM), thyroid disorders (thyroglobulin), scleroderma (PM\/Scl100), and inflammatory arthritis (proteoglycan) \\[11\\]. A search for autoantibodies that might be associated with a lowered risk of lupus was carried out by choosing specificities that were elevated in high ANA HC but were not high in SLE patients. The ratio of the mean values for the high ANA HC group to the corresponding SLE group mean was calculated, and three specificities had IgG ratios greater than 4.0: Jurkat T cells, gliadin and SK-N-SH (Figure <a href=\"#F6\" data-ref-type=\"fig\">6<\/a>). In each of these, a subset of the high ANA HC individuals showed strong reactivity while SLE patients did not have high values. For gliadin, an autoantigen associated with celiac disease, and Jurkat, a measure of anti-T cell and thymocyte antibodies, the distributions were significantly different for the three groups (*P* = 0.042 and 0.033, respectively). Although the antineuronal antibodies measured by SKNSH reactivity did not show significant differences across groups by ANOVA, a dichotomized analysis showed values greater than 400 MFI units were present only in the high ANA HC group (*P* = 0.0242). The four highest values for each of these different autoantibodies represented the same individuals.\n\nAn independent ELISA assay was carried out for thyroglobulin (TG) autoantibodies to compare with the findings on the array. Anti-TG levels of the IgG class measured by the two techniques were significantly correlated (*P* = 0.007) and values were significantly higher in the HC high ANA group (1.18 \u00b1 0.23 EU) than the HC low ANA group (0.60 \u00b1 0.07 EU; *P* = 0.030), consistent with the array results.\n\n## Gene expression profiles\n\nMean gene expression values were compared between the two HC groups defined by ANA status, and this analysis identified 95 dysregulated genes at a significance level of *P* \\<0.01. Somewhat surprisingly, the vast majority of these genes (90 out of 95) were upregulated in the ANA-negative group. Of the five genes that were upregulated in the high ANA HC group, the highest (two-fold difference) was *TGM2*, which encodes the celiac disease autoantigen transglutaminase 2.\n\nTo detect specificities that might be informative of ANA and diagnosis status, a second analysis was carried out by first comparing the SLE and high ANA HC groups to determine gene specificities that were significantly different at a significance level of *P* \\<0.001. This list was then sorted based on the values for the calculated ratio SLE\/ANA-High HC. The most highly expressed specificity in this list was *IFI27*, which was 65-fold higher in SLE than in ANA High HC. Overall, 69 genes in this list had greater than two-fold differences between SLE and ANA High HC. These 69 genes were then resorted based on differences in the ratio of ANA High HC\/ANA Low HC. The highest ratio in this set was two-fold and out of the top 10 specificities, 7 were in the Type I interferon signature (*IFITM3, MX1, IFI6, IFI44L, ISG15, OAS1, IFIT3*) and one encodes a protein that is activated by dsRNA as might be present in a viral infection (*EIF2AK2*). The specificities that most clearly showed stepwise increases in the three groups going from Low ANA HC to High ANA HC to SLE were *IFITM3* and *MX-1* (Figure <a href=\"#F7\" data-ref-type=\"fig\">7<\/a>).\n\n# Discussion\n\nDetecting autoimmune disease in early or preclinical stages is clinically important because the institution of treatment prior to the onset of organ damage has a greater chance to ameliorate or even cure the disease \\[12\\]. However, early and reliable diagnosis of lupus is a challenge, in large part due to the performance profiles of available diagnostic tests. The optimal test would be sensitive enough to detect all individuals who have a disease while at the same time delivering sufficient specificity to have reasonable predictive probability that the disease is likely. The classic screening test for SLE is the presence in serum of antinuclear antibodies (ANAs) measured by immunofluorescence staining of a cellular substrate \\[13\\]. ANA positivity is for all practical purposes required to make a diagnosis of lupus since more than 99% of patients with SLE have significant levels of this autoantibody detected at some time during the course of disease. However, since the prevalence of SLE is low, most individuals presenting to a physician with ANA positivity do not in fact have lupus and are not at high risk for developing this disease. But there are few available quantitative and objective measures to establish prognosis for an individual with a positive ANA. This contrasts to the tools that are available for determining the risk of cardiovascular disease, where multifaceted profiles including elements of personal and family history, social habits, body measurements and lab tests can generate a reasonable and very personalized risk prediction for an individual patient \\[14\\].\n\nIn practice, physicians actually do employ some profiling to assess the risk associated with ANA positivity. Young women are more likely to develop lupus than old men, for example, so ANA positivity in the former is of greater concern. The present study confirms our previous observation that female gender is a risk factor for significant ANA positivity \\[1\\]. This result is also consistent with other findings in healthy control populations including a study of 500 normal individuals in Brazil showing that ANA positivity was almost twice as prevalent in females as in males \\[15\\]. Similar findings were reported in a rural Canadian population, with the gender difference being greatest at higher ANA levels, as was also noted in the present study \\[16\\]. The enhanced female risk profile does not appear to extend to the anti-CCP antibody that is associated with RA, another female-predominant autoimmune disorder. Reasons for the association of female gender with strong ANA positivity remain obscure.\n\nAge was not correlated with ANA levels in HCs, which seems to contradict the generally-accepted hypothesis that immunosenescence is associated with increased autoantibody production due to decreased self-regulatory mechanisms. The present findings are consistent, however, with other reports \\[15\\] and suggest that in a cross-sectional analysis such as this there are many reasons for ANA positivity. The younger individuals may in fact have abnormal immune regulation that predisposes to SLE-like disease while older persons may develop autoreactivity as part of aging immune responses that do not lead to development of pathology.\n\nOther clues are available to suggest approaches to stratifying risk in the ANA-positive population. One is the well-recognized presence of other autoantibodies that accumulate prior to SLE diagnosis \\[4\\]. The prevalence of any one of these is low, however. For example, ribosomal P autoantibodies are highly specific for SLE but are present in less than one-third of patients \\[17\\]. This finding predicts that if autoantibodies are included in risk profiling, multiplexed assays will be required. One such approach that is available clinically, the ENA panel, was not useful in the high ANA HC individuals in the present study because very few positives were found. This result is consistent with our previous experience indicating that only the high ANA positives are likely to have ENA specificities \\[1\\] and even then, the prevalence of other autoantibodies in HCs is very low. The alternative approach which has been applied in the present study is to greatly expand the repertoire of autoantibodies that can be probed by use of an autoantigen array, which in other studies has been shown to provide novel insights into expressed autoantibody repertoires \\[18\\]. The array data in the present study revealed increased autoreactivity in a group of high ANA HCs. One of the elevated autoantibodies was thyroglobulin, and since autoimmune thyroid disease is probably about 10-fold more common than SLE \\[19\\], this result suggests that a significant proportion of the ANA positivity seen by rheumatologists is related to thyroid autoimmunity. Longitudinal studies suggest that thyroid autoreactivity, especially in women, may be predictive of thyroid dysfunction \\[20\\]. Autoantibodies to cartilage proteoglycan can be measured in several systemic and joint-specific rheumatic diseases including Sjogren's Syndrome, rheumatoid arthritis, lupus and ankylosing spondylitis \\[21\\], suggesting that undetected or preclinical joint inflammation may contribute to ANA positivity. The relative increase in skin autoreactivity in the high ANA HC group might be related to a relative enrichment for skin antigens on the arrays, so this finding should be interpreted with caution. However, it does raise the interesting question of whether some early autoimmune events might take place in cutaneous structures. Transient ANA positivity has been observed in patients with polymorphous light eruption \\[22\\] and exposure to sun in susceptible individuals can trigger major organ-damaging lupus \\[23\\]. The interface dermatitis that characterizes the pathology in SLE as well as in other skin disorders may precede a diagnosis of lupus \\[24\\] and is associated with activation of the Type I interferon gene signature \\[25\\]. The triggering of this set of genes in skin, however, is not limited to inflammatory disease but can also occur as part of the immune response to viruses, raising the question of whether cutaneous reactions to environmental agents in susceptible hosts might generalize to a systemic response.\n\nThe autoantibody arrays show that although the autoreactivity spectrum in SLE is broad, not all specificities are elevated. Upregulated autoantibodies to gliadin, and to T lymphocytes and neuroblastoma cells were present only in the ANA high HC group, and not in the SLE patients. This result confirms in part our previous report that gliadin autoreactivity is associated with incomplete forms of lupus that are associated with myopathies \\[26\\]. Whether these autoantibodies are actively protective and lower the risk of lupus or alternatively are predictive of other autoimmune diseases developing in these individuals will require further longitudinal investigation.\n\nUpregulated genes observed in high ANA HC individuals include some in the Type I IFN signature that are associated with SLE \\[27,28\\]. While some of these genes, notably *IFI27*, were only elevated in SLE, others such as *MX-1* showed an intermediate level in the High ANA HC group. In addition to being the most highly-upregulated of the IFN genes in our sample, other data suggest that *IFI27* is relatively more specific for lupus than at least some of the other IFN-inducible genes. A recent study demonstrated that *IFI27* is more likely to be upregulated in lupus than in another autoimmune condition, idiopathic thrombocytopenic purpura \\[29\\]. The specific functions of many of the proteins associated with IFN-related genes are obscure, but *MX-1* is closely associated with the response to the influenza virus, so upregulation of this gene in normal individuals might reflect the ubiquitous exposure to this pathogen.\n\nThe present data suggest possible components of a lupus risk profile. As in the cardiovascular risk profiling equations, gender will be a factor but in lupus the risk will be associated with females rather than males. The age category will be inverted from that of cardiovascular disease, with greater weight given to younger ages. The lupus risk is correlated with ANA levels, not just positivity, with values in the upper quartile having a two- to three-fold elevation of risk \\[1\\], so this will be an important component. Other autoantibodies that may add to risk include those that are clinically well-known like anti-dsDNA and anti-Sm, as well as other novel specificities including the skin determinants identified in the present study. On the other hand, antineuronal, anti-thyroid or gliadin autoantibodies might steer attention away from SLE towards other autoimmune disorders. Elevated expression of genes related to the Type I IFN signature is likely to add points to the risk equation.\n\nThis study has several limitations. One is the lack of information regarding use of medications, especially hormones, by the HC. Whether administration of estrogen in the form of oral contraceptives or postmenopausal replacement therapy might induce high ANA levels in a healthy individual cannot be ascertained from our data. Another limitation is the cross-sectional design which does not permit insights into changes that evolve over time. And it would be of interest to determine reactivity to foreign antigens such as infectious agents to further interpret the significance of the autoreactive responses. Ultimately, validation of risk profiles will require longitudinal studies.\n\nFinally, since SLE is a relatively rare disorder, the probability of finding a new onset patient is low even after ANA positivity has been identified. One approach to increasing the likelihood of useful results would be to follow individuals who already have some of the identified risk profile components. For example, studies could be carried out in individuals who have been sent for ANA testing for any reason. The very fact that the individual sought medical attention and that an ANA was ordered is likely to increase the pre-test probability of disease. Such an approach has shown that in the population of individuals sent for rheumatoid factor testing, the pre-test probability for RA is 17% \\[30\\].\n\n# Conclusions\n\nThis study shows that it may be possible to identify the small percentage of ANA positive individuals who are at risk for development of SLE. Characterization of protein and gene expression profiles accompanying ANA positivity has potential to enable more precise risk quantification and ultimately to identify pre-clinical stages of this disease that would allow early and definitive treatment.\n\n# Abbreviations\n\nANA: antinuclear antibody; CCP: cyclic citrullinated peptide; DRADR: Dallas Regional Autoimmune Disease Registry; ENA: extractable nuclear antibody; EU: ELISA units; FDR: first degree relative; HC: healthy controls; IFN: interferon; ILE: incomplete lupus; MFI: mean fluorescence intensity; RA: rheumatoid arthritis; SLE: systemic lupus erythematosus; TG: thyroglobulin\n\n# Competing interests\n\nNJO has equity interest in ArthroChip LLC and has received research grants from Medimmune, Novo Nordisk, Genentech\/Roche and Human Genome Sciences. DRK has received a research grant from Human Genome Sciences. All other authors declare that they have no competing interests.\n\n# Authors' contributions\n\nQZL supervised the protein and gene expression array assays and analyses. DRK organized the patient registry. JQ organized and performed laboratory assays. JZ performed the protein array assays, while YL performed data analyses for protein and gene expression array assays. VKB coordinated patient recruitment for the registry. BFC contributed to design and validation of the autoantibody array. EKW conceived of the study design and NJO assembled the patient cohorts and supervised clinical phenotyping. QZL, DK, EKW and NJO wrote the manuscript with input and consensus from all authors. All authors read and approved the final manuscript.\n\n# Supplementary Material\n\n###### Additional file 1\n\n**Components of the Autoantigen Array**. This table lists the components of the array along with sources of the autoantigens.\n\nClick here for file\n\n## Acknowledgements\n\nThis work was supported by NIH\/NIAMS P50AR0455503. We appreciate the assistance of Azza Mutwally, Alice Cox, Sarmistha Sen, Justin Ribault and Pratik Doshi for enrolling and evaluating the individuals enrolled in DRADR. Michelle Christadoss and Ferdicia Carr-Johnson processed the samples and carried out the laboratory assessments. Jessica Mayeux assisted with data analysis. Heidi Jacobe MD shared with us a set of skin-specific autoantigens for inclusion in the protein arrays, and we appreciate her input. Thanks also to Chandra Mohan MBBS, PhD, for helpful discussions and support.\n\nThe data presented in this publication have been deposited in the NCBI Gene Expression Omnibus with accession number GSE 24706 and may be accessed online \\[31\\].","meta":{"dup_signals":{"dup_doc_count":135,"dup_dump_count":41,"dup_details":{"curated_sources":2,"2023-40":3,"2022-49":1,"2022-33":2,"2022-05":1,"2021-43":1,"2020-50":1,"2020-40":1,"2020-29":1,"2020-10":1,"2020-05":1,"2019-47":1,"2019-39":1,"2017-39":1,"2017-09":15,"2016-50":1,"2016-40":2,"2016-36":18,"2016-30":13,"2015-48":3,"2015-40":2,"2015-35":2,"2015-32":3,"2015-27":3,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":4,"2014-42":10,"2014-41":3,"2014-35":3,"2014-23":3,"2014-15":4,"2024-18":1,"2017-13":1,"2015-18":3,"2015-11":3,"2015-06":2,"2014-10":3,"2013-48":3,"2013-20":2,"2024-22":1}},"file":"PMC3132017"},"subset":"pubmed_central"}
{"text":"date: 2018-09\ntitle: 3 in 5 babies not breastfed in the first hour of life Breastfeeding within an hour after birth is critical for saving newborn lives\n\n**31 July 2018 News Release Geneva** - An estimated 78 million babies \u2013 or three in five \u2013 are not breastfed within the first hour of life, putting them at higher risk of death and disease and making them less likely to continue breastfeeding, say UNICEF and WHO in a new report. Most of these babies are born in low- and middle-income countries.\n\nThe report notes that newborns who breastfeed in the first hour of life are significantly more likely to survive. Even a delay of a few hours after birth could pose life-threatening consequences. Skin-to-skin contact along with suckling at the breast stimulate the mother's production of breastmilk, including colostrum, also called the baby's 'first vaccine', which is extremely rich in nutrients and antibodies.\n\n\"When it comes to the start of breastfeeding, timing is everything. In many countries, it can even be a matter of life or death,\" says Henrietta H. Fore, UNICEF Executive Director. \"Yet each year, millions of newborns miss out on the benefits of early breastfeeding and the reasons \u2013 all too often \u2013 are things we can change. Mothers simply don't receive enough support to breastfeed within those crucial minutes after birth, even from medical personnel at health facilities.\"\n\nBreastfeeding rates within the first hour after birth are highest in Eastern and Southern Africa (65%) and lowest in East Asia and the Pacific (32%), the report says. Nearly 9 in 10 babies born in Burundi, Sri Lanka and Vanuatu are breastfed within the first hour. By contrast, only two in 10 babies born in Azerbaijan, Chad and Montenegro do so.\\*\n\n\"Breastfeeding gives children the best possible start in life,\" says Dr Tedros Adhanom Ghebreyesus, WHO Director-General. \"We must urgently scale up support to mothers \u2013 be it from family members, health care workers, employers and governments, so they can give their children the start they deserve.\"\n\nCapture the Moment, which analyzes data from 76 countries, finds that despite the importance of early initiation of breastfeeding, too many newborns are left waiting too long for different reasons, including:\n\n-   Feeding newborns food or drinks, including formula: Common practices, such as discarding colostrum, an elder feeding the baby honey or health professionals giving the newborn a specific liquid, such as sugar water or infant formula, delay a newborn's first critical contact with his or her mother.\n\n-   The rise in elective C-sections: In Egypt, caesarean section rates more than doubled between 2005 and 2014, increasing from 20% to 52%. During the same period, rates of early initiation of breastfeeding decreased from 40% to 27%. A study across 51 countries notes that early initiation rates are significantly lower among newborns delivered by caesarean section. In Egypt, only 19% of babies born by C-section were breastfed in the first hour after birth, compared to 39% of babies born by natural delivery.\n\n-   Gaps in the quality of care provided to mothers and newborns: The presence of a skilled birth attendant does not seem to affect rates of early breastfeeding, according to the report. Across 58 countries between 2005 and 2017, deliveries at health institutions grew by 18 percentage points, while early initiation rates increased by 6 percentage points. In many cases, babies are separated from their mothers immediately after birth and guidance from health workers is limited. In Serbia, the rates increased by 43 percentage points from 2010 to 2014 due to efforts to improve the care mothers received at birth.\n\nEarlier studies, cited in the report, show that newborns who began breastfeeding between two and 23 hours after birth had a 33% greater risk of dying compared with those who began breastfeeding within one hour of birth. Among newborns who started breastfeeding a day or more after birth, the risk was more than twice as high.\n\nThe report urges governments, donors and other decision-makers to adopt strong legal measures to restrict the marketing of infant formula and other breastmilk substitutes.\n\nThe WHO and UNICEF-led Global Breastfeeding Collective also released the 2018 Global Breastfeeding Scorecard, which tracks progress for breastfeeding policies and programmes. In it, they encourage countries to advance policies and programmes that help all mothers to start breastfeeding in the first hour of their child's life and to continue as long as they want.\n\nAvailable from: <http:\/\/www.who.int\/news-room\/detail\/31-07-2018-3-in-5-babies-not-breastfed-in-the-first-hour-of-life>","meta":{"dup_signals":{"dup_doc_count":172,"dup_dump_count":47,"dup_details":{"curated_sources":2,"2023-50":2,"2023-40":2,"2023-23":3,"2023-06":3,"2022-40":2,"2022-33":2,"2022-21":3,"2022-05":2,"2021-49":2,"2021-43":2,"2021-39":2,"2021-31":3,"2021-25":3,"2021-17":2,"2021-10":2,"2021-04":3,"2020-50":2,"2020-45":2,"2020-40":5,"2020-34":1,"2020-29":6,"2020-24":5,"2020-16":3,"2020-10":3,"2020-05":2,"2019-51":4,"2019-47":2,"2019-43":6,"2019-39":2,"2019-35":6,"2019-30":3,"2019-26":4,"2019-22":6,"2019-18":4,"2019-13":3,"2019-09":10,"2019-04":5,"2018-51":6,"2018-47":6,"2018-43":6,"2018-39":3,"2018-34":8,"2024-30":5,"2024-26":2,"2024-22":3,"2024-18":6,"2024-10":3}},"file":"PMC6201016"},"subset":"pubmed_central"}
{"text":"date: 2017-05\ntitle: \"DEPRESSION: LET'S TALK\" SAYS WHO, AS DEPRESSION TOPS LIST OF CAUSES OF ILL HEALTH\n\n**30 MARCH 2017 \\| GENEVA -** Depression is the leading cause of ill health and disability worldwide. According to the latest estimates from WHO, more than 300 million people are now living with depression, an increase of more than 18% between 2005 and 2015. Lack of support for people with mental disorders, coupled with a fear of stigma, prevent many from accessing the treatment they need to live healthy, productive lives.\n\nThe new estimates have been released in the lead-up to World Health Day on 7 April, the high point in WHO's year-long campaign \"Depression: let's talk\". The overall goal of the campaign is that more people with depression, everywhere in the world, both seek and get help.\n\nSaid WHO Director-General, Dr Margaret Chan: \"These new figures are a wake-up call for all countries to re-think their approaches to mental health and to treat it with the urgency that it deserves.\"\n\nOne of the first steps is to address issues around prejudice and discrimination. \"The continuing stigma associated with mental illness was the reason why we decided to name our campaign Depression: let's talk,\" said Dr Shekhar Saxena, Director of the Department of Mental Health and Substance Abuse at WHO. \"For someone living with depression, talking to a person they trust is often the first step towards treatment and recovery.\"\n\n**Urgent need for increased investment**\n\nIncreased investment is also needed. In many countries, there is no, or very little, support available for people with mental health disorders. Even in high-income countries, nearly 50% of people with depression do not get treatment. On average, just 3% of government health budgets is invested in mental health, varying from less than 1% in low-income countries to 5% in high-income countries.\n\nInvestment in mental health makes economic sense. Every US\\$ 1 invested in scaling up treatment for depression and anxiety leads to a return of US\\$ 4 in better health and ability to work. Treatment usually involves either a talking therapy or antidepressant medication or a combination of the two. Both approaches can be provided by non-specialist health-workers, following a short course of training, and using WHO's mhGAP Intervention Guide. More than 90 countries, of all income levels, have introduced or scaled-up programmes that provide treatment for depression and other mental disorders using this Intervention Guide.\n\nFailure to act is costly. According to a WHO-led study, which calculated treatment costs and health outcomes in 36 low-, middle- and high-income countries for the 15 years from 2016-2030, low levels of recognition and access to care for depression and another common mental disorder, anxiety, result in a global economic loss of a trillion US dollars every year. The losses are incurred by households, employers and governments. Households lose out financially when people cannot work. Employers suffer when employees become less productive and are unable to work. Governments have to pay higher health and welfare expenditures.\n\n**Associated health risks**\n\nWHO has identified strong links between depression and other noncommunicable disorders and diseases. Depression increases the risk of substance use disorders and diseases such as diabetes and heart disease; the opposite is also true, meaning that people with these other conditions have a higher risk of depression.\n\nDepression is also an important risk factor for suicide, which claims hundreds of thousands of lives each year. Said Dr Saxena: \"A better understanding of depression and how it can be treated, while essential, is just the beginning. What needs to follow is sustained scale-up of mental health services accessible to everyone, even the most remote populations in the world.\"\n\nDepression is a common mental illness characterized by persistent sadness and a loss of interest in activities that people normally enjoy, accompanied by an inability to carry out daily activities, for 14 days or longer.\n\nIn addition, people with depression normally have several of the following: a loss of energy; a change in appetite; sleeping more or less; anxiety; reduced concentration; indecisiveness; restlessness; feelings of worthlessness, guilt, or hopelessness; and thoughts of self-harm or suicide.\n\nAvailable from: <http:\/\/www.who.int\/mediacentre\/news\/releases\/2017\/world-health-day\/en\/>","meta":{"dup_signals":{"dup_doc_count":209,"dup_dump_count":65,"dup_details":{"curated_sources":2,"2023-50":3,"2023-40":1,"2023-23":1,"2023-14":3,"2023-06":1,"2022-49":1,"2022-40":3,"2022-33":1,"2022-27":1,"2022-21":2,"2022-05":2,"2021-49":2,"2021-43":2,"2021-39":2,"2021-31":1,"2021-25":1,"2021-21":2,"2021-17":1,"2021-10":2,"2021-04":4,"2020-50":2,"2020-45":4,"2020-40":1,"2020-34":2,"2020-29":1,"2020-24":1,"2020-16":1,"2020-10":1,"2020-05":3,"2019-47":2,"2019-39":2,"2019-35":4,"2019-30":3,"2019-26":1,"2019-22":5,"2019-18":4,"2019-13":3,"2019-09":5,"2019-04":3,"2018-51":2,"2018-47":5,"2018-43":3,"2018-39":4,"2018-34":1,"2018-30":4,"2018-26":8,"2018-22":6,"2018-17":3,"2018-13":8,"2018-09":4,"2018-05":10,"2017-51":4,"2017-47":5,"2017-43":7,"2017-39":4,"2017-34":8,"2017-30":5,"2017-26":7,"2017-22":7,"2017-17":6,"2024-26":2,"2024-22":3,"2024-18":3,"2024-10":3,"2024-30":1}},"file":"PMC5447225"},"subset":"pubmed_central"}
{"text":"abstract: Retinal neurodegeneration is an early event in the pathogenesis of diabetic retinopathy (DR). Somatostatin (SST) is an endogenous neuroprotective peptide that is downregulated in the diabetic eye. The aim of the study was to test the usefulness of topical administration of SST in preventing retinal neurodegeneration. For this purpose, rats with streptozotocin-induced diabetes mellitus (STZ-DM) were treated with either SST eye drops or vehicle for 15 days. Nondiabetic rats treated with vehicle served as a control group. Functional abnormalities were assessed by electroretinography (ERG), and neurodegeneration was assessed by measuring glial activation and the apoptotic rate. In addition, proapoptotic (FasL, Bid, and activation of caspase-8 and caspase-3) and survival signaling pathways (BclxL) were examined. Intraretinal concentrations of glutamate and its main transporter glutamate\/aspartate transporter (GLAST) were also determined. Treatment with SST eye drops prevented ERG abnormalities, glial activation, apoptosis, and the misbalance between proapoptotic and survival signaling detected in STZ-DM rats. In addition, SST eye drops inhibited glutamate accumulation in the retina and GLAST downregulation induced by diabetes mellitus. We conclude that topical administration of SST has a potent effect in preventing retinal neurodegeneration induced by diabetes mellitus. In addition, our findings open up a new preventive pharmacological strategy targeted to early stages of DR.\nauthor: Cristina Hern\u00e1ndez; Marta Garc\u00eda-Ram\u00edrez; Lidia Corraliza; Jimena Fern\u00e1ndez-Carneado; Josep Farrera-Sinfreu; Berta Ponsati; \u00c1gueda Gonz\u00e1lez-Rodr\u00edguez; \u00c1ngela M. Valverde; Rafael Sim\u00f3Corresponding author: Cristina Hern\u00e1ndez, <firstname.lastname@example.com>.\ndate: 2013-07\nreferences:\ntitle: Topical Administration of Somatostatin Prevents Retinal Neurodegeneration in Experimental Diabetes\n\nDiabetic retinopathy (DR) has been classically considered to be a microcirculatory disease of the retina. However, there is growing evidence to suggest that retinal neurodegeneration is an early event in the pathogenesis of DR that participates in the microcirculatory abnormalities that occur in DR (1\u20138). We have contributed to this field by demonstrating that the main hallmarks of retinal neurodegeneration (apoptosis and glial activation) are already present in the retinas of diabetic donors without any microcirculatory abnormalities in ophthalmoscopic examinations performed during the year before death (9\u201311).\n\nGlutamate is the major retinal excitatory neurotransmitter for the photoreceptor-bipolar-ganglion cell circuit. However, elevated glutamate levels in the retina (which result in excessive stimulation) are implicated in the so-called \"excitotoxicity,\" which leads to neurodegeneration (12). The excitotoxicity of glutamate is the result of overactivation of ionotropic glutamate receptors, which have been found to be overexpressed in rats with streptozotocin-induced diabetes mellitus (STZ-DM) (13,14). Apart from glutamate, oxidative stress (15), advanced glycation end product receptor upregulation (16,17), and renin-angiotensin system activation (18\u201320) play an essential role in retinal neurodegeneration induced by diabetes mellitus. Finally, recent evidence indicates that diabetes mellitus\u2013induced downregulation of neuroprotective factors synthesized by the retina is also involved in the neurodegenerative process of the diabetic eye (21). On these bases, it is reasonable to hypothesize that therapeutic strategies based on neuroprotection will be effective in preventing or arresting DR development.\n\nSomatostatin (SST) is one of the most important neuroprotective factors synthesized by the retina, retinal pigment epithelium (RPE) being its main source in the human eye. The human retina produces significant amounts of SST, as deduced by the strikingly high levels reported within the vitreous fluid (22,23). Besides SST, its receptors (SSTRs) are also expressed in the retina, with SSTR1 and SSTR2 being the most widely expressed (24). The production of both SST and its receptors simultaneously suggests a relevant autocrine action in the human retina. SST acts as a neuromodulator in the retina through multiple pathways, including intracellular Ca^2+^ signaling, nitric oxide function, and glutamate release from the photoreceptors. Apart from neuroprotection, SST has potent antiangiogenic properties and regulates various ion\/water transport systems (24). Therefore, SST seems to be essential in preventing both proliferative DR (PDR) and diabetic macular edema (DME).\n\nIn the early stages of DR, there is a downregulation of SST that is associated with retinal neurodegeneration (9). In fact, it has recently been reported that intravitreal administration of SST and SST analogs protects the retina from AMPA-induced neurotoxicity (25). In addition, the lower expression of SST in RPE and neuroretina is associated with a dramatic decrease of intavitreal SST levels in both DME (26) and PDR (22,23). As a result, the physiological role of SST in preventing both fluid accumulation within the retina and neovascularization could be reduced, and consequently, the development of DME and PDR would be favored. For all these reasons, SST replacement treatment can be considered a new target not only for preventing the neurodegenerative process but also for more advanced stages of DR such as DME and PDR.\n\nWhen the early stages of DR are the therapeutic target, it would be inconceivable to recommend in the clinical practice an aggressive treatment such as intravitreous injections. On the other hand, the use of eye drops has not been considered an appropriate route for the administration of drugs aimed at preventing or arresting DR because of the general assumption that they do not reach the posterior chamber of the eye. However, there is emerging evidence showing the capacity of a lot of drugs to reach the retina in pharmacologically effective concentrations, at least in animal models (27).\n\nOn this basis, the aim of the current study was to determine whether topical administration of SST prevents the retinal neurodegenerative process that occurs in STZ-induced diabetic rats. In addition, the effects of SST eye drops on apoptotic\/survival signaling pathways and retinal glutamate levels have been examined. Finally, the possibility that SST acts through preventing glutamate accumulation by upregulating glutamate\/aspartate transporter (GLAST) has also been explored.\n\n# RESEARCH DESIGN AND METHODS\n\nA total of 24 male Sprague-Dawley rats were obtained from Charles River Laboratories (Senneville, Quebec, Canada). A minimum acclimatization period of 14 days preceded treatment. STZ (60 mg\/kg) was administered to 8-week-old (*n* = 16) rats by intravenous injection via tail vein on day \u22122 prior to starting SST treatment. The remaining eight rats were injected with vehicle and served as a nondiabetic control group. Animals with blood glucose levels \\>250 mg\/dL the day after STZ administration were considered diabetic. On day \u22121, all animals were weighed and randomly assigned to treatment groups (vehicle or SST) using a computer-based randomization procedure. Randomization was performed by stratification using body weight as the parameter.\n\nSST or vehicle eye drops were administered directly onto the superior corneal surface of each eye using a syringe. One drop (20 \u00b5L) of SST (1%; 10 mg\/mL) or vehicle (20 \u00b5L of 0.9% sodium chloride) was administered once daily for 14 days. On day 15, the animals' eyes were instilled with a drop of SST or vehicle \\~1 h prior to necropsy. Therefore, the elapsed time between the onset of diabetes mellitus until necropsy was 16 days.\n\nA dose-response experiment assessing the hallmarks of retinal neurodegeneration (apoptosis and glial activation) was performed using SST eye drops at 10, 2, and 0.5 mg\/mL (<a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>).\n\nExperimental design of the dose-efficacy study\n\n![](2569tbl1)\n\nThe animals used in this study were treated in accordance with the Association for Research in Vision and Ophthalmology Statement.\n\n## Pharmacokinetic analyses.\n\nA pharmacokinetic analysis after a single topical dose of radiolabeled ^125^I-somatostatin was performed. For this purpose, seven rats were anesthetized and placed in a supine position. After they were placed in the experimental area, 10 \u00b5L (2.5 \u00b5Ci) ^125^I-somatostatin was applied in each eye with a micropipette. The animals were killed at 15, 30, and 60 min and 4, 6, 12 and 24 h after the application of the labeled SST solution. After the animals were killed, the eyes were obtained and gently washed in a volume of 20 mL physiologic saline. A small incision was performed in the rear portion of the eye ball, and the eye content was extracted. The vitreous humor, retina, and choroid-RPE were collected. The sclera was placed in different tubes for independent measurements. All the samples were weighted and counted in the \u03b3 counter in order to obtain the percentage of injected dose for each gram of tissue. Samples of blood were also obtained.\n\nIn addition, absorption of carboxyfluorecein-labeled SST (CF-SST) eye drops through the ocular structures was also evaluated in 8-week-old Sprague-Dawley rats. For this purpose, three rats were treated with vehicle eye drops in both eyes. Three rats were treated with the fluorescent label (CF) eye drops. The eye drops of CF-SST (10 mg\/mL; one drop) were administered to the right eye, and the vehicle was administered to the left eye of three rats. One animal of each group was killed at 10, 30, and 60 min after the application of the corresponding eye drop. The eyes were enucleated, fixed in 4% paraformaldehyde, submerged in optimum cutting temperature Tissue-teck, frozen in liquid nitrogen, and stored at \u221280\u00b0C until microtome sectioned. The whole eyes were cryosectioned to 50-\u03bcm sections, mounted with antifade-mounting medium with DAPI, and visualized in fluorescent and confocal microscopes (Olympus). All images were taken with the same settings.\n\n## Ophthalmological examinations.\n\nSlit lamp biomicroscopy, indirect ophthalmoscopy, and intraocular pressure measurement were performed in all animals at baseline and at days 7 and 13 posttreatment.\n\n## Electroretinography.\n\nElectroretinogram evaluations were performed on day \u20131 before treatment and on day 14 as follows: the animals were dark adapted for at least 1 h prior to electroretinography (ERG) recording and then anesthetized with isoflurane to maintain the anesthesia prior to and during the procedure. Tropicamide (1%) was applied to each eye prior to the test. A contact lens electrode was placed on the surface of each eye, and a needle electrode was placed cutaneously on the head between the two eyes. A cutaneous ground electrode was placed near the base of the tail. Carboxymethylcellulose (1%) drops were applied to the interior surface of the contact lens electrodes prior to their placement on the eyes. A topical anesthetic (proparacaine, 0.5%) was applied to the eyes. The amplitude and implicit time of the b-wave after scotopic flash stimuli were measured. b-wave amplitude and implicit time were measured as defined by the International Society for Clinical Electrophysiology of Vision (28).\n\n## Histopathology.\n\nEyes were prepared for histopathological examination by being embedded in paraffin wax, sectioned, and stained with hematoxylin-eosin. For each eye, a total of three sagittal sections (one section through the optic nerve, one section \\~500 \u00b5m medially, and another section \\~500 \u00b5m laterally from the optic nerve) were evaluated.\n\n### Assessment of glial activation.\n\nSections of 7-\u03bcm thicknesses were obtained from the paraffin blocks and fixed on highly adherent slides (Visionbiosystems; Newcastle upon Tyne, U.K.). They were desparaffined in xylol; rehydrated in 100% ethanol, 90% ethanol, and 75% ethanol; and incubated for 10 min in PBS. Unspecific unions were blocked by incubating the samples for 1 h in PBS 1% BSA and 0.05% Triton X-100. Thereafter, the primary rabbit anti-human glial fibrillar acidic protein (GFAP) antibody (Sigma, Madrid, Spain) diluted in the blocking buffer (1:100) was incubated for 36 h at 4\u00b0C. After three washings for 5 min with PBS, the sections were incubated with a secondary human IgG antibody labeled with Alexa Fluor 568 (Invitrogen, Eugene, OR) for 1 h at room temperature. The labeled sections were washed and mounted with fluorescent medium containing DAPI for identifying cell nuclei (Vector Laboratories, Burlingame, CA). Positive sections for GFAP were captured in a confocal microscope (FV1000; Olympus, Hamburg, Germany). Optical sections were obtained with a 568-nm laser for Alexa Fluor 568 and a 405-nm laser for the DAPI (image resolution 1,024 \u00d7 1,024 pixels). The degree of glial activation was evaluated by using a score system based on the extent of GFAP staining (29). This scoring system is as follows: negligible staining (score 0), M\u00fcller cell endfeet region\/ganglion cell layer (GCL) only (score 1), M\u00fcller cell endfeet region\/GCL plus a few proximal processes (score 2), M\u00fcller cell endfeet plus many processes but not extending to outer nuclear layer (ONL) (score 3), M\u00fcller cell endfeet plus processes throughout with some in the ONL (score 4), and M\u00fcller cell endfeet plus a lot of dark processes from GCL to the outer margin of ONL (score 5).\n\n### Assessment of apoptosis.\n\nThe In Situ Cell Death Detection kit (Roche Diagnostics, Mannheim, Germany) was used to evaluate the presence of apoptotic cells. Sections were deparaffined in xylol; rehydrated in 100% ethanol, 90% ethanol, and 75% ethanol; and incubated for 10 min in PBS. Autofluorescence was eliminated after incubation with 0.2% potassium permanganate for 20 min and 1% oxalic acid for 30 s before the TUNEL procedure was started. Positive and negative controls were processed at the same time. Three confocal images were recorded (\u00d740) corresponding to a surface of 317.13 \u00d7 317.13 \u03bcm for each section. Staining with propidium iodide was done to examine nucleus morphology and discard false positives. The total number of nuclei and nuclei positive for TUNEL were counted with the ImageJ program.\n\n### Proapoptotic and survival signaling.\n\nProapoptotic signaling pathways were investigated by analyzing several proapoptotic molecules (FasL, caspase-8, total Bid, truncated Bid, and active caspase-3), as well as antiapoptotic markers (BclxL), by Western blot as previously described (30).\n\n## Glutamate quantification.\n\nQuantification of glutamate was performed by liquid chromatography coupled to mass spectrometry. Chromatographic separation was performed on an Agilent 1200 series (Waldbronn, Germany) using an Ascentis Express HILIC column, 50 \u00d7 2.1 mm with 2.7 \u00b5m particle size from Supelco (Belfonte, PA), maintained at 25\u00b0C throughout the analysis, a mobile phase acetonitrile (ACN), and water (50 mmol\/L ammonium acetate) with a flow rate of 0.6 mL\/min. The volume injected was 10 \u00b5L. The mobile phase involved a gradient starting at 87% of ACN, which was maintained for 3 min. Then, from min 3 to 10 the ACN content was decreased to 20% and increased again to 87% at min 12.5. Glutamate was eluted at 6.5 min. The mass detection system was an Agilent 6410 Triple Quad (Santa Clara, CA) using positive electrospray ionization with a gas temperature of 350\u00b0C, gas flow rate of 12 L\/min, nebulizer pressure of 45 \u03c8, capillary voltage of 3,500 V, fragmentor of 135 V, and collision energy of 10 V.\n\n## GLAST expression\n\n### RNA isolation and GLAST mRNA quantification.\n\nTotal RNA was extracted from retinas with the Rneasy Mini kit with DNAase digestion (Qiagen; IZASA, Madrid, Spain). One microgram of total RNA was used for reverse transcription with TaqMan and random primer reagents (Applied Biosystems, Madrid, Spain). Real-time PCR was performed with the GLAST specific assay for exon 3\u20134 boundary Rn00570130_m1 (Applied Biosystems), and results were normalized to the expression level of \u03b2-actin (Rn00667969_m1) as a housekeeping gene. Relative quantification values were obtained using an ABI Prism 7000 SDS software (Applied Biosystems).\n\n### Immunohistochemistry for GLAST.\n\nSections of 4 \u03bcm were deparaffined in xylene and hydrated in a graded ethanol series. Then, sections were antigen retrieved (sodium citrate 10 mmol\/L, pH 6.0) and washed in PBS. Next, sections were incubated in blocking solution (2% BSA, Tween 0.05% PBS) for 1 h at room temperature followed by incubation with primary antibody anti-GLAST (1:200; Abcam, Cambridge, U.K.). After washing, sections were incubated with a fluorescent anti-rabbit Alexa Fluor 488 as a secondary antibody (Life Technologies, Madrid, Spain) in blocking solution for 1 h, washed, and mounted in Vectashield (Vector Laboratories). DAPI was used for nuclear staining. Quantification of fluorescence intensity of images was performed as previously explained for GFAP.\n\n## Statistical analysis.\n\nNormal distribution of the variables was evaluated using the Kolmogorov-Smirnov test. Data were expressed either as means \u00b1 SD or as median (range). Comparisons of continuous variables between groups were performed using the unpaired Student *t* test or Mann-Whitney *U* test. For comparisons within groups (ERG of day 14 vs. ERG of day \u22121), the paired Student *t* test was used. Levels of statistical significance were set at *P* \\< 0.05.\n\n# RESULTS\n\n## SST contained in eye drops reached the retina.\n\nThe results of pharmacokinetic study are displayed in <a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<\/a>. After topical ocular administration of ^125^I-somatostatin, the maximum postdose concentration (5% of applied dose per gram of tissue) was detected at 1 h. However, at 15 min most of the ^125^I-somatostatin had already reached the retina. This rapid absorption argues against the permeation of SST through the cornea. For further exploration of this issue, a pharmacokinetic study using eye drops containing CF-SST was performed. This study clearly showed that SST contained in the eye drops reached the retina not through the cornea but by the trans-scleral route, thus bypassing the anterior chamber ([Supplementary Fig. 1](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-0926\/-\/DC1)).\n\n## Ocular safety of SST treatment.\n\nNo significant differences in the cornea, pupil size, or intraocular pressure were observed among the three groups at baseline or during follow-up.\n\n## SST treatment prevents ERG abnormalities.\n\nERG was performed to assess retinal function. b-wave amplitude was significantly reduced at day 14 in the diabetic group treated with vehicle (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<em>A<\/em><\/a>). By contrast, in the control group and in the diabetic group treated with SST eye drops no reduction of b-wave amplitude was observed. b-wave implicit time increased at day 14 in diabetic rats treated with vehicle (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<em>B<\/em><\/a>). However, in the control group and in the diabetic group treated with SST eye drops the implicit time did not increase at day 14. Therefore, SST treatment prevented ERG abnormalities caused by diabetes mellitus.\n\n## Neurodegeneration was prevented in diabetic rats treated with SST eye drops\n\n### Glial activation.\n\nThe retinas of nondiabetic control rats revealed GFAP immunolabeling in cells and their processes in the inner layer of the retina, especially in the inner limiting membrane, and GCL (GFAP score 0\u20131) (<a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<\/a>). According to their location and morphology, these GFAP-positive cells were interpreted as being retinal astrocytes. However, a slight GFAP immunofluorescence in M\u00fcller cells could be detected. In the retina of diabetic rats treated with placebo, GFAP expression was prominent along the inner limiting membrane, in M\u00fcller cell endfeet, and in M\u00fcller cell radial fibers extending through both the inner and outer retina (GFAP score 1\u20133). Diabetic rats treated with SST eye drops presented significantly lower GFAP score (0\u20131) than diabetic rats treated with placebo (<a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<\/a>).\n\n### Retinal apoptosis.\n\nRepresentative images of the presence of apoptotic cells in the retinas of the three experimental groups are shown in <a href=\"#F4\" data-ref-type=\"fig\">Fig. 4<em>A<\/em><\/a>. Two weeks after the induction of diabetes mellitus with STZ, the whole percentage of retinal apoptotic cells, as well as the percentage of apoptotic cells in retinal layers, was significantly higher in comparison with that observed in retinas from age-matched nondiabetic controls (<a href=\"#F4\" data-ref-type=\"fig\">Fig. 4<em>B<\/em><\/a>). In all groups, apoptosis was highest in the GCL. Diabetic rats treated with SST eye drops presented a significantly lower ratio of apoptosis in all retinal layers than diabetic rats treated with placebo and similar to nondiabetic rats (<a href=\"#F4\" data-ref-type=\"fig\">Fig. 4<em>B<\/em><\/a>).\n\n### Dose response effect.\n\nThe effectiveness of SST eye drops at concentrations of 2 mg\/mL was similar to that of eye drops at 10 mg\/mL concentrations in preventing glial activation and apoptosis. However, the effect of 0.5 mg\/mL was significantly lower than that obtained using either 2 or 10 mg\/mL (<a href=\"#F5\" data-ref-type=\"fig\">Fig. 5<\/a>).\n\n### Proapoptotic and survival molecules.\n\nThe results of proapoptotic and survival molecules measured in the retina of the three rat groups studied are shown in <a href=\"#F6\" data-ref-type=\"fig\">Fig. 6<\/a>. The expression of FasL, a proapoptotic component of the death receptor apoptotic pathway, was significantly increased in neuroretinas from diabetic rats compared with control rats. The interaction of FasL with the death receptor Fas\/CD95 assembles the death-inducing signaling complex (31). This includes the recruitment of caspase-8. Activation of caspase-8, monitored by the presence of its 18-kDa active fragment, was significantly increased in neuroretinas of diabetic rats compared with controls. These data were reinforced by the detection of elevated levels of truncated Bid fragment in diabetic samples. As a result, neuroretina from diabetic rats displayed a significant activation of the executer caspase-3, monitored by the presence of its 17-kDa active fragment. Treatment with SST eye drops prevented the upregulation of all of these proapoptotic molecules.\n\nOn the other hand, a reduction in BclxL, an antiapoptotic member of the Bcl2 family, was found in diabetic retinas in comparison with control retinas. When treated with SST eye drops, BclxL expression was greatly improved (<a href=\"#F6\" data-ref-type=\"fig\">Fig. 6<\/a>).\n\n## Topical administration of SST prevents the increase of glutamate induced by diabetes mellitus.\n\nGlutamate levels (arbitrary units, median \\[range\\]) in the diabetic retinas were higher than in nondiabetic retinas (117 \\[57\u2013412\\] vs. 47 \\[27\u2013300\\]; *P* = 0.012). In diabetic rats treated with SST eye drops, glutamate concentration (40 \\[21\u2013127\\]) was significantly decreased in comparison with diabetic rats treated with vehicle (*P* = 0.021) and similar to control rats (*P* = 0.51).\n\n## Upregulation of GLAST expression after SST treatment.\n\nGene and protein expression of GLAST were downregulated in retinas of diabetic rats (<a href=\"#F7\" data-ref-type=\"fig\">Fig. 7<\/a>). In diabetic rats treated with SST, this downregulation was prevented and GLAST protein levels were even increased in comparison with nondiabetic rats (<a href=\"#F7\" data-ref-type=\"fig\">Fig. 7<\/a>).\n\n# DISCUSSION\n\nIn the current study, we provide the first evidence that topical administration of SST prevents retinal neurodegeneration in STZ-DM rats. SST eye drops prevented b-wave abnormalities in the ERG (reduction in amplitude and increase in implicit time), which are considered sensitive indicators of DR (32). In addition, topical administration of SST abrogated the characteristic hallmarks of neurodegeneration (glial activation and apoptosis) caused by short-term diabetes mellitus. In this regard, it should be noted that the degree of glial activation and apoptosis in diabetic rats treated with SST eye drops was very similar to that found in nondiabetic rats.\n\nGlial activation is a prominent event in retinal neurodegeneration. Retinal astrocytes normally express GFAP, while M\u00fcller cells do not. However, during the course of diabetes mellitus there is an aberrant expression of GFAP by M\u00fcller cells (33). Because M\u00fcller cells produce factors capable of modulating blood flow, vascular permeability, and cell survival and because their processes surround all the blood vessels in the retina, it seems that these cells play a key role in the pathogenesis of retinal microangiopathy in the diabetic eye (33,34). In this paper, we have found that SST eye drops were able to significantly prevent glial activation or, in other words, most of the aberrant expression of GFAP in M\u00fcller cells.\n\nApart from glial activation, apoptosis is the other hallmark of retinal neurodegeneration. As previously reported (35), we found a significant rate of apoptosis in retinas from STZ-induced diabetic rats in comparison with nondiabetic control rats. In addition, we found a misbalance between apoptotic and survival signaling pathways. In this regard, we demonstrated that the activation of the death receptor pathway, monitored by the elevation in the expression of FasL and the activation of caspase-8, was significantly higher in neuroretina from STZ-induced diabetic rats than in nondiabetic controls. Importantly, our data clearly show increased Bid cleavage in the neuroretina of diabetic rats. This result suggests that in the diabetic neuroretina, the apoptotic signals emerging from the death receptor pathway are amplified to additional activation of the intrinsic apoptotic signaling pathway, resulting in a stronger activation of the executer caspase-3. On the other hand, a significant downregulation of BclxL (an antiapoptotic molecule belonging to the Bcl2 family) was found in diabetic retinas. Notably, topical administration of SST was able to prevent all these abnormalities in proapoptotic\/survival signaling.\n\nApart from the prevention of apoptosis and glial activation, SST eye drops significantly reduced glutamate retinal levels. Rapid removal or inactivation of glutamate is necessary to maintain the normal function of the retina and for avoiding excitotoxicity. Elevated levels of glutamate in the retina have been found in experimental models of diabetes mellitus (36\u201338), as well as in the vitreous fluid of diabetic patients (12,39). The reasons why diabetes mellitus facilitates extracellular accumulation of glutamate include *1*) an increase of glutamate production by glial cells due to the loss of the M\u00fcller cell\u2013specific enzyme glutamine synthetase, which converts glutamate to glutamine (36,37); *2*) a reduction in the retinal ability to oxidize glutamate to \u03b1-ketoglutarate (37); *3*) the death of neurons with subsequent release of intracellular contents; and *4*) the impairment of glutamate removal from extracellular space by the glial cells (12). Regarding this later point, in the current study we provide evidence that GLAST, the main glutamate transporter expressed by M\u00fcller cells (40), is significantly decreased in STZ-induced diabetic rats. More importantly, SST eye drops not only completely prevent GLAST downregulation induced by diabetes mellitus but even lead to a significant increase in GLAST expression (mRNA and protein). These findings can contribute to our understanding of the underlying mechanisms involved in the retinal reduction of AMPA-induced neurotoxicity reported for SST and SST analogs administered intravitreally (25).\n\nThe study of the mechanisms linking neurodegeneration with early vascular abnormalities is crucial for understanding how neuroprotection could also exert beneficial effects in diabetic microangiopathy. SST may inhibit angiogenesis directly through SST receptors present on endothelial cells (41) and also indirectly through the downregulation of vascular endothelial growth factor (VEGF) (42,43) or by the inhibition of postreceptor signaling events not only of VEGF but also of other peptide growth factors such as IGF-1, epidermal growth factor, and platelet-derived growth factor (44). The potential effects of SST in abrogating microvascular impairment are beyond the scope of the current study, and specific studies on this issue are urgently needed.\n\nGiven the essential role of neurodegeneration in the pathogenesis of DR, it is reasonable to hypothesize that therapeutic strategies based on the neuroprotective effects of SST would be effective in preventing or arresting DR development. The observation that circulating growth hormone\u2013IGF-1 production is reduced by SST has been the basis for proposing systemic administration of SST analogs for treating DR. However, this concept has not been supported by clinical intervention trials. The main concerns of systemic administration of SST are as follows: *1*) the paracrine effects of SST synthesized by the retina involve SSTR subtypes other than SSTR2, which mediates growth hormone inhibition and for which octreotide presents high affinity, and *2*) SST analogs do not cross the blood-retinal barrier and would have access only where there is disruption of the blood-retinal barrier, thus limiting the amount of the drug reaching the retinal target tissues. For these reasons and given that a downregulation of retinal production of SST occurs in diabetic retina, a replacement using local administration of the natural peptide seems a reasonable approach. In this regard, the neuroprotective effects of topical administration of brimonidine and nerve growth factor have already been reported in experimental models (45\u201347). These findings open up the possibility of developing a topical therapy targeting the early stages of DR in which the use of the only currently established therapies such as laser photocoagulation or intravitreal injections of corticosteroids or anti-VEGF agents are inappropriately invasive. In addition, topical administration of drugs limits their action to the eye and minimizes the associated systemic effects, resulting in higher patient compliance (48). Therefore, topical therapies could revolutionize the care of diabetic patients (49). In this regard, a phase II-III, randomized controlled clinical trial (EUROCONDOR-278040) to assess the efficacy of SST and brimonidine administered topically to prevent or arrest DR has been approved by the European Commission in the setting of the FP7-HEALTH-2011.\n\nFinally, the route by which SST contained in the eye drops reaches the retina deserves a brief comment. The pharmacokinetic study revealed a rapid absorption of ^125^I-SST, which was unexpected for a compound that reached the retina diffusing through the cornea. The study performed using CF-SST showed that the absorption of SST eye drops was mainly by the trans-scleral route.\n\nIn summary, we provide first evidence that topical administration of SST has a potent effect in preventing the retinal neurodegenerative process that occurs in the early stages of DR. A preventive effect on the impairment of survival\/apoptotic signaling induced by diabetes mellitus and a significant reduction in glutamate-induced excitotoxicity are among the mechanisms by which SST exerts its beneficial actions.\n\n# Supplementary Material\n\n###### Supplementary Data\n\n## ACKNOWLEDGMENTS\n\nThis study was supported by grants from the Ministerio de Ciencia e Innovaci\u00f3n (SAF2012-35562 and SAF2009-08114), from the European Foundation for the Study of Diabetes, and from the 7th Framework Programme (EUROCONDOR, FP7-278040).\n\nNo potential conflicts of interest relevant to this article were reported.\n\nC.H. designed the project, led the analysis, wrote the manuscript, reviewed and edited the manuscript, and approved the final version of the manuscript. M.G.-R. and L.C. led the analysis, reviewed the manuscript, and approved the final version of the manuscript. J.F.-C., J.F.-S., and B.P. contributed to discussion, reviewed the manuscript, and approved the final version of the manuscript. A.G.-R. led the analysis, reviewed the manuscript, and approved the final version of the manuscript. A.M.V. contributed to discussion, reviewed the manuscript, and approved the final version of the manuscript. R.S. designed and coordinated the project, wrote the manuscript, reviewed and edited the manuscript, and approved the final version of the manuscript. C.H. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.\n\nThe authors thank Dr. O. Yanes (CIBERDEM) for glutamate quantification and Dr. A.R. Carvalho (Ophthalmologic Research Unit, Vall d' Hebron Research Institute) for her technical assistance in obtaining the images of pharmacokinetic analysis.\n\n# REFERENCES","meta":{"dup_signals":{"dup_doc_count":109,"dup_dump_count":47,"dup_details":{"curated_sources":2,"2021-04":1,"2019-39":1,"2019-30":1,"2019-09":1,"2018-51":1,"2018-43":1,"2018-34":1,"2018-26":2,"2018-17":3,"2018-09":3,"2018-05":1,"2017-51":1,"2017-47":3,"2017-43":2,"2017-39":3,"2017-34":1,"2017-30":5,"2017-26":2,"2017-22":2,"2017-17":5,"2017-09":4,"2017-04":6,"2016-50":3,"2016-44":4,"2016-40":3,"2016-36":3,"2016-30":3,"2016-26":3,"2016-22":3,"2016-18":3,"2016-07":2,"2015-40":1,"2015-32":2,"2015-27":1,"2015-22":3,"2015-14":2,"2014-52":3,"2014-42":3,"2014-41":3,"2014-35":1,"2014-23":1,"2021-43":1,"2017-13":2,"2015-18":3,"2015-11":2,"2015-06":1,"2024-22":1}},"file":"PMC3712066"},"subset":"pubmed_central"}
{"text":"author: J. Glenn MorrisAddress for correspondence: J. Glenn Morris, Jr., Emerging Pathogens Institute, University of Florida, PO Box 100009, Bldg 62, S Newell Dr, Gainesville, FL 32610-0009, USA; email: <firstname.lastname@example.com>\ndate: 2011-01\nreferences:\ntitle: How Safe Is Our Food?\n\nHow safe is our food? Put another way, how much illness in the United States is caused by foodborne pathogens? It sounds like a simple question. Getting a reasonable answer, however, is far from simple. The basic problem lies in the fact that only a small fraction of foodborne disease cases get reported through official (or unofficial) reporting systems. Calculating the \"real\" rate of foodborne illness requires development of models that use reported cases as a starting point to estimate underlying disease rates. Given the plethora of pathogens that can be transmitted through foodborne routes, this is a complex, and somewhat daunting, process. It is, however, necessary for assessing the safety of foods and developing strategies for disease prevention. The articles by Scallan et al. (*1*,*2*) in this issue represent the latest efforts to develop such estimates of the magnitude of foodborne illness in the United States.\n\nIn 1999, Mead et al. (*3*) published initial estimates of foodborne disease in the United States. This landmark undertaking was the first to provide a comprehensive compilation of data from a variety of sources, including the Centers for Disease Control and Prevention (CDC) and the medical literature. It resulted in the often-cited estimates that foodborne pathogens cause 76 million episodes of illness, 325,000 hospitalizations, and 5,000 deaths each year in the United States. (Hereafter, episodes of illness are referred to as illnesses.) During the past decade, these numbers have strongly driven ongoing efforts to implement or reform regulatory systems to protect the public from foodborne illness. However, some aspects of the methods have been criticized, particularly the high degree of uncertainty of particular parameters and thus of the results themselves (*4**\u2013**6*). These concerns have led to requests for CDC to repeat and update the work of Mead et al., using better methods and parameter estimates that more closely reflect current realities.\n\nNow, \u224811 years later, Scallan et al. have produced \"Sons of Mead,\" which include substantial improvements to the methods used by Mead et al. and to the quality and timeliness of data (*1*,*2*). Scallan et al. should be commended, especially for 2 specific improvements: their advanced treatment of statistical uncertainty and variability and their transparent inclusion of voluminous appendixes of data, models, and assumptions. These authors followed the same basic approach as Mead et al. but chose to report their estimates in 2 articles. In the first article, they based their estimates of illnesses caused by 24 major pathogens (e.g., *Salmonella* spp., *Escherichia coli* O157:H7) primarily on data from the Foodborne Diseases Active Surveillance Network (FoodNet) and other pathogen-specific surveillance systems. In the second article, they estimated illnesses caused by unknown (or unspecified) pathogens by subtracting illnesses caused by known pathogens from the annual estimated number of cases of acute gastroenteritis in the US population and adjusting the result by the percentage assumed to be acquired domestically through food. If these 2 estimates are combined, as they were by Mead et al., the new totals are 47.8 million foodborne illnesses, 127,839 hospitalizations, and 3,037 deaths per year in the United States.\n\nWhen one compares the 1999 and 2010 estimates (76 million vs. 47.8 million illnesses), the immediate response is to ask: Does this mean that food in this country is safer than it was 11 years ago? Unfortunately, the Scallan et al. articles do not enable us to answer this question. The methods, underlying assumptions, and parameter estimates used to generate these new numbers differ sufficiently from those used \u224811 years ago to preclude comparisons. In fact, if one looks simply at rates of overall gastrointestinal illness in the United States, based on FoodNet Population Surveys (*2*), one might infer that overall rates of acute gastrointestinal illness have increased during this period, from 0.49 episodes per person per year in 2000\u20132001, to 0.54 in 2002\u20132003, and to 0.73 in 2006\u20132007 (see \\[*7*\\] for a discussion of some methodologic issues with regard to the 2006\u20132007 survey). For the Scallan et al. articles, these 3 numbers were averaged to arrive at a rate of 0.6 episodes of acute gastroenteritis per person per year over the past decade. In contrast, Mead et al. used an estimate of 0.79 episodes of gastroenteritis per person per year, based on FoodNet data but also on older community surveys; they also used a somewhat different definition of acute gastrointestinal illness. This difference in estimated annual rates of acute gastroenteritis, when combined with a lower assumed proportion of gastroenteritis that is foodborne, explains much of the dramatic drop in total annual episodes of foodborne disease. Had Scallan et al. elected to use the 2006\u20132007 FoodNet estimate of 0.73 cases per person per year rather than use the average of 0.6 cases, their numbers would have been substantially higher and closer to the Mead et al. estimates.\n\nThus, if we can't use the Scallan estimates for comparison, is there any way to say whether food in the United States is safer now than it was 11 years ago? The best answer to this question comes from the FoodNet system (*7*), an active laboratory-based sentinel surveillance system that was established to monitor the public health impact of the 1995 US Department of Agriculture (USDA) Pathogen Reduction: Hazard Analysis and Critical Control Point (HAACP) System regulations (the first major revision of USDA food safety regulations since 1906). FoodNet provides annual data from designated sentinel surveillance sites on numbers of laboratory-diagnosed cases of 10 predominantly foodborne bacterial and parasitic pathogens; it reports actual case totals, not estimates. Despite year-to-year variability (including significant decreases in incidence of *Shigella* spp. and *E. coli* O157:H7 for 2009) (*8*), the overall trends show an initial drop in incidence of infection with the major bacterial foodborne pathogens after implementation of the 1995 USDA regulations, followed by a leveling off of incidence in subsequent years. One exception is infections caused by *Vibrio* spp., which are increasing, partly because climate change is affecting coastal environments (*9*). Bottom line: with the exception of *Vibrio* spp., things don't seem to be getting worse; however, after the initial decline since the USDA regulatory changes in 1995, one does not see evidence of sustained improvement.\n\nHow do numbers from the United States compare with those from Europe and the rest of the world? Again, differences in methods used by Scallan et al. make it difficult, if not impossible, to directly compare these numbers with those being published by other countries, including Canada, Australia, and members of the European Union (*10**\u2013**12*). Although these new estimates cannot be compared directly with previous estimates or with estimates from other countries, these articles nonetheless constitute a necessary starting point for generation of more robust and regularly updated numbers. Looking across time, use of a consistent method, with regular updating of data (ideally annually), would provide a basis for assessing the effect of changes in regulation and other interventions at a national level. Similarly, if the methods are further modified in keeping with current international discussions on standardization of foodborne disease estimates (*13*), direct comparison of US numbers with those from other countries may become possible.\n\nEstimates of the relative burden of disease caused by specific pathogens are crucial for improving our understanding of foodborne illness risks, but they are insufficient on their own. To target interventions (which are almost always food specific), illnesses must be quantified in terms of food\u2013pathogen combinations. Doing so, in turn, requires development of what have been termed food attribution data (*14**,**15*). That is, how much salmonellosis is caused by eating contaminated chicken versus eggs, beef, or pork? How often is beef, compared with produce, the source of infection with *E. coli* O157:H7? Likewise, summary statistics such as number of cases, hospitalizations, and deaths ignore at-risk subpopulations and chronic sequelae such as end-stage renal disease, congenital toxoplasmosis, and irritable bowel syndrome. As such, the World Health Organization and many industrialized countries are increasingly reporting integrated measures of disease, such as disability-adjusted life years, which more fully capture disease symptoms and severities (*13*). Furthermore, to reduce specific foodborne hazards, we need information about the many factors along the complex farm-to-table pathway that can lead to the introduction or amplification of pathogens that contaminate food. This information would also help determine feasibility and efficacy of potential interventions.\n\nAs outlined in a recent Institute of Medicine report (*16*), implementation of a modern, risk-based food safety system in the United States will ultimately require much better data and a strong analytic capacity at the federal level that cuts across current agency lines. Although we still have a long way to go to bring our food safety system into the current century, the articles by Scallan et al. are critical steps in the right direction.\n\nDr Morris is director of the Emerging Pathogens Institute and professor of medicine at the College of Medicine, University of Florida; a member of the Institute of Medicine Food and Nutrition Board; and an associate editor for CDC's Emerging Infectious Diseases journal. He has worked extensively with foodborne pathogens and served on 5 National Academy of Sciences\/Institute of Medicine expert committees dealing with food safety.\n\n# References","meta":{"dup_signals":{"dup_doc_count":257,"dup_dump_count":68,"dup_details":{"curated_sources":3,"2023-14":2,"2022-49":1,"2022-33":1,"2022-27":2,"2021-39":2,"2021-31":2,"2021-21":1,"2021-17":1,"2021-10":1,"2021-04":1,"2020-50":1,"2020-45":3,"2020-40":1,"2019-35":1,"2019-22":1,"2019-18":1,"2019-09":2,"2018-51":1,"2018-43":1,"2018-39":1,"2018-30":2,"2018-22":1,"2018-17":1,"2018-13":1,"2018-09":1,"2017-51":1,"2017-47":2,"2017-43":1,"2017-39":1,"2017-34":1,"2017-30":1,"2017-26":2,"2017-17":2,"2017-09":1,"2017-04":2,"2016-50":2,"2016-44":2,"2016-40":2,"2016-36":2,"2016-30":2,"2016-26":1,"2016-22":1,"2016-18":4,"2016-07":7,"2015-48":7,"2015-40":6,"2015-35":8,"2015-32":7,"2015-27":5,"2015-22":7,"2015-14":8,"2014-52":8,"2014-49":11,"2014-42":19,"2014-41":11,"2014-35":11,"2014-23":11,"2014-15":14,"2023-23":1,"2024-10":1,"2017-13":2,"2015-18":8,"2015-11":6,"2015-06":8,"2014-10":10,"2013-48":7,"2013-20":7,"2024-26":1}},"file":"PMC3375763"},"subset":"pubmed_central"}
{"text":"abstract: An approach using 'chemical genetics' has identified small-molecule agonists of the Hedgehog signaling pathway that may lead the way to drugs for chronic degenerative diseases.\nauthor: Jonathan B Weitzman\ndate: 2002\nreferences:\ntitle: Agonizing Hedgehog\n\nIt is a rare treat when a drug discovery program teaches us something about the biology of the process that it attempts to modulate. The paper by Maria Frank-Kamenetsky and colleagues in this issue of the *Journal of Biology*\\[1\\] presents a compelling example of how the search for therapeutics can provide powerful experimental tools and insights into fundamental biology, blurring the distinction between applied and basic research. By characterizing a small group of chemically similar agonists of the **Hedgehog** signaling pathway, Frank-Kamenetsky *et al*. have been able to propose a new model for how the **Smoothened** component of the Hedgehog-receptor complex works, and to hint at the existence of natural-ligand agonists of the Hedgehog signaling pathway (see <a href=\"#F2\" data-ref-type=\"fig\">'The bottom line'<\/a> box for a summary of their work).\n\n# Hedgehog history\n\nSignaling by the Hedgehog (Hh) family of secreted proteins plays a central role in regulating cell differentiation and tissue patterning during development \\[2\\]. The *hedgehog* gene (*hh*) was first identified by virtue of its role in the specification of positional identity during *Drosophila* embryonic segmentation, and it was subsequently found to control patterning of structures such as the eye and the abdominal cuticle. In mammals there are three *hh* homologs, called ***Sonic Hedgehog***, ***Indian Hedgehog*** and ***Desert Hedgehog*** (*Shh*, *Ihh* and *Dhh*, respectively), which have been implicated in patterning events in a range of developing tissues \\[2,3\\] (see the <a href=\"#F3\" data-ref-type=\"fig\">'Background'<\/a> box).\n\nRecently, signaling by Hh has been shown to be important for patterning of the cerebellum, where it promotes the proliferation of granule neuron precursors. A link between Hh proteins and stem-cell proliferation has raised the enticing possibility that modulating Hh signaling might be relevant for the clinical management of certain degenerative diseases. Indeed, it was recently demonstrated that Shh might be effective in treating peripheral nerve damage or degenerative brain disorders such as **Parkinson's disease**\\[4,5\\]. Perhaps not surprisingly given its role in development, misregulated Hh signaling has also been implicated in **cancer**\\[3\\]. Specifically, medulloblastoma and basal cell carcinoma (BCC) are associated with inappropriate activation of Hh signaling \\[6,7\\]. These observations motivated Frank-Kamenetsky and colleagues to search for small-molecule modulators of the Hh pathway, with the hope that **antagonists** and **agonists** might be used as drugs to treat proliferative or degenerative diseases, and that **small molecules** might prove more amenable to pharmacological delivery than the Hh-family proteins themselves.\n\n# Sending the signal\n\nHedgehog signaling challenges the way we normally think about signal transduction pathways. Biology is full of examples of extracellular ligands that bind to specific cell-surface receptors, initiating a cascade of biochemical events (often involving protein kinases) that leads to the activation of a transcription factor and the induction of a set of effector genes. But Hedgehog signaling is not so simple. Even the ligand is complicated: the Hh proteins undergo unusual processing and cleavage to generate an extracellular cholesterol-linked peptide that serves as the signaling ligand \\[2\\]. And the receptor component is far from understood.\n\nThe cellular response to Hh is controlled by two transmembrane proteins, **Patched** (Ptc) and Smoothened (Smo). The Ptc protein weaves across the cell membrane twelve times and resembles some transmembrane channels. It acts as a negative regulator of the Hh signal and has been defined as a tumor-suppressor. In contrast, Smo is a proto-oncogene and activates signaling in response to Hh ligand. The Smo protein is a seven-transmembrane receptor that resembles conventional G-protein-coupled receptors (Figure <a href=\"#F1\" data-ref-type=\"fig\">1a<\/a>).\n\nIt appears that Ptc inhibits Smo, although the precise mechanisms are unclear. Hh stimulation relieves Smo from inhibition by Ptc, leading to the generation of an intracellular signal that culminates in a nuclear transcriptional response (Figure <a href=\"#F1\" data-ref-type=\"fig\">1b<\/a>). When Ptc is removed the pathway is constitutively 'on', independent of the Hh ligand, whereas certain mutations in Smo can activate Hh signaling, bypassing Ptc regulation. A heteromeric receptor model has been proposed, in which Hh interacts directly with Ptc and thereby affects the interaction of Ptc with Smo \\[8\\]. \"But things look much more complicated than we had earlier thought,\" says Philip Beachy (Johns Hopkins University School of Medicine, Maryland, USA). \"The previous models are not tenable,\" and he suggests that alternative models of receptor function must be considered.\n\n# The power of chemical genetics\n\nFrank-Kamenetsky *et al.*\\[1\\] chose to use **chemical genetics** in an attempt to identify compounds that could interfere with the inhibition of Smo by Ptc, or could activate Smo independent of Ptc, or that might act downstream of Smo. In addition to making effective drug candidates, these molecules might also help to illuminate the complex mechanisms underlying signaling by Hh, Ptc, and Smo (see the <a href=\"#F4\" data-ref-type=\"fig\">'Behind the scenes'<\/a> box for more of the background to the work).\n\nThe high-throughput screen was elegantly simple. First, Frank-Kamenetsky *et al.* identified a cell line that responds well to Hh stimulation. The introduction of a reporter gene (encoding the firefly luciferase protein) that was turned on by Hh signaling permitted screening for compounds that block or induce signaling by monitoring the expression of the luciferase protein (using a simple luminescence test). They had previously used such a screen to isolate antagonists of Hh signaling, and had demonstrated the effectiveness of some antagonists as potential anti-tumor drugs to treat BCC \\[7\\]. Their new screen of 140,000 synthetic compounds led to the discovery of a few candidate agonists that could stimulate the reporter gene and mimic Hh activity.\n\nOnce the cell-based screen was completed, the chemists took over, synthesizing 300 derivative molecules until they found a few compounds that were related to the previous 'best' agonist but that were a thousand times more effective at eliciting a cellular response, affecting cells when applied in the nanomolar range. \"Getting more potent compounds was essential if we were to figure out where the agonists were acting\" recalls Jefferey Porter who headed the team at Curis, Inc.\n\nThen the cell biologists began again, studying the effects of the agonists on the proliferation of primary neonatal cerebellar granule neuron precursors. They monitored the incorporation of tritiated thymidine into cultured rat neurons (as a marker of DNA synthesis and hence proliferation) and were pleased to see that the agonists were as effective in this assay as the Hh protein itself. An assay using an explant of embryonic neural plate was used to confirm that the agonists could induce dose-dependent gene expression in neural precursors, just as the Shh protein does.\n\nHaving established the effects of the agonists in culture assays, the researchers then turned to an *in vivo* model, feeding the compounds to pregnant mice and following the effects on the phenotypes of embryos lacking *Shh* or *Smo*. The treated embryos displayed activated Hh signaling, demonstrating that the compounds were not toxic and could cross the placental barrier. The developmental defects of *Shh*^-\/-^ embryos were rescued by treatment with the agonist but the compound had no effect on Hh signaling in the absence of Smo.\n\n\"Once we knew that the agonists were targeting Smo, we wanted to investigate whether they bound to Smo directly and how they activated Hh signaling,\" says Porter. The cell line that was created for the screen served as a useful tool to test the effects of known antagonists on the function of the agonist. An anti-Hh blocking antibody had no effect, so the agonist must work downstream of the Hh-Ptc interaction. But the agonist was blocked by antagonists that work at the level of Smo or further downstream. \"We have similar conclusions,\" says Beachy, whose group used photo-affinity labeling and cross-linking experiments to show that small-molecule agonists and antagonists bind directly to Smo.\n\nNext, for Frank-Kamenetsky *et al.,* it was time for some careful biochemistry. Analysis of the expression of fusion proteins of Ptc or Smo showed that, unlike the Hh ligand itself, the agonist had no effect on the stability of the Ptc protein. In contrast, both Hh and the agonist could increase the stability of the Smo receptor. Immunoprecipitation experiments with radiolabeled agonist showed that the agonist must bind directly to Smo receptors, and that Hh-signaling inhibitors compete with the agonist for binding. Pharmokinetic analysis provided evidence for a single binding site competition model. Finally, Frank-Kamenetsky *et al*. exploited an oncogenic, constitutively active mutant form of Smo (Smo^act^); the agonists bound equally well to mutant and wild-type forms, whereas the antagonist bound less well to the mutant form.\n\n# Learning lessons from drugs\n\nFrank-Kamenetsky *et al*. have demonstrated convincingly that high-throughput chemical genetics can be used to isolate modulators of a developmental signaling pathway. The agonists that they have generated are efficient and apparently non-toxic mimics of Hh signals and are promising candidates for drugs for regenerative medicine. The authors work at the biotechnology company Curis Inc. (Cambridge, USA), so finding new drugs is obviously their primary objective. But the agonists also provide useful tools for probing the complex Hh-Ptc-Smo signaling pathway.\n\n\"A lot of our biological insight is driven by having specific chemical inhibitors, and many drugs are used as tools to dissect signaling systems as a substitute for genetic studies,\" says Arnon Rosenthal (Rinat Neuroscience Corp., Palo Alto, USA). Beachy agrees: \"these compounds, first the plant-derived inhibitor cyclopamine and more recently the agonists, have really helped us to understand Smoothened function.\" Recent work from Beachy's lab \\[9,10\\], demonstrating that Ptc suppresses Smo in catalytic manner, has led to speculation that Ptc may function as a transporter protein pumping natural small-molecule Smo modulators across the cell membrane. Rosenthal adds that \"good basic research always leads to better medicines, since the more we understand about the mechanisms operating in the body, the better able we are to modulate them rationally.\"\n\nThe data presented by Frank-Kamenetsky *et al*. \\[1\\] led them to propose a new model for Hh signaling based on the classic 'ternary complex model' that was developed to describe ligand-induced conformational changes of G-protein-coupled receptors \\[11\\]. According to this model, active and inactive conformations of Smo are selected by the binding of agonists or antagonists at independent sites. Furthermore, the model predicts that Smo binds to a novel effector molecule and it raises the possibility that endogenous ligands, analogous to the newly found agonist, may naturally regulate Smo activity.\n\nCheryll Tickle (University of Dundee, UK) studies the role of Hh in development and finds the possibility that there are endogenous small-molecule agonists that interact with Smoothened particularly intriguing. \"This would mean that we are missing a whole layer of control of Hedgehog signaling in our current models,\" she notes. Porter adds, \"It is tempting to speculate that endogenous molecules act directly on Smo, bypassing Ptc. That is consistent with what we know about how G-protein-coupled receptors work. But at the moment, it's pure speculation.\"\n\nThe study by Frank-Kamenetsky *et al.*\\[1\\] exploits a dazzling variety of experimental techniques to illustrate the path from high-throughput screening to compound characterization. Biochemists, cell biologists, pharmacologists and chemists have come together to demonstrate effectively that 'drug discovery' can combine all the excitement of 'real' scientific discovery with the satisfaction of isolating compounds that might be used for tomorrow's therapies.\n\n## Figures and Tables","meta":{"dup_signals":{"dup_doc_count":137,"dup_dump_count":42,"dup_details":{"curated_sources":2,"2019-09":1,"2018-51":1,"2018-43":1,"2018-22":1,"2018-05":1,"2017-39":1,"2017-34":1,"2017-26":1,"2017-22":2,"2017-17":1,"2017-09":11,"2017-04":1,"2016-50":1,"2016-44":2,"2016-40":1,"2016-36":13,"2016-30":10,"2016-22":1,"2016-18":1,"2016-07":13,"2015-48":3,"2015-40":2,"2015-35":2,"2015-32":2,"2015-27":3,"2015-22":3,"2015-14":2,"2014-52":3,"2014-49":2,"2014-42":9,"2014-41":5,"2014-35":5,"2014-23":4,"2014-15":6,"2019-22":1,"2015-18":3,"2015-11":3,"2015-06":3,"2014-10":3,"2013-48":3,"2013-20":2,"2024-18":1}},"file":"PMC137067"},"subset":"pubmed_central"}
{"text":"author: Christopher Gardner; Judith Wylie-Rosett; Samuel S. Gidding; Lyn M. Steffen; Rachel K. Johnson; Diane Reader; Alice H. Lichtenstein; [^1][^2][^3][^4][^5]\ndate: 2012-08\nreferences:\nsubtitle: : A Scientific Statement from the American Heart Association and the American Diabetes Association\ntitle: Nonnutritive Sweeteners: Current Use and Health Perspectives\n\nA 2009 American Heart Association scientific statement titled \"Dietary Sugars Intake and Cardiovascular Health\" (1) concluded that current intake of added sugars among Americans greatly exceeds discretionary calorie allowances based on the 2005 U.S. Dietary Guidelines (2). For this reason, the American Heart Association Nutrition Committee recommended population-wide reductions in added sugars intake. The present statement from the American Heart Association and the American Diabetes Association addresses the potential role of nonnutritive sweeteners (NNS) in helping Americans to adhere to this recommendation in the context of current usage and health perspectives.\n\nBy definition, NNS, otherwise referred to as very low-calorie sweeteners, artificial sweeteners, noncaloric sweeteners, and intense sweeteners, have a higher intensity of sweetness per gram than caloric sweeteners such as sucrose, corn syrups, and fruit juice concentrates. As a caloric sweetener replacement, they are added in smaller quantities; hence, they provide no or few calories. In our current food supply, NNS are widely used in thousands of beverages and other food products such as diet soft drinks, yogurts, desserts, and gum. Food manufacturers often use a blend of NNS or use a blend of sugar and NNS to improve the flavor acceptability of NNS. In developing this scientific statement, the writing group reviewed issues pertaining to NNS in the context of data on consumer attitudes, consumption patterns, appetite, hunger and energy intake, body weight, and components of cardiometabolic syndrome. The objective was to review the literature to determine whether there were adequate data to provide guidance for the use of NNS.\n\nThe focus of the statement is on the 6 NNS that are described in <a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>. Aspartame, acesulfame-K, neotame, saccharin, and sucralose are regulated as food additives by the U.S. Food and Drug Administration and therefore had to be approved as safe before being marketed. Regarding stevia, at this time, the U.S. Food and Drug Administration has not made a determination as to the Generally Recognized As Safe status, but has issued no objection letters for a number of Generally Recognized As Safe notifications for stevia sweeteners (<http:\/\/www.fda.gov\/Food\/FoodIngredientsPackaging\/GenerallyRecognizedasSafeGRAS\/GRASNotificationProgram\/default.htm>). Because all 6 of these NNS have current U.S. Food and Drug Administration approval, issues related to safety of these compounds are not addressed. In addition, the review of the literature is primarily restricted to human studies in which noncaloric sweeteners are used as a replacement for caloric sweeteners.\n\n# Literature Search\n\n## Approach\n\nThe literature search was conducted by using the following search terms: nonnutritive sweetener(s), artificial sweetener(s), noncaloric sweetener(s), diet (beverages and soft drinks), and the names of each of the 6 NNS available for use in the United States (including their brand names). The initial search by use of PubMed was limited to original research published after 2000, studies conducted in humans (primarily restricted to controlled trials and prospective cohort studies), and systematic reviews. Additional articles were identified by use of the literature cited in the original publications and review articles, and the Evidence Analysis Library of the American Dietetic Association (<http:\/\/www.adaevidencelibrary.com\/>), as well. A Google search was used to identify published marketing research related to consumer views of NNS.\n\nNonnutritive sweeteners (3\u20136)\n\n![](1798tbl1)\n\n## Summary of available literature\n\nThe literature search yielded relatively few research studies that focused on the specific objective of this scientific statement, the potential role of NNS in facilitating reduction of added sugars intake in humans. This is likely attributable to inherent complexities involved in the design and implementation of these types of studies. In particular, experimental studies designed to test the effects of NNS on health outcomes in food are particularly challenging, because replacing added sugars with NNS alters dietary composition (i.e., assuming portion size is not reduced, the relative proportions of fat, protein, and carbohydrate may be changed or the nature of the carbohydrate alone may be changed) in ways that can add potential confounding to the simple comparison of added sugars versus NNS. The majority of the human data in prospective observational studies and randomized controlled trials on the use of NNS focus specifically on diet soft drinks as a replacement\/displacement of regular soft drinks. Assessment in large prospective cohorts of the use of NNS from sources other than diet soft drinks has been limited methodologically by such factors as lack of specificity in intake data collected by food frequency questionnaires (FFQs) and lack of accurate NNS composition values in available databases. Much of the published literature on NNS addresses research conducted in animal models and evaluations of potential toxicity, neither of which were areas of focus for this statement. Hence, the review that follows is notably limited by the lack of an extensive evidence base.\n\n# Consumer Attitudes and Rationale for Using or Avoiding NNS\n\nFood and beverage industry publications provide some insights into consumers' rationale for NNS use. As expected, some of this use is tied to consumer efforts to decrease their intake of calories and caloric sweeteners. A 2012 survey of American adults by the International Food Information Council Foundation found that 51% of the respondents reported trying to limit sugar, 44% were trying to limit high-fructose corn syrup, and 29% were trying to limit low-calorie sweeteners (7). When asked to select \u22651 choices from a list of reasons for using NNS, 41% chose \"are an option for people with diabetes,\" 41% chose \"can reduce the calorie content of foods,\" 40% chose \"can play a role in weight loss or weight management,\" 30% chose \"can be part of an overall healthful diet,\" and 31% chose \"don't know enough about them to provide an answer.\" Seventy-three percent of those consuming NNS reported doing so to reduce total calories consumed.\n\nAlthough consumer demand for reduced-calorie and added-sugars products has resulted in a proliferation of new NNS products, American consumers have concerns about NNS, as well. In a Mintel survey conducted in a representative sample of Americans, the vast majority of survey respondents (81%) agreed with the statement, \"Sugar-free foods do not taste as good as those made with real sugar\" (7a). The Mintel survey also found that 64% of respondents indicated they were concerned about the safety of \"artificial\" sweeteners (7a). Survey data from the Hartman Group indicated that 44% of consumers reported they \"deliberately avoid\/reduce intake of artificial (flavors, sweeteners, colors or dyes),\" but, when asked about specific NNS, the proportion drops to 39%, 33%, and 21% for saccharin, aspartame, and sucralose, respectively. The survey did not include other NNS (8). Consumers' concerns about taste and artificial ingredients may motivate food manufacturers to promote sucralose as being made from \"real\" sugar and stevia-derived NNS as being \"natural\" and made from the leaves of the stevia plant. Consumer education will need to stress that promotion of a certain NNS as \"real\" or \"natural\" does not necessarily indicate that such products are more efficacious or safer than other NNS.\n\n# Estimates of Consumption Patterns\n\nInformation on the amounts of NNS in beverages and foods is limited and not easily accessible. Food processors and manufacturers are required to list the NNS in product ingredient lists but, with the exception of saccharin, are not required to provide the amount used in products or to release the information to federal agencies or the general public (3). Between 1999 and 2004, there was a large increase in the number of new products with NNS available in the United States, estimated to be \\>6,000 (9). Of the 6 available NNS, sucralose (found in 2,500 products), acesulfame-K (found in 1,103 products), and aspartame (found in 974 products) were the most popular (9). NNS are most commonly used in carbonated beverages (3). Estimates of consumption patterns are typically made by reporting amounts of beverage and food items containing NNS, rather than amounts of specific NNS consumed.\n\n## Nationwide Food Consumption Survey, National Health and Nutrition Examination Survey, andU.S. Department of Agriculture Economic Research Service Data\n\nMattes and Popkin (3) examined the trends in consumption of foods and beverages with NNS among Americans aged \u22652 years by use of the U.S. Department of Agriculture Nationwide Food Consumption Survey for 1965, 1977 to 1978, and 1989 to 1991 and the National Health and Nutrition Examination Survey (NHANES) for 1999 to 2000, 2001 to 2002, and 2003 to 2004. Consumption of NNS in both beverages and foods increased over time. The survey data indicate that, on any given day, 15% of the U.S. population consumed NNS in 2003 to 2004 in comparison with 3% in 1965. The proportion of the population choosing beverages with NNS remained relatively stable between 1989 and 2004 (10.1% and 10.8%, respectively), whereas the proportion consuming NNS in foods increased from 3.2% to 5.8% during that time period (3). Notably, the increase in NNS products was not accompanied by a corresponding decrease in reported consumption of products sweetened with added sugars, which indirectly suggests that NNS are not being used to displace products sweetened with added sugars. This same pattern of parallel increases in both NNS products and products with added sugars has been reported elsewhere (10,11) and is evident from per capita food availability data as compiled by U.S. Department of Agriculture Economic Research Service for regular versus diet soft drinks, with intake increasing primarily from 1980 to 1990 and then leveling off between 1990 and 2003 (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<\/a>).\n\nOne of the authors (L.M.S.) examined the prevalence of consuming NNS foods and beverages in children and adults with the use of data from NHANES 2007\u20132008. Dietary intake in NHANES was assessed by a 24-hour recall in-person interview, which presents a \"snapshot\" of foods and beverages consumed by NHANES participants on a given day*.* The most frequently reported and consumed NNS product was NNS beverages in comparison with intake of NNS foods or packets of artificial sweeteners among male and female children and adults in NHANES 2007\u20132008 (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<\/a>; L.M. Steffen, unpublished data, 2011). After further examination of NNS beverage intake by age group, the prevalence of consumption of these beverages increased across age groups, especially among females (<a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<\/a>; L.M. Steffen, unpublished data, 2011). In general, more adults were NNS beverage consumers than children or adolescents.\n\n## Other perspectives on NNS users\n\nDietary intake assessed by FFQ, as is common in observational cohort studies, provides an estimate of those who \"ever\" versus \"never\" use NNS-containing beverage or food items included in the questionnaire; this is a different perspective than that derived from 24-hour recall data as above. The prevalence of saccharin intake in 1980 (the only NNS available at that time) was assessed in a subset of the Nurses' Health Study (NHS) characterized as being free from major illnesses and other lifestyle factors that might modify weight gain. Intake was assessed by use of a 61-item FFQ completed by 31,940 women. The report indicated that the prevalence of saccharin users was 56% (12). In the Health Professionals Follow-up Study (HPFS), artificially sweetened beverage intake was assessed in 1986 and every 4 years after that during 20 years of follow-up with use of a 131-item FFQ (13). By use of a cumulative average over this time period, 54% of the men reported consuming artificially sweetened beverages at least 2 times per month. The San Antonio Heart Study enrolled a randomly selected sample from non-Hispanic white and Mexican American neighborhoods. Among 3,682 participants who were examined first between 1979 and 1988 and then again 7 to 8 years later, 48% reported some consumption of NNS in beverages that included diet soft drinks, coffee, or tea. Consumption of NNS in food items was not assessed (14).\n\n# Appetite, Hunger, and Energy Intake\n\nFor NNS-sweetened items to successfully contribute to reductions in calories from added sugars or other sources, they must also avoid causing compensatory energy intake immediately or later in the day. If choosing a diet soft drink over a regular calorie soft drink leads to a decrease of \u223c100 kcal per 8-oz serving, but later in the day causes an alteration in appetite or hunger that results in an additional 50, 100, or 200 kcal of intake, the initial \"calorie saving\" effect would be altered or reversed. Or, if choosing a diet soft drink enables an individual to simultaneously justify eating a 150-calorie snack, the calorie savings from the NNS would also be negated.\n\nMattes and Popkin (3) critically examined the evidence for effects of NNS on compensatory appetite and food intake. The 8 potential mechanisms they reviewed are listed and briefly described in <a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a>. The review concluded that the available evidence either refuted or was insufficient to refute or support each of these potential mechanisms or hypotheses for NNS increasing appetite, hunger, or energy intake (3).\n\nPotential mechanisms of effects of NNS on compensatory appetite and food intake\n\n![](1798tbl2)\n\nThe complexity in attributing the extent to which NNS consumption is compensated for by intake of other foods is fraught with methodological challenges both in controlled feeding experiments and free-living trials. According to International Food Information Council Foundation research, \\<10% of Americans can accurately estimate the number of calories they should consume in a day. Consumer education on daily energy requirements is needed, and that products containing NNS may assist in weight control when used in place of full-calorie products, if not compensated for otherwise. Controlled feeding experiments might or might not show an impact of NNS on future caloric intake, but, by definition, these are not real-world situations. Conversely, in the real world, so many factors impact food choice that attributing caloric intake after NNS consumption uniquely to that consumption may also not be true and may be confounded particularly by reverse causality; that is, the previous NNS choice might be deliberate in anticipation of a known future exposure to higher caloric meal.\n\n## Energy intake compensation\n\nVarious studies that have examined energy intake as a primary or secondary outcome have used designs that contrast beverages and foods made with caloric sweeteners versus NNS. In a meta-analysis of weight loss studies that tested the effectiveness of aspartame in reducing energy intake, data from 12 studies were pooled to address energy intake compensation (15). The weighted average of energy intake compensation in the \u223c24 hours that followed aspartame intake was 32% (i.e., 68% of the original energy deficit was uncompensated and was maintained through the subsequent period of intake). Of the 4 studies from the meta-analysis that used beverages alone, the compensation was just 15% for the subsequent 24 hours; that is, the data suggest there is less compensation in beverages than foods, resulting in a more effective net reduction in calories when replacing sweetened beverages with NNS beverages (15). The implication here is that anything \\<100% compensation results in a net reduction in 24-hour energy intake.\n\nOne of the studies included in the meta-analysis involved 24 men and women who consumed 4 different beverages, each for 4 weeks, in a crossover design. Subsequent food intake was monitored. The beverages included a full caloric beverage (sucrose) versus an NNS beverage (aspartame), provided with either an orange or raspberry flavor. Data were collected at baseline and after 4 weeks of habituation (16). The investigators observed a significant lack of energy intake compensation over the course of the day among study participants when they were consuming the full caloric beverages relative to the NNS beverages. In another study, 21 overweight adults were assigned to consume 28% of their energy intake as sucrose, mostly as beverages. After 10 weeks, there was a significant increase in their energy intake, body weight, fat mass, and blood pressure (17). These effects were not observed in a similar group of 20 overweight adults who consumed NNS in place of the sucrose, but otherwise had a similar dietary intake composition in comparison with the other group (17). In a controlled feeding experiment that involved a preload snack containing sucrose (493 kcal), aspartame (290 kcal), or stevia (290 kcal), the energy intake of subsequent meals was similar; the energy intake for the combined preload snack and later meals combined was lower for the aspartame and stevia phases versus the sucrose phase (*P* = 0.01) (18). All of these studies, therefore, observed a net reduction in energy intake for NNS use relative to the comparison condition with sucrose.\n\nNot all such studies have observed a net reduction in overall energy intake when NNS were used instead of caloric sweeteners. A 4-week trial, providing either diet soft drinks (sweetened with aspartame) or regular calorie soft drinks resulted in similar total energy intake in healthy-weight adults of both sexes (19) and in overweight women as well (20). Similarly, the results of a 25-week study conducted in adolescents suggested that normal-weight youth compensated for the energy reduction when consuming diet beverages. However, as evidence of the complexity of this issue, the same study reported that the use of NNS beverages did result in a net energy deficit in those adolescents in the highest body mass index (BMI) tertiles (a conclusion that should be tempered by awareness that the intervention group was counseled to replace sugar-sweetened beverages with diet beverages and\/or water, making it difficult to differentiate the effects of NNS versus water) (21).\n\nOverall, the limited human studies literature in this area of NNS research suggests that compensation is an important factor to consider when assessing impact on energy intake over periods of \u226524 hours. Further studies are warranted to address some of the complexities inherent in this issue, including the potential impact of being informed versus na\u00efve about the presence or absence of NNS in items being consumed. It is worth noting that, in preschool children, when energy intake is reduced by substitution of nutrient-dense, lower-calorie food choices instead of NNS (e.g., fruits and vegetables), some energy intake compensation occurs, but net energy intake remains lower over several days (22,23).\n\n## Appetite, or preference, for sweet taste\n\nExamination of the potential effects of NNS on appetite requires consideration of genetic and environmental factors that may regulate preference for intensity of sweet taste (24\u201326). One focus of such research concerns the metabolic\/hormonal responses to both caloric sweeteners and NNS to determine whether these can be linked to mechanisms that promote future weight gain or other adverse physiological consequences. Sweet taste is mediated largely by a single receptor composed of the 2 subunits, TAS1R2 and TAS1R3. Understanding how the polymorphism of this receptor gene affects preferences is important to understanding usage of NNS. Recent data in mice suggest that additional mechanisms may exist, but these have not been confirmed in humans (27). Newer animal research also suggests the possibility that sweet-taste receptors exist in the gut, triggering complex metabolic\/hormonal responses to NNS. The impact of these receptors and their metabolic consequences on obesity and diabetes mellitus in humans is just beginning to be studied (28). Although ingestion of foods containing NNS may trigger a wide array of responses related to food absorption that may impact future food consumption (29), adverse effects related to these gut receptors have yet to be identified (30,31).\n\nA preference for higher intensity of sweetness exists in humans in infancy, declines in adolescence in comparison with infancy, and then plateaus or slowly declines in young adulthood, and may or may not decline further in old age (32,33). There is significant interindividual variation in sweet- and bitter-taste preference, and this preference may be genetically determined (34\u201337). These genetic differences may impact preferences for both caloric sweeteners and NNS, which may also have a bitter-taste component. Although animal research suggests that sweet-taste preference can be conditioned through repeated exposure (38), preference for sweet taste in humans is more complex and involves many central receptor mechanisms, including but not limited to dopamine signaling (39). Critical factors such as food culture and social expectations may also have an impact (3). The presence of caloric sweeteners may also enhance taste acceptance (40).\n\n# Cardiovascular and Metabolic Health Outcomes\n\nThe potential effects of NNS on body weight, cardiometabolic variables, and diabetes mellitus\/glycemic response were examined. The evidence considered was limited to prospective studies and controlled trials conducted in humans. For the controlled trials, primary emphasis was given to study designs that involved replacing caloric sweeteners with NNS. Prospective cohort studies were also considered, but cross-sectional data were not considered. Although NNS intake may precede and lead to changes in weight or metabolic variables, the opposite is also plausible (i.e., individuals who become overweight\/obese may seek out NNS items after becoming overweight\/obese or being informed they have type 2 diabetes mellitus). Therefore, it is not possible to distinguish the temporal relationship between these by using cross-sectional studies, and, as will be discussed, it can be difficult to distinguish this relationship even in some prospective studies.\n\n## Body weight\n\nThe vast majority of data available related to NNS consumption and body weight has focused on creating an energy deficit by substituting NNS for sugars in the beverage component of the diet, with only a few intervention studies also targeting foods. Findings in both controlled intervention trials and prospective observational studies have been inconsistent.\n\nControlled intervention trials have reported that use of products with NNS results in weight loss or has negligible effects on weight. In a review of 16 trials with a primary focus on aspartame and weight control, weight loss was a primary outcome in 9 of the 16 trials (15). Beyond summarizing the overall pattern of success with weight loss in these studies, the investigators attempted to quantify the magnitude of caloric substitution required for meaningful weight loss. By their calculations, a weight loss of 0.2 kg\/week over 12 weeks was achieved from a net energy deficit of 220 kcal\/day attributable to aspartame substitutions for caloric sweeteners. Determining the net energy deficit required consideration of energy intake compensation, which was different for foods and beverages (as described previously, see <span class=\"smallcaps\">energy intake compensation<\/span>). For foods it was determined that displacing 330 kcal\/day of sugar (82.5 g), and assuming 32% compensation, would yield the 220-kcal deficit. For beverages, with a calculated 15% compensation, 260 kcal of caloric sweetener, or 70 g (the amount found in \u223c2, 12-oz soft drinks), would need to be displaced to yield the same net 220-kcal deficit.\n\nIn a study conducted with 103 subjects who were 13 to 18 years of age and who regularly consumed sugar-sweetened beverages, BMI change was the primary outcome. The randomized trial contrasted home delivery of NNS beverages (i.e., water and diet soft drinks, iced teas, lemonade, and punches) to a control group that did not receive beverages and were instructed to continue their habitual consumption. Overall, there were no significant differences in BMI changes over the 25-week period; however, in a post hoc stratified analysis of participants in the highest tertile of baseline BMI, the BMI change was significantly greater, reflecting weight loss, in the group receiving the NNS beverages (21). Another study involved a family-based randomized trial testing the impact of promoting sucralose and sucralose-containing products as substitutes for caloric sweeteners in beverages and foods (41). This study was conducted in conjunction with instructions to add 2,000 steps\/day to their habitual physical activity. One hundred families assigned to the intervention were compared with 93 families assigned to follow their habitual diet and activity patterns. After 6 months, none of the primary study outcomes (changes in BMI for age, waist circumference, or percentage of body fat) were significantly different between treatment groups, either for the children (primary target population), or their parents (secondary target population). In post hoc analyses, the investigators found a borderline significant improvement in the intervention group for the percentage of children who maintained or reduced their BMI for age.\n\nAs would be expected, controlled intervention trials involving dietary manipulations, as described above, are generally limited to relatively short durations of \u22646 months (1 exception being a 16-week intervention with aspartame-sweetened foods and beverages with a 1-year maintenance period and 2 years of follow-up; this study was included in the review of 16 trials previously described) (15,42). Longer-term perspectives are available from prospective observational studies, with the important limitation that these studies can address only associations, and not causal effects.\n\nA limited number of prospective observational studies have addressed NNS and weight, most of which assessed NNS intake by FFQ. None of the studies were designed specifically to assess associations between NNS and weight. Two studies published \\>20 years ago reported a positive association between NNS use and weight gain (12,43). However, these findings were observed by the use of limited dietary assessment methods, and in selected (i.e., not representative) study populations. A 2008 study examining the San Antonio Heart Study population reported a positive dose-response association between artificially sweetened beverage consumption (i.e., diet soft drinks, and coffee and tea with added NNS) and incidence of overweight\/obesity during a 6- to 7-year follow-up period of 5,158 adults. The association was consistent across sex and ethnicity; overall, the change in BMI was 47% greater among NNS users than nonusers after adjusting for baseline BMI and demographic and behavioral characteristics (14). A 2011 study reported findings on associations of a broad range of diet and lifestyle variables with 12- to 20-year weight change observed in 3 separate cohorts that included 120,877 U.S. women and men from the NHS, NHS II, and the HPFS. For a given unit of 1 diet soft drink serving\/day, the association with weight change was not statistically significant in the NHS, whereas there was a statistically significant but modest association with weight loss (\\<0.25 lb\/4 years) observed in NHS II and HPFS after adjusting for a broad range of demographic and lifestyle variables (44).\n\nA separate publication based on only the HPFS population focused specifically on sugar-sweetened beverage versus artificially sweetened beverage (ASB) intake and provides important insights into the opportunities and limitations of the perspective offered by prospective observational studies. Cumulative averages of intake were determined by FFQs administered every 4 years for 20 years, starting in 1986, and the main outcome of interest in this publication was incidence of type 2 diabetes mellitus. At the start of the study, in 1986, participants were asked to report any weight change in the preceding 5 years, between 1981 and 1986. The weight change data were presented separately for those who reported losing weight (14%\u201318% of the population, depending on the specific quartile of ASB intake \\[L. de Koning, personal communication, 2011\\]) and those who reported gaining weight (45%\u201351% of the population, depending on the specific quartile of ASB intake) in that time frame; the remainder reported no weight change (13). Among those who lost weight, there was a significant trend of greater weight lost for higher versus lower ASB intake quartiles (0.9 \u00b1 0.2 versus 0.5 \u00b1 0.5 kg, means \u00b1 SD, respectively, for the highest versus lowest quartile). Among those who gained weight, there was a significant trend of greater weight gained for higher versus lower ASB intake quartiles (2.6 \u00b1 4.6 versus 1.8 \u00b1 3.4 kg, respectively). This may appear contradictory; how could greater intake be associated with both greater weight loss and greater weight gain? However, this suggests there may be importantly different subsets of the population: one that successfully uses ASB for weight control and another that does not. On the other hand, the different time frames of the weight change data versus ASB intake data must also be considered; the 1981 to 1986 weight change preceded the 1986 to 2006 cumulative average ASB intake reported in the article; from a chronological perspective it is the weight change that is predicting ASB consumption, rather than vice versa (i.e., weight gain may predict subsequent increased ASB consumption). Three additional findings presented in this study further highlight the complexities involved: the following trends for baseline characteristics for the full study population, regardless of 1981 to 1986 weight change, were all statistically significantly related to ASB intake; diet quality as assessed by the Alternative Healthy Eating Index was higher, total energy intake was lower, and BMI was higher for those consuming greater amounts of ASB. Overall, the data from this study suggest there may be important subsets of the population that incorporate NNS products into their diet with different objectives or with differential success, and that there is likely substantial confounding in these types of analyses given the many dietary and health variables that correlate with NNS intake (12,43).\n\n### Assimilating results from intervention and observational studies.\n\nThe 2 sets of studies reviewed above help to demonstrate what have been and will continue to be challenges in reaching definitive conclusions about the effectiveness of NNS in supporting energy intake restriction and promoting weight loss and weight control. The short-term nature of intervention studies limits their ability to observe long-term weight change, and the typical personal\/individual support from study staff and related heightened awareness of NNS intake among study participants limit the generalizability of the findings. On the other hand, among the observational studies, the challenges of accurately assessing NNS intake, the many potential confounders, and the difficulty in determining directionality (e.g., possible reverse causality) represent important limitations. Nonetheless, the strength of intervention trials in establishing causality and the opportunity in observational studies to address long-term changes in health outcomes suggest that both approaches will continue to be useful and complementary in providing guidance regarding NNS use. Currently, available data provide, at best, modest support for a modest effect on weight with NNS use, with many important caveats to the available published findings and ample opportunity for future studies to build on past studies to more effectively address this issue.\n\n## Cardiovascular disease risk factors, cardiometabolic syndrome, and coronary heart disease\n\nSeveral prospective studies have been conducted to examine associations of NNS soft drink (i.e., diet soft drink) intake with chronic disease, including coronary heart disease (CHD), chronic kidney disease, and cardiometabolic syndrome and its individual components. In the NHS, consuming \u22652 servings of diet soft drinks per day was associated with an increased risk of CHD and chronic kidney disease in comparison with consuming \\<1 serving of diet soft drinks per month over 11 to 12 years of follow-up (45,46). Middle-aged men and women consuming <u>\\><\/u>1 servings of diet soft drinks per day had \\>30% higher risk of developing metabolic syndrome over 9 years of follow-up (47). Similar results were also observed for the components of metabolic syndrome (47). In the Northern Manhattan Study of \\>2,500 multiethnic adults with 10 years of follow-up and a cardiovascular event incidence of \u223c2%, daily diet soft drink intake was associated with an increased risk of events; regular soft drink intake was not associated with events. However, the small numbers of events, the unusually small numbers of daily consumers of diet soft drinks in this population, and the plausibility of reverse causality led the authors to caution against possible misinterpretation of the findings (48). In contrast, a recent report from the HPFS examining CHD incidence over 22 years in almost 43,000 men with 3,683 CHD events, observed a positive association between sugar-sweetened beverage intake and CHD risk, but no association with ASB (49). The authors specifically noted in their discussion that ASB consumption was probably, at least in part, attributable to response to the diagnosis of a chronic condition. Finally, in a recent study of the joint associations of consuming a prudent (healthy) or Western diet pattern with or without diet beverage consumption among young adults with \\>20 years of follow-up (CARDIA \\[Coronary Artery Risk Development In Young Adults\\] study), consumers of both a prudent (healthy) diet pattern and diet beverages had a lower risk of having elevated glucose or low high-density lipoprotein-cholesterol, but not the metabolic syndrome, than nonconsumers following a prudent diet pattern or consumers and nonconsumers following a Western diet pattern (50). In general, the literature in this area is sparse, limited to prospective observational studies, and examines NNS intake from beverages, but not foods. In addition, these observational prospective studies can only identify associations, not causal determinants, and there is often a strong plausibility of reverse causality for some of the significant associations observed.\n\n## Diabetes mellitus and glycemic response\n\nA recent publication of 20-year follow-up data from healthy men in the HPFS addressed the relationship between intake of sugar-sweetened beverages and ASB and the development of type 2 diabetes mellitus (13). For sugar-sweetened beverages, the association with developing diabetes mellitus remained significant after adjusting for potential confounders. ASB intake was significantly associated with developing type 2 diabetes mellitus in an age-adjusted analysis. However, the association was no longer significant in subsequent multivariate-adjusted analysis; the association between ASB and diabetes mellitus was largely explained by health status\/family history related to diabetes mellitus risk, preenrollment weight change, dieting, and BMI. These findings suggest that the men who consumed ABS may have done so to address a weight gain and health conditions associated with increased risk of developing diabetes mellitus.\n\nWith regard to NNS and glycemic response, 4 randomized trials that varied from 1 to 16 weeks in duration found no significant difference between the effects of NNS and various comparisons (sucrose, starch, or placebo) on standard measures of glycemic response (i.e., plasma glucose and insulin, HbA~1c~, C-peptide) and, in general, did not detect clinically relevant effects (51\u201354). One study that involved contrasting treatment arms assigned participants to either *1*) a diet consistent with American Diabetes Association guidelines (control arm) or *2*) a lower calorie experimental diet containing sucralose. The result was greater reductions in HbA~1c~, weight, and BMI for the experimental diet group (55). The experimental diet, however, differed from the control diet in fat replacer and fructose content, and sucralose content, as well, and therefore it is not possible to determine the separate contribution of the sucralose content to the study effects. The submission to the U.S. Food and Drug Administration to designate stevia extract, rebaudioside A, also known as rebiana, with Generally Recognized As Safe status noted the lack of effects on blood glucose. Information about a lack of blood glucose effects was presented to address claims that some stevia-derived compounds had a therapeutic blood glucose\u2013lowering benefit (56). Potential effects of beverages or foods containing NNS on glycemic response are likely attributable to the replacement of carbohydrate by NNS rather than any direct effect. The American Dietetic Association (renamed the Academy of Nutrition and Dietetics in 2012) concluded in a 2010 review that, in general, NNS do not affect glycemic response (do not raise postmeal glucose response or have a negligible inherent effect on glycemic response) in people with diabetes mellitus. Although the intrinsic glycemic effects of NNS are similar to other calorie-free\/carbohydrate-free products, some NNS-containing products may contain energy and carbohydrate from other sources that need to be considered (57).\n\n# Needs for Further Research\n\nBecause of the paucity of well-designed human studies that address the specific, practical, public health issues, as presented above, further research is warranted in several areas.\n\n## NNS and energy compensation\n\nThe most obvious potential benefit of NNS is the substitution of energy from added sugars, thereby facilitating both energy and carbohydrate intake reductions (with potential glycemic control benefits). However, what would appear to be a potential benefit is complicated by compensation; that is, the extent to which an immediate energy and carbohydrate reduction through NNS use is followed by a subsequent increase in energy intake (regardless of macronutrient type) beyond what would have been consumed in the absence of the use of NNS. Existing data suggest that there is partial compensation with differences in intake after consuming food and beverages containing NNS. Energy intake compensation appears to be greater after consuming foods containing NNS than that for beverages containing NNS. Future work should test and compare different food and beverage vehicles of the NNS, and compare the different NNS themselves and NNS combinations as well, in an effort to maximize potential impact.\n\n## NNS mechanisms of action and pathophysiology\n\nResearch in the area of energy compensation would be facilitated and complemented by continued research into the mechanisms of potential NNS effects. This would include, but not be limited to, increasing the body of knowledge of oral, gut, and neural receptors, and hormonal responses that contribute to alterations in food preferences, appetite regulation, and satiety that are elicited by NNS. This research should inform the selection of foods, beverages, specific NNS (or combinations), and perhaps specific population subsets for studies of compensation effects.\n\n## Randomized controlled trials using NNS to impact body weight or glycemic control\n\nWell-designed and controlled designed clinical trials are needed to determine the long-term effect of NNS on body weight regulation and glycemic control at different points in the life cycle. Weight control studies (e.g., weight loss) should be minimally 1 year in duration or longer, whereas glycemic control studies could be shorter in duration. Studies of both end points would benefit from better informed selections of the types, amounts, combinations, and vehicles of NNS delivery, as described in the previous 2 areas of suggested further research.\n\n## Assessment of NNS intake\n\nImmediate needs include updating of current food databases with accurate data for the NNS content of foods and revision of instruments used to estimate food intake to accurately capture NNS content.\n\n# Conclusions and Recommendations\n\nAt this time, there are insufficient data to determine conclusively whether the use of NNS to displace caloric sweeteners in beverages and foods reduces added sugars or carbohydrate intakes, or benefits appetite, energy balance, body weight, or cardiometabolic risk factors. Limiting added sugars is an important strategy for supporting optimal nutrition and healthy weights, as concluded in the 2009 American Heart Association scientific statement \"Dietary Sugars Intake and Cardiovascular Health\" (1). Monitoring carbohydrate intake, which includes limiting added sugars, is also a key strategy to achieve glycemic control as published in the American Diabetes Association clinical practice recommendations (58). There are some data to suggest that NNS may be used in a structured diet to replace sources of added sugars and that this substitution may result in modest energy intake reductions and weight loss. Successful reduction in energy intake requires that there is incomplete compensation of energy reduction from the use of NNS-containing beverages and\/or foods. The impact of incorporating NNS and NNS-containing beverages and foods on overall diet quality should be included in assessing the overall balance of benefits and risks. Apparent from the available literature is the paucity of data from well-designed human trials exploring the potential role of NNS in achieving and maintaining a healthy body weight and minimizing cardiometabolic risk factors.\n\nThe evidence reviewed suggests that when used judiciously, NNS could facilitate reductions in added sugars intake, thereby resulting in decreased total energy and weight loss\/weight control, and promoting beneficial effects on related metabolic parameters. However, these potential benefits will not be fully realized if there is a compensatory increase in energy intake from other sources.\n\n# Disclosures\n\nWriting group disclosures\n\n![](1798tbl3)\n\nReviewer disclosures\n\n![](1798tbl4)\n\n## Acknowledgments\n\nFor potential conflicts of interest, please see <span class=\"smallcaps\">DISCLOSURES<\/span>.\n\nThe authors thank Xia Zhou, MS, for programming, data presented in <a href=\"#F2\" data-ref-type=\"fig\">Figs. 2<\/a> and <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>, and working with Dr. Steffen.\n\n# References\n\n[^1]: The American Heart Association and the American Diabetes Association make every effort to avoid any actual or potential conflicts of interest that may arise as a result of an outside relationship or a personal, professional, or business interest of a member of the writing panel. Specifically, all members of the writing group are required to complete and submit a Disclosure Questionnaire showing all such relationships that might be perceived as real or potential conflicts of interest.\n\n[^2]: This document was approved by the American Heart Association Science Advisory and Coordinating Committee on April 18, 2012, and by the American Diabetes Association Executive Committee on April 19, 2012.\n\n[^3]: Correspondence about this article can be directed to [firstname.lastname@example.com](firstname.lastname@example.com).\n\n[^4]: This article has been copublished in *Circulation*.\n\n[^5]: \u00a9 2012 by the American Heart Association, Inc., and the American Diabetes Association.","meta":{"dup_signals":{"dup_doc_count":141,"dup_dump_count":73,"dup_details":{"curated_sources":2,"2023-23":2,"2022-40":1,"2022-21":2,"2021-31":2,"2021-17":1,"2021-10":2,"2020-45":2,"2020-40":3,"2020-34":1,"2020-29":1,"2020-24":3,"2020-16":3,"2020-05":1,"2019-47":1,"2019-43":3,"2019-35":1,"2019-30":3,"2019-26":1,"2019-22":1,"2019-18":2,"2019-04":2,"2018-51":3,"2018-47":2,"2018-43":1,"2018-39":3,"2018-34":1,"2018-30":2,"2018-22":2,"2018-17":1,"2018-13":2,"2018-09":1,"2018-05":2,"2017-51":1,"2017-47":2,"2017-43":2,"2017-39":2,"2017-34":1,"2017-30":2,"2017-26":2,"2017-22":2,"2017-17":2,"2017-09":2,"2017-04":1,"2016-50":1,"2016-44":4,"2016-40":2,"2016-36":2,"2016-30":2,"2016-26":1,"2016-22":2,"2016-18":2,"2016-07":2,"2015-48":2,"2015-35":1,"2015-32":2,"2015-27":1,"2015-22":2,"2015-14":1,"2014-52":2,"2014-49":4,"2014-42":5,"2014-41":1,"2014-35":4,"2014-23":2,"2014-15":2,"2023-50":1,"2017-13":5,"2015-18":2,"2015-11":1,"2015-06":2,"2014-10":2,"2013-48":1,"2024-18":1}},"file":"PMC3402256"},"subset":"pubmed_central"}
{"text":"author: Philip E Bourne; Leo M Chalupa\\* To whom correspondence should be addressed. E-mail: <firstname.lastname@example.com>[^1]\ndate: 2006-02\nreferences:\ntitle: Ten Simple Rules for Getting Grants\n\nThis piece follows an earlier Editorial, \"Ten Simple Rules for Getting Published\" \\[1\\], which has generated significant interest, is well read, and continues to generate a variety of positive comments. That Editorial was aimed at students in the early stages of a life of scientific paper writing. This interest has prompted us to try to help scientists in making the next academic career step\u2014becoming a young principal investigator. Leo Chalupa has joined us in putting together ten simple rules for getting grants, based on our many collective years of writing both successful and unsuccessful grants. While our grant writing efforts have been aimed mainly at United States government funding agencies, we believe the rules presented here are generic, transcending funding institutions and national boundaries.\n\nAt the present time, US funding is frequently below 10% for a given grant program. Today, more than ever, we need all the help we can get in writing successful grant proposals. We hope you find these rules useful in reaching your research career goals.\n\n# Rule 1: Be Novel, but Not Too Novel\n\nGood science begins with new and fresh ideas. The grant writing process should be a pleasure (no, we are not kidding), for it allows you to articulate those ideas to peers who have to read your grants but not necessarily your papers. Look at grant writing as an opportunity to have an impact. Feel passionate about what you are writing\u2014if you are not passionate about the work, it is probably not a good grant and is unlikely to get funded. \"Me-too\" science will not get funded when funding levels are low. On the other hand, science that is too speculative will not be supported either, particularly when funds are tight\u2014sad but true.\n\n# Rule 2: Include the Appropriate Background and Preliminary Data as Required\n\nYou need to convince reviewers that the work you propose needs to be done and that you are the best person to do it. Different granting programs require differing amounts of preliminary data. For certain programs, it can be said that the work must be essentially done before the grant is awarded, and that the funds are then used for the next phase of the research program. There is some truth in this. So where appropriate, do provide some tantalizing preliminary result, making sure to tell the reviewers what these results imply with respect to the specific aims of your proposal. In formulating the motivation for your proposal, make sure to cite all relevant work\u2014there is nothing worse than not appropriately citing the work of a reviewer! Finally, convince the reviewer that you have the technical and scientific background to perform the work as proposed.\n\n# Rule 3: Find the Appropriate Funding Mechanism, Read the Associated Request for Applications Very Carefully, and Respond Specifically to the Request\n\nMost funding organizations have specific staff to assist in finding funding opportunities, and most funding agencies have components of their Web sites designed to help investigators find the appropriate programs. Remember, programs want to give away money\u2014the jobs of the program's staff depend on it. The program staff can help you identify the best opportunities. If your grant does not fit a particular program, save your time and energy, and apply elsewhere, where there is a better programmatic fit.\n\n# Rule 4: Follow the Guidelines for Submission Very Carefully and Comply\n\nMany funding bodies will immediately triage grants that do not comply with the guidelines\u2014it saves the program time and money. This extends to all the onerous supporting material\u2014budget justification, bibliographies, etc. Get them right and keep them updated for future applications. Even if it goes to review, an inappropriately formulated application may aggravate the reviewers, and will have a negative impact even if the science is sound. Length and format are the most frequent offenders.\n\n# Rule 5: Obey the Three Cs\u2014Concise, Clear, and Complete\n\nThe grant does not have to fill the allotted page count. Your goal should be to provide a complete reckoning of what is to be done, as briefly as possible. Do not rely on supplements (which may not be allowed) or on Web sites (review may be actively discouraged since it has the potential to compromise anonymity). Specify the scope up-front and make sure it is realistic with respect to the funds requested. A common temptation for inexperienced grant writers is to propose to do too much. Such applications are usually judged as overly ambitious and consequently poorly rated.\n\n# Rule 6: Remember, Reviewers Are People, Too\n\nTypically, reviewers will have a large number of grants to review in a short period. They will easily lose concentration and miss key points of your proposal if these are buried in an overly lengthy or difficult-to-read document. Also, more than likely, not all the reviewers will be experts in your discipline. It is a skill to capture the interest of experts and nonexperts alike. Develop that skill. Unlike a paper, a grant provides more opportunity to apply literary skills. Historical perspectives, human interest, and humor can all be used judiciously in grants to good effect. Use formatting tricks (without disobeying rule 4), for example, underlining, bolding, etc., and restate your key points as appropriate. Each section can start with a summary of the key points.\n\n# Rule 7: Timing and Internal Review Are Important\n\nGive yourself the appropriate lead time. We all have different approaches to deadlines. Ideally, you should complete a draft, leave sufficient time to get feedback from colleagues, and then look at the grant again yourself with a fresh eye. Having a spectrum of scientific colleagues who are similar to the likely reviewer pool critique your grant is very valuable.\n\n# Rule 8: Know Your Grant Administrator at the Institution Funding Your Grant\n\nAt the end of the day, this person is your best advocate. How well you understand each other can make a difference. Many grant administrators have some measure (limited to complete) discretionary control over what they fund. The more they know and understand you and your work, the better your chances of success. Do not rely just on E-mail to get to know the grant administrator. Do not be intimidated. Talk to them on the telephone and at meetings where possible\u2014they want to help.\n\n# Rule 9: Become a Grant Reviewer Early in Your Career\n\nBeing on review panels will help you write better grants. Understanding why grants get triaged before complete review, how a panel reacts to a grant, what the discretionary role of program officers is, and what the role of oversight councils is provide valuable lessons for writing successful grants of your own and for giving others advice about this process.\n\n# Rule 10: Accept Rejection and Deal with It Appropriately\n\nRejection is inevitable, even for very good grants when funding levels are low. Learn to live with rejection and to respond appropriately. Do not be defensive; address each criticism head on and respond with facts and not emotional arguments. When resubmission is necessary, make it very clear to the reviewer that you understand what was wrong the first time. Indicate precisely how you have fixed the problems. In the resubmitted application, never argue with the validity of the prior review. If the grant was close to being funded the first time around, remind the reviewers of that fact by including the previous score if appropriate, and make it crystal clear why this version is much improved.\n\nThere are no previously unrevealed secrets to grant writing presented here. Rather, it is a concise picture intended to help our early career readers take the next step. If you feel like you need more detail, take a look at Kraicer's article \\[2\\]. Good luck on getting those grants.\n\n# References\n\n[^1]: Philip E. Bourne is a professor in the Department of Pharmacology, University of California San Diego, La Jolla, California, United States of America, and is Editor-in-Chief of *PLoS Computational Biology*. Leo M. Chalupa is a professor and chair in the Section of Neurobiology, Physiology, and Behavior, University of California Davis, Davis, California, United States of America.","meta":{"dup_signals":{"dup_doc_count":109,"dup_dump_count":59,"dup_details":{"curated_sources":2,"2023-23":3,"2023-06":1,"2022-33":1,"2022-27":1,"2021-39":1,"2021-10":1,"2020-40":1,"2020-10":2,"2019-26":2,"2019-22":1,"2019-13":1,"2019-09":2,"2018-47":2,"2018-43":1,"2018-34":3,"2018-26":1,"2018-22":1,"2018-09":1,"2018-05":2,"2017-39":2,"2017-34":1,"2017-30":2,"2017-26":1,"2017-22":1,"2017-17":2,"2017-09":2,"2017-04":2,"2016-50":2,"2016-44":1,"2016-40":1,"2016-36":2,"2016-30":3,"2016-26":2,"2016-22":1,"2016-18":2,"2016-07":2,"2015-48":2,"2015-40":3,"2015-35":2,"2015-32":3,"2015-27":1,"2015-22":2,"2015-14":2,"2014-52":3,"2014-49":1,"2014-42":3,"2014-41":4,"2014-35":1,"2014-23":5,"2014-15":3,"2023-50":1,"2024-22":2,"2017-13":1,"2015-18":3,"2015-11":2,"2015-06":2,"2014-10":2,"2013-48":1,"2013-20":1}},"file":"PMC1378105"},"subset":"pubmed_central"}
{"text":"abstract: A combination of mathematical modeling and direct observation of the swimming behavior of dolphin mother-calf pairs has shown how the calf can gain much of the energy required for swimming if it is positioned correctly relative to the mother, a situation that may be disrupted during the chases that result from tuna-fishing practices.\nauthor: Pete Moore\ndate: 2004\nreferences:\ntitle: Examining dolphin hydrodynamics provides clues to calf-loss during tuna fishing\n\nAnyone who has bought a can of 'dolphin-friendly' tuna knows that dolphins can be unintended victims of the tuna-fishing industry. Now, a mathematical model shows that dolphin calves can gain up to 90% of the energy required for swimming if they position themselves correctly relative to their mothers, but that the calves are in danger of being lost if this positioning breaks down \\[1\\]. As observations of dolphins in the wild support the model (see <a href=\"#F2\" data-ref-type=\"fig\">'The bottom line'<\/a> box for a summary of the findings), the new work starts to give a possible explanation why dolphin numbers remain at unnaturally low levels despite changes in the tuna-fishing industry aimed at protecting dolphin populations.\n\n# A brief history of tuna and dolphins\n\nDuring the 1970s an estimated 350,000 dolphins were killed each year as an unintended but inevitable **by-catch** of the tuna fishing in the eastern tropical Pacific Ocean (see the <a href=\"#F3\" data-ref-type=\"fig\">'Background'<\/a> box). This occurred because between 1959 and 1963 the tuna-fishing fleet switched from pole and line fishing to a technique using vast **'purse seine'** nets. Dolphins are caught because fishermen have known for a long time that tuna gather beneath schools of dolphins and very often seabirds congregate overhead - if you want to catch tuna, look for dolphins.\n\nPurse-seine fisheries involve a fast moving boat and a helicopter that search for the telltale signs of seabirds and dolphins. On arriving near a school of dolphins the main boat launches four to six speedboats that operate like sheep dogs, chasing the dolphins so that they are kept together while the main boat encircles them with a one-mile long, 400-foot deep curtain of net, the top edge of which is held on the surface by floats (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>). The chase tends to last between 15 and 30 minutes, but occasionally extends to up to an hour. Once the circle is complete, the fishermen pull a rope that draws the bottom of the net together. This captures the tuna, but also encloses the dolphins. Without assistance, the dolphins can get caught up in the net and, unable to reach the surface, drown.\n\nSince the 1980s various changes in fishing procedures have reduced dolphin mortality. Before pulling the net in, the main boat turns and, because of the way it is constructed, the circular net forms a finger-like shoot opposite the large boat. The speedboats head for this part of the net and hold it open so that the dolphins can swim out. Given that dolphins experience this procedure fairly frequently, they appear to learn what to do. Their escape is helped by the fact that the net in this area is made of a very fine mesh so that they don't get caught, and divers swim inside the net actively pulling some of the stragglers out to the open ocean.\n\nWhile the effort given to helping dolphins escape has cut deaths significantly, conservationists have been puzzled to note that dolphin numbers are nevertheless failing to recover. One possible clue to the problem came when researchers examining data collected by observers placed with the tuna fleet found that more lactating mothers were killed within the net than nursing calves. Elizabeth Edwards, a marine biologist working for the National Marine Fisheries Service in San Diego, USA, notes that \"70-80% of lactating females killed were not killed with a calf, and the findings were consistent over several years\". Edwards and her colleagues started looking to see whether calves could become separated from their mothers during fishing operations \\[2,3\\]. This would not be recorded in mortality statistics, but could radically influence the recovery of the dolphin population. \"Dolphins are very precocious animals,\" says Frank Fish of West Chester University, in West Chester PA. \"We always marvel at wildebeest and other ungulates that start moving with the herd within 20 minutes of being born, but dolphins are moving constantly, so the neonate doesn't even have the luxury of 20 minutes.\"\n\nMarine biologists studying dolphin behavior frequently see mothers and calves swimming closely together at speeds of up to 2.4 m\/sec, with young calves making little if any obvious swimming motions. The assumption is that the calf is **drafting** in its mother's **slipstream**, just as cyclists save energy by packing together during a race. The principle is widespread in biology. \"We are always amazed at how smart animals are in their ability to conserve energy - such as ducklings' use of the flow pattern in the water that the mother produces as she swims along, so that they can reduce the amount of energy they need to expend to maintain that speed,\" says Fish. But in the case of dolphins there has never been any hard science aimed at seeing if this is a lovely myth or a physical reality.\n\nEnter mathematician and aerodynamicist Daniel Weihs, who works at the Technion, Israel Institute of Technology, Haifa. He was involved in research some 25 years ago that aimed to find the speed dolphins needed to reach before they started jumping \\[4\\], and now he came back to the topic, looking for mathematical evidence of forces between mothers and calves (see the <a href=\"#F4\" data-ref-type=\"fig\">'Behind the scenes'<\/a> box for more of the rationale of the work).\n\n# Reduce complexity\n\nThe first task in making a mathematical model of a biological situation is to identify the dominant effects and simplify the issue. Once calculations are made in this simplified world it is relatively easy to add back the complications. Weihs started by assuming that dolphins are ellipsoidal in shape and that their bodies are six times as long as they are wide. Using a regular symmetrical object makes the equations much less complex, and previous studies had indicated that dolphin bodies were closely represented by an ellipsoid. On top of this, people working on torpedoes and other underwater weaponry had already developed equations that predict ellipsoid behavior in water.\n\nNext, he assumed that the water is inviscid - that is, it has no viscosity. This enabled him to employ d'Alembert's paradox. Named after eighteenth century French mathematician and physicist Jean le Rond d'Alembert, this paradox states that objects surrounded by an inviscid, incompressible liquid experience no drag as the liquid moves past. In doing this he had placed his theoretical dolphins into a simple Newtonian world where objects continue to move until influenced by some external force. \"The main point here is that any single body under the conditions we are talking about produces no forces at all if it is moving at constant speed,\" explains Weihs. The question then is, what forces occur in this environment when you have two objects moving closely together?\n\nFinally, he introduced two more assumptions: first, that the body shapes did not change during swimming; and second, that the dolphins were swimming at sufficient depth that they were not creating any surface waves. The results were clear. As the mother's body moves through the water, it pushes water sideways and outwards, and after she has moved forwards the water flows back in to fill the vacated space. \"This means that if a second body is placed close enough to the first and within the area of forward motion of water, the relative velocity it feels will be less than its absolute velocity,\" notes Weihs.\n\nWeihs' initial model showed that there were areas immediately in front of the mother dolphin, as well as to the side and behind, where a baby dolphin could swim and gain up to 90% of the force needed to move through the water from its mother. What's more, there were areas where Bernoulli attraction would actively pull a calf sideways, towards its mother.\n\n# Add in some reality\n\nLife, however, is not that simple. Any baby dolphin that tried to stay immediately ahead of its mother would have great difficulty. \"The situation,\" explains Weihs, \"would be akin to pushing a string and expecting it to stay straight - it would be too unstable.\" Again, being immediately behind the mother wouldn't work, because a real dolphin moves by forcing a jet of water backwards, by flexing its body and using its tail flukes. While the initial calculations might suggest that immediately behind the mother was a great place, common sense shows that it wouldn't. At the same time, the flexing of the mother's body means that any zone directly above or below her would be too unstable to be helpful to a calf. This leaves the two zones on either side of the mother and about two thirds of the way back as the two most stable and energetically favorable places for a youngster to hitch a lift.\n\nAway from the mathematician's calculations, real dolphins also experience drag from the viscosity of the water. Adding this back makes the energy saving even more extreme. Any drag is proportional to the square of velocity, so positioning yourself in an area of forward-moving water allows you to experience much less drag. The energy required to achieve this velocity also increases as the cube of velocity. Consequently, any 10% saving in apparent velocity leads to a nearly 30% saving in energy. \"The predicted force reduction is so substantial that it must play a major role in determining the calf's swimming position in practice,\" says Timothy Pedley, G. I. Taylor Professor of Fluid Mechanics in the Department of Applied Mathematics and Theoretical Physics at Cambridge, UK.\n\nDolphins do also rise to the surface and create surface waves. The energy used to create these waves constitutes another drag on the animals. Marine engineers calculate this as the Froude number, a non-dimensional coefficient defined as the ratio of inertial force to gravitational force. Applying this to the dolphins' situation reduces the effect of the previously mentioned gains. Consequently, swimming near to the surface presents added problems to a calf that is trying to keep up with its mother. The point of interest for the tuna-fishing scenario is that in chase situations dolphins need to be nearer to the surface, as they need to breathe more frequently.\n\nHaving determined the hydrodynamic basis for drafting, Weihs then applied his calculations to photographs taken from helicopters of mother-calf dolphin pairs swimming together in the wild. His conclusion is that in this real-life situation the calves gain between 30% and 60% of the force required to keep up with their mothers \\[1\\]. \"This is a very nice model and is almost certainly the explanation for the observations of drafting,\" comments Pedley, adding: \"it is not often that one can be so sure of the relevance of a mathematical calculation to biological phenomena.\"\n\n# Energetic advantage\n\n\"Weihs' paper establishes the basis for how drafting works between mothers and calves and indicates that drafting is really important to the mother-calf bond,\" says Edwards, from the National Marine Fisheries Service. What is now open to question is the extent to which drafting operates at the pace created in the tuna-fishing chase, when adult dolphins move by a combination of bursts of speed, leaps out of the water and brief periods of coasting. \"Reviewing the literature shows that in a chase situation the mothers will probably just run away and 'assume' that the babies can just draft along side,\" Edwards comments.\n\nEdwards would also like to refine the model to specifically address the limits to drafting, and compare these limits to the expected conditions during purse-seine tuna-fishing operations. This will help to determine whether fishery-related mother-calf separations are a likely source of unobserved dolphin mortality in the eastern tropical Pacific Ocean.\n\nSo, dolphin researchers believe Weihs' paper to be a good step forward, but as with all good research it generates a new layer of questions. \"Weihs has given us the mechanical energetics of the situation. What we now need is an understanding of how this translates into metabolic demand,\" says Fish. \"The model also needs to be developed to take in all the movements of the dolphin.\" Doing that will move the mathematics closer to the mammal, and may ultimately help to reverse the tuna-fishing-related decline in dolphin numbers.\n\n## Figures and Tables","meta":{"dup_signals":{"dup_doc_count":212,"dup_dump_count":48,"dup_details":{"curated_sources":2,"2020-10":1,"2019-47":1,"2019-39":1,"2019-26":1,"2019-13":1,"2019-09":1,"2018-47":1,"2018-43":1,"2018-39":1,"2018-30":1,"2018-05":1,"2017-39":1,"2017-34":3,"2017-26":1,"2017-22":3,"2017-17":1,"2017-09":17,"2017-04":1,"2016-50":2,"2016-44":3,"2016-40":2,"2016-36":15,"2016-30":15,"2016-22":1,"2016-18":1,"2016-07":15,"2015-48":5,"2015-40":4,"2015-35":6,"2015-32":5,"2015-27":5,"2015-22":4,"2015-14":4,"2014-52":5,"2014-49":6,"2014-42":16,"2014-41":6,"2014-35":8,"2014-23":7,"2014-15":8,"2020-40":1,"2015-18":5,"2015-11":4,"2015-06":5,"2014-10":5,"2013-48":4,"2013-20":4,"2024-18":1}},"file":"PMC416559"},"subset":"pubmed_central"}
{"text":"author: Gerard D Wright\ndate: 2010\ninstitute: 1Michael G DeGroote Institute for Infectious Disease Research and the Department of Biochemistry and Biomedical Sciences, McMaster University, 1200 Main St W., Hamilton, Canada L8N 3Z5\ntitle: Q&A: Antibiotic resistance: where does it come from and what can we do about it?\n\n# Is antibiotic resistance inevitable?\n\nYes. Historically, the discovery of the sulfa drugs in the 1930 s and the subsequent development of penicillin during World War II ushered in a new era in the treatment of infectious diseases. Infections that were common causes of death and disease in the pre-antibiotic era - rheumatic fever, syphilis, cellulitis and bacterial pneumonia - became treatable, and over the next 20 years most of the classes of antibiotics that find clinical use today were discovered and changed medicine in a profound way. The availability of antibiotics enabled revolutionary medical interventions such as cancer chemotherapy, organ transplants and essentially all major invasive surgeries from joint replacements to coronary bypass. Antibiotics, though, are unique among drugs in that their use precipitates their obsolescence. Paradoxically, these cures select for organisms that can evade them, fueling an arms race between microbes, clinicians and drug discoverers.\n\n# How do successful antibiotics work and what is the basis of resistance to them?\n\nAntibiotics target essential bacterial physiology and biochemistry, causing microbial cell death or the cessation of growth. There are five major antibiotic targets: the bacterial cell wall, the cell membrane, protein synthesis, DNA and RNA synthesis, and folic acid (vitamin B9) metabolism (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>). These bacterial targets are different or nonexistent in eukaryotic cells (including those of humans), which means that antibiotics are relatively nontoxic drugs. For example, the \u03b2-lactam antibiotics such as penicillins, cephalosporins and carbapenems block the synthesis of the bacterial cell wall. This structure is absent in higher organisms but is essential for bacterial survival. The bacterial ribosome is the target of the tetracycline, aminoglycoside, macrolide and other antibiotics, and is sufficiently different from the eukaryotic ribosome that cross-inhibition does not occur.\n\nResistance to antibiotics occurs through four general mechanisms: target modification; efflux; immunity and bypass; and enzyme-catalyzed destruction (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>). Target modification can occur through mutation of the targets themselves - for example, the topoisomerases that are the target of the fluoroquinolone antibiotics - or by the production of enzymes that modify antibiotic targets, as, for example, in ribosomal methylation. Vancomycin resistance is a version of target modification where new biosynthetic machinery is engaged to alter cell-wall structure. Efflux occurs through a large family of protein pumps that eject antibiotics from inside the cell. In immunity, antibiotics or their targets are bound by proteins that prevent the antibiotic binding to its target. Arguably, the most specific and evolved mechanism of antibiotic resistance are enzymes that recognize antibiotics and modify them in such a way as to eliminate the functional characteristics that enable them to interact with their targets. For example, \u03b2-lactamases hydrolytically cleave the core \u03b2-lactam ring that is characteristic of the class and essential to antibiotic action.\n\n# Has the problem of antibiotic resistance worsened over time?\n\nResistance to antibiotics was recorded even before the first clinical use of penicillin in the early 1940 s. In the intervening years, resistance to all classes of antibiotics has emerged, and there are no antibiotics for which resistance does not exist. There are two general strategies for resistance. One comprises mechanisms that transfer resistance vertically from a bacterium to its progeny. Examples are mutations in chromosomal genes that give rise to drug-insensitive products, such as the point mutations in the genes encoding DNA gyrase or topoisomerase IV that result in resistance to fluoroquinolone antibiotics such as ciprofloxacin. The second strategy includes the actions of genes that can be transmitted both vertically to progeny and horizontally to other bacteria, even those of different genera. These genes are located on mobile genetic elements such as plasmids, which can carry one or more resistance genes. Many of the \u03b2-lactamase genes that confer resistance to the penicillin, cephalosporin, penem and monobactam antibiotics are located on such elements, as is the glycopeptide-resistance gene cluster *vanHAX*, which provides resistance to vancomycin. The prevalence and mobility of resistance genes in previously sensitive pathogenic bacteria has now reached crisis levels in many cases because new antibiotics are no longer being developed at a rate that can keep pace with microbial evolution.\n\nIn the past two decades we have witnessed:\n\n\u2022 the rise of so-called extended spectrum \u03b2-lactamases (ESBLs), which are mutants of enzymes that previously could only inactivate penicillins but now have gained activity against many cephalosporins;\n\n\u2022 carbapenemases such as KPC and NDM-1 that inactivate all \u03b2-lactam antibiotics;\n\n\u2022 plasmid-mediated (and thus horizontally disseminated) resistance to fluoroquinolone antibiotics;\n\n\u2022 the spread of virulent MRSA (methicillin-resistant *Staphylococcus aureus*) in the community;\n\n\u2022 the rise of multi-drug resistant *Neisseria gonorrhoea*;\n\n\u2022 the emergence and global dissemination of multi-drug resistant *Acinetobacter baumannii*, *Pseudomonas aeruginosa*, *Klebsiella pneumoniae* and Enterobacteriaceae;\n\n\u2022 the spread of extensively drug resistant *Mycobacterium tuberculosis*;\n\n\u2022 the development of resistance to the two newest antibiotics to be approved for clinical use - daptomycin and linezolid.\n\nResistance is relentless and unavoidable as long as we use antibiotics.\n\n# Where does resistance come from?\n\nAntibiotic resistance is the evolutionary response to the strong selective pressure that results from exposure to these compounds. The horizontal dissemination of resistance genes into bacterial species and genera that are not themselves intrinsically resistant, as well as the maintenance of resistance mutations vertically through populations is likely to be the result of contemporary use of these drugs in the clinic and on the farm. Support for this hypothesis is the infrequency of antibiotic resistance in collections of pathogenic bacteria that pre-date the antibiotic era.\n\nNevertheless, antibiotic resistance is a natural phenomenon. It has been recognized for decades that the resistance mechanisms that have emerged in the clinic parallel those that are intrinsic to the bacteria that produce antibiotics. Recent studies of non-pathogenic soil bacteria have revealed that the majority of environmental bacteria tested are multi-drug resistant. This reflects the fact that these microbes live and have evolved in an environment where small bioactive molecules, some toxic, some benign, are plentiful and diverse. Bacteria have simply evolved to interact with them and control their biological effects. Pathogens, on the other hand, are often more virulent forms of our commensal bacteria and simply have not been exposed to the diversity and types of small molecules found in the environment; as a result, they have not required the gamut of resistance genes found in some environmental bacteria.\n\nFurthermore, the genes and proteins responsible for resistance in environmental bacteria are homologous to those found circulating in pathogens, strongly suggesting contemporary horizontal gene transfer. Opportunistic pathogens with environmental reservoirs - for example, *P. aeruginosa* and *A. baumannii* - are highly drug resistant and have a remarkable capacity to acquire new resistance genes. The environment is therefore a large reservoir of potential resistance genes: the environmental 'resistome'.\n\nGiven the vast numbers of bacteria on the planet and the massive selection pressure provided by antibiotics, the movement of antibiotic-resistance elements from benign, but resistant, microbes into previously susceptible pathogens is simply a matter of time and opportunity.\n\n# Can anything be done to slow down the emergence of resistance?\n\nAntibiotics themselves are the source of the evolutionary pressure that eventually renders them obsolete. Limiting exposure of microbes to antibiotics therefore makes good sense to reduce the opportunity for the selection and dissemination of resistance. The inappropriate use of antibiotics by clinicians and the agricultural community needs to be curtailed. Over the past several years, the medical community in particular has made concrete efforts to curb the improper use of antibiotics. The European Union has taken the lead in limiting the non-therapeutic use of antibiotics in food animals. Robust surveillance networks that span the clinic and the farm need to be supported in order to monitor the impact of resistance and the emergence of new threats in real time. In North America, efforts such as the Strategies to Address Antimicrobial Resistance Act seek to diminish antibiotic use in agriculture and improve surveillance. Furthermore, there have been several successful campaigns to educate the public on the importance of antibiotics and the proper use of these drugs. While none of these efforts is perfect, there is much to be celebrated and encouraged.\n\nThese measures all serve to reduce antibiotic use and, as a result, delay the emergence of resistance. Furthermore, by decreasing selection pressure, the opportunity for the rise of particularly clinically challenging or virulent organisms should be lessened. All strategies that reduce the incorrect use of antibiotics are welcome, but in the end new drugs will always be needed because of the inevitability of resistance.\n\nUnfortunately, in the developing world, access to antibiotics is frequently not regulated and their use in agriculture is often rampant. These facts make antibiotic stewardship especially challenging. In an era of rapid intercontinental travel, pathogens are no longer geographically contained and can move from country to country with ease. The recent examples of transcontinental spread of the severe acute respiratory syndrome (SARS) virus from Guangdong province in China to Hong Kong and then Canada in 2003, and the NDM-1 carbapenemase, which inactivates all \u03b2-lactam antibiotics and appears to have originated in the Indian subcontinent but is now found in North America, the UK and Europe, make the point.\n\n# What about new antibiotics?\n\nThe growing problem of resistance fuels a continuous need for new antibiotic drugs. The enterobacteria that produce carbapenemase are just one example of antibiotic-resistant enterobacteria. Other Gram-negative pathogens resistant to virtually all antibiotics include multi-drug resistant *A. baumannii* and *P. aeruginosa*. The expanding problem of MRSA, and the global challenge of extensively drug-resistant *M. tuberculosis* (also called extreme drug-resistant *M. tuberculosis*), require new therapies.\n\nThere are some promising new candidates on the horizon, especially for the treatment of infections caused by Gram-positive pathogens such as MRSA and enterococci. As already mentioned, two new drugs active against this microbial spectrum - daptomycin and linezolid - have been introduced in the past decade. Tigecycline, a third-generation semi-synthetic tetracycline antibiotic approved in 2005, also has activity against MRSA. The semi-synthetic glycopeptide antibiotic telavancin recently received approval in the United States and the fifth-generation cephalosporin ceftobiprole is available in some European countries and Canada. However, there are few candidates in late-stage clinical trials suitable for the problem of Gram-negative pathogens. Here, often the choice of last resort is colistin, an antibiotic discovered more than 50 years ago and seldom used in the past because of adverse affects, including kidney toxicity; however, it is now increasingly used.\n\n# Why are there so few new drugs?\n\nThere are a number of reasons, some economic, for the paucity of new antibiotics. They include challenging and shifting processes of government regulatory approval that add to the risk for the pharmaceutical industry. Furthermore, considerations of return on investment favor drugs for chronic diseases, which are taken by patients over long periods of time, often decades. In contrast, antibiotics cure disease and are taken for short periods of time.\n\nOther reasons for the decline in antibiotic discovery and development are scientific. The first wave of antibiotics discovered five decades ago have been termed the 'low hanging fruit'. Despite the discovery of numerous compounds with antibiotic properties in the years since, few have had the requisite properties to become drugs. Most antibiotics are natural products or their derivatives that have been isolated from soil bacteria. Some researchers have suggested that this source might now be exhausted.\n\nFurthermore, the promise of the genomic era and the reality of hundreds of available bacterial genomes have so far failed to deliver the hoped-for new molecular targets for antibiotics. Other new technologies, such as high-throughput screening of libraries of synthetic molecules, have not resulted in new drugs, although this may reflect a poor choice of chemical classes in the screens, emphasizing molecules more active in human biology than as antibiotics. Test compounds were often skewed in favor of small lipophilic molecules with physical properties meeting the criteria of Lipinski's Rule of 5. However, though helpful in assessing the prospect of a compound to be an orally active drug for human disease, this strategy has been shown to fail when searching for antibiotics.\n\n# So what would be suitable chemical matter for leads?\n\nWell, natural chemicals have significant advantages. Although the first antibiotics introduced into the clinic were the synthetic sulfonamides, the majority of antibiotics in current clinical use are bacterially produced natural products or their derivatives; only a few are completely synthetic in origin. The reasons for this in part reflect the history of antibiotic discovery post-penicillin, and the relative ease of discovery of suitable molecules through screening the products of soil microbes compared with libraries of synthetic compounds. Many of these 'natural' antibiotics have desirable drug-like qualities, such as good bioavailability, the ability to cross the cell membrane (and outer membrane in the case of compounds with Gram-negative activity) and the ability to evade efflux systems, and chemical structures that favor binding to vital cellular targets, supporting the idea that natural products encompass privileged structures in antibiotic drug discovery. However, the increasing difficulty of identifying new chemical compounds with equally suitable drug-like characteristics from natural sources has caused natural-product-based screening programs to fall out of favor in many pharmaceutical firms over the past few decades.\n\nInstead, the ability of parallel synthesis methods to generate hundreds of thousands of synthetic molecules suitable for modern high-throughput screening has shifted the focus in favor of synthetic molecules in commercial antibacterial drug discovery. The advantages of synthetic compounds are not insignificant: pure lead molecules can easily be produced in quantity and quality suitable for clinical trials, and are relatively easily modified to improve target affinity. However, after two to three decades of emphasis on such molecules and millions of dollars spent on high-throughput *in vitro* and cell-based screens, no new synthetic antibiotics have emerged. Linezolid, the one synthetic antibiotic to be brought to market in the past decade, was discovered using traditional medicinal chemistry in a research program with a plant-disease focus in the early 1980 s.\n\n# So does that mean natural products are best after all?\n\nThey do have great advantages, although a direct comparison of the success and failure of synthetic as against natural product libraries is unfair. Microbial natural products have evolved over millennia to interact with biological molecules, whereas the synthetic chemical libraries used in antibiotic drug-discovery screens were generally developed with a focus on eukaryotic drug-discovery campaigns, as noted earlier. Efforts to develop physical-property rules for antibiotics and to incorporate natural-product-like chemical complexity in libraries of synthetic chemicals will no doubt improve success in identifying new synthetic antibiotic leads.\n\nIronically, at the same time that the pharmaceutical industry was abandoning natural-product libraries, university researchers were making remarkable advances in understanding the molecular details of natural-product biosynthesis by bacteria. Many bacteria, especially the actinomycete group of common environmental bacteria, are prodigious producers of natural products. These are termed secondary metabolites to contrast with molecules of primary metabolism, such as carbohydrates, amino acids and so on. Secondary metabolites range in molecular weight from around 100 daltons (Da) to up to 5,000 Da and they have diverse biological activities, including induction of cell death (antibiotics such as tetracycline, vancomycin and daptomycin, and anticancer agents such as adriamycin), iron sequestration (for example, enterobactin), facilitation of cell-cell communication (\u03b3-butyrolactones), protection from oxidizing agents (phenazines), and a host of others.\n\nThe bacterial natural products that are most important as antibiotics include polyketides, such as the macrolides and tetracyclines, and non-ribosomal peptides - that is, peptides that are not synthesized on ribosomes - which include \u03b2-lactams and glycopeptides such as vancomycin. These are produced in the cell in assembly-line fashion on large dedicated enzyme platforms called, respectively, polyketide synthases and non-ribosomal peptide synthetases. Following assembly the compounds are then 'decorated' by a series of modifying enzymes, such as glycosyltransferases. The end result is a molecule of often complex structure, with multiple chiral centers and functional groups such as sugars, halogens, sulfates, acyl groups and others.\n\nIn general, bacterial genes that encode the production of natural products are clustered together in the genome, greatly facilitating analysis and prediction of biosynthetic pathways and structures. Indeed, several software packages (for example, NP. searcher) have been developed based on rules-based understanding of natural-product biosynthesis. The availability of cheap, rapid genome sequencing means that time-consuming construction and screening of gene libraries for natural-product clusters can now be bypassed. Genome sequencing has also revealed a hitherto unrealized richness in the quantity and variability of natural-product biosynthetic clusters. Sequenced genomes of bacteria of the actinomycetes class reveal 20 to 30 biosynthetic clusters in each organism. Furthermore, natural-product producing bacteria from non-soil environments are being investigated and these have already resulted in new chemical matter, suggesting that there is a fantastic wealth of untapped chemical diversity waiting to be discovered. Perhaps some of this diversity will include new antibiotic chemical scaffolds.\n\nWe are in a remarkably productive time for natural-product research that is serving to reinvigorate interest in this sector. At the same time, the application of synthetic biology approaches to this field could serve to improve issues of yield and expand chemical diversity.\n\n# Are there alternatives to new antibiotics?\n\nYes. First, existing discarded antibiotics can be re-examined. The development of daptomycin is instructive. Daptomycin was discovered by the antibiotic group at Eli Lilly in the 1980 s, but was not fully developed because of toxicity concerns. The antibiotic was obtained by researchers at Cubist in 1997 and by altering the dosing, this group was able to bring the antibiotic to market in 2003, since when it has proved highly successful in treating infections caused by Gram-positive pathogens. Certainly, there are other 'old' antibiotics discovered by the pharmaceutical industry but not developed at the time that could be resurrected as leads for new drugs.\n\nA second option is the combination of antibiotics with each other and with other drugs to improve efficacy. Infectious-disease physicians often combine antibiotics in an effort to achieve synergy, and this well-established practice has resulted in formulated drug combinations, such as co-trimoxazole (trimethoprim and sulfamethoxazole). Combination of antibiotics with non-antibiotics deserves investigation as well. Several natural products have been discovered by Satoshi Omura's group that potentiate the activities of antibiotics such as imipenem in *S. aureus* by unknown mechanisms.\n\nOther antibiotic adjuvants are inhibitors of resistance mechanisms. The tremendous commercial and clinical success of Augmentin (ampicillin together with the \u03b2-lactamase inhibitor clavulanic acid) and other similar combinations speaks to the power of such combinations. Our growing understanding of the mechanisms of resistance should fuel such approaches. Inhibitors of efflux pumps, for example, have been discovered, and though challenging to implement in organisms with multiple redundant systems, are worthy of continued investigation.\n\nFinally, other strategies orthogonal to antibiotics must be on the table. We should never forget vaccines as proven and outstanding protective agents against infectious diseases. Bacterial viruses (bacteriophages) were used extensively to treat bacterial infections in the former Soviet Union and could find new application in the face of outbreaks of multi-drug resistant bacteria, especially in settings such as hospital infections. The use of enhancers of innate immunity, such as cationic antimicrobial peptides, is also an approach worth investigating.\n\n# What is the outlook for new drugs and further resistance?\n\nWe need antibiotics to maintain our current standard of health care. As already stated, resistance is a natural evolutionary phenomenon that cannot be stopped. Through judicious use of current drugs and the development of new ones, the pace of resistance development can be controlled without impairing our ability to control disease. The need for new drugs is, however, acute. Antimicrobial stewardship alone cannot fulfill our requirement for new antibiotics.\n\nWe are in a remarkably exciting time for fundamental research in antibiotics. The rapidity of genome sequencing, the maturing of our knowledge of natural product biosynthesis, a growing understanding of the physical properties of ideal antibiotics, the development of new strategies to develop synthetic compounds with improved antibiotic properties, and the possibilities of synthetic biology combine to suggest that we are entering a highly productive period of antibiotic discovery. The challenges of moving these advances into the clinic fast enough to keep pace with resistance are significant, but with concerted effort between scientists, funders, industry, regulators and clinicians, I believe they can be overcome.\n\n# Where can I go for more information?\n\n## Articles\n\nArias CA, Murray BE: **Antibiotic-resistant bugs in the 21st century - a clinical super-challenge**. *N Engl J Med* 2009, **360:**439-443.\n\nBaquero F, Alvarez-Ortega C, Martinez JL: **Ecology and evolution of antibiotic resistance**. *Env Microbiol Rep* 2009, **1:**469-476.\n\nBoucher HW, Talbot GH, Bradley JS, Edwards JE, Gilbert D, Rice LB, Scheld M, Spellberg B, Bartlett J: **Bad bugs, no drugs: no ESKAPE! An update from the Infectious Diseases Society of America**. *Clin Infect Dis* 2009, **48:**1-12.\n\nFenical W, Jensen PR: **Developing a new resource for drug discovery: marine actinomycete bacteria**. *Nat Chem Biol* 2006, **2:**666-673.\n\nFischbach MA, Walsh CT: **Antibiotics for emerging pathogens**. *Science* 2009, **325:**1089-1093.\n\nKumarasamy KK, Toleman MA, Walsh TR, Bagaria J, Butt F, Balakrishnan R, Chaudhary U, Doumith M, Giske CG, Irfan S, Krishnan P, Kumar AV, Maharjan S, Mushtaq S, Noorie T, Paterson DL, Pearson A, Perry C, Pike R, Rao B, Ray U, Sarma JB, Sharma M, Sheridan E, Thirunarayan MA, Turton J, Upadhyay S, Warner M, Welfare W, Livermore DM, Woodford N: **Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study**. *Lancet Infect Dis* 2010, **10:**597-602.\n\nLivermore DM: **Has the era of untreatable infections arived?** *J Antimicrob Chemother* 2009, **64 Suppl 1:**i29-i36.\n\nShahid M, Sobia F, Singh A, Malik A, Khan HM, Jonas D, Hawkey PM: **Beta-lactams and beta-lactamase-inhibitors in current- or potential-clinical practice: a comprehensive update**. *Crit Rev Microbiol* 2009, **35:**81-108.\n\nWright GD: **The antibiotic resistome: the nexus of chemical and genetic diversity**. *Nat Rev Microbiol* 2007, **5:**175-186.\n\nYamazaki H, Nonaka K, Masuma R, Omura S, Tomoda H: **Xanthoradones, new potentiators of imipenem activity against methicillin-resistant Staphylococcus aureus, produced by *Penicillium radicum* FKI-3765-2: I. Taxonomy, fermentation, isolation and biological properties**. *J Antibiot (Tokyo)* 2009, **62:**431-434.\n\n## Web links\n\n**Infectious Diseases Society of America (IDSA)** <http:\/\/www.idsociety.org\/>\n\n## Acknowledgements\n\nResearch in the author's lab on antibiotic resistance is supported by a Canada Research Chair, the Canadian Institutes of Health Research and the Natural Sciences and Engineering Research Council.","meta":{"dup_signals":{"dup_doc_count":107,"dup_dump_count":65,"dup_details":{"curated_sources":2,"2023-40":1,"2023-14":1,"2022-49":1,"2022-40":1,"2022-21":1,"2021-49":2,"2021-39":1,"2021-31":1,"2021-10":1,"2020-45":1,"2020-34":1,"2020-05":1,"2019-51":1,"2019-47":3,"2019-39":1,"2019-35":1,"2019-30":1,"2019-18":2,"2019-09":1,"2019-04":2,"2018-51":1,"2018-47":2,"2018-43":1,"2018-39":2,"2018-34":2,"2018-30":1,"2018-26":1,"2018-22":1,"2018-17":1,"2018-13":2,"2018-09":1,"2018-05":2,"2017-51":1,"2017-47":1,"2017-43":2,"2017-39":3,"2017-34":2,"2017-30":2,"2017-26":3,"2017-22":4,"2017-17":2,"2017-09":4,"2017-04":3,"2016-50":1,"2016-44":3,"2016-40":3,"2016-36":2,"2016-30":3,"2016-22":1,"2016-18":1,"2015-40":1,"2015-27":1,"2015-22":2,"2014-52":2,"2014-49":1,"2014-42":1,"2014-23":1,"2023-50":1,"2017-13":4,"2015-18":1,"2015-11":1,"2014-10":2,"2013-48":1,"2013-20":2,"2024-22":1}},"file":"PMC2942819"},"subset":"pubmed_central"}
{"text":"author: Margitta Worm\ndate: 2011\ninstitute: 1Department of Dermatology and Allergy, Allergy Center Charit\u00e9, CCM, Charit\u00e9 - Universit\u00e4tsmedizin Berlin, Charit\u00e9platz 1, D-10117 Berlin, Germany\ntitle: Q&A: Food additive intolerance\n\n# Q&A: Food additive intolerance\n\n## 1) What is food additive intolerance and can you tell us what the most common symptoms are?\n\nFood additive intolerance is a non-IgE mediated food hypersensitivity. The hypersensitivity is induced by the food additives via a **direct** mast cell activation. Although the exact pathophysiology is unknown, various clinical signs are characteristic of food additive intolerance. These include cutaneous symtoms like redness of the skin, urticaria and angioedema as well as other organ related symptoms, such as dyspnea, hypotension or dizziness.\n\n## 2) What are the differences between intolerance and allergy?\n\nBoth, IgE-mediated allergy, but also food additive intolerance are mast cell dependent reactions. The release of mast cell mediators like histamine, leukotrienes and others leads to the onset of the above mentioned clinical symptoms, which cannot be distinguished concerning the underlying mechanisms. In the case of an allergic reaction, crosslinking of membrane bound IgE via an allergen is inducing mast cell degranulation. In food additive intolerance a direct action of the additive on the mast cells is proposed, however the exact mechanisms are not known.\n\n## 3) What are the most common food additives that cause intolerance?\n\nThe most common food additives to which patients are intolerant are sulfite, sodium benzoate and food colorings. In addition, histamine is often accused of inducing intolerance reactions, however its exact role as an intolerance reaction as such requires more clarification.\n\n## 4) What is the prevalence of food intolerance?\n\nIt is estimated that 0.1 - 1.5% of the population may suffer from food additive tolerance. So far the data suggest that intolerance reactions occur more frequently in older patients rather than in young children Further, it is known that drug intolerance towards acetylsalicylic acid (ASA) occurs more frequently in asthmatic patients. However, whether this is also true in respect to the prevalence of food intolerance in asthmatics remains to be determined.\n\n## 5) Is there an age dependent increase in allergy risk and what are the reasons for this?\n\nThe risk of developing an allergy to my knowledge does not solely depend on age but rather depends on the allergen and the specific exposure situation of an individual. The lifetime risk for a food allergy probably does decrease rather than increase over time. However, in the case of food additive intolerance, this decrease of the risk over time might not be true. A possible explanation for this might be a change of the gastrointestinal barrier function. In addition the presence of additional cofactors, which can trigger such reactions (intake of drugs like ACE-inhibitors, physical activity, in-take of alcohol), makes the onset of food additive intolerance in later life more likely.\n\n## 6) Are there any co-morbidities that increase the risk of being intolerant to food additives?\n\nAs mentioned above, probably patients with moderate to severe asthma are at a higher risk of being of intolerant to food additives. In addition, it is well known that patients who suffer from mastocytosis, a genetic disease where mast cells occur in increased numbers in the skin and other organs have an increased risk to develop systemic-reactions to food additives\n\n## 7) What are the current diagnostic and management strategies for food additive intolerance?\n\nTo date, the optimal management strategies for food additive intolerance include the avoidance of an increased intake of food additives in general, in particular in large amounts. For example, a meal with ripened cheese, wine and a colored dessert should be avoided. If a patient has skin and gastrointestinal symptoms, a prophylactic intake of antihistamines might be useful. Diagnostic procedures involve an elimination diet (3 - 4 weeks) followed by double blind-placebo-controlled-food challenge (DBPCFC) tests. Only if the double blind-placebo-controlled-food challenge is positive the diagnosis can be proven and dietary recommendations be made. Previous data of patients suffering from chronic urticaria has indicated a change in diet can facilitate gastrointestinal barrier recovery, which enables the patient to include certain food items step by step again over time again.\n\n## 8) Are there any difficulties in the diagnosis of food additive intolerance?\n\nThe major difficulties in the diagnosis of food additive intolerance are that the history of symptoms made by the patients might not be clear. In such cases, a symptom diary might be helpful. It is important to note that *in vivo* tests, such as the skin prick-test, and *in vitro tests* such as determination of specific IgE cannot be used to make the diagnosis. Moreover, other methods such as the cellular activation test (CAST) measuring histamine release and\/or the leukotriene pathway production can not be recommended to confirm the diagnosis. Therefore, research in this area is urgently required. This would help to improve the diagnostic methods of food additive intolerance, identify patients at risk and would support the development of new therapeutic strategies. The lack of knowledge in this field is e.g. related to the fact that food additive intolerance cannot be studied well in vitro as mast cell reactivity is different if studied in vitro versus in vivo.\n\n## 9) What does the future hold for the diagnosis and management of food additive allergies?\n\nI am sure that in the future we will be able to come up with novel diagnostic approaches. These might be based on cellular test systems and\/or a metabolomic analysis from patients' serum. In particular, mediators and their receptors from the leukotriene pathway should be considered in more detail as data from previous studies suggest that genetic polymorphisms of leukotriene receptors may be influential, as well as deviations within the leukotriene profile might be relevant. In terms of medical strategies, the exact role of the gastrointestinal barrier will need more clarification. If methods can be developed to increase the barrier function of the gut these might also offer novel approaches for food intolerant patients because less absorption or enhanced local degradation of the food additive in the gut would decrease the systemic burden.\n\n## 10) Where can I find out more?\n\nSoost S, Leynaert B, Almqvist C, Edenharter G, Zuberbier T, Worm M. Risk factors of adverse reactions to food in German adults. Clin Exp Allergy. 2009 Jul;39(7):1036-44. Epub 2009 Mar 3\n\nRoehr CC, Edenharter G, Reimann S, Ehlers I, Worm M, Zuberbier T, Niggemann B. Food allergy and non-allergic food hypersensitivity in children and adolescents. Clin Exp Allergy. 2004 Oct;34(10):1534-41\n\nZuberbier T, Edenharter G, Worm M, Ehlers I, Reimann S, Hantke T, Roehr CC, Bergmann KE, Niggemann B. Prevalence of adverse reactions to food in Germany - a population study. Allergy. 2004 Mar;59(3):338-45\n\nFiedler EM, Zuberbier T, Worm M. A combination of wheat flour, ethanol and food additives inducing FDEIA. Allergy. 2002 Nov;57(11):1090-1. No abstract available\n\nEhlers I, Hipler UC, Zuberbier T, Worm M. Ethanol as a cause of hypersensitivity reactions to alcoholic beverages. Clin Exp Allergy. 2002 Aug;32(8):1231-5\n\nWorm M, Vieth W, Ehlers I, Sterry W, Zuberbier T. Increased leukotriene production by food additives in patients with atopic dermatitis and proven food intolerance. Clin Exp Allergy. 2001 Feb;31(2):265-73.\n\nWorm M, Ehlers I, Sterry W, Zuberbier T. Clinical relevance of food additives in adult patients with atopic dermatitis. Clin Exp Allergy. 2000 Mar;30(3):407-14\n\nBrockow K, Jofer C, Behrendt H, Ring J. Anaphylaxis in patients with mastocytosis: a study on history, clinical features and risk factors in 120 patients. Allergy. 2008 Feb;63(2):226-32\n\nReese I, Zuberbier T, Bunselmeyer B, Erdmann S, Henzgen M, Fuchs T, J\u00e4ger L, Kleine-Tebbe J, Lepp U, Niggemann B, Raithel M, Saloga J, Vieths S, Werfel T. Diagnostic approach for suspected pseudoallergic reaction to food ingredients. J Dtsch Dermatol Ges. 2009 Jan;7(1):70-7. Epub 2008 Nov 24. Review\n\nBuhner S, Reese I, Kuehl F, Lochs H, Zuberbier T. Pseudoallergic reactions in chronic urticaria are associ-ated with altered gastroduodenal permeability. Allergy. 2004 Oct;59(10):1118-23","meta":{"dup_signals":{"dup_doc_count":107,"dup_dump_count":54,"dup_details":{"curated_sources":4,"2022-49":1,"2022-33":1,"2021-10":1,"2020-50":1,"2020-24":1,"2019-35":1,"2019-26":1,"2019-18":1,"2019-09":1,"2018-51":1,"2018-43":1,"2018-34":1,"2018-26":1,"2018-17":1,"2018-05":1,"2017-39":1,"2017-30":1,"2017-26":1,"2017-22":2,"2017-17":1,"2017-09":5,"2017-04":2,"2016-50":2,"2016-44":2,"2016-40":2,"2016-36":4,"2016-30":6,"2016-22":2,"2016-18":1,"2016-07":8,"2015-48":4,"2015-40":3,"2015-35":1,"2015-32":1,"2015-27":2,"2015-22":2,"2015-14":1,"2014-52":2,"2014-49":3,"2014-42":3,"2014-41":2,"2014-35":2,"2014-23":4,"2014-15":2,"2023-14":1,"2024-10":1,"2017-13":1,"2015-18":2,"2015-11":1,"2015-06":2,"2014-10":1,"2013-48":2,"2013-20":4,"2024-22":1}},"file":"PMC3247877"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  The Hedgehog (Hh) signaling pathway is vital to animal development as it mediates the differentiation of multiple cell types during embryogenesis. In adults, Hh signaling can be activated to facilitate tissue maintenance and repair. Moreover, stimulation of the Hh pathway has shown therapeutic efficacy in models of neuropathy. The underlying mechanisms of Hh signal transduction remain obscure, however: little is known about the communication between the pathway suppressor Patched (Ptc), a multipass transmembrane protein that directly binds Hh, and the pathway activator Smoothened (Smo), a protein that is related to G-protein-coupled receptors and is capable of constitutive activation in the absence of Ptc.\n  .\n  # Results\n  .\n  We have identified and characterized a synthetic non-peptidyl small molecule, Hh-Ag, that acts as an agonist of the Hh pathway. This Hh agonist promotes cell-type-specific proliferation and concentration-dependent differentiation *in vitro,* while *in utero* it rescues aspects of the Hh-signaling defect in *Sonic hedgehog*-null, but not *Smo*-null, mouse embryos. Biochemical studies with Hh-Ag, the Hh-signaling antagonist cyclopamine, and a novel Hh-signaling inhibitor Cur61414, reveal that the action of all these compounds is independent of Hh-protein ligand and of the Hh receptor Ptc, as each binds directly to Smo.\n  .\n  # Conclusions\n  .\n  Smo can have its activity modulated directly by synthetic small molecules. These studies raise the possibility that Hh signaling may be regulated by endogenous small molecules *in vivo* and provide potent compounds with which to test the therapeutic value of activating the Hh-signaling pathway in the treatment of traumatic and chronic degenerative conditions.\nauthor: Maria Frank-Kamenetsky; Xiaoyan M Zhang; Steve Bottega; Oivin Guicherit; Hynek Wichterle; Henryk Dudek; David Bumcrot; Frank Y Wang; Simon Jones; Janine Shulok; Lee L Rubin; Jeffery A Porter\ndate: 2002\ninstitute: 1Curis, Inc., 61 Moulton Street, Cambridge, MA 02138, USA; 2Columbia University, College of Physicians and Surgeons, 701 West 168 Street, New York, NY 10032, USA\nreferences:\ntitle: Small-molecule modulators of Hedgehog signaling: identification and characterization of Smoothened agonists and antagonists\n\n# Background\n\nThe *hedgehog* (*hh*) gene was identified two decades ago in *Drosophila* as a critical regulator of cell-fate determination during embryogenesis \\[1\\]. Subsequent work in several model systems has defined and characterized the *Hh* gene family that encodes highly conserved secreted signaling proteins (for review see \\[2\\]). Hedgehog (Hh) proteins are synthesized as approximately 45 kDa precursors that autoprocess in an unprecedented fashion, resulting in the covalent attachment of a cholesterol moiety to the amino-terminal half of the precursor \\[2\\]. This processed amino-terminal domain, Hh-Np, is responsible for the activation of a unique and complex signaling cascade that is essential for controlling cell fate throughout development and into adulthood \\[2\\]. In mammals there are three Hh-family proteins: Sonic (Shh), Indian (Ihh), and Desert (Dhh). Gene-targeting experiments in mice have demonstrated that the development and patterning of essentially every major organ requires input from the Hh pathway \\[2\\].\n\n*In vitro* culture systems of neuronal tissues have been used to characterize the biology of the Hh-signaling pathway. Most notably, the neural-plate explant assay has defined the concentration-dependent role that ventrally expressed Shh plays in opposing dorsally expressed bone morphogenetic proteins (BMPs) to pattern the neural tube \\[2\\]. The assay demonstrates that the Hh-signaling cascade can distinguish between small concentration differences in the Hh ligand to instruct the differentiation of specific neuronal cell types. Additional insights have been gained by utilizing cultures of postnatal cerebellar neuron precursors \\[2\\]. These studies have shown that Hh patterns the cerebellum by promoting proliferation of the granule neuron precursors. Given the role that Hh signaling plays in promoting progenitor-cell proliferation, it is not surprising that misregulation of Hh signaling has been implicated in the biology of certain cancers, in particular basal cell carcinoma (BCC) and medulloblastoma.\n\nThe Hh-signaling pathway comprises three main components: the Hh ligand; a transmembrane receptor circuit composed of the negative regulator Patched (Ptc) plus an activator, Smoothened (Smo); and finally a cytoplasmic complex that regulates the Cubitus interruptus (Ci) or Gli family of transcriptional effectors. Additional pathway components are thought to modulate the activity or subcellular distribution of these molecules \\[2\\]. There is positive and negative feedback at the transcriptional level as the *Gli1* and *Ptc1* genes are direct transcriptional targets of activation of the pathway.\n\nSmo is a seven-pass transmembrane protein with homology to G-protein-coupled receptors (GPCRs), while Ptc is a twelve-pass transmembrane protein that resembles a channel or transporter. Consistent with its role as an essential pathway inhibitor, removal of Ptc renders the Hh pathway constitutively 'on', independent of the Hh ligand. Similarly, specific point mutations in the transmembrane helices of Smo are capable of constitutively stimulating the pathway, effectively bypassing Ptc inhibition \\[3\\]. At present, a controversy surrounds the mechanism by which Ptc inhibits Smo. Although early studies suggested a simple, direct, stoichiometric regulation, more recent data support a more complicated indirect or catalytic model \\[2\\]. And although it has been demonstrated that Hh directly interacts with \\[4\\] and destabilizes \\[5\\] Ptc, the downstream molecular events remain obscure. In particular, little is known about the means by which Ptc exerts its inhibitory effect on Smo, or how Smo communicates with the cytoplasmic Ci\/Gli transcription factor complex.\n\nThrough a 'chemical genetic' approach of identifying and studying the mechanism of action of small-molecule agonists (and antagonists), we hoped to uncover some of the complexities of the Hh-signaling system. Small-molecule modulators of growth-factor pathways have proven valuable in providing enhanced understanding of the intracellular events that occur subsequent to receptor activation, and in establishing the biological functions of these pathways \\[6-8\\]. In Hh signaling, multiple insights have been gained through the use of the plant-derived Hh antagonist cyclopamine \\[9-16\\] and a recently identified synthetic small-molecule Hh-signaling inhibitor, Cur61414 \\[17\\]. Interestingly, these specific inhibitors of Hh signaling appear to function downstream of Ptc but their precise molecular target(s) and mechanism of action are unknown.\n\nAlthough genetic manipulations involving gain-of-function point mutations of Smo \\[3\\] have demonstrated that the pathway can be activated independently of Hh ligand, no small molecules with this capability have been identified. Indeed, it has proven difficult to identify small-molecule agonists of any signaling pathway activated by a protein ligand. Two examples have recently been described, however. One involved identification of a non-peptide activator of the granulocyte colony-stimulating factor (GCSF) pathway that appeared to act via receptor oligomerization \\[18\\]. Another report described a small-molecule activator of the insulin-signaling pathway that also acts at the level of the receptor \\[19\\].\n\nSince the Hh receptor, Ptc, serves to inhibit signaling, a small-molecule pathway activator would need to be capable of one of the following: first, interfering with the inhibitory effect that Ptc exerts on Smo; second, activating Smo without affecting Ptc; or third, activating the pathway downstream of Smo. Identifying small molecules with any of these activities would provide useful information concerning the details of Hh signaling and would also provide a simple means of modulating activity of the pathway *in vivo* or *in vitro*.\n\nIn this article, we show that a non-peptidyl small-molecule agonist of Hh signaling has been identified that has all the known signaling properties of the recombinant Hh protein. But this agonist, unlike Hh protein, appears to bypass the Ptc-regulatory step, by interacting directly with Smo. Furthermore, studies with the agonist and several antagonists of Hh signaling suggest that Smo can be activated or inhibited by direct interaction with small-molecule ligands. These observations suggest that the Ptc-Smo receptor circuit may incorporate native small-molecule ligands in the regulation of Hh signaling.\n\n# Results\n\n## Isolation of Hh agonists by high-throughput screening\n\nTo identify small-molecule agonists of Hh signaling, we established a mammalian-cell-based assay. After testing several cell lines for Hh-dependent induction of the target genes *Ptc1* and *Gli1*\\[2\\], we identified C3H10T1\/2 and TM3 cells as optimal responders. We then introduced into each line a plasmid containing a luciferase reporter downstream of multimerized Gli binding sites and a minimal promoter \\[20\\]. An isolated stable clone of the 10T1\/2 cell transfectants (referred to as clone S12) gave a 10-20-fold up-regulation of luciferase activity (Figure <a href=\"#F1\" data-ref-type=\"fig\">1a<\/a>) when stimulated with Hh protein \\[21\\] for 24 hours. Using this assay system, we screened 140,000 synthetic compounds at a concentrations of 2-5 \u03bcM and isolated several putative agonists. One of these molecules - Hh-Ag 1.1 (Figure <a href=\"#F1\" data-ref-type=\"fig\">1a,b<\/a>) - was studied further. Hh-Ag 1.1 exhibited half-maximal stimulation (EC~50~) at around 3 \u03bcM, and an activation maximum (A~max~) of approximately 35% compared to the Hh protein control (Figure <a href=\"#F1\" data-ref-type=\"fig\">1a<\/a>). In the presence of sub-threshold signaling levels of Hh protein (0.3 nM), the EC~50~ of Hh-Ag 1.1 was reduced to around 0.4 \u03bcM and the A~max~ approached 70% (Figure <a href=\"#F1\" data-ref-type=\"fig\">1a<\/a>).\n\nWe next tested whether expression of endogenous Hh-responsive genes was stimulated by the agonist. Using quantitative PCR, Hh-Ag 1.1 was shown clearly to elevate the expression of *Gli1* and *Ptc1* in a dose-dependent manner (Figure <a href=\"#F1\" data-ref-type=\"fig\">1c<\/a>).\n\n## Chemical modifications increase potency\n\nIn an effort to improve the potency of Hh-Ag 1.1, over 300 derivatives were synthesized and tested in the cell-based reporter assay. The relative potencies of the most active derivatives - 1.2, 1.3, 1.4 and 1.5 - are shown in Figure <a href=\"#F1\" data-ref-type=\"fig\">1d<\/a>. The most potent, Hh-Ag 1.5, had an EC~50~ of approximately 1 nM. Thus, potency was increased over 1000-fold by chemical modification. The structures of compounds 1.2 and 1.3 are shown in Figure <a href=\"#F1\" data-ref-type=\"fig\">1e<\/a>. Hh-Ag 1.2 was the most stable derivative *in vivo* and *in vitro* (data not shown) and was used for most cell-based assays. Hh-Ag 1.3 showed lower toxicity in embryonic tissue cultures (data not shown) and was used for the neural plate explant assays described below. These experiments suggest that the agonist may have many of the properties of the Hh ligand. To specifically test this, we used two established *in vitro* assay systems that detect the effects of Hh on primary neuronal precursors.\n\n## *In vitro* assay of neuronal precursors\n\n### Proliferation activity of the agonist\n\nIt has recently been shown that primary neonatal cerebellar granule neuron (CGN) precursors proliferate in response to Hh stimulation \\[2\\]. To determine whether the Hh agonist could elicit this response, we monitored \\[^3^H\\]-thymidine incorporation of cultured rat CGN precursors treated with Hh protein, Hh-Ag 1.1, Hh-Ag 1.2, or vehicle (DMSO). The original active molecule, Hh-Ag 1.1, stimulated thymidine incorporation at 5 \u03bcM, but not at 1.75 \u03bcM (Figure <a href=\"#F1\" data-ref-type=\"fig\">1f<\/a>). The extent of proliferation was around 50% of that seen with a high dose of Hh protein (50 nM). Hh-Ag 1.2 stimulated proliferation at 300 nM and 100 nM to levels comparable to those seen with Hh protein (Figure <a href=\"#F1\" data-ref-type=\"fig\">1f<\/a>). These data demonstrate that the agonists can elicit a biological response in primary cells similar to that produced by Hh protein.\n\n### Morphogenic activity of the agonist\n\nNeural progenitors within the intermediate region of the chick neural plate (Figure <a href=\"#F2\" data-ref-type=\"fig\">2a<\/a>) respond to increasing concentrations of Hh protein by adopting specific fates. The identity of these cells can be assessed by their distinct expression patterns of a set of transcription factors \\[2\\]. Three of these transcription factors - Pax7, MNR2 and Nkx2.2 - whose expression is differentially sensitive to increasing concentrations of Hh protein were assayed in response to varying concentrations of the agonist (Hh-Ag 1.3). The dorsal spinal cord marker Pax7 is normally repressed by low concentrations of Hh \\[22\\]. Pax7 expression was extinguished by 1-10 nM agonist (Figure <a href=\"#F2\" data-ref-type=\"fig\">2b,c,d,e,f<\/a>). Higher concentrations of agonist (10-200 nM) induced expression of the motor neuron progenitor marker MNR2 (Figure <a href=\"#F2\" data-ref-type=\"fig\">2b,g,h,i,j<\/a>), and yet higher concentrations (20 nM-1 \u03bcM) induced the most ventral interneuron progenitor marker Nkx2.2 (Figure <a href=\"#F2\" data-ref-type=\"fig\">2b,k,l,m,n<\/a>). This dose-dependent profile of expression closely resembles the response achieved by increasing concentrations of Hh protein \\[22-24\\], demonstrating that the Hh agonist mimics the concentration-dependent inductive activity of Hh on neural precursors.\n\n## Activity of the agonist *in vivo*\n\nTo explore the site of action of the Hh agonist within the Hh pathway, we developed an *in vivo* assay for the agonist that would allow us to test its activity in *Shh-* and *Smo*-mutant mouse embryos *in utero*. First, we compared the expression of *Ptc1* in vehicle- and agonist-treated *Ptc1*^*lacZ*\/+^ mouse embryos \\[25\\]. The *Ptc1*^*lacZ*\/+^ mouse expresses \u03b2-galactosidase under control of *Ptc1*-regulatory elements and thus reports Hh-pathway activity in mouse tissues. Hh-Ag 1.2 (Figure <a href=\"#F1\" data-ref-type=\"fig\">1e<\/a>) was chosen for study on the basis of its relatively low toxicity, long serum half-life and ability to cross the placenta (data not shown). Hh-Ag 1.2 was delivered by oral gavage to pregnant mice at 7.5 and 8.5 days post coitum (7.5 and 8.5 dpc). Embryos were collected at embryonic day (E) 9.5 and analyzed by staining with the \u03b2-galactosidase chromogenic substrate X-gal. In vehicle-treated embryos, *Ptc1* expression was confined primarily to the ventral neural tube (Figure <a href=\"#F3\" data-ref-type=\"fig\">3a,c<\/a>). In embryos treated with Hh-Ag 1.2, however, expression of *Ptc1*^*lacZ*^ was greatly extended dorsally in the neural tube and throughout the adjacent mesoderm (Figure <a href=\"#F3\" data-ref-type=\"fig\">3b,d<\/a>). These embryos also displayed open rostral neural tubes, similar to those of *Ptc1*^-\/-^ embryos. These experiments demonstrate that the agonist compound effectively activates Hh signaling *in vivo* following oral administration.\n\n### Agonist site of action *in vivo*\n\nHaving established an *in utero* assay for Hh signaling, we next investigated whether the agonist could rescue aspects of *Shh*- or *Smo*-mutant phenotypes, by monitoring *lacZ* expression in *Smo*^-\/-^*Ptc1*^*lacZ*\/+^ embryos \\[26\\] and *Ptc1* mRNA levels in *Shh*^-\/-^ embryos.\n\nPregnant mice from *Shh*^+\/-^ and *Smo*^+\/-^ intercrosses were treated by oral gavage with vehicle or agonist (15 mg\/kg) at 6.5 and 7.5 dpc. Embryos were collected at 6-8 somite stages (E8.5) when the midline defects are first detectable in both *Shh*^-\/-^ and *Smo*^-\/-^ embryos, but prior to any general retardation of growth and development \\[26,27\\]. In both *Shh*^+\/-^ and *Smo*^+\/-^*Ptc1*^*lacZ*\/+^ embryos, *Ptc1* was detected in ventral neural tube, somites and lateral plate mesoderm (Figure <a href=\"#F3\" data-ref-type=\"fig\">3e,i,m<\/a>). Treatment with the agonist dramatically enhanced and expanded the expression of *Ptc1* in these heterozygous embryos (Figure <a href=\"#F3\" data-ref-type=\"fig\">3f,j,n<\/a>). This was consistent with what we have observed in wild-type embryos (Figure <a href=\"#F3\" data-ref-type=\"fig\">3a,b,c,d<\/a>). It is worth noting that the agonist-treated embryos exhibited overgrowth of the headfolds and hindbrain, reminiscent of *Ptc1*^-\/-^ embryos (compare Figure <a href=\"#F3\" data-ref-type=\"fig\">3e,m<\/a> with <a href=\"#F3\" data-ref-type=\"fig\">3f,n<\/a>).\n\n*Shh*^-\/-^ and *Smo*^-\/-^ embryos at this stage (6-8 somites) started to show fused ventral lips of the cephalic folds, and a single continuous optic vesicle, indicating lack of a clearly defined midline (red arrow, Figure <a href=\"#F3\" data-ref-type=\"fig\">3g<\/a>, and data not shown). As expected, *Ptc1* expression was not detected in the ventral neural tube of the vehicle-treated *Shh*^-\/-^ embryos (arrowhead, Figure <a href=\"#F3\" data-ref-type=\"fig\">3g<\/a>), whereas expression was seen in lateral plate mesoderm and weakly in somites (Figure <a href=\"#F3\" data-ref-type=\"fig\">3g,k<\/a>). This is most likely due to Ihh signaling in these tissues \\[26\\]. Both Shh and Ihh signaling were dependent on Smo, however, because *Ptc1* expression could not be detected in *Smo*^-\/-^ embryos (Figure <a href=\"#F3\" data-ref-type=\"fig\">3o<\/a>).\n\nFollowing agonist treatment, we observed that the neural tube and somite expression of *Ptc1* in *Shh*^-\/-^ embryos was greater than vehicle-treated wild-type levels (compare Figure <a href=\"#F3\" data-ref-type=\"fig\">3h,l<\/a> with <a href=\"#F3\" data-ref-type=\"fig\">3e,i<\/a>). The midline defects in *Shh*^-\/-^ embryos were at least partly rescued by agonist treatment (compare Figure <a href=\"#F3\" data-ref-type=\"fig\">3g<\/a> and <a href=\"#F3\" data-ref-type=\"fig\">3h<\/a>; red arrows). Like *Shh*^+\/-^ embryos, *Shh*^-\/-^ embryos had overgrown headfolds after administration of the Hh agonist (Figure <a href=\"#F3\" data-ref-type=\"fig\">3f<\/a> and <a href=\"#F3\" data-ref-type=\"fig\">h<\/a>). In contrast, agonist treatment had no detectable effect on either morphology or *Ptc1* expression in *Smo*^-\/-^ embryos (compare Figure <a href=\"#F3\" data-ref-type=\"fig\">3o<\/a> and <a href=\"#F3\" data-ref-type=\"fig\">p<\/a>). In summary, these studies demonstrate that agonist activity *in vivo* does not depend upon Shh, but that Smo is absolutely required.\n\n## Mechanism of action\n\n### Chemical epistasis studies\n\nWe sought to determine the level at which the agonist acts in the Hh pathway, in cultured cell assays. To begin addressing this question, we used the Hh reporter cell line to conduct competition experiments between the Hh agonist and known Hh- signaling antagonists that block the pathway at different levels (Figure <a href=\"#F4\" data-ref-type=\"fig\">4a<\/a>). These include: a Hh-protein-blocking antibody, 5E1 \\[23\\]; a natural product derivative, cyclopamine \\[9,10\\] that has recently been shown to act downstream of Ptc, perhaps at the level of Smo \\[11\\]; a recently identified synthetic small-molecule inhibitor, Cur61414, which has inhibitory properties similar to cyclopamine \\[17\\]; and forskolin, an adenylate cyclase\/protein kinase A activator that is thought to block Hh signaling by stimulating degradation of members of the Gli family of transcriptional activators \\[2\\].\n\nThe Hh-blocking antibody 5E1 had no effect on pathway activation by the agonist (Figure <a href=\"#F4\" data-ref-type=\"fig\">4b<\/a>), while forskolin (Figure <a href=\"#F4\" data-ref-type=\"fig\">4c<\/a>), cyclopamine (Figure <a href=\"#F4\" data-ref-type=\"fig\">4d<\/a>) and Cur61414 (Figure <a href=\"#F4\" data-ref-type=\"fig\">4e<\/a>), were all inhibitory. The lack of inhibition by 5E1 eliminates the possibility that the small molecule agonist activates signaling indirectly via stimulation of Hh expression. Furthermore, this supports the data showing that the agonist can activate signaling in *Shh*^-\/-^ embryos (Figure <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>) and suggests that the agonist function is not only downstream of the Hh protein but also independent of the endogenous Hh-signaling modulators, Tout veloux and HIP, that act via the Hh ligand \\[2\\]. The competition experiment with forskolin showed identical inhibition curves for Hh protein and the agonist, strongly suggesting that the action of the small molecule is upstream of the protein-kinase-A-sensitive step in the pathway. In contrast, the competition experiments with cyclopamine (Figure <a href=\"#F4\" data-ref-type=\"fig\">4d<\/a>) and Cur61414 (Figure <a href=\"#F4\" data-ref-type=\"fig\">4e<\/a>) showed that Hh protein and the agonist differ in their sensitivity to these antagonists. Specifically, the agonist appears somewhat resistant to the inhibitory effect of cyclopamine and Cur61414. Identical results were seen using the slightly less active cyclopamine-related natural compound jervine, and the more potent synthetic derivative of cyclopamine, KAAD-cyclopamine (data not shown). These results argue that the agonist activates the pathway downstream of the Hh-Ptc interaction while cyclopamine, Cur61414 and the agonist may act at a similar level in the Hh-signaling cascade.\n\n### Regulation of Ptc and Smo by Hh protein and Hh agonist\n\nRecent work in *Drosophila* tissue culture has shown that endogenous Ptc and Smo proteins are differentially affected by the addition of Hh to the growth medium \\[5\\]. Ptc was destabilized, while Smo accumulated following post-translational modification. To test whether similar phenomena occur in mammalian cells with Hh protein and agonist, we generated stable cell lines expressing two epitope-tagged proteins, Ptc coupled to green fluorescent protein, Ptc-GFP, and Smo coupled to a fragment of influenza hemagglutinin, HA-Smo. Figure <a href=\"#F5\" data-ref-type=\"fig\">5a<\/a> shows an immunoprecipitation (anti-GFP) plus protein blot (anti-Ptc) analysis of extracts from these cells treated for 4, 8 and 24 hours with vehicle, 25 nM Hh protein or 0.2 \u03bcM Hh agonist (see Figure <a href=\"#F1\" data-ref-type=\"fig\">1e<\/a>; Hh-Ag 1.2). This experiment shows that Ptc-GFP appears to be destabilized by Hh protein but not by the agonist. Similar results were seen at higher doses of agonist (up to 2 \u03bcM) and in several independent lines (data not shown). These data further support the idea that Hh protein and the agonist act in distinct ways to stimulate the pathway.\n\nFigure <a href=\"#F5\" data-ref-type=\"fig\">5b<\/a> shows an immunoblot (anti-HA) of total extracts from HA-Smo-expressing cells treated for 2, 5, 8 and 20 hours with vehicle, 35 nM Hh protein or 0.5 \u03bcM Hh agonist. In contrast to the results with Ptc-GFP, incubation of cells with both Hh protein and the small-molecule agonist resulted in the apparent accumulation of HA-Smo protein after 5 hours of incubation. To test whether the accumulation of HA-Smo in response to Hh protein or the agonist required protein synthesis, a similar study was performed in the presence of cycloheximide (Figure <a href=\"#F5\" data-ref-type=\"fig\">5c<\/a>). Under these conditions, HA-Smo accumulation was detectable 5 hours after addition of either Hh protein or the agonist (Figure <a href=\"#F5\" data-ref-type=\"fig\">5c<\/a>); this result argues that the effect of Hh protein and the agonist on HA-Smo levels does not require new protein synthesis. Finally, with increasing concentrations of Hh protein and the agonist there is a clear dose-dependent increase of HA-Smo levels (Figure <a href=\"#F5\" data-ref-type=\"fig\">5d<\/a>). These effects on epitope-tagged Smo protein were observed in multiple lines (data not shown). Taken together, these data suggest that Hh protein and the agonist share the ability to stabilize Smo, but only Hh protein can destabilize Ptc. Yet the agonist is fully capable of activating the full signaling pathway.\n\n## Testing Smo as the molecular target\n\n### Binding in whole cells\n\nOur biochemistry experiments (above) show that the agonist modulates Smo levels, and thus may activate Hh signaling by directly binding Smo. To explore this possibility we tested whether a tritiated form of the agonist analog Hh-Ag 1.5 could form a complex with Smo, when Smo is transiently overexpressed in 293T cells. Figure <a href=\"#F6\" data-ref-type=\"fig\">6a<\/a> shows immunoprecipitable counts of extracts from cells incubated at 37\u00b0C for 2 hours with 5 nM \\[^3^H\\]-Hh-Ag 1.5 either in the absence (columns 1-3) or presence of competitors (columns 4-9).\n\nImmunocomplexes from untransfected control and \u03b2-adrenergic-receptor transfected cells did not contain significant counts (Figure <a href=\"#F6\" data-ref-type=\"fig\">6a<\/a>, columns 1, 2). Immunocomplexes derived from cells expressing Smo (Figure <a href=\"#F6\" data-ref-type=\"fig\">6a<\/a>, column 3) resulted in the recovery of approximately 40,000 of the 800,000 added counts, however. To test the specificity of this apparent Hh-Ag\/Smo complex, cells were incubated with 5 \u03bcM (1000-fold molar excess) of unlabeled Hh Ag 1.5 or an unlabeled, signaling-inactive but structurally similar compound, an Hh-Ag 1.1 derivative that has a two-carbon linker in place of the cyclohexane ring (Figure <a href=\"#F6\" data-ref-type=\"fig\">6a<\/a>; Hh-Ag 1.5, column 4; Hh-Ag control, column 5). The addition of the unlabeled Hh-Ag 1.5, but not the inactive Hh-Ag 1.1 derivative agonist control, resulted in the complete absence of counts in the immunocomplex. These results suggest that a stable, specific interaction can form between Smo and the Hh agonist.\n\nIt has been shown that the Hh-pathway antagonists cyclopamine and Cur61414 block signaling in a Ptc-independent manner \\[11,17\\] and therefore may act directly on Smo. Having established a binding assay for a small-molecule agonist binding to Smo-expressing cells, we next tested whether the Hh antagonists could selectively compete out binding of \\[^3^H\\]-Hh-Ag 1.5. To perform theses studies, Smo-overexpressing 293T cells were incubated for 2 hours at 37\u00b0C with 5 nM \\[^3^H\\]-Hh-Ag 1.5 in the presence of either KAAD-cyclopamine at 5 \u03bcM (Figure <a href=\"#F6\" data-ref-type=\"fig\">6a<\/a>, column 6), the related but inactive plant compound tomatadine at 5 \u03bcM (Figure <a href=\"#F6\" data-ref-type=\"fig\">6a<\/a>; Antag control 1, column 7), Cur61414 at 5 \u03bcM (Figure <a href=\"#F6\" data-ref-type=\"fig\">6a<\/a>, column 8), or a related but inactive Cur61414 derivative (Figure <a href=\"#F6\" data-ref-type=\"fig\">6a<\/a>; Antag control 2, column 9) at 5 \u03bcM. These data show that the Hh-signaling inhibitors, but not structurally related inactive compounds, can significantly compete with the binding of the Hh agonist to Smo-expressing cells. This supports the model that all of these small-molecule modulators of Hh signaling are direct ligands of Smo.\n\nWe next asked whether a derivative of the Hh agonist carrying a photoactivatable crosslinker could be coupled directly to Smo, to facilitate further biochemical characterization of the binding site. To perform this experiment we synthesized a tritiated diazirine derivative of Hh-Ag 1.2 with an EC~50~ in the cell-based assay of 35 nM (data not shown). We incubated this compound at 0.5 \u03bcM with HA-Smo- or control, GFP-transfected 293T cells and subsequently ultraviolet-irradiated them to initiate crosslinking. Fractionation by SDS-polyacrylamide gel electrophoresis and autoradiography of the resulting immunocomplexes from these cells showed crosslinking exclusively to HA-Smo, but with an efficiency of less than 1% (data not shown). This result demonstrates that a Hh-agonist derivative can be covalently crosslinked to Smo in living cells. More efficient crosslinkers are required to extend these studies, however.\n\n### Cell-free membrane-binding assays\n\nTo test whether the Hh agonist could interact with Smo *in vitro*, we transiently overexpressed murine Smo, murine Ptc, rat \u03b22-adrenergic receptor and GFP in 293T cells, harvested membranes and performed a filtration membrane-binding assay in a 96-well plate with \\[^3^H\\]-Hh-Ag 1.5 added at 2 nM. Figure <a href=\"#F6\" data-ref-type=\"fig\">6b<\/a> shows a bar graph of the bound counts from these binding assays (murine Smo, column 1; GFP, column 2; \u03b2AR, column 3; murine Ptc1, column 4; and a no-membrane plate control, column 5). The no-membrane control (column 5) was included to show the degree of non-specific binding to the filter-plate apparatus. The Smo-containing membranes (column 1) are the only samples that exhibit significant binding above that seen in the absence of membranes.\n\nTo assess the specificity of binding, we repeated the experiment in the presence and absence of a 1000-fold molar excess of unlabeled agonist (2 \u03bcM). The addition of 'cold' compound completely competed out these counts (Figure <a href=\"#F6\" data-ref-type=\"fig\">6c<\/a>, column 1 compared to column 2). To control for this observation, we added an unlabeled, inactive Hh agonist to the binding assay at 2 \u03bcM (a 1000-fold molar excess). This compound was unable to compete out the binding of \\[^3^H\\]-Hh-Ag 1.5 to the Smo-containing membranes (Figure <a href=\"#F6\" data-ref-type=\"fig\">6c<\/a>, column 3). These results argue that Smo and the agonist form a specific complex *in vitro*, as predicted by the whole cell\/immunocomplex binding assay (Figure <a href=\"#F6\" data-ref-type=\"fig\">6a<\/a>).\n\nHaving established an *in vitro* binding assay for the Hh agonist to Smo, we next tested whether the Ptc-independent Hh antagonists could selectively compete with the interaction. Binding was assayed in the presence of KAAD-cyclopamine at 10 \u03bcM (Figure <a href=\"#F6\" data-ref-type=\"fig\">6c<\/a>, column 4), tomatadine at 10 \u03bcM (Figure <a href=\"#F6\" data-ref-type=\"fig\">6c<\/a>; Antag control 1, column 5), Cur61414 at 10 \u03bcM (Figure <a href=\"#F6\" data-ref-type=\"fig\">6c<\/a>, column 6), or the inactive Cur61414 derivative (Figure <a href=\"#F6\" data-ref-type=\"fig\">6c<\/a>; Antag control 2, column 7) at 10 \u03bcM. These data show that the Hh-signaling inhibitors, but not structurally related inactive compounds, can significantly compete with the binding of the Hh agonist to Smo membranes.\n\n## Kinetics, saturation and competition binding analysis\n\nNext, we sought to generate association, dissociation and saturation-binding curves, in order to derive affinity constants for the interaction of the Hh agonist and Smo. To control for nonspecific binding we used either Cur61414 or Hh-Ag 1.5 as unlabeled competitors. Similar results were generated if control membranes (from cells transfected with GFP, \u03b2AR, or Ptc) were used to define the non-specific level (data not shown).\n\nFirst, we performed a kinetic analysis to establish the reversibility of the binding reaction and the approximate incubation time required for equilibrium binding studies. Association assays were performed at 37\u00b0C by combining 2 nM \\[^3^H\\]-Hh-Ag 1.5 with Smo-containing membranes for various times prior to harvesting and counting. Dissociation studies were initiated by adding 2 \u03bcM unlabeled Hh-Ag 1.5 after 2 hours of association. Samples were then incubated for 1-26 hours prior to harvesting and counting. Figure <a href=\"#F7\" data-ref-type=\"fig\">7a<\/a> shows the association and dissociation phases of agonist binding to Smo-containing membranes. Using the Prism GraphPad software, these data were fit to one-phase exponential association and decay curves, respectively, and gave an association t\u00bd of approximately 1 hour and a dissociation t\u00bd of approximately 10 hours. These results demonstrate that the binding of the agonist to Smo is reversible and that equilibrium binding will require binding reaction times of approximately 50 hours (five times the t\u00bd of dissociation).\n\nNext, we performed a saturation binding experiment. To establish total, nonspecific, and specific binding curves (Figure <a href=\"#F7\" data-ref-type=\"fig\">7b<\/a>), we added a range of \\[^3^H\\]-Hh-Ag 1.5 concentrations (0.01-3 nM) in the presence or absence of unlabeled Hh-Ag-1.5 at 2 \u03bcM. Identical results were seen if Cur61414 at 10 \u03bcM was used as the competitor (data not shown). On the basis of the binding kinetics, incubations were carried out for approximately 45 hours at 37\u00b0C, to allow for equilibrium to be reached. Using the Prism GraphPad software to perform non-linear regression analysis and curve fitting, we concluded that the data best fit a simple one-site binding model with a predicted K~d~ of 0.37 nM for Hh-Ag 1.5. This K~d~ is in general agreement with the EC~50~ values observed in the cell-based assay (0.37 nM as compared to 1 nM).\n\nTo further validate our binding results, we performed a competition assay using several agonist derivatives across a range of concentrations (0.01 nM to 1 \u03bcM). Figure <a href=\"#F7\" data-ref-type=\"fig\">7c<\/a> shows the competition curves for Hh-Ag 1.5, Hh-Ag 1.3, Hh-Ag 1.2, Hh-Ag 1.1, and the signaling-inactive Hh-Ag 1.1 derivative described above. With the exception of the inactive derivative, these compounds all compete out the binding of \\[^3^H\\]-Hh-Ag 1.5 (0.4 nM) to the Smo-containing membranes. These data are best fit to a single-binding-site competition model that predicts the following K~i~ values: Hh-Ag 1.5, 0.52 nM; Hh-Ag 1.3, 8.4 nM; Hh-Ag 1.2, 22 nM; and Hh-Ag 1.1, 96 nM. These K~i~ values are in general agreement with the agonist EC~50~ values in cell culture for these compounds, with the exception that the Hh-Ag 1.1 compound is not as potent in signaling assays (EC~50~ 2 \u03bcM) as its K~i~ (96 nM) might predict. This suggests an uncoupling of binding and signaling for certain agonists. Although binding affinities and signaling efficacy can correspond for certain ligand\/receptor complexes, exceptions often arise \\[28\\] because binding affinity does not necessarily measure the ability of a compound to induce an active receptor conformation. As a control for these binding studies, identical competition experiments were performed with membranes from cells transfected with GFP, or with the \u03b2-adrenergic receptor. No specific binding or apparent competition was seen under these conditions (data not shown).\n\nWe next compared binding of KAAD-cyclopamine, Cur61414 and Hh-Ag 1.5 to a constitutively active mutant of Smo (Smo^act^) or to wild-type Smo (Smo^wt^). The two Hh-signaling antagonists, KAAD-cyclopamine and Cur61414, have shown decreased potency on Smo^act^-expressing cells, leading to the speculation that they may bind this mutant form of Smo less well than the wild-type form \\[11,17\\]. We sought to determine whether the Hh agonist binds Smo^act^ with a higher affinity, an observation seen with certain ligands and constitutively active mutants of GPCRs \\[29\\]. To perform this experiment, we isolated membranes from cells transfected with a cDNA construct encoding a tryptophan-to-leucine mutation at residue 539 (W539L) of murine Smo. This oncogenic mutation has been found in human basal cell carcinoma \\[3\\] and the correspondingly mutated protein is capable of ligand-independent activation of the Hh pathway in cell-culture assays \\[11\\]. A kinetic and saturation binding assay with Smo^act^-containing membranes showed that this mutant protein binds the Hh agonist with an affinity identical to that of Smo^wt^ (data not shown).\n\nUsing Smo^act^- and Smo^wt^-containing membranes, we then performed competition binding studies by adding increasing concentrations of unlabeled Hh-Ag 1.5, KAAD-cyclopamine or Cur61414 in the presence of \\[^3^H\\]-Hh-Ag 1.5 (0.4 nM). These binding curves (Figure <a href=\"#F7\" data-ref-type=\"fig\">7d<\/a>) can be fit to a single-site competition model. Although the K~i~ for Hh-Ag 1.5 on Smo^act^-containing membranes was essentially identical to that observed for Smo^wt^-containing membranes (approximately 0.5 nM), the K~i~ values of the Hh antagonists were seven-fold higher on the Smo^act^- compared to the Smo^wt^-containing membranes for both KAAD-cyclopamine (38.3 nM versus 5.8 nM) and Cur61414 (309 nM versus 44 nM). These results strongly support the model, initially hypothesized for cyclopamine \\[11\\], that the reduced potency observed for Hh antagonists on Smo^act^-expressing cells is directly due to a reduced affinity of the antagonist for the mutated Smo protein. The agonist, on the other hand, would be predicted to bind to a site on Smo that is not affected by this gain-of-function Smo^act^ mutation.\n\n# Discussion\n\nHh signal transduction has been the focus of intense research over the past decade due to the central role it plays in development and its emerging biomedical relevance in areas ranging from regenerative medicine to oncology \\[2,30\\]. Our goal in these studies was to isolate and characterize small-molecule modulators of Hh signaling in order to understand better the regulation of pathway activation and to generate potential therapeutics. Our work shows firstly that it is possible to identify potent small molecule agonists of Hh signaling, secondly that these compounds can mimic the effects of recombinant Hh protein in multiple assays used to define the properties of Hh signaling, thirdly that these compounds act by binding directly to Smo, and finally that two Ptc-independent inhibitors of Hh signaling compete for this binding to Smo, strongly suggesting they too act directly on Smo.\n\n## Models of Smo-ligand interaction\n\nTo interpret the results of the competition binding studies, we assume that the mutation in Smo^act^, like those in constitutively activate mutants of other GPCRs \\[29\\], indirectly influences ligand binding by creating a change in the normal equilibrium between the different conformations of Smo. Thus, the mutation would not directly influence the binding pocket for either ligand. A simple two-state model (Figure <a href=\"#F8\" data-ref-type=\"fig\">8a<\/a>) predicts that the agonist (Ag, green square) and the antagonist (Ant, pink circle) compete for the same site on Smo to activate or inactivate Hh-pathway signaling. It also suggests that antagonists should bind Smo^act^ with a lower affinity than they bind Smo^wt^, while the agonist should bind with a higher affinity, as it prefers the active conformation. Such a model cannot accommodate our observations with the gain-of-function Smo mutant. Thus we introduce a ternary complex model (Figure <a href=\"#F8\" data-ref-type=\"fig\">8b<\/a>), used traditionally to describe the behavior of GPCRs in binding studies with agonist and antagonists \\[31\\], as well as constitutively active receptors \\[29\\]. The ternary complex model for Hh signaling suggests that there are two independent binding sites on Smo^wt^, one specific for the agonist and another specific for antagonists. Binding at either site would decrease the affinity for interactions at the other site (allosteric binding with high negative cooperativity). The agonist-bound form represents the normal activated state, while the antagonist-bound form is considered the inactive conformation. There are also other conformations that would not be bound, or would be transiently bound, by both ligands. A signaling pathway coupler, or effector (in blue), is proposed to bind the activated state of Smo^wt^ so as to generate a complex competent to initiate Hh signaling. Throughout the discussion the term 'coupler\/effector' is used to describe an unknown molecule that binds activated Smo in such a way as to trigger signal transduction. The model further suggests that the Smo^act^ protein resides in a stable conformation in the absence of agonist that is capable of forming an active coupler\/effector complex resistant to antagonist, but not agonist, binding.\n\nSpecifically, our data suggest that the agonist binds and stabilizes (or induces) an active signaling state of Smo while the antagonists bind and stabilize (or induce) an inactive form. Furthermore, the gain-of-function Smo mutation renders the protein less sensitive to the inhibitors, presumably because the amino-acid substitution directly stabilizes or induces an active conformation. On the basis of a simple two-state model, one might predict an increased affinity of the agonist for the mutant form, but in our studies binding of the agonist, unlike the antagonist, is not affected by the activating mutation, suggesting that a more complex model requiring two binding sites and perhaps multiple active conformations is needed to account for the observations. Thus, we propose a variation of the classic 'ternary complex model' (Figure <a href=\"#F8\" data-ref-type=\"fig\">8<\/a>), a decades-old paradigm that has provided the foundation for describing ligand induced conformational changes of GPCRs \\[31\\].\n\nBriefly, if this model is applied to Hh signaling, it proposes that, firstly, agonist and antagonists act at independent sites to select active and inactive conformations of Smo; secondly, that Smo engages an undefined coupler\/effector, when it is in its signaling state; and finally, that the gain-of-function mutant form of Smo, Smo^act^, adopts an abnormal conformation that resembles the coupler\/effector-bound signaling state of Smo with low affinity for antagonists but normal affinity for the agonist.\n\n## Activating Hh signaling through the GPCR-like Smo receptor\n\nAs a receptor class, GPCRs are considered excellent drug targets because they are often regulated through interactions with small natural ligands \\[32,33\\]. Specifically, studies of classic GPCRs, such as the \u03b2-adrenergic receptors, show that in the absence of endogenous ligand (agonists) these receptors exist in multiple interconvertible conformations that are predominately inactive \\[34\\]. Upon exposure to their natural ligands, however, the active receptor forms are preferentially stabilized, allowing them to readily engage G-protein couplers and to create signaling-competent complexes. Multiple compound classes have been isolated on the basis of their ability to compete for the binding of \u03b2-adrenergic receptors by their natural ligands. These competitors can mimic the natural ligand activity (agonists) or interfere with it (antagonists).\n\nIn addition to the binding sites for natural or endogenous agonists (orthosteric sites), many GPCRs have also been found to have allosteric sites \\[35\\]. These sites can bind natural ligands, as in the case of Zn ions and heparin for the dopamine and neurokinin receptors, respectively, or bind synthetic drugs such gallamine, in the case of the muscarinic receptors \\[35\\]. Binding of small molecules to these allosteric sites can modulate activity of a receptor without directly mimicking or competing out the interaction of ligands to the orthosteric sites. In summary, GPCRs have an array of potential regulatory binding sites, or potential drug targets.\n\nHow does Smo compare with other GPCRs with regard to the properties described above? Although there is clear structural homology between Smo and other GPCRs, endogenous ligands have yet to be discovered. Early models of Hh signaling proposed a Hh-regulated Ptc-Smo complex that directly controlled the conformation of Smo, making endogenous ligands unnecessary. But recent studies argue against this stoichiometric model \\[5,36\\], indicating that perhaps natural ligands should be considered. Furthermore, regarding G-protein-effector coupling for Smo, the results are also equivocal. Although no compelling data have been presented that directly link classic G-protein activation \\[37\\] to the canonical Hh pathway involving Ci\/Gli stimulation \\[2\\], recent studies show that under certain conditions Smo can engage G-protein subunits in a Ptc-dependent manner \\[38\\] and that G-protein-mediated cAMP modulation may underlie certain effects of Hh on neuronal tissue \\[39\\].\n\nFinally, with respect to pharmacological properties, our studies indicate that Smo behaves like a classic GPCR in many regards and that the models used to describe this large family of receptors can be applied to Hh signaling (Figure <a href=\"#F8\" data-ref-type=\"fig\">8<\/a>). Two relatively novel concepts for Hh signaling are raised by these GPCR models: firstly, the importance of considering the active Smo-coupler\/effector complexes when modeling pathway regulation, and secondly the potential for endogenous ligands in the regulation of Smo activity.\n\n## GPCR models for Smo and potential mechanisms of Ptc function\n\nA recent study in *Drosophila* suggested that Hh stimulates the pathway via Ptc degradation and, as a result, Smo is stabilized through extensive phosphorylation at the plasma membrane where it initiates signaling \\[5\\]. This led to speculation that a Ptc-regulated phosphatase may control the subcellular distribution and stability of Smo \\[5\\]. Although suggestive, the studies in *Drosophila* did not establish that phosphorylation, translocation or stabilization of Smo is required for pathway stimulation. It is plausible that some or all of these effects on Smo result from a feedback inhibition mechanism that targets the activated Smo receptor. Interestingly, while we did observe stabilization of Smo by Hh in our mammalian cell experiments (Figure <a href=\"#F5\" data-ref-type=\"fig\">5<\/a>), we did not detect Smo phosphorylation or translocation to the plasma membrane (data not shown). Perhaps the cellular (or species) context dictates the degree to which the active conformations of Smo are associated with such changes. Thus, in considering models of Ptc function based on Hh-stimulated effects it is important to consider whether the form of Smo that is being observed is the active (coupler\/effector bound) signaling state, or perhaps a downregulated and inactive form.\n\nSeveral potential points for regulation by Ptc during the formation of a Smo-coupler\/effector complex are apparent in a ternary complex model. The ideas proposed for the *Drosophila* system, in which Ptc may affect the levels of Smo or its subcellular localization, are easily accommodated. Ptc-induced instability of Smo protein would indirectly reduce the concentration of an active Smo-coupler\/effector complex. Furthermore, limiting access of Smo to its effector through targeted vesicle trafficking would prevent a signaling-competent complex from forming. Alternatively, Ptc could more directly maintain Smo in an inactive state. On the basis of our studies it is tempting to speculate that native small molecules with properties similar to our agonist and antagonists act directly on Smo in a Ptc-dependent manner. These putative endogenous Smo modulators could represent orthosteric or allosteric ligands.\n\nThe simplest model would have Ptc acting catalytically to dock a natural antagonist directly onto (or remove an agonist from) Smo. A more complex model would involve Ptc restricting the distribution of Smo such that it is forced into compartments containing the natural antagonists or lacking the natural agonists. Finally, Ptc could control the distribution of the endogenous small-molecule modulators themselves. Ptc shares sequence homology with molecules associated with vesicle trafficking and transporter activity, namely SCAP and NPC1 \\[36,40-42\\]; if it also shares the activities of these molecules, as suggested by a recent study \\[36\\], then the possibility of the existence of endogenous Smo ligands that are docked via Ptc should be explored.\n\n## Therapeutic potential of a Hh-pathway agonist\n\nVarious studies in mammals have shown that *Hh* genes are expressed in discrete areas of the adult organism and may function in the normal maintenance of mature organ systems \\[43-46\\]. In addition, the regenerative healing of vascular and skeletal tissues following acute injuries appears to be aided by re-activating the Hh-signaling cascade \\[47,48\\]. Taken together, these observations suggest that the Hh pathway may represent a point of intervention for treating certain degenerative disorders. Two recent studies in models of Parkinson's disease and peripheral nerve damage support this claim, by demonstrating that pathway activation with a Hh-protein ligand has therapeutic value \\[49,50\\]. On the basis of our current understanding of these models and the specific mechanism of action of the Hh agonists, we predict that an agonist-derivative with low toxicity and favorable pharmacokinetics would replicate these positive results. As a drug, a Hh agonist would represent an attractive alternative to an expensive Hh-protein therapeutic. Beyond the economics, for disorders of the central nervous system a small molecule with the potential to cross the blood-brain barrier would eliminate the need for injections directly into the brain, the current delivery mode for central nervous system protein therapies.\n\n# Materials and methods\n\n## Chemical libraries and medicinal chemistry\n\nThe compound libraries used in our screens were purchased from a number of commercial vendors and were primarily generated by combinatorial chemistry approaches. The Hh-agonist class was isolated from a library synthesized by Oxford Asymmetry International, now EvotecOAI. The derivatization of this compound class utilized standard procedures, the details of which will be published elsewhere.\n\n## Cultured cell line assays\n\nTM3 and C3H10T1\/2 cells (ATCC; Manassas, USA) were maintained according to the instructions of ATCC. Stable Hh-signaling reporter cell lines were established by G418 selection following transfection with a luciferase reporter plasmid \\[20\\] containing the neomycin-resistance gene. Hh signaling was monitored by plating cells at 70% confluence in growth medium. After 24 hours the cells were changed to 0.5% serum-containing medium, and Hh protein or compounds were added; 24 hours later the cells were either monitored for luciferase activity using the Luc-lite assay kit (Packard Instrument Company, Meriden, USA) or harvested for RNA isolation using an RNA isolation kit (Qiagen; Valencia, USA). RNA was subjected to quantitative RT-PCR analysis (Taqman; Applied Biosystems, Foster City, USA) utilizing *Gli1, Ptc1* and *GAPDH* primers and probes. Assays were run on a Prism 7700 instrument (ABI; Applied Biosystems).\n\n## Recombinant Hh protein\n\nThe Hh protein used in the studies described here was bacterially overexpressed amino-terminal human Shh modified at its amino-terminal cysteine by an octyl maleimide moiety \\[21\\]. This lipophilic Shh form showed comparable potency to native Shh in the cell-based reporter assay (data not shown).\n\n## Retroviral cell lines\n\nMouse *Smo* and *Ptc1* genes were introduced by a retroviral approach utilizing the pLPCX vector (Clontech; Palo Alto, USA) to limit the copy number per cell. Stable HA-Smo and Ptc-GFP lines were established by puromycin selection following infection of TM3 cells with the respective retroviruses. TM3 cells expressing both epitope-tagged Ptc and Smo were derived by infecting first with an HA-Smo construct and subsequently with a Ptc-GFP construct. The levels of Ptc were relatively low in these lines and a standard immunoprecipitation procedure followed by western blotting was required to detect the Ptc-GFP protein. The HA-Smo protein was highly expressed and was easily detected in western blots of whole cell extracts. The HA tag was sub-cloned into the *Smo* gene so that it would reside immediately after the Smo signal sequence. The GFP tag was inserted before the stop codon of the *Ptc* open reading frame.\n\n## *In utero* Hh-signaling assays\n\nGeneration of *Ptc1*^*lacZ*^, *Shh* and *Smo* mutant mice has been described previously \\[25,26\\]. *Ptc1*^*lacZ*\/+^ mice were kindly provided by Matthew Scott. *Shh*^+\/-^ and *Smo*^+\/-^ mice were kindly provided by Andrew McMahon. Agonist solution was prepared in fine suspension in 0.5% methylcellulose\/0.2% Tween 80 at 1.5 mg\/ml. Compound was administered by oral gavage to pregnant mice once a day for two days at 100 \u03bcl per 10 g body weight. Embryos were collected 24 hours later. Whole-mount *in situ* hybridization with *Ptc1* probe and X-gal staining for whole-mount \u03b2-galactosidase detection were performed as described \\[26\\]. For histology, embryos stained with X-gal were post-fixed in 4% paraformaldehyde, wax-embedded, and 20 \u03bcm sections were prepared.\n\n## Primary cerebellar cultures\n\nCerebellar neurons were dissected out of postnatal (one week) rat brains, and placed into primary cell culture. Briefly, cells were placed in 96-well plates at a density of approximately 150,000 cells per well in basal medium of Eagle (Gibco; Carlsbad, USA) supplemented with 26 mM KCl, 2 mM glutamine and 10% calf serum. Treatment agents were added once, on the first day of culture (0 DIV). Cells were left in culture until 2 DIV, when \\[^3^H\\]-thymidine was added for 5 hours. Cells were then lysed, and the incorporation of \\[^3^H\\]-thymidine was determined by scintillation counting.\n\n## Neural plate explant assay\n\nIntermediate regions of the open neural tube (i-explants) were dissected from stage 10-11 chick embryos and embedded in collagen gel \\[23\\]. Explants were cultured in Ham-F12 supplemented with 3 g\/l D-glucose, Mito Serum Extender (Collaborative Research; Bedford, USA), penicillin\/streptomycin (Gibco), 2 mM L-glutamine (Gibco) and Hh-Ag 1.3 (0.1, 1, 10, 20, 200 and 1000 nM prepared as 1000X stocks in DMSO; n = 6 explants). As a control, some explants were cultured with vehicle alone or with octylated Hh-N recombinant protein. Cultures were fixed after 22 hours, stained with mouse monoclonal antibodies against Pax7, MNR2 or rabbit polyclonal antibodies against Nkx2.2 and the number of immunoreactive cells per explant counted.\n\n## Whole cell\/immunocomplex binding assay\n\nCultured cells - 70% confluent 293T cells in 6-well plates - were either left untransfected or transfected using Fugene6 with a pCDNA3.1 construct containing HA-tagged Smo or v5-epitope tagged \u03b22AR (Invitrogen; Carlsbad, USA). After 48 hours cells were switched from 10% fetal bovine serum containing DME media to 0.5% FBS containing media supplemented with either 5 nM \\[^3^H\\]-Hh-Ag 1.5 alone or 5 nM \\[^3^H\\]-agonist plus 5 \u03bcM of various competitors (see Results). After 2 hours of incubation at 37\u00b0C, cells were washed one time with PBS and subsequently lysed in 0.5 ml of lysis\/wash buffer containing 1% NP40 in Tris-buffered saline plus an EDTA-free protease inhibitor cocktail (Roche; Indianapolis, USA) for 20 minutes at 4\u00b0C. Cell extracts were spun at 14,000 rpm in a microcentrifuge and supernatants were incubated for 40-60 minutes with either anti-HA beads (Roche) or anti-v5 antibody (Invitrogen) and Protein A beads (Pierce; Rockford, USA) to form immunocomplexes. Immunobeads were then spun down and washed three times with 0.5 ml lysis\/wash buffer per wash. The washed pellets were then resuspended in SDS sample buffer and combined with scintillation fluid. Counts per minute (cpm) for each sample were then determined in a scintillation counter (Packard topcount).\n\n## Membrane binding assays\n\nMembranes were prepared as follows. Briefly, approximately 10^8^ cells were transfected with pcDNA 3.1 constructs (Invitrogen) bearing either murine *Smo* (wild-type or W539L mutant), GFP, rat \u03b22AR (Invitrogen), or murine *Ptc* cDNAs using Fugene 6 (Roche). After 48 hours cells were harvested by scraping in PBS, centrifuged at 1,000 \u00d7 *g* for 10 minutes, and gently resuspended in around 10 ml of a 50 mM Tris pH 7.5, 250 mM sucrose buffer containing an EDTA-free protease inhibitor cocktail (Roche). This cell suspension was then placed in a nitrogen cavitation device (Parr Instrument Co, Moline, USA) and exposed to nitrogen gas (230 psi) for 10 minutes. Lysed cells were released from the device and centrifuged at 20,000 rpm in an SS34 rotor for 20 minutes at 4\u00b0C. Supernatants were discarded and the pellets were resuspended in 10% sucrose, 50 mM Tris pH 7.5, 5 mM MgCl, 1 mM EDTA solution using three 10-second pulses with a Polytron (Brinkman; Westbury, USA) at a power setting of 12. Using these membranes, filtration binding assays were performed according to previously described protocols \\[28\\]. To reduce nonspecific binding, 96-well filtration plates (fiberglass FB filters; Millipore, Bedford, USA) were pre-coated as suggested by the manufacturer with 0.5% polyethyleneimine + 0.1% BSA and then washed four times with 0.1% BSA.\n\nFor association and dissociation studies, membranes (1.5 \u03bcg total protein) were incubated in polypropylene tubes with 2 nM \\[^3^H\\]-Hh-Ag 1.5 in the presence or absence of 2 \u03bcM competitor in binding buffer (50 mM Tris pH 7.5, 5 mM MgCl, 1 mM EDTA, 0.1 % bovine serum albumin) plus EDTA-free protease inhibitor cocktail (Roche) in a final volume of 250 \u03bcl for 1-26 hours at 37\u00b0C. For saturation and competition binding analysis, membranes (1.5 \u03bcg total protein) were incubated on the plates with various concentrations of the \\[^3^H\\]-Hh-Ag 1.5 (plus and minus competitors) in binding buffer plus EDTA-free protease inhibitor cocktail (Roche) at a final volume of 1 ml for approximately 45 hours at 37\u00b0C to allow binding to reach apparent equilibrium. Binding reaction mixtures (0.2 ml for association\/dissociation studies and 0.75 ml in saturation and competition experiments) were then transferred to the pre-coated 96-well filtration plates (Millipore fiberglass FB filters), filtered and washed over a vacuum manifold with six 300 \u03bcl per well washes of binding buffer supplemented with 2% hydoxypropyl cyclodextrin (HPCD; Sigma; ST Louis, USA) + 0.1% BSA to decrease non-specific binding. Identical results were obtained if incubations were done in borosilicate glass or siliconized plastic tubes. Centrifugation assays were also performed that replicate the filtration assay results (data not shown). Additionally, these experiments showed that the extent of ligand depletion was less than 10% in these studies. Binding-kinetics experiments were performed similarly to the saturation and competition studies. All binding data were evaluated using a nonlinear regression analysis program (Prism; GraphPad; San Diego, USA). K~i~ values were calculated using the Cheng-Prusoff correction equation \\[28\\], where K~i~ = IC~50~\/1+ \\[L\\]\/K~d~, and K~d~ for Hh-Ag 1.5 was determined to be 0.37 nM by the saturation analysis.\n\n## Note added in proof\n\nRelated results demonstrating the action of cyclopamine on Smo have been reported by Beachy and colleagues \\[51,52\\].\n\n### Acknowledgments\n\nWe thank Douglas Melton, Andrew McMahon, Thomas Jessell and Phillip Beachy for comments on the manuscript. Shh-, Smo- and Ptc-transgenic lines were graciously provided by the labs of Andrew McMahon and Matthew Scott. We thank James Chen and Phillip Beachy for providing us with KAAD-cyclopamine and for sharing their observations that cyclopamine and its derivatives bind directly to Smoothened and that our agonists compete this interaction. Superior medicinal chemistry support as well as screening libraries were provided by Larry Kruse, Andy Boyd, Steven Price and the team at Evotec\/Oxford Asymmetry International.\n\n## Figures and Tables","meta":{"dup_signals":{"dup_doc_count":136,"dup_dump_count":52,"dup_details":{"curated_sources":2,"2022-21":2,"2021-49":1,"2021-10":1,"2020-45":1,"2020-34":1,"2020-16":1,"2020-10":1,"2019-51":1,"2019-43":2,"2019-30":1,"2019-22":1,"2019-09":2,"2018-51":1,"2018-43":1,"2018-22":1,"2018-05":1,"2017-39":3,"2017-30":1,"2017-26":2,"2017-22":3,"2017-17":1,"2017-09":24,"2017-04":2,"2016-50":1,"2016-44":3,"2016-40":3,"2016-36":25,"2016-30":20,"2016-26":1,"2016-22":2,"2016-18":2,"2016-07":1,"2015-48":1,"2015-40":1,"2015-35":1,"2015-32":1,"2015-27":1,"2015-22":1,"2015-14":1,"2014-52":1,"2014-49":1,"2014-42":2,"2014-41":1,"2014-35":1,"2014-23":1,"2023-23":1,"2015-11":1,"2015-06":1,"2014-10":1,"2013-48":1,"2013-20":1,"2015-18":1}},"file":"PMC137065"},"subset":"pubmed_central"}
{"text":"author: Vandana Mehta; C Balachandran; Sudhir Nayak\ndate: 2008\nreferences:\ntitle: HERPETIFORM PEMPHIGUS CLINICALLY RESEMBLING BULLOUS PEMPHIGOID\n\nHerpetiform pemphigus was first introduced by Jablonska in 1975, as a variant of pemphigus that combines the clinical features of dermatitis herpetiformis with the immunological features of pemphigus.1 Because of its rare and atypical clinical presentation, patients are often initially diagnosed as Dermatitis herpetiformis, Linear IgA bullous disease, Bullous pemphigoid or Pemphigus foliaceus. We report a case of herpetiform pemphigus in an elderly lady which was initially misdiagnosed as bullous pemphigoid, however histology and immunofluorescence helped in clinching the diagnosis.\n\nA 75-year-old woman presented with a one year history of an intensely pruritic eruption which began on her extremities and later expanded to involve the trunk with only partial response to topical corticosteroids and antihistamines. She was otherwise healthy with no accompanying constitutional symptoms and not on any other medications. Cutaneous examination revealed a symmetric polymorphous eruption of old and new lesions, comprising of erythematous urticarial papules and plaques studded with clear and cloudy vesicles (<a href=\"#F0001\" data-ref-type=\"fig\">Fig. 1<\/a>), ulcerated and crusted lesions arranged in herpetiform groups. Oral and genital mucosa were normal. Clinically a differential diagnosis of bullous pemphogoid was entertained, however a Tzanck smear from a clear vesicle showed abundant neutrophils, with only few eosinophils. Biopsy for histopathology showed mild epidermal hyperplasia with a subcorneal blister containing neutrophils and few acantholytic cells. Predominant areas of neutrophilic spongiosis with a superficial perivascular infiltrate of eosinophils and neutrophils were seen in the papillary dermis. Direct and indirect immunofluorescence demonstrated strong intercellular deposits of IgG and C3 in the upper epidermis. Based on the histology and immunofluorescence findings, the diagnosis was revised to herpetiform pemphigus and patient was started on oral steroids.\n\nPemphigus herpetiformis (PH) is recognized as a distinct subset of pemphigus by its pruritus, rarity of mucosal involvement, invariable presence of eosinophils on histology and its tendency to respond to sulfones. Various terms that have been used to describe it are *acantholytic herpetiform dermatitis*, *pemphigus controlled by sulfapyridine*, *mixed bullous disease* and *eosinophilic spongiosis in pemphigus*. Clinically patients present with severe intractable pruritus and erythematous urticarial papules and plaques with occasional vesicles in a herpetiform arrangement. Even though the clinical picture is reminiscent of dermatitis herpetiformis, the histological findings are variable and include eosinophilic spongiosis, subcorneal pustules with minimal or no apparent acantholyis.2 Though eosinophilic spongoisis is classically seen in pemphigus herpetiformis, neutrophilic spongiosis may also be encountered and should be recognized as an important diagnostic clue3 as was seen in our case. Definitive diagnosis is established by immunopathology which shows *in vivo* bound IgG deposits on the keratinocyte cell surface primarily in the upper epidermis. The auto-antibodies in most patients of PH target desmoglein 1 and because desmoglein 1 is predominantly located in the upper epidermis, there is preferential binding of IgG to the upper epidermis as was seen in our case. Why the auto-antibodies in PH and pemphigus vulgaris give rise to different clinical presentations could be explained by their preferential binding to different epitopes on the same antigen molecule.4\n\nIn our case, the presence of severe pruritus in an elderly female with blisters and absence of mucosal involvement made us think of bullous pemphigoid, however, to our surprise the histology and immunofluorescence turned out to be PH. Patient was started on oral steroids along with dapsone and is currently under follow-up.\n\n# References","meta":{"dup_signals":{"dup_doc_count":168,"dup_dump_count":7,"dup_details":{"curated_sources":2,"2015-18":27,"2015-11":24,"2015-06":26,"2014-10":21,"2013-48":28,"2013-20":25,"2017-13":1,"unknown":14}},"file":"PMC2763728"},"subset":"pubmed_central"}
{"text":"abstract: # OBJECTIVE\n  .\n  The Diabetes Prevention Program (DPP) and its Outcomes Study (DPPOS) demonstrated that either intensive lifestyle intervention or metformin could prevent type 2 diabetes in high-risk adults for at least 10 years after randomization. We report the 10-year within-trial cost-effectiveness of the interventions.\n  .\n  # RESEARCH DESIGN AND METHODS\n  .\n  Data on resource utilization, cost, and quality of life were collected prospectively. Economic analyses were performed from health system and societal perspectives.\n  .\n  # RESULTS\n  .\n  Over 10 years, the cumulative, undiscounted per capita direct medical costs of the interventions, as implemented during the DPP, were greater for lifestyle (\\$4,601) than metformin (\\$2,300) or placebo (\\$769). The cumulative direct medical costs of care outside the DPP\/DPPOS were least for lifestyle (\\$24,563 lifestyle vs. \\$25,616 metformin vs. \\$27,468 placebo). The cumulative, combined total direct medical costs were greatest for lifestyle and least for metformin (\\$29,164 lifestyle vs. \\$27,915 metformin vs. \\$28,236 placebo). The cumulative quality-adjusted life-years (QALYs) accrued over 10 years were greater for lifestyle (6.81) than metformin (6.69) or placebo (6.67). When costs and outcomes were discounted at 3%, lifestyle cost \\$10,037 per QALY, and metformin had slightly lower costs and nearly the same QALYs as placebo.\n  .\n  # CONCLUSIONS\n  .\n  Over 10 years, from a payer perspective, lifestyle was cost-effective and metformin was marginally cost-saving compared with placebo. Investment in lifestyle and metformin interventions for diabetes prevention in high-risk adults provides good value for the money spent.\nauthor: aCorresponding author: Diabetes Prevention Program Coordinating Center, <firstname.lastname@example.com>. [^1][^2][^3][^4][^5]\ndate: 2012-04\nreferences:\nsubtitle: : An intent-to-treat analysis of the DPP\/DPPOS\ntitle: The 10-Year Cost-Effectiveness of Lifestyle Intervention or Metformin for Diabetes Prevention\n\nIntensive lifestyle and metformin interventions can delay or prevent progression from impaired glucose tolerance (IGT) to type 2 diabetes (1\u20133). The Diabetes Prevention Program (DPP), a randomized controlled clinical trial, demonstrated that compared with the placebo intervention (placebo), the intensive lifestyle intervention (lifestyle) reduced the incidence of type 2 diabetes by 58%, and the metformin intervention (metformin) reduced the incidence of type 2 diabetes by 31% over 2.8 years (3). The Diabetes Prevention Program Outcomes Study (DPPOS) is a long-term follow-up of the DPP participants to investigate whether the delay in the development of diabetes observed during the DPP is sustained and to assess the long-term effects of the interventions on health.\n\nThe DPPOS has followed participants for an additional 7 years during which time participants in lifestyle and metformin were encouraged to continue those interventions, and all participants were offered a group lifestyle intervention (4). The incidence of diabetes during the 10-year average follow-up after randomization was reduced by 34% in those initially randomized to lifestyle and 18% in those initially randomized to metformin compared with placebo (4). Previously, we reported the resource utilization and costs of care in the DPP (5), and the cost per quality-adjusted life-year (QALY) gained over the 3-year timeframe of the randomized controlled clinical trial (6). We also used 3-year DPP data and a computer model to simulate the longer-term cost-effectiveness of the interventions. Although we (7) and others (8,9) suggested that lifestyle would be cost-effective or even cost-saving over the long term, one analysis suggested that it might be too expensive for health plans or a national program to implement (10). In this report, we present a within trial intent-to-treat analysis spanning the combined 10-year DPP\/DPPOS timeframe to assess the longer-term cost-effectiveness of lifestyle and metformin for diabetes prevention. All of the DPPOS clinical centers as well as the DPP Coordinating Center had institutional review board approvals. All participants gave written informed consent.\n\n# RESEARCH DESIGN AND METHODS\n\n## Interventions\n\n### DPP.\n\nThe DPP enrolled 3,234 participants with IGT and fasting hyperglycemia who were at least 25 years of age and had BMI of 24 kg\/m^2^ or higher (22 kg\/m^2^ in Asian Americans) (3). Enrollment began in July 1996 and ended in May 1999. Mean age was 51 years of age and mean BMI was 34.0 kg\/m^2^ (3). Sixty-eight percent were women, and forty-five percent were members of minority groups (3).\n\nThe goals for participants randomized to lifestyle were to achieve and maintain a weight reduction of at least 7% of initial body weight through diet and physical activity of moderate intensity, such as brisk walking, for at least 150-min per week. A 16-session core curriculum (given approximately weekly in individual participant sessions) and subsequent individual sessions (usually monthly) and group sessions with case managers were designed to reinforce the behavioral changes. The medication interventions (metformin and placebo) were initiated at a dose of 850 mg taken orally once a day. At 1 month, the dose was increased to 850-mg twice daily. Adherence was reinforced during individual quarterly visits with case managers. Standard lifestyle recommendations were provided to all groups through written information and an annual 20- to 30-min individual session that emphasized the importance of a healthy lifestyle. Mean follow-up at the end of the DPP was 3.2 years. For the purposes of this analysis, we assumed that all subjects were enrolled in the DPP for exactly 3 years.\n\n### DPP\/DPPOS bridge.\n\nAt the end of the DPP in July 2001, masked treatment was discontinued and each participant had a 1-h debriefing and closure visit during which he or she was informed of the main DPP results. In light of the proven benefits of lifestyle, all participants were offered a group-implemented 16-session lifestyle intervention between January and July 2002. Forty percent of lifestyle, fifty-eight percent of metformin, and fifty-seven percent of placebo participants attended at least one session (11). Each session lasted 1 h and was taught by one staff member. Participants received reminders for the sessions and the full packet of course materials. The original lifestyle group was offered additional lifestyle support and was not encouraged to take metformin. The original metformin group was encouraged to continue metformin and to participate in the group lifestyle intervention. Those randomized to placebo stopped placebo and were encouraged to participate in the group lifestyle intervention. For the purposes of this analysis, we assumed that year 4 represented the DPP\/DPPOS bridge.\n\n### DPPOS maintenance.\n\nThe DPPOS maintenance phase started in September 2002. All active participants were eligible for continued follow-up, and 2,766 of 3,150 (88%) enrolled (4). These included 910 from lifestyle, 924 from metformin, and 932 from placebo. During the DPPOS, the group lifestyle intervention was implemented as the Healthy Lifestyle Program (HELP) for all participants. HELP reinforced the original weight loss and physical activity goals and focused on current topics in nutrition, physical activity, stress management, and diabetes prevention. HELP consisted of four quarterly 1-h group visits. All participants received a reminder for HELP sessions. Although all participants were invited to attend all HELP sessions, many chose to attend fewer.\n\nThe DPP participants initially randomized to lifestyle were also eligible to receive two additional sessions, referred to as BOOST sessions, per year to reinvigorate their self-management behaviors for weight loss. Those randomized to metformin and placebo were excluded from BOOST sessions. The sessions were more intensive than HELP sessions and reinforced specific behavioral self-management activities (e.g., self-monitoring of fat, calories, and\/or physical activity, as well as weight checks) important for weight loss and physical activity adherence and\/or maintenance. In addition, the sessions promoted home-based behavioral self-management of weight and physical activity through the use of motivational campaigns. Lifestyle participants received reminders for each BOOST session.\n\nDuring the DPPOS, metformin participants taking study-provided metformin received an annual complete blood count and serum creatinine for drug safety monitoring. Participants were encouraged to see their personal physicians for treatment of emergent adverse events. Only metformin participants were encouraged to take metformin, and only 1% of nondiabetic participants in lifestyle and 3% of nondiabetic participants in placebo took metformin prescribed outside the study (4). For the purposes of this analysis, we assumed that years 5\u201310 represented DPPOS follow-up.\n\n### Interventions for participants who developed diabetes.\n\nParticipants identified with glucose levels diagnostic of diabetes at their 6-monthly visits were seen within 6 weeks for glucose testing to confirm the diagnosis. Participants with confirmed newly diagnosed diabetes received 1 h of individual counseling focused on self-monitoring of blood glucose, were provided with meters and test strips and encouraged to monitor their glucose levels once daily, and were maintained in their randomized intervention groups. Treatment for diabetes and surveillance for complications and comorbidities were performed by the participants' own health care providers. Medications used by the DPP participants for management of diabetes were recorded every 6 months on a drug summary form.\n\n## Costs\n\nWe calculated the total direct medical costs associated with the DPP\/DPPOS interventions over each of the 10 years after randomization ([Supplementary Table 1](http:\/\/care.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/dc11-1468\/-\/DC1)). Direct medical costs of the interventions were estimated from the resources used and unit costs adjusted to 2010 U.S. dollars ([Supplementary Table 2](http:\/\/care.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/dc11-1468\/-\/DC1)). As a sensitivity analysis, we estimated what the cost of lifestyle might have been during the DPP if it had been administered in a group format rather than individually (DPP group lifestyle intervention). We recalculated the costs of lifestyle assuming that the core curriculum and monthly follow-up visits with the lifestyle case managers, which were conducted individually during the 3 years of the DPP, were conducted as group sessions with 10 participants. Studies have shown that group intervention programs can be as effective as individual programs (12,13). Although metformin was implemented with brand name metformin (Glucophage), we assumed that it was implemented with generically priced metformin throughout the 10 years of the DPP\/DPPOS.\n\nAs previously described, we also estimated the direct medical cost of care outside the study (5). The direct medical cost of care outside the study included the costs of hospital, emergency room, urgent care, outpatient services, and telephone calls to health care providers. These were determined annually from patient self-report. They also included the cost of prescription medications. This was estimated from the number of prescription medications that participants reported taking at semiannual visits and the mean average wholesale price of a prescription medication dispensed by a large U.S. pharmacy benefit manager in 2009.\n\nDirect nonmedical costs were assessed twice, once during the DPP and once during the DPPOS, and costs were annualized. In estimating the direct nonmedical costs of the interventions, we considered the costs of food, food preparation items (blenders, cookbooks, food scales, freezers, microwave ovens, mixers, popcorn poppers, steamers, and woks), exercise classes, gym memberships, personal trainers, and exercise equipment (bicycles, exercise videos, free weights, golf clubs, home gyms, shoes, stationary bicycles, steps and treadmills) (5). We also considered the costs of transportation to study visits and to medical visits (5). The value of the time that participants spent shopping, cooking, exercising, and traveling to and attending appointments was also assessed (5). The costs of exercise were valued according to whether participants \"disliked,\" were \"neutral,\" or \"liked\" leisure time physical activity (5,14) ([Supplementary Table 2](http:\/\/care.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/dc11-1468\/-\/DC1)). Although direct nonmedical costs are not usually paid by private insurers or government health programs, we included them in our cost calculations from a societal perspective.\n\n## Outcomes\n\nWe assessed outcomes in terms of QALYs (15). QALYs measure length of life adjusted for quality of life as assessed by the health utility score. By convention, health utility scores are placed on a continuum where perfect health is assigned a value of 1.0 and health judged equivalent to death is assigned a value of 0.0. We assessed health utilities annually using the Self-Administered Quality of Well-Being Index (QWB-SA) (6). The QWB-SA is a widely used, validated, multiattribute utility model that combines preference-weighted values for symptoms and functioning to provide a health utility score. The numerical value assigned by the QWB-SA to quality of life reflects the public's judgment of the desirability of the health state. Mathematically, QALYs are calculated as the sum of the product of the number of years of life and the quality of life, measured in health utilities, in each of those years.\n\n## Perspective\n\nFor the base-case analysis, we followed the recommendations of the Panel on Cost-Effectiveness in Health in Medicine (15) and took the perspective of a health system. Thus, we included only direct medical costs in our base-case analysis. We included direct nonmedical costs excluding participant time in a sensitivity analysis from a modified societal perspective and direct nonmedical costs including participant time in a sensitivity analysis from a full societal perspective. These sensitivity analyses assessed the impact of covering the cost of the behavioral interventions implemented by the study participants on society as a whole.\n\n## Analyses\n\nThe analyses of lifestyle, metformin, and placebo were based on the design, cost, and clinical effectiveness of the interventions as implemented in the 3 years of the DPP and the 7 years of the DPPOS. For the DPP group lifestyle sensitivity analysis, we estimated what the costs of lifestyle would have been during the 3 years of DPP if the 16-session core curriculum and monthly follow-up visits with the case managers had been conducted as group sessions with 10 participants. We further assumed that outcomes would have been the same as observed for lifestyle. We excluded from the analyses the costs of the research component of the DPP\/DPPOS. All costs were expressed as year 2010 U.S. dollars ([Supplementary Table 2](http:\/\/care.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/dc11-1468\/-\/DC1)). Analyses were performed with a 10-year time horizon. Initial analyses were performed without discounting. Subsequently, both cost and health outcomes were converted to net present value using a 3% discount rate, and incremental cost-effectiveness ratios were calculated using the discounted costs and QALYs.\n\n# RESULTS\n\n## Costs\n\nThe annual undiscounted, per capita, direct medical costs of lifestyle, metformin, and placebo over 10 years are summarized in <a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a> as are the costs of the DPP group lifestyle sensitivity analysis. <a href=\"#F1\" data-ref-type=\"fig\">Figure 1<em>A<\/em><\/a> illustrates the cumulative, undiscounted, per participant intervention costs. The cumulative, undiscounted per participant cost of the lifestyle intervention as implemented in the DPP (\\$4,601) was substantially greater than the metformin intervention (\\$2,300) or the placebo intervention (\\$769). The estimated cost of the DPP group lifestyle intervention (\\$3,023) was approximately one-third less than that of the lifestyle intervention. The costs of lifestyle were substantially less during the DPPOS than during the DPP because of the change from an individual- to a group-implemented intervention and because fewer visits took place. The costs of placebo were higher during the DPPOS than during the DPP because placebo participants engaged in the group lifestyle intervention. During the DPPOS, lifestyle and metformin each cost approximately \\$140 per participant per year. The costs of the interventions during the DPP differ somewhat from those reported previously as we have added the costs of fasting glucose and glucose tolerance testing and incorporated generic pricing for metformin (5). Detailed cost calculations are presented in [Supplementary Table 1](http:\/\/care.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/dc11-1468\/-\/DC1).\n\nUndiscounted, per capita, direct medical costs of the DPP\/DPPOS interventions by intervention group and study year (\\$)\\*\n\n![](723tbl1)\n\nThe cumulative, undiscounted per capita direct medical costs of nonintervention-related medical care by intervention group and year following randomization are shown in <a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a> and <a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>B<\/em><\/a>. These are the costs of medical care received outside the DPP\/DPPOS. The direct medical costs of nonintervention-related medical care were substantially greater than the costs of the interventions, and within 3 years, the cumulative costs of nonintervention-related medical care exceeded the 10-year cumulative direct medical costs of the interventions. The cumulative per-participant direct medical costs of nonintervention-related medical care increased substantially over time. From the outset of the DPP, the per-participant costs of placebo exceeded those of lifestyle and metformin. The greater cost of nonintervention-related medical care for placebo was largely driven by greater use of outpatient and inpatient services, prescription medications, and by the greater rate of conversion to diabetes with the attendant costs of self-monitoring and laboratory tests (<a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a>). Across treatment groups, the direct medical costs of nonintervention-related medical care were 34\u201344% higher among diabetic participants compared with nondiabetic participants ([Supplementary Table 3](http:\/\/care.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/dc11-1468\/-\/DC1)). Over 10 years, cumulative, per capita nonintervention-related direct medical costs were greater by \\$1,853 and \\$2,905 for placebo compared with metformin and lifestyle, respectively.\n\nUndiscounted, per capita, direct medical costs of care outside the DPP\/DPPOS by intervention group and study year, and distribution of undiscounted, per capita, 10-year, direct medical costs of care outside the DPP\/DPPOS by intervention group and type (\\$)\n\n![](723tbl2)\n\nOver 10 years, the cumulative, undiscounted, per capita direct medical costs of the interventions were greater for participants randomized to lifestyle (\\$4,601) and metformin (\\$2,300) compared with placebo (\\$769). In contrast, the cumulative, undiscounted, per capita direct medical costs of nonintervention-related medical care (medical care received outside the DPP\/DPPOS) were greater for placebo (\\$27,468) than for metformin (\\$25,615) or lifestyle (\\$24,563). By year 10, the cumulative, undiscounted, per capita, total direct medical costs of the interventions and nonintervention-related medical care were higher for lifestyle than for placebo but were lower for metformin than for placebo (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>C<\/em><\/a>). They were also lower for the DPP group lifestyle (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>C<\/em><\/a>).\n\n[Supplementary Table 4](http:\/\/care.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/dc11-1468\/-\/DC1) summarizes the undiscounted, per capita, cumulative, 10-year direct nonmedical costs by intervention group and type defined as diet-related costs, physical activity\u2013related costs, transportation-related costs, and participant time-related costs. Diet-related costs were substantial but did not differ among intervention groups. As might be expected, physical activity\u2013related costs were greatest for lifestyle. Transportation-related costs were also substantially higher for lifestyle and metformin due to the greater number of study visits. Total diet-, physical activity-, and transportation-related costs were greatest for lifestyle but similar for metformin and placebo. Participant time contributed substantially to direct nonmedical costs. Participant time related to the interventions (time spent traveling to study visits, at study visits, and for intervention-related calls) was greater for lifestyle and metformin than for placebo. Participant time related to medical care outside of the interventions was generally greater for placebo than for metformin or lifestyle. Time spent shopping and cooking was the largest component of participant time but differed little across intervention groups. Although lifestyle subjects spent more time exercising, the adjusted value of the time they spent exercising was less than for either metformin or placebo because of their greater enjoyment of leisure time physical activity and the lower opportunity cost. The total, per capita, 10-year, direct nonmedical costs\u2014including the costs of participant time\u2014were lowest for metformin (\\$144,143) and similar for placebo and lifestyle (\\$147,043 and \\$147,493, respectively).\n\n## Health utilities\n\nEvery year after randomization, quality of life was better for lifestyle than for metformin or placebo ([Supplementary Table 3](http:\/\/care.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/dc11-1468\/-\/DC1)). Across treatment groups, quality of life was worse among participants who developed diabetes ([Supplementary Table 3](http:\/\/care.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/dc11-1468\/-\/DC1)). Since more placebo participants developed diabetes, the cumulative, undiscounted, per participant quality of well-being score gained over 10 years was greatest for lifestyle (6.81), intermediate for metformin (6.69), and least for placebo (6.67) (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>D<\/em><\/a>).\n\n## Cost-effectiveness\n\n<a href=\"#T3\" data-ref-type=\"table\">Table 3<\/a> summarizes the differences in total costs and QALYs among the treatment groups and the incremental cost-effectiveness ratios of lifestyle and metformin versus placebo, and lifestyle versus metformin. The incremental cost-effectiveness ratio is also shown for the DPP-group lifestyle versus placebo. From the health system perspective, from the modified societal perspective, and from the societal perspective, lifestyle cost more than placebo but was also more effective as assessed by the QALYs that were gained. From a health system perspective, with both costs and health outcomes discounted at 3% per year, the cost of lifestyle compared with placebo was approximately \\$10,000 per QALY gained; however, metformin had slightly lower costs and nearly the same outcome (as assessed by QALYs) as placebo. Compared with metformin, lifestyle cost more but produced better health outcomes. From a health system perspective, with both costs and health outcomes discounted at 3% per year, the cost of lifestyle compared with metformin was approximately \\$13,400 per QALY gained. DPP-group lifestyle, like metformin, was generally less expensive and more effective than placebo.\n\nDifferences in costs and QALYs and incremental cost-effectiveness ratios for lifestyle and metformin versus placebo and lifestyle versus metformin over 10 years from three alternative perspectives\n\n![](723tbl3)\n\n# CONCLUSIONS\n\nUsing 3 years of DPP data and computer simulation modeling, we and others suggested that screening for glucose intolerance in the overweight and obese population and implementing lifestyle and metformin interventions would have favorable cost-effectiveness ratios (7\u20139,16). However, one analysis suggested that lifestyle might be too expensive for health plans or a national program to implement (10). The current study, a 10-year, within-trial, intention-to-treat analysis of the DPP\/DPPOS demonstrates that lifestyle is indeed cost-effective, and metformin is marginally cost-saving or at least cost-neutral compared with placebo. Even when the direct nonmedical costs of the interventions are considered, the interventions are cost-effective. Health and social policies should support the funding of intensive lifestyle and metformin interventions for diabetes prevention in high-risk adults.\n\nWhen a new treatment is more effective and less costly than usual care, it should be widely adopted and used. Unfortunately, fewer than 1 in 5 new interventions in health and medicine are cost-saving compared with usual care (17). Published cost-effectiveness ratios\u2014that is the cost in dollars per QALY gained for prevention and treatment\u2014range from less than \\$10,000 per QALY to greater than \\$1 million per QALY with most falling between \\$10,000 and \\$50,000 per QALY. While influenza immunization has been demonstrated to be cost-saving in the Medicare population, interventions such as mammography, antihypertensive treatment, and cholesterol treatment for secondary prevention of cardiovascular disease have been estimated to cost between \\$10,000 and \\$60,000 per QALY (18). Widely implemented interventions such as dialysis for end-stage renal disease (\\$50,000 to \\$100,000 per QALY) and left ventricular assist devices (\\$500,000 to \\$1.4 million per QALY) are substantially more expensive.\n\nFrom the perspective of a health system or society, what is the value of delaying or preventing the development of type 2 diabetes? From a health system perspective, it delays or prevents the direct medical costs of diabetes including the costs of diabetes education and nutritional counseling, glucose monitoring, antihyperglycemic treatments, and surveillance and treatment of complications (19\u201321). From a societal perspective, diabetes prevention also reduces health care\u2013related costs to the individual not reimbursed by the health system and time lost from work and usual activities (19). It also improves quality of life.\n\nThe direct medical costs of diabetes are enormous. The American Diabetes Association has estimated that total per capita health care expenditures for people with diabetes are \\$11,744 per year, of which \\$6,649 is attributed to diabetes (19). This estimate likely overstates the costs of diabetes in DPP participants, since DPP participants who developed diabetes were screen-detected very early in their clinical course and had few complications. The costs of diabetes increase with duration of diabetes and with the presence of complications and comorbidities and would be expected to be lower for persons with short durations of diabetes and for those without complications (22). Patients with new clinically diagnosed diabetes have been reported to have costs 2.1 times those of individuals without diabetes, and the incremental cost of diabetes is apparent from the time of diagnosis (20). In 2000, using data from a single managed-care health plan, we estimated that the median, annual, direct medical cost of care for a man with diet-controlled type 2 diabetes with no microvascular, neuropathic, or cardiovascular risk factors or complications was approximately \\$1,700 (23). More recently, using data from approximately 7,100 type 2 diabetic patients enrolled in eight managed-care health plans participating in Translating Research Into Action for Diabetes (TRIAD), we demonstrated that median, annual, per capita, direct medical costs of care were approximately \\$2,500 for a man with recent-onset diabetes without complications or comorbidities (R. Li, personal communication). These costs of recent-onset type 2 diabetes are quite consistent with those observed during the DPP\/DPPOS. Compared with the substantial costs of diabetes, the costs of lifestyle, metformin, and DPP group lifestyle seem quite small.\n\nIt is now clear that the use of a 3-year time horizon in our previous within-trial economic analysis resulted in a higher cost per QALY gained than the current analysis, which used a 10-year time horizon (6). With a 3-year time horizon, treatment costs were higher, and the benefits of lifestyle and metformin in terms of averted diabetes were less. The costs of both lifestyle and metformin were greatest in year 1, decreased substantially in years 2 and 3, and decreased further during years 4 through 10. In contrast, much of the benefit of both lifestyle and metformin, as assessed by both cumulative, nonintervention-related direct medical costs and quality of life, occurred after 3 years of follow-up.\n\nThere are a number of limitations to our analyses. First, the DPPOS was an observational follow-up of the DPP, a randomized controlled clinical trial. It is likely that during the DPPOS, when 57% of placebo participants also attended at least one group lifestyle intervention (HELP) session, placebo was more effective than the usual care that nonstudy participants might receive (11). Thus, if real-world usual care was used for comparison, the difference between lifestyle and placebo might have been greater. The fact that 58% of metformin participants also attended at least one HELP session may also have made metformin appear more effective and cost-effective compared with lifestyle (11). Second, the costs of medical care outside the interventions appear low compared with those reported in the literature for people with diabetes (19). This likely reflects the fact that trial participants were earlier in the natural history of diabetes and were healthier than patients with diabetes in the general population. It is important to note that resource utilization and cost were assessed in an identical fashion across intervention groups so the differences among groups, which determine the incremental cost-effectiveness ratios, should be reasonably accurate. Third, in our analysis of the DPP group lifestyle intervention, we assumed that lifestyle could be implemented in a group rather than in an individual format at one-third lower cost and achieve the same outcomes. Although group-implemented lifestyle interventions have been shown to be at least as effective as individual programs for weight loss, there has not been a direct comparison of individual and group lifestyle interventions for diabetes prevention (12,13). Finally, we recognize that in assessing the direct nonmedical costs of the interventions, we have overestimated diet-related costs. Clearly, people need to eat whether or not they participate in a randomized controlled clinical trial. We did not have a good method for distinguishing study-related and nonstudy-related food consumption. Nevertheless, the data are instructive in that they indicate that there were not substantial differences in diet-related costs across intervention groups.\n\nThis 10-year, within trial, intention-to-treat economic analysis of the DPP\/DPPOS demonstrates that lifestyle, when compared with placebo, is cost-effective, and metformin is marginally cost-saving from a health system and societal perspective. If a DPP group lifestyle intervention could be delivered at one-third lower cost than the DPP lifestyle intervention and achieve the same outcomes, it would also be cost-saving or cost-effective compared with placebo. Even when compared with metformin, lifestyle was cost-effective from both a health system and societal perspective. These analyses should assist health plans and policy makers in comparing the benefit of diabetes prevention to other preventive and palliative interventions. The adoption of diabetes prevention programs by health plans and society will result in important health benefits over 10 years and represents a good value for the money spent.\n\n## Acknowledgments\n\nDuring the DPPOS, the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) of the National Institutes of Health provided funding to the clinical centers and the DPP Coordinating Center for the design and conduct of the study, and collection, management, analysis, and interpretation of the data. The Southwestern American Indian Centers were supported directly by the NIDDK, including its Intramural Research Program, and the Indian Health Service. The General Clinical Research Center Program, National Center for Research Resources, and the U.S. Department of Veterans Affairs supported data collection at many of the clinical centers. Funding was also provided by the National Institute of Child Health and Human Development; the National Institute on Aging; the National Eye Institute; the National Heart, Lung, and Blood Institute; the Office of Research on Women's Health; the National Institute on Minority Health and Health Disparities; the Centers for Disease Control and Prevention; and the American Diabetes Association.\n\nBristol-Myers Squibb and Parke-Davis provided additional funding and material support during the DPP. Lipha (Merck-Sante) provided medication and LifeScan Inc. donated materials during the DPP and the DPPOS. No other potential conflicts of interest relevant to this article were reported.\n\nW.H.H. researched data, wrote the manuscript, and is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. S.L.E. and M.B.B. researched data, conducted analyses, reviewed and edited the manuscript, and contributed to discussion. R.E.R., M.G.M., R.T.A., T.J.O., M.A.F., P.Z., and C.D.S. researched data, reviewed and edited the manuscript, and contributed to discussion.\n\nParts of this study were presented in oral form at the 71st Scientific Sessions of the American Diabetes Association, San Diego, California, 24\u201328 June 2011, and in poster form at the 47th Annual Meeting of the European Association for the Study of Diabetes, Lisbon, Portugal, 12\u201316 September 2011.\n\nThe DPP Research Group gratefully acknowledges the commitment and dedication of the participants of the DPP and the DPPOS.\n\n### APPENDIX\n\nMembers of the writing group are William H. Herman, MD, MPH^1^; Sharon L. Edelstein, ScM^2^; Robert E. Ratner, MD^3^; Maria G. Montez, RN, MSHP^4^; Ronald T. Ackermann, MD, MPH^5^; Trevor J. Orchard, MBBCh, MMedSci^6^; Mary A. Foulkes, PhD^2^; Ping Zhang, PhD^7^; Christopher D. Saudek, MD^8\u2020^; and Morton B. Brown, PhD^1^.\n\nFrom the ^1^University of Michigan, Ann Arbor, Michigan; ^2^The Biostatistics Center, The George Washington University, Rockville, Maryland; the ^3^Medstar Health Research Institute, Washington, D.C.; the ^4^University of Texas Health Science Center at San Antonio, San Antonio, Texas; the ^5^Northwestern University Feinberg School of Medicine, Chicago, Illinois; the ^6^University of Pittsburgh, Pittsburgh, Pennsylvania; the ^7^Division of Diabetes Translation, Centers for Disease Control and Prevention, Atlanta, Georgia; and ^8^The Johns Hopkins University School of Medicine, Baltimore, Maryland.\n\n\u2020Deceased.\n\n# References\n\n[^1]: Clinical trial reg. nos. NCT00038727 (DPPOS) and NCT00004992 (DPP), [clinicaltrials.gov](clinicaltrials.gov).\n\n[^2]: This article contains Supplementary Data online at <http:\/\/care.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/dc11-1468\/-\/DC1>.\n\n[^3]: A slide set summarizing this article is available online.\n\n[^4]: \\*A complete list of the members of the Diabetes Prevention Program Research Group, centers, and staff can be found in the Supplementary Data online, and members of the writing group are listed in the <span class=\"smallcaps\">appendix<\/span>.\n\n[^5]: The opinions expressed in this article are those of the investigators and do not necessarily reflect the views of the funding agencies.","meta":{"dup_signals":{"dup_doc_count":160,"dup_dump_count":78,"dup_details":{"curated_sources":2,"2023-40":1,"2022-21":3,"2021-49":4,"2021-43":1,"2021-39":1,"2021-31":4,"2021-25":1,"2021-21":2,"2021-17":3,"2021-10":3,"2021-04":3,"2020-50":4,"2020-45":1,"2020-40":1,"2020-34":1,"2020-29":2,"2020-16":1,"2020-10":1,"2020-05":1,"2019-51":1,"2019-47":4,"2019-43":7,"2019-35":1,"2019-26":1,"2019-04":1,"2018-51":1,"2018-47":2,"2018-43":1,"2018-39":1,"2018-30":2,"2018-26":1,"2018-22":1,"2018-17":1,"2018-13":1,"2018-09":1,"2018-05":1,"2017-51":1,"2017-47":3,"2017-43":1,"2017-39":3,"2017-34":2,"2017-30":1,"2017-26":2,"2017-22":1,"2017-17":2,"2017-09":3,"2017-04":3,"2016-50":3,"2016-44":3,"2016-40":3,"2016-36":3,"2016-30":4,"2016-26":1,"2016-22":2,"2016-18":1,"2016-07":2,"2015-48":2,"2015-40":1,"2015-35":1,"2015-32":1,"2015-27":2,"2015-22":2,"2015-14":2,"2014-52":2,"2014-49":4,"2014-42":5,"2014-41":2,"2014-35":4,"2014-23":5,"2014-15":1,"2023-50":1,"2017-13":2,"2015-18":2,"2015-11":2,"2015-06":2,"2014-10":3,"2013-48":1,"2024-10":1}},"file":"PMC3308273"},"subset":"pubmed_central"}
{"text":"abstract: We should not be distracted by the debate about whether or not global warming is caused by humans; whatever the answer is, we urgently need to retard or reverse the trend.\nauthor: Gregory A Petsko\ndate: 2009\ninstitute: 1Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02454-9110, USA\ntitle: A harsh climate\n\nI might as well come right out and say it: I don't care whether global warming is caused by manmade greenhouse gas emissions. And neither should you.\n\nBefore you start reaching for your laptops, iPhones, and BlackBerrys to fire off scathing emails, give me a moment to explain why I made this statement and what it really means. I bet that, when I'm through, you will agree with me.\n\nThis column is being written because of the confluence of two events. One is a meeting in Copenhagen of representatives of most of the world's nations, aimed at formulating a new global strategy for dealing with the climate crisis. The talks have ground to a halt as I write this because the group of developing countries, known as the G-77, has accused the United States and other industrialized states of forsaking the Kyoto Protocol, the current climate agreement that imposes greenhouse gas emissions on nearly every developed nation.\n\nThe second event is 'Climategate', the release of illegally hacked emails between climatologists. As an example of giving aid and comfort to the enemy, Climategate could hardly be improved on. In late November, a computer file including more than 1,000 emails either sent from or to members of the University of East Anglia's Climate Research Unit (CRU) was stolen and released on the Internet. The emails contain language that opponents of emission curbs have seized on as alleged examples of data manipulation and outright fraud on the part of climate researchers. For example, one email apparently sent by the head of the CRU, Professor Phil Jones, refers to using \"Mike's *Nature* trick of adding in the real temps to each series for the last 20 years ... to hide the decline\". The CRU is one of the leading research units on climate change, and their data had a major role in the Fourth Assessment Report of the Intergovernmental Panel on Climate Change (IPCC), released in 2007, that provided unequivocal evidence for global warming (see Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>).\n\nOf course, scientists use the word 'trick' all the time as a shorthand term for a method or algorithm, but professional skeptics rarely bother themselves with the way scientists work. It seems likely that the files were stolen in an attempt to undermine the Copenhagen talks, but my assessment is that there are so many other contentious issues in that meeting that this is a relatively minor matter for most of its participants. Nevertheless, Professor Jones has stepped down as head of the CRU pending an internal investigation. In my view, he should instead have been made to write on the blackboard 1,000 times: \"I will never put anything into an email or text message that could be embarrassing to me or to my organization if it were read by someone else, and if I don't believe this I should ask Tiger Woods.\"\n\nOne of the most sensible things I have read about the climate debate is an opinion piece by Stewart Brand in the 15 December 2009 edition of *The New York Times*. He argues that the popular depiction of the combatants as belonging to two camps, the alarmists and the skeptics, is fallacious. There are actually four sides: *denialists*, a group consisting of people with a right-wing political agenda who assert that the claim that global warming is caused by manmade emissions is a lie and is not based on sound science; *skeptics*, a group largely comprising scientists who argue that climate science, particularly large-scale modeling, is far too imperfect to form the basis of a consensus; *warners*, another group of scientists who believe that the best climate models accurately predict a looming planetary disaster and that human production of greenhouse gases is the primary cause; and *calamatists*, a collection of environmental activists whose agenda, like that of the denialists, is ideologically driven, but in the opposite direction: they have a neo-luddite view of industrialization, and believe the denialists are evil. As Brand, a self-described warner, points out, understanding from which of these camps any given argument springs is useful in distinguishing propaganda from science, and appeals to emotion from evidence-based assertions.\n\nYet even Brand misses what I think is the crucial point, the point I want to make in this column, which is that you can't win a war if you are fighting in the wrong field. And in the war over climate change, which should be fought in the field of science, the denialists and the calamatists have dragged us into battle on their turf.\n\nWhen you're in a fight with an opponent who is not above using invective and illogic, the worst mistake you can make is letting the other side define the terms of the debate. That's exactly what has happened in the argument about climate change. For decades the denialists insisted that the earth was not getting warmer. Short-term fluctuations were meaningless, they asserted. Climate modeling was worse than useless. The doomsayers were just trying to push a liberal political agenda, and so on. But after massive amounts of data were collected and analyzed by the Intergovernmental Panel on Climate Change, it became clear, on the release of their report in 2007, that no sensible person could deny that a dramatic rise in the planet's average temperature had been occurring for at least a century (see Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>). Largely thanks to Al Gore, this information also reached the general public, whose reaction even the staunchest denialists could not ignore.\n\nSo they did what clever, unprincipled losers often do: they changed the issue. Of course the earth is getting warmer, they said (blithely ignoring the fact that they had said exactly the opposite the day before), but human activities have nothing to do with it. It's entirely due to natural causes, and people who assert that manmade greenhouse gases are causing the problem are employing flawed science, deliberately distorting the facts (Climategate), and are using fear to advance the same old, tired environmental activism. Because global warming is not a manmade phenomenon, there is no scientific or political reason to limit manmade greenhouse gas emissions. Sarah Palin (why am I not surprised?) is one of the leaders of this chorus, stating recently that climate change occurs naturally \"like gravity\", while warning that reducing greenhouse gas emissions will mean \"job losses\" and \"economic costs\". (This is the same ex-Alaska governor Sarah Palin who, before becoming a national political figure, said in July of last year, \"Alaska's climate is warming. While there have been warming and cooling trends before, climatologists tell us that the current rate of warming is unprecedented within the time of human civilization. Many experts predict that Alaska, along with our northern latitude neighbors, will warm at a faster pace than any other areas, and the warming will continue for decades.\" I don't know whether to laugh at that kind of soulless opportunism or just cry.)\n\nThis strategy is actually working, to some extent. It's much harder to establish the cause of something than it is to prove that something is happening, and the data supporting manmade emissions as the leading driver of climate change are not nearly as persuasive, or as immune to challenge, as the data demonstrating the fact of global warming. And scientists, foolishly, have allowed that to become the center of the climate crisis debate. I say foolishly because, in so doing, they have given up the victory that they already won.\n\nThe denialists have conceded the fact of climate change. And here is my central point: once you admit that the earth is warming rapidly, it does not matter in the least whether that trend is due to manmade causes or not.\n\nRegardless of its origin, a rapidly changing climate is a very bad thing. We have built an entire civilization on the assumption of long-term climate stability. We grow wheat in Kansas rather than in the Yukon because Kansas has an ideal climate for growing wheat and the Yukon is too cold, and we assume that will still be the case 10 years from now. We build our cities on the coast because that is convenient for shipping goods, and we assume the coastline won't suddenly move 10 miles inland. We don't have air conditioning in many homes in northern California because we assume the average temperature won't suddenly rise by several degrees, making summer unbearably hot. We assume that England won't have a yearly climate like Lapland, even though its position on the globe might lead one to expect otherwise, because the Gulf Stream will always be there off the west coast, keeping things moderate. Every one of these assumptions fails in the event of significant global warming. One reason I prefer the term 'climate crisis' to 'climate change' or 'global warming' in discussing this problem is because our dependence on stable long-term climate patterns means that any change in those patterns represents a potential catastrophe on a planet-wide scale.\n\nIt doesn't matter what the cause of that crisis is; once you accept the fact that the crisis is coming, the only thing that matters is how to prevent it or slow it down. And the only way we have of doing that at the moment is to reduce our greenhouse gas emissions. Put another way, human activity may not even be causing the earth's temperature to rise, but human activity is the only means we have of doing something about it.\n\nA simple analogy may make this point clearer. Suppose we learned tomorrow that there was one chance in ten that a huge asteroid, recently discovered, was going to crash into the earth in 5 years, killing a billion people and raining debris in such amounts as to blot out sunlight significantly for a year. (A similar event is thought to have led to the extinction of the dinosaurs.) Would anyone in his or her right mind argue that, because we couldn't prove that human activity was responsible for the asteroid, there was no reason to hurt our economy by spending hundreds of billions of dollars firing nuclear-tipped rockets at it to destroy it or alter its course? Yet that's exactly what the denialists are trying to argue now, in the case of a climate crisis that has at least an equal probability of globally devastating consequences.\n\nTrue, our climate models can't predict with certainty that the steps being considered in Copenhagen will retard, halt or reverse the current warming trend. But they represent all we can do at the moment. If global warming is being caused primarily by greenhouse gases, as many thoughtful scientists believe, then they will do a lot. If global warming is actually caused by, say, sunspots or something similar, reduction of emissions may not do so much. But everyone agrees that they will do something, and my point is that something simply has to be done.\n\nI hope you see now why I started this essay as I did. We should not be debating whether human activity is responsible for global warming or not. Given that even the denialists and skeptics have conceded the fact of global warming, the debate should be over the most effective means of doing something about it. This means, I am afraid, not just limiting our discussion to controls on CO~2~ emissions. We need to look seriously at developing technologies for carbon sequestration, alternative fuels, and carbon-neutral technologies for transportation and energy production. Much of this will involve engineering microorganisms and plants, so genomics is going to be very important in enabling these technologies as we grapple with the crisis. I also see no escape from at least investigating ideas for geoengineering - solutions involving deliberate changing in sunlight absorption, carbon capture and temperature reduction on a continent- or planet-wide scale. My gut reaction to geoengineering is that it is a terrible idea, born as much of hubris as desperation, that should be shelved permanently because we will never have the kind of models that would guarantee beforehand that it could be done safely. But the fact is, we don't know what we don't know when it comes to such projects, and, given the severity of the climate crisis, if someone wants to propose that we should at least begin to study such solutions to determine the extent of our ignorance and the possibility that we might someday be able to employ them, I wouldn't say no.\n\nSo, the next time you find yourself in a debate with someone over the climate crisis, and they say that we shouldn't reduce CO~2~ emissions because there is no definitive proof that manmade greenhouse gases are the cause of global warming, respond by saying, \"Then if an alien race were threatening to exterminate mankind, you wouldn't do anything to try to stop them because human activities weren't the cause of the alien invasion, is that right?\" And they'll reply, \"Of course not! But this is completely different.\" And you'll say, \"No, it's not. Let me explain why.\"\n\nGiven the harsh climate that has developed around the subject of global warming, you probably won't convince them that they're wrong. But at least you'll be having the right argument.","meta":{"dup_signals":{"dup_doc_count":253,"dup_dump_count":62,"dup_details":{"curated_sources":2,"2019-51":1,"2019-47":1,"2019-39":2,"2019-30":1,"2019-26":1,"2019-22":1,"2019-18":1,"2019-13":2,"2019-04":2,"2018-47":4,"2018-39":2,"2018-34":1,"2018-30":2,"2018-26":1,"2018-22":2,"2018-17":4,"2018-13":1,"2018-09":4,"2018-05":1,"2017-51":4,"2017-47":2,"2017-43":3,"2017-39":1,"2017-34":2,"2017-30":2,"2017-26":3,"2017-22":4,"2017-17":2,"2017-09":13,"2017-04":3,"2016-50":4,"2016-44":5,"2016-40":5,"2016-36":14,"2016-30":14,"2016-26":4,"2016-22":5,"2016-18":5,"2016-07":9,"2015-48":7,"2015-40":3,"2015-35":8,"2015-32":6,"2015-27":7,"2015-22":8,"2015-14":4,"2014-52":4,"2014-49":4,"2014-42":10,"2014-41":6,"2014-35":4,"2014-23":4,"2014-15":6,"2023-14":1,"2017-13":4,"2015-18":6,"2015-11":4,"2015-06":3,"2014-10":5,"2013-48":4,"2013-20":4,"2024-18":1}},"file":"PMC2812938"},"subset":"pubmed_central"}
{"text":"abstract: Monosodium urate crystals stimulate monocytes and macrophages to release IL-1\u03b2 through the NALP3 component of the inflammasome. The effectiveness of IL-1 inhibition in hereditary autoinflammatory syndromes with mutations in the NALP3 protein suggested that IL-1 inhibition might also be effective in relieving the inflammatory manifestations of acute gout. The effectiveness of IL-1 inhibition was first evaluated in a mouse model of monosodium urate crystal-induced inflammation. IL-1 inhibition prevented peritoneal neutrophil accumulation but TNF blockade had no effect. Based on these findings, we performed a pilot, open-labeled study (trial registration number ISRCTN10862635) in 10 patients with gout who could not tolerate or had failed standard antiinflammatory therapies. All patients received 100 mg anakinra daily for 3 days. All 10 patients with acute gout responded rapidly to anakinra. No adverse effects were observed. IL-1 blockade appears to be an effective therapy for acute gouty arthritis. The clinical findings need to be confirmed in a controlled study.\nauthor: Alexander So; Thibaut De Smedt; Sylvie Revaz; J\u00fcrg Tschopp\ndate: 2007\ninstitute: 1Service of Rhumatologie, Department of Medicine, Centre Hospitalier Universtaire Vaudois and University of Lausanne, 1011 Lausanne, Switzerland; 2Apoxis SA, Avenue de S\u00e9velin 18-20, 1004 Lausanne, Switzerland; 3Institute of Biochemistry, University of Lausanne, chemin de Boveresses 155, 1066 Epalinges, Switzerland\nreferences:\ntitle: A pilot study of IL-1 inhibition by anakinra in acute gout\n\n# Introduction\n\nAcute gout is a common cause of arthritis, affecting approximately 1% of the adult population, and epidemiological evidence suggests that its prevalence is increasing \\[1\\]. Current treatments during an acute attack include nonsteroidal antiinflammatory drugs (NSAIDs), colchicine and corticosteroids. Although these agents are generally effective, they also present significant risks in patients who have pre-existing renal, cardiovascular and gastrointestinal diseases.\n\nGouty inflammation is due to monosodium urate (MSU) crystal-induced release of proinflammatory cytokines from leukocytes. Among the many cytokines implicated \\[2,3\\], IL-1 may have a special role in the inflammatory network, as MSU crystals stimulate IL-1 release by monocytes and synovial mononuclear cells \\[4\\]. The MSU crystals trigger IL-1 release through innate immune pathways, which include TLR-2 and TLR-4, found on the surface of monocytes and macrophages, as well as the 'inflammasome' complex that leads to IL-1\u03b2 activation \\[5,6\\]. The inflammasome acts as an intracellular sensor of inflammatory stimuli and regulates the activation of caspase-1. On assembly of the inflammasome, which consists of a member of the nucleotide-binding oligomerization domain-leucine rich repeat protein family (such as NALP1, NALP2, NALP3, or IPAF), the adaptor protein ASC and caspase-1 \\[7\\], caspase-1 becomes active and cleaves pro-IL-1\u03b2 to release the mature p17 form of IL-1\u03b2. Activators of the NALP3 inflammasome include ATP, the microbial cell-wall component muramyl dipeptide and bacterial RNAs \\[8-10\\]. MSU and calcium pyrophosphate dihydrate crystals directly activate the inflammasome via NALP3 in monocytes or macrophages to release active IL-1\u03b2 and cause neutrophil influx into the peritoneal space when administered by intraperitoneal injection. These responses were abrogated in ASC^-\/-^ or caspase-1^-\/-^ mice \\[6\\].\n\nSpontaneous activation of the NALP3 inflammasome due to mutations in the *NALP3* gene has been implicated in hereditary autoinflammatory syndromes such as Muckle\u2013Wells syndrome and chronic infantile neurologic cutaneous articular \\[11\\]. Affected patients respond dramatically to IL-1 inhibition \\[12,13\\], suggesting that IL-1\u03b2 plays a crucial role in the pathogenesis of inflammation in these conditions. Based on these findings, we questioned whether IL-1 inhibition may also have a beneficial effect in gouty inflammation.\n\nAs treatment with drugs currently used in acute gout is not always well tolerated or is contraindicated due to coexistent medical problems, we investigated the validity of IL-1 blockade as therapy in acute gout. We first analyzed the effects of IL-1 inhibition using the mouse peritoneal model of MSU-induced inflammation and then assessed the effects of anakinra in patients with acute gout who presented contraindications or were refractory to standard treatment in an open study.\n\n# Methods\n\n## Reagents\n\nMSU crystals were prepared based on the method described previously \\[14\\]. Briefly, 1.68 g uric acid was dissolved in 500 ml of 0.01 M NaOH and heated to 70\u00b0C. NaOH was added as required to maintain the pH between 7.1 and 7.2, and the solution was filtered and incubated at room temperature with little stirring slowly and continuously 24 hours.\n\n## Animal studies\n\nBALB\/C mice were treated intraperitoneally with PBS or 0.5 mg MSU crystals in 0.5 ml sterile PBS. Some mice were injected intraperitoneally with 200 \u03bcg anti-IL-1RI mAb (clone 35F5; BD Pharmingen, San Jose, CA, USA) or with 200 \u03bcg anti-TNF mAb (clone TN3-19.12; BD Pharmingen) or with 200 \u03bcg anakinra (Kineret; Amgen, Thousand Oaks, CA, USA) at the time of MSU injection. Mice were euthanized after 6 hours by CO~2~ exposure and the peritoneal cavities were washed with 10 ml cold PBS. The lavage fluids were analyzed for neutrophil recruitment by fluorescence-activated cell sorting using the neutrophil markers Ly-6G and CD11b (BD Pharmingen). Five mice per group were used for study.\n\n## Human studies\n\nThe diagnosis of gout arthritis was based on clinical and laboratory features. All patients fulfilled the American College of Rheumatology criteria for acute gouty arthritis \\[15\\], but all had a long previous history of either recurrent gouty attacks or tophaceous gout. Patients were treated with anakinra on an open-label basis. All subjects had either failed conventional treatment with NSAIDs, colchicine or corticosteroids for at least 48 hours or had developed significant side-effects on these drugs in the past. Patients with an active untreated infection, with uncontrolled diabetes, with uncontrolled heart or respiratory failure or with chronic renal failure with a creatinine clearance \\<30 ml\/min were not eligible. Joint infection was excluded by prior bacterial culture in all cases that underwent joint aspiration.\n\nAll patients consented to receive anakinra. Treatment was administered daily at a dose of 100 mg subcutaneously for 3 days. On starting anakinra, the NSAID or colchicine therapy was discontinued. Patients who were already on low-dose corticosteroids continued their treatment at the same dose. Clinical efficacy was assessed by clinical examination of the swollen and tender joint count as well as the patient's evaluation of the efficacy of treatment in terms of pain reduction after 3 days of therapy in comparison with their symptoms before treatment. All patients were followed up for \\>1 month. During follow-up, patients were evaluated for side-effects related to the treatment and for joint symptoms related to gout. All patients were followed by physicians responsible for this study (AS and SR). The study protocol was approved by the local institution's ethics committee and has the ISRTCN trial registration number 10862635.\n\n# Results\n\n## MSU-induced peritoneal inflammation and its inhibition by anakinra or anti-IL-1RI mAb\n\nTo study the inflammatory response to MSU crystals *in vivo*, we used a well-described model of neutrophil infiltration in the peritoneal cavity subsequent to MSU crystal administration. In previous experiments we determined that intraperitoneal administration of MSU crystals induced a dose-dependent neutrophil accumulation at the site of crystal deposition, with a plateau effect observed at 500 \u03bcg (data not shown), and this dose was therefore used in subsequent experiments. We went on to determine whether IL-1 blockade reduced the inflammatory response in the same model. Coadministration of MSU crystals with two different IL-1 inhibitors (anti-IL-1RI mAb or IL-1R antagonist (anakinra)) had similar and marked inhibitory effects on neutrophil recruitment (Figure <a href=\"#F1\" data-ref-type=\"fig\">1a<\/a>). This effect was not observed when anti-TNF blocking mAb was administered in the same fashion as the IL-1 inhibitors (Figure <a href=\"#F1\" data-ref-type=\"fig\">1b<\/a>). The differences were statistically significant in comparison with the positive MSU control.\n\nThe same dose of TNF inhibitor was effective in suppressing liver inflammation induced by concavalin A administration, a model of hepatitis dependent on TNF production (data not shown).\n\n## Effects of anakinra in gout patients\n\nThe effectiveness of IL-1 inhibition in suppressing the symptoms of acute gout was assessed in 10 patients who could not tolerate or did not respond to standard antiinflammatory treatments. A summary of the medical histories is presented in Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>. All patients responded rapidly to the drug, with the most rapid onset observed within 24 hours. In all patients, subjective symptoms of gout were greatly relieved by 48 hours after the first injection. No side-effects were observed during the study period. Clinical examination of affected joints showed complete resolution of signs of arthritis in 9\/10 patients on day 3 after initiation of treatment.\n\nClinical summary of the 10 patients studied and their response to treatment\n\n| Patient | Clinical presentation | Affected joints | Serum uric acid (normal range, 160\u2013390 \u03bcmol\/l) | Serum creatinine (normal range, 44\u201380 \u03bcmol\/l) | Hypouricemic treatment | Effect of anakinra (hours) | Patient assessment of improvement in pain (%) |\n|----|----|----|----|----|----|----|----|\n| Case 1 (female, 72 years old) | Chronic tophaceous gout, renal stones | Fingers, toes | 637 | 79 | Uricase | 36 | 70 |\n| Case 2 (male, 70 years old) | Chronic tophaceous gout | Ankle, toes | 564 | 202 | Allopurinol | 24 | 90 |\n| Case 3 (male, 72 years old) | Acute gout | Knee, ankle, foot | 482 | 121 | Allopurinol | 24 | 90 |\n| Case 4 (male, 51 years old) | Acute gout | Ankle, toe | 396 | 84 | Allopurinol | 24 | 100 |\n| Case 5 (male, 40 years old) | Acute gout | Ankle, toe | 322 | 113 | Allopurinol | 36 | 100 |\n| Case 6 (female, 72 years old) | Acute gout | Feet, toe | 572 | 72 | None | 36 | 80 |\n| Case 7 (male, 76 years old) | Acute gout | Ankle, foot | 338 | 79 | None | 36 | 100 |\n| Case 8 (male, 70 years old) | Acute gout | Wrist, elbow, hand | 779 | 406 | None | 48 | 50 |\n| Case 9 (male, 53 years old) | Chronic tophaceous gout | Elbow, finger, foot, ankle | 660 | 84 | Allopurinol | 48 | 50 |\n| Case 10 (male, 38 years old) | Acute gout | Wrist, finger | 540 | 84 | None | 24 | 60 |\n\n## Case 1\n\nA 72-year-old woman with a 13-year history of chronic tophaceous gout and hyperuricemia was treated with rasburicase. The patient previously had a severe cutaneous reaction to allopurinol, and uricosuric treatment with benzbromazone caused renal stones. During previous gout flares, medical treatment was unsatisfactory. She could only tolerate a low dose of diclofenac (50\u2013100 mg\/day), as higher doses caused gastrointestinal side-effects including one episode of gastrointestinal hemorrhage. Colchicine at 1 mg\/day provoked intolerable diarrhea, and oral corticosteroid caused severe abdominal pain.\n\nUricase treatment was commenced (14 mg intravenously daily for 5 days) and resulted in rapid lowering of the patient's uric acid levels. On the fourth day of treatment, arthritis developed in her hand and foot joints. As treatment with diclofenac during previous flares took more than 1 week to relieve her symptoms, treatment with anakinra was started. Her arthritis responded rapidly and she was able to continue the course of uricase. No acute flares were observed during the following 2 months.\n\n## Case 2\n\nA 70-year-old man with an 8-year history of chronic tophaceous gout was assessed for hypouricemic treatment. The patient's past medical history included congestive cardiac failure, severe ischemic heart disease, hypertension and renal insufficiency (serum creatinine, 202 \u03bcmol\/l; normal range, 44\u201380 \u03bcmol\/l). Previous trials of treatment with allopurinol had to be abandoned because acute gout developed after the first dose, which did not respond to small doses of NSAIDs. Higher doses of NSAIDs were contraindicated because of renal failure. Colchicine at low doses (\\<1 mg\/day) provoked rapid onset of diarrhea.\n\nThe patient was again started on a low dose of allopurinol (100 mg), and after the first dose developed acute arthritis of the right foot and ankle. Anakinra was administered for 3 days with rapid and complete resolution of signs and symptoms of arthritis. The patient continued on allopurinol 100 mg daily, and at follow-up 2 months later he had no further flare-ups while continuing on the same dose of allopurinol.\n\n## Case 3\n\nA 72-year-old man with a past history of diabetes, hypertension, renal failure and ischemic heart disease presented with polyarticular gout. Arthritis involved the knees, the right ankle and the right tarsal joints. MSU crystals were detected in the knee joint aspirate.\n\nOwing to renal impairment and a history of rectal bleeding on colchicine, treatment with oral prednisone at 30 mg daily was started with a tapering dose over 7 days. Despite steroids, the arthritis remained active and the patient could not walk. After steroids were stopped, the patient received a 3-day course of anakinra, with complete resolution of arthritis by the second day. There had been no recurrence of arthritis at follow-up 1 month later.\n\n## Case 4\n\nA 50-year-old man with a 20-year history of polyarticular gout principally involving the right ankle and big toe sought medical advice because of increasingly frequent gout attacks. He had started allopurinol but could not continue treatment because it induced flare-ups of arthritis. NSAIDs caused severe gastrointestinal pain, and colchicine at 1 mg daily was ineffective. Higher doses of colchicine provoked diarrhea. On examination, he had arthritis of the right ankle joint and MSU crystals were identified in the joint aspirate.\n\nA trial of anakinra was initiated at the same time as starting allopurinol (300 mg\/day). The patient's arthritis responded rapidly and he continued on allopurinol. He was asymptomatic 2 months later.\n\n## Case 5\n\nA 40-year-old man with a 12-year history of gout presented with recurrent podagra of the right big toe. The attacks occurred approximately once every 2 months, and responded normally to indomethacin 150 mg daily over 7 days. The patient was taking allopurinol 300 mg daily and his uric acid level was stable at 322 \u03bcmol\/l. He could not tolerate colchicine, which induced diarrhea when given at doses above 1.5 mg daily.\n\nThe patient consulted after another attack, which did not respond to indomethacin over 2 days. Joint aspiration confirmed the presence of urate crystals in the joint fluid. The patient received anakinra and his symptoms completely resolved 36 hours after the first injection. He has had no further attacks at the 6-week follow-up.\n\n## Case 6\n\nA 73-year-old female presented with acute gout involving the big toes of both feet for the first time. She had a past medical history of hypertension and knee osteoarthritis. On examination, both metatarsophalangeal-1 joints were red and swollen and tender to the touch. The joint aspirate did not yield any fluid. Serum uric acid was raised at 572 \u03bcmol\/l and serum creatinine was normal at 72 \u03bcmol\/l. A presumptive diagnosis of acute gout was made because of the patient's clinical presentation and the hyperuricemia.\n\nThe patient's symptoms had not responded to 2 days of treatment with diclofenac (100 mg daily), so anakinra was started. Her joint pains were 50% improved by 24 hours and 80% by 36 hours after the injections. No further recurrence was reported on follow-up at 1 month. The patient was started subsequently on allopurinol.\n\n## Case 7\n\nA 76-year-old man developed polyarticular gout affecting his right ankle, the right tarsal joint and both big toe joints during hospitalization for acute cholecystitis and bacterial sepsis. The patient had a past medical history of cardiovascular disease treated with low-dose aspirin and nitrates, of hypertension, of obstructive airway disease and of cirrhosis. On examination, joint inflammation was observed at the aforementioned affected sites. Aspiration of the ankle joint revealed uric acid crystals.\n\nOwing to a lack of response to NSAIDs over 48 hours, anakinra treatment was started. The patient responded after the first injection, and the symptoms and signs of gout were completely abolished 48 hours afterwards.\n\n## Case 8\n\nA 70-year-old man developed arthritis of his right wrist, elbow, hand and shoulder. The patient's past medical history included chronic renal failure, diabetes, and cardiovascular disease with atrial fibrillation. Gout was suspected because of hyperuricemia and he was treated with colchicine, which provoked rectal bleeding and a worsening of renal failure. The patient's serum urate was raised at 779 \u03bcmol\/l and his serum creatinine was 258 \u03bcmol\/l. The joint aspiration of the left elbow revealed MSU crystals.\n\nA trial of treatment with prednisone caused a decompensation of the patient's diabetes. Anakinra was started and on review, after completing the injections, the symptoms of arthritis of the knee and elbow were greatly reduced, but the right wrist and hand remained moderately painful.\n\n## Case 9\n\nA 53-year-old male presented with severe tophaceous gout of 17 years' duration. In the past, treatment with allopurinol and uricosurics had been attempted but discontinued because treatment provoked acute attacks of gout that were not well controlled by NSAIDs. Colchicine caused severe diarrhea at a dose of 1 mg\/day. There was a positive family history of gout. On examination, tophi were detected over the elbows, fingers, knees, ankles and feet, with signs of inflammation in the hands, knees and right foot. The patient's serum urate was increased at 660 \u03bcmol\/l and his renal function was normal.\n\nOwing to the patient's prior intolerance of NSAIDs and colchicine, a further attempt to start allopurinol was made under cover of anakinra, given at 100 mg daily over 3 days. On starting anakinra, the patient reported a 50% decrease in joint pains after 2 days and he started allopurinol 200 mg daily and acetametacin 90 mg daily without complications. On follow-up 1 month later, the patient had one minor attack of gout and the allopurinol was continued.\n\n## Case 10\n\nA 38-year-old male presented with painful arthritis of the left wrist. In the past, the left wrist was fractured in a road traffic accident and the patient also had a past history of gout affecting the ankles and the left knee. Aspiration of the left wrist showed MSU crystals. Colchicine (2 mg\/day) over 5 days was not effective in controlling the symptoms of gout.\n\nAnakinra was administered and there was a rapid clinical response. On follow-up at day 7 after the first injection, the left wrist was still slightly swollen and tender but the ankle and knee were asymptomatic. The patient was subsequently started on allopurinol.\n\n# Discussion\n\nMSU crystals induce tissue inflammation via multiple mechanisms. The release of monocyte-derived cytokines, triggered by urate crystals, can in turn modulate endothelial expression of adhesion molecules that enhance neutrophil recruitment to the site of inflammation \\[16\\]. Within the joint, the release of chemokines, prostanoids as well as kinins further amplifies the inflammatory response \\[17\\]. The recent finding that MSU crystals induce IL-1 release by activation of the NALP3 inflammasome led us to investigate whether IL-1 blockade could inhibit inflammation provoked by MSU. Our results from animal studies confirmed the effectiveness of IL-1 inhibition in preventing neutrophil trafficking to the peritoneum, whereas TNF inhibition did not have any effect. Indeed, TNF inhibition aggravated neutrophil influx. At present we do not have any simple explanation for the apparent increase in neutrophil accumulation in mice treated with the anti-TNF mAb. This observation was made in the two separate experiments performed with five animals per group. The anti-TNF agent employed inhibited concavalin A-induced hepatitis, demonstrating that the reagent is biologically active.\n\nThe latter results differed from those reported by Chapman and colleagues, who found that anti-TNF inhibited MSU-induced endothelial activation, as evidenced by scintigraphy, in a porcine monoarthritis model \\[16\\]. These differences may be due to the different species employed in the animal models as well as to the differences in the cytokine inhibitors tested. Apart from the inflammasome, IL-1 secretion by macrophages in contact with MSU can also be induced through a TLR-dependent mechanism \\[5\\]. In the current experiments, IL-1 inhibitors would block the effects of activation via both the TLR and the inflammasome pathways.\n\nAs proof of concept that IL-1 inhibition may be clinically effective, we performed an open-label study of anakinra treatment in patients with acute gout. All patients had acute excacerbations of longstanding gout and three had severe tophaceous gout. All the patients had either not responded adequately to standard treatment with NSAIDs, colchicine or steroids (*n* = 7) or had significant comorbidities that made use of these treatments hazardous (*n* = 4). Three patients could not continue hypouricemic treatment with allopurinol because of the acute flares provoked at the start of treatment. Only three daily injections were administered, and in all cases the symptoms of gout responded rapidly. All patients had responded within 48 hours, and in four patients (cases 2, 3, 4 and 10) the symptoms improved within 24 hours. Patients' subjective assessment of pain due to gout was positive, and the mean diminution of pain was 79% by day 3 after the first injection. Not all patients responded so well, particularly in two of the patients who had tophaceous gout. They had a moderate response, although still with \\>50% reduction of pain. As IL-1 blockade may increase the risk of infection, we excluded any patient who had proven or clinically suspected active infection. When appropriate, joint aspiration was performed to exclude septic arthritis. No treatment-related side-effects were observed during therapy and there were no infectious complications. On follow-up, three patients who had tophaceous gout reported mild joint pains but no acute flare during the first month.\n\nAlthough these findings are positive, we have to stress that this was an open-label study and confirmation of these results would require a randomized controlled trial to prove that IL-1 inhibition is effective in acute gout. If the effectiveness of IL-1 is confirmed, then a short course of IL-1 inhibition may prove to be a valid addition to the therapeutic arsenal when the physician is confronted with complicated cases of gouty arthritis.\n\n# Conclusion\n\nIn this pilot study involving 10 patients with gouty arthritis refractory to conventional therapies, anakinra given at 100 mg daily for 3 days rapidly relieved the inflammatory symptoms of gout. These results reinforce the findings that implicate IL-1\u03b2 in the pathophysiology of gout and need to be confirmed by randomized controlled trials. IL-1 inhibition may be a promising therapeutic target in crystal-induced arthritis.\n\n# Abbreviations\n\nIL = interleukin; mAb = monoclonal antibody; MSU = monosodium urate; NALP = Nacht, LRR and Pyrin domain containing protein; NSAID = nonsteroidal antiinflammatory drug; PBS = phosphate-buffered saline; TLR = Toll-like receptor; TNF = tumor necrosis factor.\n\n# Competing interests\n\nThe authors declare that they have no competing interests.\n\n# Authors' contributions\n\nThe study was conceived by AS and JT. TDS performed the animal studies. AS and SR performed the clinical study.","meta":{"dup_signals":{"dup_doc_count":108,"dup_dump_count":40,"dup_details":{"curated_sources":2,"2023-23":1,"2022-49":1,"2022-33":1,"2022-27":1,"2022-05":1,"2021-43":2,"2021-31":1,"2021-21":1,"2020-50":1,"2020-40":1,"2020-34":1,"2020-16":1,"2020-05":1,"2019-51":1,"2019-39":1,"2015-48":4,"2015-40":2,"2015-35":3,"2015-32":4,"2015-27":3,"2015-22":4,"2015-14":4,"2014-52":4,"2014-49":5,"2014-42":8,"2014-41":6,"2014-35":7,"2014-23":6,"2014-15":3,"2023-50":1,"2024-22":1,"2024-18":1,"2017-13":2,"2015-18":4,"2015-11":4,"2015-06":3,"2014-10":3,"2013-48":3,"2013-20":4,"2024-30":1}},"file":"PMC1906806"},"subset":"pubmed_central"}
{"text":"author: Carl ZimmerCarl Zimmer is a science writer based in Guilford, Connecticut, United States of America. E-mail: <email@example.com>\ndate: 2006-12\nreferences:\ntitle: The Genome: An Outsider's View\n\nThe Buddha once told a story about a king who ordered a group of blind men to be presented with an elephant. Each man touched a different part of the animal. The king then asked them what an elephant is like.\n\nThe blind men who touched the elephant's head replied, \"An elephant, your majesty, is just like a water jar.\" The blind men who touched its ear said, \"An elephant, your majesty, is just like a winnowing basket.\" The blind men who touched its tusk declared, \"An elephant, your majesty, is just like a plowshare.\" The ones who touched the trunk replied, \"An elephant, your majesty, is just like a plow pole.\" The blind men who touched the body replied, \"An elephant, your majesty, is just like a storeroom.\" The blind men who touched the foot replied, \"An elephant, your majesty, is just like a post.\" The blind men who touched the hindquarters replied, \"An elephant, your majesty, is just like a mortar.\" The blind men who touched the tail replied, \"An elephant, your majesty, is just like a pestle.\" And the blind men who touched the tuft at the end of the tail replied, \"An elephant, your majesty, is just like a broom.\"\n\nThe blind men fell into a fistfight, shouting, \"An elephant is like this, an elephant is not like that! An elephant is not like this, an elephant is like that!\" \\[1\\]\n\nI am a science writer, and my chief passion is biology. I spend time with biologists of all stripes\u2014computational biologists, paleontologists, biochemists, ecologists, and all the rest. It is a marvelous privilege. But there are times, I must confess, when I feel like I am watching a blind fistfight.\n\nOne of the first bouts I witnessed took place in the late 1990s, when I was researching the origin of whales. Whales descend from terrestrial mammals, and made the transition to water between about 50 million and 40 million years ago. In the 1990s paleontologists began unearthing a series of spectacular fossils documenting that transition, including whales with full-blown legs. Functional morphologists joined forces to investigate the transition, studying swimming otters to understand how proto-whales might have moved through the water. I spent a lot of time with scientists such as these. They are naturalists. They have to know a lot of natural history. They have to memorize the details of many species, to understand how the physiology, behavior, morphology, and ecology of each animal add up into an integrated whole. Yet these naturalists also know that they only have a slippery grasp on all of that embodied complexity.\n\nI put what I learned from those naturalists into my first book, *At the Water's Edge* \\[2\\]. As I was finishing up my manuscript, I began coming across papers in which scientists were taking a radically different approach to the question of whale origins: they were comparing the DNA of whales to that of other mammals.\n\nAt the time, molecular phylogenies were still a novelty. The computational methods for calculating them were relatively new, and scientists could only use them to compare a few gene sequences of whales and other mammals. But the conclusions from these few studies were the same: the closest living cousins of whales are hippos.\n\nThis may not seem like a big deal. It certainly didn't seem to bother the scientists who carried out the studies. They were just analyzing digital code, abstracted from the animals that carried it. The results spoke for themselves. Nevertheless, they gave paleontologists and mammalian systematists conniptions.\n\nHere's why. Hippos are artiodactyls (also known as even-toed ungulates). Other artiodactyls include cows, camels, and pigs, and goats. Zoologists have long recognized a number of anatomical features that unite artiodactyls in their own group, distinct from other hoofed mammals such as horses and rhinos. One of the most obvious hallmarks was a bone in the ankle, the astragalus. The artiodactyl astragalus has a unique double-pulley shape that allows the artiodactyl hoof to swing back and forth in a distinctive way.\n\nIn the 1990s paleontologists found a number of spectacular early whale fossils, but they had yet to find bones from the whale ankle. The bones and teeth they did find suggested that the closest relatives of whales were an extinct group of mammals called mesonychids. Mesonychids were hoofed mammals, but they did not have a double-pulley astragalus. Therefore, paleontologists concluded, they were probably not artiodactyls. And if mesonychids were not artiodactyls, then whales could not be either. And that meant that the whale\u2013hippo link had to be wrong.\n\nAs far as I could tell, this line of reasoning caused no distress among the scientists who found the hippo\u2013whale link in their genetic data. Frankly, I'm not sure they knew what an astragalus was. How many computational biologists are trained in anatomy? The shape of the mesonychid astragulus was irrelevant to them, really. To them, DNA was an overwhelmingly superior source of information. It could be analyzed precisely. It was strings of code, rather than a maddening blur of phenotype.\n\nI decided that this potential whale\u2013hippo link was important enough to mention in my book, but did so only briefly because the results were so preliminary. Still, I left the matter open, and I'm glad I did. Subsequent studies on mammal DNA continued to support the whale\u2013hippo link. And meanwhile paleontologists discovered more fossils of ancient walking whales. In 2001 Phil Gingerich and his colleagues from the University of Michigan and from Pakistan described a skeleton of the seal-like species *Rodhocetus*. Among its bones, they found an astragulus\u2014the first whale astragulus ever discovered. And lo and behold, the astragalus had a double-pulley shape \\[3\\]. The DNA, it seems, had been right all along.\n\nThis experience made a strong impression on me. I was struck by the divide between these two kinds of biologists. Each group had a profound confidence in their own sources of information, and an abiding skepticism about the other's. As I learned more about the history of modern biology, I realized that this rift did not begin in the 1990s. It was already present in the 1950s, as molecular biologists began championing their new science over more traditional ways of understanding life.\n\nHarvard University's biology department was a microcosm of this conflict. James Watson, fresh from discovering the structure of DNA, breezed into the department in 1956 with revolution on his mind. \"It was time to sweep beyond mere description of animals and plants and move into a new biology based on chemistry and physics,\" as Watson's biographer, Victor McElheny, writes \\[4\\].\n\nNeedless to say, the Harvard naturalists were not happy. Edward O. Wilson, entomologist, ecologist, and sociobiologist, pushed back hard. \"Watson, having risen to historic fame at an early age, became the Caligula of biology,\" he writes in his autobiography, *Naturalist*. \"It was foolish, we argued, to ignore principles and methodologies distinctive to the organism, population, and ecosystem, while waiting for a still formless and unproved molecular future\" \\[5\\]. The struggle only ended when Harvard's biologists agreed to split their department in two.\n\nOver the past fifty years, these factions of biologists have had a complex relationship. Some scientists have continued to carry out relatively traditional natural history work, with little need to delve into molecular (or computational) biology. Others have given little attention to natural history, focusing their efforts instead on deciphering the complexities of a membrane channel, or building new algorithms for identifying open reading frames. In some cases, biologists have bridged this divide, and the result has been a fruitful collaboration. But in other cases\u2014such as the DNA studies on whales and hippos\u2014one group moves into the other's traditional territory, sparking new conflict.\n\n\"That can't be right\" is a refrain I've often heard when computational biologists report a controversial result. Along with the whale\u2013hippo link, I can also recall the snorts that came with the first studies on human DNA suggesting that we can trace our ancestry to Africa 150,000 years ago. The picture is certainly more complex than the first \"out of Africa\" scenarios suggested, but the core of that idea has withstood further scrutiny. These days I sense a growing confidence in computational biologists, perhaps brought on by successes such as these. Many are no longer content merely to present an analysis of DNA and leave it at that. They use their findings to make sweeping statements about ecology and evolution.\n\nOne example of this new ambition was a paper published earlier this year on the evolution of cats \\[6\\]. The scientists offered a sweeping scenario for cat evolution, complete with migrations of cats out of Asia into the New World and back, along with the emergence of the major groups of felids, ranging from ocelots to bobcats to lions. The scientists based their scenario entirely on an analysis of cat DNA. They did not consider a single fossil of a cat, nor did they have a paleontologist expert on cats as a coauthor. Cat fossil experts inform me that fossils of true cats as old as 17 million years have been discovered in North America. The geneticists put the arrival of cats in North America at only 8 million years ago. Whether or not the DNA results are correct, it is striking that the report does not even mention the existence of fossils that do not fit the pattern.\n\nGenomes unquestionably contain a rich store of information, but sometimes that information can be deceptive. Recently, two paleontologists, Philip Donoghue of the University of Bristol and Mark Purnell of the University of Leicester, described how this deception can arise in the study of vertebrate evolution \\[7\\]. A number of studies have suggested that vertebrates underwent several rounds of genome duplication since they diverged from their closest invertebrate ancestors. One of these duplications may have coincided with the emergence of jawed vertebrates (gnathostomes). This link has prompted some scientists to propose that this duplication triggered a leap in complexity that set gnathostomes apart from their closest vertebrate relatives, the hagfish and lampreys. Gnathostomes not only have jaws, but also have a long list of other traits that set them apart from agnathans, including an advanced immune system, a mineralized skeleton, teeth, and fins. They are also far more diverse. Perhaps a genome duplication provided the raw material for an adaptive radiation.\n\nAs Donoghue and Purnell demonstrate, this idea only makes sense if you ignore the fossil record. Paleontologists have found a rich record of fossils documenting the gradual assembly of the gnathostome body plan. Conodonts, for example, possessed a mineralized skeleton, dentine, and enamel\u2014but nothing else that sets gnathostomes apart from agnathans. Fins evolved later, and a mineralized braincase came later still. Nowhere along the phylogeny of vertebrates is there a sudden leap of complexity in the forerunners of true gnathostomes, nor is there an abrupt leap in levels of diversity. One is left to wonder just when the alleged effects of a genome duplication occurred.\n\nTaking natural history seriously is good not only because it can steer a computational biologist away from these sorts of errors. It also opens up fascinating new questions to which computational biologists can profitably apply their skills. Consider the coelacanth, a rare, ugly fish found in the Indian Ocean. It belongs to the lobe fins, a group of fish from which we and all other land vertebrates evolved. Scientists knew about the fossils of coelacanths long before they saw one alive. The fossil record of their ancestors reaches back 400 million years, but it was not until 1938 that living coelacanths were discovered off the coast of South Africa.\n\nI present the coelacanth in the spirit of the Buddha. Is it a fish, or a just bag of digital code?\n\nEarlier this year, computational biologists published a remarkable paper about the coelacanth in *Nature* \\[8\\]. The paper had its origins in the startling discovery that certain regions of the human genome that do not code for proteins are highly conserved. Natural selection has kept these sequences much the same in humans and in other mammals. The scientists were puzzled as to how natural selection could preserve their homology, since these sequences do not encode any protein.\n\nThe scientists decided to see how widespread these regions are among animals. Searching gene databases, they discovered related genetic sequences in the coelacanth. Despite more than 400 million years separating our ancestors, the coelacanth and human regions were 80% identical. A close examination of the coelacanth segments revealed them to be transposons\u2014virus-like stretches of DNA that could insert new copies of themselves into the genome.\n\nThe authors conclude that these ultraconserved regions of the human genome descend from a transposon in the ancestors of lobe fins. In the tetrapod lineages, the transposons lost their ability to spread through the genome. Some became exapted for regulatory functions, and were preserved by natural selection. The others were eroded by mutations. In the coelacanth lineage, on the other hand, the transposons remained active.\n\nAs fascinating as these results are, I was also struck by how they were framed. The authors begin their paper by referring to the coelacanth as a \"living fossil.\" They end their paper with the conclusion that by preserving this family of transposons, the fish \"acts, in a sense, as a living molecular fossil. The remaining 99.9% of its genome, as yet unsequenced, may very well hold precious traces of additional events that helped shape our own evolution.\"\n\nThe coelacanth is a living fossil only insofar as scientists once thought it was extinct. But that is not the dictionary definition of a living fossil: an organism \"that has remained essentially unchanged from earlier geologic times\" \\[9\\]. Paleontologists have been giving the coelacanth lineage a fresh look, and they've discovered a surprising amount of morphological change \\[10\\]. Some ancient relatives of today's coelacanths were slender, eel-like creatures. Others had round, sunfish-like bodies. Today's coelacanths are probably not living fossils in an ecological sense, either. Early fossils of coelacanths are often found in sedimentary rocks formed in coastal waters. Living coelacanths, by contrast, only live deep under water.\n\nI would question whether the coelacanth is much of a molecular living fossil, either. The new research certainly shows that coelacanths preserve information in their genome that reveals some of the history of ultraconserved regions in our genome that would otherwise be lost. But that's because their transposons are still \"alive,\" while our transposons lost their ability to replicate a long time ago and have become frozen in place. In that sense, *we* are the living fossils.\n\nIf one thinks about the coelacanth's natural history as well as its genome, a new set of questions arises. Transposons are prone to mutations, which can take away their ability to insert new copies in a genome. These coelacanth transposons have been replicating for 400 million years, and now probably take up a significant fraction of the coelacanth genome. And yet they still have not diverged very much in all that time. Is natural selection conserving them? Is natural selection acting on the transposon or the coelacanth? How do these proliferating transposons affect the physiology of the coelacanth? Do they act like pathogens, or are they providing some benefit we don't yet understand? Does their expansion play any role in the ecology of living coelacanths?\n\nNone of these questions would even arise without the invaluable work of computational biologists. But if all the blind men gather together around the coelacanth, perhaps they can better understand just what this creature is.\u2002\n\nI would like to thank Pavel Penzer, Alberto Apostolico, and Concettina Guerra for inviting me to Recomb '06, where I presented a talk on which this essay is based. Michael Coates, Phil Donoghue, John Gatesy, Phil Gingerich, David Haussler, Christine Janis, Blair Van Valkenburgh, and other scientists have generously shared their thoughts about the biological elephant.\n\n# References","meta":{"dup_signals":{"dup_doc_count":155,"dup_dump_count":58,"dup_details":{"curated_sources":2,"2022-33":1,"2021-43":1,"2020-45":1,"2019-39":1,"2019-30":1,"2019-13":1,"2018-47":1,"2018-30":1,"2018-26":1,"2018-22":1,"2018-17":1,"2018-13":1,"2018-09":2,"2018-05":1,"2017-51":1,"2017-47":2,"2017-43":1,"2017-39":2,"2017-34":2,"2017-30":2,"2017-26":2,"2017-22":3,"2017-17":3,"2017-09":4,"2017-04":3,"2016-50":3,"2016-44":4,"2016-40":4,"2016-36":5,"2016-30":3,"2016-26":4,"2016-22":3,"2016-18":2,"2016-07":3,"2015-48":3,"2015-40":2,"2015-35":3,"2015-32":3,"2015-27":4,"2015-22":4,"2015-14":3,"2014-52":3,"2014-49":5,"2014-42":9,"2014-41":7,"2014-35":5,"2014-23":6,"2014-15":5,"2023-50":1,"2024-10":1,"2017-13":1,"2015-18":3,"2015-11":2,"2015-06":3,"2014-10":3,"2013-48":3,"2013-20":2,"2024-22":1}},"file":"PMC1761651"},"subset":"pubmed_central"}
{"text":"abstract: The late Quaternary megafauna extinction was a severe global-scale event. Two factors, climate change and modern humans, have received broad support as the primary drivers, but their absolute and relative importance remains controversial. To date, focus has been on the extinction chronology of individual or small groups of species, specific geographical regions or macroscale studies at very coarse geographical and taxonomic resolution, limiting the possibility of adequately testing the proposed hypotheses. We present, to our knowledge, the first global analysis of this extinction based on comprehensive country-level data on the geographical distribution of all large mammal species (more than or equal to 10 kg) that have gone globally or continentally extinct between the beginning of the Last Interglacial at 132 000 years BP and the late Holocene 1000 years BP, testing the relative roles played by glacial\u2013interglacial climate change and humans. We show that the severity of extinction is strongly tied to hominin palaeobiogeography, with at most a weak, Eurasia-specific link to climate change. This first species-level macroscale analysis at relatively high geographical resolution provides strong support for modern humans as the primary driver of the worldwide megafauna losses during the late Quaternary.\nauthor: Christopher Sandom; S\u00f8ren Faurby; Brody Sandel; Jens-Christian Svenninge-mail: <firstname.lastname@example.com>[^1]\ndate: 2014-07-22\ninstitute: Ecoinformatics and Biodiversity, Department of Bioscience, Aarhus University, Ny Munkegade 114, Aarhus C 8000, Denmark\nreferences:\ntitle: Global late Quaternary megafauna extinctions linked to humans, not climate change\n\n# Introduction\n\nDuring the Late Pleistocene and early Holocene, regions around the world suffered losses of megafauna species of a magnitude unprecedented for many millions of years \\[1\u20133\\]. Although extinctions are common in the Quaternary fossil record, such rapid and large global species losses without functional replacements are unusual. Multiple explanatory hypotheses for this global extinction event have been proposed, including climate change \\[4,5\\], the spread of modern humans (*Homo sapiens*) and related effects of hunting and habitat change \\[6\u20139\\], an extra-terrestrial impact \\[10\\] and hyper-disease \\[11\\]. With little or no evidence to support the latter two as global explanations \\[11\u201314\\], we focus here on climate change and hominin palaeobiogeography. The relative importance of climate and hominin drivers has been subject to long-standing, highly charged controversy (e.g. \\[3,15,16\\]).\n\nAlthough megafaunal extinctions occurred worldwide, most studies focus on a specific region \\[17\u201319\\], and the few global-scale studies have been geographically incomplete and\/or very coarse-grained \\[3,20\\]. In an influential review, Barnosky *et al*. \\[3\\] provided a continental breakdown of the Late Pleistocene megafauna extinctions and reported that humans were the main extinction driver in Australia and North America, and climate was the main driver for Europe, but that data were insufficient for making assessments for Africa, Asia and South America. Nogu\u00e9s-Bravo *et al*. \\[20\\] followed up by linking these genus-level continental-resolution patterns to continental-average climate change, finding extinction patterns among Africa, Eurasia and North America consistent with climate-driven extinction; however, South America strongly deviated from this trend, with the lowest climate change and the most severe genus-level extinctions. A geographically comprehensive species-level assessment of the global-scale extinction pattern remains lacking. Improvements and increased access to fossil, palaeoclimate and hominin palaeobiogegraphical records, more accurate dating and taxonomic revisions now make possible much more geographically fine-grained analysis of megafauna extinctions worldwide, namely species-level extinctions per Taxonomic Databases Working Group (TDWG) level 3 country (see Material and methods for a description of these regions).\n\n## The climate change hypothesis\n\nWe focus on temperature and precipitation contrasts between full glacial and full interglacial conditions, as these represent the strongest climate shifts during the late Quaternary. We estimated these as the Last Glacial Maximum (LGM, *ca* 21 000 years BP) to present-day (1950\u20132000) contrast and, in supplement, the Last Interglacial (LIG, *ca* 130 000 years BP) to LGM contrast. Palaeoclimatic changes can be represented both by simple macroclimatic changes through time or by climate change velocities that incorporate spatial climate gradients to estimate how fast climate moved in space, providing a direct estimate of how rapidly species would have needed to migrate to track climate \\[21,22\\]. Where climate change is severe and the topography is flat, species would typically have to travel greater distances to find suitable conditions, experiencing greater risks of extinction. As a result, high late Quaternary climate change velocity is associated with low modern species endemism in mammals, birds and amphibians worldwide, indicating greater past extinction rates in more climatically unstable regions \\[21\\]. We used mean annual temperature and annual precipitation anomaly and velocity between the LGM and the present to represent the potential severity of climate change over the study period (<a href=\"#RSPB20133254F1\" data-ref-type=\"fig\">figure 1<\/a>*d*,*e* and the electronic supplementary material, figure S1). If climate change has been an important driver of extinction, then climate change magnitude or velocity should be positively correlated with the proportion of the cumulative late Quaternary large mammal species per TDWG country that have become globally or continentally extinct during 132 000\u20131000 years BP. Later extinctions were not considered because of the strong known human effects during this period. Although our analysis is at the country scale and species may respond to more local environments, large mammals generally have large ranges \\[23\\] and within-TDWG country variation in climate change velocity is typically small (see Material and methods).\n\n## The human palaeobiogeography hypothesis\n\nHominins have migrated out of Africa in waves successively increasing the hominin-occupied area so that megafauna have co-occurred and coevolved with hominins for differing lengths of time in different regions. Possibly as early as the Late Pliocene or Early Pleistocene, *Homo erectus* and contemporary hominins expanded into southern Asia and south and western Europe \\[24\\] (<a href=\"#RSPB20133254F1\" data-ref-type=\"fig\">figure 1<\/a>*c* 'Archaic-early'). *Homo neanderthalensis* and Denisovan humans pushed these boundaries further, colonizing most of Eurasia and leaving only the very northern extremities unpopulated during interglacials \\[25\\] (<a href=\"#RSPB20133254F1\" data-ref-type=\"fig\">figure 1<\/a>*c* 'Archaic-late'). While archaic humans appear not to have inhabited the northernmost parts of Eurasia, no terrestrial megafaunal species was endemic to these parts (see the electronic supplementary material, Data S1). The residing megafauna would have encountered archaic hominins during range extensions into more southerly regions or rare archaic incursions into the north; thus, we classify this region with those occupied by archaic hominins in our main analyses (<a href=\"#RSPB20133254F1\" data-ref-type=\"fig\">figure 1<\/a>*c* 'Archaic-peripheral'). By contrast, faunas in Australia, North and South America and to some extent Japan did not have any contact with hominins until the expansion of modern humans \\[26\\] (<a href=\"#RSPB20133254F1\" data-ref-type=\"fig\">figure 1<\/a>*c* '*Homo sapiens*-only'). Late Quaternary megafauna extinctions have been hypothesized to have been more severe where modern humans were the first hominin to arrive, suddenly introducing a new and effective big-game predator into regions with megafauna naive to human hunting \\[27\\]. Conversely, extinctions have been posited to have been less severe in Africa because of long-term, gradual hominin\u2013megafauna coevolution \\[3\\]; alternatively, pre-Late Pleistocene megafauna extinctions in regions occupied by pre-*sapiens* hominins may also have contributed to fewer late Quaternary extinctions \\[27,28\\], or human populations may have been more suppressed by disease here \\[15\\]. Under this scenario, we hypothesize that the most severe extinctions occurred where modern humans were the first hominins to arrive and that the lowest extinction occurred in sub-Saharan Africa, the centre of origin and evolution for hominins overall as well as for *H. sapiens* \\[24\\] (<a href=\"#RSPB20133254F1\" data-ref-type=\"fig\">figure 1<\/a>*c* '*Homo*-origin').\n\nWe aim to evaluate the relative support for the climate change and hominin palaeobiogeography hypotheses for the late Quaternary megafauna extinctions by, to our knowledge, the first comprehensive species-level, fine-grained global macroecological analysis. We achieve this aim and find strong support for hominin palaeobiogeography as the critical factor for explaining the strong geographical variation in megafauna extinction severity worldwide.\n\n# Material and methods\n\n## Extinct species of the late Quaternary\n\nA thorough examination of the scientific literature identified 177 taxonomically accepted, extinct or continentally extirpated large mammals (more than or equal to 10 kg) that occurred during the Late Pleistocene and early\/middle Holocene (132 000\u20131000 years BP). The process was begun by gathering information on species names reported lost during the late Quaternary from review publications and original research articles followed by detailed species by species research to confirm their taxonomic validity and presence within our study period (electronic supplementary material, table S1). In some circumstances, we chose to reject uncertain species closely related to accepted species occurring over similar ranges to ensure that our extinction densities were conservative. A notable example includes the North American horses, where only two *Equus* species were accepted, *Equus ferus* and *Equus semiplicatus* (representing the stilt-legged horses), following \\[29\\]. Species were only accepted if they are recorded from a directly dated site within the time period studied. In order to be conservative, we did not accept dates based on co-occurrence with potential indicator fossils, i.e. species thought to be associated with a particular time period. As a result, the number of extinctions that are too old to be correctly dated with carbon-14 dating may be underestimated in our analysis. Species that were of uncertain taxonomy or occurred from sites thought to be within the late Quaternary, but lack a confirmed date were included in a list of potential, but uncertain late Quaternary megafauna extinctions (*n* = 27; electronic supplementary material, table S1). Of these 27 taxonomically or temporally uncertain species, we recorded regional presences for 17 of them, which were included in a supporting sensitivity analysis (electronic supplementary material, table S3). Species mass were primarily collected from Smith *et al*. \\[30\\] and supplemented with additional sources or, where required, were estimated based on masses of species in the same genus (electronic supplementary material, table S1). We defined 'large' mammals as more than or equal to 10 kg (*n* = 177 spp.). We repeated the analyses including species more than or equal to 44 kg (*n* = 154 spp.) to be consistent with much of the literature published on the megafauna extinctions \\[3\\]. Species lost in the last 1000 years were excluded because of their much clearer association to human drivers of extinction.\n\n## Extinct species distribution\n\nGeographical locations were collected for each accepted and uncertain species. Mapping was performed at the resolution of TDWG level 3 regions \\[31\\]. In total, 229 TDWG countries were included; islands, Antarctica and glaciated countries were excluded (see the electronic supplementary material for details). A species was determined to be present if an identified specimen was recorded for a given TDWG country. Location records for accepted and uncertain species were downloaded from the Global Biodiversity Information Facility (GBIF) on 29 November 2012 and supplemented with data gathered from FAUNMAP \\[32\\] and the primary literature based on searches of species names and regional reviews as listed in the electronic supplementary material, table S1. Species range maps produced by experts were accepted as valid locations. GBIF data were cleaned to remove erroneous records based on the following criteria: species recorded on a continent with no dated site on the specified continent, records associated with animals in captivity, records within the open ocean and records with latitude and longitude of zero. For continentally extinct species, locations recorded from their extant range were removed. After locations in open ocean had been removed, locations for species occurring close to the coast were moved to the nearest TDWG country to account for lower sea levels during the Last Glacial. A presence\/absence map, representing total cumulative range of each species across the study period (see the electronic supplementary material for further details), was plotted for each species (based on the electronic supplementary material, Data S1). The species distributions maps were typically discontinuous at least partly as a result of geographical gaps in the fossil record (electronic supplementary material, Data S1). These probably primarily reflect geographical bias in collection efforts as well as taphonomic bias, so we controlled for this by filling gaps in species ranges where: (i) the range was discontinuous (e.g. *Hippidion principale* and *Elephas namadicus*), (ii) there were holes within the range (e.g. Nevada, Colorado and Oklahoma for *Canis dirus*), and (iii) to fill peripheral TDWG countries on the edge of ranges (e.g. the Northern Provinces of Australia and the Guianas). For species-specific exceptions, see the electronic supplementary material. Where the gaps between records were extensive, gaps were filled on a path that best reflected environmental conditions in the regions where specimens were recovered. The primary analysis was performed including the filled records. To test the sensitivity of the results to our interpolations, the analysis was re-run using only the direct records.\n\n## Data analysis\n\nThe total number of extinct large mammals was calculated for each TDWG country (<a href=\"#RSPB20133254F1\" data-ref-type=\"fig\">figure 1<\/a>*b*). To account for naturally occurring species richness gradients and the uneven area of each TDWG country, extinction was expressed as a proportion of the total number of extinct and extant species (<a href=\"#RSPB20133254F1\" data-ref-type=\"fig\">figure 1<\/a>*a*). To remove the effects of human-driven extinctions and range contractions occurring in the last 1000 years, we estimated current extant large mammal richness for each TDWG country by accounting for the magnitude of human impact \\[33\\]. We initially created a general linear model (GLM), with a Quasi-family, logit link and variance of *\u03bc*(1 \u2212 *\u03bc*), with large mammal richness modelled as a function of continent, small mammal richness (International Union for Conservation of Nature range maps \\[34\\]) and human impact \\[33\\]. Six continents were included in our analysis; however, Asia was divided into two, tropical and temperate according to the TDWG classifications, and modelled individually. Human impact \\[33\\] was a significant predictor of large mammal diversity in North America (*p* \u2264 0.001), and temperate Asia (*p* \u2264 0.001), and approached significance in Europe (*p* = 0.062). For these regions, extant large mammal TDWG richness was estimated from this model using the lowest human impact recorded within the specific continent, and so within the predictive range of our models (electronic supplementary material, figure S11).\n\nWe focused on temperature and precipitation contrasts between the LGM, *ca* 21 000 years BP and the present-day interglacial climate, as this contrast represents the full glacial\u2013interglacial amplitude of climate change during our study period. The chronology of extinctions, however, extends across the late Quaternary, including extinctions prior to the LGM \\[3\\]. To account for this, we also explored climate change between the LIG and the LGM as a supplementary analysis (electronic supplementary material, figure S10). Importantly, temperature and precipitation anomalies are highly correlated between the LIG\u2013LGM and LGM\u2013present time periods (0.97 and 0.82, respectively), indicating spatial consistency in the severity of climate change across the late Quaternary. Replacing the LGM\u2013present climate contrasts with the LIG\u2013LGM contrasts in a supplementary analysis provides even less support for the climate change hypothesis.\n\nClimate change was measured using four variables, two measures of climate change velocity \\[21,22\\], the rate of displacement of the mean annual temperature and annual precipitation from the LGM to today, we measured the spatial gradient using a 2.5 arcminute resolution map, and two measures of climate change magnitude, LGM to present mean annual temperature anomaly and precipitation anomaly; these climate data were obtained from WorldClim at 2.5 arcminutes \\[35\\] (<a href=\"#RSPB20133254F1\" data-ref-type=\"fig\">figure 1<\/a>*d*,*e*; electronic supplementary material, figure S1). Temperature and precipitation anomaly were calculated by averaging records from the MIROC3 and CCSM models \\[36,37\\]. For each climate change variable, a standard measure was created by taking the square root of each data point and dividing by the maximum, creating scores between 0 and 1. For the measures of anomaly, absolute values were used as we are exploring the magnitude of change and not the direction. Although our analysis is at the TDWG country scale, species will probably respond to more local environments. Local-scale topography affects climate change velocities in particular and could vary considerably within region. To assess the degree of climate change velocity variation, velocity range was calculated for each TDWG country. The median velocity range per country was 16 m yr^\u22121^ for temperature and 44 m yr^\u22121^ for precipitation. This suggests that the velocity variation within each TDWG country was relatively small and unlikely to be problematic. To test for bias against climate explanatory variables measured on a continuous scale and for uncertainty in climate models, we used *k*-means clustering and a climate sensitivity test, respectively (see the electronic supplementary material).\n\n## Model selection\n\nClimate change, hominin palaeobiogeography and hominin\u2013climate combination models were constructed and compared to determine which predictor variables best describe the recorded regional extinction ratios. As velocity is calculated using climate change anomaly to avoid problems with collinearity, particularly in the temperature data, the velocity and anomaly of the same predictor variable were not included in the same climate or hominin\u2013climate combination model. For the hominin models, we explored all combinations of hominin palaeobiogeography and the optimal description was found to be lumping the three Archaic regions together (based on an ordinal analysis of the five categories that revealed significant differences between *Homo*-origin and Archaic-early, and Archaic-peripheral and *H. sapiens*-only, but not between Archaic-early and Archaic-late and Archaic-peripheral), which we refer to as Archaic-combined. This combined model also performed better than including modern human arrival time \\[38\\] (based on Akaike information criterion (AIC)). We initially constructed GLMs using an arcsine square-root transformation of the response variable to account for the non-normal distribution of the proportion data. This transformation was deemed appropriate because the nature of these binomial data were not a factor of sampling effort and we wanted to give a 50% reduction of large mammal community within a TDWG country the same weight no matter if the reduction was from four to two species or 50\u201325 to avoid recording low extinction severity in regions with low total species diversity. We used AIC to select the best climate change, hominin palaeobiogeography and hominin\u2013climate combined models. For comparison to the above approach, we also constructed a GLM using a Quasi-family, logit link and variance of *\u03bc*(1 \u2212 *\u03bc*), to account for the non-normal distribution of proportion data \\[39\\], finding the results to be consistent (see the electronic supplementary material). To assess the degree of spatial autocorrelation (SAC), we computed correlograms of GLM model residuals using the 'ncf' package in R \\[40\\], with distance classes of 500 km (electronic supplementary material, figure S3). A spatial autoregressive (SAR) model of the error type (SAR~err~ sensu \\[41\\]) was used to account for SAC in the multivariate hominin\u2013climate model and compared to the GLM using the arcsine square-root transformation. The SAR models were implemented in the R package 'spdep' \\[42\\]. The spatial weights matrix was defined by connecting each TDWG country to its four closest neighbours. This SAR model removed residual SAC (electronic supplementary material, figure S3). All analyses were performed in R.v. 2.15.1 \\[43\\].\n\n# Results\n\nOur extensive and taxonomically conservative literature review provided evidence of 177 globally or continentally extinct large mammal species (more than or equal to 10 kg; Africa 18, Asia 38, Australasia 26, Europe 19, North America 43 and South America 62; electronic supplementary material, table S1; <a href=\"#RSPB20133254F1\" data-ref-type=\"fig\">figure 1<\/a>*b*) for the period 132 000\u20131000 years BP. Of the 229 TDWG countries included in the analysis, the highest number of extinct species occurred in Texas (33 species), while Uruguay showed the highest proportion of extinct species (78%, <a href=\"#RSPB20133254F1\" data-ref-type=\"fig\">figure 1<\/a>*a*). Southern South America, southeast North America, western Europe and southern Australia emerged as extinction hotspots, with sub-Saharan Africa and southern Asia as notable cold spots (<a href=\"#RSPB20133254F1\" data-ref-type=\"fig\">figure 1<\/a>*a*). Thirty-one TDWG countries had no recorded extinctions.\n\nUsing climate change predictors only, a GLM including both temperature anomaly and precipitation velocity best described the extinction pattern, although with poor explanatory power (GLM: *F*~226~ = 29.790, *p* \\< 0.001; ). Human palaeobiogeography included as a categorical variable with three levels, *Homo*-origin, Archaic-combined and *H. sapiens*-only (<a href=\"#RSPB20133254F1\" data-ref-type=\"fig\">figure 1<\/a>*c*), had strong explanatory power (GLM: *F*~226~ = 201.240, *p* \\< 0.001; ). A full model was constructed from the predictors of the best climate and human models with interactions between human biogeography and the two climate predictors (GLM: *F*~220~ = 68.740, *p* \\< 0.001; ; electronic supplementary material, figure S2 and table S2). The full model was significantly better than either the human or climate model (climate: *p* \\< 0.001; human: *p* \\< 0.001), but explanatory power was only weakly improved over the human-only model.\n\nBecause of strong SAC in the residuals (electronic supplementary material, figure S3), the pairwise and multivariate analyses were all re-done accounting for SAC. A simultaneous autoregressive (SAR) model was used to account for SAC in the multivariate climate\u2013hominin model. The best combined SAR model included hominin palaeobiogeography, temperature anomaly and an interaction between the two, while precipitation velocity was excluded (<a href=\"#RSPB20133254F2\" data-ref-type=\"fig\">figure 2<\/a> and <a href=\"#RSPB20133254TB1\" data-ref-type=\"table\">table 1<\/a>). The model indicated significant effects of hominin palaeobiogeography, with intermediate extinctions in the Archaic-combined region and the most severe extinctions in the *H. sapiens*-only region (<a href=\"#RSPB20133254F3\" data-ref-type=\"fig\">figure 3<\/a>), temperature anomaly and their interaction. The interaction reflected the fact that the temperature anomaly effect was observed only in the Archaic-combined region (<a href=\"#RSPB20133254F2\" data-ref-type=\"fig\">figure 2<\/a>). These findings were robust to decisions related to the palaeontological information, distribution interpolation, extant species richness estimation and size threshold as well as to whether climate change was recorded as a categorical variable, was represented by the LIG\u2013LGM shift rather than the LGM\u2013present shift, and uncertainty in the climate models (electronic supplementary material, figures S4\u2013S12, tables S3\u2013S7 and see Sensitivity Analysis section).\n\nSAR model explaining global patterns in the proportion of extinct large mammals (more than or equal to 10 kg). (Est, regression coefficient; s.d., standard deviation; *Z*, test statistic; *p*, *p*-value; HS, *H. sapiens-*only region; AC, Archaic-combined region; TA, mean annual temperature anomaly between LGM and today, standardized to range between 0 and 1.)\n\n|                     | estimate | s.d.  | *Z*    | *p*     |\n|:--------------------|----------|:------|--------|---------|\n| intercept           | \u22120.353   | 0.263 | \u22121.341 | 0.18    |\n| *H. sapiens-*only   | 1.107    | 0.274 | 4.037  | \\<0.001 |\n| Archaic-combined    | 0.735    | 0.271 | 2.713  | 0.007   |\n| temperature anomaly | 1.504    | 0.739 | 2.037  | 0.042   |\n| HS \u00d7 TA             | \u22121.516   | 0.749 | \u22122.022 | 0.043   |\n| AC \u00d7 TA             | \u22121.225   | 0.752 | \u22121.630 | 0.103   |\n| pseudo *R*^2^       | 0.635    |       |        |         |\n| *p*                 | \\<0.001  |       |        |         |\n| AIC                 | \u2212272.57  |       |        |         |\n\nThis analysis illustrates that the late Quaternary megafauna extinctions were strongly linked to hominin palaeobiogeography and only weakly to glacial\u2013interglacial climate change. The relationship between hominin palaeobiogeography and extinction magnitude is striking, with universally low extinctions in sub-Saharan Africa (maximum 13%), where hominins and the megafauna have long coexisted, but widespread exceptionally high extinctions in Australia and the Americas, where modern humans were the first hominin present. Only three TDWG countries in the *H. sapiens*-only region had extinction ratios below 30%, two of which are small, coastal states in North America with missing data as a likely cause of their low extinction values. The third is Japan, but the coding of hominin\u2013palaeobiogeography for this country is potentially problematic because the country includes some endemic species that were never in contact with archaic humans, but many others with ranges that were, suggesting that the country could alternatively be coded as 'Archaic-peripheral'. Eurasia falls between these extremes with a wide variety of extinction ratios, presenting a more complicated story. Globally, human palaeobiogeography alone accounts for 64% (GLM estimate) of the variation in extinction, while temperature anomaly, in combination with precipitation velocity in the best climate-only model, had much weaker explanatory power (20%) and only showed a relation to the proportion of extinct large mammals in the Archaic-combined region in the combined model.\n\n# Discussion\n\nThe global pattern of late Quaternary megafauna extinction presents a clear picture that extinction is closely tied to the geography of human evolution and expansion and at most weakly to the severity of climate change. The pattern of extinctions closely followed the hominin paleobiogeography hypothesis with increasing severity of extinction with reduced period of hominin\u2013megafauna coevolution, notably with uniformly high extinction in areas where *H. sapiens* was the first hominin to arrive. By contrast, only in Eurasia was there a climate change signal.\n\nHigh extinction despite a relatively stable climate is most striking in South America, forming a strong contrast to sub-Saharan Africa where extinction was minimal in spite of similar glacial\u2013Holocene climate changes (<a href=\"#RSPB20133254F1\" data-ref-type=\"fig\">figure 1<\/a>*a*,*d*,*e* and the electronic supplementary material, figure S1). This relationship is particularly notable in southern South America where extinction ratios were among the highest recorded. Extinctions in the region have been linked to fluctuations between humid and dry periods and shifts in the availability of savannah and forest habitats. For instance, De Vivo & Carmignotto \\[44\\] associated a humid period in the mid-Holocene with loss of savannah and the demise of some megafauna. There is no indication, however, that these fluctuations were greater than in Africa or elsewhere \\[45\\]. Furthermore, dietary analyses of large South American herbivores such as *Eremotherium laurillardi*, *Stegomastodon waringi* and *Toxodon platensis* indicate that these extinct species were adaptable generalists, altering their diet between grazing and mixed feeding according to the habitat they occupied \\[46\\]. Additionally, other areas maintained grassland or forest throughout the whole period despite fluctuating climatic conditions \\[47\\]. The pampas region, for example, remained open throughout this time \\[47\\], but many megafauna species still disappeared, such as *Megatherium americanum*, which has been associated with human exploitation \\[48\\]. Extinctions in South America are poorly dated, but many have occurred after modern human arrival, approximately 15 000 years BP, and continued into the climatically relatively stable Holocene \\[19\\].\n\nMost Australian extinctions occurred prior to the LGM, and so outside our LGM\u2013present climate change predictor variable, just subsequent to the time humans are thought to have arrived (72\u201344 kyr BP) \\[3\\]. Australia experienced gradual drying from 400 ka that is proposed to have achieved a hydrological threshold in the last glacial; Wroe & Field \\[4\\] claim this drying drove a staggered extinction of many of the Pleistocene megafauna prior to the arrival of modern humans. Alternatively, it has been argued that modern humans caused the Australian megafauna extinctions either via fire-driven vegetation changes \\[49\\] or hunting \\[8\\]. Rule *et al*. \\[50\\] gave a detailed chronology for a dramatic decline in large mammal biomass in northeastern Australia during a period of climatic stability millennia after discernible drying events, after human arrival and prior to habitat change, supporting human-driven megafauna attrition as a driver of vegetation community change rather than the reverse (see also \\[17,51,52\\]). In agreement with this, we observe high extinction rates across Australia, but low to moderate temperature and precipitation anomalies and velocities (<a href=\"#RSPB20133254F1\" data-ref-type=\"fig\">figure 1<\/a>*a*,*d*,*e*). Additionally, LIG\u2013LGM climate change measures indicated similarly moderate to low levels of relative temperature change across this period (electronic supplementary material, figure S10).\n\nOur results also confirm earlier observations that extinctions could be severe even in relatively climatically stable regions in North America, where the vegetation changed little \\[53\\]. For instance, despite relatively little climate change and local survival of, for example, chapparel vegetation from LGM to today \\[54\\], California lost 21 megafauna species (53%, <a href=\"#RSPB20133254F1\" data-ref-type=\"fig\">figure 1<\/a>*a*,*b*) such as *Nothrotheriops shastense*, *Capromeryx minor* and *C. dirus* (see the electronic supplementary material, Data S1). In some cases, megafaunal species went extinct even though their preferred local diet has remained plentiful to the present-day, e.g. *Mammut americanum*, which fed on twigs of trees such as bald cypress (*Taxodium distichum*) in Florida \\[55\\]. *Euceratherium collinum* and *N. shastense* are further examples of species from western USA that went extinct despite the continued presence of favoured plant food sources in the region \\[56\\]. Grund *et al*. \\[57\\] highlights that the extinction pattern across North America is not consistent with climate as the main driver because species with small geographical ranges were not disproportionately affected. These megafauna patterns contrast with the global findings reported by Sandel *et al*. \\[21\\] that small-ranged amphibians, mammals (mostly small-sized) and birds are absent or scarce where Quaternary climate change has been severe. The period of extinction in North America occurred approximately 11 500\u201310 000 years BP, well within our period of measured climate change, and broadly occurred when climate change was severe, but also consistent with the arrival of modern humans \\[3\\].\n\nMartin & Wright \\[6\\] were first to argue strongly for modern humans as the likely driver of megafauna extinctions worldwide, envisioning a swift overkill at the range front of expanding modern human populations. Since the emergence of that idea, other slower extinction models have been proposed involving anthropogenic fires and human-driven habitat change \\[49,50\\]. Although strong scepticism continues about humans as the extinction driver, primarily based on an argued scarcity of archaeological evidence of megafauna kills \\[4,16\\], others have shown that that kill sites are not rarer than expected \\[58,59\\] and that prehistoric human populations had the potential to exterminate megafauna populations \\[7\\]. Our analysis further supports humans as the key driver of extinction, but also has yielded the critical finding of little association between climate change severity and the proportion of extinct large mammals, suggesting that climate change was not a pervasive cause of this global-scale extinction event. Some earlier research has indicated that late Quaternary climate change affected megafauna species population dynamics and distributions \\[60\\], similar to the well-documented effects on smaller animals and plants \\[61\\]. Such effects, however, do not explain why most megafauna species did not go extinct where climate change was most severe or during previous glacial\u2013interglacial cycles. In addition, although megafauna\u2013hominin\u2013climate interactions will have varied among species and regions, our analysis strongly supports modern human colonization of virgin territory as the more pervasive driver of extinction.\n\nThe temperature-related pattern in the Archaic-combined region is consistent with previous suggestions of climate as a major extinction driver there, especially in northern areas \\[3,60,62\\]. The relationship appears to be driven by high temperature anomaly and extinctions in northwest Eurasia and low-temperature anomaly and extinctions in southern Asia, but there are also widespread mismatches (<a href=\"#RSPB20133254F1\" data-ref-type=\"fig\">figure 1<\/a>*a*,*d*). However, the absence of a climate\u2013extinction association from the *H. sapiens*-only region is inconsistent with extinctions resulting from synergism between climatic stress and hunting pressure because the association should rather have been strongest here. Instead, this effect appears consistent with human-driven extinctions with climatic constraints on archaic human distributions in northern parts of Eurasia \\[25\\], causing these regions to function as at least partial megafaunal refugia until modern human arrival \\[63\\]. At the same time, subtropical and tropical southern Asia were earlier, more permanently, and more densely colonized by archaic humans \\[24\\] and may thus be relatively similar to sub-Saharan Africa, with low late Quaternary extinctions because of either coevolution and\/or pre-Late Pleistocene extinctions \\[28,64\\]. Such a scenario fits the extinction pattern for *Mammuthus primigenius* in Eurasia, with a range contraction towards high latitudes despite earlier temperate occurrences \\[65\\], and late survival in the high north beyond the range of modern humans, followed by sudden extinction with human arrival to these regions \\[60,63\\]. Eurasia experienced the most prolonged extinction during the Late Pleistocene with species lost before and after the LGM \\[3\\]. However, the pattern of regional climate change severity was similar between the LIG\u2013LGM and LGM\u2013present (electronic supplementary material, figure S10) so the relationship between climate change and extinction severity is expected to be consistent regardless of whether species were lost before or after the LGM.\n\nSub-Saharan Africa experienced mild late Quaternary extinctions, as would be expected with the length of megafauna\u2013hominin co-occurrence in this region. It is also worth noting that there are some indications that a number of megafauna extinctions in sub-Saharan Africa occurred earlier, in the Early- to Mid-Pleistocene, possibly as a result of early hominin hunting or competition \\[66\\]. However, Africa also experienced minimal glacial\u2013Holocene temperature change and fits the continental-scale pattern of low climate change and extinction severity that Nogu\u00e9s-Bravo *et al*. \\[20\\] described. At the same time, the area nevertheless experienced strong changes in precipitation, particularly in West Africa where extinction was exceptionally low (electronic supplementary material, figure S1; <a href=\"#RSPB20133254F1\" data-ref-type=\"fig\">figure 1<\/a>*a*). Given the already mentioned strong contrasts in extinctions between sub-Saharan Africa and South America despite similar climate changes, Africa conforms well to the extinction pattern expected from hominin palaeobiogeography.\n\nOur analysis illustrates the value of taking a macroscale approach for understanding this global-scale extinction event, particularly highlighting the numerous discrepancies between the extent of climatic change and extinction severity at a regional scale. Furthermore, our findings indicate that the current low diversity in large mammals \\[2\\] in many continental areas is an anthropogenic phenomenon, not a natural one, with important implications for nature management \\[67\\].\n\n# Supplementary Material\n\n###### Supplementary Material\n\n# Supplementary Material\n\n###### Table S1\n\n# Supplementary Material\n\n###### Supplementary Data 1\n\n# Supplementary Material\n\n###### Supplementary Data 2\n\n# Supplementary Material\n\n###### R analysis script\n\n## Acknowledgements\n\nWe acknowledge help with data collection from Lovisa Kirkeby and Brian Kerr, and human arrival time information from Anders Eriksson.\n\n## Data accessibility\n\nThe data reported in this paper are provided in the electronic supplementary material.\n\n## Funding statement\n\nWe gratefully acknowledge economic support from the 15 Juni Fonden and the European Research Council (ERC-2012-StG-310886-HISTFUNC).\n\n# References\n\n[^1]: These authors contributed equally to this study.","meta":{"dup_signals":{"dup_doc_count":122,"dup_dump_count":41,"dup_details":{"curated_sources":2,"2018-47":3,"2018-43":1,"2018-34":1,"2018-30":3,"2018-26":1,"2018-17":2,"2018-13":3,"2018-09":2,"2017-51":3,"2017-47":2,"2017-43":3,"2017-39":1,"2017-34":3,"2017-30":2,"2017-26":3,"2017-22":2,"2017-17":4,"2017-09":4,"2017-04":4,"2016-50":3,"2016-44":5,"2016-40":4,"2016-36":5,"2016-30":5,"2016-26":4,"2016-22":4,"2016-18":3,"2016-07":3,"2015-48":3,"2015-40":2,"2015-35":3,"2015-32":2,"2015-27":3,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":4,"2017-13":3,"2015-18":2,"2015-11":3,"2015-06":3}},"file":"PMC4071532"},"subset":"pubmed_central"}
{"text":"author: David S Pisetsky\ndate: 2012\ninstitute: 1Duke University Medical Center, 151G, Durham VA Medical Center, Durham, NC 27705, USA\ntitle: 4th International Forum on Rheumatoid Arthritis: Great Times Near the Great Wall\n\nBeijing, an incredible city, was the site for the 4th International Forum on Rheumatoid Arthritis (IFRA), an incredible meeting. Professor Zhan-guo Li, Professor of Medicine at Peking University and President of the Asia Pacific League Against Rheumatism, and his co-chairs, Professor Lars Klareskog, Professor of Rheumatology, Karolinska Institutet, and Professor Kazuhiko Yamamoto, Professor of Medicine, University of Tokyo, organized the meeting. They did a terrific job and created a program to showcase the excitement and dynamism of both modern China and modern rheumatology. Professor Li was a great host as hundreds of Chinese rheumatologists packed the Beijing Xinyuan Hotel to hear lectures by rheumatologists from China as well as invited speakers from around the world. I have attended few meetings where the energy was so high and the eagerness to learn so great.\n\nWhile most of the international speakers had previously visited Beijing, the meeting was my first trip to China. To say the least, I was surprised by the city's modernity. Growing up in a very frosty time of the Cold War, China - usually called Red China or Communist China in the United States - was very much a forbidden country until Nixon thawed relationships with secret diplomacy, a ping-pong tournament and a memorable visit in 1972. I remain uncertain about the city's name because, during my lifetime, Beijing has also been called both Peking and Peiping. The images I have of modern China come mostly from the telecasts of the 2008 Olympics, although the NBC commentators seemed mostly interested in street food as if restaurants did not exist. I had few preconceived ideas of the real Beijing as opposed to the attractions I read about in *1000 Places to See Before You Die*.\n\nWhile China is a very old country, Beijing is a very new city. I would say that Beijing has the ambience of a boom town - but boom town is a misnomer for a megalopolis that has a population of about 20 million people and a land area of 6,500 square miles. About half the size of Belgium, Beijing makes Chicago look like a quaint little village with its neighborhoods of row houses. High-rise apartments and office buildings are everywhere as if the only direction to grow is up. Alas, a dense haze often covers the landscape, blotting the outline of some beautiful buildings and preventing appreciation of the sheer magnitude of the construction that has marked the explosive growth of Beijing in recent years.\n\nThroughout the city, wide avenues and highways are dense with cars at all hours. Driving around for sightseeing, I was gratified (and indeed mystified) to see many cars with Buick on the name plate. Indeed, I probably saw more Buicks in Beijing than I do in Durham, North Carolina. There used to be an advert that said 'This is not your father's Buick.' I guess in China the advert says 'This is not your American's Buick.' That would be true since the car is the product of GM Shanghai, a joint venture of General Motors and CDAI. Actually, GM sells more cars in China than in the United States. Volkswagens, Hyundais and Audi are also frequent vehicles and far outnumber the bicycles and scooters I was expecting.\n\nAs a tourist attraction, the ultra-modern Birds Nest Olympics stadium with its gleaming lattice shell designed by Swiss architects now competes for attention with the ancient Great Wall, the Forbidden City and the Summer Palace. The Egg is another landmark and a great building. For those who delight in stirring architecture, I recommend Beijing Airport. Airports are not usual venues for sightseers since they call forth memories of long lines at immigration, canceled flights and lost luggage. Nevertheless, Beijing Airport is a spectacular structure that is filled with light from an undulating ceiling of windows. The outside roof is painted red, the Chinese symbol for good luck. If you go to Beijing, you of course visit the airport, perhaps discombobulated by the time-zone change. My recommendation is to rouse yourself awake and look at the building and not just stumble through it.\n\nLike China, rheumatology research is experiencing a boom and is on the go. As talks at the IFRA demonstrated clearly, modern science moves with incredible speed and is producing voluminous data at a dizzying pace. The technology keeps getting better and better, the analysis of the pathogenesis more and more precise. (Alas, while I saw the data presented by speakers from China, I will have to confine my comments to those talks that were in English. I want to make sure that my comments are reasonably correct and apologize to those whose work I cannot describe with confidence.)\n\nStill in the midst of the genetics revolution, rheumatology is entering the epigenetics revolution. Studies by Professor Steffen Gay of Zurich and Professor Gary Firestein of San Diego described extraordinary progress in unraveling epigenetic changes in fibroblasts that may endow these cells with an invasive phenotype. These changes can result from base methylation reactions that lead to long-term changes in gene function; interestingly, some epigenetic changes may be transmitted through the generations. In this talk, Professor Gay raised the possibility of therapies to undo epigenetic modifications, including some agents that I can purchase at my local GNC store. Discussing another element of gene regulation, Professor Iain McInness of Glasgow illustrated how miRNA can regulate key events in immune activation. miRNA can be a target for therapy, as illustrated by findings from Professor Gay and colleagues on the use of antagomirs in a pulmonary hypertension model.\n\nModern technology is also providing new insights into B-cell abnormalities that underpin autoimmunity in rheumatoid arthritis (RA). Presenting a study of antibodies to citrullinated proteins (ACPA), Professor Klareskog described the extraordinary commitment of synovial B cells to ACPA production, with about 30% of these cells pumping out immunoglobulin to drive events in synovial inflammation. ACPA may have some unexpected roles in pathogenesis since, as Professor Klareskog showed, antibodies to citrullinated vimentin may trigger osteoclastogenesis, perhaps driving bone loss that is so characteristic of RA. Complementing these talks was a presentation by Professor Joachim Kalden of Erlangen on the treatment of autoimmunity with bortezemib, a proteasome inhibitor that can interfere with plasma cells. In both murine models as well as early studies on patients with lupus, this agent can reduce anti-DNA levels; effects on protective antibodies are of course a concern and will be closely watched as this therapy advances.\n\nA presentation by Professor Ed Keystone of Toronto illustrated striking progress on the clinical front. Professor Keystone addressed two main topics, highlighting recent presentations from the European League Against Rheumatism meeting in Berlin. Two messages are clear. The first is that, in head-to-head trials, agents that differ in action can lead to similar clinical responses. Adalimumab and subcutaneous abatacept thus showed comparable efficacy; reference to prior trials suggests that the effects of subcutaneous abatacept may differ from those of the intravenous formulation with respect to the time course and magnitude of the response. The results of the head-to-head trials are also surprising in view of an impression that the efficacy of co-stimulatory blockade may differ from that of TNF blockade. These studies raise the question of why agents that attack fundamentally different cellular pathways act so similarly. While current terminology emphasizes the concept of targeted therapy, the action of treatments may converge so that the same bull's eye is hit even if the arrows come from different directions.\n\nA second message of Professor Keystone's talks relates to remission. As the data he presented showed, treatment of RA with current agents and strategies (that is, treat to target and tight control) can induce remission in a significant number of patients as defined by well-established outcome measures. Furthermore, as he reported in patients achieving remission with a combination approach, elimination of a biological agent does not lead to relapse in many patients, with remission sustained at least over months. In the words of Professor Keystone, this is a game changer.\n\nI think that the frequency of remission in recent clinical studies has caught many rheumatologists off guard. RA has long been conceptualized as a chronic, even relentless, disease with changes (?epigenetic) in synoviocytes leading to joint destruction even in the absence of inflammation. Interpretation of these clinical studies is, of course, dependent on the definition of remission and the extent of subclinical synovitis and radiological progression that may occur in a patient in whom arthritis seems quiescent.\n\nOutcomes researchers will have ample opportunity to debate the criteria for remission and which composite measures (Disease Activity Score, Clinical Disease Activity Index, Simplified Disease Activity Index, Boolean), and their components, are most informative and reliable. I sometimes think rheumatologists do not really know how to quantify disease activity, for either clinical or research purposes, especially when inflammation is low. In my own clinical practice, I have thought that joint tenderness is important to this determination although Professor Keystone, an expert with decades of experience, gave the nod to joint swelling as a measure of active disease. During a discussion session, I asked Professor Keystone about his approach to assess swelling. He said ballottement of joint swelling is key, showing how he squeezes a joint with his fingers to determine its character and the presence of inflammation. If rheumatologists have to rely on ballottement to make crucial decisions to start or stop drugs, I have my concerns. Given the power and elegance of today's science, some biomarker or imaging modality must be out there to help with decision-making and tell whether the fire of inflammation smolders or has been successfully doused.\n\nIn addition to its importance clinically, the phenomenon of remission should drive inquiry into disease mechanisms, providing a unique setting to explore critical events in pathogenesis. Much research in rheumatology now focuses on preclinical autoimmunity, the time prior to the onset of clinical symptoms, in which immune cell abnormalities (for example, ACPA expression, cytokine production) are revving up for their full onslaught. With remission, there is perhaps now a state of postclinical autoimmunity in which signs and symptoms abate.\n\nWhat is going on in patients with remission? Consider two examples. ACPA have a close relationship to RA, with some data, including animal studies, pointing to a direct role in joint inflammation. If ACPA titers do not change much during remission, then their role in pathogenesis is up for grabs. Perhaps during remission, ACPA specificity shifts in some subtle but significant way to nonpathogenic species. Since these antibodies emanate from long-lived plasma cells, however, I doubt such a change. Alternatively, an antigen required for immune complex formation may have disappeared during remission or, more probably, the downstream effectors have been quieted.\n\nA second example relates to epigenetics. Many current studies on the epigenetic modification of fibroblasts involve operative specimens obtained at the time of replacement joint surgery. Synoviocytes in such tissues have been subject to years (?decades) of inflammation, the assault of biomechanical stress and even a slug or two of intra-articular glucocorticoids to go along with years of disease-modifying antirheumatic drugs. Until the advent of remission, it would be difficult to know which of these changes is primary or secondary - although, as Dr Firestein showed, IL-1 can affect DNA methylation. Now, with remission, epigenetic imprinting can be studied in a new way, although obtaining samples of synovium during remission may be challenging unless techniques for easy tissue retrieval become available.\n\nThe IFRA occurred in the midst of the US presidential election although, with current politics, it seems we are always in the midst of a presidential election. Talk of 2016 is already heating up. Palin versus Clinton? Whatever the outcome of the 2012 contest - red or blue - the prospects for the National Institutes of Health budget look bleak - gray if not black. Given the drive to shrink the federal budget and blot away red ink, stagnant funding may be the best we can do although genuine cutbacks may occur. Cutbacks on research now would be a great tragedy since technology is leading to unprecedented discoveries that can be translated into treatment advance. I wish some of our politicians would have attended the IFRA to hear lectures by Chinese investigators or to speak to Chinese students whose numbers have swelled with the influx of government money. Those politicians would have seen what a commitment to building research can accomplish in terms of innovation, economic growth and the inspiration provided to young people who are anxious to discuss the effects of IL-2 on regulatory T cells or the role of STING in the immune response to DNA in lupus.\n\nAccording to de Tocqueville, the genius of America came from the pursuit of 'self-interest properly understood.' China seems to be taking a different path, however. Practically and philosophically, it seems that China wants to pursue national interest properly understood. In either system, proper understanding must balance the interests of the individual and those of the state, always a great challenge. China is an old country and America is a young one, but both are struggling to find a balance consistent with their histories and cultures. Given the importance of US and Chinese economies in the world, there will be many onlookers to this great political process.\n\nI was honored to attend the IFRA and witness the extraordinary accomplishments of China in rheumatology research. It was a truly wonderful occasion of fellowship and cooperation, and I was excited to meet and toast new friends with potently delicious Chinese wine that had a strong resemblance to what in North Carolina we call white lightning. I look forward to many more years of international cooperation as we work together to advance rheumatology research and improve the health of people of the world properly understood.\n\nGan bei!\n\n# Abbreviations\n\nACPA: antibodies to citrullinated proteins; IFRA: 4th International Forum on Rheumatoid Arthritis; IL: interleukin; miRNA: microRNA; RA: rheumatoid arthritis; STING: stimulator of interferon genes; TNF, tumor necrosis factor.\n\n# Competing interests\n\nThe author declares that they have no competing interests.\n\n# Suggested reading\n\n1\\. Barber GN: **Cytoplasmic DNA innate immune pathways**. *Immunol Rev* 2011, **243:**99-108.\n\n2\\. Boyman O, Sprent J: **The role of interleukin-2 during homeostasis and activation of the immune system**. *Nat Rev Immunol* 2012, **12:**180-190.\n\n3\\. Brock M, Samillan VJ, Trenkmann M, Schwarzwald C, Ulrich S, Gay RE, Gassmann M, Ostergaard L, Gay S, Speich R, Huber LC: **Antagomir directed against miR-20a restores functional BMPR2 signalling and prevents vascular remodeling in hypoxia-induced pulmonary hypertension**. *Eur Heart J* 2012. \\[Epub ahead of print\\]\n\n4\\. Felson DT, Smolen JS, Wells G, Zhang B, van Tuyl LH, Funovits J, Aletha D, Allaart CF, Bathon J, Bombardieri S, Brooks P, Brown A, Matucci-Cerinic M, Choi H, Combe B, de Wit M, Dougados M, Emery P, Furst D, Gomez-Reino J, Hawker G, Keystone E, Khanna D, Kirwan J, Kvien TK, Landew\u00e9 R, Listing J, Michaud K, Martin-Mola E, Montie P, *et al*.; American College of Rheumatology; European League Against Rheumatism: **American College of Rheumatology\/European League Against Rheumatism provisional definition of remission in rheumatoid arthritis for clinical trials**. *Arthritis Rheum* 2011, **63:**573-586.\n\n5\\. Harris U, Georgess D, Bang H, Bozec A, Axmann R, Ossipova E, Jakobsson PJ, Baum W, Nimmerjahn F, Szarka E, Sarmay G, Krumbholz G, Neumann E, Toes R, Scherer HU, Catrina AI, Klareskog L, Jurdic P, Schett G: **Induction of osteoclastogenesis and bone loss by human autoantibodies against citrullinated vimentin**. *J Clin Invest* 2012, **122:**1791-1802.\n\n6\\. Hughes-Austin JM, Deane KD, Derber LA, Kolfenbach JR, Zerbe GO, Sokolove J, Lahey LJ, Weisman MH, Buckner JH, Mikuls TR, O'Dell JR, Keating RM, Gregersen PK, Robinson WH, Holers VM, Norris JM: **Multiple cytokines and chemokines are associated with rheumatoid arthritis-related autoimmunity in first-degree relatives without rheumatoid arthritis: Studies of the Aetiology of Rheumatoid Arthritis (SERA)**. *Ann Rheum Dis* 2012. \\[Epub ahead of print\\]\n\n7\\. Keystone EC, Smolen J, van Riel P: **Developing an effective treatment algorithm for rheumatoid arthritis**. *Rheumatology* 2012, **51:**v48-v54.\n\n8\\. Nakano K, Whitaker JW, Boyle DL, Wang W, Firestein GS: **DNA methylome signature in rheumatoid arthritis**. *Ann Rheum Dis* 2012. \\[Epub ahead of print\\]\n\n9\\. Neubert K, Meister S, Moser K, Weisel F, Maseda D, Amann K, Wiethe C, Winkler TH, Kalden JR, Manz RA, Voll RE: **The proteasome inhibitor bortezomib depletes plasma cells and protects mice with lupus-like disease from nephritis**. *Nat Med* 2008, **14:**748-755.\n\n10\\. Sokolove J, Bromberg R, Deane KD, Lahey LJ, Derber LA, Chandra PE, Edison JD, Gilliland WR, Tibshirani RJ, Norris JM, Holers VM, Robinson WH: **Autoantibody epitope spreading in the pre-clinical phase predicts progression to rheumatoid arthritis**. *PLoS One* 2012, **7**:e35296.\n\n11\\. Studenic P, Smolen JS, Aletaha D: **Near misses of ACR\/EULAR criteria for remission: effects of patient global assessment in Boolean and indexbased definitions**. *Ann Rheum Dis* 2012, **71:**1702-1705.","meta":{"dup_signals":{"dup_doc_count":128,"dup_dump_count":39,"dup_details":{"curated_sources":2,"2022-33":1,"2021-43":1,"2021-31":1,"2021-21":1,"2021-10":1,"2020-45":1,"2020-10":1,"2017-17":1,"2017-09":10,"2017-04":1,"2016-50":1,"2016-44":1,"2016-36":10,"2016-30":9,"2016-22":1,"2016-18":1,"2016-07":11,"2015-48":3,"2015-40":3,"2015-35":3,"2015-32":2,"2015-27":3,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":4,"2014-42":8,"2014-41":6,"2014-35":3,"2014-23":6,"2014-15":4,"2023-40":1,"2015-18":3,"2015-11":2,"2015-06":3,"2014-10":3,"2013-48":2,"2013-20":4,"2024-10":1}},"file":"PMC3580510"},"subset":"pubmed_central"}
{"text":"author: David T. JonesCorresponding author: David T. Jones, <firstname.lastname@example.com>.\ndate: 2012-07\nreferences:\ntitle: Neural Networks, Cognition, and Diabetes: What Is the Connection?\n\nDiabetes has been associated not only with subtle cognitive deficits in mental speed and flexibility (1,2) but also with an increased risk for development of significant disruption in cognitive function in the form of dementia (3). In order to prevent subtle cognitive sequelae and potentially reduce the burden of dementia on an aging world population (4), a better understanding of the pathophysiological mechanisms underlying the cognitive dysfunction in diabetes is needed.\n\nThere are several factors associated with diabetes and its management beyond acute metabolic or vascular insults that are potentially involved in this cognitive disruption, a few of which will be briefly reviewed here. Hyperglycemia may affect cognitive function by altering synaptic plasticity in the brain (5), increasing levels of oxidative stress (6), and\/or subtly altering the cerebral microvasculature (7). Treatment of diabetes may lead to slight or occasionally more severe periods of hypoglycemia, which may translate to structural and metabolic alterations of the central nervous systems and subsequent cognitive dysfunction. However, it has been observed that subtle diabetes-related cognitive changes are associated with chronic hyperglycemia rather than episodes of hypoglycemia (8). The same study also observed that there was no significant difference in cognitive function between aggressively managed subjects with reduced levels of microvascular complications and those with a greater degree of microvascular complications. Other studies have observed this as well (9). This may indicate that the association between chronic hyperglycemia and subtle cognitive dysfunction is related to factors outside of microvascular complications alone. In this context, the potential impact of chronic exposure to endogenous or exogenous insulin above the normal physiological range should be critically evaluated.\n\nMild cognitive impairment is associated not only with age of diabetes onset and disease duration but also with insulin treatment (10). The risk of dementia is also highest in people with diabetes treated with insulin (11). Hyperinsulinemia in nondiabetic individuals has also been associated with cognitive impairment (12). This association is confounded by other comorbid factors, disease duration, and disease severity. However, there are several sound biological mechanisms by which prolonged hyperinsulinemia may influence central nervous system function. Not only is insulin a vasoactive substance (13), but it also inhibits \"housekeeping\" processes important for healthy brain aging (i.e., autophagy) (14) and influences processing of proteins related to Alzheimer disease (15). Prolonged exposure of the brain to higher than physiological levels of insulin may alter metabolic pathways in a manner that is deleterious to cognitive circuitry, given that this circuitry is dependent on cells influenced by these metabolic processes. These factors likely impact the changes associated with the phenomenon of \"brain insulin resistance\" observed in Alzheimer disease.\n\nIt should be noted that comorbid conditions and confounding variables such as hypertension, brain infarcts, dyslipidemia, obesity, socioeconomic status, depression, and levels of mental and physical activity must also be taken into consideration when discussing diabetes-associated cognitive dysfunction.\n\nAny factor that precipitates cognitive dysfunction must affect brain circuitry in some manner. Given this necessity, one potential strategy for elucidating the relevant pathophysiological link between diabetes and cognitive dysfunction is to isolate the factors discussed above and compare their effects not only on direct cognitive measures or on the risk of developing cognitive impairment but also on measures of the neural networks that are affected. Neural networks have been shown to be acutely altered by structural lesions causing cognitive deficits (16) and are altered in at-risk populations prior to structural changes such as atrophy (17) before clinically measurable effects on cognition. Therefore, measures of neural networks may be best suited to track the earliest effects of diabetes on brain function. In this vein, the study presented in this issue of *Diabetes* by van Duinkerken et al. (18) lays the groundwork for these investigations. This study is an extension of their work investigating the neural network correlates of cognitive deficits observed in type 1 diabetes mellitus (T1DM), in which they hypothesize that these changes are modified by the presence of microvascular disease. Previously these investigators explored functional connectivity within neural networks using magnetoencephalography (MEG) (19) and concluded that \"chronic hyperglycemia, among other factors, may negatively affect brain functioning even before microvascular damage becomes manifest.\" In the current study, the authors skillfully use functional magnetic resonance imaging (fMRI) in the absence of an experimentally predetermined task, also known as resting-state fMRI, but referred to herein as task-free fMRI (TF-fMRI). (Vemuri et al. \\[20\\] review the application of this technique in a related cognitive context.) TF-fMRI allows for greater spatial resolution and visualization of networks of synchronized activity in the brain (intrinsic connectivity networks \\[ICNs\\]) than typically achieved by MEG.\n\nThe authors used TF-fMRI to identify ICNs in 49 T1DM patients with microangiopathy (MA^+^) and 52 without microangiopathy (MA^\u2212^) compared with 48 control (CN) subjects and their relationship to various cognitive and disease variables. They found increases in connectivity in visual processing and sensorimotor networks in MA^\u2212^ not present in MA^+^ relative to CN subjects, and decreases in connectivity in attentional, auditory\/language processing, and executive control networks that progressed in degree going from CN\\>MA^\u2212^\\> MA^+^. Given that the MA^+^ subjects were significantly older and had greater depressive symptoms, higher systolic blood pressure, and an earlier disease onset and longer disease duration, a subsequent analysis was conducted matching 27 MA^+^ and 28 MA^\u2212^ on disease onset and duration. In this analysis, the increased connectivity in the MA^\u2212^ group in the visual processing and sensorimotor networks was the only statistically significant finding. It should be emphasized that the MA^+^ group did not differ from the CN group in these two networks (MA^+^ results are reported as a relative decrease to MA^\u2212^, just as the CN group would appear as a relative decrease to MA^\u2212^, both of which being primarily driven by the MA^\u2212^ group's increase in connectivity in these two networks).\n\nThis study will certainly advance the field in the search for pathophysiological mechanisms of cognitive decline in diabetes. The authors focused on a T1DM population, which may be less confounded by metabolic syndrome comorbidities than a type 2 diabetic population. They also had a relatively large sample of well-characterized subjects with expert acquisition and analysis of TF-fMRI data. The study's weaknesses lie in the inherent difficulties in this and other TF-fMRI studies. TF-fMRI is reliant on the blood oxygenation level\u2013dependent signal and the fidelity of neurovascular coupling, which may be altered by microvascular changes. However, the similar results the authors report using MEG (which is not dependent on neurovascular coupling) are reassuring. In this regard, future TF-fMRI studies may benefit from inclusion of additional neuroimaging measures of white and gray matter integrity, which will also help tease apart other etiological factors. TF-fMRI also needs a model of the organization of the brain when it is not being experimentally directed (i.e., in a \"resting state\" during a task-free experimental paradigm). This will allow for a priori hypothesis generation and also circumvent some of the difficulties of TF-fMRI experimental design and interpretation.\n\nOne common difficulty in TF-fMRI interpretation is the phenomenon of increased functional connectivity, which is typically ascribed to \"compensation.\" However, this link has not been firmly established nor is it based on a model of undirected brain activity measured by TF-fMRI. In Alzheimer dementia, the earliest functional changes detected in at-risk populations appear to be increases in functional connectivity in posterior limbic circuitry as early as the third decade of life (21). The same circuitry displays decreased functional connectivity later in life and is accompanied by increased functional connectivity in anterior\/frontal limbic pathways (22), which in turn also decreases as Alzheimer disease progresses (23). Therefore, the increases in connectivity observed in the MA^\u2212^ group studied by van Duinkerken et al. may be related to an intermediate stage of connectivity change. This is clearly evident in the networks displaying decreased connectivity in a dose-dependent manner going from CN\\>MA^\u2212^\\> MA^+^. This line of reasoning is also supported by the fact that the difference between MA^+^ and MA^\u2212^ was not present when they were matched on disease duration and age of onset. Therefore, the results of this study do not strongly implicate microvascular disease as the only, or most prominent, factor related to diabetes-associated cognitive disruption. However, this study does implicate disease severity and duration, suggesting that metabolic derangements associated with chronic exposure to either hyperglycemia and\/or hyperinsulinemia may be more prominent in the development of diabetes-associated cognitive decline.\n\nFuture studies should focus on differentiating the effects of chronic hyperglycemia and hyperinsulinemia on measures of neural network integrity. Such studies will not only advance our understanding of the pathophysiological mechanisms underlying the cognitive dysfunction in diabetes but will also refine the use of neural network metrics as biomarkers of cognition. This will facilitate tracking of neural network responses to early intervention strategies targeting key pathophysiological mechanisms before the appearance of any cognitive dysfunction, thereby refining strategies aimed at reducing the cognitive burden of diabetes.\n\n## ACKNOWLEDGMENTS\n\nNo potential conflicts of interest relevant to this article were reported.\n\n# REFERENCES","meta":{"dup_signals":{"dup_doc_count":127,"dup_dump_count":55,"dup_details":{"curated_sources":2,"2022-05":1,"2020-16":1,"2019-51":1,"2019-47":1,"2019-43":1,"2019-39":1,"2019-26":1,"2019-18":1,"2019-13":1,"2018-51":1,"2018-39":1,"2018-26":2,"2018-22":2,"2018-17":1,"2018-13":2,"2018-09":1,"2018-05":2,"2017-51":1,"2017-47":2,"2017-43":4,"2017-39":2,"2017-34":2,"2017-30":5,"2017-26":3,"2017-22":1,"2017-17":7,"2017-09":4,"2017-04":8,"2016-50":4,"2016-44":4,"2016-40":4,"2016-36":4,"2016-30":4,"2016-26":3,"2016-22":3,"2016-18":3,"2016-07":2,"2015-48":2,"2015-40":1,"2015-35":2,"2015-32":1,"2015-22":2,"2015-14":1,"2014-52":3,"2014-49":2,"2014-42":2,"2014-41":2,"2014-35":2,"2014-23":3,"2023-06":1,"2017-13":4,"2015-18":3,"2015-11":1,"2015-06":1,"2024-10":1}},"file":"PMC3379670"},"subset":"pubmed_central"}
{"text":"abstract: A report on the 11th European Conference on Computational Biology (ECCB), Basel, Switzerland, September 9-12, 2012.\nauthor: Evangelos Bellos; Lachlan JM Coin; Myrsini Kaforou\ndate: 2012\ninstitute: 1Department of Epidemiology and Biostatistics, School of Public Health, Imperial College London, St Mary's Hospital, Norfolk Place, London, W2 1PG, UK; 2Department of Genomics of Common Disease, School of Public Health, Imperial College London, The Hammersmith Hospital, Du Cane road, London, W12 0NN, UK; 3BGI-Shenzhen, Beishan Road, Shenzhen, 518083, China; 4Section of Paediatrics and Wellcome Trust Centre for Clinical Tropical Medicine, Division of Infectious Diseases, Department of Medicine, Imperial College London, St Mary's Hospital, Norfolk Place, London, W2 1PG, UK\ntitle: Bioinformatics: living on the edge\n\n# Meeting report\n\nCan music be decomposed to single notes on a piece of paper? Can life be fully understood by the function of genes in isolation? These are some of the questions that Denis Noble (University of Oxford, UK) posed in one of the more philosophical keynote addresses of this year's European Conference on Computational Biology (ECCB). Noble, a famed critic of genetic reductionism, in concert with renowned guitarist Christoph Denoth, set out to explore how synergy begets complexity both in art and in science. And in doing so, he captured a central theme of the conference: holism.\n\nFrom gene prediction to comparative genomics to protein structure prediction, the early days of post-genomic bioinformatics research were arguably characterized by a reductionist approach to understanding life. Systems biology, on the other hand, started on the opposite end of the spectrum, attempting to directly model complex behavior arising from biomolecular interplay, largely abstracted from the individual components. In this post-ENCODE era, in which tremendous progress has been made in understanding each biomolecule individually, the focus now seems to have shifted toward a middle ground: leveraging the treasure trove of information about the nodes of the network to elucidate the intricate web of interactions represented by the network edges.\n\nGiven the sustained development of next-generation sequencing technologies and their essential role in the ascendancy of genomics, it is perhaps surprising that talks on this subject were conspicuously scarce at ECCB. However, this is not to say that genomics was not pervasive throughout the conference; it was, from keynotes to posters, just indirectly so. Sequencing technologies appear to be maturing into stable tools that are ubiquitously used to obtain deep knowledge of biomolecules themselves, as well as intermolecular relationships. As a silent partner, genomics is providing both the starting point and the means for investigating networks, but without stealing the spotlight.\n\n# Genetic interactions: within genomes\n\nThe systematic identification and characterization of genome-wide genetic interactions is one area that has already been transformed by the availability of sequence data for multiple species. Colm Ryan (University of California, San Francisco, USA) compared experimentally derived epistasis maps of the fission yeast *Schizosaccharomyces pombe* and the budding yeast *Saccharomyces cerevisiae* to show that the overall level of genetic cross-talk between different biological processes tends to be more conserved than the underlying interactions. He further demonstrated how epistasis maps can be used to annotate uncharacterized genes, drawing on the observation that genes with similar genetic interaction profiles are often involved in the same functional pathway. Similar approaches may be used as a model for genetic interaction analysis in more complex organisms, in an effort to understand not only biological functions, but also the mechanisms that underlie disease phenotypes. Philipp Bucher (\u00c9cole Polytechnique F\u00e9d\u00e9rale de Lausanne, Switzerland) exploited lineage-specific whole-genome duplication events in teleost fish (including zebrafish and pufferfish lineages) to investigate the post-duplication fate of ultraconserved non-coding elements. These elements tend to be organized in large clusters around developmental genes, and were found mostly to be retained together in only one of the two genome copies. This winner-takes-all scenario suggests that ultraconserved non-coding elements operate as part of a dense cooperativity network, which could be responsible for their high levels of conservation.\n\n# Genetic interactions: between genomes\n\nIn a *tour de force* keynote presentation focusing on the social organization of fire ants (*Solenopsis invicta*), Laurent Keller (University of Lausanne, Switzerland) demonstrated that an ant's behavior is determined not only by its own genome but also by other genomes in the population. By way of example, Keller described what might be called a selfish allele in a 13.9 Mbp non-recombining region, encoding the odorant protein Gp-9, that regulates its own frequency by mediating altruistic and aggressive instincts. Michal Linial (The Hebrew University of Jerusalem, Israel) explored interactions between virus and host genomes from an evolutionary perspective. Linial demonstrated how both amino acid and codon usage of viral proteomes have adapted to maximize translational efficiency in specific host environments. Linial also showed that host-acquired genes in virus genomes tend to have fewer protein domains and shorter linker regions than their host counterparts. Understanding these evolutionary adaptations may prove essential to the effective management of viral infections.\n\n# Protein-RNA interactions\n\nThe recently published results of the ENCODE project established that at least three quarters of the human genome is capable of being transcribed. One approach to understanding the functional role of this pervasive transcription may be the identification of new RNA-binding proteins, as presented by Cristoph Dieterich (Max Delbr\u00fcck Center for Molecular Medicine, Germany). By purifying proteins that have been cross-linked to polyadenylated mRNA, followed by quantitative mass spectrometry, Dieterich identified 797 mRNA-bound proteins, one third of which were previously unknown and likely participate in post-transcriptional gene-regulation networks. Moreover, Dieterich used PAR-CLIP to sequence the mRNA-binding sites of these proteins. Mihaela Zavolan (Biozentrum, Switzerland) also reported a novel application of PAR-CLIP, in which the Argonaute component of the RISC complex was used as bait to discover non-canonical microRNA (miRNA)-target interactions. Intriguingly, Zavolan showed how a single miRNA molecule (miR-294) can target components of the chromatin-remodeling complex and prevent differentiation in embryonic stem cells, thus concluding that a handful of miRNAs may be sufficient to determine cell fate.\n\n# Protein-protein interactions\n\nAlthough protein-protein interaction (PPI) networks have been extremely valuable as a starting point for modeling cellular processes, it is well known that PPI databases have a high false-positive rate, while omitting many genuine interactions. The gold standard for the field, as described by Barry Honig (Columbia University, USA), is a three-dimensional structure of a complex that incorporates the interacting proteins, but this level of confirmation is available for less than 0.5% of all recorded PPIs. Honig presented a computationally efficient method for superimposing homologous proteins onto known structural interactions in order to generate interaction models, which are subsequently combined with co-expression and functional similarity information. This approach makes clever use of homology to known structures; however, as Chris Sander (Memorial Sloan-Kettering Cancer Center, USA) noted, only half of the well-characterized protein families in the Pfam database have a known three-dimensional structure for any of their members. To address this limitation, Sander identified co-evolving residues from multiple sequence alignments in order to predict residue-residue proximity in folded protein structures. Philip Kim (University of Toronto, Canada) illustrated the tissue-specific nature of PPI networks by monitoring how alternative splicing events impact upon them. Kim presented evidence that as much as a third of all tissue-specific alternatively spliced exons lead to differential PPI through both the creation of new and the destruction of existing interactions.\n\n# Protein-drug interactions\n\nChemogenomics is an emerging research area that is focused on identifying novel drug-protein interactions by screening libraries of small molecules against the proteome, through the computational analysis of high-throughput data. Yoshihiro Yamanishi (Kyushu University, Japan) presented a method, based on sparsity-induced binary classifiers, that incorporates data on the chemical structures of drugs, genomic information about target proteins and all known protein-ligand interactions across different protein families into a predictive model. The model can predict underlying interactions between drug chemical substructures and protein functional sites, which are involved in drug-target interaction networks. With a view to understanding and minimizing drug side effects in patients, Sayaka Mizutani (Kyoto University, Japan) presented an approach for integrating drug side effect data with drug-protein binding information, using sparse canonical correlation. This technique was used to derive highly correlated sets of drug-targeted proteins and side effects, along with the drugs that drive their correlation. Such methods can play an important role in predicting potential side-effects and their mediating pathways even at the drug design phase.\n\n# Conclusions\n\nThe opening keynote lecture of ECCB was delivered by Nobel laureate Aaron Ciechanover (Technion - Israel Institute of Technology, Israel) who described his *magnum opus*: unraveling the previously unknown cellular mechanism of protein degradation. Ciechanover outlined many aspects of ubiquitin-mediated protein degradation, including the role of E3 ubiquitin ligases in ensuring high target specificity. He further highlighted the link between dysregulation of the ubiquitin-proteasome pathway and disease. Various levels of this pathway have been identified as drug targets to treat or suspend the progression of diseases such as myeloma, leukemia and viral encephalitis. While this talk was inspiring, the fact that these discoveries were made without the assistance of high-throughput technologies or bioinformatics was sobering, and raises the question of whether computational analyses of high-throughput data can lead to the elucidation of still unknown, fundamental cellular mechanisms. In other words, when will bioinformatics have its Nobel Prize moment? The talks presented at this conference suggest that by transforming information on complex biomolecular interactions, obtained at high cellular and temporal resolution, into sequence data, bioinformaticians may be well on the way to achieving such a feat.\n\n# Abbreviations\n\nECCB: European Conference on Computational Biology; ENCODE: Encyclopedia of DNA Elements; Mbp: megabase pair; miRNA: microRNA; PAR-CLIP: photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation; PPI: protein-protein interaction; RISC: RNA-induced silencing complex.\n\n# Competing interests\n\nThe authors declare that they have no competing interests.","meta":{"dup_signals":{"dup_doc_count":104,"dup_dump_count":30,"dup_details":{"curated_sources":4,"2021-39":1,"2019-39":1,"2017-09":9,"2016-44":1,"2016-40":1,"2016-36":9,"2016-30":5,"2016-22":1,"2016-18":1,"2016-07":5,"2015-48":3,"2015-40":3,"2015-35":3,"2015-32":3,"2015-27":3,"2015-22":2,"2015-14":2,"2014-52":3,"2014-49":5,"2014-42":4,"2014-41":5,"2014-35":6,"2014-23":5,"2014-15":3,"2023-40":1,"2015-18":3,"2015-11":3,"2015-06":3,"2014-10":3,"2013-48":3}},"file":"PMC3491407"},"subset":"pubmed_central"}
{"text":"abstract: Precise regulation of \u03b2-cell function is crucial for maintaining blood glucose homeostasis. Pax6 is an essential regulator of \u03b2-cell\u2013specific factors like insulin and Glut2. Studies in the developing eye suggest that Pax6 interacts with Mitf to regulate pigment cell differentiation. Here, we show that Mitf, like Pax6, is expressed in all pancreatic endocrine cells during mouse postnatal development and in the adult islet. A *Mitf* loss-of-function mutation results in improved glucose tolerance and enhanced insulin secretion but no increase in \u03b2-cell mass in adult mice. Mutant \u03b2-cells secrete more insulin in response to glucose than wild-type cells, suggesting that Mitf is involved in regulating \u03b2-cell function. In fact, the transcription of genes critical for maintaining glucose homeostasis (insulin and *Glut2*) and \u03b2-cell formation and function (*Pax4* and *Pax6*) is significantly upregulated in *Mitf* mutant islets. The increased Pax6 expression may cause the improved \u03b2-cell function observed in *Mitf* mutant animals, as it activates insulin and Glut2 transcription. Chromatin immunoprecipitation analysis shows that Mitf binds to *Pax4* and *Pax6* regulatory regions, suggesting that Mitf represses their transcription in wild-type \u03b2-cells. We demonstrate that Mitf directly regulates *Pax6* transcription and controls \u03b2-cell function.\nauthor: Magdalena A. Mazur; Marcus Winkler; Elvira Gani\u0107; Jesper K. Colberg; Jenny K. Johansson; Hedvig Bennet; Malin Fex; Ulrike A. Nuber; Isabella ArtnerCorresponding author: Isabella Artner, <firstname.lastname@example.com>.\ndate: 2013-08\nreferences:\ntitle: Microphthalmia Transcription Factor Regulates Pancreatic \u03b2-Cell Function\n\nThe islets of Langerhans consist of \u03b1-, \u03b2-, \u03b4-, \u03b5-, and pancreatic polypeptide (PP) cells, which produce the hormones glucagon, insulin, somatostatin, ghrelin, and PP, respectively. Together these hormones regulate fuel metabolism, with insulin and glucagon being essential for glucose homeostasis. Thus, glucagon secreted from \u03b1-cells stimulates the mobilization of glucose through gluconeogenesis and glycogenolysis, while \u03b2-cell\u2013secreted insulin promotes glucose storage. Defects in \u03b1- and \u03b2-cell function play a significant role in the ability of diabetic individuals to maintain glycemic control.\n\nCharacterization of the transcription factors regulating insulin and glucagon expression has demonstrated their significance to islet cell function and endocrine cell development. For example, pancreatic and duodenal homeobox 1 (Pdx1) is crucial for pancreas development; pancreatic endodermal progenitors fail to proliferate in the absence of Pdx1, resulting in a small pancreatic rudiment (1). Pdx1 is also critical for mature \u03b2-cell activity because deletion of this factor from adult \u03b2-cells results in a severe diabetic phenotype, which is at least partially caused by reduced insulin and Glut2 expression (2). Pax6 and NeuroD1 are also required for insulin and Glut2 expression, and loss of these transcription factors affects \u03b2-cell function and endocrine cell differentiation (3,4). Significantly, mutations in *PDX1* (5), *PAX6* (6), and *NEUROD1* (7) also cause maturity-onset diabetes of the young in humans.\n\n\u03b2-Cell function and glucose responsiveness are established during late embryonic and postnatal development (8). Recently, the MafA and MafB transcription factors have been identified as key regulators of these processes because of their ability to regulate genes essential for endocrine cell function, such as insulin, glucagon, *Pdx1*, and *Glut2* (9\u201311). Embryonic \u03b2-cells initially express *MafB*, but *MafB* expression is lost in postnatal \u03b2-cells, which express *MafA* instead (12). The switch between MafB and MafA expression in \u03b2-cells is associated with the development of functional \u03b2-cells. Recent gene profiling studies have shown that MafB regulates genes required for \u03b2-cell function during embryonic \u03b2-cell differentiation while the same genes are activated by MafA in adult \u03b2-cells (12). These gene expression experiments have shown that microphthalmia transcription factor (Mitf) expression is reduced in *MafA* and *MafB* mutant embryonic pancreata (12). Mitf, a basic-helix-loop-helix-leucine-zipper transcription factor, regulates melanogenesis by activating transcription of pigment cell-specific genes in both the skin and retina (13,14). In addition, it can act as a transcriptional repressor (14,15) and controls the expression of cell survival (*Bcl2*) (16) and cell cycle regulatory (*Cdk2* and *p16\/Ink4a*) genes (17,18).\n\nIn this study, we show that Mitf is produced in the developing pancreas and adult islets. *Mitf* loss-of-function mice have lower blood glucose levels than wild-type animals in response to an intraperitoneal glucose challenge but also during nonfasted conditions. Mutant islets secrete more insulin upon exposure to high glucose concentrations, and *Mitf* mutant animals have higher circulating insulin levels in fasted conditions. Additionally, the expression of genes regulating blood glucose levels (insulin and *Glut2*) and \u03b2-cell formation and function (*Pax4* and *Pax6*) is significantly higher in *Mitf* mutant than in wild-type islets. Promoter occupancy studies show that Mitf binds to the pancreas-specific *Pax4* and *Pax6* regulatory regions, which suggests that Mitf directly regulates the transcription of these genes in \u03b2-cells and thereby modulates \u03b2-cell function.\n\n# RESEARCH DESIGN AND METHODS\n\nMice with a point mutation (C to T at nucleotide 916) in the *Mitf* gene have previously been characterized (19). The *Mitf* cloudy eye (*Mitf^ce\/ce^*) mutation results in a lack of the Mitf zipper domain due to the presence of a stop codon between the helix-loop-helix and zipper motif. This leads to the synthesis of a truncated protein incapable of DNA binding (19). *Mitf^ce\/ce^* mice have white coat color and small eyes. Heterozygous and wild-type animals both have black coat color. For distinguishing between the latter, DNA samples were sequenced (forward, ggtccattgtcttgttttatcacag; reverse, gtatccaccctctgccat). Wild-type, heterozygous, and homozygous animals are distinguished by C, C\/T, and T at bp 916 of the *Mitf* gene (<http:\/\/www.informatics.jax.org\/searches\/accession_report.cgi?id=mgi:1856532>). Wild-type and heterozygous *Mitf* animals were indistinguishable in physiological and histological studies (data not shown), and therefore the data for them are combined as \"wt\/het\" data. All animal work was approved by a local ethics committee for animal research.\n\n## Immunohistochemistry.\n\nPancreata from E15.5 and E18.5 embryos, postnatal day P0, P7, and P21, and 12 week-old mice were fixed for 2 h at 4\u00b0C in 4% paraformaldehyde in PBS, washed with PBS, dehydrated, and embedded in paraffin. Sections (6 \u03bcm) were used for immunohistochemical analysis as previously described (9).\n\n## Antibodies.\n\nPrimary antibodies used were as follows: rabbit anti-Mitf (1:1,000; gift from S. Saule, Institut Curie, Paris), rabbit anti-Mitf (1:1,000; Abcam), guinea pig anti-insulin (1:2,000; Millipore), mouse anti-glucagon (1:2,000; SIGMA), sheep anti-somatostatin (1:2,000; American Research Products), guinea pig anti-PP (1:2,000; Millipore), goat anti-ghrelin (1:500; Santa Cruz), rabbit anti-Glut2 (1:500; Chemicon International), rabbit anti-MafA (1:1,000; Bethyl Laboratories), rabbit anti-Nkx6.1 (1:1,000; \u03b2-Cell Biology Consortium), rabbit anti-Pax6 (1:1,000; Covance), mouse anti-Pax6 (1:100; Developmental Hybridoma Bank), goat anti-Pdx1 (1:1,000; \u03b2-Cell Biology Consortium), mouse anti-islet 1 (1:100; Developmental Hybridoma Bank), and mouse anti-Synaptophysin (1:100; Abcam). Secondary antibodies used were as follows: (1:500) diluted CY2, CY3, CY5 conjugated anti-mouse, -goat, -guinea pig, -rabbit, and -sheep (Jackson ImmunoResearch). DAPI (Invitrogen) was used for nuclear counterstaining (1:6,000).\n\n## Blood glucose level measurements.\n\nRandom-fed blood glucose samples were collected at a consistent time of the day (1200\u20131300 h). Fasted blood glucose levels were measured after a 6-h fasting period (800\u20131400 h) with a handheld glucometer (OneTouch; Lifescan). Mean difference between wild-type and *Mitf^ce\/ce^* was tested with Student *t* test.\n\n## Intraperitoneal glucose tolerance test and insulin secretion measurements.\n\nIntraperitoneal glucose tolerance test (IPGTT) was performed on 12-week- and 6-month-old *Mitf^ce\/ce^* and wild-type mice after overnight fasting (12 h) using an injection of 2 g glucose\/kg body wt. Blood glucose levels were measured using a handheld glucometer (OneTouch; Lifescan). Blood was collected from an incision in the distal part of the tail. Measurements were taken at 0, 5, 15, 30, 60, and 120 min after glucose administration for the IPGTT and at 0, 2, 5, and 15 min for glucose-stimulated insulin secretion studies. For the latter, blood was collected into heparin-coated tubes (Sarstedt) and the serum fractions were analyzed with a Mouse Insulin ELISA (Mercodia) according to the manufacturer's instructions.\n\n## Insulin secretion assay.\n\nIslets were isolated by collagenase digestion and handpicked under a stereomicroscope (20). Batches (*n* = 8) of three islets for each condition were kept in secretion assay buffer (SAB) (114 mmol\/L NaCl, 4.7 mmol KCl, 1.2 mmol\/L KH~2~PO~4~, 1.16 mmol\/L MgSO~4~, 20 mmol\/L HEPES, 2.5 mmol\/L CaCl~2~, 25.5 mmol\/L NaHCO~3~, and 0.2% BSA; pH 7.2) containing 2.8 mmol\/L glucose for 60 min in an incubator at 37\u00b0C. Three islets per well were transferred to a 96-well plate containing 200 \u03bcL\/well SAB but with the addition of 2.8, 8.3, and 16.7 mmol\/L glucose with or without 35 mmol\/L KCl. After transfer of all islets, the plate was placed in an incubator at 37\u00b0C. At 60 min, a buffer sample was removed for measurement of insulin by ELISA.\n\n## Glucagon secretion measurements.\n\nBlood samples from random-fed and 12-h-fasted wild-type and Mitf mutant mice were collected into heparin-coated tubes. Samples were chilled immediately and centrifuged at 4\u00b0C. Plasma aliquots were stored at \u221220\u00b0C. Glucagon concentrations were determined using a glucagon radioimmunoassay kit (Millipore). Statistical analysis was conducted using Student *t* test.\n\n## Islet isolation, RNA extraction, RT-PCR, and quantitative PCR.\n\nIslet isolation has previously been described (20). RNA from E18.5 and P7 pancreas and islets of 12 week-old mice was extracted with ToTally RNA (Ambion) or RNeasy mini kit (Qiagen). RNA concentrations were measured with a NanoDrop ND-1000 spectrophotometer (NanoDrop), and RNA quality was assessed using a Bioanalyzer (Agilent). Only samples with a RNA Integrity Number \\>7.5 were further analyzed. Quantitative PCR was performed in two steps. First, reverse transcription was carried out with SuperScript III (Invitrogen) using 500 ng RNA. For the quantitative PCR reaction, cDNA was diluted 1:200 and all assays were performed with Fast SYBR Green Master Mix on a StepOnePlus Real-Time PCR instrument (Applied Biosystems). Primer sequences are available upon request. Formation of expected PCR products was confirmed by electrophoresis and melting curve analysis. Gene expression data were normalized to the expression of the internal control gene hypoxanthine-guanine phosphoribosyltransferase (*HPRT*). Experiments were repeated at least five times\u2014each in triplicate. Raw data from real-time PCR measurements were exported from StepOne Software v2.1 and analyzed in Microsoft Excel. The data are shown as mean expression with SEM. Graphs represent the fold change in comparison with the wild-type control samples, set as 1. Mean difference was tested with Student *t* test.\n\n## Image analysis, quantification, and statistical analysis.\n\nImmunofluorescence images were collected with Zeiss Axioplan 2 imaging (Zeiss, Jena, Germany) in AxioVision Rel 4.9 software. Immunohistochemistry and quantification were performed from at least three adult wild-type and *Mitf^ce\/ce^* pancreata. \u03b2-Cell mass was assessed by quantifying the pancreatic and insulin-stained area in sections at a 720-\u03bcm interval throughout the whole pancreatic organ. The percentage of endocrine cells expressing ghrelin was assessed by quantifying Pax6 and ghrelin-producing cells at a 96 (E18.5) or 120 (P7) \u03bcm interval throughout the whole pancreas. Synaptophysin-expressing cells were measured in every 20th section of adult pancreas and normalized to the pancreatic weight. Mean differences were tested with Student *t* test; *n* \u2265 3.\n\n## Chromatin immunoprecipitation.\n\n\u03b2-Cell\u2013derived \u03b2-TC6 cells and \u03b1-cell\u2013derived \u03b1-TC6 (American Type Culture Collection) were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% calf serum. Cells were transfected with FLAG-tagged Mitf expression plasmid (True ORF cDNA clone MR227086; ORIGene) using Metafectene Pro (Biontex). Cells were harvested 48 h after transfection, fixed in 1% formaldehyde in Dulbecco's modified Eagle's medium without serum, and treated with lysis buffer (10 mmol\/L Tris-HCl, pH 8.0; 10 mmol\/L NaCl; 3 mmol\/L MgCl~2~; 1% NP-40; 1% SDS; and 0.5% sodium deoxycholate \\[DOC\\]) and sonicated four times for 5 min on high (30 s on and 30 s off) settings in a Bioruptor sonicator (Diagenode). Protein\/DNA chromatin fragments were immunoprecipitated with mouse anti-FLAG antibody (Sigma) or mouse IgG (Santa Cruz) as previously described (21).\n\nNonquantitative PCR reactions were performed using purified immunoprecipitated DNA with 0.8 \u03bcmol\/L primers specific for *Pax6^EE^* (AGGCACGTCCTGGATGTTAG and CCCCAACCTCATTCTTTTCA), *Pax6^PE^* (ACTCAGGCCTGTGGTTATGC and TCAAGAGCGAAGCTGGAAAC), *Pax4* (AGGGACAATTAGCCCCAAAC and ACAGAAGCTTTCGACCCAGA), *Runx2* (GTTGTTTGATTTGTTTTGAAGG and TTTTACCTAAAATGTGGTTTTTG), and *PEPCK* (GAGTGACACCTCACAGCTGTGG and GGCAGGCCTTTGGATCATAGCC) transcriptional control region primers. PCR products were visualized on a 1.5% agarose gel in 1\u00d7 Tris-Borate-EDTA buffer. Each chromatin immunoprecipitation (ChIP) experiment was repeated at least three times using independent chromatin preparations.\n\n## Transfection assays.\n\nThe Pax6 P0 sequence (\u22123.8 to 0.2 kb) was PCR amplified from mouse genomic DNA and inserted upstream of a promoterless firefly luciferase plasmid (pGL2basic; Promega). Human embryonic kidney (HEK)293 cells were transfected with pPax6P0-LUC, cytomegalovirus-driven Mitf, and\/or Pax6, and Renilla luciferase plasmids (phRL-CMV; Promega) using Metafectene Pro reagent (Biontex). Extracts were prepared 40\u201348\u200ah later and analyzed for firefly and Renilla luciferase activity using the Dual-Luciferase Reporter Assay System (Promega). Mean difference was tested with one-way ANOVA; *n* = 3.\n\n# RESULTS\n\n## Mitf is specifically expressed in endocrine cells in the postnatal and adult pancreas.\n\nImmunohistochemical analysis was performed to determine which cell types produce Mitf during pancreas development and in the adult. Mitf expression was not detected at E15.5 (data not shown) but was initially observed at E18.5 ([Supplementary Fig. 1](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1464\/-\/DC1)). At P0, Mitf was found in nuclei of endocrine, exocrine, and ductal cells (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>A<\/em>\u2013<em>D<\/em><\/a> and [Supplementary Fig. 1](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1464\/-\/DC1)). Postnatally, Mitf expression becomes restricted to islet endocrine cells ([Supplementary Fig. 1](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1464\/-\/DC1)). Analysis of adult wild-type pancreata showed that Mitf is expressed in all hormone-producing cells (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>E<\/em>\u2013<em>H<\/em><\/a>). Mitf expression is restricted to nuclei of insulin-producing cells, whereas it is also detected in the cytoplasm of \u223c20% glucagon cells ([Supplementary Fig. 2](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1464\/-\/DC1)).\n\n## *Mitf^ce\/ce^* mice have improved blood glucose clearance and elevated insulin secretion.\n\nBlood glucose measurements showed that *Mitf^ce\/ce^* mice had significantly lower blood glucose levels than their wild-type littermates whether they were random-fed or fasted (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<em>A<\/em><\/a>). In addition, *Mitf^ce\/ce^* animals had improved blood glucose clearance after glucose injection at 12 weeks and 6 months. Blood glucose levels stayed below 20 mmol\/L even at 15\u201330 min after the glucose injection (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<em>C<\/em> and <em>D<\/em><\/a>) and returned faster to homeostatic conditions. Glucose-stimulated serum insulin levels were measured to determine whether this improved glucose clearance is a direct result of altered \u03b2-cell function. Interestingly, *Mitf* mutant animals have significantly higher serum insulin levels in fasting conditions than wild-type animals (0 min \\[<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<em>E<\/em><\/a>\\]), which suggests that Mitf is required for proper \u03b2-cell function. In contrast, plasma glucagon levels were not significantly changed in *Mitf^ce\/ce^* animals compared with wild-type littermates in both fasted and random fed conditions (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<em>B<\/em><\/a>). Stimulation of *Mitf^ce\/ce^* and wild-type isolated islets with 16.7 mmol\/L glucose and 35 mmol\/L KCl resulted in a 60% increased insulin secretory response (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<em>F<\/em><\/a>). Our results demonstrate that *Mitf* mutant mice have enhanced glucose tolerance and are protected from high blood glucose levels by elevated insulin secretion.\n\n## Number of endocrine cells is unchanged in *Mitf^ce\/ce^* animals.\n\nImmunohistochemical analysis of wild-type and *Mitf^ce\/ce^* pancreatic sections showed no obvious changes in the appearance of pancreatic islets (<a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<em>A<\/em><\/a>). For determination of whether the elevated serum insulin levels observed in *Mitf^ce\/ce^* mice result from an increase in \u03b2-cell mass, quantitative immunohistochemical analysis was performed. The percentage of insulin-labeled wild-type and *Mitf^ce\/ce^* cell area and total islet cell area were comparable (<a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<em>B<\/em><\/a> and [Supplementary Fig. 4*C* and *D*](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1464\/-\/DC1)) suggesting that Mitf is not required for \u03b2-cell specification or regulation of \u03b2-cell mass. Insulin and *PP* mRNA levels were significantly increased in *Mitf^ce\/ce^* islets (<a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<em>C<\/em><\/a>), while ghrelin transcription was decreased to only 20% of wild-type (<a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<em>C<\/em><\/a>). Immunohistochemical quantification of ghrelin-producing cells revealed no differences in the percentage of ghrelin cells within the endocrine Pax6^+^ cell population at E18.5 and P7 ([Supplementary Fig. 3*A*\u2013*E*](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1464\/-\/DC1)), while ghrelin mRNA levels were already reduced by P7 ([Supplementary Fig. 3*F*](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1464\/-\/DC1)). In accordance with previous reports (22) ghrelin cells were almost absent in adult wild-type and *Mitf^ce\/ce^* islets, which precluded a quantitative analysis at this time point. Quantitative immunohistochemical analysis showed no significant increase in PP cell number (data not shown) in *Mitf^ce\/ce^* islets. These findings suggest that insulin, ghrelin, and PP production is altered in individual hormone-expressing cells. *Mitf^ce\/ce^* and wild-type \u03b2-cells were analyzed using electron microscopy to determine whether the increase in insulin mRNA levels results in enhanced production of insulin secretory granules. *Mitf^ce\/ce^* \u03b2-cells have the same number of insulin granules per cell, and granule morphology is comparable with the wild-type ([Supplementary Table 1](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1464\/-\/DC1) and [Supplementary Fig. 4*A* and *B*](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1464\/-\/DC1)). Our findings illustrate that Mitf is critical for proper hormone expression but not essential for endocrine cell specification and maintenance.\n\n## Gene expression of key \u03b2-cell genes is increased in *Mitf^ce\/ce^* islets.\n\nGene expression analysis and immunohistochemistry were performed to further characterize *Mitf^ce\/ce^* islets. First, we analyzed the expression level and distribution of transcription factors involved in \u03b2-cell development and\/or function. The cellular distribution of known \u03b2-cell factors Pax6, Pdx1, MafA, and Nkx6.1 was unchanged (<a href=\"#F4\" data-ref-type=\"fig\">Fig. 4<em>A<\/em>\u2013<em>H<\/em><\/a>). However, a twofold increase in *Pax6* and *Pax4* mRNA levels was observed in *Mitf^ce\/ce^* islet samples (<a href=\"#F4\" data-ref-type=\"fig\">Figs. 4<em>K<\/em><\/a> and <a href=\"#F5\" data-ref-type=\"fig\">5<\/a>). Interestingly, \u03b2-Cell\u2013specific expression of *Glut2,* a known Pax6 target gene (3) was increased twofold in mutant islets (<a href=\"#F4\" data-ref-type=\"fig\">Fig. 4<em>O<\/em><\/a>), while the transcription of other glucose-sensing, metabolizing, and insulin secretory genes was unaffected (<a href=\"#F5\" data-ref-type=\"fig\">Fig. 5<\/a>). These results suggest that Mitf is controlling \u03b2-cell function by directly regulating genes required for \u03b2-cell function and differentiation.\n\n## Mitf binds to the pancreas-specific regulatory sequences of the *Pax4* and *Pax6* genes.\n\nPrevious studies have established that Mitf acts as a transcriptional activator and repressor in the developing eye (13\u201315). We hypothesize that Mitf directly controls *Pax6* transcription, since *Pax6* mRNA levels were upregulated in *Mitf^ce\/ce^* mice (<a href=\"#F4\" data-ref-type=\"fig\">Fig. 4<em>K<\/em><\/a>) and *Mitf* overexpression prevents basic fibroblast growth factor (bFGF)-induced *Pax6* upregulation in cultured retinal pigmented epithelial cells (14,23). The mouse *Pax6* gene contains several regulatory elements that confer tissue-specific expression in the pancreas, eye, and brain (24). Among those, the P0 proximal regulatory element (PE) is required for embryonic pancreas-specific *Pax6* expression at physiological levels (25), while ectodermal expression is controlled by the EE element (EE). ChIP analysis was performed in mouse \u03b1-TC6 and \u03b2-TC6 cells transfected with FLAG-tagged Mitf to determine whether Mitf occupied the *Pax6* EE and PE sequences (*Pax6^EE^* and *Pax6^PE^*). An enrichment of both *Pax6^EE^* and *Pax6^PE^* was found using primer pairs that span the *Pax6^PE^* and *Pax6^EE^* regions compared with control sequences only after \u03b1-FLAG immunoprecipitation (<a href=\"#F6\" data-ref-type=\"fig\">Fig. 6<em>B<\/em> and <em>C<\/em><\/a>). A similar analysis demonstrated that flag-tagged Mitf also immunoprecipitates *Pax4* regulatory elements in \u03b2-TC6 cells (<a href=\"#F6\" data-ref-type=\"fig\">Fig. 6<em>B<\/em><\/a>). For further assessment of functional regulation of the *Pax6* P0 promoter, Pax6 and Mitf were cotransfected with a *Pax6P0*-driven reporter construct in HEK293 cells (<a href=\"#F6\" data-ref-type=\"fig\">Fig. 6<em>A<\/em> and <em>D<\/em><\/a>). Pax6P0 firefly luciferase (pPax6P0-LUC) activity was enhanced upon cotransfection with Pax6, while cotransfection with Mitf resulted in transcriptional repression. Cotransfection of Mitf and Pax6 resulted in loss of Mitf-mediated repression, suggesting that interactions between these factors are critical for the transcriptional control of the *Pax6P0* regulatory region. The ChIP and luciferase reporter data are complimentary and demonstrate that Mitf directly binds to and regulates *Pax6* regulatory elements, suggesting that increased *Pax6* mRNA levels in *Mitf^ce\/ce^* are a direct effect of the loss of Mitf.\n\n# DISCUSSION\n\nPrevious studies have established Mitf as a master regulator of pigment cell differentiation (rev. in 26). Our previous results have shown that Mitf expression is reduced in *MafA* and *MafB* mutant embryonic pancreata (12) suggesting that Mitf may be involved in \u03b2-cell differentiation and function. In this study, we demonstrate that Mitf is initially expressed in the pancreatic epithelium at E18.5 and that its expression becomes restricted to adult islet cells. Mitf inactivation results in altered \u03b2-cell function, due to an enhanced insulin secretory response, without changes in \u03b2-cell mass. In contrast, the transcription of genes critical for the regulation of blood glucose levels (insulin and *Glut2*) and \u03b2-cell formation and function (*Pax4* and *Pax6*) was significantly upregulated in *Mitf^ce\/ce^* islets, suggesting that Mitf is critical for adult \u03b2-cell function.\n\nDuring development, Mitf was initially detected in all pancreatic epithelial cells from E18.5 onward ([Supplementary Fig. 1](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1464\/-\/DC1)). Interestingly, *Mitf* expression increases and becomes progressively restricted to islet cells postnatally and is not detected in ductal and exocrine cells after the first weeks of birth. Our findings are supported by a gene-profiling study of Ngn3^+^ endocrine progenitor cells and their descendants, which showed that *Mitf* transcription increases postnatally, with the highest expression observed in adult islet cells (27). The late onset of *Mitf* expression and localization in endocrine cells coincides with profound morphological and physiological changes in the pancreas: endocrine cells cluster into mature islets, with \u03b2-cells in the core and other hormone-producing cells at the periphery. In addition, and perhaps most importantly, the glucose responsiveness of \u03b2-cells is established at this time (8). During specification of zebrafish melanocytes, Mitf is specifically required for late steps of differentiation (28), which also supports a role for Mitf in \u03b2-cell maturation or function.\n\nTo determine Mitf's function in the developing and adult pancreas, we analyzed a *Mitf* mutant mouse model. *Mitf^ce\/ce^* pancreata were histologically indistinguishable from wild-type littermates, \u03b2-cell area was comparable, and no changes in adult islet cell composition were observed. However, as evident from our in vitro studies on isolated islets, the insulin response to high glucose and KCl concentrations was significantly greater in *Mitf^ce\/ce^* animals than in wild-type, whereas insulin response to a stimulation with lower glucose concentrations was not significantly affected (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<em>F<\/em><\/a>). This shows that the total \u03b2-cell secretory capacity is increased in mutant mice. Interestingly, *Mitf^ce\/ce^* animals also had fasting hyperinsulinemia in the presence of slight hypoglycemia (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<em>A<\/em> and <em>E<\/em><\/a>), again showing increased insulin secretion. Plasma glucagon levels were unchanged in the fasted and fed condition (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<em>B<\/em><\/a>) suggesting that \u03b1-cell function is not affected in *Mitf^ce\/ce^* animals. After intraperitoneal glucose challenge, glucose tolerance was improved (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<em>C<\/em> and <em>D<\/em><\/a>), which also reflects increased glucose-stimulated insulin secretion. However, when glucose-stimulated insulin secretion was determined the only significant difference was observed at 0 min (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<em>E<\/em><\/a>). Mitf function in peripheral tissues may also influence glucose responsiveness and partially account for the altered glucose clearance in *Mitf^ce\/ce^*. However, thus far, no data supporting a role of Mitf in muscle, liver, and adipose glucose metabolism have been reported, and transcription in these tissues is relatively low (gene expression atlas, <http:\/\/www.ebi.ac.uk\/arrayexpress\/>). Collectively, these observations suggest that loss of *Mitf* specifically alters \u03b2-cell function by increasing glucose responsiveness and insulin secretion.\n\nGene expression analysis showed increased transcription of key \u03b2-cell genes such as *Pax4*, *Pax6*, insulin, and *Glut2*, which could contribute to the increased insulin secretory response. Thus elevated insulin mRNA levels may result in increased total insulin content and in a larger pool of insulin granules that are ready to be released, which could influence insulin secretion. However, *Mitf^ce\/ce^* \u03b2-cells have no change in the total number of insulin granules ([Supplementary Fig. 4](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1464\/-\/DC1) and [Supplementary Table 1](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db12-1464\/-\/DC1)), and the percentage of ready releasable granules stays the same suggesting that mutant \u03b2-cell function is most likely improved owing to changes in glucose sensing. Thus, elevated *Glut2* expression may positively influence this process, since it is the only glucose transporter expressed in \u03b2-cells that mediates glucose-stimulated insulin secretion (29,30).\n\nUpregulation of *Pax6* transcription may be the key to the improved \u03b2-cell function observed in *Mitf^ce\/ce^*, since it has been shown that this transcription factor regulates the expression of several \u03b2-cell genes like insulin, *Glut2*, *PC1\/3*, and glucokinase (31). Interestingly, we observed enhanced transcriptional activity of only some Pax6 target genes like insulin and *Glut2* but not others, which suggests that a twofold increase in Pax6 expression is not sufficient to augment the transcription of all Pax6 target genes in this context.\n\nLoss-of-function studies have established a critical role for Pax4 in \u03b2-cell development (32), while ectopic Pax4 expression in adult \u03b2-cells is associated with increased proliferation and protection against apoptosis (33). Mitf binds to *Pax4* regulatory elements, and *Mitf^ce\/ce^* islets have twofold higher *Pax4* mRNA levels than wild-type islets but no difference in \u03b2-cell mass and thus no difference in \u03b2-cell proliferation and specification. This may be due to the moderate upregulation of *Pax4* transcription in *Mitf^ce\/ce^* islets compared with *Pax4* overexpression models used in recent work, which has studied the effects of 15-fold increased *Pax4* (33). However, the moderately increased Pax4 expression in *Mitf^ce\/ce^* islets may still positively influence \u03b2-cell function and contribute to improved glucose clearance.\n\nPrevious studies have demonstrated that Mitf acts both as transcriptional activator and repressor (14,15,34): it activates genes required for cell pigmentation (13,14), while ectopic Mitf expression prevents *Pax6* transcription in the developing eye (14,23) and Pax6 and Mitf cooperatively repress common target genes in the retinal pigmented epithelium (15). Thus far, Mitf repressor function has only been described in the context of coexpression with Pax6 but not with other transcriptional repressors like groucho (15,35). Here, we demonstrate that Mitf binds to and represses the *Pax6 P0* regulatory region, which is required for high *Pax6* expression during pancreas development (25,36). This finding suggests that Mitf is partially responsible for inactivation of the embryonic pancreatic *Pax6 P0* promoter.\n\nIn vitro transfection and gel shift experiments have demonstrated that direct protein-protein interaction between Pax6 and Mitf results in an inactivation of both proteins (37). The Mitf^ce^ protein lacks the leucine zipper domain, which results in loss of DNA binding and reduced interaction with cofactors (19). The leucine zipper domain is also required for interactions with Pax6 (37); thus, Mitf^ce^ protein not only has lost its transcriptional activity but also fails to interact and repress Pax6 function in mutant \u03b2-cells. These in vitro studies support our observation that the expression of the newly identified direct Mitf target gene *Pax6* is elevated in *Mitf^ce\/ce^* animals and that enhanced *Pax6* levels and transcriptional activity may result in increased *Glut2*, *Pax4*, and insulin transcription.\n\nOur data identify Mitf as a novel transcriptional repressor in adult \u03b2-cells and suggest that regulation of \u03b2-cell function depends on interplay between the Mitf and Pax6 transcription factors.\n\n## ACKNOWLEDGMENTS\n\nThis work was supported by the Swedish Research Council, the JDRF, Diabetesfonden, Lindh\u00e9s, and Jeanssons Stiftelse.\n\nNo potential conflicts of interest relevant to this article were reported.\n\nM.A.M. designed research, researched data, and wrote the manuscript. M.W., E.G., and J.K.C. researched data. J.K.J. reviewed the manuscript and contributed to discussion. H.B. researched data. M.F. researched data, reviewed the manuscript, and contributed to discussion. U.A.N. reviewed the manuscript and contributed to discussion. I.A. designed research and wrote the manuscript. M.A.M. and I.A. are the guarantors of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.\n\nThe authors thank S. Saule (Institut Curie), C. Wright (Department of Cell and Developmental Biology, Vanderbilt University Medical Center), and the Beta Cell Biology Consortium (BCBC) for providing antibodies; B. Ahr\u00e9n (Department of Clinical Sciences, Lund University), H. Semb (Stem Cell Center, Lund University, and The Danish Stem Cell Center, Copenhagen University), and R. Stein (Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine) for comments on the manuscript; M. Magnusson for mouse strain maintenance; B.-M. Nilsson (Clinical Research Center, Lund University) for plasma glucagon measurements; and R. Wall\u00e9n (Lund University) for transmission electron microscopy analysis.\n\n# REFERENCES","meta":{"dup_signals":{"dup_doc_count":141,"dup_dump_count":46,"dup_details":{"curated_sources":2,"2018-26":2,"2018-22":1,"2018-17":2,"2018-13":1,"2018-09":2,"2018-05":1,"2017-51":2,"2017-47":2,"2017-43":6,"2017-39":2,"2017-34":2,"2017-30":6,"2017-26":3,"2017-22":3,"2017-17":7,"2017-09":5,"2017-04":8,"2016-50":3,"2016-44":5,"2016-40":4,"2016-36":3,"2016-30":4,"2016-26":3,"2016-22":4,"2016-18":3,"2016-07":4,"2015-48":4,"2015-40":1,"2015-35":2,"2015-32":3,"2015-27":3,"2015-22":4,"2015-14":1,"2014-52":3,"2014-49":3,"2014-42":5,"2014-41":2,"2014-35":2,"2014-23":2,"2021-25":1,"2017-13":4,"2015-18":2,"2015-11":4,"2015-06":3,"2014-10":1,"2024-22":1}},"file":"PMC3717881"},"subset":"pubmed_central"}
{"text":"abstract: Genome annotation currently tends to represent a static snapshot. Routine re-annotation, perhaps using wiki software, would help.\nauthor: Steven L Salzberg\ndate: 2007\ninstitute: 1Center for Bioinformatics and Computational Biology and Department of Computer Science, 3125 Biomolecular Sciences Building, University of Maryland, College Park, MD 20742, USA\nreferences:\ntitle: Genome re-annotation: a wiki solution?\n\nSo you think that gene you just retrieved from GenBank \\[1\\] is correct? Are you certain? If it is a eukaryotic gene, and especially if it is from an unfinished genome, there is a pretty good chance that the amino acid sequence is wrong. And depending on when the genome was sequenced and annotated, there is a chance that the description of its function is wrong too.\n\nLarge-scale genome sequencing has revolutionized biology over the past ten years, generating vast amounts of new information that has radically transformed our understanding of hundreds of species, including ourselves. Sequencing centers continue to churn out new DNA sequences for a fantastic variety of species, covering more and more of the tree of life. Along with these sequences, the centers also produce genome annotation, which includes the locations and descriptions of all identifiable genes. These gene lists are the first pictures we get of what's inside a newly sequenced genome, and they can reveal key insights into what makes an organism distinctive. Sometimes the gene lists themselves are part of the story; for example, when the human genome was published \\[2,3\\], the headline was that humans have 'only' 25,000 genes, in contrast to earlier estimates of 100,000 or more. For many microbial species, the genome helps us to understand how the organism can accomplish something particularly difficult, such as how *Deinococcus radiodurans* (to cite just one of many examples) can withstand exposure to radiation levels far in excess of what a human could tolerate \\[4\\]. With each new human pathogen, the gene list helps us determine how the organism infects humans, how it causes sickness and (sometimes) how it becomes resistant to antibiotics. For these and other reasons, the accuracy of the gene list is tremendously important.\n\n# What is genome annotation?\n\nBefore addressing the problems with annotation, I will first summarize how it is done. The process of sequencing and annotating the DNA of a bacterial species has become highly automated in recent years, but the major steps are quite similar to what was done for the very first bacterial genome, *Haemophilus influenzae*, in 1995 \\[5\\].\n\nFigure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a> shows an outline of the main steps of the whole-genome shotgun sequencing and annotation process for a bacterial genome. Similar procedures - for both sequencing and annotation - are followed for much larger genomes, including the human genome, although the details vary. The laboratory steps have not changed greatly since *H. influenzae*: they begin with DNA purification, followed by shearing the DNA into countless small fragments (the 'shotgun' step). These fragments are then cloned and sequenced from both ends and assembled, usually resulting in a set of contiguous DNA sequences (contigs) joined together into larger scaffolds. The annotation pipeline can be applied immediately to these contigs, but in projects where the genome will be finished, the annotation software is usually run later, when the gaps between contigs have been filled in.\n\nMost annotation pipelines are considerably more complex than shown in Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>, but they share the same outline. First a gene finder (such as, for bacteria, Glimmer \\[6\\] or GeneMark \\[7\\]) is run over the genome, producing a set of predicted protein-coding genes. These programs are very accurate, though not perfect. They are far more accurate than eukaryotic gene finders, however, primarily because the problem is far more difficult in eukaryotic genomes. In either case, the next step in the pipeline is to take the set of predictions and search them against one or more protein databases using BLAST \\[8\\], HMMer \\[9\\] or other programs. For each gene that has a significant match, the BLAST output can be used to assign a name and function to the protein. The accuracy of this step depends not only on the annotation software, but also on the quality of the annotations already in the database. For genes with no match, the pipeline might keep them and label them as 'hypothetical', or it might discard them based on criteria as simple as minimum length.\n\nAnnotation pipelines also run separate searches for tRNA and rRNA genes, and they may include other components as well. The pipeline software will usually take extra steps to find any genes missed by earlier steps; typically this involves running a translated search, aligning all six possible translations of the unannotated sections to a database.\n\n# Partial and draft genomes\n\nFinishing a genome - sequencing every remaining nucleotide of every chromosome and creating a gap-free assembly - is considerably slower and more expensive than the high-throughput shotgun-sequencing phase. As a result, a growing number of genomes are being released in 'draft' form and will remain in this form indefinitely. These include many bacteria and the majority of eukaryotic genomes. (In fact, only a handful of eukaryotic genomes, such as those of *Saccharomyces cerevisiae* and *Caenorhabditis elegans* but not including the human, are truly finished.)\n\nThe effect of draft genomes upon annotation is considerable: many genes will 'run off' the end of contigs or appear on two or more separate contigs. This in turn complicates the subsequent steps of annotation and is likely to lead to additional errors in assigning gene function. For example, a gene fragment is liable to match a small protein domain, and functions based on a single domain hit are not reliable. A gene that is split across two contigs might be annotated twice. Draft sequences also have much higher sequencing error rates, which can introduce erroneous stop codons in the middle of genes or improperly merge adjacent but distinct genes.\n\n# The role of GenBank\n\nOnce a genome - draft or complete - is annotated, the DNA sequence along with the annotation is normally deposited in GenBank. Countless researchers rely on GenBank \\[1\\], EMBL \\[10\\] and DDBJ \\[11\\] (which mirror one another) as their primary source for genome annotation, and for a good reason: these databases are the world's largest public repositories of genome information. GenBank now contains over 65 billion base pairs (Gbp) of sequence, up from just 2 Gbp in 1998 and 10 Gbp in 2000, and it continues to grow at an astonishing rate. If you want to find a gene, GenBank should definitely be your first stop. Yet I frequently hear claims within the bioinformatics community that the 'GenBank annotation' of a particular genome is fraught with problems, and that the speaker can fix them.\n\nIs the GenBank annotation perfect? Of course not. How good it is, though, depends on many variables, and the consumer of GenBank data would be wise to be aware of them (in other words, *caveat emptor*). The first and most important point to understand is that GenBank is not simply a database; it is also a library. A scientist who submits a sequence to GenBank is the owner of that sequence and is listed on the 'author' line in the GenBank entry. Just as with any article published in a journal, the author (and only the author) has the right to submit an erratum. Because GenBank is an electronic library, an erratum is really an update: new sequences or annotations replace the old ones, although GenBank keeps a record of the changes so that the original entry can still be retrieved if necessary. This notion of GenBank as a library (or an electronic journal) is frequently misunderstood, especially when a scientist discovers an annotation error. Even if the error is overwhelmingly obvious, the custodians of GenBank cannot simply fix it, any more than the editor of a journal can correct one of the papers published in that journal. Another way to think of this is to recognize that a 'GenBank annotation' is not 'GenBank's' annotation, but rather the annotation of whoever deposited the sequence in the first place.\n\nWhen confronted with this problem, some scientists react by suggesting that GenBank (and DDBJ and EMBL) should allow scientists to fix errors that they find. But this would quickly destroy the archival function of GenBank, as original entries would be erased over time. It would also violate the agreement that GenBank has with all its submitters that their entries belong to them and can only be changed by them. This agreement has been crucial in GenBank's near-universal acceptance by the genomics community as the central resource for DNA sequences. The idea of allowing others to alter GenBank annotation also immediately begs the question of who should be permitted to make such alterations.\n\nThis leaves us with a problem: users go to GenBank expecting to find the authoritative annotation for a genome, and what they find might be far less than that. Most genome annotation deposited in GenBank remains static for years, and many annotations have never been changed since their initial publication. Nonetheless, many scientists assume that GenBank annotation is kept up to date, and they are surprised to hear that it is not.\n\nFor example, 479 genes in the *H. influenzae* Rd genome are currently listed as hypothetical proteins. Of these, 217 have at least one extremely strong BLAST hit to another species (*E*-value \\< 10-100), which means they should at least be called 'conserved hypothetical' proteins. And 40 of these have matches to a gene with an assigned function, meaning that a re-annotation would result in these genes having a more meaningful name than 'hypothetical protein'.\n\n# Some inconvenient truths\n\nEven considering all of the issues above, one might reasonably expect that as protein databases have grown, annotation has improved and that recently annotated genomes (at least) will be of the highest quality. This is not quite true. What is true is that a BLAST search of a protein that is run today will yield far more results than it would have five or ten years ago, and these results in turn should lead to better annotation. Not all software is equally good, however, and the annotation pipelines vary considerably in their quality. There is also wide variation in the skills and experience of those operating the pipelines. Further complicating matters, some genomes are subjected to careful curation and review, whereas others receive only automated annotation. In the early days of sequencing, the sequencing teams included experts on the biology of each genome, and their manual curation dramatically improved the annotation of those species. Today that is no longer true: high-throughput sequencing centers are large, efficient factories with unique expertise in the methods necessary for sequencing, but they sometimes have very little expertise on the biology of the species they are sequencing. The inconvenient truth is that, as a result of these factors and others, some genomes are poorly annotated even today.\n\nThere are several ways in which genome annotation can be erroneous. The first and most fundamental is simply that the gene models may be wrong. Although bacterial gene-finding systems \\[6,7\\] are highly accurate, finding 98-99% of protein-coding genes in most species, they still occasionally miss genes. Their accuracy at placing the start site is a bit lower, probably closer to 90%, which is excellent but far from the perfect accuracy that some might expect. In the past, the accuracy of (bacterial) start-site prediction was closer to 80%, and many of the genomes in GenBank were predicted with earlier versions of gene finders. Note that all these accuracy figures are much lower for eukaryotic annotation. Some annotation pipelines include algorithms to adjust start sites, which can be done by looking closely at the boundaries of alignments to homologous proteins.\n\nFalse positives represent another type of erroneous annotation: when the prediction of a gene-finding program does not match any previously known protein, the annotators (or the annotation pipeline software) must decide whether or not to include that prediction in the gene list. Over the years, annotation groups have used a variety of rules to make this decision, and they have inevitably included thousands of false predictions in the publicly available genome annotation. These predictions are mostly harmless unless they result in effort being expended trying to verify them. In some cases, too, they might 'hide' functional RNA genes or true genes in a different reading frame from that of the false prediction.\n\nPerhaps the biggest problem with genome annotation is erroneous and inconsistent naming of genes. Much of this is due to the simple fact that our knowledge of genes has improved but the annotation has remained static. Thus a gene labeled 'hypothetical protein' a few years ago might now have a known function. A second problem is what's known as transitive catastrophe: the phenomenon whereby a name is transferred from one gene to another on the basis of sequence similarity (usually from a BLAST search) but where the original name is incorrect. As more genomes are annotated, and more BLAST searches are run, the name gets transferred to other proteins, and the original source of the name quickly becomes lost. It is well known in the genomics community that thousands of such transitive errors have propagated through sequence databases, and efforts are under way to try to clean up some of the mess. In the meantime, though, many genes remain incorrectly annotated.\n\nLet us consider just one example, selected more or less at random from the bacterium *H. influenzae* Rd \\[4\\]. The gene *fdxH* encodes formate dehydrogenase, \u03b2 subunit, GenBank accession number NP438180. When the genome was sequenced in 1995, this gene (encoding a 312 amino acid protein) was similar to very few other genes; even the orthologous *Escherichia coli* gene was not yet sequenced. It is very difficult today to reconstruct what the best BLAST hit was back then, but today there are 197 highly significant BLAST hits to 123 distinct species. Thus, it is pretty clear that this gene today should be well-annotated because of the multitude of highly similar proteins. Yet if we look at the list of matching proteins, we find a variety of names given, including not only the name found on NP438180 itself, but also: formate dehydrogenase-O \u03b2 subunit; formate dehydro-genase, nitrate-inducible, iron-sulfur subunit; HybA protein; formate dehydrogenase-N, Fe-S \u03b2 subunit, nitrate-inducible; hypothetical protein PaerPA_01004979; hypothetical protein Bpse11_03005113; 4Fe-4S ferredoxin, iron-sulfur binding; and Twin-arginine translocation pathway signal. Some of these names seem to be synonymous, but others clearly are not. To decide properly among them, we need to look at the source of each annotation and at the species to which it is attached.\n\n# Possible solutions\n\nSo if we can't always trust GenBank, what can we do? Clearly we cannot just ignore it. The scientific community must have a resource that contains the genes from all the species that have been sequenced. For the past 25 years, GenBank, EMBL and DDBJ have been enormously successful at providing these data. The pace of sequencing has changed the rules of the game, however: sequencing centers are pouring out genomes, annotating them rapidly and moving on. An archive of these annotations may be useful, but a static archive is insufficient.\n\nOne part of the solution is obvious: annotation must be regularly re-computed using the latest databases and software. For a small number of model organisms, this is already happening, but these species represent a tiny proportion of all known genes. Simply re-running an automated pipeline on all genomes is not sufficient, though, because that would over-write many of the carefully curated, manually annotated genes that have been produced in the past. Unfortunately, there is no standard label attached to such genes, so there is no way for an automated pipeline to know that they should be trusted. Therefore, we also need to launch an effort to start identifying those genes that are well annotated and, beyond that, to start recording the evidence used to annotate each gene.\n\nAnother solution is to create a new, expanded database that can display all the alternative annotations for any locus in a genome. If this were available, then scientists could be provided with links from any gene to alternative or overlapping gene predictions as well as alternative gene names. Along with each annotation could be a link to the evidence supporting it; for example, the date of a BLAST search or a citation to experiments contained in a journal article.\n\n# A wiki solution?\n\nVarious members of the genomics community have considered these and other solutions, but so far none have emerged as the standard. Several new databases have been developed with alternative genome annotation, or with re-annotation (for example, the TIGR Comprehensive Microbial Resource \\[12\\]), but none of them has attracted nearly as much web traffic as GenBank or the other databases at NCBI \\[13\\]. The difficulties in changing this system are many: first, for example, there are some genomes for which GenBank is still the best source, and second, if another, better source of annotation exists, how is someone to discover it?\n\nA relatively new model of sharing expertise through the Internet might offer a solution. This model is the 'wiki': a shared resource that anyone can edit. This open-editing framework for websites and data was first introduced in 1995, and it was initially viewed with skepticism by many in the Internet community, who argued that wiki-based websites would be filled with unreliable, inaccurate information. But the success of the online encyclopedia Wikipedia \\[14\\] has demonstrated that, despite the skeptics, a wiki site can be accurate, up-to-date and incredibly useful. Genome annotation has many of the same features of an encyclopedia: the information required to produce it is broad-based and the expertise is scattered around the scientific community in a very wide range of laboratories, most of whom are not connected to genome projects. I therefore propose that a 'genome wiki' might provide just the solution we need for genome annotation. A wiki would allow the community of experts to work out the best name for each gene, to indicate uncertainty where appropriate and to discuss alternative annotations. Although wikis will not (and should not) supplant well-curated model-organism databases, for the majority of species they might represent our best chance for creating accurate, up-to-date genome annotation.\n\nWhether or not a genome wiki emerges, we will probably need an archival repository of annotation for many years to come. The international database consortium represented by GenBank, EMBL and DDBJ has served that purpose remarkably well for a long time and will continue to do so. Despite this success, the genomics community needs an accurate, continually updated source of genome annotation for every species, and we can hope that a solution to this problem will emerge in the near future.\n\n## Figures and Tables","meta":{"dup_signals":{"dup_doc_count":207,"dup_dump_count":62,"dup_details":{"curated_sources":2,"2022-33":1,"2022-21":1,"2021-49":1,"2021-04":1,"2020-24":1,"2020-05":1,"2019-51":1,"2019-47":1,"2019-39":1,"2019-26":1,"2019-18":1,"2019-09":1,"2019-04":1,"2018-51":2,"2018-47":1,"2018-43":1,"2018-30":1,"2018-17":1,"2018-13":1,"2018-09":2,"2018-05":2,"2017-51":2,"2017-47":1,"2017-39":3,"2017-34":2,"2017-30":1,"2017-26":1,"2017-22":1,"2017-17":2,"2017-09":11,"2017-04":2,"2016-50":2,"2016-44":2,"2016-40":2,"2016-36":11,"2016-30":8,"2016-22":1,"2016-18":1,"2016-07":8,"2015-48":5,"2015-40":4,"2015-35":5,"2015-32":5,"2015-27":6,"2015-22":6,"2015-14":4,"2014-52":5,"2014-49":7,"2014-42":12,"2014-41":8,"2014-35":7,"2014-23":7,"2014-15":3,"2023-50":1,"2017-13":2,"2015-18":6,"2015-11":5,"2015-06":5,"2014-10":5,"2013-48":5,"2013-20":6,"2024-26":1}},"file":"PMC1839116"},"subset":"pubmed_central"}
{"text":"author: Enzo Bonora; Jaakko TuomilehtoCorresponding authors: Enzo Bonora, <email@example.com>, and Jaakko Tuomilehto, <firstname.lastname@example.com>.\ndate: 2011-05\nreferences:\ntitle: The Pros and Cons of Diagnosing Diabetes With A1C\n\nAn International Expert Committee was convened in 2008 by the American Diabetes Association (ADA), the European Association for the Study of Diabetes, and the International Diabetes Federation to consider the means for diagnosing diabetes in nonpregnant individuals, with particular focus on the possibility to indicate A1C as an alternative if not a better tool (1). After reviewing the available literature and a thorough discussion on the advantages and the limits of previous diagnostic strategies (essentially based on fasting glucose assessment) and the considered alternative approach (based on A1C measurement), a consensus was reached that the latter (i.e., A1C) should be included among diagnostic tools for diabetes and, with the exception of a number of clinical conditions, should even be preferred in diabetes diagnosis in nonpregnant adults.\n\nThe main conclusion of the International Expert Committee was implemented in the most recent clinical recommendations issued by the ADA. However, in these guidelines, A1C is indicated as a diagnostic tool alternative but not superior to blood glucose, leaving to the health care professional the decision about what test to use in an individual.\n\nThe World Health Organization is currently examining the proposal made by the International Expert Committee and is carefully addressing the controversial issues still remaining, most of which have been the subject of letters to the editor and articles recently published in the literature. Nevertheless, the use of A1C for diagnosing diabetes is rapidly becoming a reality in many Western countries.\n\nIn the text that follows, one of us (E.B.) will present the main points supporting A1C (pros) and the other (J.T.) will illustrate the main counterpoints challenging A1C (cons) as the primary tool for diabetes diagnosis. The text has been prepared in full coordination and the final conclusions represent the opinion of both authors. <a href=\"#T1\" data-ref-type=\"table\">Tables 1<\/a> and <a href=\"#T2\" data-ref-type=\"table\">2<\/a> summarize the pros and cons.\n\n# PROs\n\n## A1C captures chronic hyperglycemia better than two assessments of fasting or 2-h oral glucose tolerance test plasma glucose\n\nDiabetes has been diagnosed for decades with fasting plasma glucose (FPG) assessment or, much less frequently, with an oral glucose tolerance test (OGTT). Hyperglycemia as the biochemical hallmark of diabetes is unquestionable. However, fasting and 2-h OGTT gauge just a moment of a single day. In addition, the two assessments required to confirm diagnosis might be fallacious in describing a chronic and complex clinical condition. In this respect, there is no doubt that a biochemical or clinical parameter describing the extent of a biological phenomenon over a long period provides a more robust indicator of glycemia than a parameter describing it in the short term or in a given moment only. Accordingly, there are some good examples in medicine: urinary albumin excretion rate provides more reliable information on the presence and the degree of microalbuminuria than spot urinary albumin-to-creatinine ratio; serum IGF-I is definitely more efficacious than serum growth hormone when monitoring patients with acromegaly, etc.\n\nLabeling a person with a diagnosis of diabetes has several psychological and legal implications and requires a robust and reliable approach. The measurement of A1C equals the assessment of hundreds (virtually thousands) of fasting glucose levels and also captures postprandial glucose peaks; therefore, it is a more robust and reliable measurement than FPG and\/or 2-h OGTT plasma glucose. This is particularly valid when FPG oscillates above and below the cut point of 126 mg\/dL or 2-h plasma glucose (PG) oscillates above and below the cut point of 200 mg\/dL. Of note, the 2-h PG had poor reproducibility. From a clinical standpoint, having an FPG of 120 or 130 mg\/dL or having a 2-h PG of 185 or 215 is virtually the same, but from the patient's perspective (perception of having a disease, psychological well-being, health insurance, recognition of particular benefits, or imposition of certain limitations, etc.), it makes a substantial difference. Therefore, a diagnostic tool gauging chronic rather than spot hyperglycemia is certainly preferable.\n\n## A1C is better associated with chronic complications than FPG\n\nDifferent from National Diabetes Data Group criteria, which were essentially based on distribution of glucose levels within the general population, the 1997 ADA criteria (and the subsequently recommended World Health Organization criteria) established diabetic glycemic levels by means of their association with retinopathy, the most exclusive and specific diabetes complication. Various observational studies documented that an increased prevalence of nonproliferative diabetic retinopathy can be observed with fasting glucose levels around 7.0 mmol\/L (126 mg\/dL) and 2-h PG around 11.1 mmol\/L (200 mg\/dL). Interestingly, the same studies documented that retinopathy increased with A1C levels around 6.5% (2\u20134). These results were confirmed in a more recent study including almost 30,000 subjects recruited in several countries. Such study clearly showed that prevalent retinopathy started to increase in the A1C category of 6.5\u20137.0% (5). Therefore, a cut point of A1C for diagnosing diabetes with an approach similar to the one used with FPG and 2-h PG is available (and indeed already was available in older studies).\n\nIt is well known that cardiovascular disease (CVD) is the most frequent chronic complication of diabetes, with incidence rates 5- to 10-fold higher than with microvascular disease. For this reason, the association of A1C with CVD can be considered a major issue when discussing the potential use of A1C for diagnosing diabetes. In this regard, it is worth mentioning that, in the general population, FPG is a poor marker of future CVD events, whereas 2-h OGTT and A1C are good predictors (6,7).\n\n## Fasting is not needed for A1C assessment and no acute perturbations (e.g., stress, diet, exercise) affect A1C\n\nPlasma glucose levels are not stable but rather vary throughout the day, mainly in postprandial periods. Although it is believed that fasting glucose levels are reproducible across days, a number of acute perturbations of glucose homeostasis have been described. Acute stress can increase endogenous glucose production substantially and impair glucose utilization. People who are worried about blood sampling or experience a stressful situation in the hours preceding blood sampling can have an increase in fasting glucose concentration. On the contrary, exercise can decrease glucose levels, and an evening or early-morning session of physical exercise can affect the level of fasting glycemia. Moreover, most individuals do not pay attention to the request or are not asked to consume a diet with at least 200 g carbohydrate in the days before testing glucose. Some individuals do not abstain from food in the 8 h before testing, thus arriving to the laboratory in the postabsorptive rather than fasting condition. In addition, smoking or taking certain medications can adversely affect fasting glucose. The lack of appropriate preparation for glucose testing makes FPG less reliable for diabetes diagnosis, with results sometimes falsely elevated and sometimes apparently normal. On the contrary, A1C is not influenced by acute perturbations or insufficient fasting. Indeed, A1C can be measured anytime, irrespective of fasting or feeding.\n\n## A1C has a greater pre-analytical stability than plasma glucose\n\nEven when preparation to glucose testing is optimal, plasma glucose values may still be misleading because of pre-analytical instability. In fact, tubes for blood collection do not always contain antiglycolytic substances, and even when they do, significant glucose consumption occurs in blood cells in the first 1\u20132 h after sampling because glycolysis is inhibited in its more distal steps by NaF or other preservatives. As long as the sample is not processed and plasma and blood cells are separated by centrifugation, a significant glucose loss is observed. In this regard, it must be emphasized that, quite often, blood samples reach the laboratory and are processed hours after withdrawal. Consistently, glucose concentration decreases 5\u20137% (on average \u223c0.5 mmol\/L) per hour and even more rapidly in cases of high ambient temperature (8,9). In such cases, glucose levels can show results lower than they are and diabetes diagnosis can be missed. It has been estimated that pre-analytical variability of FPG is 5\u201310%. On the contrary, pre-analytical variability of A1C is negligible. As for analytical variability, it is superimposable for glucose and A1C, being \u223c2%.\n\n## Standardization of A1C assay is not inferior to standardization of glucose assay\n\nOne of the main concerns surrounding A1C and raising perplexities on its use for diabetes diagnosis is the poor standardization of the assay. Quite surprisingly, the same concerns and perplexities do not extend to A1C use for diabetes monitoring despite the understanding that only when A1C is aligned to the Diabetes Control and Complications Trial (DCCT)\/UK Prospective Diabetes Study (UKPDS) standard should the recommended target be pursued (in general \\<7%). A great effort was made in the U.S. and other countries to make reproducible A1C across laboratories with an effective standardization program. Such a program has been recently completed and is being implemented worldwide to provide more reliable information to physicians who monitor diabetic patients (10). The standardization is expected to minimize laboratory biases and is a prerequisite to use A1C not only for monitoring but also for diagnosing diabetes.\n\nAlthough it is generally believed that glucose assay is highly reproducible across laboratories, this is not true. A recent survey conducted in 6,000 U.S. laboratories clearly documented a significant bias in glucose assessment in as many as 41% of them, yielding a misclassification of glucose tolerance in 12% of subjects (11). Therefore, the argument that A1C cannot be used for diabetes diagnosis because of poor standardization is no longer tenable.\n\n## Biological variability of A1C is lower than that for FPG\n\nWhen the same subjects have two assessments of the available glucose-related parameters, the correlation is stronger among the individual A1C measurements than among the FPG or 2-h PG measurements. The coefficients of variation of A1C, FPG, and 2-h PG are 3.6, 5.7, and 16.6%, respectively (12). This reflects of course both biological and analytical variability. However, although the latter was similar for A1C and FPG (\u223c2%), biological variability of A1C was severalfold lower than that of FPG (\\<1 vs. \u223c4%) (13). This finding confirms that the two required assessments of FPG to diagnose diabetes can provide quite unreliable information, whereas A1C, especially if measured twice as recommended, provides more robust clinical information.\n\n## Individual susceptibility to glycation might be an additional benefit of A1C assessment\n\nIt is a common clinical finding that many subjects have an A1C value lower or higher than expected when examining their daily glycemic profiles. Using the DCCT database, McCarter et al. (14) calculated the hemoglobin glycation index (HGI) as the difference between observed and predicted A1C level and identified categories of patients with low, moderate, or high HGI. Most interestingly, they found that subjects with high HGI had a greater risk of developing retinopathy and nephropathy, even when they had good glucose control, and that subjects with lower HGI had a low incidence of microangiopathy despite high mean blood glucose levels. This finding demonstrates that A1C assessment might provide not only information on chronic hyperglycemia but also a measure of whole-body susceptibility of protein glycation and, therefore, risks of diabetes complications that are more strictly related to this pathogenic mechanism.\n\n## Using the same biomarker for diagnosing and monitoring diabetes might be an advantage\n\nA1C is used to monitor diabetes and to establish the degree of metabolic control. Deviation from individualized A1C targets prompts physicians to modify treatment strategies with lifestyle intervention and\/or drug titration or changes. The use of A1C for diagnosing diabetes has the advantage that, in subjects with A1C \u22656.5% (i.e., diabetes), baseline A1C is already measured and deviation from target is immediately available (no A1C measurement as a second step after FPG assessment). In subjects with A1C of 6.00\u20136.49% (i.e., high risk of diabetes), an effective prevention strategy can be immediately undertaken with the awareness that a single A1C is definitely more reliable than a single FPG to stratify the risk of the disease. Yet, in subjects with A1C of 5.50\u20135.99% plus other diabetes risk factors (e.g., central obesity, atherogenic dyslipidemia, hypertension, and\/or metabolic syndrome), counseling can be immediately offered because diabetes risk is substantial, and single A1C assessment is definitely more reliable than single FPG to capture chronically high-normal glucose levels.\n\nPertinent to this issue is the firm belief that the implementation of the standardization of A1C assay would proceed more rapidly worldwide if A1C were to also be used for diagnosing diabetes. A1C assessment is crucial for diabetes monitoring, and establishing the individual A1C target definitely requires that the parameter is International Federation of Clinical Chemistry (IFCC) standardized and DCCT aligned. In fact, the A1C target and the deviation from it in the single patient remain totally uncertain when the laboratory provides A1C data that are not aligned to standard.\n\n## Cost of the assay: savings or no savings?\n\nOne of the major concerns raised by critics of the use of A1C for diagnosing diabetes is the higher cost of the assay when compared with FPG. There is no doubt that from an analytical point of view (cost of reagents and equipment), FPG is cheaper than A1C. However, other considerations about cost should be made. FPG assessment requires overnight fasting, whereas A1C can be assessed any time. This means that a person could go or could be driven by a relative\/friend to the laboratory, even during lunch or in the late afternoon, avoiding loss of work hours. It is also possible to collect blood for A1C assessment in the evening and hand it to the laboratory in the following days. Yet, in subjects with FPG \u22657 mmol\/L (\u2265126 mg\/dL), A1C assessment would be needed the next few days as a second step in a newly diagnosed diabetes workup. On the contrary, when A1C assessment yields a value \u22656.5%, the second step required to initiate diabetes monitoring after diagnosis would be completed, with a substantial savings of both analytical and nonanalytical costs. On the other hand, when using FPG to screen for diabetes and finding a value in the range of 5.6\u20136.9 mmol\/L (100\u2013125 mg\/dL; impaired fasting glucose), an OGTT is frequently prescribed (mainly in Europe and less frequently in the U.S.) to establish glucose tolerance. This test requires hours in the laboratory, with additional analytical and nonanalytical costs. In such cases, which represent a sizable portion of the general population, A1C rather than FPG would provide an immediate diabetes diagnosis or a valuable risk stratification (15) without supplementary testing.\n\n## Impact of changing the diagnostic laboratory parameter on epidemiology of diabetes\n\nA further critique to the program of moving from FPG to A1C for diabetes diagnosis comes from people who state that epidemiology of the disease is based on FPG and that the scenario would change if A1C were used instead of FPG. A recent report based on the U.S. population (16) showed that the use of A1C rather than FPG would not significantly change diabetes prevalence and that the categorization would not change in as many as 97.7% of subjects. Moreover, this study showed that half of the subjects with FPG \u22657 mmol\/L (\u2265126 mg\/dL) had an A1C value in the 6.00\u20136.49% range, thus deserving strict monitoring and an intervention. In this regard, however, it should be emphasized that any comparison of A1C with FPG (or 2-h OGTT PG) is equivocal because a true gold standard is not available. FPG, which in classic studies relating glucose parameters (including A1C) to retinopathy was measured just one time and with less than optimal pre-analytical and analytical procedures, cannot be taken as the gold standard. Therefore, any study examining sensitivity and specificity of A1C for diagnosing diabetes suffers from these limitations and is questionable. At present, the gold standard is probably the combination of FPG, 2-h PG, and A1C assessments with optimal pre-analytical, analytical, and standardized procedures and confirmatory testing for all parameters. This is not feasible on a large-scale basis and cannot be recommended. A1C seems to be a reasonable approach for all reasons discussed above (summarized in <a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>).\n\nReasons to prefer A1C compared with plasma glucose determination for diagnosing diabetes\n\n|  |\n|----|\n| Chronic hyperglycemia is captured by A1C but not by FPG (even when repeated twice). |\n| Microangiopathic complications (retinopathy) are associated with A1C as strongly as with FPG. |\n| A1C is better related to cardiovascular disease than FPG. |\n| Fasting is not needed for A1C assessment. |\n| No acute perturbations (e.g., stress, diet, exercise, smoking) affect A1C. |\n| A1C has a greater pre-analytical stability than blood glucose. |\n| A1C has an analytical variability not inferior to blood glucose. |\n| Standardization of A1C assay is not inferior to blood glucose assay. |\n| Biological variability of A1C is lower than FPG and 2-h OGTT PG. |\n| Individual susceptibility to protein glycation might be caught by A1C. |\n| A1C can be used concomitantly for diagnosing and initiating diabetes monitoring. |\n| Diabetes assessment with A1C assay is not necessarily greater than with glucose assessment. |\n\n# CONs\n\n## Diabetes is clinically defined by high blood glucose and not by glycation of proteins\n\nThe introduction of A1C as the diagnostic tool for diabetes, in particular, if this parameter is considered the primary tool, will lead to a major change in the pathophysiological paradigm that defines the syndrome called \"diabetes.\" So far, diabetes has been defined as \"a clinical condition of elevated glucose concentration in blood\". High A1C represents high glycation of proteins in the body, which is a substantially different biochemical abnormality, although it is certainly secondary to high blood glucose. In medicine, it is important to pay attention to primary phenomena before emphasizing the secondary ones. Moreover, high A1C is only observed subsequently to an increase in blood glucose, but there are few data on how long the delay is. Regardless of the length of this delay (weeks, months), diagnosis of diabetes using A1C would occur later than with blood glucose assessment. In many cases, such a delay might have negative clinical consequences.\n\n## A1C is a poor marker of important pathophysiological abnormalities featuring diabetes\n\nOGTT and 2-h post-glucose levels do reflect the pathophysiology behind diabetes better than any other glycemic parameter, since they provide information on what occurs in the postprandial state, when glucose levels are at the highest levels during the day and when the health of the pancreatic \u03b2-cell is essential. On the contrary, fasting glucose is the least informative among glycemic parameters, since in most subjects, it corresponds to the lowest glucose level during the day and it reflects the long nocturnal period when there is no intake of food and no particular stress for \u03b2-cells. However, humans spend most of their time in postprandial or postabsorptive states that are deranged in diabetes. A1C is a poor indicator of what occurs in the postprandial state. A1C captures only chronic hyperglycemia, but it will miss acute hyperglycemia. Normal blood glucose levels 2 h after glucose load indicates a good \u03b2-cell capacity, whereas high 2-h OGTT glucose levels document an impairment of \u03b2-cell function (17). This means that only 2-h OGTT PG can provide reliable information on the key pathophysiological defect of diabetes, also providing advice regarding the correct therapy to overcome it. This can be compared with ambulatory blood pressure monitoring (ABPM), where the main features predicting cardiovascular events are not only the long-term average blood pressure but the daily variation in blood pressure (especially the lack of a physiological nocturnal dip). Thus, ABPM is clinically useful in finding out blood pressure patterns, not estimating the long-term average. Recently, the Insulin Resistance Atherosclerosis Study (IRAS) showed that A1C is a weaker correlate of insulin resistance and insulin secretion in studies of metabolism compared with FPG and 2-h PG (18).\n\n## A1C has a poor sensitivity in diabetes diagnosis and would change the epidemiology of diabetes\n\nDiabetes diagnosis based on A1C misses a large proportion of asymptomatic early cases of diabetes that can only be identified by the OGTT. According to a recent Chinese study, A1C sensitivity is inferior compared with fasting blood glucose at the population level (19). Also, people with impaired glucose tolerance (IGT), in whom the efficacy of diabetes prevention has been unequivocally proven (20), cannot be detected by A1C.\n\nEpidemiological studies carried out in the general population showed that A1C and plasma glucose (FPG and\/or 2-h OGTT) identify partially different groups of diabetic subjects (21). A1C \u22656.5% identifies \\~30\u201340% of previously undiagnosed patients with diabetes (16). A larger percentage is detected by FPG (\\~50%) and 2-h PG (\\~90%). These findings are based on several recent studies, including the 2003\u20132006 NHANES (30% of diabetic individuals detected by A1C \u22656.5%, 46% by FPG \u2265126 mg\/dL, and 90% by 2-h PG \u2265200 mg\/dL) (18) and the IRAS (32, 45, and 87%, respectively) (18). In Qingdao, China, the \u22656.5% A1C cut point detects 30% of individuals with diabetes according to 2003 ADA criteria (19). In Chennai, India, however, A1C \u22656.5% detects 78% of individuals with newly diagnosed diabetes according to these criteria (22). In the IRAS, A1C of 5.7\u20136.4% predicted type 2 diabetes better with increasing BMI, and there were significant ethnic differences in the performance of A1C of 5.7\u20136.4% to detect diabetes (18). The ethnic differences in A1C compared with glucose measurements were also well demonstrated in the Diabetes Prevention Program population (23) and in a recent multiethnic database by Christensen et al. (24) that showed that there are no systematic interpretations as to why a shift to an A1C-based diagnosis for diabetes has substantially different consequences for diabetes prevalence across ethnic groups and populations.\n\n## 2-h Glucose level and IGT are stronger predictors of CVD than A1C\n\nBecause high glucose is toxic and causes many types of tissue damage, any indicator of hyperglycemia is predictive of diabetes complications. In the general population, FPG is a poor marker of mortality and future CVD events, whereas 2-h PG and A1C are better predictors (8,10,25\u201327). When analyzed jointly, only 2-h PG remains a statistically significant predictor of mortality and CVD (28,29). The findings regarding associations of FPG, 2-h PG, and A1C with retinopathy from the Pima Indians in the ADA 1997 report describing diagnostic thresholds of each glycemic parameter were derived by univariate analyses, and the multivariate analysis aiming at identifying the best glycemic parameters for diagnosis has never been reported. One of the main issues is that people with IGT have \\~40% increased mortality compared with normoglycemic people, and these individuals cannot be identified by measuring FPG or A1C. In addition, lifestyle intervention has been shown to prevent the progression from IGT to diabetes and also reduce their mortality risk to the level observed among normoglycemic people (30,31). Such prevention trial evidence does not exist for A1C or FPG, and this evidence should not be forgotten when deciding the approaches to identify intermediate hyperglycemia. Moreover, these results indicate that early intervention is effective in reducing mortality in people with IGT, and therefore, we should attempt to make the diagnosis of hyperglycemia as early as possible.\n\n## Fasting is not essential to identify perturbation in glucose metabolism\n\nMeasuring blood glucose in the fasting state in nondiabetic individuals is probably the least efficient way to identify early signs of perturbations in glucose metabolism. Because excessive postprandial glucose excursions are marking the first signs of abnormal glucose regulation and they also seem to best predict cardiovascular outcome, fasting is not really the central issue. It is likely that fasting has been overemphasized in diagnosing type 2 diabetes. We may pay attention to approaches used in the diagnosis of high blood pressure that also vary markedly during the day, but despite this variation, we are able to identify individuals with hypertension, even though measurements are not restricted to certain hours of the day but are done at any time.\n\n## Standardization of A1C assay is very poor and standardization of glucose assay is easier to implement\n\nInaccuracies in measurement and poor standardization of A1C assays are still a common problem, even in Western countries. Although a less than perfect standardization also exists for plasma glucose, this assay might be more easily aligned to a standard than A1C. Such programs now exist in the U.S., Japan, and Sweden, but there is still a long way to a global standardization of the A1C assays. Actually, all glycemic assessments require confirmation to make the diagnosis of diabetes correctly, mainly to avoid errors in sample handling and laboratory procedures.\n\n## A1C assay is unreliable and cannot be used in many subjects\n\nAbnormal hemoglobin traits are not uncommon in many regions of the world, and they significantly interfere with A1C assay (32), leading to spurious results. Also, there are several clinical conditions that influence erythrocyte turnover (e.g., malaria, chronic anemia, major blood loss, hemolysis, uremia, pregnancy, smoking, and various infections) that are responsible for misleading A1C data. Still, we are aware of ethnic differences in the relation between blood glucose and A1C levels (33) as well as an effect of aging. If different cut points regarding all these conditions need to be considered, A1C cannot be easily used to diagnose diabetes.\n\n## Within-day biological variability of plasma glucose might unveil disturbance of glucose metabolism\n\nBiological variability in plasma glucose reflects our daily patterns of diet, physical and mental activity, sleep, etc., and also depends on possible pathophysiological processes that may underlie type 2 diabetes. By definition, postprandial, and also 2-h PG, vary more than FPG. In this regard, A1C, which does not have any substantial biological variability, provides little information on pathophysiological processes leading to type 2 diabetes. The variability in A1C is entirely due to other phenomena, not pathophysiological disturbances.\n\n## Individual susceptibility to glycation of hemoglobin is not relevant to diabetes diagnosis\n\nThe HGI was calculated in patients with type 1 diabetes from the DCCT (17). This parameter is not relevant to the diagnosis of diabetes in the general population, in which 99% of subjects have A1C levels definitely lower than patients with type 1 diabetes. Subjects with high HGI had a greater risk of developing retinopathy and nephropathy, even when they had good glucose control (i.e., FPG was not very high), whereas subjects with lower HGI had a very low incidence of microangiopathy despite high mean blood glucose levels. This finding indicates that postprandial glucose excursions must have been very high in the former and very low in the latter. A1C reflects high mean exposure to glucose but not glucose fluctuations during the day. Unfortunately, in this analysis with HGI, postprandial glucose excursions and daily glucose variability were not taken into account.\n\n## Using the same biomarker for diagnosing and monitoring diabetes might not have positive effects only\n\nThis approach may be useful, but it also may lead to problems in two ways. First, people who have diabetes (based on their glucose values) will remain undiagnosed and untreated, since they are considered \"nondiabetic\" according to their A1C. Also, if the intermediate level of A1C (6.00\u20136.49 or 5.70\u20136.49%) was used to predict diabetes, it performed less well than impaired fasting glucose and\/or IGT (18). Whereas the 6.5% A1C threshold misses a large percentage of previously undiagnosed diabetes, its clinical consequences remain unknown. It is important to recognize this problem. One obvious consequence is that with a less sensitive test, individuals who fall below the threshold are not considered in cardiovascular management algorithms as high-risk individuals and are probably treated less effectively for other risk factors.\n\nSecond, a large proportion of newly diagnosed diabetic patients based on current glucose criteria have A1C \\<6.5%. In the Finnish Diabetes Prevention Study, the sensitivity of A1C \u22656.5% to diagnose diabetes was only 39%, i.e., 61% of newly diagnosed case subjects had A1C \\<6.5% (34). If this same threshold were to be used for treatment, these patients would not be accepted to be treated, even though their glucose levels were twice the glucose threshold for diabetes. This would also mean that in 61% of high-risk people who were regularly monitored for diabetes, the actual diagnosis would have been delayed\u2014for how long, we do not know, since diabetic people were referred to antidiabetic therapy based on their high glucose values.\n\n## Cost of the assay: glucose is unquestionably cheaper than A1C\n\nWhichever way we calculate the assay costs, A1C assay is more expensive than glucose assay, and it will thus remain so despite the speculative claim that the cost of A1C assay will become less expensive when used more extensively. In addition, many individuals at high risk of diabetes would need other laboratory tests that require fasting (e.g., lipid profile, hepatic profile, etc.), and therefore adding a glucose determination to the panel is not really a major issue. Also, the vast majority of laboratories in primary care collect samples in the morning, and they do not operate after \"working hours.\" This makes the claim that A1C can be measured \"any time of the day\" rather theoretical.\n\nIn a large part of the world, A1C is not available, and its cost is so high that it is meaningless to even discuss whether it should be given a priority over simple and inexpensive glucose measurements. This step would divide the world into two categories: developed societies in which diabetes diagnosis is made with A1C and less developed societies (between and within countries) in which diabetes diagnosis is made with plasma glucose: such a division should be avoided. It would add to the inequities in health and health care.<a href=\"#T2\" data-ref-type=\"table\"><\/a>\n\nReasons not to prefer A1C compared with plasma glucose determination for diagnosing diabetes\n\n|  |\n|----|\n| Diabetes is clinically defined by high blood glucose and not by glycation of proteins. |\n| A1C is a poor marker of important pathophysiological abnormalities featuring diabetes. |\n| A1C has a poor sensitivity in diabetes diagnosis and would change the epidemiology of diabetes. |\n| 2-h glucose level and IGT are stronger predictors of CVD than A1C. |\n| Fasting is not essential to identify perturbation in glucose metabolism. |\n| Standardization of A1C assay is poor, even in Western countries, and standardization of glucose assay would be easier to implement. |\n| In many subjects, A1C assay is unreliable and cannot be used. |\n| A1C has significant differences in various ethnic groups, which are poorly understood and characterized. |\n| Within-days biological variability of plasma glucose might unveil disturbance of glucose metabolism. |\n| Individual susceptibility to glycation of hemoglobin is not relevant to diabetes diagnosis. |\n| Using the same biomarker for diagnosing and monitoring diabetes might have negative effects. |\n| Cost of the assay: glucose is unquestionably cheaper than A1C, and A1C assay is not available on a large scale in most of the countries. |\n| A1C levels vary not only according to glycemia, but also to erythrocyte turnover rates (e.g., hemoglobinopathies, malaria, anemia, blood loss) as well as other factors. |\n| Correlation between A1C and FPG is \\~0.85%, which means that as many as 30% of the variation in FPG is not explained by A1C and vice versa. |\n| Nothing is known about changes in A1C during the development of diabetes. |\n| A1C levels of 6.0\u20136.5% do not predict diabetes as effectively as FPG and 2-h PG (OGTT). |\n| Sensitivity of A1C to detect diabetes defined by the OGTT is \\<50%; thus, the majority of diabetic individuals will remain undiagnosed if A1C is used. |\n| The levels of A1C predicting future retinopathy, nephropathy, etc., in the population is not well established (\\<6.5%?). |\n| No diabetes prevention trials have selected their populations based on A1C. |\n| Using A1C will delay the diagnosis of diabetes in \\~60% of incident cases. |\n\n# CONCLUSIONS\n\nThere is no doubt that hyperglycemia is the biochemical hallmark of diabetes and is a prerequisite for diagnosis. In this respect, moving from blood glucose to A1C might sound like a sort of heresy. There is also no doubt that all epidemiological data based on blood glucose assessment might be considered less important if the disease were mainly diagnosed with A1C. This might create confusion, disappointment, anxiety, and concern in all who lived a glucocentric existence. Partly rewarding would be the fact that, in several clinical conditions, A1C could not be used and blood glucose assessment would remain the standard diagnostic procedure. In all other conditions (most subjects), A1C could become the reference method, provided that its assay be aligned to international standards. Also mandatory is that the cost of assay declines and becomes affordable in less developed societies. Longitudinal studies should also reassure us about the relative benignity of clinical conditions in which A1C is below the diagnostic threshold of 6.5% but FPG and\/or 2-h OGTT PG are above the thresholds of 7 or 11 mmol\/L, respectively. This is currently one of the most relevant worries related to potentially missing diagnosis.\n\nGlucose assessment is familiar and cheaper, but A1C seems to provide several advantages, especially in a scenario in which OGTT is rarely used and never repeated as a confirmatory testing. Perhaps accepting a double diagnostic approach in which both blood glucose and A1C do coexist as diagnostic tools is reasonable. In the meantime, epidemiological and clinical studies will hopefully provide further data to better understand whether the current recommendations to replace FPG with A1C are well founded.\n\nWe agree that the research and debate on the pros and cons of using A1C versus glucose assay as a diagnostic tool for diabetes should continue in a constructive manner until a larger and truly evidence-based consensus is reached.\n\n## Acknowledgments\n\nNo potential conflicts of interest relevant to this article were reported.\n\n# References","meta":{"dup_signals":{"dup_doc_count":119,"dup_dump_count":57,"dup_details":{"curated_sources":2,"2023-50":2,"2022-40":1,"2021-49":1,"2021-25":2,"2021-21":2,"2021-17":2,"2020-29":1,"2020-24":2,"2019-43":4,"2019-39":1,"2019-35":2,"2019-09":1,"2019-04":2,"2018-47":3,"2018-39":1,"2018-30":1,"2018-22":3,"2018-13":2,"2018-05":1,"2017-51":2,"2017-43":3,"2017-39":1,"2017-34":2,"2017-30":1,"2017-26":3,"2017-22":1,"2017-17":3,"2017-09":3,"2017-04":3,"2016-50":2,"2016-44":3,"2016-40":2,"2016-36":2,"2016-30":2,"2016-26":2,"2016-22":2,"2016-18":2,"2016-07":2,"2015-48":2,"2015-40":2,"2015-35":2,"2015-32":2,"2015-27":2,"2015-22":2,"2015-14":2,"2014-52":2,"2014-49":2,"2014-42":5,"2014-41":3,"2014-35":4,"2014-23":1,"2024-10":1,"2017-13":3,"2015-18":2,"2015-11":2,"2015-06":2,"2024-22":1}},"file":"PMC3632159"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  The frequency of outpatient diagnostic errors is challenging to determine due to varying error definitions and the need to review data across multiple providers and care settings over time. We estimated the frequency of diagnostic errors in the US adult population by synthesising data from three previous studies of clinic-based populations that used conceptually similar definitions of diagnostic error.\n  .\n  # Methods\n  .\n  Data sources included two previous studies that used electronic triggers, or algorithms, to detect unusual patterns of return visits after an initial primary care visit or lack of follow-up of abnormal clinical findings related to colorectal cancer, both suggestive of diagnostic errors. A third study examined consecutive cases of lung cancer. In all three studies, diagnostic errors were confirmed through chart review and defined as missed opportunities to make a timely or correct diagnosis based on available evidence. We extrapolated the frequency of diagnostic error obtained from our studies to the US adult population, using the primary care study to estimate rates of diagnostic error for acute conditions (and exacerbations of existing conditions) and the two cancer studies to conservatively estimate rates of missed diagnosis of colorectal and lung cancer (as proxies for other serious chronic conditions).\n  .\n  # Results\n  .\n  Combining estimates from the three studies yielded a rate of outpatient diagnostic errors of 5.08%, or approximately 12 million US adults every year. Based upon previous work, we estimate that about half of these errors could potentially be harmful.\n  .\n  # Conclusions\n  .\n  Our population-based estimate suggests that diagnostic errors affect at least 1 in 20 US adults. This foundational evidence should encourage policymakers, healthcare organisations and researchers to start measuring and reducing diagnostic errors.\nauthor: Hardeep Singh; Ashley N D Meyer; Eric J ThomasCorrespondence to Dr Hardeep Singh, VA Medical Center (152), 2002 Holcombe Blvd, VAMC 152, Houston, TX 77030, USA; <firstname.lastname@example.com>\ndate: 2014-09\ninstitute: 1Houston Veterans Affairs Center for Innovations in Quality, Effectiveness and Safety, Michael E. DeBakey Veterans Affairs Medical Center and the Section of Health Services Research, Department of Medicine, Baylor College of Medicine, Houston, Texas, USA; 2Division of General Medicine, Department of Medicine, University of Texasat Houston, Memorial Hermann Center for Healthcare Quality and Safety, University of Texas Medical School at Houston, Houston, Texas, USA\nreferences:\ntitle: The frequency of diagnostic errors in outpatient care: estimations from three large observational studies involving US adult populations\n\n# Background\n\nDiagnostic errors pose an important threat to healthcare quality and safety in outpatient settings.1 2 The frequency of outpatient diagnostic errors is unknown,3 due in part to varying definitions across studies and to the challenge of detecting these errors, which typically emerge across multiple episodes of care over time. A recent review by Graber discussed the use of several methods to study diagnostic errors, including autopsies, case reviews, surveys, incident reporting, standardised patients, second reviews and malpractice claims.3 However, most of these methods have limited use in determining the frequency of diagnostic error in outpatient medical practice.\n\nPopulation-based estimates of diagnostic error could help prioritise ongoing patient safety efforts. We aimed to estimate the frequency of outpatient diagnostic errors in the US adult population by synthesising data from three previous studies of clinic-based populations that used conceptually similar definitions of diagnostic error.4\u20136\n\n# Methods\n\nData sources included two previous studies that used electronic triggers, or algorithms, to detect unusual patterns of return visits (primary care study) or lack of follow-up of abnormal clinical findings for colorectal cancer (CRC) (colon cancer study), both suggestive of diagnostic errors.4 6 A third study examined consecutive cases of lung cancer in two institutions (lung cancer study).5 In all three studies, diagnostic errors were confirmed through chart review and defined as missed opportunities to make a timely or correct diagnosis based on available evidence. The criteria for diagnostic errors were comparable across the three studies and excluded atypical presentations and appropriate decisions to watch and wait.\n\nThe triggers in the primary care study6 were based on the occurrence of unexpected return visits and were therefore selective for misdiagnosis of acute conditions and exacerbations of chronic conditions. Trigger 1 was a primary care index visit followed by unplanned hospitalisation within 14\u2005days; trigger 2 was a primary care index visit followed by one or more unscheduled visit(s) within 14\u2005days. Control visits did not meet either criterion. We considered for inclusion all primary care visits (n=212\u2005165 visits and 81\u2005483 unique patients) in two large health systems between 1 October 2006 and 30 September 2007. For the record review, we reviewed all trigger 1 records from one of the sites (n=220 records) and randomly chose the rest from the second site, yielding a reviewed sample of 674 records out of the total of 1086 visits selected. Maintaining a record sampling ratio from each health system that was consistent with trigger 1, we reviewed 669 of 14\u2005777 visits selected by trigger 2 and 614 of 193\u2005810 control non-triggered visits. Errors in triggered and non-triggered (control) cases were judged to have occurred when adequate data to suggest the final, correct diagnosis existed at an earlier primary care visit, or if documented abnormal findings at the visit should have prompted additional evaluation that would have revealed the correct diagnosis. Determination of errors was made on retrospective review by two independent raters. In extrapolating error rates to all primary care patients at our study sites, we assumed that rates of error in randomly selected samples of triggered and non-triggered cases were applicable to the entire cohort of triggered and non-triggered records in each clinic population.\n\nAlthough we presumed that the types of errors detected in the primary care study were likely to account for the majority of outpatient diagnostic errors, we also aimed to estimate the rate of errors related to initial diagnosis of less common but serious chronic conditions. Unlike misdiagnosis of acute conditions, these errors may not become apparent for weeks or months. As a proxy for such errors in outpatient care, we obtained frequency estimates for missed and delayed diagnosis of CRC and lung cancer, represented in the two cancer studies,4 5 respectively. These conditions were selected due to their relative frequency in several types of prior observational studies related to diagnostic errors.5 7\u201312 In both of our studies, diagnostic errors were characterised by lack of timely follow-up on specific 'red flags' or 'alarm' features5 10 13 of cancer in patients who were eventually diagnosed with either cancer. For instance, for CRC this was operationally defined as lack of follow-up 60\u2005days after documentation of hematochezia (bright red blood per rectum), positive faecal occult blood test and iron deficiency anaemia. For lung cancer, we defined lack of follow-up as failure to initiate further investigation or an appropriate action within 7\u2005days of a documented 'red flag' such as an abnormal chest X-ray. None of these data overlapped with the primary care study. We assumed that failure to follow-up on these 'red flags' accounted for all diagnostic errors associated with these types of cancer.\n\nWe extrapolated the frequency of diagnostic error obtained from our studies to the US adult population,14 using the primary care study as a proxy to estimate rates of diagnostic error for acute conditions (and exacerbations of existing conditions) and the two cancer studies to conservatively estimate rates of missed diagnosis of CRC and lung cancer (as proxies for other serious chronic conditions). Because of the heterogeneity of the conditions we included, we assumed that they represented the spectrum of diseases at risk for outpatient diagnostic errors.\n\n# Results\n\nIn the primary care study, samples of triggered visits and control visits were reviewed to determine the percentages of visits that contained errors. These percentages were extrapolated to the larger triggered and non-triggered samples. For trigger 1, 141 errors were found in 674 visits reviewed, yielding an error rate of 20.9%. Extrapolating to all 1086 trigger 1 visits yielded an estimate of 227.2 errors. For trigger 2, 36 errors were found in 669 visits reviewed, yielding an error rate of 5.4%. Extrapolating to all 14\u2005777 trigger 2 visits yielded an estimate of 795.2 errors. Finally, for the control visits, 13 errors were found in 614 visits reviewed, yielding an error rate of 2.1%. Extrapolating to all 193\u2005810 control visits yielded an estimate of 4103.5 errors. Thus, we estimated that 5126 errors would have occurred across the three groups. We then divided this figure by the number of unique primary care patients in the initial cohort (81\u2005483) and arrived at an estimated error rate of 6.29%. Because approximately 80.5% of US adults seek outpatient care annually,15 the same rate when applied to all US adults gives an estimate of 5.06% (see <a href=\"#BMJQS2013002627F1\" data-ref-type=\"fig\">figure 1<\/a>).\n\nThe colon cancer study estimated errors in CRC diagnosis in 26 out of 291\u2005773 patients seen in outpatient care (0.009%). Extrapolating to the US adult population that seeks outpatient care, we estimated 0.007% of all adults would have errors related to CRC per year. In the lung cancer study, we found 127 errors in 587 cases of lung cancer, yielding a 21.64% error rate. Extrapolating to the 80.5% of the US adult population that seeks outpatient care and the 0.072% of the population that becomes newly diagnosed with lung cancer each year,16 we estimate 0.013% of all adults would have errors related to lung cancer per year.\n\nCombining estimates from the three studies yields a rate of outpatient diagnostic errors of 5.08%, or approximately 12 million US adults every year. Based on our previous work,17 we estimated that about one-half of errors would have the potential to lead to severe harm.\n\n# Discussion\n\nAlthough diagnostic errors are difficult to define and measure, there is mounting evidence of their importance and frequency across several types of conditions.3 7 11 18 Applying aggregate diagnostic error rates from three previous studies conducted by our group, we estimate that approximately 12 million adults in the USA could experience outpatient diagnostic errors each year. Our estimates are generally consistent with data from the general public about diagnostic errors. A recent telephone survey of a random probability sample of over 2000 US adults suggested that more than one in 10 respondents reported a 'diagnostic mishap'.19 Similarly, a survey of 726 paediatricians revealed that more than half (54%) reported making a diagnostic error at least once or twice per month.20 Thus, other recent data corroborate our finding that diagnostic errors are common.\n\nPrevious estimations of the frequency of diagnostic error largely relied on expert opinion. For example, Elstein estimated errors occur in about 10\u201315% of diagnoses.21 As noted by Graber, many methods to study diagnostic errors have limited use in calculating the frequency of the problem. For example, autopsy rates have declined,22 the number of self-reports remains low and malpractice claims data are prone to bias and are non-representative.11 20 23\u201325 On the other hand, population-based studies have relied on medical record reviews, which are generally a gold standard for determining diagnostic error.26 Our electronic trigger approach facilitated selective record reviews, thus making the error determination process more efficient than either consecutive or random record reviews. Our estimate, based on a large sample size, is likely to be the most robust one thus far to address the frequency of diagnostic error in routine outpatient practice.\n\nAlthough it is unknown how many patients will be harmed from diagnostic errors, our previous work17 suggests that about one-half of diagnostic errors have the potential to lead to severe harm. While this is only an estimate and does not imply all those affected will actually have harm, this risk potentially translates to about 6\u2005million outpatients per year. Additionally, while the contribution of the two cancers to the overall estimation was small, we believed it was important to include them to show their relative contribution. This is because delayed cancer diagnosis is believed to be one of the most harmful and costly types of diagnostic error in the outpatient setting and its significance has become apparent not only in malpractice claims but also in retrospective studies of consecutive cancer cases, surveys and studies of failures to follow-up abnormal test results.5 7\u201312 In many of these studies, lung cancer and CRC are the most common cancers for which diagnosis is delayed.\n\nOur estimates are limited by our assumptions of generalisability. To keep the definition of diagnostic error uniform across studies, we only used data from our own work. Other studies have used different methods and definitions of diagnostic errors and we believed that this would make our extrapolations weaker. Similar to other methods to study medical errors, our trigger was not sensitive enough to capture all types of diagnostic errors in outpatient settings. Our triggers did not adequately capture errors in diagnosis of uncommon diseases, some of which might evolve over a prolonged period of time in the outpatient setting. Our studies also might not have adequately addressed other types of common chronic conditions that could be vulnerable to diagnostic error.27 For example, a recent study from the UK suggests that opportunities to diagnose chronic obstructive pulmonary disease at an earlier stage are being missed in the majority of cases.28 While it is tempting to do so, we did not extrapolate our chronic condition proxy estimates to all types of cancer or other types of chronic diseases because it would not be possible to do this accurately. Lastly, chart reviews inherently would miss errors due to documentation-related issues and this false negative rate is largely unknown. All of these limitations thus suggest that we underestimated the frequency of diagnostic errors and frequency could be well over 5%.\n\nIn conclusion, we estimate the frequency of diagnostic error to be at least 5% in US outpatient adults, a number that entails a substantial patient safety risk. This population-based estimate should provide a foundation for policymakers, healthcare organisations and researchers to strengthen efforts to measure and reduce diagnostic errors.\n\nWe thank Annie Bradford, PhD for assistance with medical editing.\n\n# References","meta":{"dup_signals":{"dup_doc_count":169,"dup_dump_count":63,"dup_details":{"curated_sources":2,"2023-50":5,"2023-40":4,"2023-23":2,"2023-14":5,"2023-06":4,"2022-49":2,"2022-40":2,"2022-33":9,"2022-27":8,"2022-21":6,"2022-05":6,"2021-49":6,"2021-43":1,"2021-39":6,"2021-25":3,"2021-21":4,"2021-17":5,"2021-10":3,"2021-04":2,"2020-50":5,"2020-45":2,"2020-40":1,"2020-34":4,"2020-29":1,"2020-24":1,"2020-16":1,"2020-10":1,"2020-05":1,"2019-51":2,"2019-47":1,"2019-43":2,"2019-39":1,"2019-35":1,"2019-30":1,"2019-26":3,"2019-22":3,"2019-18":2,"2019-09":1,"2019-04":2,"2018-51":2,"2018-47":2,"2018-39":1,"2018-34":1,"2018-26":3,"2018-22":1,"2018-09":2,"2017-51":2,"2017-47":3,"2017-43":2,"2017-39":2,"2017-34":1,"2017-30":2,"2017-26":2,"2017-22":3,"2017-17":2,"2017-09":3,"2024-30":3,"2024-26":1,"2024-22":2,"2024-18":3,"2024-10":3,"2017-13":1,"2015-18":1}},"file":"PMC4145460"},"subset":"pubmed_central"}
{"text":"author: Bob Weinhold\ndate: 2006-03\ntitle: Epigenetics: The Science of Change\n\nFor nearly a century after the term \"epigenetics\" first surfaced on the printed page, researchers, physicians, and others poked around in the dark crevices of the gene, trying to untangle the clues that suggested gene function could be altered by more than just changes in sequence. Today, a wide variety of illnesses, behaviors, and other health indicators already have some level of evidence linking them with epigenetic mechanisms, including cancers of almost all types, cognitive dysfunction, and respiratory, cardiovascular, reproductive, autoimmune, and neurobehavioral illnesses. Known or suspected drivers behind epigenetic processes include many agents, including heavy metals, pesticides, diesel exhaust, tobacco smoke, polycyclic aromatic hydrocarbons, hormones, radioactivity, viruses, bacteria, and basic nutrients.\n\nIn the past five years, and especially in the past year or two, several groundbreaking studies have focused fresh attention on epigenetics. Interest has been enhanced as it has become clear that understanding epigenetics and epigenomics\u2014the genomewide distribution of epigenetic changes\u2014will be essential in work related to many other topics requiring a thorough understanding of all aspects of genetics, such as stem cells, cloning, aging, synthetic biology, species conservation, evolution, and agriculture.\n\n# Multiple Mechanisms\n\nThe word \"epigenetic\" literally means \"in addition to changes in genetic sequence.\" The term has evolved to include any process that alters gene activity without changing the DNA sequence, and leads to modifications that can be transmitted to daughter cells (although experiments show that some epigenetic changes can be reversed). There likely will continue to be debate over exactly what the term means and what it covers.\n\nMany types of epigenetic processes have been identified\u2014they include methylation, acetylation, phosphorylation, ubiquitylation, and sumolyation. Other epigenetic mechanisms and considerations are likely to surface as work proceeds. Epigenetic processes are natural and essential to many organism functions, but if they occur improperly, there can be major adverse health and behavioral effects.\n\nPerhaps the best known epigenetic process, in part because it has been easiest to study with existing technology, is DNA methylation. This is the addition or removal of a methyl group (CH~3~), predominantly where cytosine bases occur consecutively. DNA methylation was first confirmed to occur in human cancer in 1983, and has since been observed in many other illnesses and health conditions.\n\nAnother significant epigenetic process is chromatin modification. Chromatin is the complex of proteins (histones) and DNA that is tightly bundled to fit into the nucleus. The complex can be modified by substances such as acetyl groups (the process called acetylation), enzymes, and some forms of RNA such as microRNAs and small interfering RNAs. This modification alters chromatin structure to influence gene expression. In general, tightly folded chromatin tends to be shut down, or not expressed, while more open chromatin is functional, or expressed.\n\nOne effect of such processes is imprinting. In genetics, imprinting describes the condition where one of the two alleles of a typical gene pair is silenced by an epigenetic process such as methylation or acetylation. This becomes a problem if the expressed allele is damaged or contains a variant that increases the organism's vulnerability to microbes, toxic agents, or other harmful substances. Imprinting was first identified in 1910 in corn, and first confirmed in mammals in 1991.\n\nResearchers have identified about 80 human genes that can be imprinted, although that number is subject to debate since the strength of the evidence varies. That approximate number isn't likely to rise much in years to come, writes a team including Ian Morison, a senior research fellow in the Cancer Genetics Laboratory at New Zealand's University of Otago, in the August 2005 *Trends in Genetics*. Others in the field disagree. Randy Jirtle, a professor of radiation oncology at Duke University Medical Center, and his colleagues estimated in the June 2005 issue of *Genome Research* that there could be about 600 imprinted genes in mice; in an October 2005 interview Jirtle said he's anticipating a similar tally for humans, even though the known imprintable genes of mice and people have an overlap of only about 35%.\n\n# Links to Disease\n\nAmong all the epigenetics research conducted so far, the most extensively studied disease is cancer, and the evidence linking epigenetic processes with cancer is becoming \"extremely compelling,\" says Peter Jones, director of the University of Southern California's Norris Comprehensive Cancer Center. Halfway around the world, Toshikazu Ushijima is of the same mind. The chief of the Carcinogenesis Division of Japan's National Cancer Center Research Institute says epigenetic mechanisms are one of the five most important considerations in the cancer field, and they account for one-third to one-half of known genetic alterations.\n\nMany other health issues have drawn attention. Epigenetic immune system effects occur, and can be reversed, according to research published in the November\u2013December 2005 issue of the *Journal of Proteome Research* by Nilamadhab Mishra, an assistant professor of rheumatology at the Wake Forest University School of Medicine, and his colleagues. The team says it's the first to establish a specific link between aberrant histone modification and mechanisms underlying lupus-like symptoms in mice, and they confirmed that a drug in the research stage, trichostatin A, could reverse the modifications. The drug appears to reset the aberrant histone modification by correcting hypoacetylation at two histone sites.\n\nLupus has also been a focus of Bruce Richardson, chief of the Rheumatology Section at the Ann Arbor Veterans Affairs Medical Center and a professor at the University of Michigan Medical School. In studies published in the May\u2013August 2004 issue of *International Reviews of Immunology* and the October 2003 issue of *Clinical Immunology*, he noted that pharmaceuticals such as the heart drug pro-cainamide and the antihypertensive agent hydralazine cause lupus in some people, and demonstrated that lupus-like disease in mice exposed to these drugs is linked with DNA methylation alterations and interruption of signaling pathways similar to those in people.\n\n# Substantial Changes\n\nMost epigenetic modification, by whatever mechanism, is believed to be erased with each new generation, during gameto-genesis and after fertilization. However, one of the more startling reports published in 2005 challenges this belief and suggests that epigenetic changes may endure in at least four subsequent generations of organisms.\n\nMichael Skinner, a professor of molecular biosciences and director of the Center for Reproductive Biology at Washington State University, and his team described in the 3 June 2005 issue of *Science* how they briefly exposed pregnant rats to individual relatively high levels of the insecticide methoxychlor and the fungicide vinclozolin, and documented effects such as decreased sperm production and increased male infertility in the male pups. Digging for more information, they found altered DNA methylation of two genes. As they continued the experiment, they discovered the adverse effects lasted in about 90% of the males in all four subsequent generations they followed, with no additional pesticide exposures.\n\nThe findings are not known to have been reproduced. If they are reproducible, however, it could \"provide a new paradigm for disease etiology and basic mechanisms in toxicology and evolution not previously appreciated,\" says Skinner. He and his colleagues are conducting follow-up studies, assessing many other genes and looking at other effects such as breast and skin tumors, kidney degeneration, and blood defects.\n\nOther studies have found that epigenetic effects occur not just in the womb, but over the full course of a human life span. Manel Esteller, director of the Cancer Epigenetics Laboratory at the Spanish National Cancer Center in Madrid, and his colleagues evaluated 40 pairs of identical twins, ranging in age from 3 to 74, and found a striking trend, described in the 26 July 2005 issue of *Proceedings of the National Academy of Sciences*. Younger twin pairs and those who shared similar lifestyles and spent more years together had very similar DNA methylation and histone acetylation patterns. But older twins, especially those who had different lifestyles and had spent fewer years of their lives together, had much different patterns in many different tissues, such as lymphocytes, epithelial mouth cells, intra-abdominal fat, and selected muscles.\n\nAs one example, the researchers found four times as many differentially expressed genes between a pair of 50-year-old twins compared to 3-year-old twins, and the 50-year-old twin with more DNA hypomethylation and histone hyperacetylation (the epigenetic changes usually associated with transcriptional activity) had the higher number of overexpressed genes. The degree of epigenetic change therefore was directly linked with the degree of change in genetic function.\n\nSometimes the effects of epigenetic mechanisms show up in living color. Changes in the pigmentation of mouse pup fur, ranging from yellow to brown, were directly tied to supplementation of the pregnant mother's diet with vitamin B~12~, folic acid, choline, and betaine, according to studies by Jirtle and Robert Waterland published in August 2003 (issue 15) in *Molecular and Cellular Biology*. The color changes were directly linked to alterations in DNA methylation. In a study forthcoming in the April 2006 issue of *EHP*, Jirtle and his colleagues also induced these alterations through maternal ingestion of genistein, the major phytoestrogen in soy, at doses comparable to those a human might receive from a high-soy diet. The methylation changes furthermore appeared to protect the mouse offspring against obesity in adulthood, although there are hints that genistein may also cause health problems, via additive or synergistic effects on DNA methylation, when it interacts with other substances such as folic acid.\n\n# Other Drivers of Change\n\nSubstances aren't the only sources of epigenetic changes. The licking, grooming, and nursing methods that mother rats use with their pups can affect the long-term behavior of their offspring, and those results can be tied to changes in DNA methylation and histone acetylation at a glucocorticoid receptor gene promoter in the pup's hippocampus. This finding was published in the August 2004 issue of *Nature Neuroscience* by Moshe Szyf, a professor in McGill University's Department of Pharmacology and Therapeutics, and his colleagues. In the same study, the researchers found that the effects weren't written in stone; giving the drug trichostatin A to older pups could help reverse the effects of poor maternal care received when they were younger. In the 6 June 2003 *Journal of Biological Chemistry* and the 23 November 2005 *Journal of Neuroscience*, Szyf and many of the same colleagues also demonstrated that giving the amino acid <span class=\"smallcaps\">l<\/span>-methionine to older pups could negate the benefits of high-quality maternal care received when they were younger.\n\nAlong with behavior, mental health may be affected by epigenetic changes, says Arturas Petronis, head of the Krembil Family Epigenetics Laboratory at the Centre for Addiction and Mental Health in Toronto. His lab is among the first in the world, and still one of only a few, to study links between epigenetics and psychiatry. He and his colleagues are conducting large-scale studies investigating links between schizophrenia and aberrant methylation, and he says understanding epigenetic mechanisms is one of the highest priorities in human disease biology research. \"We really need some radical revision of key principles of the traditional genetic research program,\" he says. \"Epigenetics brings a new perspective on the old problem and new analytical tools that will help to test the epigenetic theory.\" He suggests that more emphasis is needed on studying non-Mendelian processes in diseases such as schizophrenia, asthma, multiple sclerosis, and diabetes.\n\nThe past decade has also been productive in developing strong links between aberrant DNA methylation and aging, says Jean-Pierre Issa, a professor of medicine at The University of Texas M.D. Anderson Cancer Center. He presented information on aging and epigenetic effects at a November 2005 conference titled \"Environmental Epigenomics, Imprinting, and Disease Susceptibility,\" held in Durham, North Carolina, and sponsored in part by the NIEHS. Some of the strongest, decade-old evidence shows progressive increases in DNA methylation in aging colon tissues, and more recent evidence links hypermethylation with atherosclerosis. Altered, age-related methylation has also been found in tissues in the stomach, esophagus, liver, kidney, and bladder, as well as the tissue types studied by Esteller. Much of Issa's current work focuses on the links between epigenetic processes, aging, the environment, and cancer, and possible ways to therapeutically reverse methylation linked with cancer.\n\n# Current and Future Quandaries\n\nThe accumulated evidence indicates that many genes, diseases, and environmental substances are part of the epigenetics picture. However, the evidence is still far too thin to form a basis for any overarching theories about which substances and which target genes are most likely to mediate adverse effects of the environment on diseases, says Melanie Ehrlich, a biochemistry professor at the Tulane University School of Medicine and Tulane Cancer Center who has been conducting research on the topic for more than two decades.\n\nThat sense of uncertainty generally leaves epigenetics out of the regulatory picture. \"It's \\[too early\\] to actually use it at the moment,\" says Julian Preston, acting associate director for health at the EPA's National Health and Environmental Effects Research Laboratory. But Preston says the agency already relies more on its improving understanding of mechanistic processes, including epigenetics, and there is a clear effort within the EPA to expand genomics efforts both within the agency and with others with whom the agency works.\n\nAt the FDA, scientists are investigating many drugs that function through epigenetic mechanisms (although as spokes-woman Christine Parker notes, the agency bases its approvals on results of clinical trials, not consideration of the mechanism by which a drug works). One such drug, azacitidine, has been approved for use in the United States to treat myelodysplastic syndrome, a blood disease that can progress to leukemia. The drug turns on genes that had been shut off by methylation. The drug's epigenetic function doesn't make it a \"miracle drug,\" however. Trials indicate it benefits only 15% of those who take it, and a high percentage of people suffer serious side effects, including nausea (71%), anemia (70%), vomiting (54%), and fever (52%).\n\nEhrlich points out that azacitidine also has effects at the molecular level\u2014such as inhibiting DNA replication and apoptosis\u2014that may be part of its therapeutic benefits. The drug's mixed results might also be explained in part by a study published in the October 2004 issue of *Cancer Cell* by Andrew Feinberg, director of the Johns Hopkins University Center for Epigenetics in Common Human Disease, and his colleagues. They found that each of two tested drugs, trichostatin A and 5-aza-2\u2032-deoxycytidine (which is related to azacitidine), can turn on hundreds of genes while also turning off hundreds of others. If that finding holds in other studies, it suggests one key reason why it is so difficult to create a drug that doesn't cause unintended side effects.\n\n# Public and Private\n\nDespite the potentially huge role that epigenetics may play in human disease, investment in this area of study remains tiny compared to that devoted to traditional genetics work. Several efforts to change that are under way.\n\nIn Europe, the Human Epigenome Project was officially launched in 2003 by the Wellcome Trust Sanger Institute, Epigenomics AG, and the Centre National de G\u00e9notypage. The group's focus is on DNA methylation research tied to chromosomes 6, 13, 20, and 22. They may be joined soon by organizations in Germany and India, where scientists plan to work on chromosomes 21 and X, respectively, says Sanger senior investigator Stephan Beck.\n\nBut comprehensively studying all the epigenetic and epigenomic factors related to a multitude of diseases and health conditions will take much more work. \"A \\[comprehensive\\] Human Epigenome Project is a lot more complicated than a Human Genome Project,\" Jones says. \"There's only one genome, \\[but\\] an epigenome varies in each and every tissue.\" The Human Genome Project was a worldwide effort that took more than a decade and billions of dollars to complete.\n\nJones and Robert Martienssen addressed some of the complexities of a comprehensive, worldwide Human Epigenome Project in the 15 December 2005 issue of *Cancer Research*. Reporting on a June 2005 workshop convened by the American Association for Cancer Research, they concluded that, despite all the looming difficulties, such a project is essential, and the technology is sufficiently advanced to begin.\n\n\"I think it's going to happen a lot sooner than I thought just a year or so ago,\" Jirtle says. A group of researchers has already started the footwork to launch a U.S. complement to the European Human Epigenome Project effort \\[see box, p. A165\\].\n\nOther efforts are gaining ground. Another European group, the Epigenome Network of Excellence, took off in June 2004. This information exchange network includes members in the public and private sectors spread throughout ten Western European countries. Their objectives are to coordinate research, provide mentors, and encourage dialogue via their website. And in Asia, a conference held 7\u201310 November 2005 in Tokyo, \"Genome-Wide Epigenetics 2005,\" was dedicated in large part to facilitating a coordinated epigenomics research effort in Japan and possibly all of Asia, says Ushijima, one of the conference's organizers.\n\nIn the United States, the National Cancer Institute and the National Human Genome Research Institute formally kicked off a major effort 13 December 2005 that will include epigenomic work. The pilot project of The Cancer Genome Atlas, funded by \\$50 million each from the two institutes, is designed to lay the groundwork for comprehensive study of genomic factors related to human cancer. The initial three-year effort is expected to focus on just two or three of the more than 200 cancers known to exist, but if it's successful in developing methods and technologies, the number of cancers evaluated could then expand. If a high number of cancer genes are eventually scrutinized, the effort would be the equivalent of thousands of Human Genome Projects.\n\nTo help push the boundaries further, the NIEHS and the National Cancer Institute are in the midst of awarding grants totaling \\$3.75 million to study a wide range of epigenetic topics, such as identification of high-risk populations, dietary influences on cancer, and detailed study of numerous specific mechanisms linking environmental agents with epigenetic mechanisms and resulting disease. The dozen or so recipients are expected to launch their projects by fall 2006.\n\nThe NIEHS has also begun to integrate epigenomics projects into its research portfolio over the past five to six years. \"It's an emerging area that's very important,\" says Frederick Tyson, a program administrator in the NIEHS Division of Extramural Research and Training. And epigenetics is likely to be one of the half dozen or so most important considerations as NIEHS proceeds with its Environmental Genome Project, according to institute director David Schwartz.\n\nThe DNA Methylation Society, a professional group, has been growing slowly but steadily over the past decade, says founder and current vice president Ehrlich. As part of its efforts, the society launched a journal, *Epigenetics*, in January 2006 with the goal of covering a full spectrum of epigenetic considerations\u2014medical, nutritional, psychological, behavioral\u2014in any organism. Such groups are a valuable rallying point for this field, Jirtle says. He himself slowly worked his way into epigenetics from an initial cancer focus, and his segue is typical of many. \"If you study epigenetics, you don't have a home; we come from all different fields,\" he says.\n\nInterest in the private sector is also picking up. For instance, Epigenomics AG, with offices in Berlin and Seattle, is working on early detection and diagnosis of cancer and endometriosis (for which there is limited evidence of an epigenetic component), as well as development of products to predict effectiveness of drugs to treat these diseases. Founded in 1998, and now with about 150 employees, the company is focusing on DNA methylation mechanisms, and is working with companies such as Abbott Laboratories, Johnson & Johnson, Philip Morris, Roche Diagnostics, Pfizer, and AstraZeneca. CEO Oliver Schacht says the surging interest in this field is typified by the difference between the 2004 American Association for Cancer Research conference, which had half a dozen or so talks or posters on epigenetics, and the 2005 event, which had about 200.\n\n# Tool Time\n\nIf epigenetic work is to continue breaking new ground, many observers say technology will need to continue advancing. Jones and Martienssen note in their paper that there must be additional improvements in high-throughput technologies, analytical techniques, computational capability, mechanistic studies, and bioinformatic strategies. They also say there is a need for basics such as standardized reagents and a consistent supply of antibodies for testing.\n\nPreston agrees with many of these ideas, and says there is also a need to develop a comprehensive tally of all proteins in the cell and to get better protein modification information. He says universities are recognizing the demand for the talents needed to solve epigenomics problems, and are increasing their efforts to cover these topics in various ways, especially at the graduate school level.\n\nOther groups are doing their part by creating tools to further the field. All the imprinted genes identified so far are tracked in complementary efforts by Morison's and Jirtle's groups and the Mammalian Genetics Unit of the U.K. Medical Research Council. The European managers of the DNA Methylation Database have assembled a compendium of known DNA methylations that, although not comprehensive, still provides a useful tool for researchers investigating the roughly 22,000 human genes.\n\nKunio Shiota, a professor of cellular biochemistry at the University of Tokyo and one of the co-organizers of the November 2005 Tokyo conference, says epigenetic advances will rely in part on a range of processes that are slowly becoming familiar to more researchers\u2014massively parallel signature sequencing (MPSS), chromatin immunoprecipitation microarray analysis (ChIP-chip), DNA adenine methyltransferase identification (Dam-ID), protein binding microarrays (PBM), DNA immunoprecipitation microarray analysis (DIP-chip), and more. Someday, he says, these terms could become fully as familiar as MRI and EKG.\n\nThe rapidly growing acceptance of epigenetics, a century after it first surfaced, is a huge step forward, in Jirtle's opinion. \"We've done virtually nothing so far,\" he says. \"I'm biased, but the tip of the iceberg is genomics and single-nucleotide polymorphisms. The bottom of the iceberg is epigenetics.\"\n\n# Resources\n\n**Professional Organizations and Projects**\n\n-   DNA Methylation Society (international) <http:\/\/www.dnamethsoc.com\/main.htm>\n\n-   Epigenome Network of Excellence (Europe) <http:\/\/www.epigenome-noe.net>\n\n-   Human Epigenome Project (Europe) <http:\/\/www.epigenome.org>\n\n**Journal**\n\nEpigenetics\n\n<http:\/\/www.landesbioscience.com\/journals\/epigenetics\/>\n\n**DNA Methylation Database**\n\n<http:\/\/www.methdb.de\/front.html>\n\n**Imprinted Gene Databases**\n\n-   <http:\/\/igc.otago.ac.nz\/home.html>\n\n-   <http:\/\/www.geneimprint.com\/databases\/?c=clist>\n\n-   <http:\/\/www.mgu.har.mrc.ac.uk\/research\/imprinting\/>\n\n# U.S. Human Epigenome Project\n\nIn December 2005 a group of 40 international scientists publicly proposed a U.S. Human Epigenome Project to complement a European project of the same name launched in 2003. Group member Andrew Feinberg, a geneticist at the Johns Hopkins University School of Medicine, says, \"We're hoping to see how this idea takes hold. There is this ocean of information that is largely unexplored.\"\n\nThe goal of the U.S. project will be to comprehensively map methylation and histone modifications \u2014the two main classes of epigenetic modifications\u2014in a diverse set of normal tissues. These epigenomes would then serve as a reference for comparison with diseased tissues, revealing epigenetic causes of disease. Project organizers are now compiling a detailed proposal, with budget estimates and a timeline.\n\nAlthough both the U.S. and European projects ultimately aim to map all genes, the U.S. effort will look at different tissue and cell types than the European effort, and will also look at model organisms like yeast and the fly. The two groups are already working closely together in planning their projects to avoid redundancies, and this cooperation will likely continue.\n\nUnderstanding cancer would be one long-term goal for the U.S. project, but epigenetics\u2014changes in gene expression heritable from cell to daughter cell without changes in DNA sequence\u2014transcends any one disease. \"It has profound implications in aging, neurological disorders, and child development,\" says Peter Jones, another group member and director of the Norris Comprehensive Cancer Center at the University of Southern California. Jones and his colleagues argue that the importance of epigenetics in human disease, together with the maturing of technologies for mapping epigenetic changes, make a human epigenome project both critical and feasible.\n\nEpigenetics, says cancer biologist Jean-Pierre Issa of The University of Texas M.D. Anderson Cancer Center, could prove more important than genetics for understanding environmental causes of disease. \"Cancer, atherosclerosis, Alzheimer's disease \\[are all\\] acquired diseases where the environment very likely plays an important role,\" he points out. \"And there's much more potential for the epigenome to be affected \u2026 than the genome itself. It's just more fluid and more easy to be the culprit.\" **\u2013 Ken Garber**","meta":{"dup_signals":{"dup_doc_count":123,"dup_dump_count":25,"dup_details":{"curated_sources":2,"2022-40":1,"2022-21":1,"2016-18":4,"2016-07":5,"2015-48":6,"2015-40":3,"2015-35":4,"2015-32":5,"2015-27":4,"2015-22":5,"2015-14":4,"2014-52":5,"2014-49":8,"2014-42":8,"2014-41":7,"2014-35":9,"2014-23":4,"2014-15":7,"2023-14":1,"2015-18":5,"2015-11":5,"2015-06":4,"2014-10":6,"2013-48":6,"2013-20":4}},"file":"PMC1392256"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  Factors of IT adoption have largely been discussed in the literature. However, existing frameworks (such as TAM or TTF) are failing to include one important aspect, the interaction between user and task.\n  .\n  # Method\n  .\n  Based on a literature study and a case study, we developed the FITT framework to help analyse the socio-organisational-technical factors that influence IT adoption in a health care setting.\n  .\n  # Results\n  .\n  Our FITT framework (\"**F**it between **I**ndividuals, **T**ask and **T**echnology\") is based on the idea that IT adoption in a clinical environment depends on the fit between the attributes of the individual users (e.g. computer anxiety, motivation), attributes of the technology (e.g. usability, functionality, performance), and attributes of the clinical tasks and processes (e.g. organisation, task complexity). We used this framework in the retrospective analysis of a three-year case study, describing the adoption of a nursing documentation system in various departments in a German University Hospital. We will show how the FITT framework helped analyzing the process of IT adoption during an IT implementation: we were able to describe every found IT adoption problem with regard to the three fit dimensions, and any intervention on the fit can be described with regard to the three objects of the FITT framework (individual, task, technology). We also derive facilitators and barriers to IT adoption of clinical information systems.\n  .\n  # Conclusion\n  .\n  This work should support a better understanding of the reasons for IT adoption failures and therefore enable better prepared and more successful IT introduction projects. We will discuss, however, that from a more epistemological point of view, it may be difficult or even impossible to analyse the complex and interacting factors that predict success or failure of IT projects in a socio-technical environment.\nauthor: Elske Ammenwerth; Carola Iller; Cornelia Mahler\ndate: 2006\ninstitute: 1Institute for Health Information Systems, UMIT \u2013 University for Health Sciences, Medical Informatics and Technology, Hall in Tyrol, Austria; 2Institute for Educational Science, University of Heidelberg, Germany; 3Dept. of Psychiatry, University Hospitals of Heidelberg, Germany\nreferences:\ntitle: IT-adoption and the interaction of task, technology and individuals: a fit framework and a case study\n\n# Background\n\nIt is hard to imagine health care without Information and Communication Technology (ICT). Information technology in health care has existed for about four decades, and has gained widespread usage. Electronic patient records offer health care professionals access to vast amounts of patient-related information; decision support systems support clinical actions; and knowledge servers allow direct access to state-of-the-art clinical knowledge to support evidence-based medical practice \\[1\\].\n\nIntroduction of ICT can radically affect health care organisation and health care delivery and outcome. It is evident that the use of modern ICT offers tremendous opportunities to support health care professionals and to increase the efficiency, effectiveness and appropriateness of care \\[2,3\\].\n\nHowever, not all projects introducing IT in health care are successful. It is estimated that up to 60 \u2013 70% of all software projects fail (e.g. \\[4\\]), leading to enormous loss of money within healthcare and also to loss of confidence on IT from the side of users and managers.\n\nIt is interesting to recognize that the same IT system can be seen as success by one department or professional group, but as a failure or at least as problematic by another department or professional group. Various interconnected factors seem to exist that influence success or failure. In fact, the notion of success and failure has been largely discussed in the literature in the last years. We will not try to repeat the overall discussion here, but just refer to some good references (\\[5-11\\]).\n\nWhat we observe in any case is that the objective effects of the same IT system can largely differ in different settings. This is not surprising if we understand information systems as technical systems embedded in a social-organizational environment (see also \\[12\\]). The technology we are introducing in different clinical settings can be largely equal (e.g. the same PACS software in various radiological departments). But the socio-organizational setting may be quite different (e.g. different organization of workflow, different patient profiles, different motivation of staff, different management support, different IT history etc.), leading to different adoption processes of the same IT system, and thus to different effects (e.g. increased efficiency on one ward, user boycott on the other ward).\n\nWhat does this mean for a systematic IT management in hospitals? We argue that it would be helpful to know more about the factors influencing IT adoption, success and failure, and to be able to predict the effects in a certain setting.\n\nTherefore, at least two questions arise which should be answered by medical informatics research:\n\n1\\. What are the \"socio-organizational\" factors that influence adoption of an IT system in a given socio-organizational context?\n\n2\\. Based on the answers to question 1: Is there any way to predict the effects of an IT system in a certain context?\n\n## The aim of this paper\n\nThe aim of this paper is to present an approach to answer the first question. Based on a literature study, we will present a framework (the FITT framework) to better analyse the socio-organisational-technical factors that influence IT adoption. We will present the application of this framework in the analysis of a case study, describing the adoption of a nursing documentation system in several departments of a German University Hospital.\n\nWith regard to the second question, we will argue that from some more philosophical point of view, the exact prediction of success and failure may not be possible at all.\n\n## Previous work on IT adoption\n\nAnalysis of the factors influencing adoption (and thus also success and failure) of IT systems in health care has been an issue in research for many years. We will define IT adoption as follows, based on the discussion in \\[13\\]: for voluntary used system, IT adoption is reflected in the usage of the IT system; for mandatory used systems, IT adoption is reflected in the overall user acceptance. In the next paragraph, we will analyse some research results on factors for IT adoption, focussing on general valid frameworks.\n\nAnalysing the concept of information system (IS) success, DeLone \\[5\\] developed an **information success model** for management information systems. This model describes that the effects of IT on the user (the individual impact) and thus on the overall organization depends on the use and the user satisfaction. Those two aspects themselves depend on the quality of the IT system and the quality of the information in this system (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>). This model was used to structure a broad literature review, but seems not to be further validated. The authors discuss that IS success is a multidimensional construct based on the interaction of factors, and that a corresponding measurement instrument should therefore include not only the described criteria, but also their interaction.\n\nThe information success model is quite interesting as it describes the *interaction* of various factors. However, its shortcoming seems to be the isolated focus on IT quality and system quality, indicating that only the system's quality itself determines the overall impact. This does not help to explain why the same IT system can be adopted in a different way, and have rather different effects, in various settings.\n\nThe **technology acceptance model (TAM)** of Davis \\[14\\] tries to analyse why users adopt or reject a system. It defines the constructs \"perceived ease of use\" and \"perceived usefulness\" to predict attitude towards using and actual system use. Both factors themselves depend on features of the system (Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>).\n\nWhile trying to verify his model by questioning 112 users of one company, Davis \\[14\\] could partly confirm the expected links in his model. In his discussion, he stresses that this model is only usable for voluntary use of IT system, and that further factors should be included in his model, such as extrinsic motivation, user experiences with the system, and characteristics of the task to be supported by IT (e.g. complexity of a task).\n\nThis TAM model was adopted and extended by other researchers such as \\[15,16\\] and \\[17\\]. For example, Dixon \\[16\\] extended it to the **Information Technology Adoption Model (ITAM)**. He tried to refine the \"system design features\" of the TAM model by describing that an IT system has requirements (such as required IT knowledge of the users, or necessary technical infrastructure) that must be matched with the knowledge and skills of the users and with the available technical infrastructure. He called this \"fit\" and argued that perceived usefulness and perceived ease of use are not dependent on the system design features, but on this *fit* of user and system design features. The paper stays unclear whether the ITAM model was more formally validated. It is also unclear why those points already discussed as missing by Davis \\[14\\] (such as extrinsic motivation or task characteristics) were not included.\n\nAll of the presented models seem to concentrate rather strongly on individual attribute of the users and of the technology, neglecting attributes of the clinical environment and of the supported clinical tasks that in our opinion are of high importance to understand IT adoption processes. ITAM is however interesting as it introduced the *notion of fit*, explaining that it is not individual attributes which are important, but the quality of fit between e.g. IT complexity and IT knowledge.\n\nThe idea of fit is more comprehensively elaborated in the **task-technology-fit model (TTF)** of Goodhue \\[8,13,18\\]. He takes into account not only technology and user, but he also considers the complexity of the clinical tasks which have to be supported by an IT system. He examines the influence of the three factors \u2013 individual abilities, technology characteristics, and task requirements \u2013 on performance and on user evaluation of IT systems, highlighting the significance of the interaction (fit) of those three factors (Figure <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>). He argues that TTF (task-technology fit, or more correct task-individual-technology fit, as explained by \\[13\\]) is the extent to which technology functionality matches task requirements and individual abilities. Goodhue argues that user evaluation is a sufficient surrogate of TTF, and that it is appropriate for both mandatory and voluntary used IT system. The TTF model was used in the area of management information systems, and many of the proposed links within the model could be validated in studies in various studies with hundreds of users.\n\nTTF extends the other described models by concentrating on the fit. IT also includes the object of clinical task (e.g. task complexity, organization of tasks, interdependence with other tasks) to be supported by IT. However, TTF only focuses on the fit between user and technology, and between task and technology (see Figure <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>). It does not consider the interaction of user and task \u2013 which is, however, in our opinion an important success factor for IT introduction projects. For example, introduction projects may fail because nurses are not sufficiently motivated for nursing process documentation at all, independent of the tool used, or physicians may not be motivated to do a complete order entry themselves, instead of ordering a nurse to complete the order, because of the additional time it will take them. In addition, TTF and derived models do not reflect on the dynamics of introduction projects. Attributes of users, task and technology frequently change over time in a clinical environment, and thus also their interaction and their fit change.\n\nHowever, the notion of fit has been found useful in many other studies, too. For example, Folz-Murphy \\[19\\] described problems of the fit between user requirements and available IT functionality. Zigurs \\[20\\] examined the fit between task and technology in the area of group supports systems. Dishaw et.al. \\[21\\] extended the TTF \u2013 combined with the TAM model \u2013 with the construct of computer self-efficacy. With reference to the domain specific of users abilities the developed model of Dishaw et.al. also implied a relation between the attributes of user and task. The idea of fit seems thus to be helpful in various contexts.\n\nOverall, the presented approaches present a good basis for the analysis of the IT adoption; however, all of them show some limitations.\n\nBases on this analysis of the literature, we will now present a framework of fit between individuals, task and technology (FITT framework), taking into account the process-oriented character of an IT introduction. We will use our framework in a retrospective analysis of a corresponding case study.\n\n# Methods: The FITT framework\n\nBased on the literature review, we found it useful to use the interaction (fit) of users, tasks and technology as the basis to better understand IT adoptions.\n\nOur FITT framework (\"**F**it between **I**ndividuals, **T**ask and **T**echnology\") is based on the idea that IT adoption in a clinical environment depends on the fit between the attributes of the users (e.g. computer anxiety, motivation), of the attributes of the technology (e.g. usability, functionality, performance), and of the attributes of the clinical tasks and processes (e.g. organisation, task complexity) (Figure <a href=\"#F4\" data-ref-type=\"fig\">4<\/a>).\n\nAn \"Individual\" can represent an individual user or a user group. \"Technology\" can stand for the interaction of various tools needed to accomplish a given tasks (e.g. hardware, software, network). But the technology does not only comprise computer-based tools, but all tools used by the individuals to execute the tasks, therefore including also paper-based tools. \"Task\" comprises the wholeness of tasks and working processes that have to be completed (e.g. nursing documentation, order entry etc.) by the user and that are supported by the given technology.\n\nMany researchers focus on the aspect of \"organisation\". Organisational aspects in our model are either part of the individual aspect (individuals work in various roles and various groups in an organization), or they are considered in the task aspect (the clinical tasks and processes are organized in a given way, with defined responsibilities).\n\nThe objective of IT management can now be defined as reaching an optimal fit between technology, user and task. This means that e.g. user involvement in the selection process or a good user support can improve the fit between the three aspects. Individuals must therefore be *sufficiently* motivated and knowledgeable to execute a certain task. The technology must offer *sufficient* functionality and performance to support a given clinical task. And the user must be *sufficiently* trained to use a given technology adequately. An insufficient fit will probably lead to problems during implementation projects.\n\nThe **quality of fit** depends on the attributes of the objects. The following list presents some examples on attributes that affect the various fit dimensions:\n\n\u2022 Attributes on individual level: IT knowledge, motivation and interest in the task to be completed, flexibility and openness to new ways of working, team culture, organizational context, cooperation within a team, and politics within an organisation.\n\n\u2022 Attributes on task level: Organisation of the tasks to be completed, activities and their interdependence, complexity of tasks.\n\n\u2022 Attributes on technology level: Stability and usability of a software or hardware tool, costs of a tool, functionality, available technical infrastructure, integration of tools, availability of tools in a certain clinical situation.\n\nIn order to influence and improve the fit, management can *directly influence* those attributes of task, individual, and technology. For example, a reorganization of documentation processes may improve the fit between task and technology; training sessions for users may improve the fit between technology and individuals; a software update may influence both the fit between technology and task (e.g. new functionality being implemented) and between individual and technology (e.g. usability being improved). Here are some examples for possible deliberate **interventions** on the three objects to influence and optimize the fit:\n\n\u2022 Intervention on the individual level: user involvement in system selection and introduction (change management), user training sessions, good user support, motivation by the management (leadership issues).\n\n\u2022 Intervention on the task level: Reorganisation of task and working processes (e.g. new ways for order entry), clarification of the responsibilities (e.g. for nursing documentation).\n\n\u2022 Intervention on the technology level: Hardware and software updates, redesign of paper-based forms, network upgrade.\n\nBesides the direct interventions on the three objects, there are also external factors that may influence the fit, but which cannot easily be controlled by the IT management. The following list presents examples for those **external influencing factors**:\n\n\u2022 Intervention on the individual level: Staff changes (e.g. reducing IT knowledge), workload of staff (e.g. reducing time for IT use), changes of hospital strategy (e.g. IT is now seen to contribute to competitiveness of the hospital).\n\n\u2022 Intervention on the task level: Rising complexity of the task (e.g. by new legal documentation requirements), general organisational changes in the organisation, changes in patient profiles.\n\n\u2022 Intervention on the technology level: New software standards, new technological achievements.\n\nDue to those external factors, there will never be a complete static situation with regard to the three fit dimension and therefore to IT adoption. The external factors can improve or deteriorate the fit, while the deliberate interventions of IT management will be aimed at steadily improving the fit. There may only be a partly stable situation where the positive and negative changes are mostly balanced. It is helpful to describe this fit management and fit dynamics as a **loop-back system** (Figure <a href=\"#F5\" data-ref-type=\"fig\">5<\/a>).\n\nThe overall aim is to have an optimal fit to allow an easy IT adoption. As described, the fit model allows us to describe what we can do to influence and balance the fit. The larger the difference between the actual fit and the planned fit, the higher the problems during an IT introduction. For example, low fit between users and technology may lead to user frustration and finally to user boycott if no interventions (e.g. IT training sessions) are organized.\n\nWe assume that this basic theoretical approach can help analyzing the process of IT adoption during an IT implementation project in a clinical environment in the following ways (Figure <a href=\"#F6\" data-ref-type=\"fig\">6<\/a>):\n\n1\\. Any disruptions during an introduction project can be described and analysed with regard to the disruption in one of the **three fit dimensions** (task-technology, technology-individual, or individual-task). This should help plan projects, as problems can be anticipated in advance, or can help to analyse problems in a project retrospectively in order to learn from them.\n\n2\\. Any intervention that is taken to improve a project, to make it successful, can be analysed and described with regard to one of the **three objects** (task, individual, or technology). Any of those interventions on the objects will thereby indirectly affect the fit dimensions.\n\nWe will now present a case study where the FITT framework was applied in a retrospective analysis, to show how it can help describe and analyse an implementation project.\n\n# Reanalysis of a case study: IT adoption and FITT framework in a German university hospital\n\nA computer-based nursing process documentation system was introduced on several wards of the University Hospitals of Heidelberg between 1998 and 2001. This introduction was accompanied by various evaluation activities which among others investigated the following aspects:\n\n\u2022 General computer knowledge and attitudes to computers in nursing before, during and after system introduction.\n\n\u2022 Nurses acceptance of the nursing care process (the task to be supported by the IT) before, during and after system introduction.\n\n\u2022 User satisfaction with the nursing documentation system before, during and after introduction.\n\n\u2022 Quality of nursing documentation before, during and after system introduction.\n\n\u2022 Overall affects of the nursing documentation systems on nursing workflow.\n\nThese evaluations were done e.g. based on standardized and validated psychometric questionnaires (given to all nurses, with return rates around 80%), standardized documentation quality audits (analysing the nursing records of 20 patients per ward at three points of time), and focus group interviews with 1 \u2013 2 nurses per ward and with nursing and project management. Methods and results of the evaluation studies have been published e.g. in \\[22-25\\]. More details on all studies can be found in the corresponding German research reports \\[26-28\\] as well as in \\[29\\].\n\nIn general, the evaluation results showed high user acceptance of the IT system, and positive effects e.g. on documentation quality. A detailed analysis, however, showed differences in the reactions of the wards with regard to the new IT system. On one (somatic) ward, user acceptance was much lower than on the other wards, and several problems during IT introduction occurred here. On this ward (ward C), user acceptance was very low shortly after the introduction, and remained rather low even months after it (Figure <a href=\"#F7\" data-ref-type=\"fig\">7<\/a>).\n\nThe FITT framework was used to analyse the differences on the wards, the process of IT adoption, and the effects of interventions taken by the project and IT management to improve IT adoption. This analysis was based on the available results of the already mentioned various specific evaluation studies.\n\nIn this paragraph, we will present the result of this analysis for two somatic and two psychiatric wards. As already discussed, three of them showed a quick IT adoption, one of them showed a more problematic introduction (Figure <a href=\"#F7\" data-ref-type=\"fig\">7<\/a>). A complete report of this analysis has been published in a German project report \\[27\\].\n\nAll wards had used a paper-based documentation system prior to IT introduction which was now in part replaced by a computer-based system. This new IT system covered all steps of the nursing process (nursing anamnesis, care planning, documentation and evaluation of care \u2013 for a detailed explanation of the nursing process, see e.g. \\[30\\]). However, all functionalities were only used on the psychiatric wards where all steps of the nursing process were documented. The documentation on the somatic wards concentrated on the documentation of nursing anamnesis, care planning, nursing tasks, and omitting the evaluation of care. Nursing notes were written on all wards in the IT system.\n\n## Dermatological ward\n\nThe dermatological ward had 20 beds, around 12 nurses and a mean length of stay of about 10 days in 2000. The IT system was introduced in Sept. 2000. Questionnaires and documentation analysis were conducted three months before IT introduction and again in Dec. 2000 and in June 2001. A focus group interview study was conducted in February 2002.\n\nThe analysis on this ward found a rather uncomplicated and quick adoption of the new IT system. We will present the reanalysis of this case on the three fit dimensions:\n\n\u2022 **Fit between individuals and task**: This fit was mostly uncomplicated from the very beginning. Both ward managers and nurses stated in the interviews that they were convinced of the necessity of a high-quality nursing documentation, for legal reasons and for the reputation of nursing. The nursing process was mostly well accepted, as the questionnaires showed. Documentation analysis and interviews confirmed that nursing documentation was more complete after IT introduction than before. However, as the intensive documentation audits showed, not all steps of the nursing process were well documented, and the documentation was in part not adequately adapted to the individual patient.\n\n\u2022 **Fit between individuals and technology**: This fit was uncomplicated from the very beginning. The young, motivated team with high IT skills had no problems in learning the new technology. Computer acceptance and computer security levels were found to be high from the very beginning both in the questionnaires and the interviews.\n\n\u2022 **Fit between task and technology**: This fit was a bit problematic at the beginning, as the documentation analysis showed. The pre-defined nursing care plans offered by the IT system were at first not sufficiently adapted to the need of this ward. In addition, the computer equipment was first insufficient (to small number of computers, too slow hardware) to support a timely documentation process. Because documentation has always been done in the ward headquarters, no mobile or bedside computers were found necessary.\n\nSummarizing, on the dermatologic ward, we found a good individual-technology fit after the IT introduction. The individual-task as well as the task-technology fit were not optimal at the very beginning (Figure <a href=\"#F8\" data-ref-type=\"fig\">8<\/a>).\n\nIn order to improve the problematic fit dimensions, project management intervened as follows during the introduction period:\n\n\u2022 **Intervention with regard to task**: None.\n\n\u2022 **Intervention with regard to user**: Several onsite discussion to increase nurses' knowledge of the nursing process and how to correctly use pre-defined standardized nursing care plans, to increase fit between individual and task.\n\n\u2022 **Intervention with regard to technology**: The pre-defined standardized nursing care plans were refined, to improve adaptation to the individual patient; hardware was updated and extended, thereby increasing fit between task and technology.\n\nThose interventions seem to have improved the fit. The nurses judge the support of documentation by the software and hardware equipment as rather good after two years both in the interviews as well in the standardized questionnaires. The documentation analysis also show an improvement in documentation quality.\n\n## Paediatric wards\n\nThe paediatric ward had 15 beds, around 13 nurses, and a mean length of stay about 5 days in 2000. The nursing documentation system was introduced in Oct. 2000. Questionnaires and documentation analysis were conducted three months before IT introduction and again in Jan. 2001 and in July 2001. A focus group interview study was conducted in February 2002.\n\nCompared to the other wards this ward showed rather low user satisfaction values with the nursing documentation system during the introduction phase. An analysis structured according to the FITT framework showed several problematic areas:\n\n\u2022 **Fit between individuals and task**: The detailed documentation audits showed that nursing documentation was incomplete both before and after IT introduction (for details, see \\[24\\]). The documentation audits showed that the amount of documentation rose heavily during IT introduction, but documentation quality did not increase in the same manner (e.g. inadequate adoption of standardized nursing care plans to the individual patient). User attitudes with regard to the nursing care process strongly declined after IT introduction (details e.g. in \\[23\\]). In questionnaires and interviews, users complained about high time efforts for documentation. These and other results indicated that the fit between individuals and task may have already been problematic before IT introduction, and now deteriorated after IT introduction, as the new IT tool forced a more complete documentation, without bringing obvious benefits to the nurses.\n\n\u2022 **Fit between individuals and technology**: Validated questionnaires as well as focus group interviews showed some initial problems handling the new hardware and software. As the questionnaires showed, the users were rather unfamiliar with computers in the beginning. Some of the users were not too enthusiastic to learn the new IT system. However, the general attitudes with regard to computers in nursing were comparable to the other wards and on a medium level at the beginning. All in all, there were only some smaller problems in this fit on this dimension.\n\n\u2022 **Fit between task and technology**: This fit was found to be very problematic. Focus group interviews with users and managers revealed that in the beginning the software was not optimally customized. For example, the predefined nursing care plans in the software were found to be insufficiently adapted to the patients of this ward (a problem comparable to the dermatological ward). Also, the functionality and performance of the system was judged to be insufficient in some parts. For example, the repeated documentation of one item during a longer time period was not well supported. A big problem was also that no mobile computers or bedside terminals were available, which disturbed the common documentation workflow \u2013 while the nurses on this ward were used to documenting at least some aspects at the patients bedside, this had not been reflected by adequate hardware equipment in the introduction phase. From the users point of view, this all led to high and unnecessary time efforts for documentation.\n\nSummarizing, on this ward, all three fit dimensions were disturbed in the introduction period (Figure <a href=\"#F9\" data-ref-type=\"fig\">9<\/a>). Therefore it is not surprising that we found rather low user satisfaction (e.g., about half of the users wanted to stop using the software after three months) during this period.\n\nDue to these problems, project management decided on the following interventions which we have structured according to the FITT framework:\n\n\u2022 **Intervention with regard to task**: The workflow for documentation was reorganized, e.g. the number of items which needed to be documented were reduced, and some intermediate paper-based documentation was allowed to react on the missing mobile tools. This improved the individual-task as well as the task-technology fit.\n\n\u2022 **Intervention with regard to user**: Onsite training to refresh knowledge on nursing process and nursing documentation helped to increase the individual-task fit. Further individual training sessions with regard to computers in general and the software were organized. This helped increase the fit between individual and technology.\n\n\u2022 **Intervention with regard to the technology**: Missing functionality was implemented, erroneous functions were corrected, and hardware was updated to increase the performance, thus increasing the fit between task and technology.\n\nAll those interventions affected the three fit dimensions differently. The repetition of the quantitative evaluation about 9 months after implementation indicated a clear improvement in user satisfaction, reflecting in our opinion an improvement in the fit, which was also supported by the interview study. In addition, in the documentation analysis, the amount of documentation was now found to be reduced.\n\n## Psychiatric wards\n\nAs both psychiatric wards were found to be rather similar in IT adoption, they will be discussed here together. The wards had 21 resp. 28 beds and around 19 resp. 17 nurses. Mean length of stay was around 21 resp. 14 days in 2000. The nursing documentation system was introduced in Nov. 1998 resp. Nov. 1999. Questionnaires and documentation analysis were conducted three months before IT introduction, in Febr. 99 resp. March 2000, and again in Aug 2000. A focus group interview study was conducted in February 2002 with nurses from both wards. Both wards had long-term experience with paper-based documentation of the nursing process.\n\nBoth wards showed a mostly uncomplicated IT adoption:\n\n\u2022 **Fit between individuals and task**: This fit was uncomplicated from the very beginning. Nursing documentation and nursing process were highly accepted by ward management and nurses, as reflected in the questionnaires and interviews. Documentation analysis found high quality and completeness of documentation, even when some parts still appeared to be too standardized.\n\n\u2022 **Fit between individuals and technology**: Nurses were motivated to work with the new system. At the beginning, some nurses were not very IT experienced and had some initial problems, but computer acceptance scores were nevertheless high. User confidence and security in working with the IT system was found to be rather high.\n\n\u2022 **Fit between task and technology**: In the beginning, performance and functionality of the IT system were regarded as insufficient by the nurses. Also the quality of the predefined nursing care plans were found weak. Nurses felt that the system was not very useful to support nursing documentation in the first months.\n\nSummarizing, on these wards, the fit dimensions were rather good, with some problems only in the fit between task and technology (this comparable to the other wards) (Figure <a href=\"#F10\" data-ref-type=\"fig\">10<\/a>).\n\nProject management decided on the following interventions that we have structured according to the FITT framework:\n\n\u2022 **Intervention with regard to task**: None.\n\n\u2022 **Intervention with regard to user**: Some individual computer support was offered to increase fit between technology and individual.\n\n\u2022 **Intervention with regard to the technology**: Missing functionality was implemented, erroneous functions were corrected, and hardware was updated to increase the performance, therefore increasing the fit between task and technology.\n\nThese interventions helped to optimize the fit. The evaluations after several months and even years after implementation showed high user satisfaction and an improvement in nursing documentation quality, although some functions of the system were still being criticised for not being adequately adapted to the specific needs of a psychiatric ward.\n\n# Results: Facilitators and barriers to IT adoption\n\nBased on the result of the analysis of our different study wards, we will now collect the factors that seem to represent facilitators and barriers to adoption of a computer-based nursing documentation system. Based on the assumption that IT adoption depends on the fit between individual, task and technology, we found indicators in the reanalysis of our case study that affect IT adoption of nursing documentation systems (formulated in the way that the \"higher\/better\" the attribute, the easier IT adoption):\n\n\u2022 Relevant attributes of **individuals**: Commitment to nursing process as basis for nursing, commitment to nursing care planning, commitment to written nursing documentation, commitment to own professional nursing role (IT as professional tool), acceptance of computers in general, acceptance of computers in nursing, computer skills, typing skills (may be correlated with computers skills), general computer knowledge in years, age of nurses (may be correlated with computer knowledge), professional experience (may be correlated with age), number and motivation of key-users, overall motivation of wards to introduce the system, climate of support and trust within the nursing team, quality management skills of nurses, low expectations with regard to computers and nursing documentation, low number of staff members and work load of ward, low staff fluctuation, low number of part-time staff, night watches and nursing trainees on the ward, commitment to standardisation of nursing tasks (IT as support, or IT reducing individuality of nursing).\n\n\u2022 Relevant attributes of the **task of nursing documentation**: Low complexity, amount and level of detail of documentation, clear organization, clearly structured place and time of documentation, quality of implemented predefined nursing care plans, low number of nursing tasks that have to be documented in each shift, low use of documentation (e.g. once per shift), long length of stay of patients, low complexity of patient profiles (children, adults), high use of documentation by other health care professionals, available time during routine work to learn the system, no parallel redundant use of different documentation media (IT, paper), clear agreements with regard to organisation of documentation, availability of nursing standards from other wards or earlier projects, high degree of standardisation of nursing.\n\n\u2022 Relevant attributes of the **technology**: Quality and amount of functionality of software, usability and user friendliness of software, stability and flexibility of software, quality and performance of hardware and network, availability of sufficient number of computers, availability of mobile computers, clear version and update management.\n\nBased on our analysis, the following interventions and external factors can be found which may have a positive influence on the fit between individuals, technology and tasks (mostly corresponding to the \"active interventions\" in Figure <a href=\"#F5\" data-ref-type=\"fig\">5<\/a>) and therefore on IT adoption:\n\n\u2022 Positively affecting **individual-technology fit**: IT training sessions, positive external norms (e.g. computers belong to nursing), high computer acceptance by nursing management, high motivation and training of key users, intensive user support, step-wise implementation of functionality (instead of all at one point of time), reduction of nursing workload during the introduction phase (e.g. by additional staff).\n\n\u2022 Positively affecting **individual-task fit**: Efficient training sessions on the nursing process, high acceptance of nursing process by nursing management, high external norms (e.g. nursing is an own profession), clarification of responsibilities within nursing documentation, reorganization and restructuring of nursing documentation processes, clarification of the sensible amount of nursing documentation (to avoid over-documentation), increased use of predefined nursing care plans, step-wise introduction of nursing process.\n\n\u2022 Positively affecting **task-technology fit**: Reorganisation and restructuring of nursing documentation, local adaptation of predefined nursing care plans, update of software functionality enabling the reflection of the tasks characteristics for a ward, increase in number and availability of computers, introduction of mobile tools in case the tasks make this necessary.\n\nPlease note that the interventions do in fact directly influence attributes of individual, technology, or task, thereby only indirectly influencing one or two of the three fit dimensions. For example, by organizing additional training session, we can improve the IT skills (attributes) of the individuals, and thereby also indirectly influence the individual-technology fit.\n\nThis analysis should highlight that \u2013 even for this rather restricted case study and the limited focus on nursing documentation \u2013 a variety of factors can be found that influence the fit between individuals, technology and task and therefore IT adoption. This supports the often discussed fact that success and failure is a rather complex and multi-dimensional construct.\n\n# Discussion\n\nIn this paper, we presented \u2013 based on an analysis of other IT adoption frameworks from the literature \u2013 a framework of the interaction between individual, technology and tasks (the FITT framework). This framework was used to support a structured retrospective analysis of the introduction of a nursing documentation system in a German University Hospital. The detailed analysis of the case study showed common features, but also differences of IT adoption of the wards which could be easily reflected and analysed based on the FITT framework.\n\nThe FITT framework focuses on the significance of the optimal interaction (fit) of individual user, technology, and task. The fit between the attributes is more important than the individual attributes themselves. For example, IT skills of the users are not sufficient for the success of an introduction \u2013 rather, they must match the requirements by the IT software (e.g. software complexity).\n\nIn our case study, the clear structure with three objects and three fit dimensions helped us reflect on the different reactions of the wards we had found in the evaluation studies, on the problems which occurred during introduction, and on the interventions of project management. We did not find any aspect that we could not easily structure within this FITT framework. This however can not be regarded as formal proof of completeness of our framework.\n\nThe idea of fit has been introduced by other authors before, such as \\[16\\] or \\[18\\]. Nancy Levensen discussed in here keynote at the Information Technology in Health Care Conference (ITHC 2004) in Portland that system failure often depends on failures in the interaction between components, not on the quality of the components themselves that often do not present problems from an isolated point of view. Southon \\[31\\] discussed the fit between organization, technology and user skills. Lundberg \\[32\\] examined the interaction between actors (staff), artefacts (technology) and working processes during a PACS installation. And Palvia \\[33\\] analysed the significance of the factors task, technologies, user, and organisation during a system introduction.\n\nBut none of those previous and the other analysed authors, to our knowledge, noticed the important interaction between user and task. There are many examples e.g. in the area of nursing documentation systems or computerized physician order entry where the users were not motivated to do a certain task \u2013 independent of the quality and functionality of the IT tool that was introduced! The reason why this interaction is often overlooked seems simple: In many cases, IT introduction is accompanied by organizational changes (e.g., when CPOE is introduced, a much higher documentation burden is suddenly put on the physicians), often leading to low user satisfaction or even user boycott (see example in \\[34\\]). These problems are then often attributed to the IT system (suspecting a low fit between IT and user or between IT and task). But in fact the problems are mostly coming from a more fundamental ill-acceptance of the new task to be done, thus reflecting a low fit between user and task! For an example of this ill-acceptance, see the detailed analysis of a CPOE introduction by Massaro \\[35\\].\n\nManagement of fit can be regarded as a loop back system, reflecting that the fit is never really stable, but that it is changing based on external factors or deliberate interventions, making fit management a constant and complex task for the whole life cycle of an IT system.\n\nIn our case, all wards showed specific and time-dependent IT adoption processes while starting to work with computer-based nursing documentation system. Unfortunately, we could not analyse this process in detail, as the measurement points of the various studies were too different and irregular (the available data came from four partly independent evaluation studies). A more refined analysis would have needed an analysis in regular short intervals. Thus, all our presentations with regard to the dynamic of the IT adoption within the wards must be taken with care \u2013 e.g., in Figure <a href=\"#F7\" data-ref-type=\"fig\">7<\/a>, we do not know whether there have not been ups or down of user satisfaction in between the two measurement points.\n\nAll wards are now working rather successfully with the computer-based nursing documentation system, however, still the fit is not in complete balance, as further changes are steadily occurring (e.g. new staff members need to be trained, new documentation standards have to be implemented, software errors lead to updates, refinement of organization of nursing documentation, new documentation guidelines leading to training session etc.). We expect that managing the fit balance is a continuous task which is never really completed, and can be described as a loop-back system (cp. Figure <a href=\"#F5\" data-ref-type=\"fig\">5<\/a>).\n\nOur case study shows the complexity of a broad introduction of an IT system in various settings: Individuals and tasks are rather different in the various settings, requiring high flexibility of the IT system and individual IT introduction and support activities to get the best fit for each ward. This helps to explain why we can find different IT adoption even when the same software and hardware is introduced.\n\nInterestingly, it seems that the involved users often are not able to distinguish between the various fit dimensions. For example, the nurses in our study partly expressed the opinion that the new software system was not usable. Detailed analysis revealed that at least some problems were based on the miss-fit between user and task \u2013 and not on a miss-fit between user and technology.\n\nIn this context, we want to stress the significance of the user in IT introduction projects. Investing in user training and user support can have positive effects on both individual-task and individual-technology fit. In addition, user involvement in system design and selection helps to build more adequate systems, therefore also improving the task-technology fit. And, as Goodhue \\[8\\] shows, user evaluation is a good surrogate for the overall fit, explaining partly why user acceptance studies have found so widespread use (which should not be understood as to underestimate the significance of objective performance measures).\n\nIn case we accept the FITT framework as a point of basic understanding of IT adoption \u2013 what are the following steps in ongoing development of this framework? Some may argue that the first logical step would now be to develop measurement instruments for the fit (as e.g. Goodhue \\[18\\] tries for his task-technology fit) which in fact seems necessary. The second step could then be to quantify the factors influencing the fit, to allow better and quantifiable prediction and planning of successful IT adoption \u2013 e.g. if we knew that IT skills explain around 65% of the variability of fit between IT and users, then we may consider it useful to invest in training session. Comparable quantitative oriented research on factors was e.g. done by \\[23,36\\] or \\[37\\]. To achieve this goal, we would have to compile a complete list of attributes of tasks, technology and individual influencing the fit. However, this research approach would only make sense from a realistic (or positivistic) point of view where we expect that an absolute reality exists, where objects have attributes which we can unambiguously measured.\n\nFrom another perspective that is often called relativistic or constructivistic, this approach may be seen as misleading, as no absolute and measurable reality is thought to exist. In any case, we are dealing here with people whose reactions to given inputs cannot be precisely predicted \u2013 as von F\u00f6rster \\[38\\] would put it, socio-technical systems are non-trivial systems. From this point of view, the significance of the various factors influencing the fit can only be analysed based on the background of a given setting, with all of the political, organizational and individual history influencing the fit. The isolated analysis of say four factors (out of an unknown but probably very large number of existing factors) can never lead to a significant and comprehensive insight into what is going on in a given context. And a generalisation of interactions between these limited numbers of factors could never reflect all possible settings and would thus not be useful. Summarizing, from a more epistemological point of view, it may be difficult or even impossible to analyse the complex and interacting factors that influence the fit.\n\nWithout adopting any specific research paradigm here, we would like to argue that research on factors influencing the fit (or IT adoption in general) must try to generalize from individual cases. We must come to a valid measurement instrument for the fit dimensions. And we are convinced that independent of the setting, there may be some priority of factors (e.g. in our case, we found IT skills to be less important than acceptance of the nursing process on all wards). Even such a rough priority list would help IT managers to optimize planning and directing of IT systems in clinical context \u2013 without trying to quantify the individual impacts and their interactions, which in fact does not seem helpful. Research in this direction has been done e.g. by \\[39\\] or \\[40\\].\n\nIn any case, at the moment, the FITT framework presents a straight-forward analytic framework to describe and analyse IT adoption case studies. It is innovative in the sense that it clearly describes the objects and their interactions affecting the fit, understanding fit management as a loop-back system.\n\nThe FITT framework was based on the retrospective analysis of the adoption of a nursing documentation system on four wards over 3 years. With regard to the broad literature we have discussed in the beginning, showing the usefulness of the notion of fit in various settings, we expect the FITT framework to be valid also for other IT types in other settings \u2013 but this still needs to be verified.\n\nThe framework should now continue to be refined and also balanced to other adoption theories. And, as said, we need a valid quantitative measurement instrument for the three fit dimensions. The description of the dynamics of change can be improved by introducing a time axis into the framework. After further refinement and validation of this theoretical approach, we expect an even better support for the planning and evaluation of IT introduction projects.\n\n# Conclusion\n\nIn this paper, we presented \u2013 based on an analysis of other IT adoption frameworks from the literature \u2013 a framework of the interaction between individual, technology and tasks (the FITT framework). This framework was successfully used to support a structured retrospective analysis of the introduction of a nursing documentation system in a German University Hospital. Based on this case study, we derived facilitators and barriers to IT adoption of clinical information systems. This work should support a better understanding of the reasons for IT introduction failures and therefore enable a better prepared and more successful IT introduction projects.\n\n# Competing interests\n\nThe author(s) declare that they have no competing interests.\n\n# Authors' contributions\n\nThe case study was planned and executed by EA. CI participated in the qualitative part of the case study, CM in the quantitative part of the study. The FITT framework was developed by EA and CI together. The paper was written by EA with the support of CI and CM.\n\n# Pre-publication history\n\nThe pre-publication history for this paper can be accessed here:\n\n<http:\/\/www.biomedcentral.com\/1472-6947\/6\/3\/prepub>\n\n### Acknowledgements\n\nWe would like to thank all the staff from the various study wards as well as the large project team for their long-time dedication to this research project. A preliminary version of this paper was presented at the conference \"Information Technology in Health Care (ITCH 2004)\" in Portland in September 2004 \\[41\\].","meta":{"dup_signals":{"dup_doc_count":141,"dup_dump_count":63,"dup_details":{"curated_sources":4,"2023-14":2,"2022-49":1,"2022-40":2,"2022-21":2,"2021-43":1,"2021-17":1,"2021-04":1,"2020-40":1,"2020-10":1,"2019-51":1,"2019-43":1,"2019-35":1,"2019-30":1,"2019-18":2,"2019-13":2,"2019-09":1,"2019-04":1,"2018-51":2,"2018-47":1,"2018-43":1,"2018-39":1,"2018-34":3,"2018-30":2,"2018-26":1,"2018-22":1,"2018-17":2,"2018-13":2,"2018-09":1,"2018-05":1,"2017-51":2,"2017-47":1,"2017-43":1,"2017-39":1,"2017-34":2,"2017-30":1,"2017-26":2,"2017-22":11,"2017-17":9,"2017-09":3,"2017-04":2,"2016-50":2,"2016-44":2,"2016-40":4,"2016-36":8,"2016-30":10,"2016-22":1,"2015-40":2,"2015-35":1,"2015-22":3,"2014-52":3,"2014-35":1,"2014-23":2,"2014-15":1,"2023-40":1,"2024-10":3,"2017-13":6,"2015-18":4,"2015-11":1,"2015-06":1,"2014-10":2,"2013-48":2,"2013-20":1,"2024-26":1}},"file":"PMC1352353"},"subset":"pubmed_central"}
{"text":"abstract: Dynamic positron emission tomography (PET) imaging was performed using sequential tracer injections (\\[^15^O\\]H~2~O, \\[^11^C\\]3-O-methylglucose \\[3-OMG\\], and \\[^18^F\\]fluorodeoxyglucose \\[FDG\\]) to quantify, respectively, skeletal muscle tissue perfusion (glucose delivery), kinetics of bidirectional glucose transport, and glucose phosphorylation to interrogate the individual contribution and interaction among these steps in muscle insulin resistance (IR) in type 2 diabetes (T2D). PET imaging was performed in normal weight nondiabetic subjects (NW) (*n* = 5), obese nondiabetic subjects (OB) (*n* = 6), and obese subjects with T2D (*n* = 7) during fasting conditions and separately during a 6-h euglycemic insulin infusion at 40 mU\u00b7m^\u22122^\u00b7min^\u22121^. Tissue tracer activities were derived specifically within the soleus muscle with PET images and magnetic resonance imaging. During fasting, NW, OB, and T2D subjects had similar \\[^11^C\\]3-OMG and \\[^18^F\\]FDG uptake despite group differences for tissue perfusion. During insulin-stimulated conditions, IR was clearly evident in T2D (*P* \\< 0.01), and \\[^18^F\\]FDG uptake by muscle was inversely correlated with systemic IR (*P* \\< 0.001). The increase in insulin-stimulated glucose transport was less (*P* \\< 0.01) in T2D (twofold) than in NW (sevenfold) or OB (sixfold) subjects. The fractional phosphorylation of \\[^18^F\\]FDG during insulin infusion was also significantly lower in T2D (*P* \\< 0.01). Dynamic triple-tracer PET imaging indicates that skeletal muscle IR in T2D involves a severe impairment of glucose transport and additional impairment in the efficiency of glucose phosphorylation.\nauthor: Bret H. Goodpaster; Alessandra Bertoldo; Jason M. Ng; Koichiro Azuma; R. Richard Pencek; Carol Kelley; Julie C. Price; Claudio Cobelli; David E. KelleyCorresponding author: Bret H. Goodpaster, <email@example.com>.\ndate: 2014-03\nreferences:\ntitle: Interactions Among Glucose Delivery, Transport, and Phosphorylation That Underlie Skeletal Muscle Insulin Resistance in Obesity and Type 2 Diabetes: Studies With Dynamic PET Imaging\n\n# Introduction\n\nThe rate of glucose uptake into skeletal muscle during insulin-stimulated conditions is a strong determinant of systemic insulin sensitivity (and insulin resistance \\[IR\\]). Much of the control of glucose uptake into skeletal muscle is located at the steps of glucose delivery, transport, and phosphorylation (1\u20134). Among these loci of metabolic control, stimulation of glucose transport mediated by translocation of GLUT4 in response to insulin is crucial (5\u20137). The small accumulation of glucose within insulin-stimulated muscle has provided supporting evidence that defects in glucose transport are primarily responsible for skeletal muscle IR (4,8\u201310). The small intracellular accumulation of glucose, however, could also be consistent with defects in glucose delivery (11). Studies using positron emission tomography (PET) have revealed that most of the insulin-stimulated control of glucose uptake in lean, nondiabetic human skeletal muscle is nearly equally distributed between glucose delivery and transport (12). The relative contribution of glucose delivery and transport within skeletal muscle in obesity and type 2 diabetes (T2D) remains uncertain.\n\nDefects in glucose phosphorylation in response to insulin can also potentially contribute to IR. Animal models suggest that glucose phosphorylation capacity may limit glucose uptake when the capacity for glucose transport is high (13,14), although genetic manipulation of the glucose phosphorylation capacity by hexokinase overexpression appears to be less influential during insulin stimulation (1,14). In dynamic PET imaging studies quantifying rates of glucose delivery, transport, and phosphorylation using \\[^15^O\\]H~2~O, \\[^11^C\\]3-O-methylglucose (3-OMG), and \\[^18^F\\]fluorodeoxyglucose (FDG), respectively, Bertoldo et al. (12) reported that glucose phosphorylation represented an additional locus of control governing insulin-stimulated rates of glucose uptake in skeletal muscle of lean, healthy subjects. It is not clear, however, the degree to which skeletal muscle IR in obesity or T2D can be attributed to the control of glucose transport and phosphorylation.\n\nThe purpose of the current clinical investigation was to use dynamic in vivo PET imaging to dissect out potential loci of skeletal muscle IR in obesity and T2D. We used the triple-tracer method developed by Bertoldo et al. (12) to quantify rates of glucose delivery, transport, and phosphorylation during fasting (basal) and insulin-stimulated conditions. The present study provides novel in vivo quantitative information regarding the contribution and interaction of these proximal steps to skeletal muscle IR.\n\n# Research Design and Methods\n\n## Research Volunteers\n\nInformed, written consent was obtained from all participants, and the protocol was approved by the University of Pittsburgh Institutional Review Board. Three groups of research volunteers were recruited by advertisement: normal weight, nondiabetic (NW); overweight or obese nondiabetic (OB); and patients with T2D. NW is considered the control group in this study. The clinical characteristics are shown in <a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>; groups were well matched for age and sex distribution, and OB and T2D groups were closely matched for BMI. Blood lipoprotein levels were similar among groups, although plasma triglycerides tended to be higher in T2D. All participants had a medical examination to verify good health. Participants with T2D were treated with lifestyle measures alone or with metformin, which was withheld 3 days before the metabolic studies.\n\nClinical characteristics\n\n![](1058tbl1)\n\n## Metabolic Studies\n\nAll participants underwent two sessions of triple-tracer PET imaging: one during fasting conditions (basal) and another, in random order, during steady-state insulin-stimulated conditions (insulin), as described below. On the evening before a study, research volunteers were admitted to the University of Pittsburgh Clinical Translational Research Center and fasted overnight. In the morning, catheters were placed in an antecubital vein for infusions and in a radial artery for blood sampling. During basal studies, volunteers did not receive insulin or dextrose infusions. During insulin studies, insulin was infused for 6 h at 40 mU \u22c5 m^\u22122^ \u22c5 min^\u22121^, arterial glucose was measured at 5-min intervals, and euglycemia was maintained by an adjustable infusion of 20% dextrose. Arterial glucose was measured with a YSI glucose analyzer (Yellow Springs, OH). Plasma insulin was measured by radioimmunoassay, and plasma fatty acids were measured by colorimetric enzymatic assay (Wako NEFA C test kit; Wako Chemicals, Richmond, VA).\n\n## PET and Magnetic Resonance Imaging\n\nPET and magnetic resonance imaging (MRI) was performed at the University of Pittsburgh PET Center and the Magnetic Resonance Research Center using procedures previously described (12). Briefly, a Siemens\/CTI ECAT HR+ PET scanner was used in the three-dimensional imaging mode (63 parallel planes; axial field-of-view = 15.2 cm, slice width = 2.4 mm), and the final reconstructed PET image resolution was \u223c6 mm. Participants were positioned in the PET scanner with the midcalf area in the center of the field.\n\n### \\[^15^O\\]H~2~O Imaging\n\n\\[^15^O\\]H~2~O was given as an \u223c30-mCi bolus, and arterial blood sampling was obtained with a Siemens liquid activity monitor. PET scanning began with the injection and lasted 3 min (18 frames of 10 s duration each). PET data were corrected for radioactive decay and scatter (15). During insulin studies, the insulin infusion was given for at least 2 h before PET imaging.\n\n### \\[^11^C\\]3-OMG Imaging\n\n\\[^11^C\\]3-OMG was administered as a slow bolus of \u223c5 mCi \u223c30 min after completing PET imaging of \\[^15^O\\]H~2~O, and dynamic PET scan was started (90 min; 36 frames; 8 of 15 s duration, 8 of 30 s duration, 4 of 1 min duration, and 16 of 5 min duration). Arterial \\[^11^C\\]3-OMG was measured in 0.5-mL samples obtained manually (10 samples every 6 s, 8 samples every 15 s, 7 samples every 1 min, 10 samples every 5 min, and 3 samples every 10 min). Blood samples were immediately centrifuged, and 200 \u03bcL of plasma was removed for immediate \\[^11^C\\] counting with a COBRA Auto-Gamma model 5003 gamma counter (Packard Instruments, Meriden, CT).\n\n### \\[^18^F\\]FDG Imaging\n\nTwo hours after injection of \\[^11^C\\]3-OMG, \u223c6 mCi \\[^18^F\\]FDG was given as a slow intravenous bolus, and dynamic PET scanning was started (90 min; 28 frames; 8 of 30 s duration, 8 of 60 s duration, 8 of 4 min duration, and 4 of 8 min duration). Arterial blood draws were made for determining \\[^18^F\\]FDG activity (10 \u00d7 6 s, 8 \u00d7 15 s, 7 \u00d7 1 min, 10 \u00d7 5 min, and 3 \u00d7 10 min). Blood was immediately centrifuged, and 100 \u03bcL of plasma was used for \\[^18^F\\] counting (\\>350 KeV).\n\n### MRI of Skeletal Muscle\n\nA T1-weighted MRI of the midcalf was obtained on the same day as the PET imaging session and consisted of a series of T1-weighted images (acquisition parameters: axial plane, repetition time = 31 ms, echo time = 15 ms, flip angle = 10\u00b0, field of view = 40 \u00d7 20 cm, matrix size = 256 \u00d7 192 \u00d7 120, partition thickness = 1.5 mm, contiguous partitions, 1 average, total acquisition time = 12.75 min) acquired for high-resolution anatomy. The MRIs were aligned to PET images by a previously described method (16,17). Briefly, summation PET images were created from the frames of the initial 15 min of the scanning period and coregistered with MRI so that precise anatomical alignment was achieved.\n\n## Measuring Tissue Activity in PET Images\n\nA region of interest (ROI) was placed over soleus muscles on the summation PET images and MRIs, and these ROIs were applied to each frame (18 for \\[^15^O\\]H~2~O, 36 for \\[^11^C\\]3-OMG, and 28 for \\[^18^F\\]FDG) across 58 of the 68 planes of each leg, omitting the proximal and distal 5 planes to reduce the influence of scatter. Tracer activity within an ROI was converted to radioactivity concentration (\u00b5Ci\/mL) using an empiric phantom-based calibration factor (\u00b5Ci\/mL\/PET counts per pixel).\n\n## Compartmental Modeling\n\n### \\[^15^O\\]H~2~O\n\nThe model of Kety (18) was used to estimate tissue perfusion of skeletal muscle.\n\n### \\[^11^C\\]3-OMG\n\nCompartmental modeling of \\[^11^C\\]3-OMG kinetics was performed, as previously described in detail (19,20). Briefly, this model has three compartments (plasma and two tissue pools) with four rate constants as shown in <a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>A<\/em><\/a>. The rate constants (mL\/mL\/min) and (min^\u22121^) describe a reversible exchange between plasma and the first tissue compartment and are ascribed physiologically to an exchange of \\[^11^C\\]3-OMG between the capillary and the muscle interstitial space. The rate constants (min^\u22121^) and (min^\u22121^) describe a reversible exchange between the first and second tissue compartments and are ascribed physiologically to bidirectional transmembrane glucose transport. From these rate constants, an overall partition coefficient V~d~ for uptake of \\[^11^C\\]3-OMG from plasma to tissue can be calculated and is divisible into a partition coefficient for the first compartment, muscle interstitial space V~ic~, and a partition coefficient for the tissue compartment V~ec~, as shown in <a href=\"#equ1\" data-ref-type=\"disp-formula\">Eq. 1<\/a>:\n\n![](1058equ1.jpg)\n\n### \\[^18^F\\]FDG\n\nCompartmental modeling of \\[^18^F\\]FDG kinetics was performed as previously described (21\u201323). Briefly, a model of three compartments with five rate constants was used that is similar to that described for \\[^11^C\\]3-OMG but with an additional tissue compartment and rate constant to represent phosphorylation of \\[^18^F\\]FDG. In this article, to avoid confusion with the rate constants used for \\[^11^C\\]3-OMG, those pertaining to \\[^18^F\\]FDG are designated with an asterisk (k~n~\\*). Thus, and describe the delivery of \\[^18^F\\]-FDG from the capillary to the muscle interstitial space and reserve process, respectively; and describe inward and outward transport of \\[^18^F\\]FDG; and describes the phosphorylation of \\[^18^F\\]FDG. From the five rate constants, the fractional uptake of \\[^18^F\\]FDG can be determined, as represented by the macroparameter K (mL\/mL\/min) shown in <a href=\"#equ2\" data-ref-type=\"disp-formula\">Eq. 2<\/a>:\n\n![](1058equ2.jpg)\n\n## Glucose\n\nA specific glucose tracer was not used in this study; instead, a glucose model was developed from the kinetics determined using \\[^11^C\\]3-OMG and \\[^18^F\\]FDG, as previously described (12,21\u201325). Rate constants from compartmental modeling of \\[^11^C\\]3-OMG were used for that of glucose, and a value for the rate constant of glucose phosphorylation was additionally derived from the lumped constant (LC), as shown in <a href=\"#equ3\" data-ref-type=\"disp-formula\">Eq. 3<\/a>:\n\n![](1058equ3.jpg)\n\nFrom glucose k~1~\u2013k~5~ parameters, intracellular glucose concentration (Ce) was estimated, using <a href=\"#equ4\" data-ref-type=\"disp-formula\">Eq. 4<\/a> (16):\n\n![](1058equ4.jpg)\n\nFollowing the theory of metabolic control analysis, the control coefficients (CC) of delivery, transmembrane transport, and phosphorylation were calculated as shown in <a href=\"#equ5\" data-ref-type=\"disp-formula\">Eq. 5<\/a> (17,26,27):\n\n![](1058equ5.jpg)\n\nThe higher the value of a control coefficient, the more that this process is rate limiting and according a locus of control.\n\n## Statistics\n\nData are presented as mean \u00b1 SEM. One-way ANOVA was used to examine for significance of differences among subject groups. One-way repeated-measures ANOVA was used to compare basal and insulin group parameters. A *P* \\< 0.05 was considered significant.\n\n# Results\n\n## Systemic and Muscle Insulin Sensitivity\n\nThe clinical characteristics of the research subjects are presented in <a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>, and the rates of exogenous glucose infusion during insulin infusion clamp studies are shown in <a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>A<\/em><\/a>. Compared with NW, both OB and T2D manifested systemic IR, which was most severe in T2D (*P* \\< 0.001). Fasting glucose values were significantly different among T2D (144.0 \u00b1 13.0 mg\/dL), NW (96.5 \u00b1 1.8 mg\/dL), and OB (94.4 \u00b1 1.5 mg\/dL) (*P* \\< 0.05). Insulin levels were not significantly different among the three groups at fasting (NW 9.81 \u00b1 0.94, OB 18.01 \u00b1 3.54, T2D 18.39 \u00b1 1.80 \u00b5U\/mL) or insulin stimulation (NW 87.95 \u00b1 2.18, OB 102.30 \u00b1 8.89, T2D 102.52 \u00b1 6.02 \u00b5U\/mL). Free fatty acids were also not significantly different between the groups at fasting (NW 592 \u00b1 65, OB 577 \u00b1 30, T2D 532 \u00b1 63 \u00b5mol\/L) or insulin stimulation (NW 106 \u00b1 33, OB 127 \u00b1 31, T2D 167 \u00b1 35 \u00b5U\/mL). Skeletal muscle (soleus) insulin sensitivity, as measured by K, the macroscopic PET imaging parameter for \\[^18^F\\]FDG uptake, was strongly correlated with respective glucose infusion rates as shown in <a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>B<\/em><\/a>.\n\n## Tissue Perfusion (\\[^15^O\\]H~2~O)\n\nTissue perfusion in soleus muscle was lower (*P* \\< 0.01) in OB (0.0194 \u00b1 0.0055 mL\/min-mL) and T2D (0.0125 \u00b1 0.0022 mL\/min-mL) than in NW (0.0316 \u00b1 0.0139 mL\/min-mL) during basal conditions. During insulin infusion, muscle tissue perfusion did not change significantly from basal conditions in any group. The k~1~ parameter obtained from \\[^11^C\\]3-OMG modeling, which describes tracer delivery from plasma to tissue, has values that are concordant with those obtained using \\[^15^O\\]H~2~O, a tracer that is used to measure solely tissue perfusion kinetics. This concordance of k~1~ values for these two tracers is consistent with earlier findings (12,20,28).\n\n## Tissue Uptake of \\[^11^C\\]3-OMG\n\nThe mean group soleus muscle tissue time\u2013activity curves for \\[^11^C\\]3-OMG are shown in <a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<em>A<\/em> and <em>B<\/em><\/a> for basal and insulin-stimulated conditions, respectively. Using these data in conjunction with arterial plasma time\u2013activity data, tissue data were modeled to estimate parameters describing both delivery (plasma to extracellular space) and bidirectional glucose transport for \\[^11^C\\]3-OMG, and these parameters of the tracer kinetics that underlie the shapes of the tissue\u2013activity curves are shown in <a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a>. The kinetic parameter of great interest for this investigation of muscle IR is k~3~, which described the inward transport for \\[^11^C\\]3-OMG, a nonmetabolized glucose analog that has nearly identical affinity as glucose for glucose transporters (25). Across groups, the basal values for k~3~ were similar (<a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<\/a>). During insulin infusion, k~3~ increased sevenfold in NW participants, which is similar to earlier findings in lean nondiabetic subjects (12,19). In the OB subjects, the group mean value for k~3~ increased sixfold over the basal condition, which is a similar response to that found in NW. However, in T2D, the group mean value for k~3~ during insulin-stimulated conditions was significantly lower than in NW or OB (*P* \\< 0.01), and although the value increased during the insulin condition compared with the basal condition, the change was a twofold increase, which was a blunted response to insulin compared with the changes observed in the NW and OB nondiabetic groups. Because the transport of \\[^11^C\\]3-OMG is bidirectional, the higher tissue activities and k~3~ values in the NW and OB groups were associated with higher k~4~ values (and greater changes in k~4~ during insulin compared with basal conditions). As well, the higher efficiency of inward glucose transport during insulin-stimulated conditions was associated with reduced values for k~2~, the parameter describing efflux from tissue (extracellular space) to plasma. In T2D, the insulin-stimulated versus basal changes in k~2~ and k~4~ were blunted compared with NW and OB. The insulin-resistant response of glucose transport in T2D is also evident within the volume of distribution parameters for \\[^11^C\\]3-OMG (<a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a>), which reveals that although there was evidence of soleus response to insulin in T2D compared with basal conditions, the amplitude of this response was attenuated in T2D, indicating that impaired insulin stimulation of glucose transport contributes strongly to muscle IR.\n\nKinetic parameters for \\[^11^C\\]3-OMG\n\n![](1058tbl2)\n\n## Tissue Uptake of \\[^18^F\\]FDG\n\nThe key difference between \\[^11^C\\]3-OMG and \\[^18^F\\]FDG is that the latter glucose analog can be a substrate for hexokinase and undergo phosphorylation, which over the short time course of these PET imaging studies causes \\[^18^F\\]FDG-6-P to be formed and irreversibly trapped within soleus muscle. The soleus muscle time\u2013tissue activity curves for \\[^18^F\\]FDG during basal and insulin-stimulated conditions are shown in <a href=\"#F4\" data-ref-type=\"fig\">Fig. 4<em>A<\/em> and <em>B<\/em><\/a>. During basal conditions, there was close similarities across groups, but during insulin-stimulated conditions, marked group differences became evident with sharply diminished tissue activity in T2D and a discernibly altered shape. For the goals of this investigation, to study the potential contribution of glucose phosphorylation to IR, the arterial and tissue activity for \\[^18^F\\]FDG were used to determine K, the macroscopic parameter of irreversible tracer uptake. As described in the <span class=\"smallcaps\">research design and methods<\/span>, K was used in conjunction with kinetic parameters for \\[^11^C\\]3-OMG and with the lumped constant to estimate the kinetics of actual glucose delivery, bidirectional transport, and phosphorylation. The kinetic parameters for \\[^18^F\\]FDG are presented in [Supplementary Table 1](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db13-1249\/-\/DC1). These data will not be further discussed here other than to note that the findings across groups and between basal and insulin studies are similar to those for \\[^11^C\\]3-OMG, with differences reflecting known analog effects for \\[^18^F\\]FDG (as reflected in the lumped constant) and with the addition of k~5~, the kinetic parameter ascribed to the formation of \\[^18^F\\]FDG-6-P (<a href=\"#F5\" data-ref-type=\"fig\">Fig. 5<\/a>).\n\nThe partition coefficient V~ec~ for glucose in the tissue extracellular compartment relative to plasma did not change from the basal to the insulin conditions. The partition coefficient for glucose in the tissue intracellular compartment relative to plasma increased during the insulin condition (*P* \\< 0.05) in NW and OB but not in T2D. was then used to calculate the intracellular glucose values when multiplied by the plasma glucose levels. Intracellular glucose levels were not significantly different at basal conditions, but under insulin stimulation, NW had significantly higher intracellular glucose levels than T2D (NW 33.7 \u00b1 4.8, OB 31.3 \u00b1 12.5, T2D 10.1 \u00b1 2.0 mg\/dL; *P* \\< 0.05).\n\n## Control Coefficient for Glucose Skeletal Muscle Uptake\n\nThe rate constants describing glucose kinetics were used to calculate the control coefficient for delivery (CC~delivery~), transport (CC~transport~), and phosphorylation (CC~phosphor~) during the basal and insulin conditions are presented in <a href=\"#F6\" data-ref-type=\"fig\">Fig. 6<\/a>. Glucose transport and phosphorylation (hexokinase) largely account for control of glucose kinetics in skeletal muscle during basal conditions. Of these, transport exerted stronger control than phosphorylation. Thus, glucose delivery was not found to substantially control glucose uptake into skeletal muscle during basal conditions. This pattern was consistent across all groups. For NW subjects, this pattern of distribution of control shifted during insulin conditions such that glucose delivery and transport contributed nearly equally to the control of glucose uptake and together accounted for 90% of control, whereas phosphorylation was calculated to manifest only the remaining 10% of rate control for the kinetics of glucose uptake. This was in contrast to that observed for T2D in which insulin-stimulated glucose uptake was predominantly, but not exclusively controlled, that is, limited by glucose transport. Thus, the distribution of insulin-mediated glucose uptake was vastly different in T2D muscle.\n\n# Discussion\n\nTo our knowledge, this study is the first to use three tracers (\\[^15^O\\]H~2~O, \\[^11^C\\]3-OMG, and \\[^18^F\\]FDG) during dynamic PET imaging to ascertain separate and specific defects in glucose metabolism in obesity and T2D. The primary findings were that lower insulin-stimulated glucose transport accounts for a quantifiable and substantial degree of IR in skeletal muscle of T2D and that additional defects in glucose phosphorylation characterize the IR of T2D.\n\nFollowing an overnight fast, neither glucose transport nor phosphorylation was different in the skeletal muscle of OB or T2D compared with lean, healthy muscle. The predominant control of glucose uptake in the fasting state was at the level of glucose transport across all study groups, with some additional control posited by glucose phosphorylation. Glucose delivery exerted only negligible control, although this was quantitatively lower in muscle in T2D. The lower basal muscle blood flow in diabetes could perhaps reflect a lower capillary density or perfusion (29). These observations are consistent with other studies showing that basal glucose uptake is not impaired in IR and diabetes (30) and that glucose transport is the rate-limiting step in basal glucose uptake (30).\n\nThe glucose infusion rates in response to physiological levels of insulin were lower for OB subjects, and this systemic IR was more severe in T2D, which was strongly associated with the macroscopic index of muscle glucose flux assessed by PET. The lower rates of glucose transport and fractional phosphorylation in OB than in NW, although not as markedly low as in T2D, clearly indicate that skeletal muscle IR in OB, along with normal fasting glycemia, helps to define the prediabetic state. This is consistent with values reported in earlier studies in normal weight subjects using similar protocols (12,19). However, despite the nearly 50% lower systemic glucose infusion rates in OB subjects, their lower insulin-stimulated increase in glucose transport was not statistically different, suggesting that obesity alone has less dramatic effects of inward glucose transport despite fairly marked systemic IR. The small number of subjects could have limited our ability to quantify the obesity-related defects in glucose transport, particularly given the heterogeneity of insulin sensitivity in obesity. Moreover, the systemic insulin sensitivity could have been confounded by hepatic IR, which again, highlights the utility of PET to quantify skeletal muscle glucose metabolism. We also note that a lean, T2D group was not included, which could have further quantified the effects of both obesity and T2D. Additionally, we recognize that even though our sex ratios were similar, there could be sex-specific differences in metabolic disease that may have influenced the findings. In stark contrast, skeletal muscle in T2D had much lower fractional glucose transport rates and intracellular glucose levels. These results are consistent with lower muscle GLUT4 content and translocation in T2D (30\u201333) and with the slight accumulation of free glucose in response to insulin (4,8,10), all of which have been interpreted as defects in glucose transport and comprising a major limitation to glucose uptake in response to insulin. Although bidirectional glucose transport within muscle can be assessed directly in vitro, the in vivo glucose transport also depends on delivery to the tissue. The time\u2013tissue activity curves for 3-OMG reflect an integration of these two reversible processes (12,20,28), which the current study distinguished using sequential imaging with \\[^15^O\\]H~2~O and \\[^11^C\\]3-OMG. Insulin-stimulated rates of muscle tissue perfusion were similar across subject groups and are similar to those reported previously for normal weight subjects (34). These rates corresponded very well with kinetic values for tissue perfusion attributed to k~1~ ascertained for \\[^11^C\\]3-OMG and \\[^18^F\\]FDG. Rates of tissue perfusion did not change significantly in response to insulin compared with fasting. This does not discount the hemodynamic effects of insulin to stimulate capillary recruitment, as has been demonstrated (35,36), which is beyond the spatial resolution of PET. Improving spatial resolution with PET may provide additional insight into the dynamic interaction between glucose delivery and transport in IR. NW subjects in the current study had a redistribution of control of glucose uptake from predominantly glucose transport in the fasting state to greater control by delivery in response to insulin, which was not observed for muscle in OB or T2D subjects. Thus, the greater control of glucose uptake by insulin in muscle of NW subjects is consistent with in vivo studies in rodents, indicating that insulin-stimulated glucose uptake is constrained by delivery (11,14,37). This is also in accord with a major defect in insulin-stimulated glucose transport in diabetic muscle in the current study.\n\nThe insulin-stimulated fractional phosphorylation of glucose was significantly lower in muscle in diabetes. Thus, after accounting for glucose delivery and lower glucose transport in diabetes ascertained specifically with \\[^11^C\\]3-OMG, an additional defect in glucose phosphorylation exists in insulin-resistant diabetic muscle. This defect was quite pronounced in diabetic muscle and was not as obvious in muscle of OB subjects. This finding could reflect the heterogeneity or wider range in the severity of IR of obesity and suggests that obesity per se does not unequivocally cause defects in skeletal muscle glucose metabolism but, nevertheless, contributes to the underlying progression of IR eventually causing T2D. Dynamic PET imaging performed using \\[^18^F\\]FDG indicated that glucose phosphorylation does not exert strong rate control on glucose uptake during insulin-stimulated conditions in muscle of NW subjects but still distinguishes skeletal muscle IR in T2D. This is similar to a previous article that used dynamic PET studies modeling \\[^18^F\\]FDG uptake kinetics to observe a major defect in glucose transmembrane transport with an additional, albeit smaller, defect in glucose phosphorylation (23). The current study extends these findings in two very important ways. First, the use of multiple PET tracers in the same subject has allowed us to separate and quantify the proximal steps of glucose transport and phosphorylation with considerably less dependence on mathematical modeling of kinetic data. Second, we have now more clearly shown for the first time using these multiple PET tracers that glucose phosphorylation is impaired in skeletal muscle in T2D after accounting for defects in glucose transport. This is consistent with reports in animal models demonstrating additional defects in insulin-stimulated glucose phosphorylation (1,11,13,14,38\u201340). Both GLUT4 and hexokinase are coordinately regulated (41), and both track strongly with oxidative capacity and capillary density in muscle, all of which have been consistently demonstrated to be affected in T2D (30,31,33,42).\n\nIn summary, dynamic PET imaging of skeletal muscle glucose metabolism reveals a major defect in glucose transport, which defines much of the severity of IR of T2D, and that an additional impairment within the efficiency of glucose phosphorylation is present. In contrast to lean, healthy skeletal muscle in which the control of insulin-stimulated glucose uptake is more broadly distributed across each of the proximal steps of glucose delivery, transport, and phosphorylation, the rate-limiting control of glucose metabolism in muscle in T2D is posited mostly at glucose transport, with smaller control being manifest at glucose delivery and phosphorylation. These triple-tracer PET imaging data have important implications for clinical treatment of T2D, and these methodologies could be valuable in determining whether these defects persist at higher doses of insulin and which specific control points may be rectified with insulin-sensitizing interventions.\n\n# Supplementary Material\n\n###### Supplementary Data\n\n## Article Information\n\n**Acknowledgments.** The authors acknowledge the efforts and cooperation of the research volunteers and the support from the staffs of the University of Pittsburgh General Clinical Research Center and the PET Center.\n\n**Funding.** These studies were supported by the National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases (NIH-NIDDK) (DK-60555-02), by the University of Pittsburgh General Clinical Research Center (5MO1-RR-00056), and by the Obesity and Nutrition Research Center (NIH-NIDDK, P30-DK-46204). This work was also supported by NIH grant EB-01975.\n\n**Duality of Interest.** R.R.P. is currently employed by Amylin Pharmaceuticals. D.E.K. is currently employed by Merck Sharp & Dohme Corp. No other potential conflicts of interest relevant to this article were reported.\n\n**Author Contributions.** B.H.G. supervised the project and wrote the manuscript. A.B. performed the modeling analysis. J.M.N. researched the data, contributed to the discussion, and reviewed and edited the manuscript. K.A., R.R.P., and C.K. researched the data. J.C.P. and C.C. contributed to the modeling analysis. D.E.K. contributed to the discussion and reviewed and edited the manuscript. B.H.G. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.\n\n# References","meta":{"dup_signals":{"dup_doc_count":144,"dup_dump_count":48,"dup_details":{"curated_sources":2,"2019-51":1,"2019-47":1,"2019-30":1,"2018-39":1,"2018-30":1,"2018-26":1,"2018-22":1,"2018-17":1,"2018-13":2,"2018-09":3,"2018-05":2,"2017-47":5,"2017-43":6,"2017-39":3,"2017-34":2,"2017-30":9,"2017-26":2,"2017-22":3,"2017-17":11,"2017-09":4,"2017-04":12,"2016-50":4,"2016-44":4,"2016-40":4,"2016-36":4,"2016-30":3,"2016-26":4,"2016-22":4,"2016-18":3,"2016-07":3,"2015-48":3,"2015-40":1,"2015-35":2,"2015-32":1,"2015-27":1,"2015-22":2,"2015-14":1,"2014-52":3,"2014-49":2,"2014-42":2,"2014-41":4,"2014-35":1,"2023-06":1,"2017-13":4,"2015-18":3,"2015-11":2,"2015-06":3,"2024-10":1}},"file":"PMC3931396"},"subset":"pubmed_central"}
{"text":"abstract: **Objective** To examine how change in level of physical activity after middle age influences mortality and to compare it with the effect of smoking cessation.\n  .\n  **Design** Population based cohort study with follow-up over 35 years.\n  .\n  **Setting** Municipality of Uppsala, Sweden.\n  .\n  **Participants** 2205 men aged 50 in 1970-3 who were re-examined at ages 60, 70, 77, and 82 years.\n  .\n  **Main outcome measure** Total (all cause) mortality.\n  .\n  **Results** The absolute mortality rate was 27.1, 23.6, and 18.4 per 1000 person years in the groups with low, medium, and high physical activity, respectively. The relative rate reduction attributable to high physical activity was 32% for low and 22% for medium physical activity. Men who increased their physical activity level between the ages of 50 and 60 continued to have a higher mortality rate during the first five years of follow-up (adjusted hazard ratio 2.64, 95% confidence interval 1.32 to 5.27, compared with unchanged high physical activity). After 10 years of follow-up their increased physical activity was associated with reduced mortality to the level of men with unchanged high physical activity (1.10, 0.87 to 1.38). The reduction in mortality associated with increased physical activity (0.51, 0.26 to 0.97, compared with unchanged low physical activity) was similar to that associated with smoking cessation (0.64, 0.53 to 0.78, compared with continued smoking).\n  .\n  **Conclusions** Increased physical activity in middle age is eventually followed by a reduction in mortality to the same level as seen among men with constantly high physical activity. This reduction is comparable with that associated with smoking cessation.\nauthor: Liisa Byberg; H\u00e5kan Melhus; Rolf Gedeborg; Johan Sundstr\u00f6m; Anders Ahlbom; Bj\u00f6rn Zethelius; Lars G Berglund; Alicja Wolk; Karl Micha\u00eblssonCorrespondence to: L Byberg <email@example.com>\ndate: 2009\nreferences:\ntitle: Total mortality after changes in leisure time physical activity in 50 year old men: 35 year follow-up of population based cohort\n\n# Introduction\n\nDespite the known hazards of physical inactivity, it continues to be a major health problem.1 Physical inactivity is associated with increased incidence rates of obesity, diabetes, cardiovascular diseases, osteoporosis, and cancer.1 2 3 4 Short term randomised controlled trials in young to middle aged adults have shown a healthier risk profile with exercise.4 5 It is therefore recommended that adults engage in at least 30 minutes of moderate physical activity preferably on all days of the week.5 6 7 Adherence to these guidelines is associated with half the risk of mortality seen in sedentary people.8 Nevertheless, about half of all middle aged men in the West do not take part in regular physical activity.1 2 Whereas being physically inactive in younger years seems detrimental, we do not know whether an increase in exercise level later in life reduces mortality rates. If the impact on mortality could be compared with the effects of other changes in lifestyle habits it would be easier to communicate this potential health benefit.\n\nIn prospective studies that have examined changes in leisure time physical activity level in relation to mortality,9 10 11 12 13 14 15 few have considered the possibility of additional changes in physical activity behaviours during follow-up.13 14 15 Furthermore, the impact on mortality in relation to age at the time of the activity changes and the time elapsed since the changes occurred have not been established.\n\nWe investigated the impact of physical activity on total (all cause) mortality from middle to old age, taking into account changes in activity during follow-up. We studied this in a population based cohort of men aged 50 at baseline who were followed up for more than 35 years and investigated on five occasions. We also compared the effect of an increased level of physical activity on the mortality rate with that achieved by cessation of smoking.\n\n# Methods\n\nThe present investigation used data from the population based Uppsala longitudinal study of adult men (ULSAM, [www.pubcare.uu.se\/ULSAM](www.pubcare.uu.se\/ULSAM)). Figure 1 shows the study population <a href=\"#fig1\" data-ref-type=\"fig\"><\/a>. In 1970 all 2841 men born in 1920-4 and living in the municipality of Uppsala, Sweden, were invited to attend a health survey (survey 1).16 Of those invited, 2322 men (82%) aged 49-51 participated. Participants in survey 1 were invited for reinvestigations at ages 60, 70, and 77 (surveys 2-4). Participants in surveys 3 and 4 were invited for reinvestigation at age 82 (survey 5).\n\nWe restricted our study population to the 2205 men who had responded to the questions regarding leisure time physical activity in the self administered questionnaire in survey 1. Identical questions were asked at each survey:\n\n1.  1 Do you spend most of your time reading, watching TV, going to the cinema, or engaging in other, mostly sedentary activities?\n\n2.  2 Do you often go walking or cycling for pleasure?\n\n3.  3 Do you engage in any active recreational sports or heavy gardening at least 3 hours every week?\n\n4.  4 Do you regularly engage in hard physical training or competitive sport?\n\nWe considered the two highest activity categories, questions 3 and 4, together as only 5% of the participants reported hard physical training. Participants answering yes to question 1 were categorised as low activity; 2 as medium; and 3 and 4 as high. The questionnaire has been used and validated in other studies and has also been shown to have a high reliability over time.17 18 19 20\n\nChanges in the physical activity level between the first and second survey were considered in four categories: unchanged low (low or medium activity at both surveys), unchanged high (high activity at both surveys), decreased (high in survey 1, low or medium in survey 2), and increased (low or medium in survey 1, high in survey 2).\n\nBased on job titles retrieved from the national censuses from 1970 and 1980, we categorised occupational physical activity as sedentary, light, medium, high, unknown, and none (unemployed).21\n\nHeight and weight were measured at each survey and body mass index (BMI, weight (kg)\/height (m)^2^) calculated. Obesity was defined as BMI \u226530. Systolic and diastolic blood pressure in the supine position was measured by standard procedures. Information on antihypertensive medication was retrieved by questionnaire, as was information on smoking habits. Smoking status was categorised as current smoker, former smoker, or never smoker. Changes in smoking status between two surveys were categorised as continued smoking, smoking cessation, and never smoking. Total fasting serum cholesterol concentration was measured with enzymatic techniques. The presence or absence of diabetes mellitus was determined at each survey according to the guidelines current at that time. Information on perceived health (classified as not good or good) and alcohol use at surveys 2 and 4 (classified according to the Michigan alcoholism screening test as abstainer, normal, suspected alcohol dependence22) was retrieved from the questionnaires. The census from 1970 provided information on educational level. Socioeconomic group at survey 1 was interview based and categorised as low, medium, and high.23 By linkage to the national hospital discharge register, we collected information on any musculoskeletal, neurological, or psychiatric disorder. The prevalence of each of these disorders and of diabetes was \\<1.2% at baseline.\n\nUsing the individual 10 digit personal identification number given to all Swedish citizens,24 we traced the entire cohort in the continuously updated Swedish National Population Register until the date of death or 31 December 2006. Our main outcome was total (all cause) mortality. There was no loss to follow-up.\n\n## Statistical analyses\n\nWe used Cox's proportional hazards regression with delayed entry to estimate hazard ratios with 95% confidence intervals and survival curves for the association between physical activity and mortality. The values of physical activity, as well as covariate information, were updated by data from each subsequent visit. The last observed value carried forward technique was used for missing data. The non-response frequencies for physical activity were 130, 89, 68, and 0 at surveys 2-5, respectively. Table 1 shows details of the sensitivity analyses.<a href=\"#tbl1\" data-ref-type=\"table\"><\/a>\n\n\u2002Sensitivity analysis, showing hazard ratios and 95% confidence intervals for each model\n\n| Physical activity category | Model 1 | Model 2 |\n|----|----|----|\n| **Last observed value carried forward\\*** |  |  |\n| High | 1.00\u2020 | 1.00\u2020 |\n| Medium | 1.32 (1.18 to 1.49) | 1.24 (1.10 to 1.39) |\n| Low | 1.75 (1.48 to 2.06) | 1.41 (1.19 to 1.67) |\n| **Lowest possible level\u2021** |  |  |\n| High | 1.00\u2020 | 1.00\u2020 |\n| Medium | 1.31 (1.16 to 1.48) | 1.22 (1.08 to 1.38) |\n| Low | 1.73 (1.50 to 2.00) | 1.50 (1.29 to 1.74) |\n| **Highest possible level\u2021** |  |  |\n| High | 1.00\u2020 | 1.00\u2020 |\n| Medium | 1.22 (1.09 to 1.37) | 1.14 (1.02 to 1.29) |\n| Low | 1.71 (1.45 to 2.02) | 1.36 (1.15 to 1.62) |\n| **Excluding men with missing values\u00a7** |  |  |\n| High | 1.00\u2020 | 1.00\u2020 |\n| Medium | 1.29 (1.14 to 1.46) | 1.20 (1.06 to 1.36) |\n| Low | 1.87 (1.57 to 2.23) | 1.44 (1.20 to 1.72) |\n| **Excluding men with missing survey participation\u00b6** |  |  |\n| High | 1.00\u2020 | 1.00\u2020 |\n| Medium | 1.59 (1.32 to 1.92) | 1.43 (1.18 to 1.74) |\n| Low | 2.04 (1.56 to 2.65) | 1.66 (1.27 to 2.17) |\n\nModel 1 includes physical activity as exposure. Model 2 includes physical activity, smoking status, obesity status, height and weight (both continuous); self perceived health (not good, good); physical activity at work (sedentary, light, medium, high, unknown, unemployed); diabetes mellitus, and any musculoskeletal, neurological, or psychiatric disorders (yes, no for each disorder); and alcohol use (abstainer, normal, suspected alcohol dependence).\n\n\\* Missing values imputed by last observed value carried forward method.\n\n\u2020Reference category.\n\n\u2021Additional error was introduced to imputed values by assigning them lowest or highest possible level.\n\n\u00a7Excludes all men with at least one missing observation; 5396 observations remained, representing 1918 participants and 1174 deaths; observation time was 52 593 person years.\n\n\u00b6Includes only men who attended all possible surveys before death; 3188 observations remained, representing 895 participants and 525 deaths; observation time was 22 814 person years.\n\nThe proportional hazards assumptions for the Cox models were confirmed formally by Shoenfeld's tests, and graphically by comparing Nelson-Aalen plots. We considered one crude and two multivariable models. The first multivariable model included smoking status (current, former, never); weight and height (both continuous); self perceived health (good, not good); physical activity at work in 1970 and 1980 (sedentary, light, medium, high, unknown; or unemployed); alcohol use at ages 60 and 77 years (abstainer, normal, suspected alcohol dependence); and presence or absence of obesity, diabetes mellitus, and any musculoskeletal, neurological, or psychiatric disorder as covariates. The second multivariable model additionally included systolic and diastolic blood pressure, antihypertensive drugs, total serum cholesterol concentration, educational level, and socioeconomic group.\n\nWe assessed the relative excess risk due to interaction, a measure of biological interaction,25 from a Cox's proportional hazards model as described previously.26 27\n\nRemaining life expectancy was calculated as the area under the survival curve using the trapezoidal rule approximation for the integral between the start of the study and the end of follow-up. We calculated differences in remaining life expectancy between exposure levels with 95% confidence intervals for the differences using the bootstrap centile method with 10\u2009000 replications.\n\nIn analyses of changes in physical activity level between the ages of 50 and 60, we partitioned follow-up time from date of examination in the second survey into five year intervals to account for non-proportional hazards.\n\nStatistical analyses were performed with Stata\/SE 10.1 (StataCorp, College Station, TX), SAS 9.1 (SAS Institute, Cary, NC, USA), and R (R Foundation for Statistical Computing, Vienna, Austria, 2007).\n\n# Results\n\nTable 2 shows baseline characteristics by level of physical activity at age 50.<a href=\"#tbl2\" data-ref-type=\"table\"><\/a> Almost half of the men reported a high level of physical activity, corresponding to at least three hours of recreational sports or heavy gardening a week, 36% reported medium activity, corresponding to walks and cycling; and 15% were sedentary. At the end of follow-up, we had 61\u2009456 person years and 1329 men (60%) had died.\n\n\u2002Characteristics at baseline (survey 1) according to physical activity at age 50. Values are means (SD) or number (percentage) of men\n\n| Variable and category | Physical activity level |  |  |\n|----|----|----|----|\n|  | Low (n=325) | Moderate (n=802) | High (n=1078) |\n| Age (years) | 49.6 (0.6) | 49.6 (0.6) | 49.7 (0.6) |\n| Weight (kg) | 78.7 (12.8) | 77.3 (11.1) | 77.8 (10.6) |\n| Height (cm) | 175.9 (6.1) | 176.0 (6.0) | 176.5 (5.8) |\n| Body mass index (kg\/m^2^) | 25.4 (3.7) | 24.9 (3.2) | 25.0 (3.1) |\n| No (%) obese\\* | 40 (12.3) | 41 (5.1) | 62 (5.8) |\n| Supine systolic blood pressure (mm Hg) | 133 (19) | 134 (18) | 132 (17) |\n| Supine diastolic blood pressure (mm Hg) | 83 (12) | 84 (11) | 83 (11) |\n| No (%) taking antihypertensive drugs | 18 (5.5) | 38 (4.7) | 37 (3.4) |\n| Total serum cholesterol (mmol\/l) | 6.95 (1.32) | 6.93 (1.33) | 6.83 (1.30) |\n| No (%) with self rated good health | 242 (74.5) | 641 (79.9) | 902 (83.7) |\n| No (%) by smoking status: |  |  |  |\n| \u2003Never smoker | 66 (20.3) | 196 (24.4) | 296 (27.5) |\n| \u2003Former smoker | 61 (18.8) | 181 (22.6) | 281 (26.1) |\n| \u2003Current smoker | 198 (60.9) | 425 (53.0) | 501 (46.5) |\n| No (%) by occupational physical activity in 1970: |  |  |  |\n| \u2003Sedentary | 33 (10.2) | 86 (10.7) | 139 (12.9) |\n| \u2003Light | 101 (31.1) | 255 (31.8) | 380 (35.3) |\n| \u2003Medium | 39 (12.0) | 100 (12.5) | 126 (11.7) |\n| \u2003High | 36 (11.1) | 86 (10.7) | 111 (10.3) |\n| \u2003Unemployed\/unknown | 116 (35.7) | 275 (34.3) | 322 (29.9) |\n\n\\*BMI \u226530 kg\/m^2^.\n\nThe absolute mortality rate was 27.1, 23.6, and 18.4 per 1000 person years for men with low, medium, and high physical activity. The relative rate reduction attributable to high physical activity was 32% compared with low activity and 22% compared with medium activity. The relative mortality rates were highest among sedentary men and lowest among the most active men (table 3, fig 2)<a href=\"#fig2\" data-ref-type=\"fig\"><\/a> <a href=\"#tbl3\" data-ref-type=\"table\"><\/a>. This trend was also apparent in each category of smoking (not shown). The estimated effect of sedentary behaviour on mortality was similar to the effect of smoking and obesity (table 3).<a href=\"#tbl3\" data-ref-type=\"table\"><\/a> Adjustment for covariates attenuated the associations.\n\n\u2002Hazard ratio (HR) and 95% confidence intervals of total mortality for physical activity level in leisure time, smoking, and obesity status using time updated and baseline data\n\n| Exposure and category | Time at risk (person years) | No of deaths | Hazard ratio (95% CI) |  |  |\n|----|----|----|----|----|----|\n|  |  |  | Model 1 | Model 2 | Model 3 |\n| **Time updated model\\*** |  |  |  |  |  |\n| Physical activity: |  |  |  |  |  |\n| \u2003High | 28 790 | 531 | 1.00\u2020 | 1.00\u2020 | 1.00\u2020 |\n| \u2003Medium | 25 371 | 600 | 1.32 (1.18 to 1.49) | 1.24 (1.10 to 1.39) | 1.22 (1.08 to 1.37) |\n| \u2003Low | 7306 | 198 | 1.75 (1.48 to 2.06) | 1.41 (1.19 to 1.67) | 1.40 (1.18 to 1.66) |\n| Smoking status: |  |  |  |  |  |\n| \u2003Never | 20 657 | 465 | 1.00\u2020 | 1.00\u2020 | 1.00\u2020 |\n| \u2003Former | 19 420 | 373 | 1.18 (1.02 to 1.36) | 1.15 (1.00 to 1.32) | 1.10 (0.96 to 1.27) |\n| \u2003Current | 21 389 | 491 | 2.09 (1.82 to 2.39) | 1.98 (1.72 to 2.28) | 1.90 (1.64 to 2.19) |\n| Obesity status: |  |  |  |  |  |\n| \u2003Non-obese | 56 620 | 1157 | 1.00\u2020 | 1.00\u2020 | 1.00\u2020 |\n| \u2003Obese | 4847 | 172 | 1.52 (1.29 to 1.78) | 1.49 (1.19 to 1.86) | 1.51 (1.21 to 1.90) |\n| **Baseline model\u2021** |  |  |  |  |  |\n| Physical activity: |  |  |  |  |  |\n| \u2003High | 30 930 | 617 | 1.00\u2020 | 1.00\u2020 | 1.00\u2020 |\n| \u2003Medium | 21 850 | 504 | 1.20 (1.07 to 1.35) | 1.16 (1.03 to 1.31) | 1.14 (1.01 to 1.28) |\n| \u2003Low | 8687 | 213 | 1.30 (1.12 to 1.52) | 1.14 (0.98 to 1.34) | 1.13 (0.96 to 1.33) |\n| Smoking status: |  |  |  |  |  |\n| \u2003Never | 16 626 | 266 | 1.00\u2020 | 1.00\u2020 | 1.00\u2020 |\n| \u2003Former | 15 446 | 291 | 1.21 (1.03 to 1.43) | 1.22 (1.03 to 1.44) | 1.20 (1.01 to 1.41) |\n| \u2003Current | 29 395 | 777 | 1.87 (1.63 to 2.16) | 1.88 (1.63 to 2.17) | 1.94 (1.67 to 2.24) |\n| Obesity status: |  |  |  |  |  |\n| \u2003Non-obese | 58 032 | 1220 | 1.00\u2020 | 1.00\u2020 | 1.00\u2020 |\n| \u2003Obese | 3434 | 114 | 1.81 (1.50 to 1.20) | 1.30 (1.01 to 1.68) | 1.26 (0.97 to 1.62) |\n\nModel 1 includes one exposure only. Model 2 includes physical activity, smoking status, and obesity status; and as covariates height and weight (both continuous); self perceived health (not good, good); physical activity at work (sedentary, light, medium, high, unknown, unemployed); diabetes mellitus and any musculoskeletal, neurological, or psychiatric disorders (yes, no for each disorder); and alcohol use (abstainer, normal, suspected alcohol dependence). Model 3 as for model 2 but with addition of systolic and diastolic blood pressure, use of antihypertensive drugs, total serum cholesterol concentrations, educational level, and socioeconomic group.\n\n\\*Based on time updated data and mortality follow-up.\n\n\u2020Reference category.\n\n\u2021Based on baseline data and mortality follow-up.\n\nThe models in which we used time updated variables showed higher estimates of the effect of physical activity than models with baseline data (table 3).<a href=\"#tbl3\" data-ref-type=\"table\"><\/a>\n\nWe converted the differences in death rates into differences in remaining life expectancies from age 50. Men who reported high levels of physical activity were expected to live 3.8 (95% confidence interval 2.7 to 4.8) years longer than sedentary men and 1.8 (1.3 to 2.2) years longer than men who reported medium levels of physical activity. After adjustment for potential confounding variables the corresponding remaining life expectancies were 2.3 (1.3 to 3.3) and 1.1 (0.6 to 1.6) years, respectively.\n\n## Changes in physical activity level\n\nWe investigated the influence on mortality of changes in physical activity between the ages of 50 and 60 in the 1759 men who participated in the first and second survey. From the date of examination in survey 2, we studied 34\u2009020 person years and 998 men died. At the age of 60 about 27% (n=474) still reported high physical activity, 11% (n=194) had increased and 24% (n=414) had reduced levels, and 38% (n=677) reported unchanged low activity. Men who changed their physical activity level could not be distinguished from men with unchanged activity based on baseline clinical characteristics (not shown).\n\nChanges in physical activity between the ages of 50 and 60 were associated with mortality (fig 3)<a href=\"#fig3\" data-ref-type=\"fig\"><\/a>. After 10 years of follow-up the mortality rate did not differ between men who had increased their physical activity and men who were at an unchanged high level (table 4).<a href=\"#tbl4\" data-ref-type=\"table\"><\/a> This is also illustrated by the parallel failure curves after about age 70 (fig 3).<a href=\"#fig3\" data-ref-type=\"fig\"><\/a> During the first five years of follow-up, however, the mortality rate was 2.6 times higher in men who had increased their level of physical activity compared with men whose physical activity was high but unchanged (table 3).<a href=\"#tbl3\" data-ref-type=\"table\"><\/a> Adjustment for smoking, obesity, and other covariates attenuated the estimates but did not change the interpretation of the results.\n\n\u2002 Hazard ratios with 95% confidence intervals for total mortality after different follow-up periods by changes in physical activity between ages of 50 and 60\n\n| Follow-up period and physical activity level | Follow-up (person years) | No of deaths | Hazard ratio (95% CI) |  |\n|----|----|----|----|----|\n|  |  |  | Model 1 | Model 2 |\n| **Unrestricted** |  |  |  |  |\n| Unchanged high | 10 508 | 249 | 1.00\\* | 1.00\\* |\n| Increased from low or moderate to high | 4862 | 154 | 1.27 (1.04 to 1.55) | 1.21 (0.99 to 1.48) |\n| Reduced from high to low or moderate | 7020 | 233 | 1.53 (1.28 to 1.83) | 1.35 (1.12 to 1.61) |\n| Unchanged low or moderate | 11 629 | 362 | 1.47 (1.25 to 1.73) | 1.33 (1.12 to 1.56) |\n| **Deaths after 5 years of follow-up** |  |  |  |  |\n| Unchanged high | 10 464 | 234 | 1.00\\* | 1.00\\* |\n| Increased from low or moderate to high | 4813 | 136 | 1.18 (0.95 to 1.45) | 1.13 (0.91 to 1.39) |\n| Reduced from high to low or moderate | 6952 | 211 | 1.51 (1.25 to 1.82) | 1.33 (1.10 to 1.61) |\n| Unchanged low or moderate | 11 542 | 325 | 1.46 (1.23 to 1.73) | 1.31 (1.11 to 1.56) |\n| **Deaths after 10 years of follow-up** |  |  |  |  |\n| Unchanged high | 10 190 | 198 | 1.00\\* | 1.00\\* |\n| Increased from low or moderate to high | 4649 | 115 | 1.14 (0.90 to 1.43) | 1.10 (0.87 to 1.38) |\n| Reduced from high to low or moderate | 6694 | 178 | 1.60 (1.30 to 1.96) | 1.43 (1.17 to 1.76) |\n| Unchanged low or moderate | 11 064 | 263 | 1.49 (1.24 to 1.79) | 1.36 (1.13 to 1.64) |\n| **Deaths within 5 years of follow-up** |  |  |  |  |\n| Unchanged high | 2340 | 15 | 1.00\\* | 1.00\\* |\n| Increased from low or moderate to high | 929 | 18 | 3.05 (1.54 to 6.06) | 2.64 (1.32 to 5.27) |\n| Reduced from high to low or moderate | 2028 | 22 | 1.66 (0.86 to 3.20) | 1.50 (0.77 to 2.92) |\n| Unchanged low or moderate | 3287 | 37 | 1.75 (0.96 to 3.19) | 1.58 (0.86 to 2.90) |\n| **Deaths within 10 years of follow-up** |  |  |  |  |\n| Unchanged high | 4589 | 51 | 1.00\\* | 1.00\\* |\n| Increased from low or moderate to high | 1783 | 39 | 1.98 (1.30 to 3.00) | 1.70 (1.12 to 2.60) |\n| Reduced from high to low or moderate | 3874 | 55 | 1.25 (0.86 to 1.83) | 1.10 (0.74 to 1.61) |\n| Unchanged low or moderate | 6326 | 99 | 1.43 (1.02 to 2.01) | 1.23 (0.87 to 1.74) |\n\nModel 1 includes time updated variable change in physical activity as exposure. Model 2 includes time updated variable change in physical activity as exposure and covariates: smoking status, obesity status, height and weight, self perceived health, physical activity at work, diabetes mellitus, any musculoskeletal, neurological, or psychiatric disorders, and alcohol use.\n\n\\*Reference category.\n\nEighteen of the 154 deaths (12%) among men with increased physical activity occurred within the first five years of follow-up and 21 men died during the subsequent five years. This group with increased mortality during the first 10 years after an increase in physical activity could not be distinguished on the basis of differences in the cardiovascular risk factor profile either at age 50 or 60. Exclusion of men with diagnosis of or echocardiography signs of cardiovascular disease at the age of 50 or 60 did not change any of the observed associations. Neither was there any difference in causes of death between this group and others. Men who maintained a low level of physical activity and men who decreased their level had higher mortality rates than those with unchanged high physical activity, irrespective of the length of follow-up (table 4, fig 3).<a href=\"#tbl4\" data-ref-type=\"table\"><\/a> <a href=\"#fig3\" data-ref-type=\"fig\"><\/a>\n\nWe studied men who increased their physical activity level in more detail. The mortality rate after more than 10 years of follow-up was halved in men who had increased their physical activity to a high level both from a medium (adjusted hazard ratio 0.58, 0.39 to 0.87) and a low (0.51, 0.26 to 0.97) level. There was no evident reduction in mortality rate among men who increased their physical activity from low to medium (0.81, 0.51 to 1.31). As a comparison, smoking cessation was associated with a 40% lower mortality rate after more than 10 years of follow-up compared with current smoking (0.64, 0.53 to 0.78). Men who had never smoked had a 60% lower mortality rate than current smokers (0.42, 0.33 to 0.52).\n\nThere was no evidence of biological interaction between increased physical activity and smoking cessation in their effects on mortality; the relative excess risk due to interaction was \u22120.40 (\u22121.30 to 0.50).\n\n# Discussion\n\n## Principal findings\n\nIncreased levels of physical activity in middle age have an effect on mortality. After a 10 year period of increased physical activity the excess mortality seen in inactive men was reduced to the same levels of mortality as seen in physically active men. There is, however, a period of at least 5 years before this risk reduction during which the risk is higher. The halved mortality rate after 10 years of follow-up after increased physical activity between the ages of 50 and 60 (compared with continued inactivity) was similar to that seen after smoking cessation (compared with continued smoking).\n\n## Strengths and weaknesses of the study\n\nThe main strengths of our study include the population based design, the long follow-up from a time before the men reached ages with a high mortality rate, the repeated measurements, and the objective and complete information on mortality. Our analyses also took into account changes over time in potential confounders including smoking, obesity, self perceived health, and morbidity and in classic risk factors for mortality including hypertension and total cholesterol concentration. We also adjusted our estimates for socioeconomic group and educational level.\n\nThe main limitation of our study is its restriction to men and the crude assessment of physical activity by questionnaire, with risk of misclassification possibly leading to underestimation of the results.28 There is, however, no ideal test to measure physical activity,29 either by questionnaire or by more objective assessments. We used a questionnaire created in collaboration with the Swedish National Institute of Public Health that estimates physical activity over the life cycle.30 Although our categories indexing physical activity should be regarded as reflecting common perceptions or patterns of physical activity rather than precise measures of levels, the levels estimated from these four simple questions are positively associated with physiological measurements of fitness,16 17 skeletal muscle capillary density,31 and pedometer results,32 indicating that these levels do represent physical activity. Simple questionnaires are well suited for the study of changed behaviour in large epidemiological studies,28 and the repetition of identical questions at each survey is one of the main strengths of our study.\n\nThe last observed value carried forward technique could introduce bias, despite being the preferred imputation method.33 As results from sensitivity analysis were consistent with the main results, it seems unlikely that this imputation method introduced bias. In our multivariable models, the adjustment for variables that can be regarded not only as confounders but also as intermediates on the causal pathway\u2014such as perceived health, body weight, and diabetes\u2014might also have conservatively biased our estimates.\n\n## Strengths and weaknesses in relation to other studies\n\nMany previous studies have used baseline data and changes in activity to examine whether physical activity influences mortality, without applying a time dependent approach in their analyses.9 10 11 12 34 35 Our associations between baseline data on physical activity and mortality rates were generally weaker than when we took into account changes within individuals in the level of physical activity during follow-up, further emphasising the importance of using time updated variables when behaviour such as physical activity is studied in relation to clinical outcomes.36 Time dependent life table analysis for estimation of remaining life expectancy among 50 year old men in the Framingham Heart Study showed 3.9-4.1 years longer life expectancy for men reporting high physical activity and 1.6-1.8 years longer life expectancy for men reporting medium physical activity, compared with the sedentary group.13 14 We obtained estimates of similar magnitude.\n\nTotal mortality was substantially influenced by the length of time since the change in physical activity took place. One recent study that used time dependent analysis to investigate change in physical activity in relation to mortality in men and women aged 19 to over 90 years found an association between change in physical activity and mortality in men under the age of 70.15 In that study, however, the direction of change, the age when the change in physical activity occurred, and the time that had elapsed since the change remained unclear.\n\nA change in physical activity in the lower activity range might reflect a change in sedentary behaviour rather than in actual physical activity. Recent research showed that sedentary behaviour and physical activity were independently associated with obesity.37 Future research should aim to separate these two effects.\n\nAs measurement of physical activity is crude and difficult to compare between studies, we wanted to compare the effect of physical activity on mortality with the effect of smoking. Self reported smoking has a high validity,38 and smoking is probably the most well established modifiable behavioural risk factor for premature death.39 The graded inverse association between physical activity level and mortality was independent of and present in each category of smoking status. The impact of increased physical activity on mortality was on par with the effect of smoking cessation. A recent study in women found a comparable reduction in mortality after smoking cessation.40\n\nOur observational results might differ from those from meta-analyses of clinical trials because such trials tend to be conducted in high risk populations and are designed so that the exposure has a low measurement error and that the probability of confounding is reduced. The effect we observed after increased physical activity, however, is larger than the effect of a 1 mmol\/l reduction in cholesterol concentration (18% reduction of total mortality)41 and the effect of treatment with any antihypertensive drug (10% reduction of total mortality).42\n\n## Possible explanations\n\nWe previously showed that an increase in the level of physical activity in the present cohort was associated with beneficial changes in the metabolic profile, including a decrease in body weight and in blood concentrations of insulin, proinsulin, glucose, and lipids.43 An improved metabolic profile reduces the prevalence of metabolic syndrome, obesity, and cardiovascular disease. This is one suggested pathway whereby physical activity is thought to reduce mortality.13 34 The effect of hypertension and high total cholesterol concentration on mortality is well established, but in our analysis the association between physical activity and mortality was independent of these. Other suggested mechanisms for the effect of physical activity on mortality include changes in inflammatory and hormonal responses and gut mobility44 and effects on the nervous systems, which influence neuromuscular and brain function.45 All these mechanisms might be important for the progression of many diseases and general wellbeing. In older people physical activity is also important for maintaining muscle strength, postural balance,46 47 functional independence, and health related quality of life.48 The effect of physical activity on the risk of premature death might be modulated by genetic differences.49 Recent results in Swedish twins, however, argue against genetics as a cause of the association between physical activity and mortality.50 In addition, inactive twins have been reported to have shorter telomere lengths corresponding to higher biological age by up to 10 years compared with their more active co-twin.51\n\n## Implications, unanswered questions, and future research\n\nWithin the first five years after the investigation at the age of 60 we observed an increased risk associated with increased physical activity compared with men with unchanged high activity. Given the small numbers of deaths we are reluctant to place a strong emphasis on this increased risk, especially as mortality was not higher than mortality in men who continued to be sedentary. Although there might not be a true increase in mortality soon after increased physical activity, there is nonetheless no immediate benefit. This has also been observed in a community based cluster randomised trial in older people in whom participation in an exercise programme had no effect on mortality during the two year follow-up.52 There was, however, a much larger than expected improvement in health related quality of life after the intervention, suggesting that increased activity might have early health benefits that are collectively too small to have an impact on total mortality.\n\nOur results could have important implications in the planning of clinical trials as these would need to be long enough to extend beyond the period of no benefit and to take into consideration the ethical implications of a possible increase in short term risk. Different follow-up times might also yield different results in observational studies.\n\nIt is difficult to quantify the least amount of physical activity needed for a beneficial effect on mortality in our study. Our dose-response relations suggest that the more activity the better, but on the basis of our data it is not possible to determine whether the recommended minimum amount of physical activity was achieved or not.5 6 7\n\nWe could not identify any characteristics distinguishing those who died shortly after taking up physical activity. A healthy survivor effect cannot be excluded as survival until age 60 was necessary for this analysis. Further research should investigate whether and to what extent increased physical activity affects mortality in the period soon after the change. In addition, the effects in other age groups and in women need to be studied. Mechanisms and reasons for an active choice to change physical activity are not fully understood.\n\n## Conclusion\n\nIn conclusion, we have shown a graded reduction in total mortality risk with increasing physical activity level in men. Increased physical activity in middle age increases longevity after an induction period of up to 10 years of no benefit. An increase in physical activity has the same impact on lowering mortality rate in the long term as smoking cessation. Thus, efforts for promotion of physical activity, even among middle aged and older men, are important.\n\n### What is already known on this topic\n\n1.  Physical activity is beneficial for health and prolongs life among younger individuals\n\n2.  Inactivity and resulting disease remains a major health problem\n\n### What this study adds\n\n1.  There is a dose dependent association between level of physical activity and total mortality in middle aged men\n\n2.  Increased physical activity prolongs life among middle aged and older men, though there might be a period of 5-10 years before an effect is seen on total mortality\n\n3.  The effect on mortality after increased physical activity is on par with that of smoking cessation\n\n**Cite this as:** *BMJ* 2009;338:b688","meta":{"dup_signals":{"dup_doc_count":118,"dup_dump_count":72,"dup_details":{"curated_sources":2,"2023-50":2,"2023-40":2,"2023-06":2,"2022-49":3,"2022-40":1,"2022-27":1,"2022-21":2,"2022-05":2,"2021-49":1,"2021-43":2,"2021-39":1,"2021-21":1,"2021-17":1,"2021-10":2,"2020-45":1,"2020-40":1,"2020-34":1,"2020-24":1,"2020-10":1,"2020-05":1,"2019-51":1,"2019-39":1,"2019-35":1,"2019-30":1,"2019-26":2,"2019-22":1,"2019-18":1,"2019-09":1,"2019-04":1,"2018-51":1,"2018-43":1,"2018-39":1,"2018-34":1,"2018-26":1,"2018-22":1,"2018-17":1,"2018-09":1,"2018-05":1,"2017-47":2,"2017-43":2,"2017-34":3,"2017-26":6,"2017-22":1,"2017-17":2,"2016-50":1,"2016-44":2,"2016-40":1,"2016-36":3,"2016-30":2,"2016-26":1,"2016-22":2,"2016-18":2,"2016-07":1,"2015-48":2,"2015-40":1,"2015-35":1,"2015-32":1,"2015-27":2,"2015-22":3,"2015-14":2,"2014-52":2,"2014-49":1,"2014-42":2,"2014-41":2,"2014-35":2,"2014-23":3,"2024-10":1,"2017-13":5,"2015-18":2,"2015-11":2,"2015-06":2,"2024-18":1}},"file":"PMC2654773"},"subset":"pubmed_central"}
{"text":"abstract: Data recently published in *BMC Biology* provide insights into the normal physiological function of c-*myc* in the development and regeneration of the mammary gland and indicate a key role in epithelial cell proliferation, elaboration of ductal alveoli, and the biosynthetic capacity and milk production of the mature organ.\n  .\n  See related research article <http:\/\/www.biomedcentral.com\/1741-7007\/7\/63>\nauthor: Nicole M Sodir; Gerard I Evan\ndate: 2009\ninstitute: 1Department of Pathology and Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA 94143-0502, USA\nreferences:\ntitle: Nursing some sense out of Myc\n\nThe three Myc proteins, c-Myc, L-Myc and N-Myc, are all basic helix-loop-helix (bHLH)-Zip transcription factors that coordinate cell proliferation, cell cycle progression, cell growth, metabolism, differentiation and tissue remodeling, as well as a variety of protective checkpoint mechanisms such as growth arrest and apoptosis. Myc proteins form heterodimers with their bHLH-Zip partner Max, trans-activating genes by binding at canonical E-Box CAC^G^\/~A~TG recognition elements in target gene promoters and repressing other genes through interactions with the zinc finger protein Miz-1 and\/or recruitment of the DNA methyl-transferase corepressor Dnmt3a (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>) \\[1\\]. Expression array, SAGE, chromatin IP, promoter scanning and whole cell proteomic approaches identify thousands of Myc target genes with diverse roles in virtually every aspect of cell and tissue behavior, including growth, metabolism, cell cycle, differentiation, telomere maintenance, DNA damage and repair, intracellular membrane transport, cytoskeleton, cytokine production, angiogenesis, invasion and apoptosis \\[2\\] (see also <http:\/\/www.myc-cancer-gene.org\/site\/mycTargetDB.asp>).\n\nIn normal cells expression of Myc proteins is tightly regulated, either by developmental cues or, in the case of c-Myc expression in adult regenerative tissues, by mitogenic stimulation. In the absence of such proactive induction, short-lived Myc mRNAs and proteins are rapidly cleared from cells, which then default back to their non-proliferative state. This tight control of Myc expression is defective in almost all cancers, either because of deregulating mutations in the Myc genes themselves, or through the relentless induction of Myc expression by upstream oncogenic signals such as the Wnt\/\u03b2-catenin, Notch or RTK\/Ras pathways. Moreover, ectopic activation of Myc is sufficient in many adult tissues to engage, co ordinate and maintain the diverse intracellular (cell growth, cell cycle progression, biosynthetic metabolism, ribogenesis and translation) and extracellular (release of cytokines and chemokines, recruitment of inflammatory cells, extensive stromal remodeling, invasion and angiogenesis) processes that somatic cells require for their orderly expansion (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>). Myc's highly pleiotropic effects are mirrored by its wide range of gene targets, estimated to encompass some 20% of all vertebrate genes \\[2\\].\n\nIt is notable that the role of Myc as a pivotal coordinator of cell proliferation is common to almost all adult tissues. Since different tissues vary widely in their architecture, regenerative capacity and the risks of infection and oncogenesis, many of Myc's potential target genes are likely to be regulated in a context-dependent fashion; that is, the extent of their control by Myc (and, hence, the actual execution of their cognate biological programs) is dependent on other factors such as cell type and history, local environment and circumstance.\n\n# Effects of Myc inhibition\/knockout on normal tissues and cells\n\nA pressing question in Myc biology is whether or not Myc function is essential for somatic cell proliferation. Myc proteins exert very broad but relatively subtle effects on cells. Their impact on expression of individual genes is generally modest and there is no known gene whose expres sion is dependent solely on Myc: rather, Myc appears to modify the efficiency with which more bespoke transcription factors regulate their targets, in part through its general impact on chromatin architecture and accessibility \\[3\\]. Myc is therefore unlikely to be essential for any single transcriptional program but may nonetheless act as the essential coordinator that integrates all the disparate transcriptional growth programs into a coherent strategic whole. Unfortunately, the experimental data remain unclear on the requirement for Myc function in cell prolifera tion, one complication being functional complementation between different members of the Myc family, principally c- and N-Myc since L-Myc has much weaker intrinsic transcriptional activity \\[4\\]. These may be constitutively co-expressed or induced to compensate for any deficit in their siblings. c-*myc*-deficient mice die by embryonic day 10.5 (E10.5) with overt hematopoietic, vascular and placental defects \\[5\\]. While this indicates a critical and non-redundant role for c-*myc* in developmental angiogenesis and erythropoiesis, the consequent embryonic lethality obscures any obligate role c-*myc* might have in tissues beyond this developmental stage. However, it is certain that c-*myc* is also required for proliferation and elaboration of many other somatic tissues (see below). Germ-line knockouts of N-*myc* in mice also die *in utero* around E11.5 from profound defects in multiple tissues, including the central and peripheral nervous systems and multiple epithelial tissues \\[6\\]. L-Myc-deficient mice, by contrast, show no overt pheno type \\[7\\]. Rat1 fibroblast cells do not detectably express L-Myc and N-Myc and, when somatically deleted for c-*myc*, remain viable but exhibit dramatically slower proliferative rates and severe cell cycle defects. Loss of c-*myc* compro mises multiple phases of the cell cycle, slowing progression through both G1 and G2 and depressing and delaying activities of all cyclin Cdk complexes. A similar failure to proliferate is also evident in primary mouse embryo fibroblasts derived from mice in which c-myc has been con dition ally deleted.\n\nMore recently, both conditional deletion and dominant negative inhibition have been used to investigate *in vivo* the roles of Myc proteins in adult tissues. Surprisingly, initial studies in skin \\[8\\] and gut \\[9\\] suggested that deletion of c-*myc* had little impact on maintenance of either regenerative tissue or hematopoietic stem cells \\[10\\]. In the latter case at least, such independence from c-Myc is due to co-expression of N-Myc, consistent with known degeneracy in c-Myc and N-Myc function. More recent conditional deletion studies in the gastrointestinal (GI) tract, however, suggest that the apparent lack of requirement for c-Myc in tissue homeostasis may be an artifact of relatively low penetrance of expression of the Cre recombinase that excises the c-*myc* gene, since driving the Cre expression from a more powerful and pervasive promoter induces profound and lethal attrition of intes tinal crypts \\[11\\]. In addition, systemic expression *in vivo* of a dominant interfering Myc bHLHL-Zip dimerization domain mutant (Omomyc) that efficiently blocks c-Myc, N-Myc and L-Myc-dependent transcriptional activation triggers a marked reduction of proliferation in tissues with rapid turnover (for example intestinal epithelium, skin, bone marrow, testis) but has no discernible acute effect on adult organs with low proliferative indices (for example pancreas, kidney, liver, heart or lung). Strikingly, this massive reduction in proliferation rate of regenerative tissues in such Omomyc-expressing animals is not accompanied by any cell death or loss of tissue integrity: consequently, such systemic Myc inhibition has no pathological negative impact on the animals, even over extended periods (4 to 12 weeks). More over, the phenotypic effects of such Myc inhibition are completely and rapidly reversible upon subsequent Omomyc downregulation \\[12\\].\n\nIn a recent study published in *BMC Biology*, Stoelzle *et al.* \\[13\\] investigate the specific physiological role of c-*myc* in the mammary gland by conditional gene deletion using Cre-loxP recombinase technology. Cre (Cyclization Recombination) is a sequence-specific DNA recombinase that is used to excise stretches of DNA flanked either side by loxP (Locus of X-over P1) DNA sequences. Expression of Cre under the control of a tissue-specific promoter can then be used to delete specific gene sequences in a tissue-specific manner. Whey acidic protein *(WAP)iCre* transgenic mice were used to delete the *loxP*-flanked c-*myc* locus specifically in luminal alveolar cells, starting at mid-pregnancy and continuing throughout lactation. Affected *c-myc*^*fl*\/*fl*^*;WAPiCre*^+^ mothers exhibited a marked deficit in milk production and capacity to nurse their young, associated with a failure of terminal end buds to elaborate alveoli within the lactating mammary gland. Since the *WAP* promoter is triggered during first pregnancy, parous *c-myc*^*fl*\/*fl*^*;WAPiCre*^+^ start subsequent pregnancies with c-myc already deleted. During such pregnancies, mice show dramatic deficits in mammary proliferation and maturation, although the resulting hypoplastic tissues differentiate normally. This proliferative deficit is mirrored by the markedly reduced efficiency of mammary tissue regeneration when parous *c-myc*^*fl*\/*fl*^*;WAPiCre*^+^ mammary tissue was transplanted into cleared fat pads of immuno-compromised recipient mice.\n\nWhile such data suggest that proliferation of mammary epithelial progenitor cells is still possible in the absence of c-*myc*, albeit at reduced efficiency, the usual uncertainties of Cre-loxP technology obtain. The progressive appearance of the signature restriction fragment induced by Cre deletion of c-*myc* clearly indicates accumulation of cells with deleted c-Myc, but it is less clear what proportion of cells retain either one or both functional c-*myc* allele(s). No surrogate marker of recombination was used to estimate deletion efficiency and, as the authors point out, c-Myc immunohistochemistry remains a capricious and insensitive arbiter of recombination efficiency. Any remaining c-*myc*-competent cells would be expected to contribute variably to initial growth and subsequent regeneration of mammary tissues and their uneven representation might explain the partial capacity of parous *c-myc*^*fl*\/*fl*^*;WAPiCre*^+^ mammary tissue to reconstitute the gland in na\u00efve recipients, albeit at reduced efficacy.\n\nIt is notable that c-*myc*-deleted mutant glands exhibit alveolar cells with condensed ER and secretory capacity and reduced levels of ribosomal RNA and milk proteins. They are also far less efficient at producing the principal milk proteins \u03b1-lactalbumin and \u03b2-casein. Taken together, this indicates an important role for c-*myc* in establishing, and perhaps maintaining, biosynthetic output of the mammary alveolar cells. Such data recall c-myc deletion in intestinal epithelium, which generates c-Myc-deficient crypt progenitor cells that are not only smaller, cycle more slowly and enter mitosis with decreased cell size, but also exhibit reduced biosynthetic activity relative to their wild-type counterparts. Thus, studies in both mammary and intestinal epithelium concur that c-Myc is important for cells to establish the necessary biosynthetic capacity to progress expeditiously through the cell cycle. However, whether this indicates that c-Myc also plays a role in *maintaining* the specialized protein synthetic capacity of differentiated mammary epithelial cells is unclear: it is equally plausible that absence of c-Myc during the ontogeny of these secretory cells left them with irremediable structural deficits in intracellular architecture that preclude efficient production of milk proteins. Indeed, this latter notion would be most consistent with the selective deficiency such cells show in production of milk proteins, which presumably requires the unusual secretory architecture of mature mammary alveolar cells but not other 'housekeeping' polypeptides like \u03b2-actin and GAPDH, which do not. Distinguishing between the two possibilities awaits studies in which deleted Myc function is subsequently restored in established mammary epithelium to ascertain whether or not this reinstates biosynthetic capacity, either immediately or during subsequent pregnancy-induced re-growth.\n\n# Myc as a target in cancer therapy\n\nThe pivotal, and largely non-redundant, role Myc appears to play in coordinating proliferation of tumor cells has reignited interest in it as a possible target for cancer therapy. This notion is strengthened by the widespread deregulation of Myc observed in human cancers and by emerging evidence that inactivation of Myc function can suppress both initiation and evolution of cancers in multiple tissue types and trigger the rapid and complete regression of established tumors, even in cancers where Myc itself is not the 'driving' oncogene \\[12\\]. However, since Myc is also required for the proliferation of many normal tissues, there have always been concerns that Myc inhibition might be accompanied by catastrophic side effects in normal tissues. Recently, however, such concerns have been largely allayed by data indicating that the impact of systemic Myc inhibition, at least over the relatively short term, is only mild and completely reversible. The studies by Stoelzle *et al.* \\[13\\] offer additional reassurance by demon stra ting that inhibition of Myc is not toxic *per se* for established, adult mammary tissue, although clearly care would need to be taken in administering any Myc inhibitor during periods of mammary tissue regeneration since this might lead to transient, and perhaps permanent, mammary gland dysfunction. For the moment, pharmacological inhibitors remain a pipe dream. However, advances in drug design, strategy and delivery may some day allow us to target the non-redundant processes that really make tumors tick. Of these, Myc is an obvious and provocative candidate.","meta":{"dup_signals":{"dup_doc_count":129,"dup_dump_count":40,"dup_details":{"curated_sources":2,"2023-14":1,"2022-27":1,"2020-34":1,"2020-10":1,"2019-47":1,"2019-13":1,"2018-51":1,"2018-43":1,"2018-34":1,"2018-05":1,"2017-39":1,"2017-30":1,"2017-22":1,"2017-09":11,"2016-50":1,"2016-44":2,"2016-36":11,"2016-30":13,"2015-48":3,"2015-40":3,"2015-35":2,"2015-32":3,"2015-27":1,"2015-22":3,"2015-14":2,"2014-52":4,"2014-49":6,"2014-42":8,"2014-41":5,"2014-35":6,"2014-23":5,"2014-15":4,"2023-40":1,"2015-18":3,"2015-11":3,"2015-06":3,"2014-10":3,"2013-48":4,"2013-20":3,"2024-30":1}},"file":"PMC2776920"},"subset":"pubmed_central"}
{"text":"date: 2013-04\ntitle: In This Issue of *Diabetes*\n\nEdited by Helaine E. Resnick, PhD, MPH\n\n# A Case for the Development of DPP-4 Inhibitors for Stroke Prevention in Diabetes?\n\nThe study by Darsalia et al. shows that linagliptin, a drug currently in use against type 2 diabetes, may also protect against stroke. In this issue of *Diabetes* (**p. 1289**), the investigators studied the antistroke efficacy of linagliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor, in type 2 diabetic mice. Type 2 diabetes is a risk factor for stroke, and stroke patients with diabetes have a greater risk of stroke recurrence and mortality than nondiabetic stroke patients. Glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) agonists are in clinical use against type 2 diabetes. GLP-1R activation can also occur by prolonging the half-life of endogenous GLP-1 via inhibition of DPP-4. In a first set of experiments, 25-week-old obese diabetic mice were treated with either 10 mg\/kg\/body weight linagliptin per day or 2 mg\/kg\/body weight sulfonylurea glimepiride per day or vehicle for 7 weeks. Glimepiride was chosen because it does not affect the incretin system. At 4 weeks into the treatment, stroke was induced by transient middle cerebral artery occlusion. In a second set of experiments, 10-week-old control mice were treated with linagliptin, glimepiride, or vehicle for 7 weeks, similar to the first experiment. Again, all mice were subjected to stroke at 4 weeks. Linagliptin was efficacious against stroke in both type 2 diabetic mice and control mice. However, glimepiride showed antistroke efficacy only in control mice. The investigators propose that glimepiride's neuroprotective effects are mediated by increased insulin secretion, whereas the efficacy of linagliptin is glucose independent and likely involves GLP-1. These results suggest that the development of DPP-4 inhibitors for stroke prevention and treatment in patients with diabetes, as well as in nondiabetic high-risk patients, may be warranted. \u2014 *Laura Gehl, PhD*\n\nDarsalia et al. The DPP-4 inhibitor linagliptin counteracts stroke in the normal and diabetic mouse brain: a comparison with glimepiride. Diabetes 2013;62:1289\u20131296\n\n# Questioning the Use of Resveratrol for Managing Obesity-Related Metabolic Complications\n\nData in this issue of *Diabetes* (**p. 1186**) raise questions about the utility of resveratrol as a nutritional supplement in metabolic disorders. The study by Poulsen et al. explored the use of resveratrol in obese human subjects. The polyphenolic compound resveratrol, which is prevalent in nature, has been seen for years as a potential tool in managing obesity and obesity-related complications. In rodent models, resveratrol has been shown to protect against diet-induced metabolic abnormalities, and resveratrol is currently sold as an over-the-counter human nutritional supplement. Yet very few data are available to support the use of resveratrol in humans. Poulsen et al. conducted a randomized, placebo-controlled, double-blinded, parallel-group trial, in which 24 obese men with no other health problems were randomly assigned to resveratrol or placebo treatment for 4 weeks. Subjects in the resveratrol group received 1,500 mg *trans*-resveratrol daily for the 4-week period. Insulin sensitivity was assessed by hyperinsulinemic-euglycemic clamp. Subjects were clamped at a blood glucose level of \u223c5 mmol\/L with an insulin infusion of 0.5 mU\/kg\/min. Insulin levels increased from \u223c75 pmol\/L in the basal period to \u223c315 pmol\/L in the clamp period, and no significant differences were observed between the two groups for either period. Similarly, resveratrol failed to have a significant effect on fasting glucose, fasting insulin, blood pressure, resting energy expenditure, or inflammatory and metabolic biomarkers. Although the high incidence of obesity and type 2 diabetes calls for novel approaches to obesity management, these results do not support the use of resveratrol as a nutritional supplement for obese humans without a defined metabolic defect. \u2014 *Laura Gehl, PhD*\n\nPoulsen et al. High-dose resveratrol supplementation in obese men: an investigator-initiated, randomized, placebo-controlled clinical trial of substrate metabolism, insulin sensitivity, and body composition. Diabetes 2013;62:1186\u20131195\n\n# Blocking A\u03b2Improves Insulin Sensitivity in AD Mouse Model\n\nIn this issue of *Diabetes* (**p. 1159**), Zhang et al. found that not only does amyloid-\u03b2 (A\u03b2) induce hepatic insulin resistance in vivo but that neutralizing A\u03b2 also improves insulin sensitivity in an Alzheimer disease (AD) mouse model. In previous research, this group reported that in vitro A\u03b2 induces insulin resistance via JAK\/STAT3\/SOCS-1 signaling. The new series of experiments confirmed that those results hold in vivo. Treatment with A\u03b242 in C57BL\/6J mice showed both increased fasting blood glucose levels and markedly reduced insulin sensitivity, without significant change in body weight. Further demonstrating the effects of A\u03b2, the researchers treated APPswe\/PSEN1dE9 (APP\/PS1) mice with control IgG or 6C8 or 1H3 anti-A\u03b2 neutralizing antibodies. After 4 months of intraperitoneal injections, mice treated with either 6C8 or 1H3 anti-A\u03b2 antibodies showed significantly decreased fasting blood glucose levels. After 9 months, mice treated with 6C8 anti-A\u03b2 antibody showed both decreased fasting blood glucose levels and improved insulin sensitivity. Further experiments in APP\/PS1 mice explored the mechanism of insulin resistance and showed that treatment with A\u03b242 caused upregulation of SOCS-1 and activation of JAK2\/STAT3 signaling in the liver. Neutralization of A\u03b2 with 1H3 anti-A\u03b2 antibody treatment inhibited the JAK2\/STAT3\/SOCS-1 signaling pathway. Expanding on this, the researchers used RNAi to knockdown JAK2 in APP\/PS1 mice; consequently, JAK2\/STAT3\/SOCS1 signaling was inhibited, and both insulin sensitivity and glucose metabolism improved. This new report adds to the body of knowledge about the relationship between type 2 diabetes and AD and suggests that reducing A\u03b2 levels or inhibiting JAK2 activity has potential as therapeutic interventions for insulin resistance and type 2 diabetes. \u2014 *Deborah Elbaum, MD*\n\nZhang et al. Amyloid-\u03b2 induces hepatic insulin resistance in vivo via JAK2. Diabetes 2013;62:1159\u20131166\n\n# Rac1 Regulates Contraction-Induced Glucose Uptake in Skeletal Muscle\n\nIn this issue of *Diabetes* (**p. 1139**), Sylow et al. establish that Ras-related C3 botulinum toxin substrate 1 (Rac1) is not only activated by muscle contraction but that it is also necessary for contraction-induced skeletal muscle glucose uptake. By quantifying the GTP-bound Rac1 immediately after exercise, the investigators demonstrated that Rac1 is activated by exercise in both mice and humans. Confirmatory experiments in mice showed Rac1 to be essential in contraction-induced glucose uptake. Soleus and extensor digitorum longus (EDL) muscles were incubated with one of two Rac1 inhibitors, NSC23766 or Rac1 inhibitor II. Treatment with either inhibitor decreased contraction-induced glucose uptake in the muscle. Treatment with NSC23766 reduced glucose uptake by \u223c55% in both soleus and EDL muscle, and the corresponding decreases after treatment with Rac1 inhibitor II were 58% in soleus muscle and 22% in EDL. Transgenic mice with a muscle-specific inducible Rac1 knockout also showed decreased contraction-induced glucose uptake. Further experiments showed Rac1 activation to be independent of AMPK; neither targeted AMPK kinase activity deletion nor AICAR-induced AMPK activation altered normal Rac1 activation. This established that Rac1 is not activated downstream of AMPK. Rac1 is already known to be important for insulin-induced GLUT4 translocation; this series of experiments introduces and establishes its importance in exercise-induced skeletal muscle glucose uptake. As Rac1 activation appears to be either independent or upstream of AMPK activity (or both), further studies are needed to help elucidate this signal transduction pathway. In the future, pharmacological activators of Rac1 might prove to be novel therapies for the control of blood glucose levels. \u2014 *Deborah Elbaum, MD*\n\nSylow et al. Rac1 is a novel regulator of contraction-stimulated glucose uptake in skeletal muscle. Diabetes 2013;62:1139\u20131151","meta":{"dup_signals":{"dup_doc_count":131,"dup_dump_count":42,"dup_details":{"curated_sources":2,"2020-10":1,"2018-26":2,"2018-22":1,"2018-17":2,"2018-05":4,"2017-47":2,"2017-43":6,"2017-39":3,"2017-34":2,"2017-30":6,"2017-26":3,"2017-22":3,"2017-17":9,"2017-09":5,"2017-04":8,"2016-50":3,"2016-44":5,"2016-40":3,"2016-36":3,"2016-30":3,"2016-26":3,"2016-22":3,"2016-18":2,"2015-48":3,"2015-40":2,"2015-35":2,"2015-32":2,"2015-27":3,"2015-22":3,"2015-14":1,"2014-52":3,"2014-49":2,"2014-42":4,"2014-41":2,"2014-35":3,"2014-23":3,"2023-23":1,"2017-13":4,"2015-18":2,"2015-11":3,"2015-06":3,"2024-26":1}},"file":"PMC3609576"},"subset":"pubmed_central"}
{"text":"abstract: I believed that genomics was like a tsunami: a force that, when it crested, would change everything.\nauthor: Gregory A Petsko\ndate: 2005\ninstitute: 1Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02454-9110, USA\ntitle: Tsunami\n\nIt began at the bottom of the ocean, off the west coast of Indonesia. From there it spread outward, silently, invisible under the surface of the water. When it came ashore, in forty countries, some as far as 4,000 miles from where it started, it killed upwards of 150,000 people. There have been deadlier catastrophes - the earthquake in China in the mid-1970s killed a quarter of a million in that country, and the cyclone that devastated Bangladesh in the decade before that is thought to have caused half a million deaths - but none that involved so many different nations scattered across so much of the earth's surface. The tsunami spawned by the magnitude 9.0 earthquake of 26 December 2004 was perhaps the first truly global natural disaster in modern history: a third of the world's countries were directly affected. The worldwide scope of the destruction reminds us of something that genomics is also starting to make clear: that we are all truly one people, that national and racial differences are artificial and insignificant compared to the common bond of our humanity. Despite all our technological prowess and environmental hubris, we also have yet another grim reminder that Nature, not Man, is still boss of this planet.\n\nThe word 'tsunami' comes from the Japanese words for harbor (tsu) and wave (nami). It refers to a series of giant undersea waves that travel at high velocity for very long distances, and that crest when they hit a shoreline in the form of a devastating surge, sometimes as much as 30 meters high. Tsunamis are often called 'tidal waves' but that's a misnomer: the phenomenon has nothing to do with the tides. It has its origins, like everything else that involves the earth's surface, in plate tectonics.\n\nIt is hard to imagine that the theory of plate tectonics, which is at the heart of all modern geological science, is only a hundred years old and was not widely accepted until the 1970s. Schoolchildren had noticed for hundreds of years that the facing shapes of South America and Africa could be fitted neatly together like pieces of a jigsaw puzzle to make a single entity (Francis Bacon had noticed it in 1620 but drew no conclusion), but it wasn't until 1908 that the amateur American geologist Frank Bursley Taylor proposed that the continents had once slid around and that this motion might have thrust up the world's mountain chains. His theory was taken up by the German planetary astronomer-turned-meteorologist Alfred Wegener, who in 1912 proposed that all the world's continents had once been part of a single giant landmass he called Pangaea, which had split apart in a process of lateral motion that was still continuing. Traditional geologists attacked both Wegener and his ideas viciously, and it wasn't until the decade after his death (he froze to death on a scientific expedition in Greenland in 1930) that the great English geologist Arthur Holmes provided an explanation for how Wegener's motion could occur. In a textbook published in 1944 he speculated that heat caused by the decay of radioactive elements in the earth's crust could produce powerful convection currents that could slide the continents around on the earth's surface. He has probably as good a claim as anyone to be the father of the modern view of continental drift, although, curiously, he often expressed skepticism about his own theory. Harry Hess, a Princeton University geologist, figured out in the 1950s that there were two large plates of land under the floor of the Atlantic Ocean and that their relative motion was responsible for the topography and geology of the seafloor. Finally, in 1963, Cambridge University geophysicist Drummond Matthews and his graduate student Fred Vine used magnetic readings to prove that the seafloor and the continents were in motion. (Canadian geologist Lawrence Morley came up with the same result at the same time but his paper was rejected by the *Journal of Geophysical Research*.) J. Tuzo Wilson of Toronto showed at about the same time how plate tectonics could explain the behavior of the ocean floor at mid-ocean ridges. Still, even in the 1970s, many textbooks of geology continued to dismiss plate tectonics as physically impossible.\n\nToday we know that the surface of the earth is composed of about a dozen large plates and almost two dozen smaller ones, all moving in different directions. Where they grind against one another (regions geologists call 'subduction zones'), the tremendous force can be released either slowly and steadily, giving rise to thermally active regions like Iceland, or sporadically and violently, giving rise to earthquakes and volcanic eruptions. That is what happened on 27 August 1883, when subduction along the Java Trench, where the Indo-Australian plate is moving under the Indonesian Island chain, caused the explosive eruption of the volcano on the Indonesian island of Krakatoa that in turn generated waves that reached 41 meters in height, destroying 165 coastal towns and villages along the Sunda Strait between the islands of Java and Sumatra and killing 36,417 people. (Hollywood made a movie about this great disaster in 1969: *Krakatoa*, *East of Java*. In case you are ever tempted to equate Hollywood productions with history, let me point out that Krakatoa is west of Java.) That is probably what happened in 1648 B.C., when the entire Minoan civilization on the island of Crete was wiped out, in a single day, as the result of a tsunami created by the explosive eruption of the volcano on Santorini.\n\nAnd that is what happened on 26 December 2004, when the Indian plate slid underneath the Burma plate (a subduction zone), driving a 6oo-mile long piece of the earth's crust 20 to 50 feet upwards on the floor of the Indian Ocean. This sudden rise in the seafloor displaced an enormous volume of water - exactly as if the ocean were a swimming pool and someone had just dropped a large block of concrete into it. The displacement spread outward in all directions, like the ripples that would spread from that block. But because the event occurred underwater, the displacement traveled underwater until it encountered the sharply rising seafloor on the edge of an island or continent. When the undersea waves hit this obstacle, they were pushed straight up, compressed into walls of water that surged over the landmass.\n\nThe speed of a tsunami depends on the square of the depth of the ocean: the deeper the water, the faster the displacement travels. The Indonesian tsunami formed in deep water, which meant that the wave velocity reached upwards of 800 km\/h, the speed of a commercial jet aircraft. When they encounter the shallow depths of a coastline the speed of tsunami waves slows to perhaps 45 km\/h, still fast enough to do tremendous damage. At top speed it took the tsunami less than 7 hours to cross the Indian Ocean and reach the east coast of Africa, where the waves came ashore in Somalia and killed 150 people who cannot possibly have understood that the power that was destroying them had been spawned more than 3,000 miles away.\n\nLike the ripples from that dropped block of concrete, a tsunami is actually a series of waves, usually spaced about 15-20 minutes apart, with troughs in between them. To the observer on shore, the approach of a tsunami often begins with a rapid receding of the shoreline, much further out than normal. This is followed, 8-10 minutes later, by the first wave, which surges onto the shore, often traveling a half a mile or more inland. As the cycle repeats, the first wave recedes, carrying anything loose back out to sea. Then the next wave hits. In Thailand, Sri Lanka and Indonesia, where the worst damage occurred, many people who survived the impact of the first wave were swept out to sea as it receded, or were killed by one of the surges that followed. But what fascinates me the most about tsunamis is that, until they reach land, they are practically unnoticeable on the surface of the ocean. Their amplitude in deep water is often only a meter or less. An ocean liner or a fishing vessel would pass right over them, completely unaware that underneath it a force was racing onward that, when it surfaced, could obliterate an entire country.\n\nI've been thinking a lot about that sort of thing recently, because it seems to me that it's a pretty good metaphor for what is going on in science. I started writing this column because I believed that genomics was like a tsunami: a force that, when it crested, would change everything, and I wanted to have an excuse to think about what that would mean. The true impact of the genomics revolution is only starting to be apparent now, and it's very different from what it was predicted to be when the Human Genome Project began in the late 1980s. It has not had a significant impact on human health yet - the disease genes that have been discovered have generally come from specific individual research programs, and pharmacogenomics has initially focused on polymorphisms in genes that were already identified before the human genome was sequenced. Genomics has produced technology, such as cDNA microarrays and mass spectroscopy-based proteomics, that is likely to play a major role in diagnostics in the near future, but not necessarily in treatment. No, the major effects, which are now rolling across biology like a series of waves, are cultural.\n\nBecause of genomics, data gathering and analysis is now valued highly - in some instances above hypothesis-driven research. Because of genomics, targeted big science projects, such as structural genomics, that aim to produce easily appreciated results (usually in the form of large amounts of data), are consuming a large chunk of funding that would otherwise go to individual investigator-initiated basic research. Because of genomics, there is a perception in some quarters that when you have analyzed something you have understood it; mathematical modeling of biological processes is beginning to become a substitute for experimental probing. Because of genomics, a kind of mysticism is creeping back into biology: we use terms, like 'systems biology' and 'emergent properties', that have echoes of vitalism in them -almost as though we are starting to believe that we cannot explain the behavior of living systems in terms of the physics and chemistry of their component parts. We can argue about whether this is good or bad for our field. We can argue about how we should react to it. But we cannot ignore it. None of this could have been appreciated in 1990. It was all moving beneath the surface, moving rapidly and inexorably and now it is upon us. Until the next tsunami comes along, genomics, like molecular biology before it, will change our scientific world whether we like it or not.\n\nSo one of the lessons I take from what happened on 26 December is the folly of believing that things will remain as they are today. We go about our lives and our careers unaware of the great forces that move, unseen, beneath the usual tide of events. Until they crest they are almost undetectable, so we don't talk about them or plan for them. And once they do crest, they can change our careers and our lives in a very short time. It would seem that, of all the qualities we need to survive and thrive in an unpredictable world, flexibility - adaptability if you will - might just be the most valuable. The other lesson is a practical one. If you're standing on shore, looking out at what seems to be a perfectly calm ocean, and you suddenly see the shoreline receding rapidly, exposing the sea bottom much farther out than usual, turn around and run like hell away from the water. You need to get a half mile inland; a mile would be better. You have, if you're lucky, maybe 10 minutes.\n\n## Figures and Tables","meta":{"dup_signals":{"dup_doc_count":111,"dup_dump_count":44,"dup_details":{"curated_sources":2,"2019-51":1,"2019-22":1,"2019-09":1,"2019-04":1,"2018-51":1,"2018-47":1,"2018-43":1,"2018-34":1,"2018-30":2,"2018-13":2,"2017-47":1,"2017-34":1,"2017-26":1,"2017-22":1,"2017-09":9,"2016-44":1,"2016-40":1,"2016-36":10,"2016-30":8,"2016-22":1,"2016-18":1,"2016-07":6,"2015-48":2,"2015-35":2,"2015-32":2,"2015-27":2,"2015-22":2,"2015-14":2,"2014-52":2,"2014-49":4,"2014-42":5,"2014-41":3,"2014-35":4,"2014-23":3,"2014-15":3,"2023-23":1,"2017-13":3,"2015-18":4,"2015-11":2,"2015-06":2,"2014-10":2,"2013-48":2,"2013-20":3,"2024-18":1}},"file":"PMC551523"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  The clinical learning environment is a complex social entity that influences student learning outcomes in the clinical setting. Students can experience the clinical learning environment as being both facilitative and obstructive to their learning. The clinical environment may be a source of stress, creating feelings of fear and anxiety which in turn affect the students' responses to learning. Equally, the environment can enhance learning if experienced positively.\n  .\n  # Objectives\n  .\n  This study described pupil enrolled nurses' experiences of facilitative and obstructive factors in military and public health clinical learning settings.\n  .\n  # Method\n  .\n  Using a qualitative, contextual, exploratory descriptive design, three focus group interviews were conducted until data saturation was reached amongst pupil enrolled nurses in a military School of Nursing.\n  .\n  # Results\n  .\n  Data analysed provided evidence that acceptance by clinical staff and affordance of self-directed learning facilitated learning. Students felt safe to practise when they were supported by the clinical staff. They felt a sense of belonging when the staff showed an interest in and welcomed them. Learning was obstructed when students were met with condescending comments. Wearing of a military uniform in the public hospital and horizontal violence obstructed learning in the clinical learning environment.\n  .\n  # Conclusion\n  .\n  Students cannot have effective clinical preparation if the environment is not conducive to and supportive of clinical learning, The study shows that military nursing students experience unique challenges as they are trained in two professions that are hierarchical in nature. The students experienced both facilitating and obstructing factors to their learning during their clinical practice. Clinical staff should be made aware of factors which can impact on students' learning. Policies need to be developed for supporting students in the clinical learning environment.\nauthor: Eucebious Lekalakala-Mokgele; Ernestine M. Caka**Correspondence to:** Eucebious Lekalakala-Mokgele **Email:**<firstname.lastname@example.com>**Postal address:** PO Box 72, Medunsa 0204, South Africa[^1]\ndate: 2015-03-31\nreferences:\ntitle: Facilitative and obstructive factors in the clinical learning environment: Experiences of pupil enrolled nurses\n\n# Introduction\n\nMilitary health nurse training differs from general nurse training in South Africa. Nurses are usually trained in private or public schools and colleges of nursing for a period of two years (for enrolled nurses) and four years (for comprehensive basic nursing) (South African Nursing Council 1975; 1993a; 1993b). The military in South Africa train their own enrolled nurses within the military schools of nursing for two years as pupil enrolled nurses (PEN), although under the same South African Nursing Council regulation 2175 for the training of regular enrolled nurses (South African Nursing Council 1993a). The military PEN commence with a six-month basic military training to prepare them as soldiers first before they embark on a two-year nursing programme, on completion of which they are registered as enrolled nurses.\n\nMaginnis and Croxon (2007:218) insist that the clinical placement of students needs to prepare them adequately for practice in an environment that is constantly changing. Whilst nurses are generally trained in corresponding private and public clinical learning environments (CLEs), military nurses receive their own clinical learning experiences in the military health and public health institutions, for diverse clinical placement and to be exposed to different health care experiences that will equip them to nurse holistically (Purdie, Sheward & Gifford 2008:315).\n\nThis arrangement of placing military students in public health institutions was made because military hospitals were initially used only as a sickbay (a room for treating sick and injured soldiers) and for visiting the doctor on an outpatient basis. They therefore lacked diverse clinical exposure for their students. These students, including PEN, have to experience different challenges in two diverse CLEs. In the military health system only members of the military (various ranks) and their family members may be admitted as patients, whereas the public health sector accommodates civilian patients from all of the different spheres of South African society (Caka & Lekalakala-Mokgele 2013:2).\n\n## Problem statement\n\nThe CLE constitutes a clinical setting which offers the students an opportunity to practise the necessary skills to enable them provide patient care (Cheraghi *et al*. 2008:26). The uniqueness of the CLE in the military health service means that the experiences of the PENs in the military are different from those in the public sector. Firstly, their workplace learning takes place in a military milieu where power ranks are more pronounced. It is where they may have to treat high-ranking personnel as their patients and have to also contend with high-ranking clinical staff who are often also trained in military nursing and military culture. Secondly, because of the limited number of clinical areas in the military health service, students are allocated to public hospitals where they take care of civilian patients under different conditions than exist in the military setting.\n\nLiterature reveals that there are both facilitative and obstructive factors to students' learning within this environment (L\u00f6fmark & Wikblad 2001:49). The researcher's personal experience of five years as an educator in the military health service, as well as her concern about the need to understand the dynamics that facilitate or obstruct learning, led to initiation of this project in order to investigate the experiences of PEN in the military health service. This has not been done previously in South Africa and it was not known how these PEN would describe their experiences in these two diverse clinical settings. The following research question was asked: 'How do military PEN experience their clinical learning environment?'\n\n## Definition of key concepts\n\n### Clinical learning environment\n\nIn this study, the CLE is a learning environment accredited by the South African Nursing Council where students acquire the knowledge and skills to manage both military patients of various ranks and their family members. It also includes the public health sector which accommodate civilian patients from all spheres of South African society.\n\n### Military health setting\n\nThis is a formal setting for the development of various skills, including clinical skills, that will prepare PEN in times of war and defence of the country. PEN are trained to care for sick or injured soldiers and their families in the line of battle.\n\n### Public health setting\n\nA setting that mobilises local, State, national and international resources to ensure optimal conditions for people's health. It comprises preventive, curative and rehabilitative services (Basu, Jina & Naidoo 2008:7).\n\n### Pupil enrolled nurses\n\nThis term refers to persons undergoing a two-year programme at an approved nursing school, who have complied with the prescribed conditions and have furnished the prescribed particulars as required by section 24 *x* of the *Nursing Act* 50 of 1978 (Republic of South Africa 1978).\n\n## Literature review\n\nLiterature suggests that nursing students' clinical placement experiences are often fraught with longitudinal and multidimensional challenges (Clare *et al*. 2003). These challenges include a lack of support of students (from both the CLE staff and their facilitators), which can lead to missed opportunities in terms of teachable moments (Newton, Billet & Ockerby 2009). Reports suggest that nurses deal more with documentation than patient care \u2013 the feeling that there is a lack of interpersonal relationships between students, staff members and the patients is also documented (Pearcey & Draper 2008:595). The CLE is even recognised as a source of stress that creates feelings of fear and anxiety, which in turn affect the students' responses to learning (Chesser-Smyth 2005:320).\n\nAccording to Levett-Jones and Lathlean (2009:348) students can experience the CLEs as belligerent if they are constantly criticised and judged harshly by clinical staff. For many students, clinical placements are typified by a feeling of alienation and a lack of belonging (Goh & Watt 2003:14; Newton *et al*. 2009:633). These behaviours can be obstructive to learning. According to L\u00f6fmark and Wikblad (2001:46), the CLE is obstructive to learning when there is no student\u2013supervisor relationship and\/or when supervisors make condescending comments, are irritated or not interested in students and do not allow students to take part in patient care activities.\n\nViolence is another factor that is obstructive to learning. Carley and Mackway-Jones (2005:26) suggest that nursing profession is hierarchical and is embedded in the military system. This hierarchy results in student nurses experiencing 'horizontal violence' (Curtis, Bowen & Reid 2007:156), with junior members in the profession being bullied, ignored and disrespected.\n\nFacilitative factors are considered to be those that promote learning. Literature has identified multiple such factors, amongst others a sense of belonging and acceptance. A number of affective consequences are said to influence the student's sense of belonging, including feeling safe, comfortable, satisfied and happy (Levett-Jones & Lathlean 2008:103). A sense of belonging is underpinned by a need for affirmation, friendship and being part of the group (Friedkin 2004:410). When students realise that they are not part of the collective, they may present with low self-esteem and unhappiness, which may result in a diminished learning process (Baumeister, Twenge & Nuss 2002:817). It is thus important to inculcate a sense of belonging that produces happy group members, because students who feel ostracised by their peers or those in authority will not function optimally (Tabane & Human-Vogel 2010:504).\n\nAccording to L\u00f6fmark and Wikblad (2001:47), being allowed to take responsibility and to work independently, having opportunities to practise tasks and receive feedback, collaborating with staff and supervising others, gaining an overview of the setting and a sense of control, are all factors considered to facilitate learning in the CLE. Levett-Jones *et al*. (2007:211) assert that a feeling of acceptance, being valued and being involved with others are some of the attributes of belonging. The purpose of this article is to explore and describe PEN experiences of facilitative and obstructive factors in clinical learning in two diverse settings.\n\n# Research methods and design\n\n## Design\n\nAn explorative, descriptive, contextual qualitative research approach was used in this study to explore and describe the experiences of PEN in the military health CLE and within public health settings.\n\n## Population and sample\n\nThere was a total of 30 PEN in the studied institution at the time of collecting data for this study, which included PEN in their second year of training. The second-year PEN have been in training for over a year and have been more exposed to the various clinical learning situations, including placements in public hospitals. Criteria for inclusion in this study were that participants should be registered as PEN in the Military Nursing College, must be in their second year of study and should have been allocated to the military health CLE and public hospitals. The final sample size, using the eligibility criteria, was determined by data saturation (Streubert & Carpenter 2011:90).\n\n## Data collection method\n\nData were collected by means of focus group interviews. A total of 19 students participated in the study voluntarily. Three focus group sessions were conducted, with two groups consisting of six members and a third group consisting of seven members. An expert in the facilitation of focus groups was approached to act as a moderator for the sessions. Appointment of the moderator was important, as the researcher was a facilitator in the nursing college and known to the students, so this allowed participants to participate freely in the study. The moderator was a specialist psychiatric nurse skilled and experienced in focus group management. She is also an expert in qualitative research. The researcher made arrangements for the venue (a classroom in the Military Nursing College) in which to conduct interviews. The researcher also clarified the process of focus group interviews to the participants, as well as the expectations of the moderator during the interviews and how the interviews would be conducted. Data were recorded using a tape recorder with the permission of participants and field notes were handwritten by the researcher to capture actual discussions (Polit & Beck 2005:307). Participants' reactions and attitudes were also observed and noted throughout the interviews. Data were collected until saturation was reached (Streubert & Carpenter 2011:90\u201391).\n\n## Data analysis\n\nThe data were analysed according to Henning's four-step method (2004:126). Tape-recorded interviews were listened to as soon as the interviews had been completed and these were then transcribed verbatim. The researcher recorded and organised the data on note cards and audiotapes were labelled to ease data retrieval. Cross-checking of data with each note card was performed to keep track of each piece of data collected. Units were then organised into a system derived from the data. After the data were organised they were ready to be categorised into themes and sub-themes. The researcher and co-coder created codes from the data independently; extracted codes were then compared for similarities and differences. Where differences occurred these were resolved, leading to emergence of the themes and sub-themes presented in the findings. The co-coder was a specialist in analysing and coding qualitative studies.\n\n## Context of the study\n\nThe study was conducted in a Military Nursing College, of which there are three in South Africa. This college is situated in an urban area in one of the nine provinces in South Africa. A quiet classroom in one particular college was organised by the researcher in which to collect data without distraction.\n\n# Ethical considerations\n\nApproval to conduct this study was obtained from the Ethics Committee of the Faculty of Health Sciences of North-West University (Potchefstroom Campus), the Military Ethics Committee and the Officer Commanding of 3 Military Hospital. A written letter of request was given to the students, inviting them to participate in the study. Participants were informed in full about the study and were asked to provide written consent to participate. They were informed that the research did not form part of their work and that they could withdraw from participation at any time during the process without being disadvantaged in any way. All participants were assured that confidentiality would be maintained as only the researcher had access to the audiotaped material. Audiotapes were kept locked in a safe place and erased after the research was completed. The use of real names was avoided.\n\n## Trustworthiness\n\nTrustworthiness was maintained by using strategies of credibility, transferability, dependability and confirmability (Lincoln & Guba 1985:290). The research methodology and research context were described in detail to enable interested researchers to transfer these to other studies. The results and findings of the research process, including raw data, field notes, data reduction and analysis products, and theoretical notes relating to trustworthiness were kept as an audit trail of what transpired during the research process to ensure confirmability. Dependability was achieved through clear description of the exact methods of data collection, analysis and interpretation. The appointment of an independent coder with field research expertise also assisted in ensuring dependability. Prolonged engagement with the data assisted the researcher in identifying recurring patterns and themes, thus ensuring credibility.\n\n# Results\n\nThis study set out to describe PEN experiences of facilitative and obstructive factors in clinical learning in two diverse settings. Two main themes emerged from the analysis: factors facilitating learning and those obstructing learning. Self-directed learning and acceptance by staff were experienced as facilitating PEN learning, whilst non-acceptance by staff, wearing of military uniform in public hospitals and workplace violence were experienced as obstructive to learning.\n\n## Factors facilitating learning\n\n### Self-directed learning\n\nThe limited number of learning opportunities in the military health setting afforded students ample time to practise procedures on these few available opportunities and to be in control of their own learning. The ability to control their own learning was perceived as empowering: they could make decisions and enjoy learning as they did not feel pressured to complete tasks but could work at their own pace. This practice made students feel that they are trusted to make judgements about patient care, which facilitated learning. This was represented in the following excerpts:\n\n> 'Being a military nurse works at advantage because we are only serving military members and their family, so we don't have too much pressure on us, we learn at our own pace and given time to learn all of those stuff.' (PEN 1)\n>\n> 'They give us the opportunity to find solutions for patients' problems on our own and to make good judgements.' (PEN 8)\n>\n> 'Here at the military \u2026 we are given topics to prepare and present, we look for information ourselves; in that way we get to know what is happening in the ward.' (PEN 14)\n\n### Acceptance by staff\n\nAcceptance is another facilitating factor experienced by the students in this study. They expressed that they felt accepted by the doctors and nurses in the public CLE as they received good guidance and supervision from them in the wards of the public hospitals. This created confidence and competency in implementing nursing procedures, as the supervisor was always around. The following excerpts validate this:\n\n> 'In the public hospital they accept us as part of the team, and the doctors will explain everything about the patient. We will do everything under supervision of the doctor.' (PEN 2)\n>\n> 'In the provincial hospital the sister calls you and teaches you the procedure.' (PEN 7)\n>\n> 'When we are in the outside hospitals and there are procedures that they want us to learn more about, they will call us to watch.' (PEN 10)\n\n## Factors obstructive to learning\n\n### Non-acceptance by staff\n\nOne respondent said that they did not always feel accepted, and was made to feel that they were always in the way, particularly in the military CLE:\n\n> 'Sometimes they will become angry, saying students are just causing traffic. How could I learn when they don't accept us?' (PEN 11)\n\n### Wearing military uniform in public health settings\n\nWearing military uniform when allocated to public hospitals was perceived as being obstructive to learning. PEN explained that they felt marginalised when referred to as soldiers and not nurses by some staff members. Their perception was that they were easily blamed for everything that went wrong because they wore a military uniform. The participants also alleged that some staff members often made condescending remarks about them and caused them a lot of embarrassment. Patient discomfort was also cited. Their views were stated as follows:\n\n> 'Sometimes in public hospitals we feel bad as we wear military uniform, because they will call us soldiers and sometimes securities. They are sometimes not nice.' (PEN 9)\n>\n> 'Maybe something was not done, they will say \"it's the soldiers\"; it is always a pain for us, they call us soldiers and not nurses. So it's not nice as this embarrasses us.' (PEN 2)\n>\n> 'Patients are also not comfortable when we have to do procedures on them. They do not see us as nurses, they call us soldiers because of our uniform.' (PEN 15)\n\n### Workplace violence\n\nParticipants expressed that they were often subjected to different forms of violence. These were mostly non-physical in nature, in the form of shouting, bad mouthing, being blamed and felt punished if one person was in the wrong. Some felt that military rules are applied in nursing and perceived this in a negative light:\n\n> 'I just want to add on that issue of the shouting, wherever you go as long as you in the department they will shout.' (PEN 5)\n>\n> 'They call us, but when they call the college, stating how bad we are. The only thing we will hear from them, is how lazy we are, behind the staff nurses' back, they tell us we work more than them. They talk about us to the staff nurses, when those staff nurse are not there they talk about them.' (PEN 16)\n>\n> 'One of the old nurses forgot to take out the temperature, I mean, the thermometer, then when we were supposed to change the patient, eh, to give the patient a bed pan that thermometer fell and it broke, the glass was all over the bed. They blamed the students, even though we came after 3 \\[*pm*\\], they blame the students. If anything goes wrong they blame the students.' (PEN 13)\n>\n> 'He started swearing at me, verbally abusing me. I then called the police. I opened a case of assault.' (PEN 14)\n>\n> 'The military rules are applied in nursing and it is very destructive. When one student makes a mistake, everybody is punished.' (PEN 6)\n\nOne of the participants in this study was physically assaulted by a high-ranking member whilst carrying out his nursing duties:\n\n> 'A rank is misused in the military; I was physically assaulted by a high-ranking member whilst carrying out my nursing duties.' (PEN 19)\n\n# Discussion\n\nFindings show that the students experienced the CLE as both facilitating and obstructive to their learning. Compared to the military setting, students experienced the public CLE to be more facilitative as they felt empowered, could self-direct their clinical learning and had support from the clinical staff. Sil\u00e9n and Uhlin (2008:462) are of the opinion that 'students' feelings of being in charge and having a genuine impact on the learning situations are crucial for their desire to take responsibility'. Compton (2005:36) asserts that there is a strong linkage between self-directed learning and positive psychology, and that attributes such as perceived control and satisfaction are important for self-directed learning to occur. Turner (2007) concurs that 'self-directed learners demonstrate greater awareness of their responsibility in making learning meaningful and monitoring themselves', thus making them more effective learners and social beings. According to L\u00f6fmark and Wikblad (2001:49), these positive attributes are said to be facilitating factors for learning in the clinical setting; these authors showed in their study that students value independence as a facilitating factor.\n\nThe second finding was that some participants experienced acceptance in the public setting where the staff was supportive and willing to supervise them. Several authors posit that it is important that students fit in and are accepted as part of the group during clinical placement, as this kind of welcoming environment is a source of active participation and provides learning opportunities (Hartigan-Rogers *et al*. 2007:1; Levett-Jones & Lathlean 2009:348; Nash, Lemcke & Sacre 2009:48). Staff members need to be available, friendly and willing to teach in order to construct a climate conducive to learning (Croxon & Maginnis 2009:240).\n\nIn their study, Levett-Jones and Lathlean (2008:107) found that 'students felt more empowered and enabled to capitalise on the available learning opportunities when they felt they had a legitimate place in the nursing team'; conversely, if not accepted, they can find it difficult to learn. Individuals' perceptions about their participation and level of acceptance in the group influence their sense of belonging (Tabane & Human-Vogel 2010:493). This sense of acceptance must be fostered, as learners who feel ostracised by those in authority may present with low self-esteem and unhappiness. If the PEN (learners) feel that they are not part of the collective, their functioning may not be optimal, resulting in a diminished learning process (Baumeister *et al*. 2002; Tabane & Human-Vogel 2010:504). According to Chesser-Smyth (2005:320), the morale of students can be boosted if they are welcomed, respected and valued and, as a result, they become more eager to learn.\n\nIt is also evident that students are not always accepted in the military CLE. Such behaviour is identified as being obstructive to learning (Hathorn, Machtmes & Tillman 2009:229).\n\nStudies have shown that a lack of support in the CLE can be a source of anxiety and stress for students (Elliott 2002:72; Levett-Jones & Lathlean 2008:109; Papastavrou *et al*. 2009:176; Shipton 2002:244). Literature cites different reasons for a lack of support for students in the clinical setting, including registered nurses seeing facilitation of learning or supervision as extra work for them and the unavailability of staff (Clarke, Gibb & Ramprogus 2003:106; Mongwe 2001:4).\n\nThe wearing of military uniform was perceived as obstructive to learning by participants in the study. They claimed that the uniform caused them embarrassment, that they often felt marginalised and were blamed for multiple wrongs regarding patient care. Studies have shown that CLEs can be very traumatic for students when they are exposed to harsh remarks and made to feel like outsiders (Hathorn *et al*. 2009:242; Levett-Jones & Lathlean 2008:105; Rush, McCracken & Talley 2009:315).\n\nThe hierarchy within both CLEs made the PEN experience of clinical learning more challenging and was perceived as being obstructive to learning.\n\nThis study shows that participants were exposed to some form of workplace violence in the CLE. Workplace violence has many names in nursing literature. According to Mendez (2011:2), the phenomenon is known as horizontal violence, lateral violence, bullying, and intrapersonal workplace aggression. I the context of nursing, lateral violence has been defined as 'nurse-to-nurse aggression' (Griffin 2004:258). According to the literature, lateral violence finds expression in physical assault, psychological abuse and non-physical violence, such as verbal abuse, mobbing, gossip, jealousy, blaming, yelling, insulting, name-calling, lack of respect, bullying and intimidation, backstabbing, unfair criticism and defamatory statements about individuals or groups, whether it comes from nurses or other healthcare professionals (Bartholomew 2006:4\u20135; Brooks & Phillips 2013:9; Edwards & O'Connell 2007:27; Felblinger 2009; Gimeno *et al*. 2012: 30; Nachreiner *et al*. 2007:674)\n\nHorizontal violence is defined as '\\[*r*\\]epeated, offensive, abusive, intimidating, or insulting behavior, abuse of power, or unfair sanctions that makes recipients upset and feel humiliated, vulnerable, or threatened, creating stress and undermining their self-confidence' (Vessey, DeMarco & Budin, as quoted in Sellers *et al*. 2009\u20132010:21). According to King-Jones (2011:80), horizontal violence includes forms of non-physical intergroup conflict that manifest in overt and covert behaviours of hostility ranging from intimidating body language to sarcastic comments, fault finding, belittling gestures, discourtesy, disinterest and discouragement, amongst other behaviours.\n\nPEN were also subjected to physical violence. One of the participants was physically assaulted by a high-ranking member who was not satisfied with the treatment he received. According to Thomas and Burk (2009:229), this conduct can affect the working environment of the demoralised students and impede their learning. Experiences that have been described by PEN are related to the two very different roles \u2013 being a soldier and being a nurse \u2013 which causes the students to experience conflict. Fundamentally, one role is about force, aggression and violence whereas the other is about caring for the sick and the injured. It is not established if they have been prepared for these dual role.\n\n# Limitations\n\nThere are three military nursing colleges in South Africa and the findings of the study are limited to the opinions of only one group of second-year students at one of these. It is therefore important to conduct future studies on students in other colleges.\n\n# Conclusion\n\nThis study indicates that students experience both facilitating and obstructing factors to their learning during their clinical practice. The findings indicate that learning in the clinical setting is facilitated when students take responsibility and control of their learning and their self-confidence increases. Students feel safe to practise when they are supported by the clinical staff. They feel that they belong when the staff shows interest in them and welcomes them.\n\nThe study also showed that learning is obstructed when students are not accepted and are met with condescending comments or are blamed and embarrassed by the clinical staff. The lack of supervision in the military setting further obstructed their learning. Wearing of military uniform during the public clinical learning seemed to be problematic for the PEN. It was also evident that students experienced some forms of violence in the CLEs. Violence in any form is not conducive to learning and will be obstructive. Students cannot effectively be prepared clinically if the environment is not conducive to and supportive of clinical learning. Whilst research in this setting brought the experiences of military PEN to the fore, similar studies on non-military PEN may be useful in order to compare similarities and differences, with a view to using such information to improve the teaching and learning environment of students.\n\n## Recommendations\n\nBased on the conclusions of the study, it is recommended that students be allowed to take responsibility and initiative for their learning, as this can promote learning. It is also recommended that PEN should not be compelled to wear military uniform in public hospitals and should be allowed to dress in similar uniforms to their counterparts, so that they are not treated differently when allocated to the CLE of public hospitals. Clinical staff should be made aware of the impact that condescending remarks may have on students' learning. They should also recognise the need to support actively and welcome students so as to enhance the working environment. Policies on managing violence in the clinical area need to be developed, with specific attention on how this may affect students, particularly when they are dealing with high-ranked officers. There is also a need to prepare students for the dual role of being a nurse and a soldier so that they are able to experience the CLEs in a positive light.\n\n## Acknowledgements\n\nThe authors would like to thank the Medical Research Council for funding the study.\n\n## Competing interests\n\nThe authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article.\n\n## Authors' contributions\n\nE.M.C. (The South African Military Nursing College) conducted the study for her master's dissertation and provided input on the manuscript during the review process. E.L-M. (University of Limpopo) assumed the major responsibility for getting the article written and then corrected and refined the manuscript.\n\n# References\n\n[^1]: **How to cite this article:** Lekalakala-Mokgele, E. & Caka, E.M., 2015, 'Facilitative and obstructive factors in the clinical learning environment: Experiences of pupil enrolled nurses', *Curationis* 38(1), Art. \\#1263, 7 pages. <http:\/\/dx.doi.org\/10.4102\/curationis.v38i1.1263>","meta":{"dup_signals":{"dup_doc_count":131,"dup_dump_count":28,"dup_details":{"curated_sources":2,"2023-23":1,"2022-21":1,"2018-26":2,"2018-22":2,"2018-17":6,"2018-13":2,"2018-09":4,"2018-05":3,"2017-51":2,"2017-47":4,"2017-43":4,"2017-39":4,"2017-34":4,"2017-30":4,"2017-26":4,"2017-22":4,"2017-17":6,"2017-09":6,"2017-04":7,"2016-50":9,"2016-44":4,"2016-40":8,"2016-36":9,"2016-30":7,"2016-26":9,"2016-22":7,"2023-40":1,"2017-13":5}},"file":"PMC6091680"},"subset":"pubmed_central"}
{"text":"abstract: In 1959 Ernst Mayr challenged the relevance of mathematical models to evolutionary studies and was answered by JBS Haldane in a witty and convincing essay. Fifty years on, I conclude that the importance of mathematics has in fact increased and will continue to do so.\nauthor: James F Crow\ndate: 2009\ninstitute: 1Genetics Laboratory, University of Wisconsin, 425 G Henry Mall, Madison, WI 53706-1574, USA\nreferences:\ntitle: Mayr, mathematics and the study of evolution\n\nIn 1959 Ernst Mayr (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>) flung down the gauntlet \\[1\\] at the feet of the three great population geneticists RA Fisher, Sewall Wright and JBS Haldane (Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>): \"But what, precisely,\" he said, \"has been the contribution of this mathematical school to the evolutionary theory, if I may be permitted to ask such a provocative question?\" His skepticism arose in part from the fact that the mathematical theory at the time had little to say about speciation, Mayr's major interest. But his criticism was more broadly addressed to the utility of the entire approach. A particular focus was the simplification that he called \"beanbag genetics\", in which \"Evolutionary genetics was essentially presented as an input or output of genes, as the adding of certain beans to a beanbag and the withdrawing of others.\" \\[1\\].\n\nMayr was, however, criticizing textbook simplifications, rather than the actual work of the three pioneers. Far from treating gene frequency changes as analogous to the consequence of beans jostling at random in a bag, both Fisher and Wright considered gene interactions in detail. Fisher (Figure <a href=\"#F2\" data-ref-type=\"fig\">2a<\/a>) showed that, despite interactions between genes, natural selection acts on the additive component of the genetic variance. It is as if nature were familiar with least squares. The beanbag criticism was particularly inappropriate for Wright (Figure <a href=\"#F2\" data-ref-type=\"fig\">2b<\/a>), who specifically devised his 'shifting balance' theory as a way for a population to go from one harmonious gene combination (Mayr would say \"integrated genotype\") to another when intermediates were disadvantageous.\n\nWho was to answer Mayr's criticism? Fisher was already dead, and in any case preferred attack to defense, and Wright was too gentle \u2013 though admittedly not always when Mayr was involved: returning from Italy where he had received the prestigious Balzan Prize in 1984, Wright told me that the value of the prize was considerably diminished when he discovered that Mayr had won it the year before. In the event, however, it was Haldane (Figure <a href=\"#F2\" data-ref-type=\"fig\">2c<\/a>) who took up the challenge. And he did it with flair and gusto. The result was \"A defense of beanbag genetics\" \\[2\\]. This was Haldane at his best \u2013 witty, spirited, informed, interesting and convincing.\n\nBut the larger question remains: what indeed has been the contribution of mathematical theory to evolution? Mathematics is not central to evolution in the way it has been in theoretical physics. Solid advances have been made without using mathematics, much being due to Mayr himself \\[3\\]. And these continue. Yet, I shall argue that mathematical ideas have made important, and often essential, contributions, and still do. Many concepts that are now established were arrived at mathematically, although their origins have since been forgotten.\n\nFor example, the idea that polymorphisms become stabilized in populations because heterozygotes are at an advantage is now found in elementary textbooks, but Fisher was the first to formulate it. Loss of heterozygosity with inbreeding is also textbook knowledge, but it was not clear until Wright developed the theory and invented a simple algorithm for quantifying it. Similarly, the idea that the impact of mutation on the population depends on the mutation rate rather than the magnitude of the mutant effect is now taken for granted, but that was not known until Haldane showed it mathematically. One final example is the inheritance of the ABO blood groups, which was in doubt from the time of their discovery at the turn of the twentieth century until Bernstein's mathematical population analysis in 1924 \\[4\\]. All of these applications used only elementary methods, and they must have been known to Mayr. Often, concepts that were developed mathematically were later explained in intuitive, non-mathematical ways. HJ Muller was particularly inventive in finding such explanations. But the mathematical derivation usually came first. It's a lot easier to find an intuitive explanation when you already know the answer.\n\nIronically, Mayr himself unwittingly provided an especially compelling argument for mathematical analysis. His theory of \"genetic revolutions\" assumed that from a well integrated population, genetic drift in a small founder offshoot will sometimes produce a population with a new set of genotypes integrated in a new way. Intuitively, a small founder population seemed a particularly unlikely place to find a new favorable gene combination, and this was indeed shown to be the case in a very detailed mathematical analysis by Barton and Charlesworth \\[5\\]. If Mayr had had more respect for mathematical population genetics, he never would have made what most theorists regard as the mistake of proposing that small founder populations are a likely source of major evolutionary changes by genetic drift.\n\nRecent mathematical work has gone well beyond that of the three pioneers. Partly this is due to skilled mathematicians entering the field and bringing new techniques with them; especially noteworthy are stochastic processes. Second, and perhaps more important, is the extensive use of computers. Often you can use a computer to get by without deep mathematical knowledge. An additional influence is the explosive growth of molecular data, which lend themselves to mathematical treatment. In the first half of the twentieth century, population genetics and evolution had a beautiful theory, but there were very limited opportunities to apply it. Now the situation is reversed. Molecular data accumulate too fast to be assimilated.\n\nWhat are some of the newer developments in evolution that are owed to mathematical theory? Here are a few.\n\n# Neutral theory, molecular clocks and selective sweep\n\nOne striking result in the post-Mayr period was Motoo Kimura's neutral theory, independently developed in 1968 by him and by Jack King and Thomas Jukes \\[6\\]. These writers shocked the biological world by arguing that the bulk of molecular evolution is due to selectively neutral mutations driven by the mutation process rather than selection. I think it would please Mayr that the general idea \u2013 that the rate of evolution in the population is equal to the rate of mutation in a single individual \u2013 can be derived by simple reasoning using school mathematics. Yet, in order to apply the idea, we need to know how long a time period must be observed. This depends on how long it takes for a lucky new mutant to increase in frequency and completely replace its predecessors. That is not a simple problem and requires sophisticated theory. Kimura solved it using a diffusion model (see \\[6\\]). When selection and migration are taken into account, the theory is much more complicated.\n\nOne contribution of the neutral theory has been to provide a rationale for a molecular clock. Essentially, all our estimates of evolution rates depend on the assumption that the molecular changes used in constructing the clock are mutation-driven. The near constancy of average mutation rates permits reasonably accurate time estimates. Fortunately, enough of the DNA does not have an obvious function and can reasonably be supposed to be evolving by neutral kinetics, or near enough so that the neutral theory can be used in practice. And the experimenter can choose genomic regions most likely to behave in a neutral manner.\n\nA second important attribute of the neutral theory is that it supplies a natural null hypothesis for the study of selection. And yet another outgrowth of the neutral theory is the view that much of the molecular polymorphism in natural populations is effectively neutral. This is especially useful now that variation in the frequencies of single-nucleotide polymorphisms (SNPs) is easily observed.\n\nThe various measures that are used to quantify genetic variability are outgrowths of population genetics theory. One striking result of such theory is the realization that all of the worldwide human population is descended from Africa, and moreover from a small area within Africa. The evidence for this striking conclusion is that molecular variance is greater in African peoples than elsewhere. The molecular clock can be used as one measure of the time taken during various human migrations and, of course, *Homo sapiens* is not the only species that can be studied in this way.\n\nAnother outgrowth of population thinking is the 'selective sweep'. A new favorable allele arises by mutation, spreads through the population and becomes fixed at a rate that is determined mainly by how favorable it is. A consequence of this fixation is that neutral or weakly selected alleles linked to the locus are swept along with it. Because of this, there is a region on either side of the selected locus that is deficient in genetic variability. Such regions of reduced variability are footprints of a selective sweep in the past and, remarkably, provide evidence for events that occurred long ago and which can no longer be observed. Although the basic idea is simple and requires no mathematics, an assessment of how much the variability is reduced and the linkage distance over which the reduced variability occurs depend on mathematical theory.\n\n# Mathematics and the computation of family relationships\n\nAn area of biology in which mathematics, and especially computers, have become absolutely essential is systematics, Ernst Mayr's own field. Formerly, assessing species relationships and building phylogenetic trees based mainly on morphological differences was a matter of intuition and judgment. Systematists often disagreed, sometimes violently. Then came the DNA revolution. A mammalian DNA sequence supplies billions of bits of information, thus for the first time providing an opportunity for a procedure independent of personal judgments \\[7\\]. In recent decades the methods have steadily improved. The preferred procedures, such as Fisher's maximum likelihood, required a great deal of computation, and for a while this meant that large phylogenies were out of computer range. This is no longer true. Computers are now much faster, so their speed is no longer a limitation. Standards have increased in another way, too. It is now *de rigueur* to do statistical tests of significance of the tree structure and parts thereof. Many of these involve permutation methods, which have the merit of requiring minimum assumptions. They are computation-intensive, but with modern computers this is no longer an impediment.\n\nOne striking example from such studies, which came as a complete surprise to classical systematists, is the close relationship of the elephant to the shrew. Another example is in primates. For many decades the relationship of chimpanzee, gorilla and man has been uncertain. Molecular analysis of DNA sequences, using the newly developed theory, has shown that our closest relatives are chimpanzees. Furthermore, that we and the chimpanzees are 99% identical at the DNA level came as a surprise to many. Equally surprisingly, we share some 90% of our DNA with mice, rabbits, dogs, horses and elephants. Yet this is no surprise to those acquainted with the neutral theory. These numbers are fully consistent with expectations based on mutation rates and the times involved. Finally, there is now help available in the form of computer programs that can work out phylogenies and display the information graphically (see \\[7\\]). These not only eliminate a lot of tedious work, but place advanced methods in the hands of relative novices.\n\n# Coalescence and speciation\n\nFinally, there has been a major theoretical advance, coalescent theory \\[8\\]. Instead of looking forward in time, this method looks backward. Any two alleles or homologous nucleotides are ultimately derived from a single one; that is, looking backward, they coalesce. This has been the subject of extensive theoretical work in recent years. One problem for which coalescent theory provided at least an approximate answer is the question of whether there was any mating between our ancestors and contemporary Neanderthals. Small amounts of admixture are not ruled out, but coalescent theory has shown that any substantial intermating is very unlikely, as discussed by John Wakeley \\[8\\]. My other examples have been relatively simple, but this one isn't, as is apparent from this discussion. It involves a great deal of algebra. Another example, also given by Wakeley, is evidence for a selective sweep in *Drosophila simulans* \\[8\\].\n\nUntil recently, mathematical theory had contributed little to the study of speciation. Mayr emphasized allopatric speciation and the prevailing model, due to Dobzhansky and Muller \\[9\\], prevailed. Recent mathematical studies \\[10\\] support it and favor the view that speciation genes correspond to normal genes, selected for their effects within the species. Furthermore, there is evidence that these genes evolve rapidly. Thus, hybrid incompatibility is a by-product of ordinary selection in geographically isolated populations. There is no evidence that random drift plays an important part \\[9\\], so Mayr's 'genetic revolution' and similar ideas have little support. Yet it is important to point out that, aside from this, Mayr has usually been right \\[3\\]. The field of mathematical studies of speciation is barely started; it will surely increase.\n\n# Looking to the future\n\nI have given only a few examples of the part that mathematical theory has played in evolution studies. There are many more, but these, I hope, constitute a convincing sample of the importance of mathematics in population genetics and evolution. I do not intend to imply that all evolutionary study need be mathematical and theory-driven. Much exciting evolutionary biology is done in the Mayr non-mathematical tradition \\[3\\]. For example, 'evo-devo' studies, looking at changes in development during evolution, have produced exciting results while largely ignoring population genetics. Another non-mathematical example is horizontal gene transfer brought about by transposable elements, which is especially important in the evolution of microorganisms. There is also abundant evidence for increases in genetic complexity by the accumulation of small duplications. And, as always, a lot of morphological and behavioral evolution is interesting in and of itself. Yet, my guess is that as these subjects become more quantitative, population genetic theory will play an increasing role.\n\nThe rise of molecular methods has led to an increase in the importance of mathematics in population genetics and evolution. The abundance of data that require mathematical analysis has greatly increased. At the time of Mayr's challenge, evolution had a beautiful theory but very few opportunities to apply it. Now the situation is reversed: data appear faster than existing theory can deal with them. That mathematics will play an increasingly important evolutionary role in the near future seems clear.\n\n# Envoi\n\nI think these examples show not only that mathematical theory is helpful, but that it is often essential. I don't know what Ernst would say today. He might have had a change of mind, but I doubt it. Knowing how much he enjoyed arguing, I suspect he would be quite critical of much that I have written. Unfortunately, although he lived to be 100, he was not immortal and died in 2005. Were he still alive, I would surely hear from him and whatever his opinions, he would not keep them to himself. He would have enjoyed an argument, preferably over a glass of sherry. And so would I.\n\n### Acknowledgements\n\nI am indebted to Bret Payseur for reading the manuscript and offering some very useful suggestions.","meta":{"dup_signals":{"dup_doc_count":149,"dup_dump_count":43,"dup_details":{"curated_sources":4,"2020-40":1,"2020-10":1,"2019-47":1,"2019-35":1,"2019-26":1,"2019-13":1,"2019-04":1,"2018-47":1,"2018-39":1,"2018-13":1,"2017-47":1,"2017-34":1,"2017-22":1,"2017-09":14,"2016-50":1,"2016-44":1,"2016-40":1,"2016-36":13,"2016-30":11,"2016-22":1,"2016-07":14,"2015-48":3,"2015-40":2,"2015-35":3,"2015-32":3,"2015-27":3,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":4,"2014-42":9,"2014-41":5,"2014-35":6,"2014-23":4,"2014-15":6,"2021-04":1,"2015-18":3,"2015-11":2,"2015-06":3,"2014-10":3,"2013-48":3,"2013-20":3,"2017-13":1}},"file":"PMC2687772"},"subset":"pubmed_central"}
{"text":"abstract: # Introduction\n  .\n  Neutrophils and monocytes play an important role in overt inflammation in chronic inflammatory joint diseases such as rheumatoid arthritis (RA). The sympathetic nervous system (SNS) inhibits many neutrophil\/monocyte functions and macrophage tumor necrosis factor (TNF), but because of the loss of sympathetic nerve fibers in inflamed tissue, sympathetic control is attenuated. In this study, we focused on noradrenergic and TNF regulation of human neutrophil peptides 1-3 (HNP1-3), which are proinflammatory bactericidal \u03b1-defensins.\n  .\n  # Methods\n  .\n  Synovial tissue and cells were obtained from patients with RA and osteoarthritis (OA). By using immunohistochemistry and immunofluorescence, HNP1-3 were tracked in the tissue. With synovial cell-culture experiments and ELISA, effects of norepinephrine, TNF, and cortisol on HNP1-3 were detected.\n  .\n  # Results\n  .\n  HNP1-3 were abundantly expressed in the synovial lining and adjacent sublining area but not in deeper layers of synovial tissue. The human \u03b2-defensin-2, used as control, was hardly detectable in the tissue and in supernatants. HNP1-3 double-stained with neutrophils but not with macrophages, fibroblasts, T\/B lymphocytes, and mast cells. Norepinephrine dose-dependently decreased HNP1-3 levels from RA and OA cells. TNF also inhibited HNP1-3 levels from OA but not from RA cells. Cortisol inhibited HNP1-3 levels only in OA patients. A combination of norepinephrine and cortisol did not show additive or synergistic effects.\n  .\n  # Conclusions\n  .\n  This study demonstrated an inhibitory effect of norepinephrine on HNP1-3 of mixed synovial cells. In light of these findings, the loss of sympathetic nerve fibers with low resting norepinephrine levels might also augment the inflammatory process through HNP1-3.\nauthor: Birgit Riepl; Susanne Gr\u00e4ssel; Reiner Wiest; Martin Fleck; Rainer H Straub\ndate: 2010\ninstitute: 1Laboratory of Experimental Rheumatology and Neuroendocrino-Immunology, Division of Rheumatology, Department of Internal Medicine I, University Hospital, F.J. Strauss Allee 11, 93053 Regensburg, Germany; 2Department of Orthopedic Surgery, University Hospital Regensburg, Asklepios Clinic Bad Abbach, Kaiser-Karl-V.-Allee 3, 93077 Bad Abbach, Germany\nreferences:\ntitle: Tumor necrosis factor and norepinephrine lower the levels of human neutrophil peptides 1-3 secretion by mixed synovial tissue cultures in osteoarthritis and rheumatoid arthritis\n\n# Introduction\n\nRheumatoid arthritis (RA) is a chronic joint disease leading to severe erosions of adjacent bone, which are not observed in patients with osteoarthritis (OA). Although an inflammatory process is present in OA synovial tissue, RA patients demonstrate a higher state of synovial tissue inflammation compared with OA patients. In the pathophysiology of RA, T cells, B cells, macrophages, fibroblasts, and osteoclasts play dominant roles. In addition, neutrophils are important mediators of tissue inflammation in RA, and neutrophils are the most abundant cell type in the synovial fluid \\[1\\]. Neutrophil production of proteases, reactive oxygen species, S100 proteins, cytokines, chemokines, and complement stimulate inflammation \\[2\\]. Fc-gamma receptors on neutrophils can bind immune complexes that can perpetuate the inflammatory process \\[2\\]. In addition, neutrophils produce important antimicrobial proteins, such as defensins \\[3\\].\n\nA large number of defensins and defensin-like peptides have been reported in many organisms. As of March 2010, 363 entries had been recorded in a defensin database \\[4\\]. One distinguishes \u03b1-defensins (in neutrophils) from \u03b2-defensins (in other cells). Human neutrophils contain four \u03b1-defensins (HNP-1 to HNP-4) \\[5\\]. HNPs are unique to neutrophils and account for \\~99% of the total \u03b1 defensin content of these cells \\[5\\]. HNPs exert chemotactic, immunomodulating, and cytotoxic effects and participate in inflammation \\[5\\]. In contrast, human \u03b2-defensins have been described mainly in epithelial cells but also in leukocytes, heart, skeletal muscle, testis, keratinocytes, tonsil, placenta, and other tissues \\[4\\]. Defensins are expressed and released on bacterial stimuli involving the Toll-like receptors and, in addition, other stimuli, such as cytokines \\[6\\]. Besides microbicidal activities, defensins can stimulate TNF secretion from macrophages, as recently reported \\[7\\].\n\nIn tissues of patients with RA and OA, the investigation of defensins has recently begun. One report demonstrated the presence of HNP1 3 positive cells in synovial tissue of healthy subjects and in patients with suppurative arthritis, osteoarthritis (OA), and RA \\[8\\]. However, regulation of HNP1-3 in these diseases (for example, by cytokines) has not yet been investigated. Another report found an association of HNP1-3 in synovial fluid of patients with RA and severe erosive joint disease \\[9\\]. These studies clearly demonstrate that neutrophil defensins are present in inflamed tissues of patients with RA and OA. Some reports also demonstrated that defensins can be produced by dendritic cells and monocytes \\[10,11\\], but this has not been demonstrated in synovial tissue or cells of RA and OA patients.\n\nFor several years, we have been interested in the role of the sympathetic nervous system (SNS) in OA, RA, and experimental arthritis \\[12,13\\]. We have been attracted by effects of the SNS on neutrophils and monocytes, but effects on defensin secretion are presently not known. It is recognized that activation of the SNS enhances neutrophil and monocyte mobilization, leading to increased numbers of circulating neutrophils and monocytes, called the first line of defense \\[14-16\\]. Such a mechanism might increase the numbers of neutrophils and monocytes that enter inflamed tissue at sites of leaky endothelial structures (leakiness is important because catecholamines inhibit neutrophil and monocyte attachment to normal endothelium). However, once neutrophils and monocytes have entered inflamed tissue, the major neurotransmitter of sympathetic nerve fibers, norepinephrine, inhibits several neutrophil and monocyte functions. For example, norepinephrine decreases migration \\[17,18\\], oxygen radical production \\[19\\], phagocytosis \\[18\\], and bactericidal activity \\[20\\]. These inhibitory influences were present only when norepinephrine appeared at high concentrations (via \u03b2~2~-adrenergic receptors). This might be quite different at low concentrations when norepinephrine exerts its effects via \u03b1-adrenoceptors \\[21-23\\].\n\nThus, in the presence of sympathetic nerve fibers in the tissue, norepinephrine would inhibit many proinflammatory activities of neutrophils and monocytes via \u03b2~2~-adrenoceptors, which might also play a role in synovial tissue of patients with OA and RA. However, in inflamed synovial tissue of patients with RA, sympathetic nerve fibers are lost and replaced by catecholamine-producing cells \\[24\\]. The remaining catecholamine concentrations are low in the synovial tissue, leading to concentrations suitable only for \u03b1-adrenergic signaling \\[24\\]. Although norepinephrine through \u03b1-adrenergic signaling stimulates proinflammatory factors in neutrophils and macrophages \\[21-23\\], these low concentrations may well play a proinflammatory role in inflamed tissue.\n\nBy using synovial cells of patients with RA, we investigated the effect of norepinephrine on abundance of the proinflammatory bactericidal proteins HNP1-3. Experiments were also carried out in cells from OA patients because possible differences might represent one factor for the differential effects on bone (erosions in RA versus formation of new bone in OA). For comparison, we investigated the human defensin \u03b2-defensin 2 (HBD 2). TNF was used as another important proinflammatory stimulus to influence defensin secretion for comparison. In addition, the study aimed to investigate the influence of cortisol alone or together with norepinephrine because cooperative antiinflammatory effects of cortisol and norepinephrine have been described \\[25\\].\n\n# Materials and methods\n\n## Patients\n\nIn this investigation, we included 10 patients with OA and seven patients with RA. Diagnosis of RA was based on the established criteria according to the American College of Rheumatology (formerly, the American Rheumatism Association) \\[26\\]. The characteristics of patients are given in Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>. Erythrocyte sedimentation rate and serum levels of C-reactive protein were measured by using standard techniques.\n\nPatient characteristics\n\n|  | Osteoarthritis | Rheumatoid arthritis |\n|----|----|----|\n| Number | 10 | 7 |\n| Age (years) | 62.8 \u00b1 3.2 | 57.0 \u00b1 4.0 |\n| Sex (f\/m) | 6\/4 | 4\/3 |\n| C-reactive protein (mg\/L) | 5.1 \u00b1 1.3 | 25.9 \u00b1 14.1^\\#^ |\n| Erythrocyte sedimentation rate (mm) | 11.5 \u00b1 1.5 | 27.6 \u00b1 3.1\\* |\n| Lining layer thickness, cells | 2.0 \u00b1 0.5 | 2.5 \u00b1 0.2 |\n| Cellular density (all cells), cells\/mm^2^ | 364 \u00b1 169 | 635 \u00b1 329 |\n| T-cell density, cells\/mm^2^ | 1.1 \u00b1 0.1 | 1.9 \u00b1 0.5 |\n| Macrophage density, cells\/mm^2^ | 55 \u00b1 38 | 52 \u00b1 16 |\n| Medication |  |  |\n| \u2003Prednisolone (%) | N.A. | 86.0 |\n| \u2003Daily prednisolone (mg\/day) | N.A. | 6.1 \u00b1 1.7 |\n| \u2003Methotrexate (%) | N.A. | 71.0 |\n| \u2003Leflunomide (%) | N.A. | 14.0 |\n| \u2003Nonsteroidal antiinflammatory drugs (%) | 70.0 | 71.0 |\n\nData are given as mean \u00b1 SEM. \\**P* \\< 0.05; ^\\#^*P* \\< 0.005 for the comparison with osteoarthritis. Abbreviations: N.A., not applicable.\n\nThe study was approved by the Ethics Committee of the University of Regensburg. Patients were informed about the purpose of the study and gave written consent.\n\n## Synovial tissue preparation, isolation, and culture of primary mixed synovial cells\n\nOA and RA patients underwent elective knee-joint replacement surgery. Synovial tissue samples were obtained immediately after opening the knee-joint capsule, preparation of which was described \\[24\\]. In brief, a piece of synovial tissue of \u22649 cm^2^ was dissected. A larger piece of the synovial tissue was used to isolate mixed synovial cells (for culture experiments, see later). Approximately eight pieces of the same synovial area were used for immunohistochemistry and immunofluorescence, which were fixed for 12 to 24 hours in phosphate-buffered saline (PBS) containing 4% formaldehyde and then incubated in PBS with 20% sucrose for 12 to 24 hours. Thereafter, they were placed in protective freezing medium and quick-frozen (Tissue Tek; Sakura Finetek, Zoeterwoude, The Netherlands). All tissue samples were stored at 80\u00b0C.\n\nFor culture experiments, mixed synovial cells were isolated during the morning hours by enzymatic digestion for 1 to 2 hours at 37\u00b0C by using Liberase (Roche Applied Science, Mannheim, Germany). At approximately 2 to 4 p.m., the synovial cells were resuspended in RPMI 1640 medium (Sigma, Taufkirchen, Germany), supplemented with 1% penicillin\/streptomycin (Life Technologies, Inc., Paisley, U.K.) and 0.1% amphotericin B (Bristol-Myers Squibb, Munich, Germany). In total, 2.5 \u00d7 10^5^ isolated synovial cells of OA or RA patients were incubated for 24 hours in the presence of 200 \u03bcM <span class=\"smallcaps\">L<\/span>-ascorbic acid, together with norepinephrine, TNF, cortisol, or norepinephrine plus cortisol in indicated concentrations (all substances from Sigma, Steinheim, Germany).\n\nThe percentage of different types of synovial cells was tested by specific antibodies against prolyl 4 hydroxylase (for the synoviocyte type B, fibroblasts; Calbiochem, Bad Soden, Germany), CD163 (synoviocyte type A, macrophages; Dako, Hamburg, Germany), CD3 (T cells; Dako), CD19 (B lymphocytes; Dako) neutrophils (elastase; Fitzgerald Industries Int. Inc., Acton, MA, USA), and mast cells (tryptase; Abcam, Cambridge, UK). In preliminary experiments with primary early-culture mixed synoviocytes, we detected that \\~37% were positive for prolyl 4 hydroxylase, 26% for CD163, 12% for CD3, 5% for CD19, 10% for elastase, and \\<1% for tryptase.\n\n## Immunohistochemistry and double immunofluorescence\n\nApproximately ten 5-\u03bcm sections were cut from the frozen tissue blocks. For immunohistochemistry with alkaline phosphatase as the enzyme system, sections were blocked with 20% acetic acid for 20 minutes at 4\u00b0C. Sections were further blocked with 10% bovine serum albumin, 10% fetal calf serum, and 10% chicken serum for 45 minutes (all from Sigma). Then, sections were incubated with either monoclonal mouse anti-human antibodies against HNP1-3 (BMA Biomedicals, Augst, Switzerland, no. T 1034; dilution, 1:1,000; these antibodies recognize HNP-1 to -3) or polyclonal rabbit anti-human antibodies against HBD-2 (Biologo, Kronshagen, Germany, no. DEF002; dilution, 1:100). Both primary antibodies were incubated overnight at 4\u00b0C. After intensive washing with phosphate-buffered saline, sections were incubated with secondary antibodies for 1 hour at room temperature (for HNP1-3, goat anti-mouse coupled to alkaline phosphatase; Dako, no. D0486; dilution, 1:100; for HBD-2, goat anti-rabbit coupled to alkaline phosphatase, Dako, no. D0487; dilution, 1:100). The sections were developed by using BCIP\/NBT substrate (Dako, K0598). We controlled specific staining by using irrelevant primary antibodies or serum or by omitting the primary antibody.\n\nDouble immunofluorescence was carried out with fixed frozen tissue and a similar blocking procedure to that mentioned earlier. For HNP1-3 and HBD-2, the primary antibodies of BMA Biomedicals and Biologo were used in the same dilution as given earlier. For double staining, respective antibodies were used against activated macrophages (CD163, Dako), T lymphocytes (CD3, Dako), fibroblasts (prolyl-4 hydroxylase, Dako), B lymphocytes (CD19, Dako), mast cells (tryptase; Abcam, Cambridge, UK), and neutrophils (elastase; Fitzgerald Industries International; and Lab Vision NeoMarkers via Thermo Scientific, Dreieich, Germany). Fluorescent staining of positive cells was achieved by incubating the sections with respective secondary Alexa Fluor 488 and 546 antibodies or F(ab')~2~ fragments (Molecular Probes via Invitrogen, Karlsruhe, Germany). Nuclei were stained with Vectashield mounting medium with DAPI (Vector Laboratories via Biozol, Eching, Germany). We controlled specific staining by using irrelevant primary antibodies or serum or by omitting the primary antibody.\n\n## Determination of HNP1-3 and HBD-2 in supernatants of synovial cells\n\nHNP1-3 were measured with commercially available ELISA (HyCult Biotechnology, Uden, The Netherlands; this ELISA recognizes HNP-1 to -3). The detection limit of this assay was 20 pg\/ml. Intra- and interassay coefficient of variation were \\<10%.\n\nWith respect to HBD-2, a new ELISA was established by using two commercially available antibodies (capture: Biologo, no. DEF002, polyclonal rabbit anti-human antibodies; dilution, 1:1,000; detection, R&D Systems, Wiesbaden, Germany; AF2758, polyclonal goat anti-human antibodies; dilution, 1:200). After overnight coating with the capture antibody at 4\u00b0C, extensive washing, and blocking with 10% fetal calf serum, 100 \u03bcl of standard (recombinant HBD-2; Dianova, Hamburg, Germany, no. CYT-26732) or 100 \u03bcl supernatant of synovial cells was incubated for 2 hours at room temperature. After extensive washing, the detection antibody was added for another hour (at room temperature). After extensive washing, a rabbit anti-goat tertiary antibody was used, coupled to biotin (Dako, Hamburg, Germany; no. E0466). By using streptavidin coupled to horseradish peroxidase and tetramethylbenzidine (TMB) as the substrate, the ELISA was developed. The detection limit of this assay was \\~8 pg\/ml. Intra- and interassay coefficients of variation were \\<15%.\n\n## Superfusion technique of synovial tissue\n\nAs described in detail earlier \\[24\\], we used a microsuperfusion-chamber apparatus to superfuse pieces of synovial tissue with culture medium. This technique allows the determination of spontaneous defensin release directly from fresh synovial tissue. The superfusion chambers had a volume of \\~80 \u03bcl. Super-fusion was performed for 6 hours at a tempera-ture of 37\u00b0C and a flow rate of 66 \u03bcl\/min. Synovial tissue pieces had a standard size of 5 \u03bcm in diameter with a precision biopsy punch (Stiefel, Offenbach, Germany). Every hour, superfusate was collected to measure HNP1-3 and HBD-2, as described earlier.\n\n## Presentation of the data and statistical analysis\n\nAll data are given as mean \u00b1 SEM. Box plots give the 10^th^, 75^th^, 50^th^ (median), 25^th^, and 10^th^ percentile. Group medians were compared by using the nonparametric Mann-Whitney test (SPSS\/PC, Advanced Statistics, V15.0, SPSS Inc., Chicago, IL, USA). A value of *P* \\< 0.05 was the significance level.\n\n# Results\n\n## Immunohistochemical localization of HNP1-3 and HBD-2 and double immunofluorescence\n\nTo study the localization of defensins, immunohistochemistry of the synovial lining and sublining area was performed in patients with OA and RA. A representative staining of an RA patient is given in Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>. HNP1 3 were detected mainly in the synovial lining and in the directly adjacent sublining area (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>, left panels). HNP1-3 were present in most of the RA patients and OA patients. This was quite different for HBD-2, which was rarely detectable in RA and OA patients. The staining in Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>, right panels, is a rare example of an RA patient in whom HBD-2-positive cells were detected in the sublining zone.\n\nIn further extensive double immunofluorescence studies, we provide evidence that HNP1-3 were colocalized to elastase-positive neutrophils (Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>, left panels). HNP1-3 were not detected in macrophages, fibroblasts, T lymphocytes, B lymphocytes, or mast cells. Similar to that in immunohistochemistry, HBD-2 was rarely detected with immunofluorescence. In these cases, HBD-2 was colocalized with prolyl-4 of fibroblasts and CD19 of B lymphocytes (Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>, right panels). No similar colocalization was observed for macrophages, T lymphocytes, neutrophils, or mast cells.\n\n## HNP1-3 and HBD-2 from synovial tissue and synovial cells\n\nTo study the release of HNP1-3 or HBD-2, the protein was detected in the superfusate of synovial tissue. Synovial tissue released HNP1-3, which was particularly evident in RA patients (Figure <a href=\"#F3\" data-ref-type=\"fig\">3A<\/a>). The slow decline of superfusate concentration is typical for a washout of the protein and not for a decrease in secretion or production (Figure <a href=\"#F3\" data-ref-type=\"fig\">3a<\/a>). Compatible with the findings in immunohistochemistry, HBD-2 superfusate levels were lower, as substantiated in RA and OA patients (Figure <a href=\"#F3\" data-ref-type=\"fig\">3b<\/a>).\n\nOn a very different time scale, levels of defensins were studied in supernatants of cultured mixed synovial cells (Figure <a href=\"#F3\" data-ref-type=\"fig\">3c<\/a>). The levels of HNP1-3 increased over time, whereas HBD-2 was barely detectable (Figure <a href=\"#F3\" data-ref-type=\"fig\">3c<\/a>, compare detection limits with those in Figure <a href=\"#F3\" data-ref-type=\"fig\">3a, b<\/a>). Because HNP1-3 were detectable by ELISA, further functional studies included only these peptides. We studied early HNP1-3 appearance within 24 hours to minimize a possible effect by necrosis or apoptosis of these cells.\n\n## Density of neutrophils in synovial tissues of patients with OA and RA\n\nThe presence of HNP1-3 prompted us to study the density of elastase-positive neutrophils in synovial tissues of patients with OA and RA. The density of neutrophils was higher in RA as compared with OA patients (Figure <a href=\"#F3\" data-ref-type=\"fig\">3d<\/a>). The relatively low density of neutrophils is due to the fact that neutrophils were present mainly in the lining and adjacent sublining area but not in deeper layers of the synovial tissue, which were also considered in calculating tissue density (see legend in Figure <a href=\"#F3\" data-ref-type=\"fig\">3d<\/a>).\n\n## Influence of TNF, norepinephrine, and cortisol on levels of HNP1-3\n\nTo study important factors that might influence HNP1-3 levels, such as proinflammatory cytokines like TNF, this cytokine was used in culture experiments with mixed synoviocytes. TNF slightly decreased HNP1-3 levels in the supernatant of OA cells, which was significant at 1 ng\/ml (Figure <a href=\"#F4\" data-ref-type=\"fig\">4<\/a>). In RA synoviocytes, TNF did not exert a similar U-formed dose-response effect because no significant differences were observed (Figure <a href=\"#F4\" data-ref-type=\"fig\">4<\/a>).\n\nIn contrast, norepinephrine decreased supernatant HNP1-3 levels produced by mixed synovial tissue cultures in both OA and RA patients (Figure <a href=\"#F5\" data-ref-type=\"fig\">5a, b<\/a>). It is obvious that this inhibition was present only at high concentrations when norepinephrine exerts its effects mainly via \u03b2-adrenoceptors (Figure <a href=\"#F5\" data-ref-type=\"fig\">5a, b<\/a>).\n\nIn OA patients, cortisol at high concentrations of 10^-6^ *M* reduced HNP1-3 concentrations (Figure <a href=\"#F5\" data-ref-type=\"fig\">5c<\/a>), which was not observed in RA patients (Figure <a href=\"#F5\" data-ref-type=\"fig\">5d<\/a>). In addition, the combination of cortisol plus norepinephrine led to a decrease of HNP1-3 levels produced by mixed synovial tissue cultures in OA and also in RA patients (Figure <a href=\"#F5\" data-ref-type=\"fig\">5c, d<\/a>). However, the combined effect of the two hormones did not exceed the individual effects of norepinephrine alone.\n\n# Discussion\n\nThis study demonstrates the presence of two human defensins in the synovial tissue of patients with OA and RA. The \u03b1-defensin HNP1-3 and the \u03b2-defensin HBD-2 were present in synovial tissue, whereas HNP1-3 was undoubtedly allocated to neutrophils (we have not tested for dendritic cells). HBD-2 was found in a small number of fibroblasts and B lymphocytes. Although HNP1-3 were easily detectable by using histologic techniques or in culture experiments, HBD 2 was barely visible in tissue and measurable in supernatants. This study further demonstrated that norepinephrine inhibited HNP1 3 in both RA and OA patients' mixed synovial cell cultures, and cortisol and TNF slightly inhibited this \u03b1-defensin only in OA patients. This investigation adds to the understanding of how the SNS might influence HNP1-3 in chronic inflammatory joint diseases.\n\nIn a previous report, the presence of HNP1 3-positive cells was demonstrated in the synovial lining of healthy subjects and in patients with suppurative arthritis, OA, and RA \\[8\\]. However, these authors did not study the regulation of HNP1-3, which was a particular aspect in the present work. Another study reported very high synovial fluid levels of HNP1 3 in RA patients, associated with more-severe erosions \\[9\\]. Because we used the same ELISA mentioned in the latter study, we were surprised that our superfusate and supernatant levels of HNP1-3 were lower. We interpret these earlier findings of Bokareva *et al.* \\[9\\] insofar as HNP1-3 accumulate in the synovial fluid, which has been demonstrated for other factors as well (for example, estrogens \\[27\\]). Because we have not studied synovial fluid levels, we cannot directly compare the results of the two studies. However, discrepant findings might also depend on increased abundance of neutrophils in synovial fluid, as compared with synovial tissue.\n\nAlthough HNP1-3 defensins have been reported to be expressed by granulocytes in synovial tissue \\[8\\], it was not clear whether these proteins are actually released in the tissue. By using the superfusion approach, we were able to demonstrate that these proteins are released by few granulocytes in synovial tissue. In the present study, HNP1-3 is produced in higher amounts in RA than in OA, which might reflect the overall greater inflammation in patients with chronic RA. This is particularly relevant because RA patients were treated with prednisolone and antiproliferative drugs that should have reduced proinflammatory factors like HNP1-3. HNP1-3-positive cells were elastase-positive neutrophils, and we can exclude that other cells such as macrophages, fibroblasts, T and B lymphocytes, or mast cells stain positive for this protein. However, we have not tested dendritic cells that can also produce HNP1-3 \\[10,11\\]. The relatively low number of neutrophils in the tissue might be a consequence of rapid migration into the synovial fluid (\\>95% are neutrophils).\n\nThe mentioned first study of Paulsen *et al.* also reported on HBD-2 \\[8\\], which was not found in their samples investigated (they found the defensins HBD-3, CAP37, and LL37). We confirmed that immunohistologic detection of HBD-2 is a rare event (found in only one patient with RA and in one with OA). Nevertheless, this RA patient had numerous positive cells that allowed double staining, revealing double-positive fibroblasts and B lympho-cytes. Because HBD-2 levels were below the detection limit in most superfusate and supernatant samples, functional experiments with TNF and hormones aimed only to investigate the \u03b1-defensin HNP1-3. Further studies corroborated that neutrophils are present in OA and RA tissue, and that the density of these cells is higher in RA than in OA patients. It is known that the SNS has a strong influence on neutrophils (but also on monocytes), so we were particularly interested in the functional effects of the SNS on HNP1-3.\n\nThe major neurotransmitter of sympathetic nerve fibers, norepinephrine, inhibits several cellular functions. For example, norepinephrine decreases migration \\[17,18\\], oxygen radical production \\[19\\], phagocytosis \\[18\\], and bactericidal activity \\[20\\]. These inhibitory influences on neutrophils (but also on monocytes) were present only when norepinephrine appeared at high concentrations (via \u03b2~2~-adrenergic receptors). This might be quite different at low concentrations, when norepinephrine exerts its effects via \u03b1-adrenoceptors \\[21-23\\]. Our present study supports the inhibitory influence of norepinephrine, focusing on the \u03b1-defensin HNP1-3. In both RA and OA patients, norepinephrine dose-dependently decreased supernatant levels of HNP1-3 from mixed synovial tissue cultures, which reached the significance level only at high concentrations. These norepinephrine effects were stronger when compared with cortisol alone, which did not influence HNP1-3 in RA synovial cells. In addition, the combination of norepinephrine plus cortisol did not materialize in an additive or even synergistic effect. These results confirm the overall inhibitory effect of norepinephrine on cellular functions, given that concentrations of this neurotransmitter are high enough. At low concentrations of 10^-9^ *M* norepinephrine, we did not observe opposite effects that would indicate a proinflammatory influence on mixed synovial tissue cultures via \u03b1-adrenoceptors.\n\nBecause TNF is an important proinflammatory molecule in chronic inflammatory joint diseases, the effect of this cytokine on HNP1-3 levels was tested in this study. It was known that \u03b2-defensins such as HBD-2 can be stimulated by TNF in epithelial cells and astrocytes \\[28,29\\], but, to our knowledge, effects of TNF on HNP1-3 were not investigated (especially not in OA\/RA material). TNF decreased HNP1-3 supernatant levels in OA mixed synovial cultures, and the dose-response curve was U-shaped with a maximum inhibition at 1.0 ng TNF\/ml. TNF did not exert similar effects in RA cell cultures, which may be related to already high TNF levels in these cell preparations (receptor desensitization). Because, conversely, HNP1-3 can stimulate TNF secretion from macrophages, as recently reported \\[7\\], the TNF-induced inhibition of HNP1-3 might be part of a negative-feedback loop that prevents overshooting of innate immune responses. From this point of view, we hypothesize that HNP1-3 precedes TNF in the stimulatory cascade of innate immunity events. This would be an important aspect because TNF has been installed at the forefront of the innate immunity cascade in RA \\[30\\].\n\n# Conclusions\n\nIn conclusion, HNP1-3 is present in neutrophils, and as compared with HBD-2, it is abundantly present in chronically inflamed synovial tissue of patients with RA and OA. Because HNP1-3 levels are decreased by norepinephrine in mixed synovial-cell cultures, this study demonstrates that the SNS might inhibit neutrophil (but also monocyte) function *in vivo*. Sympathetic nerve fibers are lost in inflamed tissue \\[24,31\\], so concentrations of this neurotransmitter are probably too low to induce antiinflammatory activities. A similar situation exists for TNF, which is also decreased by norepinephrine \\[32\\]. HNP1-3 can stimulate secretion of TNF \\[7\\], so the loss of sympathetic nerve fibers with low concentrations of norepinephrine might have double-negative effects, possibly increasing both local HNP1-3 and TNF levels. This scenario can be relevant in overt inflammation of the tissue when monocytes (HNP1-3, TNF), macrophages (TNF), and neutrophils (HNP1-3) play a decisive role.\n\n# Competing interests\n\nThe authors declare that they have no competing interests.\n\n# Authors' contributions\n\nBR and SG participated in the concept and design and acquisition of data. RW and MF participated in interpretation of data and in drafting and revising the article. RHS participated in the concept and design, analysis and interpretation of data, and drafting and revising the article.\n\n## Acknowledgements\n\nThe authors thank Dr. Sven Anders and Dr. Joachim Grifka for kindly supplying synovial tissue.\n\nThis study was supported by a grant from the Deutsche Forschungsgemeinschaft (Research Unit FOR696).","meta":{"dup_signals":{"dup_doc_count":125,"dup_dump_count":37,"dup_details":{"curated_sources":2,"2022-27":1,"2020-16":1,"2020-05":1,"2019-39":1,"2019-30":1,"2019-22":1,"2019-13":1,"2019-04":1,"2018-09":1,"2017-43":1,"2017-22":1,"2017-09":14,"2016-44":1,"2016-36":14,"2016-30":12,"2015-48":3,"2015-40":3,"2015-35":3,"2015-32":3,"2015-27":3,"2015-22":3,"2015-14":2,"2014-52":3,"2014-49":4,"2014-42":9,"2014-41":5,"2014-35":2,"2014-23":5,"2014-15":4,"2022-49":1,"2015-18":3,"2015-11":3,"2015-06":3,"2014-10":3,"2013-48":3,"2013-20":2,"2024-10":1}},"file":"PMC2911901"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  Consciousness poses two main problems. The first is understanding the conditions that determine to what extent a system has conscious experience. For instance, why is our consciousness generated by certain parts of our brain, such as the thalamocortical system, and not by other parts, such as the cerebellum? And why are we conscious during wakefulness and much less so during dreamless sleep? The second problem is understanding the conditions that determine what kind of consciousness a system has. For example, why do specific parts of the brain contribute specific qualities to our conscious experience, such as vision and audition?\n  .\n  # Presentation of the hypothesis\n  .\n  This paper presents a theory about what consciousness is and how it can be measured. According to the theory, consciousness corresponds to the capacity of a system to integrate information. This claim is motivated by two key phenomenological properties of consciousness: differentiation \u2013 the availability of a very large number of conscious experiences; and integration \u2013 the unity of each such experience. The theory states that the quantity of consciousness available to a system can be measured as the \u03a6 value of a complex of elements. \u03a6 is the amount of causally effective information that can be integrated across the informational weakest link of a subset of elements. A complex is a subset of elements with \u03a6\\>0 that is not part of a subset of higher \u03a6. The theory also claims that the quality of consciousness is determined by the informational relationships among the elements of a complex, which are specified by the values of effective information among them. Finally, each particular conscious experience is specified by the value, at any given time, of the variables mediating informational interactions among the elements of a complex.\n  .\n  # Testing the hypothesis\n  .\n  The information integration theory accounts, in a principled manner, for several neurobiological observations concerning consciousness. As shown here, these include the association of consciousness with certain neural systems rather than with others; the fact that neural processes underlying consciousness can influence or be influenced by neural processes that remain unconscious; the reduction of consciousness during dreamless sleep and generalized seizures; and the time requirements on neural interactions that support consciousness.\n  .\n  # Implications of the hypothesis\n  .\n  The theory entails that consciousness is a fundamental quantity, that it is graded, that it is present in infants and animals, and that it should be possible to build conscious artifacts.\nauthor: Giulio Tononi\ndate: 2004\ninstitute: 1Department of Psychiatry, University of Wisconsin, Madison, USA\nreferences:\ntitle: An information integration theory of consciousness\n\n# Background\n\nConsciousness is everything we experience. Think of it as what abandons us every night when we fall into dreamless sleep and returns the next morning when we wake up \\[1\\]. Without consciousness, as far as we are concerned, there would be neither an external world nor our own selves: there would be nothing at all. To understand consciousness, two main problems need to be addressed. \\[2,3\\]. The *first problem* is to understand the conditions that determine to what extent a system has consciousness. For example, why is it that certain parts of the brain are important for conscious experience, whereas others, equally rich in neurons and connections, are not? And why are we conscious during wakefulness or dreaming sleep, but much less so during dreamless sleep, even if the brain remains highly active? The *second problem* is to understand the conditions that determine what kind of consciousness a system has. For example, what determines the specific and seemingly irreducible quality of the different modalities (e.g. vision, audition, pain), submodalities (e.g. visual color and motion), and dimensions (e.g. blue and red) that characterize our conscious experience? Why do colors look the way they do, and different from the way music sounds, or pain feels? Solving the first problem means that we would know to what extent a physical system can generate consciousness \u2013 the *quantity* or level of consciousness. Solving the second problem means that we would know what kind of consciousness it generates \u2013 the *quality* or content of consciousness.\n\n# Presentation of the hypothesis\n\n## The first problem: What determines to what extent a system has conscious experience?\n\nWe all know that our own consciousness waxes when we awaken and wanes when we fall asleep. We may also know first-hand that we can \"lose consciousness\" after receiving a blow on the head, or after taking certain drugs, such as general anesthetics. Thus, everyday experience indicates that consciousness has a physical substrate, and that that physical substrate must be working in the proper way for us to be fully conscious. It also prompts us to ask, more generally, what may be the conditions that determine to what extent consciousness is present. For example, are newborn babies conscious, and to what extent? Are animals conscious? If so, are some animals more conscious than others? And can they feel pain? Can a conscious artifact be constructed with non-neural ingredients? Is a person with akinetic mutism \u2013 awake with eyes open, but mute, immobile, and nearly unresponsive \u2013 conscious or not? And how much consciousness is there during sleepwalking or psychomotor seizures? It would seem that, to address these questions and obtain a genuine understanding of consciousness, empirical studies must be complemented by a theoretical analysis.\n\n### Consciousness as information integration\n\nThe theory presented here claims that consciousness has to do with the capacity to integrate information. This claim may not seem self-evident, perhaps because, being endowed with consciousness for most of our existence, we take it for granted. To gain some perspective, it is useful to resort to some thought experiments that illustrate key properties of subjective experience: its informativeness, its unity, and its spatio-temporal scale.\n\n#### Information\n\nConsider the following thought experiment. You are facing a blank screen that is alternately on and off, and you have been instructed to say \"light\" when the screen turns on and \"dark\" when it turns off. A photodiode \u2013 a very simple light-sensitive device \u2013 has also been placed in front of the screen, and is set up to beep when the screen emits light and to stay silent when the screen does not. The first problem of consciousness boils down to this. When you differentiate between the screen being on or off, you have the conscious experience of \"seeing\" light or dark. The photodiode can also differentiate between the screen being on or off, but presumably it does not consciously \"see\" light and dark. What is the key difference between you and the photodiode that makes you \"see\" light consciously? (see Appendix, i)\n\nAccording to the theory, the key difference between you and the photodiode has to do with how much *information* is generated when that differentiation is made. Information is classically defined as reduction of uncertainty among a number of alternatives outcomes when one of them occurs \\[4\\]. It can be measured by the entropy function, which is the weighted sum of the logarithm of the probability (p) of alternatives outcomes (i): H = - \u03a3p~i~log~2~p~i~. Thus, tossing a fair coin and obtaining heads corresponds to 1 bit of information, because there are just two alternatives; throwing a fair die yields log~2~(6) \u2248 2.59 bits of information, because there are six equally likely alternatives (H decreases if some of the outcomes are more likely than others, as would be the case with a loaded die).\n\nWhen the blank screen turns on, the photodiode enters one of its two possible alternative states and beeps. As with the coin, this corresponds to 1 bit of information. However, when you see the blank screen turn on, the state you enter, unlike the photodiode, is one out of an extraordinarily large number of possible states. That is, the photodiode's repertoire is minimally differentiated, while yours is immensely so. It is not difficult to see this. For example, imagine that, instead of turning homogeneously on, the screen were to display at random every frame from every movie that was or could ever be produced. Without any effort, each of these frames would cause you to enter a different state and \"see\" a different image. This means that when you enter the particular state (\"seeing light\") you rule out not just \"dark\", but an extraordinarily large number of alternative possibilities. Whether you think or not of the bewildering number of alternatives (and you typically don't), this corresponds to an extraordinary amount of information (see Appendix, ii). This point is so simple that its importance has been overlooked.\n\n#### Integration\n\nWhile the ability to differentiate among a very large number of states is a major difference between you and the lowly photodiode, by itself it is not enough to account for the presence of conscious experience. To see why, consider an idealized one megapixel digital camera, whose sensor chip is essentially a collection of one million photodiodes. Even if each photodiode in the sensor chip were just binary, the camera as such could differentiate among 2^1,000,000^ states, an immense number, corresponding to 1,000,000 bits of information. Indeed, the camera would easily enter a different state for every frame from every movie that was or could ever be produced. Yet nobody would believe that the camera is conscious. What is the key difference between you and the camera?\n\nAccording to the theory, the key difference between you and the camera has to do with *information integration*. From the perspective of an external observer, the camera chip can certainly enter a very large number of different states, as could easily be demonstrated by presenting it with all possible input signals. However, the sensor chip can be considered just as well as a collection of one million photodiodes with a repertoire of two states each, rather than as a single integrated system with a repertoire of 2^1,000,000^ states. This is because, due to the absence of interactions among the photodiodes within the sensory chip, the state of each element is causally independent of that of the other elements, and no information can be integrated among them. Indeed, if the sensor chip were literally cut down into its individual photodiodes, the performance of the camera would not change at all.\n\nBy contrast, the repertoire of states available to you cannot be subdivided into the repertoire of states available to independent components. This is because, due to the multitude of causal interactions among the elements of your brain, the state of each element is causally dependent on that of other elements, which is why information can be integrated among them. Indeed, unlike disconnecting the photodiodes in a camera sensor, disconnecting the elements of your brain that underlie consciousness has disastrous effects. The integration of information in conscious experience is evident phenomenologically: when you consciously \"see\" a certain image, that image is experienced as an integrated whole and cannot be subdivided into component images that are experienced independently. For example, no matter how hard you try, for example, you cannot experience colors independent of shapes, or the left half of the visual field of view independently of the right half. And indeed, the only way to do so is to physically split the brain in two to prevent information integration between the two hemispheres. But then, such split-brain operations yield two separate subjects of conscious experience, each of them having a smaller repertoire of available states and more limited performance \\[5\\].\n\n#### Spatio-temporal characteristics\n\nFinally, it is important to appreciate that conscious experience unfolds at a characteristic spatio-temporal scale. For instance, it flows in time at a characteristic speed and cannot be much faster or much slower. No matter how hard you try, you cannot speed up experience to follow a move accelerated a hundred times, not can you slow it down if the movie has decelerated. Studies of how a percept is progressively specified and stabilized \u2013 a process called microgenesis \u2013 indicate that it takes up to 100\u2013200 milliseconds to develop a fully formed sensory experience, and that the surfacing of a conscious thought may take even longer \\[6\\]. In fact, the emergence of a visual percept is somewhat similar to the developing of a photographic print: first there is just the awareness that something has changed, then that it is something visual rather than, say, auditory, later some elementary features become apparent, such as motion, localization, and rough size, then colors and shapes emerge, followed by the formation of a full object and its recognition \u2013 a sequence that clearly goes from less to more differentiated \\[6\\]. Other evidence indicates that a single conscious moment does not extend beyond 2\u20133 seconds \\[7\\]. While it is arguable whether conscious experience unfolds more akin to a series of discrete snapshots or to a continuous flow, its time scale is certainly comprised between these lower and upper limits. Thus, a phenomenological analysis indicates that consciousness has to do with the ability to integrate a large amount of information, and that such integration occurs at a characteristic spatio-temporal scale.\n\n### Measuring the capacity to integrate information: The \u03a6 of a complex\n\nIf consciousness corresponds to the capacity to integrate information, then a physical system should be able to generate consciousness to the extent that it has a large repertoire of available states (information), yet it cannot be decomposed into a collection of causally independent subsystems (integration). How can one identify such an integrated system, and how can one measure its repertoire of available states \\[2,8\\]?\n\nAs was mentioned above, to measure the repertoire of states that are available to a system, one can use the entropy function, but this way of measuring information is completely insensitive to whether the information is integrated. Thus, measuring entropy would not allow us to distinguish between one million photodiodes with a repertoire of two states each, and a single integrated system with a repertoire of 2^1,000,000^ states. To measure information integration, it is essential to know whether a set of elements constitute a causally integrated system, or they can be broken down into a number of independent or quasi-independent subsets among which no information can be integrated.\n\nTo see how one can achieve this goal, consider an extremely simplified system constituted of a set of elements. To make matters slightly more concrete, assume that we are dealing with a neural system. Each element could represent, for instance, a group of locally interconnected neurons that share inputs and outputs, such as a cortical minicolumn. Assume further that each element can go through discrete activity states, corresponding to different firing levels, each of which lasts for a few hundred milliseconds. Finally, for the present purposes, let us imagine that the system is disconnected from external inputs, just as the brain is virtually disconnected from the environment when it is dreaming.\n\n#### Effective information\n\nConsider now a subset S of elements taken from such a system, and the diagram of causal interactions among them (Fig. <a href=\"#F1\" data-ref-type=\"fig\">1a<\/a>). We want to measure the information generated when S enters a particular state out of its repertoire, but only to the extent that such information can be integrated, i.e. each state results from causal interactions within the system. How can one do so? One way is to divide S into two complementary parts A and B, and evaluate the responses of B that can be caused by all possible inputs originating from A. In neural terms, we try out all possible combinations of firing patterns as outputs from A, and establish how differentiated is the repertoire of firing patterns they produce in B. In information-theoretical terms, we give maximum entropy to the outputs from A (A^Hmax^), i.e. we substitute its elements with independent noise sources, and we determine the entropy of the responses of B that can be induced by inputs from A. Specifically, we define the *effective information* between A and B as EI(A\u2192B) = MI(A^Hmax^;B). Here MI(A;B) = H(A) + H(B) - H(AB) stands for mutual information, a measure of the entropy or information shared between a source (A) and a target (B). Note that since A is substituted by independent noise sources, there are no causal effects of B on A; therefore the entropy shared by B and A is necessarily due to causal effects of A on B. Moreover, EI(A\u2192B) measures all possible effects of A on B, not just those that are observed if the system were left to itself. Also, EI(A\u2192B) and EI(B\u2192A) in general are not symmetric. Finally, note that the value of EI(A\u2192B) is bounded by A^Hmax^ and B^Hmax^, whichever is less. In summary, to measure EI(B\u2192A), one needs to apply maximum entropy to the outputs from B, and determine the entropy of the responses of B that are induced by inputs from A. It should be apparent from the definition that EI(A\u2192B) will be high if the connections between A and B are strong and specialized, such that different outputs from A will induce different firing patterns in B. On the other hand, EI(A\u2192B) will be low or zero if the connections between A and B are such that different outputs from A produce scarce effects, or if the effect is always the same. For a given bipartition of a subset, then, the sum of the effective information for both directions is indicated as EI(A ![](1471-2202-5-42-i1.gif) B) = EI(A\u2192B) + EI(B\u2192A). Thus, EI(A ![](1471-2202-5-42-i1.gif) B) measures the repertoire of possible causal effects of A on B and of B on A.\n\n#### Information integration\n\nBased on the notion of effective information for a bipartition, we can assess how much information can be integrated within a system of elements. To this end, we note that a subset S of elements cannot integrate any information (as a subset) if there is a way to partition S in two parts A and B such that EI(A ![](1471-2202-5-42-i1.gif) B) = 0 (Fig. <a href=\"#F1\" data-ref-type=\"fig\">1b<\/a>, vertical bipartition). In such a case, in fact, we would clearly be dealing with at least two causally independent subsets, rather than with a single, integrated subset. This is exactly what would happen with the photodiodes making up the sensor of a digital camera: perturbing the state of some of the photodiodes would make no difference to the state of the others. Similarly, a subset can integrate little information if there is a way to partition it in two parts A and B such that EI(A ![](1471-2202-5-42-i1.gif) B) is low: the effective information across that bipartition is the limiting factor on the subset's information integration capacity. Therefore in order to measure the information integration capacity of a subset S, we should search for the bipartition(s) of S for which EI(A ![](1471-2202-5-42-i1.gif) B) reaches a minimum (the informational \"weakest link\").\" Since EI(A ![](1471-2202-5-42-i1.gif) B) is necessarily bounded by the maximum entropy available to A or B, min{EI(A ![](1471-2202-5-42-i1.gif) B)}, to be comparable over bipartitions, should be normalized by H^max^(A ![](1471-2202-5-42-i1.gif) B) = min{H^max^(A); H^max^(B)}, the maximum information capacity for each bipartition. The *minimum information bipartition* ^MIB^A ![](1471-2202-5-42-i1.gif) B of subset S \u2013 its 'weakest link' \u2013 is its bipartition for which the normalized effective information reaches a minimum, corresponding to min{EI(A ![](1471-2202-5-42-i1.gif) B)\/H^max^(A ![](1471-2202-5-42-i1.gif) B)}. The *information integration* for subset S, or \u03a6(S), is simply the (non-normalized) value of EI(A ![](1471-2202-5-42-i1.gif) B) for the minimum information bipartition: \u03a6(S) = EI(^MIB^A ![](1471-2202-5-42-i1.gif) B). The symbol \u03a6 is meant to indicate that the information (the vertical bar \"I\") is integrated within a single entity (the circle \"O\", see Appendix, iii).\n\n#### Complexes\n\nWe are now in a position to establish which subsets are actually capable of integrating information, and how much of it (Fig. <a href=\"#F1\" data-ref-type=\"fig\">1c<\/a>). To do so, we consider every possible subset S of m elements out of the n elements of a system, starting with subsets of two elements (m = 2) and ending with a subset corresponding to the entire system (m = n). For each of them, we measure the value of \u03a6, and rank them from highest to lowest. Finally, we discard all those subsets that are included in larger subsets having higher \u03a6 (since they are merely parts of a larger whole). What we are left with are *complexes* \u2013 individual entities that can integrate information. Specifically, a *complex* is a subset S having \u03a6\\>0 that is not included within a larger subset having higher \u03a6. For a complex, and only for a complex, it is appropriate to say that, when it enters a particular state out if its repertoire, it generates and amount of integrated information corresponding to its \u03a6 value. Of the complexes that make up a given system, the one with the maximum value of \u03a6(S) is called the *main complex* (the maximum is taken over all combinations of m\\>1 out of n elements of the system). Some properties of complexes worth pointing out are, for instance, that a complex can be causally connected to elements that are not part of it (the input and output elements of a complex are called *ports-in* and *ports-out*, respectively). Also, the same element can belong to more than one complex, and complexes can overlap.\n\nIn summary, a system can be analyzed to identify its complexes \u2013 those subsets of elements that can integrate information, and each complex will have an associated value of \u03a6 \u2013 the amount of information it can integrate (see Appendix, iv). To the extent that consciousness corresponds to the capacity to integrate information, complexes are the \"subjects\" of experience, being the locus where information can be integrated. Since information can only be integrated *within* a complex and not outside its boundaries, consciousness as information integration is necessarily subjective, private, and related to a single point of view or perspective \\[1,9\\]. It follows that elements that are part of a complex contribute to its conscious experience, while elements that are not part of it do not, even though they may be connected to it and exchange information with it through ports-in and ports-out.\n\n#### Information integration over space and time\n\nThe \u03a6 value of a complex is dependent on both spatial and temporal scales that determine what counts as a state of the underlying system. In general, there will be a \"grain size\", in both space and time, at which \u03a6 reaches a maximum. In the brain, for example, synchronous firing of heavily interconnected groups of neurons sharing inputs and outputs, such as cortical minicolumns, may produce significant effects in the rest of the brain, while asynchronous firing of various combinations of individual neurons may be less effective. Thus, \u03a6 values may be higher when considering as elements cortical minicolumns rather than individual neurons, even if their number is lower. On the other hand, \u03a6 values would be extremely low with elements the size of brain areas. Time wise, \u03a6 values in the brain are likely to show a maximum between tens and hundreds of milliseconds. It is clear, for example, that if one were to stimulate one half of the brain by inducing many different firing patterns, and examine what effects this produces on the other half, no stimulation pattern would produce any effect whatsoever after just a tenth of a millisecond, and \u03a6 would be equal to zero. After say 100 milliseconds, however, there is enough time for differential effects to be manifested, and \u03a6 would grow. On the other hand, given the duration of conduction delays and of postsynaptic currents, much longer intervals are not going to increase \u03a6 values. Indeed, a neural system will soon settle down into states that become progressively more independent of the stimulation. Thus, the search for complexes of maximum \u03a6 should occur over subsets at critical spatial and temporal scales.\n\nTo recapitulate, the theory claims that consciousness corresponds to the capacity to integrate information. This capacity, corresponding to the *quantity* of consciousness, is given by the \u03a6 value of a complex. \u03a6 is the amount of effective information that can be exchanged across the minimum information bipartition of a complex. A complex is a subset of elements with \u03a6\\>0 and with no inclusive subset of higher \u03a6. The spatial and temporal scales defining the elements of a complex and the time course of their interactions are those that jointly maximize \u03a6.\n\n## The second problem: What determines the kind of consciousness a system has?\n\nEven if we were reasonably sure that a system is conscious, it is not immediately obvious what kind of consciousness it would have. As was mentioned early on, our own consciousness comes in specific and seemingly irreducible qualities, exemplified by different modalities (e.g. vision, audition, pain), submodalities (e.g. visual color and motion), and dimensions (e.g. blue and red). What determines that colors look the way they do, and different from the way music sounds, or pain feels? And why can we not even imagine what a \"sixth\" sense would feel like? Or consider the conscious experience of others. Does a gifted musician experience the sound of an orchestra the same way you do, or is his experience richer? And what about bats \\[10\\]? Assuming that they are conscious, how do they experience the world they sense through echolocation? Is their experience of the world vision-like, audition-like, or completely alien to us? Unless we accept that the kind of consciousness a system has is arbitrary, there must be some necessary and sufficient conditions that determine exactly what kind of experiences it can have. This is the second problem of consciousness.\n\nWhile it may not be obvious how best to address this problem, we do know that, just as the quantity of our consciousness depends on the proper functioning of a physical substrate \u2013 the brain, so does the quality of consciousness. Consider for example the acquisition of new discriminatory abilities, such as becoming expert at wine tasting. Careful studies have shown that we do not learn to distinguish among a large number of different wines merely by attaching the appropriate labels to different sensations that we had had all along. Rather, it seems that we actually enlarge and refine the set of sensations triggered by tasting wines. Similar observations have been made by people who, for professional reasons, learn to discriminate among perfumes, colors, sounds, tactile sensations, and so on. Or consider perceptual learning during development. While infants experience more than just a \"buzzing confusion\", there is no doubt that perceptual abilities undergo considerable refinement \u2013 just consider what your favorite red wine must have tasted like when all you had experienced was milk and water.\n\nThese examples indicate that the quality and repertoire of our conscious experience can change as a result of learning. What matters here is that such perceptual learning depends upon specific changes in the physical substrate of our consciousness \u2013 notably a refinement and rearranging of connections patterns among neurons in appropriate parts of the thalamocortical system (e.g \\[11\\]). Further evidence for a strict association between the quality of conscious experience and brain organization comes from countless neurological studies. Thus, we know that damage to certain parts of the cerebral cortex forever eliminates our ability to perceive visual motion, while leaving the rest of our consciousness seemingly intact. By contrast, damage to other parts selectively eliminates our ability to perceive colors. \\[12\\]. There is obviously something about the organization of those cortical areas that makes them contribute different qualities \u2013 visual motion and color \u2013 to conscious experience. In this regard, it is especially important that the same cortical lesion that eliminates the ability to perceive color or motion also eliminates the ability to remember, imagine, and dream in color or motion. By contrast, lesions of the retina, while making us blind, do not prevent us from remembering, imagining, and dreaming in color (unless they are congenital). Thus, it is something having to do with the organization of certain cortical areas \u2013 and not with their inputs from the sensory periphery \u2013 that determines the quality of conscious experiences we *can* have. What is this something?\n\n### Characterizing the quality of consciousness as a space of informational relationships: The effective information matrix\n\nAccording to the theory, just as the quantity of consciousness associated with a complex is determined by the amount of information that can be integrated among its elements, the quality of its consciousness is determined by the informational relationships that causally link its elements \\[13\\]. That is, the way information can be integrated within a complex determines not only how much consciousness is has, but also what kind of consciousness. More precisely, the theory claims that the elements of a complex constitute the dimensions of an abstract relational space, the *qualia space*. The values of effective information among the elements of a complex, by defining the relationships among these dimensions, specify the structure of this space (in a simplified, Cartesian analogue, each element is a Cartesian axis, and the effective information values between elements define the angles between the axes, see Appendix, v). This relational space is sufficient to specify the quality of conscious experience. Thus, the reason why certain cortical areas contribute to conscious experience of color and other parts to that of visual motion has to do with differences in the informational relationships both *within* each area and *between* each area and the rest of the main complex. By contrast, the informational relationships that exist outside the main complex \u2013 including those involving sensory afferents \u2013 do not contribute either to the quantity or to the quality of consciousness.\n\nTo exemplify, consider two very simple linear systems of four elements each (Fig. <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>). Fig. <a href=\"#F2\" data-ref-type=\"fig\">2a<\/a> shows the diagram of causal interactions for the two systems. The system on the left is organized as a divergent digraph: element number 1 sends connections of equal strength to the other three elements. The analysis of complexes shows that this system forms a single complex having a \u03a6 value of 10 bits. The system on the right is organized as a chain: element number 1 is connected to 2, which is connected to 3, which is connected to 4. This system also constitutes a single complex having a \u03a6 value of 10 bits. Fig. <a href=\"#F2\" data-ref-type=\"fig\">2b<\/a> shows the effective information matrix for both complexes. This contains the values of EI between each subset of elements and every other subset, corresponding to all informational relationships among the elements (the first row shows the values in one direction, the second row in the reciprocal direction). The elements themselves define the dimensions of the qualia space of each complex, in this case four. The effective information matrix defines the relational structure of the space. This can be thought of as a kind of topology, in that the entries in the matrix can be considered to represent how close such dimensions are to each other (see Appendix, vi). It is apparent that, despite the identical value of \u03a6 and the same number of dimensions, the informational relationships that define the space are different for the two complexes. For example, the divergent complex has many more zero entries, while the chain complex has one entry (subset {1 3} to subset {2 4}) that is twice as strong as all other non-zero entries.\n\nThese two examples are purely meant to illustrate how the space of informational relationships within a complex can be captured by the effective information matrix, and how that space can differ for two complexes having similar amounts of \u03a6 and the same number of dimensions. Of course, for a complex having high values of \u03a6, such as the one underlying our own consciousness, qualia space would be extraordinarily large and intricately structured. Nevertheless, it is a central claim of the theory that the structure of phenomenological relationships should reflect directly that of informational relationships. For example, the conscious experiences of blue and red appear irreducible (red is not simply less of blue). They may therefore correspond to different dimensions of qualia space (different elements of the complex). We also know that, as different as blue and red may be subjectively, they are much closer to each other than they are, say, to the blaring of a trumpet. EI values between the neuronal groups underlying the respective dimensions should behave accordingly, being higher between visual elements than between visual and auditory elements. As to the specific quality of different modalities and submodalities, the theory predicts that they are due to differences in the set of informational relationships *within* the respective cortical areas and *between* each area and the rest of the main complex. For example, areas that are organized topographically and areas that are organized according to a \"winner takes all\" arrangement should contribute different kinds of experiences. Another prediction is that changes in the quality and repertoire of sensations as a result of perceptual learning would also correspond to a refinement of the informational relationships within and between the appropriate cortical areas belonging to the main complex. By contrast, the theory predicts that informational relationships outside a complex \u2013 including those among sensory afferents \u2013 should not contribute directly to the quality of conscious experience of that complex. Of course, sensory afferents, sensory organs, and ultimately the nature and statistics of external stimuli, play an essential role in shaping the informational relationships among the elements of the main complex \u2013 but such role is an indirect and historical one \u2013 played out through evolution, development, and learning \\[14\\] (see Appendix, vii).\n\n### Specifying each conscious experience: The state of the interaction variables\n\nAccording to the theory, once the quantity and quality of conscious experience that a complex *can* have are specified, the particular conscious state or experience that the complex *will* have at any given time is specified by the activity state of its elements at that time (in a Cartesian analogue, if each element of the complex corresponds to an axis of qualia space, and effective information values between elements define the angles between the axes specifying the structure of the space, then the activity state of each element provides a coordinate along its axis, and each conscious state is defined by the set of all its coordinates). The relevant activity variables are those that mediate the informational relationships among the elements, that is, those that mediate effective information. For example, if the elements are local groups of neurons, then the relevant variables are their firing patterns over tens to hundreds of milliseconds.\n\nThe state of a complex at different times can be represented schematically by a state diagram as in Fig. <a href=\"#F2\" data-ref-type=\"fig\">2c<\/a> (for the divergent complex on the left and the chain complex on the right). Each column in the state diagram shows the activity values of all elements of a complex (here between 0 and 1). Different conscious states correspond to different patterns of activity distributed over *all* the elements of a complex, with no contribution from elements outside the complex. Each conscious state can thus be thought of as a different point in the multidimensional qualia space defined by the effective information matrix of a complex (see Appendix, viii). Therefore, a succession or flow of conscious states over time can be thought of as a trajectory of points in qualia space. The state diagram also illustrates some states that have particular significance (second to fifth column). These are the states with just one active element, and all other elements silent (or active at some baseline level). It is not clear whether such highly selective states can be achieved within a large neural complex of high \u03a6, such as that one that is postulated to underlie human consciousness. To the extent that this is possible, such highly selective states would represent the closest approximation to experiencing that element's specific contribution to consciousness \u2013 its quality or \"quale\". However, because of the differences in the qualia space between the two complexes, the same state over the four elements would correspond to different experiences (and mean different things) for the two complexes. It should also be emphasized that, in every case, it is the activity state of *all* elements of the complex that defines a given conscious state, and both active and inactive elements count.\n\nTo recapitulate, the theory claims that the quality of consciousness associated with a complex is determined by its effective information matrix. The effective information matrix specifies all informational relationships among the elements of a complex. The values of the variables mediating informational interactions among the elements of a complex specify the particular conscious experience at any given time.\n\n# Testing the hypothesis\n\n## Consciousness, information integration, and the brain\n\nBased on a phenomenological analysis, we have argued that consciousness corresponds to the capacity to integrate information. We have then considered how such capacity can be measured, and we have developed a theoretical framework for consciousness as information integration. We will now consider several neuroanatomical or neurophysiological factors that are known to influence consciousness. After briefly discussing the empirical evidence, we will use simplified computer models to illustrate how these neuroanatomical and neurophysiological factors influence information integration. As we shall see, the information integration theory not only fits empirical observations reasonably well, but offers a principled explanation for them.\n\n### Consciousness is generated by a distributed thalamocortical network that is at once specialized and integrated\n\nAncient Greek philosophers disputed whether the seat of consciousness was in the lungs, in the heart, or in the brain. The brain's pre-eminence is now undisputed, and scientists are trying to establish which specific parts of the brain are important. For example, it is well established that the spinal cord is not essential for our conscious experience, as paraplegic individuals with high spinal transactions are fully conscious. Conversely, a well-functioning thalamocortical system is essential for consciousness \\[15\\]. Opinions differ, however, about the contribution of certain cortical areas \\[1,16-21\\]. Studies of comatose or vegetative patients indicate that a global loss of consciousness is usually caused by lesions that impair multiple sectors of the thalamocortical system, or at least their ability to work together as a system. \\[22-24\\]. By contrast, selective lesions of individual thalamocortical areas impair different submodalities of conscious experience, such as the perception of color or of faces \\[25\\]. Electrophysiological and imaging studies also indicate that neural activity that correlates with conscious experience is widely distributed over the cortex (e.g \\[20,26-29\\]). It would seem, therefore, that the neural substrate of consciousness is a distributed thalamocortical network, and that there is no single cortical area where it all comes together (see Appendix, ix).\n\nThe fact that consciousness as we know it is generated by the thalamocortical system fits well with the information integration theory, since what we know about its organization appears ideally suited to the integration of information. On the information side, the thalamocortical system comprises a large number of elements that are functionally specialized, becoming activated in different circumstances. \\[12,30\\]. Thus, the cerebral cortex is subdivided into systems dealing with different functions, such as vision, audition, motor control, planning, and many others. Each system in turn is subdivided into specialized areas, for example different visual areas are activated by shape, color, and motion. Within an area, different groups of neurons are further specialized, e.g. by responding to different directions of motion. On the integration side, the specialized elements of the thalamocortical system are linked by an extended network of intra- and inter-areal connections that permit rapid and effective interactions within and between areas \\[31-35\\]. In this way, thalamocortical neuronal groups are kept ready to respond, at multiple spatial and temporal scales, to activity changes in nearby and distant thalamocortical areas. As suggested by the regular finding of neurons showing multimodal responses that change depending on the context \\[36,37\\], the capacity of the thalamocortical system to integrate information is probably greatly enhanced by nonlinear switching mechanisms, such as gain modulation or synchronization, that can modify mappings between brain areas dynamically \\[34,38-40\\]. In summary, the thalamocortical system is organized in a way that appears to emphasize at once both functional specialization and functional integration.\n\nAs shown by computer simulations, systems of neural elements whose connectivity jointly satisfies the requirements for functional specialization and for functional integration are well suited to integrating information. Fig. <a href=\"#F3\" data-ref-type=\"fig\">3a<\/a> shows a representative connection matrix obtained by optimizing for \u03a6 starting from random connection weights. A graph-theoretical analysis indicates that connection matrices yielding the highest values of information integration (\u03a6 = 74 bits) share two key characteristics \\[8\\]. First, connection patterns are different for different elements, ensuring functional specialization. Second, all elements can be reached from all other elements of the network, ensuring functional integration. Thus, simulated systems having maximum \u03a6 appear to require both functional specialization and functional integration. In fact, if functional specialization is lost by replacing the heterogeneous connectivity with a homogeneous one, or if functional integration is lost by rearranging the connections to form small modules, the value of \u03a6 decreases considerably (Fig <a href=\"#F3\" data-ref-type=\"fig\">3b,3c<\/a>). Further simulations show that it is possible to construct a large complex of high \u03a6 by joining smaller complexes through reciprocal connections \\[8\\]. In the thalamocortical system, reciprocal connections linking topographically organized areas may be especially effective with respect to information integration. In summary, the coexistence of functional specialization and functional integration, epitomized by the thalamocortical system \\[30\\], is associated with high values of \u03a6.\n\n### Other brain regions with comparable numbers of neurons, such as the cerebellum, do not contribute to conscious experience\n\nConsider now the cerebellum. This brain region contains more neurons than the cerebral cortex, has huge numbers of synapses, and receives mapped inputs from the environment and controls several outputs. However, in striking contrast to the thalamocortical system, lesions or ablations indicate that the direct contribution of the cerebellum to conscious experience is minimal. Why is this the case?\n\nAccording to the theory, the reason lies with the organization of cerebellar connections, which is radically different from that of the thalamocortical system and is not well suited to information integration. Specifically, the organization of the connections is such that individual patches of cerebellar cortex tend to be activated independently of one another, with little interaction possible between distant patches \\[41,42\\]. This suggests that cerebellar connections may not be organized so as to generate a large complex of high \u03a6, but rather to give rise to many small complexes each with a low value of \u03a6. Such an organization seems to be highly suited for both the learning and the rapid, effortless execution of informationally insulated subroutines.\n\nThis concept is illustrated in Fig. <a href=\"#F4\" data-ref-type=\"fig\">4a<\/a>, which shows a strongly *modular* network, consisting of three modules of eight strongly interconnected elements each. This network yields \u03a6 = 20 bits for each of its three modules, which form the system's three complexes. This example indicates that, irrespective of how many elements and connections are present in a neural structure, if that structure is organized in a strongly modular manner with little interactions among modules, complex size and \u03a6 values are necessarily low. According to the information integration theory, this is the reason why these systems, although computationally very sophisticated, contribute little to consciousness. It is also the reason why there is no conscious experience associated with hypothalamic and brainstem circuits that regulate important physiological variables, such as blood pressure.\n\n### Subcortical centers can control consciousness by modulating the readiness of the thalamocortical system without contributing directly to it\n\nIt has been known for a long time that lesions in the reticular formation of the brainstem can produce unconsciousness and coma. Conversely, stimulating the reticular formation can arouse a comatose animal and activate the thalamocortical system, making it ready to respond to stimuli \\[43\\]. Groups of neurons within the reticular formation are characterized by diffuse projections to many areas of the brain. Many such groups release neuromodulators such as acetylcholine, histamine, noradrenaline, serotonin, dopamine, and glutamate (acting on metabotropic receptors) and can have extremely widespread effects on both neural excitability and plasticity \\[44\\]. However, it would seem that the reticular formation, while necessary for the normal functioning of the thalamocortical system and therefore for the occurrence of conscious experience, may not contribute much in terms of specific dimensions of consciousness \u2013 it may work mostly like an external on-switch or as a transient booster of thalamocortical firing.\n\nSuch a role can be explained readily in terms of information integration. As shown in Fig. <a href=\"#F4\" data-ref-type=\"fig\">4b<\/a>, neural elements that have widespread and effective connections to a main complex of high \u03a6 may nevertheless remain informationally excluded from it. Instead, they are part of a larger complex having a much lower value of \u03a6.\n\n### Neural activity in sensory afferents to the thalamocortical system can determine what we experience without contributing directly to it\n\nWhat we see usually depends on the activity patterns that occur in the retina and that are relayed to the brain. However, many observations suggest that retinal activity does not contribute directly to conscious experience. Retinal cells surely can tell light from dark and convey that information to visual cortex, but their rapidly shifting firing patterns do not correspond well with what we perceive. For example, during blinks and eye movements retinal activity changes dramatically, but visual perception does not. The retina has a blind spot at the exit of the optic nerve where there are no photoreceptors, and it has low spatial resolution and no color sensitivity at the periphery of the visual field, but we are not aware of any of this. More importantly, lesioning the retina does not prevent conscious visual experiences. For example, a person who becomes retinally blind as an adult continues to have vivid visual images and dreams. Conversely, stimulating the retina during sleep by keeping the eyes open and presenting various visual inputs does not yield any visual experience and does not affect visual dreams. Why is it that retinal activity usually determines what we see through its action on thalamocortical circuits, but does not contribute directly to conscious experience?\n\nAs shown in Fig. <a href=\"#F4\" data-ref-type=\"fig\">4c<\/a>, adding or removing multiple, segregated incoming pathways does not change the composition of the main complex, and causes little change in its \u03a6. While the incoming pathways do participate in a larger complex together with the elements of the main complex, the \u03a6 value of this larger complex is very low, being limited by the effective information between each afferent pathway and its port in at the main complex. Thus, input pathways providing powerful inputs to a complex add nothing to the information it integrates if their effects are entirely accounted for by ports-in.\n\n### Neural activity in motor efferents from the thalamocortical system, while producing varied behavioral outputs, does not contribute directly to conscious experience\n\nIn neurological practice, as well as in everyday life, we tend to associate consciousness with the presence of a diverse behavioral repertoire. For example, if we ask a lot of different questions and for each of them we obtain an appropriate answer, we generally infer that a person is conscious. Such a criterion is not unreasonable in terms of information integration, given that a wide behavioral repertoire is usually indicative of a large repertoire of internal states that are available to an integrated system. However, it appears that neural activity in motor pathways, which is necessary to bring about such diverse behavioral responses, does not in itself contribute to consciousness. For example, patients with the locked-in syndrome, who are completely paralyzed except for the ability to gaze upwards, are fully conscious. Similarly, while we are completely paralyzed during dreams, consciousness is not impaired by the absence of behavior. Even lesions of central motor areas do not impair consciousness.\n\nWhy is it that neurons in motor pathways, which can produce a large repertoire of different outputs and thereby relay a large amount of information about different conscious states, do not contribute directly to consciousness? As shown in Fig. <a href=\"#F4\" data-ref-type=\"fig\">4d<\/a>, adding or removing multiple, segregated outgoing pathways to a main complex does not change the composition of the main complex, and does not change its \u03a6 value. Like incoming pathways, outgoing pathways do participate in a larger complex together with the elements of the main complex, but the \u03a6 value of this larger complex is very low, being limited by the effective information between each port-out of the main complex and its effector targets.\n\n### Neural processes in cortico-subcortico-cortical loops, while important in the production and sequencing of action, thought, and language, do not contribute directly to conscious experience\n\nAnother set of neural structures that may not contribute directly to conscious experience are subcortical structures such as the basal ganglia. The basal ganglia are large nuclei that contain many circuits arranged in parallel, some implicated in motor and oculomotor control, others, such as the dorsolateral prefrontal circuit, in cognitive functions, and others, such as the lateral orbitofrontal and anterior cingulate circuits, in social behavior, motivation, and emotion \\[45\\]. Each basal ganglia circuit originates in layer V of the cortex, and through a last step in the thalamus, returns to the cortex, not far from where the circuit started \\[46\\]. Similarly arranged cortico-ponto-cerebello-thalamo-cortical loops also exist. Why is it that these complicated neural structures, which are tightly connected to the thalamocortical system at both ends, do not seem to provide much direct contribution to conscious experience? (see Appendix, x)\n\nAs shown in Fig. <a href=\"#F4\" data-ref-type=\"fig\">4e<\/a>, the addition of many parallel cycles also generally does not change the composition of the main complex, although \u03a6 values can be altered (see Appendix, xi). Instead, the elements of the main complex and of the connected cycles form a joint complex that can only integrate the limited amount of information exchanged within each cycle. Thus, subcortical cycles or loops implement specialized subroutines that are capable of influencing the states of the main thalamocortical complex without joining it. Such informationally insulated cortico-subcortical loops could constitute the neural substrates for many unconscious processes that can affect and be affected by conscious experience \\[3,47\\]. It is likely that new informationally insulated loops can be created through learning and repetition. For example, when first performing a new task, we are conscious of every detail of it, we make mistakes, are slow, and must make an effort. When we have learned the task well, we perform it better, faster, and with less effort, but we are also less aware of it. As suggested by imaging results, a large number of neocortical regions are involved when we first perform a task. With practice, activation is reduced or shifts to different circuits \\[48\\]. According to the theory, during the early trials, performing the task involves many regions of the main complex, while later certain aspects of the task are delegated to neural circuits, including subcortical ones, that are informationally insulated.\n\n### Many neural processes within the thalamocortical system may also influence conscious experience without contributing directly to it\n\nEven within the thalamocortical system proper, a substantial proportion of neural activity does not appear to contribute directly to conscious experience. For example, what we see and hear requires elaborate computational processes dealing with figure-ground segregation, depth perception, object recognition, and language parsing, many of which take place in the thalamocortical system. Yet we are not aware of all this diligent buzzing: we just *see* objects, separated from the background and laid out in space, and know what they are, or *hear* words, nicely separated from each other, and know what they mean. As an example, take binocular rivalry, where the two eyes view two different images, but we perceive consciously just one image at a time, alternating in sequence. Recordings in monkeys have shown that the activity of visual neurons in certain cortical areas, such as the inferotemporal cortex, follows faithfully what the subject perceives consciously. However, in other areas, such as primary visual cortex, there are many neurons that respond to the stimulus presented to the eye, whether or not the subject is perceiving it \\[49\\]. Neuromagnetic studies in humans have shown that neural activity correlated with a stimulus that is not being consciously perceived can be recorded in many cortical areas, including the front of the brain. \\[26\\]. Why does the firing of many cortical neurons carrying out the computational processes that enable object recognition (or language parsing) not correspond to anything conscious?\n\nThe situation is similar on the executive side of consciousness. When we plan to do or say something, we are vaguely conscious of what we intend, and presumably these intentions are reflected in specific firing patterns of certain neuronal groups. Our vague intentions are then translated almost miraculously into the right words, and strung together to form a syntactically correct sentence that conveys what we meant to say. And yet again, we are not at all conscious of the complicated processing that is needed to carry out our intentions, much of which takes place in the cortex. What determines whether the firing of neurons within the thalamocortical system contributes directly to consciousness or not? According to the information integration theory, the same considerations that apply to input and output circuits and to cortico-subcortico-cortical loops also apply to circuits and loops contained entirely within the thalamocortical system. Thus, the theory predicts that activity within certain cortical circuits does not contribute to consciousness because such circuits implement informationally insulated loops that remain outside of the main thalamocortical complex. At this stage, however, it is hard to say precisely which cortical circuits may be informationally insulated. Are primary sensory cortices organized like massive afferent pathways to a main complex \"higher up\" in the cortical hierarchy? Is much of prefrontal cortex organized like a massive efferent pathway? Do certain cortical areas, such as those belonging to the dorsal visual stream, remain partly segregated from the main complex? Do interactions *within* a cortico-thalamic minicolumn qualify as intrinsic mini-loops that support the main complex without being part of it? Unfortunately, answering these questions and properly testing the predictions of the theory requires a much better understanding of cortical neuroanatomy than is presently available \\[50,51\\].\n\n### Consciousness can be split if the thalamocortical system is split\n\nStudies of split-brain patients, whose corpus callosum was sectioned for therapeutic reasons, show that each hemisphere has its own, private conscious experience. The dominant, linguistically competent hemisphere does not seem to suffer a major impairment of consciousness after the operation. The non-dominant hemisphere, although it loses some important abilities and its residual capacities are harder to assess, also appears to be conscious. \\[5\\]. Some information, e.g. emotional arousal, seems to be shared across the hemispheres, probably thanks to subcortical common inputs.\n\nViewing consciousness as information integration suggests straightforward explanations for these puzzling observations. Consider the simplified model in Fig. <a href=\"#F5\" data-ref-type=\"fig\">5a<\/a>. A main complex having high \u03a6 includes two sets of elements (\"hemispheres\") having similar internal architecture that are joined by \"callosal\" connections (top panel). When the callosal connections are cut (bottom panel), the single main complex splits and is replaced by two smaller complexes corresponding to the two hemispheres. There is also a complex, of much lower \u03a6, which includes both hemispheres and a \"subcortical\" element that provide them with common input. Thus, there is a sense in which the two hemispheres still form an integrated entity, but the information they share is minimal (see Appendix, xii).\n\n### Some parts of the thalamocortical system may contribute to conscious experience at one time and not at another\n\nUntil now, we have considered structural aspects of the organization of the nervous system that, according to the information integration theory, explain why certain parts of the brain contribute directly to consciousness and others do not, or much less so. In addition to neuroanatomical factors, neurophysiological factors are also important in determining to what extent a given neural structure can integrate information. For example, anatomical connections between brain regions may or may not be functional, depending on both pathological or physiological factors. Functional disconnections between certain parts of the brain and others are thought to play a role in psychiatric conversion and dissociative disorders, may occur during dreaming, and may be implicated in conditions such as hypnosis. Thus, functional disconnections, just like anatomical disconnections, may lead to a restriction of the neural substrate of consciousness.\n\nIt is also likely that certain attentional phenomena may correspond to changes in the neural substrate of consciousness. For example, when one is absorbed in thought, or focused exclusively on a given sensory modality, such as vision, the neural substrate of consciousness may not be the same as when we are diffusely monitoring the environment. Phenomena such as the attentional blink, where a fixed sensory input may at times make it to consciousness and at times not, may also be due to changes in functional connectivity: access to the main thalamocortical complex may be enabled or not based on dynamics intrinsic to the complex \\[52\\]. Phenomena such as binocular rivalry may also be related, at least in part, to dynamic changes in the composition of the main thalamocortical complex caused by transient changes in functional connectivity \\[53\\]. At present, however, it is still not easy to determine whether a particular group of neurons is excluded from the main complex because of hard-wired anatomical constraints, or is transiently disconnected due to functional changes.\n\nFigure <a href=\"#F5\" data-ref-type=\"fig\">5b<\/a> (top panel) shows a simple model obtained by taking three subsets of elements of (relatively) high \u03a6 and connecting them through reciprocal connections. Specifically, the first subset, which stands for supramodal areas of the brain, is reciprocally connected to the second and third subsets, which stand for visual and auditory areas, respectively. In this idealized example, the visual and auditory subsets are not connected directly among themselves. As one can see, the three subsets thus connected form a single main complex having a \u03a6 value of 61 bits. In the bottom panel, the auditory subset has been disconnected, in a functional sense, by mimicking a profound deactivation of its elements. The result is that the main complex shrinks and the auditory subset ends up outside the main complex. Note, however, that in this particular case the value of \u03a6 changes very little (57 bits), indicating that it might be possible for the borders of the main complex to change dynamically while the amount of consciousness is not substantially altered. What would change, of course, would be the configuration of the space of informational relationships. These simulations suggest that attentional mechanisms may work both by changing neuronal firing rates, and therefore saliency within qualia space, as well as by modifying neuronal readiness to fire, and therefore the boundaries of the main complex and of qualia space itself. This is why the set of elements underlying consciousness is not static, but can be considered to form a \"*dynamic complex*\" or \"*dynamic core*\" \\[1,9\\].\n\n### Depending on certain neurophysiological parameters, the same thalamocortical network can generate much or little conscious experience\n\nAnother example of the importance of neurophysiological parameters is provided by sleep \u2013 the most familiar of the alterations of consciousness, and yet one of the most striking. Upon awakening from dreamless sleep, we have the peculiar impression that for a while we were not there at all nor, as far as we are concerned, was the rest of the world. This everyday observation tells us vividly that consciousness can come and go, grow and shrink. Indeed, if we did not sleep, it might be hard to imagine that consciousness is not a given, but depends somehow on the way our brain is functioning. The loss of consciousness between falling asleep and waking up is relative, rather than absolute. \\[54\\]. Thus, careful studies of mental activity reported immediately after awakening have shown that some degree of consciousness is maintained during much of sleep. Many awakenings, especially from rapid eye movement (REM) sleep, yield dream reports, and dreams can be at times as vivid and intensely conscious as waking experiences. Dream-like consciousness also occurs during various phases of slow wave sleep, especially at sleep onset and during the last part of the night. Nevertheless, a certain proportion of awakenings do not yield any dream report, suggesting a marked reduction of consciousness. Such \"empty\" awakenings typically occur during the deepest stages of slow wave sleep (stages 3 and 4), especially during the first half of the night.\n\nWhich neurophysiological parameters are responsible for the remarkable changes in the quantity and quality of conscious experience that occur during sleep? We know for certain that the brain does not simply shut off during sleep. During REM sleep, for example, neural activity is as high, if not higher, than during wakefulness, and EEG recordings show low-voltage fast-activity. This EEG pattern is known as \"activated\" because cortical neurons, being steadily depolarized and close to their firing threshold, are ready to respond to incoming inputs. Given these similarities, it is perhaps not surprising that consciousness should be present during both states. Changes in the quality of consciousness, however, do occur, and they correspond closely to relative changes in the activation of different brain areas. \\[54\\].\n\nDuring slow wave sleep, average firing rates of cortical neurons are also similar to those observed during quiet wakefulness. However, due to changes in the level of certain neuromodulators, virtually all cortical neurons engage in slow oscillations at around 1 Hz, which are reflected in slow waves in the EEG \\[55\\]. Slow oscillations consist of a depolarized phase, during which the membrane potential of cortical neurons is close to firing threshold and spontaneous firing rates are similar to quiet wakefulness, and of a hyperpolarized phase, during which neurons become silent and are further away from firing threshold. From the perspective of information integration, a reduction in the readiness to respond to stimuli during the hyperpolarization phase of the slow oscillation would imply a reduction of consciousness. It would be as if we were watching very short fragments of a movie interspersed with repeated unconscious \"blanks\" in which we cannot see, think, or remember anything, and therefore have little to report. A similar kind of unreadiness to respond, associated with profound hyperpolarization, is found in deep anesthesia, another condition where consciousness is impaired. Studies using transcranial magnetic stimulation in conjunction with high-density EEG are currently testing how response readiness changes during the sleep waking cycle.\n\nFrom the perspective of information integration, a reduction of consciousness during certain phases of sleep would occur even if the brain remained capable of responding to perturbations, provided its response were to lack differentiation. This prediction is borne out by detailed computer models of a portion of the visual thalamocortical system (Hill and Tononi, in preparation). According to these simulations, in the waking mode different perturbations of the thalamocortical network yield specific responses. In the sleep mode, instead, the network becomes bistable: specific effects of different perturbations are quickly washed out and their propagation impeded: the whole network transitions into the depolarized or into the hyperpolarized phase of the slow oscillation \u2013 a stereotypic response that is observed irrespective of the particular perturbation (see Appendix, xiii). And of course, this bistability is also evident in the spontaneous behavior of the network: during each slow oscillation, cortical neurons are either all firing or all silent, with little freedom in between. In summary, these simulations indicate that, even if the anatomical connectivity of a complex stays the same, a change in key parameters governing the readiness of neurons to respond and the differentiation of their responses may alter radically the \u03a6 value of the complex, with corresponding consequences on consciousness.\n\n### Conscious experience and time requirements\n\nConsciousness not only requires a neural substrate with appropriate anatomical structure and appropriate physiological parameters: it also needs time. As was mentioned earlier, studies of how a percept is progressively specified and stabilized indicate that it takes up to 100\u2013200 milliseconds to develop a fully formed sensory experience, and that the surfacing of a conscious thought may take even longer. Experiments in which the somatosensory areas of the cerebral cortex were stimulated directly indicate that low intensity stimuli must be sustained for up to 500 milliseconds to produce a conscious sensation \\[56\\]. Multi-unit recordings in the primary visual cortex of monkeys show that, after a stimulus is presented, the firing rate of many neurons increases irrespective of whether the animal reports seeing a figure or not. After 80\u2013100 milliseconds, however, their discharge accurately predicts the conscious detection of the figure. Thus, the firing of the same cortical neurons may correlate with consciousness at certain times, but not at other times \\[57\\]. What determines when the firing of the same cortical neurons contributes to conscious experience and when it does not? And why may it take up to hundreds of milliseconds before a conscious experience is generated?\n\nThe theory predicts that the time requirements for the generation of conscious experience in the brain emerge directly from the time requirements for the build-up of effective interactions among the elements of the main complex. As was mentioned above, if one were to perturb half of the elements of the main complex for less than a millisecond, no perturbations would produce any effect on the other half within this time window, and \u03a6 would be equal to zero. After say 100 milliseconds, however, there is enough time for differential effects to be manifested, and \u03a6 should grow. This prediction is confirmed by results obtained using large-scale computer simulations of the thalamocortical system, where the time course of causal interactions and functional integration can be studied in detail \\[38,58,59\\], Hill and Tononi, unpublished results). For example, in a model including nine functionally segregated visual areas, the time it takes for functionally specialized neurons located in several different areas to interact constructively and produce a specific, correlated firing pattern is at least 80 milliseconds \\[38\\]. These correlated firing patterns last for several hundred milliseconds. After one or more seconds, however, the network settles into spontaneous activity states that are largely independent of previous perturbations. Thus, the characteristic time scale for maximally differentiated responses in thalamocortical networks appears to be comprised between a few tens of milliseconds and a few seconds at the most.\n\nIn summary, the time scale of neurophysiological interactions needed to integrate information among distant cortical regions appears to be consistent with that required by psychophysical observations (microgenesis), by stimulation experiments, and by recording experiments.\n\n# Summary: seeing blue\n\nThe previous examples show that the information integration theory is consistent with several empirical observations concerning the neural substrate of consciousness. Moreover, they show that the theory can provide a principled account of why consciousness is associated with certain parts of the brain rather than with others, and with certain global modes of functioning more than with others. To recapitulate the main tenets of the theory, it may be useful to reconsider the initial thought experiment.\n\nImagine again that you are comfortably facing a blank screen that is alternately on and off. When the screen turns on, you see a homogenous blue field, indeed for the sake of the argument we assume that you are having a \"pure\" perception of blue, unencumbered by extraneous percepts or thoughts (perhaps as can be achieved in certain meditative states). As you have been instructed, you signal your perception of blue by pushing a button. Now consider an extremely simplified scenario of the neural events that might accompany your seeing blue. When the screen turns on, a volley of activity propagates through the visual afferent pathways, involving successive stages such as retinal short wavelength cones, blue-yellow opponents cells, color constant cells, and so on. Eventually, this volley of activity in the visual afferent pathways leads to the firing of some neuronal groups in color-selective areas of the temporal lobe that, on empirical grounds, are our best bet for the neural correlate of blue: i) their activity correlates well with your perception of blue whether you see, imagine, or dream blue, in a way that is as stable and as susceptible to illusions as your perception of blue; ii) their microstimulation leads to the perception of blue; and iii) their selective lesion makes you unable to perceive blue. Let us assume, then, that these neuronal groups quickly increase their firing, and within a few tens of milliseconds they reach and then maintain increased levels of firing (see Appendix, xiv). We also assume that, at the same time, neuronal groups in neighboring cortical areas go on firing at a baseline level, largely unaffected by the blue light. These include neuronal groups in other visual areas that are selective for shape or movement; neuronal groups in auditory area that are selective for tones; and many others. On the other hand, the volley of activity originating in the retina does not exhaust itself by generating sustained activity in the color areas of the temporal lobe. Part of the volley proceeds at great speed and activates efferent motor pathways, which cause you to push the signaling button. Another part activates cortico-subcortico-cortical loops in your prefrontal cortex and basal ganglia, which almost make you speak the word \"blue\" aloud. In the meantime, many other parts of the brain are buzzing along, unaffected by what is going on in the visual system: cerebellar circuits are actively stabilizing your posture and gaze, and hypothalamic-brainstem circuits are actively stabilizing your blood pressure. What components in this simplified neural scenario are essential for your conscious experience of blue, and why?\n\nThe information integration theory makes several claims that lead to associated predictions. A first claim is that the neural substrate of consciousness as we know it is a complex of high \u03a6 that is capable of integrating a large amount of information \u2013 the main complex. Therefore, *whether* a group of neurons contributes directly to consciousness is a function of its belonging to the main complex or not. In this example, the theory would predict that blue-selective neurons in some high-level color area should be *inside* the main complex; on the other hand, blue-sensitive neurons in afferent visual pathways, neurons in efferent pathways mediating the button-pressing response, neurons in cortico-subcortico-cortical and intracortical loops mediating subvocalization of the word \"blue\", neurons in the cerebellum controlling posture and neurons in hypothalamic-brainstem circuits controlling blood pressure should be *outside*. This even though these neurons may be equally active when you see blue, and even though some of them may be connected to elements of the main complex. In principle, joint microstimulation and recording experiments, and to some extent an analysis of patterns of synchronization, could determine participation in the main complex and test this prediction. The theory also predicts that blue-selective neurons in the main complex contribute to the conscious experience of blue only if their activation is sufficiently strong or sustained that they can make a difference, in informational terms, to the rest of the complex. Additional predictions are that, if a group of neurons that is normally part of the main complex becomes informationally disconnected from it, as could occur through attentional effects or in certain phases of sleep, the same group of neurons, firing in exactly the same way, would not contribute to consciousness. Moreover, according to the theory, the other groups of neurons within the main complex are essential to our conscious experience of blue even if, as in this example, they are not activated. This is not difficult to see. Imagine that, starting from an intact main complex, we were to remove one element after another, except for the active, blue-selective one. If an inactive element contributing to \"seeing red\" were removed, blue would not be experienced as blue anymore, but as some less differentiated color, perhaps not unlike those experienced by certain dichromats. If further elements of the main complex were removed, including those contributing to shapes, to sounds, to thoughts and so forth, one would soon drop to such a low level of consciousness that \"seeing blue\" would become meaningless: the \"feeling\" (and meaning) of the quale \"blue\" would have been eroded down to nothing. Indeed, while the remaining neural circuits may still be able to discriminate blue from other colors, they would do so very much as a photodiode does (see Appendix, xv).\n\nA second claim of the theory is that the quality of consciousness is determined by the informational relationships within the main complex. Therefore, *how* a group of neurons contributes to consciousness is a function of its informational relationships inside the complex and not outside of it. In this example, blue-selective neurons within the main complex have become blue-selective no doubt thanks to the inputs received from the appropriate afferent pathways, and ultimately because of some aspects of the statistics of the environment and the resulting plastic changes throughout the brain. However, the theory predicts that their present firing contributes the quale \"blue\" exclusively because of their informational relationships within the main complex. If connections *outside* the main complex were to be manipulated, including the afferent color pathways, the experience elicited by activating the blue-selective neurons within the complex would stay the same. Conversely, if the relationships *inside* the main complex were to change, as could be done by changing the pattern of connections within the color-selective area and with the rest of the complex, so would the conscious experience of blue. That is, activating the same neurons would produce a different conscious experience.\n\n# Implications of the hypothesis\n\nTo conclude, it is worth mentioning some of the implications that derive from the information integration theory of consciousness. At the most general level, the theory has ontological implications. It takes its start from phenomenology and, by making a critical use of thought experiments, it argues that subjective experience is one and the same thing as a system's capacity to integrate information. In this view, experience, that is, information integration, is a fundamental quantity, just as mass, charge or energy are. It follows that any physical system has subjective experience to the extent that it is capable of integrating information, irrespective of what it is made of. Thus, an intriguing implication of the theory is that it should be possible to construct conscious artifacts by endowing them with a complex of high \u03a6. Moreover, it should be possible to design the quality of their conscious experience by appropriately structuring their effective information matrix.\n\nIt also follows that consciousness is not an all-or-none property, but it is graded: to varying degrees, it should exist in most natural (and artificial) systems. Because the conditions needed to build complexes of high \u03a6 are apparently not easy to achieve, however, correspondingly high levels of experience are probably available to only a few kinds of systems, primarily complex brains containing the right type of architecture for maximizing functional specialization and integration. A related implication is that consciousness should also exist, to varying degrees, at multiple spatial and temporal scales. However, it is likely that, in most systems, there are privileged spatial and temporal scales at which information integration reaches a maximum.\n\nConsciousness is characterized here as a disposition or *potentiality* \u2013 in this case as the potential differentiation of a system's responses to all possible perturbations, yet it is undeniably *actual*. Consider another thought experiment: you could be in a coma for days, awaken to consciousness for just one second, and revert to a coma. As long as your thalamocortical system can function well for that one second, you will be conscious. That is, a system does not have to explore its repertoire of states to be conscious, or to know how conscious it is supposed to be: what counts is only that the repertoire is potentially available. While this may sound strange, fundamental quantities associated with physical systems can also be characterized as dispositions or potentialities, yet have actual effects. For example, mass can be characterized as a potentiality \u2013 say the resistance that a body would offer to acceleration by a force \u2013 yet it exerts undeniable effects, such as attracting other masses. This too has intriguing implications. For example, because in this view consciousness corresponds to the potential of an integrated system to enter a large number of states by way of causal interactions within it, experience is present as long as such potential is present, whether or not the system's elements are activated. Thus, the theory predicts that a brain where no neurons were activated, but were kept ready to respond in a differentiated manner to different perturbations, would be conscious (perhaps that nothing was going on). Also, because consciousness is a property of a system, not of a state, the state the system is in only determines which particular experience becomes actual at any given time, and not whether experience is present. Thus, a brain where each neuron were microstimulated to fire as an exact replica of your brain, but where synaptic interactions had been blocked, would be unconscious.\n\nThe theory predicts that consciousness depends exclusively on the ability of a system to integrate information, whether or not it has a strong sense of self, language, emotion, a body, or is immersed in an environment, contrary to some common intuitions. This prediction is consistent with the preservation of consciousness during REM sleep, when both input and output signals from and to the body are markedly reduced. Transient inactivation of brain areas mediating the sense of self, language, and emotion could assess this prediction in a more cogent manner.\n\nNevertheless, the theory recognizes that these same factors are important historically because they favor the development of neural circuits forming a main complex of high \u03a6. For example, the ability of a system to integrate information grows as that system incorporates statistical regularities from its environment and learns \\[14\\]. In this sense, the emergence of consciousness in biological systems is predicated on a long evolutionary history, on individual development, and on experience-dependent change in neural connectivity. Indeed, the theory also suggests that consciousness provides an adaptive advantage and may have evolved precisely because it is identical with the ability to integrate a lot of information in a short period of time. If such information is about the environment, the implication is that, the more an animal is conscious, the larger the number of variables it can take into account jointly to guide its behavior.\n\nAnother implication of the theory is that the presence and extent of consciousness can be determined, in principle, also in cases in which we have no verbal report, such as infants or animals, or in neurological conditions such as coma and vegetative states, minimally conscious states, akinetic mutism, psychomotor seizures, and sleepwalking. In practice, of course, measuring \u03a6 accurately in such systems will not be easy, but approximations and informed guesses are certainly conceivable.\n\nAt present, the validity of this theoretical framework and the plausibility of its implications rest on its ability to account, in a coherent manner, for some basic phenomenological observations and for some elementary but puzzling facts about the relationship between consciousness and the brain. Experimental developments, especially of ways to stimulate and record concurrently the activity of broad regions of the brain, should permit stringent tests of some of the theory's predictions. Equally important will be the development of realistic, large-scale models of the anatomical organization of the brain. These models should allow a more rigorous measurement of how the capacity to integrate information relates to different brain structures and certain neurophysiological parameters \\[38,50,59\\]. Finally, the theoretical framework presented here aims primarily at understanding the necessary and sufficient conditions that determine the quantity and quality of consciousness at the most general level. Further theoretical developments will be required to address several issues that are central to the study of consciousness in a biological and psychological context, such as the relationship of consciousness to memory and language, higher order aspects of consciousness \\[60,61\\], and its relationship to the self) \\[62\\]. Undoubtedly, a full understanding of how the brain generates human consciousness remains a formidable task. However, if experimental investigations can be complemented by a principled theoretical approach, it may not lay beyond the reach of science.\n\n# Appendix\n\ni\\. The problem can also be posed in neural terms. When we see light, certain neurons in the retina turn on, as do other neurons higher up in the brain. Based on what we know, the activity of neurons in the retina is not directly associated with conscious experience of light and dark \u2013 they behave just like biological photodiodes that signal to higher centers. Somewhere in those higher centers, however, there seem to be some neurons whose activity is indeed tightly correlated with the conscious experience of light and dark. What is special about these higher neurons?\n\nii\\. Note that this information has nothing to do with how complicated the scene is, or how many different objects it appears to contain, but only with the number of alternative outcomes.\n\niii\\. This quantity is also called ^*MIB*^*complexity*, for minimum information bipartition complexity. Note that, in most cases, the bipartitions for which the normalized value of EI will be at a minimum, everything else being equal, will be bipartitions that cut the system in two halves, i.e. midpartitions \\[2\\].\n\niv\\. Complexes can also be defined using mutual information instead of effective information, by exploiting the endogenous sources of variance that may exist in an isolated system \\[8\\]. A related measure could be constructed using the formalism of \u03b5-machines \\[63\\]. \u03a6 would then be related to the H^\u03bc^ of the minimal \u03b5-machine capable of reproducing the causal structure of a process, i.e. of the \u03b5-machine that cannot be decomposed into a collection of lower H^\u03bc^ \u03b5-machines.\n\nv\\. An elementary description of the qualia space is given by the author in \\[9\\], chapter 13.\n\nvi\\. While the entries in the matrix contain all the relevant informational relationships defining this space, they do not reveal necessarily how the space is organized in an economical and explicit manner. This may be done by employing procedures akin to multidimensional scaling although, since the matrix is asymmetrical and involves high-order terms (among subsets of elements), this may not be easy. Satisfactorily mapping the phenomenological differences between modalities, submodalities and dimensions onto the structure of qualia space will require that we thoroughly characterize and understand the latter.\n\nvii\\. Of course, sensory afferents usually play a role in determining which particular conscious experience we have at any given time (they better do so, if experience is to have an adaptive relationship to the environment). Nevertheless, particular experiences can be triggered even when we are disconnected from the environment, as in dreams.\n\nviii\\. Note also that a \"pure\" sensation of blue defines a point in this N-dimensional qualia space as much as the experience of a busy city street, full of different objects, of sounds, smells, associations, and reflections defines another point.\n\nix\\. However, certain areas such as the posterior cingulate cortex and precuneus, some lateral parietal areas, and associated paramedian thalamic nuclei, may constitute strategic crossroads for coordinating the interactions among different sensory maps and frames of reference concerning the body and the environment. Bilateral lesions to such areas may lead to a virtual breakdown of information integration in the thalamocortical system \\[22,24\\]. A global, persistent disruption of consciousness can also be produced by focal lesions of paramedian mesodiencephalic structures, which include the intralaminar thalamic nuclei. Most likely, such focal lesions are catastrophic because the strategic location and connectivity of paramedian structures ensure that distributed cortico-thalamic loops can work together as a system.\n\nx\\. Statements about the lack of direct contributions to consciousness of basal ganglia loops need to be qualified due to the difficulty of evaluating the precise effects of their selective inactivation, as well as to the unreliability of introspective assessments about the richness of one's experience, especially after brain lesions. Similar considerations apply to brain structures not discussed here, such as the claustrum, the amygdala, and the basal forebrain.\n\nxi\\. A similar kind of analysis could be applied to other neurological disconnection syndromes.\n\nxii\\. An explanation in terms of reduced degrees of freedom may also apply to loss of consciousness in absence and other seizures, during which neural activity is extremely high and near-synchronous over many cortical regions (Tononi, unpublished results).\n\nxiii\\. While we do not yet have such a tight case for the neural correlate of blue, we are close to it with motion sensitive cells in area MT and in somatosensory cortex, at least in monkeys \\[64\\].\n\nxv\\. In this sense, a particular conscious experience, its meaning, and the underlying informational relationships within a complex end up being one and the same thing. Such internalistic, relationally defined meanings generally relate to and ultimately derive from entities in the world. To the extent that the brain has a long evolutionary history and is shaped by experience, it is clear that internally specified meanings (and conscious states) bear an adaptive relationship to what is out there.\n\n### Acknowledgements\n\nI thank Chiara Cirelli, Lice Ghilardi, Sean Hill, Marcello Massimini, and Olaf Sporns for helpful discussions.\n\n## Figures and Tables","meta":{"dup_signals":{"dup_doc_count":149,"dup_dump_count":76,"dup_details":{"curated_sources":3,"2023-50":2,"2023-23":1,"2023-14":1,"2022-49":2,"2022-33":2,"2022-21":2,"2022-05":1,"2021-43":1,"2021-39":1,"2021-25":2,"2021-17":3,"2021-04":1,"2020-45":2,"2020-40":1,"2020-29":1,"2020-16":1,"2020-10":2,"2020-05":1,"2019-47":2,"2019-39":1,"2019-35":1,"2019-30":1,"2019-22":3,"2019-13":1,"2019-09":1,"2019-04":1,"2018-51":2,"2018-47":1,"2018-43":2,"2018-39":4,"2018-34":1,"2018-30":1,"2018-26":3,"2018-17":1,"2018-13":1,"2018-09":2,"2018-05":1,"2017-47":1,"2017-39":1,"2017-34":2,"2017-30":1,"2017-26":2,"2017-22":5,"2017-17":5,"2017-09":5,"2017-04":2,"2016-50":2,"2016-44":3,"2016-40":3,"2016-36":3,"2016-30":8,"2016-26":1,"2016-22":1,"2016-18":1,"2016-07":1,"2015-40":2,"2015-35":1,"2015-22":3,"2015-14":1,"2014-52":3,"2014-49":1,"2014-42":1,"2014-41":1,"2014-35":3,"2014-23":4,"2014-15":2,"2024-22":2,"2024-18":1,"2017-13":6,"2015-18":2,"2015-11":1,"2015-06":2,"2014-10":1,"2013-48":2,"2013-20":2,"2024-30":1}},"file":"PMC543470"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  The majority of South African aging population are women, who spend late adulthood experiencing natural menopause. Despite the government spending billions of rand on different services for ageing women, menopausal challenges to African women still receive little attention.\n  .\n  # Objectives\n  .\n  The aim of the study was to explore and describe the perceptions of African women regarding natural menopause, in order to propose recommendations for health and social support systems for women in Mamelodi, Tshwane district.\n  .\n  # Method\n  .\n  A qualitative, exploratory, descriptive and contextual design was used to conduct the study. The population of the study consisted of menopausal women, between the ages 45 and 60 years or more, visiting the clinics for collection of chronic medication and other health assessment. Individual face-to-face interviews were conducted, using a semi-structured interview guide to collect data. Tesch's method of qualitative data analysis was used in the study.\n  .\n  # Results\n  .\n  The main theme that emerged from the study was 'attitude toward menopause', which was supported by cultural beliefs and experience. The African menopausal women expressed the importance of health support systems that will meet their needs within their context.\n  .\n  # Conclusion\n  .\n  Women's health programs and educational health information at facilities should include menopausal education to promote and improve health of all African menopausal women during their adulthood. There is a need to establish a women's health support group network within communities to share menopausal experiences with peers. The training and education curriculum of healthcare providers should include detailed menopause in order to provide comprehensive, congruent care.\nauthor: Gloria N. Makuwa; Steppies R. Rikhotso; Fhumulani M. Mulaudzi**Correspondence to:** Gloria Makuwa, **Email:**<email@example.com>**Postal address:** 1499 Kaallaagte Street, Pretoria 0186, South Africa[^1]\ndate: 2015-12-17\nreferences:\ntitle: The perceptions of African women regarding natural menopause in Mamelodi, Tshwane district\n\n# Introduction\n\nMenopause is a normal, natural phenomenon for all ageing women, irrespective of culture or country of origin (Ramakuela et al. 2014:1). Rabiee, Nasirie and Zafarqandie (2014:4) explained menopause as a complex and multidimensional biological, social and psychological phenomenon for women between, on average, the ages of 45 and 60 (or more) years.\n\nTo promote women's health during middle age and beyond, it is necessary to understand that menopause is a natural ageing process that all women undergo because of reduced production of reproductive hormones, oestrogen and progesterone (Lobo 2012; Rahman, Salehin & Iqbal 2011:188). There are a multitude of factors to be considered for menopause besides the women's age; these include biological, socioeconomic and lifestyle factors. Rabiee et al. (2014:1) argued about the factors which affect the transitional period in a woman's life and its relation to sexual desire in some women.\n\nAccording to Norwitz and Schorge (2010:63), women experience menopause in a universal manner, as all bodily signs and symptoms are influenced by factors such as an ageing endocrine system leading to decline in ovarian function, a decrease in oestrogen that causes hot flushes, night sweats and vaginal dryness. However, the actual experience and attitude of women on menopause is individualistic. The attitude of women is ominously influenced by sociocultural factors \u2013 race, culture and ethnicity \u2013 as well as multicultural factors (Rapaport 2015:1). Agwu, Umeora and Ejikeme (2008:217) argued differently that not all women experienced menopause the same way, as their perceptions depend on their individual and sociocultural backgrounds.\n\nIn multicultural South Africa, health beliefs and practices vary within different communities. Menopause is viewed by some African women as the freedom from monthly menstruation and the transition period toward permanent infertility (Helman 2007:47; Ramakuela et al. 2014:2). Helman (2007:48) added that in the Zulu culture, for example, women who are in menopause are viewed as clean and can enter into the field or cows' kraal without ruining the crops or causing sickness to the cattle. Ande et al. (2011:300) and Ozuzu-Nwaiwu (2007:34) support the view that most African women's cultures accept menopause as a natural ageing process caused by a supernatural effects, resulting in African women rarely refusing hormone replacement therapy (HRT) (Ande et al. 2011:300; Ozuzu-Nwaiwu 2007:34), because of inadequate reproductive health-related knowledge about the phenomenon.\n\nSome African women's cultures perceive menopause as being a negative life event and a 'disease-entrapment process' resulting from cessation of menstruation, causing illness or poison in the woman's body system. Limited literature has been conducted in the South African context to understand the perceptions of African women regarding menopause (Ozuzu-Nwaiwu 2007:35). As a result, the study was conducted to explore and describe the perceptions of African women regarding natural menopause in Mamelodi, Tshwane district.\n\n## Problem statement\n\nWomen in multicultural South Africa perceive natural menopause differently as it is influenced by geographical location, cultural beliefs, socialisation, female reproductive health knowledge deficit and societal practices. Ramakuela et al. (2014:1) alluded that menopause in a rural village of Limpopo Province is determined by cultural beliefs because menopause is considered to be a taboo and\/or sensitive discussion topic, meaning that women remain silent for fear of embarrassment (Agwu et al. 2008:220). Mustafa and Sabir (2012:176) revealed that other cultures, for example, Western women, report menopausal symptoms and receive assistance and support as they have reproductive health-related knowledge, including regarding ageing process. The cultural silence held by some African menopausal women may result in health-related problems because of inadequate knowledge. Approximately 51% of the South African population are women, with an average age of 55.2 years (Statistics South Africa 2012:3) and it is of great concern that few studies have been conducted that focus on understanding the cultural perceptions of South African women regarding reproductive health knowledge on natural menopause.\n\nCurrently, African women who are experiencing menopause reported health problems related to menopause only if they had a serious illness accompanied by unexplained pains, irritability, unusual emotional behaviour or unexplained signs and symptoms. The failure of some menopausal women to openly discuss and share information and reproductive health-related knowledge regarding natural menopause may lead to misunderstandings about menopause. There have been only a few studies done which are related to the perceptions of women regarding natural menopause.\n\n### Aim and objective of the study\n\nThe aim of the study was to explore and describe the perceptions of women regarding natural menopause in order to propose recommendations for healthcare providers to provide reproductive health-related knowledge regarding human developmental stages for all women in Tshwane district.\n\n## Definition of concepts\n\n### Perceptions\n\nThe receiving, selecting, organising and interpreting of sensory information or stimuli by combining them with the results of previous experience to produce a meaningful picture of one's world (Lantos 2010:388). It refers to how natural menopausal African women describe the experiences and feelings of being in the transitional period of their life, between the ages 45 to 60 years.\n\n### African woman\n\nAn 'African woman' is an adult woman who bears children and has a specified occupation or role in a family context such as a wife and\/or mother or grandmother (Stevenson & Waite 2011:1659). In this study, it refers to an African woman between the ages of 46 and 55, experiencing natural menopause and residing in Mamelodi, Tshwane district. The African woman's lifestyle and way of life is to focus on child bearing and caring for their large family (Hall et al. 2007:107).\n\n### Menopause\n\nThis refers to the condition designed by nature and regarded as a normal ageing process (Ramakuela et al. 2014:1), whereby the woman's monthly menstrual periods ceases. It is normally diagnosed retrospectively a year following secondary amenorrhoea in woman of 45 years of age or older.\n\n### Significance of study\n\nThe findings of this study may assist in providing African women with health-related knowledge regarding menopause and contribute to the body of knowledge in nursing and other health professionals to provide comprehensive and congruent care to menopausal women of different African cultures. This could be achieved by sharing information through training institutions for the inclusion of the curriculum on women's health, focusing on menopause. The findings will further inform the authorities to provide assistance to ageing African women for the establishment of health support groups in the community of Mamelodi in Tshwane district, where menopausal women share their menopausal experiences with peers.\n\n# Research design\n\nThe research design is the overall plan for conducting research that will be applied in an attempt to address the research questions, including specifications for enhancing the study's integrity (De Vos et al. 2011:309). A qualitative, explorative, descriptive and contextual design was used.\n\n## Research approach and method\n\nThe design enhanced a systematic and interactive approach exploring life experiences of human participants to attach meaning (Polit & Beck 2012:16). The explorative aspect of the study was to explore the dimensions and relationships between the phenomena that were studied (Polit & Beck 2012:727); and the existing perceptions of African women regarding natural menopause were discovered through interviews and observations.\n\nDescriptive study, according to Botma et al. (2010:194) is a study that is based on the general premises of naturalistic inquiry. The perceptions of African women regarding natural menopause were described within the context of the study setting. Contextual research is based on findings valid within the time, space and value context in which the study was being conducted (Polit & Beck 2012:732), in this case, in Mamelodi, Tshwane district.\n\nData were collected from African women who were at the menopausal stage of between the ages of 45 and 60 years, using semi-structured, individual interviews in Mamelodi, Tshwane district.\n\n### Context of the study\n\nThis article is written based on a mini-dissertation submitted for a Master's degree at the University of Pretoria. The study was conducted in two Mamelodi clinics in Tshwane district.\n\n### Population, sample and sampling technique\n\nAfrican menopausal women were recruited during day clinic visits for minor ailments, routine body check-ups and for chronic medication collection, through the assistance of the clinic professional nurses at the setting. The purposive-convenience sampling technique was used to sample the participants (Botma et al. 2010:125). It is purposive as the researcher selected a representative sample of African menopausal women, namely, those between the ages of 45 and 60 years old.\n\nThe participants were purposively handpicked from the population (patients at the clinic) based on the researcher's knowledge and judgement. It is also convenient, as the African menopausal women selected were only those who were at the clinic on the day of data collection, as assisted by the clinic professional nurses. The sample was drawn from the population according to the sample inclusion criteria, as described below.\n\n### Inclusion criteria\n\nThe inclusion criteria refer to those criteria that specify the population characteristics to be included in the study, that is, the eligibility criteria (Polit & Beck 2012:274). The sampling criteria for this study were:\n\n-   An ageing woman between 45 and 60 years old.\n\n-   Must have had no menstrual periods for the past 12 months.\n\n-   Must be attending the clinic services in Mamelodi, Tshwane district.\n\n-   Must reside in Mamelodi, Tshwane district.\n\n-   Must be willing to share their perceptions regarding menopause with the researcher voluntarily and without any coercion.\n\n### Data collection\n\nThe semi-structured, one-to-one individual interview method was used in the study to produce rich data as the participants were able to reflect and describe their perceptions regarding natural menopause in their own words.\n\n### Accesses to participants\n\nThe majority of the women were at the clinic to collect their chronic medication and, coincidentally, they met the inclusion criteria for the study. The main questions were asked by the researcher as guided by the interview guide and some probing questions followed. A digital recorder was used to record the interviews; and field notes were taken for non-verbal cues through observation by the researcher. Data were collected until data saturation was reached with the 18th participant, that is, when the menopausal women were providing information similar to the information already gathered from previous participants.\n\n### Data treatment\n\nData analysis is a process of bringing order and meaning to a large amount of data into smaller, manageable segments that can be retrieved and revised (De Vos et al. 2011:397). Data were analysed according to Tesch' s (1990) eight steps of data analysis, as follows:\n\n1.  All data collected through interviews, digital recorder and observations were transformed into written text with the date, time and the place where the interview took place. Field notes were also typed and labelled.\n\n2.  The researcher read through the data several times to obtain a general sense of the information and to reflect on its overall meaning.\n\n3.  Several transcripts were read through and highlighted or segments (phrases) in them.\n\n4.  A list was compiled of all the topics that came to the researcher's mind.\n\n5.  The researcher used the compiled list to analyse the transcripts by looking for segments (phrases) from the transcripts that fit the topics.\n\n6.  All the segments that fitted a particular topic were put together and were given descriptive names as subthemes.\n\n7.  The subthemes were sorted and grouped together, then given descriptive names as themes.\n\n8.  Digital recorder interviews were transcribed verbatim, then analysed and clustered into themes, categories and subcategories.\n\n# Ethical considerations\n\nThe study obtained ethical clearance from the University of Pretoria's Ethics Committee (protocol number 32\/2014), the Ethical Committee of the Gauteng Department of Health, Tshwane district and the Nursing management of the two clinics in Mamelodi, Tshwane district. The recruited participants were provided with information leaflets and the researcher explained the aims, objectives and significance of the study. They were reassured that no harm would be experienced, either emotionally, physically or psychologically. The participants were given informed consent forms to sign voluntarily and without coercion. Confidentiality and anonymity were maintained throughout the report (De Vos et al. 2011:116; Polit & Beck 2012:177) and transcripts and recordings were kept in a locked safe by the researcher.\n\n# Trustworthiness\n\nThe following criteria were used and applied throughout the study to ensure trustworthiness: credibility, dependability, transferability and confirmability (Botma et al. 2010:232):\n\n-   **Credibility** was enhanced by using member checking and peer examination. The researcher conducted semi-structured, one-to-one individual interviews until data saturation was reached, using digital recorder and field notes to capture accurate data. The data collected were continuously checked with the participants to confirm that transcriptions were true descriptions and interpretations of every participant's own perception and experience. Field notes and the digital recording were given to the supervisor to check the allocated themes.\n\n-   **Dependability** refers to the findings that will not change if a similar study with the same participants and in a similar context were done at a different time (Polit & Beck 2012:585). The researcher used individual semi-structured, one-to-one individual interview method for all the participants. A digital recorder and field notes were used to ensure an audit trail. Systematic documentation was created by the researcher for the purpose of the audit trail that was available and accessible to the supervisors as proof of all the decisions made throughout the study. The data were analysed and an agreement was reached by the researcher and supervisors with regard to identification of themes.\n\n-   **Transferability** was ensured by the researcher by providing thick description of context, the type of sampling method and the characteristics of participants, data collection and analysis.\n\n-   **Confirmability** refers to the objectivity or neutrality of the data; there must be an agreement between two or more independent people regarding the data's relevance and its meaning (Polit & Beck 2012:585). The researcher and the supervisor reduced data to themes after consensus had been reached.\n\n# Results and discussion\n\nThe main theme that emerged from the study was: attitude toward natural menopause.\n\n## Attitude toward natural menopause\n\nThis theme exposed the attitude of women regarding natural menopause based on their culture, experience and knowledge. Adequate knowledge and better understanding about menopause by women leads to a positive attitude toward this natural stage in life. If there is misunderstanding about reproductive health-related knowledge on menopause, some women may misunderstand the feeling of being in the menopausal state, such as mood swings and irritability, resulting in inadequate acceptance of this normal phenomenon in ageing women and may thus be perceived in a negative light (Adewuyi & Akinade 2010:1778).\n\n### Positive attitude\n\nMost participants in the study regarded menopause as a natural process of ageing and life changes. The participants understood natural menopause as a cessation of the menstrual circle, resulting in no more reproduction or falling pregnant. The study conducted by Im et al. (2008:548) revealed that most women had a positive attitude toward natural menopause.\n\nAfrican women above 60 years who experienced natural menopause for a longer period, perceived it positively. According to Ramakuela et al. (2014:2), menopause was accepted as a normal phenomenon and menopausal women felt happy about the post-reproductive life stage. Some of the participants said:\n\n> 'I think menopause is the time when you become old and you are no longer going to fall pregnant again. It is a natural change of life and ageing. We are living a good life like children. You no longer have a problem of buying some pads. I really enjoy this stage. You can sleep without clothes and not being disturbed.' (File C\/Participant 7, 60 years old)\n>\n> 'I feel alright with the stage of menopause. I must admit that now is the time I did not start of being in the stage of menopause \\[*sic*\\] but I started there, way back, as a lady. Now is the time to be in this stage because everything has its time and I cannot be ever young.' (File C\/Participant 17, 58 years old)\n>\n> 'I am growing old. The age is catching up with me but I am just happy because I do not have my periods or buy any pads.' (File D\/Participant 1, 63 years old)\n\nAs noted from the quotes above, African women with experience, knowledge and who are above 60 years of age accept natural menopause as a normal ageing process. Sociocultural beliefs had no influence on the freedom to discuss natural menopause openly with the researcher, who was not only a woman but also above 50 years of age and within the class of African menopausal women. The African women discussed natural menopause freely without any signs of embarrassment and accepted the physiological changes occurring in their bodies without complaints.\n\n### Negative attitude\n\nAlthough most of the participants accepted menopause as a natural process of ageing, some participants who were below the age of 60 were negative, due to about the changes occurring in their bodies after cessation of menstruation. These women highlighted their lack of knowledge about natural menopause because of cultural beliefs, which restricted them from talking about it, as it is regarded as taboo. One participant said:\n\n> 'I do not have enough information about menopause. It is a difficult stage because you do not have the experience and knowledge of what to expect. I do not understand why there are changes in my body or life as whole.' (File D\/Participant 11, 52 years old)\n\nThe quote above indicates that the participant (52 years old) did not understand the changes occurring in her body and lacked knowledge about the physiological processes associated with female ageing. Below, the quote from another participant:\n\n> 'At least, when you were menstruating every month, it prevented you from being ill. But now, my menstruation is no longer coming out and causes a lot of diseases. I am person who is having a lot of blood. Since I stopped to have my monthly period, my blood pressure is high. It can be a problem; you can maybe have \u2026 womb cancer. There are lots of diseases which can occur in your body.' (File C\/Participant 15, 49 years old)\n\nParticipants below 60 years of age believed that the 'blood that is not coming out' (File D\/Participant 11, 53 years old) accumulates in their body and cause health problems. The women presented the following signs and symptoms as caused by menopause: headache, backache, painful joints and generalised body pain. Pain was the most common menopausal symptom linked with blood 'that does not come out', as shown in the quote below. One of the participants said:\n\n> 'I think it is this blood that does not flow, that does not come out. I do not know where this blood goes. I am thinking that maybe it is accumulation of blood in the body that causes pains because the blood does not manufacture babies any longer and no more functioning \\[*sic*\\].' (File D\/Participant 11, 53 years old)\n\nSome of the participants in the study related the body changes to changes in their hormones and retained blood resulting in weight gain and skin pigmentation, as revealed below:\n\n> 'My skin and body size have changes. The face is dark and wrinkled and not shiny like when I was young. My skin is dry and itching even if I have washed; I think is the hormones and ageing.' (File D\/Participant 4, 55 years old)\n>\n> 'I gained weight. My face has pimples because of the \"blood that does not come out.\"' (File C\/Participant 11, 53 years old)\n\nThe explanation above indicates that some women lack knowledge about menopause and have contradictory sentiments. This was supported by the study conducted by Mustafa and Sabir (2012:175), who stated that women believe menopause is a natural condition that affects them in a negative manner. The study conducted by Agwu et al. (2008:219) alluded to the fact that some women believed that abstinence from intercourse precipitates menopause and heralds its symptoms.\n\nA widowed participant said:\n\n> 'I think menopause is to grow up but my elder sisters, who are older than me, are still menstruating. They are having their husbands. So, I think when you are menstruating, you sleep with a man. I am not sleeping with a man, that it is why menstruation has stopped. The blood stays in your body makes you sick, it must come out every month. It stays under the stomach and at the back and make you feel pains.' (File D\/Participant 4, 46 years old)\n\nThis indicates that some of the women below 60 years of age do not understand the causes of menopause because of limited experience and inadequate reproductive health-related knowledge during their menopausal stage and education development regarding menopause and procreation. It is supported by the quote below, which implies that menopause was regarded as taboo to discuss with an elder; there was no sharing of knowledge and experience.\n\nOne participant said:\n\n> 'I have so many complaints and my mother has six children, but I do not remember her complaining about heat and other diseases. What makes me feel bad is that I did not have my own children.' (File D\/Participant 12, 48 years old)\n\nThe study revealed that the attitude of some African menopausal women was negative. Most of the women who expressed a negative attitude toward menopause were below 60 years of age and had limited knowledge related to reproductive health-related knowledge about the natural ageing part of menopause. The women of 60 years old and above understood and accepted menopause as a natural phenomenon in life and part of ageing. This indicates that they accepted menopause more positively than women below the age of 50 years (Robak-Cho\u0142ubek et al. 2014:666).\n\nThe findings of the study provide insight on African women's perception regarding natural menopause. The more aged the African women, the more knowledgeable and experienced about natural menopause they become. This study revealed that 'young' African menopausal women (below the age of 50 years) lack adequate reproductive health-related knowledge about physiological human developmental stages (Robak-Cho\u0142ubek et al. 2014:666). Inexperience and inadequate health-related knowledge on menopause negatively affects the understanding and perception of natural menopause. African menopausal women need support systems from all relevant stakeholders, including the government, to reduce the cost of curative measures when these women are chronically ill.\n\n# Limitations of the study\n\nThe findings of this study were obtained from menopausal women, between the ages 45 and 60 years, in Mamelodi, Tshwane district, therefore they cannot be generalised as these were the perceptions of the women in this particular setting. More studies on women's cultural perceptions on natural menopause need to be done in South Africa to explore and describe their attitude regarding this natural phenomenon.\n\n# Recommendations\n\nThe second objective of the study was to propose recommendations for healthcare providers in all healthcare settings to provide reproductive health-related knowledge regarding human developmental stages for all women in Tshwane district. The participants in this study suggested that they need to be furnished with reproductive health-related knowledge during the visit to healthcare settings. Based on the findings of the study, the researcher has recommendations to clinical practice, nursing education and nursing research with regard to natural menopause.\n\n## Clinical practice\n\nHealthcare providers should provide health education on menopause to women who visit the clinic for consultations and follow-up on chronic medications. The researcher further recommends that Gauteng Province government's health department, in collaboration with Tshwane district, provide clinic services for menopausal women, which will be managed by their peers to reduce embarrassment and allow for open, free discussion.\n\n## Health education\n\nTshwane district should include reproductive health and human development, especially female reproductive development, in health education for all female clients in community health clinics. The health education of African women should also include a memorandum of understanding (MoU) with the Tshwane district and the clinics within its jurisdiction.\n\n## Nursing research\n\nThe researcher recommends that further studies be conducted on natural menopause for all African menopausal women. The researchers should collaborate with ageing African women in projects which relate to their health issues in order to promote their cooperation and understanding about health-related matters of the ageing African female population.\n\n# Conclusion\n\nThe perceptions of women regarding natural menopause revealed that menopause is viewed according to the attitude of the participants, which is influenced by their cultural beliefs and reproductive health knowledge related to menopause, as well as women's ageing process and development. Some African menopausal women perceived menopause as positive whilst others perceived it as negative, depending on the understanding of the participant respectively. There is a lack of information sharing and limited knowledge about natural menopause because of some cultural sensitivity of reproductive topics and fear of embarrassment. It seems as if the Tshwane district clinics do not provide certain services to women in menopause. These are support systems, such as social networking, and equal opportunities for education in reproductive health and menopause (Thekkedath & Joseph 2009:19). Peer groups for menopausal women in Mamelodi, Tshwane district could be a positive step toward health and wellbeing.\n\nThere is a need for community health nurses and other healthcare providers to provide menopausal services such as seminars and health education on menopause to women to reduce the health problems that are related to this phenomenon, by preparing the women in an open, free environment and sharing this natural stage with others. One of the functions of nursing is teaching or educating the public through health education, therefore all nurses should educate the clients at the clinic level as well as during home visits. The South African Nursing Council should ensure that all aspects which are currently overlooked when caring for patients and their families because of changed diet-related life styles which impact on human physiological processes, are somehow revived in the practice of nursing.\n\n## Acknowledgements\n\nThe authors would like to thank all the participants for providing and sharing information freely.\n\n## Competing interests\n\nThe authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article.\n\n## Authors' contributions\n\nG.N.M. (University of Pretoria) conducted the whole study, guided by S.R.R. (University of Pretoria) for literature sources and writing. S.R.R. further drafted the article for G.N.M. to follow as a neophyte author. Literature search and corrections from reviews were addressed by S.R.R. F.M.M (University of Pretoria) provided general, overall support and guidance as an experienced and knowledgeable senior academic, ensuring that the article was completed within timeframe.\n\n# References\n\n[^1]: **How to cite this article:** Rikhotso, S.R., Makuwa, G.N. & Mulaudzi, F.M., 2015, 'The perceptions of African women regarding natural menopause in Mamelodi, Tshwane district', *Curationis* 38(2), Art. \\#1531, 7 pages. <http:\/\/dx.doi.org\/10.4102\/curationis.v38i2.1531>","meta":{"dup_signals":{"dup_doc_count":103,"dup_dump_count":33,"dup_details":{"curated_sources":2,"2020-05":1,"2019-35":2,"2019-13":1,"2019-04":1,"2018-47":1,"2018-39":1,"2018-30":1,"2018-26":1,"2018-22":3,"2018-17":3,"2018-13":2,"2018-09":4,"2018-05":1,"2017-51":2,"2017-47":4,"2017-43":2,"2017-39":4,"2017-34":2,"2017-30":4,"2017-26":2,"2017-22":3,"2017-17":4,"2017-09":5,"2017-04":6,"2016-50":6,"2016-44":4,"2016-40":3,"2016-36":6,"2016-30":4,"2016-26":6,"2016-22":5,"2023-06":1,"2017-13":6}},"file":"PMC6091694"},"subset":"pubmed_central"}
{"text":"abstract: **Objective** To assess the association between consumption of fried foods and risk of coronary heart disease.\n  .\n  **Design** Prospective cohort study.\n  .\n  **Setting** Spanish cohort of the European Prospective Investigation into Cancer and Nutrition.\n  .\n  **Participants** 40\u2009757 adults aged 29-69 and free of coronary heart disease at baseline (1992-6), followed up until 2004.\n  .\n  **Main outcome measures** Coronary heart disease events and vital status identified by record linkage with hospital discharge registers, population based registers of myocardial infarction, and mortality registers.\n  .\n  **Results** During a median follow-up of 11 years, 606 coronary heart disease events and 1135 deaths from all causes occurred. Compared with being in the first (lowest) quarter of fried food consumption, the multivariate hazard ratio of coronary heart disease in the second quarter was 1.15 (95% confidence interval 0.91 to 1.45), in the third quarter was 1.07 (0.83 to 1.38), and in the fourth quarter was 1.08 (0.82 to 1.43; P for trend 0.74). The results did not vary between those who used olive oil for frying and those who used sunflower oil. Likewise, no association was observed between fried food consumption and all cause mortality: multivariate hazard ratio for the highest versus the lowest quarter of fried food consumption was 0.93 (95% confidence interval 0.77 to 1.14; P for trend 0.98).\n  .\n  **Conclusion** In Spain, a Mediterranean country where olive or sunflower oil is used for frying, the consumption of fried foods was not associated with coronary heart disease or with all cause mortality.\nauthor: Pilar Guallar-Castill\u00f3n; Fernando Rodr\u00edguez-Artalejo; Esther Lopez-Garcia; Luz M Le\u00f3n-Mu\u00f1oz; Pilar Amiano; Eva Ardanaz; Larraitz Arriola; Aurelio Barricarte; Genevieve Buckland; Mar\u00eda-Dolores Chirlaque; Miren Dorronsoro; Jos\u00e9-Mar\u00eda Huerta; Nerea Larra\u00f1aga; Pilar Marin; Carmen Mart\u00ednez; Esther Molina; Carmen Navarro; J Ram\u00f3n Quir\u00f3s; Laudina Rodr\u00edguez; Mar\u00eda Jos\u00e9 Sanchez; Carlos A Gonz\u00e1lez; Conchi Moreno-IribasCorrespondence to: P Guallar-Castill\u00f3n <email@example.com>\ndate: 2012\nreferences:\ntitle: Consumption of fried foods and risk of coronary heart disease: Spanish cohort of the European Prospective Investigation into Cancer and Nutrition study\n\n# Introduction\n\nFrying is one of the most commonly used methods for cooking in Western countries. When food is fried its nutritional content changes\u2014the food loses water and takes up fat, increasing its energy density.1 2 Frying modifies both the foods and the frying medium because oils deteriorate during frying, especially when reused, through the processes of oxidation and hydrogenation, leading to a loss of unsaturated fats and an increase in trans fats.1 Thus fried food absorbs degradation products of the frying oil.3 At the same time, frying may also improve palatability by making food crunchy.4 5\n\nFried foods have been associated with various cardiovascular risk factors in cross sectional studies. In Spain, the Pizarra study of 1226 adults showed that consumption of food fried with reused oils was associated with a higher prevalence of arterial hypertension.6 Furthermore, a cross sectional analysis of the Spanish cohort of the European Prospective Investigation into Cancer and Nutrition (EPIC) study concluded that consumption of fried foods was positively associated with general and central obesity.7 Similarly, in the SUN (Seguimiento University of Navarra) cohort in Spain, more frequent consumption of fried foods at baseline was associated with a higher risk of becoming overweight or obese during a six year follow-up.8 Lastly, in another cross sectional study in 2090 Italian adults, consumption of fried food was associated with lower high density lipoprotein cholesterol levels and a larger waist circumference.9\n\nOnly a few studies have evaluated the effect of fried foods on risk of cardiovascular disease. A case-control study in Costa Rica found no association between consumption of fried foods and risk of non-fatal acute myocardial infarction.10 Another case-control study, INTERHEART, observed a positive association between fried foods and acute myocardial infarction.11\n\nTherefore, although interest in the associations of dietary patterns and specific cooking techniques with risk of disease is increasing,12 little is known about the health effects of fried foods. We evaluated prospectively the association between consumption of fried foods and the risk of coronary heart disease in the EPIC-Spain cohort.\n\n# Methods\n\nThe methods of the EPIC project have been reported elsewhere.7 13 14 15 16 For the present analysis we used the data from the Spanish cohort of EPIC, which included 41\u2009438 healthy adults (15\u2009632 men), aged 29-69. Study participants were recruited between 1992 and 1996 in five Spanish regions. Three of these regions are located in the north (Asturias, Gipuzkoa, and Navarra) and two in the south, including the Mediterranean shore (Granada and Murcia). Selection of these regions aimed to maximise variability in diet because food and nutrient intake has traditionally been different between the north and the south of Spain and between the inner regions and coastal areas. To increase variability in diet, participants were also recruited in urban and rural areas from various educational and social sectors. Study participants included mostly blood donors, civil servants, and the general population. Participation rates varied from 55% to 60% between regions. The exposures of interest were assessed at baseline, and participants were followed up until 31 December 2004. All participants gave written informed consent before enrolment.\n\n## Assessment of food consumption\n\nUsing a computerised dietary history questionnaire,17 previously validated in Spain18 19 20 and administered by trained interviewers, we obtained data on usual food consumption for each participant. We used household measures, standard units, and a collection of 35 sets of photographs of simple foods, mixed foods, and drinks to quantify the portion of each food (g\/day) consumed. Each interview on dietary history lasted about 40-50 minutes.\n\nParticipants were asked about food consumed in a typical week during the previous 12 months. Foods consumed at least twice a month were recorded. The dietary history questionnaire recorded information on up to 662 different foods, including those consumed individually and those consumed as part of 203 recipes and regional dishes. Of these foods, 212 were fried. We calculated energy and nutrient intake using the EPIC food composition table.21\n\nInformation on cooking methods was collected as part of the dietary history questionnaire. The same food may be cooked by several methods as part of a single recipe. For example, in the preparation of paella (a typical Spanish recipe), rice is both fried and boiled. Thus, fried food was defined as food for which frying was the only cooking method used. Such food could be deep fried or pan fried and it could be battered, crumbed, or saut\u00e9ed. To estimate the absorption of fat from frying, we used an absorption coefficient for each fried food.18 We also recorded the type of oil used for dressing, cooking, and frying.\n\n## Assessment of non-dietary variables\n\nInformation on demographic variables, educational level, smoking, and physical activity was obtained through interview at recruitment. Physical activity was assessed with a validated questionnaire that included activity at work, at home, and during leisure time.22 Participants were also asked if they had diabetes mellitus, hypercholesterolaemia, hypertension, cancer, or angina, or had experienced myocardial infarction or stroke. In addition they were asked to report their menopausal status and any use of oral contraceptives or hormone replacement therapy. Trained observers used standardised procedures to measure weight, height, and waist circumference. The body mass index was calculated as weight (kg) divided by height squared (m^2^).\n\n## Ascertainment and validation of coronary heart disease\n\nAt baseline, prevalent coronary heart disease was self reported. During follow-up, we ascertained incident coronary heart disease with a telephone questionnaire (at three years after recruitment) and through record linkage with three sources of information: hospital discharge databases, population based myocardial infarction registries (available in Murcia, Navarra, and Gipuzkoa), and regional mortality registries and the national mortality database (managed by the National Statistical Institute, Madrid, Spain), which provided information on date and cause of death.\n\nCoronary heart disease was classified according to the *International Classification of Diseases*, ninth revision (ICD-9 codes 410-414) and 10th revision (ICD-10 codes I20-I25). A team of trained doctors and nurses validated coronary heart disease events against hospital records and autopsy reports.\n\nCoronary heart disease events were classified on the basis of symptoms, signs, biomarkers, and findings on electrocardiogram or at autopsy, according to standard criteria.23 A definite coronary heart disease event was defined as one meeting all relevant criteria for a fatal or non-fatal acute myocardial infarction, or angina requiring revascularisation (coronary artery bypass graft or percutaneous transluminal coronary angioplasty). We defined probable and possible acute coronary heart disease events as cases where electrocardiogram findings were non-specific or the biomarkers were equivocal or missing.13\n\n## Statistical analysis\n\nOf the 41\u2009438 participants in the cohort, we excluded the following from the analyses: 193 for having coronary heart disease at baseline; 167 for an implausibly high or low dietary consumption, defined as 3 standard deviations either way from the cohort mean (\\<788 kcal\/day or \\>5710 kcal\/day); and 321 for lacking data on important variables such as date of coronary heart disease event (n=12), smoking (n=22), diabetes mellitus (n=71), hypercholesterolaemia (n=197), and hypertension (n=60). Thus the analyses were carried out on 40\u2009757 participants.\n\nWe used Cox regression to obtain hazard ratios for coronary heart disease according to sex specific quarters of fried food consumption. The quarter (first) with lowest consumption was used as reference. In the Cox models, age was the underlying time variable, with entry time defined as the participant's age at recruitment and exit time as the age at the coronary heart disease event, death, or 31 December 2004, whichever came first. To reduce violations of the proportional hazards assumption we also stratified models by age at recruitment (five year groups), sex, and centre. We built three models, with progressive adjustment for potential confounders to determine their influence on the results. Model 1 was adjusted for energy intake and model 2 was additionally adjusted for educational level, smoking, physical activity (at work, at home, and during leisure time), diabetes mellitus, hyperlipidaemia, cancer, oral contraceptive use, menopause, hormone replacement therapy, ethanol intake, and consumption of non-fried foods (vegetables, fruits, nuts, dairy products, meat, and fish). Model 3 was additionally adjusted for possible mediators of the study association reported in the literature: body mass index, waist circumference, and hypertension. We categorised continuous variables into quarters, except for age, which was analysed as a continuous variable. The P for linear trend was calculated by modeling the quarters for fried food as a continuous variable. Moreover, we also calculated hazard ratios of coronary heart disease and of all cause mortality for a 100 g increase in fried food consumption analysed as a continuous variable.\n\nNew analyses were done after censoring the first two years of follow-up and after excluding those who reported a change in their diet during the previous year. To test whether the study association varied with type of oil used for frying (olive oil versus sunflower oil or other vegetable oils), we used the likelihood ratio test, which compared models with and without interaction terms (products of the quarters for fried food and type of oil). We also used the same procedure to test whether results varied by sex.\n\nStatistical significance was set at P\\<0.05. The analyses were carried out with SAS, version 9.1 for Windows. This manuscript follows the recommendations of the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) initiative.24\n\n# Results\n\nAn average 138 g of fried food was consumed daily, including 14 g of oil used for frying. About 7% of the total amount of food consumed was fried. Fried food consumption ranged from 0-817 g\/day for men and 0-657 g\/day for women. Sixty two per cent of participants used olive oil for frying and the rest used sunflower oil or other vegetable oils.\n\nParticipants with a higher fried food intake were younger, of a higher level of education, more often smokers, and less often sedentary at work (table 1<a href=\"#tbl1\" data-ref-type=\"table\"><\/a>). Moreover, they had a lower prevalence of diabetes mellitus and hyperlipidaemia, a higher energy and ethanol intake, and a lower consumption of non-fried vegetables, fruit, dried fruit and nuts, milk products, and non-fried fish (table 2<a href=\"#tbl2\" data-ref-type=\"table\"><\/a>). They also had a lower frequency of diagnosed hypertension. Among women, the frequency of menopausal status decreased with the consumption of fried food (table 2).\n\n\u2002Consumption of fried foods, sociodemographic variables, and lifestyles of study participants at baseline, by quarters of fried food consumption. Values are means unless stated otherwise\n\n| Characteristics | Quarters of fried food consumption |  |  |  |\n|----|----|----|----|----|\n|  | 1 (lowest) | 2 | 3 | 4 (highest) |\n| **Consumption of fried foods** |  |  |  |  |\n| Total amount of fried food consumed (g\/day) | 47.0 | 105.7 | 158.4 | 249.6 |\n| Fried fish (g\/day) | 12.6 | 28.8 | 40.6 | 53.6 |\n| Fried meat (g\/day) | 8.3 | 21.7 | 36.2 | 59.9 |\n| Fried potatoes (g\/day) | 9.3 | 21.6 | 33.3 | 57.7 |\n| Fried eggs (g\/day) | 4.0 | 10.7 | 17.1 | 28.0 |\n| **Sociodemographic variables** |  |  |  |  |\n| Age (years) | 50.4 | 49.5 | 48.9 | 48.3 |\n| Educational level (%): |  |  |  |  |\n| \u2003No formal education | 41.9 | 36.4 | 31.7 | 27.6 |\n| \u2003Primary education | 31.6 | 35.9 | 40.8 | 46.6 |\n| \u2003Secondary education | 12.6 | 14.2 | 16.0 | 16.0 |\n| \u2003University education | 13.3 | 12.8 | 10.8 | 9.0 |\n| **Lifestyle** |  |  |  |  |\n| Smoking (%): |  |  |  |  |\n| \u2003Never smoker | 58.3 | 59.3 | 59.5 | 59.7 |\n| \u2003Current smoker | 22.5 | 22.5 | 23.7 | 24.2 |\n| \u2003Former smoker | 19.2 | 18.2 | 16.8 | 16.1 |\n| Physical activity at work (%): |  |  |  |  |\n| \u2003Sedentary | 22.8 | 22.1 | 20.4 | 18.8 |\n| \u2003Standing | 62.1 | 64.2 | 63.7 | 63.4 |\n| \u2003Manual labour | 6.2 | 8.5 | 10.3 | 12.3 |\n| \u2003Heavy manual labour | 2.2 | 1.9 | 2.1 | 2.5 |\n| \u2003Does not work | 3.8 | 3.3 | 3.6 | 3.1 |\n| Physical activity at home (METs h\/week) | 68.6 | 69.8 | 70.0 | 68.1 |\n| Physical activity in leisure time (METs h\/week) | 28.7 | 28.2 | 28.6 | 28.4 |\n\nMET=metabolic equivalents. Range of quarters of fried food consumption in g\/day for men: first quarter (0-112), second quarter (113-176), third quarter (177-246), and fourth quarter (247-817), and for women: first quarter (0-59), second quarter (60-103), third quarter (104-154), and fourth quarter (155-657).\n\n\u2002Morbidity, menopausal status, use of hormone treatment, consumption of non-fried food, and potential mediators of study association among study participants at baseline, by quarters of fried food consumption. Values are means unless stated otherwise\n\n| Characteristics | Quarters of fried food consumption |  |  |  |\n|----|----|----|----|----|\n|  | 1 (lowest) | 2 | 3 | 4 (highest) |\n| Diseases diagnosed: |  |  |  |  |\n| \u2003Diabetes mellitus (%) | 7.5 | 5.1 | 3.9 | 3.2 |\n| \u2003Hyperlipidaemia (%) | 24.8 | 20.5 | 18.5 | 16.7 |\n| \u2003Cancer (%) | 1.0 | 0.9 | 0.8 | 0.8 |\n| Menopause (%)\\* | 42.2 | 36.9 | 33.0 | 29.3 |\n| Hormone treatment (%)\\*: |  |  |  |  |\n| \u2003Oral contraceptives | 24.7 | 26.4 | 26.6 | 27.4 |\n| \u2003Hormone substitution therapy | 6.4 | 6.2 | 5.5 | 5.6 |\n| Other nutrients and foods consumed: |  |  |  |  |\n| \u2003Total energy (kcal\/day) | 1920.1 | 2133.0 | 2294.8 | 2566.6 |\n| \u2003Ethanol (g\/day) | 11.5 | 14.5 | 17.0 | 20.4 |\n| \u2003Non-fried vegetables (g\/day) | 255.0 | 241.1 | 232.3 | 237.4 |\n| \u2003Fruit (g\/day) | 336.6 | 322.0 | 315.3 | 311.2 |\n| \u2003Dried fruit and nuts (g\/day) | 5.0 | 4.6 | 4.2 | 3.9 |\n| \u2003Milk products (g\/day) | 304.7 | 298.1 | 296.9 | 292.0 |\n| \u2003Non-fried meat (g\/day) | 93.9 | 98.0 | 97.9 | 96.2 |\n| \u2003Non-fried fish (g\/day) | 37.2 | 29.6 | 26.0 | 23.8 |\n| Mediators of study association: |  |  |  |  |\n| \u2003Body mass index (kg\/m^2^) | 28.5 | 28.3 | 28.1 | 28.1 |\n| \u2003Waist circumference (cm) | 92.8 | 92.0 | 91.4 | 91.4 |\n| \u2003Hypertension (%) | 23.7 | 20.9 | 18.4 | 17.4 |\n\nSee table 1 for range (g\/day) of quarters of fried food consumption for men and for women.\n\n\\*Calculated for women.\n\nDuring a median follow-up of 11 years, 606 definite coronary heart disease events occurred (466 myocardial infarctions and 140 anginas requiring revascularisation). There were also 712 definite, possible, or probable coronary heart disease events and 1135 deaths from all causes.\n\nFor definite coronary heart disease events, no association with fried food consumption was observed in analyses adjusting for energy intake, age, sex, and centre (table 3<a href=\"#tbl3\" data-ref-type=\"table\"><\/a>, model 1). The hazard ratio of coronary heart disease for the highest versus the lowest quarter of fried food consumption was 0.94 (95% confidence interval 0.72 to 1.23; P for trend 0.52). Similar results were obtained after additional adjustment for the main confounders (table 3, model 2): 1.11 (0.84 to 1.46; P for trend 0.60). Finally, no association was observed between fried food and incident coronary heart disease after adjusting for possible mediators such as body mass index, waist circumference, and hypertension (table 3, model 3). Compared with the first (lowest) quarter of fried food consumption, the multivariate hazard ratios of coronary heart disease were 1.15 (0.91 to 1.45) in the second quarter, 1.07 (0.83 to 1.38) in the third quarter, and 1.08 (0.82 to 1.43) in the fourth quarter (P for trend 0.74). Lastly, after full adjustment, a 100 g increase in the consumption of fried food did not show an association with risk of coronary heart disease (hazard ratio 1.00, 0.90 to 1.11). The results were similar after censoring the first two years of follow-up and after excluding those who reported a change in their diet during the previous year. The results did not vary between those who used olive oil for frying and those who used sunflower oil or other vegetable oils (P for interaction 0.22), or between the sexes (P for interaction 0.19).\n\n\u2002Association between fried food consumption and incidence of definite coronary heart disease and all cause mortality during 11 years of follow-up in Spanish cohort of European Prospective Investigation into Cancer and Nutrition\n\n| Variables | Quarters of fried food consumption |  |  |  | P for trend | For 100 g increase of fried food |\n|----|----|----|----|----|----|----|\n|  | 1 (lowest) | 2 | 3 | 4 (highest) |  |  |\n| Coronary heart disease: |  |  |  |  |  |  |\n| \u2003Total No | 10 188 | 10 190 | 10 190 | 10 189 |  |  |\n| \u2003Person years | 109 578 | 110 394 | 110 943 | 111 762 |  |  |\n| \u2003No of events | 154 | 163 | 150 | 139 |  |  |\n| \u2003Model 1\\*: hazard ratios (95% CI) | 1 | 1.05 (0.84 to 1.33) | 0.97 (0.76 to 1.24) | 0.94 (0.72 to 1.23) | 0.52 | 0.95 (0.86 to 1.06) |\n| \u2003Model 2\u2020: hazard ratios (95% CI) | 1 | 1.15 (0.91 to 1.46) | 1.08 (0.84 to 1.39) | 1.11 (0.84 to 1.46) | 0.60 | 1.01 (0.91 to 1.12) |\n| \u2003Model 3\u2021: hazard ratios (95% CI) | 1 | 1.15 (0.91 to 1.45) | 1.07 (0.83 to 1.38) | 1.08 (0.82 to 1.43) | 0.74 | 1.00 (0.90 to 1.11) |\n| All cause mortality: |  |  |  |  |  |  |\n| \u2003Total No | 10 188 | 10 190 | 10 190 | 10 189 |  |  |\n| \u2003Person years | 110 135 | 111 019 | 111 560 | 112 342 |  |  |\n| \u2003No of deaths | 328 | 276 | 273 | 258 |  |  |\n| \u2003Model 1\\*: hazard ratios (95% CI) | 1 | 0.90 (0.76 to 1.07) | 0.91 (0.77 to 1.09) | 0.89 (0.73 to 1.08) | 0.26 | 0.98 (0.91 to 1.06) |\n| \u2003Model 2\u2020: hazard ratios (95% CI) | 1 | 0.94 (0.79 to 1.11) | 0.95 (0.80 to 1.14) | 0.94 (0.77 to 1.15) | 0.60 | 1.00 (0.92 to 1.08) |\n| \u2003Model 3\u2021: hazard ratios (95% CI) | 1 | 0.94 (0.79 to 1.11) | 0.95 (0.79 to 1.13) | 0.93 (0.77 to 1.14) | 0.98 | 1.00 (0.92 to 1.08) |\n\nQuarters are sex specific. See table 1 for range (g\/day) of quarters of fried food consumption for men and for women.\n\n\\*Adjusted for age, sex, centre (Asturias, Gipuzkoa, Granada, Murcia, or Navarra), and energy intake (quarters).\n\n\u2020Adjusted for variables in model 1 and for ethanol consumption (quarters among consumers), educational level (no formal education, primary, vocational, secondary, university education), smoking (never, current, former), physical activity at work (sedentary, standing, manual labour, heavy manual labour, no work), physical activity at home (metabolic equivalents h\/week in quarters), physical activity in leisure time (metabolic equivalents h\/week in quarters), diabetes mellitus, hyperlipidaemia, cancer, oral contraceptives, menopause, hormone replacement therapy, and consumption of fruit (quarters), nuts (quarters), and dairy products (quarters), and non-fried foods (quarters): vegetables, meat, and fish.\n\n\u2021Adjusted for variables in model 2 and for body mass index (\\<25 and \u226525, \\<30 and \u226530, \\<35 and \u226535), waist circumference (quarters), and hypertension.\n\nSimilar results were also obtained in the combined analysis of definite, possible, and probable coronary heart disease events. Compared with the first (lowest) quarter of fried food consumption, the multivariate hazard ratio of coronary heart disease in the fully adjusted model (model 3) was 1.08 (0.88 to 1.34) for the second quarter, 1.06 (0.84 to 1.33) for the third quarter, and 1.04 (0.81 to 1.34) for the fourth quarter (P for trend 0.83).\n\nFried food consumption also failed to be associated with all cause mortality. Compared with the lowest quarter of fried food consumption, those in the highest quarter had a hazard ratio of 0.93 (0.77 to 1.14; P for trend 0.98). Moreover, no association was observed between a 100 g increase in intake of fried food and total mortality (1.00, 0.92 to 1.08; table 3, model 3).\n\nOf the total amount of fried food consumed, 24% (34 g\/day) was fish, 22% (31 g\/day) meat, 21% (30 g\/day) potatoes, and 11% (15 g\/day) eggs. No association was observed between each of these fried food groups and incident coronary heart disease (table 4<a href=\"#tbl4\" data-ref-type=\"table\"><\/a>). Comparing the highest and lowest quarters, the multivariate hazard ratio for the fully adjusted model was 1.13 (0.89 to 1.44; P for trend 0.26) for fried fish, 1.09 (0.82 to 1.43; P for trend 0.32) for fried meat, 0.90 (0.70 to 1.15; P for trend 0.45) for fried potatoes, and 0.87 (0.68 to 1.13; P for trend 0.30) for fried eggs. These fried foods also failed to show an association with all cause mortality (table 4).\n\n\u2002Association between consumption of main types of fried food and incidence of definite coronary heart disease and all cause mortality during 11 years of follow-up in Spanish cohort of European Prospective Investigation into Cancer and Nutrition\n\n| Variables | Quarters of fried food consumption |  |  |  | P for trend | For 100 g increase of fried food |\n|----|----|----|----|----|----|----|\n|  | 1 (lowest) | 2 | 3 | 4 (highest) |  |  |\n| **Coronary heart disease** |  |  |  |  |  |  |\n| Fried fish: |  |  |  |  |  |  |\n| \u2003No of events | 144 | 136 | 149 | 177 | \u2014 | \u2014 |\n| \u2003Hazard ratio (95% CI)\\* | 1 | 0.99 (0.78 to 1.26) | 1.06 (0.83 to 1.35) | 1.13 (0.89 to 1.44) | 0.26 | 1.03 (0.82 to 1.29) |\n| Fried meat: |  |  |  |  |  |  |\n| \u2003No of events | 137 | 158 | 174 | 137 | \u2014 | \u2014 |\n| \u2003Hazard ratio (95% CI)\\* | 1 | 1.17 (0.93 to 1.49) | 1.40 (1.09 to 1.79) | 1.09 (0.82 to 1.43) | 0.32 | 1.05 (0.82 to 1.35) |\n| Fried potatoes: |  |  |  |  |  |  |\n| \u2003No of events | 188 | 151 | 138 | 129 | \u2014 | \u2014 |\n| \u2003Hazard ratio (95% CI)\\* | 1 | 0.92 (0.74 to 1.15) | 0.95 (0.75 to 1.19) | 0.90 (0.70 to 1.15) | 0.45 | 0.91 (0.71 to 1.17) |\n| Fried eggs: |  |  |  |  |  |  |\n| \u2003No of events | 184 | 151 | 145 | 126 | \u2014 | \u2014 |\n| \u2003Hazard ratio (95% CI)\\* | 1 | 0.99 (0.80 to 1.24) | 0.95 (0.76 to 1.20) | 0.87 (0.68 to 1.13) | 0.30 | 0.84 (0.51 to 1.40) |\n| **All cause mortality** |  |  |  |  |  |  |\n| Fried fish: |  |  |  |  |  |  |\n| \u2003No of events | 297 | 276 | 271 | 291 | \u2014 | \u2014 |\n| \u2003Hazard ratio (95% CI)\\* | 1 | 0.99 (0.84 to 1.17) | 0.97 (0.81 to 1.15) | 0.92 (0.77 to 1.10) | 0.34 | 0.84 (0.69 to 1.01) |\n| Fried meat: |  |  |  |  |  |  |\n| \u2003No of events | 346 | 246 | 264 | 279 | \u2014 | \u2014 |\n| \u2003Hazard ratio (95% CI)\\* | 1 | 0.80 (0.67 to 0.95) | 0.90 (0.75 to 1.08) | 0.94 (0.78 to 1.14) | 0.72 | 0.96 (0.79 to 1.16) |\n| Fried potatoes: |  |  |  |  |  |  |\n| \u2003No of events | 353 | 273 | 249 | 260 | \u2014 | \u2014 |\n| \u2003Hazard ratio (95% CI)\\* | 1 | 0.90 (0.77 to 1.06) | 0.92 (0.78 to 1.09) | 0.99 (0.83 to 1.18) | 0.87 | 1.10 (0.91 to 1.32) |\n| Fried eggs: |  |  |  |  |  |  |\n| \u2003No of events | 350 | 265 | 245 | 275 | \u2014 | \u2014 |\n| \u2003Hazard ratio (95% CI)\\* | 1 | 0.96 (0.81 to 1.13) | 0.90 (0.75 to 1.06) | 1.05 (0.87 to 1.25) | 0.90 | 1.11 (0.76 to1.64) |\n\n\\*Adjusted the same as model 3 in table 3.\n\nRange of quarters of fried food consumption in g\/day: fish consumption for men: first quarter (0-15), second quarter (16-35), third quarter (36-61), and fourth quarter (62-439), and for women: first quarter (0-8), second quarter (9-23), third quarter (24-40), and fourth quarter (41-346); meat consumption for men: first quarter (0-12), second quarter (13-34), third quarter (35-65), and fourth quarter (66-384), and for women: first quarter (0-2), second quarter (3-15), third quarter (16-36), and fourth quarter (37-347); potato consumption for men: first quarter (0-12), second quarter (13-33), third quarter (34-59), fourth quarter (60-560), and for women: first quarter (0-3), second quarter (4-17), third quarter (18-34), and fourth quarter (35-282); egg consumption for men: first quarter (0-6), second quarter (7-15), third quarter (16-30), fourth quarter (31-176), and for women: first quarter (0-0), second quarter (1-8), third quarter (9-17), and fourth quarter (18-123).\n\n# Discussion\n\nThis analysis of the Spanish cohort of the European Prospective Investigation into Cancer and Nutrition found no association between consumption of fried food and risk of coronary heart disease or all cause mortality.\n\n## Comparison with other studies\n\nFew studies have evaluated this association. Our results are consistent with those of a case-control study of non-fatal myocardial infarction in Costa Rica, which obtained an odds ratio of 1.06 (0.59 to 1.91; P for trend 0.65) for the highest versus lowest fifth of fried food consumption.10 However, in the Costa Rican investigation, palm and partially hydrogenated soy oil were used for frying, whereas in this study mainly olive and sunflower oils\u2014the most commonly used oils in Mediterranean countries\u2014were used.\n\nOur results differ from those of the INTERHEART study, where fried food consumption was associated with a higher risk of acute myocardial infarction.11 However, some methodological differences limit the comparison of results. For instance, INTERHEART did not assess quantitatively the intake of fried food; moreover, it recorded the consumption of only nine fried foods and it did not report the type of oil used for frying.\n\nIn a cross sectional analysis of the Multi Ethnic Study of Atherosclerosis (MESA), no association was found between consumption of fried fish and subclinical atherosclerosis, assessed by the thickness of intima-media in the common carotid. Nor was fried fish consumption associated with significant changes in serum levels of total cholesterol, low density lipoprotein cholesterol, high density lipoprotein cholesterol, or tryglicerides.25 The Cardiovascular Health Study, a cohort of 3919 older people (\u226565 years) followed for nine years, also did not find an association of consumption of fried fish or fish sandwiches with mortality from coronary heart disease or cardiac arrhythmia, or with incidence of non-fatal acute myocardial infarction.26\n\n## Possible mechanisms of the study association\n\nThe frying process is complex and as yet is not well understood. Laboratory investigations show that fried foods may act through many mechanisms, such that the resulting effect on coronary heart disease is difficult to anticipate. Frying changes the quality and composition of food. For example, frying usually augments the fat content, and for some foods, such as potatoes, the dietary fibre is increased owing to the formation of resistant starch.1 Frying can specifically increase the amount of trans fatty acids in foods.27 The amount depends on the frying technique (deep or pan frying), the degree of thermal degradation of the oil, the type of food, and, above all, the type of oil (the degree of oil unsaturation increases the formation of trans fatty acids).28 However, some foods such as certain types of fish show minimal variations in the content of trans fatty acids after frying, regardless of whether olive oil or sunflower oil is used.29 On the other hand, the use of solid fats for frying can also increase the content of trans fatty acids.\n\nThe association between consumption of fried foods and endothelial damage is controversial. In a cross sectional analysis of the Multi Ethnic Study of Atherosclerosis (MESA), consumption of fried fish was inversely associated with the level of soluble intercellular adhesion molecule-1, a marker of endothelial activation. The authors did not expect this finding, since it was not observed for consumption of non-fried fish.30 In contrast, meals rich in oils previously used for deep frying in fast food restaurants have been associated with endothelial damage.31 In another study, however, foods rich in oils previously used for frying and high in oxidation products increased postprandial triglyceridaemia but did not affect endothelial function.32\n\nOther mechanisms may be operating during the frying process. For example, in an in vitro study fried eggs increased the production of angiotensin converting enzyme inhibitory peptides more than boiled eggs did.33 On the other hand, frying also increases the levels of cholesterol oxidation products34 and reduces paraoxonase activity, an enzyme that inhibits oxidation of low density lipoprotein cholesterol.35\n\nThis analysis of the EPIC-Spain cohort suggests that the above mentioned mechanisms may offset each other in the general population, with the result that frying with mainly olive oil or sunflower oil is not associated with a higher risk of coronary heart disease. Frying with other types of fats may still be harmful.\n\n## Limitations and strengths of the study\n\nThis study has some limitations. Firstly, diet was measured only at baseline. Secondly, some residual confounding cannot be ruled out, since diabetes mellitus, hypertension, and hypercholesterolaemia were self reported. In Spain, however, evidence exists of the diagnostic validity of self reported diabetes mellitus and hypertension.36 37 Thirdly, some degree of under-reporting of fried food consumption is possible, given that people perceive it as unhealthy; and non-differential under-reporting would bias the hazard ratio towards 1, hiding a possible association. Fourthly, we do not know the type of frying procedure (deep or pan) carried out, the extent that oils were reused, and the time and temperature used for frying. It is possible that these factors are differentially associated with coronary heart disease. Fifthly, we cannot separate the effect of a specific food from the effect of the cooking method used. For instance, we cannot separate the effect of frying with oil (for example, olive oil) from the cardioprotective effect of omega 3 fatty acids in fish. Sixthly, results are based on what people ate between 1992 and 1996. Unfortunately, since then no studies have been published on the consumption of fried food by the Spanish population. Data from household consumption surveys, however, indicate that the macronutrient intake has not changed materially from 1991 to 200638; also, overall adherence to the Mediterranean pattern seems fairly stable in the period 1998 to 2005,39 although an increase in the consumption of non-alcoholic beverages and dairy products and a decrease in consumption of potatoes and legumes has been observed over the past two decades.38 Finally, although average consumption of fried food in the highest quarter was fairly high, our results cannot rule out that a fried food intake higher than that observed in this study is associated with coronary heart disease.\n\nThis analysis also has important strengths, such as the use of a validated dietary history with quantitative information on the consumption of a large number of fried foods, the large sample size, long follow-up, and adjustment for numerous potential confounders.\n\n## Conclusions\n\nIn a Mediterranean country where olive and sunflower oils are the most commonly used fats for frying, and where large amounts of fried foods are consumed both at and away from home, no association was observed between fried food consumption and the risk of coronary heart disease or death.\n\nOur results are directly applicable only to Mediterranean countries with frying methods similar to those in Spain. Firstly, oil (mainly olive and sunflower) rather than solid fat is used for frying in Spain. It is well established that olive oil is less prone to oxidation than other edible oils or fats.40 41 Secondly, consumption of fried foods in Spain is not a proxy for fast food intake. Fast foods are generally prepared by deep frying with oils used several times, and are consumed mostly away from home. In Spain, fried foods are consumed both at and away from home, and both deep frying and pan frying are used. Moreover, we can assume that oil is not reused many times for foods consumed at home; however, the cardiovascular effects of food fried with overly reused oils merit further research. Finally, consumption of fried snacks high in salt is fairly low in Spain, whereas in other countries such as the United States they provide an important percentage of energy intake.42\n\n### What is already known on this topic\n\n1.  The consumption of fried food has been associated with some cardiovascular risk factors, such as hypertension, obesity, and low levels of high density lipoprotein cholesterol\n\n2.  The association between fried food consumption and coronary heart disease has been evaluated in some case-control studies, with heterogeneous results\n\n### What this study adds\n\n1.  In Spain, a Mediterranean country that uses olive or sunflower oil for frying, the consumption of fried foods was not associated with risk of coronary heart disease or all cause mortality\n\n2.  Frying with other types of fats, reusing oils several times, or consuming fried snacks high in salt may still be harmful\n\nCite this as: *BMJ* 2012;344:e363","meta":{"dup_signals":{"dup_doc_count":120,"dup_dump_count":59,"dup_details":{"curated_sources":2,"2023-50":2,"2023-23":1,"2022-27":2,"2022-05":1,"2021-49":1,"2021-25":1,"2021-17":1,"2021-10":1,"2020-34":1,"2020-24":1,"2020-10":1,"2019-51":1,"2019-35":1,"2019-22":1,"2019-18":1,"2019-13":1,"2019-09":1,"2019-04":1,"2018-47":1,"2018-39":2,"2018-17":2,"2018-09":1,"2018-05":1,"2017-47":2,"2017-43":2,"2017-39":1,"2017-34":1,"2017-30":1,"2017-26":2,"2017-22":2,"2017-17":1,"2017-09":1,"2016-50":1,"2016-44":2,"2016-40":1,"2016-36":3,"2016-30":3,"2016-26":2,"2016-22":3,"2016-18":2,"2016-07":3,"2015-48":3,"2015-40":2,"2015-35":3,"2015-32":2,"2015-27":3,"2015-22":3,"2015-14":2,"2014-52":3,"2014-49":4,"2014-42":8,"2014-41":3,"2014-35":5,"2014-23":5,"2017-13":3,"2015-18":3,"2015-11":3,"2015-06":2,"2024-22":1}},"file":"PMC3265571"},"subset":"pubmed_central"}
{"text":"date: 2014-01-10\ntitle: Findings of research misconduct.\n\n# Findings of Research Misconduct\n\n**Notice Number:** NOT-OD-14-039\n\n**Key Dates  \nRelease Date:** January 9, 2014\n\n**Related Announcements  \n** None\n\nNone\n\n**Issued by  \n** Department of Health and Human Services ([DHHS](http:\/\/www.hhs.gov\/))**  \n**\n\nPurpose\n\nNotice is hereby given that the Office of Research Integrity (ORI) has taken final action in the following case:\n\nBaoyan Xu, M.D., Ph.D., National Heart, Lung, and Blood Institute, National Institutes of Health: Based on allegations made by readers of a published paper,^[1](#ftn1)^ additional review by the National Institutes of Health (NIH) and ORI, and a limited admission by the Respondent that \\`\\`some better looking strips were repeatedly used as representatives for several times \\[sic\\],' ORI found that Dr. Baoyan Xu, formerly a Postdoctoral Fellow, Hematology Branch, Systems Biology Center, National Heart, Lung, and Blood Institute (NHLBI), NIH, and currently at the Institute of Infectious Diseases, Southwest Hospital, Third Military Medical University, Chonqing, China, engaged in research misconduct in research supported by intramural research at NHLBI, NIH.\n\nThe questioned research involves a Western blot analysis of IgM and IgG antibodies from Chinese subjects in patients with non-A-E hepatitis and control subjects to test reactivity towards a newly discovered virus. Analysis of Figure 6 of the published paper and Figure S4 of the online supplemental information identified thirteen pairs of Western blot bands which had a common origin yet were labeled as from different subjects and usually as detecting a different class of immunoglobulin. Specifically the following pairs were shown to match using forensically useful tools in Photoshop. Each represent a falsification in one or both of the figures as indicated in the table:\n\n| Identity of Strips | Located in:           |\n|--------------------|-----------------------|\n| A1 IgM\/ F1 IgG     | Fig. 6 & Fig. S4      |\n| B6 IgM\/E1 IgM      | Fig. 6 & Fig. S4      |\n| D7 IgM\/A11 IgG     | Fig. 6 & Fig. S4      |\n| G3 IgM\/H4IgG       | Fig. S4               |\n| H9 IgM\/F4 IgG      | Fig. S4               |\n| A4 IgM\/E2 IgG      | Fig. S4               |\n| A5 IgM\/B9 IgM      | Fig. S4               |\n| C9 IgG\/C6 IgM      | Fig. S4               |\n| D11 IgM\/H11 IgG    | Fig. S4               |\n| D5 IgM\/A1 IgG      | Fig. S4               |\n| A10 1GM\/F7 IgG     | Fig. S4               |\n| C11 1gM\/E9 IgG     | Fig. 6                |\n| F3 IgG\/E9 IgM      | F3 in S4\/E9 in Fig. 6 |\n\nThe Respondent agreed to correction of Figures 6 and S4 of the PNAS paper.\n\nDr. Xu has entered into a Voluntary Settlement Agreement and has voluntarily agreed for a period of three (3) years, beginning on December 6, 2013:\n\n\\(1\\) That prior to the submission of an application for U.S. Public Health Service (PHS) support (including NIH support) for a research project on which the Respondent's participation is proposed, and prior to Respondent's participation in any capacity on PHS-supported research, Respondent shall ensure that a plan for supervision of her duties is submitted to ORI for approval; the supervision plan must be designed to ensure the scientific integrity of Respondent's research contribution; Respondent agrees that she shall not participate in any PHS-supported research until such a supervision plan is submitted to and approved by ORI; Respondent agrees to maintain responsibility for compliance with the agreed-upon supervision plan;\n\n\\(2\\) That any institution employing her shall submit, in conjunction with each application for PHS funds, or report, manuscript, or abstract involving PHS-supported research in which Respondent is involved, a certification to ORI that the data provided by Respondent are based on actual experiments or are otherwise legitimately derived, that the data procedures, and methodology are accurately reported in the application, report, manuscript, or abstract, and that the text in such submission is her own or properly cites the source of copied language and ideas; and\n\n\\(3\\) To exclude herself voluntarily from serving in any advisory capacity to PHS including, but not limited to, service on any PHS advisory committee, board, and\/or peer review committee, or as a consultant.\n\n^1^Xu, B., Zhi, N., Hu, G., Wan, Z., Zheng, X., Liu, X., Wong, S., Kajigaya, S., Zhao, K., Mao, Q., & Young, N.S. \\`\\`Hybrid DNA virus in Chinese patients with seronegative hepatitis discovered by deep sequencing.' Proc. Natl. Acad. Sci. (US) 110(25):10264-10269; hereafter referred to as the PNAS paper.'\n\nInquiries\n\nPlease direct all inquiries to:**  \n  \n** David E. Wright, Ph.D.**  \n** Director**  \n** Office of Research Integrity**  \n** 1101 Wootton Parkway, Suite 750**  \n** Rockville, MD 20852**  \n** Telephone: 240-453-8800","meta":{"dup_signals":{"dup_doc_count":104,"dup_dump_count":34,"dup_details":{"curated_sources":2,"2020-45":1,"2017-30":1,"2017-22":1,"2017-17":1,"2017-09":2,"2017-04":1,"2016-50":1,"2016-44":2,"2016-40":3,"2016-36":2,"2016-30":3,"2016-26":3,"2016-22":3,"2016-18":2,"2016-07":4,"2015-48":4,"2015-40":5,"2015-35":5,"2015-32":4,"2015-27":4,"2015-22":4,"2015-14":4,"2014-52":4,"2014-49":4,"2014-42":5,"2014-41":5,"2014-35":4,"2014-23":3,"2014-15":2,"2021-31":1,"2015-18":4,"2015-11":4,"2015-06":4,"2014-10":2}},"file":"PMC4259754"},"subset":"pubmed_central"}
{"text":"abstract: Manganese dioxide nanofibers with length ranged from 0.1 to 1 \u03bcm and a diameter of about 4\u20136 nm were prepared by a chemical precipitation method. Composite electrodes for electrochemical supercapacitors were fabricated by impregnation of the manganese dioxide nanofibers and multiwalled carbon nanotubes (MWCNT) into porous Ni plaque current collectors. Obtained composite electrodes, containing 85% of manganese dioxide and 15 mass% of MWCNT, as a conductive additive, with total mass loading of 7\u201315 mg cm^\u22122^, showed a capacitive behavior in 0.5-M Na~2~SO~4~ solutions. The decrease in stirring time during precipitation of the nanofibers resulted in reduced agglomeration and higher specific capacitance (SC). The highest SC of 185 F g^\u22121^ was obtained at a scan rate of 2 mV s^\u22121^ for mass loading of 7 mg cm^\u22122^. The SC decreased with increasing scan rate and increasing electrode mass.\nauthor: Jun Li; QuanMin Yang; Igor Zhitomirsky\ndate: 2010\ninstitute: 1Department of Materials Science and Engineering, McMaster University, 1280 Main Street West, Hamilton, ON, L8S 4L7, Canada; 2Vale Inco Limited, Mississauga, ON, L5K 1Z9, Canada\nreferences:\ntitle: Composite Electrodes for Electrochemical Supercapacitors\n\n# Introduction\n\nPorous Ni materials, such as plaques \\[1\\] and foams \\[2\\], are widely used in industry for the fabrication of electrodes for rechargeable batteries. Nanostructured active materials are impregnated chemically or electrochemically into the porous Ni structures, which are used as current collectors \\[3\\]. Ni plaques are current collectors of choice for battery applications demanding high power and reliability, along with long cycle life, such as batteries for aerospace and railway applications, power tools and some portable electronics \\[1\\]. The pore size of Ni plaques is smaller compared to that of foams. The smaller pore size decreases the distance for electrons to travel from the current collector into the active material during cell discharge and consequently improves the discharge rate characteristics for high power applications \\[3\\]. Significant advances in the development of Ni plaques were achieved by the use of filamentary Ni particles with high surface area, which improved contact with active materials \\[4\\].\n\nA new wave of interest in the application of porous Ni materials is related to the development of electrochemical supercapacitors (ES) \\[5\\]. ES can complement or replace batteries in electrical energy storage applications when high-power delivery is required \\[6\\]. Manganese dioxides with various crystalline structures are important materials for electrodes of ES \\[7\\]. The interest in the application of manganese dioxide in ES is related to low cost of this material, which exhibits high SC in environmentally friendly aqueous electrolytes \\[8\\].\n\nThe charging mechanism of manganese dioxide electrodes of ES is described by the following reaction \\[9\\]:\n\n![](1556-276X-5-512-i1.gif)\n\nwhere A^+^ = Li^+^, Na^+^, K^+^, H^+^. Equation 1 indicates that high electronic and ionic conductivity of the electrode material are important in order to utilize the high theoretical capacitance (1,370 F g^\u22121^) of manganese dioxide \\[10\\]. Composite \\[11\\] and thin film electrodes \\[12\\] were developed and investigated. The properties of MnO~2~ are influenced by crystalline structure, particle size, porosity and surface area \\[13\\]. A complicating factor in the application of MnO~2~ in ES is low electronic conductivity of this material. This problem can be addressed by the use of advanced current collectors, such as Ni plaques.\n\nIn a previous investigation \\[14\\], manganese dioxide nanofibers were prepared by a chemical precipitation method and utilized for the fabrication of composite manganese dioxide\u2014MWCNT films by electrophoretic deposition. The composite films deposited on stainless steel foils showed high SC in a voltage window of 1.0 V. However, the SC decreased significantly with increasing film thickness. The results presented below indicated that relatively high SC can be achieved at high active material loading using Ni plaques as current collectors. In this approach, the high surface area and porous structure of the Ni plaques provided improved electrical contact of the current collector with active material and enabled good electrolyte access to the active material. We presented experimental results on the fabrication of composite electrodes, investigation into electrode microstructure and electrochemical behavior.\n\n# Experimental Procedures\n\nManganese dioxide nanofibers were prepared by the method described in the previous investigation \\[14\\]. In this approach, precipitation was performed by the reduction of 0.2-M KMnO~4~ (Aldrich) solutions with ethanol using the following reaction:\n\n![](1556-276X-5-512-i2.gif)\n\nMWCNT were provided by Arkema company. The average diameter of MWCNT was \\~15 nm and length \\~0.5 \u03bcm. Ni plaques with mass of 0.1 mg cm^\u22122^ were provided by Inco company and impregnated with a manganese dioxide slurry containing 15 mass% of MWCNT.\n\nXRD studies were performed with a diffractometer (Nicolet I2) using monochromatic Cu K\u03b1 radiation at a scanning speed of 0.5 deg min^\u22121^. TGA and DTA of the manganese dioxide nanofibers were carried out in air at a heating rate of 5 \u00b0C\/min using a thermoanalyzer (Netzsch STA-409). Electron microscopy investigations were performed using a JEOL 2010F transmission electron microscope and a JEOL JSM-7000F scanning electron microscope equipped with energy-dispersive spectroscopy.\n\nCapacitive behavior of the electrodes was studied using a potentiostat (PARSTAT 2273, Princeton Applied Research) controlled by a computer using a PowerSuite electrochemical software. Electrochemical studies were performed using a standard three-electrode cell containing 0.5 M-Na~2~SO~4~ aqueous solution degassed with purified nitrogen gas. The surface area of the working electrodes was 1 cm^2^. The counter electrode was a platinum gauze, and the reference electrode was a standard calomel electrode (SCE). Cyclic voltammetry (CV) studies were performed within a potential range of 0\u20131.0 V versus SCE at scan rates of 2\u2013100 mV s^\u22121^. The SC was calculated using half the integrated area of the CV curve to obtain the charge (*Q*), and subsequently dividing the charge by the mass of the active material (*m*) and the width of the potential window (\u0394*V*):\n\n![](1556-276X-5-512-i3.gif)\n\nImpedance spectroscopy investigations were performed in the frequency range of 0.1 Hz\u2013100 kHz at amplitude voltage of 5 mV. Simulations of the impedance behavior were performed on the basis of the equivalent-circuit models using ZsimpWin 3.10 commercial software.\n\n# Results and Discussion\n\nIn the previous investigation \\[14\\], manganese dioxide nanofibers were prepared by the reduction in KMnO~4~ solutions with ethanol, followed by stirring of obtained suspensions during 20 h, and then washing and drying of the suspension. As an extension of the previous investigation, the stirring time was varied in the range of 1\u201320 h. Figure <a href=\"#F1\" data-ref-type=\"fig\">1a<\/a>, <a href=\"#F1\" data-ref-type=\"fig\">1b<\/a> show typical TEM images of the nanofibers obtained after 1 h of stirring. The diameter of the nanofibers was 4\u20136 nm and length 0.1\u20131 \u03bcm. It was found that the nanofibers were not individual crystals but were composed of small nanoparticles with typical size less than 5 nm. The diameter of the nanofibers was non-uniform (Fig. <a href=\"#F1\" data-ref-type=\"fig\">1b<\/a>). The increase in the stirring time from 1 to 20 h resulted in partial agglomeration of the nanofibers, which formed bundles (Fig. <a href=\"#F1\" data-ref-type=\"fig\">1c<\/a>), containing individual nanofibers. This result was in a good agreement with the results of investigation into other nanomaterials \\[15\\], which indicated that stirring of nanoparticles can promote agglomeration.\n\nXRD studies (Fig. <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>) showed changes in the diffraction patterns of the nanofibers with increasing stirring time. The XRD pattern of the nanofibers stirred for 1 h showed XRD peaks of birnessite, corresponding to the JCPDS file 87-1497. According to the literature, birnessite has a two-dimensional layered structure that consists of edge-shared MnO~6~ octahedra with cation and water molecules occupying the interlayer regions \\[16\\]. The birnessite formula is generally expressed as A~*x*~MnO~2+*y*~(H~2~O)~*z*~, in which A represents an alkali metal cation. The average oxidation state of Mn usually falls between 3.6 and 3.8, which represents a predominance of Mn^4+^ with minor amounts of Mn^3+^ and Mn^2+^\\[16\\]. The intensity of the peaks of the birnessite phase decreased with increasing stirring time, indicating lower crystallinity. After 20 h of stirring, the XRD pattern showed very small broad peaks. It was suggested that the nanofibers contained crystalline and amorphous phases.\n\nIt was found that the precipitated manganese dioxide nanofibers contained potassium and adsorbed water. EDS studies showed the K\/Mn atomic ratio of 0.16 \u00b1 0.03 and 0.14 \u00b1 0.03 for nanofibers stirred during 1 and 20 h, respectively. Figure <a href=\"#F3\" data-ref-type=\"fig\">3<\/a> shows TGA and DTA data for the nanofibers stirred during 1 h. The TGA studies of the powders showed the mass loss related to the dehydration at temperatures below 400 \u00b0C. Corresponding DTA data showed a broad endotherm at \\~150 \u00b0C. Small mass gain in the range of 400\u2013420 \u00b0C in TGA data and corresponding DTA exothermic peak can be attributed to oxidation of non-stoichiometric manganese dioxide. The mass loss in the range of 880\u2013900 \u00b0C and corresponding endotherm can be attributed to reduction of manganese dioxide in agreement with the literature data \\[17\\]. Similar behavior was observed for the nanofibers stirred during 20 h.\n\nThe slurries containing manganese dioxide nanofibers and MWCNT were used for the impregnation of Ni plaques. Figure <a href=\"#F4\" data-ref-type=\"fig\">4a<\/a> shows a schematic of the cross-section of a Ni plaque, which consists of a perforated Ni foil and sintered Ni particles. The voids between the particles provide a space for the loading of the plaques with an active material. Figure <a href=\"#F4\" data-ref-type=\"fig\">4b<\/a>, <a href=\"#F4\" data-ref-type=\"fig\">4c<\/a> show SEM images of the surface of a Ni plaque at different magnifications. The plaques exhibited porous microstructure with pore size in the range of 1\u201350 \u03bcm (Fig. <a href=\"#F4\" data-ref-type=\"fig\">4b<\/a>). The SEM image obtained at higher magnification (Fig. <a href=\"#F4\" data-ref-type=\"fig\">4c<\/a>) showed small particle size of Ni particles in the range of 0.5\u20133 \u03bcm, which formed a conductive porous matrix. It was suggested that high surface area, small particle size, porosity and conductivity of Ni plaques are beneficial for application in ES. The SEM image of the impregnated material showed porous fibrous microstructure (Fig. <a href=\"#F5\" data-ref-type=\"fig\">5<\/a>) containing manganese dioxide nanofibers and MWCNT.\n\nIn the previous investigation \\[14\\], it was found that manganese dioxide nanofibers and MWCNT formed a porous fibrous network, which was beneficial for the electrolyte access to the active material. However, the SC of the films deposited on metal foil substrates decreased significantly with increasing film mass from 50 to 300 \u03bcg cm^\u22122^. In contrast, the results presented below showed that relatively high SC can be obtained for active material loading of 7\u201315 mg cm^\u22122^ using Ni plaque current collectors. In this approach, porous Ni current collectors \\[2\\] provided improved contact with active material, whereas MWCNT was used as a conductive additive.\n\nFigure <a href=\"#F6\" data-ref-type=\"fig\">6<\/a> shows typical CVs for the composite electrode with material loading of 7 mg cm^\u22122^. Within the potential range of 0\u20131.0 V versus SCE, the composite electrode exhibited capacitive-like current-potential responses, indicated by the box shape of the CVs. It is clear from the Fig. <a href=\"#F6\" data-ref-type=\"fig\">6<\/a> that there are no redox peaks in the range between 0 and 1.0 V. Figure <a href=\"#F7\" data-ref-type=\"fig\">7<\/a> shows SC at different scan rates for the composite electrodes. The SC decreased with increasing scan rate due to the diffusion limitations in pores. The electrodes prepared using powders stirred during 1 h showed higher SC compared to the powders stirred during 20 h. This can be attributed to low agglomeration of the powders stirred during 1 h. This result indicated that further optimization of the powder processing conditions can be beneficial for the improvement in electrode performance. The highest SC of 185 F g^\u22121^ was achieved at a scan rate of 2 mV s^\u22121^ for material loading of 7 mg cm^\u22122^. However, the SC decreased with increasing material loading (Fig. <a href=\"#F8\" data-ref-type=\"fig\">8<\/a>). The reduction in the SC is especially evident at a scan rate of 100 mV s^\u22121^. Turning again to the results of the previous investigation \\[14\\], it is seen that due to the use of Ni plaque current collectors, the material loading can be increased by 1\u20132 orders of magnitude, and relatively high SC can be obtained. Moreover, the SC (Fig. <a href=\"#F8\" data-ref-type=\"fig\">8<\/a>) at a scan rate of 100 mV s^\u22121^ for the 7 mg cm^\u22122^ loading of Ni plaque (SC = 111 F g^\u22121^) was higher compared to the SC of the 0.24 mg cm^\u22122^ film (SC = 82 F g^\u22121^) deposited on a foil substrate \\[14\\]. The high SC at high scan rates is important for the fabrication of efficient ES \\[6\\].\n\nFigure <a href=\"#F9\" data-ref-type=\"fig\">9<\/a> shows charge\u2013discharge behaviour for the composite electrode with material loading of 10 mg cm^\u22122^. The charge\u2013discharge curves obtained at different current densities were nearly linear and indicated good capacitive behavior in agreement with box shape CVs shown in Fig. <a href=\"#F6\" data-ref-type=\"fig\">6<\/a>. Figure <a href=\"#F10\" data-ref-type=\"fig\">10<\/a> shows impedance spectroscopy data for the sample of the same mass. The equivalent circuit of ES was discussed in the literature and included RC transmission line, describing the porous electrode \\[5\\]. C~*n*~ elements represent double-layer capacitance and pseudo capacitance, whereas R~*n*~ elements represent electrolyte resistance in pores, Faradaic resistance and equivalent series resistance of the electrodes \\[18\\]. Conway and Pell described the impedance of porous electrode using 5-element (*n* = 5) circuit \\[5\\]. A constant phase impedance (CPE) element, rather than a pure capacitance *C*, was used in another investigation \\[19\\]. The CPE element describes a 'leaking' capacitor with microscopic roughness of the surface and capacitance dispersion of interfacial origin. The equivalent circuits should allow an optimum representation of the measured spectra with a minimum set of model parameters. Good agreement of simulated and measured data (Fig. <a href=\"#F10\" data-ref-type=\"fig\">10<\/a>) was found for the equivalent circuit similar to that proposed for composite ruthenium oxide-graphite electrodes \\[20\\]. In this circuit, *C*~1~ and *R*~1~ described double-layer capacitance and charge transfer resistance, respectively. *Q*~2~ described CPE impedance of porous electrode, and *R*~2~ represented electrolyte resistance. The high frequency *Z*\u2032 value of the complex impedance *Z* = *Z*\u2032 \u2212 *iZ*\u2033 showed that *R* = *R*~1~ + *R*~2~ is about 0.4 Ohm for the sample with an area of 1 cm^2^. Relatively low resistance *R* is beneficial for high power ES \\[18\\].\n\n# Conclusions\n\nManganese dioxide nanofibers with length ranged from 0.1 to 1 \u03bcm and a diameter of about 4\u20136 nm were prepared by a chemical precipitation method. Composite electrodes for ES, containing two different fibrous materials, were fabricated by impregnation of slurries of the manganese dioxide nanofibers and MWCNT, as a conductive additive, into porous Ni plaque current collectors. The composite electrodes with total mass loading of 7\u201315 mg cm^\u22122^ showed good capacitive behavior in the 0.5 M Na~2~SO~4~ solutions. The reduction in stirring time of the precipitated nanofibers resulted in lower agglomeration and higher SC. The highest SC of 185 F g^\u22121^ was obtained at a scan rate of 2 mV s^\u22121^ for materials loading of 7 mg cm^\u22122^. Testing results indicated that Ni plaques are promising current collector materials for application in ES.\n\n## Acknowledgments\n\nThe authors gratefully acknowledge the financial support of the Natural Sciences and Engineering Research Council of Canada.\n\n### Open Access\n\nThis article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.","meta":{"dup_signals":{"dup_doc_count":101,"dup_dump_count":26,"dup_details":{"curated_sources":4,"2022-33":1,"2021-10":1,"2020-29":1,"2019-51":1,"2019-43":1,"2015-48":4,"2015-40":3,"2015-35":5,"2015-32":2,"2015-27":4,"2015-22":5,"2015-14":1,"2014-52":5,"2014-49":5,"2014-42":5,"2014-41":6,"2014-35":7,"2014-23":8,"2014-15":3,"2023-40":1,"2015-18":5,"2015-11":4,"2015-06":5,"2014-10":4,"2013-48":5,"2013-20":5}},"file":"PMC2894092"},"subset":"pubmed_central"}
{"text":"abstract: A report on the American Society for Biochemistry and Molecular Biology symposium 'Transcriptional Regulation by Chromatin and RNA polymerase II', Lake Tahoe, USA, 29 October-1 November 2004.\nauthor: Bing Li; Chun Ruan; Jerry L Workman\ndate: 2005\ninstitute: 1Stowers Institute for Medical Research, 1,000 East 50th Street, Kansas City, MO 64110, USA; 2The Huck Institute of the Life Sciences, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA\ntitle: Histones: should I stay or should I go?\n\nThis year's American Society for Biochemistry and Molecular Biology symposium on transcription covered a wide variety of topics ranging from chromatin regulation, through the initiation of transcription and elongation process during transcription by RNA polymerase II (Pol II) to the roles of signaling in transcription and development. This report focuses on the sessions on chromatin, which led to many insightful discussions as a consequence of the rapid advances in this field over the past few years.\n\n# Direct and indirect functions of histone modifications\n\nA central role for chromatin in regulating processes such as transcription and replication is now widely recognized. It is generally believed that the dynamic regulation of chromatin structure makes use of a diverse repertoire of post-translational histone modifications, ATP-dependent chromatin remodeling and histone-variant exchange. Prevailing views on histone modification and its consequences for the modulation of chromatin dynamics, as proposed in the 'histone code hypothesis', suggest that combinations of covalent modifications of specific histone residues comprise a structural and chemical 'code' that can be recognized by other protein modules which then regulate DNA accessibility and function.\n\nTony Kouzarides (University of Cambridge, UK) presented an interesting case that strongly supports this hypothesis. His group has found that methylation of lysine 20 of histone H4 in the fission yeast *Schizosaccharomyces pombe* is mediated by a novel protein, Set9, that contains a SET domain, a feature that characterizes a subset of chromatin modulators. Unlike other cases of histone lysine methylation, H4 Lys20 methylation appears not to be involved in regulating transcription or the formation of heterochromatin. Instead, it plays a pivotal role in the DNA-damage response pathway. Loss-of-function Set9 mutants and histone H4 K20R (lysine to arginine) mutants prematurely proceed to mitosis by skipping through the checkpoint that can cause arrest between G2 and M phases, even if they have unrepaired DNA. Kouzarides reported that Set9 is required for phosphorylation of the cell-cycle checkpoint protein Crb2, a homolog of the mammalian p53-binding protein 53BP1, and its recruitment to DNA double-strand breaks. He proposed that Crb2 might bind to methylated histone through its Tudor domain, which is present in many RNA-binding proteins. He made the interesting point that the H4 Lys20 methylation itself was not induced upon DNA damage and thus must be present already. Given that Crb2 binds to methylated H4 only at double-strand breaks, it was suggested that high-order structural change in chromatin elicited by DNA damage might be crucial for exposing the buried modified histone tails to their binding partners.\n\nRecent systematic proteomic studies have identified the physical location of a number of novel histone modifications, and Michael Cosgrove (Johns Hopkins School of Medicine, Baltimore, USA) called our attention to certain scenarios in which dynamic chromatin regulation might not be explained by the histone code hypothesis alone. Cosgrove has mapped known histone modifications onto the crystal structure of the nucleosome core particle and he pointed out that a good proportion of them lie in the globular domains of the core histones. In fact, many modifications are positioned at the nucleosome lateral surface, where they would be likely to affect histone-DNA interactions. From a structural perspective he suggested that these modifications might have direct roles in fine tuning histone-DNA contacts. Moreover, for these modifications to be made to pre-assembled nucleosomes, a dramatic conformational change would be required to make specific residues accessible to the modifying enzyme.\n\nCosgrove proposed a 'regulated nucleosome mobility' model, in which the mobility of nucleosomes is dictated by the affinity of histone octamers for DNA, which is in turn highly regulated by the concerted action of ATP-dependent remodeling factors and histone-modifying enzymes. In this model, remodeling and histone modification can mutually influence each other; this differs from the predictions of the histone-code hypothesis that histone modifications are established first and then direct later remodeling events. The eventual changes in nucleosome mobility can result from either remodeling or covalent modifications or combinations of both. Cosgrove cited genetic evidence to support this argument, noting that some *Sin* (Swi-independent) mutations in histones, which can bypass the requirements for Swi\/Snf chromatin-remodeling enzymes or the histone acetyl transferase Gcn5 for transcription, coincide with several modified residues located at histone-DNA contact regions, suggesting that it is nucleosome mobility that matters.\n\n# Nucleosome displacement in transcription\n\nSeveral presentations at the meeting shed new light on the relationship between nucleosome occupancy and transcriptional activity. In a poster presentation, Cheol-Koo Lee of Jason Lieb's laboratory (University of North Carolina, Chapel Hill, USA) described their work on determining relative nucleosome occupancy throughout the *Saccharomyces cerevisiae* genome. Using chromatin immunoprecipitation (ChIP) assays in combination with DNA microarrays that cover the entire yeast genome at a 1 kilobase (kb) resolution, they found that immunoprecipitating Myc-tagged histone H4 and histone H3 preferentially pulls down more coding DNA than non-coding DNA. This suggests that nucleosomes are depleted from active regulatory elements. By comparing their data to transcription-profile datasets, Lee and colleagues found that the extent of nucleosome occupancy at gene promoters is inversely proportional to the rate of gene transcription. Moreover, when growth conditions are altered, nucleosome occupancy on promoters undergoes some dramatic changes as gene-expression patterns change, but the same inverse correlation still applies. Interestingly, this universal pattern of nucleosome distribution does not appear to rely on specific histone tails, as Lee and colleagues observed similar results in yeast strains bearing various tailless histone mutations.\n\nKevin Struhl (Harvard Medical School, Boston, USA) reached a similar conclusion - that there is an inverse correlation between nucleosome occupancy and transcription - for the yeast *GAL1* promoter. He also reported that heat inactivation of the Pol II carboxy-terminal domain kinase Kin28 caused a reduction of Pol II occupancy at promoters and coding regions, and a corresponding increase in H3 occupancy of the same regions. This suggests a tight link between the presence of Pol II and histone loss. Using an artificial gene containing a *GAL1* promoter driving a long coding sequence, Struhl and colleagues were able to monitor the last wave of Pol II traveling along the gene after inactivation of the promoter. They discovered that as Pol II transcribed through the gene, histones were immediately deposited onto the DNA behind the polymerase, thus restoring the chromatin structure.\n\nStruhl also presented biochemical data suggesting that promoter DNA may resist nucleosome formation. He and his colleagues reconstituted nucleosomes with yeast genomic DNA *in vitro*, isolated the free DNA from the assembled nucleosomes, and subsequently hybridized it to DNA microarrays of the whole yeast genome. They found that 75-80% of promoter regions were half as likely to reconstitute nucleosomes than were coding regions. This suggests that intrinsic properties of promoter DNA sequence may contribute to nucleosomal distribution in a manner independent of transcription.\n\nDavid Gross (Louisiana State University Health Sciences Center, New Orleans, USA) has also observed apparent histone eviction in yet another model system. Heat-shock activation of the *S. cerevisiae* *HSP82* gene coincided with loss of nucleosomes from its promoter and coding regions. He claimed that dissociation of histones at *HSP82* was not caused by the global effects of heat shock, as two genes (*YAR1*, *CIN2*) adjacent to *HSP82* did not lose histones. Unlike the scenarios described by Lee, Gross observed a transient hyperacetylation of H2A, H3 and H4 tails at promoter regions prior to the loss of histones; the functional consequence of this is still unclear, however. Most importantly, in this case transcription *per se* does not seem to be the direct cause of these dramatic changes in the chromatin. Deletion of the TATA box reduced *HSP82* transcription to a much lower level than that observed with a promoter mutation that weakens binding of the transcriptional activator heat-shock factor (HSF). Nevertheless, the domain-wide histone eviction is almost unaffected in the TATA mutant but is abolished when HSF can no longer efficiently bind to the upstream sequence. This is in marked contrast to Struhl's results, where transcription seems to have an active role. What causes histone dissociation at *HSP82* is largely unknown; dissociation does, however, seem to be independent of some prominent chromatin remodelers such as Swi\/Snf, Gcn5, the histone methylase Set1 and elongation factor Paf1.\n\nFrom studies to determine which factors might be directly responsible for nucleosome loss, Melissa Adkins of Jessica Tyler's lab (University of Colorado Health Sciences Center, Denver, USA) provided evidence that the histone H3\/H4 chaperone Asf1 mediates the loss of nucleosomes at the *PHO5* and *PHO8* promoters upon activation and that histone loss was essential for transcriptional activation of these two yeast genes. This observation implies that in this case histone eviction might not be driven by the Pol II machinery traveling along the DNA; rather, it might be a prerequisite for Pol II transcription to occur after the activator (Pho4) has bound. Adkins also reported that nucleosomes were reassembled onto the *PHO5* promoter during gene repression and that the protein Spt6 might be involved in this reassembly. This is consistent with the known role of Spt6 in repressing faulty transcription from cryptic start sites in coding regions. Unsurprisingly, however, Asf1 cannot be the single answer for all genes because it seems to be unnecessary for the activation of *GAL1* and *HSP82*.\n\nThe apparent discrepancies among these reports may very well reflect the diversity and subtlety of chromatin regulation at different genes, or we may simply be missing some important links in our understanding. It is clear, however, that great strides have been made in establishing the paradigm that assembly and disassembly of nucleosomes occurs in a very dynamic manner even within a single cell cycle.","meta":{"dup_signals":{"dup_doc_count":122,"dup_dump_count":43,"dup_details":{"curated_sources":4,"2019-26":1,"2019-13":1,"2019-04":2,"2018-47":1,"2018-43":1,"2018-34":1,"2018-30":2,"2018-13":2,"2017-47":1,"2017-34":1,"2017-26":1,"2017-22":1,"2017-09":9,"2016-44":1,"2016-40":1,"2016-36":9,"2016-30":6,"2016-22":1,"2016-18":1,"2016-07":3,"2015-48":3,"2015-40":2,"2015-35":3,"2015-32":3,"2015-27":3,"2015-22":3,"2015-14":1,"2014-52":3,"2014-49":5,"2014-42":7,"2014-41":6,"2014-35":4,"2014-23":4,"2014-15":4,"2019-35":1,"2024-10":1,"2015-18":3,"2015-11":3,"2015-06":3,"2014-10":3,"2013-48":3,"2013-20":3,"2024-30":1}},"file":"PMC551529"},"subset":"pubmed_central"}
{"text":"abstract: A report from Microbial Genomes, a joint conference of the American Society for Microbiology and the Institute for Genomic Research, Monterey, California, USA, 28-31 January, 2001.\nauthor: Gary K Schoolnik\ndate: 2001\ninstitute: 1Departments of Medicine and of Microbiology and Immunology, Stanford Medical School, Beckman Center, Stanford, CA 94305, USA. E-mail: email@example.com\ntitle: The accelerating convergence of genomics and microbiology\n\nThe rapid use of available genome sequences by biologists was brilliantly evident in many of this meeting's plenary lectures. The most pervasive theme was the relationship between genome sequence and adaptation of a microbe to a specialized niche. It is particularly instructive in this regard to contrast the largest and smallest bacterial genomes discussed.\n\nThe complex and resourceful life style of Myxobacteria as free-living soil organisms (Dale Kaiser, Stanford University, USA) includes organized social behavior during food-seeking excursions and, in periods of scarcity, the formation of fruiting bodies containing spores that can be carried by the wind to richer habitats. Fruiting body development has attracted the attention of developmental biologists because it provides an opportunity to study spatial differentiation in a genetically tractable and relatively simple organism. Recent information about the surprisingly small size of the human genome notwithstanding, it is reasonable to assume that complexity and genome size should be related. And so it seems to be: Barry Goldman (Monsanto, St Louis, USA) reported that *Myxococcus xanthus* has a genome of 9.5 Mb, making it the largest bacterial genome so far sequenced, and containing an estimated 7,500 open reading frames (ORFs).\n\nBy contrast, the genome of the obligate aphid endosymbiont *Buchnera aphidicola*, discussed by Siv Andersson (Uppsala University, Sweden), comprises only 583 ORFs, of which 579 are related to *Escherichia coli* genes. Thus, *B. aphidicola* probably evolved from a genetically well-endowed, free-living ancestor and, while occupying its niche within the cytoplasm of a eukaryotic cell, drastically contracted its genome size. Insight into how this occurred comes from comparison of the sequences of two closely related *Buchnera* strains that co-evolved with their corresponding aphid hosts over the past 50 million years. On average, one gene has been eliminated each 5-10 million years. As the size of a genome is the sum of the rates at which genes are lost and gained, the on-going loss of *Buchnera* DNA was not compensated for by the acquisition of horizontally transferred DNA or by the duplication of indigenous genes. *Buchnera* probably illustrates a late stage in a multistep evolutionary process from free-living microbe, through the intermediate stages of facultative and then obligate intracellular parasitism, to its current status as an endosymbiont.\n\nSome (but not all) microbiologists consider the cytoplasm to be an extreme environment and thus inhabited by extremophiles. None would doubt, however, that the capacity of, or indeed the requirement for, *Halobacterium* sp. NRC-1 to grow in 3-5 M NaCl would so qualify. The completion of its sequence, reported by Shiladitya DasSarma (University of Massachusetts, Amherst, USA), shows that *Halobacterium* contains 91 copies of insertion sequences and that its 2,682 ORFs are distributed between one large chromosome and two related minichromosomes. Transposition of its many IS repetitive elements would provide a mechanism for genetic plasticity through the fusion of smaller replicons. In turn, this may have led to the acquisition of essential genes by megaplasmids and hence to the possibility of competition between plasmid and chromosome. A second undisputed adaptation to an extreme environment is the capacity of hyperthermophilic archaea to grow at high temperature. One such organism, *Pyrococcus furiosus*, grows optimally at 100?C and is resistant to ionizing radiation owing, in part, to its capacity to reassemble chromosome fragments. Jocelyn DiRuggiero (University of Maryland Biotechnology Institute, Baltimore, USA) discussed the initiation of microarray hybridization experiments to identify genes that are differentially expressed during exposure of this organism to ? irradiation and to sublethal temperatures.\n\nThe oceans encompass surprisingly diverse environments, where different depths within the water column provide distinctive habitats within which adaptive evolution occurs. One such depth-dependent variable is light. Edward DeLong and colleagues (Monterey Bay Aquarium Research Institute, California, USA) used large-insert bacterial artificial chromosome (BAC) libraries to characterize uncultured marine bacterioplankton. Among the genes recovered was the coding sequence for a bacterial rhodopsin. Although this gene was found in the genome of an uncultivated ? proteobacterium, it is most similar in amino-acid sequence to archaeal rhodopsins, indicating its possible acquisition by horizontal gene transfer. Expression of this gene in *E. coli* and functional analysis of the corresponding protein showed a ?~max~ corresponding to the wavelength of light available in the upper water column. By contrast, deeper-water rhodopsins were found to be blue-shifted, indicating that spectral tuning had occurred during the evolution of this protein family within bacteria living at different depths.\n\nEvolutionary divergence by closely related strains living at different ocean depths is nowhere better demonstrated than in *Prochlorococcus*, a marine cyanobacterium whose photosynthetic activity contributes significantly to primary production in the oceans. Gabrielle Rocap (Massachusetts Institute of Technology, Cambridge, USA) contrasted the sequence of two highly related strains of *Prochlorococcus* that exhibit strikingly different optimal light levels that adapt one for growth at the ocean surface and the other for growth in a deep water environment. Despite their closely related 16S rRNA sequences, the two genomes are substantially different with respect to size, GC content and codon usage. The strain living in the nitrogen-poor waters of the ocean surface lacks genes for nitrite and nitrate reductases, whereas the strain living in nitrogen-rich, deeper water possesses genes predicted to confer both activities. Thus, the sequencing of closely related strains living in different environments led to the identification of two genomic 'eco-types' that reflect both the constraints and advantages of their respective niches. How this diversity arose will be a matter of much future study, but surely one such mechanism is lateral gene transfer between natural populations of marine bacteria, as discussed by John Paul (University of South Florida, St Petersburg, USA).\n\nThree fascinating genome sequence works-in-progress were presented. Steven Slater (Cereon Genomics LLC, Cambridge, USA) discussed *Agrobacterium tumefaciens*, a plant pathogen and the etiological agent of crown gall disease; Martin Odom (DuPont, Wilmington, USA) discussed the status and initial findings of the *Methylomonas* project, a highly specialized aerobic eubacterium that can use only C-1 compounds (methanol\/methane) for carbon and energy; and Malcolm Gardner (The Institute for Genome Research (TIGR), Rockville, USA) discussed *Theileria parva*, a tick-borne intracellular protozoan parasite of domesticated ruminants that induces malignant transformation of lymphocytes leading to fatal lymphosarcomas in afflicted animals.\n\nThe use of genome sequences to study microbial pathogenesis and epidemiology or to develop new vaccines and diagnostic assays was the focus of five talks. I presented my laboratory's microarray expression-profiling study of *Mycobacterium tuberculosis* growing within the macrophage phagosome and its transcriptional response to the nutrients and reactive oxygen and nitrogen intermediates that characterize this niche. Timothy Palzkill (Baylor College of Medicine, Houston, USA) described a phage-display method to express protein products corresponding to each of the 1,030 ORFs of the *Treponema pallidum* genome. This is a particularly worthwhile goal because this bacterium, the agent of syphilis, cannot be propagated *in vitro* and there is a compelling need for improved serological tests. The emerging drug resistance of *Staphylococcus aureus* is a major threat to all hospitalized patients and thus it was encouraging to learn that Uwe von Ahsen and colleagues (Intercell GmBH, Vienna, Austria) had used patients' sera and *in vivo* and *in vitro* methods to display all the proteins encoded by the staphylococcal genome, to identify genes coding for antigenic proteins expressed during infection and accessible to the immune system.\n\nThe movement of a genetically diverse infectious agent through a population can be tracked with the highest resolution by the combined use of conventional and molecular epidemiology. For the latter, Andreas Duesterhoeft (Qiagen GmBH, Dusseldorf, Germany) described a method for genotyping microorganisms by detecting single-nucleotide polymorphisms, and its use to study the epidemiology of tuberculosis. Genetic diversity can also occur during a chronic infection and can lead to mutants that are particularly well adapted to the host; these may be more virulent or more able to persist. Evgeni Sokurenko (University of Washington, Seattle, USA) addressed this important phenomenon by describing a method that can identify point mutations in total genomic DNA and is suitable for the analysis of genetic variability in bacteria isolated from infected tissues.\n\nGenomics now and in the foreseeable future will both require and spawn novel analytical, data-mining and sequencing tools. Sung-Hou Kim (University of California, Berkeley, USA) forcefully admonished attendees that a crucial distinction exists between protein functions that are predicted from homology searches of databases, the proven role of a protein within a cellular pathway, and its molecular function as discerned by structure-function analysis. This is not merely the gap between genomics and proteomics - and the need to bridge it - as the latter term generally refers only to the 'proteome', that is, the complete protein expression capacity of the genome. Rather, as discussed by Kim, it will probably require innovative use of the rapidly expanding crystal-structure database to compile an encyclopedia of all protein folds and the development of tools that identify the set of folds that comprise the inferred protein products of newly sequenced genomes.","meta":{"dup_signals":{"dup_doc_count":127,"dup_dump_count":40,"dup_details":{"curated_sources":2,"2022-33":1,"2022-05":1,"2021-04":1,"2020-24":1,"2019-47":1,"2019-04":1,"2018-51":1,"2018-43":1,"2018-34":1,"2018-22":1,"2018-09":1,"2017-47":1,"2017-34":1,"2017-26":1,"2017-17":1,"2016-07":1,"2015-48":5,"2015-40":2,"2015-35":4,"2015-32":5,"2015-27":4,"2015-22":5,"2015-14":4,"2014-52":5,"2014-49":10,"2014-42":9,"2014-41":6,"2014-35":5,"2014-23":8,"2014-15":6,"2023-14":1,"2024-10":1,"2017-13":1,"2015-18":5,"2015-11":4,"2015-06":5,"2014-10":4,"2013-48":4,"2013-20":5,"2024-26":1}},"file":"PMC138923"},"subset":"pubmed_central"}
{"text":"abstract: Macrophages play an important role in modulating the immune function of the human body, while foam cells differentiated from macrophages with subsequent fatty streak formation play a key role in atherosclerosis. We hypothesized that nanotopography modulates the behavior and function of macrophages and foam cells without bioactive agent. In the present study, nanodot arrays ranging from 10\u2010 to 200\u2010nm were used to evaluate the growth and function of macrophages and foam cells. In the quantitative analysis, the cell adhesion area in macrophages increased with 10- to 50-nm nanodot arrays compared to the flat surface, while it decreased with 100- and 200-nm nanodot arrays. A similar trend of adhesion was observed in foam cells. Immunostaining, specific to vinculin and actin filaments, indicated that a 50-nm surface promoted cell adhesion and cytoskeleton organization. On the contrary, 200-nm surfaces hindered cell adhesion and cytoskeleton organization. Further, based on quantitative real-time polymerase chain reaction data, expression of inflammatory genes was upregulated for the 100- and 200-nm surfaces in macrophages and foam cells. This suggests that nanodots of 100\u2010\u2009and 200\u2010nm triggered immune inflammatory stress response. In summary, nanotopography controls cell morphology, adhesions, and proliferation. By adjusting the nanodot diameter, we could modulate the growth and expression of function-related genes in the macrophages and foam cell system. The nanotopography-mediated control of cell growth and morphology provides potential insight for designing cardiovascular implants.\nauthor: Mohammed Mohiuddin; Hsu-An Pan; Yao-Ching Hung; Guewha Steven Huang\ndate: 2012\ninstitute: 1Department of Material Science and Engineering, National Chiao Tung University, 1001 University Road, Hsinchu 300, Taiwan; 2Section of Gynecologic Oncology, Department of Obstetrics and Gynecology, China Medical University and Hospital, 91 Hsueh Shih Road, Taichung 404, Taiwan; 3College of Medicine, China Medical University, Taichung 404, Taiwan\nreferences:\ntitle: Control of growth and inflammatory response of macrophages and foam cells with nanotopography\n\n# Background\n\nRecent fabrication of nanostructured materials with different surface properties has generated a great deal of interest for developing implant materials, i.e., cardiovascular, dental, orthopedic, percutaneous, subcutaneous, and auditory \\[1-5\\]. The interface between nanostructured materials and biological tissues is likely to vary dependent upon the surface properties of the nanomaterial. Understanding the degree of toxicity induced by the unique cellular interaction of nanostructured materials is a major concern before utilization in biomedical applications \\[6-8\\]. Therefore, fabricating biocompatible materials which are designed to perform specific functions within living organisms has become a key component for generating nanodevices for biomedical applications, including implants.\n\nMacrophages play a critical role during innate and acquired immune responses through the phagocytosis of foreign material. During an immune response, macrophages are typically the first cell type to respond and will secrete proteins (cytokines and chemokines) in order to recruit more immune cells to the site of injury. Atherosclerosis is a pathological process that takes place in the major arteries and is the underlying cause of heart attacks, stroke, and peripheral artery disease. The earliest detectable lesions, called fatty streaks, contain macrophage foam cells that are derived from recruited monocytes. The formation of these foam cells correlates to inflammatory responses \\[9-11\\]. In particular, immune cells such as monocytes and macrophages play a key role in mediating host tissue response to implants in the foreign body reaction.One study demonstrated that the macrophage receptor with collagenous structure (MARCO) displayed limited expression in healthy cells but increased in expression around the synovial fluid following hip replacements \\[12\\]. This study indicated that the presence of a foreign body can generate an immune response, and the continued presence of the foreign body can potentially lead to macrophage buildup and production of foam cells.\n\nRecent reports have shown that microscaled landscapes are able to direct shape and migration of cultured cells. When cultured on ridges and grooves of nanoscale dimensions, cells migrate more extensively to the ridges than into the grooves. Cell shape is aligned and extended in the direction of the groove \\[13\\]. Osteoblasts grown on a fibrous matrix composed of multiwalled carbon nanofibers (100-nm in diameter) exhibit increased proliferation compared to those on flat glass surfaces \\[14-16\\]. Nanodots larger than 100-nm in diameter induced an apoptosis-like morphology for NIH-3T3 fibroblast cells \\[17\\]. Breast epithelial cells proliferate and form multicellular spheroids on interwoven polyamide fibers fabricated using electrospinning polymer solution onto a glass slide \\[18\\]. A 3-D nanofibrillar surface covalently modified with tenascin-C-derived peptides enhances neuronal growth *in vitro*\\[19\\]. The cardiomyoblast H9c2 shows induced cell adhesion and cytoskeleton organization on nanodot arrays smaller than 50-nm \\[20\\].\n\nRecently, arrays of nanodots with defined diameter and depth have been fabricated using aluminum nanopores as a template during oxidation of tantalum thin films \\[21\\]. The pore size of aluminum oxide is controllable and uniformly distributed; the depth of dots depends on the voltage applied; thus, it can serve as a convenient mold to fabricate tantalum into a nanodot array of specific diameter and depth. The structure containing nanodots of uniform size could serve as a comparable nanolandscape to probe cellular response at the molecular level. Although many implant surface topographies are commercially available, there is generally a lack of detailed comparative histological studies at the nano-interface that document how these surfaces interact with living cells, in particular immune cells. In the present study, different sizes of nanodot arrays ranging from 10-\u2009to 200-nm were used to evaluate the growth and inflammatory response of macrophages and foam cells.\n\n# Methods\n\n## Chemicals\n\nDulbecco's modified Eagle medium (DMEM), FBS, antibiotics, and all other tissue culture reagents were obtained from Gibco (Life Technologies, Carlsbad, CA, USA). Glutaraldehyde and osmium tetroxide were purchased from Electron Microscopy Sciences (Hatfield, PA, USA). Anti-vinculin mouse antibody was purchased from Abcam (Cambridge, MA, USA). Alexa Fluor 594 phalloidin and Alexa Fluor 488 goat anti-mouse IgG were purchased from Invitrogen (Carlsbad, CA, USA). Trypsin was purchased from Sigma-Aldrich (St. Louis, MO, USA). CuSO4, KBr, thiobarbituric acid, trichloroacetic acid, and other commonly used chemicals were purchased from Sigma-Aldrich or Merck (Whitehouse Station, NJ, USA).\n\n## Isolation of mouse peritoneal macrophages and formation of foam cells\n\nResident peritoneal macrophages were isolated and cultured from five mice (20 g each) and were washed once with DMEM and once with DMEM containing 10% fetal bovine serum. The cells were seeded on to different nanodot arrays, ranging from 10-\u2009to 200-nm, in DMEM containing 10% fetal bovine serum and 100 pg\/ml penicillin and cultured for 3 to 4 h at 37\u00b0C in an incubator containing 5% CO~2~ with 90% humidity. The nonadherent cells were removed, and the monolayers were then placed in DMEM containing 10% fetal bovine serum supplemented with 100 \u03bcg\/ml oxidized low-density lipoprotein (ox-LDL) or acetyl LDL; plates were further incubated for an additional 72 h.\n\n## Oil red O staining\n\nMonolayers of foam cells prepared on nanodot surface were fixed with 10% formaldehyde in phosphate-buffered saline (PBS) (pH 7.4) for 10 min at room temperature, then stained with Oil Red O, and counterstained with hematoxylin for 10 min \\[22\\].\n\n## Fabrication and characterization of nanodot arrays\n\nNanodot arrays were fabricated by anode aluminum oxide processing as described previously \\[21\\]. A tantalum nitride (TaN) thin film of 150-nm thickness was sputtered onto a 6-in. silicon wafer followed by deposition of a 3-\u03bcm-thick aluminum onto the top of a TaN layer. Anodization was carried out in 1.8 M sulfuric acid at 5 V for the 10-nm nanodot array or in 0.3 M oxalic acid at 25, 60, and 100 V for 50-, 100-, and 200-nm nanodot arrays, respectively. Porous anodic alumina was formed during the anodic oxidation. The underlying TaN layer was oxidized into tantalum oxide nanodots using the alumina nanopores as a template. The porous alumina was removed by immersing in 5% (*w*\/*v*) H~3~PO~4~ overnight.\n\nThe dot diameters were 15\u2009\u00b1\u20092.8-nm, 58.1\u2009\u00b1\u20095.6-nm, 95.4\u2009\u00b1\u20099.2-nm, and 211.5\u2009\u00b1\u200930.6-nm for the 10-, 50-, 100-, and 200-nm dot arrays. The average heights were 11.3\u2009\u00b1\u20092.5-nm, 51.3\u2009\u00b1\u20095.5-nm, 101.1\u2009\u00b1\u200910.3-nm, and 154.2\u2009\u00b1\u200927.8-nm, respectively. Dot-to-dot distances were 22.8\u2009\u00b1\u20094.6-nm, 61.3\u2009\u00b1\u20096.4-nm, 108.1\u2009\u00b1\u20092.3-nm, and 194.2\u2009\u00b1\u200915.1-nm, respectively. The dimension and homogeneity of nanodot arrays were measured and calculated from images taken using a JEOL JSM-6500 TFE-SEM (JEOL Ltd., Akishima, Tokyo, Japan).\n\n## The cell viability assay\n\nCells were harvested and fixed with 4% formaldehyde in PBS for 30 min followed by PBS wash for three times. The membrane was permeated by incubating in 0.1% Triton X-100 for 10 min, followed by PBS wash for three times. The sample was incubated with 4\u2032,6-diamidino-2-phenylindole (DAPI) and phalloidin for 15 min at room temperature. The samples were mounted and imaged using a Leica TSC SP2 confocal microscope (Leica Microsystems Ltd., Milton Keynes, UK). The number of viable cells was counted using ImageJ software (National Institutes of Health, Bethesda, MA, USA) and expressed in terms of cell density.\n\n## Scanning electron microscopy\n\nThe harvested cells were fixed with 1% glutaraldehyde in PBS at 4\u00b0C for 20 min, followed by post-fixation in 1% osmium tetroxide for 30 min. Dehydration was performed through a series of ethanol concentrations (10-min incubation each in 50%, 60%, 70%, 80%, 90%, 95%, and 100% ethanol) and air-dried. The specimen was sputter-coated with platinum and examined using a JEOL JSM-6500 TFE-SEM at an accelerating voltage of 5 keV.\n\n## Immunostaining\n\nCells were harvested and fixed with 4% paraformaldehyde in PBS for 15 min followed by PBS wash for three times. Cell membrane was permeated by incubating in 0.1% Triton X-100 for 10 min, followed by PBS wash for three times, and blocked by 1% bovine serum albumin (BSA) in PBS for 1 h and PBS wash for three times. The sample was incubated with anti-vinculin antibody (properly diluted in 1% BSA) and phalloidin for 1 h, followed by incubation with Alexa Fluor 488 goat anti-mouse antibody for 1 h, and then followed by PBS wash for three times. Samples were mounted and imaged by using a Leica TSC SP2 confocal microscope.\n\n## Quantitative real-time RT-PCR\n\nTotal RNA was extracted from macrophages and foam cells using TRI-reagent (Talron Biotech, Rehovot, Israel) according to the manufacturer's specifications. The RNA was isolated using chloroform extraction and isopropanol precipitation. The crude RNA extract was immediately purified with an RNeasy Mini Kit (Qiagen, Venlo, Netherlands) to remove impurities and unwanted organics. Purified RNA was resuspended in DEPC water and quantified by OD~260~. The OD~260~ to OD~280~ ratio usually exceeded 2.0 at this stage. For cDNA synthesis, 1 \u03bcg of total RNA was annealed with 1 \u03bcg of oligo-dT primer, followed by reverse transcription using Super Script\u00ae III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) in a total volume of 50 \u03bcl. Between 0.2 and 0.5 \u03bcl of the reverse transcription reactions was used for quantitative polymerase chain reaction (qPCR) using SYBR Green I on an iCycler iQ5 (Bio-Rad Laboratories, Hercules, CA, USA). Cycling conditions were as follows: 1\u00d7 (5 min at 95\u00b0C) and 50\u00d7 (20 s at 95\u00b0C, 20 s at 55\u00b0C, and 40 s at 72\u00b0C). Fluorescence was measured after each 72\u00b0C step. Expression levels were obtained as threshold cycles (Ct), which were determined by the iCycler iQ Detection System software (Bio-Rad Laboratories, Hercules, CA, USA). Relative transcript quantities were calculated using the \u0394\u0394Ct method. Glyceraldehydes-3-phosphate dehydrogenase (GAPDH) was used as a reference gene and was amplified from the same cDNA samples. Due to the difference in threshold cycles of the sample mRNA relative to the GAPDH, mRNA was defined as \u0394Ct. The difference between the \u0394Ct of the untreated control and the \u0394Ct of the SMF-treated sample was defined as \u0394\u0394Ct. The fold change in mRNA expression was expressed as 2^\u0394\u0394Ct^. The results were expressed as the mean\u2009\u00b1\u2009standard deviation (SD) of six experiments.\n\n# Results and discussion\n\n## Nanotopography-modulated morphology and cell spread of macrophages and foam cells\n\nTo characterize how the macrophages and foam cells interact with the aforementioned nanodot arrays, the cells were cultured for 72 h on nanodot arrays of different diameters including flat substrate as a control. The morphological appearance of adhered cells was imaged by both optical image microscopy and scanning electron microscopy (SEM) (Figures <a href=\"#F1\" data-ref-type=\"fig\">1<\/a> and <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>). Macrophages and foam cells grown on the flat surface and 10-nm nanodot arrays exhibited flat and extended conformation during the course of 3 days. Cells grown on the 50-nm nanodot arrays showed more extended morphology than those on the flat surface with apparently larger surface area for each cell. Cells grown on the 100-nm nanodot arrays exhibited a distorted morphology with shrinking surface area and increased length of lamellipodia. Apoptosis-like appearance and reduction in surface area with extended lamellipodia were seen with cells seeded on the 200-nm nanodot arrays.\n\nFormation of focal adhesions reflected by the attachment of filopodia and lamellipodia to the substratum indicates healthy growth for cultured cells. SEM images showed that the lamellar body of migrating cells, seeded on 50-nm nanodot arrays, exhibited wide and thick characters with a large number of filopodia (Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>). Cells seeded on flat and 10-nm nanodot arrays showed comparable lamellipodia. However, the cells seeded on 100- and 200-nm nanodot arrays were mounted with extended length and narrow-size lamellipodia. Figure <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>a shows the correlation between cell spread area and dot size for macrophages and foam cells . Cell surface area representing the percent adhesion area of viable cells relative to cells cultured on a flat surface was calculated and plotted against the nanodot diameter. Based on quantitative analysis, the cell spread area of macrophages increased significantly between 21.6% and 37.9% with increasing dot sizes of 10-\u2009to 50-nm, respectively. Interestingly, as dot diameter increased from 100- to 200-nm, there was a significant reduction in cell surface area of 22.7% and 43.2%, respectively. A similar biphasic trend, increasing cell surface area from 10- to 50-nm and decreasing from 100-\u2009to 200-nm, was observed with foam cells. This trend of change correlated with qualitative analysis as shown in Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>. Although the precise reason for such differential growth pattern is not known, since increasing the dot diameter above 100-nm stimulates an apoptosis-like growth and a significant reduction in the surface area, these results demonstrate that nanodot arrays larger than 100-nm are less biocompatible to cells.\n\n## Nanotopography-modulated cell adhesion and cytoskeleton organization of macrophages and foam cells\n\nTo evaluate cell adhesion and cytoskeleton reorganization, immunostaining specific to vinculin and actin filaments was performed on nanodot arrays (Figure <a href=\"#F4\" data-ref-type=\"fig\">4<\/a>). The amount of vinculin staining in foam cells was significantly less than that in macrophages for all nanodot sizes. Foam cells had fewer focal adhesion molecules than macrophages when grown on nanodot arrays and might have the destiny of apoptosis. Vinculin staining was well distributed for macrophages grown on the flat surface and on the 10- to 100-nm nanodot arrays, with the highest density of vinculin for cells grown on 50-nm nanodot arrays. Nevertheless, the amount of vinculin staining decreased for cells grown on 200-nm nanodot arrays (Figure <a href=\"#F4\" data-ref-type=\"fig\">4<\/a>). For foam cells, vinculin staining had the same trend as that for macrophages: increasing from flat to 50-nm nanodot arrays, becoming gradually lost for cells grown on 100-nm nanodot arrays, and completely disappearing for 200-nm nanodot arrays.\n\nImmunostaining of actin filaments indicated a well-organized cytoskeleton in macrophages grown on flat, 10-, and 50-nm nanodot arrays, but it is gradually lost for 100-nm nanodot arrays and has completely disappeared for 200-nm nanodot arrays. For foam cells, cytoskeleton arrangement had the same trend as macrophages: increasing from flat to 50-nm nanodot arrays, becoming gradually lost for cells grown on 100-nm nanodot arrays, and completely disappearing for 200-nm nanodot arrays.\n\nImmunostaining indicated that nanodot arrays in the range of 10- to 100-nm promoted cell adhesion and cytoskeleton organization for macrophages and foam cells (Figure <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>b). Best adhesion occurred at 50-nm nanodots, whereas nanodots of 200-nm retarded the formation of focal adhesions and inhibited the organization of the cytoskeleton.\n\n## Nanotopography-modulated cell density\n\nTo evaluate the viability of macrophages and foam cells on varied nanodot arrays, cells were seeded on nanodot arrays, ranging from 10-\u2009to 200-nm including flat control. Macrophages and foam cells were cultured for 72 h, and then, DAPI staining was performed to verify viable cells on each nanodot array and flat surface (Figure <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>c). For macrophages, compared to the flat surface, there were 10.6%, 149.1%, and 26.5% increases in the number of viable cells for 10-, 50-, and 100-nm nanodot arrays, respectively, but 41.2% reduction was observed on 200-nm nanodot arrays. For foam cells, a 110% increase in the number of viable cells was observed for 50-nm nanodot arrays, and 28.6% reduction occurred for 200-nm nanodot arrays.\n\n## Effect of nanotopography on the expression of genes related to inflammation and circulatory repair\n\nThe cytokine gene expression profiles related to inflammation of macrophages and foam cells cultured on nanodot arrays were measured at 72 h, using qPCR. The cytokines examined were TNF-\u03b1, IL-6, IL-10, CCL-2, and CCL-3 (Figure <a href=\"#F5\" data-ref-type=\"fig\">5<\/a>a,b). In addition, genes important for the development of the circulatory system and repair were also evaluated (Figure <a href=\"#F5\" data-ref-type=\"fig\">5<\/a>c,d): PAI-1, VEGF, and PECAM.\n\nTNF-\u03b1 is a pro-inflammatory cytokine and is responsible for activation and positive regulation of the NF\u03baB pathway, which is a key regulator of the immune response \\[23\\]. There was a five-fold increase in TNF-\u03b1 found at 50-nm nanodot arrays in macrophages, while a five- to six-fold increase was demonstrated in foam cells at 50- to 100-nm nanodot arrays (Figure <a href=\"#F5\" data-ref-type=\"fig\">5<\/a>a,b). Similarly, IL-6 encodes a pro-inflammatory cytokine that is critical for activating an acute inflammatory response and is responsible for recruiting adaptive immune cells to the site of injury or infection. There was a significant three-fold increase of IL-6 in macrophages cultured on 200-nm nanodot arrays, while there was about a one-fold increase at 50- to 100-nm nanodot arrays. In contrast, the foam cells responded differently than the macrophages, with a three- to five-fold increase of IL-6 when cultured on 10- to 100-nm nanodot arrays and a two-fold increase for 200-nm nanodot arrays. In comparison, IL-10 is an anti-inflammatory cytokine responsible for blocking the synthesis of pro-inflammatory cytokines and negatively regulates NF\u03baB activation. There was an increasing trend of IL-10 gene expression in the macrophages and the foam cells (Figure <a href=\"#F5\" data-ref-type=\"fig\">5<\/a>a,b). For the CCL-2 chemokine, which functions to recruit monocytes and other immune cells, there was no significant change in any of the macrophage topography conditions. However, in foam cells, there was a 2.5-fold increase at 200-nm nanodot arrays, while there was less than one-fold increase at 10- to 100-nm nanodot arrays (Figure <a href=\"#F5\" data-ref-type=\"fig\">5<\/a>b). Another common chemokine is CCL-3. This protein is responsible for recruiting and activating leukocytes to aid in an immune response. Figure <a href=\"#F5\" data-ref-type=\"fig\">5<\/a>a showed less than 1% increase in CCL-3 in macrophages at 10- to 200-nm nanodot arrays, while a six-fold increase at 200-nm nanodot arrays in foam cells was observed (Figure <a href=\"#F5\" data-ref-type=\"fig\">5<\/a>b).\n\nIn addition to the immune response, genes associated with circulatory repair were also investigated. PAI-1 is a gene involved in the prevention of blood clots. There was a five-fold increase in PAI-1 observed at 200-nm nanodot arrays in macrophages (Figure <a href=\"#F5\" data-ref-type=\"fig\">5<\/a>c), whereas foam cells displayed an inconsistent pattern of increase of PAI-1 (Figure <a href=\"#F5\" data-ref-type=\"fig\">5<\/a>d). Furthermore, VEGF is known to play a key role in the creation of new blood vessels during development as well as repair during injury. There was an inconsistent increase in VEGF for the macrophages, while foam cells demonstrated a significant increase at the 50-nm topography (Figure <a href=\"#F5\" data-ref-type=\"fig\">5<\/a>d). PECAM is responsible for removing old neutrophils from the site of injury and preventing buildup of immune cell debris. In the macrophages, there was no change in PECAM genes; however, the foam cells displayed significant increases for the 50- to 200-nm topographies. In summary, the differences in relative gene expressions shown here are topography induced and normalized to flat surface.\n\nWhen evaluating the gene expression trends observed in the macrophages, there was a highly significant increase in TNF-\u03b1 expression for the 50-nm topography. For all of the topographies, there was limited impact on the expression of CCL-3 and CCL-2, whereas, there was a mild elevation in IL-6 expression. Taken together, there was a limited induction of an inflammatory response for these topographies. The expression of PAI-1, VEGF, and PECAM were also evaluated, and there were low levels of expression for all genes in all topographies except for the 200-nm topography, which displayed significant increases in PAI-1 expression. Since PAI-1 helps to prevent blood clots and the buildup of cells, in conjunction with data from the gene expression studies where limited immune responses were observed, the 200-nm topography might assist in the prevention of foam cell formation.\n\nWhen evaluating the foam cells directly, there were high levels of the early-response pro-inflammatory cytokines IL-6 and TNF-\u03b1 for the 50- and 100-nm topographies with mild increases in expression for the 10- and 200-nm topographies. Furthermore, the 200-nm topography demonstrated high levels of expression for the pro-inflammatory cytokine CCL-3. In addition, when the circulatory repair genes were assessed, the 100-nm topography demonstrated a significant increase in the PECAM gene which functions to remove leukocyte debris from cells. Since foam cells are derived from macrophages that are not cleared from the system, this 100-nm topography could greatly aid in the prevention or removal of foam cells from the body.\n\nAfter 72 h, macrophages showed an increase in inflammatory gene expression on 10- and 200-nm nanodot arrays, while the difference was not significant compared to the flat. Foam cells showed the most inflammatory gene expression on 200-nm nanodot arrays. The common acute inflammation gene expression of CCL-3 responded significantly to the topography of 100- and 200-nm nanodot arrays for foam cells. However, the macrophages showed the most acute inflammation on the 10-nm nanodot arrays. Thus, the topographical effect on the PAI-1 gene expression was difficult to discern.\n\nRecently, it has been shown that three-dimensional surface topography (size, shape, and surface texture) is one of the most important parameters that influence cellular reactions \\[24,25\\]. Other studies have demonstrated that the difference in cellular response correlates with a modulation of the concentration of serum proteins on the surface \\[26,27\\]. Many studies have shown that cell biomaterial interactions can activate macrophages which results in the synthesis of pro-inflammatory agents such as TNF\u03b1, IFN\u03b3, IL-1, and IL-6 \\[28,29\\] Most likely, the surface properties, such as material surface chemistry and topography, can modulate the expression of pro-inflammatory cytokines and chemokines by macrophages in a time-dependent manner \\[30\\].\n\nAlthough many studies have investigated cellular reaction to different surface patterns, the behavior of immune cells, such as macrophages and foam cells, cultured on different diameters of nanodots has not been studied thoroughly. Our study suggests that topography may modulate the phenotypes of macrophages and foam cells in the context of foreign body response. The response to topography in the form of nanodot arrays in the range of 10-\u2009to 200-nm has revealed a distinctive pattern, and topography indeed affects cell morphology, density, adhesion, and cytokine expression compared to flat controls. The changes in cell morphology are observed in four different sizes of nanodot arrays, indicating that the findings in this study are topography-mediated. Using topography-induced change in macrophages and foam cell behavior, it is possible to influence phenotypic response, such as cell activation, motility, and maturation in the foreign body response. While there is a mild induction of the inflammatory response for the 100- and 200-nm topographies, these growth conditions also supported expressions of genes that would be responsible in the prevention of foam cell formation and the removal of foam cells, suggesting potential benefits. Furthermore, it might be possible to treat the cells with antioxidants or other anti-inflammatory mediators to prevent the inflammatory response while benefiting from the increase in PAI-1 and PECAM.\n\n# Conclusion\n\nWe have shown topologic modulation of cell growth, cell density, cell spreading area, and immune functions. Our results demonstrated that 50-nm nanodots displayed a biocompatible surface compared to 100- and 200-nm nanodots in terms of macrophage and foam cell growth. In addition, based on qPCR data, 100- and 200-nm surface-induced inflammatory gene expression in macrophages and foam cells suggest that nanostructured materials (100- and 200-nm) trigger the immune inflammatory stress response. The role of topography in modulating implant tissue reaction would require further elucidation. This study suggests that nanotopography may be beneficial for the design of cardiovascular implants.\n\n# Competing interests\n\nThe authors declare that they have no competing interests.\n\n# Authors' contributions\n\nMM and HAP are responsible for concept and design. MM carried out the experiments, analyzed the data, and wrote the paper under the close supervision of YCH and GSH. All authors read and approved the final manuscript.\n\n# Authors' information\n\nMM and HAP are doctoral degree students at the Department of Material Science and Engineering, National Chiao Tung University. GSH is a professor at the Department of Material Science and Engineering, National Chiao Tung University. YCH is a professor at the Department of Obstetrics and Gynecology, China Medical University and Hospital and also professor at the College of Medicine, China Medical University.\n\n## Acknowledgment\n\nThis study was supported in part by the National Science Counsel Grant 100-2923-B-009-001-MY3 and by the 'Aim for the Top University Plan of the National Chiao' Tung University and Ministry of Education, Taiwan, R.O.C.","meta":{"dup_signals":{"dup_doc_count":106,"dup_dump_count":24,"dup_details":{"curated_sources":2,"2022-40":1,"2019-51":1,"2016-07":1,"2015-48":5,"2015-40":4,"2015-35":5,"2015-32":4,"2015-27":3,"2015-22":5,"2015-14":5,"2014-52":4,"2014-49":5,"2014-42":12,"2014-41":6,"2014-35":5,"2014-23":7,"2014-15":2,"2023-40":1,"2015-18":5,"2015-11":5,"2015-06":5,"2014-10":4,"2013-48":4,"2013-20":5}},"file":"PMC3507756"},"subset":"pubmed_central"}
{"text":"abstract: A report of the seventh annual meeting on Systems Biology: Global Regulation of Gene Expression, 23-27 March 2010, Cold Spring Harbor, USA.\nauthor: Manolis Kellis; John L Rinn\ndate: 2010\ninstitute: 1Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02142, USA; 2Computer Science and Artificial Intelligence Laboratory, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; 3Department of Pathology, Beth Israel Deaconess Medical Center, Boston, MA 02215, USA; 4Department of Pathology, Harvard Medical School, Boston, MA 02115, USA\ntitle: Sequences to systems\n\n# Meeting report\n\nFrom a chemical standpoint, a cell can seem as a densely intertwined mess of molecules, but their emergent systems-level properties exhibit structure, coordination and order, leading to the orchestrated complexity we call life. As the seventh annual meeting on Systems Biology: Global Regulation of Gene Expression at the Cold Spring Harbor Laboratory showcased, systems biology aims to bridge the vast gap of knowledge between the pairwise molecular interactions of individual cellular components and the large-scale behavior of cellular circuits that result from their synergism. The talks given at the meeting exemplified a new definition of systems biology, where genomic technologies can revolutionize our understanding of classical problems by enabling molecular interactions to be linked to systems behavior in a mechanistic way.\n\nA large number of definitions have been proposed for systems biology, including 'the study of a single pathway by someone with an attention-deficit disorder' humorously offered by Irv Bernstein and quoted in a keynote address by Steve Henikoff (Fred Hutchinson Cancer Research Center, Seattle, USA). Henikoff went on to demonstrate that Trithorax and Polycomb response elements that trigger cellular differentiation could be recognized by their patterns of histone turnover. He argued that histone variant H2A.Z is mutually exclusive with DNA methylation, drawing a novel connection between histones, their dynamics and the epigenetic inheritance of DNA methylation.\n\nThe importance of epigenetic information in mediating the stable inheritance of cellular states was underscored in the context of human disease by the second keynote speaker, Kevin Struhl (Havard Medical School, Boston, USA). By dissecting the transcriptional circuits responding to Src signaling that can yield stably transformed cells, Struhl uncovered a feedback loop mediated by the transcription factor NF\u03baB that is sustainable for many generations in the absence of further signaling. Struhl went further to demonstrate that this pathway is targetable using FDA-approved drugs, opening up novel avenues of cancer therapeutics.\n\n# Parsing the genome\n\nThe study of any system begins with a comprehensive list of parts and their interactions, and so a major theme of the meeting was the mapping of the building blocks of gene regulation: the control regions, the DNA words within them and their sentence structure.\n\nOn one hand, several talks focused on defining sites of transcriptional factor binding, histones and sites of DNA methylation at ever-increasing resolution and complexity. For example, two talks by Barbara Wold (California Institute of Technology) and Stephen Tapscott (Fred Hutchinson Cancer Research Center) presented data revealing thousands of binding sites for the master regulator of muscle differentiation MyoD, concluding that numerous additional functions and or combinatorics result in cell-fate decisions such as muscle differentiation. One of us (MK), presented a new algorithm specifically for revealing biologically meaningful combinations of chromatin marks, and their corresponding chromatin states, that reflect rich information about the underlying biological function of the associated genomic regions. The algorithms have distinguished more than 50 chromatin states, including 11 different classes of promoter regions, associated with different functional categories, and several classes of enhancer regions associated with different expression levels for downstream genes.\n\nOn the other hand, several talks focused on the DNA sequences predictive of regulatory activity. Eric Mendenhall (Massachusetts General Hospital, Boston, USA) presented remarkable data that distinct types of DNA sequences can recruit key chromatin factors such as Polycomb. Through multiple transgenic studies Mendenhall showed that he could insert DNA sequence motifs, even those 'that have never seen Polycomb before', into the mouse genome that subsequently recruited Polycomb binding, suggesting a model of sequence-based recruitment of this complex. Eileen Furlong (European Molecular Biology Laboratory, Heidelberg, Germany) demonstrated the importance of combinatorial binding events in determining cell fate decisions, using a support vector machine to model transcription factor binding data and reveal the *cis*-regulatory motifs and motif combinations that define mesodermal fate in *Drosophila melanogaster*. A major conclusion was clear evidence that a single expression pattern could result from several different combinatorial codes of transcription factors and their dynamics. Arend Sidow (Stanford University, Stanford, USA) presented interesting insight into what determines nucleosome occupancy during the differentiation of distinct blood cell types both *in vitro* and *in vivo*. He presented clear evidence that *in vitro* nucleosomes associate with sequence-specific motifs, such as GC motifs, and exclude others, such as AT sites, whereas *in vivo* these sequence preferences are largely overridden. Moreover, Sidow demonstrated that nucleosome positioning is globally remodeled across different states of cellular differentiation.\n\nCollectively these studies demonstrate the power of chromatin-level and DNA-level information for predicting regulatory activity, the importance of word combinations and mark combinations, and the interplay of static sequences and dynamic cell-type specific changes in defining the processes of differentiation and, more generally, gene regulation.\n\n# Unconventional interactions of the secondary type\n\nThe late Ed Lewis famously said 'Progress will still need to be driven by the logic of genetics and by further increases in abstraction'. This sentiment was echoed at this year's meeting, most prominently in the unveiling of numerous unconventional protein-DNA and protein-RNA interactions.\n\nA dramatic example was presented by Heng Zhu (Johns Hopkins Medical Center, Baltimore, USA), who has used human proteome arrays to map global DNA-binding interactions. His analysis identified a striking and atypical interaction between kinases and DNA. Specifically, Zhu identified a carboxy-terminal domain of the kinase ERK2 that binds to DNA with nanomolar affinity and through this interaction serves as a transcriptional repressor. He also presented initial evidence for more than 300 unconventional DNA-binding proteins, including unusual suspects such as RNA-binding proteins and mitochondrial proteins.\n\nHoward Chang (Stanford University, Stanford, USA) presented a comprehensive study demonstrating modular binding of the large intergenic noncoding RNA (lincRNA) HOTAIR to chromatin-modifying complexes such as PRC2, the histone demethylase LSD1, and COREST, a DNA-binding protein. Chang presented a model, based on functional assays, whereby HOTAIR binds all three of these unconventional RNA-binding proteins through a different specific structured domain and in this way can guide them specifically to their target genomic loci. Thus, lincRNAs could serve as adaptors to facilitate the formation of specific RNA-protein interactions. One of us (JR) presented evidence that such mediation is a global phenomenon that extends beyond HOTAIR and includes hundreds of lincRNAs interacting with chromatin-modifying complexes and other DNA-binding proteins involved in cancer. Overall, the genome could be replete with unorthodox RNA-binding interactions serving as RNA adaptors to facilitate the establishment and or maintenance of specific epigenetic states. Tom Gingeras (Cold Spring Harbor Laboratory, New York, USA) expanded the horizons of unorthodox RNA functions, presenting a rich diversity of noncoding RNA molecules that localize to distinct cellular compartments and are derived from unconventional processes, emphasizing the further need for abstraction in our understanding of the mammalian transcriptome and the potential regulatory roles of noncoding RNAs.\n\n# Close encounters of the three-dimensional type\n\nAt this year's meeting the genome came to life in three dimensions, as a result of 'proximity ligation' techniques that define the three-dimensional neighbors of a given genomic sequence relative to all others in the genome. This reveals potentially important regulatory interactions mediated by DNA looping and compartmentalization.\n\nJob Dekker (University of Massachusetts, Worcester, USA) described work using several variants of chromatin-conformation capture (3C) to reveal these interactions. Dekker and his colleagues have used carbon-copy 3C (5C) to resolve the three-dimensional structure of the beta-globin locus in both the activated and repressed states. He proposed a model by which a large genomic-linker region serves to open up or compact the locus upon activation or repression, respectively. Erez Lieberman-Aiden (Broad Institute, Cambridge, USA) has expanded the technique to take on the entire genome, and reported that megabases of DNA fold into a dense, highly organized, and knot-free structure known as a 'fractal globule', thus providing a glimpse of the three-dimensional genome.\n\nUsing a different proximity ligation approach called ChIA-PET, Yijun Ruan (Genome Institute of Singapore, Singapore) has revealed three-dimensional genomic architecture in the context of transcriptional regulation. Ruan specifically demonstrated tertiary DNA interactions mediated by transcription factors and RNA polymerase II. An interesting connection to this finding was presented by Martin Hemberg (Children's Hospital, Harvard Medical School, Boston, USA) where he described that numerous enhancers, which often result from long-range DNA interactions, are associated with RNA polymerase II and are transcribed at low levels.\n\nCollectively these studies have added a new dimension to systems biology and provide novel insights into the three-dimensional architecture of the human genome.\n\n# Systematic evolution\n\nThe evolutionary dimension of gene regulation was addressed in regard to the selective pressures and plasticity of regulatory regions and elements. Ollie Rando (University of Massachusetts, Worcester, USA) presented an evolutionary analysis of nucleosome positioning in 12 ascomycete species. Perhaps most notably, the nucleosome positioning corresponded to specific gene sets, such as mitochondrial genes or splicing genes, and differed in physiologically relevant ways in different species.\n\nContinuing the theme of evolution and binding localization, Mike Snyder (Stanford University, Stanford, USA) presented an unprecedented dataset comprising the binding sites for several key transcription factors across ten different human individuals. Snyder showed that genetic variation between individuals had resulted in the loss and gain of binding sites for key transcriptional players such as RNA polymerase II and the transcription factor NF\u03baB. Thus, individual variation results in unique binding patterns of key factors, which should provide potential insights for personalized medicine and disease susceptibility.\n\nAlexander Stark (IMP, Vienna, Austria) presented comparative work on the DNA binding of transcriptional regulators of mesoderm development in six *Drosophila* species at a range of evolutionary distances from each other. He showed that not only were the locations of the peaks representing binding-site occupancy conserved, but also the intensity of the peaks, suggesting that lower-occupancy sites are also functional and are selectively maintained. He presented evidence that these sites were subsequently occupied at later developmental stages, suggesting that lower occupancy may be associated with priming for subsequent activation.\n\n# Cell circuits and dynamics\n\nThe ultimate goal in systems biology is to define cellular circuits and their resulting cellular outputs. This year's meeting reported unprecedented progress in mapping cell circuits in response to specific stimuli, perturbing them and monitoring their cellular responses. A comprehensive example was presented by Aviv Regev (Broad Institute, Cambridge, USA), who described work with a model mammalian cell system - mouse dendritic cells - which trigger distinct inflammatory or antiviral responses depending on the input stimuli. Regev's strategy was to monitor the complete transcriptional response to each stimulus, identify the key regulators (transcription factors, chromatin-modifying complexes and RNA-binding proteins) in each pathway, and then individually perturb the actions of hundreds of these regulators via stable gene knockdown and monitor the resulting transcriptional changes. This not only identified numerous new regulators across these critical cellular pathways, but also revealed the cooption of cell-cycle circuits in the antiviral response. This approach to systems biology could also be applied to numerous other cell-based models.\n\nUnderlying seemingly stable cell circuits are multiple additional layers of modulation, which include both stochastic single-cell variability in transcript levels and regulated processes of post-transcriptional inhibition and splicing. Alexander Van Oudenaarden (Massachusetts Institute of Technology, Cambridge, USA) demonstrated that cell-fate decisions in *Caenorhabditis elegans* intestinal specification arise from an underlying process of variable transcription levels. These stochastic processes resulted in threshold-dependent ON or OFF decisions of master regulators that guide cellular differentiation. David Bartel (Whitehead Institute, Cambridge, USA) described how the dramatic effects of post-transcriptional regulation by microRNAs (miRNAs) can result from even the slightest fine-tuning of transcript levels and translation efficiency. In a potential reversal of the dominant dogma in miRNA regulation, he also presented evidence from mammalian cells that for two miRNAs at least, the major effect of targeting appears to be transcript degradation rather than translational inhibition. Chris Burge (Massachusetts Institute of Technology, Cambridge, USA) presented studies that revealed a remarkable complexity in mRNA transcript splicing and how these isoforms are altered in cancer.\n\nCollectively, this year's meeting revealed new insight into genomic sequences, binding sites and structures that has moved systematic study of gene regulation to new dimensions of both understanding and further abstraction in systems biology.","meta":{"dup_signals":{"dup_doc_count":122,"dup_dump_count":45,"dup_details":{"curated_sources":4,"2023-14":1,"2021-39":1,"2020-40":1,"2020-29":1,"2019-47":1,"2019-35":1,"2019-26":1,"2019-13":1,"2019-04":2,"2018-47":2,"2018-43":1,"2018-34":1,"2018-22":1,"2018-05":1,"2017-39":2,"2017-22":2,"2017-17":1,"2017-09":8,"2016-40":1,"2016-36":10,"2016-30":6,"2016-22":1,"2016-18":1,"2015-48":3,"2015-40":2,"2015-35":3,"2015-32":3,"2015-27":3,"2015-22":2,"2015-14":3,"2014-52":3,"2014-49":5,"2014-42":9,"2014-41":4,"2014-35":5,"2014-23":4,"2014-15":2,"2023-50":1,"2015-18":3,"2015-11":3,"2015-06":3,"2014-10":3,"2013-48":3,"2013-20":2,"2024-26":1}},"file":"PMC2898084"},"subset":"pubmed_central"}
{"text":"abstract: A look into the future of a biologist: daily activities governed by presidential mandates and acts.\nauthor: Gregory A Petsko\ndate: 2007\ninstitute: 1Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02454-9110, USA\ntitle: A day in the life of a genome biologist in the not-too-distant future\n\n**Megalopolis, United States of Western Hemisphere, 18 June 2273.** The following fragment was uncovered in an underwater archaeological dig, on the site now generally believed to be the location of the almost mythical engineering school, Screwloose La Tech. Ever since global warming led to the flooding of the entire Eastern Seaboard of North America in 2138, artifacts from the Cambrian Period, as the period of the great universities in Cambridge is called, have been very hard to come by. This document is therefore of great historical significance. Nothing is known about the writer, except that similarities of style suggest that he may actually have been the same as the master of the *Genome Biology* columns, whose work has recently been challenged as fraudulent.\n\nPursuant to Federal Paperwork Reduction Act 3,671 of 2016, herewith is submitted daily activities report for Saturday, 3 June 2030.\n\n**7:44 am to 8:02 am.** Arrived shuttle parking lot number 4, located 11.25 miles from campus. Turned off motor on electric car and detached mandatory passive restraint devices (seat belt, shoulder harness, neck brace, ankle restraints and wrist locks) before exiting. Plugged battery cord into recharger built into parking meter. Inserted \\$255 in five dollar coins to cover daily parking and battery charging costs. \\[Personal note: must remember to get more five dollar coins from bank. Ever since university raised off-campus parking fees for faculty to \\$41,000 per year, have been lugging around more bags of coins than an armored car.\\] Waited 30 minutes for shuttle bus to campus.\n\n**8:46 am to 9:27 am.** Arrived at office. Couldn't unlock door because minor head cold has altered appearance of eyes and eyeball-scanner security device couldn't recognize them. Waited 30 minutes for campus swat team to respond to security alarm and unlock door.\n\n**9:28 am to 11:11 am.** Turned on computer and checked email. Only 1,459 new messages since last night. Ran new Microsoft anti-spam program, which automatically deleted 832 of them, including message from mother. Ran newer Microsoft anti-spam bug fixer, which restored message from mother and other 831 junk messages, plus 204 new junk messages it found from somewhere. Switched to personal examination of messages. Found that only two messages actually were important; all others were spam, including message from mother.\n\n**11:12 am to 11:54 am.** Surprise inspection by agents from Government Office for Toxicity Checking and Hazard Assessment (GOTCHA). Examined all labs-on-chips at every student desk-bench. Found four minor violations (two chips were not dusted and the dust from two others was not properly collected and stored). Ever since scientific responsibility for wrongdoing (SCREW) mandate of 2020, signed into law by President George IJK Bush IV, scientist responsible for laboratory must pay fines for any GOTCHA violations immediately from personal funds. Presented debit card to agents for fines of \\$4,400 (\\$1,100 per violation). Oh well, still a lot better than last week.\n\n**11:55 am to 12:39 pm.** Met with two students from advanced genomic, cell, molecular and psychological neurosociobiology course. Listened to complaints about their midterm grade. Students argued that grade of A+++, well below class average, severely hampered their ability to gain admission to medical school and therefore constituted grievous psychological harm. Threatened to inflict equivalent grievous bodily harm unless I changed grades. Ever since mandatory American national interpersonal armament concealment (MANIAC) statute law, signed by President George LMN Bush V, required that all citizens carry concealed weapons at all times, such student-faculty disputes have become easier to resolve. Started reaching for pocket grenade launcher but then considered that odds of 2-against-1 dictated non-violent response. Changed grades to class average of Super-A+++++++.\n\n**12:40 pm to 12:45 am.** Ate lunch, which as always consisted of trans-fat sandwich on tobacco leaf bread. Paused to reflect how amazing that earlier generations of scientists actually considered this unhealthy - exactly the opposite of what careful research has now shown.\n\n**12:45 pm to 1:56 am.** Reviewed results of latest genome sequencing project in the lab. Sequence of gerbil genome, begun at 12 noon, now complete. Immediately entered file of results into Microsoft Office 2025, which immediately crashed after erasing file. Restored file from backup microdot and relaunched program, which proceeded to write paper describing results, including changing spelling of my name to something it liked better. Attempted to change name back only to be told by the program that it was smarter than me, always would be smarter than me, and that I should go away and let my betters do their job. \\[Personal note: visit district court and change spelling of my name to that used by program\\].\n\n**1:56 am to 2:39 pm.** Submitted finished manuscript, with Bill Gates III first author, as he is on all papers everywhere, to *Nature Gerbil*, *Gerbil Cell*, *Gerbil Genome Biology*, *PloS Gerbil*, and *Current Gerbil Biology*. (Ever since famous Supreme Court decision in case of J Craig Venter *vs Nature*, *Science* and *Cell*, scientists have had the right to submit their papers simultaneously to as many journals as they like, and to have the same paper published in up to five journals at once.) Since the Harold Varmus overthrow of capitolism (HAVOC) Act of 2011 made it illegal for journals to reject papers or to make a profit, all journals now accept all papers automatically so paper was immediately accepted by all five. Should appear on-line sometime in 2037 if the backlog doesn't get worse.\n\n**2:39 am to 3:55 pm.** Attended mandatory daily sensitivity training workshop, required of all faculty by the Office of Self-Esteem, which has controlled all US universities since the Political Correctness Wars of 2014. Was reminded yet again that any idea, no matter how idiotic, must be respected since to do otherwise would stifle student creativity and self-expression.\n\n**3:55 pm to 4:00 pm.** Thought about meaning of results of research.\n\n**4:00 pm to 6:15 pm.** Realized I had forgotten to check e-mail for several hours. Found 2,651 new messages. Not bad. Deleted all except offer from ex-Nigerian government official who wants to give me \\$24 billion in return for promise to think happy thoughts about him. Must look into this tomorrow.\n\n**6:15 pm to 7 pm.** Packed up to go home. Waited 45 minutes for shuttle bus to parking lot. While waiting, checked latest news on iPhone. Still no progress on ending war in Iraq.","meta":{"dup_signals":{"dup_doc_count":114,"dup_dump_count":46,"dup_details":{"curated_sources":2,"2023-14":1,"2022-21":1,"2021-43":1,"2020-40":1,"2019-39":1,"2019-26":1,"2019-13":1,"2019-04":1,"2018-47":1,"2018-39":1,"2018-30":1,"2018-22":1,"2018-05":1,"2017-39":1,"2017-26":1,"2017-09":6,"2016-50":1,"2016-40":1,"2016-36":8,"2016-30":6,"2016-22":1,"2016-18":1,"2016-07":4,"2015-48":3,"2015-40":2,"2015-35":3,"2015-32":3,"2015-27":3,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":4,"2014-42":7,"2014-41":4,"2014-35":3,"2014-23":4,"2014-15":5,"2023-50":1,"2017-13":1,"2015-18":3,"2015-11":2,"2015-06":3,"2014-10":2,"2013-48":3,"2013-20":3,"2024-26":1}},"file":"PMC1868945"},"subset":"pubmed_central"}
{"text":"date: 2000\ntitle: Draft Heidelberg Collaboration Statement of Purpose (22\/9\/2000)\n\n*Warning - this is an unedited draft, which will be replaced by a final version mid of October \\[will be posted at this URL\\].*\n\nFollowing a meeting convened to discuss the EU-funded MedCERTAIN project on improving information quality on the Internet, a broad group of individuals from 4 continents and 21 countries, drawn from a wide range of sectors including industry, leading Web sites, governmental and intergovernmental bodies, academia, consumer organisations, standards and ethical code bodies and publishers agree that:\n\n1\\. Despite the enormous value for the public, patients and professionals of the health and medical Internet, the participants are concerned about its potential for harm. However, it is also important not to limit freedom of expression and use of the internet for health related purposes.\n\n2\\. The participants wish to explore in an ongoing collaborative effort opportunities for helping people, patients and professionals to identify health information useful to them, addressing certain key issues, including:\n\n-   What is an appropriate way to describe the characteristics of the full range of Internet health and medical sites ?\n\n-   How can the descriptions be summarised as metadata for different purposes and users of health information ?\n\n-   How do the identity, training and other factors (eg. self reporting vs. external audit) influence the description and summary ?\n\n-   Can criteria be developed to indicate quality for different users and purposes ?\n\n-   How can the potential of information and communications technology, such as the MedCERTAIN project, be harnessed in a consistent and cost-effective way ?\n\n-   What are the legal and other implications of trustmarks and other devices or mechanisms to denote quality ?\n\n-   How to establish and motivate a worldwide, representative network of evaluators?\n\n-   What are the areas of potential overlap with other initiatives and organisations?\n\n3\\. The participants resolve to work together, building on experience and expertise of this group, key quality improvement initiatives such as Discern, eHealth ethics code, HONcode, Hi-Ethics code, OMNI and others, and working towards a methodological clarification and resolution of the above and other related issues in the near future, providing constructive support to the MedCERTAIN and other projects and continuing to build together trust on the health Internet.\n\n*Participants:*\n\nLucas M. Bachmann (Horten Zentrum f\u00fcr praxisorientierte Forschung und Wissenstransfer, Switzerland), Carl R. Blesius (MedCERTAIN, Germany), Markus Blume (mymedia GmbH, Germany), Carl J. Brandt (NetDoktor.com, Denmark), Dan Brickley (MedCERTAIN, United Kingdom), Alejandro Cacherosky (Buenos Aires Health Secretariat, Argentina), Ken Campell (Leukaemia Research Fund, United Kingdom), Richard Cleland (Federal Trade Comission, U.S.A.), Phil Cross (MedCERTAIN, United Kingdom), Elenice de Castro (PAHO\/WHO Pan American Health Organization \/ World Health Organization, Brazil), Guy de Roy (Ordre des Medecins Conseil National, Belgium), Tony Delamothe (BMJ, United Kingdom), Martin D. Denz (Swiss Medical Informatics Association \/ Universit\u00e4tsspital Z\u00fcrich, Switzerland), Persephone Doupi (Erasmus University, Netherlands), Joan Dzenowagis (WHO World Health Organization, Switzerland), Christer Edling (The Swedish Society of Medicine, Sweden), Gunther Eysenbach (University of Heidelberg \/ MedCERTAIN, Germany), Gerard Freriks (TNO, Netherlands), Franz Fr\u00fchwald (Internationales B\u00fcro der \u00d6sterreichischen \u00c4rztekammer, Austria), Lisa Gray (BIOME, United Kingdom), Pelle Gustafsson (Swedish Medical Association, Sweden), Gerhard Heine (European Commission, Luxembourg), Katrin H\u00f6rner (Arztpartner \/ Almeda, Germany), Robert Hsiung (University of Chicago, U.S.A.), Jostein Ingulfsen (Norwegian Board of Health, Norway), Thomas Isenberg (German Association of Consumer Organisations - Arbeitsgemeinschaft der Verbraucherverb\u00e4nde, Germany), Edward Jacob (KNMG, Netherlands), Alex R. Jadad (Canadian Cochrane Centre, Canada), Jacobo Kelber (ensalud.net, Mexico), Hugo Kitzinger (mymedia GmbH, Germany), Inge Kokot (Deutsches Gr\u00fcnes Kreuz e.V., Germany), Hans-Joachim Koubenec (Stiftung Warentest, Germany), Michel Labrecque (Universit\u00e9 Laval Qu\u00e9bec, Canada, Canada), Kristian Lampe (Finnish Office for Health Technology Assessment \/ MedCERTAIN, Finland), Stephane Lejeune (Swiss Cancer League, Switzerland), Leonard B. Lerer (INSEAD, France), Odile Leroy (PasteurMed, France), Nicolas Lienert (Medgate AG, Switzerland), P\u00e5l Lindstr\u00f6m (LocusMedicus, Sweden), S\u00e1ndor Lipp (Hungarian Ministry of Health, Hungary), Leena Lodenius (Finnish Duodecim Medical Society, Finland), Antti Malmivaara (Finnish Institute of Occupational Health, Finland), Miquel Angela Mayer Pujadas (Official Medical College of Barcelona, Spain), Peter Mills (EncycloMedica Ltd, United Kingdom), Cesar Molinero (Planet Medica, Belgium), Marc Muret (Z\u00fcrcher Aerzte f\u00fcr Klassische Hom\u00f6opathie, Switzerland), Wolfgang Nagel (GesundheitScout24 GmbH, Germany), Tim Nater (Health on the Net Foundation HON, Switzerland), Joerg Nitzsche (Zentralbibiliothek f\u00fcr Medizin Koeln, Germany), Debra O\u00b4Connor (La Trobe University, Australia), Gert Purkert (Arztpartner \/ Almeda, Germany), Ramon Sarrias Ramis (Official Medical College of Barcelona, Spain), Christine Reuter (A MedWorld AG, Germany), Ahmad Risk (Internet Healthcare Coalition, United Kingdom), Carl B\u00e9n\u00e9dict Roth (GetWellness, Switzerland), Sebastian Schmid (Arztpartner \/ Almeda, Germany), Christiane Schmitz (Heinrich Heine Universit\u00e4t, Germany), Luk Schoonbaert (Belgacom Multimedia Ventures, Belgium), Frank Schuler (gfp Kommunikation K\u00f6ln, Germany), Ulrich Schwanke (DocCheck, Germany), Sasha Shepperd (Imperial College School of Medicine, United Kingdom), Myra Sidrassi (MedCERTAIN, Germany), Chris Sigouin (McMaster University, Canada), Chris Silagy (Monash Medical Centre, Australia), Denise Silber (Internet Healthcare Coalition, U.S.A), Martin Sonderegger (Universit\u00e4tsspital Z\u00fcrich, Switzerland), Anke Steckelberg (Universit\u00e4t Hamburg, Germany), Frederik Tautz (Heinrich Heine Universit\u00e4t, Germany), Nicolas P. Terry (Center for Health Law Studies, St. Louis University, U.S.A.), Christian Thomeczek (\u00c4rztliche Zentralstelle Qualit\u00e4tssicherung, Germany), Wouter Tukker, (Praha Communications, Czech Republic), Gerard H. van der Zanden (The Netherlands Institute for Health Promotion and Disease Prevention NIGZ, Netherlands), Georg von Below (Swiss Medical Assoc.\/FMH, Switzerland), Frank von Danwitz (Deutsches Diabetes-Forschungsinstitut, Germany), C.-Peter Waegemann (Medical Records Institute, U.S.A.), Thomas Wetter (Dept. Of Medical Informatics, Universit\u00e4t Heidelberg, Germany), Petra Wilson, (European Commission, Belgium), Margaret A. Winker (JAMA, U.S.A.), Jeremy Wyatt (University College London, United Kingdom), Gabriel Yihune (MedCERTAIN, Germany)","meta":{"dup_signals":{"dup_doc_count":111,"dup_dump_count":67,"dup_details":{"curated_sources":2,"2023-23":1,"2023-14":1,"2023-06":1,"2021-43":1,"2021-39":1,"2021-31":1,"2020-34":1,"2020-24":1,"2020-10":1,"2019-47":1,"2019-35":1,"2019-26":1,"2019-18":1,"2018-51":1,"2018-39":1,"2018-34":1,"2018-30":1,"2018-26":1,"2018-22":1,"2018-17":1,"2018-13":1,"2018-09":1,"2018-05":1,"2017-51":1,"2017-47":1,"2017-43":1,"2017-39":1,"2017-34":1,"2017-30":1,"2017-26":2,"2017-22":1,"2017-17":2,"2017-09":2,"2017-04":2,"2016-50":2,"2016-44":2,"2016-40":2,"2016-36":2,"2016-30":2,"2016-26":2,"2016-22":1,"2016-18":2,"2016-07":2,"2015-48":2,"2015-35":2,"2015-32":2,"2015-27":2,"2015-22":2,"2015-14":2,"2014-52":2,"2014-49":3,"2014-42":7,"2014-41":2,"2014-35":4,"2014-23":2,"2014-15":3,"2023-40":1,"2024-30":2,"2024-26":1,"2024-10":2,"2017-13":1,"2015-18":2,"2015-11":2,"2015-06":2,"2014-10":2,"2013-48":2,"2013-20":2}},"file":"PMC1761870"},"subset":"pubmed_central"}
{"text":"abstract: Does identifying genetic differences between groups itself constitute an act of racism?\nauthor: Gregory A Petsko\ndate: 2004\ninstitute: 1Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02454-9110, USA\ntitle: Color blind\n\nBy the time you read this, the result of the 2004 US Presidential election will be known - assuming, of course, that there isn't a repeat of the travesty of 2000, when the Supreme Court of the United States, by a vote that split along the lines of which political party had appointed which particular judge, awarded the election to George W. Bush by the slimmest of margins (less than 1,000 votes) in the state of which his brother was governor. Contested ballots notwithstanding, I never cease to be amazed when I cast my vote on election day. After the cacophony of the seemingly endless campaign, on election day a curious quiet descends on the country. No bands play. No troops march. People waiting in line to vote tend to speak in hushed tones, almost as though they were in church. I didn't see a single cell phone in use. The transfer of power in what is arguably the most powerful nation in the history of the world happens softly, like a whisper in the dark. That's the wonder of democracy.\n\nContrary to what many Americans think, democracy wasn't invented here. It has a long history, going back at least to ancient Greece. And, especially if we have another disputed election this time, I would have trouble arguing that it was perfected here either. But if not the practice of democracy, I think the concept of democracy may have reached its zenith on these shores, in the city of Philadelphia, in the month of June, in the year 1776. At that time and place, Thomas Jefferson composed these words for the document in which the thirteen former British colonies declared their independence: \"all men are created equal\". As a weapon in a war, which it was, that phrase was powerful. It was meant to convince and to confound, and it did both. Then it developed a life of its own, and began to reverberate around the world like the sound of an exploding volcano. It echoed in the French Revolution thirteen years later. It could be heard in 1848, the year of revolutions in Europe. It was still echoing more than a hundred years later when India broke from Britain. It can still be heard today, as loud and potent as ever. Not bad for an idea that is biological nonsense.\n\nBasketball star Michael Jordan could jump through the roof of a building; I never could. Footballer David Beckham can bend a corner kick around a goalpost; I can barely kick a soccer ball straight. I can't run a marathon in under three hours and I never would be able to no matter how hard and long I trained. Neither could most people, nor do they expect to. Implicit in the meritocracy of sport is the idea that, although practice and coaching are important, there are vast differences in talent among individuals. Such limitations are understood in other areas as well: most people cannot do high-level mathematics and probably never could no matter how long they studied. I took piano lessons for five years, but Horowitz is a pianist; I'm not, and never will be. We all know these things, and accept them. But taken literally, \"all men are created equal\" could imply that such facts aren't true. Of course, the phrase was never meant to refer to physical abilities, or talent for music or abstract reasoning. It meant that all men (alas, the Founding Fathers probably really did mean only men - there were no Founding Mothers to set them straight) should be equal in the eyes of the law, and the government. Since I live in a country where the wealthy and influential often receive preferential treatment by both, I understand that this is an ideal not always achieved in practice.\n\nBut the extreme interpretation is always there, in any democracy. It's responsible for the contempt of 'the elite' or 'intellectuals' that one hears, especially around election time. It underlies our iconoclastic tendencies. Somewhere, in some inner place that is hard to reach, a lot of us harbor the secret belief that we're just as good as anybody, in just about everything. Maybe this delusion is harmless, but I doubt it. I think it contributes to the streak of anti-intellectualism that has a foothold in Western culture right now. I think it also makes it easier for populists and demagogues to coax people into voting against their self-interests. But even if I'm wrong about these consequences, there is one area in which the notion of absolute equality is certainly dangerous: the area of medicine.\n\nThis month, a report appeared in *The New England Journal of Medicine* (**351**:2049-2055; issue of 11 November 2004) entitled \"Combination of Isosorbide Dinitrate and Hydralazine in Blacks with Heart Failure\". The article was written by Anne L. Taylor MD, Susan Ziesche RN, and a number of other authors, on behalf of the African-American Heart Failure Trial Investigators. The paper presents the results of a study of 1,050 black patients with advanced heart failure. Half received a placebo, while the other half received BiDil, a combination of two generic drugs (isosorbide dinitrate, which stimulates the production of nitric oxide, and hydralazine, an antioxidant and vasodilator that appears to prevent the nitric oxide from deactivating). The results were so striking that the study was terminated prematurely so that all remaining patients could be given BiDil. Over the two years of the study, 6.2% of the patients given the drug died, compared with 10.2% of those given the standard treatment, a 43% improvement. First hospitalizations were also reduced by 33%. Ray Gibbons, a cardiologist at the Mayo Clinic and spokesman for the American Heart Association, says \"When you have this level of risk reduction in a high-stakes disease like heart failure, it's at least a home run.\" The study was initiated by NitroMed, Inc., a biotechnology company in Lexington, Massachusetts. Combined use of the two generic drugs would allow NitroMed to receive a new patent for the joint formulation. An earlier trial of the mixture, by Dr Jay Cohn of the University of Minnesota in the 1980s, had been disappointing: in a test against the general population, the drugs fared worse than angiotensin converting enzyme (ACE) inhibitors. But Dr Cohn reanalyzed the data when it became clear years later that one reason African-Americans die from heart failure at 2.5 times the rate of Caucasians was because ACE inhibitors appear to be much less effective in black patients than in non-blacks. Sure enough, in the 395 black patients in the original study, the drug combination appeared better than the standard therapy. The new trial, exclusively in African-Americans, was begun as a consequence.\n\nSo why isn't everybody cheering? Many are, including the Association of Black Cardiologists, who cosponsored the trial. But others are not. In a perspective in the same issue of *The New England Journal of Medicine*, Dr M Gregg Bloche of Georgetown University Law Center asks, \"Are we moving into a new era of race-based therapeutics?\" He believes the answer is yes, especially since the US Food and Drug Administration has already indicated that if the drug is approved, it will be labeled as indicated for patients of African-American descent, an unprecedented situation. (Although at least 29 drugs are known to have different efficacies between blacks and Caucasians, none of them is labeled in this way.) JudyAnn Bigby, director of the Office for Women, Family and Community Programs at Brigham and Women's Hospital in Boston, fears that \"if people get one little inkling that there's a biological basis to race, we could potentially lose ground in understanding racial differences in disease. Biology could be an excuse for not looking at the social basis\" of diseases like heart disease, which is known to be affected by stress, income, diet and access to health care, among other non-genetic factors. \"You have the federal government giving its imprimatur, its stamp of approval, to using race as a biological category,\" warns Jonathan Kahn, of Hamline University, who wrote a paper in the *Yale Journal of Health Policy, Law, and Ethics* on the danger of approving BiDil. \"To my mind, it's the road to hell being paved with good intentions.\"\n\nThis concern is understandable given the sorry history of the US when it comes to matters of race. Yet we have known for half a century that sickle-cell anemia is overwhelmingly a disease of blacks, that Tay-Sachs Disease is overwhelmingly a disease of Ashkenazi Jews, and that cystic fibrosis is overwhelmingly a disease of Caucasians. Somehow that knowledge has not set us on the road to hell. Why not? I think that main reason is that 'race' in these diseases is only a surrogate marker: in each case the specific genetic abnormality is known. Blacks do not suffer disproportionally from sickle-cell anemia because they are genetically inferior to whites, they suffer from it because the sickle cell mutation is more commonly found in people of African origin, and because the presence of the mutation in such individuals appears to confer protection against infection by the malaria parasite. But there are 'non-blacks' who have the same mutation and suffer from the same disease. Race isn't the issue; the issue is who has the relevant genetic trait. Once the affected gene is known, anybody can be tested for the disease-causing mutation. Race becomes irrelevant.\n\nBiDil works in African-Americans because there is some gene - possibly more than one, but probably no more than a few - that differs between most people of African-American descent and those of, say, Indo-European descent. The tools of genomics can be used to find that gene or genes, and when that is done it will be possible to test everyone who suffers from heart failure for the mutations that confer susceptibility to BiDil therapy. When that happens, it will certainly become clear that many non-blacks would also benefit from BiDil. Since this would increase the market for the drug considerably above, say, the 375,000 African-Americans who suffer from moderate to severe heart failure, it would be in the interest of NitroMed to support the search for those genetic differences. Then the drug could be labeled the right way: according to its molecular targets, not the color of the patient's skin.\n\nLast spring, The Magic Theater in San Francisco presented a play by Cassandra Medley entitled *'Relativity'*. It concerns an African-American woman scientist who is asked by her mother to disprove that all racial groups are genetically similar (you can read a synopsis of it in Sandra Soo-Jin Lee's thoughtful review in *PloS Biology* 2004, **2**:1263-1264). The play raises the question: does identifying genetic differences between groups itself constitute an act of racism? Obviously I don't think so, but I do realize that any statement of differences between races can never be neutral. That is why we must shift the discussion away from color and towards those things that make us different as individuals, not as members of some group.\n\nGenomic data indicate that all humans share about 99.9% of our genetic make-up. As more data accumulate and it becomes clear that the concept of race is meaningless biologically, we should be able to agree that all 'men' are almost equal biologically, and to shift our focus once and for all to those genetic differences that are meaningful, such as those that determine our susceptibility to disease and our responses - beneficial and adverse - to therapy. One of the great future benefits of the Human Genome Project may be that, in years to come, when someone is described as being a credit to their race, it will be understood by everyone that what is meant is the human race.","meta":{"dup_signals":{"dup_doc_count":118,"dup_dump_count":42,"dup_details":{"curated_sources":2,"2023-14":1,"2022-21":1,"2021-43":1,"2020-24":1,"2018-51":1,"2018-43":1,"2018-34":1,"2018-13":1,"2017-47":1,"2017-34":1,"2017-30":1,"2017-26":1,"2017-22":1,"2017-09":10,"2016-44":1,"2016-40":1,"2016-36":10,"2016-30":8,"2016-22":1,"2016-18":1,"2016-07":7,"2015-48":3,"2015-40":2,"2015-35":3,"2015-32":3,"2015-27":3,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":5,"2014-42":6,"2014-41":2,"2014-35":4,"2014-23":4,"2014-15":3,"2023-50":1,"2015-18":3,"2015-11":3,"2015-06":3,"2014-10":2,"2013-48":3,"2013-20":2}},"file":"PMC545786"},"subset":"pubmed_central"}
{"text":"date: 2004\ntitle: Editorial\n\nFor most of the history of scientific publications, readers paid to access research articles through subscriptions; and now they also have the option of paying one-time fees for downloading over the web. But having to pay for access limits how many people can read, use and cite research. By contrast, from its inception, *Journal of Biology* has published its research articles under the open access conditions promoted by its publisher, BioMed Central. Articles are universally and freely available online to everyone, to read, download, and pass on to others, as described in BioMed Central's Open Access Charter \\[<http:\/\/www.biomedcentral.com\/info\/about\/charter>\\]. Now, *Journal of Biology* takes an additional step towards the publishing model adopted by the other journals in BioMed Central's stable. From July 2004, authors of research articles that are accepted for publication will be asked to pay an article-processing charge of US\\$1,500, to cover the costs associated with making their article available.\n\nOnce authors have paid this fee, open access articles are freely and universally accessible online and can be read by anyone at no cost. Authors hold copyright for their work and allow anyone to reproduce and disseminate the article, provided that it is correctly cited and no errors are introduced. And a copy of the full text of each open access article is immediately archived in a central archive, such as PubMed Central \\[<http:\/\/www.pubmedcentral.org>\\].\n\nOpen access has four great benefits. First, authors are assured that their work is disseminated to the widest possible audience. Second, the information available to researchers is not limited by their library's budget - or their nation's wealth. Third, the widespread availability and central archiving of research articles enhances literature searching and facilitates meta-analyses of data. And fourth, the results of publicly funded research become accessible to all taxpayers, not just those with access to a specialist library.\n\nAuthors of research articles are asked to pay a processing charge if their research is accepted for publication (further details of what the charge covers and its rationale are available online \\[<http:\/\/www.biomedcentral.com\/info\/about\/apcfaq>\\]). Authors from resource-poor countries have their charges waived; and in fact most authors are not individually responsible for paying the charge, as many institutions are 'members' of BioMed Central \\[<http:\/\/www.biomedcentral.com\/inst\/>\\], paying an annual fee that covers the charges for all authors at that institution. The 450 or so current members include the World Health Organization, the US National Institutes of Health, and all UK universities - as well as the institutes housing the vast majority of *Journal of Biology*'s published authors.\n\nMany funding agencies have realized the importance of open access publishing and have specified that their grants may be used directly to pay article-processing charges. Although some authors may consider US\\$1,500 expensive, *Journal of Biology* does not levy additional page or color charges; to put this in perspective, the cost of including a single color figure in an article in *Cell* is US\\$1,000 \\[<http:\/\/www.cell.com\/misc\/page?page=authors>\\].\n\nSeveral journals now offer free access to their articles online, but this is different from open access (as defined by the Bethesda Declaration \\[<http:\/\/www.earlham.edu\/~peters\/fos\/bethesda.htm>\\]) - in particular if the journal delays free access for 6-12 months, or has a copyright policy that does not allow dissemination of the work. But *Journal of Biology* is not alone in providing open access funded by article-processing charges. BioMed Central currently publishes nearly 100 journals using this model \\[<http:\/\/www.biomedcentral.com\/info\/about\/apcjournals>\\]; and the Public Library of Science already publishes *PLoS Biology* and will this year launch *PLoS Medicine*, both with an article-processing charge of US\\$1,500 per accepted article \\[<http:\/\/www.plos.org>\\]. As these open access journals can testify, the need to pay article-processing charges does not seem to dissuade authors from submitting articles to a journal; indeed, it may do the opposite, perhaps because it reassures authors of the journal's plans for longevity and the stability of its business model.\n\nArticle-processing charges will allow *Journal of Biology* to continue to provide its exceptional research articles under open access, immediately free to all readers. We believe that the more important the research, the more important that it be immediately and completely freely available. We hope you agree, and that you show your support by submitting your next important research article to *Journal of Biology*.","meta":{"dup_signals":{"dup_doc_count":116,"dup_dump_count":44,"dup_details":{"curated_sources":2,"2020-10":1,"2019-47":1,"2019-39":1,"2019-26":1,"2019-09":1,"2018-47":1,"2018-39":1,"2018-30":1,"2018-05":1,"2017-39":1,"2017-30":1,"2017-26":1,"2017-17":1,"2017-09":5,"2016-44":2,"2016-40":1,"2016-36":6,"2016-30":5,"2016-22":1,"2016-18":1,"2016-07":7,"2015-48":3,"2015-40":3,"2015-35":3,"2015-32":3,"2015-27":3,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":3,"2014-42":8,"2014-41":4,"2014-35":5,"2014-23":3,"2014-15":5,"2020-40":1,"2017-13":1,"2015-18":3,"2015-11":3,"2015-06":3,"2014-10":3,"2013-48":3,"2013-20":3,"2024-18":1}},"file":"PMC416561"},"subset":"pubmed_central"}
{"text":"abstract: Bioinformatics-based searches for correlations between subcellular localization and pI or charge distribution of proteins have failed to detect meaningful correlations. Recent work published in *BMC Biology* finds that a physicochemical metric of charge distribution correlates better with subcellular pH than does pI.\n  .\n  See research article <http:\/\/www.biomedcentral.com\/1741-7007\/7\/69>\nauthor: Bertrand Garcia-Moreno\ndate: 2009\ninstitute: 1Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218, USA\nreferences:\ntitle: Adaptations of proteins to cellular and subcellular pH\n\nThe need for tight regulation of intracellular pH is one of the most important and constant organizing principles of living systems. It is essential because the fundamental energy transduction machinery of cells runs mainly on H^+^ gradients and proton-coupled electron transfer reactions. It is also necessary because pH determines the charge state of weak acids and bases (for example, side chains of Asp, Glu, His, Lys and Arg, and so on), and the charge state in turn affects many physical and physiological properties of biological molecules, especially proteins and RNAs (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>). Regulation of intracellular pH is so central to the living state that even the most primitive cells capable of energy transduction must have been able to control intracellular pH by expelling H^+^ produced from the hydrolysis of organic compounds.\n\nHow tightly is pH regulated in cells and organisms? Consider that the normal pH of human arterial blood is 7.40. Depression below pH 7.35 leads to acidosis, a condition that requires medical attention, and elevation to pH 7.50 results in alkalosis, responsible for the unpleasant symptoms of altitude sickness. Similarly small changes of 0.1 pH units in intracellular pH or within subcellular compartments can have physiological consequences. What is remarkable is that despite the need for very tight regulation, the pH in various cellular compartments varies significantly. In organs such as the human stomach, it can be extreme.\n\nIn cells, pH is nearly neutral in the cytoplasm, in the endoplasmic reticulum and in mitochondria. It is more acidic in vacuoles, lysosomes (as low as pH 5) and in the Golgi. It is more basic in the nucleus and in peroxisomes (as high as pH 8) \\[1\\]. Proteins can harness these differences in cellular and subcellular pH for physiological purposes. For example, the influenza virus requires exposure to the slightly acidic conditions in the lysosome to become activated \\[2\\]. Human hemoglobin acts similarly as a pH sensor that targets exercised tissue for delivery of oxygen by responding to local acidity \\[2\\].\n\nThe tight regulation of cellular and subcellular pH might imply that charged residues of proteins have been tailored for structural or functional purposes under specific conditions of pH. For example, the isoelectric point (pI) of proteins, which describes the balance between acidic and basic residues, might have co-evolved with the pH of the organelles in which they exist to enable or to optimize function \\[3\\]. Learning to recognize these adaptations would be useful for the annotation of proteomes and for understanding protein function and evolution. Previous bioinformatics-based searches for correlations between subcellular localization and pI or charge distribution have yielded interesting nuggets but have failed to detect a meaningful correlation \\[4\\]. The problem is that the pI is a relatively insensitive global metric of the number and types of ionizable groups; the distribution of pI values in a proteome is inherently bimodal owing to the normal differences in the p*K*~a~ (the acid dissociation constant) of the dominant acidic (Asp and Glu) and basic (Lys and Arg) residues \\[4,5\\]. Now Chan and Warwicker \\[5\\] have examined the correlation of intracellular pH with calculated electrostatic contributions to stability. Using this more physical metric of charge distribution, they found that the average pH of maximal stability for proteins in a subcellular compartment correlates better with subcellular pH than does pI. The properties of histidine residues underlie this correlation.\n\n# Effects of charge distribution on properties of proteins\n\nIdentifying further meaningful correlations between subcellular localization or subcellular pH with the number, types, distribution and properties of ionizable groups in proteins, starting from either sequence or structure, will be interesting but challenging. The problem is that the charge properties of proteins have evolved under pressure to satisfy a large number of physical and biological constraints (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>). Trying to identify trends or adaptations by focusing on one or two among the many constraints is akin to examining a piece of music through statistical analysis of musical notes or their values (that is, their duration), ignoring all other attributes of the music. Averaged over their entire oeuvre, the works of Bach and Beethoven might indeed show statistically significant differences in their predilection for some notes, but it is unlikely that this will be reflected in any individual piece, that the level of discrimination will be sufficient to identify a composer from among a list of hundreds, or that this approach will improve our understanding or appreciation of music. A meaningful search for adaptations of proteins to subcellular pH should consider simultaneously some of the following structural, biological and solution properties of proteins, all of which are influenced by charged residues.\n\n## Spatial localization\n\nThe distribution of charges on a protein can be biased by the protein's location within a subcellular compartment. For example, membrane proteins tend to be more basic than cytosolic ones \\[4\\]. This might be an adaptation to facilitate interactions between positive charges on the protein and the predominantly negatively charged polar head groups of membrane phospholipids. However, to illustrate how deceptive general trends can be, consider that some proteins that interact strongly and irreversibly with membranes actually use Ca^2+^ bridges that require post-translational modifications that add negative charges to the proteins \\[2\\].\n\n## Stability\n\nThe pH dependence of stability of proteins is governed by differences in p*K*~a~ values of ionizable groups in folded and unfolded forms. Using structure-based continuum electrostatics calculations, Alexov \\[6\\] showed previously that the pI and the pH optimum for stability can be quite different. Some proteins, such as proteases in the lysosome, are clearly adapted for maximal stability and activity in the relatively low pH of this compartment. Chan and Warwicker \\[5\\] show that this correlation of the pH of maximal stability with intracellular pH is only evident when the properties of many proteins within a subcellular compartment are averaged. Their study illustrates the power of quantitative physicochemical approaches in the analysis of proteomes. The effectiveness of this approach is also illustrated by other studies of charge contributions to the stability of thermophilic proteins. Proteins from thermophilic organisms usually have a higher number of charges and ion pairs than their mesophilic homologs \\[7\\]. An analysis of the number and types of charges would conclude that charges in thermophilic proteins were selected to enhance the stability of proteins through Coulomb interactions. However, a structure-based study of electrostatic contributions to stability using a physicochemical model failed to detect any correlation between the excess charges in thermophilic proteins and increased stability \\[7\\].\n\n## Solubility\n\nSolubility is a critical factor in the evolution of protein sequences and folds \\[8\\]. In general, charges in globular proteins are surrounded by charges of the opposite sign \\[9\\]. This would seem to reflect evolutionary tuning of surface charges to maximize stabilizing Coulomb interactions in the folded state, but it is more likely to be an adaptation to enhance solubility. Charges affect the solubility of proteins; therefore, the adaptation of charges to tune any property of a protein for a specific subcellular pH could affect solubility. Because there is no theory of protein solubility, it is impossible to determine how a specific distribution of charges affects solubility. In general, solubility correlates with pI (proteins tend to be least soluble at their pI); therefore, given the lack of correlation between pI and subcellular pH, solubility probably does not correlate well with subcellular pH. A large number of ionizable residues, especially basic ones, can enhance the solubility of a protein. However, unexpected factors can also influence solubility. For example, the solubility of folded proteins is affected by stability; a large energy gap between the fully folded, soluble form and partially unfolded, less soluble forms is needed to prevent population of aggregation-prone states. Solubility can in fact be affected by single mutations that do not alter the number of charges in the protein.\n\n## Interactions\n\nCharges are essential both to prevent and to stabilize complexes of proteins with macromolecules and with small molecules. A correlation has been found between the pH optimum of stability of monomeric proteins and of their complexes \\[10\\]. This suggests that pH-dependent properties of the monomers and of the complexes coevolved at the same pH. The presence of a specific constellation of charges for functional interactions can bias the distribution and properties of charges in proteins. For example, regions of high density of positive charge are usually found in the faces of proteins that interact with nucleic acids. Any correlation between nuclear pH and the distribution of charges in nuclear proteins in a eukaryotic cell is likely to be a secondary consequence of these functional adaptations.\n\n## Enzymatic activity\n\nThe ionizable groups that control enzymatic catalysis usually titrate with highly perturbed p*K*~a~ values tuned for catalysis under the appropriate pH conditions. Just one of these groups with perturbed p*K*~a~ can have a dramatic influence on the pH dependence of protein stability \\[11\\]. This might be the reason that the pH optimum of enzymatic reactions is, in general, not correlated with the pH of maximum stability. The adaptation of some proteins to subcellular pH might involve tuning of the p*K*~a~ values of active-site residues. There are no computational tools that reproduce the properties of internal ionizable groups accurately, and so this essential adaptation to subcellular pH cannot yet be examined quantitatively.\n\n## Environmental conditions\n\nPrevious attempts to identify protein adaptations to subcellular pH have not considered that charges and their contribution to stability, solubility, dynamics, conformation, function, and so on are sensitive to physical variables (temperature, pressure), and to the chemical composition of their milieu (osmolytes, ionic composition, metabolites). Chan and Warwicker \\[5\\] point out that differences in the distribution of charges in proteins from extremophiles and mesophiles illustrate the important roles of charges in proteins for adaptation to specific environmental conditions. The influence of the ionic milieu on adaptations to intracellular pH deserves special mention because subcellular pH is coupled to ion homeostasis (that is, changes in intracellular pH are coupled to changes in concentration of other ions). Protein adaptations to unique ionic environments are already known. For example, hemoglobin, which is regulated physiologically by the large anion 2,3-bis-phosphglycerate, has a cleft with a high concentration of positively charged residues where the di-anion binds \\[2\\]. Another example is that of extracellular proteins or proteins that exist in vacuoles or other compartments that can have high Ca^2+^ concentrations. These proteins might be expected to be more acidic to maximize interactions of Asp and Glu residues with Ca^2+^ to enhance stability or for other purposes. Attempts to identify protein adaptations to subcellular pH should consider how the ionic milieu characteristic of a subcellular compartment might have influenced these adaptations.\n\n## Buffer capacity\n\nThe regulation of subcellular pH is partly achieved through the buffer capacity of metabolites and macromolecules. This is the reason that a correlation of protein pI with subcellular pH has been sought. However, the ability of a protein to act as a buffer in a subcellular environment will depend not just on the number of ionizable groups and their p*K*~a~ values, but also on the concentration of protein in the compartment. A meaningful correlation between pI and subcellular pH might yet be found if protein concentration and compartment volume were taken into consideration.\n\n## Biological considerations\n\nThe distribution of charges in proteins can be biased by a number of biological factors that are not linked to intra-cellular pH in an obvious way. Evolutionary history, mutational bias, AT nucleotide bias, level of gene expression and optimization of translational efficiency are some factors that will have to be examined closely.\n\n## Physicochemical considerations\n\nChan and Warwicker \\[5\\] have raised other interesting issues that will have to be considered when searching for protein adaptations to subcellular pH. The adaptations might not be tuned for function in the steady state intracellular pH, but rather to the transient changes in pH that might be experienced in an intracellular compartment. Chan and Warwicker also made the interesting suggestion that the dependence of the pH of maximum stability on subcellular pH might reflect the need to minimize spontaneous fluctuations in H^+^ concentration within a compartment \\[5\\]. There is clear need for a more detailed inventory of H^+^ in subcellular compartments based on the known number and calculated p*K*~a~ of weak acids and bases, and on emerging data of protein localization and concentrations. There is also need for improved understanding of the diffusion properties of protons in the osmotically complex intracellular environment, where there is no water that is not under the influence of solute, and where, formally speaking, the concept of pH is not valid.\n\n# Conclusions\n\nMany examples of regulatory adaptations of proteins for stability or function at a specific pH are known. If charges in proteins have indeed been optimized for specific functional purposes, quite possibly they also display structural adaptations to specific subcellular conditions of pH and ionic composition. These adaptations will not be easily identified from bioinformatics analysis of proteomes using global metrics of charge distribution (such as pI). Although in general, the mean properties of proteins might be less informative than the deviations from the mean, in practice the only correlations that have been found with intra-cellular pH are with electrostatic properties calculated with physical models, averaged over many different types of proteins in a given subcellular compartment. To identify further adaptations of proteins to subcellular pH it will be useful to analyze proteomes with physicochemical models, and to consider simultaneously many of the physical and biological constraints that guided the evolution and the adaptation of proteins to the pH and ionic properties of their physiological milieu. This more integrative and physical approach might begin to reveal how different elements are combined in harmony to constitute the symphony of the cell.","meta":{"dup_signals":{"dup_doc_count":130,"dup_dump_count":49,"dup_details":{"curated_sources":2,"2021-10":1,"2020-50":1,"2020-40":1,"2020-29":1,"2020-16":1,"2020-05":1,"2019-51":1,"2019-43":1,"2019-35":1,"2019-26":1,"2019-13":1,"2019-04":1,"2018-47":1,"2018-39":1,"2018-13":1,"2017-47":1,"2017-34":1,"2017-26":1,"2017-09":12,"2016-50":1,"2016-44":1,"2016-40":1,"2016-36":11,"2016-30":11,"2016-22":1,"2016-18":1,"2015-48":3,"2015-40":3,"2015-35":3,"2015-32":3,"2015-27":2,"2015-22":2,"2015-14":3,"2014-52":3,"2014-49":2,"2014-42":7,"2014-41":5,"2014-35":5,"2014-23":3,"2014-15":5,"2021-21":1,"2017-13":1,"2015-18":3,"2015-11":3,"2015-06":3,"2014-10":4,"2013-48":3,"2013-20":3,"2024-26":1}},"file":"PMC2804283"},"subset":"pubmed_central"}
{"text":"abstract: Hally Mahler and colleagues evaluate a six-week voluntary medical male circumcision campaign in Iringa province of Tanzania, providing a model for matching supply with demand for services and showing that high-volume circumcisions can be performed without compromising client safety.\nauthor: Hally R. Mahler; Baldwin Kileo; Kelly Curran; Marya Plotkin; Tigistu Adamu; Augustino Hellar; Sifuni Koshuma; Simeon Nyabenda; Michael Machaku; Mainza Lukobo-Durrell; Delivette Castor; Emmanuel Njeuhmeli; Bennett Fimbo\\* E-mail: <firstname.lastname@example.com>[^1]\ndate: 2011-11\ninstitute: 1Jhpiego\/Tanzania, Dar es Salaam, Tanzania; 2Jhpiego, Baltimore, Maryland, United States of America; 3Ministry of Health, Iringa Region, Tanzania; 4Office of HIV and AIDS, United States Agency for International Development, Washington, District of Columbia, United States of America; 5Ministry of Health and Social Welfare, Dar es Salaam, Tanzania; Centers for Disease Control and Prevention, United States of America\nreferences:\ntitle: Voluntary Medical Male Circumcision: Matching Demand and Supply with Quality and Efficiency in a High-Volume Campaign in Iringa Region, Tanzania\n\n# Summary Points\n\n-   The government of Tanzania has adopted voluntary medical male circumcision (VMMC) as an important component of its HIV prevention strategy and aims to reach 2.8 million uncircumcised men within the next three years.\n\n-   In June and July 2010, a six-week VMMC campaign in Tanzania's Iringa Region performed 10,352 circumcisions.\n\n-   Strategies adopted by the campaign to generate demand included the widespread dissemination of messages focused on the provision of free VMMC by specially trained health care providers and on the HIV prevention benefits of VMMC.\n\n-   Clinical efficiency was improved through, for example, the use of multiple beds in an assembly line, and the efficient use of staff time through task shifting and task sharing.\n\n-   The experiences of this campaign suggest that high-volume VMMC can be performed without compromising client safety, and provide a model for matching supply and demand for VMMC services elsewhere.\n\n# Introduction\n\nSeveral randomized controlled trials have demonstrated the safety and efficacy of voluntary medical male circumcision (VMMC) in HIV transmission prevention among heterosexual men \\[1\\]\u2013\\[3\\]. Consequently, in 2007, the World Health Organization (WHO) and the Joint United Nations Programme on HIV\/AIDS (UNAIDS) recommended that countries with a high prevalence of HIV and a low prevalence of male circumcision scale up VMMC within their comprehensive HIV prevention programming \\[4\\]. WHO currently recommends a minimum package for VMMC services that includes group and individual education on VMMC, HIV risk reduction, and other male sexual and reproductive health issues; condom promotion and provision; HIV testing and screening; treatment of sexually transmitted infections (STIs) and physiological abnormalities; and provision of VMMC under local anesthesia with postoperative observation and two to three postoperative visits (two, seven, and 42 days postoperatively) \\[5\\]. WHO guidance on optimizing the volume and efficiency of VMMC services \\[6\\] recommends the use of multiple surgical beds per surgical team, \"task shifting\" the surgical procedure from surgeon to nurses and\/or clinical officers, \"task sharing\" less complex steps of the circumcision procedure to lower credentialed but highly trained health care cadres, and the use of forceps-guided surgical techniques, alcohol gel scrubs between surgical cases, and electrocautery for hemostasis.\n\nIn 2009, the government of Tanzania adopted WHO's recommendation to scale up VMMC \\[4\\]. Tanzania has adult HIV and male circumcision prevalences of 5.7% and 67%, respectively \\[7\\]. Regional variations in both male circumcision and HIV prevalence exist that correlate inversely. Religious, educational, ethnic, and cultural factors and differences in the proportion of people living in urban and rural settings may explain some of the regional differences in male circumcision coverage. Notably, a national situation assessment on VMMC reported that 93% of respondents in traditionally non-circumcising areas of Tanzania would take their sons to be circumcised if the services were available, and identified the major deterrents to seeking VMMC as financial costs and having no family or cultural history of circumcision \\[8\\].\n\nIn 2010, the government of Tanzania set a goal of 80% VMMC coverage in its draft proposal \"National Strategy for Scaling Up Male Circumcision for HIV Prevention\" \\[9\\]. To achieve this goal, approximately four million circumcisions must be completed in the next five years, 2.8 million of them during the initial three-year implementation stage. The Tanzanian strategy for VMMC scale-up prioritizes eight regions of relatively high HIV and low male circumcision prevalence, with men aged 10\u201324 and 25\u201334 years as the primary and secondary priority groups, respectively. The strategy permits task shifting and specifies that VMMC services should be free of charge within the public sector.\n\nIringa Region, a largely rural region with a population of 1.9 million, has the highest adult HIV prevalence in Tanzania (15.7%) and relatively low circumcision coverage (29%) \\[7\\]. As part of the national strategy, the five-year target for VMMC in Iringa Region is 264,990 circumcisions \\[9\\]; modeling estimates suggest that one HIV infection will be averted for every 4.5 VMMCs performed in this region \\[10\\]. Iringa Region was one of the first Tanzanian regions to adopt VMMC as a part of its HIV prevention program \\[11\\]. The Iringa regional health authorities, in collaboration with the United States President's Emergency Plan for AIDS Relief (PEPFAR) and the Maternal and Child Health Integrated Program (MCHIP), a project funded by the United States Agency for International Development (USAID) and managed by Jhpiego (an affiliate of Johns Hopkins University), launched the region's VMMC program in September 2009. By April 2010, five health facilities were providing routine, closely monitored VMMC services that covered three of the region's eight districts.\n\nIn June 2010, the Iringa Region VMMC program undertook its first VMMC campaign. In this case study, we describe the campaign's approach to service delivery and the factors that influenced its quality, efficiency, and safety, and we discuss how the experiences of this campaign might serve as a model for future VMMC campaigns in the Iringa Region and elsewhere.\n\n# Description of the Campaign\n\nThe Iringa Region VMMC campaign was conducted in three districts of the region between June 21 and July 31, 2010, to coincide with school leave, the end of the harvest season, and Iringa's cool season (previous formative assessment revealed strong community preference for VMMC during the cool season \\[12\\]). VMMC services were provided Monday to Friday from 8:00 to 17:00, and Saturdays from 8:00 to 13:00, and were coordinated by committees and teams acting at regional, district, and site levels.\n\nAt the regional level, a committee of key stakeholders, which was formed three months before the campaign and included regional health and administrative authorities, campaign managers, and monitoring and evaluation experts, provided campaign oversight. The committee supervised VMMC facilities, provided quality assurance of services, and oversaw the dispensing of regular supplies of HIV test kits, condoms, and other consumable commodities such as sutures, gloves, lidocaine, and antibiotics provided by MCHIP. MCHIP also provided additional surgical beds and instruments, and other infrastructure items to the facilities providing VMMC services during the campaign.\n\nAt the district level, demand creation subcommittees were composed of district officials, health facility staff, and international and community-based organizations. These subcommittees, which were tasked with recruiting clients and sensitizing community political and administrative leaders, met several times before and during the VMMC campaign.\n\nFinally, each campaign site had a management team composed of the health facility's medical officer in charge, who was responsible for overall service delivery and quality at the site, and a site campaign manager, who was responsible for daily reporting on and oversight of the VMMC service. Every evening during the campaign, site managers participated in a debriefing session with campaign headquarters, during which problems with campaign implementation, shortages of commodities, issues related to client demand, and AEs were addressed.\n\nThese three levels of organization worked together to ensure the efficiency, quality, and safety of the campaign. In particular, as described below, they followed the guidance provided by WHO for using human resources efficiently, for maximizing the throughput of clients, for detecting AEs, and for creating a minimum VMMC package \\[5\\],\\[6\\]. Demand creation innovations were added to this package by MCHIP to match supply with demand.\n\n## Human Resources\n\nThe regional campaign committee developed a human resources plan that considered the WHO guidance for improving efficiencies, the number of surgical bays available, the expected client load, and available counseling and clinical staff. In this assembly-line service model, every four beds at a site required one circumcising surgeon, four bed nurses, one or two equipment and commodities runners, an equipment cleaner, and two HIV counselors. Regardless of the number of beds, each site also required a receptionist, an autoclave operator, a janitor, and a data manager. To reduce burnout, the clinical staff on the VMMC team, who were dedicated fully to VMMC throughout the campaign, rotated between roles (surgeon, bed nurse, and counselor). In addition, the most productive \"surgeons\" rotated among sites to encourage a spirit of collegiality. Other motivators for providers included T-shirts, daily meals, and overtime pay equivalent to US\\$7.00 per day. The Ministry of Health and Social Welfare paid staff salaries, with the exception of six counselors working for local non-governmental organizations, who were funded through other PEPFAR-supported programs. Finally, to ensure efficient and high-quality service provision, all the nurses (who constituted 80% of the campaign's VMMC providers, including \"surgeons\"), clinical officers, and physicians involved in the campaign were trained using the WHO\/UNAIDS\/Jhpiego male circumcision manual \\[5\\], and were provided with comprehensive on-site mentoring by a proficient VMMC physician.\n\n## Demand Creation\n\nSeveral approaches were taken to create demand for VMMC during the campaign. Through the district-based demand creation subcommittees, community-based organizations received a one-day training session designed to enable the promotion of the campaign by peer educators and outreach workers through activities such as traditional theater, small group sessions, one-to-one peer education, and speeches to community groups. These community workers received no additional compensation or incentive for adding the topic of VMMC to their usual activities. Brochures and other print materials were distributed that targeted specific audiences including adolescents and their guardians, female partners of potential VMMC clients, and men aged 18 years and above. In addition, three radio advertisements promoting the campaign ran eight times per day on regional radio stations, and regional officials appeared on local chat and health-related programs in the weeks leading up to the campaign. Finally, during the campaign, the regional health authorities arranged for announcements about the availability of services to be made across facility catchment areas. Based on the national situation assessment, in which cost was identified as a barrier to services \\[8\\], the demand creation messages emphasized that VMMC services were now free and would be performed by specially trained health care providers. Additional messages focused on the HIV prevention benefits of VMMC. Demand creation activities were scaled back by the fourth week of the campaign because of overwhelming demand, and midway through the campaign all sites began scheduling clients 1\u20132 weeks in advance, except for clients traveling long distances, who were given same-day services.\n\n## Increasing Client Volume\n\nAll of the efficiency considerations recommended by WHO were adopted for increasing client volume, with the exception of electrocautery (which was not endorsed for VMMC services in Tanzania) and the use of disposable surgical instruments kits. Specifically, time-saving surgical techniques, such as the forceps-guided method of circumcision, were used, and the number of surgical kits was doubled at sites without an autoclave to minimize the interruption of services caused by transfer of kits to autoclave-equipped sites for processing. To reduce client congestion during initial and follow-up visits and increase efficiency, the campaign provided additional trained counselors to prepare a large number of clients for the surgery; added tents and other temporary structures at VMMC sites (<a href=\"#pmed-1001131-g001\" data-ref-type=\"fig\">Figure 1<\/a>) to create additional space for counseling, postoperative follow-up, and data entry; scheduled clients up to two weeks in advance of surgery; paid special attention to maintaining the motivation of participating health care workers; and developed campaign-specific data monitoring tools.\n\n## Quality and Safety\n\nAs stipulated in WHO guidance, HIV testing was offered to all clients on an opt-out basis during individual counseling. Consent for HIV testing was verbal (the standard of care in Tanzania); a guardian's consent was required for clients under the age of 18. VMMC clients who tested HIV-positive received circumcision if they were eligible for surgery (eligibility was based upon their overall health and the absence of physiological abnormalities of the penis or STIs that would otherwise preclude them) and gave consent (during the campaign, all clients provided written consent for surgery), but were counseled about the lack of HIV prevention benefits for HIV-infected men and the increased risk of transmitting HIV to sexual partners during postoperative healing. All HIV-positive clients were referred to HIV care and treatment services, which were available at each circumcising site; clients with STIs were referred to STI treatment services at the same health facility and counseled to return for circumcision after their treatment was complete; and clients with physiological abnormalities were referred to the district or regional urologist.\n\nInfection prevention quality standards were applied at each of the campaign facilities, and the definitions for AEs in the WHO\/UNAIDS\/Jhpiego \"Manual for Male Circumcision under Local Anaesthesia\" were used to monitor AEs during the campaign \\[5\\] During counseling, the VMMC team emphasized the importance of clients returning for their postoperative reviews, AEs were described to clients to encourage reporting, and each client received details of a 24-hour emergency phone number staffed by VMMC team clinicians. All AEs were recorded using the standard WHO form \\[5\\] and were discussed during the site managers' nightly debriefs. The regional committee received weekly briefings on the occurrence of severe AEs and looked for trends to determine whether there might be cause for concern at any one site.\n\nThroughout the campaign, client-level data on service delivery and AEs were entered daily by data clerks within each health facility team into an electronic web-based data system designed for intensive monitoring and feedback. This system allowed each site management team to receive nightly reports of clients prepared, clients circumcised, AEs, and other key client data. The Johns Hopkins University Institutional Review Board and the Tanzanian Ministry of Health and Social Welfare approved the use of these data for this case study.\n\n# Achievements of the Campaign\n\nDuring the six weeks of the VMMC campaign, six circumcising teams of 16 individuals, plus site managers, drivers, and data clerks (140 participants in total) circumcised 10,352 adolescent and adult males, 1.72 times the campaign's target of 6,000 men. The average number of clients served per week increased as the campaign progressed (<a href=\"#pmed-1001131-g002\" data-ref-type=\"fig\">Figure 2<\/a>), and all the VMMC sites served more than 1,700 clients during the campaign period; Ngome Health Centre, which dedicated a large space to the VMMC campaign, achieved 2,781 circumcisions with eight beds and two surgical teams (<a href=\"#pmed-1001131-t001\" data-ref-type=\"table\">Table 1<\/a>). Staff retention was high throughout the campaign. Overall, 50% of clients came from outside facility catchment areas (defined as further than 15 km from the VMMC site), which suggests that clients were willing to travel great distances for free and safe services. However, there was substantial variability across sites: the percentage of clients coming from outside facility catchment areas ranged from 0.3% at Mafinga District Hospital to 68% at Tosamaganga Hospital (<a href=\"#pmed-1001131-t001\" data-ref-type=\"table\">Table 1<\/a>). Postoperative return rates ranged from 67% to 80% of clients for the two-day follow-up, and from 64% to 77% of clients for the seven-day follow-up (<a href=\"#pmed-1001131-g003\" data-ref-type=\"fig\">Figure 3<\/a>). Finally, almost 93% of the clients reached during the campaign were between 10 and 24 years old (<a href=\"#pmed-1001131-g004\" data-ref-type=\"fig\">Figure 4<\/a>). Lugoda Hospital, which is on a tea plantation, had the highest percentage of clients in the 20- to 34-year-old age range (43%), presumably because of the large population of adult tea workers in its catchment area.\n\n10.1371\/journal.pmed.1001131.t001\n\n###### VMMCs performed during the campaign, by site.\n\n<img src=\"pmed.1001131.t001\" id=\"pmed-1001131-t001-1\" \/>\n\n| Site | District | Characteristics of Site | Number of Beds | Number of Surgical Teams | Total VMMCs | VMMC Clients from Outside the Catchment Area<a href=\"#nt101\" data-ref-type=\"table-fn\">a<\/a> |  |\n|:---|:--:|:--:|:--:|:--:|:--:|:--:|:--:|\n|  |  |  |  |  |  | *N* | Percent |\n| Iringa Regional Hospital | Iringa Municipal | Urban; regional referral hospital | 4 | 1 | 1,784 | 1,114 | 62% |\n| Ngome Health Centre | Iringa Municipal | Urban; collaboration with Iringa Regional Hospital to serve overflow of clients | 8 | 2 | 2,781 | 843 | 46% |\n| Lugoda Hospital | Mufindi | Rural; services aimed primarily at tea plantation workers | 4 | 1 | 1,847 | 6 | 0.3% |\n| Mafinga District Hospital | Mufindi | Peri-urban; district referral hospital | 5 | 1<a href=\"#nt102\" data-ref-type=\"table-fn\">b<\/a> | 1,896 | 1,874 | 33% |\n| Tosamaganga Hospital | Iringa Rural | Rural; large, well-established, and well-utilized facility | 4 | 1 | 2,044 | 1,394 | 68% |\n| **Total** | **\u2014** | **\u2014** | **25** | **6** | **10,352** | **5,231** | **50%** |\n\nCatchment area defined as within 15 km of the facility.\n\nWith extra nurse.\n\nThe overall AE rate during the campaign was less than 1% (<a href=\"#pmed-1001131-t002\" data-ref-type=\"table\">Table 2<\/a>). Eighty percent of AEs were minor or moderate; only 20% were categorized as severe. The most common intra-operative AE was bleeding; swelling and infection were the most frequent postoperative AEs. All AEs were treated and clients were healed or healing by the end of the campaign.\n\n10.1371\/journal.pmed.1001131.t002\n\n###### Adverse events during the campaign.\n\n<img src=\"pmed.1001131.t002\" id=\"pmed-1001131-t002-2\" \/>\n\n| AE | Occurrence |  | Severity |  | Total AEs |\n|:---|:--:|:--:|:--:|:--:|:--:|\n|  | Intra-operative | Postoperative | Mild\/Moderate | Severe |  |\n| Damage to penis | 1 |  |  | 1 | 1 |\n| Excess skin removal | 3 |  |  | 3 | 3 |\n| Excessive bleeding | 4 |  |  | 4 | 4 |\n| Swelling of the penis or scrotum |  | 28 | 15 | 13 | 28 |\n| Infection |  | 64 | 64 |  | 64 |\n| Total | 8 | 92 | 79 | 21 | 100 |\n\nHIV testing uptake was virtually universal (99%), and the overall HIV prevalence was less than 1% for the campaign clients, although it increased with age (<a href=\"#pmed-1001131-t003\" data-ref-type=\"table\">Table 3<\/a>). Of the 74 individuals who tested HIV-positive, all went on to be circumcised. Twenty-two STI cases were identified during the campaign; all clients with STIs were referred for treatment and subsequently circumcised.\n\n10.1371\/journal.pmed.1001131.t003\n\n###### HIV testing, by age group.\n\n<img src=\"pmed.1001131.t003\" id=\"pmed-1001131-t003-3\" \/>\n\n| Result     |   Age   |         |         |         |       |\n|:-----------|:-------:|:-------:|:-------:|:-------:|:-----:|\n|            | 10\u201314 y | 15\u201319 y | 20\u201324 y | 25\u201334 y | \u226535 y |\n| Negative   |  2,199  |  5,616  |  1,754  |   565   |  134  |\n| Positive   |   10    |   10    |    9    |   31    |  14   |\n| Not tested |    0    |    5    |    5    |    0    |   0   |\n| Total      |  2,209  |  5,631  |  1,768  |   596   |  148  |\n\n# Lessons Learned\n\n## Efficiency\n\nThe data collected during the Iringa Region campaign indicate that, using the efficiency model adopted by the campaign, a four-bed\/one surgeon facility can circumcise up to 60 clients and an eight-bed\/two surgeon facility can achieve 120 circumcisions per day over a six-week period, and that the efficiency of VMMC service provision can increase over time. Importantly, the experiences gained during this campaign indicate that, by transferring some VMMC providers from larger sites to smaller sites, it is possible to provide a high-volume service at small sites without greatly impacting on the provision of normal health services provided at these sites. The data presented in this case study also suggest that supply and demand for VMMC can be matched by focusing on community-driven demand, and by ensuring efficient site-level client flow by adding counselors as needed and by expanding the space available for VMMC with tents, careful scheduling, detailed logistics, and the adoption of surgical efficiencies.\n\n## Quality\n\nThe long distances traveled by many clients to receive services during the campaign (some clients stated that they traveled as far as 100 km) was unanticipated and suggests that a well-motivated population will travel long distances to VMMC sites during campaigns. However, the willingness to travel long distances may also reflect a need for anonymity or client perceptions of service quality. For example, Iringa Regional Hospital's high \"long-distance\" caseload may have been related to client perceptions that a referral facility would provide higher quality services, a possibility that warrants further investigation. The high uptake of HIV testing seen during the campaign is not unusual\u2014HIV testing acceptability is generally very high in Tanzania\u2014but may have been magnified by a low perception of HIV risk among young (pre-sexual) clients, by the decreasing stigma associated with an HIV-positive status in the region, or by the knowledge that HIV care and treatment services were available at the circumcising sites.\n\n## Safety\n\nAE rates fell below those of normal service delivery (from just under 2% to 1%) during the campaign period \\[13\\]. This suggests that VMMC providers might become more proficient during high-volume campaigns, although increased oversight and supervision, training by highly qualified VMMC mentors, and quality improvement exercises undertaken during the campaign period may also have helped reduce AE rates.\n\n# Challenges\n\nThis case study highlights several major challenges for future high-volume VMMC campaigns. For example, it suggests that ways will need to be found to improve the participation of older male clients. Only 24% of clients served during the Iringa Region campaign were older than 20 years, and previous modeling has shown that for the greatest immediate public health impact, VMMC should cover the sexually active population \\[10\\]. Other challenges for future campaigns that are revealed by this case study include the possibility that demand may be higher than anticipated, that there may be insufficient sites providing VMMC services in rural regions, and that the procurement of commodities that are not readily available may require considerable lead times. Furthermore, the case study illustrates how, in many developing countries, surgical instruments, disposable commodities, and pharmaceuticals may need to be imported from abroad, and reusable surgical instruments may have to be transported long distances over difficult terrain to and from facilities with autoclaves. Finally, it draws attention to how infrastructure issues, electricity outages, and the geographic terrain may pose additional challenges in many developing countries.\n\n# Conclusions\n\nThe Iringa Region experience shows that VMMC service delivery can be provided to large numbers of men efficiently without compromising quality of service and client safety through a campaign mode of service delivery implemented almost exclusively in the public sector. Although there are considerable challenges associated with implementing such campaigns, they are not insurmountable, as this case study illustrates. Moreover, with contextualization, we suggest that similar campaigns could be replicated in other settings in east and southern Africa where VMMC for HIV prevention has been prioritized.\n\nNotably, since the completion of the Iringa Region campaign, expansion of VMMC services to the remaining five districts of the Iringa Region has become a priority. In December 2010, a three-week campaign that coincided with the school holidays resulted in nearly 3,000 clients being circumcised. By April 2011, all districts in the Iringa Region were offering VMMC and, between June 20 and August 13, 2011, another eight-week campaign served 31,046 VMMC clients across the Iringa Region.\n\nThe authors would like to acknowledge the following people who played important roles in the implementation of the campaign and synthesis of campaign lessons learned: Ezekial Mpuya, Paul Luvanda, Menrad Dimoso, Caroline Kiame, Leonard Ndeki, Sekasua Mndeme, Rajabu Muhombolage, Benny Lugoe, Flora Hezwa, and the health providers of Iringa Region. We also wish to thank the Tanzania National AIDS Control Programme and USAID\/Tanzania.\n\nThe campaign data were presented as a poster at the 18th Conference on Retroviruses and Opportunistic Infections in February 2011 and in an oral poster presentation at the 6th International AIDS Society Conference on HIV Pathogenesis, Treatment and Prevention in July 2011.\n\n# References\n\n## Abbreviations\n\nAE\n\n:   adverse event\n\nMCHIP\n\n:   Maternal and Child Health Integrated Program\n\nPEPFAR\n\n:   United States President's Emergency Plan for AIDS Relief\n\nSTI\n\n:   sexually transmitted infection\n\nUNAIDS\n\n:   Joint United Nations Programme on HIV\/AIDS\n\nUSAID\n\n:   United States Agency for International Development\n\nVMMC\n\n:   voluntary medical male circumcision\n\nWHO\n\n:   World Health Organization\n\n[^1]: Conceived and designed the experiments: HM BK KC MP TA AH EN BF. Analyzed the data: HM BK MP MM MLD BF. Wrote the first draft of the manuscript: HM. Contributed to the writing of the manuscript: HM BK KC MP TA AH SK SN MM MLD DC EN BF. [ICMJE](http:\/\/www.icmje.org\/) criteria for authorship read and met: HM BK KC MP TA AH SK SN MM MLD DC EN BF. Agree with manuscript results and conclusions: HM BK KC MP TA AH SK SN MM MLD DC EN BF.","meta":{"dup_signals":{"dup_doc_count":162,"dup_dump_count":38,"dup_details":{"curated_sources":4,"2022-49":1,"2022-40":1,"2022-05":1,"2021-43":1,"2021-21":1,"2021-17":1,"2020-45":1,"2020-05":1,"2019-47":2,"2019-43":1,"2019-30":1,"2017-09":10,"2016-36":3,"2016-30":5,"2016-26":1,"2016-22":1,"2016-18":2,"2016-07":7,"2015-48":4,"2015-40":3,"2015-35":7,"2015-32":8,"2015-27":7,"2015-22":5,"2015-14":3,"2014-52":8,"2014-49":4,"2014-42":10,"2014-41":12,"2014-35":9,"2014-23":9,"2014-15":6,"2023-23":1,"2024-18":1,"2015-18":9,"2015-11":5,"2014-10":5,"2024-30":1}},"file":"PMC3226544"},"subset":"pubmed_central"}
{"text":"abstract: The use of quantitative metrics to gauge the impact of scholarly publications, authors, and disciplines is predicated on the availability of reliable usage and annotation data. Citation and download counts are widely available from digital libraries. However, current annotation systems rely on proprietary labels, refer to journals but not articles or authors, and are manually curated. To address these limitations, we propose a social framework based on crowdsourced annotations of scholars, designed to keep up with the rapidly evolving disciplinary and interdisciplinary landscape. We describe a system called Scholarometer, which provides a service to scholars by computing citation-based impact measures. This creates an incentive for users to provide disciplinary annotations of authors, which in turn can be used to compute disciplinary metrics. We first present the system architecture and several heuristics to deal with noisy bibliographic and annotation data. We report on data sharing and interactive visualization services enabled by Scholarometer. Usage statistics, illustrating the data collected and shared through the framework, suggest that the proposed crowdsourcing approach can be successful. Secondly, we illustrate how the disciplinary bibliometric indicators elicited by Scholarometer allow us to implement for the first time a universal impact measure proposed in the literature. Our evaluation suggests that this metric provides an effective means for comparing scholarly impact across disciplinary boundaries.\nauthor: Jasleen Kaur; Diep Thi Hoang; Xiaoling Sun; Lino Possamai; Mohsen JafariAsbagh; Snehal Patil; Filippo Menczer\\* E-mail: <firstname.lastname@example.com>[^1][^2]\ndate: 2012\ninstitute: 1 Center for Complex Networks and Systems Research, School of Informatics & Computing, Indiana University, Bloomington, United States of America; 2 University of Engineering and Technology, Vietnam National University, Hanoi, Vietnam; 3 Department of Computer Science and Technology, Dalian University of Technology, China; 4 Department of Pure and Applied Mathematics, University of Padua, Padua, Italy; The Centre for Research and Technology, Hellas, Greece\nreferences:\ntitle: Scholarometer: A Social Framework for Analyzing Impact across Disciplines\n\n# Introduction\n\nMany disciplinary communities have sought to address the need to organize, categorize, and retrieve the articles that populate their respective online libraries and repositories. Unfortunately, the great promise of such mechanisms is hindered by the fact that disciplinary categories, as an organizing principle, do not accommodate the trend toward interdisciplinary scholarship and the continual emergence of new disciplines. An initial step towards a solution comes in the form of journal indices, such as those supported by Thomson-Reuters as part of their Journal Citation Reports (JCR) and Web of Science (WoS) commercial products. Systems like the Web of Science, and similar discipline classifications such as MeSH for life sciences, PACS for physics, and ACM CCS for computing, are based on a *top-down* approach in which the ontology is maintained by dedicated curators. However, as disciplines evolve through novel discoveries and interdisciplinary collaborations, semantic predicates associated with these ontologies may become increasingly vague and less informative and will fail to identify the interdisciplinary work occurring at the granularity level of articles and the new areas that emerge at the disciplinary boundaries.\n\nThe \"Web Science\" paradigm suggests an alternative approach. Rather than attempting to match new scientific production to predefined categories, it would be useful to facilitate semantic evolution by empowering scholars to annotate each other's work. This *bottom-up* approach has already been adopted in popular systems such as Bibsonomy.org \\[1\\], Mendeley \\[2\\], and many others \\[3\\], \\[4\\]. There are at least three benefits to such a *crowdsourcing* model: (i) dynamic classification that scales with the growth of the number of authors, articles, and specializations \\[5\\]; (ii) flexibility to capture emergent interdisciplinary fields compared to hierarchical taxonomy \\[6\\]; and (iii) emergence of structure and consensus from the shared vocabularies of interdisciplinary collaborators \\[7\\], \\[8\\].\n\nDisciplinary boundaries create similar hurdles for measuring scholarly impact, although these hurdles are relegated more to standards and practices. For example, the fields of history and physics have very different publishing patterns and standards of collaboration. A historian may work for years to publish a solitary work while an experimental physicist may co-author numerous articles during the same time period. How do we compare scholars across fields?\n\nRadicchi *et al.* \\[9\\] found that citations follow a universal distribution across disciplines when certain discipline-specific statistical quantities are taken into account. Thus, they reasoned, one could construct a universal impact measure to compare authors across disciplines. However, such discipline specific statistics are not available from established bibliometric sources. The crowdsourcing model described above has the added advantage that when combined with citation information about the authors, it can enable the collection of statistical data necessary for the computation of cross-disciplinary impact metrics.\n\nWhat we envisage is crowdsourcing the knowledge of community members in a scenario similar to those explored in citizen science \\[10\\] and games with a purpose \\[11\\]. Users would provide disciplinary annotations in exchange for access to citation data obtained from querying bibliographic services (e.g., Google Scholar, CiteSeer, Scopus, and Web of Science). The combined annotation and citation data could then be freely shared with the public. In practice, the idea is to provide a social client interface to an extant Web source of scholarly data, allowing users to perform academic impact analysis based on author queries. This means the data will originate from two general sources: (i) citation data will be collected from public and private sources online, and (ii) users will annotate authors with discipline tags.\n\nScholarometer is a social tool for scholarly services developed at Indiana University, with the dual aim of exploring the crowdsourcing approach for disciplinary annotations and cross-disciplinary impact metrics \\[12\\]. These two aims are closely related and mutually reinforcing. The annotations enable the collection of discipline specific statistics, and therefore the computation of universal impact metrics. In turn, the service provided to users by computing these metrics works as an incentive for the users to provide the annotations.\n\nThe goal of this paper is to detail the design and implementation of the Scholarometer tool. We present visualization and data exchange services that are fueled by the data crowdsourced through Scholarometer. We also outline the computation of both disciplinary and universal rankings of authors enabled by this data. In particular, we make the following contributions:\n\n-   We present the architecture, user interface, and data model used in the design and implementation of the Scholarometer system. We discuss several heuristics employed to deal with the noisy nature of both bibliographic data and user-supplied annotations (<a href=\"#s2\" data-ref-type=\"sec\">Materials and Methods<\/a> section).\n\n-   As an illustration of potential applications of crowdsourced scholarly data, we report on data sharing and interactive visualization services. These applications suggest that the crowdsourcing framework yields a meaningful classification scheme for authors and their disciplinary interactions (Data Sharing and Visualization section).\n\n-   By leveraging socially collected discipline statistics, we implement the so-called \"universal -index\" proposed by Radicchi *et al.* \\[9\\]. This is the first implementation that makes the metric publicly available. We show that user-provided tags provide stable disciplinary coverage, and that the universal -index can be a reliable indicator for comparing the scholarly impact of individual authors across different disciplines (<a href=\"#s3\" data-ref-type=\"sec\">Results<\/a> section).\n\n## Background\n\nTools exist for both citation analysis (e.g., Publish or Perish \\[13\\]) and social management of bibliographic records (e.g., Mendeley \\[2\\]). To our knowledge, Scholarometer is the first system that attempts to couple these two functions with the goal of achieving a synergy between disciplinary annotations and universal impact metrics \\[12\\].\n\nThe extraction of bibliographic information from online repositories is not new. Bibliographic management tools such as BibDesk offer robust search of online resources and digital libraries like PubMed \\[14\\]; users can import objects into Connotea using Digital Object Identifiers (DOI) \\[3\\]; and Zotero can spider through and collect bibliographic information from webpages \\[15\\]. These and many other bibliographic management tools are compared in Wikipedia \\[16\\].\n\nSocial tagging of scholarly work is not a new idea, either \\[3\\], \\[4\\], \\[17\\]. In the folksonomies that result from these social tagging systems, tags are assigned to papers. In Scholarometer, users tag authors instead. This makes it possible to collect disciplinary annotations in a more convenient way for the users. Further, we emphasize the use of scholarly disciplines in the annotations, as discussed in the sectionUser Iterface.\n\nWe have chosen to use Google Scholar as the citation database for our research. Web of Science, Scopus, and Microsoft Academic Search are possible alternatives \\[18\\], but Google Scholar has the advantage of being free and comprehensive, claiming to cover articles, theses, books, abstracts, and other scholarly literature from all areas of research \\[19\\]. Our preliminary analysis suggests that Google Scholar has a higher coverage than Microsoft Academic Search \\[20\\].\n\nAn important goal of the proposed annotation crowdsourcing platform is to enable the computation of scholarly impact. Bibliometrics is the use of statistical methods to analyze scholarly data and identify patterns of authorship, publication, and use. Constitutive of bibliometrics is citation analysis, used to measure the impact or influence of authors and papers in a particular field. There is a plenitude of citation measures. Some (e.g., Hirsch's -index \\[21\\]) balance productivity and impact by trading off between number of publications and number of citations; others seek to apportion the proper weight for highly cited publications (e.g., Egghe's -index \\[22\\]) or apportion citations fairly for papers with multiple authors (e.g., Schreiber's -index \\[23\\]); and still others (e.g., Radicchi *et al.*' s universal -index \\[9\\]) attempt to quantitatively compare the impact of authors across disciplines. Scholarometer implements multiple citation measures including -index, -index, -index and universal -index. We compare the -index with the universal -index, as discussed in the section Impact Analysis and Universality.\n\nScholarometer's crowdsourcing method, in which annotation data is generated by users in exchange for a service, is grounded in prior work as well. Amazon's Mechanical Turk \\[24\\], Wikipedia \\[25\\], and GalaxyZoo \\[10\\] are popular examples of crowdsourcing. This technology coordinates the application of human intelligence as a stopgap in problems that computers are unable to solve. For example, people may prove more capable at describing or judging certain objects such as a picture or piece of music. The ESP 'game with a purpose' \\[11\\] is another forerunner of Scholarometer, in which users generate useful annotation data in exchange for entertainment \\[26\\], \\[27\\].\n\n# Materials and Methods\n\nIn this section we outline the main features of the Scholarometer system, available at scholarometer.indiana.edu.\n\n## Architecture\n\nAny citation analysis tool can only be as good as its data source. As mentioned earlier, Scholarometer uses Google Scholar as a data source, which provides freely accessible publication and citation data to users without requiring a subscription. Google Scholar provides excellent coverage, in many cases better than the Web of Science \u2014 especially in disciplines such as computer science, which is dominated by conference proceedings, and some social sciences, dominated by books. Nevertheless, Google Scholar is based on automatic crawling, parsing, and indexing algorithms, and therefore its data is subject to noise, errors, and incomplete or outdated citation information. The data collected from Google Scholar comprises the number of papers by an author along with their citation counts and publication years. Alternative sources, such as Microsoft Academic Search (academic.research.microsoft.com) or CiteSeer (citeseerx.ist.psu.edu), can provide the same data for the queried author. Therefore, the system architecture and design that we describe below are independent of the data source.\n\nDue to the lack of an API to access Google Scholar data, a server-based implementation would violate Google Scholar's policy about crawling result pages, extracting data (by scraping\/parsing) and making such data available outside of the Google Scholar service. Indeed, server-based applications that sit between the user and Google Scholar are often disabled, as Google Scholar restricts the number of requests coming from a particular IP address. Workarounds such as configurable proxies are not desirable solutions as they also appear to violate policy. We further excluded Ajax technology due to the same origin policy for JavaScript, and the gadget approach because it would render the tool dependent on a particular data source. We turned to a client-based approach, but ruled out a stand-alone application (such as Publish or Perish) for portability reasons. These design considerations led us to a browser extension approach, which is platform and system independent and, to the best of our knowledge, in compliance with Google's terms of service.\n\nIn keeping with the above considerations, Scholarometer is implemented as a smart browser extension, through which the user queries the source, annotates the results, and shares with the Scholarometer community only annotation metadata from the users and public citation data. We emphasize that Scholarometer does not store a copy of a subset of the Google Scholar database. In particular, the records returned to the users from Google Scholar are not stored. The data that our system collects from the users comprises of publication year, number of citations, and number of authors for each article. This information is open for the users to share with the community.\n\nThe architecture and workflow of Scholarometer is illustrated in <a href=\"#pone-0043235-g001\" data-ref-type=\"fig\">Figure 1<\/a>. There are six steps: (1) The user enters a query and discipline tags for an author into a search form provided by the browser extension. (2) The browser extension forwards the query to Google Scholar. (3) Google Scholar returns the query results to the browser. (4) The browser extension then forwards the results to the Scholarometer server. This parses the results to extract citation and other metadata, which is then inserted into the database, along with annotation metadata. (5) The Scholarometer server sends to the client browser the bibliographic records and impact measures for the queried author(s). (6) Finally, the client browser renders the data in an interactive way. The user views results in a new browser tab and can perform advanced actions such as sorting, filtering, deleting, and merging records.\n\n## User Interface\n\nThe Scholarometer tool has two interfaces for communicating with users: one in the browser extension for entering queries and tags, the other in the main browser window for presenting and manipulating bibliographic data and citation analysis results. The browser extension is available in two versions: one for the Firefox browser hosted at the Mozilla Firefox Add-ons site, and one for Chrome browser hosted at the Google Chrome Web store (scholarometer.indiana.edu\/download.html). The Firefox interface is illustrated in <a href=\"#pone-0043235-g002\" data-ref-type=\"fig\">Figure 2<\/a>.\n\nThe query interface in the browser extension is designed to identify one or more authors and retrieve their articles. The default interface hides many advanced features and simplifies the common case of a single author uniquely identified by name. Advanced interfaces are available with explicit Boolean operators for multiple authors or ambiguous names, with controls for filtering subject areas and languages, and with additional keyword fields.\n\nTagging a queried author with disciplinary annotations is a key requirement of the extension interface. We considered two possibilities for the set of usable tags. One is the use of a predefined, controlled vocabulary. This closed approach has the advantage of producing \"clean\" labels, but the limitation of disallowing the bottom-up, user-driven tracking of new and emerging disciplines, which is a crucial goal of our project. At the other extreme, the open approach of free tagging addresses the latter goal but opens the door to all kinds of noise, from misspelled keywords to the use of non-disciplinary labels that can be useful to a particular individual but not necessarily to the community \u2014 think of tags such as \"ToRead,\" \"MyOwn,\" \"UK,\" and so on. We therefore aimed for a compromise solution in our design. The user must enter at least *one* annotation from a controlled hierarchical ontology of disciplines, and can enter any free tags without additional constraints. The predefined labels are the set of subject categories extracted from the three major Thomson-Reuters citation indices (*Science Expanded*, *Social Science*, and *Arts & Humanities*). This way each queried author is associated in the Scholarometer database with at least one established subject category and one of the three high-level classes. In addition, we provide an autocomplete feature to make it easy for users to enter discipline tags and reuse tags from other users, thus decreasing the frequency of misspellings.\n\nThe interface in the main browser window is designed to facilitate the manipulation and cleaning of the results, to visualize how the impact measures are calculated, and to expose annotations from other users for the same author(s). The output screen is divided into three panels:\n\n1.  A filter panel with two modules. One module is for pruning the set of articles based on the publication year or the number of citations. The second module is for limiting the set of articles to selected name variations or co-authors.\n\n2.  The list of articles, with utilities for live searching and for alternating between a simplified and an extended view, as well as links to external resources. This panel also has remove and merge utilities to correct two common sources of noise in Google Scholar results: articles written by homonymous authors and different versions of the same paper.\n\n3.  A citation analysis panel reporting impact measures. As discussed in the section sec:background, many impact measures have been proposed, and it is infeasible to implement them all. Since a single measure can only capture some aspect of scientific evaluation, a good citation analysis tool should incorporate a set of measures that capture different features, such as highly cited publications, co-authorship, and different citation practices. To this end we have implemented Hirsch's -index \\[21\\], Egghe's -index \\[22\\], Schreiber's -index \\[28\\], and Radicchi *et al.*' s index that we call \\[9\\]. Note that this is the first implementation of the universal -index available to the public, which is enabled by the joint availability of annotation and citation data, as explained in detail in the section hf. The citation analysis panel displays values for each discipline tag of an author, along with percentiles. Finally, the panel shows two plots illustrating the citation distribution and publications per year. All the data in the citation analysis panel is dynamically generated and updated in response to any filter, merge or delete action performed in the other panels.\n\nTo provide additional incentives for users to submit more queries, thus contributing more annotation data, we offer the functionality of exporting bibliographic records from the main browser window. Publication data can be exported individually or in bulk into formats commonly used by reference management tools and scholarly data sharing services. At present, Scholarometer supports the following formats: (BIB), RefMan (RIS), EndNote (ENW), comma-separated values (CSV), tab-separated values (XLS), and BibJSON \\[29\\]. Note that the publication data is generated dynamically at query time and not stored on our server, except for a temporary cache. Since the data provider (Google Scholar in our case) makes the bibliographic records freely accessible to end users and not our service, it is up to the users to save this information for local use or for sharing more broadly. Data that can be exported also includes citation counts and disciplinary annotations. This helps users who plan to share the data via scholarly tagging systems, such as BibSonomy, and facilitates the propagation of socially-vetted tags.\n\n## Query Management Heuristics\n\nThe data that we collect comes from users, so it is naturally noisy and subject to various issues. We propose several heuristics to deal with these sources of noise. <a href=\"#pone-0043235-g003\" data-ref-type=\"fig\">Figure 3<\/a> illustrates how these heuristics are integrated into Scholarometer's query manager.\n\nWe employ a blacklist to prevent spammers from polluting our database. An example is the fictitious author \"Ike Antkare,\" fabricated to highlight the vulnerability of online sources of citation data \\[30\\]. When a query matches a name from the blacklist, the system generates an error message. Fraudulent names are manually added to the blacklist by system administrators.\n\nA critical challenge for bibliometric services is that author names are often ambiguous. Ambiguous names lead to biased impact metrics. The problem is amplified when names are collected from heterogeneous sources, including crowdsourced annotations. This is the case in the Scholarometer system, which cross-correlates author names in user queries with those retrieved from bibliographic data. A component of the Scholarometer system therefore attempts to detect an ambiguous name at query time. When an author name is deemed ambiguous, the user is prompted to refine the query. This design aims at decreasing noise in the database and limiting inaccurate impact analysis.\n\nOur first attempt to deal with ambiguous author names deployed a simple heuristic rule based on citation counts associated with name variations \\[12\\]. With the increasing popularity of the tool, the number of authors in the Scholarometer system has grown significantly, revealing that many ambiguous names were undetected. We thus introduced a supervised learning approach to detect ambiguous names at query time, based on a combination of features. We extended the original heuristics by exploring a feature that measures the consistency among the topics associated with publication metadata, with the help of crowdsourced discipline annotations. The accuracy is about 75% \\[31\\].\n\nWork on the ambiguous name detection problem is ongoing. We are currently exploring the incorporation of additional features into the classifier. One new class of features under study is based on metadata consistency. We developed a two-step method to capture the consistency between coauthor, title and venue metadata across publications. Authors are likely to collaborate with a certain group of authors, write papers with related titles, and publish papers in similar journals or conferences. The metadata associated with these publications by the same author should be consistent. Another new feature is the consistency between topics associated with publication metadata and discipline annotations crowdsourced from the users. By combining all these features, the accuracy reaches almost 80% \\[20\\].\n\nSince there is no established way to uniquely identify authors (the ORCID initiative is under development \\[32\\]), we use a signature of the query as an author identifier. Keywords used in queries contribute to the generation of unique identifiers. To reduce duplicate author records, the system uses the following rules when a query is submitted (see <a href=\"#pone-0043235-g003\" data-ref-type=\"fig\">Figure 3<\/a>):\n\n1.  If the author name is already present in the database, the system prompts the user to make a selection from a list of names provided along with citation metadata.\n\n2.  If the user chooses someone from the list, the system updates the information for the author rather than creating a new record.\n\n3.  If the user does not choose a name from the list, but an author generated from an identical query is present in the database, the user is prompted to use additional keywords to disambiguate the query.\n\n4.  If the user does not choose a name from the list, and an identical author is not present in the database, a new record for the author is created.\n\nA second issue is the arbitrary nature of uncontrolled discipline annotations. As mentioned earlier, free tags can be noisy, ambiguous, or duplicated. We employ manual and automatic techniques to deal with noisy annotations. We found different types of noise in our tag collection. First, some users misunderstand the tagging request, and utilize author names instead of discipline names as tags. Second, misspelled disciplines names are common, resulting in a duplication of existing tags. Third, some users adopt acronyms without checking if an extended version of the discipline name already exists (e.g., \"hci\" vs. \"human computer interaction\"). Finally, people may abuse the tool, using non-sensical or random tags, e.g., the first discipline starting with the letter 'a.'\n\nSome of these issues can be dealt with automatically by (i) checking if a tag corresponds to an author name present in the database, and (ii) ordering all tags in lexicographical order and calculating the edit distance within a window of neighboring tags. We employ the Damerau-Levenshtein (DL) distance \\[33\\] to this end. All the pairs of tags whose DL distance is less than a fixed threshold (currently 2) are flagged. The tag with lower use in each pair is merged into the one with higher use, with manual supervision. A tool developed for internal use allows system administrators to perform these tasks, as well as to remove junk tags and manually merge tags where appropriate, e.g., a user generated tag such as \"AI\" that duplicates the predefined \"computer science, artificial intelligence\" discipline.\n\nFinally, we need a way to estimate the reliability of crowdsourced discipline tags. We view each query as a vote for the discipline tags of the queried author. For example, a query that tags Einstein with \"physics\" and \"philosophy\" generates a vote for (Einstein, \"physics\") and a vote for (Einstein, \"philosophy\"). The number of votes together with the number of tags can be used to determine heuristically which tags are *reliable* for each author. The intuition is that the more tags an author has, the greater the possible confusion, and proportionally the greater the number of votes necessary for a tag signal to rise above the noise. To derive a heuristic according to this intuition, we simulated a model in which votes are assigned to tags for an author by randomly drawing from the overall distribution of votes. Suppose that an author has tags that have received votes collectively, and a new vote is assigned to tag resulting in votes for out of a total of votes. Note that increases if . We can measure the change in entropy corresponding to this new vote, where . According to our intuition, tag is reliable if , i.e., the confusion as measured by the entropy decreases as a result of the number of votes accrued by . We simulated this model 1000 times, using a total of 1000 tags and stopping each run when . We then averaged the change in entropy corresponding to the combination of and values for each new vote. In <a href=\"#pone-0043235-g004\" data-ref-type=\"fig\">Figure 4<\/a> we show that according to this model, the number of votes necessary to make a tag reliable grows slowly with the number of tags . We chose to adopt a heuristic threshold: a tag is deemed reliable for an author with tags if it has votes.\n\n## Data Sharing and Visualization\n\nScholarometer provides several ways to share the crowdsourced data with the research community, and to explore the data through interactive visualizations.\n\nThe API (scholarometer.indiana.edu\/data.html) makes the data collected by Scholarometer available. It also makes it easy to integrate citation-based impact analysis data and annotations into other applications. It exposes information about authors, disciplines, and relationships among authors and among disciplines.\n\nThe Widget provides an easy and customizable way to embed a dynamically updated citation analysis report into any website. The results screen in the main browser window includes a special \"widget\" button (see <a href=\"#pone-0043235-g002\" data-ref-type=\"fig\">Figure 2<\/a>) leading to code and instructions to embed the citation analysis report. The widget feature can also be accessed from the home page (scholarometer.indiana.edu\/cgi-bin\/widget.cgi).\n\nScholarometer also publishes crowdsourced data according to the basic principles of \"Linked Data\" \\[34\\] (scholarometer.indiana.edu\/data.html). The aim is to make information about authors and disciplines based on citation analysis available on the Semantic Web. Linked Data is a style of publishing and interlinking structured data on the Semantic Web. Data is described and linked using a language called RDF (Resource Description Framework). Users can use generic RDF browsers (e.g., Tabulator, Disco, OpenLink Browser), RDF crawlers (e.g., SWSE, Swoogle), and query agents (e.g., SemWeb Client Library, SWIC) to explore the data. We assign URIs (Uniform Resource Identifiers) to authors and disciplines and implement an HTTP mechanism called \"content negotiation\" to provide an HTML representation in addition to the RDF representation of a resource. Linked Data encourages interlinks between different data sources, which enable Semantic Web browsers and crawlers to navigate between them. As illustrated in <a href=\"#pone-0043235-g005\" data-ref-type=\"fig\">Figure 5<\/a>, Scholarometer RDF links primarily point to DBpedia, DBLP, Freebase and Opencyc data sets using the owl:sameAs property, which indicates that two URIs refer to the same thing.\n\nOne way to explore the quality of the annotations obtained through the crowdsourcing approach employed by the Scholarometer system is to map the interdisciplinary collaborations implicit in the tags. Since an author can be tagged with multiple disciplines, we can interpret such an annotation as an indicator of a link between these disciplines. For example, if many users tag many authors with both \"mathematics\" and \"economics\" tags, we can infer that these disciplines are strongly related, even though they belong to different branches of the JCR \u2014 science and social sciences, respectively. <a href=\"#pone-0043235-g006\" data-ref-type=\"fig\">Figure 6<\/a> presents a network that visualizes the relationships between the tags in Scholarometer based on the number of authors annotated with each tag. The nodes in the network represent disciplines. Each node's area is proportional to the number of authors in the corresponding discipline, i.e., the total number of authors tagged with that discipline. Nodes corresponding to JCR categories are colored based on the ISI citation indices: blue for science, red for social sciences, and orange for arts and humanities. User-defined disciplines are represented by gray nodes. We see a predominance of scholarly data in the sciences based on current Scholarometer usage. The presence of large gray nodes underlines the limits of the JCR classification. Edges represent interdisciplinary collaborations, as induced by author annotations. We represent each discipline as a vector of authors, where each coordinate is the number of votes assigning the corresponding author to a discipline. An edge connecting two disciplines has a weight equal to the cosine similarity between the two vectors. The more common authors who are tagged with both disciplines, the stronger the weight. The interactive visualization also displays the top authors in a discipline and their impact metrics when a user hovers over the node. The layout of the network is obtained by Fruchterman and Reingold's force-directed algorithm \\[35\\], so that related disciplines are more likely to be near each other. The plausible map of science that results from our annotations, as illustrated by the highlighted areas in <a href=\"#pone-0043235-g006\" data-ref-type=\"fig\">Figure 6<\/a>, suggests that the crowdsourcing framework yields a meaningful classification scheme for authors and their disciplinary interactions.\n\nAlong with interactive discipline network, Scholarometer also provides interactive visualizations of author networks. Starting from an author submitted in a query, the author network displays similar authors. An author is represented as a vector of discipline tags, weighted by votes. Author nodes are connected by an edge weighed by the cosine similarity between the corresponding vectors. Authors are therefore deemed similar if they are tagged similarly. These visualizations can help identify potential referees, members of program committees and grant panels, collaborators, and so on. Such a scenario is illustrated in <a href=\"#pone-0043235-g007\" data-ref-type=\"fig\">Figure 7<\/a>. Author nodes are colored based on their predominant reliable tag. The tooltip of a node in the network displays the corresponding author's impact metrics.\n\n# Results\n\n## Data Analysis\n\nThe Scholarometer system was first released in November 2009. At the time of this writing the Scholarometer database has collected information about 1.9 million articles by 26 thousand authors in 1,200 disciplines. There are about 90 thousand annotations, or tag-author pairs. Once we apply the heuristics described in the section heuristics, we reduce these numbers to about 1.4 million articles by about 21 thousand reliable authors with about 34 thousand reliable annotations into about 900 reliable disciplines. Naturally this folksonomy grows and evolves daily as Scholarometer handles new queries. The growth in the numbers of discipline tags, authors, and queries is charted in <a href=\"#pone-0043235-g008\" data-ref-type=\"fig\">Figure 8<\/a>, illustrating an initial phase of exponential growth followed by a steady linear regime. <a href=\"#pone-0043235-g009\" data-ref-type=\"fig\">Figure 9<\/a> (top) displays the dynamics of the top 20 discipline tags, based on number of authors. To better illustrate the proportions of authors in the various disciplines, the ratios are plotted in <a href=\"#pone-0043235-g009\" data-ref-type=\"fig\">Figure 9<\/a> (bottom). We observe that the Scholarometer database was initially dominated by computing-related disciplines, due to the publicity received by the tool in the computer and information science community. Over time, the collection has become more uniform and the coverage of various disciplines has grown.\n\nVarious statistics for authors and disciplines are available on the Scholarometer website (scholarometer.indiana.edu\/explore.html). The annotation data enables us to derive rankings for authors \u2014 both universal and disciplinary \u2014 based on impact metrics. <a href=\"#pone-0043235-t001\" data-ref-type=\"table\">Table 1<\/a> shows the universal rankings of top authors by , , , and respectively. We can see that compared to the -index, the -index favors authors such as D.R. Cox and A. Shleifer, with books that have received very high numbers (thousands) of citations. The -index favors authors with many top publications that are single-authored; M. Friedman and physics nobel prize winner S. Weinberg are good examples. The universal metric (discussed next in section hf) brings to the top some authors whose citations are not as numerous in absolute terms, but who are leaders in their respective fields \u2014 nobel prize winner in economics P. Krugman and C.R. Sunstein from law are good examples.\n\n10.1371\/journal.pone.0043235.t001\n\n###### Top authors according to various impact measures (based on values as of January 2012).\n\n<img src=\"pone.0043235.t001\" id=\"pone-0043235-t001-1\" \/>\n\n|     |              |               |             |              |\n|:----|:------------:|:-------------:|:-----------:|:------------:|\n| 1   |   JN Ihle    |    DR Cox     |   S Freud   |   S Freud    |\n| 2   |  WC Willett  | GM Whitesides | M Friedman  |  N Chomsky   |\n| 3   | MJ Stampfer  |    JN Ihle    | P Bourdieu  |   S Kumar    |\n| 4   |  M Friedman  |    W Zhang    |  SH Snyder  |   W Zhang    |\n| 5   |   W Zhang    |    S Freud    |  E Witten   | CR Sunstein  |\n| 6   |  SH Snyder   |   LA Zadeh    | JE Stiglitz |   R Langer   |\n| 7   |    Y Sun     |  A Shleifer   | S Weinberg  |   E Witten   |\n| 8   |   S Freud    |  P Bourdieu   |  N Chomsky  |  P Krugman   |\n| 9   | B Vogelstein |   N Chomsky   |  HA Simon   |  P Bourdieu  |\n| 10  |   S Kumar    |  T Maniatis   |  RA Posner  | JL Goldstein |\n\n<a href=\"#pone-0043235-t002\" data-ref-type=\"table\">Table 2<\/a> shows an example ranking of top authors in a particular discipline (\"computer science, artificial intelligence\") by the same impact measures. Once again we observe that the -index ranks higher authors of books and other very highly cited publications, such as D.E. Knuth and S. Haykin. D. Dubois and L.A. Zadeh have many top cited single-authored articles and as a result are highly ranked by . Finally, in the ranking by we see that some authors with high are replaced by other well-known researchers in artificial intelligence, such as J. Kleinberg, O. Faugeras, and A. Halevy. The metric is discussed in more detail next.\n\n10.1371\/journal.pone.0043235.t002\n\n###### Top authors tagged with \"computer science, artificial intelligence\" according to various impact measures (as of January 2012).\n\n<img src=\"pone.0043235.t002\" id=\"pone-0043235-t002-2\" \/>\n\n|     |             |             |             |             |\n|:----|:-----------:|:-----------:|:-----------:|:-----------:|\n| 1   | DE Goldberg |  LA Zadeh   |  LA Zadeh   |  LA Zadeh   |\n| 2   |   S Thrun   | DE Goldberg |  DE Knuth   | NR Jennings |\n| 3   | NR Jennings | AL Barabasi | DE Goldberg | AL Barabasi |\n| 4   |  D Dubois   |  DE Knuth   |  D Dubois   | A Zisserman |\n| 5   |  LA Zadeh   |  S Haykin   |   S Thrun   | I Horrocks  |\n| 6   | A Zisserman | NR Jennings | NR Jennings |  J Peters   |\n| 7   | AL Barabasi |  G Salton   |   H Prade   | J Kleinberg |\n| 8   |   H Prade   |  M Dorigo   | JY Halpern  | O Faugeras  |\n| 9   |  DE Knuth   | A Zisserman | A Zisserman |   S Thrun   |\n| 10  | I Horrocks  |  D Dubois   |  MY Vardi   |  A Halevy   |\n\n## Impact Analysis and Universality\n\nThe universal -index, which we refer to as , was proposed by Radicchi *et al.* \\[9\\]. For each discipline tag and year, we maintain statistics about the average number of papers written by authors in that discipline and in that year, and about the average number of citations to papers written in that discipline and in that year. When we receive a query about an author in a certain discipline, we update these statistics. Following Radicchi *et al.*, we rescale the number of citations of each paper. This is done by dividing by (for the discipline of the author and the year of the paper). Papers are then ranked by the rescaled number of citations . Similarly, we divide the resulting rank of each paper by (again for the given discipline and year). The universal value for the author is defined as the maximum rescaled rank such that each of the top articles have at least rescaled citations each.\n\nNote that an author tagged with several disciplines will have multiple values, one per discipline. Since different disciplines have different citation patterns, an author should only pay attention to values in disciplines that s\/he knows to be appropriate.\n\nSince the discipline\/year statistics depend on the annotations we collect from queries, they are subject to noise and may take a while to converge. Once the statistics are reliable, one should in theory be able to compare the impact of authors in different disciplines. Given the dependence of on and , we have looked at the convergence and stability properties of these rescaling factors \\[12\\]. The relative change in the values of and for all tags (in a particular year) was close to zero, suggesting that the rescaling factors converge quickly and are quite stable.\n\nWe have already shown in <a href=\"#pone-0043235-t001\" data-ref-type=\"table\">Tables 1<\/a> and <a href=\"#pone-0043235-t002\" data-ref-type=\"table\">2<\/a> how identifies top authors in their respective fields. To show how also allows to compare the impact of authors across disciplines, let us consider the example of two authors, S.H. Snyder in neurosciences and H. Garcia-Molina in computer science. Their impact cannot be compared based on the -index as the two disciplines have different numbers of authors, publications, and citation patterns. Indeed, Snyder has (global rank 6) while Garcia-Molina has (global rank 60), suggesting that the former has a greater impact than the latter in absolute terms. However, when we compare the two based on the universal -index, we find them in an effective tie for global rank 11 (). Indeed, both authors are equally successful (ranked first) in their respective fields.\n\nFor a quantitative evaluation of the universality of and metrics, we follow the approach from Radicchi *et al.* to observe whether top authors from different areas are equally represented. To this end, let us compare the distribution of top authors based on the three JCR indices. We have approximately 20,000 reliable author-discipline annotations in *science* disciplines, 6,300 in the *social sciences*, and 1,750 in *arts & humanities*. Given such an unbalanced set, we sample 1,000 random authors from each set of disciplines. The authors in the sample are ranked by and metrics, and the top 100 are selected based on each metric. This process is repeated 1,000 times. The resulting distributions of category tags for the top 100 authors are shown in <a href=\"#pone-0043235-g010\" data-ref-type=\"fig\">Figure 10<\/a>. The distribution based on displays a clear bias toward science disciplines (45%), followed by social sciences (33%) and the least represented arts & humanities (22%). The metric is not as biased, preserving a much better balance (36%, 28%, 36% respectively). This supports the universality claim, suggesting that the impact of authors in different disciplines can be compared in a more meaningful way using the metric.\n\nAnother way to verify that is a more universal citation impact metric than is to look at the distribution of impact values across disciplines. While we expect different authors within a discipline to have different impact, a universal metric should make different disciplines comparable. Let us therefore consider the average values of and across the top 250 disciplines based on number of authors. A more universal measure should have a smaller variance across disciplines. However, the values of tend to be smaller than those of , therefore to compare the variances let us define the normalized of discipline as , where is the average of authors in discipline and is the average across all disciplines. The normalized , , is defined analogously. The respective variances are and . A Levene test of equality of variances reveals that the difference is very significant (). We conclude that makes it more fair to compare impact in different disciplines. An illustration is presented in <a href=\"#pone-0043235-t003\" data-ref-type=\"table\">Table 3<\/a>. The top disciplines based on average are dominated by life sciences, with a few exceptions such as theoretical physics. The life sciences tend to have more authors who publish a lot compared to other disciplines. Top tags based on average have greater diversity, ranging from biology to geosciences, materials science, and atmospheric sciences.\n\n10.1371\/journal.pone.0043235.t003\n\n###### Top disciplines based on and values, defined in the text as averages across all authors tagged with discipline .\n\n<img src=\"pone.0043235.t003\" id=\"pone-0043235-t003-3\" \/>\n\n|  | Discipline |  | Discipline |  |\n|:---|:--:|:--:|:--:|:--:|\n| 1 | hematology | 35 | ophthalmology | 1.93 |\n| 2 | obesity | 34 | geosciences, multidisciplinary | 1.75 |\n| 3 | physics, theoretical | 33 | neuroimaging | 1.72 |\n| 4 | gastroenterology & hepatology | 32 | materials science, multidisciplinary | 1.71 |\n| 5 | immunology | 31 | clinical neurology | 1.70 |\n| 6 | biostatistics | 30 | meteorology & atmospheric sciences | 1.68 |\n| 7 | medicine | 29 | geochemistry & geophysics | 1.63 |\n| 8 | nutrition & dietetics | 29 | radiology, nuclear medicine & medical imaging | 1.62 |\n| 9 | medicine, research & experimental | 29 | pathology | 1.60 |\n| 10 | neuroimaging | 27 | psychology, experimental | 1.59 |\n\nWe considered disciplines with at least 20 authors (as of April 2012).\n\n# Conclusions\n\n## Summary\n\nWe introduced a Web Science approach to gather scholarly metadata. We presented Scholarometer, a social Web tool that leverages crowdsourced scholarly annotations with many potential applications, such as bibliographic data management, citation analysis, science mapping, and scientific trend tracking. We discussed a browser-based architecture and implementation for the Scholarometer tool, affording platform and source independence while complying with the usage policy of Google Scholar and coping with the noisy nature of the crowdsourced data. We outlined disambiguation algorithms to deal with the challenge of common author names, by incorporating a classifier into the query manager.\n\nWe found evidence that the crowdsourcing approach can yield a coherent emergent classification of scholarly output. The annotation and citation metadata that we collect is shared with the research community via an API and linked open data. By combining a visualization of disciplinary networks with lists of high-impact authors into an interactive application, the Scholarometer system can be a powerful resource to explore relevant scholars and disciplines. Interactive author networks can help one identify influential authors in one's discipline or in interdisciplinary or emerging areas.\n\nWe outlined several citation-based impact metrics that are computed by the Scholarometer tool, including the first implementation of the universal -index. We also found that the statistics collected by our social tool make the metric more appropriate compared to the original -index for comparing the impact of authors across disciplinary boundaries.\n\n## Future Work\n\nOf course, as the crowdsourced database grows, our data for each discipline will become more representative and our measures more reliable.\n\nAdditional metrics can be implemented, for instance universal ones based on percentiles \\[36\\] and the successive -index for groups \\[37\\], which could be used to rank department-like units. We also plan to compute *temporal metrics*, i.e., to track an author's impact backward in time. This would allow to compare authors at the same stage in their career, even if they are not contemporary.\n\nStudies of co-authorship patterns in conjunction with citation patterns might help further characterize the structure and evolution of disciplines. Moreover, by tracking the spikes in the popularity of disciplines, we plan to explore trends in scientific fields, in particular how disciplines emerge and die over time.\n\nPart of the work presented in this paper was performed while Xiaoling Sun and Lino Possamai were visiting the Center for Complex Networks and Systems Research (cnets.indiana.edu) at the Indiana University School of Informatics and Computing. We are grateful to Geoffrey Fox, Alessandro Flammini, Santo Fortunato, Hongfei Lin, Filippo Radicchi, Jim Pitman, Ron Larsen, Johan Bollen, Stasa Milojevic, two anonymous referees, and all the members of the Networks and agents Network (cnets.indiana.edu\/groups\/nan) for helpful suggestions and discussions.\n\n# References\n\n[^1]: **Competing Interests:** The authors have declared that no competing interests exist.\n\n[^2]: Conceived and designed the experiments: DH JK XS FM. Performed the experiments: JK DH XS LP MJ SP. Analyzed the data: JK DH XS FM. Contributed reagents\/materials\/analysis tools: JK DH XS LP MJ SP FM. Wrote the paper: JK XS FM.","meta":{"dup_signals":{"dup_doc_count":148,"dup_dump_count":46,"dup_details":{"curated_sources":2,"2022-49":1,"2022-40":1,"2022-05":2,"2021-31":1,"2020-50":1,"2020-45":1,"2019-51":1,"2019-09":1,"2018-47":2,"2018-22":1,"2018-13":1,"2017-43":2,"2017-30":1,"2017-26":2,"2017-22":7,"2017-17":7,"2017-09":2,"2017-04":2,"2016-40":1,"2016-36":1,"2016-30":4,"2016-26":1,"2016-18":1,"2016-07":4,"2015-48":9,"2015-40":3,"2015-35":6,"2015-32":4,"2015-27":5,"2015-22":4,"2015-14":5,"2014-52":6,"2014-49":3,"2014-42":3,"2014-41":6,"2014-35":11,"2014-23":7,"2014-15":5,"2023-14":1,"2017-13":2,"2015-18":4,"2015-11":4,"2015-06":2,"2014-10":4,"2013-48":3,"2024-22":1}},"file":"PMC3440403"},"subset":"pubmed_central"}
{"text":"abstract: Magnetic resonance spectroscopy offers a broad range of noninvasive analytical methods for investigating metabolism in vivo. Of these, the magnetization-transfer (MT) techniques permit the estimation of the unidirectional fluxes associated with metabolic exchange reactions. Phosphorus (^31^P) MT measurements can be used to examine the bioenergetic reactions of the creatine-kinase system and the ATP synthesis\/hydrolysis cycle. Observations from our group and others suggest that the inorganic phosphate (P~i~) \u2192 ATP flux in skeletal muscle may be modulated by certain conditions, including aging, insulin resistance, and diabetes, and may reflect inherent alterations in mitochondrial metabolism. However, such effects on the P~i~ \u2192 ATP flux are not universally observed under conditions in which mitochondrial function, assessed by other techniques, is impaired, and recent articles have raised concerns about the absolute magnitude of the measured reaction rates. As the application of ^31^P-MT techniques becomes more widespread, this article reviews the methodology and outlines our experience with its implementation in a variety of models in vivo. Also discussed are potential limitations of the technique, complementary methods for assessing oxidative metabolism, and whether the P~i~ \u2192 ATP flux is a viable biomarker of metabolic function in vivo*.*\nauthor: Douglas E. Befroy; Douglas L. Rothman; Kitt Falk Petersen; Gerald I. ShulmanCorresponding author: Douglas E. Befroy, <firstname.lastname@example.com>.\ndate: 2012-11\nreferences:\ntitle: ^31^P-Magnetization Transfer Magnetic Resonance Spectroscopy Measurements of In Vivo Metabolism\n\nBecause of its versatility, magnetic resonance spectroscopy (MRS) has emerged as a powerful modality for studying tissue-specific intracellular metabolism in vivo. Several metabolically relevant nuclei, including hydrogen (^1^H), carbon (^13^C), and phosphorus (^31^P), are magnetic resonance (MR)-visible, permitting the detection of a range of metabolites in a variety of tissues. The noninvasive nature of the technique allows repeated measurements from the same location, so that the response to an acute intervention (e.g., exercise, insulin stimulation) or longer-term treatment (e.g., diet manipulation, drug therapy) can be assessed. Using more sophisticated MRS techniques, discrete reaction rates can also be estimated using ^13^C-MRS to monitor the metabolic fate of ^13^C-labeled substrates or by using magnetization-transfer (MT) techniques to monitor the unidirectional fluxes associated with metabolic exchange reactions. Of these, ^31^P-MT methods offer the ability to examine the key bioenergetic reactions of the creatine kinase (CK) equilibrium (ATP \u21c6 phosphocreatine \\[PCr\\] exchange) and the ATP synthesis\/hydrolysis cycle (ATP \u21c6 inorganic phosphate \\[P~i~\\] exchange). Particular interest has been generated by the observations from our group and others that unidirectional P~i~ \u2192 ATP flux in muscle may be modulated in certain disease states, including insulin resistance and diabetes, that may reflect alterations in mitochondrial metabolism. However, disparate results have been reported by different groups, and recent articles have raised concerns about the absolute magnitude of the measured reaction rates. Given that many clinical MR scanners now have the capability of performing ^31^P-MT experiments and the implementation of the method is becoming more widespread, we would like to offer our perspective on the application of the technique in vivo. In this article, we review our experience using the ^31^P-MT technique to measure P~i~ \u2192 ATP flux in a variety of models, describe limitations of the methodology, assess alternative and complementary methods of estimating cellular bioenergetics, and discuss the validity of using the technique as a biomarker of metabolic function in vivo.\n\n# THE MT TECHNIQUE\n\nThe MT techniques work on the principle that if the MR signal of an exchanging metabolite is perturbed, the transfer of this effect to its exchange partner can be detected, while leaving the underlying chemical equilibrium unaffected. The perturbation may either eliminate (saturate) or invert the signal of the initial metabolite of interest and is accomplished by applying a frequency-selective saturation or inversion pulse prior to acquiring the MR spectrum. These approaches give rise to the saturation-transfer (ST) and inversion-transfer techniques, respectively. For in vivo studies, the ST technique is typically used because of ease of calibration of an effective frequency-selective saturation pulse. On in vivo MR systems, particularly those used for human studies, the accurate, short-duration inversion pulses required for the inversion-transfer technique are difficult to implement due to inherent limitations in radiofrequency field homogeneity and the high-power requirements of such pulses. The ST effect from the terminal (\u03b3) phosphate group of ATP to PCr due to the CK equilibrium and to P~i~ due to ATP synthesis\/hydrolysis in skeletal muscle is shown in the ^31^P spectra in <a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<\/a>. The magnitude of the effect (e.g., \u0394PCr or \u0394P~i~) is proportional to the kinetics of exchange. Underlying assumptions for the application of the technique are that the metabolites of interest have distinct peaks in the MR spectrum, that exchange is at steady state (i.e., that the metabolite concentrations do not change during the experiment), and that exchange is rapid with respect to the lifetime of the MR signal.\n\nThe ATP synthesis\/hydrolysis cycle is a phosphate-exchange reaction under the chemical equilibrium shown in Eq. <a href=\"#e1\" data-ref-type=\"disp-formula\">1<\/a>, where *k*~f~ and *k*~r~ are the forward and reverse rate constants. From simple reaction kinetics, the forward reaction (P~i~ \u2192 ATP flux) rate is equivalent to the forward rate constant multiplied by the concentrations of ADP and P~i~ (Eq. <a href=\"#e2\" data-ref-type=\"disp-formula\">2<\/a>). At steady state, the tissue concentration of ADP remains constant, and this relationship can be simplified to generate a pseudo\u2013first-order rate constant of exchange for P~i~ (*k*\u2032~f~) that incorporates the effects of the ADP concentration (Eq. <a href=\"#e3\" data-ref-type=\"disp-formula\">3<\/a>). During the ST experiment (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<\/a>), \u03b3ATP saturation causes a reduction in the observed P~i~ signal (M\u2032) from its equilibrium value (M~0~) due to the effects of phosphate exchange (i.e., *k*\u2032~f~) and the influence of the effective MR relaxation time (T~1~\u2032) of P~i~ (Eq. <a href=\"#e4\" data-ref-type=\"disp-formula\">4<\/a>).\n\nEquation <a href=\"#e4\" data-ref-type=\"disp-formula\">4<\/a> requires that the following conditions are met: that there is full equilibration between the sites of ATP utilization (i.e., in the cytosol) and ATP production (mitochondria and cytosol); that across this system, ATP production \u2261 utilization; that the saturation pulse fully nulls the \u03b3ATP signal and has minimal off-resonance effects; and that the detected compartment (i.e., cytosolic P~i~) is fully MR-visible and not participating in competing reactions. A more detailed description of the effects of chemical exchange on the observed MR signal and the derivation of these equations is beyond the scope of this article but can be found in the classic articles describing MT from McConnell (1) and Forsen and Hoffman (2).\n\nFrom a practical perspective, the estimation of P~i~ \u2192 ATP flux therefore requires the determination of three parameters: *1*) calculation of the ST effect (M\u2032\/M~0~) from \u03b3ATP to P~i~; *2*) calibration of the T~1~ of P~i~ under conditions of \u03b3-ATP saturation (T~1~\u2032); and *3*) determination of the intracellular P~i~ concentration. The unidirectional (P~i~ \u2192 ATP) flux that contributes to the exchange can then be calculated according to Eq. <a href=\"#e3\" data-ref-type=\"disp-formula\">3<\/a>. To ensure that a study is fully described, we recommend that all of these parameters are reported when publishing data acquired using the ^31^P-ST technique. Two experimental approaches are possible: in the progressive saturation experiment, M\u2032 and T~1~\u2032 are determined simultaneously by acquiring a series of spectra in which the duration of the saturation pulse is gradually lengthened, and the extent of the ST effect increases until steady state is achieved. Alternatively, M\u2032 can be determined from a single spectrum in which the duration of the saturation pulse is sufficient (i.e., several times T~1~\u2032) to establish the new steady-state MR equilibrium, with T~1~\u2032 determined by a separate experiment. An inversion-recovery calibration with \u03b3ATP saturation during the repetition time (T~R~) and throughout the inversion delay will typically be performed to estimate T~1~\u2032. The intracellular concentration of P~i~ can be determined from a fully relaxed ^31^P spectrum (where T~R~ \\>\\> T~1~, and all metabolites are detected with equal sensitivity) with reference to ATP as an internal standard. Imperfections in the frequency specificity of the saturation pulse can lead to off-resonance effects modulating the intensity of the peak of interest. Therefore, M~0~ is usually determined under control saturation conditions with the saturation pulse irradiating a downfield frequency from P~i~ that mirrors the separation between the \u03b3ATP and P~i~ peaks. These effects can be significant, particularly at lower MR field strengths in which the frequency separation between the exchanging metabolites is relatively small (3,4). The performance and off-resonance effects of a saturation pulse are also influenced by its duration and therefore may not be consistent between the variable saturation pulse lengths of the progressive saturation experiment. We have found that this tends to lead to an underestimate of T~1~\u2032 and prefer the steady-state style experiment, in which pulse performance is equivalent among all scans.\n\n# PRACTICAL CONSIDERATIONS FOR THE IMPLEMENTATION OF ^31^P-ST IN VIVO\n\nBecause rates of P~i~ \u2192 ATP flux determined using the ^31^P-ST experiment incorporate the measurement of three independent parameters (M\u2032\/M~0,~ T~1~\u2032, and \\[P~i~\\]), it is important to estimate these parameters as accurately as possible. High signal\/noise ^31^P spectra are required, particularly for the determination of M\u2032\/M~0~, in which a relatively small decrement (\u223c20%) in the magnitude of the P~i~ resonance, present in relatively low concentration (\u223c2 mmol\/L) compared with the other ^31^P metabolites, is detected. Therefore, a stable experimental setup is required that minimizes or eliminates movement of the region of interest. Inadvertent contractions may perturb the maintenance of metabolic steady state in muscle and should be prevented. For animal experiments, these conditions are best satisfied by performing the ST experiment under light anesthesia. To ensure precision in the estimate of M\u2032\/ M~0~ in our studies, we repeat the measurement multiple times to confirm experimental stability.\n\nThe duration of the experiment will be dependent on the tissue of interest, species, and field strength of the MR system used. Our ^31^P-ST experiments in human muscle take \u223c60 min on a 4 Tesla system, whereas 4\u20136 h are required for an ST experiment in mouse hindlimb at 9.4 Tesla due to the relatively small mass of muscle tissue available. The overall length of the ST experiment is dominated by the estimation of T~1~\u2032, which requires the acquisition of several spectra to characterize the evolution of the MR signal. This calibration can be shortened by obtaining fewer points, although this will also decrease the accuracy of the calibration. Faster methods are available that allow the estimation of T~1~ by varying the extent to which the MR signal is excited (variable flip-angle methods) (5); however, the implementation of these methods in vivo can be problematic because very accurate excitation pulses are required for precise estimation of the T~1~ relaxation time.\n\n# BIOLOGICAL APPLICATIONS OF ^31^P-ST TO MEASURE P~i~ \u2192 ATP FLUX\n\nThe biological feasibility of using the ST technique to assess P~i~ \u2192 ATP flux was initially demonstrated in cell suspensions of *Escherichia coli* (6). Subsequent experiments in yeast cell suspensions (7) and implementation of the technique in the isolated perfused heart (8,9) and perfused liver (10) preparations demonstrated the applicability of the technique in a variety of in vitro systems. Translation of the methodology to in vivo applications was first performed in rat brain (11) followed by studies in the exercising rat hindlimb in vivo (12) and semi-invasive studies of dog (13) and lamb heart (14). We sought to add in vivo estimates of P~i~ \u2192 ATP flux by ^31^P-ST to complement ^13^C-MRS methods we were developing to assess rates of substrate oxidation via the tricarboxylic acid cycle (TCA cycle flux) in resting skeletal muscle. We initially performed proof-of-principle studies in rat hindlimb (15,16) followed by studies in human muscle to validate the methods (17) and examine how these metabolic fluxes were modulated by aging (18), insulin resistance (19,20), and endurance exercise training (21). We have also taken advantage of transgenic mouse models to examine how overt genetic modulations of mitochondrial content (22) and metabolism may influence the flux measured by the ^31^P-ST technique (23; D.E.B. and G.I.S., unpublished observations). More recently, there has been a rapid expansion in the number of studies using the ^31^P-ST technique in vivo, with applications examining the effects of insulin stimulation (24,25), insulin resistance (26\u201328), diabetes (29,30), MELAS (mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes) syndrome (31), and acromegaly (32) on resting muscle P~i~ \u2192 ATP flux. Pilot studies have been reported in human liver (33,34), and validation experiments have been performed in human (35,36) and rat (37) brain.\n\n# LIMITATIONS OF THE ^31^P-ST TECHNIQUE\n\nThe early studies examining the applications of the ^31^P-ST technique in vitro observed that under certain conditions, the measured P~i~ \u2192 ATP flux was significantly greater than that predicted from oxygen consumption measurements, assuming the theoretical phosphate-to-oxygen (P:O) ratio for mitochondrial oxidative phosphorylation of \u223c3. This phenomenon was demonstrated not only in yeast (7,38) but also in isolated perfused heart (39) and liver (10). The study by Alger et al. (7) noted that an appreciable P~i~ \u2192 ATP flux could be detected in yeast under anaerobic conditions that was insensitive to the mitochondrial inhibitor oligomycin, suggesting that ATP synthesis via glycolysis was also detected by ^31^P-ST and may augment the flux observed under aerobic conditions. Studies using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoglycerate kinase (PGK) in solution at concentrations and conditions designed to mimic those found in the Langendorff perfused heart confirmed that these enzymes could catalyze a significant P~i~ \u2192 ATP flux (40). Subsequent experiments using iodoacetate to inhibit ATP generation via the GAPDH\/PGK enzyme couple demonstrated that these enzymes were contributing significantly to the measured P~i~ \u2192 ATP flux rates in these systems (10,38,39) potentially via catalyzing P~i~ \u21c6 ATP exchange. With the GAPDH\/PGK contribution eliminated, the apparent P:O ratio was normalized to \u223c2 to 3, below the theoretical limit (38,39). It has also been demonstrated using radiolabeled phosphate that near equilibrium P~i~ \u21c6 ATP exchange can occur within isolated mitochondria at low respiration rates (41), either at the level of the ATP synthase complex or the adenine-nucleotide translocase (ANT). At higher respiration rates, the reverse flux (ATP \u2192 P~i~) was significantly decreased, leading to a switch from reversible P~i~ \u21c6 ATP exchange to unidirectional ATP production by the mitochondrion. This observation is consistent with the normalization of P:O at higher workloads in heart (14,39) and exercising rat muscle in vivo (12). Overexpression of the F~1~F~0~ ATP synthase in *E. coli* had no effect on the P~i~ \u2192 ATP flux measured by ^31^P-ST (42) under aerobic conditions. In contrast, overexpression of the ANT in yeast oxidizing glucose led to a significant increase in P~i~ \u2192 ATP flux (43), indicating a role for the ANT in catalyzing P~i~ \u21c6 ATP exchange.\n\n# INFLUENCE OF NEAR-EQUILIBRIUM P~i~ \u21c6 ATP EXCHANGE ON IN VIVO STUDIES USING ^31^P-ST\n\nRecent articles have expressed concern over the contribution of unidentified P~i~ \u21c6 ATP exchange mechanisms to absolute rates of P~i~ \u2192 ATP flux measured in vivo using ^31^P-ST, highlighting the discrepancy between estimated P:O ratios and querying whether this unidirectional flux reflects mitochondrial activity (44\u201346). In addition to putative contributions from exchange via GAPDH\/PGK (45) and at the mitochondrion, Balaban and Koretsky (46) suggest that small metabolite pools, undetectable in the ^31^P-MR spectrum due to their low concentration, could be irradiated during the ST experiment, undergo exchange with P~i~, and contribute to the observed MT effect. These concerns are certainly valid in terms of deconvoluting the specific mechanisms that contribute to the P~i~ \u2192 ATP flux and quantifying absolute rates of net (i.e., forward flux minus reverse flux) mitochondrial ATP synthesis.\n\nWhen considering the extent to which glycolytic ATP production or GAPDH\/PGK exchange may contribute to the observed rates of P~i~ \u2192 ATP flux measured by the ^31^P-ST experiment in vivo, it is worth noting that observations from these in vitro studies may not translate directly to those in an intact organisms due to several factors. Firstly, absolute rates of P~i~ \u2192 ATP flux measured in vitro in both cell preparations and isolated organs are significantly higher than those measured in vivo (<a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>). These in vitro systems are often studied under conditions in which glucose is the sole or primary substrate, which may enhance the contribution of glycolytic metabolism to the measured P~i~ \u2192 ATP flux and play a role in the erroneously high P:O ratios observed in these preparations. Furthermore, there are substantial differences in the expression of glycolytic enzymes between tissues and different species. The expression\/activity levels for PGK, which exhibits a high flux control coefficient for ATP \u21c6 P~i~ exchange (47), can vary by an order of magnitude among human tissues and between the same tissue in humans and rodents (<a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a>). Resting skeletal muscle, for example, derives most of its energy from fat oxidation rather than glycolysis, and this is reflected in the relative PGK activity where heart <u>\u2248<\/u> brain \\>\\> liver \\> muscle. PGK activity in most human tissues is significantly lower than in the counterpart tissue in rats and substantially lower than that in mice, reflecting the greater reliance on glycolytic metabolism in rodents. In brain, despite the high PGK activity and significant role for glycolysis, close to theoretical P:O ratios have been reported (35,37). The extrapolation of metabolic mechanisms between in vitro and in vivo preparations, from one tissue to another or between different species, is therefore not straightforward, and we urge caution when interpreting the ramifications of an observation in a particular preparation.\n\nAbsolute P~i~ \u2192 ATP flux rates reported in the literature for different in vitro and in vivo preparations\n\n![](2669tbl1)\n\nProtein expression\/activity (arbitrary units) of PGK1 isoform between different tissues and species\n\n![](2669tbl2)\n\nIn the following section, we will describe our experiences with the ^31^P-ST technique in a variety of systems, which along with studies from other laboratories, support the notion that P~i~ \u2192 ATP flux responds to perturbations in oxidative metabolism and, when supported by complementary methods, can be used as a biomarker of in vivo mitochondrial activity. Detailed descriptions of the experimental procedures we have used can be found in the cited literature. It is worth noting that although different MR systems were used, we implemented comparable ^31^P-ST protocols for all of our human and rodent studies. Saturation-pulse lengths, sequence repetition times, and the delays for the inversion-recovery calibration were optimized according to the inherent T~1~\u2032 of P~i~ on each MR system, and appropriate coil geometries were chosen for the region of interest.\n\nThe most compelling evidence we have obtained that demonstrates that the P~i~ \u2192 ATP flux measured by ^31^P-ST responds to perturbations in skeletal muscle oxidative function is from transgenic mouse models with direct manipulation of genes that modulate mitochondrial content and metabolism. Mice with muscle-specific overexpression of peroxisome proliferator\u2013activated receptor \u03b3 (PPAR\u03b3) coactivator 1\u03b1 (PGC1\u03b1), a regulator of oxidative metabolism, exhibit increased mitochondrial density and enhanced expression and protein content of genes involved in oxidative phosphorylation. P~i~ \u2192 ATP flux in the muscle of these mice was increased \u223c40% (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<\/a>), and a concomitant increase in fatty-acid oxidation was detected in isolated soleus muscle strips (22). It is interesting to note that short-term (3 weeks) high-fat feeding enhanced muscle P~i~ \u2192 ATP flux in both wild-type and PGC1\u03b1 overexpressing mice (22) (<a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<\/a>). This phenomenon is supported by data obtained from rats fed a high-fat diet for 2.5 weeks in which the in vivo oxidative capacity of muscle (assessed using the ^31^P-MRS PCr recovery method) was increased as well as mitochondrial DNA content and the oxidative capacity of isolated mitochondria (48), presumably to increase the ability to oxidize the excess of available lipid. Significantly, this enhancement in mitochondrial capacity had dissipated after 25 weeks of high-fat feeding, despite a sustained elevation in mitochondrial DNA, which corresponds with the decreased muscle P~i~ \u2192 ATP flux observed in rats fed a high-fat diet for longer (10-week) duration (49). Uncoupling protein 3 (UCP3) is a mitochondrial membrane protein found in skeletal muscle that has high homology with UCP1, which is found in brown adipose tissue and mediates thermogenesis by dissipating the H^+^ electrochemical gradient across the inner mitochondrial membrane. We observed that in vivo skeletal muscle P~i~ \u2192 ATP flux was increased in UCP3 knockout mice (23); conversely, muscle-specific overexpression of UCP3 led to a decreased flux (D.E.B. and G.I.S., unpublished observations) \\[<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<\/a>, data were acquired using the same MR protocols as described in Choi et al. (22)\\]. These observations are supported by in vitro data. The ATP\/ADP ratio was increased in mitochondria isolated from the muscle of UCP3-knockout mice, and State III (ADP-stimulated) respiration rates were increased (50). Palmitate and pyruvate oxidation have been observed to be elevated in soleus muscle strips isolated from UCP3 overexpressing mice (51) accompanied by an increased in State IV (nonstimulated) respiration rates in mitochondria (52) and whole-body energy expenditure (53).\n\nTo more fully characterize mitochondrial oxidative function in skeletal muscle in vivo, we use estimates of P~i~ \u2192 ATP flux assessed by ^31^P-ST in conjunction with direct measurements of substrate oxidation via the TCA cycle using dynamic ^13^C-MRS. A thorough analysis of this technique is beyond the scope of this article and can be found in the literature (20,54), but a brief description is warranted. Rates of TCA cycle flux in muscle can be estimated by infusing ^13^C-labeled acetate and using ^13^C-MRS to monitor ^13^C-label incorporation into the muscle glutamate pool, which acts as a surrogate for the intermediates of the TCA cycle. Kinetic analysis of the time courses of ^13^C glutamate enrichment can be accomplished using computer-modeling techniques to fit the data to a metabolic model of the TCA cycle, and rates of acetate oxidation and total TCA cycle flux can be estimated. These studies use pharmacy-prepared, sterile infusates of acetate, a naturally occurring endogenous substrate, that have been positionally enriched with the MR-visible stable isotope of carbon (^13^C). Concentrations of plasma acetate during the infusion remain relatively low (\u223c1 mmol\/L) to avoid mass action effects of the substrate that could otherwise influence metabolism. As such, these studies can be accomplished under conditions that minimize the perturbation of resting muscle metabolism, are minimally invasive, and very safe.\n\nWe found that both P~i~ \u2192 ATP flux, measured by ^31^P-ST, and TCA cycle flux, measured by ^13^C-MRS, were both reduced by \u223c40% in the calf muscles of healthy, lean, elderly individuals (\u223c70 years of age) with respect to healthy, young (\u223c27 years), control subjects who were matched for BMI and physical activity (<a href=\"#F4\" data-ref-type=\"fig\">Fig. 4<\/a>) (18). Similarly, using the same ^13^C- and ^31^P-MRS methods, both TCA cycle and P~i~ \u2192 ATP flux were reduced by \u223c30% in healthy, young, lean, insulin-resistant (IR) offspring of type 2 diabetic patients (IR offspring) compared with age-BMI-activity\u2013matched insulin-sensitive (IS) control subjects (IS subjects) (19,20) (<a href=\"#F5\" data-ref-type=\"fig\">Fig. 5<\/a>). In vitro analysis of muscle biopsy samples from a similar cohort of IR offspring (55) revealed a comparable \u223c35% reduction in mitochondrial density, as assessed by electron microscopy, as well as decreased content of proteins associated with oxidative phosphorylation (cytochrome *c* oxidase-1, succinate dehydrogenase, and pyruvate dehydrogenase) compared with matched IS subjects. This reduction in mitochondrial content and function in the young, lean IR offspring was consistent with a reduction in the ratio of slow-twitch (oxidative) muscle fibers to fast-twitch (glycolytic) fibers (19).\n\nWe have also examined muscle-specific P~i~ \u2192 ATP flux in different muscle groups of the calf using a localized version of the ^31^P-ST method to restrict the measurement to either the gastrocnemius or soleus muscle compartments (<a href=\"#F6\" data-ref-type=\"fig\">Fig. 6<\/a>). Muscle-specific P~i~ \u2192 ATP flux, measured in healthy volunteers, was significantly faster in the soleus muscle compared with the gastrocnemius (*V*~Pi\\ \u2192\\ ATP~ = 8.12 \u00b1 0.86 vs. 4.97 \u00b1 1.08 mmol\/g\/min) (56). This disparity in P~i~ \u2192 ATP flux is consistent with the differences in mitochondrial number, oxidative enzyme content, and oxygen consumption expected for the more oxidative phenotype of the soleus than the gastrocnemius (57\u201361).\n\nWe have used the combination of ^31^P-ST assessment of P~i~ \u2192 ATP flux and ^13^C-MRS measurement of TCA cycle flux to noninvasively assess energetic efficiency in skeletal muscle in both humans and rodents (16,17). Mitochondrial uncoupling stimulated by acute 2,4-dinitrophenol (DNP) or chronic thyroid hormone (T3) administration are known to lead to an increase in oxygen consumption (16,62\u201366). Consistent with this mechanism of action, DNP and T3 treatment in rats caused substantial increases in muscle TCA cycle flux, yet the P~i~ \u2192 ATP flux was unaffected, reflecting decreased mitochondrial efficiency (16). This observation was supported by a lack of effect of either T3 or DNP on the ATPase activity of isolated mitochondria or any detectable differences in muscle GAPDH\/PGK exchange rates assessed by the deuterated glucose technique. Similar decreases in mitochondrial energetic efficiency using the same in vivo ^13^C-\/^31^P-MRS methods have also been observed in human skeletal muscle in response to T3 treatment (17).\n\nThese data indicate that rates of resting muscle P~i~ \u2192 ATP flux and substrate oxidation via the TCA cycle determined using ^31^P- and ^13^C-MRS techniques appear to track qualitatively with modulations in mitochondrial content and the extent of mitochondrial uncoupling. Assuming that three molecules of O~2~ are ultimately consumed per C~2~ unit oxidized by the TCA cycle and that the P:O ratio is \u223c3 (these approximations hold whether the substrate source is fatty acids or glucose), rates of muscle P~i~ \u2192 ATP flux are estimated to be \u223c1.6 \u03bcmol\/g\/min. Therefore, absolute rates of ATP production measured using the ^31^P-ST technique (\u223c7.5 \u03bcmol\/g\/min) are still significantly higher than those calculated from TCA cycle activity. It is worth noting that this discrepancy in ATP production (\u223c4.7-fold) is far lower than that estimated from measurements of oxygen consumption by arterio\/venous (A\/V) difference, near infrared spectroscopy (NIRS), and ^15^O~2~ positron emission tomography (44). This is because rates of oxygen consumption calculated from TCA cycle flux data (\u223c0.29 \u03bcmol(O~2~)\/g\/min) exceed those calculated by NIRS and positron emission tomography by 3\u201310-fold and are at the upper limit of literature values reported using A\/V difference methods. We believe that phenotypic differences in the muscle group(s) examined using the different techniques contribute, in part, to these discrepancies in rates of O~2~ consumption and may be exacerbated by experimental considerations for each method. For example, A\/V difference methods tend to sample blood-oxygen concentration across an entire limb in which other tissues may contribute to the measured O~2~ consumption. Accurate estimates of muscle mass\/volume are also required to determination rates of O~2~ utilization. NIRS techniques sample from a relatively superficial region of interest that may not exhibit comparable rates of O~2~ consumption to the bulk of the tissue. We feel that the most relevant methodological comparison is between the TCA cycle flux and ^31^P-ST because both ^13^C and ^31^P data were acquired from comparable regions of interest in the calf muscles. As discussed above, we believe that the disparity in absolute rates of ATP production estimated by these methods in resting muscle is likely because of near equilibrium P~i~ \u21c6 ATP exchange at the level of the mitochondria, with only a minor contribution from exchange catalyzed by GAPDH\/PGK.\n\nData from other groups also support the notion that in vivo estimates of P~i~ \u2192 ATP flux correlate with tissue oxidative metabolism. Brindle et al. (12) examined the effects of electrically stimulated contractions on rat muscle using ^31^P-ST and observed a linear response of P~i~ \u2192 ATP flux to increases in muscle workload. An earlier study in perfused muscle established that muscle oxygen consumption is also proportional to workload (67). Comparing the data from these studies, despite P~i~ \u21c6 ATP exchange contributing to the measured unidirectional P~i~ \u2192 ATP flux at rest and leading to an overestimation in the P:O ratio in quiescent muscle, at higher workloads the P:O ratio trended toward the theoretical value. Although the increases in P~i~ \u2192 ATP flux and oxygen consumption due to muscle contraction were not directly proportional, the ^31^P-ST measurement appeared to track oxygen utilization and therefore respond to increases in mitochondrial activity. In the rat brain, by modulating the depth of anesthesia, P~i~ \u2192 ATP flux was found to be tightly correlated with the cerebral activity level and the extent of energy demand (37). Similarly, rates of P~i~ \u2192 ATP flux measured in the human visual cortex (35) and monkey brain (68) agree well with predicted values calculated from cerebral metabolic rates of glucose or oxygen consumption or from MRS measurements of TCA cycle flux.\n\n# METABOLIC IMPLICATIONS OF DIFFERENCES IN RESTING MUSCLE P~i~ \u2192 ATP FLUX\n\nIn contracting muscle, numerous studies have demonstrated that mitochondrial ATP production is likely to be demand-driven and regulated through substrate-level feedback via ADP and P~i~, both of which increase in response to contractile activity. The factors that regulate mitochondrial activity in resting muscle in vivo are less clear, in which any underlying metabolic perturbations are likely to be more subtle (and therefore difficult to detect experimentally). In this section, we discuss the metabolic implications that stem from our observations of reduced rates of basal P~i~ \u2192 ATP flux in the muscle of elderly individuals and IR offspring.\n\nUnder basal conditions, protein synthesis and the maintenance of ion homeostasis are likely to be the major cellular processes that consume ATP. There is evidence to suggest that rates of protein synthesis are decreased in the muscle of elderly subjects (69,70) and that pH and ion homeostasis may be disrupted (71,72). Although blunted increases in protein synthesis in response to insulin stimulation have been reported in individuals with type 2 diabetes (73), basal rates of muscle protein synthesis appear to be unaffected (74). Decreased content and activity of the Na^+^\/K^+^-ATPase, a transmembrane ion pump that maintains the cellular membrane homeostasis, has been reported in type 2 diabetic patients (75), obese IR individuals (76), and in response to high-fat feeding (77). The activity of this protein may contribute significantly to basal energy requirements in muscle as well as other tissues (78). These observations match with the decreases in TCA cycle and ATP flux that we observe in the muscle of elderly and IR offspring; however, the implication of cause and effect is unclear. Although a mechanism of demand-driven regulation of resting ATP flux is plausible, it is also feasible that protein synthesis may have been downregulated and\/or ion homeostasis disrupted due to the decreased availability of ATP.\n\nInterestingly, under resting conditions in which intracellular concentrations of both ADP and P~i~ are low, it has been suggested that the cellular redox potential (i.e., availability of NADH) may also exert respiratory control (79,80). Our observations of decreased P~i~ \u2192 ATP flux in the muscle of elderly and IR individuals may be a result of the decreased TCA cycle activity, which reduces the availability of NADH for oxidation by the electron transport chain.\n\nA recent study by Lim et al. (30) has suggested that the decreased rates of muscle ATP turnover observed in type 2 diabetic subjects may be due to reduced rates of glucose uptake and glycogen synthesis. We do not believe that such effects are likely to play a significant role in our studies of the elderly or IR offspring because glucose disposal in resting muscle under fasting conditions will be very low and of insufficient magnitude to exert an effect on oxidative rates.\n\n# COMPARISON OF ^31^P-ST WITH OTHER METHODS FOR ESTIMATING IN VIVO MITOCHONDRIAL METABOLISM IN SKELETAL MUSCLE\n\nAs described above, we have implemented ^31^P-ST measurements of P~i~ \u2192 ATP flux in conjunction with ^13^C-MRS estimates of TCA cycle flux to assess muscle mitochondrial metabolism in vivo. Other groups have complemented their ^31^P-ST measurements with dynamic ^31^P-MRS to estimate muscle mitochondrial oxidative capacity from the kinetics of PCr repletion following a bout of exercise, a method that has been validated in humans and rat models (81). This functional estimate of oxidative capacity has been shown to correlate well with whole-body maximal oxygen consumption (*<span class=\"smallcaps\">V<\/span>*<span class=\"smallcaps\">o<\/span>~2max~) and estimates of mitochondrial capacity, enzyme expression\/content and respiratory function of isolated mitochondria, determined in vitro using biopsy samples (82\u201385). Muscle oxidative capacity determined by PCr recovery has been demonstrated to be reduced in type 2 diabetic patients (86,87), although this finding is not universal (88,89). Interestingly, in a rat model of mitochondrial dysfunction, induced by chronic inhibition of complex 1 of the electron transport chain, hindlimb muscle oxidative capacity determined by PCr recovery was significantly decreased, whereas resting P~i~ \u2192 ATP flux was unaffected (90). Similarly, in human subjects with a genetic defect in insulin-receptor signaling, oxidative capacity of the vastus lateralis was decreased versus healthy controls, whereas resting P~i~ \u2192 ATP in the calf muscle was unchanged (26). However, it is unclear whether valid comparisons of the methods can be made in this study because each technique was performed in different muscle compartments where the metabolic phenotype is likely to differ significantly. The disagreement between findings with PCr recovery and ^31^P ST in these studies has been used to query the ST method and whether it reflects oxidative activity in skeletal muscle. However, an alternate interpretation, which we support, is that the two measurements assess different aspects of mitochondrial function: total muscle oxidative capacity versus resting energy production, that are not necessarily proportional due to intracellular regulation of mitochondrial function.\n\nAlthough the PCr recovery method seems to provide an excellent estimate of functional mitochondrial\/oxidative capacity, several factors concerning the effective application of the method should be considered for those unfamiliar with the technique. The kinetics of PCr repletion following exercise are usually characterized by fitting the recovery to a monoexponential and assuming the postexercise ATP generation is entirely oxidative (91). However, bi- or multiexponential behavior has been observed in response to high-intensity exercise. This response may be a consequence of tissue heterogeneity or differences in fiber recruitment during exercise, although a contribution of glycolytic ATP production has also been suggested (92,93). Intracellular acidification, typically encountered during high-intensity exercise protocols, appears to slow the rate of PCr recovery potentially due to direct effects on mitochondrial respiration (94,95), the use of ATP for H^+^ pumping to restore pH (84,96), or via modulating the CK equilibrium (97\u201399). The pH dependence of PCr recovery rates may also reflect selective recruitment of muscle fibers for low- versus high-intensity contractions (100).\n\nThe rate of muscle PCr depletion monitored by ^31^P-MRS during limb ischemia induced by circulatory occlusion has also been used as an estimate of resting ATP use. A comparison of the ^31^P-MRS methods has been performed in the calf muscles by Schmid et al. (101), who observed correlations among muscle P~i~ \u2192 ATP flux, the decline in PCr induced by cuff ischemia, and the time constant of PCr recovery (\u03c4~PCr~) following exercise protocol during ischemia. The correlation among these parameters would seem to suggest that each reflects oxidative activity; however, we urge caution over the interpretation of these data due to methodological uncertainties in this study. Reservations include the lack of control over end-exercise pH, which, as described above, has been demonstrated to modulate the kinetics of PCr recovery, and the use of a plantar flexion exercise protocol in combination with a relatively large diameter coil for ^31^P signal detection. Plantar flexion involves preferential activation of the gastrocnemius muscle groups, which will exhibit enhanced PCr depletion compared with the soleus and influence the observed PCr recovery kinetics. In contrast, the region of interest for the P~i~ \u2192 ATP flux and ischemic PCr utilization measurements performed in resting muscle will correspond to a weighted average of the soleus and gastrocnemius muscle groups. The relative contribution of deeper lying muscle groups in this study will be particularly exacerbated by the relatively large-diameter coil (10 cm) used for ^31^P signal detection. The use of a smaller-diameter ^31^P coil would act as a rudimentary method of signal localization by confining signal detection to the superficial muscle groups and ensure that the sampled volume was equivalent for each ^31^P technique. Finally, we also have concerns over whether ischemic conditions are a valid metabolic environment in which to assess resting ATP turnover.\n\nIn vitro analyses of muscle biopsy samples may also offer additional insight into mitochondrial metabolism. Mitochondrial density, mitochondrial DNA copy number, the expression and content of mitochondrial proteins, as well as the respiratory function of isolated mitochondria or skinned muscle fibers provide a theoretical estimate of the maximal mitochondrial capacity and have been used as indirect markers of mitochondrial function. As mentioned above, the functional estimate of mitochondrial metabolism determined using the PCr recovery technique correlates with many of these theoretical estimates (82\u201385), reflecting that these methods are all biomarkers of mitochondrial capacity rather than resting or intermediate mitochondrial function. Taken together, these studies highlight that these techniques are likely measuring different aspects of mitochondrial metabolism, resting activity versus maximal capacity, and to more fully characterize the metabolic effects of an intervention or disease, multiple parameters of oxidative function, ideally a combination of resting function, maximal capacity, and the response to an intermediate metabolic demand, should be assessed.\n\nPerforming in vitro analyses of mitochondrial metabolism from muscle biopsy samples therefore enables an array of parameters to be assessed, and the experimental procedures involved are highly sensitive and can typically be accomplished in most laboratories. However, a number of caveats should be considered: the biopsy itself is an invasive measurement and is extracted from a small region of tissue that may not accurately represent the entire muscle. Repeated sampling, although feasible, will occur from slightly different regions of tissue that may not contain the same muscle fiber-type composition. The integrated network of metabolic processes that exist in vivo are no longer present, and assays usually provide an estimate of the theoretical maximal capacity without the limitations imposed by regulatory mechanisms, substrate availability, or product inhibition. Normalization of data can also be problematic because different internal reference standards (total protein content, total mitochondrial protein content, specific mitochondrial proteins) are often used. The MR methods described in this article offer the ability to estimate mitochondrial function in vivo, in which regulatory mechanisms remain intact and the same region of tissue can be sampled noninvasively and repeatedly, although this is obviously constrained by the availability of an MR system. The PCr recovery method is relatively straightforward to implement on many scanners and can yield estimates of functional muscle mitochondrial capacity, provided an appropriate contraction protocol is selected, particularly when studying populations that may have impaired muscle metabolism. The TCA cycle flux measurement using ^13^C-MRS to monitor the oxidation of ^13^C-labeled substrates is considerably more complex to implement and generally requires customized MR hardware\/software as well as specialized metabolic modeling analyses. Additional constraints are provided by the length of the study (\u223c2 to 3 h within the scanner), which may be prohibitive for certain subjects, and the overall cost of the experiment due to the need for ^13^C-labeled substrates, extensive MR system time, and personnel. However, this method probably provides the most accurate estimate of resting mitochondrial activity in vivo.\n\n# CONCLUSION\n\nIn conclusion, MT MRS techniques offer a unique ability to examine the unidirectional fluxes that contribute to metabolic exchange reactions. The P~i~ \u2192 ATP flux associated with the ATP synthesis\/hydrolysis cycle is of particular interest because it is a key bioenergetic process supporting the energy demands of a tissue. Although the ^31^P-ST experiment measures all metabolic contributions to the P~i~ \u2192 ATP flux, including glycolytic ATP production and near-equilibrium exchange at the level of GAPDH\/PGK and\/or the mitochondrion, experiments in a variety of in vivo systems suggest that the flux does respond to perturbations in mitochondrial metabolism. Different experimental techniques to assess muscle metabolism in vivo (e.g. P~i~ \u2192 ATP flux by ^31^P-ST, oxidative capacity by PCr recovery using dynamic ^31^P-MRS, and substrate oxidation by dynamic ^13^C-MRS) likely assess different parameters of mitochondrial function. Therefore, to more fully characterize oxidative function in vivo, we recommend the use of complementary analytical techniques to obtain a multiparametric assessment of mitochondrial metabolism.\n\n## ACKNOWLEDGMENTS\n\nThis work was supported by grants from the U.S. Public Health Service (R01-DK-49230, R01-DK-40936, R24-DK-085638, U24-DK-059635, P30-DK-45735, and R01-AG-23686) and a Distinguished Clinical Scientist Award (to K.F.P.) from the American Diabetes Association.\n\nNo potential conflicts of interest relevant to this article were reported.\n\nD.E.B., D.L.R., K.F.P., and G.I.S. contributed to the writing of this article. D.E.B. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.\n\n# REFERENCES","meta":{"dup_signals":{"dup_doc_count":156,"dup_dump_count":48,"dup_details":{"curated_sources":2,"2023-50":2,"2023-40":1,"2020-34":1,"2019-13":1,"2018-51":1,"2018-39":2,"2018-26":1,"2018-22":4,"2018-17":1,"2018-13":5,"2018-05":1,"2017-51":4,"2017-47":2,"2017-43":6,"2017-39":2,"2017-34":3,"2017-30":5,"2017-26":5,"2017-22":2,"2017-17":8,"2017-09":6,"2017-04":9,"2016-50":5,"2016-44":6,"2016-40":7,"2016-36":6,"2016-30":7,"2016-26":6,"2016-22":6,"2016-18":2,"2016-07":3,"2015-48":2,"2015-40":1,"2015-35":3,"2015-32":1,"2015-27":2,"2015-22":2,"2015-14":2,"2014-52":3,"2014-49":1,"2014-41":1,"2014-35":1,"2024-30":2,"2024-26":2,"2017-13":5,"2015-18":3,"2015-11":2,"2015-06":1}},"file":"PMC3478545"},"subset":"pubmed_central"}
{"text":"author: Philip E BournePhilip E. Bourne is Editor-in-Chief of *PLoS Computational Biology*. E-mail: <email@example.com>\ndate: 2005-10\ntitle: Ten Simple Rules for Getting Published\n\nThe student council (<http:\/\/www.iscbsc.org\/>) of the International Society for Computational Biology asked me to present my thoughts on getting published in the field of computational biology at the Intelligent Systems in Molecular Biology conference held in Detroit in late June of 2005. Close to 200 bright young souls (and a few not so young) crammed into a small room for what proved to be a wonderful interchange among a group of whom approximately one-half had yet to publish their first paper. The advice I gave that day I have modified and present as ten rules for getting published.\n\n# Rule 1: Read many papers, and learn from both the good and the bad work of others.\n\nIt is never too early to become a critic. Journal clubs, where you critique a paper as a group, are excellent for having this kind of dialogue. Reading at least two papers a day in detail (not just in your area of research) and thinking about their quality will also help. Being well read has another potential major benefit\u2014it facilitates a more objective view of one's own work. It is too easy after many late nights spent in front of a computer screen and\/or laboratory bench to convince yourself that your work is the best invention since sliced bread. More than likely it is not, and your mentor is prone to falling into the same trap, hence rule 2.\n\n# Rule 2: The more objective you can be about your work, the better that work will ultimately become.\n\nAlas, some scientists will never be objective about their own work, and will never make the best scientists\u2014learn objectivity early, the editors and reviewers have.\n\n# Rule 3: Good editors and reviewers will be objective about your work.\n\nThe quality of the editorial board is an early indicator of the review process. Look at the masthead of the journal in which you plan to publish. Outstanding editors demand and get outstanding reviews. Put your energy into improving the quality of the manuscript *before submission*. Ideally, the reviews will improve your paper. But they will not get to imparting that advice if there are fundamental flaws.\n\n# Rule 4: If you do not write well in the English language, take lessons early; it will be invaluable later.\n\nThis is not just about grammar, but more importantly comprehension. The best papers are those in which complex ideas are expressed in a way that those who are less than immersed in the field can understand. Have you noticed that the most renowned scientists often give the most logical and simply stated yet stimulating lectures? This extends to their written work as well. Note that writing clearly is valuable, even if your ultimate career does not hinge on producing good scientific papers in English language journals. Submitted papers that are not clearly written in good English, unless the science is truly outstanding, are often rejected or at best slow to publish since they require extensive copyediting.\n\n# Rule 5: Learn to live with rejection.\n\nA failure to be objective can make rejection harder to take, and you will be rejected. Scientific careers are full of rejection, even for the best scientists. The correct response to a paper being rejected or requiring major revision is to listen to the reviewers and respond in an objective, not subjective, manner. Reviews reflect how your paper is being judged\u2014learn to live with it. If reviewers are unanimous about the poor quality of the paper, move on\u2014in virtually all cases, they are right. If they request a major revision, do it and address every point they raise both in your cover letter and through obvious revisions to the text. Multiple rounds of revision are painful for all those concerned and slow the publishing process.\n\n# Rule 6: The ingredients of good science are obvious\u2014novelty of research topic, comprehensive coverage of the relevant literature, good data, good analysis including strong statistical support, and a thought-provoking discussion. The ingredients of good science reporting are obvious\u2014good organization, the appropriate use of tables and figures, the right length, writing to the intended audience\u2014do not ignore the obvious.\n\nBe objective about these ingredients when you review the first draft, and do not rely on your mentor. Get a candid opinion by having the paper read by colleagues without a vested interest in the work, including those not directly involved in the topic area.\n\n# Rule 7: Start writing the paper the day you have the idea of what questions to pursue.\n\nSome would argue that this places too much emphasis on publishing, but it could also be argued that it helps define scope and facilitates hypothesis-driven science. The temptation of novice authors is to try to include everything they know in a paper. Your thesis is\/was your kitchen sink. Your papers should be concise, and impart as much information as possible in the least number of words. Be familiar with the guide to authors and follow it, the editors and reviewers do. Maintain a good bibliographic database as you go, and read the papers in it.\n\n# Rule 8: Become a reviewer early in your career.\n\nReviewing other papers will help you write better papers. To start, work with your mentors; have them give you papers they are reviewing and do the first cut at the review (most mentors will be happy to do this). Then, go through the final review that gets sent in by your mentor, and where allowed, as is true of this journal, look at the reviews others have written. This will provide an important perspective on the quality of your reviews and, hopefully, allow you to see your own work in a more objective way. You will also come to understand the review process and the quality of reviews, which is an important ingredient in deciding where to send your paper.\n\n# Rule 9: Decide early on where to try to publish your paper.\n\nThis will define the form and level of detail and assumed novelty of the work you are doing. Many journals have a presubmission enquiry system available\u2014use it. Even before the paper is written, get a sense of the novelty of the work, and whether a specific journal will be interested.\n\n# Rule 10: Quality is everything.\n\nIt is better to publish one paper in a quality journal than multiple papers in lesser journals. Increasingly, it is harder to hide the impact of your papers; tools like Google Scholar and the ISI Web of Science are being used by tenure committees and employers to define metrics for the quality of your work. It used to be that just the journal name was used as a metric. In the digital world, everyone knows if a paper has little impact. Try to publish in journals that have high impact factors; chances are your paper will have high impact, too, if accepted.\n\nWhen you are long gone, your scientific legacy is, in large part, the literature you left behind and the impact it represents. I hope these ten simple rules can help you leave behind something future generations of scientists will admire.","meta":{"dup_signals":{"dup_doc_count":143,"dup_dump_count":73,"dup_details":{"curated_sources":2,"2023-40":2,"2023-23":2,"2023-06":2,"2022-49":2,"2022-40":1,"2022-33":1,"2022-21":3,"2022-05":1,"2021-49":1,"2021-43":1,"2021-39":1,"2021-31":1,"2021-17":1,"2021-10":1,"2021-04":1,"2020-34":1,"2020-29":3,"2019-35":1,"2019-26":1,"2019-22":1,"2019-13":2,"2019-09":1,"2019-04":2,"2018-47":2,"2018-43":1,"2018-34":1,"2018-30":2,"2018-26":1,"2018-17":1,"2018-13":1,"2018-09":3,"2018-05":2,"2017-51":3,"2017-47":4,"2017-43":3,"2017-39":3,"2017-34":4,"2017-30":3,"2017-26":4,"2017-22":2,"2017-17":4,"2017-09":4,"2017-04":2,"2016-50":3,"2016-44":1,"2016-40":2,"2016-36":1,"2016-30":4,"2016-26":1,"2016-18":1,"2016-07":1,"2015-48":1,"2015-40":2,"2015-35":1,"2015-32":1,"2015-27":1,"2015-22":2,"2015-14":1,"2014-52":1,"2014-49":4,"2014-42":3,"2014-41":3,"2014-35":2,"2014-23":3,"2014-15":4,"2023-50":1,"2024-22":4,"2024-18":1,"2017-13":3,"2015-18":2,"2015-11":1,"2015-06":1,"2013-48":2}},"file":"PMC1274296"},"subset":"pubmed_central"}
{"text":"author: F Barone; M Bombardieri; J Spencer; P Isaacson; F Humby; P Morgan; S Challacombe; G Valesini; C Pitzalis\ndate: 2007\ninstitute: 1Department of Rheumatology, GKT School of Medicine, London, UK; 2Department of Immunology, GKT School of Medicine, London, UK; 3Department of Immunology, UCL, London, UK; 4Department of Oral Pathology and Medicine, GKT School of Medicine, London, UK; 5Department of Reumatologia, La Sapienza, Roma, Italy\ntitle: CXCL13, CCL21 and CXCL12 are upregulated in mucosal-associated lymphoid tissue lymphomas in patients with Sjorgen's syndrome and cooperate in the maintenance of the immune response and malignant cell survival\n\n# Background\n\nWe previously demonstrated in minor salivary glands of patients with Sjogren's syndrome (SS-mSGs) that a true phenomenon of ectopic lymphoneogenesis takes places, with the formation of germinal centres (GCs), in association with the ectopic expression of the lymphoid chemokines (CKs) CXCL13 and CCL21. In Sjogren's syndrome major salivary glands (SS-MSGs) these structures are known to support clonal immunoglobulin heavy chain gene rearrangements and somatic hypermutation, favouring expansion and selection of autoreactive B-cell clones, clonally related to lymphomatous B-cell clones (responsible for lymphoma development in Sjogren's syndrome (SS)). The involvement of lymphoid CKs and CXCL12 in the development of lymphoid malignancies has been demonstrated. At present, however, no data on the expression of these CKs nor their cellular source within SS-MSGs and mucosal-associated lymphoid tissue lymphomas (MALT-Ls) from patients with SS have been described.\n\n# Objective\n\nTo assess the specific contribution of lymphoid CKs in the organization of lymphoid proliferation within SS-MSGs and SS-MALT-Ls.\n\n# Methods\n\nWe studied 12 SS-mSGs, four SS-MSGs with lympho-epithelial lesion and 20 SS-MSGs with non-Hodgkin B-cell MALT-Ls. In order to define the histological organization of the lymphoid infiltrate (reactive versus malignant areas) and identify the B-cell subpopulations infiltrating the glands, immunohistochemistry was carried out for cellular (CD21, bcl-2, bcl-6, IgD, CD20, CD3, CD68) and vascular markers (CD31, PNAd). On sequential sections, digital images for CXCL13, CCL21 and CXCL12 were analysed by thresholding positive staining in detected areas of interest. Digital image analysis estimated CK's volume fraction areas as a ratio between CXCL13, CCL21 and CXCL12 positive areas over malignant or reactive areas. CK's producing cells were identified by double staining on sequential sections. Mononuclear cells were isolated from two SS-MALT-L parotids following enzymatic digestion and stained for CD19, CD38, CD27, CD24, IgM, IgD, CD10, CD5, CD3, CD4, CD69, CD45RO, CXCR5, CXCR4 and CCR7. Finally, mRNA from total SS-MSGs and MALT-Ls and isolated B cells and T cells from MALT-Ls were analysed by RT-PCR for the transcript levels of CXCL13, CCL21 and CXCL12.\n\n# Results\n\nReactive areas, characterized by T-cell\/B-cell segregation, CD20^+^IgD^+^bcl-2^+^ follicular B cells, presence of follicular dendritic cell networks in GCs and high endothelial venule formation were detected in 100% of MALT-Ls. A B-cell population (CD20^+^IgD^-^bcl-2^+^) characterized by nuclear abnormalities and monocytoid appearance was consistently observed within the proliferating ducts and identified as the B-cell malignant component. FACS analysis on isolated MALT-L mononuclear cells showed B cells in diverse maturative stages (transitional, mature and memory B cells) and a B-cell population CD19^pos^IgD^low^CD24^neg^CD27^low\/neg^CD5^neg^CD10^neg^ not detectable in the controls. Ectopic expression of CXCL13 and CCL21 was observed in 100% of SS-MSGs and MALT-Ls. A significant increase in CXCL13's volume fraction analysis was observed both in SS-MSGs and SS-MALT-L reactive areas, as compared with SS-mSG follicular areas (*P* \\< 0.05 and *P* \\< 0.05, respectively). A strong difference in CXCL13 within the MALT-L reactive area compared with malignant areas was detected (*P* \\< 0.001). CCL21 was significantly increased in MALT-Ls compared with both SS-mSGs and SS-MSGs and was mainly confined to the T-cell area. CXCL12 was strongly expressed by ductal epithelial cells, vessels and reactive areas in SS-mSGs, SS-MSGs and MALT-Ls. Interestingly a significant increase in CXCL12 expression on MALT-L malignant areas as compared with reactive areas in SS-mSGs and SS-MSGs was detected (*P* \\< 0.01 and *P* \\< 0.05). RT-PCR analysis showed increased CXCL13 and CCL21 level in SS-MSGs compared with MALT-Ls and SS-mSGs, while strong upregulation in CXCL12 transcript in MALT-Ls as compared with SS-mSGs and SS-MSGs was detected.\n\nCXCL13, CCL21 and CXCL12 were detected on CD68^+^ cells by immunohistochemistry, while CD20 and CD3, CXCL13 and CCL21 double staining and mRNA analysis on MALT-extracted T and B lymphocytes showed negligible expression of the two CKs in MALT-L-extracted lymphocytes. Interestingly we identified strong CXCL12 expression on CD19^+^ MALT-L isolated cells both at the protein and mRNA level and on ductal epithelial cells in close contact with the malignant B-cell infiltration. In agreement with this increase, we demonstrated a significant downregulation of CXCR4 on MALT-L-isolated B cells, likely to be the result of CXCR4 internalization upon CXCL12 ligation.\n\n# Conclusion\n\nIn SS-MALT-Ls a strong upregulation of the lymphoid CKs CXCL13 and CCL21 takes place and is associated with the organization of the reactive areas involved in the maintenance of the autoimmune process within the malignancy. These findings support a contributory role for CXCL13 and CCL21 in the pathogenesis of MALT lymphomas in SS. These results imply that the CK\/CK^-^ receptor axis is functional in MALT-L malignant cells, and suggest that the glandular microenvironment, B-cell receptor signalling upon antigen engagement and autocrine signals from the same malignant population concur in MALT lymphomagenesis, triggering local activation of malignant B cells and favouring their survival and expansion.","meta":{"dup_signals":{"dup_doc_count":117,"dup_dump_count":48,"dup_details":{"curated_sources":2,"2023-14":1,"2022-49":1,"2022-33":1,"2021-39":1,"2021-25":1,"2021-10":1,"2020-40":1,"2020-29":1,"2020-05":1,"2019-39":1,"2019-30":1,"2019-22":1,"2019-13":1,"2018-51":1,"2018-43":1,"2018-34":1,"2018-22":1,"2018-05":1,"2017-39":1,"2017-30":1,"2017-22":1,"2017-09":6,"2016-44":2,"2016-40":1,"2016-36":6,"2016-30":6,"2015-48":3,"2015-40":3,"2015-35":3,"2015-32":2,"2015-27":3,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":4,"2014-42":9,"2014-41":5,"2014-35":3,"2014-23":6,"2014-15":2,"2023-50":1,"2015-18":3,"2015-11":3,"2015-06":3,"2014-10":3,"2013-48":3,"2013-20":4,"2024-26":1}},"file":"PMC4061941"},"subset":"pubmed_central"}
{"text":"abstract: A report on the 2004 Keystone Symposium 'Human Genome Sequence Variation and the Inherited Basis of Common Disease', Breckenridge, USA, 8-13 January 2004.\nauthor: Evan E Eichler; Kelly A Frazer\ndate: 2004\ninstitute: 1Department of Genetics, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106, USA; 2Perlegen Sciences, 2021 Stierlin Court, Mountain View, CA 94043, USA\ntitle: The nature, pattern and function of human sequence variation\n\nThe Keystone Symposia on Human Genome Sequence Variation and the Inherited Basis of Common Disease and Quantitative Genetics in Model Organisms were held concurrently at the Beaver Run Resort this year. The dual nature of the meeting created a unique atmosphere in which participants were encouraged to cross-attend sessions. Indeed, several sessions were held jointly between the two meetings, creating the opportunity for interaction among a larger body of scientists than typically attend a Keystone meeting. This unusual format encouraged cross-fertilization of ideas among population, quantitative and human geneticists as well as epidemiologists and genome scientists. This report is dedicated primarily to the proceedings within the Human Sequence Variation sessions.\n\n# Polymorphisms and haplotype mapping\n\nTwo presentations confronted the prevailing hypothesis that common diseases are likely to be due solely to common genetic variants, such as those that can be identified by mapping frequent polymorphisms. Aravinda Chakravarti (Johns Hopkins University, Baltimore, USA) challenged the community by raising, once again, the unsettling specter that rare variants might in fact underlie a significant fraction of common disease. He presented data from his longstanding work on the complex genetics of Hirschsprung Disease. The data strongly suggest that both common and rare single-nucleotide polymorphisms (SNPs) should be considered if the true nature of a complex genetic disease is to be understood. Similarly, David Altshuler (Massachusetts General Hospital, Boston, USA) described a genetic analysis of diabetes. He showed preliminary results suggesting that mitochondrial DNA is involved in type II diabetes and hypothesized that primary alterations in mitochondrial oxidative phosphorylation pathways contribute to type II diabetes. Both common and rare variants are equally important in this case.\n\nSeveral presentations focused on the way that the structure of the human genome can be understood as being made up of haplotype blocks, and on how this may be useful as well as its biological origin and significance. Mark Daly (Massachusetts Institute of Technology, Cambridge, USA) provided insight into the progress and potential early fruits of the human HapMap project, which aims to produce a complete human haplotype map. A central question is how well the current (200,000 genotyped SNPs) and projected (500,000 SNPs by April 2004) haplotype maps recapitulate existing genetic variation within the human population. Extrapolating from existing data, Daly concluded that a density of one SNP marker every 5 kilobases should be sufficient to tag around 80% of human variation. Andy Clark (Cornell University, Ithaca, USA) stressed the need to impute missing genotypes on the basis of flanking SNP information. He presented a powerful Bayesian approach, which used the linkage disequilibrium between SNP pairs from a region of high marker density to infer the genotypes of missing data. He suggested that with a sufficient density of markers, information about the haplotype phase (the paternal or maternal origin) is unnecessary. David Goldstein (University College London, UK) similarly emphasized the need to identify hidden SNPs and further suggested that when the minor allele frequency drops below 7% there would be insufficient power to predict a hidden SNP within a second sample (based on a sample size of 64 individuals). His simulation studies predicted a loss in performance when the SNP density drops below one SNP per 6 kilobases.\n\nOne of the highlights of the meeting was the analysis by Peter Donnelly (University of Oxford, UK) of the landscape of fine-scale recombination. He presented a composite likelihood approach with which to estimate rates of recombination between pairs of SNPs. Using polymorphism data to infer properties of fine-scale recombination in a well-studied 10 megabase region of chromosome 20, he showed that the level of recombination varied as much as three orders of magnitude. His results suggest that 80% of all recombination occurs in around 25% of the sequence. While no clear sequence properties of 'hotspots' and 'coldspots' of recombination emerged, in general coldspots were found to be larger than hotspots, and hotspots tend to locate outside genes. There was a general agreement among several speakers that the only convincing correlation with the boundaries of some, but not all, haplotype blocks is increased recombination frequency.\n\nOne of the anticipated uses of SNPs and information about haplotype block structure is to improve the power of association studies for human genetic disease. K.F. outlined a strategy to tackle complex genetic traits using high-throughput methods of genotyping. Based on an analysis of a large number of individuals (around 1,000) for genetic variation and plasma concentration of low-density lipoprotein (LDL), the importance of well-characterized case-control samples and replica testing of pooled samples becomes very evident. Richard Lifton (Yale University, New Haven, USA) discussed the genetic determinants of hypertension and metabolic syndrome. He used families at extreme points of the phenotypic range - extreme hypotension and extreme hypertension - to identify 15 genes associated with this disease; 14 of these are involved in the renin-angiotension system, causing either increased or decreased Na^+^ reabsorption. Lifton argued that this is the reason that drugs targeting salt absorption are superior in the treatment of hypertension. A similar success story was echoed by Stephen O'Brien (National Cancer Institute, Frederick, USA) in a detailed study of a cohort of more than 1,000 individuals with acquired immunodeficiency syndrome (AIDS) who have been clinically monitored for over 10 years. His research has identified and\/or confirmed 15 genes (including the immune-cell surface molecules CD4, CD5, RANTES, HLA class I, and others) that affect infection with human immunodeficiency virus (HIV-1) and disease progression. Finally, David Hunter (Harvard Medical School, Boston, USA) discussed the role of gene-environment interactions in common disease. He described several examples where dietary and medical advice should be dependent on genotype information, including the finding that the *APOE*4 allele, which has been linked to both Alzheimer's disease and hypertension, is more associated with cognitive decline in individuals with uncontrolled hypertension than in individuals with controlled hypertension.\n\n# Model organisms\n\nIn a joint session between the two concurrent meetings, a series of talks focused on how model organisms might be used to move from genotype to phenotype or function. Keith Davies (Paradigm Genetics, Research Triangle Park, North Carolina, USA) described an industrial-level high-throughput transgenic facility that focuses on the systematic collection of phenotype information from *Arabidopsis*. Genotype-phenotype correlation data for over 16,000 *Arabidopsis* genes (including previously unknown genes) is in progress. Using a transposon-tagging system to detect open reading frames (ORFs) in yeast, Michael Snyder (Yale University, New Haven, USA) described the systematic experimental verification of genes in the yeast genome. He emphasized the need to annotate genes, as well as transcription-factor binding sites, experimentally, and not to rely strictly on *in silico* analyses. In contrast, Eric Lander (Whitehead Institute, Cambridge, USA) showed the power of comparative whole-genome sequence analysis of yeast genomes to systematically identify genes, regulatory elements and processes of genome evolution. His analysis of four yeast genomes predicted 5,695 'real' genes. Similar analyses between multiple mammalian genomes are revealing many unexplained conserved elements and \"the spectacular state of ignorance\" in the area of functional genomics.\n\nA particularly novel aspect of this meeting was the emphasis on inherited patterns of gene expression. Several studies have shown that natural genetic variation can cause significant differences in gene expression, suggesting that phenotypic variation can result not only from coding variation but also from regulatory variation that affects gene expression. To study the genetic architecture of natural variation in gene expression, Leonid Kruglyak (Fred Hutchinson Cancer Research Center, Seattle, USA) conducted a linkage analysis of genome-wide expression patterns in a cross between a laboratory and a wild strain of *Saccharomyces cerevisisae*. Over 1,500 genes were differentially expressed between the parental strains. These loci fell into two categories: *cis*-acting modulators of single genes (around 20%) and *trans*-acting modulators of many genes (around 80%). Surprisingly, analysis of the *trans*-acting loci by molecular function did not show an enrichment of transcription factors. Kevin White (Yale University School of Medicine, New Haven, USA) described the evolution of gene expression in *Drosophila*. He addressed the question, \"If evolution was played several times under similar conditions would it repeat itself?\" *Drosophila* from 27 inbred strains (with 20 males and 20 females of each strain) were divided into six populations. Three populations were raised in a hypoxic environment and three in a normoxic environment for several generations. When gene expression patterns were compared between strains in the two oxygen levels, 195 (53%) of the 368 genes that showed greater than a two-fold expression difference had evolved in all six populations, suggesting that some genes are more prone to change their expression levels, perhaps due to selective pressures.\n\nStephanie Monks (University of Washington\/Rosetta Inpharmatics, Seattle, USA) discussed the genetics of gene expression in mice. She established a genetic map of expression for 111 F2 mice resulting from a C57BL\/J6 \u00d7 DBA\/2J cross. For each of the F2 mice, RNA was isolated from the liver and 23,574 genes were assayed for expression using arrays. The study demonstrated that the distribution of quantitative trait loci (QTLs) controlling gene expression is non-random in the genome. Justin Fay (Washington University, St Louis, USA) addressed the question of whether or not transcriptional variation has functional consequences. Nine isolates of *S. cerevisiae* were grown in rich media in the presence or absence of copper sulfate. Two strains with demonstrated resistance to copper sulfate showed a reduced growth rate, and two different strains produced rust colored colonies. Gene expression differences correlated with resistance were enriched for oxidative stress and the unfolded protein response, while those related to coloration were almost exclusively in the methionine\/sulfur assimilation pathway.\n\nIn general, the pattern and nature of human genome sequence variation was the primary focus of the meeting, although due diligence was given to insights that could be gleaned from excellent studies from model organisms such as *Drosophila*, mouse, yeast and *Arabidopsis*. The scope of presentations was significantly more 'global' than in past meetings, due in part to the near-completion of the human genome, improvements in genotyping and the amount of analyzed sequence data now available. The wider range of topics appealed to a broader base of biologists. During the course of this meeting, a number of the 'usual' questions emerged. What is the contribution of rare versus common SNPs to the molecular basis for complex genetic disease? What density of SNP markers is sufficient for discriminating disease associations? Is there functional significance to haplotype block structures? How soon will geneticists routinely be able to resolve the genetic basis of complex disease? While there were no final answers to these and other questions, it was clear that significant advances are being made in these directions.","meta":{"dup_signals":{"dup_doc_count":120,"dup_dump_count":39,"dup_details":{"curated_sources":4,"2019-09":1,"2018-51":2,"2018-43":2,"2018-34":1,"2018-30":1,"2018-13":1,"2017-47":1,"2017-34":1,"2017-26":1,"2017-22":1,"2017-09":10,"2016-44":1,"2016-40":1,"2016-36":10,"2016-30":9,"2016-22":1,"2016-18":1,"2016-07":7,"2015-48":3,"2015-40":2,"2015-35":3,"2015-32":3,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":3,"2014-42":10,"2014-41":3,"2014-35":4,"2014-23":3,"2014-15":2,"2019-26":1,"2015-18":3,"2015-11":3,"2015-06":3,"2014-10":3,"2013-48":3,"2013-20":2,"2024-18":1}},"file":"PMC395779"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  Gestational Diabetes Mellitus (GDM) increases risks for type 2 diabetes and weight management is recommended to reduce the risk. Conventional dietary recommendations (energy-restricted, low fat) have limited success in women with previous GDM. The effect of lowering Glycaemic Index (GI) in managing glycaemic variables and body weight in women post-GDM is unknown.\n  .\n  # Objective\n  .\n  To evaluate the effects of conventional dietary recommendations administered with and without additional low-GI education, in the management of glucose tolerance and body weight in Asian women with previous GDM.\n  .\n  # Method\n  .\n  Seventy seven Asian, non-diabetic women with previous GDM, between 20- 40y were randomised into Conventional healthy dietary recommendation (CHDR) and low GI (LGI) groups. CHDR received conventional dietary recommendations only (energy restricted, low in fat and refined sugars, high-fibre). LGI group received advice on lowering GI in addition. Fasting and 2-h post-load blood glucose after 75 g oral glucose tolerance test (2HPP) were measured at baseline and 6 months after intervention. Anthropometry and dietary intake were assessed at baseline, three and six months after intervention. The study is registered at the Malaysian National Medical Research Register (NMRR) with Research ID: 5183.\n  .\n  # Results\n  .\n  After 6 months, significant reductions in body weight, BMI and waist-to-hip ratio were observed only in LGI group (P\\<0.05). Mean BMI changes were significantly different between groups (LGI vs. CHDR: -0.6 vs. 0 kg\/m^2^, P= 0.03). More subjects achieved weight loss \u22655% in LGI compared to CHDR group (33% vs. 8%, P=0.01). Changes in 2HPP were significantly different between groups (LGI vs. CHDR: median (IQR): -0.2(2.8) vs. +0.8 (2.0) mmol\/L, P=0.025). Subjects with baseline fasting insulin\u22652 \u03bcIU\/ml had greater 2HPP reductions in LGI group compared to those in the CHDR group (\u22121.9\u00b10.42 vs. +1.31\u00b11.4 mmol\/L, P\\<0.001). After 6 months, LGI group diets showed significantly lower GI (57\u00b15 vs. 64\u00b16, P\\<0.001), GL (122\u00b133 vs. 142\u00b135, P=0.04) and higher fibre content (17\u00b14 vs.13\u00b14 g, P\\<0.001). Caloric intakes were comparable between groups.\n  .\n  # Conclusion\n  .\n  In women post-GDM, lowering GI of healthy diets resulted in significant improvements in glucose tolerance and body weight reduction as compared to conventional low-fat diets with similar energy prescription.\nauthor: Sangeetha Shyam; Fatimah Arshad; Rohana Abdul Ghani; Norasyikin A Wahab; Nik Shanita Safii; Mohd Yusof Barakatun Nisak; Karuthan Chinna; Nor Azmi Kamaruddin\ndate: 2013\ninstitute: 1School of Post Graduate Studies and Research, International Medical University, Kuala Lumpur, Malaysia; 2Department of Nutrition and Dietetics, International Medical University, Kuala Lumpur, Malaysia; 3Endocrine Unit, Department of Medicine, Faculty of Medicine, Universiti Kebangsaan Malaysia (National University of Malaysia), Kuala Lumpur, Malaysia; 4Dietetics Program, School of Healthcare Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia; 5Department of Nutrition & Dietetics, Faculty of Medicine & Health Sciences, Universiti Putra Malaysia, Serdang, Malaysia; 6Epidemiology and Biostatistics Unit, Department of Social and Preventive Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia\nreferences:\ntitle: Low glycaemic index diets improve glucose tolerance and body weight in women with previous history of gestational diabetes: a six months randomized trial\n\n# Background\n\nHistory of gestational diabetes mellitus (GDM) is a non-modifiable risk factor for developing type 2 diabetes mellitus (T2DM) \\[1,2\\]. Frequently, women with prior GDM exhibit tendencies for central obesity, insulin resistance and glucose intolerance \\[3\\]. Thirty-three to 50% of GDM women develop overt T2DM five years after delivery \\[4\\]. At ten years postpartum, 35\u201360% of GDM women are known to develop type 2 DM \\[3\\]. Hence GDM represents a challenging group to institute effective intervention \\[5\\].\n\nIntensive lifestyle intervention has shown to be effective in reducing risk for T2DM in high risk individuals \\[6,7\\]. Conventional diets for primary prevention of T2DM consistently support energy-restricted, low-fat, and high\u2013complex carbohydrate regime to achieve weight loss \\[8,9\\]. However, such interventions have shown limited success in reducing postpartum weight in women after GDM \\[10\\].\n\nMeanwhile, emerging evidence emphasizes the role of insulin secretion and insulin resistance in body weight regulation \\[11\\]. Therefore, dietary factors that influence these parameters would include the type of carbohydrate defined by its glycaemic index (GI) and its overall glycaemic potential described by its glycaemic load (GL, i.e. sum of the product of GI and available carbohydrate amounts for all foods consumed in the diet \\[12\\]) are known to modulate weight loss, especially in hyper-insulinaemic women \\[13,14\\]. Low GI diets also improve glycaemic control in Asian diabetics \\[15\\] and reduce risk for T2DM \\[16\\]. However the effect of lowering dietary GI in women with prior GDM is currently unknown. This study aimed to evaluate the effect of adding low GI dietary advice to conventional dietary prescription on glycaemic variables and body weight in comparison to conventional dietary prescription among women with previous GDM.\n\n# Methods\n\nThis study was conducted at the endocrine clinic of a tertiary hospital. The project was approved by the Ethic and Research Review Committees of the institutions involved, in-line with national regulations and according to the International Harmonization Consensus on Good Clinical Practice. The study is registered at the Malaysian National Medical Research Register with Research ID: 5183. The trial was carried out according to Consolidated Standards of Reporting Trials (CONSORT) guidelines.\n\n## Inclusion and exclusion criteria\n\nHealthy women in the age group of 20\u201340 years old, with a history of GDM (defined according to WHO criteria, \\[17\\]), and high risks for developing T2DM were included. While GDM itself is a non-modifiable risk factor for T2DM, certain modifiable risk factors that increase the risk in this group of subjects have been identified. These include central obesity, higher postpartum body mass index (BMI), fasting blood sugar (FBS), 2 h post-load blood glucose (2HPP) and energy intake \\[18,19\\]. Hence preventive interventions would be especially beneficial to subjects who have high risks as indentified by the inclusion criteria of the study. We therefore defined high risk as satisfying one of the following four conditions: BMI\\>23 kg\/m^2^, or waist circumference\\> 80 cm \\[20\\], or impaired glucose tolerance (IGT) defined as 2HPP after 75 g screening oral glucose tolerance test (OGTT) \u22657.8 and\\<11.1 mmol\/L or impaired fasting glucose (IFG) defined as FBS\\>5.6 mmol\/L or a family history of T2DM. Exclusion criteria were the diagnosis of T2DM (FBS\u22657.0 mmol\/L, or 2HPP\u226511.1 mmol\/l, \\[17\\], or presence of other health complications and usage of drugs affecting body weight and glucose control. Written informed consent was obtained from all participants. The women were enrolled into the trial at a minimum of two months after their last GDM delivery.\n\n## Measurement of outcome variables\n\nThe primary end point of this study was 2HPP after a 75 g oral glucose tolerance test was administered. FBS, fasting serum insulin (INS) and anthropometric measures were the secondary endpoints investigated.\n\nBody weight rounded to 0.1 kg was measured in light clothing, after emptying pockets and without footwear, using digital weighing scales (Model: BWB-800A, Tanita Corporation, Tokyo, Japan). Height without footwear was measured using the wall mounted stadiometer (Model No. 206, SECA, Hamburg, Germany), rounded to 0.1 cm to calculate BMI. BMI was calculated as the ratio of weight in kg to the square of height in m^2^. Waist and hip circumference in cm was measured as per WHO guidelines \\[21\\] and waist-to-hip ratio was calculated. All anthropometric measurements were done by a single researcher to avoid bias. Anthropometric measurements were made at baseline, three and six months after intervention.\n\nBlood samples were collected after a 12-h fasting, and analysed for FBS and insulin. A 75 g OGTT was administered and 2HPP samples were obtained. Blood glucose was analysed using the G6PD\/Hexokinase method as per routine medical centre laboratory protocol. Serum samples for INS were frozen at \u221220\u00b0C until batch processed for analysis. INS was analysed with chemiluminescent enzyme-labelled immunometric assay, using Immulite 2000 automated analyser (Diagnostic Products Corporation, Los Angeles, USA). Blood glucose and INS values were measured at baseline and 6 months after intervention.\n\nDietary intake was assessed with 3-day dietary records. The subjects were asked to report intake for any 2 weekdays and one weekend day for each visit. Subjects were trained to use the diet records at the screening visit and pictures of household measures were provided to assist the subjects with recording the amounts. Diet records were collected at baseline, three and six months after intervention. All food records were reviewed with subjects during their follow up visits by the research nutritionist to ensure completeness of entries.\n\nGL by definition is the \"product of the GI of foods and its carbohydrate content\" \\[22\\]. GL for individual food items in the dietary record was calculated as the product of diet GI and carbohydrate intake divided by 100 \\[23\\]. Dietary GL represents both the quantity and the quality of carbohydrate in diet \\[22\\] and was calculated as sum total of GL of foods consumed in the day \\[24\\]. Diet GI was calculated using the formula Diet GI= Diet GL\u00d7100\/amount of carbohydrate in the diet \\[24\\].\n\nA Microsoft Excel-based food composition database and diet intake calculator- \"DietPLUS Version 3\" was built for this study and was used for dietary analysis \\[25\\]. Due to lack of availability of local GI values, GI values for numerous Malaysian foods needed to be matched or estimated. Hence a systematic GI value assignment to foods in the Malaysian food composition table to similar foods published internationally was undertaken. To improve the accuracy of this GI-matching process, the GI assignment was based on previously published methodology \\[26\\]. The international GI and GL database and its updated version the online database of University of Sydney was used for this purpose \\[27,28\\]. The methodology of adding glycaemic index and glycaemic load functionality to the Malaysian food composition database has been previously published \\[25\\].\n\nInternational Physical activity short Questionnaire (IPAQ-short) was used to assess physical activity levels of the subjects \\[29\\].\n\n## Randomisation\n\nAll eligible subjects (n=77) were randomized strictly according to a computerized allocation (1:1) list generated using randomisation software \\[30\\]; by an individual from outside this research group. The researchers were unaware of the sizes of blocks used. Participants were randomized to either a conventional healthy dietary recommendation (CHDR) or the \"CHDR+Low GI\" group (LGI).\n\n## Dietary intervention\n\nDetails on the educational intervention and tools used have been published earlier \\[31\\]. However for the sake of completeness a brief description is provided here. The aim of the nutrition education was to achieve and maintain a 5-7% reduction in body weight if BMI\\>23 and maintain current weight if BMI\\<23 during the one year complete trial period. The nutrition intervention aimed to achieve this objective through establishing two diets that were similar in energy and macronutrient content but with different dietary GI. Individual energy requirement was calculated by multiplying basal metabolic rate (BMR) by an appropriate activity factor \\[32\\] based on reported physical activity pattern and occupation. Harris Benedict method was used to calculate BMR \\[32\\]. If the subject had a BMI\\>23 and if she was not breast feeding, the energy prescription was reduced by 500 Kcal. Subjects with BMI\\< 23 or those breastfeeding an infant\\<6 mo of age were prescribed their energy requirement (energy prescribed=energy requirement). All EP was rounded to the nearest hundred. Energy prescription was capped at 1800 Kcal\/day. Each subject was given an individualized dietary sheet with the recommended number of servings of each food group per day according to their energy prescription. Subjects were taught meal planning based on their individualized energy prescription and a sample one day menu was provided during the intervention visit. Concepts of serving size, number of servings and food exchange groups were taught to both groups of subjects. These dietary prescriptions were indicative in nature as the study subjects were \"out-patients\" and intervention was educational in nature.\n\nPrimary nutrition educational intervention was administered through a structured one-to-one session with the research nutritionist, at baseline. Conceptual guidance to achieve conventional recommended diet (CHDR) low in fat and refined sugars, and high in fibre was provided using the 5M framework proposed by the Malaysian Ministry of Health ( minimize salt, sugar, oil, more fruit and vegetables) \\[33\\]. Subjects were encouraged to indulge in moderate physical activity for 30min, at least five times a week.\n\nThe LGI group in addition to the above conventional recommendations also received a GI-education component that taught subjects to substitute high GI foods with low GI options. Staple foods primarily determine dietary GI \\[34\\], hence, GI-education focused on the strategies to select low-GI options for high GI rice, bread and breakfast cereals. Subjects were advised to restrict rice intake to once per day since most locally available rice varieties were high in GI \\[35\\]. They were encouraged to choose lower GI foods like spaghetti, noodles or multi-grain bread at other times. Subjects were not required to memorize numerical GI values of foods. However a list of foods that classified foods as high, moderate or low GI was provided to aid making choices. The LGI subjects were asked to include one low GI food at each meal.\n\nColour coded (pink for LGI and blue for CHDR) take-home booklets were also provided to the subjects as added reference. To improve retention of participants in the trial two electronic interactions (short messaging service (SMS) or e-mail, as per the subject's preference) per month was established. These contacts were similar between the groups in terms of frequency and content of delivery.\n\n## Sample size calculation\n\nSample size was calculated to detect at the end of one year, a 1.5 mmol\/L difference in the postprandial blood glucose, the primary end point for this study. This difference is thought to be of clinical significance \\[36\\] between the diet treatments, with a power of 80%. This was based on the assumption that the standard deviation of the response variable is 1.5 mmol\/L \\[37\\]. A total of 34 subjects, 17 per group, were required for this study. Taking into account the higher attrition rates in preventive trials \\[38\\], and reported reluctance among women post-GDM to participate \\[5\\], we started with a bigger sample size.\n\n## Statistical analysis\n\nStatistical analyses were performed using IBM SPSS (Version 19, Somers, NY, USA). The effect of the respective dietary interventions on anthropometric measurements and glycaemic variables at the end of 6 months was investigated. Primary analysis included intent to treat analysis of anthropometric data and glycaemic variables. Missing data was imputed conservatively with the last observation carried forward technique. A secondary analysis was carried out using the completers' analysis. For the dietary data analysis the baseline values and a mean of the intake at three and six months visit was used. A sub-analysis to determine the differential response to the dietary intervention in subjects with varying fasting insulin levels was carried out. Fasting insulin value of 2 \u03bcIU\/ml was chosen to be the cut-off for this analysis since it was approximately the natural median INS value for our subjects\n\nThe statistical significance standard was set at 5%. Data normality was tested using the Shapiro\u2013Wilks test. Data is presented as Mean \u00b1 SD, unless stated otherwise. Differences between groups were assessed using independent samples *t* test and changes in outcomes before and after intervention were analysed using paired *t* tests. When the test assumptions were not met, Mann\u2013Whitney U-test test for two samples was used to compare values between groups and Wilcoxon Sign test was used to compare the changes in outcome within the group. Effect size values were computed to compare the effects of the diet treatments \\[14\\]. Effect sizes between 0.2-0.5, 0.5-0.8 and \\>0.8 were taken to denote \"small\", \"moderate\" and \"large\" changes in outcomes \\[14\\].\n\nLinear regression analysis was used to study the role of energy intake, macronutrient intakes, dietary fibre, GI and GL as predictor variables on changes in body weight. In modelling 2HPP, in addition to the above mentioned predictors (dietary variables), change in body weight was included. In modelling FBS, in addition to dietary variables mentioned above, changes in body weight and 2HPP were included. A sub-analysis was done grouping subjects with INS\\<2 \u03bcIU\/L and \u22652 \u03bcIU\/L in studying the models as attempted above. We also wanted to test if the effect of GI on 2HPP was mediated by changes in weight, among the subjects with INS\u22652 \u03bcIU\/L. In this test the Sobel's Method was used \\[39\\].\n\n# Results\n\nSeventy-seven subjects were recruited out of whom a total of 62 (29 in CHDR and 33 in LGI group) completed the 6 months on the trial (refer to Figure\u00a0<a href=\"#F1\" data-ref-type=\"fig\">1<\/a>). The two groups had comparable baseline characteristics as shown in Table\u00a0<a href=\"#T1\" data-ref-type=\"table\">1<\/a>.\n\nBaseline characteristics of subjects (Mean\u00b1SD)\n\n| **Characteristic** | **LGI** | **CHDR** | **P**^**1**^ **Value** |\n|:---|:--:|:--:|:--:|\n| Age (y) | 30.9\u00b14.3 | 31.5\u00b14.5 | 0.999 |\n| No. of Pregnancies\\* | 2\u00b12 | 2\u00b11 | 0.245 |\n| No. of GDM Pregnancies\\* | 1\u00b10 | 1\u00b11 | 0.891 |\n| Parity\\* | 2\u00b12 | 2\u00b12 | 0.671 |\n| Duration since Last GDM delivery (mo)\\* | 6\u00b13 | 5\u00b12 | 0.246 |\n| Weight (kg) | 65.3\u00b111.5 | 64.6\u00b112.5 | 0.794 |\n| BMI (kg\/m^2^) | 26.4\u00b14.6 | 26.3\u00b14.6 | 0.952 |\n| % subjects with BMI\\>23 (Overweight) | 74.4 | 71.1 | 0.802 |\n| % subjects with BMI\\>25 | 59 | 63.2 | 0.816 |\n| % subjects with BMI\\>27.5 (Obese) | 38.5 | 47.5 | 0.494 |\n| % subjects breast feeding | 10.3 | 21.1 | 0.420 |\n| Waist Circumference (cm) | 83.2\u00b18.5 | 82.7\u00b19.6 | 0.789 |\n| Waist: Hip Ratio | 0.81\u00b10.06 | 0.80\u00b10.05 | 0.608 |\n| Fasting blood glucose(mmol\/L) | 4.7\u00b10.54 | 4.8\u00b10.53 | 0.405 |\n| 2 h post 75 g OGTT blood glucose (mmol\/L) | 6.8\u00b11.6 | 6.2\u00b11.4 | 0.067 |\n\nLegend: *LGI* Low GI Group, *CHDR* Conventional healthy Dietary Recommendation Group.\n\n\\*Expressed as median\u00b1interquartile range, P^1^ Value: test between groups.\n\n## Changes in anthropometry\n\nIntent to treat (ITT) analysis showed significant reductions from baseline to six months in body weight (P=0.018), BMI (P=0.008), waist circumference (P\\<0.001) and waist-hip-ratio (P=0.02) in LGI group. In CHDR group there was a significant reduction in waist circumference (P=0.048) only (Refer Table\u00a0<a href=\"#T2\" data-ref-type=\"table\">2<\/a>). The effect sizes for the changes in weight, BMI, waist circumference and waist-hip-ratio from baseline to six months in the LGI group were 0.40, 0.45, 0.70 and 0.38, respectively which are moderate to large. The effect sizes for the changes in the same variables in the CHDR group were merely 0.02, 0.02, 0.33 and 0.22, respectively.\n\nAnthropometric outcomes during the course of the study (Mean\u00b1SD)\n\n| **Outcome** | **LGI** |  |  | **CHDR** |  |  |\n|:---|:--:|:--:|:--:|:--:|:--:|:--:|\n|  | **baseline** | **3 months** | **6 months** | **baseline** | **3 months** | **6 months** |\n| Weight (Kg) | 65.3\u00b111.5^a^ | 64.6\u00b111.5^b^ | 64.0\u00b111.7^ab^ | 64.6\u00b112.5 | 64.7\u00b112.6 | 64.5\u00b113.0 |\n| BMI(kg\/m2) | 26.4\u00b14.6^a^ | 26.0\u00b14.7^a^ | 25.8\u00b14.7^a^ | 26.3\u00b14.6 | 26.4\u00b14.7 | 26.3\u00b14.8 |\n| WC (cm) | 83.2\u00b18.5 ^a^ | 83.0\u00b110.4^b^ | 80.5\u00b19.1^ab^ | 82.7\u00b19.6 ^a^ | 82.5\u00b110.8 | 81.5\u00b110.1^a^ |\n| WHR | 0.81\u00b10.06^a^ | 0.80 \u00b10.06 | 0.79 \u00b10.05^a^ | 0.80\u00b10.05 | 0.80\u00b10.05 | 0.79\u00b10.05 |\n\nLegend: *LGI* Low GI Group, *CHDR* Conventional healthy Dietary Recommendation Group, *BMI* Body mass index, *WC* waist circumference *WHR* waist hip ratio.\n\nFor each group, values in a row with similar alphabetical superscripts show significantly different means in paired tests.\n\nAs shown in Figure\u00a0<a href=\"#F2\" data-ref-type=\"fig\">2<\/a>, a greater number of subjects achieved a percentage weight loss\u22655% in LGI as compared to CHDR Group (33% vs. 8%, P=0.01)\n\n## Changes in glycaemic variables\n\nThe subjects had normoglycaemia or dysglycaemia (impaired fasting glucose\/ impaired glucose tolerance) as tested by 75 g OGTT at baseline. FBS and 2HPP levels in the two groups were comparable at baseline (See, Table\u00a0<a href=\"#T3\" data-ref-type=\"table\">3<\/a>).\n\nChanges from baseline in glycaemic values (Mean\u00b1SD)\n\n| \u00a0 | **LGI** |  |  | **CHDR** |  |  |\n|:---|:--:|:--:|:--:|:--:|:--:|:--:|\n| \u00a0 | **Baseline** | **6 months** | **P**^**1**^ | **Baseline** | **6 months** | **P**^**1**^ |\n| FBS (mmol\/L) | 4.7\u00b10.53 | 5.0\u00b11.2 | 0.060 | 4.8\u00b10.53 | 4.9\u00b10.53 | 0.111 |\n| \u2206FBS (mmol\/L)\\* | \u22120. 2 (0.6) |  |  | 0.1(0.6) |  |  |\n| 2HPP (mmol\/L) | 6.8\u00b11.6 | 6.8\u00b12.8 | 0.960 | 6.2\u00b11.4 | 6.8\u00b11.6 | 0.010 |\n| \u22062HPP (mmol\/L)\\* | \u22120.2 (2.8) ^a^ |  |  | 0.8 (2.0)^a^ |  |  |\n\nLegend: *LGI* Low GI Group, *CHDR* Conventional healthy Dietary Recommendation Group. *FBS* Fasting blood sugar, *2HPP* 2 hour post load blood glucose after 75 g OGTT. P1: Significance of changes within groups.\n\n\\* Data presented as median (interquartile range).\n\n^a^ Difference between groups significant (P=0.025).\n\nFrom baseline to 6 months, the changes in fasting blood sugar were not significant in both groups (See, Table\u00a0<a href=\"#T3\" data-ref-type=\"table\">3<\/a>). There was no significant change in median 2HPP in the LGI group (\u22120.2 (IQR: 2.8) mmol\/L, P= 0.960). In the CHDR group, there was a significant increase in median 2HPP (+0.8 (IQR: 2.0) mmol\/L, P=0.01). The changes in 2HPP were significantly different between groups (P=0.025, See, Table\u00a0<a href=\"#T3\" data-ref-type=\"table\">3<\/a>).\n\nResults obtained from per-protocol analysis for changes in anthropometric and glycaemic outcomes were similar to the respective analysis done using ITT (data for per-protocol analysis not shown here).\n\n## Baseline glucose tolerance status and response to dietary treatment\n\nThe anthropometric and glycaemic changes among subjects varying in baseline glucose tolerance status are shown in the following Table\u00a0<a href=\"#T4\" data-ref-type=\"table\">4<\/a>. There was a slight increase in weight and 2HPP blood glucose among subjects with dysglycaemia in CHDR group but in the LGI group there was a slight decrease in these two measurements. Among subjects with dysglycaemia at baseline 37.5% of subjects in CHDR (n= 3\/8) and 64.3% in LGI (n=9\/14) reverted to normoglycaemia (RR: 1.5, P= 0.378).\n\nAnthropometric and glycaemic changes in subjects with varying glucose tolerance status during the six months study period (Mean\u00b1SD)\n\n| **Baseline glucose tolerance status** | **Normoglycaemia** |  |  | **Dysglycaemic subjects** |  |  |\n|:---|:--:|:--:|:--:|:--:|:--:|:--:|\n| **Outcome changes** | **CHDR** | **LGI** | **P** | **CHDR** | **LGI** | **P** |\n| **Weight (kg)** | \u22120.2\u00b12.72 | \u22121.93\u00b13.48\\* | 0.446 | 0.5\u00b12.97 | \u22120.7\u00b13.37 | 0.446 |\n| **BMI (kg\/m2)** | \u22120.08\u00b11.04 | \u22120.76\u00b11.41\\* | 0.077 | 0.25\u00b11.2 | \u22120.6\u00b11.54 | 0.230 |\n| **WC (cm)** | \u22121.33\u00b13.34 | \u22123.72\u00b14.24\\*\\* | 0.046 | \u22122.14\u00b14.53 | \u22122.67\u00b13.66\\* | 0.786 |\n| **WHR** | \u22120.01\u00b10.04 | \u22120.02\u00b10.005 | 0.427 | \u22120.02\u00b10.05 | \u22120.03\u00b10.03\\* | 0.282 |\n| **FBS (mmol\/L)** | 0.15\u00b10.41 | 0.31\u00b10.53\\* | 0.290 | 0.04\u00b10.45 | 0.49\u00b11.7 | 0.507 |\n| **2HPP (mmol\/L)** | 0.88\u00b11.2\\*\\*\\* | 0.24\u00b12.04 | 0.216 | 0.47\u00b12.22 | \u22120.49\u00b14.05 | 0.572 |\n\nLegend: *LGI* Low GI Group, *CHDR* Conventional healthy Dietary Recommendation Group. *BMI* Body mass index, *WC* waist circumference, *WHR* waist hip ratio, *FBS* Fasting blood sugar, *2HPP* 2 hour post load blood glucose after 75g OGTT.\n\nP values show the significance of difference in changes between groups.\n\nValues with asterisks denote significant changes in paired-tests, \\*P\u22640.05, \\*\\*P\u22640.01, \\*\\*\\*P\u22640.001.\n\n## Fasting insulin levels and response to dietary treatment\n\nOut of the 77 subjects, 44 (57.1%) had INS\\<2 \u03bcIU\/L and 33 (42.9%) had INS\u22652 \u03bcIU\/L. The percentage of subjects with INS\\<2 \u03bcIU\/L in the LGI and CHDR groups were 61.5 and 52.6% respectively. This difference between the groups was not significant (P=0.228).\n\nAmong the subjects with INS\\<2 \u03bcIU\/L, there was no significant difference in the anthropometric and glycaemic changes for both LGI and CHDR groups (data not shown).\n\nAs shown in Figure\u00a0<a href=\"#F3\" data-ref-type=\"fig\">3<\/a>, among subjects with baseline INS\u22652 uIU\/ml the percentage weight loss was higher in the LGI group compared to the CHDR group (\u22123.1% vs. + 0.2%, P=0.09).\n\nFor 2HPP, LGI subjects with baseline INS\u22652 \u03bcIU\/ml, recorded a significant reduction (\u22121.9\u00b10.42 mmol\/L, P=0.002) while the 2HPP levels increased (1.31\u00b1 1.4, P=0.004) among similar subjects in the CHDR group (See Figure\u00a0<a href=\"#F4\" data-ref-type=\"fig\">4<\/a>). The level of 2HPP changes between the groups was significant (P \\<0.001). A sizable increase in 2HPP levels among subjects with baseline INS\u22652 \u03bcIU\/ml, was noted among those in the highest GI quartile (P value for trend =0.019, See Figure\u00a0<a href=\"#F5\" data-ref-type=\"fig\">5<\/a>). There was no significant association between GI and changes in 2HPP among subjects with baseline INS\\<2 \u03bcIU\/ml (P=0.576, See Figure\u00a0<a href=\"#F5\" data-ref-type=\"fig\">5<\/a>).\n\nChanges in FBS were however not significant within or between diet groups in such subjects (data not shown).\n\n## Changes in dietary intake\n\nFood records were collected from the subjects at baseline, at 3 months and at 6 months. Food records from 34 subjects in LGI group and 31 subjects in CHDR group were used in the analysis. The results of the dietary analysis are presented in Table\u00a0<a href=\"#T5\" data-ref-type=\"table\">5<\/a>. The after intervention values are the average between three and six month values.\n\nDietary intake of subjects before and after intervention (Mean \u00b1 SD)\n\n| **Dietary outcome** | **LGI** |  |  | **CHDR** |  |  | **P values for difference in changes between groups** |\n|:---|:--:|:--:|:--:|:--:|:--:|:--:|:--:|\n|  | **Baseline** | **After intervention** | **P**^**1**^ | **Baseline** | **After intervention** | **P**^**1**^ |  |\n| Calories | 1804\u00b1495 | 1660\u00b1368 | 0.075 | 1721\u00b1 491 | 1612\u00b1352 | 0.171 | 0.970 |\n| CHO g | 245\u00b175 | 206\u00b152 | 0.002 | 225\u00b168 | 220\u00b148 | 0.627 | 0.224 |\n| CHO en% | 55\u00b19 | 51\u00b15\\*\\* | 0.050 | 53\u00b17 | 55\u00b17\\*\\* | 0.240 | 0.065 |\n| Protein g | 66\u00b121 | 69\u00b115 | 0.323 | 70\u00b126 | 63\u00b121 | 0.119 | 0.191 |\n| Protein en% | 15\u00b14 | 20\u00b16 | \\<0.001 | 16\u00b15 | 19\u00b17 | 0.124 | 0.400 |\n| Fat g | 62\u00b123 | 58\u00b118 | 0.418 | 60\u00b120 | 53\u00b116 | 0.064 | 0.695 |\n| fat en% | 29\u00b18 | 28\u00b15 | 0.368 | 30\u00b16 | 25\u00b17 | 0.004 | 0.177 |\n| Dietary Fibre (g) | 13\u00b15 | 17\u00b14\\*\\*\\* | 0.001 | 13\u00b15 | 13\u00b14\\*\\*\\* | 0.583 | 0.022 |\n| Glycaemic Index | 62\u00b15 | 57\u00b15\\*\\*\\* | 0.003 | 62\u00b17 | 64\u00b16\\*\\*\\* | 0.4 | 0.033 |\n| Glycaemic Load | 152\u00b141 | 122\u00b133\\* | \\<0.001 | 142\u00b142 | 141\u00b135\\* | 0.854 | 0.085 |\n\n*LGI* Low GI Group, *CHDR* Conventional Healthy Dietary Recommendation Group.\n\nP^1-^ P values for changes within groups.\n\n\\*Values significantly different between groups - (P\\<0.05), \\*\\*Values significantly different between groups (P=\\<0.01). \\*\\*\\*Values significantly different between groups (P=\\<0.001).\n\nBaseline dietary intakes were comparable between groups. In the LGI group, mean percentage of calories from carbohydrate, amount of carbohydrates, diet GI and GL decreased significantly from baseline, while percentage of energy from protein and dietary fibre increased significantly. The only significant dietary change from baseline in the CHDR group was the reduction in the percentage of energy from fat. After six months, mean percentage of calories from carbohydrate, GI and GL were significantly lower in the LGI compared to the CHDR group. Fibre intake was significantly higher in the LGI group. During the study period, only changes in dietary glycaemic index and dietary fibre intakes were significantly different between groups (Refer Table\u00a0<a href=\"#T5\" data-ref-type=\"table\">5<\/a>).\n\nBased on the results from regression analysis, mean GL during the six months after intervention was the single independent predictor of weight loss in subjects (R^2^= 13.5%, standardized B= 0.37, P= 0.003). Weight change was the single independent predictor of changes in 2HPP (R^2^= 7.4%, standardized B= 0.272, P= 0.02). Change in 2HPP was the independent predictor of change in FBS (R^2^=32. 7%, standardized B= 0.572 P\\< 0.001).\n\nIn subjects with INS\u22652 \u03bcIU\/L, mean GI primarily explained changes in 2HPP mean (R^2^=43. 8%, standardized B= \u22120.662 P\\< 0.001), with reduction in 2HPP being caused by a reduction in GI. Further analysis with block regression showed that mean GI explained 43.8% of the change in 2HPP while weight change explained only an additional of 3.9% (P=0.227) in these subjects. The Sobel's method was used to further determine if the effect of GI on 2HPP was mediated through changes in weight. The results showed that mediation effect was small and not significant in subjects with INS\u22652 \u03bcIU\/L (Indirect effect vs. Direct effect: unstandardized B= \u22120.05 vs. 0.196, P values 0.716 vs. \\<0.001).\n\n## Physical activity and adherence\n\nPhysical activity levels remained comparable between the groups at baseline (LGI vs. CHDR: median (IQR):608 (2727) vs. 900(2304) MET.min\/week, P=0.143) and at the end of six months study period (933 (1403) vs.965 (857) MET.min\/week, P= 0.908). Dietary adherence (self reported and calculated, data not shown here) were statistically comparable between the groups.\n\n# Discussion\n\nThe current study was designed to investigate the effects of adding low-GI nutrition education to conventional healthy dietary advice in subjects with previous history of GDM. Baseline characteristics were well matched in terms of socio-demographic and metabolic variables. Subjects in the LGI group of this study lost an average of 1.3 kg compared to the 0.1 kg loss in the CHDR arm, after six months on the trial. These findings are in agreement with the Cochrane review which concluded that subjects on low GI diets lost an average of 1 kg more than subjects on high GI diets, in trials that lasted up to 6 months in duration \\[40\\]. In this study, more post-GDM subjects in LGI group achieved a moderate 5% weight loss (P=0.01). Hence, low GI diets aid weight loss in the study population of women with previous GDM as compared to conventional healthy diets with similar energy prescriptions, during a 6 month period.\n\nModerate weight loss in the range of 5-10% has been proven to improve cardio-metabolic risk in high risk subjects \\[41\\]. Furthermore, every 1 kg weight reduction achieved in the first year of the Diabetes Prevention Program (DPP) translated into a 16% reduction in risk for conversion to T2DM \\[42\\]. Hence by promoting moderate weight loss in the group of post-GDM subjects, LGI diets may contribute effectively to lowering their cardio-metabolic risks.\n\nCHDR subjects had a significant increase of 0.6 mmol\/L from the baseline 2HPP levels, after six months' intervention. The magnitude of this change described by its effect size is \"moderate\" and remains a cause for concern in GDM women who are known to be at an increased risk for development of glucose intolerance \\[3\\]. Therefore, conventional healthy diets that emphasize on calorie and fat control do not benefit these women with prior GDM, in terms of improving glucose tolerance.\n\nThe LGI group, on the contrary, had maintained their 2HPP levels during the same period of study. As an increase in 2HPP predominantly represents glucose intolerance \\[17\\], lowering of 2HPP within these subjects by LGI diet would lead to the reduction of the prevalence of abnormal glucose tolerance. This line of thought finds further support in the higher rate of reversal to normoglycaemia observed among IGT subjects in LGI group after six months (50% vs. 40%, P= 0.689.). Furthermore, post-challenge plasma glucose spikes are thought to be more strongly associated with risk for atherosclerosis than FBS in a cohort at risk for diabetes \\[43\\]. Hence, the improvement in glucose tolerance could delay or prevent the subsequent development of T2DM and its complications in this high risk group of subjects.\n\nThere is a paucity of data on the normal fasting insulin range for young healthy Malaysian women. A Malaysian study among 30 healthy volunteers reported a median fasting insulin level of 4.7 \u03bcIU\/ml with a central 95% range of 2.1 to 12.1 \u03bcIU\/ml \\[44\\]. Baseline INS in 18\/38 subjects in CHDR group and 15\/39 subjects in LGI group were \\<2 \u03bcIU\/ml. Given that women with prior GDM have innate tendencies towards insulin resistance \\[45\\], one would expect our study population to have higher fasting insulin levels. It was unexpected that 57% of our subjects would have INS less than the 2.1 \u03bcIU\/ml. However, Farhan et al., (2012) also found the mean fasting insulin values, of a small group (n=10) of Austrian GDM women, at 3 month postpartum to be 1.63 \u03bcIU\/ml. One possible explanation for this occurrence is that only 43% of our subjects could be classified as being obese (BMI\\>27.5) \\[46\\], which could have resulted in the normoglycaemic state represented in the fasting blood glucose and normal or low INS. The median BMI of this study group was 26, and hence these subjects could be defined only as being mildly overweight based on the WHO criteria \\[47\\]. This finding suggests that despite their inherent insulin resistance accentuated by pregnancy, these subjects were at a very early stage of pre-diabetes, making early detection and intervention necessary if not paramount. We acknowledge that a large cross sectional study of healthy Malaysian individuals is necessary to determine normal reference fasting insulin range for this population.\n\nThe dietary intervention resulted in significant different outcomes in subjects with baseline INS\u22652 \u03bcIU\/ml with respect to anthropometry and 2HPP. For subjects with baseline INS\u22652 \u03bcIU\/ml the greatest improvement in glucose tolerance was noted among subjects in the lowest quartile for dietary GI at 6 months. Furthermore these subjects also achieved more weight loss when on LGI diets. These findings are consistent with the 6 months finding from the CALERIE study which reported that women with higher postprandial insulinaemic response lost more weight on low GI diets after 6 months on intervention \\[14\\]. However the mean baseline fasting insulin levels in the CALERIE subjects was 11.9 \u03bcIU\/ml, which was much higher than the mean for this study group.\n\nAdditionally, among subjects with higher baseline INS, the LGI group observed a significant reduction in the 2HPP while the 2HPP levels increased in the CHDR group. This difference in 2HPP changes between groups was statistically significant (Mean \u2206(LGI-CHDR) =2.4 mmol\/L, P=0.004). Since interventions that reduced 2-h plasma glucose by \\>0.84 mmol\/L are thought to halve the risk for incidence of T2DM \\[36\\], LGI diets may be highly recommended for post-GDM women with higher fasting insulin levels. Furthermore, in subjects with INS\u22652 \u03bcIU\/L the reduction in 2HPP brought about by lowering dietary GI was not primarily mediated through body weight loss. This mechanism of action is of special interest in post-GDM women, who have little success in attaining moderate weight loss compared to other high risk groups \\[1\\].\n\nThe favourable anthropometric and glycaemic responses to low GI intervention in those with higher insulin levels (as against low or normal insulin secretors) can be explained by the exaggerated responses to increase in GI demonstrated among hyperinsulinaemic (insulin resistant) subjects \\[14\\]. While we do not possess the postprandial insulin values for our subjects to classify them as having typical hyperinsulinaemic response to a glucose challenge, an increase in fasting insulin concentrations is also associated with lower liver insulin clearance, which conserves insulin and contributes to hyperinsulinaemia \\[48\\]. Recent studies further corroborate the findings that dietary GI may have varying effects depending on individual metabolic functioning of the \"adipo-insular axis\" \\[49\\].\n\nA trend for increasing weight and 2HPP blood glucose was observed in subjects with dysglycaemia within the CHDR group. Furthermore a higher likelihood of conversion to normoglycaemia among dysglycaemic subjects was observed in the LGI group. In the current trial, 37.5% and 64.3% of subjects with dysglycaemia at baseline, returned to normoglycaemia at 6 months in CHDR and LGI groups respectively. The percentage of dysglycaemic subjects returning to normoglycaemia in the LGI arm of our study was comparable to the results from lifestyle intervention in China that also used acarbose and metformin \\[50\\]. Hence, LGI diets could be more effective in managing glycaemia among hyperinsulinaemic, dysglycaemic women with a previous history of GDM.\n\nWe acknowledge the following limitations to the study. This study is limited by the fact that the dietary intake including GI and GL were calculated based on reported intakes, though attempts were made to ensure completeness of reporting. The 3-day food record was chosen to evaluate dietary data. This was done so since this tool is the least intrusive of diet recording techniques and was hence best suited to this group of subjects with many recognized barriers to participate in preventive interventions \\[5\\]. To improve the accuracy of the reporting, subjects were trained to use the three day food records at the screening visit and assisted by illustrations of household measures to aid in recording amounts consumed. Secondly, fifty- two percent of the subjects were identified as under-reporters based on a EI:BMR\\<1.2, \\[51\\]) at baseline. However, these observations were within the estimates for prevalence of under-reporting among overweight and obese women \\[52\\]. Furthermore, the percentage of under-reporters in LGI and CHDR groups were comparable between groups (p=0.405). Furthermore, excluding the under-reporters did not alter the statistical significance observed in the dietary intake among subjects. Food records also do not capture details of food processing and other factors affecting dietary variables, including GI. Nevertheless, outcome changes in the LGI and CHDR groups were consistent with previous reports for similar dietary intake comparisons \\[34,35\\], thereby adding credence to the dietary data obtained from the subjects.\n\nSelf-reported dietary data are considered acceptable as indicators of dietary quality including percentage contribution to energy from macronutrients \\[53\\]. Dietary GI, being a measure of quality could also be expected to be minimally affected by underreporting. However, it is felt that underreporting may have also contributed to the smaller differences in GL observed between the groups.\n\nIt is also acknowledged that the clinically significant 10 point difference in dietary GI between the groups \\[54\\], could not be achieved after 6 months of intervention. Also the dietary GI for the LGI group was actually in the intermediate GI category (diet GI less than sixty \\[16\\]). However, more subjects in LGI had intermediate GI (62 vs. 21%), while more subjects in the CHDR group were in the high GI category (79 vs. 37%). Asian trials that achieved comparable difference in GI of around six units between groups, have also documented significant beneficial effects in terms of reductions in waist circumference, fasting blood sugar and glycaemic control in diabetic subjects or those with impaired fasting glucose \\[34,35\\]. This study was able to demonstrate that moderate reductions in GI of seven units had beneficial effects on the anthropometric and 2HPP outcomes of Asian post-GDM women with postpartum normoglycaemia, IFG or IGT.\n\nWe concede that neither group reached the universal 25-30 g dietary fibre intake recommendation. Achieving this target in the Malaysian environment has been recognized as a challenge \\[55\\]. However, it should be noted that LGI group had a significantly higher fibre intake as compared to the CHDR group in this study (17 vs. 13 g, P\\<0.001). Thus, adding GI education to CHDR improved dietary fibre intakes in subjects which could not be achieved with CHDR. Such additional benefits in dietary quality when using low GI diets in the Asian population has also been documented earlier \\[56\\].\n\nWe do not possess formal data on satisfaction of the subjects with the two dietary interventions. However, self-reported adherence and calculated adherence to energy and fat intake prescriptions and drop-out rates were comparable between the groups. Thus it may be appropriate to consider that acceptance to both interventions may also be similar.\n\nThe assay employed to measure INS samples in this study, failed to analyse samples with INS \\<2 \u03bcIU\/ml. Hence the INS cut-off used to group subjects to conduct the sub-analysis to investigate the differential effect of the diets in subjects with varying insulin levels can be considered arbitrary. However, the varied metabolic response to the two diets provides a strong evidence for the added benefit of lowering GI for subjects with higher insulin levels.\n\n# Conclusions\n\nThis six months intervention study showed that lowering the dietary GI of conventional diets improved glucose tolerance and reduced body weight, in comparison to the conventional diet in women with previous history of GDM. Subjects with higher fasting insulin level in the low GI group demonstrated the most positive effects in the improvement of their dysglycaemia and weight loss.\n\n# Abbreviations\n\n2HPP: 2-h post 75 g OGTT blood glucose; BMR: Basal metabolic rate; FBS: Fasting blood sugar; GDM: Gestational diabetes mellitus; GI: Glycaemic index; GL: Glycaemic load; INS: Fasting serum insulin; OGTT: Oral glucose tolerance test; T2DM: Type 2 diabetes mellitus.\n\n# Competing interests\n\nThe authors have no potential conflict of interests to declare.\n\n# Authors' contributions\n\nNAK and FA conceived the project, designed the study, and were involved in data interpretation and editing of the manuscript. SS and RAG helped with study design, data collection, data analysis, interpretation and wrote the manuscript. NAW was involved in study design, data collection, and editing the manuscript. BNMY and NSS participated in designing the study, data interpretation and editing of the manuscript. KC was involved in study design, data analysis and interpretation and editing of the manuscript. All authors read and approved the final manuscript.\n\n## Acknowledgements\n\nGardenia Bakeries, KL provided vouchers for low- GI Breakthru Bread for subjects in the Low GI group. This project was funded by an internal research grant from International Medical University, KL, Malaysia.","meta":{"dup_signals":{"dup_doc_count":132,"dup_dump_count":38,"dup_details":{"curated_sources":2,"2023-14":1,"2022-49":1,"2021-49":1,"2021-10":1,"2020-34":1,"2019-47":1,"2019-39":1,"2017-17":1,"2017-09":24,"2017-04":1,"2016-50":1,"2016-44":1,"2016-40":1,"2016-36":1,"2016-30":1,"2016-22":1,"2016-18":1,"2015-48":4,"2015-40":1,"2015-35":4,"2015-32":4,"2015-27":4,"2015-22":4,"2015-14":4,"2014-52":4,"2014-49":4,"2014-42":11,"2014-41":5,"2014-35":8,"2014-23":6,"2014-15":6,"2023-50":1,"2015-18":4,"2015-11":3,"2015-06":4,"2014-10":4,"2013-48":4,"2024-26":1}},"file":"PMC3671161"},"subset":"pubmed_central"}
{"text":"abstract: Although most epidemic human infectious diseases are caused by recently introduced pathogens, cospeciation of parasite and host is commonplace for endemic infections. Occasional host infidelity, however, provides the endemic parasite with an opportunity to survive the potential extinction of its host. Such infidelity may account for the survival of certain types of human lice, and it is currently exemplified by viruses such as HIV.\nauthor: Robin A Weiss\ndate: 2009\ninstitute: 1Division of Infection and Immunity, University College London, 46 Cleveland Street, London W1T 4JF\nreferences:\ntitle: Apes, lice and prehistory\n\nHans Zinsser's *Rats*, *Lice and History* \\[1\\] is a classic in microbiology. Written in 1934 and subtitled *The biography of a bacillus*, it tells the tale of that dreaded disease typhus, its reservoir in rats and its transmission among humans by lice. Here, I discuss how we may in the course of prehistory have acquired the lice, and how other infections may, like the typhus bacillus, come to be shared by us and the animal species with which we are in close contact. It is a tale of infidelity that I shall begin with the recent research on lice of David Reed and colleagues \\[2,3\\] and of Mark Stoneking's group \\[4\\] who, on the basis of phylogenetic analysis, have speculated that we may have acquired a clade of head lice from another hominid species and pubic lice from gorillas; they have also suggested that lice might help determine the date when humans adopted clothing. I shall examine this unfolding story in the context of what we know about microbial infections, and will look at the promiscuity of viruses through the lens of modern molecular technology; and I will add my own speculation on why naked apes have pubic hair.\n\nLice are small, wingless insects that cannot live independently from their hosts (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>). They are frequent parasites of birds and mammals, each host species having its own type of louse. Humans harbor three kinds of these ectoparasites: head lice, body lice and pubic lice. For much of human history and prehistory blood-sucking lice have been so prevalent that they became part of our everyday language. We speak about feeling lousy and admonish our friends for nit-picking. Nits are the eggs of lice, expertly cemented onto hair shafts, as many parents know from painstakingly combing them out of their children's hair. Grooming among monkeys and apes is not only a means of social bonding but also a useful way of controlling nits and lice (Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>).\n\n# Family heirlooms and new acquisitions\n\nWriting on infections of humankind, Tony McMichael and I \\[5\\] have called those that cospeciated with their hosts 'family heirlooms' and those that crossed over from other hosts in recent evolutionary time 'new acquisitions'. The new acquisitions were initially derived from zoonotic infections but have flourished as self-sustaining infections in the human population. They have diverged from their progenitors in the original host: for example, measles is now distinct from rindepest. The majority of zoonoses, however, remain in their animal reservoirs and, so far as their sojourn in humans goes \u2013 even with limited human-to-human transfer (as with Ebola or severe acute respiratory syndrome (SARS)) \u2013 we can regard them as 'temporary exhibits'.\n\nHuman DNA might show 98% similarity to that of chimps, but we share less than 50% of our microbes and parasites with them. Ashford \\[6\\] argues that the great apes became more specialized forest dwellers at the same time that early hominids explored the savannah, and that human gut parasites resemble those of omnivorous baboons more than those of chimps because humans, like baboons but unlike chimps, are omnivorous. Further opportunities for horizontal crossover of microbes and parasites from animals to humans arose when humans spread out of Africa. When we domesticated ruminants, and animals such as dogs, cats and rats 'domesticated' us for the rich pickings around human habitation, we acquired many infections from our new neighbors \\[5,7\\]. Thus a shared habitat, rather than a shared ancestry, is important for the acquisition of many infections.\n\nMost human pandemic infections were acquired horizontally very recently on the evolutionary timescale, even though diseases such as typhus, measles and smallpox first occurred in prehistoric times. These new acquisitions originate from evolutionarily distant host species. The influenza pandemic of 1918\u20131919 came from birds, and the 1968 influenza strain could be an avian-porcine recombinant. Today's novel viral infections are more likely to originate from exotic species than from animals that were domesticated long ago \\[5\\]. The market for 'bushmeat' has led to Ebola virus outbreaks in Africa from butchering primates, and to the SARS outbreak in China from eating small carnivores, such as civet cats. The primary reservoirs of the Ebola filovirus, the SARS coronavirus and the Nipah paramyxovirus, however, seem to be in fruit bats (flying foxes).\n\nLice and nits have been found in textiles, hair and combs excavated from archaeological sites \\[1,8\\]. Given that the closest relative of the human head and body louse, *Pediculus humanus*, is *P. schaeffi*, which infests chimpanzees, one might assume that human and chimp lice have cospeciated with their hosts as family heirlooms ever since they diverged from a common ancestor. This requires, however, that the divergence among hosts and parasites approximates to the same timescale. After all, the closest relative to human immunodeficiency virus type 1 (HIV-1) is the simian immunodeficiency virus of chimpanzees (SIVcpz), but it would be facile to suggest that HIV-1 co-evolved with humans, because molecular clock estimates place the most recent common ancestor of the pandemic form of HIV-1 at 75\u2013100 years ago \\[9,10\\], and this is likely to be close to the time of the species crossover event. HIV-1 has invaded humans at least three times (groups M, N and O), and such is the power of modern forensic DNA virology that the precise location in Cameroon has been mapped for the chimpanzees carrying the SIVcpz most closely related to group M \\[11,12\\].\n\n# Origins of head and body lice\n\nUsing nuclear and mitochondrial DNA markers, Reed *et al*. \\[2\\] estimated the divergence of chimp and human *Pediculus* lice at 5.5 million years ago (MYA) and provided evidence of cospeciation with their hosts. A recent revision to 4.1 MYA for the most recent common ancestor of chimps and humans \\[13\\] may require a similar adjustment of the louse molecular clock. What is more remarkable, however, is that Reed *et al*. \\[2\\] found that human lice split into two quite distinct clades, A and B, about 1.18 MYA. There is a worldwide clade (which includes both head and body lice) and a New World clade (exclusively head lice). So how can humans harbor two clades of louse that diverged from each other over one million years ago, when that separation is tenfold older than the emergence of *Homo sapiens*?\n\nThe answer, Reed *et al*. \\[2\\] suggested, is that the separation took place around the time of divergence of the ancestors of modern humans from *Homo erectus*. These two hominid lineages then co-existed for about one million years until the demise of *H. erectus*. When modern humans radiated across Asia they might have had contact with *H. erectus*, just as in more recent millennia *H. sapiens* met *H. neanderthalensis* in Europe, as dramatized by the Nobel laureate William Golding \\[14\\]. There is no evidence that different human species interbred, but they may well have exchanged ectoparasites. Thus, the New World clade of head louse may have crossed horizontally from *H. erectus* to *H. sapiens* within the last 100,000 years.\n\nZinsser \\[1\\] noted that the hair of ancient Peruvian mummies and the scalps of pre-Columbian Native Americans contained nits or lice. Recent DNA analysis of lice from similar remains indicates that they belong to the worldwide clade A, so this clade must have been present in pre-Columbian American populations \\[15\\]. A third clade of head lice has been delineated in Ethiopia and Nepal and this clade, C, diverged from clades A and B about 2 MYA \\[16\\]. If Reed *et al*. \\[2\\] were correct to postulate that clade B came from *H. erectus*, one must wonder in which hominid population might clade C lice have maintained their separate identity.\n\n# Lice and clothing\n\nHead and body lice used to be designated *Pediculus capitis* and *P. corporis* but they are now known to belong to the same species, *P. humanus* x\\[16,17\\]. Fifty years ago Levene and Dobzhansky \\[18\\] showed that head lice could be trained or adapted to become the rather larger body lice by attaching them to the body in small pill boxes. As we celebrate the 150th anniversary of Darwin's *Origin of Species* we might recall that it was Theodosius Dobzhansky, an eminent evolutionary biologist and a Russian Orthodox Christian, who in 1973 famously challenged creationists by declaring that \"Nothing in biology makes sense except in the light of evolution.\" His research on lice was no exception.\n\nKittler *et al*. \\[4\\] initially reckoned that body lice diverged from head lice approximately 70,000 years ago, but they later increased this estimate to 107,000 years ago by correcting an error concerning the original outlier sequence. They postulated that this date of about 100,000 years ago coincided with or followed soon after the origin of clothing, because the naked human body is an inhospitable place for lice to breed. Head lice feed on the scalp and breed in hair, whereas body lice feed on the skin but breed in clothing.\n\nMore extensive phylogenetic analyses \\[16,17\\] indicate that body lice evolved from head lice several times within the worldwide clade A, as they are found in many branches of the cladisitic tree. Multiple derivations of body lice from head lice had already been considered by Zinsser \\[1\\], and it makes good sense if one considers that clothing was not a single invention. Wearing animal pelts fur-side next to the skin would have provided a suitable place for lice to breed before fabrics were developed with the inventions of spinning and weaving.\n\nIn 17th and 18th century Europe, most of the aristocracy and gentry shaved their hair and wore wigs. Had this custom arisen to protect them from lice as Zinsser \\[1\\] suggests? Not according to Samuel Pepys' diary, as he complained more than once about his wig being infested: \"Thence to my barbers, to have my periwig cleared of its nits.\" I wonder if they were head or body lice \u2013 is a wig hair or clothing?\n\n# Lice and nudity\n\nWhy naked apes are naked and when we 'lost' our hair has long been disputed, as discussed by Desmond Morris in *The Naked Ape* \\[19\\]. Rantala \\[20\\] suggested that nakedness could have had a selective advantage to rid the body of lice and other ectoparasites, a view also championed by Pagel and Bodmer \\[21\\], who added that being seen to be free of lice would be a fitness indicator and a good mating strategy. I am rather drawn to the theory first postulated by Alister Hardy \\[22\\] that humans evolved through an aquatic stage, although most anthropologists disparage this hypothesis. Yet Ashford \\[6\\] points out that several parasites specific to humans, such as three *Schistosoma* species, depend for their transmission on our entering water, which again distinguishes us from the great apes.\n\n# Pubic lice\n\nPubic lice are commonly called crabs because of their appearance (Figure <a href=\"#F1\" data-ref-type=\"fig\">1c<\/a>). They belong to a different genus, *Pthirus* (a misspelling of *Phthirus* dating back to Linnaeus), from head and body lice. On the basis of morphology, human *Pthirus pubis* is closely related to the gorilla louse, *Pthirus gorillae*. In a recent paper David Reed \\[3\\] chose a punning Miltonian title, \"Pair of lice lost or parasites regained\", because it poses the conundrum of whether all the great apes had variants of both *Pediculus* and *Pthirus* and lost one or the other, or whether humans gained *Pthirus* in addition to *Pediculus*.\n\nMolecular phylogeny indicates that human pubic lice diverged from gorilla lice as recently as 3.3 MYA \\[3\\], whereas the chimp and human host lineage split from the gorilla lineage at least 7 MYA (Figure <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>). Thus, it seems clear that humans acquired pubic lice horizontally, possibly at the time of the *Pthirus* species' split and probably directly from gorillas. Because they were already adapted to the coarse body hair of the gorilla, crabs would have found a suitable niche in human pubic hair. Indeed, the diameter of the hair is most likely the key rather than the pubic region, because pediatric infestation of *P. pubis* is well documented: the crab is then found on the eyelashes of the infant.\n\n# Origin of pubic hair\n\nReed *et al*. \\[3\\] suggest that the most recent common ancestor of the genera *Pediculus* and *Pthirus* was about 12.5 MYA, which is earlier than the estimated divergence of gorillas and the chimpanzee-human lineage. So was there duplication and separation of lice in the African anthropoid ape lineage, where they could have occupied separate ecological niches, rather as human head lice and pubic lice do today? Although this is an intriguing hypothesis, I was having difficulty envisioning a clear separation of habitats between the groin and other parts of our ancient common ancestor. My 'eureka moment' came, appropriately enough, in the shower: although naked apes have pubic hair, surely our hairy cousins don't?\n\nHow could I test my hypothesis? I knew that there was a stuffed chimpanzee in the Grant Zoological Museum at University College London and I called in on the way to my laboratory. Alas, he was a juvenile, which left the question open. A brisk walk across Regent's Park to inspect the adult gorillas in their splendid new pavilion at London Zoo strengthened my suspicion, and this was later confirmed by a visit to the chimpanzees at Whipsnade Zoo north of London. Indeed, as I noted previously \\[23\\], all the species of apes, Old World monkeys and New World monkeys seem to be less hairy in the pubic region than elsewhere; fur is present but it is short and fine.\n\nWhy do adult humans sport a thick bush of wiry pubic hair, uniquely among primates? It must surely be because we are otherwise naked. It probably serves both a visual and an odorous function, because hair aids the distribution of apocrine scent secretion, like our less visually stunning axillary hair. Unlike a beard, pubic hair is not sexually dimorphic, yet it is a feature of sexual maturation. No wonder that pubic lice are said to be the most contagious of all sexually acquired infections. Which came first, nakedness or pubic hair? I would postulate that the development of pubic hair was a consequence of the visible nakedness elsewhere on the body. Perhaps the acquisition of *P. pubis* 3.3 MYA provides the clue to when hominids developed thick pubic hair, rather as the evolution of body lice is thought to be broadly contemporaneous to the development of clothing.\n\nIt is noteworthy that the prevalence of infestation by pubic lice seems to be decreasing among women and men who remove their pubic hair using 'bikini wax', rather as the Renaissance painters discreetly depilated classical female nudes. A study from The General Infirmary, Leeds, UK, records the increasing predilection among attendants at a clinic for sexually transmitted infections to undertake extensive pubic hair waxing known as the 'Brazilian', leaving only a thin strip of hair. Armstrong and Wilson \\[24\\] noted a significant fall in the incidence of pubic lice among patients, although gonorrhea and chlamydia increased over the same period. Thus, there may be a health benefit to this emerging sexual lifestyle, and this finding would also lend support to the notion that nakedness protects humans from ectoparasites.\n\n# Humans as repositories of ancient infections\n\nGiven that humans are *nouveaux riches* regarding our collection of infections \\[5\\], can we infer further examples of infections that could have come from other hominid species or from more distantly related primates? The infections for which we have the most accurate evolutionary record are the endogenous retroviruses that have invaded host DNA. Some 8% of the human genome represents 'fossil' integrated proviruses. The human lineage accumulated several thousand retroviral genomes after the split between New World and Old World primates, but only a handful since we diverged from chimpanzees.\n\nParasites that have tightly cospeciated with their hosts are, of course, in grave danger of extinction when that host becomes an endangered species. But the 'smart' parasite would gain a whole new lease of evolutionary opportunity if it engaged in occasional 'infidelity', analogous to mutation in DNA replication. A DNA lineage that does not mutate would have an extraordinarily slow rate of evolution, whereas a super-mutator without repair would provide little opportunity for natural selection before the genotype changed further. Thus, a low mutation rate within broad fidelity of DNA replication allows for both inheritance and evolution. Likewise, total cospeciation dooms the parasite to the fate of the host, whereas the ability to move horizontally to closely related hosts would provide flexibility. Parasite jumping between related hosts might occur more frequently than realized because it might easily be overlooked.\n\nBy this reckoning, the New World clade of head lice formerly faithful to the *H. erectus* lineage \\[2\\] adopted modern humans in the nick of time. HIV-1 might well be another successful case of jumping off a sinking ship, because its former host is not likely to survive for many generations longer in the wild. Now that HIV-1 has adopted a new host species, it is enjoying a most successful adaptive radiation and has already colonized around 60 million humans (25 million of whom have died from AIDS). Such crossover events, however, are relatively rare, and only one of the three ape-to-human transfers of HIV-1 has taken off to cause the AIDS pandemic \\[11\\]. It pays the host to place barriers known as restriction factors in the path of potential pathogens. If a species barrier is not recognized by the new invader or is successfully circumvented, the infection can be more virulent in the new host.\n\nRegarding the intestinal parasites and ectoparasites that specialize in human infestation, Ashford \\[6\\] pointed out that we not only house two kinds of closely related lice (he meant *Pediculus* head and body lice), but also two species of *Cimex* bedbugs, two of *Demodex* mites and two of *Taenia* tapeworms. He therefore asked if we were once two separate populations that rejoined after a long separation, but Ashford did not have DNA sequences and molecular clock estimates available to him. Can we now view this phenomenon in the same way as the lice, as one of the pair of parasites cospeciating with *H. sapiens* and the other jumping from non-ancestral archaic humans to the modern human lineage? It would be intriguing to conduct similar molecular phylogenies of Ashford's other pairs.\n\nAshford \\[6\\] finished by stating: \"Over to the microbiologists: What do the bacteria, viruses and fungi tell us?\" There are ample examples both of cospeciation and horizontal transmission. The malaria parasite *Plasmodium falciparum* exemplifies a complex cospeciation between the parasite, its human host and its mosquito vector. *Anopheles gambiae* has also coevolved to be a specialist feeder on humans; by contrast, the other human malaria parasite, *P. vivax*, has an origin in South East Asian monkeys and is transmitted by the more promiscuous *Culex* species. Might *vivax* malaria have first adapted to *H. erectus* as an intermediate between monkeys and humans?\n\nAs a virologist, I have felt stimulated \\[23\\] to take up Ashford's challenge to consider pairs of related human viruses. HIV-1 and HIV-2 are very recent 20th century arrivals, from chimpanzees and sooty mangabey monkeys, respectively. With human T-cell lymphotropic viruses types 1 and 2 (HTLV-1 and HTLV-2), there have been multiple introductions of HTLV-1 from African and Asian monkeys and apes \\[25\\]. The provenance of HTLV-2 is puzzling, as its reservoir is in indigenous American populations and in African pygmies, which are as far apart in *H. sapiens* as one can find. Humans have five distinct polyoma viruses and multiple genital papilloma virus types. The different clades of hepatitis C virus (HCV) have deep roots but there are no known animal relatives to provide an anchor or time calibration. It would be fascinating to learn whether archaic humans harbored HCV variants, but as HCV has an RNA genome there is not much hope of gaining direct evidence by sequence amplification from ancient specimens.\n\nAll members of the herpesvirus family are thought to have strictly cospeciated with their hosts, though I have my doubts. The closest relatives to human herpes simplex virus (an \u03b1 herpesvirus), cytomegalovirus (\u03b2) and Epstein-Barr virus (\u03b3), seem to be those in the chimpanzee. Several simian species have a pair of distinct rhadinoviruses, whereas so far humans are only known to harbor one, Kaposi's sarcoma herpesvirus. On the other hand, humans have two herpes simplex viruses (HSVs), types 1 and 2. Phylogenetic analysis \\[26\\] indicates that HSV-1 and HSV-2 are further apart from each other than HSV-1 is from its chimp ortholog \\[27\\]. So where did HSV-2 come from? From its estimated age of divergence from the chimp-human HSV-1 lineage, I would place a bet on horizontal transmission from gorillas or possibly from orang-utans \\[23\\].\n\nIs it a coincidence that three human parasites acquired horizontally from great apes, namely pubic lice, HIV-1 and speculatively HSV-2, are sexually transmitted? Now, before one conjures up a King Kong scenario, it should be noted that predators can pick up parasites from their prey. The close contact involved in human ancestors butchering gorillas could have enabled *Pthirus* to jump hosts, rather as bushmeat slaughter practices probably led SIVcpz and other retroviruses to invade humans from chimpanzees in modern times \\[11,25\\].\n\n# Humans as a source of infections\n\nWith the advent of globalization, previously isolated human populations lost ninetenths of their number to the infections introduced by intrepid migrants and invaders \\[7\\]. Hernan Cortes conquered the mighty Aztec empire thanks to smallpox and measles, which the invaders inadvertently introduced to the disease-naive New World peoples \\[5\\]. The subsequent population crash was severe; so few indigenous people survived that the lucrative African slave trade was established in order to provide labor in the plantations. This pattern of export of Old World diseases was repeated in South America, Australia and Oceania. As Charles Darwin remarked in his diary on the voyage of the Beagle, \"Wherever the European has trod death seems to pursue the Aboriginal.\"\n\nIt is plausible, then, that modern humans could have transmitted lethal infectious diseases to archaic human species. *H. erectus* might have given us a clade of head lice, but *H. sapiens* may have conveyed more deadly infections to *H. erectus* and later to *H. neanderthalensis*. Hard evidence is lacking, and prehistoric legends seldom tell the tale from the point of view of the vanquished, although Golding did through the eyes of the Neanderthals \\[14\\]. To paraphrase Darwin, wherever modern humans trod during the Pleistocene era, death seemed to pursue archaic humans. There is also a danger today that the surviving great apes may be subjected to a *coup de grace* from human infections transmitted through jungle safaris and ecotourism.\n\nCross-species virulence is well known. Myxamotosis resident in American cotton-tail rabbits devastated the European rabbit, and the disappearance of red squirrels in Britain wherever American gray squirrels occur is probably due to a pox virus rather than direct competition for habitat. Similarly, the \u03b1-herpesviruses that have cospeciated with Indian and African elephants cause nothing more severe than cold sores in their natural host but each seems to be lethal to the other when the two species are unnaturally housed together in zoos \\[28\\]. SIVcpz has little effect on chimpanzees but HIV-1 causes AIDS in humans. Thus, it might pay the host to carry a fairly harmless parasite if that infection is lethal to the host's competitors.\n\n# Epilog\n\nBody lice, and occasionally head and pubic lice, transmit bacterial diseases: typhus (*Rickettsia prowazekii*) \\[29\\], trench fever (*Bartonella quintana*) and relapsing fever (*Borellia recurrentis*). The lice themselves succumb to typhus infection but pass the Rickettsia in their feces, which the human then scratches into the skin. Typhus is known as 'war fever' and 'jail fever' because it appears in conditions where lice thrive. At the end of *Rats*, *Lice and History* Zinsser \\[1\\] wrote \"Typhus is not dead. It will live on for centuries and will break into the open whenever human stupidity and brutality give it a chance.\" Sadly, Zinsser's words were prophetic. Within a few years, typhus became the major slayer in the Nazi concentration camps; typhus broke out among Rwandan refugees in Burundi in 1994, in Bosnia in 1995 and most recently in Goma.\n\nHuman brutality is another feature shared with chimpanzees that has survived in the human lineage \\[30\\]. It is too bad that we are not closer to the pygmy chimp (bonobo), which evolved a means of conflict resolution between troupes through alpha females engaging in lesbian sex. But that is another story.\n\n### Acknowledgements\n\nI am grateful to Tim Harrison and David Reed for commenting on a draft of this paper.","meta":{"dup_signals":{"dup_doc_count":103,"dup_dump_count":29,"dup_details":{"curated_sources":1,"2023-50":2,"2023-14":1,"2022-49":1,"2022-40":1,"2022-21":1,"2021-49":1,"2021-31":1,"2021-21":2,"2020-40":1,"2020-10":1,"2019-47":1,"2019-39":1,"2017-30":1,"2016-36":1,"2016-07":16,"2015-48":4,"2015-40":2,"2015-35":4,"2015-32":4,"2015-27":4,"2015-22":6,"2015-14":3,"2014-52":4,"2014-49":5,"2014-42":13,"2014-41":5,"2014-35":5,"2014-23":5,"2014-15":6}},"file":"PMC2687769"},"subset":"pubmed_central"}
{"text":"author: Pabulo Henrique Rampelotto\ndate: 2013-08-07\ninstitute: Interdisciplinary Center for Biotechnology Research, Federal University of Pampa, Ant\u00f4nio Trilha Avenue, P.O.Box 1847, 97300-000, S\u00e3o Gabriel\u2013RS, Brazil; E-Mail: <firstname.lastname@example.com>\ntitle: Extremophiles and Extreme Environments\n\nOver the last decades, scientists have been intrigued by the fascinating organisms that inhabit extreme environments. Such organisms, known as extremophiles, thrive in habitats which for other terrestrial life-forms are intolerably hostile or even lethal. They thrive in extreme hot niches, ice, and salt solutions, as well as acid and alkaline conditions; some may grow in toxic waste, organic solvents, heavy metals, or in several other habitats that were previously considered inhospitable for life. Extremophiles have been found depths of 6.7 km inside the Earth's crust, more than 10 km deep inside the ocean\u2014at pressures of up to 110 MPa; from extreme acid (pH 0) to extreme basic conditions (pH 12.8); and from hydrothermal vents at 122 \u00b0C to frozen sea water, at \u221220 \u00b0C. For every extreme environmental condition investigated, a variety of organisms have shown that they not only can tolerate these conditions, but that they also often require those conditions for survival.\n\nThey are classified according to the conditions in which they grow: As thermophiles and hyperthermophiles (organisms growing at high or very high temperatures, respectively), psychrophiles (organisms that grow best at low temperatures), acidophiles and alkaliphiles (organisms optimally adapted to acidic or basic pH values, respectively), barophiles (organisms that grow best under pressure), and halophiles (organisms that require NaCl for growth). In addition, these organisms are normally polyextremophiles, being adapted to live in habitats where various physicochemical parameters reach extreme values. For example, many hot springs are acid or alkaline at the same time, and usually rich in metal content; the deep ocean is generally cold, oligotrophic (very low nutrient content), and exposed to high pressure; and several hypersaline lakes are very alkaline.\n\nExtremophiles may be divided into two broad categories: extremophilic organisms which require one or more extreme conditions in order to grow, and extremotolerant organisms which can tolerate extreme values of one or more physicochemical parameters though growing optimally at \"normal\" conditions.\n\nExtremophiles include members of all three domains of life, *i.e.*, bacteria, archaea, and eukarya. Most extremophiles are microorganisms (and a high proportion of these are archaea), but this group also includes eukaryotes such as protists (e.g., algae, fungi and protozoa) and multicellular organisms.\n\nArchaea is the main group to thrive in extreme environments. Although members of this group are generally less versatile than bacteria and eukaryotes, they are generally quite skilled in adapting to different extreme conditions, holding frequently extremophily records. Some archaea are among the most hyperthermophilic, acidophilic, alkaliphilic, and halophilic microorganisms known. For example, the archaeal *Methanopyrus kandleri* strain 116 grows at 122 \u00b0C (252 \u00b0F, the highest recorded temperature), while the genus *Picrophilus* (e.g., *Picrophilus torridus*) include the most acidophilic organisms currently known, with the ability to grow at a pH of 0.06.\n\nAmong bacteria, the best adapted group to various extreme conditions is the cyanobacteria. They often form microbial mats with other bacteria, from Antarctic ice to continental hot springs. Cyanobacteria can also develop in hypersaline and alkaline lakes, support high metal concentrations and tolerate xerophilic conditions (*i.e.*, low availability of water), forming endolithic communities in desertic regions. However, cyanobacteria are rarely found in acidic environments at pH values lower than 5\u20136.\n\nAmong eukaryotes, fungi (alone or in symbiosis with cyanobacteria or algae forming lichens) are the most versatile and ecologically successful phylogenetic lineage. With the exception of hyperthermophily, they adapt well to extreme environments. Fungi live in acidic and metal-enriched waters from mining regions, alkaline conditions, hot and cold deserts, the deep ocean and in hypersaline regions such as the Dead Sea. Nevertheless, in terms of high resistance to extreme conditions, one of the most impressive eukaryotic polyextremophiles is the tardigrade, a microscopic invertebrate. Tardigrades can go into a hibernation mode, called the tun state, whereby it can survive temperatures from \u2212272 \u00b0C (1 \u00b0C above absolute zero!) to 151 \u00b0C, vacuum conditions (imposing extreme dehydration), pressure of 6,000 atm as well as exposure to X-rays and gamma-rays. Furthermore, even active tardigrades show tolerance to some extreme environments such as extreme low temperature and high doses of radiation.\n\nIn general, the phylogenetic diversity of extremophiles is high and very complex to study. Some orders or genera contain only extremophiles, whereas other orders or genera contain both extremophiles and non-extremophiles. Interestingly, extremophiles adapted to the same extreme condition may be broadly dispersed in the phylogenetic tree of life. This is the case for different psychrophiles or barophiles, for which members may be found dispersed in the three domains of life. There are also groups of organisms belonging to the same phylogenetic family that have adapted to very diverse extreme or moderately extreme conditions.\n\nOver the last few decades, the fast development of molecular biology techniques has led to significant advances in the field, allowing us to investigate intriguing questions on the nature of extremophiles with unprecedented precision. In particular, new high-throughput DNA sequencing technologies have revolutionized how we explore extreme microbiology, revealing microbial ecosystems with unexpectedly high levels of diversity and complexity. Nevertheless, a thorough knowledge of the physiology of organisms in culture is essential to complement genomic or transcriptomic studies and cannot be replaced by any other approach. Consequently, the combination of improved traditional methods of isolation\/cultivation and modern culture-independent techniques may be considered the best approach towards a better understanding of how microorganisms survive and function in such extreme environments.\n\nBased on such technological advances, the study of extremophiles has provided, over the last few years, ground-breaking discoveries that challenge the paradigms of modern biology and make us rethink intriguing questions such as \"what is life?\", \"what are the limits of life?\", and \"what are the fundamental features of life?\". These findings have made the study of life in extreme environments one of the most exciting areas of research, and can tell us much about the fundaments of life.\n\nThe mechanisms by which different organisms adapt to extreme environments provide a unique perspective on the fundamental characteristics of biological processes, such as the biochemical limits to macromolecular stability and the genetic instructions for constructing macromolecules that stabilize in one or more extreme conditions. These organisms present a wide and versatile metabolic diversity coupled with extraordinary physiological capacities to colonize extreme environments. In addition to the familiar metabolic pathway of photosynthesis, extremophiles possess metabolisms based upon methane, sulfur, and even iron.\n\nAlthough the molecular strategies employed for survival in such environments are still not fully clarified, it is known that these organisms have adapted biomolecules and peculiar biochemical pathways which are of great interest for biotechnological purposes. Their stability and activity at extreme conditions make them useful alternatives to labile mesophilic molecules. This is particularly true for their enzymes, which remain catalytically active under extremes of temperature, salinity, pH, and solvent conditions. Interestingly, some of these enzymes display polyextremophilicity (*i.e.,* stability and activity in more than one extreme condition) that make their wide use in industrial biotechnology possible.\n\nFrom an evolutionary and phylogenetic perspective, an important achievement that has emerged from studies involving extremophiles is that some of these organisms form a cluster on the base of the tree of life. Many extremophiles, in particular the hyperthermophiles, lie close to the \"universal ancestor\" of all organisms on Earth. For this reason, extremophiles are critical for evolutionary studies related to the origins of life. It is also important to point out that the third domain of life, the archaea, was discovered partly due to the first studies on extremophiles, with profound consequences for evolutionary biology.\n\nFurthermore, the study of extreme environments has become a key area of research for astrobiology. Understanding the biology of extremophiles and their ecosystems permits developing hypotheses regarding the conditions required for the origin and evolution of life elsewhere in the universe. Consequently, extremophiles may be considered as model organisms when exploring the existence of extraterrestrial life in planets and moons of the Solar System and beyond. For example, the microorganisms discovered in ice cores recovered from the depth of the Lake Vostok and other perennially subglacial lakes from Antarctica may serve as models for the search of life in the Jupiter's moon Europa. Microbial ecosystems found in extreme environments like the Atacama Desert, the Antarctic Dry Valleys and the Rio Tinto may be analogous to potential life forms adapted to Martian conditions. Likewise, hyperthermophilic microorganisms present in hot springs, hydrothermal vents and other sites heated by volcanic activity in terrestrial or marine areas may resemble potential life forms existing in other extraterrestrial environments. Recently, the introduction of novel techniques such as Raman spectroscopy into the search of life signs using extremophilic organisms as models has open further perspectives that might be very useful in astrobiology.\n\nWith these groundbreaking discoveries and recent advances in the world of exthemophiles, which have profound implications for different branches of life sciences, our knowledge about the biosphere has grown and the putative boundaries of life have expanded. However, despite the latest advances we are just at the beginning of exploring and characterizing the world of extremophiles. This special issue discusses several aspects of these fascinating organisms, exploring their habitats, biodiversity, ecology, evolution, genetics, biochemistry, and biotechnological applications in a collection of exciting reviews and original articles written by leading experts and research groups in the field. I would like to thank the authors and co-authors for submitting such interesting contributions. I also thank the Editorial Office and numerous reviewers for their valuable assistance in reviewing the manuscripts.\n\n## Conflict of Interest\n\nThe authors declare no conflict of interest.","meta":{"dup_signals":{"dup_doc_count":140,"dup_dump_count":37,"dup_details":{"curated_sources":2,"2018-09":1,"2017-47":1,"2017-39":1,"2017-30":3,"2017-22":1,"2017-17":2,"2017-09":2,"2017-04":3,"2016-50":3,"2016-44":4,"2016-40":4,"2016-36":4,"2016-30":4,"2016-26":4,"2016-22":3,"2016-07":4,"2015-48":3,"2015-40":3,"2015-35":5,"2015-32":5,"2015-27":3,"2015-22":5,"2015-14":5,"2014-52":4,"2014-49":6,"2014-42":11,"2014-41":7,"2014-35":5,"2014-23":8,"2014-15":4,"2018-17":1,"2015-18":5,"2015-11":5,"2015-06":4,"2014-10":2,"2013-48":2,"2017-13":1}},"file":"PMC4187170"},"subset":"pubmed_central"}
{"text":"abstract: Advances in sequencing technology have led to a sharp decrease in the cost of 'data generation'. But is this sufficient to ensure cost-effective and efficient 'knowledge generation'?\nauthor: Andrea Sboner; Xinmeng Jasmine Mu; Dov Greenbaum; Raymond K Auerbach; Mark B Gerstein\ndate: 2011\ninstitute: 1Program in Computational Biology and Bioinformatics, Yale University, New Haven, CT 06520, USA; 2Department of Molecular Biology and Biophysics, Yale University, New Haven, CT 06520, USA; 3Sanford T Colb & Co. Intellectual Property Law, 4 Shaar Hagai, Marmorek, Rehovot, 76122, Israel; 4Center for Law and the Biosciences, Stanford Law School, Stanford University, Stanford, CA 94305, USA; 5Sholar in Residence Center for Health Law, Bioethics and Health Policy Kiryat Ono College, 55000, Isreal; 6Department of Computer Science, Yale University, New Haven, CT 06520, USA\nreferences:\ntitle: The real cost of sequencing: higher than you think!\n\nIn recent years, a technological revolution has enabled a series of advances in our knowledge of complex biological processes from development to disease, largely fueled by massively parallel sequencing technology, or next-generation sequencing (NGS) \\[1,2\\]. Applications of NGS include studies of entire genomes (whole-genome sequencing), investigations of smaller functional portions of the genome (exome sequencing), and analysis of the transcribed genome (RNA-seq) and protein-DNA binding sites (ChIP-seq). We are still in the early days of this 'revolution', with new technological improvements expected to be introduced in the next few years: longer reads, faster processing, and a growing number of more 'exotic' experimental applications that can benefit from sequencing technologies, such as the chromatin conformation capture family of experiments to reveal a structural view of the genome, and methods such as GRO-seq and CLIP-seq, and several others, to study protein-RNA binding \\[3-5\\].\n\nAdvances in sequencing technologies paved the way for launching the \\$1,000 genome challenge in 2005 \\[6-8\\], an almost impossible goal to imagine at the time. In fact, the cost of sequencing the first human genome was about \\$3 billion \\[9\\], and it took several international institutes, hundreds of researchers and 13 years to complete. However, in the past few years the cost of sequencing has declined exponentially: James Watson's genome was completed for less than \\$1 million \\[10\\]; by 2009 the cost for a whole-genome sequence dropped to \\$100,000 \\[11\\]. Hence, today, a mere 10 years after the completion of the first draft of the human genome, the goal of the \\$1,000 genome seems surprisingly close, and it is now conceivable that this will be a step towards even cheaper genomes. Nevertheless, it has become clear that the act of sequencing DNA (or cDNA) is only one aspect of a more complex story (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>).\n\n# Cost of sequencing versus cost of computation\n\nThe National Human Genome Research Institute (NHGRI) has tracked the cost of sequencing in the centers it funds. Analyzing the data revealed a stunning picture \\[12\\]. From 2008, the cost of sequencing dropped faster than what would have been expected from Moore's law (a term used to describe a trend in the computer industry). Moore's law states that the number of transistors of an integrated circuit doubles approximately every 2 years, but it is also applicable to several other digital electronic devices \\[13,14\\]. This implies that while we will be able to generate more and more sequence bases at a fixed cost, we will soon lack the facilities to store, process, analyze and maintain the data generated (Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>). However, the NHGRI survey ignores other cost components, including the following 'non-production' activities: quality assessment\/control for sequencing projects, technology development to improve sequencing pipelines, development of bioinformatics\/computational tools to improve sequencing pipelines or to improve downstream sequence analysis, management of individual sequencing projects, informatics equipment, and data analysis downstream of initial data processing (for example, sequence assembly, sequence alignments, identifying variants and interpretation of results) \\[12\\]. These are obviously integral to any sequencing project, and need to be accounted for in the overall costs. Moreover, the economic impact of these activities is likely to play a different role depending on the users: the costs of streamlining the sequencing pipeline can affect data providers in different ways, whereas downstream analysis costs have a similar impact irrespective of the investigators.\n\nHere we highlight the relevance and important of these often unaccounted costs using examples from a typical whole-genome sequence and RNA-seq project (Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>) We consider the associated costs incurred during a four-step process: (i) sample collection and experimental design, (ii) sequencing the sample, (iii) data reduction and management, and (iv) downstream analyses (that is, the complex analyses enabled by NGS and by the experimental design) (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>, Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>).\n\nEstimates of a whole-genome sequence and an RNA-seq experiment using Illumina HiSeq 2000 machine\n\n<table>\n<thead>\n<tr>\n<th><\/th>\n<th><\/th>\n<th colspan=\"3\" style=\"text-align: center;\">Whole genome sequencing<\/th>\n<th><\/th>\n<th colspan=\"3\" style=\"text-align: center;\">RNA-Seq<\/th>\n<\/tr>\n<tr>\n<th><\/th>\n<th><\/th>\n<th style=\"text-align: center;\">2011 cost<\/th>\n<th style=\"text-align: center;\">output<\/th>\n<th style=\"text-align: center;\">2011 time<\/th>\n<th><\/th>\n<th style=\"text-align: center;\">2011 cost<\/th>\n<th style=\"text-align: center;\">output<\/th>\n<th style=\"text-align: center;\">2011 time<\/th>\n<\/tr>\n<\/thead>\n<tbody>\n<tr>\n<td style=\"text-align: left;\"><strong>Sample collection and experimental design<\/strong><\/td>\n<td style=\"text-align: left;\">from blood samples (easy to collect) to brain tissue (hard to collect)<\/td>\n<td style=\"text-align: center;\">~$100 onwards<\/td>\n<td><\/td>\n<td style=\"text-align: center;\">from a few hours to several days<\/td>\n<td colspan=\"4\" style=\"text-align: center;\">same as for whole genome sequencing<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\"><strong>Sequencing<\/strong><\/td>\n<td style=\"text-align: left;\">library preparation + running the sequencer (whole dual flow cell)<\/td>\n<td style=\"text-align: center;\">~$6500 = ~$500 + ~$6000<\/td>\n<td style=\"text-align: center;\">~380 M reads\/lane; 1 individual: ~1140 M total reads (~3 lanes for a 30 \u00d7 coverage); ~250 Gb (intermediate files)<\/td>\n<td style=\"text-align: center;\">~11-12 day<\/td>\n<td style=\"text-align: center;\">library preparation + running the sequencer (whole dual flow cell)<\/td>\n<td style=\"text-align: center;\">~$3300 = ~$300 + ~$3000<\/td>\n<td style=\"text-align: center;\">~380 M reads\/lane<\/td>\n<td style=\"text-align: center;\">~12-14 day<\/td>\n<\/tr>\n<tr>\n<td><\/td>\n<td style=\"text-align: left;\"><strong><em>Data storage, low-level processing<\/em><\/strong><\/td>\n<td><\/td>\n<td><\/td>\n<td><\/td>\n<td><\/td>\n<td><\/td>\n<td><\/td>\n<td><\/td>\n<\/tr>\n<tr>\n<td><\/td>\n<td style=\"text-align: left;\">Alignment (transfer* and storing raw data + mapping)<\/td>\n<td style=\"text-align: center;\">~$40 = ~$33 + ~$7<\/td>\n<td style=\"text-align: center;\">300 Gb (BAM file)<\/td>\n<td style=\"text-align: center;\">~1\/2 day *** (including transferring 250 Gb FASTQ ~7.5 hrs)<\/td>\n<td style=\"text-align: center;\">Alignment (transfer* and storing raw data + mapping)<\/td>\n<td style=\"text-align: center;\">~$5 = ~$3 + ~$2<\/td>\n<td style=\"text-align: center;\">~30 Gb (BAM); ~22 Gb (MRF)<\/td>\n<td style=\"text-align: center;\">&lt; 2 hrs ***<\/td>\n<\/tr>\n<tr>\n<td><\/td>\n<td style=\"text-align: left;\">(data transfer and storage for 10 days)*; **<\/td>\n<td style=\"text-align: center;\">~$40<\/td>\n<td><\/td>\n<td style=\"text-align: center;\">~8.5 hrs<\/td>\n<td style=\"text-align: center;\">(data transfer and storage for 10 days)*; **<\/td>\n<td style=\"text-align: center;\">&lt; $4<\/td>\n<td><\/td>\n<td style=\"text-align: center;\">&lt; 1 hr<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\"><strong>Data reduction and management<\/strong><\/td>\n<td style=\"text-align: left;\"><strong><em>High-level summaries***<\/em><\/strong><\/td>\n<td><\/td>\n<td><\/td>\n<td><\/td>\n<td><\/td>\n<td><\/td>\n<td><\/td>\n<td><\/td>\n<\/tr>\n<tr>\n<td><\/td>\n<td style=\"text-align: left;\">SNP calling (compute + transfer out)<\/td>\n<td style=\"text-align: center;\">&lt; $5 = ~$4 + ~$0.60<\/td>\n<td style=\"text-align: center;\">&lt; 1 Gb<\/td>\n<td style=\"text-align: center;\">~3 hrs<\/td>\n<td style=\"text-align: center;\">Gene and exon<br \/>\nexpression quantification<\/td>\n<td style=\"text-align: center;\">&lt; $1<\/td>\n<td style=\"text-align: center;\">&lt; 1 M<\/td>\n<td style=\"text-align: center;\">&lt; 1 hr (1 CPU)<\/td>\n<\/tr>\n<tr>\n<td><\/td>\n<td style=\"text-align: left;\">Indel calling (compute + transfer out)<\/td>\n<td style=\"text-align: center;\">&lt; $35 = ~$32 + ~$0.60<\/td>\n<td style=\"text-align: center;\">&lt; 1 Gb<\/td>\n<td style=\"text-align: center;\">~1 day<\/td>\n<td style=\"text-align: center;\">Isoform quantification<\/td>\n<td style=\"text-align: center;\">~$6<\/td>\n<td style=\"text-align: center;\">&lt; 1 M<\/td>\n<td style=\"text-align: center;\">~4 h<\/td>\n<\/tr>\n<tr>\n<td><\/td>\n<td style=\"text-align: left;\">SV calling (compute + transfer out)<\/td>\n<td style=\"text-align: center;\">&lt; $35 = ~$32 + ~$0.60<\/td>\n<td style=\"text-align: center;\">&lt; 1 Gb<\/td>\n<td style=\"text-align: center;\">~1 day<\/td>\n<td><\/td>\n<td><\/td>\n<td><\/td>\n<td><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\"><strong>Downstream analyses<\/strong><\/td>\n<td><\/td>\n<td style=\"text-align: center;\">&gt; $100 K<\/td>\n<td style=\"text-align: center;\">~310 Gb<\/td>\n<td style=\"text-align: center;\">months<\/td>\n<td><\/td>\n<td style=\"text-align: center;\">&gt; $100 K<\/td>\n<td style=\"text-align: center;\">~30 Gb<\/td>\n<td style=\"text-align: center;\">months<\/td>\n<\/tr>\n<tr>\n<td colspan=\"2\" style=\"text-align: left;\"><strong>Total of sequencing, data management and reduction<\/strong><\/td>\n<td style=\"text-align: center;\"><strong>~$6500<\/strong><\/td>\n<td style=\"text-align: center;\"><strong>~310 Gb<\/strong><\/td>\n<td style=\"text-align: center;\"><strong>~15 days<\/strong><\/td>\n<td><\/td>\n<td style=\"text-align: center;\"><strong>~3500<\/strong><\/td>\n<td style=\"text-align: center;\"><strong>~30 Gb<\/strong><\/td>\n<td style=\"text-align: center;\"><strong>~12-14 days<\/strong><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n^a^Assuming a 10 MB\/s transfer rate. ^b^The cost of transferring 300 GB (BAM file) if the mapping is performed locally. ^c^Sixteen CPUs were used for all calculations, unless specified. BAM, Binary Sequence Alignment\/Map; CPU, central processing unit; GB, gigabyte; MB, megabyte; SNP, single nucleotide polymorphism; \\~, approximately. The table gives an estimation of the current costs of a whole-genome sequencing experiment and a functional genomic experiment (RNA-seq) using an Illumina HiSeq 2000 machine. The cost of sequencing is based on that reported by the Center for Cancer Research \\[53\\]. It is assumed that the same human sample is used for the genome sequence as well as for the RNA-seq experiment. In this scenario, a group of four technicians and bioinformaticians can run the entire pipeline. The costs related to data processing are estimated by considering all tasks performed in the Amazon Web Services 'cloud' environment. Pricing is based on a 'US standard' of the S3 Amazon Services (storage) \\[54\\] and a cost of \\$0.68 per hour (US, East Virginia) for the use of the Amazon EC2 (computation) \\[55\\] (July 2011). See Additional file <a href=\"#S1\" data-ref-type=\"supplementary-material\">1<\/a> for a version of this table in a colored layout for easier reading.\n\n# Experimental design and sample collection\n\nAs the cost of producing sequence reads decreased, more and more applications have been designed to exploit this technology. In addition to 'traditional' whole genome and exome sequencing, and traditional functional genomics experiments, such as transcriptome analysis (small and long RNA-seq) and chromatin immunoprecipitation (ChIP-seq), other applications of NGS have been designed: array-capture followed by sequencing (for example, enriching for transposable elements), fosmid pools sequencing (to facilitate haplotype phasing) \\[15\\], metagenomics sequencing, bisulfite sequencing and methylated DNA immunoprecipitation (MeDIP-Seq) to study DNA methylation patterns \\[16,17\\]. Some of these cutting-edge experimental designs require complex molecular and cellular biology experiments, such as chromosome sorting, to prepare the library for sequencing, thus adding considerably to the overall experimental costs.\n\nIn addition to the experimental design, the nature of the sample used can significantly alter the overall budget for a sequencing project. In the past, cell lines were the preferred biological material to sequence for obvious reasons: unlimited availability, well-known properties, and they are ideal for calibration and comparison with more traditional techniques. The advantages of these cell lines outweighed the noise introduced by the artifacts of immortalized cell lines. With an improved and more reliable sequencing technology, the questions addressed by researchers have also become more challenging, ranging from assessing human genetic variability to understanding the underlying biology of complex diseases, necessitating more time-consuming collection of clinically relevant samples, For example, studying 'trios' (that is, father, mother and child) to search for rare variants requires a considerable effort in terms of coordinating the experimental design compared with collecting blood samples from normal healthy volunteers. Similarly, the collection of matched cancer and normal frozen tissues needs proper standardized protocols to obtain high-quality biological material for sequencing. Working with a standard cell line, such as HeLa, requires less effort than working with brain tissue samples (where the dissection takes about 8 h and requires at least two people; Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>).\n\nIf clinically relevant samples from patients are to be used, then we must consider the costs associated with protecting patients' rights. As more genomes are sequenced and we become better at identifying personal information from them, the protection of the raw and analyzed data is crucial to avoid compromising even traditional research studies because patients are wary of donating their samples for research. Therefore, appropriate sample collection, informed consent procedures and the development of infrastructure and security required to protect patients' data need to be considered. Institutional review boards (IRBs) or ethics committees are often charged with overseeing these tasks. However, IRBs may constrain or limit the nature of whole-genome sequencing projects in light of concerns over downstream usage of genomic data. When faced with high-risk studies, IRBs may prevent a researcher from conducting a particular study, or request that the investigator seek approval from governmental agencies, potentially resulting in bureaucratic delays, which should be factored in to project budgets in advance.\n\nFurthermore, technological advancements over the course of the research project, while providing exciting new avenues of research, might effectively render the initial informed consent agreement moot, either requiring the acquisition of a new consent or the curtailment of a promising research program due to lack of consent.\n\nOnce private data are collected there are significant costs in maintaining a secure and functioning archival system. Compared with a similar heavy-data scenario, such as picture archiving and communication systems (PACS) in radiology departments, the costs could easily reach a few million US dollars \\[18\\]. This becomes even more complicated if the laboratory storing the data also wants to provide secure access to a wider audience over the web in terms of the actual cost of the infrastructure and software, and also the man-hours necessary to maintain the system updated \\[19\\].\n\n# Sequencing the sample\n\nSequencing entails the preparation of the library from the sample and running the sequencer to generate sequence reads. Streamlining the sequencing pipeline is an important aspect, especially for large-scale data-producing centers (for example, genomics centers, core sequencing centers at universities), which can benefit the most by leveraging economies of scale. However, the solutions implemented by large-scale data producers are different from those required by smaller centers. Standardized sample collection and identification procedures, and library preparation according to best practices, are the mainstay of large-scale data producers, where the main effort is to optimize and automate the entire process for many sequencing projects. This approach ensures high-quality and reproducible data, but it also requires a large-scale infrastructure. The main challenges are to accommodate the needs of multiple users, to achieve a low down-time of the sequencers, to adapt to new reagents and experimental protocols quickly, and to reorganize the efforts of technicians based on newer, faster machines in an efficient manner. These issues can be addressed in large-scale centers by using robots to automate the process. It is likely that the initial costs of implementing this infrastructure will be partially offset by the sequencing 'running' costs, which are decreasing.\n\nAt the opposite end of the spectrum are smaller groups and individual investigators, perhaps with only one sequencer. They do not need a complex infrastructure to successfully complete their sequencing process, and the current solutions offered by the vendors are sufficient. In this scenario, a major burden is represented by the acquisition of the technical expertise to run the sequencer and keep up with the new protocols.\n\n# Data reduction and management\n\nThe basic output of a sequencing run is the set of sequence reads (in our example, about 6 billion sequence reads corresponding to approximately 600 billion nucleotides - approximately 187 \u00d7 coverage of the human genome; Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>). Typically, this information is captured, rather inefficiently, in FASTQ files, which include the quality scores of each base. Three types of computation need to be distinguished: the first aims to efficiently manage this wealth of data by generating compressed data structures of the low-level raw data that can be easily accessed; the second aims at automatically extracting high-level summaries from these low-level raw data; and the third includes all downstream analyses that one can devise.\n\n## Data management: storage and transfer\n\nIn the first category, the alignment of those reads to a reference is the initial computational step. Different strategies can be adopted, but the final result is the assignment of the reads to their genomic location (while mapping to a known reference is the simplest procedure, some strategies may perform *de novo* assembly of the reads; we believe that this comprises a small fraction of experiments). Thus, files including mapped and unmapped reads can be considered the basic archival unit of an NGS experiment, since one can perform all the downstream analyses starting from the mapped reads, as well as extract all the reads and use a different alignment approach. Clearly, efficient compression strategies (such as Binary Sequence Alignment\/Map (BAM) \\[20\\]) are desperately needed to reduce the size of those files. The amount of raw data generated will increase as sequencers will provide longer reads in the future. Indeed, the massive amounts of data constitute a major burden from several viewpoints. The capacity for storing and archiving the data is increasing at a slower pace (that is, following Moore's law) than sequencing throughput, suggesting that re-sequencing a sample is more cost-effective than keeping the data archived. However, this solution is justifiable only when working with immortalized cell lines or model organisms. It is not an option with very valuable samples, such as clinical tissues or limited amounts of antibodies.\n\nLarge datasets also pose a problem in terms of data transfer. Computational biologists have been taking full advantage of the 'open-world' paradigm, where datasets are freely shared among investigators via centralized repositories, such as Gene Expression Omnibus, ArrayExpress, UniProt and others \\[21-23\\]. However, this practice is challenged by the vast amount of data that would need to be piped through the existing networking infrastructure for uploading and downloading the data to and from those repositories. For example, at a network bandwidth of approximately 10 megabytes (MB)\/s, it would require approximately 8.5 h to transfer 300 gigabytes (GB) \\[24\\]. Recently, a reference-based compression algorithm (CRAM) has been proposed that addresses both issues of storing and transferring data \\[25\\]. Inspired by video compression, this method currently achieves a considerable reduction in storage space required: 10-fold to 30-fold. Briefly, it keeps only the differences with respect to a reference and considers the relative location of a read based on the location of the previous read. Moreover, unmapped reads are assembled 'on the fly' to achieve even higher compression rates; this is a strategy that might also lead to novel discoveries. An irreducible fraction of reads, however, will not be mapped. This observation leads to a paradox: most of the disk space is required to store 'noise' (that is, the unmapped reads; obviously, this is an oversimplification, as not all unmapped reads can be considered as noise and some might actually lead to new discoveries). In the future, by identifying what information we can afford to lose, we could achieve higher levels of compression (approximately 200-fold), helping us to catch up with the decreased cost of sequencing \\[26\\].\n\nFollowing our example, data storage, transfer and mapping would cost about \\$40 for about 12 h of compute work for whole-genome sequencing, or about \\$5 and a few hours for RNA-seq (Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>).\n\n## Data reduction: high-level summaries\n\nThe second computational category regards the processing of the reads in order to obtain meaningful high-level summaries. For example Variant Call Format (VCF) files \\[27\\] describe genomic variants, including SNPs, short indels, structural variations (deletions, duplications and, more recently, inversions and translocations), Browser Extensible Data (BED) files report binding sites for ChIP-seq experiments, and tab-delimited files can provide RPKM values quantifying gene and exon expressions \\[28\\]. Here, it is clear what types of output one aims to get (VCF files, BED files, and so on), although there is not yet a fixed set of computational algorithms to achieve this goal. The creation of these high-level summaries is analogous to what is commonly performed in other fields. For example, protein structures are now encoded in Protein Data Bank (PDB) files \\[29\\]. Very few people would analyze the raw diffraction images from which the PDB files were generated. Similarly, raw digital images acquired with modern smart phones undergo a tremendous level of processing to produce an MPEG4 compressed video that can be immediately shared with friends; and no one needs the raw high-definition images to enjoy their friends' pictures.\n\nThese high-level summaries are likely to become the most valuable piece of information from sequencing experiments. Many researchers interested in genomic variants would need only the VCF files, which are considerably smaller than the whole set of mapped reads: about 170 MB for approximately 3 million SNPs, about 8 MB for 300,000 indels, and about 0.2 MB for approximately 1,500 structural variations. Similarly, the files with expression levels from RNA-seq or binding sites from ChIP-seq are quite small: about 0.5 MB for approximately 25,000 genes (RNA-seq), about 4 MB for approximately 230,000 exons (RNA-seq), and about 3 MB for approximately 80,000 binding sites. The generation of these files takes about 1 day and about \\$35 each for whole-genome sequencing and a few hours and about \\$6 for RNA-seq (Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>).\n\n## Complexity of data reduction: hybrid solutions\n\nIn addition to their smaller, almost negligible size, high-level summaries also have the advantage of reducing the potential privacy issues related to NGS data, which are more revealing of the underlying individual and include the potential for a full characterization. Although this solution seems attainable for most final users, it is not viable for researchers developing the computational tools to mine the data, who would still need the raw sequence data. A hybrid solution is to use compressed intermediate summaries, such as Mapped Read Format (MRF) \\[30\\]. Similar to CRAM, MRF focuses on encoding the location of the reads. In addition, it can protect the most sensitive information (that is, the sequences) by separating them from the alignment information. This has the advantage of enabling all the primary analyses where only the read locations are needed, such as quantifying expression levels, and of considerably reducing the file size.\n\nThe lack of agreed standards is a problem for data reduction approaches. One of the main benefits of centralized repositories has been to define standardized metadata and data formats that proved to be quite useful for the final users of those data. However, it has also introduced an additional burden to the data providers to generate those metadata and prepare the data submission in the proper format. A 'broker' who has expertise in data processing can efficiently deal with all these aspects, and its costs should thus be taken into account, especially for smaller groups.\n\n# To the cloud!\n\nWith the majority of computational tools for NGS data analyses being open source, computational-related costs are typically only hardware infrastructure (data storage and computational power) and human resources to properly set up the computational environment and run the processing pipeline. There are currently three solutions for this type of computational analysis: subscribing to sequencing-as-a-service offerings by sequencing companies, processing the data in-house, and using cloud computing services.\n\nA few sequencing companies, such as Complete Genomics, DNANexus and Spiral Genetics among others, provide data reduction services, some also following whole-genome DNA sequencing. These data reduction pipelines are proprietary NGS data processing facilities and their cost to the customer is minimal in addition to the sequencing service (a few thousand dollars per genome). The nature of the data processing pipeline confers customers less control on the quality, reproducibility and other details of the data reduction pipeline.\n\nA common aspect for analyzing the vast amount of data is the use of parallel computing. Typically, this would entail the use of a computer cluster, a facility not available to everyone, which requires a significant amount of investment that easily reaches thousands or millions of dollars. While large-scale centers might benefit from economies of scale, smaller institutions or individual laboratories might be priced out of these facilities.\n\nAn alternative to building in-house hardware infrastructures is to resort to commercialized cloud computing services or computation-as-a-service \\[24,31\\]. In this scenario, users 'rent' computational power to perform the analysis over the Internet. Amazon.com was one of the first to offer these general-purpose cloud services with the Amazon Web Services. Several service providers now offer solutions based on this computing paradigm, including Microsoft Windows Azure, The Rackspace Cloud and the US Federal Government, among others \\[31-34\\]. This solution might appeal to smaller groups as well as large-scale genomics centers. Smaller groups can delegate the burden of implementing, maintaining and running complex infrastructures and security protocols to the service providers while concentrating on obtaining meaningful results from their data. Similar to our illustrative example reported in Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>, Langmead and colleagues \\[35\\] were able to re-align a human genome and detect SNPs for a cost of the cloud use of approximately \\$113: \\$28 for transferring and storing the raw data, the remaining \\$85 to run the analyses on 320 central processing units in about 2.5 h. Larger organizations, which might already have their own computer clusters, can still make use of these services. Indeed, some service providers allow 'hybrid' solutions where private resources can be securely extended into the cloud. This solution is particularly useful to address 'spikes' in computer usage (that is, when simultaneous requests for computational resources exceed the local cluster capacity).\n\nAnother advantage of cloud computing is that standardized computational tools can be made available through the cloud such that even casual users can execute their analysis using state-of-the-art approaches (Galaxy, Myrna, JCVI BioLinux, and so on \\[31,36-39\\]). Although cloud computing seems quite attractive, there are other aspects that one must consider. First, cloud computing is particularly effective when a lot of computation, such as molecular dynamic simulations, is required. However, in genomics, processing is tightly linked to the large amount of data. Transferring massive amounts of data to the cloud may be time consuming and prohibitively expensive for small laboratories in research institutions with standard network bandwidth (approximately 10 MB\/s). Moreover, by transferring the data to the cloud, one would create a redundancy that is not efficient, unless the output from the sequencers is directly transferred to the cloud. Reference-based compression can ameliorate some of these issues. A 10-fold reduction of a 300 GB BAM file (whole-genome sequence; Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>) will reduce the overall cost to approximately \\$4 for transferring the data to the cloud and storing them for 10 days, in addition to reducing the transfer time considerably (approximately 1 h). Second, ensuring that data from human subjects remains private is important. Guaranteeing that the sequence data are handled according to the individual consent and protected in accordance with the various regulations is mandatory, particularly when using government funding. Although some companies already provide tools to deal with these issues in the cloud, the legal aspects of handling genomic data are still in flux. For example, in the USA, depending on the source of genetic information, Health Insurance Portability and Accountability Act (HIPAA) rules may or may not fully apply (that is, direct-to-consumer genomics companies probably do not need to comply with HIPAA privacy protections). Moreover, it is not clear whether the Fourth Amendment right against search and seizure for genomic data is maintained in the cloud.\n\nA solution to the privacy issues may be that governmental funding agencies, such as the National Institutes of Health in the USA, create a 'private' cloud environment where a researcher can conduct his\/her genomics research in accordance with the legal framework. This approach may also help in lowering the share of the agency's budget spent on funding the computational infrastructure of various research organizations. In this scenario, an important aspect is the definition of a shared common legal and ethical framework if data are shared internationally.\n\n# Downstream analyses\n\nWhether the sequencing and initial data analyses are carried out externally or in house, researchers have to face the downstream analyses of NGS tailored to specific research projects. Hundreds of tools have been developed to unravel the complexity of biological mechanisms hidden in the sequence reads. Whole-genome DNA sequencing enables analyses such as looking for natural selection in genomic elements \\[40\\], investigating demographic history changes by comparing with the Neanderthal genome \\[41\\], and studying cross-species conservation, recombination hotspots and gene conversion \\[42\\]. It also allows us to perform phasing (family-, population- or physical-based) and construct diploid personal genomes \\[43\\]. A single RNA-seq experiment can be exploited to investigate not only gene expression levels, but also transcript expression levels, to identify differentially expressed 'elements' \\[44\\]. It can further provide information about novel transcriptionally active regions or novel splice sites \\[45\\], identify the presence of chimeric transcripts \\[46,47\\], and investigate the extent of RNA editing or allele-specific expression \\[48-50\\]. Moreover, the integration of multiple assays allows researchers to address more complicated questions with both a resolution and a breadth that was unforeseeable only a few years ago. For example, combining genotyping information (perhaps obtained from whole-genome sequencing) and RNA-seq data, it is possible to investigate expression quantitative trait loci \\[49,50\\]. Analyzing and combining ChIP-seq experiments allows the determination of putative functional elements, such as enhancers and promoters, and the generation of regulatory networks to study their static and dynamic aspects.\n\nIn contrast to the cost of hardware, the cost of human resources is hardly quantifiable at the current state of the art. No streamlined, standardized approach is yet available for the users, either an experienced or a casual one. Hence, considerable efforts are required to properly install, configure and run the computational pipeline to perform the data reduction as well as the more complex data analyses described here.\n\nSome computational frameworks, such as Galaxy, provide users with several tools to perform data analysis \\[36\\]. However, as pointed out by the Galaxy developers in the 'Know what your doing' section of the framework \\[51\\]: 'There is no such thing (yet) as an automated gearshift in (...)' (replace (...) with your favorite analysis: for example, short read mapping, splice-junction identification). Moreover, the time and effort considered for the data analysis pipeline in Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a> includes only the estimation of actual computing time (that is, 'machine time'). There is an unpredictable amount of extra 'human time' spent on comparing and choosing suitable and reasonable software tools, learning how to install, configure and execute them, estimating the effects of tuning the parameters, interfacing input\/output formats for serial modules in the pipeline, and debugging and streamlining. Sometimes, this latter phase is more time consuming than the actual processing time.\n\nFinally, as pointed out by Elaine Mardis, the interpretation of the results, in many cases, requires a multidisciplinary team to make sense of all the primary data \\[52\\]. Linking a genetic variation to a phenotype, especially a clinically relevant one, requires a lot of expertise and effort, first to identify highly confident variants; second, to estimate their functional impact; third, to select from among the functional ones those that are correlated to the phenotype. None of these steps can be carried out in an automated fashion. This, in turn, further supports why no streamlined data analysis pipeline currently exists and the needs to customize and tailor the process to each research project. A team of bioinformaticians, statisticians, geneticists, biologists and physicians is required to translate the information in the primary data into useful knowledge to understand the impact of genomic variants in biological systems; this can often take weeks or months of extensive experimental validations using animal models or cell lines.\n\n# Future scenarios\n\nThe impressive advances made possible by the introduction of NGS are rapidly pushing the scientific community toward uncharted territories. NGS is currently quite expensive and mainly tailored to a research setting. However, the rapid decrease in its basic operational costs will make their utilization affordable by a larger group of investigators and in a diagnostic setting. New experimental protocols, new equipment tailored to interrogate smaller portions of the genome (for example, Ion Torrent Personal Genome Machine PGM, Illumina MiSeq) and new applications of sequencing will further broaden the user base of these technologies. In turn, this means that more 'challenging' samples will be analyzed with more complex experiments thus increasing the fraction of cost due to sample collection and experimental design (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>).\n\nThe cost of sequencing is decreasing rapidly (that is, faster than Moore's law; Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>) in contrast to storage, which is decreasing in line with Moore's law. The bottleneck represented by data storage, maintenance and transfer should be addressed by better data compression algorithms, and by standardized high-level summary data. These summaries should be sufficiently expressive as to enable additional analyses and to be easily integrated with other data sets for comparative analysis. However, this approach would tackle only one aspect of the NGS data complexity. The full interpretation of the primary data is going to constitute a major expense (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>).\n\nIn conclusion, the rapid decrease in the cost of 'data generation' has not been matched by a comparable decrease in the cost of the computational infrastructure required to mine the data. Cloud computing is one promising direction for smaller groups and large-scale organizations to address some of the underlying issues, with the former clearly benefiting the most. However, careful considerations about privacy of the data and network bandwidth must be taken into account. The major burden, however, will be represented by the downstream analysis and interpretation of the results. The bioinformatics and computational biology community needs to design and develop better computational algorithms and approaches to speed up the 'knowledge generation' pipeline, taking advantage of the possibilities opened up by the cloud. More automated and more reliable tools to process and analyze NGS data are certainly needed and constitute essential steps towards the realization of personalized genomics and medicine, one of the main challenges of the 21st century.\n\n# Abbreviations\n\nBAM: Binary Sequence Alignment\/Map; BED: Browser Extensible Data; CRAM: compression algorithm; GB: gigabyte; HIPAA: Health Insurance Portability and Accountability Act; IRB: institutional review board; MB: megabyte; MRF: Mapped Read Format; NGS: next-generation sequencing; NHGRI: National Human Genome Research Institute; PDB: program database; SNP: single nucleotide polymorphism; VCF: Variant Call Format.\n\n# Competing interests\n\nThe authors declare that they have no competing interests.\n\n# Supplementary Material\n\n###### Additional file 1\n\n**Table S1: Estimates of a whole-genome sequence and an RNA-seq experiment using Illumina HiSeq 2000 machine**. A version of Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a> with colored layout for improved ease of reading.\n\nClick here for file\n\n## Acknowledgements\n\nWe would like to thank Naoki Kitabayashi, Cristina Sisu, Joel Rozowsky, Alexej Abyzov, Yuka Imamura Kawasawa and Jing Leng for useful discussions. We would like to acknowledge the National Institutes of Health for funding.\n\nThe opinions of Dov Greenbaum expressed in this piece are his own and do not necessarily represent, nor should they be imputed to represent, the opinion of his law firm, any of its employees, or its clients \\[53-55\\].","meta":{"dup_signals":{"dup_doc_count":173,"dup_dump_count":44,"dup_details":{"curated_sources":2,"2023-23":2,"2022-05":1,"2021-49":1,"2021-21":1,"2021-17":1,"2021-04":1,"2020-40":1,"2020-29":1,"2020-24":2,"2019-47":1,"2017-17":1,"2017-09":18,"2017-04":2,"2016-50":2,"2016-44":2,"2016-40":2,"2016-36":16,"2016-30":11,"2016-22":1,"2016-18":1,"2016-07":10,"2015-48":4,"2015-40":2,"2015-35":4,"2015-32":4,"2015-27":4,"2015-22":4,"2015-14":3,"2014-52":3,"2014-49":5,"2014-42":10,"2014-41":6,"2014-35":7,"2014-23":7,"2014-15":4,"2023-40":1,"2024-26":2,"2024-10":1,"2015-18":4,"2015-11":4,"2015-06":3,"2014-10":4,"2013-48":4,"2013-20":3}},"file":"PMC3245608"},"subset":"pubmed_central"}
{"text":"abstract: # Objective\n  .\n  Early detection and early treatment are of vital importance to the successful treatment of various cancers. The development of a novel screening method that is as economical and non-invasive as the faecal occult blood test (FOBT) for early detection of colorectal cancer (CRC) is needed. A study was undertaken using canine scent detection to determine whether odour material can become an effective tool in CRC screening.\n  .\n  # Design\n  .\n  Exhaled breath and watery stool samples were obtained from patients with CRC and from healthy controls prior to colonoscopy. Each test group consisted of one sample from a patient with CRC and four control samples from volunteers without cancer. These five samples were randomly and separately placed into five boxes. A Labrador retriever specially trained in scent detection of cancer and a handler cooperated in the tests. The dog first smelled a standard breath sample from a patient with CRC, then smelled each sample station and sat down in front of the station in which a cancer scent was detected.\n  .\n  # Results\n  .\n  33 and 37 groups of breath and watery stool samples, respectively, were tested. Among patients with CRC and controls, the sensitivity of canine scent detection of breath samples compared with conventional diagnosis by colonoscopy was 0.91 and the specificity was 0.99. The sensitivity of canine scent detection of stool samples was 0.97 and the specificity was 0.99. The accuracy of canine scent detection was high even for early cancer. Canine scent detection was not confounded by current smoking, benign colorectal disease or inflammatory disease.\n  .\n  # Conclusions\n  .\n  This study shows that a specific cancer scent does indeed exist and that cancer-specific chemical compounds may be circulating throughout the body. These odour materials may become effective tools in CRC screening. In the future, studies designed to identify cancer-specific volatile organic compounds will be important for the development of new methods for early detection of CRC.\nauthor: Hideto Sonoda; Shunji Kohnoe; Tetsuro Yamazato; Yuji Satoh; Gouki Morizono; Kentaro Shikata; Makoto Morita; Akihiro Watanabe; Masaru Morita; Yoshihiro Kakeji; Fumio Inoue; Yoshihiko Maehara**Correspondence to** Dr Hideto Sonoda, Department of Surgery and Science Graduate School of Medicine Kyushu University 3-1-1 Midashi,Higashi-ku, Fukuoka 812-8582, Japan; <firstname.lastname@example.com>\ndate: 2011-01-31\ninstitute: 1Department of Surgery and Science, Kyushu University at Fukuoka, Fukuoka, Japan; 2Department of Internal Medicine, Arita Kyoritsu Hospital at Arita, Saga, Japan; 3St. Sugar Cancer Sniffing Dog Training Center at Minamibousou, Chiba, Japan; 4Department of General Surgery, Arita Kyoritsu Hospital at Arita, Saga, Japan; 5Department of Internal Medicine, Fukuoka Dental College Hospital at Fukuoka, Fukuoka, Japan; 6Department of General Surgery, Fukuoka Dental College Hospital at Fukuoka, Fukuoka, Japan\nreferences:\ntitle: Colorectal cancer screening with odour material by canine scent detection\n\n###### Significance of this study\n\n# What is already known about this subject?\n\n-   Canine olfactory detection of cancer has been reported for melanoma as well as bladder, lung, breast and ovarian cancer.\n\n-   It is reported that dogs can be trained to distinguish breath samples of patients with lung and breast cancer from those of control volunteers and to distinguish ovarian cancer tissues from control tissues with high accuracy.\n\n-   Several volatile organic compounds have been identified as candidate substances for early detection of cancer using gas chromatography\/mass spectroscopy in the exhaled breath of patients with lung and breast cancer; while these data are promising, they are preliminary findings that should be interpreted with caution.\n\n# What are the new findings?\n\n-   In patients with colorectal cancer (CRC) and controls, the sensitivity of canine scent detection of breath samples compared with conventional diagnosis by colonoscopy was 0.91 and the specificity was 0.99.\n\n-   The sensitivity of canine scent detection of watery stool samples was 0.97 and the specificity was 0.99.\n\n-   The accuracy of canine scent detection was even higher for early-stage cancers.\n\n-   Canine scent detection was not confounded by current smoking, benign colorectal disease, inflammatory disease or the presence of human haemoglobin or transferrin.\n\n# How might it impact on clinical practice in the foreseeable future?\n\n-   Odour materials may become effective tools in CRC screening. In the future, studies designed to identify cancer-specific volatile organic compounds will be important for the development of new methods for early detection of CRC.\n\n-   Identification of cancer-specific volatile organic compounds will lead to solving the biological character of cancer.\n\n# Introduction\n\nDespite rapid progress in examination technology and therapy for colorectal cancer (CRC), this disease constitutes a significant proportion of the global burden of cancer morbidity and mortality.1 Multiple professional organisations have recommended screening for CRC1\u20135 and many previous studies have demonstrated the cost-effectiveness of CRC screening.6\u201311 Moreover, patients with familial adenomatous polyposis or Lynch syndrome are recommended to undergo frequent colonoscopy to reduce the morbidity and mortality associated with CRC.12\u201315 Early detection and early treatment are critical for the successful treatment of cancer and are excellent means for reducing both the economic burden and mortality. CRC therefore represents an excellent example of the value of early detection.\n\nThe faecal occult blood test (FOBT) is currently the most economic and non-invasive screening method for CRC, and many patients with positive results subsequently undergo total colonoscopy leading to a reduced incidence and mortality of CRC.2 3 6 16 However, the positive predictive value of FOBT for CRC is approximately 10%,17\u201320 16 21 considering the impact of closer examination by colonoscopy or barium enema. The development of a novel more effective CRC screening method that is as economical and non-invasive as FOBT is therefore needed.\n\nThere have been several anecdotal reports of canine scent detection of skin cancer.22 23 Moreover, Willis *et al* reported that dogs can distinguish urine from patients with bladder cancer with a mean success rate of 41%,24 while McCulloch *et al* reported that ordinary household dogs can be trained to distinguish breath samples of patients with lung and breast cancer from those of control volunteers with high accuracy (sensitivity and specificity of 0.99 and 0.99 in lung cancer and 0.88 and 0.98 in breast cancer, respectively).25 Horvath *et al* reported that, among ovarian cancer tissues and control tissues, the sensitivity was 100% and the specificity was 97.5%.26\n\nIn the present study we have confirmed the accuracy of canine scent detection of breath samples and evaluated canine scent detection of watery stool samples from patients with CRC. We also examined whether the diagnostic performance of dogs is affected by age, smoking, disease stage, cancer site, inflammation or bleeding in patients with cancer or control individuals.\n\n# Methods\n\n## Patient and control sample donors\n\nPatients were enrolled from 20 June 2008 to 20 May 2009 at the Fukuoka Dental College Medical and Dental Hospital and Arita Kyoritsu Hospital. To prepare for colonoscopy, subjects ingested 2\u2005l of a balanced electrolyte and polyethylene glycol 4000 solution (Niflec, Ajinomoto Pharma, Tokyo, Japan). Patients were required to be \\>20\u2005years old. Patients and controls completed a questionnaire about factors that could influence volatile molecules in the breath or watery stool samples including age, physical symptoms (eg, abdominal pain or distention, bloody faeces, constipation, diarrhoea, body weight loss and abdominal tumour), history of cancer treatment, present use of anticoagulants and smoking within the previous 2\u2005weeks. Colorectal diseases diagnosed primarily by colonoscopy were recorded in detail and pathological examination of biopsy samples was performed if necessary. Patients who had undergone cancer surgery within the previous year, those who did not undergo examination for cancer recurrence despite having undergone cancer surgery more than 5\u2005years previously and those currently receiving chemotherapy were excluded. A serial number was written on each sample at the time of collection to identify individual information.\n\n## Breath sampling\n\nBreath samples were acquired from the beginning to the end of exhalation. Each subject exhaled between 100 and 200\u2005ml into a breath sampling bag (Otsuka Pharmaceutical Company, Tokyo, Japan). The bags were then fitted with their end caps and sealed in ordinary grocery store Ziploc-style bags at 4\u00b0C until presentation to the dog. Only one sample was collected from each participant.\n\n## Watery stool sampling\n\nA 50\u2005ml watery stool sample was obtained by suction during colonoscopy using a trachea aspirator kit (Sumitomo Bakelite Company, Tokyo, Japan) between the rectum and descending colon and preserved at \u221280\u00b0C until filtration. The sample was filtered with qualitative filter paper No. 2 (diameter 110\u2005mm; Tokyo Roshi Kaisha, Tokyo, Japan) and further filtered by Millex GP filter (pore size 0.22\u2005\u03bcm, diameter 33\u2005mm, non-sterile type; Millipore Co, Corrigiwohill, Co. Cork, Ireland). Next, 1\u2005ml of filtrated watery stool samples was enclosed in a 5\u2005ml brown glass screw cap bottle, sealed in an ordinary grocery store small Ziploc-style bag and enclosed in a 50\u2005ml polypropylene screw cap tube and kept at \u221220\u00b0C until presentation to the dog. Only one sample was collected from each participant.\n\n## Dog and training\n\nA specially trained 8-year-old female black Labrador retriever from the St Sugar Cancer Sniffing Dog Training Center (Chiba, Japan) participated in the study. The dog was trained for water rescue beginning in 2003 and then began training as a cancer detection dog in 2005.\n\nEach cancer detection training session was considered complete when the dog could correctly distinguish between breath samples from a cancer patient and four controls consecutively in dozens of trials. The fundamental training method was a reward-based approach in which the correct behaviour is rewarded by simultaneous play with a tennis ball. The breath samples used in the training steps beginning in 2005 were collected from several hundred patients with cancer and about 500 healthy volunteers recruited using the internet.\n\nIn the first training step in 2005, breath samples from one patient with oesophageal cancer and four controls were used. The samples were removed from the breath-sampling bag and placed into a paper cup which was then covered with a paper filter for 2\u2005days. The five sample cups were placed on the floor by the handler. First, the dog smelled the standard breath sample from the patient with oesophageal cancer and then tried to identify the cup containing the oesophageal cancer scent from among the five sample cups. If the dog correctly identified the cancer sample it was rewarded with a tennis ball. A similar training session was conducted using breath samples from patients with lung cancer on the following day and was performed again using breath samples from patients with gastric cancer 2\u2005days later.\n\nIn the second step the breath-sampling bag was used with the end cap on. Breath-sampling bags containing oesophageal, lung or gastric cancer scents originating from the same patient in the first step were chosen randomly for the 1-month training period.\n\nIn the third step the dog first smelled the breath sample from one type of cancer as a standard sample and then tried to identify the bag with the scent of another cancer. The training was then continued by adding samples of other types of cancers as well as controls.\n\nThe dog was already able to detect the following types of cancer in patient breath samples prior to initiation of the present study: oesophageal cancer, breast cancer, lung cancer, gastric cancer, pancreatic cancer, hepatocellular carcinoma, cholangiocarcinoma, colorectal cancer, prostate cancer, uterine cancer, ovarian cancer and bladder cancer.\n\n## Training and test room\n\nThe training and testing of the dog were conducted in a 4.8\u2005m\u00d78.0\u2005m room with vinyl tiling and overhead fluorescent and natural window lighting. The room was not climate controlled and average ambient temperatures during the study (November 2008 through June 2009) ranged from 9\u00b0C to 20\u00b0C. At the end of each study day the floor was wiped with water and a damp mop.\n\n## Sample stations\n\nFive sample stations were positioned on the floor of the room in a single straight line spaced 52\u2005cm apart. Each station consisted of a light blue painted wooden storage container measuring 27\u2005cm wide, 30\u2005cm long and 20\u2005cm high. Each station also contained a wall 10\u2005cm deep in which the breath sample bags or the watery stool sample bottles were placed. The top of each box was covered with metal netting to prevent the dog from directly contacting the samples.\n\n## Breath and watery stool sample locations\n\nIn a single trial, the dog passed and sniffed each of the five stations (one cancer sample and four control samples). Both patient and control samples were replaced with new samples after the dog had sniffed all five sample stations. Two to four trials were performed on each day of testing.\n\n## Personnel\n\nThe dog handler called the dog into the room and encouraged the dog to sniff the stations with the command 'Search!' after smelling the standard breath sample from the patient with CRC at each test. No other people were in the room.\n\n## Classification of the dog's response\n\nCorrect responses were (1) indication by sitting down in front of a sample station containing a cancer sample (a true positive in sensitivity calculations): and (2) sniffing but not indicating on a control sample (true negative). Incorrect responses were (1) indicating on a control sample (false positive); (2) sniffing but not indicating on a cancer sample (false negative); and (3) hesitation, considered to be an incomplete reaction to either cancer or control samples (either false positive or false negative depending on whether hesitation was on a cancer or control sample).\n\n## Testing\n\nTests were conducted from 13 November 2008 to 15 June 2009 because the dog's concentration tends to decrease during the hot summer season. For each test, completely new samples were used for both cases and controls; thus, each sample was only used a single time. For each trial we used a random number table to determine the location of the sample being tested in the line-up. Before the tests the sample was placed in either a new bag or new bottle to avoid contamination of the smell under preservation, and the number described in the sample was converted from the serial number into the test number at the same time. The laboratory assistants replaced the samples. The identity of the samples was blinded to the dog, the handler and the laboratory assistants, thereby making the study double-blind. One station contained a cancer sample and the remaining four stations contained control samples. At the tests the dog smelled the standard breath sample of CRC origin, after which the dog passed the side of some boxes and sat down in front of the station determined to contain a cancer scent, and then the handler noted the result on an answer card on which the test number was written. The card was transmitted to Fukuoka Dental College by fax. After submission the answer was explained to the handler and the dog as soon as possible because the correct behaviour was rewarded with a tennis ball. The cards were collected by mail. During the tests the dog did not experience any adverse events, injuries or illness.\n\n## Occult blood test of watery stool samples\n\nThe ordinary FOBT kit, Quick Chaser Occult Blood Test Kit (Mizuho Medy, Saga, Japan) was used for simultaneous detection of human haemoglobin and human transferrin in watery stool samples. Watery stool samples were mixed with phosphate buffered saline and reacted on the test plate. The test result was judged by a laboratory medical technologist and a physician. The result was defined as positive when human haemoglobin and\/or transferrin was positive.\n\n## Statistical analysis\n\nAssociations between questionnaire variables before colonoscopy were tested by the Welch t test or \u03c7^2^ test. All statistical differences of p\\<0.05 were deemed significant. CRC staging was based on Union Internationale Contre le Cancer (UICC) criteria.\n\nDuring testing, experimenters monitored each trial and recorded their observations on a digital versatile disc. The entire dog performance dataset was audited for accuracy by comparing answer cards with digital versatile disc records.\n\nDiagnostic accuracy was calculated as sensitivity and specificity of the dog's indication of samples compared with the true diagnosis confirmed by colonoscopy. Thus, the sensitivity (or the true positive rate) of the tests is the proportion of cancer samples correctly identified by the dog while the specificity (or the true negative rate) is the proportion of control samples negatively indicated by the dog.\n\n# Results\n\nForty-eight samples were obtained from patients with CRC, 203 samples from volunteers with no history of cancer and 55 samples from patients with a previous history of cancer. For breath tests, 33 samples from patients with CRC and 132 samples from control volunteers were used and, for watery stool tests, 37 samples were used from patients with CRC and 148 samples from control volunteers. Each sample was only used once during testing. The background characteristics were well matched between cancer patients and control individuals, except for age (<a href=\"#tbl1\" data-ref-type=\"table\">table 1<\/a>). Approximately half of the controls (47% of breath samples, 50% of watery stool samples) had colorectal polyps and a small proportion of the controls (6.1% of breath samples, 10.5% of watery stool samples) had bleeding or inflammatory colorectal disease, including ischaemic colitis, non-peculiar colitis or ulcer, ulcerative colitis, diverticular bleeding, mesenteric panniculitis and chronic appendicitis (<a href=\"#tbl2\" data-ref-type=\"table\">table 2<\/a>).\n\nBackground characteristics of participants\n\n|  | Breath samples |  |  | Watery stool samples |  |  |\n|----|----|----|----|----|----|----|\n|  | Cancer | Control |  | Cancer | Control |  |\n|  | (n=33) | (n=132) | p Value | (n=37) | (n=148) | p Value |\n| Gender (male) | 15 | 64 | 0.75 | 16 | 86 | 0.10 |\n| Age (years) | 71.0 (53\u201395) | 65.0 (25\u201391) | 0.001 | 70.2 (41\u201395) | 64.6 (25\u201391) | 0.035 |\n| Complaint before colonoscopy |  |  |  |  |  |  |\n| \u2003Abdominal pain or distention | 6 | 20 | 0.67 | 10 | 22 | 0.08 |\n| \u2003Bloody faeces | 5 | 19 | 0.91 | 6 | 19 | 0.59 |\n| \u2003Constipation | 6 | 24 | 1.0 | 7 | 24 | 0.69 |\n| \u2003Diarrhoea | 3 | 8 | 0.53 | 4 | 14 | 0.95 |\n| \u2003Body weight loss | 2 | 8 | 0.68 | 3 | 4 | 0.29 |\n| \u2003Abdominal tumour | 2 | 1 | 0.19 | 2 | 1 | 0.19 |\n| \u2003No complaint | 4 | 21 | 0.59 | 3 | 28 | 0.18 |\n| \u2003Past history of colorectal disease | 1 | 20 | 0.11 | 2 | 22 | 0.13 |\n| Anticoagulant therapy | 1 | 14 | 0.31 | 1 | 18 | 0.16 |\n| Smoked within 2\u2005weeks | 3 | 28 | 0.18 | 4 | 32 | 0.21 |\n\nDisease in control cases\n\n|  | Breath samples |  | Watery stool samples |  |\n|----|----|----|----|----|\n|  | Number | Rate | Number | Rate |\n| Polyps | 62 | 47.0% | 74 | 50.0% |\n| Diverticula | 24 | 18.2% | 44 | 29.7% |\n| Haemorrhoids | 4 | 3.0% | 7 | 4.7% |\n| Ischaemic colitis | 3 | 2.3% | 4 | 2.7% |\n| Non-peculiar colitis or ulcers | 2 | 1.5% | 4 | 2.7% |\n| Ulcerative colitis | 2 | 1.5% | 4 | 2.7% |\n| Diverticular bleeding | 0 | 0% | 1 | 0.6% |\n| Mesenteric panniculitis | 1 | 0.8% | 1 | 0.6% |\n| Infectious colitis | 0 | 0% | 1 | 0.6% |\n| Chronic appendicitis | 0 | 0% | 1 | 0.6% |\n| Lynch syndrome | 1 | 0.8% | 1 | 0.6% |\n\nMultiple answers were allowed for benign disease.\n\n## Diagnostic accuracy: sensitivity and specificity in testing\n\nTo calculate sensitivity and specificity we designated the dog's response to each sample sniffed as the unit of analysis (defined above). Comparison of the judgement of the canine scent in the breath or watery stool samples with a colonoscopy-confirmed conventional diagnosis among cancer patients and controls gave an overall sensitivity for canine scent detection of 0.91 and 0.97, respectively, while the overall specificity was 0.99 and 0.99, respectively (<a href=\"#tbl3\" data-ref-type=\"table\">table 3<\/a>).\n\nAccuracy of canine scent detection by site and stage of cancer\n\n| Site\/stage | Breath samples |  |  |  |  |  | Watery stool samples |  |  |  |  |  |\n|----|----|----|----|----|----|----|----|----|----|----|----|----|\n|  | 0 | I | II | III | IV | Total | 0 | I | II | III | IV | Total |\n| Appendix |  |  | 1\/1 |  |  | 1\/1 |  |  | 1\/1 |  |  | 1\/1 |\n| Caecum |  | 1\/1 |  |  |  | 1\/1 |  | 1\/1 |  |  |  | 1\/1 |\n| Ascending | 1\/1 |  | 3\/3 | 4\/5<a href=\"#table-fn1\" data-ref-type=\"table-fn\">*<\/a> | 2\/2 | 10\/11<a href=\"#table-fn1\" data-ref-type=\"table-fn\">*<\/a> | 1\/1 | 1\/1 | 3\/3 | 5\/5 | 2\/2 | 12\/12 |\n| Transverse | 1\/1 |  |  |  |  | 1\/1 | 1\/1 |  | 1\/1 |  |  | 2\/2 |\n| Descending |  |  | 1\/1 | 1\/1 |  | 2\/2 |  |  | 1\/1 | 1\/1 |  | 2\/2 |\n| Sigmoid | 1\/1 | 4\/5 | 1\/1 |  | 1\/1 | 7\/8 | 2\/2 | 2\/2 | 1\/1 |  | 1\/1 | 6\/6 |\n| Rectum | 1\/1 | 1\/2 | 1\/1 | 3\/3 | 4\/4 | 10\/11 | 2\/2 | 1\/2 | 2\/2 | 4\/4 | 3\/3 | 12\/13 |\n| R+S |  |  |  | 1\/1 |  | 1\/1 |  |  |  | 1\/1 |  | 1\/1 |\n| Total | 4\/4 | 6\/8 | 7\/7 | 9\/10<a href=\"#table-fn1\" data-ref-type=\"table-fn\">*<\/a> | 7\/7 | 33\/36<a href=\"#table-fn1\" data-ref-type=\"table-fn\">*<\/a> | 6\/6 | 5\/6 | 9\/9 | 11\/11 | 6\/6 | 37\/38 |\n| Sensitivity | 1 | 0.67 | 1 | 0.89 | 1 | 0.91 | 1 | 0.8 | 1 | 1 | 1 | 0.97 |\n| Specificity | 1 | 0.92 | 1 | 1 | 1 | 0.99 | 1 | 0.95 | 1 | 1 | 1 | 0.99 |\n\nNumber of true positives\/total test number are shown.\n\nHesitation occurred.\n\nR+S, double rectal and sigmoid colon cancer.\n\nDuring the course of 74 tests, four discrepancies occurred between colonoscopy and the canine scent detection results (1\/38 watery stool tests and 3\/36 breath tests). The discrepancies occurred in the 22nd, 26th, 33rd and 45th tests. Moreover, these discrepancies occurred in any of four tests on the aforementioned test days. Interestingly, the watery stool sample that yielded a discrepancy came from an individual whose breath sample also resulted in a discrepancy. Samples from the three patients who did not have colon cancer but whose samples yielded positive results in the watery stool and\/or breath tests did not result in discrepancies on the other tests. These three patients showed no signs of cancer during the 24\u2005months following sampling.\n\n## Comparison between FOBT results and the dog's judgement\n\nThe results of watery stool tests and conventional FOBTs were compared to determine whether canine scent judgement relied on blood. No correlation between canine scent judgement and FOBT results was observed in patients with cancer (p=0.51) or controls (p=0.68, <a href=\"#tbl4\" data-ref-type=\"table\">table 4<\/a>). The sensitivity and specificity of FOBT with respect to CRC detection was 0.70 and 0.85, respectively.\n\nRelationship between canine scent detection and human haemoglobin and transferrin\n\n| Pathological judgement | Judgement by dog | Judgement by faecal occult blood test |  | p Value |\n|----|----|----|----|----|\n|  |  | Positive | Negative |  |\n| Cancer | Positive | 25 | 11 | 0.51 |\n|  | Negative | 1 | 0 |  |\n| Control | Positive | 0 | 1 | 0.68 |\n|  | Negative | 22 | 125 |  |\n\n## Mixture test of watery stool samples\n\nTo determine whether a specific cancer scent exists or if a particular natural scent vanishes in patients with cancer, we combined five watery stool samples into single mixture samples. One of the mixture samples was composed of one sample of cancer origin and four control samples, while four mixture samples consisted of five control samples each. Tests were performed three times as described above. Cancer types included stage IIIa ascending colon cancer, stage II transverse colon cancer and stage 0 rectal cancer. The results of all three tests were correct.\n\n# Discussion\n\nWe used the excellent ability of dogs to distinguish between different scents to examine whether odour materials can be used in the diagnosis of CRC. This study represents the first step towards the development of an early detection system using odour materials from patients with CRC.\n\nMcCulloch *et al* previously reported the use of canine scent judgement of breath samples for distinguishing between controls and patients with lung and breast cancer.25 We therefore evaluated canine scent detection of CRC first with breath samples and then with watery stool samples, which has not been previously reported. We hypothesised that the watery stool sample scent might be strong because the samples were collected from nearer to the cancer than were the breath samples. Conversely, the watery stool sample scents might have been masked by various other scents such as short-chain fatty acids and sulfides present in the stool. Moreover, we were able to compare scent detection with the FOBT by using the same stool sample and to inspect it more quantitatively than using breath samples. Indeed, despite various disrupting scents, the watery stool sample tests were superior to the breath sample tests.\n\nWe compared the accuracy of canine scent judgement with colonoscopic diagnosis using breath samples and watery stool samples from patients with CRC and controls. For breath tests, the sensitivity and specificity was 0.91 and 0.99, respectively and, for watery stool tests, the sensitivity and specificity was 0.97 and 0.99, respectively. Moreover, canine scent judgement even appeared to be highly accurate for early-stage CRC.\n\nCanine scent judgement was not confounded by current smoking, benign colorectal polyps, inflammation or infection. A statistically significant difference in age distribution was noted between cancer patients and controls (p=0.001 for breath tests, p\\<0.05 for watery stool tests). We therefore reanalysed samples from patients and controls aged \\<80\u2005years. In this subset analysis, no correlation between age and cancer status was observed (p=0.09 for 19 breath tests, p=0.93 for 24 watery stool tests). The sensitivity and specificity in this subset analysis for breath tests was 0.95 and 0.99, respectively, while the sensitivity and specificity were both 1.00 for watery stool tests.\n\nThere were four discrepancies among the 74 tests including one of 38 watery stool tests and three of 36 breath tests between the colonoscopy and canine cancer detection results. It is difficult to specify the cause of these discrepancies owing to their limited number. The discrepancies occurred in the 22nd, 26th, 33rd and 45th tests, not necessarily in the early tests. Moreover, these discrepancies occurred in any of the four tests on the aforementioned test days, not necessarily in those taken late in the day. No correlation between the discrepancies and training effect or fatigue was suspected. Interestingly, the watery stool sample that yielded a discrepancy came from an individual whose breath sample also resulted in a discrepancy. Moreover, when the watery stool test was repeated with different controls, the dog hesitated a few times and then barely detected the rectal cancer sample, leading to negative results in both the breath and watery stool tests. It is thought that some cancers are difficult to detect by canine scent.\n\nNo correlation between canine scent judgement and human haemoglobin and transferrin was observed. Thus, canine scent judgement does not appear to depend on human haemoglobin or transferrin scents. All canine scent judgements of watery stool sample mixture tests were correct, indicating that a specific cancer scent indeed exists. The volatile organic compounds (VOCs) detected during canine scent judgement presumably occur early in the pathogenesis of CRC. As canine scent judgement can be used on both breath samples and watery stool samples, these chemical compounds may be circulating throughout the body.\n\nTo determine whether a specific cancer scent exists or if a particular natural scent disappears in patients with cancer, mixtures that included watery stool samples were tested. Tests were performed three times as described above. The results of all three tests were correct, thereby suggesting that a specific cancer scent indeed exists.\n\nCanine scent judgement for distinguishing between cancer patients and controls has been reported for other cancer types.24\u201326 We obtained breath and watery stool samples from patients with breast, stomach and prostate cancer who also underwent colonoscopy. We also tested these samples using breath samples from patients with CRC as the standard. Canine scent judgement yielded correct answers for these cancers as well (data not shown), suggesting that common scents may exist among various cancer types. This is consistent with the results of McCulloch *et al* which demonstrated the utility of canine scent detection for lung and breast cancer25; however, these results are not inconsistent with the existence of cancer-specific scents.\n\nIt may be difficult to introduce canine scent judgement into clinical practice owing to the expense and time required for the dog trainer and for dog education. Scent ability and concentration vary between different dogs and also within the same dog on different days. Moreover, each dog can only conduct tests for a maximum of 10\u2005years. It is therefore necessary to identify the cancer-specific VOCs detected by dogs and to develop an early cancer detection sensor that can be substituted for canine scent judgement.\n\nBreath VOCs have been reported as biomarkers of other gastrointestinal disorders such as inflammatory bowel disease, ulcerative colitis and necrotising enterocolitis.27\u201330 However, these tests have not been widely accepted, largely due to technical difficulties in measuring the pressure of exhaled VOCs, the large variations in food intake, the variability in the required time for bacterial colonisation and the poor predictive value of a positive test.\n\nSeveral VOCs have been identified as candidate substances for early detection of cancer using gas chromatography\/mass spectroscopy (GC\/MS) in the exhaled breath of patients with lung and breast cancer31\u201338; while these data are promising, they are preliminary findings that should be interpreted with caution. We intend to evaluate these VOCs by chemical analysis and with cancer detection dogs in the near future. Identification of cancer-specific VOCs will lead to solving the biological character of cancer. We hope that the results of the present study will provide encouragement for the development of cancer detection and solving the biological character of cancer using odour material.\n\nThe authors thank Tomoe Fujino and Mika Ohnishi for their valuable technical assistance. The sponsor of the trial had no role in the design or conduct of the study, or in the analysis of the data. The corresponding author had full access to the data and had final responsibility for the decision to submit the data for publication. No compensation was given to subjects for providing breath samples or watery stool samples.\n\n# References","meta":{"dup_signals":{"dup_doc_count":209,"dup_dump_count":86,"dup_details":{"curated_sources":2,"2023-40":3,"2023-14":1,"2023-06":2,"2022-49":1,"2022-27":3,"2022-05":1,"2021-49":3,"2021-43":1,"2021-39":1,"2021-31":1,"2021-25":3,"2021-21":2,"2021-17":3,"2021-10":1,"2021-04":2,"2020-40":2,"2020-34":1,"2020-29":2,"2020-24":1,"2020-16":1,"2020-05":3,"2019-51":2,"2019-47":1,"2019-43":3,"2019-35":3,"2019-30":2,"2019-26":2,"2019-22":1,"2019-18":3,"2019-13":1,"2019-09":1,"2019-04":2,"2018-47":3,"2018-39":3,"2018-34":2,"2018-30":2,"2018-26":3,"2018-22":1,"2018-17":2,"2018-13":3,"2018-09":2,"2018-05":2,"2017-51":3,"2017-47":5,"2017-43":2,"2017-39":4,"2017-34":2,"2017-30":5,"2017-22":4,"2017-17":4,"2017-09":4,"2017-04":2,"2016-50":2,"2016-44":3,"2016-40":3,"2016-36":3,"2016-30":3,"2016-26":2,"2016-22":3,"2016-18":3,"2016-07":2,"2015-48":3,"2015-40":2,"2015-35":2,"2015-32":2,"2015-27":2,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":4,"2014-42":5,"2014-41":4,"2014-35":1,"2014-23":3,"2014-15":2,"2024-30":2,"2024-26":1,"2024-22":3,"2024-18":1,"2017-13":7,"2015-18":3,"2015-11":2,"2015-06":2,"2014-10":2,"2013-48":2,"2013-20":2}},"file":"PMC3095480"},"subset":"pubmed_central"}
{"text":"abstract: Increases in weight have been associated with corresponding increases in insulin resistance in postmenopausal women. Although estrogen has significant impact on body fat and body fat distribution, the cellular mechanisms that influence this process are not yet known. We measured adipose tissue fatty acid (FA) storage and FA storage factors in 12 premenopausal and 11 postmenopausal women matched for age and body composition. Postmenopausal women had lower postprandial FA oxidation (indirect calorimetry), greater meal FA, and direct free FA (FFA) storage than premenopausal women, including two-fold greater meal FA storage in the femoral depot. The fed\/fasted activities of adipose tissue lipoprotein lipase were not significantly different between premenopausal and postmenopausal women. In contrast, adipocyte acyl-CoA synthetase and diacylglycerol acyltransferase activities in postmenopausal women were significantly upregulated and were positively correlated with direct FFA storage rates. These findings suggest that the propensity for subcutaneous adipose tissue FA storage is increased in postmenopausal women, more so from changes in adipocyte FA storage factors than from adipose tissue lipoprotein lipase activity. Our results suggest that female sex steroids, most likely estrogen, have important effects on adipose tissue FA storage and FA oxidation that could promote fat gain in postmenopausal women.\nauthor: Sylvia Santosa; Michael D. JensenCorresponding author: Michael D. Jensen, <email@example.com>\ndate: 2013-03\nreferences:\ntitle: Adipocyte Fatty Acid Storage Factors Enhance Subcutaneous Fat Storage in Postmenopausal Women\n\nIncreases in body weight are associated with greater risks of type 2 diabetes (1\u20133), and weight gain in postmenopausal women are of special concern (4). Estrogen has remarkable effects on body fat distribution, and the decreased sex hormone production after menopause is associated with increased total body fat (5,6), especially in the central\/abdominal region (7,8). Hormone replacement therapy in early menopause may mitigate these changes in body composition and may decrease central adiposity (9). Despite the strong evidence that female sex steroids have a major influence on total body fat and body fat distribution, the cellular mechanisms mediating these effects are unknown. To address this lack of understanding, we measured the storage of dietary fatty acids (FAs) and circulating free fatty acids (FFAs) into adipose tissue in premenopausal and postmenopausal women carefully matched for age and body composition. These physiological measures were combined with measures of the adipose tissue content of a number of proteins\/enzymes required for adipocyte FA storage.\n\nThe FA stored in adipose tissue originates primarily from triglyceride-rich lipoproteins (i.e., chylomicrons and VLDL); however, there is also a component of FA redistribution via the direct FFA reuptake pathway. FFAs are the products of adipose tissue lipolysis, which is virtually the sole source of FFA in the postabsorptive state. Triglyceride-rich lipoproteins require lipoprotein lipase (LPL) to liberate the FA from the glycerol backbone of the triglyceride molecule. Regardless of whether FAs are derived from triglyceride-rich lipoproteins or circulating FFA, the FA can enter the adipocyte either via a passive (flip-flop) mechanism or via protein facilitated diffusion (10). Once inside the adipocyte, FA must undergo a series of enzymatic reactions to be stored as triglyceride. Although it is known that estrogen can modulate LPL activity by suppressing gene transcription (11), little else is understood about whether estrogen affects adipocyte storage steps, including protein-facilitated transport and the enzymes needed for triglyceride synthesis.\n\nWe performed quantitative measures of meal-derived FA and direct FFA storage in adipose tissue and integrated these physiological assessments with information regarding some key factors that regulate cellular storage of FA as triglycerides. We focused on the FA transport protein (CD36), acyl-CoA synthetase (ACS) activity, and diacylglycerol acyltransferase (DGAT) activity. These FA storage factors are involved in different tiers of adipocyte FA storage. CD36 is a cell-surface glycoprotein that facilitates FA transport into cells (12). The ACS enzymes catalyze the activation of FA to their CoA derivatives (13). The final step in FA storage as triglycerides is catalyzed by DGAT (14,15). By integrating the physiological and cellular information regarding adipocyte FA storage, we are able to provide novel insights into the effects of estrogen on systemic and regional adipose tissue metabolism in humans. The unique differences in FA storage between premenopausal and postmenopausal women we observe contribute toward our understanding of body fat patterning in women.\n\n# RESEARCH DESIGN AND METHODS\n\n## Subjects.\n\nEleven women who had undergone menopause naturally (at least 3 years from last menstrual period) or surgically (at least 2 years from salpingo-ophorectomy) participated in the research study. For the purposes of this report, we refer to this group as postmenopausal. To be included in the study, women could not have been using hormone replacement therapy for at least 2 years. Thirteen premenopausal women with normal serum estrogen concentrations (premenopausal) were recruited as age-matched and BMI-matched controls. All participants were healthy and weight was stable (\u00b11.0 kg for \\> 2 months before the study). Participants were excluded if they had diabetes, anemia, or were using antidepressants or other medications that could affect FA metabolism. Written informed consent was obtained from all participants. The study was approved by the Institutional Review Board of the Mayo Clinic.\n\n## Materials.\n\n\\[1-^14^C\\]palmitate and \\[9,10-^3^H\\]triolein were purchased from NEN Life Science Products (PerkinElmer, Boston, MA). ^2^H~2~O and \\[U-^13^C\\]palmitate (both 99 atom percent pure) were purchased from Isotec (Miamisburg, OH).\n\n## Study design.\n\nAll studies were conducted in the Mayo Clinical Research Unit. Before their inpatient study visit, total body water and body composition were measured. Participants were provided with all meals for 5 days immediately before their inpatient study day to ensure that they were in comparable and stable nutritional states (16). Participants then were admitted for their inpatient study stay. The protocol for the inpatient study days has been described elsewhere (16). Briefly, after an overnight stay, volunteers consumed an experimental meal containing \\[^3^H\\]triolein. Blood samples were collected and indirect calorimetry (DeltaTrac, Yorba Linda, CA) was performed hourly. Urine was collected over a 24-h period for nitrogen and ^3^H~2~O excretion as part of meal fat oxidation measurements. After a second consecutive overnight visit, FFA tracers were given to measure direct FFA storage rates. Abdominal and femoral adipose tissue biopsies were performed on the first day at 1400 h (1 h after lunch) and on the second day at 30 min after the bolus infusion of \\[1-^14^C\\]palmitate. Blood samples for plasma catecholamine concentrations were collected at 0, 300, and 1,440 min during the study day.\n\n## Assays and methods.\n\nPlasma triglyceride concentrations and ^3^H content in chylomicron and nonchylomicron fractions were measured as previously described (17). Urinary nitrogen was measured using an Analox GM7 Fast Enzymatic Metabolite Analyzer (Analox Instruments, Lunenburg, MA). Plasma glucose was measured using a glucose analyzer (Beckman Instruments, Fullerton, CA) and plasma catecholamine concentrations were measured by reversed phase high-performance liquid chromatography (18). Plasma triglyceride concentrations were measured using a microfluorometric assay (19). Plasma insulin concentrations were measured using chemiluminescent assays on an automated immunoassay system (Assay and DxI, Beckman Instruments, Chaska, MN). Plasma estrogen concentrations were measured via liquid chromatography\/mass spectrometry.\n\n### Body composition.\n\nFat-free mass, total body mass, and leg fat mass were measured via dual-energy X-ray absorptiometry (Lunar iDXA, GE Healthcare, Madison, WI) (20). Abdominal subcutaneous and visceral adipose tissue areas were measured by combining data from a single-slice abdominal computed tomography scan at the L2\u2013L3 level with the dual-energy X-ray absorptiometry-measured total abdominal fat content, as previously described (21).\n\n### Substrate oxidation.\n\nParticipants fasted for 12 h before measuring resting energy expenditure, as previously described (16). Measurements were taken 15 min hourly from 0 to 360 min after the consumption of the experimental meal. Carbohydrate and fat oxidation at each time point were calculated and total oxidation was determined as area under the curve (AUC) (22).\n\n### Fatty acid metabolism studies.\n\nMeal FA storage and oxidation were determined as previously described (16). Briefly, 50 \u00b5Ci \\[9,10-^3^H\\]triolein was sonicated with an Ensure Plus meal that provided 40% of individually measured resting energy expenditure (23). Participants consumed the meal at 0800 h and quadruplicate 50-\u00b5L samples of the test meal were counted on a liquid scintillation counter to determine the exact amount of \\[^3^H\\]triolein consumed. Meals with the same macronutrient composition as those provided during the week before admission were consumed at lunch (1300 h) and dinner (1800 h). ^3^H~2~O concentration in body water was measured in urine collected after a 24-h void, as previously described (16). A continuous infusion of \\[U-^13^C\\]palmitate to measure FFA flux (24) combined with a bolus infusion of \\[1-^14^C\\]palmitate was administered to measure direct adipose tissue FFA storage rates (25). Abdominal and femoral adipose tissue biopsies were performed using sterile technique and local anesthesia at 6 h and 24 h after experimental meal: the 24-h biopsy was timed to occur 30 min after the ^14^C bolus (16). After lipid extraction via collagenase digestion (26), adipocyte ^3^H and ^14^C lipid-specific activity (dpm\/g lipid) were measured as previously described (27).\n\n### Adipose tissue analysis.\n\nFat cell size was measured using photomicrographs (28), LPL activity was measured using the approach of Nilsson-Ehle and Schotz (29), ACS (30) and DGAT (15) activity were determined using enzymatic assays, and CD36 quantification was measured using sandwich ELISA (31).\n\n## Calculations, data analysis, and statistics.\n\nCalculations are described in detail elsewhere (16). Because the means of data expression can impact data interpretation (32), we provide two approaches in an attempt to address different perspectives. We use the per unit lipid expression when the question relates to whether one body fat depot competes better for the available FAs than another depot. However, when examining the factors that regulate tissue FA storage rates at a cellular level, we express the data per 1,000 adipocytes. The Shapiro-Wilk test was performed for goodness of fit and data that were not normally distributed were log-transformed. Unpaired *t* tests were applied for between-group comparisons. A mixed model 2 level and mixed model 3 level ANOVA with post hoc least-squares means tests were used to determine group differences using time and depot as within-group variables. If a relationship was apparent, then a Pearson correlation was used; otherwise, correlations in postmenopausal and premenopausal groups were determined using Spearman rank test. All data are presented as mean \u00b1 SEM and were analyzed using JMP 9.0 (SAS Institute, Cary, NC). Statistical significance was defined as *P* \\< 0.05.\n\n# RESULTS\n\n## Subject characteristics.\n\nThe two groups of women were matched for age, BMI, and body composition (<a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>). The average and range of BMI were similar in both the postmenopausal and premenopausal groups (21.6\u201338.3 kg\/m^2^). Abdominal fat cell size was smaller (*P* \\< 0.001) than thigh fat cell size in both groups and, by design, serum estrogen concentrations were greater in premenopausal than in postmenopausal women.\n\nSubject characteristics\n\n![](775tbl1)\n\nThere were no differences in baseline insulin or fasting plasma glucose concentrations between groups. Despite comparable plasma glucose concentrations, the daytime plasma insulin concentrations, as measured by the AUC, was greater (*P* = 0.008) in postmenopausal women than in premenopausal women (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<\/a>). The average (mean of three samples) plasma epinephrine concentrations were 49 \u00b1 9 and 43 \u00b1 11 pg\/mL (postmenopausal versus premenopausal; *P* = not significant) and plasma norepinephrine concentrations were 219 \u00b1 17 and 176 \u00b1 15 pg\/mL (postmenopausal versus premenopausal; *P* = not significant). Overnight postabsorptive plasma palmitate concentrations and flux were not different in the two groups (<a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>).\n\nThe AUC of nonchylomicron triglyceride concentrations in postmenopausal women tended to be greater (*P* = 0.05) than in premenopausal women (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<\/a>). There were no significant differences between premenopausal and postmenopausal groups in AUC of chylomicron triglyceride concentrations (*P* = 0.17) or specific activity (*P* = 0.15), suggesting that meal FA absorption\/transport into the circulation was similar in the two groups.\n\n## Substrate and meal FA oxidation.\n\nThe AUC of total FA oxidation measured by hourly indirect calorimetry over the 6 h after the experimental meal was greater (*P* = 0.04) in premenopausal women than postmenopausal women (<a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a>). In contrast, meal FA oxidation (^3^H~2~O generation) over 24 h was not different between groups. Overnight basal metabolic rate and respiratory exchange ratio were similar between groups (<a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a>) and within individuals between study days, indicating that the participants were in a metabolically steady state.\n\nEnergy and FA metabolism\n\n![](775tbl2)\n\n## Regional meal FA storage.\n\nThere was a group \u00d7 depot effect (*P* = 0.01) in 24-h meal FA storage (mg meal FA\u22c5g lipid^\u22121^), with a greater difference between abdominal and femoral meal FA storage in postmenopausal than in premenopausal women. Postmenopausal women stored more (*P* = 0.0002) meal FA in the femoral than abdominal depot (<a href=\"#T3\" data-ref-type=\"table\">Table 3<\/a>). In contrast, meal FA storage in premenopausal women was not significantly different between depots. Postmenopausal women also stored more (*P* = 0.001) meal FA per gram of lipid in the femoral region than premenopausal women.\n\nAdipose tissue characteristics\n\n![](775tbl3)\n\nA greater percentage of meal FA was stored in lower body subcutaneous adipose tissue (LBSQ; *P* = 0.001) in postmenopausal than premenopausal women. There was a trend (*P* = 0.05) for a greater proportion of meal fat to be stored in upper body subcutaneous adipose tissue (UBSQ) of postmenopausal versus premenopausal women. In total, postmenopausal women stored a greater (*P* = 0.002) proportion of meal FA in subcutaneous fat than premenopausal women.\n\n## Direct adipocyte FFA storage.\n\nRates of FFA storage are presented as \u00b5mol\u22c5kg adipose tissue^\u22121^\u22c5min^\u22121^ for physiological interpretation and as pmol\u22c51,000 cells^\u22121^\u22c5min^\u22121^ to permit us to address mechanisms at the cellular level. At the physiological level, there was a group \u00d7 depot effect (*P* = 0.047) in rate of direct FFA storage (\u00b5mol\u22c5kg adipose tissue^\u22121^\u22c5min^\u22121^). Rates of direct FFA storage were greater (*P* = 0.02) in femoral adipose tissue of postmenopausal than premenopausal women. The rates of direct FFA storage were greater (*P* = 0.0005) in femoral than abdominal adipose tissue only in postmenopausal women. At the cellular level, rates of FFA storage (pmol\u22c51,000 cells^\u22121^\u22c5min^\u22121^) were greater in the femoral than abdominal fat for both premenopausal (*P* = 0.009) and postmenopausal (*P* = 0.0001) women. Although the proportion of FFA stored in UBSQ and LBSQ depots via the direct pathway was not significantly different in postmenopausal women, premenopausal women stored a greater proportion (*P* = 0.01) of FFA in UBSQ than in LBSQ.\n\n## Adipose-specific effectors of FA storage\n\n### LPL activity.\n\nLPL activity was significantly greater in femoral than abdominal fat in both the fasted and fed state in both postmenopausal and premenopausal women (<a href=\"#T3\" data-ref-type=\"table\">Table 3<\/a>). However, the difference between abdominal and femoral LPL activity was not as marked in the fed state as indicated by a time \u00d7 depot effect (*P* = 0.003). In addition, the change in LPL activity between depots across time was different between groups as indicated by a time \u00d7 group \u00d7 depot effect (*P* = 0.04). This was explained by a greater increase in abdominal LPL activity from the fasted to the fed state in postmenopausal than in premenopausal women (<a href=\"#T3\" data-ref-type=\"table\">Table 3<\/a>).\n\n### LPL activity as a predictor of meal FA storage.\n\nBecause fed LPL activity correlates with fractional meal FA storage in young, lean, healthy men and women (33), we examined this relationship in this study (<a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<\/a>). The correlation between fed abdominal LPL activity and the proportion of meal FA stored in UBSQ was significant (*r* = 0.66; *P* = 0.03) in postmenopausal women, and nearly so in premenopausal women (*r* = 0.57; *P* = 0.06). Fed femoral LPL activity correlated (*P* = 0.60; *P* = 0.04) with the proportion of meal FA stored in LBSQ fat only in premenopausal women.\n\n### Adipocyte FA storage factors.\n\nOvernight postabsorptive adipose tissue CD36 content and ACS and DGAT activity data are presented in <a href=\"#T3\" data-ref-type=\"table\">Table 3<\/a> and are expressed in both physiological (per milligram of lipid) and cellular (per 1,000 cells) terms.\n\nAt both the physiological (*P* = 0.002 for abdominal; *P* = 0.007 for femoral) and cellular levels (*P* = 0.005 for abdominal; *P* = 0.03 femoral), DGAT activity was greater in postmenopausal than in premenopausal women. At the cellular level, abdominal ACS activity was greater (*P* = 0.04) in postmenopausal than in premenopausal women. There were no significant differences in regional CD36 content (per 1,000 cells) in postmenopausal versus premenopausal women.\n\nAt the physiological level, femoral ACS activity was greater (*P* = 0.01 and *P* = 0.001, respectively) than abdominal ACS activity in both postmenopausal and premenopausal women. In premenopausal women only, CD36 content per milligram of lipid was greater (*P* = 0.04) in femoral than in abdominal fat. ACS and DGAT activity and CD36 content per 1,000 cells of fat were greater (*P* \\< 0.02 for all) in femoral than in abdominal fat for premenopausal and postmenopausal women.\n\n### Adipocyte proteins versus direct FFA storage rates.\n\nFor postmenopausal women, but not for premenopausal women, the activity of ACS, DGAT, and CD36 content were all significantly associated with rate of FFA storage (per 1,000 cells) in abdominal (\u03c1 = 0.75 and *P* \\< 0.01 for all) and femoral (\u03c1 = 0.74 and *P* = 0.01; \u03c1 = 0.85 and *P* = 0.0008; \u03c1 = 0.63 and *P* = 0.04, respectively) fat (<a href=\"#F4\" data-ref-type=\"fig\">Fig. 4<\/a>). No significant associations were found between adipocyte proteins and FFA storage in premenopausal women. There were no significant physiological (per milligram of lipid) relationships between ACS and DGAT activity or between CD36 content and the rates of FFA storage per kilogram of adipose tissue. No significant associations were found between adipocyte proteins and FFA storage in premenopausal women.\n\n### Relationship between adipocyte FA storage factors.\n\nFor abdominal fat, ACS activity, DGAT activity, and CD36 content were correlated with each other in postmenopausal women (ACS versus DGAT: \u03c1 = 0.77 and *P* = 0.005; ACS versus CD36: \u03c1 = 0.69 and *P* = 0.02; DGAT versus CD36: \u03c1 = 0.78 and *P* = 0.005). Similarly, in femoral fat of postmenopausal women, ACS activity was correlated with both CD36 content and DGAT activity (\u03c1 = 0.71 and *P* = 0.02 for both). There were no relationships between CD36 content and ACS and DGAT activity in the abdominal or femoral depots in premenopausal women.\n\n# DISCUSSION\n\nAlthough there are large amounts of descriptive data regarding the effects of estrogen on body fat distribution, the underlying cellular mechanisms remain unknown. We examined the regulation of fat storage pathways in vivo in postmenopausal and age-matched and body composition\u2013matched premenopausal women. We found that meal FA storage in subcutaneous fat was greater in postmenopausal than in premenopausal women. This difference was especially evident in the femoral depot, where meal FA storage in postmenopausal women was double that of premenopausal women. Rates of direct FFA storage also were greater in postmenopausal versus premenopausal women, although differences were only significant in the femoral region. At the level of the adipocyte, CD36 content and ACS and DGAT activity are highly coregulated in postmenopausal women, and these FA storage factors were significantly related to FFA storage. Our results imply that the sex steroid deficiency of menopause increases CD36 content and ACS and DGAT activity in such a manner as to facilitate greater adipose tissue FA storage.\n\nAs expected, we observed that plasma insulin and triglycerides were somewhat greater in postmenopausal women. Although the contribution of changing fat distribution versus hormonal changes with menopause to insulin and triglyceride responses are unclear, other investigators also have observed higher fasting insulin and triglyceride concentrations in postmenopausal women (34\u201337). Abdominal adipose tissue LPL activity in the fed state was greater in postmenopausal women. The somewhat lower LPL activity in estrogen-sufficient women is consistent with findings from other groups (38,39). Homma et al. (11) found a sequence on the LPL promoter that responds to estrogen by suppressing gene transcription in vitro. Our finding that an association between femoral LPL activity and meal FA storage was present in premenopausal, but not in postmenopausal women, suggests that the upregulated femoral LPL activity in postmenopausal women is no longer rate-limiting for dietary FA storage in this depot, at least at this amount of dietary fat intake.\n\nWe also found that postmenopausal women had lower fat oxidation than premenopausal women. Whether estrogen alters fat oxidation is controversial. Although some have found that postmenopausal women treated with estrogen increase fat oxidation (40,41), others have not observed differences in fat oxidation between postmenopausal women who were estrogen-deficient versus estrogen-sufficient (42,43). Our findings of reduced postprandial FA oxidation in postmenopausal women are consistent with the findings of Lwin et al. (41). Regarding mechanism, it has been reported that ovariectomized mice administered estrogen upregulate the skeletal muscle expression of peroxisome proliferator\u2013activated receptor-\u03b4 and peroxisome proliferator\u2013activated receptor-\u03b1 (44,45), which should increase fat oxidation.\n\nTo our knowledge, this study is the first to measure a series of adipocyte FA storage factors in premenopausal and postmenopausal women. At the physiological and cellular levels, both abdominal DGAT activity and femoral DGAT activity were most strikingly different between postmenopausal and premenopausal women; DGAT activity was 25\u201340% higher in the former. Although some adipocyte FA storage factors were not significantly different between groups, all factors we measured were greater in postmenopausal women. In both abdominal and femoral depots, each adipocyte FA storage factor we measured was significantly correlated with direct FFA storage, but only in postmenopausal women. These correlations indicate a global increase in fat storage tendencies that is not specific to abdominal or femoral fat.\n\nWe cannot determine from this study whether reduced ability to oxidize fat promotes greater fat storage or whether increases in fat storage limits fat oxidation. The greater FA storage tendencies of postmenopausal women may suggest that adipose tissue diverts fat from oxidative pathways. The greater propensity toward meal and direct FFA storage in the adipose tissue of postmenopausal women was accompanied by decreases in total FA oxidation over a 6-h postprandial interval. We note, however, that meal fat oxidation (from the experimental breakfast meal) was not different between groups. This may point toward a shift of FA oxidation away from FFA and toward meal sources, which would account for the greater adipose storage of dietary fat without reductions in meal FA oxidation. Our finding of greater direct FFA storage, albeit in the postabsorptive state, is consistent with this interpretation. The combination of greater adipose tissue FA storage and greater postprandial carbohydrate oxidation in postmenopausal women theoretically could promote greater food intake to maintain glycogen stores. In this scenario, the slightly greater food intake over time could promote weight gain in the form of excess body fat.\n\nA particular strength of this study is that FA storage is examined in the context of numerous adipocyte FA storage factors, including LPL activity. If we examined only LPL activity, then we may have come to the false conclusion that only abdominal adipocyte FA storage increases with female hypogonadism. By measuring abdominal and femoral ACS and DGAT activity, as well as CD36 content, we were able to document that the overall adipocyte FA storage machinery is increased in postmenopausal women. Because our volunteers were matched for body composition and age, these differences are most likely attributable to differences in sex steroid concentrations, not other factors. However, we cannot be sure whether the differences are caused solely by lower estrogen or by a combination of low estrogen and progesterone concentrations after menopause. Another issue to consider is that differences in insulin concentrations between premenopausal and postmenopausal women may have influenced adipocyte FA storage pathways. Insulin increases the expression of adipose ACS mRNA in vitro (46,47), and human adipose tissue DGAT mRNA expression is greater in those with increased insulin sensitivity (48). Furthermore, DGAT activity is stimulated by insulin and glucose (49). In contrast, insulin does not appear to stimulate CD36 translocation (50). Thus, the greater daytime insulinemia in postmenopausal women may have impacted ACS and DGAT activity. We did not find statistically associations between the 24-h AUC of insulin concentrations and the adipogenic protein activities and content (data not shown), suggesting that the observed differences between premenopausal and postmenopausal women were influenced more so by differences in sex steroids than insulin.\n\nWe hypothesize that the effects we documented are more likely made chronic by estrogen deficiency, because premenopausal women had 10-fold greater estrogen concentrations than postmenopausal women. Furthermore, the study was designed to isolate the effects of estrogen from age and the well-described shifts in total and regional body fat distributions that occur with menopause by matching our participants for these variables.\n\nIn summary, our findings indicate that overall fat storage tendencies are increased in postmenopausal women. This finding is significant because weight gain is associated with greater risk of diabetes (1\u20133). The increase in fat storage stems from somewhat greater LPL activity and significantly greater content of adipocyte FA storage factors. It is possible that the upregulation in proteins associated with FA storage capacity in postmenopausal women contributes to the decrease in postprandial total fat oxidation. Whether the differences in FA storage between premenopausal and postmenopausal women are attributable to the effects of estrogen or the combination of estrogen, progesterone, and other factors, such as changing insulin concentrations, remains to be elucidated.\n\n## ACKNOWLEDGMENTS\n\nThis work was supported by grant NCRR 1UL1 RR024150, National Institutes of Health grants DK-45343 and DK-50456, and by ADA grant 7-06-DCS-03. S.S. received fellowship funding from the Natural Sciences and Engineering Research Council of Canada and incentive funding from the Canadian Diabetes Association.\n\nNo potential conflicts of interest relevant to this article were reported.\n\nS.S. contributed to the study design, performed the studies, researched data, and wrote the manuscript. M.D.J. designed and oversaw the study, reviewed the manuscript, and edited the manuscript. M.D.J. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.\n\nThe authors are indebted to the research volunteers for their participation. The authors also are grateful to Barbara Norby and Carley Vrieze for assistance with nursing care and adipose biopsies. They also thank Christy Allred, Debra Harteneck, Darlene Lucas, and Lendia Zhou for assay development and performance. All persons listed herein are affiliated with the Mayo Clinic, Rochester, Minnesota.\n\nThe meal FA metabolism data were presented at the Canadian Obesity Network Meeting, and the direct FFA data were presented at the 71st Scientific Sessions of the American Diabetes Association, San Diego, California, 24\u201328 June 2011.\n\n# REFERENCES","meta":{"dup_signals":{"dup_doc_count":125,"dup_dump_count":55,"dup_details":{"curated_sources":2,"2022-05":1,"2021-43":1,"2021-21":1,"2019-43":2,"2019-22":2,"2018-43":1,"2018-30":2,"2018-26":1,"2018-17":1,"2018-13":2,"2018-09":1,"2018-05":1,"2017-51":1,"2017-47":3,"2017-43":2,"2017-39":1,"2017-34":2,"2017-30":3,"2017-26":3,"2017-17":5,"2017-09":4,"2017-04":4,"2016-50":2,"2016-44":4,"2016-40":2,"2016-36":3,"2016-30":3,"2016-26":2,"2016-22":2,"2016-18":2,"2016-07":3,"2015-48":3,"2015-40":2,"2015-35":1,"2015-32":1,"2015-27":3,"2015-22":4,"2015-14":1,"2014-52":3,"2014-49":1,"2014-42":4,"2014-41":5,"2014-35":4,"2014-23":3,"2014-15":2,"2022-40":1,"2024-26":2,"2024-22":1,"2024-10":1,"2017-13":2,"2015-18":3,"2015-11":3,"2015-06":3,"2014-10":2,"2013-48":1}},"file":"PMC3581212"},"subset":"pubmed_central"}
{"text":"date: 2013-08\ntitle: Highlights From the Latest in Diabetes Research\n\n# The Relationship Between Obesity and Vitamin D Deficiency: Causation Determined?\n\nMultiple investigations have evaluated the relationship between obesity and vitamin D deficiency, two modifiable factors that contribute to the risk of several chronic diseases. While such studies have identified a strong association between these phenotypes, it has been difficult to determine causality and the direction of the association. Through a series of sophisticated genetic analyses including bi-directional Mendelian randomization (MR), results from Vimaleswaran et al. have highlighted potentially important insights regarding the link between obesity, as assessed by BMI, and vitamin D deficiency. Using meta-analysis approaches in approximately 42,000 participants of D-CarDia, the authors observed that each unit increase in BMI was associated with a 1.15% decrease in 25-hydroxyvitamin D \\[25(OH)D\\]. In order to infer causality, their findings were extended by MR using previously established BMI-related (*n* = 12) and vitamin D\u2013related (*n* = 4) single nucleotide polymorphisms in order to generate allele scores for the analyses. These analyses revealed that while the BMI allele score was significantly associated with reduced 25(OH)D levels (-0.06% \\[95% CI -0.10 to -0.02%\\], *P* = 0.004), neither the vitamin D synthesis or metabolism allele scores were associated with BMI (0.01 \\[-0.17 to 0.20\\], *P* = 0.88 and 0.17 \\[-0.02 to 0.35\\], *P* = 0.08, respectively). Using the instrumental variable (IV) ratio method, it was observed that each 10% increase in BMI led to a 4.2% decrease in 25(OH)D levels. In contrast, IV analyses did not provide evidence that 25(OH)D levels significantly affected BMI. Follow-up analyses using data from the Genetic Investigation of Anthropometric Traits (GIANT) consortium confirmed the lack of association between vitamin D allele scores and BMI. Together, these results suggest that an increase in obesity results in lower vitamin D status but that vitamin D concentration has little or no impact on the development of obesity. \u2014 *Joshua P. Lewis, PhD*\n\n**Vimaleswaran et al. Causal relationship between obesity and vitamin D status: bi-directional Mendelian randomization analysis of multiple cohorts. PLoS Med 2013;10:e1001383**\n\n# Defective Insulin Signaling: A Common Feature of Diabetes and Alzheimer's Disease?\n\nObesity and aging are at the root of considerable public health concern in many developed countries: In many industrialized nations, there has been a marked increase in the prevalence of obesity over the last several decades, a trend that is occurring in tandem with the \"graying\" of many of the same populations. The prevalence of type 2 diabetes has increased in parallel with trends in obesity; a similar pattern is occurring for both mild cognitive impairment and Alzheimer's disease (AD) in relation to the growing number of older adults in many developed countries. There has been some research from Bomfin et al. suggesting a link between diabetes and decrements in cognition, including AD. Insulin resistance (IR) is a cardinal feature of type 2 diabetes, and there is evidence supporting the idea that IR is present in AD. Findings from recent studies suggesting that brain insulin signaling is defective in AD raise obvious questions regarding the possibility that insulin signaling defects may play a role in the development of both diabetes and AD. Building on the findings showing that amyloid-\u03b2 peptide (A\u03b2) oligomers (A\u03b2Os) (synaptotoxins that collect in the AD brain) result in the loss of insulin receptors from neuronal tissues, new data suggest that A\u03b2Os also activate the Jun NH~2~-terminal kinase\/tumor necrosis factor-\u03b1 (JNK\/TNF-\u03b1) pathway and induce insulin receptor substrate (IRS)-1 phosphorylation in hippocampal neurons. Both JNK activation and IRS-1 phosphorylation are also observed in diabetes, indicating that pathologic mechanisms related to insulin signaling may be shared in the two conditions. The neuronal pathologies that were induced by A\u03b2Os in these experiments were prevented with exenatide. In Tg mice with a brain condition that models AD, exenatide blocked defective insulin signaling and improved cognition. Assuming that impaired insulin signaling is in the causal pathway for AD, exploration of the role of glucagon-like peptide 1 receptors may be ripe for future investigation. It is interesting to consider the idea that two of the most pressing chronic conditions affecting developed nations could be treated with the same therapeutic agent. Despite these intriguing results, it should be stressed that the new findings could merely reflect an association of two common diseases. \u2014 *Helaine E. Resnick, PhD, MPH*\n\n**Bomfin et al. An anti-diabetes agent protects the mouse brain from defective insulin signaling caused by Alzheimer's disease\u2013associated A\u03b2 oligomers. J Clin Invest 2012;122:1339\u20131353**\n\n# Betatropin: A New Hormone Regulating \u03b2-Cell Replication and Mass\n\nAlthough the defects underpinning type 1 and type 2 diabetes differ in fundamental ways, both conditions benefit from treatments that result in increased \u03b2-cell mass. Although there is a rapid increase in \u03b2-cell replication in the embryonic and neonatal stages of development, the rate of increase slows substantially in later life. However, the rate of \u03b2-cell replication can increase in response to certain challenges in late life, including gestation, pancreatic injury, and insulin resistance. The fact that both \u03b2-cell number and mass can increase in response to certain challenges raises the possibility to harness and direct mechanisms that favor \u03b2-cell proliferation in a manner that could have therapeutic implications. Although focusing on circulating factors is an appealing approach to this problem, targeting currently identified hormones is limited by the lack of specificity of these factors or their modest impact on \u03b2-cell proliferation. New work by Yi et al. provides intriguing data on a newly identified hormone that has potential therapeutic implications. In a series of experiments using a new mouse model in which \u03b2-cell replication can be induced, the investigators identified a gene that encodes a protein that is expressed in liver and fat. Importantly, this protein was found to be elevated in insulin resistance and to specifically induce pancreatic \u03b2-cell proliferation and increased \u03b2-cell mass. In addition to demonstrating its impact on \u03b2-cell mass, the newly published results indicate that insulin-resistant mice expressing the new protein experienced significant improvements in glucose tolerance. The investigators named this new hormone betatrophin. Although its mechanism of action remains unknown, the authors suggest that in the future, this hormone could serve as a companion to existing diabetes therapies or perhaps even replace the need for exogenous insulin. \u2014 *Helaine E. Resnick, PhD, MPH*\n\n**Yi et al. Betatrophin: a hormone that controls pancreatic \u03b2 cell proliferation. Cell 2013;153:747\u2013758**\n\n# New Loci Associated With Birth Weight Identify Genetic Links Between Intrauterine Growth and Adult Height and Metabolism\n\nFetal growth and birth weight are associated with the development of adult-onset metabolic diseases. Both environmental insults (i.e., maternal nutrition, smoking, and drug use) and genetic factors have been implicated in alterations in intrauterine growth and development. A recent report by Horikoshi et al. investigates the genetic factors affecting birth weight through an expanded meta-analysis genome-wide association study (GWAS) aimed at identifying genetic loci that also correspond with adult height and metabolic traits. The expanded meta-analysis GWAS of birth weight (26,836 subjects of European descent from 18 studies) identified four new loci associated with birth weight and confirmed three previously identified loci. Single nucleotide polymorphisms in genes in two of the loci, *ADCY5* and *CDKAL1*, were previously shown to be associated with type 2 diabetes. At both loci, the alleles associated with lower birth weight were also associated with increased risk for type 2 diabetes, with evidence for impaired insulin secretion in the adult population. These results are consistent with a reduction in birth weight due to reduced insulin concentration, which manifests as type 2 diabetes in adulthood. The Gly389-encoding allele of a missense mutation in the *ADRB1* locus (Arg389Gly) was associated with lower birth weight and lower systolic blood pressure in adulthood. Finally, the birth weight\u2013lowering alleles at two of the birth weight\u2013associated loci, *HMGA2* and *LCORL*, were also associated with lower adult height. This study not only identifies genetic links between intrauterine growth and adult height and metabolism, it also sheds new light on the biological pathways and mechanisms influencing these traits and development of disease. Dissecting these biological pathways and the relationships between the intrauterine and adult presentation of disease will allow for early, personalized prevention and treatment. \u2014 *Coleen M. Damcott, PhD*\n\n**Horikoshi et al. New loci associated with birth weight identify genetic links between intrauterine growth and adult height and metabolism. Nat Genet 2013;45:76\u201382**","meta":{"dup_signals":{"dup_doc_count":123,"dup_dump_count":46,"dup_details":{"curated_sources":2,"2023-06":1,"2021-10":1,"2020-05":1,"2018-30":1,"2018-26":2,"2018-17":3,"2018-13":1,"2018-05":4,"2017-47":1,"2017-43":7,"2017-39":1,"2017-34":3,"2017-30":6,"2017-26":3,"2017-22":1,"2017-17":8,"2017-09":3,"2017-04":9,"2016-50":3,"2016-44":3,"2016-40":3,"2016-36":2,"2016-30":3,"2016-26":3,"2016-22":3,"2016-18":1,"2016-07":2,"2015-48":3,"2015-40":3,"2015-35":2,"2015-32":1,"2015-27":1,"2015-22":3,"2015-14":3,"2014-52":2,"2014-49":1,"2014-42":4,"2014-41":3,"2014-35":2,"2014-23":2,"2023-14":1,"2017-13":3,"2015-18":3,"2015-11":2,"2015-06":2,"2024-22":1}},"file":"PMC3717868"},"subset":"pubmed_central"}
{"text":"date: 2018\ntitle: More than 90% of the world's children breathe toxic air every day\n\n**29 October 2018 News Release Geneva** - Every day around 93% of the world's children under the age of 15 years (1.8 billion children) breathe air that is so polluted it puts their health and development at serious risk. Tragically, many of them die: WHO estimates that in 2016, 600,000 children died from acute lower respiratory infections caused by polluted air.\n\nA new WHO report on Air pollution and child health: Prescribing clean air examines the heavy toll of both ambient (outside) and household air pollution on the health of the world's children, particularly in low- and middle-income countries. The report is being launched on the eve of WHO's first ever Global Conference on Air Pollution and Health.\n\nIt reveals that when pregnant women are exposed to polluted air, they are more likely to give birth prematurely, and have small, low birth-weight children. Air pollution also impacts neurodevelopment and cognitive ability and can trigger asthma, and childhood cancer. Children who have been exposed to high levels of air pollution may be at greater risk for chronic diseases such as cardiovascular disease later in life.\n\n\"Polluted air is poisoning millions of children and ruining their lives,\" says Dr Tedros Adhanom Ghebreyesus, WHO Director-General. \"This is inexcusable. Every child should be able to breathe clean air so they can grow and fulfil their full potential.\"\n\nOne reason why children are particularly vulnerable to the effects of air pollution is that they breathe more rapidly than adults and so absorb more pollutants.\n\nThey also live closer to the ground, where some pollutants reach peak concentrations \u2013 at a time when their brains and bodies are still developing.\n\nNewborns and young children are also more susceptible to household air pollution in homes that regularly use polluting fuels and technologies for cooking, heating and lighting\n\n\"Air Pollution is stunting our children's brains, affecting their health in more ways than we suspected. But there are many straight-forward ways to reduce emissions of dangerous pollutants,\" says Dr Maria Neira, Director, Department of Public Health, Environmental and Social Determinants of Health at WHO.\n\n\"WHO is supporting implementation of health-wise policy measures like accelerating the switch to clean cooking and heating fuels and technologies, promoting the use of cleaner transport, energy-efficient housing and urban planning. We are preparing the ground for low emission power generation, cleaner, safer industrial technologies and better municipal waste management, \" she added.\n\n# Key findings\n\n-   Air pollution affects neurodevelopment, leading to lower cognitive test outcomes, negatively affecting mental and motor development.\n\n-   Air pollution is damaging children's lung function, even at lower levels of exposures\n\n-   Globally, 93% of the world's children under 15 years of age are exposed to ambient fine particulate matter (PM2.5) levels above WHO air quality guidelines, which include the 630 million of children under 5 years of age, and 1.8 billion of children under 15 years\n\n-   In low- and middle-income countries around the world, 98% of all children under 5 are exposed to PM2.5 levels above WHO air quality guidelines. In comparison, in high-income countries, 52% of children under 5 are exposed to levels above WHO air quality guidelines.\n\n-   More than 40% of the world's population \u2013 which includes 1 billion children under 15 - is exposed to high levels of household air pollution from mainly cooking with polluting technologies and fuels.\n\n-   About 600'000 deaths in children under 15 years of age were attributed to the joint effects of ambient and household air pollution in 2016.\n\n-   Together, household air pollution from cooking and ambient (outside) air pollution cause more than 50% of acute lower respiratory infections in children under 5 years of age in low- and middle-income countries.\n\n-   Air pollution is one of the leading threats to child health, accounting for almost 1 in 10 deaths in children under five years of age.\n\nWHO's First Global Conference on Air Pollution and Health, which opens in Geneva on Tuesday 30 October will provide the opportunity for world leaders; ministers of health, energy, and environment; mayors; heads of intergovernmental organizations; scientists and others to commit to act against this serious health threat, which shortens the lives of around 7 million people each year. Actions should include:\n\n-   Action by the health sector to inform, educate, provide resources to health professionals, and engage in inter-sectoral policy making.\n\n-   Implementation of policies to reduce air pollution: All countries should work towards meeting WHO global air quality guidelines to enhance the health and safety of children. To achieve this, governments should adopt such measures as reducing the over-dependence on fossil fuels in the global energy mix, investing in improvements in energy efficiency and facilitating the uptake of renewable energy sources. Better waste management can reduce the amount of waste that is burned within communities and thereby reducing 'community air pollution'. The exclusive use of clean technologies and fuels for household cooking, heating and lighting activities can drastically improve the air quality within homes and in the surrounding community.\n\n-   Steps to minimize children's exposure to polluted air: Schools and playgrounds should be located away from major sources of air pollution like busy roads, factories and power plants.\n\nAvailable from: <http:\/\/www.who.int\/news-room\/detail\/29-10-2018-more-than-90-of-the-world%E2%80%99s-children-breathe-toxic-air-every-day>","meta":{"dup_signals":{"dup_doc_count":181,"dup_dump_count":42,"dup_details":{"curated_sources":2,"2023-50":2,"2023-40":1,"2023-23":1,"2023-14":2,"2023-06":1,"2022-49":2,"2022-27":2,"2022-21":2,"2022-05":5,"2021-49":2,"2021-39":2,"2021-25":1,"2021-21":1,"2021-17":3,"2021-04":4,"2020-45":2,"2020-40":3,"2020-34":1,"2020-29":3,"2020-24":3,"2020-16":2,"2020-10":2,"2020-05":2,"2019-51":2,"2019-47":3,"2019-43":4,"2019-39":2,"2019-35":7,"2019-30":15,"2019-26":6,"2019-22":11,"2019-18":12,"2019-13":14,"2019-09":6,"2019-04":20,"2018-51":2,"2018-47":16,"2024-30":2,"2024-26":2,"2024-22":2,"2024-18":2,"2024-10":2}},"file":"PMC6344660"},"subset":"pubmed_central"}
{"text":"abstract: A report on the conference 'Systems Genomics 2008', Heidelberg, Germany, 2-3 May 2008.\nauthor: Rainer Pepperkok; Stefan Wiemann\ndate: 2008\ninstitute: 1Cell Biology and Cell Biophysics Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany; 2Division of Molecular Genome Analysis, German Cancer Research Center, Im Neuenheimer Feld 580, 69120 Heidelberg, Germany\ntitle: Integrating systems biology with clinical research\n\nHigh-throughput techniques in genomics, proteomics, and cell biology hold the promise of systems-level analyses to elucidate fundamental biological principles and to understand and predict the behavior of cellular systems in health and disease. With this challenge in mind, the recent Systems Genomics 2008 conference <http:\/\/www.dkfz.de\/mga\/SG2008> in Heidelberg brought together researchers in the fields of genomics, functional genomics, and systems biology to discuss the latest technological developments and their possible implications for clinical research.\n\n# Systems and biological networks\n\nTwo keynote presentations dealt with the understanding of interaction networks, and with the implications of such knowledge for developing new therapeutic strategies in cancer. Peter Sorger (Harvard Medical School, Boston, USA) highlighted the central role of stochastic processes in the fate of cells and of the importance of determining them quantitatively. He and his colleagues have shown that, even in a visually homogeneous cell population, not all cells react the same way in response to a specific perturbation. While the causes of such an effect are complicated enough for binary decisions like apoptosis, differentiation or cell division, they will be even more so for continuous events such as signaling and cell migration. Such processes need to be quantitatively analyzed at single-cell resolution - by live-cell imaging or flow cytometry, for example. Neither genomics nor proteomics approaches can be carried out at this level of resolution yet.\n\nYossi Yarden (Weizmann Institute of Science, Rehovot, Israel) discussed the robustness of biological systems as one prerequisite for cell survival in an unfriendly environment.\n\nTaking the ErbB family of growth factor receptors and breast cancer as an example, he showed that such systems have evolved to withstand perturbations such as those induced by common therapies. He suggested that compensatory pathways provide the plasticity needed to confer drug resistance, and that this would be responsible for the long-term failure of many therapies. However, despite the fitness of cells to deal with such common perturbations, he claimed that unusual types of perturbations would render the system fragile and should re-establish drug potency. For example, targeting of ErbB2 with two different antibodies should more efficiently attract natural killer cells to the tumor cells, and combination of chemotherapy with monoclonal antibodies would better remove this receptor from the cell surface and result in reduced signaling. Cancer cells should then not be able to compensate for such uncommon perturbations, resulting in a much enhanced therapeutic response. A detailed knowledge of the signaling pathways and networks involved would define the optimal treatment regime, depending on the status of the cancer cell in terms of key signaling factors.\n\n# High-throughput technologies\n\nMuch of our current knowledge about genes and their expression is based on the approximately 7 million expressed sequence tag (EST) sequences in the UniGene database. Sumio Sugano (University of Tokyo, Japan) introduced the next-generation DNA sequencers, such as those marketed by Illumina, ABI, and Roche, which promise to determine more than 10 million sequences from just one experiment. With this remarkable technology, genes, promoters and transcription start sites will in future be able to be mapped in single cell types with unprecedented precision. Sugano showed the preliminary results of such an unbiased approach, where an Illumina sequencer had been used to map transcription start sites and transcripts. He concluded that some start sites are indeed cell-type specific and that the huge number of tags generated permits fine-grained analysis of gene expression. But in every million cDNAs captured and sequenced by these techniques, gene expression turns out to range from a few transcripts to thousands of transcripts from the same gene. Sugano pointed out that with the depth of data achievable with next-generation sequencing, sparse transcription cannot be distinguished from what could be termed 'transcriptional noise'. There are no clear cutoffs, which complicates the detection of rarely expressed genes, and especially of intergenic and antisense transcription.\n\nCaroline Shamu (Harvard Medical School, Boston, USA) discussed the many challenges associated with currently fashionable genome-scale screening by RNA interference (RNAi) using small interfering RNAs (siRNAs). She reported on projects where high-throughput transfection methods such as reverse transfection are combined with a conventional plate-and-assay design and high-content read-out to conduct more than 20 large-scale primary screens in different human and mouse cell lines. In her talk she concentrated on technical issues of RNAi screening in her central facility, stressing the importance of spending enough effort to make the assay robust, and to work on plate designs in order to circumvent edge and plate effects as these hamper data analysis. Once these issues are addressed, RNAi seems to be rather robust, as she screened for phenotypes in cancer, infectious diseases, neurobiology, and stem-cell biology, utilizing a number of different cell lines in combination with diverse transfection reagents and siRNA concentrations. While initial RNAi screens had mostly been done with plate readers, data acquisition is increasingly shifting towards high-content screening microscopy. Dorit Arlt (German Cancer Research Center, Heidelberg, Germany) reported that RNAi is also ideal for identifying functional interaction networks of genes. She presented data where knock-down of a single network component did not have a phenotype itself, yet the parallel perturbation of two or more genes did, thus revealing their functional interactions with the network. First she established a literature network of cell-cycle regulation consisting of the ErbB receptor family, AKT1 and MEK1 signaling intermediates, estrogen receptor alpha and Myc transcription factors, and cyclins D1 and E1 as well as cyclin-dependent kinases Cdk1, 4 and 6 as effector molecules. The input was epidermal growth factor (EGF), and the phosphorylation state of the retinoblatoma (Rb) protein was measured in response to siRNA treatments. She systematically perturbed the network components alone and in combinations to identify critical components in the regulation of that network. Indeed, she found novel edges in that network, most of which indicated feedback regulations, for example from cyclin D1 to AKT1 and MEK1. There was a common feeling that such screens will unravel the molecular mechanisms of cellular processes and potentially define major targets for interventions to cure human diseases. But it also became clear that such experiments take months rather than days, which needs to be improved.\n\nThe complementation of functional gene-interaction experiments with information on physical protein-protein interactions is a logical next step in the generation of protein networks. Using tandem affinity purification (TAP), Anne-Claude Gavin (EMBL, Heidelberg, Germany) and her collaborators have found that at least 80% of the proteins in yeast exert their function in complexes with other proteins. She stressed the point that protein complexes are, in general, highly dynamic structures, and often the same proteins are components of several protein complexes. To fully understand the modularity of the proteome in all its dynamics and stoichiometry will thus be a true challenge for the coming years.\n\nTwo array-based platforms were discussed as tools for qualitative and quantitative proteomics. The nucleic acid programmable protein array (NAPPA) presented by Joshua LaBaer (Harvard Institute of Proteomics, Boston, USA) enables the *in situ* production of large numbers of different protein probes with a success rate of greater than 90%. For use in this system, comprehensive collections of expression plasmids harboring the protein-coding regions of genes are being established at Harvard (in the Flexgene project) and by an international project (the ORFeome Collaboration). LaBaer described how NAPPA arrays have been used to generate protein-protein interaction maps, to test for serum-responsive proteins in the *Pseudomonas* proteome, and to detect tumor-associated antigens as a way of monitoring responses to cancer therapy. On the quantitative side, protein microarrays consisting of spotted protein lysates or antibodies tagged with Odyssey IRDye 680 or IRDye 800 were introduced by Ulrike Korf (DKFZ, Heidelberg, Germany). Detection of signals in the near infrared led to low background, low variability between samples and a high dynamic range. The highly parallel setup of these arrays enabled the dynamics of activation of the kinase ERK after stimulation with erythropoietin to be quantified in cell lines, for example. A problem in applying this method on the genome scale is the availability of high-quality antibodies that must be highly specific for their respective targets.\n\n# Challenges for computational biology and data integration\n\nWays of automating the analysis of complex phenotypes such as cellular morphology will be needed to speed up the type of screens described above. Advanced methods of image and data analysis for evaluating cellular morphology were described by Wolfgang Huber (European Bioinformatics Institute (EBI), Cambridge, UK), who showed that supervised learning approaches are able to quantify the occurrence of a number of cellular morphological phenotypes in an unbiased manner. Known complex cellular phenotypes are first user defined in a limited number of 'teaching images'. Automated analysis algorithms then recognize these phenotypes and quantify their occurrence in automatically acquired microscope images. Using this approach, Huber and his collaborators have established a cell-morphological phenoprint of the human genome by siRNA-based screens assayed by high-content microscopy.\n\nIn regard to data integration, Henning Hermjakob (EBI, Cambridge, UK), who has been involved in developing Human Protein Organization standards for reporting and data collection, stressed the necessity of common standards as prerequisites for efficient data exchange. Given the rapidly increasing number of huge and diverse datasets being generated in the 'multi-omics' sciences, the proper analysis and, even more, the integration of data depends on annotation with enough information to enable researchers to evaluate and understand how the data have been collected and for what purposes they can be sensibly exploited. Reporting guidelines and data-exchange formats from many research communities are in existence, for example, MIAME, MIAPE, and MIACA for microarray, proteomics and cellular assay data, respectively. Hermjakob noted that, unfortunately, many of these guidelines are not yet in general use by the scientific community. Harmonization of the different guidelines to enable the integration of multi-omics data, a prerequisite for systems biology, also remains a challenge.\n\nWith human genome sequencing now entering the era of '1,000 genomes' and our personal genomes coming within reach, Rolf Apweiler (EBI, Cambridge, UK) reported on the ongoing implementation of the Human Proteome Project as a natural next step. This project aims to catalog the parts list of all proteins, splice variants, and modifications. Apweiler stressed the need for appropriate technologies, cooperation, data sharing and integration in order to tackle the individual proteomes of cells, tissues, and organisms during growth and development. Compared to the 'mere' 3 billion bases of the human genome, a definitive catalog of the expression pattern of each and every protein in a human being appears to be a Herculean task.\n\n# Taking it into the clinic\n\nThe clinical session focused on the impact of genomics on two major human disease areas, cardiomyopathies and cancer. Norbert Frey (University of Heidelberg, Germany) described examples of candidate gene approaches for dilated cardiomyopathy, starting from an *in silico* identification of potential effectors, and leading via ORFeome resources to *in vitro* and *in vivo* validation in zebrafish and mouse models. Frey and his colleagues have identified one gene that is specifically expressed in the heart and localizes to the Z-discs of sarcomeres. When this gene was knocked-down in zebrafish, severe cardiomyopathic phenotypes were seen. This and other genes identified in this study are currently being screened for mutations in patients with cardiomyopathy with the aim of improving diagnosis.\n\nAlexander Marm\u00e9 (University of T\u00fcbingen, Germany) raised the question of what impact genomics had already had on the prognosis and treatment of cancer patients. In breast cancer, the age of the patient, tumor grading, and the receptor status (estrogen receptor, progesterone receptor or ErbB2) are currently utilized to decide on a therapeutic regime. But a decision based on so few biological markers is often of little benefit. A number of gene signatures for breast cancer have already been approved (for example, Oncotype DX (Genomic Health) and MammaPrint (Agendia)) or are in clinical testing (for example, H3E-MC-S080). They are based on sets of molecular and non-molecular predictive factors and should permit tailored therapies, according to Marm\u00e9. They should be better suited to a fine-grained stratification of patients, allowing personalized therapies and decreasing the likelihood of overtreating or wrongly treating patients.\n\nA complex interplay between tumor and stromal cells was highlighted by Daniel Mertens (University Hospital, Ulm, Germany). He and his collaborators found that chronic lymphatic leukemia cells quickly die in culture unless they are co-cultured with nurse-like stroma cells. Testing which factors convey the survival message to the tumor cells, they found IL-4 and CD40 to be most effective. The finding was then validated in samples from patients. This stresses the importance of paracrine signals for the growth and survival of tumor cells, and emphasizes the need to study cancer cells within their complex environment.\n\nAs the meeting showed, one major challenge is the need for cooperation between different disciplines to push forward and exploit the 'omics' sciences. \"We are all looking at the same elephant, just from different angles\", says Yarden. \"It could turn out in the end though that it had been an octopus all the time\", adds Sorger. Acquiring knowledge at the systems level raises the hope that a more comprehensive understanding of cells and tissues in health and disease will open up new avenues for the treatment of patients.\n\n### Acknowledgements\n\nThis meeting report is dedicated to Annemarie Poustka, a pioneer in genomics and genome biology and one of the organizers of Systems Genomics 2008, who died on 3 May, 2008.","meta":{"dup_signals":{"dup_doc_count":143,"dup_dump_count":43,"dup_details":{"curated_sources":2,"2022-21":1,"2021-49":1,"2020-24":1,"2019-47":1,"2019-13":1,"2018-51":1,"2018-43":1,"2018-34":1,"2018-30":1,"2018-13":1,"2017-47":1,"2017-34":1,"2017-22":1,"2017-09":12,"2016-44":2,"2016-40":1,"2016-36":11,"2016-30":9,"2016-22":1,"2016-18":1,"2016-07":1,"2015-48":4,"2015-40":1,"2015-35":3,"2015-32":4,"2015-27":4,"2015-22":3,"2015-14":3,"2014-52":4,"2014-49":4,"2014-42":9,"2014-41":6,"2014-35":7,"2014-23":6,"2014-15":5,"2022-40":1,"2015-18":4,"2015-11":3,"2015-06":4,"2014-10":5,"2013-48":5,"2013-20":4,"2024-10":1}},"file":"PMC2530878"},"subset":"pubmed_central"}
{"text":"abstract: Postural tachycardia syndrome (POTS), characterized by orthostatic tachycardia in the absence of orthostatic hypotension, has been the focus of increasing clinical interest over the last 15 years 1. Patients with POTS complain of symptoms of tachycardia, exercise intolerance, lightheadedness, extreme fatigue, headache and mental clouding. Patients with POTS demonstrate a heart rate increase of \u226530 bpm with prolonged standing (5-30 minutes), often have high levels of upright plasma norepinephrine (reflecting sympathetic nervous system activation), and many patients have a low blood volume. POTS can be associated with a high degree of functional disability. Therapies aimed at correcting the hypovolemia and the autonomic imbalance may help relieve the severity of the symptoms. This review outlines the present understanding of the pathophysiology, diagnosis, and management of POTS.\nauthor: Satish R RajAddress for correspondence: Satish R Raj MD MSCI, AA3228 Medical Center North, Vanderbilt University, 1161 21st Avenue South Nashville, TN, 37232-2195, USA. E-mail: <firstname.lastname@example.com>\ndate: 2006\nreferences:\ntitle: The Postural Tachycardia Syndrome (POTS): Pathophysiology, Diagnosis & Management\n\n# Introduction\n\nPostural tachycardia syndrome (POTS), characterized by orthostatic tachycardia in the absence of orthostatic hypotension, has been the focus of increasing clinical interest over the last 15 years \\[1\\]. Patients with POTS complain of symptoms of tachycardia, exercise intolerance, lightheadedness, extreme fatigue, headache and mental clouding. This disorder is not new \\[2\\], but has gone by many different names over the last 150 years, including mitral valve prolapse syndrome, neurocirculatory asthenia, orthostatic tachycardia, and orthostatic intolerance \\[3,4\\]. An advantage of the name postural tachycardia syndrome (POTS) is that it focuses attention on the sympathetic activation which characterizes the disorder. This review outlines the present understanding of the pathophysiology, diagnosis, and management of POTS.\n\n# Physiology of Upright Posture\n\nAssumption of the upright posture requires prompt physiological adaptation to gravity. There is an instantaneous descent of \\~500 ml of blood from the thorax to the lower abdomen, buttocks, and legs. In addition, there is a 10-25% shift of plasma volume out of the vasculature and into the interstitial tissue \\[5\\]. This shift decreases venous return to the heart, resulting in a transient decline in both arterial pressure and cardiac filling. This has the effect of reducing the pressure on the baroreceptors, triggering a compensatory sympathetic activation that results in an increase in heart rate and systemic vasoconstriction (countering the initial decline in blood pressure). Hence, assumption of upright posture results in a 10-20 beat per minute increase in heart rate, a negligible change in systolic blood pressure, and a \\~5 mmHg increase in diastolic blood pressure.\n\n# Pathophysiology of Orthostatic Dysregulation\n\nFailure of the regulatory mechanism to respond properly may lead to either ***orthostatic hypotension***, as is seen in autonomic failure, or ***orthostatic tachycardia***, as is seen in POTS. Orthostatic hypotension is defined as a fall in pressure on standing of more than 20\/10 mmHg. However, it is common in patients with autonomic failure for the decline to be much greater than this, which may result in loss of consciousness soon after standing. On the other hand, in POTS, blood pressure is typically maintained on standing or may even increase. Heart rate rises more than 30 bpm and symptoms reminiscent of impaired cerebral perfusion may develop.\n\n# Clinical Presentation of Postural Tachycardia Syndrome (POTS)\n\n## Diagnostic Criteria & Common Clinical Features\n\nPOTS is defined (<a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>) as the presence of symptoms of orthostatic intolerance for at least 6 months accompanied by a heart rate increase of at least 30 beats\/min within 5-30 minutes of assuming an upright posture. This should occur in the absence of orthostatic hypotension (a fall in blood pressure \\>20\/10 mmHg). The syndrome must occur in the absence of prolonged bed rest, medications that impair autonomic regulation (such as vasodilators, diuretics, antidepressants or anxiolytic agents), or any other chronic debilitating disorders that might cause tachycardia (such as dehydration, anemia or hyperthyroidism). It is important to recognize that this syndrome is typically disabling. Hence, the mere observation of orthostatic tachycardia is not, by itself, sufficient to make the diagnosis of POTS.\n\nSymptoms include mental clouding (\"brain fog\"), blurred or tunneled vision, shortness of breath, palpitation, tremulousness, chest discomfort, headache, lightheadedness and nausea. While pre-syncope is common in these patients, only a minority (\\~30%) actually pass out. The chest pains are almost never due to coronary artery obstruction, but are sometimes associated with electrocardiographic changes in the inferior leads, particularly when upright \\[6\\].\n\nMany patients complain of significant exercise intolerance and extreme fatigue. Even activities of daily living, such as bathing or housework, may greatly exacerbate symptoms with resultant fatigue. This can pose significant limitations on their functional capacity.\n\nThe disorder primarily affects women of child-bearing age. The female:male ratio is 4:1. The reason for the strong female predominance is not known, but it should be noted that orthostatic tolerance is reduced in normal healthy females \\[7\\]. Others disorders such as autoimmune diseases and irritable bowel syndrome are seen commonly in patients with POTS, and also have higher prevalence in women.\n\nPatients frequently report that their symptoms began following acute stressors such as pregnancy, major surgery, or a presumed viral illness, but in others cases, symptoms develop more insidiously. About 80% of female patients report an exacerbation of symptoms in the pre-menstrual phase of their ovulatory cycle (unpublished data). Gazit et al. have also reported an association between joint hypermobility and POTS \\[8\\]. Many patients have bowel irregularities and have been co-diagnosed with irritable bowel syndrome, and some have abnormalities of sudomotor regulation \\[9\\].\n\n## Psychological Profile in POTS\n\nPatients with POTS are sometimes clinically diagnosed as having anxiety disorders such as panic disorder. Indeed, patients demonstrate elevated scores on the Beck Anxiety Inventory \\[10\\] (23\u00b110 vs. 7\u00b18; P\\<0.001), a commonly used instrument that quantifies the magnitude of anxiety symptoms \\[11\\]. Unfortunately, this questionnaire includes somatic anxiety symptoms (such as palpitation) which can result from a hyperadrenergic state such as is seen in POTS. When a newer, cognitive-based measure of anxiety (the Anxiety Sensitivity Index \\[12\\]) is used, there was a trend toward less anxiety in the patients with POTS than the general population (15\u00b110 vs. 19\u00b19; P=0.063) \\[11\\]. Thus, much of the anxiety attributed to patients with POTS might be due to a misinterpretation of their physical symptoms.\n\nWe did find that patients with POTS often have diminished attention and concentration compared to matched healthy volunteers \\[11\\]. Using the Inattention score from the Connors Adult ADHD Rating Scale \\[13\\], the patients with POTS scored significantly higher than did the normal control subjects.\n\n## Physical Findings in POTS\n\nThe most striking physical feature of POTS is the severe tachycardia that develops on standing from a supine position. Blood pressure and heart rate must be measured in both postures and should be taken not only immediately after standing but also at 2, 5 and 10 minutes as occasional patients have a delayed tachycardia \\[14\\]. Normal subjects commonly develop a transient tachycardia within the 1st minute of standing that should not be mistaken for POTS. A sustained heart rate increase \u226530 beats per minute is considered diagnostic of orthostatic tachycardia (<a href=\"#F1\" data-ref-type=\"fig\">Figure 1<\/a>). The systolic blood pressure should not fall by more than 20 mmHg, and in many cases it will actually increase with standing. Recent data suggests that there may be a significant circadian variability in the orthostatic tachycardia seen in patients with POTS \\[15\\]. In a cohort of 17 patients with POTS, the orthostatic tachycardia was greater in the morning than in the evening (38\u00b14 bpm vs. 27\u00b13 bpm; P\\<0.001), while there was no diurnal difference in the orthostatic change in blood pressure. These data suggest that to optimize diagnostic sensitivity, postural vital signs should be performed in the morning.\n\nCardiac auscultation may reveal a murmur of mitral valve prolapse, but significant mitral regurgitation is unusual. A striking physical feature of POTS is the dependant acrocyanosis that occurs in 40-50% of patients with POTS (<a href=\"#F2\" data-ref-type=\"fig\">Figure 2<\/a>). These patients experience a dark red-blue discoloration of their legs, which are cold to the touch. This can extend from the feet to above the level of the knees. The reasons underlying this phenomenon are not clear. The current data suggest that the problem is not due to increased pooling in the venous capacitance vessels, but rather due to decreased blood flow in the skin \\[16,17\\].\n\n## Laboratory Abnormalities in POTS\n\nSome authors advocate the use head-up tilt table testing as a standardized method to assess an individual's response to a change in posture \\[1\\]. The patient is positioned on a standard tilt table and following baseline measurements of blood pressure and heart rate, the patient is inclined to a 70-degree head-up angle. Blood pressure and heart rate are then measured either continuously or at least every 12 minutes. The orthostatic tachycardia is often measured in a similar fashion to the standing test, with a similar threshold used to diagnose orthostatic tachycardia (an increase of \u226530 bpm) \\[1\\]. However, the physiology in response to passive standing on a tilt table (with the legs still) is not the same as\"active standing\" where the patient must support their own weight and maintain their balance. The latter requires use of the \"skeletal muscle pump\" and mimics real life, while the tilt table does not. For this reason Streeten et al. use similar criteria for orthostatic tachycardia (\\>27 bpm), but only with active standing \\[18\\]. In a recent study, we compared the orthostatic heart rate response of these 2 methods, and found that the tilt table test was associated with an increased orthostatic tachycardia in both patients with POTS and control subjects \\[19\\]. While both tests were sensitive for the diagnosis of POTS with a 30 bpm threshold for orthostatic tachycardia, the stand test had a specificity of 79% compared to only 23% for the tilt table test.\n\nPOTS patients should have only sinus tachycardia. An electrocardiogram should be done routinely to rule out the presence of an accessory bypass tract or any abnormalities of cardiac conduction. A Holter monitor might prove useful to exclude a re-entrant dysrhythmia, especially if the patient gives a history of paroxysmal tachycardia with a sudden onset and sudden offset. Other tests such as echocardiograms are only required in individual cases when there is doubt about the structural integrity of the heart.\n\nWe often measure plasma norepinephrine levels in both a supine and standing position (at least 15 minutes in each position prior to blood sampling). The supine norepinephrine is often high normal in patients with POTS, while the upright norepinephrine is usually elevated (\\>600 pg\/ml), a reflection of the exaggerated neural sympathetic tone that is present in these patients while upright.\n\nTests of autonomic nervous system function typically show intact or exaggerated autonomic reflex responses. These patients often have preserved vagal function as reflected by their sinus arrhythmia ratio in response to deep breathing. They often have a vigorous pressor response to the Valsalva maneuver, with an exaggerated blood pressure recovery and overshoot both before and after release \\[20\\].\n\nThe blood volume is low in many patients with POTS \\[5\\]. This can be objectively assessed with nuclear medicine tests to directly measure either the plasma volume or the red cell volume. This knowledge may help to focus the treatment plan.\n\nSome patients with POTS have co-existent complaints of episodic flushing. In about half of these cases there is an associated mast cell activation disorder \\[20\\]. This can be diagnosed by collecting urine from individual 2-4 hour voids following a severe flushing spell for determination of methylhistamines.\n\n# Differential Diagnosis\n\nThe clinical picture of POTS can be confused with pheochromocytoma because of the paroxysms of hyperadrenergic symptoms. Patients with pheochromocytoma are more likely to have symptoms while lying down than POTS patients, and often have much higher plasma norepinephrine levels. The diagnosis of pheochromocytoma is made by assessment of plasma or urinary metanephrines \\[21\\].\n\nThere is commonly some confusion between neurally mediated syncope and POTS. There is a clinical overlap between the 2 disorders, such that about 30% of patients with POTS also have neurally mediated syncope. Nonetheless, most patients with POTS do not faint.\n\nAlmost all patients with POTS also have associated fatigue. The reasons are not entirely clear. In some patients, but not all, the fatigue improves with pharmacological control of the orthostatic tachycardia. Some patients with POTS have symptomatic overlap with chronic fatigue syndrome.\n\n# Pathophysiology of POTS\n\nTachycardia and asthenia on standing is a final common pathway of many pathophysiological processes. POTS is therefore best viewed as a syndrome rather than a disease. Many disorders with a common key clinical presentation (the orthostatic tachycardia) have been described. Over the last decade, much has been learned about specific forms or sub-types within POTS, although a simple test to categorize the individual patient remains elusive. We discuss here the common POTS phenotypes including neuropathic POTS and central hyperadrenergic POTS (<a href=\"#F3\" data-ref-type=\"fig\">Figure 3<\/a>).\n\n## Neuropathic POTS\n\nConsidering that POTS patients have high plasma NE levels, it would seem paradoxical that a neuropathy is proposed as an underlying process. Yet some of them have a form of dysautonomia, with preferential denervation of sympathetic nerves innvervating the lower limbs \\[22-24\\]. There have been several findings consistent with this hypothesis. The results of sudomotor axon reflex testing \\[22\\] and galvanic skin stimulation \\[23\\] support this as well as skin biopsy results \\[25\\]. Further, these patients have been found to be hypersensitive to infusions of norepinephrine and phenylephrine into veins of the foot, despite high circulating plasma norepinephrine concentrations \\[24\\]. This suggests that there is a denervation hypersensitivity of the leg veins. Using a segmental norepinephrine spillover approach, Jacob et al. \\[26\\] demonstrated that patients with POTS had normal sympathetic neuronal norepinephrine release in their arms, but less norepinephrine release (and thus less sympathetic activation) in their lower body.\n\n## Hypovolemia & Blood Volume Regulation\n\nMany patients with POTS have low plasma volumes \\[27,28\\], but not all. To determine if hypovolemia existed in an unselected group of POTS patients, we studied 15 patients with POTS (not selected for blood volume) and 14 control subjects \\[5\\]. Plasma volume was measured using ^131^I labeled human serum albumin using a dye dilution technique, and compared to the predicted blood volume for each individual, based upon their height, weight, and gender. As can be seen in <a href=\"#F4\" data-ref-type=\"fig\">Figure 4<\/a>, the control subjects did not have a significant plasma volume deficit (0.8\u00b12.5%). In contrast, the patients with POTS had a plasma volume deficit of 12.8\u00b12.0% (P\\<0.001).\n\nThe renin-angiotensin-aldosterone system plays a key role in the neurohormonal regulation of plasma volume in humans. Plasma renin activity and angiotensin II would be expected to increase in response to hypovolemia in order to promote blood volume expansion. Angiotensin II promotes sodium and water retention directly by stimulating sodium resorption in the proximal tubules, and indirectly by stimulating aldosterone secretion.\n\nPatients with orthostatic tachycardia who were also hypovolemic have low levels of standing plasma renin activity and aldosterone compared to normovolemic patients \\[21,22\\]. This is true in both supine (190\u00b1140 pM vs. 380\u00b1230 pM; P=0.017) and upright posture (480\u00b1290 pM vs. 810\u00b1370 pM; P=0.019). One would have expected a compensatory increase in both plasma renin activity and aldosterone given the hypovolemia in these patients. This low level of plasma renin activity and aldosterone is a paradox that remains unexplained. These data suggest that abnormalities in the renin-angiotensin-aldosterone axis might have a role in the pathophysiology of POTS by contributing to hypovolemia and impaired sodium retention. Such hypovolemia could be accounted for by a neuropathic process involving the kidney. A significant modulator of renin release is the sympathetic nervous system. Thus perturbations in the renin-aldosterone system might result from partial sympathetic denervation involving the kidney.\n\n## Central Hyperadrenergic POTS\n\nAs a part of the definition, POTS is associated with a hyperadrenergic state (<a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>). In many such cases, the hyperadrenergic state is secondary to a partial dysautonomia or hypovolemia. There are some cases, however, in which the primary underlying problem seems to be excessive sympathetic discharge. These patients often have extremely high levels of upright norepinephrine. While we require the upright norepinephrine level to be \\>600 pg\/ml for the diagnosis of POTS, the hyperadrenergic subgroup often has upright norepinephrine level \\>1000 pg\/ml and it is occasionally \\>2000 pg\/ml. These patients sometimes have large increases in blood pressure on standing, indicating that baroreflex buffering is somehow impaired.\n\nCentral hyperadrenergic POTS in its most florid form is much less common than neuropathic POTS, comprising only \\~10% of patients. Thus therapy in these cases usually targets a decrease in sympathetic tone both centrally and peripherally.\n\nCentral sympatholytics such as methyldopa or clonidine can be used. Peripheral beta-adrenergic blockade may be better tolerated by these patients than by those with neuropathic POTS.\n\n## Norepinephrine Transporter Deficiency\n\nA specific genetic abnormality has been identified in a kindred with hyperadrenergic POTS \\[30\\]. These individuals have a single point mutation in the norepinephrine transporter (NET). The resultant inability to adequately clear norepinephrine produces a state of excessive sympathetic activation in response to a variety of sympathetic stimuli. While rare, this mutation has taught us much about the importance of a functional NET.\n\nAlthough functional NET mutations might be infrequent, pharmacological NET inhibition is very common. Many antidepressant and attention deficit medications work at least in part through inhibition of NET. This includes traditional drugs such as tricyclic antidepressants, and newer medications which are pure NET inhibitors (e.g. atomoxetine or reboxetine). Both we \\[31\\] and others \\[32\\] have found that pharmacological NET inhibition can recreate an orthostatic tachycardia phenotype in susceptible healthy volunteer subjects. Yohimbine, a central alpha-2 antagonist that will also increase synaptic norepinephrine, can also cause orthostatic tachycardia \\[33\\].\n\n## Mast Cell Activation\n\nSome patients with POTS have co-existent mast cell activation. These patients have episodic flushing and abnormal increases in urine methylhistamine (the primary urinary metabolite of histamine) \\[20\\]. Methylhistamine should ideally be measured in 2 hour aliquots at the time of a flushing episode and not just in a random 24 hour period. Other associated symptoms include shortness of breath, headache, lightheadedness, excessive diuresis, and gastrointestinal symptoms such as diarrhea, nausea, and vomiting. Flushing can be triggered by long-term standing, exercise, premenstrual cycle, meals, and sexual intercourse. These patients often have a hyperadrenergic response to posture, with both orthostatic tachycardia and hypertension. They demonstrate a vigorous sympathetic vasopressor response during the Valsalva maneuver with a blood pressure overshoot in late phase II and an exaggerated phase IV blood pressure overshoot. It is not clear if mast cell activation, releasing vasoactive mediators, represents the primary event in these patients or if sympathetic activation, through release of norepinephrine, neuropeptide Y and ATP, is the cause of mast cell activation \\[34\\].\n\nIn these patients, beta-adrenergic antagonists can actually trigger an episode and worsen symptoms. Centrally acting agents to decrease the sympathetic nervous system discharge (e.g. methyldopa or clonidine) may prove effective. Alternatively, treatment could target mast cell mediators with a combination of antihistamines (H1- and H2-antagonists) and with the cautious use of non-steroidal agents (high dose aspirin) in refractory cases.\n\n# Non-Pharmacological Treatment of POTS\n\nNo therapy is successful for all patients with POTS. Initial efforts should focus on identifying and treating any reversible causes. Potentially contributory medications (especially vasodilators, diuretics, and drugs that inhibit NET) should be withdrawn. If a patient has been through prolonged bedrest, their symptoms will gradually improve as they recondition themselves to upright posture. Treatment should be optimized for any chronic disease that is present. If there is clear evidence of a re-entrant supraventricular arrhythmia, then this should be treated, including with radiofrequency ablation as appropriate. However, radiofrequency sinus node modification for the sinus tachycardia of POTS is not recommended. This often makes the patient's symptoms worse (and occasionally the patient becomes pacemaker dependent). Specific therapies are summarized in <a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a>.\n\nIt is important to educate the patient about the nature of the disorder. The patient should avoid aggravating factors such as dehydration, and extreme heat. In order to ensure adequate hydration, we ask our patients to consume 8-10 cups of water daily and to rapidly drink 16 fl oz of water to lower their heart rates \\[35\\]. In addition, they are asked to aggressively increase their sodium intake up to 200 mEq\/day. This is often hard to achieve without NaCl tablets 1 gm\/tablet TID with meals. Elastic support hose can help to minimize the degree of peripheral venous pooling and enhance venous return. We recommend 30-40 mmHg of counter-pressure and they should come up to the waist. If the stockings are only knee-high, a line of edema can form just above the stockings. Their use can be limited by their tolerability as the stockings can be hot, itchy and uncomfortable. Exercise (both aerobic and resistance training) is also encouraged and has been shown to be beneficial \\[36\\]. In addition to reversing any \"deconditioning\", this intervention can also increase blood volume. Vigorous exercise may acutely worsen symptoms and may even result in prolonged fatigue. It is important that patients start slowly and remain within range of their \"target heart rate\" in the early stages to avoid symptoms that might discourage further exercise.\n\nAcute blood volume expansion is effective at controlling the heart rate and acutely improving symptoms. Jacob et al. \\[37\\] found that 1 liter of physiological saline infused intravenously over 1 hour decreased the orthostatic tachycardia from 33\u00b15 bpm before the infusion to 15\u00b13 bpm immediately following the infusion. The physiological saline was more effective at heart rate control than were treatments with either an alpha-1 agonist or an alpha-2 agonist. This treatment is not practical on a day to day basis as a medical setting is required to insert the intravenous catheter and infuse the saline. Recently, there have been reports of patients having regular saline infusions, typically 1 liter of normal saline every other day or every day. Many report an improvement in symptoms. However, there are not yet objective data to substantiate such benefit. Further, there is a risk of vascular access complications or infection. At this time, such therapy for patients with POTS should be considered cautiously.\n\n# Pharmacological Treatment of POTS\n\nNo medicines are approved by the United States Food and Drug Administration for the treatment of POTS. Thus all agents are used for this disorder are \"off label\". Furthermore, there are no pharmacological agents that have been tested in a long-term properly powered randomized clinical trial.\n\nIn patients in whom the presence of hypovolemia is either known or strongly suspected, fludrocortisone (an aldosterone analogue) is often used. Through enhanced sodium retention, it should expand the plasma volume, although there is a paucity of data regarding the exact mechanisms of action. Although fairly well tolerated, side effects can include hypokalemia, hypomagnesemia, worsening headaches, acne, and fluid retention with edema. Another volume expanding agent that may be helpful for short-term use is oral vasopressin (DDAVP). This agent causes the kidney to retain free water, but not sodium. Potential side effects include hyponatremia, edema and headache. Erythropoietin has occasionally proven useful in patients with POTS who are refractory to other forms of therapy. While the primary mode of action is likely an increase in intravascular volume via its increase in red cell mass, erythropoietin also appears to have a direct vasoconstrictive effect, possibly through enhanced red cell mediated nitric oxide scavenging \\[38\\]. Treatment with erythropoietin has many drawbacks including the significant expense and the need for subcutaneous administration.\n\nCentral sympatholytic medications are often useful and well tolerated in patients with the central hyperadrenergic form of POTS, but may not be as well tolerated in neuropathic POTS. Clonidine is an alpha 2 agonist that acts centrally to decrease sympathetic nervous system tone. Clonidine, at doses of 0.05 mg to 0.2 mg PO BID, can stabilize heart rate and blood pressure in patients with a large amount of postganglionic sympathetic involvement. Unfortunately, it can also cause drowsiness, fatigue and worsen the mental clouding of some patients. Methyldopa, a false neurotransmitter, is sometimes more successful in controlling symptoms in these patients at doses of 125 mg to 250 mg PO TID \\[39\\].\n\nWhen used in low doses, beta-adrenergic antagonists can be useful. We typical use propranolol 10-20 mg PO BID-QID. While this dose range is small, such doses can often have a significant impact on heart rate control, and higher doses are often not tolerated due to hypotension and fatigue.\n\nSince a failure of vascular resistance may be an integral part of neuropathic POTS, vasoconstrictors such as midodrine (an alpha-1 agonist) can be employed \\[40\\]. Some patients cannot tolerate this agent due to the unpleasant sensation of scalp tingling or goosebumps. Midodrine can also cause hypertension.\n\nWe recently reported that an unselected group of patients seen in our inpatient research unit were given a trial of the acetylcholinesterase inhibitor pyridostigmine. By increasing the levels of synaptic acetylcholine at both the autonomic ganglia and the peripheral muscarinic parasympathetic receptors, pyridostigmine significantly restrained the heart rate in response to standing in our patients with POTS. We prescribe pyridostigmine 30mg to 60 mg PO TID alone or in combination with low dose propranolol. Pyridostigmine can enhance bowel motility, so it is not always well tolerated in patients with diarrhea-predominant irritable bowel syndrome symptoms.\n\nWhile most of the treatments discussed above have focused on the control of heart rate, many patients are also greatly troubled by mental clouding. Modafinil, a stimulant whose mechanism is not yet clear, has been used in some patients with resulting improvement in alertness. However, caution is advised as it may aggravate the orthostatic tachycardia \\[41\\].\n\n# Conclusions\n\nPOTS is a disorder of the autonomic nervous system in which many symptoms can be treated. The cardinal manifestation is symptomatic orthostatic tachycardia. The disorder can produce substantial disability among otherwise healthy people. Patients with POTS demonstrate a heart rate increase of \u226530 bpm with prolonged standing (5-30 minutes), often have high levels of upright plasma norepinephrine, and many patients have a low blood volume. Therapies aimed at correcting the hypovolemia and the autonomic imbalance may help relieve the severity of the symptoms. Continued research is vital to better understand this disorder and to differentiate its various subtypes.\n\n***Funding:*** Supported in part by National Institute of Health grants 2P01 HL56693, K23 RR020783 and M01 RR00095\n\nCriteria for the Postural Tachycardia Syndrome\n\n![](ipej060084-05)\n\nTreatments for the Postural Tachycardia Syndrome\n\n![](ipej060084-06a)\n\n![](ipej060084-06b)\n\nNaCl - Table salt; PO - by mouth; OD - once daily; BID - twice daily; TID - three times daily; QID - four times daily; IV - intravenous","meta":{"dup_signals":{"dup_doc_count":103,"dup_dump_count":49,"dup_details":{"curated_sources":2,"2019-04":2,"2018-51":1,"2018-43":2,"2018-39":1,"2018-34":1,"2018-30":2,"2018-26":1,"2018-22":1,"2018-17":4,"2018-13":1,"2018-09":4,"2017-51":2,"2017-47":3,"2017-43":1,"2017-39":5,"2017-34":2,"2017-30":4,"2017-26":2,"2017-22":1,"2017-17":1,"2017-09":2,"2017-04":1,"2016-50":1,"2016-44":2,"2016-40":2,"2016-36":2,"2016-30":2,"2016-22":2,"2016-18":2,"2016-07":2,"2015-48":2,"2015-40":2,"2015-35":2,"2015-32":2,"2015-27":2,"2015-22":2,"2015-14":2,"2014-49":2,"2014-42":6,"2014-41":3,"2014-35":3,"2014-23":4,"2014-15":2,"2019-22":1,"2015-18":2,"2015-11":2,"2014-10":1,"2013-48":1,"2017-13":1}},"file":"PMC1501099"},"subset":"pubmed_central"}
{"text":"abstract: Interpretation of clinical trials to alter the decline in \u03b2-cell function after diagnosis of type 1 diabetes depends on a robust understanding of the natural history of disease. Combining data from the Type 1 Diabetes TrialNet studies, we describe the natural history of \u03b2-cell function from shortly after diagnosis through 2 years post study randomization, assess the degree of variability between patients, and investigate factors that may be related to C-peptide preservation or loss. We found that 93% of individuals have detectable C-peptide 2 years from diagnosis. In 11% of subjects, there was no significant fall from baseline by 2 years. There was a biphasic decline in C-peptide; the C-peptide slope was \u22120.0245 pmol\/mL\/month (95% CI \u22120.0271 to \u22120.0215) through the first 12 months and \u22120.0079 (\u22120.0113 to \u22120.0050) from 12 to 24 months (*P* \\< 0.001). This pattern of fall in C-peptide over time has implications for understanding trial results in which effects of therapy are most pronounced early and raises the possibility that there are time-dependent differences in pathophysiology. The robust data on the C-peptide obtained under clinical trial conditions should be used in planning and interpretation of clinical trials.\nauthor: Carla J. Greenbaum; Craig A. Beam; David Boulware; Stephen E. Gitelman; Peter A. Gottlieb; Kevan C. Herold; John M. Lachin; Paula McGee; Jerry P. Palmer; Mark D. Pescovitz; Heidi Krause-Steinrauf; Jay S. Skyler; Jay M. Sosenko; Corresponding author: Carla J. Greenbaum, <firstname.lastname@example.com>.\ndate: 2012-08\nreferences:\nsubtitle: : Evidence of at Least Two Distinct Phases From Composite Type 1 Diabetes TrialNet Data\ntitle: Fall in C-Peptide During First 2 Years From Diagnosis\n\nThe natural history of type 1 diabetes (T1D) is arguably better understood than that of many other autoimmune diseases, with decades of studies describing the disease course before and after diagnosis. In addition to providing the backdrop for studies to understand the etiopathology of T1D, such information is critical for the design and interpretation of clinical trials to alter the progression of the disease.\n\nThe generally understood picture postdiagnosis is of an inevitable fall in \u03b2-cell function within a few years after diagnosis, and many studies point to variables that affect the rate of fall, such as glycemic control, HLA type, age at diagnosis, and BMI.\n\nThere are several reasons to reexamine the natural history of \u03b2-cell function. In recent years, changes in diabetes management and consequent improvement of glycemic control have occurred. In addition, several studies point to a changing HLA distribution in patients with diabetes (1,2), and the overall population generally has had an increase in BMI (3,4). More provocative, there are increasing reports of persistence of C-peptide in individuals long after diagnosis (5) and emerging information from autopsy studies (6) that suggest that the current paradigm should be reconsidered. Furthermore, careful scrutiny of placebo groups from published clinical trials demonstrates major inconsistencies in changes in C-peptide over time between studies, which may be attributable to characteristics of the study populations, procedures used to assess \u03b2-cell function, and\/or analytic methods used for reporting data.\n\nType 1 Diabetes TrialNet is an international, multicenter clinical trial network designed to alter the course of T1D, either before diagnosis (prevention) or after clinical diagnosis, with the aim of prolonging \u03b2-cell functional survival. As of 2011, TrialNet had acquired data up to 24 months from diagnosis from three clinical trials in individuals with recently diagnosed T1D (7\u20139). These studies all had similar entry criteria, standardized approaches to diabetes management, and defined protocols for assessment of \u03b2-cell function that are highly reproducible (10). In this analysis, we take advantage of this robust dataset to describe the natural history of \u03b2-cell function in individuals through 2 years postrandomization, assess the degree of variability between patients, and investigate factors that may be related to C-peptide at and postdiagnosis.\n\n# RESEARCH DESIGN AND METHODS\n\nData from 191 subjects were included in this analysis, including all subjects from one study in which the intervention had no effect on \u03b2-cell function (7) and the placebo-treated subjects from two other studies (8,9). Inclusion of the actively treated subjects from the negative study had no appreciable impact on the results (see below). All subjects or their parents gave written informed consent and assent as appropriate prior to participation in these studies. Pertinent study entry criteria for randomization include peak C-peptide during mixed-meal tolerance test (MMTT) \u22650.2 pmol\/mL, positive for at least one diabetes autoantibody, diagnosis of T1D within the previous 100 days, and age 7\u201345 years. To ensure metabolic stability, no MMTTs were performed before 21 days after diagnosis. The mean (median) time from diagnosis to MMTT was 79 (55) days. The study protocol called for subjects to be contacted by the study team every 2 weeks to evaluate and make recommendations to ensure tight glycemic control. The mean HbA~1c~ at 1 and 2 years was 7.3 (\u00b11.5) and 7.6% (\u00b11.5), respectively.\n\n## MMTT.\n\nAs previously described (10), subjects underwent a 2- or 4-h MMTT under the following circumstances: tests were started before 10 <span class=\"smallcaps\">a.m.<\/span>, fasting glucose was required to be 70\u2013200 mg\/dL, and long-acting insulin was permitted but no short acting insulin (including no pump bolus) was allowed within 2 h before start of test. A standard preparation of fat, carbohydrate, and protein (Boost-HP; Nestle Health Care Nutrition, Inc.) was used at a dose of 6 mg\/kg to a maximum of 360 mL.\n\n## Analysis.\n\nFull details of analyses are presented in the [Supplementary Material](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db11-1538\/-\/DC1). Key points are summarized here.\n\nC-peptide values recorded as below \"lower limit of detection\" (LLD) were assigned the value of one-half the LLD. C-peptide area under the curve (AUC) was calculated using the trapezoidal method. Areas were then divided by the time period of the test, 120 or 240 min as indicated. Undetectable C-peptide on MMTT was defined as all timed values on the MMTT below the LLD.\n\n## Maintenance of C-peptide.\n\nSubjects were classified as having maintained C-peptide over time if there was no change from baseline to each of the time points after baseline. To account for statistical variation in C-peptide measurements, we used three definitions of maintained C-peptide that are suggested by two published studies. All three definitions are similar in that they each consider no change or an increase from baseline to represent a positive response. They differ in the amount of decrease they allow for a subject to still be classified as having maintained C-peptide:\n\n1.  \"Percentage Change\" definition of maintenance of C-peptide: Subject whose follow-up C-peptide value is no more than 7.5% below baseline. This was defined as \"responder\" to therapy in work published by Herold et al. (11).\n\n2.  \"Inter-test Variability\" definition of maintenance of C-peptide: The MMTT\/GST (glucagon stimulation test) (10) was a test-retest study comparing C-peptide measurements between the MMTT and the GST. In the MMTT\/GST study, individuals underwent two MMTT within a 1-month period. We used the MMTT data from that study to estimate inter-test variability of C-peptide determined by MMTT. In the current analysis, someone whose change from baseline was either nonnegative or if negative, was no more than 1 inter-test SD below baseline, was defined as having maintained C-peptide.\n\n3.  \"CV\" definition of maintenance of C-peptide: Using MMTT data from the MMTT\/GST study, the median coefficient of variation (CV) among the subjects in that study was determined. In the current analysis, the CV for each subject was computed between the baseline and each of the time points (see [Supplementary Material](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db11-1538\/-\/DC1) for details). A subject was then classified as having maintained C-peptide whenever his or her change from baseline was either nonnegative or if negative, represented a CV less than the median CV found in the MMTT\/GST study.\n\nWe also evaluated the occurrence of an increase in C-peptide that is beyond the amount expected from simple inter-test variability (i.e., an increase of at least 1 inter-test SD from baseline).\n\nAssociations between C-peptide measures with baseline covariates were assessed with Spearman correlation for continuous covariates and with ANOVA for categorical covariates. The time course of the proportion of subjects with detectable C-peptide values and the proportion of subjects with peak C-peptide \u22650.2 pmol\/mL were estimated with the Kaplan-Meier method.\n\nThe relationship of C-peptide across time with baseline covariates was evaluated with mixed linear models and, thus, slopes relating C-peptide and time were estimated for each individual subject. Slopes of C-peptide versus time were estimated for subgroups using the mean slope of individuals in that group and the estimates displayed in fan plots. The joint influence of baseline covariates on the longitudinal change in C-peptide was assessed with multivariable models. Only covariates that were found significant (*P* \\< 0.05) in univariable analysis were included in the multivariable models. Piecewise linear regression was used to test the data-driven hypothesis that the slope of C-peptide AUC was the same before and after 1 year postbaseline (12). Slopes unadjusted for covariates were compared between the three end points (fasting, peak, and AUC) using the multivariate analysis described in the [Supplementary Material](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db11-1538\/-\/DC1).\n\nOur study combined data from placebo- and active-treated subjects from the negative Type 1 Diabetes TrialNet MMF\/DZB study to increase the number of observations. To evaluate whether inclusion of the actively treated MMF\/DZB subjects significantly altered our findings, we replicated the multivariate analyses using a variable to indicate treatment in the MMF\/DZB study. This variable was never statistically significant at the *P* \\< 0.05 level. In addition, the novel observation of a biphasic fall of C-peptide (described below) was present with or without the actively treated subjects.\n\n# RESULTS\n\nThe demographic and baseline characteristics of the 191 subjects included in this analysis are shown in <a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>. Correlations of baseline characteristics with the peak and AUC C-peptide values from their baseline MMTT are also noted in <a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>. Baseline variables that were significantly associated with both peak and AUC C-peptide included age, presence of another autoimmune disease, HbA~1c~, insulin dose (units\/kg\/day), BMI and BMI *z* score, micro\u2013insulin autoantibody (mIAA) positivity, mIAA titer, and basophils.\n\nDemographic and baseline characteristics and correlations with peak and AUC C-peptide from baseline MMTT\n\n![](2066tbl1)\n\nAt entry, all individuals were required to have a peak C-peptide \u22650.2 pmol\/mL. Over time, as shown in <a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<\/a>, only 1% of individuals had undetectable C-peptide at 12 months after study entry and 7% had undetectable C-peptide at 24 months. Post hoc analysis of Diabetes Control and Complications Trial (DCCT) data has suggested that individuals with a peak C-peptide on MMTT \u22650.2 pmol\/mL were less likely to have progression of retinopathy and severe hypoglycemia than those with less than that value (13). We therefore evaluated the time until individuals reached this value. As shown in <a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<\/a>, 88% of individuals continued to have peak C-peptide \u22650.2 pmol\/mL at 12 months of follow-up and 66% at 24 months.\n\nWe also evaluated changes of three end points from the MMTT (fasting C-peptide, peak C-peptide, and AUC C-peptide) during the 2 years of the study. Comparisons between peak, AUC, and change from baseline using values from the 4- and 2-h tests showed extremely high concordance ([Supplementary Table 1](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db11-1538\/-\/DC1)). Since, by protocol, there were fewer 4-h tests performed, results from the 2-h MMTT were used in subsequent analyses. The slope of the regression of each end point on time, measured in months from the start of the study, was determined. Fasting C-peptide fell at a rate of \u22120.0076 \u00b1 0.0007, peak C-peptide at \u22120.0222 \u00b1 0.0010, and AUC C-peptide at \u22120.0172 \u00b1 0.0007 pmol\/mL\/month. These slopes were each significantly different from each other (*P* \\< 0.001), with fasting C-peptide having the smallest change over time ([Supplementary Table 2](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db11-1538\/-\/DC1)).\n\nExamination of the plots of the mean AUC C-peptide over time suggested a biphasic decline with differences in the slope before and after 12 months (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<\/a>). This difference was not explained by inclusion of individuals who lost C-peptide during the study period because truncating either those subjects or their data points below the LLD did not affect this pattern. We further explored this data-driven hypothesis using piecewise linear regression and found that the decline after 12 months was significantly slower than that seen during the first 12 months and that this was not affected by age. The slope of the AUC C-peptide before 12 months was estimated to be \u22120.0245 pmol\/mL\/month (95% CI \u22120.0271 to \u22120.0215) as compared with a slope of \u22120.0079 (\u22120.0113 to \u22120.0050) from 12 to 24 months (*P* \\< 0.001). In a similar manner, the slope of the peak C-peptide before 12 months was \u22120.0318 (\u22120.0357 to \u22120.0278) as compared with a slope of \u22120.0089 (\u22120.0136 to \u22120.0046) from 12 to 24 months (*P* \\< 0.001). The slope of fasting C-peptide before 12 months was \u22120.03322 (\u22120.03984 to \u22120.02659) and after 12 months was \u22120.00867 (\u22120.01626 to \u22120.00108) (*P* \\< 0.001).\n\nInspection of each individual's C-peptide data over time shows the variability in slopes between individuals ([Supplementary Fig. 1](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db11-1538\/-\/DC1)). We then evaluated baseline factors that may contribute to these differences in C-peptide slope ([Supplementary Table 3](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db11-1538\/-\/DC1)). Notable variables not found to be associated with C-peptide over time included Tanner stage and class II HLA type categorized as DR3 or DR4 and not DQB1\\*0602 or otherwise. Those variables found to be significantly associated were then evaluated in a multivariable model. As shown in <a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a>, only age and number of basophils impacted C-peptide over time whether AUC, peak, or fasting C-peptide was used. Additional baseline variables significantly associated with stimulated measures only (AUC and peak) were BMI *z* score and insulin dose.\n\nMultivariable analysis of baseline characteristics associated with fasting, peak, and AUC C-peptide over time\n\n![](2066tbl2)\n\nWe further explored the effect of age on C-peptide over time by grouping individuals according to quartiles according to age, taking the mean slope estimated by the multivariable model of AUC C-peptide. As shown in <a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<\/a>, the youngest children (aged 7.7\u201312.3 years) started with lower C-peptide values at their initial MMTT compared with the other subjects. The three older quartiles (aged 12.4\u201346.1 years) were not different at their initial MMTT. In contrast, the slopes of C-peptide over time showed remarkable concordance in children among the three youngest groups, distinct from that of subjects in the oldest quartile.\n\nAdditional exploratory analyses, such as considering the C-peptide in relationship to the simultaneous glucose value, neither improved the model nor changed the impact of previously identified variables on the outcome measure.\n\nWe then evaluated time-related patterns of C-peptide preservation, using the classification of subjects as having maintained C-peptide or not, separately for each of the three definitions introduced in <a href=\"#s2\" data-ref-type=\"sec\"><span class=\"smallcaps\">research design and methods<\/span><\/a>. As shown in <a href=\"#T3\" data-ref-type=\"table\">Table 3<\/a>, large numbers of subjects could be considered as having maintained C-peptide by any definition if all tests are taken into consideration. When limited to comparing the baseline with the 1- or 2-year values alone, 33 (17%) and 21 (11%) maintained C-peptide as defined by the CV definition, respectively, with similar values for the other definitions as described in <a href=\"#s2\" data-ref-type=\"sec\"><span class=\"smallcaps\">research design and methods<\/span><\/a>. Multivariable analysis found that age significantly increases, and the presence of islet cell antigen (ICA)512 antibody significantly decreases, the probability of having maintained C-peptide by the CV definition at 2 years (odds ratio age 1.067 \\[95% CI 1.026\u20131.109\\]; ICA512 0.329 \\[0.162\u20130.667\\]).\n\nMaintenance of C-peptide\n\n![](2066tbl3)\n\nIn a similar way, we also investigated time-related patterns of subjects who had strictly an increase in C-peptide of at least 1 inter-test difference SD. There were 55 of 191 subjects who experienced at least one such increase during the 2-year period. In a multivariable model, baseline predictors that enhanced the probability of this increase were increased age, GAD65 antibody positivity, and Tanner stage. Those that decreased the probability of an increase in C-peptide were insulin dose and mIAA and ICA512 positivity and titers.\n\n# DISCUSSION\n\nLoss of insulin secretion is the fundamental defect of T1D, with natural history studies showing a decline before and continuing after clinical diagnosis until a complete absence of endogenous secretion occurs. This natural history serves as the underlying rationale for studies to halt \u03b2-cell destruction. Slowing or stopping this loss of insulin secretion prior to diagnosis would delay or prevent clinical onset of disease. Retention of endogenous insulin secretion after diagnosis has been associated with clinical benefits as shown by the DCCT, in which those with preserved function obtained the benefit of intensive therapy (e.g., reduction of retinopathy) with less risk of severe hypoglycemia (14), and in islet transplant studies, in which there is a reduction in hypoglycemia even when insulin independence is not achieved (15).\n\nOur data show that 93% of individuals who started with C-peptide \u22650.2 pmol\/mL have detectable C-peptide 2 years from diagnosis, and \u223c66% are above the DCCT C-peptide threshold associated with clinical benefit. Furthermore, in contrast to both peak and AUC measures, there is limited change in fasting C-peptide during 2 years from study entry, suggesting that use of nonstimulated C-peptide as an outcome in clinical trials would make it a difficult end point to demonstrate benefit of an intervention therapy.\n\nMost important, our data indicate that the rate of fall is not constant from study entry during the next 2 years. Our data show a biphasic fall during this time; however, with the limited number of fixed time points studied, we cannot exclude the possibility that the true relationship of C-peptide over time is polyphasic. In this regard, it is worthwhile to recall that the baseline MMTT in TrialNet studies is not performed until at least 21 days after diagnosis and that the median time from diagnosis for the initial studies was 55 days; thus, we have no information about the rate of fall from the time of diagnosis to the start of our study. The rate of fall in C-peptide from \u223c2 months until 14 months from diagnosis (e.g., 0\u201312 months from study start) is distinct from the rate of fall from 14 to 26 months after diagnosis (e.g., 12\u201324 months after study start). This data suggests that there may be metabolic or immunological factors that differ soon after diagnosis rather than later. This hypothesis would need to be explored further. With the caveat that MMTT- and oral glucose tolerance test (OGTT)-stimulated C-peptide are not equivalent (16), it is of interest to note that Diabetes Prevention Trial\u2013Type 1 (DPT-1) data show little change in OGTT-stimulated C-peptide during the 30 months before diagnosis, while a marked fall in peak C-peptide occurred during the peridiagnosis period (17,18). Further insights are likely to come from measuring C-peptide responses under standardized conditions in individuals as they progress from pre- to postdiagnosis. It is also important to note that for these studies, virtually all patients had T1D diagnosed in the community versus the diagnosis of usually asymptomatic T1D from surveillance OGTTs every 6 months in DPT-1 and TrialNet Natural History and Prevention studies (19\u201321). How the fall in C-peptide would compare after these two very different methods of diagnosis is unknown but of major interest.\n\nIt is important to consider these data in the context of results of clinical trials that have preserved \u03b2-cell function. In these studies, the differences between the slopes (i.e., rate of fall) of C-peptide between placebo and treatment groups were most pronounced early, with the slopes appearing similar after this initial period (8,9,22,23). The data presented in our analysis raise two hypotheses: *1*) the intervention may only affect the metabolic or immunologic factors soon after diagnosis or *2*) the rate of fall further from diagnosis may be too flat to detect an effect of intervention.\n\nOur data also emphasize the variation in the rate of fall of C-peptide over time and demonstrate that more than one-third of subjects were \"nonprogressors\" experiencing an increase, no change, or fall within the median CV of the MMTT at least once in the 2 years of study. Approximately 1 in 6 subjects meet this definition at 1 year and \\>1 in 10 subjects at 2 years after randomization. As a consequence, one must be cautious about interpretation of small trials or in drawing clinical conclusions from a single phase 2 study.\n\nSimilar to previous studies, we found that age, insulin use at baseline, and BMI *z* score at baseline are significantly associated with AUC and peak C-peptide over time in multivariable analysis (24\u201326). It is notable that variables such as class II HLA type as defined with three categories, Tanner stage, and antibodies (number, positive or not, and titer) that have been reported in previous studies to influence rate of \u03b2-cell function decline (27\u201330) were not significantly associated with AUC or peak C-peptide over time in our multivariable model. Further work to define the role of genotype in disease progression is ongoing, including more detailed analysis of HLA class I and II.\n\nWe were particularly interested in age both as a consideration for future study design and to explore the question about whether there are differences in disease progression across the ages studied. The rate of decline of C-peptide was similar in individuals aged 7 to 21 and greater than in subjects older than 21 years. However, the youngest subjects (aged 7 to 12) started with lower C-peptide than older subjects aged 12 to 46 years. This observation is consistent with clinical impressions that younger individuals frequently have low levels of C-peptide but does not support the idea that disease progression is different in younger versus older children or that pubertal status influences the disease process. A major caveat to this conclusion, however, is that the youngest subjects in this study were age 7. Thus, our data do not permit any conclusions about younger children. The different baseline C-peptide level in the youngest group is consistent with the use of an age-dependent percentile determination of intravenous glucose tolerance test data that was used for assessing risk in DPT-1 (31). Unfortunately, little information is available on normal stimulated C-peptide values in young children, a gap that limits our ability to interpret the data in individuals with disease. Our data clearly suggest differences in disease progression between children and adults, pointing to areas ripe for further investigation and also emphasizing that a larger sample size may be required to demonstrate a clinical effect of a drug if only adults are studied. These older patients were diagnosed as T1D by their providers. How their decline in \u03b2-cell function would compare with so-called late autoimmune diabetes in adult patients is unknown but also very important for potential intervention trials.\n\nThe novel observation of the association of basophils with change in C-peptide over time was unexpected. Indeed, we initially evaluated white blood cell differentials to examine how lymphopenia might relate to clinical course. While historically conceptualized as related to mast cells whose release of interleukin (IL)-4 and IL-13 may affect allergy and protection from parasites, as recently reviewed (32), basophils have multiple roles in the immune response, including antibody production, serving as antigen presenting cells, and amplifying the memory response among others. Since higher basophil levels were associated with a less rapid fall in C-peptide over time, it is tempting to speculate that this association is based on a compensatory response to autoimmunity\u2014attempting to deviate cells via IL-4 cytokine release. However, this association may also have occurred by chance and requires replication in another dataset.\n\nTrialNet studies were conducted at a limited number of clinical centers, with close monitoring of glycemic control, using a standardized approach to MMTT (with regard to meals, insulin dosing, baseline glucose value, and time of day), and centralized laboratories with quality assurance programs. As such, combined data from these studies provide a contemporaneous description of the natural history of C-peptide under clinical trial conditions during the first 2 years after randomization in a context by which effects of intervention can be considered and future studies designed. Since hyperglycemia itself has been associated with poor \u03b2-cell function (14), the rate of fall seen in these studies in which individuals had a mean HbA~1c~ of 6.5% on entry and 7.6% at 2 years may not reflect the natural history of disease under usual clinical care. In addition, many of the observations reported must be considered hypothesis generating and require confirmation in additional datasets. To this end, TrialNet will soon use data from two additional studies: one using GAD65-alum (33) and the other involving canakinumb to test the associations observed in the trials used for the current analyses. These studies are being conducted under identical circumstances with the notable exception that subjects as young as age 3 are included in the GAD65 study, thus providing comparative data in this important age-group.\n\n# Supplementary Material\n\n###### Supplementary Data\n\n## ACKNOWLEDGMENTS\n\nThe sponsor of the trial was the Type 1 Diabetes TrialNet Study Group. Type 1 Diabetes TrialNet Study Group is a clinical trials network funded by the National Institutes of Health (NIH) through the National Institute of Diabetes and Digestive and Kidney Diseases, the National Institute of Allergy and Infectious Diseases, and the *Eunice Kennedy Shriver* National Institute of Child Health and Human Development, through Cooperative Agreements U01-DK-061010, U01-DK-061016, U01-DK-061034, U01-DK-061036, U01-DK-061040, U01-DK-061041, U01-DK-061042, U01-DK-061055, U01-DK-061058, U01-DK-084565, U01-DK-085453, U01-DK-085461, U01-DK-085463, U01-DK-085466, U01-DK-085499, U01-DK-085505, U01-DK-085509, and Contract HHSN267200800019C, the National Center for Research Resources, through Clinical Translational Science Awards UL1-RR-024131, UL1-RR-024139, UL1-RR-024153, UL1-RR-024975, UL1-RR-024982, UL1-RR-025744, UL1-RR-025761, UL1-RR-025780, UL1-RR-029890, UL1-RR-031986, and General Clinical Research Center Award M01-RR-00400, the Juvenile Diabetes Research Foundation International (JDRF), and the American Diabetes Association (ADA).\n\nNo potential conflicts of interest relevant to this article were reported.\n\nThe contents of this article are solely the responsibility of the authors and do not necessarily represent the official views of the NIH, JDRF, or ADA.\n\nAll authors are members of the Type 1 Diabetes TrialNet Study Group and, as such, contributed to data used in this article. C.J.G. and C.A.B. wrote the manuscript. D.B., S.E.G., P.A.G., K.C.H., J.M.L., P.M., J.P.P., M.D.P., H.K.-S., J.S.S., and J.M.S. contributed to discussion and reviewed and edited the manuscript. C.J.G. and C.A.B. are the guarantors of this work and, as such, had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.\n\n# REFERENCES","meta":{"dup_signals":{"dup_doc_count":102,"dup_dump_count":57,"dup_details":{"curated_sources":2,"2022-05":1,"2021-49":1,"2021-43":1,"2021-31":1,"2021-25":1,"2021-21":1,"2021-04":2,"2020-50":1,"2020-45":2,"2020-05":1,"2019-47":1,"2019-39":1,"2019-35":2,"2019-30":2,"2019-22":1,"2019-18":1,"2019-04":1,"2018-51":1,"2018-39":1,"2018-26":1,"2018-17":1,"2018-09":1,"2018-05":1,"2017-47":2,"2017-43":2,"2017-39":3,"2017-34":1,"2017-30":4,"2017-26":2,"2017-22":2,"2017-17":5,"2017-09":3,"2017-04":5,"2016-50":2,"2016-44":3,"2016-40":3,"2016-36":3,"2016-30":2,"2016-26":2,"2016-22":2,"2016-18":1,"2016-07":1,"2015-48":2,"2015-35":1,"2015-32":1,"2015-27":2,"2015-22":2,"2015-14":2,"2014-52":2,"2014-42":1,"2014-41":1,"2014-35":3,"2023-23":1,"2017-13":2,"2015-18":3,"2015-11":1,"2015-06":1}},"file":"PMC3402330"},"subset":"pubmed_central"}
{"text":"abstract: **Objective** To elicit women's responses to information about the nature and extent of overdiagnosis in mammography screening (detecting disease that would not present clinically during the woman's lifetime) and explore how awareness of overdiagnosis might influence attitudes and intentions about screening.\n  .\n  **Design** Qualitative study using focus groups that included a presentation explaining overdiagnosis, incorporating different published estimates of its rate (1\u201310%, 30%, 50%) and information on the mortality benefit of screening, with guided group discussions\n  .\n  **Setting** Sydney, Australia\n  .\n  **Participants** Fifty women aged 40\u201379 years with no personal history of breast cancer and with varying levels of education and participation in screening.\n  .\n  **Results** Prior awareness of breast cancer overdiagnosis was minimal. Women generally reacted with surprise, but most came to understand the issue. Responses to overdiagnosis and the different estimates of its magnitude were diverse. The highest estimate (50%) made some women perceive a need for more careful personal decision making about screening. In contrast, the lower and intermediate estimates (1\u201310% and 30%) had limited impact on attitudes and intentions, with many women remaining committed to screening. For some women, the information raised concerns, not about whether to screen but whether to treat a screen detected cancer or consider alternative approaches (such as watchful waiting). Information preferences varied: many women considered it important to take overdiagnosis into account and make informed choices about whether to have screening, but many wanted to be encouraged to be screened.\n  .\n  **Conclusions** Women from a range of socioeconomic backgrounds could comprehend the issue of overdiagnosis in mammography screening, and they generally valued information about it. Effects on screening intentions may depend heavily on the rate of overdiagnosis. Overdiagnosis will be new and counterintuitive for many people and may influence screening and treatment decisions in unintended ways, underscoring the need for careful communication.\nauthor: Jolyn Hersch; Jesse Jansen; Alexandra Barratt; Les Irwig; Nehmat Houssami; Kirsten Howard; Haryana Dhillon; Kirsten McCafferyCorrespondence to: K McCaffery, Screening and Test Evaluation Program (STEP), School of Public Health, Level 1, Edward Ford Building, A27, University of Sydney NSW 2006, Australia <email@example.com>\ndate: 2013\nreferences:\ntitle: Women's views on overdiagnosis in breast cancer screening: a qualitative study\n\n# Extra material supplied by the author\n\nAppendix 1: Examples of slides from audiovisual presentation to participants\n\nClick here for additional data file.\n\nAppendix 2: Details of estimates of rate of overdiagnosis in mammography screening used in study\n\nClick here for additional data file.\n\nAppendix 3: Key items from questionnaires administered at the start and end of each focus group\n\nClick here for additional data file.\n\nAppendix 4: Written definitions of overdiagnosis given by participants\n\nClick here for additional data file.\n\n# Introduction\n\nMammography screening is meant to reduce breast cancer mortality through early diagnosis and treatment of disease that would otherwise threaten life. However, in some cases a screening mammogram detects a cancer that would have neither caused death nor presented clinically during the woman's lifetime,1 meaning she would never have acquired a cancer diagnosis had she not attended screening.2 This is termed overdiagnosis or overdetection. Overdiagnosis and the resulting overtreatment of inconsequential disease are considered the most serious and important harms associated with early detection of cancer through screening,1 2 3 generating morbidity that adds to the societal burden of cancer in medical, psychological, and economic terms.4 An overdiagnosed person is unlikely to benefit and may be harmed physically and emotionally by diagnostic procedures and treatments. The implications of the diagnosis may extend to female relatives, who are consequently classified as being at higher risk.5 6\n\nThe problems of overdiagnosis and overtreatment, both in cancer screening1 and in the broader healthcare context,7 are receiving increasing attention, and an independent review took place in 2012 in the United Kingdom8 to assess whether the national breast screening programme needed to be changed in light of the latest evidence on benefits and harms, including overdiagnosis. Quantifying overdiagnosis in mammography screening remains a challenge\u2014all estimates are subject to bias, and findings vary over a wide range amid continuing methodological debate.9 10 11 Recent studies have suggested that the overdiagnosed proportion of breast cancers (invasive and non-invasive) diagnosed in women invited for screening is likely to be around 19% to 35%,8 12 13 14 15 whereas other estimates have been substantially lower16 17 or higher.18 19\n\nDebate persists regarding the appropriateness of informing women about the risk of overdiagnosis before they decide to screen.20 Many argue that women considering screening should be aware of benefits and harms, including overdiagnosis, so they can make informed decisions about whether to participate.1 14 21 22 23 24 25 However, others are wary of the possibility that explicit information on the downsides of screening might dissuade people from participating.20 26 Materials distributed by breast screening programmes worldwide generally do not explain overdiagnosis,3 5 27 28 29 and consequently the public is largely unaware of the issue.30 31 32 Two mammography decision aids (evidence based information resources to support decision making) have briefly introduced the concept of overdiagnosis via an optional hyperlink33 or appendix.34 However, the information was probably not sufficient to convey the full implications of overdiagnosis and overtreatment, including the number of women affected. In a New Zealand study,35 a citizens' deliberation method was used to educate a panel of 11 women about mammography benefits and harms, of whom 10 then changed their minds to vote against government provision of screening for women aged 40\u201349. Although overdiagnosis was referred to repeatedly during group deliberations about screening policy,35 it remains unclear how women respond to overdiagnosis on an individual level and how they might integrate this information with their personal attitudes in deciding about their own screening participation. There is also a lack of evidence about how sensitive preferences might be to the varying estimates of overdiagnosis.1\n\nMammography screening involves large numbers of women worldwide, with participants annually numbering over 840\u00a0000 in Australia36 and 2.1 million in the UK.37 The Australian mammography programme is similar to those in other developed countries: women are invited to a screening service whose leaflets emphasise the benefit without mentioning overdiagnosis (such as [www.bsnsw.org.au\/publications\/early-detection-is-vital](http:\/\/www.bsnsw.org.au\/publications\/early-detection-is-vital)). To develop optimal strategies for communicating about overdiagnosis to women considering screening, better understanding of women's perspectives on the issue is needed,38 including how views may vary according to the magnitude of overdiagnosis. We conducted a qualitative study to elicit women's responses to information about overdiagnosis in mammography screening. We employed qualitative methods, which have been used successfully in previous research into women's views of screening, their reasoning about participation, and the ideas shaping these decisions.39 40 A qualitative approach was well suited to the goals of this study which aimed to generate insights into women's conceptual understanding and interpretation of information on overdiagnosis; to explore women's evaluation of overdiagnosis including whether and how different estimates influenced screening attitudes and intentions; and to investigate views about provision of such information to the community.\n\n# Method\n\n## Design\n\nWe used focus groups to explore how women integrated information about overdiagnosis (a topic likely to be new to them) with their existing understanding of breast screening (or \"schema\")41 and how they might use the information when making their personal screening decisions. Choosing a qualitative design involving face to face contact with participants ensured we could be confident that we communicated the information effectively. Women were able to ask questions to seek clarification, meaning that we could check their understanding and identify sources of confusion as part of the research process. Collecting data through focus groups rather than individual interviews gave more control of conversation topics to the participants because it allowed participants to bring up and discuss among themselves, in their own language, relevant points beyond those anticipated by the researchers.42 Another useful feature of focus groups for this research was the opportunity for participants to hear each other's views and exchange ideas, thereby helping to clarify individual understanding and attitudes.43 Furthermore, the presence of group members with contrasting opinions often led participants to elaborate their own point of view in greater depth.42\n\nAll study procedures were approved by the University of Sydney Human Research Ethics Committee. Participants gave written consent prior to participation.\n\n## Participant selection and recruitment\n\nWe recruited a community sample of women for whom making decisions about participating in mammography screening was relevant. Eligible women were aged 40\u201379 years, spanning the broad age range in which women commonly have screening. In Australia, women aged 50\u201369 are routinely invited for free biennial mammograms through the national screening programme, while women aged 40\u201349 or over 70 are eligible for screening on request but not actively invited.\n\nWe selected Sydney suburbs that varied in socioeconomic status and approached potential participants by randomly sampling households in these areas using the electronic telephone directory. Recruitment was carried out via telephone by the Hunter Valley Research Foundation (HVRF), an independent non-profit organisation with extensive experience running community surveys and recruiting people into health research. To gain a diverse range of perspectives, we used purposive sampling to ensure inclusion of women with varying levels of education (that is, with and without post-school qualifications) and prior participation in screening (including low and high screening women in all age groups). We excluded women not fluent in English and women who had ever personally been diagnosed with breast cancer. Trained HVRF interviewers used a short series of questions to determine eligibility and compiled a database of women who had consented to contact by the researchers. We mailed study information to these women and then telephoned them to address any questions and invite them to participate.\n\nOf 118 women who were contacted and eligible, 78 (66%) consented to participate. We scheduled focus group sessions to best accommodate women's availability. Recruitment to new groups continued until the team decided we had achieved thematic saturation as indicated by data redundancy\u2014that is, when participants no longer raised themes not already elicited (n=50).44 45\n\n## Conducting the focus groups\n\nWe conducted eight age-stratified focus groups, each comprising four to nine women, from July to September 2011. Sessions were convened in neutral focus group facilities distributed across three Sydney locations that were convenient for participants rather than in a clinical or academic setting. Each session comprised several parts: introduction and consent, baseline questionnaire, warm-up discussion, a detailed presentation on breast screening and overdiagnosis which was divided into sections and interspersed with guided discussions about the content, and a final questionnaire. Sessions lasted about two hours and were facilitated by a pair of female moderators (JH and either JJ or KM, all researchers in public health and psychology). Each participant was paid \\$A150 to reimburse her for her time and to cover travel costs. We provided all participants with a written list of organisations from which they could access further information about breast screening if desired.\n\n## Presentation and discussion content\n\nAt each session, JH gave an audiovisual presentation that had been developed and reviewed by a multidisciplinary expert team of researchers and clinicians (see appendix 1 of supplementary material on bmj.com for example slides). The presentation explained overdiagnosis using a consumer friendly format (such as combining pictures with text and using plain language as much as possible). We presented a range of published estimates of the rate of overdiagnosis in mammography screening (1\u201310%, 30%, and 50% of breast cancers diagnosed in women participating regularly in screening), based on a thorough literature review (see appendix 2 on bmj.com for further details on these estimates). To put overdiagnosis in context, we also provided age stratified, evidence based information on the benefit in terms of breast cancer mortality associated with mammography screening in Australia.46 In line with evidence based recommendations,47 we illustrated cumulative outcomes in the form of natural frequencies using diagrams to show the absolute number of breast cancer deaths among 1000 screened and 1000 unscreened women, and the number of cases overdiagnosed in the screened cohort. Thorough pilot testing of the study materials and procedure among women in the relevant age range resulted in various refinements (such as replacing the term \"overdiagnosis\" with \"overdetection\" to avoid confusion with misdiagnosis).\n\nIn the group discussions, women shared their responses to the information presented and how it made them feel about screening, as well as views on different strategies for communicating about screening. Box 1 summarises the presentation content and types of questions we used to guide the discussions. At the beginning of each focus group we made it clear that we would be talking about reasons why women might choose to have screening or not to have screening, and highlighted that we would not try to persuade them one way or another. We emphasised our neutrality throughout by repeating statements of this kind, by not labelling the figures presented (such as number of overdiagnosed cases) as large or small, and by not interpreting as positive or negative any views expressed by participants. Throughout the presentation, we strongly encouraged women to request clarification as often as they needed.\n\n### Box 1: Focus group presentation topics and key discussion questions\n\n-   Introduction to breast cancer screening and treatment\n\n-   Breast cancer mortality benefit of screening, with example animation of woman gaining benefit\n\n-   Mortality benefit in numbers (breast cancer deaths averted per 1000 women screened)\n\n-   Introduction to overdetection, with example animation of woman experiencing overdetection\n\n    1.  Have you ever heard of overdetection or overdiagnosis in screening?\n\n    2.  After seeing and hearing this information about overdetection in breast screening, what are your thoughts or feelings? How does it make you feel about having breast screening?\n\n-   Overdetection in numbers (cases overdetected per 1000 women screened)\u201430% estimate\\*\n\n    1.  Given this rate of overdetection, how does it make you feel about having screening?\n\n-   Range in published overdetection estimates\n\n-   Overdetection in numbers: 1\u201310% estimate and 50% estimate\n\n    1.  If overdetection were \\[10% or less \/ 50%\\], how would you feel about having screening?\n\n    2.  Does it make a difference to you whether overdetection is 1\u201310% or 30% or 50%?\n\n    3.  Is overdetection something that you would have liked to have known about before?\n\n    4.  There are different approaches to telling people about screening. Currently the main approach is to encourage people to be screened; another is to give people information about pros and cons and let them decide. Which approach would you prefer, and why?\n\n## Data capture, coding, and analysis of qualitative data\n\nFocus group discussions were audio recorded and transcribed verbatim. Data were managed using NVivo 9 software. We conducted a thematic analysis, aiming to identify a set of main themes that captured the diverse views and feelings expressed. After the first few focus groups, the group moderators (JH, JJ, and KM) began the analysis by independently documenting and then discussing together the most salient observations from the group discussions. These impressions, together with the research questions that shaped the group discussion guide, formed the basis of the initial coding framework.\n\nTwo researchers (JH and JJ) independently reviewed all transcripts, developed codes in an iterative process, and applied these to the data to identify patterns of ideas. Using constant comparison,44 48 we continually looked for similarity and differences in the data and our coding within and across transcripts. By comparing each part of the data with the rest of the data, we established analytical categories and selected key overarching concepts. Throughout this process we met regularly with KM and the research team to discuss the framework with which to interpret the data.\n\n## Quantitative measures\n\nWe administered brief written questionnaires at the start and end of each focus group, including demographics, preferred involvement in decision making about screening (control preferences scale),49 and items to assess basic background knowledge and understanding of content presented (see appendix 3 on bmj.com for key items from questionnaires 1 and 2).\n\n## Follow-up telephone interviews\n\nJH conducted a brief telephone interview with a randomly selected third of the focus group participants in each age category (n=18) at one to four weeks after their session to identify whether views about overdiagnosis had changed and to elicit any responses that women might prefer to express privately. We asked whether the women had any further thoughts or feelings, since their focus group, about the information presented and their screening intentions. As the views expressed during follow-up telephone interviews presented no new data further to the focus group discussions, they were not analysed further.\n\n# Results\n\n## Sample characteristics\n\nFifty women participated in eight focus groups. Participants had diverse cultural and education backgrounds (table 1<a href=\"#tbl1\" data-ref-type=\"table\"><\/a>). Each age stratum included women who had participated regularly in screening and others who had not. Three quarters of women in the target 50\u201369 year old cohort and 60% of older women knew they were eligible for free screening, whereas two thirds of women in their 40s either believed they were too young to access the programme or were unsure. About 80% of respondents desired active involvement in decisions about their breast screening participation.\n\n\u2002Characteristics of women in focus groups about mammography screening and overdiagnosis (n=50)\n\n| Characteristics                           | No (%) of women |\n|-------------------------------------------|-----------------|\n| Age (years):                              |                 |\n| \u200340\u201349                                    | 19 (38)         |\n| \u200350\u201369                                    | 16 (32)         |\n| \u200370\u201379                                    | 15 (30)         |\n| Mammography screening history\\*:          |                 |\n| \u2003Screened at least once                   | 31 (62)         |\n| \u2003Never screened (age \\<50 years)          | 12 (24)         |\n| \u2003Never screened (age \\>50 years)          | 7 (14)          |\n| Education\u2020:                               |                 |\n| \u2003No formal qualifications                 | 4 (8)           |\n| \u2003Intermediate school certificate          | 11 (22)         |\n| \u2003Higher school certificate                | 9 (18)          |\n| \u2003Diploma or trade certificate             | 12 (24)         |\n| \u2003University degree                        | 13 (26)         |\n| Country of birth:                         |                 |\n| \u2003Australia                                | 27 (54)         |\n| \u2003England or Scotland                      | 4 (8)           |\n| \u2003China                                    | 4 (8)           |\n| \u2003Other                                    | 15 (30)         |\n| Marital status\u2020:                          |                 |\n| \u2003Married or living with partner           | 36 (72)         |\n| \u2003Single, separated, or widowed            | 13 (26)         |\n| No of children\u2020:                          |                 |\n| \u20030                                        | 11 (22)         |\n| \u20031                                        | 7 (14)          |\n| \u20032\u20136                                      | 31 (62)         |\n| Employment\u2020:                              |                 |\n| \u2003Retired                                  | 15 (30)         |\n| \u2003Working full time                        | 14 (28)         |\n| \u2003Working part time                        | 13 (26)         |\n| \u2003Not in paid work                         | 7 (14)          |\n| Preferred role in screening decision\u2020:    |                 |\n| \u2003I decide alone                           | 13 (26)         |\n| \u2003I decide after consulting doctor         | 12 (24)         |\n| \u2003Doctor and I share equally in deciding   | 14 (28)         |\n| \u2003Doctor decides after consulting or alone | 8 (16)          |\n\n\\*Although the Australian national mammography screening programme is available to women over 40 years old with no upper age limit (so all study participants were eligible), the programme actively recruits women for biennial screening between the ages of 50 and 69 in accordance with current recommendations.\n\n\u2020Missing data for between one and three participants.\n\nTo illuminate the study findings presented below, we have selected representative participant quotations that illustrate both typical responses and the diversity of views expressed. The examples are labelled by participant number, age, and screening history, and are drawn from women with a variety of education levels.\n\n## The concept of overdiagnosis: women's understanding and reactions\n\n### Understanding of the concept of overdiagnosis\n\nPrior awareness of overdiagnosis was limited to only a few women who had heard of it in the context of prostate cancer. The idea of overdiagnosis occurring in breast cancer screening was surprising and challenged women's beliefs about breast cancer generally being a serious and dangerous disease:\n\n1.  \"In your brain, breast cancer\u2014if it isn't treated\u2014is fatal. So I've never ever heard of that before, and I think most women wouldn't have.\" (Participant 26, age 55 years, 3 screens)\n\n2.  \"That there are okay cancers, it's something that we haven't heard of before. It's very new to me... here it is... in the statistics, but we don't hear about it, not even from our doctors.\" (Participant 15, age 47, 1 screen).\n\nBy the end of the session, most women gained a reasonable understanding of the concept of overdiagnosis, as shown by these examples of women paraphrasing the concept of indolent or \"slow growing\" cancers that do not threaten life:\n\n1.  \"Sometimes it doesn't matter if you've got \\[breast cancer\\], it wouldn't make any difference, you're still going to live as long.\" (Participant 27, age 57, 0 screens)\n\n2.  \"It could be dormant for years and years, and you wouldn't know.\" (Participant 49, age 78, 4 screens).\n\nWe also assessed participants' conceptual knowledge about overdiagnosis via two items in the written questionnaire administered at the end of each focus group. As shown in table 2<a href=\"#tbl2\" data-ref-type=\"table\"><\/a>, most of the participants' written responses indicated understanding of the key point that screening increases breast cancer incidence through the detection of non-threatening cancers. We marked women's definitions of overdetection strictly according to the following scoring criteria. To score full marks, the response had to indicate that (*a*) a person has cancer, and (*b*) it may not cause harm or treatment may not be necessary. The \"partially correct\" scoring category covered responses that ranged from those indicating some limited understanding (such as \"finding a lump that is benign\") to those that were close to achieving full marks (such as \"when there is a cancer that is detected, but may have not changed if it was not detected\"). The complete set of written definitions given by participants is provided in appendix 4 on bmj.com.\n\n\u2002Participants' responses to written knowledge items completed at the end of focus group session about mammography screening and overdiagnosis (n=50)\n\n| Item | No (%) of women |\n|----|----|\n| Who is more likely to be diagnosed with breast cancer? (multiple choice): |  |\n| \u2003Women who have screening mammograms (correct answer) | 35 (70) |\n| \u2003Women who do not have screening mammograms | 10 (20) |\n| \u2003Unsure | 5 (10) |\n| What is meant by the term \"overdetection\"? (free response question): |  |\n| \u2003Full marks | 30 (60) |\n| \u2003Partially correct | 12 (24) |\n| \u2003Incorrect | 8 (16) |\n\nThere were many questions around the methods used to identify overdiagnosis. Women often had trouble grasping how it is even possible to know that overdiagnosis is occurring:\n\n1.  \"How do we know that things are being overdetected? How do we know that there are some cancers that move more quickly or become more malignant than others? ... Overdiagnosis assumes that these women, who have been overdiagnosed, have a cancer that is not necessarily aggressive ... Who determines what's an aggressive and a non-aggressive cancer?\" (Participant 33, age 62, 5 or more screens).\n\nMany women expressed surprise or disbelief at the current limitations we described in distinguishing between cancers that do require treatment and those that may not. There was some prior awareness that cancers are graded and staged after biopsy, and our assertion that this process still cannot accurately predict the future course of every diagnosed cancer (and the need for treatment) conflicted with women's expectations:\n\n1.  \"If you have a lump ... don't they do a biopsy to check whether it is cancerous or not first? Yes and if it is, they would be able to tell you whether they are aggressive or not, right?\" (Participant 11, age 46, 2 screens).\n\nWomen also struggled with the fact that overdiagnosis cannot be identified at the level of individual patients\u2014so that even if a given case is \"slow growing,\" there is no way to know for sure that it will remain non-threatening throughout the woman's lifetime and thus represents an instance of overdiagnosis:\n\n1.  \"How do they find out? ... Have they studied a slow growing breast cancer to see how long it goes for or how do they know that?\" (Participant 24, age 52, 1 screen).\n\nBox 2 summarises the main issues around overdiagnosis that caused confusion or required clarification during the focus groups.\n\n#### Box 2: Summary of main points of confusion about overdiagnosis, based on women's questions\n\n-   How do you know that there are cancers that will not progress fast enough to cause the woman any harm in her lifetime?\n\n-   Why is it not possible to distinguish these non-threatening cancers from the potentially dangerous ones that require treatment, even after biopsy or surgical removal of the cancer?\n\n-   How can overdiagnosis be quantified if we cannot identify the individual overdiagnosed cases?\n\n-   Why does overdiagnosis matter?\n\n### Reactions to learning about overdiagnosis\n\nFor some women, learning about overdiagnosis prompted concern about the implications in terms of the undesirable psychological and physical consequences of experiencing a cancer diagnosis and enduring treatment unnecessarily:\n\n1.  \"It could cause a lot of unnecessary stress and heartache to the person.\" (Participant 42, age 72, 3 screens)\n\n2.  \"So you have to go through all the rigmarole of chemotherapy, radiation, all that sort of thing, and it may not have been necessary, and those in themselves are hard to go through.\" (Participant 09, age 45, 0 screens).\n\nIn contrast, a few women reacted quite defensively to the concept of overdiagnosis or questioned why it mattered. They disagreed with the notion that it could be considered a bad outcome to find a cancer and have treatment that was ultimately not needed. These women perceived \"overdetection\" as a value laden term, labelling the phenomenon as negative without real justification. There was also some concern about the potential to upset women who had previously accepted invitations to screening:\n\n1.  \"I think the terms 'overtreatment' and 'overdiagnosis' are negative, they're loaded terms, and I don't think it's a necessarily negative occurrence. ... Whatever kind of cancer it is\u2014from vanilla nothing through to instant killer\u2014finding that, can that be a negative? I don't think so.\" (Participant 10, age 45, 1 screen)\n\n2.  \"To me it's not 'over' ... I don't believe in 'overservicing' ... I think this is a terrible word to use because I think it's a bullying word that people think they've been pushed into doing something that's not relevant.\" (Participant 05, age 43, 1 screen).\n\nIn several groups, women speculated about ulterior motives behind overdiagnosis research. For example, some wondered whether our presentation might be part of a government plan to reduce spending on breast screening, using overdiagnosis as justification:\n\n1.  \"I think what they're trying to do here is cut out breast screening ... or cut out the funding. ... I would hate to see the funding cut for mammograms. I think it's really important that we keep it up, if that's what all this is about.\" (Participant 34, age 67, 5 or more screens).\n\nIn responding to our introduction to the concept of overdiagnosis, only one woman explicitly asked for quantitative information about its frequency of occurrence.\n\n## The impact of overdiagnosis information on decision making\n\n### Implications of overdiagnosis for decisions about screening\n\nExploring how awareness of overdiagnosis affected women's feelings about screening was one of our key objectives for the focus group discussions. We found that a few participants, especially younger women with limited screening experience, quickly interpreted the possibility of overdiagnosis and overtreatment as a distinctly negative, off-putting factor in the screening \"equation\":\n\n1.  \"Turns me off further ... I'm just adding the things that we've learnt together \u2026 and all the doctors not knowing if it is malignant or not, you add them all together and you think, 'Why bother?'\" (Participant 09, age 45, 0 screens)\n\n2.  \"I don't know, makes me a bit scared now to go for a mammogram.\" (Participant 12, age 46, 0 screens).\n\nAfter explaining and discussing the concept of overdiagnosis, we moved on to its rate, starting with the 30% estimate. Although this figure clearly surprised some women and raised a few momentary doubts, most women found this level of overdiagnosis acceptable and considered it to be outweighed by the possible benefits of early detection. Ultimately, the 30% estimate had limited impact on women's existing views and screening intentions:\n\n1.  \"I'm surprised there is overdetection, but I don't think it will really change my view.\" (Participant 13, age 47, 0 screens)\n\n2.  \"It's probably a high number I suppose ... but then, personally, I still think I would prefer to be screened than not.\" (Participant 38, age 70, 5 or more screens)\n\n3.  \"I still think the deaths are high, so I prefer taking the risk of \\[being\\] 'overdetected' than ... risk of dying.\" (Participant 15, age 47, 1 screen).\n\nThe 1\u201310% overdiagnosis estimate was generally perceived as negligible and nothing to worry about. It did not seem to be sufficient to influence screening decisions but rather tended to confirm women's confidence in screening:\n\n1.  \"The 1\u201310 is like nothing.\" (Participant 24, age 52, 1 screen)\n\n2.  \"If it's in the 10% range, then I will say, 'Yeah, just go ahead and do it.'\" (Participant 11, age 46, 2 screens).\n\nBy contrast, the 50% overdiagnosis estimate was seen as extremely high. Considering this rate elicited emotional reactions, some bordering on disbelief, and raised concerns about the potential burden of unnecessary treatment affecting so many women:\n\n1.  \"Fifty per cent, that's a huge number. \u2026 I do voluntary work at \\[hospital\\], and \\[the patients having cancer treatment\\] look dreadful and they feel dreadful. ... I hate to think of 50% of people going through all that awful treatment for no reason.\" (Participant 42, age 72, 3 screens).\n\nThe figure<a href=\"#fig1\" data-ref-type=\"fig\"><\/a> illustrates a range of ways in which women responded to the 50% overdiagnosis scenario. Some women were prompted to reconsider whether screening was likely to be worthwhile for them personally\u2014that is, taking into account their own perceived risk factors for breast cancer (such as family history, breast feeding, the pill, breast self examination, lifestyle, stress). The discussions suggested that if the rate of overdiagnosis was about 50%, some participants might decline screening altogether. More commonly, women suggested that they might delay screening (particularly younger women) or be less concerned or rigorous in ensuring they attended within the recommended two year interval. Notably, women often presented this as a relatively minor change in their screening intentions. For one woman, the 50% overdiagnosis estimate had the effect of undermining her confidence in mammography as a screening tool and stimulating a search for other screening modalities. However, some women indicated that the information did not at all affect their feelings that mammography screening was worthwhile. A few declared that they would still participate in screening regardless of an even higher overdetection risk, and even while interpreting 50% as a high rate in itself.\n\nAcross the sample, women made a clear distinction between the 30% and 50% estimates. Responses suggested that an overdetection rate of 50% might deter some women from screening at all, and for many others it would at least necessitate more careful thought about whether and when to screen. In other words, the higher rate would affect the decision making process even if it might not change women's ultimate behaviour:\n\n1.  \"If there's more chance that you would have all the treatment and it was unnecessary, you'd be less likely to have the screening, I would say.\" (Participant 30, age 61, 0 screens)\n\n2.  \"It does make a difference. ... I'd think about it a lot more with the 50%, whereas 30% I'd probably do it more easily.\" (Participant 24, age 52, 1 screen).\n\nWe also observed a relationship between previous screening attendance and the extent to which information about overdiagnosis seemed to influence perceptions of screening. Regular screeners tended not to alter their positive screening intentions after learning about overdiagnosis, whereas women with less screening experience seemed more concerned about the issue.\n\n### Implications of overdiagnosis beyond screening decisions\n\nWomen valued screening as an opportunity to gain a sense of reassurance that everything was all right. They recognised that, even in light of overdiagnosis, they still had good chances of going through screening over many years without having any cancer diagnosed. Therefore, many women did not seem motivated to make a well informed and carefully considered decision about whether to have screening. Instead they chose to defer engaging in thorough deliberation until they were faced with the relatively unlikely event of being diagnosed with cancer:\n\n1.  \"I think screening is ... something you go through to have a peace of mind. But as to ... after that, if you are diagnosed, then you have to weigh and look at the various options that you have.\" (Participant 11, age 46, 2 screens).\n\nSome women said they would still have screening but felt that, if they were subsequently diagnosed with breast cancer, treatment (or at least adjuvant therapies beyond surgery) may not be worth while if overdiagnosis were 50%. Rather than seeing treatment as the immediate logical consequence of a diagnosis, they would want to consider \"watchful waiting\" or alternative therapies. At the very least, women anticipated having more thorough discussions and desired greater involvement with their doctors to explore all their management options:\n\n1.  \"If you were diagnosed you'd really go into what treatment involves ...you'd want to make a really informed decision ... \\[but\\] I probably would still have the breast screens and then make the decision after.\" (Participant 26, age 55, 3 screens)\n\n2.  \"I'll still go \\[for screening\\]. ... Surgery's okay too, but ... anything else being needed, any chemicals to be put through my body ... then that's where I stop and think and look at the statistics.\" (Participant 15, age 47, 1 screen)\n\n3.  \"It doesn't turn me off the screening, but I would certainly, probably second-think the treatment. Not that I wouldn't get it done, I probably may take a bit longer to think about it.\" (Participant 16, age 47, 0 screens).\n\nThere were suggestions that awareness of overdiagnosis (that is, knowing that not all cancers progress and need treatment) might help newly diagnosed women to feel less afraid and more hopeful about their prognosis. Some participants felt that this knowledge might also give such women more of a sense of control and reduce the pressure to follow a predetermined disease management pathway.\n\nImportantly though, it was also suggested that awareness of overdiagnosis could worsen the experience of having cancer by making women feel less able to cope with the challenges of unpleasant treatment. As such, it was considered perhaps better for the woman concerned to believe the cancer to be life threatening and treatment crucial:\n\n1.  \"That 50% is not good information ... because if I was told ... I've got cancer, I won't want to have in the back of my mind, 'Oh well, it might not be, and I'm going through all this and my family's all going bananas for nothing.'\" (Participant 42, age 72, 3 screens).\n\n## How to communicate overdiagnosis information to women\n\n### Personal preferences regarding screening information\n\nSome respondents stated that the information about overdetection would not make any difference to their own screening views or behaviour and therefore it did not matter whether they had this information or not:\n\n1.  \"For me ... if I'm going to do it, I don't care about \\[the overdetection rate\\].\" (Participant 35, age 69, 5 or more screens).\n\nOthers felt that it was good to know about overdiagnosis in order to be able to make informed decisions about screening. This included women who felt that their own screening decision would change and those who thought that it would not:\n\n1.  \"I really think it's good to know the downside of overdetection. ... That information \\[is\\] not discouraging me to ... screen, but it just made me feel I have knowledge. ... I feel it's really good.\" (Participant 18, age 49, 0 screens).\n\nSome participants thought that making decisions was more difficult when there was a lot of information to go through, or they lacked confidence in their ability to understand numerical information (such as the chances of experiencing benefit or harm from screening). These women often preferred to trust recommendations from their doctor or the government:\n\n1.  \"It's confusing, especially if you're not mathematical, like me.\" (Participant 34, age 67, 5 or more screens)\n\n2.  \"I just want \\[my doctor\\] to say, 'You should go, you must go ... this is where you go.'\" (Participant 12, age 46, 0 screens).\n\nMany respondents were in favour of being encouraged to screen, whereas some preferred to be given balanced information and left to make an individual choice. Some women simultaneously endorsed both of these approaches:\n\n1.  \"I'd like to be encouraged to have screening. I'd also like to be armed with all the information.\" (Participant 33, age 62, 5 or more screens).\n\n### Views on providing information to the community\n\nMany women favoured full, balanced information as the most ethical approach to communicating about screening. Some were surprised that they had not heard about overdetection before and argued that up-to-date information about it ought to be more widely available. Again, this included women who remained positive about undergoing screening:\n\n1.  \"I think if you've got the information, it should be provided.\" (Participant 16, age 47, 0 screens)\n\n2.  \"I would actually say that women are entitled to know. ... How you act upon it is probably neither here nor there. It's not a bad thing, perhaps, if that reminder \\[letter every two years\\] comes with this information attached to it\u2014or any new information, for that matter, that might arise.\" (Participant 17, age 48, 1 screen).\n\nOn the other hand, women recognised the challenge posed by trying to communicate the complex information from the focus group presentation in an alternative format that would be accessible and more practical. Furthermore, some women felt that changing the conventional pro screening message to include more balanced information could unleash widespread public confusion:\n\n1.  \"They're going to confuse the people. ... All this hard work they've been doing for years, encouraging people to have mammograms ... now you are saying today, 'If you want to you can do it, but see it's your choice, see the percentages and you make your own decision.'... Years and years saying, drumming in, 'You should have this every two years,'... and now you come out and say, 'Look, they are the percentages, think about it if you're going to go and do it.' You are confusing people.\" (Participant 35, age 69, 5 or more screens).\n\nSome women who said they personally were not put off by the overdiagnosis information nonetheless expressed concern that it would dissuade others from screening. In their view, this could lead to more breast cancer deaths and compromise the gains that screening had achieved for women's health. A couple of participants suggested providing overdiagnosis information to women only if and when they were diagnosed with breast cancer rather than before screening. Some participants took an intermediate position, suggesting that the amount of information women required before screening would vary according to personal preferences and that this variation should be accommodated somehow:\n\n1.  \"I don't think a vast wad of information should be pushed on everyone willy-nilly, but people should be made aware that the information is there if they would like it ... this might help you make a decision. But I'd want to be made aware that there was information that I can and should look at, should I choose to.\" (Participant 29, age 59, 5 or more screens).\n\n# Discussion\n\n## Principal findings\n\nIn this community sample of women eligible for mammography screening, awareness of overdiagnosis of breast cancer was minimal, consistent with earlier quantitative survey research.31 32 However, most participants were able to understand the issue after the explanation we presented and the opportunity to have their questions addressed. Although virtually everyone reacted to the information about overdiagnosis with surprise, effects on women's screening attitudes were diverse. Some women expressed a commitment to screening that was unaffected by learning about the possibility of overdiagnosis, while others wanted to take this information into account and give further thought to their screening choices. The screening intentions expressed by these women depended heavily on the magnitude of overdiagnosis, with the highest estimate (50%) having a distinctly greater impact than both the lower and intermediate estimates (1\u201310% and 30%). Most participants felt that this information was important and should be available to enable women to make informed choices, although many also wanted to be encouraged to have screening.\n\nImportantly, for some participants the information on overdiagnosis caused uncertainty not about whether to have screening but rather whether to have standard treatment for a screen detected cancer or consider alternative approaches (such as watchful waiting or unproven therapies). There was also the suggestion that, for women who were treated for breast cancer, being aware of the possibility of overdiagnosis might make it more difficult to endure unpleasant side effects because of uncertainty about whether treatment was really needed. Learning about overdiagnosis in mammography raised interest in seeking alternative forms of breast screening (such as ultrasound scanning) either instead of or in addition to mammography. Several participants expressed concern about other women inappropriately being deterred from screening altogether because of overdiagnosis. Moreover, some were suspicious about an agenda behind overdiagnosis research\u2014including our own study\u2014fearing a government plan to cut funding for mammography screening.\n\n## Strengths and limitations\n\nThe strategy for communicating about breast cancer screening in the UK is currently under review. By examining lay women's perspectives on overdiagnosis, this study makes an important and timely contribution to the debate about the possible consequences and value of providing overdiagnosis information to women invited for screening.\n\nThe focus group presentation was carefully developed to be both accurate (that is, evidence based data) and understandable (such as innovative illustrations of the concepts), and was expert reviewed and extensively pilot tested. We presented the cumulative probabilities of different screening outcomes using natural frequencies, in line with evidence based recommendations.47 We explored responses to a diverse range of overdiagnosis estimates, reflecting the full body of existing literature, because the amount of overdiagnosis is contested and all estimation methods are subject to bias.9 The qualitative design is a strength of this exploratory study because it allowed unanticipated findings to emerge (such as the impact on decision making about accepting treatment for detected cancer). Encouraging women to ask questions also provided new insights into points of confusion.\n\nWe tried to minimise volunteer bias by recruiting directly from the general community, and our provision of financial compensation helped us to achieve a broad sample including women from diverse socioeconomic backgrounds who varied in age, education, prior screening, and ethnicity. However, as the sample was not designed to be statistically representative, we cannot conclude that our findings reflect general population views. We stressed our neutrality at the start of each session to mitigate against \"socially desirable\" comments in favour of screening.\n\nTo avoid overloading women with information, we did not talk about the scientific uncertainty surrounding estimates of the mortality benefit, nor did we discuss in detail the chances of other relevant screening outcomes (such as false positives and false negatives). Discussing these additional points would have provided a more comprehensive picture of factors relevant to screening and the decision making process, and might have influenced the study results. However, further increasing the complexity of the information provided would have limited the study's capacity to address the key research questions concerning overdiagnosis. It is possible that our choice of presenting the benefit information first may have created a \"framing\" effect resulting in more positive attitudes, but finding positive attitudes to cancer screening is consistent with previous literature.31 32 34\n\nIn comparison with earlier attempts to introduce the concept of breast cancer overdiagnosis to women,31 33 34 this study involved a much more thorough explanation of the issue and its magnitude, albeit in a more resource intensive manner. The purpose of the focus groups was not to imitate a \"real life\" setting but to allow us to explore the process through which women understood and responded to information about overdiagnosis, thereby providing valuable insight into women's attitudes. In our diverse sample (including women with low education and non-English speaking backgrounds) most participants' written definitions of overdetection showed full (60%) or partial (24%) understanding of the concept. However, some participants (several of whom, though reasonably fluent in English, were from other language backgrounds) did not demonstrate a clear understanding of overdiagnosis, which limited our ability to accurately gather their views about it. For such women, an individual counselling and interview session may be a more appropriate way to explain the issues and examine their responses. Importantly, in this study we asked women about their own screening intentions as opposed to their views on national screening policy.35 Earlier qualitative research with women diagnosed through screening found that they considered it important to take into account, when making screening decisions, the possibility of being diagnosed with an ultimately harmless condition.25 Our study showed that this view, expressed by breast cancer patients whose perspective on screening would have been coloured by their personal experience of cancer, was shared by many women in the general invited population.\n\n## Implications and future research\n\nThis study provides important insights into lay interpretations of information about overdiagnosis, with implications regarding how best to inform people about the issue. Firstly, the magnitude of overdiagnosis matters, with many women indicating that their screening behaviour may change depending on the trade-off between the chances of benefit and harm.50 The wide variation in published estimates (1% to 50%) is therefore problematic. Additionally, the suggestions from some women that overdiagnosis would be relevant to their decisions only if they were actually diagnosed with a screen detected cancer reveal a concerning misconception that a screening mammogram is a separable event from the cascade of investigation and intervention that may be triggered by an abnormal result. This highlights the need to explain clearly to women that once cancer is detected, evidence based treatment is virtually always indicated because potentially threatening cases cannot be differentiated from those representing overdiagnosis. Communicators must also take care not to create a situation in which women think they can avoid overdiagnosis by choosing a different screening method (for which evidence is limited) or by supplementing screening mammography with additional testing, which may increase the potential for overdiagnosis. Thus the ethical imperative to provide information about overdiagnosis must be balanced with the responsibility to address misconceptions that may lead to problems in clinical practice.\n\nAnother important point to consider in developing strategies for communication about overdiagnosis relates to understanding how people interact with new information. A theoretical framework used in the field of education known as schema theory41 highlights that the way people engage with new material is influenced by their \"schema\" (existing topic knowledge and expectations). In the case of mammography screening, attitudes tend to be strongly favourable, which is partly a function of the historical and cultural context. Women's attitudes will have been shaped through the information presented to them directly by screening services as well as their broader experience of many years of widespread public health campaigns focused on promoting the benefits of screening without explaining the harm of overdiagnosis.27 29 32 The very existence of overdiagnosis runs contrary to prevailing notions of cancer and screening. Therefore it is reasonable to expect that new messages about overdiagnosis will not necessarily be immediately understood by everyone and may provoke confusion or scepticism, as we found to some extent in our study. Rather than wishing to be informed in detail about the range of possible outcomes of screening, some participants expressed a preference to receive more basic information together with encouragement to have screening. Such preferences are consistent with the \"consider an offer\" approach,51 in which screening is offered with summarised key information and people are assisted to evaluate whether taking up the offer is right for them. At the same time, people can be encouraged to consider whether they want to access more detailed information that may help them make a choice.51 Public views and information needs may change over time as more balanced screening information and increased awareness of harms contribute to changing the schema of expectations people have about screening. Moreover, the diversity we found in women's responses to overdiagnosis supports the need for information providers to tackle the challenge of developing ways to deliver screening information flexibly rather than assuming that women's needs are homogenous.\n\nGiven that doctors' recommendations are likely to influence screening behaviour,52 one avenue for future research may be to examine the perspectives of relevant clinicians, both in terms of their views on overdiagnosis and their responses to women's attitudes as reported here. However, in countries such as Australia and the UK women are invited directly by the screening service and attend without necessarily having any discussion with a healthcare provider. Therefore, priorities for future research are to investigate whether a meaningful explanation of overdiagnosis can be condensed into a format that could be distributed with screening invitations, and to assess the impact of such information on screening intentions and participation. Also critical is clarification of the magnitude of overdiagnosis, though it is evident that such data cannot necessarily resolve whether screening benefits outweigh harms for any individual as this ultimately requires a value judgement.5 46 50 The question remains, how do we best inform women and support them to make informed decisions about mammography screening using the available evidence.23\n\n## Conclusions\n\nOverdiagnosis was unfamiliar to a diverse sample of women eligible for mammography screening in Australia. None the less, we found it was possible to explain the issue in such a way that most participants understood the concept and its implications. Women's screening preferences were sensitive to the estimated level of overdiagnosis, underscoring the need for urgent efforts to clarify this issue. Critically, women differed in the way they evaluated the relevance of overdiagnosis in making their personal decisions about screening. Some felt that the new information was unlikely to change their existing attitudes and intentions; others considered it important to take the risk of overdiagnosis into account, particularly at higher estimates. Many women expressed a preference for this information to be more widely available. However, their responses to being informed about overdiagnosis also revealed a range of potentially unwanted consequences that communicators must address. Finally, the diversity of views elicited in this study highlights the inappropriateness of making screening policy and communication decisions based on speculative assumptions about what women want and think regarding overdiagnosis. Instead, it is vital that research efforts continue to better understand women's values about overdiagnosis, to clarify its influence on their behaviour, and to determine the optimal methods to support informed choice in mammography screening.\n\n### What is already known on this topic\n\n1.  Mammography screening for breast cancer carries a risk of overdiagnosis (detection of disease that would not present clinically within the woman's lifetime), but estimates of the magnitude of this risk vary widely\n\n2.  Public awareness about breast cancer overdiagnosis is low, and there is little evidence about how women respond to the concept and extent of overdiagnosis and how they might use this information in making personal choices about screening\n\n### What this study adds\n\n1.  Evaluations of the relevance of overdiagnosis depended heavily on the different estimates of magnitude: the 50% estimate generated substantial concern, whereas the 1\u201310% and 30% estimates were considered more acceptable levels of risk\n\n2.  Women's responses were diverse: some indicated that they would take the risk of overdiagnosis into account when making screening decisions, whereas others felt that their attitudes and intentions to screen were unaffected by the new information\n\n3.  Learning about overdiagnosis raised interest in alternative screening methods as well as concern about whether and how to treat screen detected cancers; hence great care is needed in communicating this information as it may influence screening and treatment decisions in unintended ways\n\nCite this as: *BMJ* 2013;346:f158","meta":{"dup_signals":{"dup_doc_count":134,"dup_dump_count":63,"dup_details":{"curated_sources":2,"2023-40":2,"2023-14":1,"2023-06":1,"2022-49":1,"2022-27":2,"2022-21":2,"2022-05":2,"2021-49":2,"2021-43":1,"2021-39":3,"2021-31":3,"2021-25":1,"2021-04":1,"2020-50":2,"2020-34":1,"2020-24":1,"2020-16":2,"2020-10":1,"2019-47":1,"2019-39":1,"2019-26":3,"2019-18":1,"2019-09":1,"2018-51":1,"2018-43":1,"2018-39":1,"2018-30":1,"2018-22":1,"2018-13":1,"2018-05":1,"2017-47":3,"2017-43":1,"2017-34":1,"2017-26":2,"2017-22":1,"2017-17":1,"2016-44":2,"2016-36":2,"2016-30":3,"2016-26":1,"2016-22":3,"2016-18":3,"2016-07":3,"2015-48":3,"2015-40":2,"2015-35":3,"2015-32":2,"2015-27":3,"2015-22":3,"2015-14":2,"2014-52":3,"2014-49":2,"2014-42":7,"2014-41":6,"2014-35":6,"2014-23":7,"2024-22":1,"2024-10":1,"2017-13":3,"2015-18":2,"2015-11":3,"2015-06":3,"2024-26":1}},"file":"PMC3552499"},"subset":"pubmed_central"}
{"text":"abstract: Cotyledonoid dissecting leiomyoma of the uterus is a recently described rare variant of benign uterine leiomyoma. We report a case of cotyledonoid dissecting leiomyoma in a 52 year old woman who presented with menorrhagia and abdominal pain. An ultrasound scan showed a bulky uterus and a cystic heterogenous mass near the left ovary. At hysterectomy, the left broad ligament mass was removed. This was continuous with an ill-defined nodular area in the myometrial fundus. Microscopy revealed a benign smooth muscle proliferation in the myometrium that extended beyond the uterus and into the broad ligament. The lesion appeared to be dissecting the myometrial fibres and showed areas of oedema, hyalinisation and perinodular hydropic change. Cellular atypia, mitoses and coagulative necrosis were absent. The patient is alive and well 18 months after surgery. It is important to recognize this benign and unusual appearing variant of leiomyoma in order to prevent inappropriate treatment.\nauthor: Annikka Weissferdt; Madhavi B Maheshwari; Gabrielle P Downey; Terence P Rollason; Raji Ganesan\ndate: 2007\ninstitute: 1Department of Histopathology, Birmingham Women's Hospital, Metchley Park Road, Edgbaston, B15 2TG, UK; 2Department of Histopathology, Sandwell and West Birmingham Hospitals, Dudley Road, Birmingham, B18 7QH, UK; 3Department of Obstectrics and Gynaecology, Sandwell and West Birmingham Hospitals, Dudley Road, Birmingham, B18 7QH, UK\nreferences:\ntitle: Cotyledonoid dissecting leiomyoma of the uterus: a case report\n\n# Background\n\nUterine leiomyomas can present in a variety of unusual growth patterns. One of the most unusual variants is called cotyledonoid dissecting leiomyoma or Sternberg tumour \\[1\\]. These tumours have been described to occur in women of the reproductive age group \\[1\\]. They tend to arise in the subserosal myometrium of the lateral aspect of the uterus especially the uterine cornu and extend into the broad ligament and pelvic cavity. The exophytic component has characteristic spongy, bulbous protuberances over the surface that appear congested and red, resembling placental tissue. The intramural component dissects into the fascicles of the myometrium in an irregular fashion. There usually is extensive hydropic degeneration resulting in the characteristic pattern of perinodular hydropic degeneration. Cellular atypia, mitoses and coagulative tumour necrosis are typically absent \\[1\\]. There often is confusion between this lesion and smooth muscle tumours that have somewhat similar pathologic features eg. intravascular leiomyomatosis. In addition, the worrying appearances of the gross specimen are often mistaken for malignant or non-uterine lesions that may result in overtreatment. To date, no malignant behaviour or recurrence has been described in these lesions with the longest follow-up period amounting to 41 years \\[1\\].\n\n# Case report\n\nA 52 year old woman, gravida 5, para 5, presented with a 5 months history of menorrhagia, abdominal pain and tiredness. A transvaginal ultrasound scan showed a bulky uterus with an irregular and thickened endometrium of 14 mm. A 10 mm well rounded and echo poor area was also noted in the endometrium. In the region of the left ovary there was a cystic lesion that had thick irregular borders and solid components measuring 40 mm in maximum dimension. The right ovary was unremarkable. The CA 125 level was 12 units\/ml. Since the left ovarian lesion was reported as suspicious the patient underwent surgery. At the time of laparotomy, a bulky uterus was seen and a spongy lesion was noted in the region of the left ovary. A total abdominal hysterectomy with left salpingo-oophorectomy was performed and the patient is alive and well 18 months after the operation.\n\n# Methods\n\nTissue sections were fixed in 10% formalin, embedded in paraffin and stained with haematoxylin-eosin. Immunohistochemical staining with the streptavidin-biotin peroxidase detection system was performed using a Ventana automated immunostainer (Ventana, Tucson, Arizona). The antibodies smooth muscle actin (1A4, 1:400, Dakopatts, Glostrup, Denmark), desmin (D33, 1:100, Dakopatts, Glostrup, Denmark), CD10 (56C6, NEAT (Ventana kit), Ventana, Tucson, Arizona), HMB45 (HMB45, 1:50, Dakopatts, Glostrup, Denmark) and CD31 (1A1O, 1:40, Novocastra, Newcastle upon Tyne, UK) were used.\n\n# Results\n\nMacroscopic examination of the specimen revealed a corpus and cervix 110 \u00d7 60 \u00d7 50 mm with an attached left fallopian tube 30 mm in length and left ovary 20 \u00d7 15 \u00d7 10 mm. A large mass was present in the left broad ligament in the region of the left ovary measuring 60 \u00d7 40 \u00d7 30 mm. This mass had a red, spongy, cystic and gelatinous cut surface reminiscent of placental tissue and was distinct from the ovary and fallopian tube but apparently continuous with an ill-defined nodular area in the myometrium of the uterine fundus (Fig. <a href=\"#F1\" data-ref-type=\"fig\">1a<\/a>). This nodular area also appeared to be continuous with a submucosal leiomyoma measuring 20 \u00d7 20 \u00d7 10 mm.\n\nHistological examination showed that the submucosal leiomyoma was composed of bland smooth muscle cells growing in broad, sweeping fascicles with no cellular atypia, abnormal cellularity or increased mitotic activity (up to 1 mitosis per 10 high power fields). Immunohistochemically, the constituent cells were positive with smooth muscle actin and desmin (Fig. <a href=\"#F1\" data-ref-type=\"fig\">1b<\/a>) while CD10, CD31 and HMB45 were negative. This leiomyoma was continuous with the ill-defined nodular area identified macroscopically. Here, the tumour cells were noted to grow into the surrounding myometrium in a dissecting pattern (Fig. <a href=\"#F2\" data-ref-type=\"fig\">2a<\/a>). Furthermore, prominent hydropic changes amounting to perinodular hydropic degeneration were present (Fig. <a href=\"#F2\" data-ref-type=\"fig\">2b<\/a>) and the lesion extended beyond the confines of the uterus and into the broad ligament where these features were exaggerated. The fascicles of muscle appeared disorganized but there was no evidence of coagulative necrosis. Multiple congested blood vessels were present throughout the lesion but there was no intravascular growth. The fallopian tube, ovary, endometrium and cervix were unremarkable.\n\n# Discussion\n\nUterine smooth muscle tumours are known to exhibit a wide variety of growth patterns. One unusual variant is the cotyledonoid dissecting leiomyoma of the uterus or \"Sternberg tumour\" first described in 1996 by Roth et al. \\[1\\]. Including our current case, only 10 cases have been described in the English literature to date \\[1-6\\]. The case presented here demonstrated the typical features described for these tumours: an exophytic component of bulbous (cotyledonoid) smooth muscle grossly resembling placental tissue protruding from the uterine surface near the region of the cornu and extending into the the broad ligament. This exophytic mass was in continuity with an intramural component dissecting the surrounding myometrium in a sinuous pattern. The neoplastic smooth muscle cells formed disorganized fascicles in contrast to the organized pattern seen in conventional leiomyoma but did not show any evidence of nuclear atypia, increased mitotic activity or coagulative tumour necrosis. Areas of oedema and perinodular hydropic degeneration were present. The lesion was highly vascular containing multiple congested blood vessels but intravascular growth was absent.\n\nDescription of new variants of any lesion comes with attendant issues regarding nomenclature. Apart from the cotyledonoid dissecting leiomyoma described by Roth et al. \\[1\\] there are closely related variants demonstrating similar pathologic features. This similarity is mirrored in the terminology. A variant called cotyledonoid leiomyoma was described by Roth and Reed \\[7\\] in 2000. This lesion resembled cotyledonoid dissecting leiomyoma but lacked a parent intramural component. The same authors described another related variant called intramural dissecting leiomyoma \\[8\\]. This lesion shows intramural dissection but lacks the extrauterine component seen in cotyledonoid dissecting leiomyoma. Another smooth muscle tumour was described showing the characteristics of cotyledonoid dissecting leiomyoma with features of intravascular leiomyomatosis \\[9\\]. It was proposed that this variant is called \"cotyledonoid hydropic intravenous leiomyomatosis\". Whereas cotyledonoid dissecting leiomyoma is a benign lesion that has shown no malignant behaviour or recurrence on follow up \\[1\\], the prognosis for intravenous leiomyomatosis is different. Although intravenous leiomyomatosis is also regarded as a benign variant of smooth muscle tumour, the vascular invasion may extend as far as the extrauterine pelvic veins, the inferior vena cava and even the right heart \\[10-12\\]. Pulmonary metastases have also been reported \\[13\\] and the rate of recurrence and progressive growth is higher than in typical leiomyoma \\[14\\].\n\nTherefore, care should be taken not to confuse these new variants of smooth muscle tumours with unusual growth patterns. This is especially important for tumours with different prognostic implications like the proposed \"cotyledonoid hydropic intravenous leiomyomatosis\" \\[9\\]. Careful assessment of the pathologic features will be necessary to prevent confusion and resulting misdiagnosis.\n\nIn summary, cotyledonoid dissecting leiomyoma is a rare variant of benign leiomyoma that is closely related to and shares some features with other and similarly named leiomyoma variants. Not only can its bizarre gross appearance lead to a mistaken diagnosis of a malignant neoplasm, but it should also be distinguished from other unusual leiomyoma variants, especially to exclude intravascular spread, for appropriate classification and potential prognostic implications.\n\n# Competing interests\n\nThe author(s) declare that they have no competing interests.\n\n# Authors' contributions\n\nAW drafted the manuscript. RG and TR helped to draft the manuscript. MM and GD contributed the clinical background.\n\nAll authors read and approved the final manuscript.","meta":{"dup_signals":{"dup_doc_count":101,"dup_dump_count":29,"dup_details":{"curated_sources":3,"2023-50":2,"2023-14":1,"2022-27":1,"2021-43":1,"2020-29":1,"2020-10":1,"2019-43":1,"2019-39":1,"2015-48":4,"2015-40":3,"2015-35":4,"2015-32":4,"2015-27":3,"2015-22":3,"2015-14":4,"2014-52":4,"2014-49":4,"2014-42":10,"2014-41":6,"2014-35":4,"2014-23":7,"2014-15":5,"2015-18":4,"2015-11":4,"2015-06":4,"2014-10":4,"2013-48":4,"2013-20":3,"2024-22":1}},"file":"PMC1904435"},"subset":"pubmed_central"}
{"text":"date: 2019-09\ntitle: WORLD HUNGER IS STILL NOT GOING DOWN AFTER THREE YEARS AND OBESITY IS STILL GROWING \u2013 UN REPORT\n\n**15 July 2019** - An estimated 820 million people did not have enough to eat in 2018, up from 811 million in the previous year, which is the third year of increase in a row. This underscores the immense challenge of achieving the Sustainable Development Goal of Zero Hunger by 2030, says a new edition of the annual The State of Food Security and Nutrition in the World report released today.\n\nThe pace of progress in halving the number of children who are stunted and in reducing the number of babies born with low birth weight is too slow, which also puts the SDG 2 nutrition targets further out of reach, according to the report.\n\nAt the same time, adding to these challenges, overweight and obesity continue to increase in all regions, particularly among school-age children and adults.\n\nThe chances of being food insecure are higher for women than men in every continent, with the largest gap in Latin America.\n\n\"Our actions to tackle these troubling trends will have to be bolder, not only in scale but also in terms of multisectoral collaboration,\" the heads of the United Nations' Food and Agriculture Organization (FAO), the International Fund for Agricultural Development (IFAD), the UN Children's Fund (UNICEF), the World Food Programme (WFP) and the World Health Organization (WHO) urged in their joint foreword to the report.\n\nHunger is increasing in many countries where economic growth is lagging, particularly in middle-income countries and those that rely heavily on international primary commodity trade. The annual UN report also found that income inequality is rising in many of the countries where hunger is on the rise, making it even more difficult for the poor, vulnerable or marginalized to cope with economic slowdowns and downturns.\n\n\"We must foster pro-poor and inclusive structural transformation focusing on people and placing communities at the centre to reduce economic vulnerabilities and set ourselves on track to ending hunger, food insecurity and all forms of malnutrition,\" the UN leaders said.\n\n**Slow progress in Africa and Asia**\n\nThe situation is most alarming in Africa, as the region has the highest rates of hunger in the world and which are continuing to slowly but steadily rise in almost all subregions. In Eastern Africa in particular, close to a third of the population (30.8 percent) is undernourished. In addition to climate and conflict, economic slowdowns and downturns are driving the rise. Since 2011, almost half the countries where rising hunger occurred due to economic slowdowns or stagnation were in Africa.\n\nThe largest number of undernourished people (more than 500 million) live in Asia, mostly in southern Asian countries. Together, Africa and Asia bear the greatest share of all forms of malnutrition, accounting for more than nine out of ten of all stunted children and over nine out of ten of all wasted children worldwide. In southern Asia and sub-Saharan Africa, one child in three is stunted.\n\nIn addition to the challenges of stunting and wasting, Asia and Africa are also home to nearly three-quarters of all overweight children worldwide, largely driven by consumption of unhealthy diets.\n\n**Going beyond hunger**\n\nThis year's report introduces a new indicator for measuring food insecurity at different levels of severity and monitoring progress towards SDG 2: the prevalence of moderate or severe food insecurity. This indicator is based on data obtained directly from people in surveys about their access to food in the last 12 months, using the Food Insecurity Experience Scale (FIES). People experiencing moderate food insecurity face uncertainties about their ability to obtain food and have had to reduce the quality and\/or quantity of food they eat to get by.\n\nThe report estimates that over 2 billion people, mostly in low- and middle-income countries, do not have regular access to safe, nutritious and sufficient food. But irregular access is also a challenge for high-income countries, including 8 percent of the population in Northern America and Europe. This calls for a profound transformation of food systems to provide sustainably-produced healthy diets for a growing world population.\n\n**Key facts and figures**\n\nNumber of hungry people in the world in 2018: 821.6 million (or 1 in 9 people)\n\nin Asia: 513.9 million\n\nin Africa: 256.1million\n\nin Latin America and the Caribbean: 42.5 million\n\nNumber of moderately or severely food insecure: 2 billion (26.4%)\n\nBabies born with low birth weight: 20.5 million (one in seven)\n\nChildren under 5 affected by stunting (low height-for-age): 148.9 million (21.9%)\n\nChildren under 5 affected by wasting (low weight-for-height): 49.5 million (7.3%)\n\nChildren under 5 who are overweight (high weight-for-height): 40 million (5.9%)\n\nSchool-age children and adolescents who are overweight: 338 million\n\nAdults who are obese: 672 million (13% or 1 in 8 adults)\n\nNote to editors\n\nThe heads of agencies issuing today's report are: Jos\u00e9 Graziano da Silva, Director-General of FAO; Gilbert F. Houngbo, President of IFAD; Henrietta H. Fore, Executive Director of UNICEF; David Beasley, Executive Director of WFP; and Tedros Adhanom Ghebreyesus, Director-General of WHO.\n\nThe report is part of tracking progress towards Sustainable Development Goal 2 Zero Hunger, which aims to end hunger, promote food security and end all forms of malnutrition by 2030.\n\nThe 2017 report identified three factors behind the recent rise in hunger: conflict, climate and economic slowdowns. This year's report focuses on the role of economic slowdowns and downturns in food security and nutrition.\n\nReaders should avoid comparing Prevalence of Undernourishment numbers across different editions of the report because the entire data set is reviewed and revised, including possible backward revisions, before each publication. This ensures that the latest report takes into account any new information received since the release of the previous edition.\n\nAvailable from: <https:\/\/www.who.int\/news-room\/detail\/15-07-2019-world-hunger-is-still-not-going-down-after-three-years-and-obesity-is-still-growing-un-report>","meta":{"dup_signals":{"dup_doc_count":156,"dup_dump_count":37,"dup_details":{"curated_sources":2,"2023-50":3,"2023-40":4,"2023-14":2,"2023-06":5,"2022-49":4,"2022-40":5,"2022-33":2,"2022-27":2,"2022-21":1,"2021-49":3,"2021-43":3,"2021-39":1,"2021-31":2,"2021-25":1,"2021-21":1,"2021-17":4,"2021-10":2,"2021-04":2,"2020-50":4,"2020-45":3,"2020-40":3,"2020-34":3,"2020-29":6,"2020-24":5,"2020-16":6,"2020-10":4,"2020-05":7,"2019-51":15,"2019-47":3,"2019-43":10,"2019-39":4,"2019-35":29,"2024-26":1,"2024-22":1,"2024-18":1,"2024-10":1,"2024-30":1}},"file":"PMC6790494"},"subset":"pubmed_central"}
{"text":"abstract: # Objective\n  .\n  The role of breakfast energy in total daily energy intake is a matter of debate. Acute feeding experiments demonstrated that high breakfast energy leads to greater overall intake supported by cross-sectional data of a free-living population. On the other hand, a large intraindividual analysis has indicated that a high proportion of breakfast to overall intake is associated with lower daily energy intake. To evaluate these apparently contradictory results in greater detail both ways of analysis were applied to the same data set of dietary records.\n  .\n  # Methods\n  .\n  On an intraindividual basis total daily energy intake was related to the absolute values of breakfast energy intake or to the ratio of breakfast to overall intake, respectively. Food intake of 280 obese and 100 normal weight subjects was analyzed who recorded over 10 (obese) or 14 (normal weight) consecutive days, respectively.\n  .\n  # Results\n  .\n  Increasing breakfast energy was associated with greater overall intake in normal weight and obese subjects. The increasing ratio of breakfast to total daily energy intake was associated with a significant reduction of overall intake on days where post-breakfast energy was significantly reduced. Correlational and multiple regression analysis support the concept that absolute breakfast calories have the strongest influence on daily energy intake.\n  .\n  # Conclusion\n  .\n  Reduced breakfast energy intake is associated with lower total daily intake. The influence of the ratio of breakfast to overall energy intake largely depends on the post-breakfast rather than breakfast intake pattern. Therefore, overweight and obese subjects should consider the reduction of breakfast calories as a simple option to improve their daily energy balance.\nauthor: Volker Schusdziarra; Margit Hausmann; Claudia Wittke; Johanna Mittermeier; Marietta Kellner; Aline Naumann; Stefan Wagenpfeil; Johannes Erdmann\ndate: 2011\ninstitute: 1Else-Kr\u00f6ner-Fresenius Center of Nutritional Medicine, Technical University of Munich, Ismaninger Stra\u00dfe 22, 81675 Munich, Germany; 2Institute of Medical Statistics and Epidemiology, Technical University of Munich, Ismaninger Stra\u00dfe 22, 81675 Munich, Germany; 3Graduate School of Information Science in Health Technical University of Munich, Ismaninger Stra\u00dfe 22, 81675 Munich, Germany\nreferences:\ntitle: Impact of breakfast on daily energy intake - an analysis of absolute versus relative breakfast calories\n\n# Introduction\n\nThe role of breakfast consumption in daily energy intake is a matter of debate. In acute feeding experiments Cotton et al. have demonstrated that an energy-rich breakfast does not induce subsequent under-eating to compensate for the extra calories consumed in comparison to a day that started with a breakfast containing 45% less energy \\[1,2\\]. Furthermore, a cross-sectional food intake analysis of young adults in Bogalusa has shown that breakfast skipping was associated with substantially lower daily energy intake \\[3\\]. On the other hand, the intraindividual analysis of eating habits of free-living subjects has demonstrated that an increasing percentage of breakfast to overall energy intake is associated with lower daily energy intake \\[4,5\\].\n\nThe results of the studies of Cotton et al. \\[1,2\\] and Nicklas et al. \\[3\\] are apparently contradictory to the data of de Castro \\[4,5\\]. However, a direct comparison of these studies is difficult due to the substantially different methodological approach. The analysis is based either on absolute breakfast calories in relation to total daily intake \\[1-3\\] under laboratory or free-living conditions or on the percentage of breakfast to total calories in relation to total daily intake in free living subjects examined intraindividually \\[4,5\\]. In this latter group it remains unknown whether the higher ratio of breakfast to total daily energy intake is due to increased breakfast or below average intake of the subsequent meals or a combination of both. This, however, is of interest in the perspective of dietary counselling.\n\nIn the present study we have analysed the intraindividual relationship between breakfast and total daily energy intake in a group of 280 obese and 100 normal weight subjects. In both groups data were analyzed on the basis of 1. absolute breakfast energy intake and 2. the ratio of breakfast to overall energy intake, respectively. A detailed analysis of total daily and single meal food intake has been reported recently for both groups \\[6-10\\]. The overall energy intake by solid food and beverages was comparable to the age- and gender-related groups of the recent German National Nutrition Survey II \\[11\\].\n\n# Research design and methods\n\nA total of 280 overweight and obese patients and 100 normal weight subjects were evaluated. The patients contacted the outpatient clinic of the department of nutritional medicine for the treatment of their weight problem. All had maintained their weight for at least 12 months without any period of intentional or spontaneous weight loss. The normal weight subjects were recruited by advertisement and the selection was made according to age and gender to match the obese group as much as possible. All examinations were performed according to the guidelines of the ethical committee of the Technical University of Munich and in accordance to the principles of the Declaration of Helsinki.\n\nThe demographic characteristics are summarized in Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>. As part of our routine treatment program all patients had to complete a food diary over 10 consecutive days prior to the start of therapy. They were instructed to record in as detailed a manner as possible every item that they either eat or drank, the time they consumed it, the amount they ate and how the food was prepared. To improve the motivation to accurately report their habitual food intake patients were told that an accurate recording of their usual intake improves individually adapted changes of their personal eating habits during subsequent therapy. They were told that the most preferred food items of their usual diet would remain unchanged as much and as long as possible to prevent underreporting especially of snacks with high energy density (ED).\n\nDemographic characteristics of study population (mean \u00b1 SEM)\n\n|                   | Obese      | Normal weight |\n|-------------------|------------|---------------|\n| n                 | 280        | 100           |\n| Sex (female\/male) | 205\/75     | 67\/33         |\n| Age (years)       | 45 \u00b1 0.85  | 42 \u00b1 0.2      |\n| Body weight (kg)  | 108 \u00b1 3.4  | 67 \u00b1 0.3      |\n| BMI (kg\/m^2^)     | 36.6 \u00b1 0.2 | 22.5 \u00b1 0.1    |\n\nThey were informed that record keeping can produce a change in nutrient intake and they should not try to alter their normal food intake since this could be disadvantageous for the subsequent recommendations during dietary counseling. They should try to record at the time of meal ingestion as much and as often as possible to minimize problems of correctly remembering details and especially the quantity of ingested food items. They should use a scale as often as possible. They were told that the quantity of recorded food items is extremely important for the ensuing therapy. All patients were handed out a booklet with examples of characteristic portion sizes in case of eating out. The same information was given to the normal weight subjects with the exception that they were asked to record intake 14 days.\n\n## Data analysis\n\nDietary protocols were calculated with the scientific program PRODI 4.5 Expert (Kluthe, Freiburg, Germany). Main meals were divided into breakfast, lunch and dinner. Patients were told to identify main meals as breakfast, lunch and dinner specifically in their records and to indicate the respective time periods. All food items ingested between breakfast and lunch were considered as morning snack (MS). The afternoon snack (AS) comprised the time period between lunch and dinner and the evening snack (ES) considered all food items following dinner. Energy containing beverages were recorded separately. A detailed analysis of the food intake data has recently been reported \\[6-10\\].\n\nTo assess the intraindividual relationship between breakfast and total daily energy intake each individual's intake was ranked in ascending order on the basis of each day's absolute breakfast energy intake (B kcal). Thus, the lowest value of breakfast energy intake represents the mean value of each individual's lowest intake irrespective of the chronological order of recording. Similarly, data were analyzed on the basis of each individual's ratio of breakfast to total daily energy intake (%B\/T kcal), respectively.\n\nFor statistical analysis t-test for paired data was employed, where appropriate correction for multiple testing was made according to Bonferroni. Pearson correlation analysis was performed between total daily energy intake and that during breakfast. Multiple regression analysis using stepwise forward and backward variable selection was used to examine the influence of the independent variable breakfast energy, and the respective ratio of this parameter to the corresponding overall intake data on the dependent variable daily energy intake. P-values of 0.05 or less were considered significant. All data were analyzed by using SPSS (version 17.0).\n\n# Results\n\nIn ***obese*** subjects the average solid food breakfast energy intake on each individual's lowest day was 121 \u00b1 7.1 kcal and it increased up to 606 \u00b1 19.0 kcal. In part this high degree of variability in energy intake is due to occasional breakfast skipping.\n\nThe corresponding data of energy intake during the other meals and overall daily intake is shown in Figure <a href=\"#F1\" data-ref-type=\"fig\">1a<\/a>. Lunch and dinner intakes were around 550 kcal on all days independent of breakfast energy intake. During the morning snack energy intake decreased significantly on the 2 days with highest breakfast intake while afternoon and evening snack intakes remained unchanged, respectively. Total energy intake rose by \\~400 kcal which largely corresponds to the change of breakfast intake.\n\nThe analysis with consideration of energy containing beverages led to a parallel shift of breakfast and overall energy intake (Figure <a href=\"#F2\" data-ref-type=\"fig\">2a<\/a>).\n\nThe analysis on the basis of breakfast energy as percentage of total daily energy intake is shown in Figure <a href=\"#F1\" data-ref-type=\"fig\">1b<\/a>. With the progressively increasing ratio breakfast calories rose from 134 \u00b1 7.9 to 559 \u00b1 18.2 kcal. Energy intake during all single meals and overall intake decreased steadily. Dinner, snacks and overall intake were significantly lower on the 3 days with the highest ratio, respectively while lunch intake was significantly lower during 5 days (Figure <a href=\"#F1\" data-ref-type=\"fig\">1b<\/a>). When beverages were included in the analysis the relationship between breakfast and overall intake remained similar (Figure <a href=\"#F2\" data-ref-type=\"fig\">2b<\/a>). The comparison of overall daily intake between the 2 modalities of analysis showed significantly higher energy intake on the 4 days with the lowest ratio compared to the corresponding 4 days with the lowest absolute breakfast intake. Additionally the 3 days with the highest ratio had a significantly lower intake (Figures <a href=\"#F1\" data-ref-type=\"fig\">1a<\/a> and <a href=\"#F1\" data-ref-type=\"fig\">1b<\/a>).\n\nIn the ***normal weight*** group the range of daily breakfast energy intake extended from 134 \u00b1 12.8 to 674 \u00b1 24.6 kcal (Figure <a href=\"#F3\" data-ref-type=\"fig\">3a<\/a>). Similar to the obese subjects occasional breakfast skipping is also relevant in the normal weight group. Lunch and dinner energy intake remained constant at 500 to 550 kcal and similarly no significant changes were observed for the three snacks. Total energy intake increased significantly by 500 kcal\/d. Similar to the obese group inclusion of beverages leads to a parallel shift of energy intake (Figure <a href=\"#F4\" data-ref-type=\"fig\">4a<\/a>).\n\nWhen the analysis is based on the ratio of breakfast to overall intake breakfast energy was in the range of 152 to 611 kcal (Figure <a href=\"#F3\" data-ref-type=\"fig\">3b<\/a>). A significant decrease was observed at lunch, dinner, morning snack and overall daily intake, respectively. During the 5 days with the lowest ratio whole day energy intake was significantly higher compared to the corresponding days of whole intake based on absolute breakfast calories. On the other hand, the 5 days with the highest ratio were significantly lower (Figures <a href=\"#F3\" data-ref-type=\"fig\">3a<\/a> and <a href=\"#F3\" data-ref-type=\"fig\">3b<\/a>). The addition of beverages to the analysis led to a parallel shift of energy intake without substantially affecting the relationship between breakfast and overall intake (Figuer <a href=\"#F4\" data-ref-type=\"fig\">4b<\/a>).\n\nThe major food groups responsible for the significant increase of breakfast energy intake between the lowest and highest day (obese\/normal weight) were: bread (145\/126 kcal), eggs 21\/33 kcal), cake (51\/132 kcal), yogurt (9\/11 kcal), cheese (57\/84 kcal), sausages (70\/- kcal), marmalade (22\/34 kcal), butter (37\/42 kcal). Other food groups had per se no significant changes but the additive effect was responsible for the remaining increase of energy intake.\n\n## - correlations and regression analysis\n\nBreakfast energy intake correlated positively and significantly with daily energy intake in both normal weight and obese subjects (Table <a href=\"#T2\" data-ref-type=\"table\">2<\/a>). In the obese group the ratio of breakfast to overall energy intake was also positively correlated with daily energy intake. The correlation coefficients, however, were much smaller than those for the absolute intake data. In the normal weight group the ratio of energy intake was inversely and significantly correlated with daily energy intake.\n\nPearson correlation coefficients for the relationship between the various parameters of breakfast intake with daily energy intake in normal weight and obese subjects.\n\n|                         | Obese |          | Normal weight |          |\n|-------------------------|-------|----------|---------------|----------|\n| Parameters of breakfast | r     | p        | r             | p        |\n| kcal                    | 0.477 | \\<0.0001 | 0.453         | \\<0.0001 |\n| % B\/T kcal              | 0.122 | \\<0.0001 | -0.081        | 0.002    |\n\nThe multiple regression analysis (Table <a href=\"#T3\" data-ref-type=\"table\">3<\/a>) demonstrated in obese subjects a significant positive influence of breakfast energy while the ratio was inversely related to daily energy intake. In the normal weight group breakfast energy and the ratio of breakfast to overall energy intake were significantly related to daily energy intake, respectively. In both groups the strongest influence had absolute breakfast energy intake.\n\nVariable selection during multiple linear regression analysis with the independent variables breakfast energy (kcal), % B\/T kcal and total daily energy intake as the dependent variable in *obese and normal weight subjects*.\n\n|                | Obese              |          | Normal weight      |          |\n|----------------|--------------------|----------|--------------------|----------|\n|                | Std. coefficient \u03b2 | p        | Std. coefficient \u03b2 | p        |\n| kcal breakfast | 0.861              | \\<0.0001 | 1.386              | \\<0.0001 |\n| % B\/T kcal     | -0.354             | \\<0.0001 | -1.181             | \\<0.0001 |\n\n# Discussion\n\nThe impact of breakfast on total daily energy intake is a matter of debate. In an experimental setting Cotton et al. \\[1,2\\] have shown in normal weight subjects that an energy-rich breakfast leads to greater daily energy intake indicating that compensatory undereating during subsequent meals does not occur. This notion is supported by an analysis of food intake in young adults in Bogalusa \\[3\\]. It must be kept in mind though that these findings are based on a cross-sectional analysis and accordingly these data represent interindividual differences.\n\nUp to the author's knowledge the present data demonstrate for the first time this relationship in a free-living population of normal weight as well as obese subjects thereby confirming the earlier experimental data of Cotton and colleagues. When analysed intraindividually whole day energy intake increases proportionally to breakfast calories while other meals of the day remain largely unchanged. The only exception is a small reduction of the morning snack on the two days with the highest breakfast energy consumption in the obese group.\n\nIn a different approach de Castro \\[4,5\\] has demonstrated that a high ratio of breakfast to whole day energy intake is associated with lower overall intakes. The present data also confirm these previous findings. An increased ratio of breakfast to whole day energy intake can be due to an augmented consumption during breakfast or to a reduced intake during all subsequent meals or a mixture of both. The direct comparison of both ways of analysis show that the range of breakfast energy intake is largely similar.\n\nThus, the significant reduction of daily energy intake on days with a high B\/T ratio is mainly the result of below average intakes during the other meals. Moreover, it should be noted that a low B\/T ratio is associated with significantly greater overall intake compared to the days with a low absolute intake.\n\nFor the purpose of reducing daily energy intake in obesity prevention or treatment, a reduction of breakfast calories can be helpful. High breakfast energy intake is not automatically associated with low whole day intake. On these days substantially reduced energy consumption during post-breakfast meals would be necessary.\n\nWith regard to obesity treatment the role of breakfast intake is controversial. Several cross-sectional studies have indicated that breakfast intake is inversely associated with body weight \\[12-16\\]. In contrast a large longitudinal study came to the conclusion that a high percentage of breakfast energy is associated with lower weight gain \\[17\\]. It must be kept in mind though, that at the beginning of this study a high percentage of breakfast energy was associated not only with the highest overall intake but also with the highest body weight which is in contrast to the results of other cross-sectional studies \\[12-16\\]. Unfortunately food intake was assessed only at baseline but not at follow-up. Thus, lower weight gain could be due to a change of eating habits leading to reduced breakfast and overall energy intake over the three years of follow-up.\n\nMoreover, data of the National Weight Control Registry (NWCR) \\[18\\] have shown that the majority (78%) regularly eats breakfast which has been suggested to be an important factor for successful maintenance of weight loss. It is important to note, however, that this is not necessarily a causal relationship since the other 22% are also successful weight loss maintainers, despite skipping breakfast regularly or occasionally, respectively. In the present analysis 5 out of 280 obese subjects (1.8%) never had breakfast. 180 patients never skipped breakfast and the other 95 occasionally skipped breakfast. All groups had a similar BMI and total daily energy intake was not different. In the normal weight group total daily energy intake of those with regular breakfast consumption (n = 68) was significantly higher (1826 \u00b1 18.4 kcal\/d) compared to the 32 subjects who skipped breakfast occasionally (1691 \u00b1 31.5 kcal\/d; p \\< 0.001) while body weight was not different between these 2 groups, which indicates that energy expenditure has to be considered as well. In this context it is noteworthy that the regular breakfast eaters in the NWCR reported more physical activity.\n\nOn the other hand, the only interventional study of the role of breakfast in the treatment of obesity came to mixed results \\[19\\]. Baseline breakfast eaters lost more weight when assigned to the no-breakfast treatment group. On the other hand, baseline breakfast skippers lost more weight when assigned to the breakfast-eating treatment group. It should be kept in mind though, that the study population was very small and these very interesting data clearly deserve confirmation in a larger population.\n\nIn conclusion the present data demonstrate that higher energy intake at breakfast is highly associated with greater whole day energy intake in normal weight and obese subjects. Therefore low energy intake at breakfast can be helpful to lower daily intake and improve the energy balance during treatment of obesity Whether or not this approach really favours weight loss has to be examined in further interventional studies. At present prevailing data are rather equivocal.\n\n# Competing interests\n\nThe authors declare that they have no competing interests.\n\n# Authors' contributions\n\nVS and JE were involved in the study design, data acquisition, data analysis, statistics and manuscript preparation. MH participated in study design; data acquisition and data analysis. CW, JM, MK carried out data acquisition and data analysis. AN and SW performed data analysis and statistics. All authors have read and approved the final manuscript.","meta":{"dup_signals":{"dup_doc_count":127,"dup_dump_count":57,"dup_details":{"curated_sources":3,"2023-50":2,"2023-23":1,"2022-40":1,"2022-33":1,"2022-27":2,"2022-05":2,"2021-39":1,"2021-25":1,"2021-10":1,"2021-04":1,"2020-45":1,"2020-34":1,"2020-24":1,"2020-10":1,"2020-05":1,"2019-51":1,"2019-43":1,"2019-35":1,"2019-30":1,"2019-22":1,"2019-18":1,"2019-13":1,"2019-09":1,"2018-51":1,"2018-43":1,"2018-39":1,"2018-30":1,"2018-26":2,"2018-22":1,"2018-17":1,"2018-13":1,"2018-09":2,"2018-05":2,"2017-51":2,"2017-47":3,"2017-43":2,"2017-39":4,"2017-34":3,"2017-30":3,"2017-26":3,"2017-22":2,"2017-17":3,"2017-09":23,"2017-04":2,"2016-50":2,"2016-44":6,"2016-40":5,"2016-36":6,"2016-30":6,"2016-22":1,"2016-18":1,"2016-07":1,"2015-27":1,"2024-18":2,"2017-13":2,"2015-18":1,"2024-22":1}},"file":"PMC3034667"},"subset":"pubmed_central"}
{"text":"date: 2017-06\ntitle: NEW HEPATITIS DATA HIGHLIGHT NEED FOR URGENT GLOBAL RESPONSE\n\n**21 APRIL 2017 \\| GENEVA, AMSTERDAM** - New WHO data reveal that an estimated 325 million people worldwide are living with chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infection. The WHO Global hepatitis report, 2017 indicates that the large majority of these people lack access to life-saving testing and treatment. As a result, millions of people are at risk of a slow progression to chronic liver disease, cancer, and death.\n\n\"Viral hepatitis is now recognized as a major public health challenge that requires an urgent response,\" said Dr Margaret Chan, WHO Director-General. \"Vaccines and medicines to tackle hepatitis exist, and WHO is committed to helping ensure these tools reach all those who need them.\"\n\n# Increasing mortality, new infections\n\nViral hepatitis caused 1.34 million deaths in 2015, a number comparable to deaths caused by tuberculosis and HIV. But while mortality from tuberculosis and HIV has been declining, deaths from hepatitis are on the increase.\n\nApproximately 1.75 million people were newly infected with HCV in 2015, bringing the global total of people living with hepatitis C to 71 million.\n\nAlthough overall deaths from hepatitis are increasing, new infections of HBV are falling, thanks to increased coverage of HBV vaccination among children. Globally, 84% of children born in 2015 received the 3 recommended doses of hepatitis B vaccine. Between the pre-vaccine era (which, according to the year of introduction can range from the 1980s to the early 2000s) and 2015, the proportion of children under 5 years of age with new infections\n\nfell from 4.7% to 1.3%. However, an estimated 257 million people, mostly adults born before the introduction of the HBV vaccine, were living with chronic hepatitis B infection in 2015.\n\n# Epidemics in regions and \"hotspots\"\n\nHepatitis B levels vary widely across WHO regions with the WHO African Region and WHO Western Pacific Region sharing the greatest burden.\n\nWHO Western Pacific Region: 6.2% of population (115 million people)\n\nWHO African Region: 6.1% of population (60 million people)\n\nWHO Eastern Mediterranean Region: 3.3% of population (21 million people)\n\nWHO South-East Asia Region: 2% of population (39 million people)\n\nWHO European Region: 1.6% of population (15 million people)\n\nWHO Region of the Americas: 0.7% of population (7 million people)\n\nToday, unsafe injections in health care settings and injecting drug use are considered to be the most common routes of HCV transmissions. HCV prevalence by WHO region is:\n\nWHO Eastern Mediterranean Region: 2.3% of population (15 million people)\n\nWHO European Region: 1.5% of population (14 million people)\n\nWHO African Region: 1% of population (11 million people)\n\nWHO Region of the Americas: 1% of population (7 million people)\n\nWHO Western Pacific Region: 1% of population (14 million people)\n\nWHO South-East Asia Region: 0.5% of population (10 million people)\n\n# Treatment access is low\n\nThere is currently no vaccine against HCV, and access to treatment for HBV and HCV is still low.\n\nWHO's Global Health Sector Strategy on viral hepatitis aims to test 90% and treat 80% of people with HBV and HCV by 2030.\n\nThe report notes that just 9% of all HBV infections and 20% of all HCV infections were diagnosed in 2015. An even smaller fraction \u2013 8% of those diagnosed with HBV infection (1.7 million people) were on treatment, and only 7% of those diagnosed with HCV infection (1.1 million people) had started curative treatment during that year.\n\nHBV infection requires lifelong treatment, and WHO currently recommends the medicine tenofovir, already widely used in HIV treatment. Hepatitis C can be cured within a relatively short time using the highly effective direct-acting antivirals (DAAs).\n\n\"We are still at an early stage of the viral hepatitis response, but the way forward looks promising,\" said Dr Gottfried Hirnschall, Director of WHO's Department of HIV and the Global Hepatitis Programme. \"More countries are making hepatitis services available for people in need \u2013 a diagnostic test costs less than US\\$ 1 and the cure for hepatitis C can be below US\\$ 200. But the data clearly highlight the urgency with which we must address the remaining gaps in testing and treatment.\"\n\n# Country progress\n\nWHO's Global hepatitis report, 2017 demonstrates that despite challenges, some countries are taking successful steps to scale-up hepatitis services.\n\nChina achieved high coverage (96%) for the timely birth dose of HBV vaccines, and reached the hepatitis B control goal of less than 1% prevalence in children under the age of 5 in 2015. Mongolia improved uptake of hepatitis treatment by including HBV and HCV medicines in its National Health Insurance scheme, which covers 98% of its population. In Egypt, generic competition has reduced the price of a 3-month cure for hepatitis C, from US\\$ 900 in 2015, to less than US\\$ 200 in 2016. Today in Pakistan, the same course costs as little as US\\$ 100.\n\nImproving access to hepatitis C cure received a boost at the end of March 2017, when WHO prequalified the generic active pharmaceutical ingredient of sofosbuvir. This step will enable more countries to produce affordable hepatitis medicines.\n\n# Baseline for elimination\n\nWHO's Global hepatitis report, 2017 aims to provide a starting point for hepatitis elimination by indicating baseline statistics on HBV and HCV infections, including mortality, and coverage levels of key interventions. Hepatitis B and C \u2013 the 2 main types out of 5 different hepatitis infections \u2013 are responsible for 96% of overall hepatitis mortality.\n\nBrazil and the World Hepatitis Alliance. It will be held on 1\u20133 November 2017 in S\u00e3o Paulo, Brazil.\n\nAvailable from: <http:\/\/www.who.int\/mediacentre\/news\/releases\/2017\/global-hepatitis-report\/en\/>","meta":{"dup_signals":{"dup_doc_count":163,"dup_dump_count":57,"dup_details":{"curated_sources":2,"2023-50":3,"2023-40":1,"2023-14":1,"2023-06":1,"2022-49":2,"2022-40":1,"2022-27":1,"2022-21":2,"2022-05":2,"2021-49":1,"2021-43":3,"2021-39":2,"2021-31":1,"2021-25":1,"2021-21":2,"2021-10":2,"2021-04":2,"2020-50":1,"2020-40":2,"2020-34":2,"2020-29":2,"2020-24":1,"2020-16":5,"2020-05":2,"2019-51":1,"2019-47":1,"2019-35":2,"2019-30":2,"2019-22":1,"2019-18":2,"2019-13":2,"2019-09":3,"2019-04":4,"2018-51":5,"2018-47":2,"2018-43":3,"2018-39":7,"2018-34":3,"2018-30":3,"2018-26":4,"2018-22":1,"2018-17":4,"2018-13":5,"2018-09":3,"2018-05":7,"2017-51":2,"2017-47":4,"2017-43":3,"2017-39":3,"2017-34":3,"2017-30":8,"2017-26":5,"2017-22":8,"2017-17":12,"2024-26":1,"2024-18":3,"2024-30":1}},"file":"PMC5541197"},"subset":"pubmed_central"}
{"text":"author: Thomas C Erren; Philip E Bourne\\* To whom correspondence should be addressed. E-mail: <firstname.lastname@example.com>\ndate: 2007-05\nreferences:\ntitle: Ten Simple Rules for a Good Poster Presentation\n\nPosters are a key component of communicating your science and an important element in a successful scientific career. Posters, while delivering the same high-quality science, offer a different medium from either oral presentations \\[1\\] or published papers \\[2\\], and should be treated accordingly. Posters should be considered a snapshot of your work intended to engage colleagues in a dialog about the work, or, if you are not present, to be a summary that will encourage the reader to want to learn more. Many a lifelong collaboration \\[3\\] has begun in front of a poster board. Here are ten simple rules for maximizing the return on the time-consuming process of preparing and presenting an effective poster.\n\n# Rule 1: Define the Purpose\n\nThe purpose will vary depending on the status and nature of the work being presented, as well as the intent. Some posters are designed to be used again and again; for example, those making conference attendees aware of a shared resource. Others will likely be used once at a conference and then be relegated to the wall in the laboratory. Before you start preparing the poster, ask yourself the following questions: What do you want the person passing by your poster to do? Engage in a discussion about the content? Learn enough to go off and want to try something for themselves? Want to collaborate? All the above, or none of the above but something else? Style your poster accordingly.\n\n# Rule 2: Sell Your Work in Ten Seconds\n\nSome conferences will present hundreds of posters; you will need to fight for attention. The first impressions of your poster, and to a lesser extent what you might say when standing in front of it, are crucial. It is analogous to being in an elevator and having a few seconds to peak someone's interest before they get off. The sad truth is that you have to sell your work. One approach is to pose your work as addressing a decisive question, which you then address as best you can. Once you have posed the question, which may well also be the motivation for the study, the focus of your poster should be on addressing that question in a clear and concise way.\n\n# Rule 3: The Title Is Important\n\nThe title is a good way to sell your work. It may be the only thing the conference attendee sees before they reach your poster. The title should make them want to come and visit. The title might pose a decisive question, define the scope of the study, or hint at a new finding. Above all, the title should be short and comprehensible to a broad audience. The title is your equivalent of a newspaper headline\u2014short, sharp, and compelling.\n\n# Rule 4: Poster Acceptance Means Nothing\n\nDo not take the acceptance of a poster as an endorsement of your work. Conferences need attendees to be financially viable. Many attendees who are there on grants cannot justify attending a conference unless they present. There are a small number of speaking slots compared with attendees. How to solve the dilemma? Enter posters; this way everyone can present. In other words, your poster has not been endorsed, just accepted. To get endorsement from your peers, do good science and present it well on the poster.\n\n# Rule 5: Many of the Rules for Writing a Good Paper Apply to Posters, Too\n\nIdentify your audience and provide the appropriate scope and depth of content. If the conference includes nonspecialists, cater to them. Just as the abstract of a paper needs to be a succinct summary of the motivation, hypothesis to be tested, major results, and conclusions, so does your poster.\n\n# Rule 6: Good Posters Have Unique Features Not Pertinent to Papers\n\nThe amount of material presented in a paper far outweighs what is presented on a poster. A poster requires you to distill the work, yet not lose the message or the logical flow. Posters need to be viewed from a distance, but can take advantage of your presence. Posters can be used as a distribution medium for copies of associated papers, supplementary information, and other handouts. Posters allow you to be more speculative. Often only the titles or at most the abstracts of posters can be considered published; that is, widely distributed. Mostly, they may never be seen again. There is the opportunity to say more than you would in the traditional literature, which for all intents and purposes will be part of the immutable record. Take advantage of these unique features.\n\n# Rule 7: Layout and Format Are Critical\n\nPop musician Keith Richards put the matter well in an interview with *Der Spiegel* \\[4\\]: \"If you are a painter, then the most important thing is the bare canvas. A good painter will never cover all the space but will always leave some blank. My canvas is silence.\" Your canvas as poster presenter is also white space. Guide the passerby's eyes from one succinct frame to another in a logical fashion from beginning to end. Unlike the literature, which is linear by virtue of one page following another, the reader of a poster is free to wander over the pages as if they are tacked to the poster board in a random order. Guide the reader with arrows, numbering, or whatever else makes sense in getting them to move from one logical step to another. Try to do this guiding in an unusual and eye-catching way. Look for appropriate layouts in the posters of others and adopt some of their approaches. Finally, never use less than a size 24 point font, and make sure the main points can be read at eye level.\n\n# Rule 8: Content Is Important, but Keep It Concise\n\nEverything on the poster should help convey the message. The text must conform to the norms of sound scientific reporting: clarity, precision of expression, and economy of words. The latter is particularly important for posters because of their inherent space limitations. Use of first-rate pictorial material to illustrate a poster can sometimes transform what would otherwise be a bewildering mass of complex data into a coherent and convincing story. One carefully produced chart or graph often says more than hundreds of words. Use graphics for \"clear portrayal of complexity\" \\[5\\], not to impress (and possibly bewilder) viewers with complex artistry. Allow a figure to be viewed in both a superficial and a detailed way. For example, a large table might have bold swaths of color indicating relative contributions from different categories, and the smaller text in the table would provide gritty details for those who want them. Likewise, a graph could provide a bold trend line (with its interpretation clearly and concisely stated), and also have many detailed points with error bars. Have a clear and obvious set of conclusions\u2014after the abstract, this is where the passerby's eyes will wander. Only then will they go to the results, followed by the methods.\n\n# Rule 9: Posters Should Have Your Personality\n\nA poster is a different medium from a paper, which is conventionally dry and impersonal. Think of your poster as an extension of your personality. Use it to draw the passerby to take a closer look or to want to talk to you. Scientific collaboration often starts for reasons other than the shared scientific interest, such as a personal interest. A photo of you on the poster not only helps someone find you at the conference when you are not at the poster, it can also be used to illustrate a hobby or an interest that can open a conversation.\n\n# Rule 10: The Impact of a Poster Happens Both During and After the Poster Session\n\nWhen the considerable effort of making a poster is done, do not blow it on presentation day by failing to have the poster achieve maximum impact. This requires the right presenter\u2013audience interaction. Work to get a crowd by being engaging; one engaged viewer will attract others. Don't badger people, let them read. Be ready with Rule 2. Work all the audience at once, do not leave visitors waiting for your attention. Make eye contact with every visitor.\n\nMake it easy for a conference attendee to contact you afterward. Have copies of relevant papers on hand as well as copies of the poster on standard-sized paper. For work that is more mature, have the poster online and make the URL available as a handout. Have your e-mail and other demographics clearly displayed. Follow up with people who come to the poster by having a signup sheet.\n\nThe visitor is more likely to remember you than the content of your poster. Make yourself easy to remember. As the host of the work presented on the poster, be attentive, open, and curious, and self-confident but never arrogant and aggressive. Leave the visitors space and time\u2014they can \"travel\" through your poster at their own discretion and pace. If a visitor asks a question, talk simply and openly about the work. This is likely your opportunity to get feedback on the work before it goes to publication. Better to be tripped up in front of your poster than by a reviewer of the manuscript.\n\nGood posters and their presentations can improve your reputation, both within and outside your working group and institution, and may also contribute to a certain scientific freedom. Poster prizes count when peers look at your resume.\n\nThese ten rules will hopefully help you in preparing better posters. For a more humorous view on what not to do in preparing a poster, see \\[6\\], and for further information, including the opportunity to practice your German, see \\[7\\].\u2002\n\nThomas Erren's contributions to this piece are based on \\[7\\] and were stimulated by exchanges with Michael Jacobsen. Thanks also to Steven E. Brenner for useful input.\n\n# References","meta":{"dup_signals":{"dup_doc_count":245,"dup_dump_count":81,"dup_details":{"curated_sources":2,"2023-40":1,"2023-14":1,"2022-49":2,"2022-33":1,"2022-27":1,"2022-21":1,"2022-05":1,"2021-43":1,"2021-39":1,"2021-31":2,"2021-25":2,"2021-21":2,"2020-50":1,"2020-40":4,"2020-34":1,"2020-29":2,"2020-24":1,"2020-16":2,"2020-05":2,"2019-51":1,"2019-47":1,"2019-43":1,"2019-35":1,"2019-26":1,"2019-18":1,"2019-09":2,"2018-51":1,"2018-47":1,"2018-43":1,"2018-39":2,"2018-34":1,"2018-26":2,"2018-17":2,"2018-13":1,"2018-09":1,"2017-51":1,"2017-47":2,"2017-43":1,"2017-39":1,"2017-34":2,"2017-26":3,"2017-22":2,"2017-17":3,"2017-09":7,"2017-04":3,"2016-50":4,"2016-44":2,"2016-40":3,"2016-36":4,"2016-30":4,"2016-26":3,"2016-22":3,"2016-18":2,"2016-07":3,"2015-48":4,"2015-40":7,"2015-35":6,"2015-32":5,"2015-27":5,"2015-22":5,"2015-14":5,"2014-52":4,"2014-49":8,"2014-42":16,"2014-41":9,"2014-35":10,"2014-23":9,"2014-15":13,"2023-50":1,"2024-26":1,"2024-22":1,"2024-18":2,"2024-10":2,"2017-13":3,"2015-18":6,"2015-11":6,"2015-06":2,"2014-10":5,"2013-48":3,"2013-20":2,"2024-30":1}},"file":"PMC1876493"},"subset":"pubmed_central"}
{"text":"abstract: The presence of mammary glands is the defining morphological feature of mammals. The recent assembly of the bovine genome and a report in *Genome Biology* that links the milk and lactation data of bovine and other mammalian genomes will help biologists investigate this economically and medically important feature.\nauthor: Anthony V Capuco; R Michael Akers\ndate: 2009\ninstitute: 1USDA-ARS, Bovine Functional Genomics Laboratory, Powder Mill, BARC-East, Beltsville, MD 20705, USA; 2Virginia Polytechnic Institute and State University, Department of Dairy Science, 2470 Litton-Reaves Hall, Blacksburg, VA 24061, USA\nreferences:\ntitle: The origin and evolution of lactation\n\n# Minireview\n\nThe mammary gland has been a pivotal feature in the evolution and taxonomic classification of animal species, and it has even had a role in the acceptance of evolutionary theory. The presence and secretory capacity of the mammary gland provided the basis for the taxonomic grouping of species into the class Mammalia more than two centuries ago; and Darwin's explanation of how lactation may have evolved satisfied an early challenge to his theory of evolution by natural selection \\[1\\]. The challenge was that evolution of lactation was not feasible, because a neonate could not obtain a survival benefit from consuming the chance secretion of a rudimentary cutaneous gland. In response, Darwin hypothesized that mammary glands evolved from cutaneous glands that were contained within the brood pouches in which some fish and other marine species keep their eggs, and provided nourishment and thus a survival advantage to eggs of ancestral species. Two hundred years after Darwin's birth, the theory of evolution by natural selection remains a cornerstone of biology, as it has withstood this and other challenges. However, it is now clear that the mammary gland did not evolve from a brood pouch \\[1\\].\n\nMilk nourishes the neonate and helps to establish immunological and endocrine competence in the offspring. The nutrient composition of milk varies dramatically across species, and it can also be strongly influenced by the stage of lactation. For example, the fat content of milk may be as high as 60% in seals and negligible during early lactation in wallabies \\[2,3\\]. Furthermore, milk in the tammar wallaby (*Macropus eugenii*) changes from a very dilute secretion containing primarily carbohydrate during early lactation to a more energy-dense milk that contains substantial quantities of protein and fat during later phases of lactation. Thus, the details of lactation have evolved to meet the diverse reproductive and environmental demands of different species. About 10,000 years ago, the domestication of plant and animal species led to the Neolithic Revolution, with its changes in societal interactions and the evolution of civilization. Milk and dairy products were tightly coupled to this cultural evolution, and dairying (then and now) provides an important source of food and fiber throughout the world.\n\nSequencing and assembly of the bovine genome, establishment of mammary transcriptome and proteome libraries, the discovery of single nucleotide polymorphisms \\[4\\], and discoveries and developments to come, are providing important tools for agricultural scientists to investigate the biology of lactation and to adopt genotype-based breeding schemes to select for desired traits. Moreover, comparative genomic studies enable evaluation of lactation across numerous and diverse mammalian species. Regardless of the perceived target species of this research, such knowledge improves our understanding of mammary gland biology and is applicable to normal and pathological states. Danielle Lemay and colleagues, in a recent report in *Genome Biology* \\[5\\], have taken this important step towards greater understanding through comparative genomics.\n\n# Evolution of lactation\n\nLactation appears to be an ancient reproductive feature that pre-dates the origin of mammals. A cogent theory for the evolution of the mammary gland and lactation has been provided by Olav Oftedal \\[1\\]. The features of current mammals were gradually accrued through radiations of synapsid ancestors, and the mammary gland is hypothesized to have evolved from apocrine-like glands associated with hair follicles (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>). Oftedal suggests that these glands evolved from providing primarily moisture and antimicrobials to parchment-shelled eggs to the role of supplying nutrients for offspring. Fossil evidence indicates that some of the therapsids and the mammalia-formes, which were present during the Triassic period more than 200 million years ago, produced a nutrient-rich milk-like secretion.\n\nThe capacity to supply fluid and perhaps nutrients to eggs would be promoted and enhanced by incorporation of antimicrobials into the fluid. These may have been antimicrobials already produced in the skin, as in amphibian skin, and evolutionary pressure would probably have fostered the incorporation of molecules such as lysozyme and iron-binding proteins into the secretion, components that are prevalent in milk. The disaccharide lactose (galactose \u03b21\u20134 glucose) is contained in all milks, except for those of some marine mammals. Its synthesis is catalyzed in the mammary gland by lactose synthetase, an enzyme that is a complex of \u03b21\u20134-galactosyl transferase and the regulatory subunit \u03b1-lactalbumin. Because \u03b1-lactalbumin evolved from lysozyme before the division of amniotes into synapsids and sauropsids (see Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>), the capacity to produce lactose was an ancient trait that preceded its utility in milk synthesis. It is likely that early milks primarily contained antimicrobial oligosaccharides and the prevalence of lactose as a component of milk arose only when \u03b1-lactalbumin was produced in sufficient quantity.\n\nWith the synthesis of lactose, these modified secretions would have provided nutrients to the egg. The evolution of the casein family of milk proteins in particular would provide calcium, phosphate and protein to hatchlings. Fossil records suggest that caseins were present during the Triassic, because the extensive bone and tooth development evident in the relevant species at stages before independent feeding would have required delivery of ample calcium. Given this evolutionary scenario, the composition of mammary secretions during early lactation in monotremes and marsupials is likely to be similar to that of the primitive milk of mammalian predecessors. The milk then converts to a more nutrient-rich source during later stages of lactation. The evolution of placenta-based reproduction displaced the function of milk as a source of water and nutrients for the egg, leading to secretion of a complex milk throughout lactation in eutherians (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>).\n\nMilk also enhances the survival of offspring by satisfying other needs, for example, by promoting immunological competence and endocrine maturation in the neonate \\[6,7\\]. In this regard, milk seems to provide for the immediate and long-term needs of the offspring. These needs can be highly species-specific. There are also behavioral and 'psychological' aspects of suckling and nurturing between mother (dam) and offspring that produce bonds that promote neonate survival. This is an aspect of lactation that is independent of the chemical and physical characteristics of milk.\n\n# Systemic and local control of mammary function\n\nMammary gland development and function is subject to systemic and local control. In placental mammals, our understanding of this regulation has been advanced by decades of scientific inquiry, using physiological, molecular and genomic tools. In these mammals, development of the mammary gland during gestation generates abundant alveolar secretory cells. Differentiation of the secretory cells and the onset of copious milk synthesis and secretion are regulated to coincide with parturition. The combined effects of positive endocrine stimulators (prolactin, insulin, glucocorticoids, growth hormone and estradiol) are kept in check by the overriding negative influence of progesterone \\[8\\]. The decline in progesterone at parturition largely determines the onset of copious milk secretion, but regulation in marsupials differs from that in eutherians. The reproductive cycle in marsupials is characterized by a short gestation and a long lactation, during which the female will nurse offspring of different ages. Lactation in the tammar wallaby has been studied and, consistent with the marsupial reproductive strategy, is found to be insensitive to inhibition by progesterone \\[2,9\\]. The tammar wallaby offspring (joey) is born in an immature state at 26 days gestation. At birth it remains attached to the nipple for a period, during which it obtains a dilute, carbohydrate-rich milk. However, the composition of the milk changes significantly during lactaton to meet the demands of the developing joey. Moreover, the tammar has asynchronous concurrent lactation, during which the dam provides milk of differing composition from adjacent glands to feed two offspring of different ages and nutritional needs. This provides an example of local regulation of lactation.\n\nAnother clear example of local regulation is provided by lactation in the Cape fur seal (*Arctocephalus pusillus pusillus*), which is characterized by short suckling periods (2\u20133 days) on shore and lengthy foraging periods (about 20 days) at sea, during which maternal nutrient stores are replenished. In most eutherian species, milk secretion decreases in the absence of suckling, and this is accompanied by an increase in apoptosis and mammary involution, seemingly promoted by feedback inhibition from components of the unused milk. Lactation in the Cape seal has uncoupled the apoptotic response from decreased milk synthesis, so that the mammary gland simply shuts down during the long foraging periods and resumes secretion when suckling is resumed. The local factor recently implicated in this process is the milk protein \u03b1-lactalbumin. The \u03b1-lactalbumin in this group of seals (the otariid pinnipeds) apparently cannot promote apoptosis (or lactose synthesis) \\[10\\].\n\n# Features of milk and mammary genes (the 'lactome')\n\nLemay *et al*. \\[5\\] used the *Bos taurus* genome sequence (draft 3.1, August 2006) and expression libraries derived from tissue obtained during various stages of mammary development and lactation status to identify unique milk proteins and mammary-related proteins. With the exception of four milk-protein gene clusters (casein genes, immunoglobulin genes, fibrinogen genes and genes encoding proteins of the milk fat globule), they found that milk-protein genes do not cluster with each other, but rather tend to cluster with other lactation genes. They also did not cluster by developmental stage or gene duplication, suggesting that these genes clustered to facilitate coordinate gene expression.\n\nThe bovine genome was compared with six other mammalian genomes: human, dog, mouse and rat (eutherians), opossum (marsupial) and platypus (monotreme). In general, milk and mammary genes were more conserved and seemed to evolve more slowly than others in the bovine genome, despite selective breeding for milk production. This supports the hypothesis that lactation has evolved to minimize the energy cost to the dam while maximizing survival of the neonate, thus promoting survival of the maternal-offspring pair. The most divergent proteins in the lactome were those with nutritional or immunological attributes, suggesting continuing selection of these genes to meet nutritional and pathogen challenges that are incurred by diverse environments and reproductive strategies. The most conserved genes were those for proteins of the milk fat globule membrane, confirming the essentiality of this mechanism for milk-fat secretion and indicating that the diversity in milk fat may be due to altered efficiency in secretion, not to inherent changes in the secretory process. Diversity in milk composition could not be explained by diversity of the encoded milk proteins; and although gene duplication may contribute to species variation, this is not a major determinant. Thus, other regulatory mechanisms must be involved. For example, on the basis of analysis of the opossum genome, Mikkelsen *et al*. \\[11\\] concluded that most of the genomic diversity between marsupials and placental mammals comes from non-coding sequences. These, or other factors that regulate the partitioning of nutrients, the interaction between mammary gland and supporting organs, or mammary gland metabolism, may be primary determinants of milk composition.\n\nExpansion of comparative studies to include additional non-placental species and inclusion of non-coding regions of the genome is certain to provide additional insight into the regulation of mammary gland function and milk composition. For example, a systematic study of the role of microRNAs in mammary development and lactation is likely to be a fruitful area of investigation. Because no single species can provide an ample and sufficient model for the physiology of another, and because the potential gain in knowledge from comparative studies is great, the research community should not be species-centric. Continued research in mammary gland biology that incorporates comparative genomic and physiological studies of animals with varied and extreme adaptations to lactation will be necessary to provide insights into the development and regulation of mammary gland function, as well as the probable evolution of these processes.\n\nBovine genome coverage in BioMed Central:\n\n**[The cattle genome reveals its secrets](http:\/\/jbiol.com\/content\/8\/4\/36):** *Burt DW*\n\n**[The origin and evolution of lactation](http:\/\/jbiol.com\/content\/8\/4\/37):** *Capuco AV, Akers RM*\n\n**[Back to Bermuda: how is science best served?](http:\/\/genomebiology.com\/2009\/10\/4\/105):** *Church DM, Hillier LW*","meta":{"dup_signals":{"dup_doc_count":153,"dup_dump_count":32,"dup_details":{"curated_sources":2,"2022-49":1,"2022-33":1,"2020-40":1,"2020-10":1,"2019-51":1,"2019-47":1,"2019-39":1,"2019-35":1,"2017-04":1,"2015-48":6,"2015-40":6,"2015-35":7,"2015-32":6,"2015-27":6,"2015-22":7,"2015-14":5,"2014-52":6,"2014-49":5,"2014-42":18,"2014-41":7,"2014-35":8,"2014-23":9,"2014-15":7,"2023-14":1,"2017-13":1,"2015-18":5,"2015-11":6,"2015-06":7,"2014-10":9,"2013-48":5,"2013-20":4,"2024-10":1}},"file":"PMC2688910"},"subset":"pubmed_central"}
{"text":"abstract: Given that the effectiveness of genome-wide association studies for finding disease genes is being debated, an alternative would be to look for diseases linked to inherited metabolic disorders.\nauthor: Gregory A Petsko\ndate: 2009\ninstitute: 1Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02454-9110, USA\ntitle: Guilt by association\n\nIt's a favorite device of politicians who wish to smear a rival candidate. The late, unlamented Democratic Senator Joseph McCarthy employed it, successfully, against leftists whose careers he wished to destroy. The Republicans tried, unsuccessfully, to use it against Barack Obama. In its simplest form, it involves branding someone a Communist, or a terrorist, or a criminal, because they have family or friends, or possibly just casual acquaintances, who are Communists, or terrorists, or criminals. It's called guilt by association.\n\nIt's been in the news lately because it's also a favorite tactic of genome biologists, but in this case its purposes are not sinister. Scientifically, it goes by the name of genome-wide association studies, though guilt by association is just as apt. It's an attempt to find connections between simple changes in the coding sequence of genes and the risk for developing complex diseases. A product of the human genome sequence (and, one could almost say, a means of ensuring job security for the hordes of sequencers who were responsible for that project), genome-wide association studies represent the first comprehensive attempt by the genomics community to demonstrate a big payoff, in terms of benefits to human health, for the enormous amounts that were spent on that original project.\n\nIf there were world enough and time, as Andrew Marvel (or was it Francis Collins?) would say, we would perform such studies simply by sequencing the complete genomes of large cohorts of people with, say, type II diabetes, or lung cancer, or schizophrenia, or Alzheimer's disease, and then letting the computational folks sift through the resulting reams of data to sort out the varying combinations of simple spelling mistakes in many different genes that give rise to autism, or stroke, and so on - and it's quite likely that, when sequencing costs come down sufficiently, this is exactly what we will do. In the meantime, though, the effort is more restricted.\n\nThe current approach relies on data from the International Human Haplotype Mapping (HapMap) Project, which aims to determine the prevalence of common polymorphisms in the human genome, and on the fact that genetic variance at one locus can predict with high probability genetic variance at an adjacent locus, typically over distances of 30,000 base pairs of DNA, making it possible to map the common variability - and, as we shall see, the key word here is 'common' - associated with the risk of a given disease simply by genotyping approximately 500,000 judiciously chosen markers in the genome of several thousand case subjects and comparing the frequency of those markers with genotypes of control subjects. Consequently, it has become relatively routine to identify common variants (for example, those that are present in more than 5% of the population) that confer not a certainty but rather a risk of disease, typically with odds ratios of 1.2 to 5.0.\n\nBut now, in a series of articles in the 15 April 2009 online issue of the *New England Journal of Medicine*, a debate is taking place between proponents of this approach and those who argue that it will never succeed in revealing the genetic basis for complex, polygenic disorders. The reason for this scrutiny is the failure of most of the ongoing studies to find any convincing link with common diseases. It had been expected that the risk of getting cancer, diabetes, and so on would be largely controlled by a relatively small number of common variants, each of which conferred a significant risk, but only a small number of disease-associated variants have been found thus far, and, with a few exceptions, the risk they seem to confer is quite small. As Hardy and Singleton frame the question in one of four papers on the subject, \"...discussion has centered on evaluating how far such studies will take us in understanding the risks and causes of disease - and thus the time and resources that should be invested in genotyping more case subjects with any one disease to garner what many see as diminishing genetic returns.\"\n\nBecause they rely on the HapMap, current studies identify loci, not specific genes. Moreover, we currently have haplo-type maps only from single nucleotide polymorphisms (SNPs) present in at least 5% of chromosomes of each of just three groups of defined ancestry: Yoruban, Northern and Western European, and Asian (Chinese and Japanese), so by definition the markers are for rather common variants (present at \\> 5% frequency) in the human population. Underlying the project at present, then, is the assumption that common diseases are associated with common variations. A further assumption is that, even if individual alleles have only a small effect on one's risk for a disease, each contribution is large enough that a manageably small number will sum to a significant effect. But what if each contribution is very small?\n\nIn a companion perspective, Goldstein argues that very many very small contributions is exactly the case and questions the wisdom of continuing this strategy. He points out that there are examples of its successful application: \"For example, when exposed to the anti-HIV drug abacavir, a hypersensitivity reaction develops in more than half the carriers of the HLA-B\\*5701 allele, whereas such a reaction occurs in less than 5% of patients without this allele. Similarly, just three common variants are sufficient to explain 14% of the population variation in HIV-1 viral load.\" But, he continues, \"with traits such as height or type 2 diabetes, it seems that an inordinate number of common SNPs would be needed to account for a sizable fraction of heritability... The apparently modest effect of common variation on most human diseases and related traits probably reflects the efficiency of natural selection in prohibiting increases in disease-associated variants in the population.\" In other words, common diseases might well be caused by many different combinations of a large number - probably hundreds - of very rare variants, which would even eliminate the utility of the SNP hunt in identifying pathways leading to disease. \"In pointing at everything,\" Goldstein writes, \"genetics would point at nothing.\"\n\nSimilar concerns are expressed by Kraft and Hunter in a companion piece (although they favor continuing the common variant hunt). \"First, the relative risks that are found to be conferred by common risk genotypes account for only a small proportion of the sibling recurrence risk (or the risk that a sibling will also have the disease of interest). Second, in multivariate analyses of large epidemiologic data sets in which a family history of a disease is a risk factor, the inclusion of data regarding which subjects carry the known associated variants only minimally reduces the risk associated with a family history of the disease. Third, in the case of diseases that have been the focus of several genome-wide association studies, some alleles have been detected more than once, but each study has identified multiple alleles that were not identified in other studies, suggesting that many more alleles remain to be discovered. These factors suggest that many, rather than few, variant risk alleles are responsible for the majority of the inherited risk of each common disease.\"\n\nOne truly surprising result from the studies thus far is that the majority of loci identified as associated with disease risk do not map to the coding regions of individual genes. Instead, they possibly affect either the splicing of the messenger RNA or the sequences of microRNAs that regulate gene expression. Deducing the effects of non-coding changes on the level of active protein(s) in the cell is simply not possible from first principles at the moment; it will require huge experimental efforts in multiple laboratories.\n\nSo all this really seems very discouraging, but I have a modest proposal for a somewhat altered approach that I think could yield exciting results rapidly. The problem with most fishing expeditions, which is what genome-wide association studies are, is that one is never sure that one is fishing where the fish are. My proposal is to focus on where we know there are fish (or, to use another analogy, to look for the keys under the lamppost because that's where the light is). That seems unlikely to provide new information, but hear me out. I think the mistake we're making is in looking at the association between SNPs, many of which mean little or nothing, and disease. What we should be doing is looking at the association between diseases.\n\nThere are literally hundreds of inherited metabolic disorders, most of which are autosomal recessive - they require mutation in both copies of the gene in question to produce the disease. In many cases there are dozens or even more than a hundred known alleles in the gene in question, any two of which suffice. Carriers for these diseases have just a single variant and are usually free from symptoms of the disease. But it is slowly becoming clear that for at least some of the inborn errors of metabolism, the carriers are at altered risk for something else. It may be that a carrier has a reduced risk for an infectious disease, but often I think a problem with a metabolic enzyme will produce haploinsufficiency in some pathway that is involved in a very different disorder. Thus, carriers for the recessive, lysosomal storage disorder Gaucher disease are almost an order of magnitude more likely to develop Parkinson's disease. Is the connection through lysosomal dysfunction? Maybe, and that's testable: it suggests that carriers for other lysosomal storage diseases such as Niemann-Pick, Tay-Sachs, Anderson-Fabray, and Pompe's diseases should also be at increased risk for Parkinson's and perhaps other neurological disorders.\n\nThat's exactly the sort of thing a genome-wide association study could determine, and it would shed valuable light on the causes of a class of common diseases. I think it's almost guaranteed to turn up things, because metabolism is tied into all the other pathways in the cell, and because by definition the carrier alleles for a recessive disorder are mutations that must have some definite effect on the expression or function or stability of the protein in question.\n\nIn short, I think we should look at rare diseases for less rare genetic loci (the carrier frequency for Gaucher's is estimated to be 1 in 100 in the general population, and around in 1 in 20 among Ashkenazi Jews) that we know have physiological consequences, and ask whether they are associated with the risk for other, more common diseases. I think that's where the interesting connections are most likely to be found, at least until we can sequence lots of whole genomes very cheaply. After all, in a real criminal case, the police usually focus on suspects they know are likely to be guilty, because they have already been proven guilty of other things in the past. That's guilt by association, to be sure, but it tends to work.","meta":{"dup_signals":{"dup_doc_count":123,"dup_dump_count":47,"dup_details":{"curated_sources":2,"2021-10":1,"2020-24":1,"2019-30":1,"2019-22":1,"2019-09":1,"2018-51":2,"2018-39":1,"2018-30":1,"2018-17":1,"2017-51":1,"2017-47":1,"2017-39":1,"2017-34":1,"2017-26":1,"2017-17":1,"2017-09":7,"2016-50":1,"2016-44":1,"2016-40":2,"2016-36":9,"2016-30":7,"2016-22":1,"2016-18":1,"2016-07":4,"2015-48":3,"2015-40":3,"2015-35":4,"2015-32":4,"2015-27":3,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":3,"2014-42":7,"2014-41":4,"2014-35":3,"2014-23":5,"2014-15":5,"2023-50":1,"2017-13":1,"2015-18":3,"2015-11":3,"2015-06":3,"2014-10":2,"2013-48":3,"2013-20":2,"2024-26":1}},"file":"PMC2688918"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  Energy drink consumption has continued to gain in popularity since the 1997 debut of Red Bull, the current leader in the energy drink market. Although energy drinks are targeted to young adult consumers, there has been little research regarding energy drink consumption patterns among college students in the United States. The purpose of this study was to determine energy drink consumption patterns among college students, prevalence and frequency of energy drink use for six situations, namely for insufficient sleep, to increase energy (in general), while studying, driving long periods of time, drinking with alcohol while partying, and to treat a hangover, and prevalence of adverse side effects and energy drink use dose effects among college energy drink users.\n  .\n  # Methods\n  .\n  Based on the responses from a 32 member college student focus group and a field test, a 19 item survey was used to assess energy drink consumption patterns of 496 randomly surveyed college students attending a state university in the Central Atlantic region of the United States.\n  .\n  # Results\n  .\n  Fifty one percent of participants (*n* = 253) reported consuming greater than one energy drink each month in an average month for the current semester (defined as energy drink user). The majority of users consumed energy drinks for insufficient sleep (67%), to increase energy (65%), and to drink with alcohol while partying (54%). The majority of users consumed one energy drink to treat most situations although using three or more was a common practice to drink with alcohol while partying (49%). Weekly jolt and crash episodes were experienced by 29% of users, 22% reported ever having headaches, and 19% heart palpitations from consuming energy drinks. There was a significant dose effect only for jolt and crash episodes.\n  .\n  # Conclusion\n  .\n  Using energy drinks is a popular practice among college students for a variety of situations. Although for the majority of situations assessed, users consumed one energy drink with a reported frequency of 1 \u2013 4 days per month, many users consumed three or more when combining with alcohol while partying. Further, side effects from consuming energy drinks are fairly common, and a significant dose effect was found with jolt and crash episodes. Future research should identify if college students recognize the amounts of caffeine that are present in the wide variety of caffeine-containing products that they are consuming, the amounts of caffeine that they are consuming in various situations, and the physical side effects associated with caffeine consumption.\nauthor: Brenda M Malinauskas; Victor G Aeby; Reginald F Overton; Tracy Carpenter-Aeby; Kimberly Barber-Heidal\ndate: 2007\ninstitute: 1Department of Nutrition and Dietetics, East Carolina University, Greenville, North Carolina, USA; 2Department of Health Education and Promotion, East Carolina University, Greenville, North Carolina, USA; 3Department of Health, Physical Education, Recreation, and Dance, Virginia State University, Petersburg, Virginia, USA; 4School of Social Work, East Carolina University, Greenville, North Carolina, USA\nreferences:\ntitle: A survey of energy drink consumption patterns among college students\n\n# Background\n\nEnergy drink consumption has continued to gain popularity since the 1997 debut of Red Bull, the current leader in the energy drink market \\[1\\]. More than 500 new energy drinks were launched worldwide in 2006 and beverage companies are reaping the financial rewards of the 5.7 billion dollar energy drink industry \\[1\\]. Energy drinks, including Red Bull, Amp, Monster, Rock Star, Rip It, Full Throttle, and Cocaine, are designed to give the consumer a \"jolt\" of energy provided by the combination of stimulants and \"energy boosters\" that they provide, including caffeine, herbal extracts such as guarana, ginseng, and ginkgo biloba, B vitamins, amino acids such as taurine, amino acid derivatives such as carnitine, and sugar derivatives, including glucuronalactone and ribose \\[1\\]. Energy drinks typically contain 80 to 141 mg of caffeine per 8 ounces, the equivalent of five ounces of coffee or two 12-ounce cans of caffeinated soft drink such as Mountain Dew, Coca Cola, Pepsi Cola or Dr. Pepper \\[2\\]. Energy drinks have sugar-containing and sugar-free versions. For example, Monster Energy provides 24 grams of sugar per 8 ounces (12% sugar concentration) and Rip It A'Tomic Pom provides 33 grams (14% concentration) \\[3,4\\]. Similar to the booming energy drink market, the size of the energy drink container has increased over 300-fold; Monster energy offers consumers a 23 ounces option \\[3\\].\n\nDo energy drinks provide the consumer an extra burst of energy as the advertisements would have you believe? Yes, they do. Smit and colleagues found that energy drinks, as compared to placebo, had energizing effects among 18 to 55 year old participants, with effects being strongest 30 to 60 minutes after consumption and sustained at least 90 minutes \\[5\\]. Caffeine was found to be the primary constituent responsible for these effects. Although there is no human requirement for caffeine, even low doses of caffeine (12.5 to 100 mg) improve cognitive performance and mood \\[6\\]. However, caffeine has been found to have detrimental health consequences. Riesenhuber and colleagues found that the caffeine (but not taurine) in energy drinks promotes diuresis and natriuresis \\[7\\]. Further, acute caffeine consumption reduces insulin sensitivity \\[8\\] and increases mean arterial blood pressure \\[9\\]. High caffeine consumption is associated with chronic daily headaches, particularly among young women (age \\< 40 years) and among those with chronic episodic headaches and of recent onset (\\< 2 years) \\[10\\]. Central nervous system, cardiovascular, gastrointestinal, and renal dysfunction have been associated with chronic caffeine ingestion \\[11\\]. In sum, the caffeine in energy drinks will provide the consumer the desirable effects of increased alertness, improved memory, and enhanced mood. However, caffeine can have harmful physical consequences.\n\nAlthough energy drinks are targeted to the 18 to 35 year old consumer \\[12\\], there has been little research regarding energy drink consumption patterns among young adults in the United States. The purpose of this study was to determine (1) energy drink consumption patterns among college students, (2) prevalence and frequency of energy drink use for six situations, namely for insufficient sleep, to increase energy (in general), while studying, driving long periods of time, drinking with alcohol while partying, and to treat a hangover, (3) and prevalence of adverse side effects and energy drink use dose effects among college energy drink users.\n\n# Methods\n\nA Registered Dietitian and a Health Educator designed a questionnaire that assessed consumption patterns of energy drinks among college students. We initially interviewed a focus group of 32 college students who were enrolled in a senior-level course. We asked these students open-ended questions regarding situations in which college students use energy drinks, the most common energy drinks college students were using, frequency patterns (average number of energy drinks consumed for each situation the focus group identified and the average number of times per month throughout a semester students use energy drinks for each situation), and side effects from using energy drinks.\n\nBased on the focus group responses we developed a 19-item questionnaire. Questions 1 and 2 assessed demographic information (age and sex). Question 3 was a screening question, used to identify energy drink users, and asked \"in an average month for the current semester do you drink more than one energy drink per month?\" If a participant indicated \"no\", then they were instructed to skip the remaining questions in the survey and return the questionnaire to the research assistant. Participants who indicated \"yes\" to Question 3 were instructed to continue the survey, which assessed the type of energy drink usually consumed (regular or sugar-free), side effects associated with energy drink use (jolt and crash episodes, headaches, heart palpitations), and six situations for energy drink use (insufficient sleep, needing more energy (in general), studying for an exam or to complete a major course project, driving a car for a long time, drinking with alcohol while partying, and to treat a hangover).\n\nFor the purpose of this study, a jolt and crash episode was in reference to a feeling of increased alertness and energy (the jolt) followed by a sudden drop in energy (the crash) that occurs in response to using energy drinks.\n\nEach of the six situation questions had two follow up questions that assessed the average number of energy drinks consumed for that situation (for example, how many energy drinks do you drink at one time following a night of not getting enough sleep?) and the average number of times per month for the current semester the student consumes energy drinks for that situation.\n\nTo provide a frame of reference regarding what constituted an energy drink, the introduction of the questionnaire included examples of energy drinks that were popular on the campus and in social establishments in the immediate geographic region when the survey was administered, these included Red Bull, Rock Star, Amp, and Full Throttle. The questionnaire was field tested among 10 randomly chosen students who were in a public location on campus. The questionnaire took approximately two minutes to complete and modifications to the questionnaire were not necessary based on the field test responses.\n\nFrom mid-November to the first week of December 2006, 11 trained research assistants (undergraduate and graduate college students) recruited students at a single college from public locations across campus to participate in the study. The research assistants first ensured that those they approached were students at the university and that the student had not previously completed the questionnaire.\n\nThe institution is a state university, located in the Central Atlantic region of the United States. The fall 2006 enrollment statistics indicate an undergraduate enrollment of approximately 18,000 undergraduate and 6,000 graduate students, 85% of undergraduates were 18 to 24 years of age, 12% were 25 to 40 years of age and 3% 41 years of age and older \\[13\\]. Further, 92% of undergraduates attended school full-time whereas the majority (60%) of graduate students attended part time. In regard to ethnicity of the student body, 76% were non-Hispanic White, 16% non-Hispanic Black, 2% Asian, 2% Hispanic, 2% unknown, \\< 1% American Indian, and \\< 1% non-resident alien.. Sixty two percent of the total student body is female \\[13\\].\n\nTo diversify our sample, research assistants varied the time of day and days of the week during weekdays to recruit participants. In compliance with the university's Institutional Review Board for Research with Human Subjects (University and Medical Center Institutional Review Board number 06-0718), students were informed of the study protocol and those willing to participate anonymously completed the self-administered questionnaire. The project was carried out in compliance with the Helsinki Declaration.\n\nAnalyses were performed using JMP IN^\u00ae^ software \\[14\\]. Descriptive statistics included means, standard deviations, 95% confidence intervals, and frequency distributions. Pearson *\u03c7*^2^ was used to evaluate differences in frequency distribution of responses. An alpha level of .05 was used for all statistical tests.\n\n# Results\n\nA total of 496 participants, aged 21.5 \u00b1 3.7 years (95% *CI* 21.3, 21.8) completed the questionnaire. In regard to the first research question, energy drink consumption patterns among college students, 51% of participants (*n* = 253) reported drinking greater than one energy drink each month in an average month for the current semester, with significantly more female (53%) than male (42%) energy drink users reported, \u03c7^2^ (1, *N* = 496) = 6.46, *p* = .01. Seventy four percent (*n* = 187) of the 253 users drank sugar-containing versions with significantly more females (35%) than males (12%) drinking sugar-free versions, \u03c7^2^ (1, *N* = 247) = 16.56, *p* \\< .01.\n\nEnergy drink consumption patterns of college energy drink users for the six situations assessed are reported in Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>. Insufficient sleep was the most common reason to drink energy drinks, as indicated by 67% of energy drink users. The majority of users consumed energy drinks to increase their energy (65%) and to drink with alcohol while partying (54%). Fifty percent drank while studying or completing a major course project, 45% while driving a car for a long period of time, and 17% to treat a hangover. There were no significant differences in use of energy drinks for the six situations assessed by sex, as reported in Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>.\n\nSituations of energy drink use among college energy drink users in an average month for the current semester\n\n| Situation | % of females | % of males | \u03c7^2^ | *p* (sex) |\n|----|----|----|----|----|\n| Insufficient sleep^a^ | 67 | 68 | 0.17 | .68 |\n| Need energy (in general)^a^ | 62 | 69 | 1.27 | .26 |\n| Studying or major project^b^ | 46 | 56 | 2.22 | .14 |\n| Driving car for long period of time^a^ | 40 | 51 | 3.01 | .08 |\n| Mix with alcohol while partying^a^ | 57 | 50 | 1.33 | .25 |\n| Treat hangover^a^ | 16 | 18 | 0.18 | .67 |\n\n^a^*n* = 146 females, 107 males, \u03c7^2^ (1, *N* = 253); ^b^*n* = 145 females, 104 males, \u03c7^2^ (1, *N* = 249).\n\nIn regard to the second research question, the percent of users drinking one, two, and three or more energy drinks by situation are reported in Table <a href=\"#T2\" data-ref-type=\"table\">2<\/a>. The majority of energy drink users consumed one to treat a hangover, for insufficient sleep, to increase energy, and while driving a car for a long period of time. Using three or more was a common practice (49% of users) to drink with alcohol while partying. The percent of users drinking energy drinks 1 \u2013 4, 5 \u2013 10, and 11 or more days in an average month for the current semester are also reported in Table <a href=\"#T2\" data-ref-type=\"table\">2<\/a>. For the six situations assessed, the majority of users (73% to 86%) consumed energy drinks 1 \u2013 4 days in a month.\n\n% of college energy drink users^a^ reporting amount and frequency of energy drink consumption by situation in an average month for the current semester\n\n|  |  | Energy drinks consumed |  |  | Days per month |  |  |\n|----|----|----|----|----|----|----|----|\n|  |  |  |  |  |  |  |  |\n| Situation | *n* | 1 | 2 | \u22653 | 1 \u2013 4 | 5 \u2013 10 | \u226511 |\n| Insufficient sleep | 169 | 64 | 22 | 14 | 74 | 18 | 8 |\n| Need energy (in general) | 165 | 63 | 21 | 16 | 74 | 18 | 8 |\n| Studying or major project | 125 | 50 | 36 | 14 | 85 | 10 | 5 |\n| Driving car for long period of time | 114 | 63 | 23 | 14 | 86 | 9 | 5 |\n| Mix with alcohol while partying | 136 | 27 | 24 | 49 | 73 | 18 | 9 |\n| Treat hangover | 42 | 74 | 10 | 16 | 74 | 14 | 12 |\n\n^a^*n =* 253 college energy drink users.\n\nTo further identify relationships between the six situations of energy drink use and energy drink consumption patterns, we summed the number of situations for reported energy drink use and compared this to the maximum number of energy drinks consumed for any of the six situations. These results are reported in Table <a href=\"#T3\" data-ref-type=\"table\">3<\/a>. By sum of situation categories, 16% to 20% of energy drink users consumed energy drinks for a total of one to five of the six situations, 7% consumed energy drinks for a total of all six. As total situations increased so did the maximum energy drink consumption for at least one situation. For example, 40% to 81% of energy drink users who reported a total of three or more situations consumed three or more energy drinks for at least one situation, whereas 29% of those with a total of one situation and 18% of those with a total of two consumed three or more for at least one situation.\n\nMaximum number of energy drinks consumed for any of six situations by sum of situations^a^ in an average month for the current semester among college energy drink users\n\n|  |  | Maximum energy drink intake for any situation |  |  |\n|----|----|----|----|----|\n|  |  |  |  |  |\n| Parameter | *n* | 1 | 2 | \u22653 |\n| % of 1 situation responders | 45 | 56 | 15 | 29 |\n| % of 2 situation responders | 38 | 55 | 27 | 18 |\n| % of 3 situation responders | 45 | 33 | 27 | 40 |\n| % of 4 situation responders | 46 | 20 | 37 | 43 |\n| % of 5 situation responders | 43 | 5 | 32 | 63 |\n| % of 6 situation responders | 16 | 6 | 13 | 81 |\n\nNote: \u03c7^2^ (10, *N* = 233) = 53.43, *p* \\< .01.\n\n^a^Sum of six situations reported energy drink use.\n\nRegarding the third research question, weekly jolt and crash episodes were experienced by 29% of users, 22% reported ever having headaches and 19% heart palpitations from consuming energy drinks, which did not differ significantly by sex, \u03c7^2^ (1, *N* = 253) \\< 0.01, *p* = .97 for jolt and crash episodes, \u03c7^2^ (1, *N* = 234) = 0.37, *p* = .54 for heart palpitations, \u03c7^2^ (1, *N* = 252) = 0.45, *p* = .50 for headaches. The data for side effects by energy drinks consumed are reported in Table <a href=\"#T4\" data-ref-type=\"table\">4<\/a>. There was a significant dose effect for jolt and crash episodes but not for heart palpitations or headaches. For example, 57% of energy drink users who reported experiencing weekly jolt and crash episodes also consumed three or more energy drinks for at least one situation, whereas 35% of those who denied jolt and crash episodes consumed three or more.\n\nSide effects by maximum number of energy drinks consumed for any of six situations in an average month for the current semester among college energy drink users\n\n|  |  | Maximum energy drink intake |  |  |  |  |\n|----|----|----|----|----|----|----|\n| Parameter | *n* | 1 | 2 | \u22653 | \u03c7^2^ | *P* (yes versus no) |\n| Weekly jolt and crash episodes | 253 |  |  |  | 19.10 | \\< .01 |\n| \u2003% of yes responders | 74 | 12 | 31 | 57 |  |  |\n| \u2003% of no responders | 159 | 40 | 25 | 35 |  |  |\n| Ever having heart palpitations | 215 |  |  |  | 4.77 | .09 |\n| \u2003% of yes responders | 40 | 20 | 22 | 58 |  |  |\n| \u2003% of no responders | 175 | 34 | 27 | 39 |  |  |\n| Ever having headaches | 232 |  |  |  | 1.24 | .54 |\n| \u2003% of yes responders | 51 | 27 | 24 | 49 |  |  |\n| \u2003% of no responders | 181 | 33 | 27 | 40 |  |  |\n\n# Discussion\n\nEnergy drinks are marketed to young adults and marketing efforts may be particularly appealing among college students. For example, Cocaine energy drink, with a Cut Cocaine variety, has been marketed as a \"legal alternative\" to the class A drug \\[15\\]. On April 4, 2007, the Food and Drug Administration issued a warning to Drink Reboot, the firm that markets Cocaine, citing numerous marketing violations, including promoting this product as a street drug alternative \\[15\\]. Red Bull energy drink is reportedly a \"functional beverage\" that was designed to increase physical and mental performance and \"is appropriate to drink during sports, while driving, and during leisure activities\" \\[16\\] whereas Monster energy provides a \"double shot of our killer energy brew. It's a wicked mega hit that delivers twice the buzz of a regular energy drink...\" \\[3\\]. The purpose of this study was to identify energy drink consumption patterns and side effects associated with consumption of energy drinks among college students. We found that energy drink consumption is a popular practice among college students, particularly if the student has had insufficient sleep, if they need more energy in general, while studying for exams or working on major course projects and while driving an automobile for a long period of time.\n\nImprovements in mental functioning are of interest among college students, many who suffer from sleep deprivation. The American College Health Association reported that 71% of college students whom they surveyed reported insufficient sleep and not feeling rested for at least five of the past seven days \\[17\\]. Sleep deprivation is associated with selecting less difficult cognitive tasks and college students who have sleep difficulties report a greater frequency of stress \\[18,19\\]. Findings from our study support the premise that college students use energy drinks to treat sleep deprivation and while studying for exams or completing major course projects. On the other hand, caffeine consumption has not been found to affect academic performance among college students \\[20\\].\n\nThe primary ingredient in energy drinks that has a cognitive stimulating effect is the caffeine \\[5\\], whereas high sugar content (18% concentration) does not improve reaction times slowed by sleep deprivation \\[21\\]. Further, the combination of caffeine and taurine has no effect on short-term memory \\[9\\]. Although low doses of caffeine (12.5 to 50 mg) have been found to improve cognitive performance and mood \\[6\\] and 200 mg doses have been found to improve cognitive task speed and accuracy and increase alertness among young adults \\[22\\], the amount of caffeine provided in energy drinks can easily far exceed the amount necessary to promote cognitive functioning \\[23\\]. This is especially true if a student is consuming 16- or 23-ounce cans or multiple cans of energy drinks for a given situation. Although we did not assess the size of the energy drink cans that participants normally consumed, results from our study indicate that in some situations while students are consuming energy drinks, the amount of caffeine that they consume can exceed the amount needed simply to promote cognitive stimulation. For example, 50% of energy drink users in our study drank two or more energy drinks while studying for an exam or working on a major course project, and 36% to 37% drank two or more following insufficient sleep, when they needed energy throughout the day, or while driving an automobile for a long period of time. Further, drinking multiple energy drinks with alcohol was a popular practice among 73% of energy drink users. The practice of consuming greater amounts of caffeine while socializing has also been documented among American youth \\[24\\] and an alcoholic setting is considered by many college students a primary locus to socialize and to meet people \\[25\\].\n\nResults from the present study indicate that female and male college students are using energy drinks in a similar fashion. Whereas we found a greater prevalence of energy drink consumption and greater use of sugar-free varieties of energy drink use among females, we identified no situation differences nor prevalence of side effects from consuming energy drinks between sexes.\n\nThere are a number of limitations to this study that deserve discussion. First, in an effort to ensure the survey instrument could be completed quickly, we collected limited demographic information. Based on the descriptive statistics regarding age, we primarily had undergraduate participants and a slightly greater percentage of male participants as compared to the sex distribution at the university. On the other hand, random sampling throughout the weekdays and times of the day at central locations throughout campus was an advantage to the study design. Additionally, this is a rural state university with a fairly homogenous student body. Second, the data collected was self-reported. In particular, frequency patterns of energy drink intake were asked by situation and were treated as independent and distinct events, which may not have been the case. For example, and energy drink user may consume energy drinks because they had not gotten enough sleep and because they were studying for an exam. As a result, assessment of energy drink consumption may have been overestimated for each of the situation events. On the other hand, the results from this study provide important and novel information regarding energy drink consumption habits among college students. Of particular importance is the finding that using energy drinks for a number of situations is common among college students and that those who use energy drinks for three or more of the situations that we assessed tended to drink three or more energy drinks for at least one situation. Further, side effects of jolt and crash episodes, heart palpitations, and headaches are fairly common, as reported by approximately 25% of users, and there is a significant dose effect of energy drink consumption and jolt and crash episodes.\n\n# Conclusion\n\nUsing energy drinks is a popular practice among college students, as we found that 51% of 496 college students surveyed reported drinking greater than one energy drink each month. Among college energy drink users, consuming energy drinks is particularly popular for insufficient sleep, when one needs more energy in general, to drink with alcohol while partying, and when studying for an exam or completing a major course project. Drinking three or more for a given situation occurs more frequently among those who consume energy drinks for three or more of the six situations that were assessed. Side effects of consuming energy drinks, including experiencing jolt and crash episodes, hear palpitations, and headaches occur in many energy drink users. However, a dose effect was found only for jolt and crash episodes. Further research should identify if students recognize the amounts of caffeine that are present in the wide variety of caffeine-containing products they consume, the amounts of caffeine that are consumed in various situations, and the physical side effects associated with caffeine consumption.\n\n# Competing interests\n\nThe author(s) declare that they have no competing interests.\n\n# Authors' contributions\n\nBMM participated in the study design, performed the statistical analysis, and drafted the manuscript. VGA conceived of the study and drafted the manuscript. AJC assisted with statistical analysis and helped to draft the manuscript. RFO, TCA, and KBH participated in coordination and data collection and helped to draft the manuscript. All authors read and approved the final manuscript.","meta":{"dup_signals":{"dup_doc_count":126,"dup_dump_count":63,"dup_details":{"curated_sources":2,"2023-40":1,"2023-23":1,"2022-49":1,"2022-33":1,"2022-27":1,"2022-05":1,"2021-39":1,"2021-31":4,"2021-21":1,"2021-17":2,"2021-10":1,"2021-04":1,"2020-50":1,"2020-45":3,"2020-34":2,"2020-29":1,"2020-24":1,"2020-16":1,"2020-10":1,"2020-05":1,"2019-51":1,"2019-47":1,"2019-43":1,"2019-35":1,"2019-26":1,"2019-18":1,"2019-09":1,"2018-51":1,"2018-47":1,"2018-43":2,"2018-39":1,"2018-34":2,"2018-30":1,"2018-26":1,"2018-22":1,"2018-17":1,"2018-13":1,"2018-09":3,"2018-05":3,"2017-51":2,"2017-47":3,"2017-43":2,"2017-39":3,"2017-34":3,"2017-30":2,"2017-26":3,"2017-22":2,"2017-17":2,"2017-09":14,"2017-04":3,"2016-50":2,"2016-44":1,"2016-40":3,"2016-36":2,"2016-30":2,"2016-22":1,"2016-18":1,"2016-07":14,"2023-50":1,"2024-22":3,"2024-18":1,"2017-13":1,"2024-26":1}},"file":"PMC2206048"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  A nutritional assessment method that is quick and easy to conduct would be extremely useful in a complex emergency, where currently there is no agreed practical and acceptable method. Hair pluckability has been suggested to be a useful method of assessing protein nutritional status. The aim was to investigate the reliability of the trichotillometer and to explore the effects of patient characteristics on hair epilation force.\n  .\n  # Methods\n  .\n  Three observers plucked hair from twelve participants to investigate the within- and between-observer reliability. To investigate the effect of patient characteristics on hair pluckability, 12 black African and 12 white volunteers were recruited. Participants completed a short questionnaire to provide basic information on their characteristics and hair.\n  .\n  # Results\n  .\n  Mean hair pluckability measurements for the 12 participants obtained by the three observers (39.5 g, 41.2 g and 32.7 g) were significantly different (p \\< 0.001). Significant variation between patients was also found (p \\< 0.001). None of the patient characteristics significantly affected hair pluckability, with the exception of age, although this relationship was not consistent.\n  .\n  # Conclusion\n  .\n  Due to significant variation in measurements, hair pluckability does not appear to be a reliable method for assessing adult nutritional status. Hair pluckability could be a useful method of nutritional assessment in complex humanitarian emergencies but only if the reliability was improved.\nauthor: Laura A Wyness; Geraldine McNeill; Gordon J Prescott\ndate: 2007\ninstitute: 1Department of Public Health, University of Aberdeen, Medical School, Foresterhill, Aberdeen AB25 2ZD, UK; 2Department of Environmental and Occupational Medicine, Liberty Safe Work Research Centre, Foresterhill Road, Aberdeen AB25 2ZP, UK\nreferences:\ntitle: Trichotillometry: the reliability and practicality of hair pluckability as a method of nutritional assessment\n\n# Background\n\nComplex emergencies are usually characterised by large numbers of displaced persons and overwhelming need for shelter, water, sanitation and health care commonly due to extreme insecurity. Time and resources available during a complex emergency are often extremely limited, so a nutritional assessment method that is quick and easy to conduct is required. Mid-upper arm circumference and weight can be (and are) used in complex emergencies, although there remains some debate regarding the appropriate cut-off measurements to indicate nutritional status.\n\nOne of the signs of protein-energy malnutrition is hair that can be plucked easily \\[1\\]. Protein is the main component of hair; forming between 65 to 95% of the hair by weight \\[2\\]. During protein deficiency, blood proteins are maintained at the expense of structural protein; therefore structural proteins are an earlier indicator of protein insufficiency \\[3\\]. The hair root shows a prompt response to protein deficiency. Studies have shown that crash dieting, where the diet is severely constrained over a short period, can precipitate hair loss \\[4,5\\]. Chronic starvation, especially marasmus, is also associated with diffuse hair loss \\[6\\] and hair that is easy to pluck \\[7,8\\]. Chase *et al*. (1981) found patients with kwashiorkor or marasmic kwashiorkor had slightly decreased epilation force than patients with \"pure\" marasmus \\[9\\]. This should be borne in mind when assessing malnourished individuals. Further investigation of this issue was beyond the scope of this paper. In complex emergencies inexpensive and simple methods for screening for malnutrition and for assessing changes in nutritional status are required.\n\nTwo studies of measurement of hair pluckability as a possible measure of nutritional status have been reported \\[9,10\\]. In these studies, a hand-held spring dynamometer, or \"trichotillometer\" was used to measure the force in grams that was required to pluck individual hairs from the scalp. Chase *et al*. (1981) \\[9\\] measured the hair epilation force of 17 malnourished and 16 well-nourished hospitalized patients using the trichotillometer. Epilation force was found to be significantly correlated with weight, triceps skinfold thickness, arm muscle circumference and some laboratory assessments of nutritional status. Smelser *et al*. (1982) \\[10\\] determined the usefulness of trichotillometry under field conditions by measuring hair epilation force of 69 subjects at a hospital in Nigeria. Epilation force was found to be significantly correlated with weight-for-height, serum albumin and mid arm muscle circumference. The studies \\[9,10\\] both concluded that the trichotillometer might be a useful method for assessing protein nutritional status. However, to be useful in the field the method needs to have low within- and between-observer variation and low intra-individual variation. The present study was therefore conducted to investigate the reliability of hair pluckability as measured by a trichotillometer.\n\n# Methods\n\n## Study subjects\n\nData was collected for the survey investigating the reliability of the trichotillometer from staff and students at the University of Aberdeen. The inclusion criteria for the study were that participants were aged 18 years or over at the time of the study, and that they had hair of adequate length to be plucked, which covered the area on the top of the scalp.\n\nData was also collected for the comparison survey investigating the effect of participant characteristics on the epilation force required to pluck hair. The initial inclusion criteria for this study were identical to the reliability study described above. Recruitment of black African volunteers was conducted first. White volunteers were then recruited among staff and students at the University of Aberdeen and chosen to balance the black African participants according to gender and age within two years.\n\nSample size calculations for both proposed studies were performed using the statistical program \"nQuery Advisor\" (version 4.0), using data from two previous studies \\[9,10\\], to determine the required number of participants to detect a difference of 1 standard deviation (SD) assuming a power of 80%. These studies found differences in means of 21.2 g and 10.8 g between malnourished and control subjects (with SD of between 9.5 g and 11.8 g) at the 5% level of significance using unpaired t-tests \\[9,10\\]. Measurements from ten individuals were required to test the within-observer reliability and three observers were required to test the between-observer reliability. Ethical approval was obtained from Grampian Research Ethics Committee for both studies.\n\n## Data collection and measurement technique\n\nAll participants completed a short questionnaire to provide basic information on gender, age and ethnicity. Data were also collected about the colour and length of each participant's hair, and whether the hair was currently dyed, permed or if any styling products were applied on the day hair pluckability measurements were collected.\n\nInstructions from the developer of the trichotillometer, Prof. C.L. Krumdieck, were provided and adhered to by the observers. The trichotillometer has an arbitrary scale (units) which can be converted to grams using a calibrated scale. In this study one unit was equivalent to 2.5 g.\n\nIn both studies ten hairs from the top of each participant's head were epilated individually. Measurements typically took five minutes for ten hairs. The number of broken hairs and the number of malfunctions of the ratchet mechanism of the trichotillometer were recorded. If required additional hairs were epilated until a sample of ten hairs was achieved. Measurements obtained by three observers were used to assess the between-observer reliability. Measurements obtained from each observer were used to assess within-observer reliability.\n\n## Statistical analysis\n\nAnalysis of the data was conducted using SPSS, version 13. A two-way ANOVA was used to investigate the variation in hair pluckability due to differences between observers and between participants. Comparisons between the measurements obtained in groups with different participant characteristics were made using the independent t-test, a one-way ANOVA or Kruskal-Wallis test as appropriate.\n\n# Results\n\nOf the 12 participants included in the reliability study seven were female. Seven were of white British ethnicity and five were of white, non-British ethnicity. One participant did not report her age. The mean age of the other 11 participants was 35.3 years (SD = 9.1 years). The measurements within an individual were generally normally distributed although the distributions were slightly skewed for some individuals.\n\n## Observer variation\n\nThe differences in measurement between the three observers can be seen in Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>. The mean (SD) hair pluckability measurements for observer 1, 39.5 g (10.9 g), and 2, 41.4 g (13.4 g), were similar, whereas observer 3 obtained a significantly lower mean hair pluckability, 32.7 g (10.0 g). For observers 1, 2 and 3, the average ranges of individual hair pluckability measures and mean coefficients of variation (CV) were 30.5 g, 32.5 g and 28.0 g and 23.7%, 25.8% and 25.4%, respectively.\n\n## Participant variation\n\nLarge differences in hair pluckability measurements across the 12 participants were observed (Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>). Some participants had similar measurements for all 10 hairs plucked.\n\n## Two-way analysis\n\nA two-way ANOVA was carried out to investigate the variation in hair pluckability due to observer and participant effects. While there were some individuals with skewed measurements, the residuals from this analysis were normally distributed satisfying the relevant assumptions. Variances were homogeneous in the model with an interaction effect. Significant differences in hair pluckability between observers (p \\< 0.001) and between participants (p \\< 0.001) were found as well as a significant interaction effect (p = 0.001). This indicated that the differences between individuals in terms of hair pluckability were not independent of the observer.\n\nThe effect size for the observers (partial eta squared = 0.282) and the participants (partial eta squared = 0.141) can be classified as large using Cohen's 1988 criterion \\[11\\]. Post-hoc comparisons using the Sheffe's test indicated that the mean hair pluckability for observer 3 was significantly lower than the mean obtained by observers 1 and 2 (p \\< 0.001). Observers 1 and 2 did not differ significantly (p = 0.261).\n\n## Comparative study\n\nThe basic characteristics of the participants included in the comparative study and the results of the statistical analysis on the data are shown in Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>. Hair pluckability was not significantly correlated with age. However, when participants were grouped into three age groups, a post hoc comparison using Sheffe's test indicated that the mean pluckability for the age group 30\u201340 years was significantly lower than the mean score for the age groups 19\u201329 years and 41\u201352 years. Therefore, there was no consistent relationship between hair pluckability and age in these data.\n\nComparative study sample characteristics\n\n| Participant characteristic | Black African n = 12 | White n = 12 | Hair pluckability Mean (SD) | Test statistic (P value) |\n|----|----|----|----|----|\n| Ethnicity (number): |  |  |  |  |\n| \u2003Black African | 12 | 0 | 39.9 (10.8) | 0.89 (0.372)^1^ |\n| \u2003White Australian | 0 | 1 | 38.7 (11.0) |  |\n| \u2003White British | 0 | 9 | 38.7 (11.0) |  |\n| \u2003White German | 0 | 1 | 38.7 (11.0) |  |\n| \u2003White Italian | 0 | 1 | 38.7 (11.0) |  |\n| Gender (number) |  |  |  |  |\n| \u2003Male | 4 | 4 | 40.9 (11.2) | 1.58 (0.115)^1^ |\n| \u2003Female | 8 | 8 | 38.5 (10.7) |  |\n| Age (Mean (SD)) | 35.3 (9.1)^2^ | 36.7 (9.1) | 36.0 (9.1) | 0.083 (0.208)^3^ |\n| Age groups |  |  |  |  |\n| \u200319\u201329 y | 4 | 2 | 41.6 (12.5) | 10.38 (\\<0.001)^4^ |\n| \u200330\u201340 y | 3 | 6 | 35.4 (9.5) |  |\n| \u200341\u201352 y | 4 | 4 | 41.7 (10.0) |  |\n| Age groups |  |  |  |  |\n| \u200319\u201336 y | 6 | 6 | 38.4 (11.6) | 1.39 (0.187)^1^ |\n| \u2003\\>36 y | 5 | 6 | 40.2 (10.1) |  |\n| Hair color (number): |  |  |  |  |\n| \u2003Black | 11 | 1 | 39.7 (10.9) | 0.157 (0.525)^5^ |\n| \u2003Brown | 0 | 8 | 38.9 (10.9) |  |\n| \u2003Blonde | 0 | 2 | 38.9 (10.9) |  |\n| \u2003Grey | 1 | 0 | 38.9 (10.9) |  |\n| \u2003Red | 0 | 1 | 38.9 (10.9) |  |\n| Hair length (number): |  |  |  |  |\n| \u2003Shorter than ear length | 8 | 8 | 37.5 (32.5, 45.0)^6^ | 1.09 (0.579)^5^ |\n| \u2003Between chin and shoulder | 3 | 2 | 37.8 (29.9, 47.5)^6^ |  |\n| \u2003Longer than shoulder | 1 | 2 | 42.6 (29.7, 50.1)^6^ |  |\n| Hair currently permed |  |  |  |  |\n| \u2003Yes | 7 | 0 | 40.2 (10.2) | 0.78 (0.436)^1^ |\n| \u2003No | 4 | 12 | 38.9 (11.2) |  |\n| Hair currently dyed |  |  |  |  |\n| \u2003Yes | 2 | 7 | 41.4 (10.5) | 1.90 (0.058)^1^ |\n| \u2003No | 10 | 4 | 38.4 (11.0) |  |\n| Currently have hair products on hair |  |  |  |  |\n| \u2003Yes | 7 | 6 | 39.5 (10.9) | 0.25 (0.804)^1^ |\n| \u2003No | 4 | 6 | 39.1 (11.0) |  |\n| Broken hair |  |  |  |  |\n| \u2003\u22651 | 3 | 1 | 41.4 (10.7) | -1.36 (0.177)^1^ |\n| \u2003None | 9 | 11 | 38.9 (10.9) |  |\n\n^1^Independent t-test, ^2^N = 11 as one participant did not report their age, ^3^Spearman's correlation, ^4^One-way ANOVA, ^5^Kruskal Wallis test, ^6^Median (Interquartile range)\n\n# Discussion\n\nThe trichotillometer was previously reported to be a good method for assessing nutritional status in adults \\[9,10\\]. Although the effect of acute stress on hair pluckability using the trichotillometer \\[9\\] and the usefulness of the trichotillometer under field conditions \\[10\\] have been previously investigated, the reliability and the effect of various individuals' characteristics on hair pluckability have not been investigated. The observations presented here are consistent with Chase *et al*. \\[9\\] who found the average observed mean hair pluckability (SD) among well-nourished patients, when plucking ten individual hairs was 36.0 g (12.4 g).\n\nVariation between observers is not desirable since similar measurements should be obtained by all observers who use the same instrument on the same individual. This study found two of the observers reported similar results and one reported significantly lower measurements by, on average 8.7 g, which in clinical terms is quite a large difference. It is possible that a variation of this magnitude would effect the classification of the nutritional status of an individual.\n\nVariation within observers is also not desirable and, from a clinical perspective, this mean variation of close to 30 g within an observer is large. This wide variation in individual hair pluckability measurements indicates that an adequate amount of individual hairs need to be plucked in order to obtain a reproducible and representative mean hair pluckability measurement.\n\nVariation between participants for reasons unrelated to nutritional status would not be a welcome finding, as this could obscure differences between individuals with different nutritional status. This study found significant differences between the participant's hair pluckability measurements. There was also wide variation in the ten measurements obtained for each participant as indicated by relatively large coefficient of variation (CV). A CV of \\>30% is often considered as large experimental variability \\[12\\]. This may be due to biological variation in hair pluckability as the force required to pluck hair is likely to vary throughout the different stages of growth. A balance between the time available and the number of hairs required to be plucked in order to obtain a valid measurement needs to be found.\n\nA significant interaction effect was found, indicating that the differences between individuals in terms of hair pluckability were not independent of the observer. This is not desirable since the tool should be able to discriminate between individuals regardless of the observer.\n\nNo significant difference in hair pluckability between black African and white adults or between males and females was found. However, since the number of male participants was small (n = 8), a larger study may be required to ensure the results are generalisable to a wider population. There was no consistent relationship between hair pluckability and age. One result suggested that younger and older age groups required a larger force to pluck hair than the middle age group of 30\u201340 year olds. A larger study would be required to verify this finding due to the relatively small number of participants in this study. No significant difference in hair pluckability was identified with hair colour or length, even when the categories were combined to increase the number of participants in the smaller groups.\n\nAll three observers were given the same instructions on how to use the trichotillometer, and were allowed to practice obtaining measurements until they felt confident using it. Due to the availability of the observers and participants, observers may have taken measurements on a participant on different days. However, all measurements were obtained from all three observers within three days of each other. This may be a source of variation as the participant may have used hair products for one measurement and not another. However, it is unlikely to have had an effect as no significant association was found between hair pluckability and the use of hair products. Although none of the participants in this study objected to having ten hairs pulled out, the acceptability to some individuals may be lower, for example due to a lower pain threshold or traditional beliefs not permitting the removal of hair, as in some African cultures.\n\n# Conclusion\n\nIn conclusion, one classification scale can be used when assessing hair pluckability for all adults as no significant differences were identified with any of characteristics of the participants. However, the differences found between the measurements obtained by each observer and wide variations in measurements within observers suggest that the trichotillometer is not a reliable method for assessing adult nutritional status. If the reliability of the trichotillometer could be improved, for example, by adjusting the design, hair pluckability would be an extremely useful method of nutritional assessment during a complex emergency, or a situation where savings on time and resources need to be made.\n\n# Competing interests\n\nThe author(s) declare that they have no competing interests.\n\n# Authors' contributions\n\nLAW collected and analysed the data and wrote the manuscript. GM provided advice on the study design and collection of the data, and critically revised the manuscript. GJP provided advice on the collection and analysis of the data, and critically revised the manuscript. All authors read and approved the final manuscript.\n\n### Acknowledgements\n\nThe authors would like to thank Professor Carlos L Krumdieck for supplying the trichotillometer and Shona Fielding, and Kirsten Harrild from the University of Aberdeen, for assisting with the data collection. Thanks are also due to the participants who volunteered from \"The Aberdeen African Women's Group\", the \"Fountain of Love\" church, and the University of Aberdeen.","meta":{"dup_signals":{"dup_doc_count":117,"dup_dump_count":45,"dup_details":{"curated_sources":12,"2022-21":1,"2021-49":1,"2020-40":1,"2019-35":1,"2019-18":1,"2019-09":1,"2018-51":1,"2018-43":1,"2018-34":1,"2018-26":1,"2018-17":1,"2018-13":1,"2017-47":1,"2017-34":1,"2017-26":1,"2017-17":1,"2017-09":13,"2017-04":1,"2016-50":1,"2016-44":1,"2016-40":1,"2016-36":1,"2016-22":1,"2016-18":1,"2015-48":3,"2015-40":1,"2015-35":3,"2015-32":2,"2015-27":3,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":3,"2014-42":8,"2014-41":5,"2014-35":6,"2014-23":5,"2014-15":3,"2023-14":1,"2015-18":2,"2015-11":3,"2015-06":3,"2014-10":3,"2013-48":3,"2013-20":3}},"file":"PMC1868034"},"subset":"pubmed_central"}
{"text":"abstract: Gastric bypass surgery leads to marked improvements in glucose tolerance and insulin sensitivity in obese type 2 diabetes (T2D); the impact on glucose fluxes in response to a physiological stimulus, such as a mixed meal test (MTT), has not been determined. We administered an MTT to 12 obese T2D patients and 15 obese nondiabetic (ND) subjects before and 1 year after surgery (10 T2D and 11 ND) using the double-tracer technique and modeling of \u03b2-cell function. In both groups postsurgery, tracer-derived appearance of oral glucose was biphasic, a rapid increase followed by a sharp drop, a pattern that was mirrored by postprandial glucose levels and insulin secretion. In diabetic patients, surgery lowered fasting and postprandial glucose levels, peripheral insulin sensitivity increased in proportion to weight loss (\u223c30%), and \u03b2-cell glucose sensitivity doubled but did not normalize (compared with 21 nonsurgical obese and lean controls). Endogenous glucose production, however, was less suppressed during the MMT as the combined result of a relative hyperglucagonemia and the rapid fall in plasma glucose and insulin levels. We conclude that in T2D, bypass surgery changes the postprandial response to a dumping-like pattern and improves glucose tolerance, \u03b2-cell function, and peripheral insulin sensitivity but worsens endogenous glucose output in response to a physiological stimulus.\nauthor: Stefania Camastra; Elza Muscelli; Amalia Gastaldelli; Jens J. Holst; Brenno Astiarraga; Simona Baldi; Monica Nannipieri; Demetrio Ciociaro; Marco Anselmino; Andrea Mari; Ele FerranniniCorresponding author: Stefania Camastra, <firstname.lastname@example.com>.\ndate: 2013-11\nreferences:\ntitle: Long-Term Effects of Bariatric Surgery on Meal Disposal and \u03b2-Cell Function in Diabetic and Nondiabetic Patients\n\nMounting evidence supports bariatric surgery as a powerful intervention to induce remission in patients with type 2 diabetes (T2D) (1,2) and to prevent or delay incident T2D (3). This has engendered enthusiasm for bariatric surgery as a treatment for T2D (4) and has encouraged a broadening of the BMI range as an indication for surgery in diabetic patients (5).\n\nAlthough weight loss and, in the early postoperative period, caloric deficit certainly make a contribution to improve glucose tolerance, surgery itself may trigger weight-independent mechanisms eventually translating into favorable metabolic effects. This postulate is based on early animal studies (6) and, in humans, on evidence that metabolic changes sometimes precede sizeable weight loss or are disproportionate to the amount of weight lost (7). In this regard, there is evidence that different bariatric procedures (e.g., Roux-en-Y gastric bypass \\[RYGB\\], biliopancreatic diversion, and sleeve gastrectomy) may engage putative weight-independent mechanisms to different extents or involve altogether different mechanisms (8,9).\n\nA number of previous studies have documented the effects of the most popular bariatric operation, RYGB, on glycemic control and incretin hormones (10\u201324), and mechanistic studies have explored the ability of RYGB to enhance insulin action and \u03b2-cell function. The great majority of these studies have used methods based on fasting measurements (e.g., homeostasis model assessments), oral glucose tolerance test\u2013based surrogate indices of insulin sensitivity and \u03b2-cell function, or euglycemic-hyperinsulinemic clamp settings (14,16\u201321). A recent study (24) has taken a more physiological approach by comparing the impact of RYGB and gastric banding on the disposition of a mixed meal, with the use of a double-tracer technique, in nondiabetic (ND) subjects studied before and shortly after the operation (\u223c20 weeks). In the current study of morbidly obese patients with T2D, we aimed at measuring the impact of RYGB on chief physiologic determinants of meal disposal long after surgery (when body weight and metabolic adaptation have stabilized) and assessing their relation to weight loss and the attendant changes in the hormonal milieu.\n\n# RESEARCH DESIGN AND METHODS\n\n## Subjects.\n\nWe studied 12 morbidly obese patients with T2D and 15 sex- and BMI-matched morbidly obese ND patients. Diabetes was newly diagnosed in three patients, whereas in the other nine patients, diabetes duration was 3.9 \u00b1 1.2 years (range 1\u201310). HbA~1c~ was 7.2 \u00b1 0.4% (55 \u00b1 5 mmol\/mol); six patients were being treated by diet alone and six by oral hypoglycemic agents (three by metformin alone and three by metformin plus a sulfonylurea). Antidiabetic medication was discontinued 1 week before the metabolic studies. These 27 subjects all underwent laparoscopic RYGB; 10 T2D and 11 ND patients were restudied 1 year after surgery. Two control groups were included, consisting of 7 lean healthy volunteers and 14 obese ND volunteers whose BMI was matched to that of the RYGB patients at 1 year postsurgery. Thus, a total of 69 complete metabolic studies were performed.\n\nThis study was approved by the local ethics committee. The nature and purpose of the study were carefully explained to all participants before they provided written consent to participate.\n\n## Study design.\n\nAt baseline and follow-up, subjects received a mixed meal test (MTT) with a double-tracer protocol. In brief, after an overnight (12-h) fast, subjects were admitted to our Clinical Research Unit at 8:00 <span class=\"smallcaps\">a.m.<\/span>, and a polyethylene cannula was inserted into an antecubital vein for the infusion of all test substances. A second catheter was inserted retrogradely into an ipsilateral wrist vein on the dorsum of the hand for blood sampling, and the hand was kept in a heated box at 65\u00b0C to achieve arterialization of venous blood. Baseline blood samples were drawn to measure plasma glucose, insulin, C-peptide, glucagon, glucagon-like peptide 1 (GLP-1), and gastric inhibitory polypeptide (GIP) concentrations and tracer enrichments. The MTT consisted of 75 g glucose in 150 mL water, 40 g parmesan cheese, and one 50-g egg (509 kcal; 16% protein, 28% fat, and 56% carbohydrate). The glucose solution was drunk after the solid component of the meal was consumed over a period of 10 min. The oral glucose drink was enriched with 1-\\[^2^H\\]glucose in order to trace glucose absorption. A primed (28 \u00b5mol \u22c5 kg^\u22121^ \u00d7 \\[fasting plasma glucose\\]\/5) continuous (0.28 \u00b5mol \u22c5 min^\u22121^ \u22c5 kg^\u22121^) infusion of 6,6-\\[^2^H~2~\\]glucose was started (at time \u2013120 min, or at \u2013180 min in the T2D patients) via the antecubital vein catheter and continued until the end of the study. During the last 20 min of the basal equilibration period (at times \u201320, \u201310, and 0 min), blood samples were obtained for the determination of plasma glucose and hormone concentrations and tracer enrichments (when isotopic steady state was achieved). After the basal equilibration period, the meal was consumed over \u223c10 min. Plasma samples for the determination of plasma glucose and hormone concentrations and glucose tracer enrichments were obtained at 15, 30, 45, 60, 90, 120, 150, 180, 240, and 300 min after meal ingestion.\n\n## Surgical procedure.\n\nIn subjects undergoing laparoscopic RYGB, a small proximal gastric pouch of 30 mL was created with several firings of a linear stapling endocutter; the jejunum was divided 120 cm distal to the ligament of Treitz, and a 2-cm end-to-side gastrojejunostomy was performed by using a hand-sewn technique. A side-to-side jejuno-jejunostomy was then created, 150 cm distal to the gastrojejunostomy (17).\n\n## Analytical procedures.\n\nPlasma glucose was measured by the glucose-oxidase technique (Analox GM-9) and plasma insulin and C-peptide by electrochemiluminescence (on a COBAS e411 instrument; Roche, Indianapolis, IN). Plasma triglycerides and serum HDL cholesterol were assayed in duplicate by standard spectrophotometric methods on a Synchron Clinical System CX4 (Beckman Instruments, Fullerton, CA). 6,6-\\[^2^H~2~\\]glucose and 1-\\[^2^H\\]glucose enrichments were measured by gas chromatography\u2013mass spectrometry as described previously (25). Plasma glucagon was assayed by radioimmunoassay (Millipore Corporation, Billerica, MA). Plasma GLP-1 and GIP were assayed by radioimmunoassay as described previously (26). No significant cross-reactivity was declared by kit manufacturers. The sensitivity ranges are 0.003 nmol\/L and 1.39 pmol\/L for the C-peptide and insulin assay, respectively, 18.5 pg\/mL for glucagon, and 1.5 pmol\/L for GLP-1.\n\n## Data analysis.\n\nFat-free mass was estimated with the use of electric bioimpedance on a Tanita scale; of note, this method has been validated in very obese individuals against the deuterated water technique (27). Endogenous glucose production (EGP) in the fasting state and throughout the absorptive period and rate of appearance of the oral glucose component (RaO) of the meal were calculated from the time course of the plasma tracer\/tracee ratio of 6,6-\\[^2^H~2~\\]glucose and 1-\\[^2^H\\]glucose by two-compartment modeling, as previously described (25). Insulin sensitivity was calculated as the mean metabolic clearance rate of glucose (MCR~G~) during the 5 h of the MTT (from 6,6-\\[^2^H~2~\\]glucose kinetics) divided by the mean plasma insulin concentration over the same time interval.\n\n\u03b2-Cell function was quantitated by mathematical modeling of the plasma C-peptide response, as previously described (28). In brief, the model consists of three blocks: *1*) a model for fitting the glucose concentration profile, the purpose of which is to smooth and interpolate plasma glucose concentrations; *2*) a model describing the dependence of insulin (or C-peptide) secretion on glucose concentration; and *3*) a model of C-peptide kinetics, i.e., the two-exponential model proposed by Van Cauter et al. (29), in which the model parameters are individually adjusted to the subject's anthropometric data. The main parameter of the model is \u03b2-cell glucose sensitivity (\u03b2-GS), which is calculated as the mean slope of the dose-response function (i.e., relationship between insulin secretion rates \\[ISRs\\] and plasma glucose concentrations during corresponding times of the MTT). The model also yields an estimate of glucose rate sensitivity, which is the insulin secretory response to the rate of change in plasma glucose concentrations, and total insulin output, which is the total amount of insulin released over the 5 h of the meal (28). Plasma insulin clearance (MCR~I~) was estimated as the ratio of fasting ISR to the fasting plasma insulin concentration.\n\nThe prehepatic insulin-to-glucagon molar concentration ratio was estimated by the following formula: {ISR\\[*t*\\]\/hPF + \\[I(*t*)\\]}\/{\\[Glg(t)\\] \u00d7 \\[1 + MCR~Glg~\/hPF\\]}, where ISR(*t*) is the ISR at time *t*, hPF is hepatic plasma flow, \\[I(*t*)\\] and \\[Glg(*t*)\\] are the measured peripheral plasma concentrations of insulin and glucagon at time *t*, and MCR~Glg~ is the metabolic clearance rate of glucagon. hPF was estimated by multiplying the cardiac index (3.2 L \u22c5 min^\u20131^ \u22c5 m^\u22122^) (30) by a plasma-to-blood ratio of 0.6, and by assuming that hepatic blood flow is 30% of cardiac index (=0.576 L \u22c5 min^\u22121^ \u22c5 m^\u22122^) (31). MCR~Glg~ was taken to be 0.537 L \u22c5 min^\u22121^ \u22c5 m^\u22122^ (32).\n\nThe product of mean EGP and mean plasma insulin levels during the meal was taken as an index of hepatic insulin resistance. Areas under time-concentration curves (AUCs) were calculated by the trapezium rule.\n\n## Statistical analysis.\n\nAll data are given as the mean \u00b1 SEM; parameters that were nonnormally distributed are presented as the median (interquartile range). Mann-Whitney test was used to compare group values, whereas surgery-induced changes were tested by the Wilcoxon signed rank test. Time series were analyzed by ANOVA for repeated measures; for these tests, variables with skewed distribution were log transformed. Group differences over time series were analyzed by two-way ANOVA for repeated measures. Simple associations were tested by calculating the Spearman rank correlation coefficient (*\u03c1*). Statistical analyses were performed using JMP7.0 and StatView5.0. *P* \u2264 0.05 was considered statistically significant.\n\n# RESULTS\n\n## Baseline.\n\nThe four subject groups were matched for sex and age; T2D patients were older and had moderate fasting hyperglycemia (<a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>). After meal ingestion, plasma glucose excursions were similar in lean and obese controls and ND surgical patients, whereas T2D patients showed marked hyperglycemia (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>A<\/em><\/a>). Fasting and postprandial insulin concentrations were higher in all three obese groups than in lean controls (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>B<\/em><\/a> and <a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a>). Fasting ISR generally increased with obesity as did the total insulin output over the 5 h of the test (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>C<\/em><\/a> and <a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a>). In T2D patients, total insulin output was not different from that of lean controls despite the hyperglycemia. Consequently, \u03b2-GS was markedly impaired in this group (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>D<\/em><\/a> and <a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a>).\n\nAnthropometric and metabolic parameters\n\n![](3709tbl1)\n\nGlucose metabolism and insulin secretion\n\n![](3709tbl2)\n\nOn the MTT, the RaO was similar in all groups, both in time course (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>E<\/em><\/a>) and amount (averaging 51, 52, 50, and 62 g over 5 h in lean control subjects, obese control subjects, ND surgical patients, and T2D surgical patients, respectively) (<a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a>). In contrast, fasting EGP was higher in each obese group (700 \\[277\\], 771 \\[242\\], and 764 \\[395\\] \u00b5mol\/min) than in lean controls (580 \\[51\\] \u00b5mol \u22c5 min^\u22121^) and remained higher during the MTT despite the postmeal hyperinsulinemia (AUC~EGP~ = 79 \\[46\\], 100 \\[56\\], and 100 \\[97\\] mmol vs. 61 \\[15\\] of controls). The difference in EGP time course between each of the obese groups and lean controls (confirmed by repeated-measures ANOVA, *P* \\< 0.001 for all) was evident after the initial nadir (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>F<\/em><\/a>). In the whole dataset, both fasting EGP and AUC~EGP~ were positively related to BMI (*r* = 0.48, *P* = 0.001, and *r* = 0.30, *P* = 0.05, respectively). Plasma glucose clearance rose during the MTT in all groups; its mean value over 5 h was reduced in both surgical groups, especially in T2D patients, in comparison with lean controls despite the hyperinsulinemia (<a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a>). As a consequence, insulin sensitivity, as the insulin-mediated MCR~G~, was lower in both obese groups and severely impaired in T2D patients (<a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a>).\n\nIn all obese groups, fasting plasma glucagon was increased, whereas the meal-induced glucagon increments were blunted. Fasting GLP-1 and GIP levels were increased only in T2D patients, as was AUC~GIP~ (<a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a>).\n\n## After surgery.\n\nAt 1 year, ND and T2D patients had lost 31 and 35% of their initial body weight, respectively, in an approximate ratio of 1:3 between fat-free mass and fat mass (<a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>). In T2D patients, HbA~1c~ had dropped by an average 1.5%, and all patients were off antidiabetic drug treatment; of them, two patients had impaired glucose tolerance and one of these two also had impaired fasting glucose (5.9 mmol\/L). Four patients experienced dumping-like symptoms (tachycardia, sweating, nausea, and diarrhea) after meal ingestion.\n\nOn the MTT, the postsurgery plasma glucose and insulin profiles were grossly altered in both ND and T2D subjects, with a large excursion peaking at 60 min followed by a sharp drop to basal levels or below (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<\/a>). In the T2D group, the glucose and insulin AUCs were significantly smaller than preoperatively (<a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a>). The time course of insulin secretion ran parallel to that of plasma glucose (<a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<\/a>); fasting insulin secretion decreased in both groups and total insulin output decreased in the ND group (<a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a>). When viewing insulin secretion in the context of the corresponding plasma glucose levels, \u03b2-GS was significantly reduced in the ND group postsurgery (although still within the range of the obese control group), and \u223c100% increased in T2D patients (although still largely below control values) (<a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<\/a>). The small decline in \u03b2-GS in ND subjects could be due to chronically reduced carbohydrate intake or could simply be time related. Insulin sensitivity improved significantly in both groups (by two-way ANOVA from repeated measures, *F* = 10.5, *P* \\< 0.005) to levels similar to those of the obese controls. The estimates of insulin sensitivity fell along the overall relationship describing the association of insulin resistance with BMI, and the surgically induced changes in insulin sensitivity were correlated with the corresponding changes in body weight (<a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a> and <a href=\"#F4\" data-ref-type=\"fig\">Fig. 4<\/a>).\n\nIn both ND and T2D subjects postsurgery, the rate of appearance of oral glucose was similar in total amount (<a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a>) but distorted in time course, in a manner resembling the plasma glucose curves closely (<a href=\"#F5\" data-ref-type=\"fig\">Fig. 5<\/a>). Thus, most oral glucose was absorbed during the first hour after meal ingestion, as the AUC calculated in the first 60 min showed (AUC pre- vs. postsurgery: 122 \\[63\\] vs. 186 \\[37\\] mmol in ND and 129 \\[81\\] vs. 190 \\[94\\] mmol in T2D, *P* \\< 0.04 vs. postsurgery for both). EGP was unchanged in ND as well as T2D patients in the fasting state but rebounded significantly above presurgery values during meal absorption, particularly over the second half of the postcibal period when plasma glucose concentrations were back to basal or below. The EGP time course was increased in both groups (with no significant difference between T2D and ND patients, *P* = 0.74), whether expressed in absolute terms (<a href=\"#F5\" data-ref-type=\"fig\">Fig. 5<\/a>) or normalized by fat-free mass (*F* = 133.9, *P* \\< 0.0001) (<a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a>).\n\nFasting glucagon levels dropped in both ND and T2D, but the response to the meal (as the incremental AUC) was greatly enhanced in both groups (<a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a> and <a href=\"#F6\" data-ref-type=\"fig\">Fig. 6<\/a>). After surgery, the estimated prehepatic insulin-to-glucagon molar concentration ratio was higher than presurgery (especially in T2D patients) for the first 80\u2013100 min but then fell below preoperative levels for the remainder of the MTT in both groups ([Supplementary Fig. 1](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db13-0321\/-\/DC1)).\n\nThe GLP-1, but not the GIP, response was increased in ND as well as T2D; the time profile of the GLP-1 and GIP responses showed a sharper early rise and a rapid drop thereafter (<a href=\"#F6\" data-ref-type=\"fig\">Fig. 6<\/a>).\n\nMCR~I~, calculated as the ratio of fasting insulin output to fasting peripheral plasma insulin concentrations, was slightly reduced in the obese groups as compared with the lean controls and was significantly increased postsurgery in both ND and T2D (two-way ANOVA).\n\n# DISCUSSION\n\nBefore the operation, the metabolic response to the mixed meal in the ND obese subjects was characterized by insulin resistance of glucose disposal and insulin hypersecretion, with preserved \u03b2-GS and rate sensitivity, i.e., the metabolic picture of obesity. In the T2D patients, insulin resistance was worse, and both \u03b2-GS and rate sensitivity were markedly impaired, thereby accounting for the hyperglycemia. Whereas the rate of appearance of oral glucose was similar in time course and total amount in all groups, in obese and T2D patients, EGP was higher at baseline and was incompletely suppressed during the meal, in some proportion to the degree of obesity, despite the hyperinsulinemia, thereby manifesting liver insulin resistance. In the T2D patients, the blunted rise in the prehepatic insulin-to-glucagon ratio during the first 2 h postmeal ([Supplementary Fig. 1](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db13-0321\/-\/DC1)) likely contributed to the elevated EGP.\n\nOne year after RYGB, at a time when drastic weight loss had occurred and body weight had stabilized, fasting plasma glucose was similar in ND and diabetic patients, but in the latter, 2-h glucose concentrations were significantly higher (5.93 \u00b1 0.53 vs. 4.23 \u00b1 0.31 mmol\/L, *P* = 0.02), and mean glucose levels during the 5-h meal tended to be higher (<a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a>), than in ND surgical patients. Insulin sensitivity improved in both surgical groups to levels close to those of the BMI-matched control group, i.e., in rough proportion to the weight loss and without reaching the values of the lean control subjects (<a href=\"#F4\" data-ref-type=\"fig\">Fig. 4<\/a>). This result, obtained by tracer analysis of a dynamic test such as the MTT, confirms the findings of steady-state (clamp) measurements of insulin sensitivity (17), namely, that RYGB does not potentiate insulin action beyond, or independenty of, the effect on weight loss. \u03b2-GS worsened slightly in the ND surgical patients, whereas it doubled in the diabetic patients, in whom, however, it remained markedly inferior to that of BMI-matched ND subjects. This outcome prevailed despite the large increase in GLP-1 response, a consistent change after bariatric surgery (10\u201317), even as long as 10 years after surgery (33), which is quantitatively related to enhanced insulin secretion (19). In both groups, rate sensitivity improved postsurgery, likely reflecting the rapid plasma glucose excursions as well as, at least in part, the heightened GLP-1 surge ([Supplementary Fig. 2*A*](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db13-0321\/-\/DC1)). The latter finding is compatible with evidence that a rapid rather than delayed delivery of insulin improves glucose tolerance irrespective of the degree of insulin resistance (34), and may explain why meal tolerance in our patients was preserved 1 year after surgery despite the abnormal \u03b2-GS.\n\nThus, in T2D patients, recovery of both insulin sensitivity and glucose sensitivity was sizeable but incomplete, leaving behind a trace of glucose intolerance. These results confirm previous findings in both ND and T2D subjects, using a liquid formula meal and homeostasis model assessment of insulin resistance as a surrogate index of insulin resistance (35), or an oral glucose tolerance test (18). Further increments in both functions may occur if more weight is lost or as removal of the toxic effects of hyperglycemia continues; on the other hand, diabetes may relapse if weight is regained, if insulin resistance otherwise worsens, or if the disease itself should progress. In addition, as previously shown, in T2D patients, the outcome of glucose tolerance at 1 year post-RYGB may be better or worse than in the present series of patients depending on the initial degree of \u03b2-cell dysfunction (18).\n\nAs found by Bradley et al. (24) in ND subjects, RYGB drastically changed the shape of the glucose and hormonal responses to the meal, with a markedly biphasic pattern reminiscent of a dumping syndrome. Glucose fluxes confirmed that this was the consequence of the altered delivery of gastric contents to the peripheral circulation. A somewhat unexpected consequence of the altered pattern of transit of alimentary glucose was the reduced suppression of endogenous glucose release during the meal. Under euglycemic clamp conditions, hepatic and peripheral insulin resistance are typically somewhat interrelated and change consensually (36). A mixed meal, however, creates a hormonal makeover by stimulating both insulin and glucagon secretion, especially in diabetic patients in whom glucagon responses are exaggerated (37). In our surgical patients, both fasting insulin and fasting glucagon concentrations fell significantly postsurgery; correspondingly, fasting EGP did not change after the operation. After the meal, the prehepatic insulin-to-glucagon molar ratio normally rises in a time course roughly parallel to that of plasma glucose concentrations and ISRs; in T2D patients, the ratio shows the blunted initial rise followed by a sustained increase typical of the insulin secretory response ([Supplementary Fig. 1](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db13-0321\/-\/DC1)). After surgery, however, plasma glucagon rose sharply and remained above basal levels throughout the absorption period both in ND and T2D patients (<a href=\"#F6\" data-ref-type=\"fig\">Fig. 6<\/a>), such that the prehepatic insulin-to-glucagon ratio, after peaking at \u223c1 h postmeal, dropped rapidly to levels below presurgery ([Supplementary Fig. 1](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db13-0321\/-\/DC1)), in phase with the lower plasma glucose levels. Thus, the raised postmeal EGP was the integrated liver response to lower insulin, higher glucagon, and falling glucose levels. The correlation between the calculated index of hepatic insulin resistance and the glucagon response supports the role of the relative hyperglucagonemia ([Supplementary Fig. 2*B*](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db13-0321\/-\/DC1)). It should be noted that in the cited study in ND subjects (24), the time course of EGP was similar to ours but the glucagon response was rather flat and unchanged from presurgery. This difference may be due to the use of a smaller meal (\u223c300 kcal with only 9 g of protein compared with 500 kcal in the current study) and\/or the shorter time interval from surgery (22 weeks), a time at which patients are typically still losing weight. The stimulus for the meal-induced hyperglucagonemia 1 year after surgery, which has been noted before (15), remains undetermined. Diminished paracrine control of \u03b1-cell activity by insulin (38) seems unlikely as the glucagon excursions in our patients were essentially synchronous with those of plasma insulin. Potential mechanisms are enhanced postprandial neural stimulation (39), overstimulation of glucagon release by GIP (40) or GLP-2 (41), and cosecretion of glucagon and GLP-1\/GIP by intestinal cells (42). In our data, the postsurgery time courses of glucagon and GIP were in phase, lending some support to an effect of GIP on \u03b1-cells. It should also be considered that the standard glucagon assay has limitations in terms of its ability to discriminate between pancreatic glucagon and other sources (43).\n\nAlthough gastric emptying was not measured in the present studies, our previous work in post-RYGB patients (using a scintigraphic technique) has confirmed that the operation causes accelerated gastric emptying even 14\u201326 months later (44); this result is fully compatible with the accelerated appearance of oral glucose in the present series.\n\nIn summary, the long-term outcome of RYGB is a comparable weight reduction and a proportional improvement in insulin sensitivity in ND and well-controlled, recent-onset diabetic patients. In both, delivery of oral glucose to the peripheral circulation is maintained in quantity but strikingly changed in time course, with a \"dumping\" pattern resulting from the modified anatomy. Glucose tolerance is preserved in ND and much improved in diabetic patients; in the latter, a detectable degree of glucose intolerance persists. In diabetic patients, \u03b2-cell function improves presumably as a combined result of reversal of glucose toxicity, lower insulin secretory burden, and incretin-mediated potentiation, but \u03b2-GS remains subnormal. This may predispose patients to recurrent diabetes. The surgery-induced change in glucose delivery triggers not only a heightened GLP-1 response but also an exaggerated glucagon response synchronous with the glucose excursions, which likely contributes to maintain euglycemia through elevated rates of EGP.\n\n## ACKNOWLEDGMENTS\n\nThis work was aided in part by a grant from the Italian Ministry of Education, University and Research (PRIN 2010329EKE).\n\nNo potential conflicts of interest relevant to this article were reported.\n\nS.C. designed the study, performed the in vivo studies, analyzed the data, and wrote the manuscript. E.M. performed the in vivo studies and contributed to the discussion. A.G. and D.C. analyzed the tracer data. J.J.H. was responsible for hormone measurements and contributed to the discussion. B.A. and M.N. performed the in vivo studies. S.B. was responsible for laboratory measurements. M.A. operated on the patients. A.M. analyzed the \u03b2-cell function data. E.F. contributed to the design of the study, analyzed the data, and wrote the manuscript. S.C. and E.F. are the guarantors of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.\n\nThe authors are grateful to Dr. Laura Manca (University of Pisa) for help with the statistical analysis.\n\n# REFERENCES","meta":{"dup_signals":{"dup_doc_count":115,"dup_dump_count":47,"dup_details":{"curated_sources":2,"2021-21":1,"2021-17":3,"2021-10":1,"2019-47":1,"2019-04":1,"2018-26":2,"2018-22":1,"2018-17":3,"2018-09":1,"2018-05":2,"2017-51":1,"2017-47":2,"2017-43":4,"2017-39":2,"2017-34":2,"2017-30":3,"2017-26":3,"2017-22":2,"2017-17":5,"2017-09":4,"2017-04":6,"2016-50":4,"2016-44":5,"2016-40":5,"2016-36":5,"2016-30":5,"2016-26":3,"2016-22":5,"2016-18":2,"2016-07":1,"2015-48":1,"2015-35":1,"2015-32":1,"2015-27":1,"2015-22":1,"2015-14":2,"2014-52":2,"2014-49":1,"2014-41":3,"2014-35":1,"2014-23":3,"2023-23":1,"2017-13":4,"2015-18":2,"2015-11":2,"2015-06":1,"2024-22":1}},"file":"PMC3806605"},"subset":"pubmed_central"}
{"text":"abstract: A report from the Abcam genome stability conference 'Maintenance of Genome Stability', Jolly Beach Resort, Antigua, 8-11 March 2010.\nauthor: Kyle M Miller\ndate: 2010\ninstitute: 1The Gurdon Institute and Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK\ntitle: Advances in understanding genome maintenance\n\n'Maintenance of Genome Stability' is a biennial meeting that brings together, in a fantastic venue, diverse researchers working on how the integrity of genomes is maintained. Topics included DNA repair pathways, replication and recombination, and common themes included how these processes are regulated during the cell cycle, in the context of their chromatin or genomic location, and their involvement in cancer. Here, I summarize some highlights of the meeting, which included large-scale genomic studies, work on post-translational modifications in genome maintenance, and insights into new mechanisms and proteins involved in DNA repair pathways, telomeres and cancer.\n\n# Mass spectrometry proteomics\n\nPost-translational modifications in DNA damage signaling was a common theme throughout the meeting. Jiri Bartek (Centre for Genotoxic Stress Research, Copenhagen, Denmark) presented a genome-wide proteomics screen, using quantitative mass spectrometry (stable isotope labeling with amino acids in cell culture, SILAC), for phosphorylations of nuclear proteins that change following DNA damage in human cells. Over 7,000 phosphorylation sites were detected, with 2,000 being novel and not described in other proteomic screens. The power of this screen was that it analyzed the temporal regulation of phosphorylations after DNA damage. To accomplish this, cells were analyzed at time zero (no damage) and then at 5 minutes, 20 minutes, 1 hour and 8 hours after DNA damage. Taking into account only those phosphorylations detected at all time points, almost 600 phosphorylations were found to change by over twofold. Induced phosphorylations and dephosphorylations were detected. Phosphorylations that increased at early time points after DNA damage were enriched in substrates for the kinase ataxia-telangiectasia mutated (ATM). Looking at the amino acid sequences surrounding the phosphorylation sites, a new phosphorylation consensus sequence, SxxQ, was determined that does not seem to be mediated by any known kinase. This dataset is very likely to be a useful resource for researchers interested in phosphorylation in the DNA damage response (DDR).\n\nPost-translational modifications other than phosphorylation are also important for the DDR, and it is clear that ubiquitylation has a key role in DNA damage signaling. Work from Jiri Lukas' group (Center for Genotoxic Stress Research, Copenhagen, Denmark) and others have added strong evidence for this notion by identifying two ubiquitin E3 ligases, RNF8 and RNF168, as important regulators of DNA damage signaling. To gain insights into the substrates of these two enzymes, Lukas' group performed quantitative mass spectrometry (SILAC) of 6 \u00d7 His-Flag-Ub-containing complexes from cells lacking RNF8 or RNF168. Many DDR proteins were found to be ubiquitylated in an RNF8- or RNF168-dependent manner. Of the histones, only histone H2A and histone H2AX were purified and verified as substrates. Therefore, ubiquitin not only decorates the chromatin around a double-strand break (DSB) but also is conjugated to many signaling proteins, which again highlights the importance of this modification in the DDR. An understanding of how ubiquitylation affects DNA repair and signaling proteins to bring about proper repair is guaranteed to be a continued topic of research.\n\nChromatin is an important component in promoting genomic and epigenomic stability. Anja Groth (BRIC, University of Copenhagen, Denmark) discussed recent findings from her group showing the histone modifications on histones H3 and H4 that are associated with the histone chaperone Asf1b in S phase or in response to replication impaired by hydroxyurea (HU). Complexes of Asf1b containing histones H3 and H4 were purified and quantified by mass spectrometry. Groth described how all Asf1b-bound H4 molecules were acetylated on Lys5 and Lys12; histone H3 was 30% acetylated on Lys18 and 20% acetylated on Lys14; and H3 was also mono-methylated at Lys9. Acetylation of H3 on Lys56 was barely detectable, suggesting that this mark is not a general mark of histones in S phase. During replication stress (HU), methylation of H3 on Lys9 (H3K9me1) increased significantly in Asf1b complexes. Thus, this epigenetic mark is deregulated after replication stress, and Asf1b seems to have a role in sequestering H3 molecules containing this mark. As H3K9me1 can affect epigenetic silencing, this work suggests that replication stress could affect epigenomic integrity through Asf1b.\n\n# Site-specific DNA double-strand breaks\n\nThe DNA damage field has long awaited the arrival of a robust system for studying site-specific DSBs in mammalian cells. This wait might be over because two research groups revealed such systems. Gaelle Legube (University of Toulouse, France) showed a new site-specific DSB system that uses a rare cutting restriction enzyme that is regulatable. The break sites are found throughout the genome and can be analyzed simultaneously. Chromatin immunoprecipitation microarray (ChIP-chip) analysis demonstrated robust phospho-histone H2AX (\u03b3H2AX) formation at many predicted genomic break sites. Legube found that large \u03b3H2AX domains formed bidirectionally from the DNA breaks but were not uniform or symmetrical, suggesting genomic boundaries that limit \u03b3H2AX spreading. Active transcription still occurred in \u03b3H2AX domains, although the level of \u03b3H2AX was reduced around transcription start sites, possibly because of increased histone dynamics.\n\nRoger Greenberg (University of Pennsylvania, Philadelphia, USA) presented data from a system that uses an integrated Lac operator (LacO) array that is positioned approximately 4 kb upstream of a transcriptionally active site, which can be monitored in real time for DSB responses and nascent transcript formation. In this system, a LacI-FokI nuclease fusion protein is expressed that site-specifically binds the LacO array and creates site-specific damage. This is a great tool to look at the effects of DNA damage on transcription. Indeed, Greenberg's group found that, following DNA damage, transcription was rapidly halted, with a concomitant compaction of the genomic region due to DSB-induced inhibition of RNA polymerase II elongation. These effects were all dependent on the DDR kinase ATM. The transcriptional silencing occurred *in cis*, as creation of DNA damage outside this region did not affect transcription from the reporter locus. Silencing was rapidly reversible following DSB repair, as were all the effects described. Finally, ubiquitylated histone H2A was detected at this region and its deubiquitylation by the ubiquitin-specific peptidase 16 (USP16) was required for resumption of transcription following DNA repair. Thus, DNA damage within multiple kilobases of a transcriptionally active locus results in transcriptional repression that is dependent on ATM and ubiquitin.\n\n# Novel proteins and processes in genome maintenance\n\n## Deubiquitylation in the DNA damage response\n\nAs ubiquitin conjugation has been linked with DNA damage surveillance and repair, systems must be present to modulate or counteract these pathways. Daniel Durocher's group (Samuel Lunenfeld Research Institute, Toronto, Canada) has pioneered the use of genome-wide screens using small interfering RNA (siRNA) to look microscopically at how knockdown of individual genes affects DNA damage signaling and protein localization at DNA damage sites. Durocher's group previously identified two ubiquitin E3 ligase enzymes, RNF8 and RNF168, that are required for DNA repair through their combined ubiquitylation of substrates, including histones, at sites of DNA damage. Durocher's group has now performed a siRNA screen targeting the human deubiquitylating enzymes (DUBs) to analyze how these enzymes can affect ubiquitin conjugates at DNA damage sites. Daniel Durocher presented in a talk his groups findings from this screen that revealed several candidate genes whose knockdown resulted in the accumulation of ubiquitin conjugates at unrepaired DNA damage. Thus, these data show that systems do indeed exist that function to deubiquitylate substrates at sites of DNA damage. Defining the DUBs and their key targets that function in the DDR, as well as determining how ubiquitin affects these pathways, are important questions in the field. As the ubiquitin pathway is extremely complex, answers to these questions will undoubtedly uncover many surprises and interesting biology that will be important in advancing our understanding of the role of ubiquitin in genome maintenance.\n\n## Fanconi anemia\n\nFanconi anemia (FA) is a rare genetic disorder resulting in an increased incidence of some cancers and heightened cellular sensitivity to inter-strand DNA cross-linking agents. John Rouse (University of Dundee, UK) and Josef Jiricny (University of Zurich, Switzerland) introduced a new FA-associated protein, REND1\/KIAA1018, which functions as a 5' flap endonuclease and an exonuclease in the repair of inter-strand crosslinks. Following DNA damage, this protein forms foci by binding the ubiquitin-conjugated form of Fanconi anemia group D2 protein (FANCD2), revealing that it functions downstream of FANCD2. Loss of REND1\/KIAA1018 resulted in hypersensitivity to inter-strand DNA crosslinks, showing that it is a *bona fide* participant of the FA pathway. Whether mutations in REND1\/KIAA1018 are found in human patients suffering from Fanconi anemia is yet to be determined but this work suggests that there may be additional, as-yet-unidentified proteins that function in this complex repair pathway, which is essential for maintaining genome stability.\n\n## Telomeres and cancer\n\nAneuploidy is prominent in many cancers and it has been hypothesized that tetraploidy initializes these events. Dysfunctional telomeres are also a common initiating event in cancer but a link between these two processes has not yet been established. Titia de Lange (Rockefeller University, New York, USA) presented data showing how telomere dysfunction can cause endoreduplication, resulting in tetraploidy in p53-deficient cells. Deprotection of telomeres by the absence of the telomere-associated Pot1a\/b proteins resulted in cell cycle arrest, which was dependent on the protein ataxia-telangiectasia and Rad3-related (ATR), and tetraploidy. Using a powerful microscopy technique, fluorescence ubiquitination cell cycle indicator (FUCCI), de Lange showed *in vivo* data of these cells degrading geminin and re-expressing the origin licensing factor Cdt1 in the absence of mitosis, resulting in endoreduplication. When telomere end-protection was reintroduced, these tetraploid cells could resume growth. This mechanism of tetraploidization is not limited to telomere deprotection because DNA damage can result in the same outcome. Thus, these data reveal how telomeres and DNA damage can result in tetraploidy, a cellular state seen in cancer and implicated in tumorigenesis.\n\nThis successful conference series has become a must-attend meeting in the field of genome stability. Although details are unavailable for 2012, this meeting is sure to supply great science to people who are interested in the mechanisms that govern genome integrity.\n\n## Acknowledgements\n\nI thank the Wellcome Trust for providing my funding to attend this meeting.","meta":{"dup_signals":{"dup_doc_count":106,"dup_dump_count":45,"dup_details":{"curated_sources":4,"2022-21":1,"2021-49":1,"2019-43":1,"2019-26":1,"2019-18":1,"2019-09":2,"2018-51":1,"2018-47":1,"2018-39":1,"2018-30":2,"2018-13":1,"2017-47":1,"2017-34":1,"2017-22":1,"2017-09":9,"2016-44":1,"2016-40":1,"2016-36":7,"2016-30":9,"2016-22":1,"2016-18":1,"2016-07":5,"2015-48":2,"2015-40":2,"2015-35":2,"2015-32":2,"2015-27":2,"2015-22":2,"2015-14":2,"2014-52":2,"2014-49":1,"2014-42":7,"2014-41":2,"2014-35":3,"2014-23":4,"2014-15":2,"2023-23":1,"2024-10":1,"2015-18":2,"2015-11":2,"2015-06":2,"2014-10":3,"2013-48":2,"2013-20":3,"2024-26":1}},"file":"PMC2884538"},"subset":"pubmed_central"}
{"text":"abstract: Neutrophil extracellular chromatin traps (NETs) are a recently described mechanism of innate immune responses to bacteria and fungi. Evidence indicates that NETs are induced by inflammation, that they contribute to diverse disease pathologies, and that they associate with bactericidal substances. Genomic DNA is released in NETs, leading to a cell death that has been labeled NETosis. Although NETosis clearly differs from apoptosis, the classical form of cell death, recent experiments indicate a connection between NETosis and autophagy. The regulated deployment of NETs may require covalent modification of histones, the basic DNA-binding proteins that organize chromatin in the cell's nucleus and within NETs. Histone modification by peptidylarginine deiminase 4 (PAD4) is necessary for NET release. The functions of additional histone modifications, however, remain to be tested.\nauthor: Indira Neeli; Marko Radic\ndate: 2012\ninstitute: 1Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, 858 Madison Avenue, Memphis, TN 38163, USA\nreferences:\ntitle: Knotting the NETs: Analyzing histone modifications in neutrophil extracellular traps\n\nSee related research by Liu *et al.*, <http:\/\/arthritis-research.com\/content\/14\/1\/R25>\n\nLess than a decade since their discovery, neutrophil extracellular traps (NETs) remain in the headlines. Initially, interest focused on the structure of extracellular NET chromatin and its capacity to capture and damage bacteria. Soon, however, researchers began to see the implications of extracellular chromatin for the development of autoimmune diseases. One quintessential autoimmune disease, systemic lupus erythematosus (SLE), is known to arise together with autoantibodies to DNA and chromatin, although the immediate trigger for the production of these autoantibodies is unclear. A connection between NETs and autoimmunity was made by discovering that histones, a set of proteins that act as a structural harness for DNA in chromatin, are modified by peptidylarginine deiminase 4 (PAD4), an enzyme that converts arginines to citrullines. Researchers had long suspected that autoantigen modifications could provide the initial stimuli in autoimmunity because subtle alterations in a protein's primary sequence can break tolerance. PAD4 is implicated in the development of rheumatoid arthritis (RA) because the most reliable clinical test for RA uses the detection of anti-citrulline antibodies in the sera of patients.\n\nIn a sophisticated set of experiments reported in the previous issue of *Arthritis Research & Therapy*, Liu and colleagues \\[1\\] accomplished an extensive inventory of post-translational modifications in NET histones. The researchers induced NETs from human neutrophils, as well as two cell lines that assume neutrophil-like characteristics, and used a panel of 40 commercially available antisera to identify histone modifications that arise in parallel with NETs. Stimuli that were used to elicit NET release also induced histone H3 and H4 citrullination in human neutrophils and the EPRO cell line. However, other modifications such as histone H4 lysine 20 methylation and H4 lysine 16 acetylation showed inconsistent results in neutrophils versus the EPRO cells. To survey histone modifications, Liu and colleagues \\[1\\] confronted technical difficulties in that histone amino terminal tails contain the highest concentration of histone modifications yet are also highly susceptible to proteases secreted by activated neutrophils \\[2,3\\]. The histone tails act as flexible tethers that organize chromatin into higher-order structures. Interestingly, purified NETs failed to induce an immune response in mice, although a subset of SLE sera reacted strongly with citrullinated histone H3 \\[1\\]. Therefore, mechanisms that regulate histone modification deserve further attention.\n\nNeeli and colleagues \\[4\\] were the first to identify citrullinated histone H3 in NETs, a discovery that was confirmed by others \\[5\\]. Neeli and colleagues \\[4\\] provided a second important insight, namely that PAD4-citrullinated histone H3 is a reliable marker of inflammation. Thus, it became clear that the release of NETs is not an 'accident' caused by a barrage of proteases and reactive oxygen species unleashed from neutrophils. Instead, production of NETs requires enzymatic activity and input from neutrophil surface receptors and the cytoskeleton \\[6\\]. By analyzing PAD4-deficient mice, Li and colleagues \\[7\\] demonstrated that PAD4 is essential for the production of NETs in response to bacterial infections. The regulation of PAD4 activity thus moved to the forefront of the research on NETs.\n\nIt is now clear that NET release takes advantage of NADPH (nicotinamide adenine dinucleotide phosphate) oxidase and the main granule proteases to trigger and construct the extended chromatin network \\[3,8\\]. In addition, myeloperoxidase is found in NETs after their release from the cells, and this enzyme and its products are the main components in NETs that kill bacteria \\[9\\]. In a notable study from the labs of Banchereau and Pascual \\[10\\], it was reported that SLE neutrophils are poised to undergo NETosis upon stimulation with anti-ribonucleo-protein autoantibodies and that NETs released by these neutrophils contain LL37 and HMGB-1, well-known stimulators of immune responses. In subsequent analyses using sera from patients with connective tissue disease, anti-citrullinated histone antibodies were observed in Felty's syndrome, a rare disorder that shares serologic features with RA and SLE, whereas such autoantibodies were infrequent in SLE and RA \\[11\\]. These findings indicate that the process of NETosis is highly relevant to the development of human autoimmune responses, although a direct cause and effect may not connect the release of NETs to the production of autoantibodies.\n\nThe detailed characterization of NET histone modifications, as accomplished by Liu and colleagues \\[1\\], invites speculations about the possible functions of these modifications. Several questions deserve further study: Will NET histone modifications, such as methylation, acetylation, and citrullination, be found to participate in gene regulation that sets the stage for NET release? Will the primary function of histone modifications turn out to be the decondensation of nuclear chromatin that is required for NETs expand to their optimal size and internal structure? Alternatively, NET histone modifications may serve non-traditional purposes. For example, certain modifications may anchor other NET components such as elastase, LL37, or myeloperoxidase to the chromatin meshwork. Unique modifications in NETs may attract phagocytes and stimulate them to ingest the trapped microorganisms. Other histone modifications may activate or dampen the inflammatory response by acting on innate pattern recognition receptors. The answers to these questions will, no doubt, keep research on NETs in leading immunology and microbiology journals for years to come.\n\n# Abbreviations\n\nNET: neutrophil extracellular trap; PAD4: peptidylarginine deiminase 4; RA: rheumatoid arthritis; SLE: systemic lupus erythematosus.\n\n# Competing interests\n\nThe authors declare that they have no competing interests.\n\n# Authors' contributions\n\nBoth authors contributed to the writing and editing of the text.\n\n## Acknowledgements\n\nThe authors acknowledge the research support from the Lupus Research Institute of New York and the Dana Foundation for Human Immunology. We apologize for our highly focused and therefore incomplete survey of research accomplishments in NET research.","meta":{"dup_signals":{"dup_doc_count":119,"dup_dump_count":37,"dup_details":{"curated_sources":2,"2021-39":1,"2021-04":1,"2020-45":1,"2020-34":1,"2017-34":1,"2017-22":1,"2017-09":8,"2016-50":1,"2016-44":1,"2016-40":2,"2016-36":8,"2016-30":9,"2016-26":1,"2016-18":1,"2016-07":9,"2015-48":3,"2015-40":2,"2015-35":3,"2015-32":3,"2015-27":3,"2015-22":4,"2015-14":2,"2014-52":2,"2014-49":5,"2014-42":6,"2014-41":4,"2014-35":4,"2014-23":6,"2014-15":4,"2023-40":1,"2015-18":3,"2015-11":3,"2015-06":3,"2014-10":3,"2013-48":3,"2013-20":3,"2024-18":1}},"file":"PMC3446426"},"subset":"pubmed_central"}
{"text":"date: 2017\ntitle: Marijuana associated with three-fold risk of death from hypertension\n\n# introduction\n\nMarijuana use is associated with a three-fold risk of death from hypertension, according to research published recently in the European Journal of Preventive Cardiology.\n\n'Steps are being taken towards legalisation and decriminalisation of marijuana in the United States, and rates of recreational marijuana use may increase substantially as a result', said lead author Barbara A Yankey, a PhD student in the School of Public Health, Georgia State University, Atlanta, US. 'However, there is little research on the impact of marijuana use on cardiovascular and cerebrovascular mortality.'\n\nIn the absence of longitudinal data on marijuana use, the researchers designed a retrospective follow-up study of NHANES (National Health and Nutrition Examination Survey) involving participants aged 20 years and older. In 2005\u20132006, participants were asked if they had ever used marijuana. Those who answered 'yes' were considered marijuana users. Participants reported the age when they first tried marijuana and this was subtracted from their current age to calculate the duration of use.\n\nInformation on marijuana use was merged with mortality data in 2011 from the National Centre for Health Statistics. The researchers estimated the associations of marijuana use and duration of use with death from hypertension, heart disease and cerebrovascular disease, controlling for cigarette use and demographic variables including gender, age and ethnicity. Death from hypertension included multiple causes such as primary hypertension and hypertensive renal disease.\n\nAmong a total of 1 213 participants, 34% used neither marijuana nor cigarettes, 21% used only marijuana, 20% used marijuana and smoked cigarettes, 16% used marijuana and were past-smokers, 5% were past-smokers and 4% only smoked cigarettes. The average duration of marijuana use was 11.5 years.\n\nMarijuana users had a higher risk of dying from hypertension. Compared to non-users, marijuana users had a 3.42-times higher risk of death from hypertension and a 1.04 greater risk for each year of use. There was no association between marijuana use and death from heart disease or cerebrovascular disease.\n\nMs Yankey pointed out that there were limitations to the way marijuana use was estimated. For example, it cannot be certain that participants used marijuana continuously since they first tried it.\n\nShe said: 'Our results suggest a possible risk of hypertension mortality from marijuana use. This is not surprising since marijuana is known to have a number of effects on the cardiovascular system. Marijuana stimulates the sympathetic nervous system, leading to increases in heart rate, blood pressure and oxygen demand. Emergency rooms have reported cases of angina and heart attacks after marijuana use.'\n\n'We found higher estimated cardiovascular risks associated with marijuana use than cigarette smoking', said Ms Yankey. 'This indicates that marijuana use may carry even heavier consequences on the cardiovascular system than that already established for cigarette smoking. However, the number of smokers in our study was small and this needs to be examined in a larger study.'\n\n'Needless to say, the detrimental effects of marijuana on brain function far exceed that of cigarette smoking', she added. Ms Yankey said it was crucial to understand the effects of marijuana on health so that policy makers and individuals could make informed decisions.\n\nShe said: 'Support for liberal marijuana use is partly due to claims that it is beneficial and possibly not harmful to health. With the impending increase in recreational marijuana use it is important to establish whether any health benefits outweigh the potential health, social and economic risks. If marijuana use is implicated in cardiovascular diseases and deaths, then it rests on the health community and policy makers to protect the public.'\n\nSource: European Society of Cardiology Press Office","meta":{"dup_signals":{"dup_doc_count":265,"dup_dump_count":63,"dup_details":{"curated_sources":2,"2023-50":4,"2023-40":4,"2023-23":3,"2023-14":6,"2023-06":2,"2022-49":6,"2022-40":2,"2022-33":1,"2022-27":6,"2022-21":1,"2022-05":2,"2021-49":1,"2021-43":4,"2021-39":9,"2021-31":2,"2021-25":4,"2021-21":3,"2021-17":4,"2021-10":1,"2021-04":4,"2020-50":2,"2020-45":6,"2020-40":4,"2020-34":4,"2020-29":4,"2020-24":2,"2020-16":1,"2020-10":7,"2020-05":2,"2019-51":6,"2019-47":4,"2019-43":5,"2019-39":7,"2019-35":7,"2019-30":1,"2019-26":10,"2019-22":4,"2019-18":4,"2019-13":3,"2019-09":4,"2019-04":3,"2018-51":4,"2018-47":5,"2018-43":6,"2018-39":4,"2018-34":7,"2018-30":7,"2018-26":3,"2018-22":4,"2018-17":5,"2018-13":5,"2018-09":7,"2018-05":6,"2017-51":7,"2017-47":4,"2017-43":10,"2017-39":3,"2017-34":8,"2024-26":4,"2024-22":2,"2024-18":1,"2024-10":1,"2024-30":1}},"file":"PMC5730726"},"subset":"pubmed_central"}
{"text":"abstract: # Introduction\n  .\n  The goals of this study were to examine the oxemic regulation of Wnt signaling to explore whether Wnt signaling accelerates the age-related degeneration of nucleus pulposus cells, and if so, to define the mechanism underlying this effect. We investigated the expression of *Klotho*, a newly identified antiaging gene, and whether its regulation is attributable to the suppression of Wnt signaling.\n  .\n  # Methods\n  .\n  Rat nucleus pulposus cells were cultured under normoxic (21% O~2~) or hypoxic (2% O~2~) conditions, and the expression and promoter activity of Wnt signaling and *Klotho* were evaluated. The effect of Klotho protein was examined with transfection experiments, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, senescence-associated \u03b2-galactosidase staining, and cell-cycle analysis. To determine the methylation status of the Klotho promoter region, bisulfite genomic sequencing analysis was performed. Its relation with the activation of Wnt signaling was assessed. We also examined whether the expression of Klotho could block the effects of pathological Wnt expression in nucleus pulposus cells.\n  .\n  # Results\n  .\n  Nucleus pulposus cells exhibited increased \u03b2-catenin mRNA and protein under the hypoxic condition. Klotho protein was expressed *in vivo*, and protein and messenger RNA expression decreased under the hypoxic condition. Klotho treatment decreased cell proliferation and induced the quiescence of nucleus pulposus cells. In addition, Klotho treatment inhibited expression of \u03b2-catenin gene and protein compared with untreated control cells.\n  .\n  # Conclusions\n  .\n  These data indicate that Wnt signaling and Klotho form a negative-feedback loop in nucleus pulposus cells. These results suggest that the expression of Klotho is regulated by the balance between upregulation and downregulation of Wnt signaling.\nauthor: Akihiko Hiyama; Fumiyuki Arai; Daisuke Sakai; Katsuya Yokoyama; Joji Mochida\ndate: 2012\ninstitute: 1Department of Orthopaedic Surgery, Surgical Science, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan; 2Research Center for Regenerative Medicine, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan\nreferences:\ntitle: The effects of oxygen tension and antiaging factor *Klotho* on Wnt signaling in nucleus pulposus cells\n\n# Introduction\n\nRegenerative therapy for intervertebral disc degeneration has been recently reported \\[1-3\\]. Cell-based therapies for tissue regeneration offer an attractive alternative to current conservative, surgical, pharmaceutical, or gene-therapy interventions. However, to clarify the mechanism underlying low-back pain, the molecular mechanisms involved in intervertebral disc degeneration must be identified.\n\nWnt\/\u03b2-catenin (hereafter called Wnt) signaling is thought to be involved in the maintenance and destruction of bone and cartilage. Dysregulation of members of this signaling family has been described in osteoarthritis \\[4-6\\]. Wnts are secreted glycoproteins crucial for the development and homeostatic renewal of many tissues and for chondrocyte and osteoblast development. In the presence of Wnt ligands, Wnts activate a number of different signaling pathways via distinct receptors and downstream effectors that mediate effects on gene transcription \\[7-9\\]. Although Wnt signals regulate the balance between catabolic factors and anabolic factors in intervertebral discs \\[10,11\\], their regulation (upstream or downstream) in nucleus pulposus cells and the corresponding signaling mechanisms are unknown.\n\nOne of the primary causes of intervertebral disc degeneration is thought to be failure of the nutrient supply to intervertebral disc cells because of structural changes to the cartilage endplate \\[12\\]. The O~2~ levels in the nucleus pulposus may be 1% to 5%, and disc-cell metabolism can vary with O~2~ concentration. The activity of disc cells is very sensitive to changes in extracellular oxygen and pH. However, a little information is known about the effect of oxygen tension on nucleus pulposus cells \\[13\\]. Accordingly, more data are needed to determine whether a low oxygen tension is beneficial or detrimental in the culture of nucleus pulposus cells.\n\nThe jelly-like nucleus pulposus (notochord) in the middle of the disc is composed of proteoglycan and functions to disperse the normal loading forces experienced by the spine, acting as a shock absorber to maintain the trunk. However, changes in proteoglycan concentration during age-related disc degeneration are of critical importance. During embryogenesis of the intervertebral disc cells, the cells of the notochord play a critical role in initiating tissue formation and may be directly responsible for development of the nucleus pulposus. In some species, including humans, notochordal cells may eventually be lost and are replaced by chondrocyte-like cells \\[14,15\\]. By the age of 60 to 80 years, the intervertebral disc is composed entirely of fiber \\[16,17\\]. Accordingly, aging is another risk factor for intervertebral disc degeneration. During degenerative disc disease, loss of disc cells, limited proteoglycan synthesis, and a shift toward synthesis of a fibrotic matrix occur. *Klotho*, a newly identified antiaging gene, has attracted recent attention. The *Klotho* gene encodes a single-pass transmembrane protein. *Klotho* is predominantly expressed in the kidney, but it is also expressed in the brain, parathyroid gland, and heart of normal subjects \\[18-20\\]. The *Klotho* gene plays a critical role in regulating aging and in the development of age-related diseases in mammals. Loss of *Klotho* can result in multiple aging-like phenotypes \\[21,22\\], and conversely, the overexpression of *Klotho* in *Klotho*^-\/-^ mice extends their life span \\[23\\]. *Klotho* gene polymorphisms in humans are associated with pathophysiologic bone loss with aging \\[24\\], spondylosis \\[25\\], osteocalcin levels \\[26\\], and bone mineral density \\[27\\]. However, no reports exist of the expression of Klotho protein in the intervertebral disc. Moreover, factors responsible for age-associated impairment of intervertebral disc are poorly understood.\n\nIn the previous study, we focused on assessing the relation among oxygen tension, klotho, and Wnt signaling and attempted to examine the biologic mechanisms (upstream or downstream) of Wnt signaling in nucleus pulposus cells. The Wnt-signal downstream promoter may be induced by oxygen tension or an age-related gene. Therefore, the purpose of the present investigation was threefold: to determine how Wnt signaling in nucleus pulposus cells is regulated by oxygen tension; to determine the effects of the aging-suppressor gene *Klotho* on Wnt signaling; and to investigate the relation between disc degeneration (maintenance of cell proliferation and matrix synthesis) and *Klotho* expression. These findings provide new insight into the regulation and maintenance of cell proliferation and matrix synthesis by *Klotho* and Wnt signaling cross-talk in nucleus pulposus cells.\n\n# Materials and methods\n\nAll animal experiments were performed with approval from Tokai University animal study institutional review board.\n\n## Reagents and plasmids\n\nPlasmids were kindly provided by Dr. Raymond Poon (Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada) (plasmid encoding wild-type (WT) \u03b2-catenin and the backbone), Ilsa Rovira (Cardiology Branch, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD, USA) (plasmid encoding Flag-tagged full-length Klotho) \\[28\\], Dr. Young Han Lee (Institute of Biomedical Science and Technology, Konkuk University Hospital, Seoul, South Korea) (plasmids encoding luciferase-tagged Klotho constructs, Del-1, Del-2, Del-3, and Del-4) \\[29\\], Dr. Michael C. Naski (University of Texas Health Science Center at San Antonio, San Antonio, TX, USA) (plasmid encoding luciferase-tagged aggrecan) \\[30\\], Dr. Sean A. McCarthy (University of Michigan, Ann Arbor, MI, USA) (plasmid encoding Dickkopf-1 (Dkk1), Dkk2, Dkk3, and Dkk4 expression vectors or backbone vector), and Dr. Yoshihiko Yamada (Laboratory of Developmental Biology and Anomalies, National Institute of Dental Research, Bethesda, MD, USA) (plasmid encoding luciferase-tagged type-II collagen) \\[31\\]. The constitutively active (CA)-GSK3\u03b2 (no. 14754) and dominant-negative (DN) GSK3\u03b2 (no. 14755) were purchased from Addgene (Cambridge, MA, USA). Topflash (optimal Tcf-binding site) and Fopflash (a promoter with a mutated Tcf-binding site) were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY, USA). \u03b2-Catenin small interfering RNA (siRNA) (no. sc-29209) and control siRNA duplexes were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). In the Klotho constructs, the TCF\/LEF binding motifs within the klotho promoter were analyzed by using the Web-based tool for predicting transcription factor-binding sites in DNA sequences, Transcription Element Search System (TESS). As an internal transfection control, we used the empty vector pGL4.74 (Promega, Fitchburg, WI, USA) containing the *Renilla reniformis* luciferase genes. The amount of transfected plasmid, the pretransfection period after seeding, and the posttransfection period before harvesting were optimized for rat nucleus pulposus cells by using the pSV\u03b2-galactosidase plasmid (Promega). We used 6-bromoindirubin-3'-oxime (BIO) (no. 361550; Calbiochem, San Diego, CA, USA) to examine the activity of the Wnt signaling activity. BIO is a cell-permeable, highly potent, selective, reversible, and ATP-competitive specific inhibitor of GSK-3\u03b1\/\u03b2 activity \\[32\\]. Recombinant Klotho protein (\u03b1-Klotho) (no. 100-53) was purchased from Peprotech (Rocky Hill, NJ, USA).\n\n## Isolation of intervertebral disc cells\n\nIn total, 64 (32 female and 32 male) 12-week-old Sprague-Dawley rats were used for this study. None of the variables differed between females and males. Nucleus pulposus cells were isolated by using methods reported by Hiyama *et al.*, 2007 \\[1\\]. In brief, the rats were euthanized by injection of an excess of pentobarbital sodium (100 mg\/kg) (Nembutal; Abbott Laboratories, Abbott Park, IL, USA). The spinal column was removed under aseptic conditions, and the lumbar intervertebral discs were separated under microscopy. The gel-like nucleus pulposus was separated from the annulus fibrosus. The obtained nucleus pulposus tissue was digested in a mixture of 0.01% trypsin and allowed to digest at 37\u00b0C for 15 minutes. The digested tissue was passed through a cell strainer (BD Biosciences, San Jose, CA, USA) with a pore size of 100 \u03bcm and was washed twice with phosphate-buffered saline (PBS; Gibco, Invitrogen, Carlsbad, CA, USA). The isolated cells were maintained in Dulbecco modified Eagle medium (DMEM; Gibco, Invitrogen) and 10% fetal bovine serum (FBS; Gibco, Invitrogen) supplemented with antibiotics (1% penicillin\/streptomycin) at 37\u00b0C in a humidified atmosphere of 5% CO~2~. When confluent, the nucleus pulposus cells were harvested and subcultured in 10-cm dishes. Cells were then counted and plated at the appropriate density. Because cells obtained from the rat intervertebral disc tissues were variable in morphology until passage 2 to 3, we used low-passage (\\< 3) cells cultured in monolayers for all experiments.\n\n## Cell culture in hypoxia\n\nTo induce hypoxia, nucleus pulposus cells were cultured in a mixture of 2% O~2~, 5% CO~2~, and 93% N~2~ for 24 hours. The plating density chosen was dependent on the requirements of the individual assays. The concentration of oxygen and the duration of the incubation period chosen for this study were based on the *in vitro* studies of Risbud *et al.* \\[33\\] and on information generated on the oxemic status of the disc *in vivo*.\n\n## Immunofluorescence staining\n\nNucleus pulposus cells were plated in flat-bottom 96-well plates (5 \u00d7 10^3^ cells\/well) and maintained in conditions of normoxia or hypoxia for 24 hours. After this treatment, cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 (vol\/vol) in PBS for 10 minutes, blocked with PBS containing 5% FBS, and incubated overnight at 4\u00b0C with antibodies against \u03b2-catenin (1:200 dilution; Cell Signaling Technology, Danvers, MA, USA). After washing, the cells were incubated with anti-rabbit IgG Alexa Fluor 594 (red) and\/or anti-goat Alexa Fluor 488 secondary (green) antibodies (Invitrogen) at a dilution of 1:50 and 10 \u03bcM 4',6-diamidino-2-phenylindole (DAPI) for 1 hour at room temperature for nuclear staining. The samples were observed with a fluorescence microscope connected to a digital imaging system. Similar experiments were performed to assess the expression of anti-rabbit-Klotho (1:200 dilution; Abcam, Cambridge, UK) and anti-goat-Klotho (1:50 dilution; Santa Cruz Biotechnology). Negative controls without the primary antibody were prepared.\n\n## Immunohistologic studies\n\nFreshly isolated rat spines were immediately fixed in 4% paraformaldehyde in PBS, decalcified, embedded in paraffin, sectioned, and assessed by histology and then immunostaining. Sagittal sections were deparaffinized in xylene, rehydrated through a graded ethanol series, and stained with hematoxylin. To localize Klotho, sections were incubated with anti-Klotho antibody (ab75023; Abcam) in 2% bovine serum albumin (BSA) in PBS at a dilution of 1:200 at 4\u00b0C overnight. After thoroughly washing the sections, the bound primary antibody was detected with a biotinylated universal secondary antibody (Vector Laboratories, Burlingame, CA, USA) at a dilution of 1:20 for 10 minutes at room temperature. Sections were incubated with a streptavidin\/peroxidase complex for 5 minutes and washed with PBS, and the color was developed by using 3'-3-diaminobenzidine (DAB) (VECTASTAIN Universal Quick Kit; Vector Laboratories) and peroxidase DAB substrate kit (Vector), according to the manufacturer's protocol. Negative controls without the primary antibody (anti-Klotho) were prepared.\n\n## 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay\n\nTo measure disc-cell proliferation, a modified MTT assay was carried out as previously described \\[26\\]. In brief, exponentially growing nucleus pulposus cells were seeded in 24-well plates at 1.5 \u00d7 10^4^ cells\/well. After treatment with Klotho (100 ng\/ml), MTT diluted in serum-free DMEM was added to the culture medium to a final concentration of 0.5 mg\/ml. No data showed the physiological protein concentration of Klotho in the nucleus pulposus cells. We therefore chose to use the concentration of Klotho protein (100 ng\/ml) referred to in a previous study \\[34\\]. At the end of the incubation period (2 hours at 37\u00b0C), the medium was removed, and the precipitated formazan crystals were solubilized in dimethyl sulfoxide (DMSO). Product formation was measured by reading the absorbance at 590 nm by using a microplate reader (Pharmacia).\n\n## Staining for senescence-associated \u03b2-galactosidase (\u03b2-gal)\n\nTo stain nucleus pulposus cells for senescence-associated \u03b2-gal activity, we used a cell-senescence histochemical staining kit (CS0030; Sigma-Aldrich, St. Louis, MO, USA) according to the previously described protocol \\[26\\]. Cells were treated for 24 hours with or without 100 ng\/ml Klotho. To quantify the results, a minimum of 100 cells spanning five different microscopy fields were scored for staining, and results represent the mean \u00b1 standard deviation (SD).\n\n## Cell-cycle analysis by flow cytometry\n\nNucleus pulposus cells were grown in 24-well plates under a humidified 5% CO~2~ atmosphere at 37\u00b0C. The seeding density was 5 \u00d7 10^4^ cells\/ml. The cells were allowed to adhere for 24 hours in medium containing 2% FBS. The culture medium in each flask was then replaced with medium containing 0.5% FBS. Klotho (100 ng\/ml) was added to this medium as a concentrated stock solution dissolved in DMSO. After an additional incubation period of 24 or 48 hours, the cell-cycle distribution of the nucleus pulposus cells was analyzed with flow cytometry after DNA staining with propidium iodide by using the CycleTEST PLUS kit (BD Pharmingen, San Diego, CA, USA). CELLQuest (BD Pharmingen) and ModFit LT (BD Pharmingen) software packages were used for cell acquisition and analysis. Each plot represents the analysis of 10,000 events. The histograms present typical results, and the percentages of cells in G~1~, S, and G~2~\/M cell-cycle phases are shown as the means of triplicate measurements. The results from three individual experiments are shown.\n\n## Total RNA extraction and real-time reverse transcriptase (RT)-PCR analysis\n\nTotal RNA was extracted from rat nucleus pulposus and annulus fibrosus cells or tissues by using the TRIzol RNA isolation protocol (Invitrogen). Before elution from the column, RNA was treated with RNase-free DNAse I. Total RNA (100 ng) was used as a template for the real-time PCR analyses. The cDNA was synthesized by the reverse transcription of mRNA by using the protocol described previously (26). The real-time PCR analyses were performed in triplicate by using 96-well plates with the Fast SYBR Green Master Mix (Applied Biosystems, Carlsbad, CA, USA). Two microliters of cDNA per sample was used as the template for real-time PCR: 1 \u03bcl of forward primer and 1 \u03bcl of reverse primer were added to 20 \u03bcl SYBR Green Master Mix. PCR reactions were performed in an Applied Biosystems 7500 Fast system, according to the manufacturer's instructions. All primers for *\u03b2-catenin*, *Klotho*, and *aggrecan* were synthesized by Takara Bio, Inc. (Tokyo, Japan): *\u03b2-catenin* (NCBI number: AF_121265.1): forward, 5'-GCCAGTGGATTCCGTACTGT-3' and reverse, 5'-GAGCTTGCTTTCCTGATTGC-3'; *Klotho* (NCBI number: NM_031336.1): forward, 5'-CGATGTTCGTGACAGCCAATG-3' and reverse, 5'-GTTGATGCCGTCCAACACGTAG-3'; and *aggrecan* (NCBI number: NM_022190.1): forward, 5'-TCCGCTGGTCTGATGGACAC-3' and reverse, 5'-CCAGATCATCACTACGCAGTCCTC-3'. To normalize each sample, a control gene (GAPDH) was used, and the arbitrary intensity threshold (C~t~) of amplification was computed. The expression scores were obtained by the \u0394\u0394C~t~ calculation method.\n\n## Gene-suppression studies by small interfering RNA (siRNA)\n\nWe silenced \u03b2-catenin expression in nucleus pulposus cells by using small interfering RNA (siRNA) technology. In brief, nucleus pulposus cells were transferred to 24-well plates at a density of 6 \u00d7 10^4^ cells\/well 1 day before transfection. The next day, cells were treated with \u03b2-catenin siRNA or control siRNA duplexes at a final concentration of 100 to 500 ng by using Lipofectamine 2000. Cells also received Klotho promoter constructs and the pGL4.74 plasmid at the time of transfection. Six hours after transfection, the medium was replaced with complete growth medium, and the cells were allowed to recover for 18 hours. Cells were then cultured for 24 hours, and luciferase activity was measured.\n\n## Methylation analysis\n\nGenomic DNA was extracted from cells not treated with BIO and BIO-treated cells by using a FastPure DNA Kit (Takara Bio), and bisulfite treatment of genomic DNA was performed by using MethylEasy Xceed (Human Genetic Signatures) and following the manufacturers' instructions. The bisulfite-treated DNA was amplified by PCR with Takara *Taq* Hot Start Version (Takara Bio) and T-Vector pMD20 (Takara Bio) for cytosine-phosphate-guanosine (CpG)-rich regions around the rat *Klotho* gene. PCR products amplified by using Takara LA Taq HS were subcloned into T-Vector pMD20, and sequence analysis was performed. All methylation studies were performed by bisulfite modification of DNA, which converts all unmethylated CpG sites to uracil-phosphate-guanosine, leaving methylated CpGs intact. The DNA was then amplified with the following gene-specific primers: *Klotho*, forward, 5'-TGATGTGGGGATATTTTAGGA-3', and reverse, 5'-CAACAAATACAACRACAACAAA-3'. After sequencing of the amplified DNA, methylated CpGs were identified with visual inspection of the sequencing traces in the electropherograms and by comparison with sequencing traces of BIO-treated cell gene sequences relative to the reference in the control cell sequence.\n\n## Western blotting analysis\n\nAfter treatment, nucleus pulposus cells were immediately washed 3 times with ice-cold PBS and solubilized in lysis buffer. Proteins were prepared by using the CellLytic NuCLEAR extraction kit (Sigma-Aldrich). All wash buffers and the final resuspension buffer included \u00d7 1 protease inhibitor cocktail (Roche, Switzerland), NaF (5 m*M*), and Na~3~VO~4~ (200 \u03bc*M*). Total lysates (30 to 60 \u03bcg of protein\/lane) were subjected to electrophoresis by using 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad, Hercules, CA, USA). The resolved proteins were transferred electrophoretically to nitrocellulose membrane \"blots\". The blots were blocked with 5% BSA in TBST (50 m*M* Tris, pH 7.6, 150 m*M* NaCl, 0.1% Tween 20) and incubated overnight at 4\u00b0C in 5% BSA in TBST with anti-\u03b2-catenin (1:1,000; Cell Signaling) or anti-Klotho (1:1,000; Abcam). Immunolabeling was detected with electrochemiluminescence reagent (Amersham Biosciences, Piscataway, NJ, USA). Quantification of Western blots was performed by using Image J pixel analysis (NIH Image software). Data from Western blots are presented as band density normalized to the loading control (actin).\n\n## Transfections and dual luciferase assay\n\nNucleus pulposus cells were transferred to 24-well plates at a density of 6 \u00d7 10^4^ cells\/well 1 day before transfection. In several experiments, cells were cotransfected with 100 to 500 ng of expression plasmids or the backbone vector, along with reporter plasmids. Lipofectamine 2000 (Invitrogen) was used as a transfection reagent. After 24 hours, cells were maintained in either the hypoxic or the normoxic condition. Twenty-four hours after treatment, the cells were harvested, and a Dual-Luciferase reporter assay system (Promega) was used for the sequential measurements of firefly and *Renilla* luciferase activities. The results were normalized for transfection efficiency and are expressed as a relative ratio of luciferase to pGL4.74 activities (denoted as relative activity). To check the transfection efficiency, nucleus pulposus cells were transfected with plasmid encoding green fluorescent protein; the transfection efficiency for nucleus pulposus cells was 60% to 70%. In addition, the overexpression or silencing efficiency of the targets was confirmed at the mRNA level or protein level. The luciferase activities and relative ratios were quantified by using a Turner Designs Luminometer Model TD-20\/20 instrument (Promega).\n\n## Statistical analysis\n\nTypically, data were compiled from at least three independent triplicate experiments, each performed on separate cultures and on separate occasions. We calculated and have displayed the responses as fold change (over the untreated control). Data are presented as mean \u00b1 SD. Comparisons of data between groups were performed by using the Student *t* test or ANOVA for assessing variance. Statistical significance (*P* \\< 0.05) is denoted with an asterisk.\n\n# Results\n\n## Oxygen tension directly regulates Wnt signaling in nucleus pulposus cells\n\nWe first examined whether oxygen tension activates the transcriptional activity of the Wnt signal pathway by using the TCF- and \u03b2-catenin-dependent Topflash reporter plasmid in nucleus pulposus cells. Figure <a href=\"#F1\" data-ref-type=\"fig\">1A<\/a> and <a href=\"#F1\" data-ref-type=\"fig\">1B<\/a> shows that under the hypoxic condition, Topflash activity increased significantly by 1.3- to 1.5-fold in nucleus pulposus cells after 24 hours of incubation (*P* \\< 0.05). Nucleus pulposus cells cotransfected with the WT-\u03b2-catenin expression plasmid, or treated with a different concentration of BIO, showed a dose-dependent increase in the activity of Topflash. Fopflash promoter (a promoter with a mutated TCF-binding site) activity was unresponsive to oxygen tension (data not shown). We next measured the relative expression level of \u03b2-catenin mRNA under normoxic and hypoxic conditions. We analyzed mRNA expression with real-time RT-PCR. The \u03b2-catenin mRNA level was threefold higher in nucleus pulposus cells cultured under the hypoxic (2% O~2~) compared with the normoxic condition (21% O~2~) (*P* \\< 0.05) (Figure <a href=\"#F1\" data-ref-type=\"fig\">1C<\/a>). To confirm the dependence of \u03b2-catenin expression on oxygen tension, as shown in Figure <a href=\"#F1\" data-ref-type=\"fig\">1D<\/a>, we showed that total \u03b2-catenin level and the nuclear translocation of \u03b2-catenin increased to a greater extent in nucleus pulposus cells cultured under the hypoxic condition than in cells cultured under the normoxic condition. In the hypoxic environment, a significant increase in the levels of \u03b2-catenin protein was seen (Figure <a href=\"#F1\" data-ref-type=\"fig\">1D<\/a>; arrows). To explore the premise that oxygen tension regulates \u03b2-catenin protein expression, nucleus pulposus cells were treated with BIO under normoxic and hypoxic conditions. The expression of \u03b2-catenin protein in nucleus pulposus cells was induced by hypoxia (Figure <a href=\"#F1\" data-ref-type=\"fig\">1E<\/a>).\n\n## Expression of Klotho by intervertebral disc\n\nTo examine whether Klotho is involved in intervertebral disc differentiation, we first determined the expression profiles of Klotho in intervertebral discs. Expression of the Klotho protein and mRNA was analyzed by immunostaining and real-time PCR. We immunostained discs in sagittal sections from 3-week-old (Figure <a href=\"#F2\" data-ref-type=\"fig\">2A, a-d, i<\/a>) and 12-week-old (Figure <a href=\"#F2\" data-ref-type=\"fig\">2A, e-h, j<\/a>) rats by using an antibody to Klotho. Klotho was expressed in cells of the nucleus pulposus (Figure <a href=\"#F2\" data-ref-type=\"fig\">2A, d<\/a>), but not of the annulus fibrosus (Figure <a href=\"#F2\" data-ref-type=\"fig\">2A, c<\/a>), in the 3-week-old rat discs. Klotho expression was higher in the annulus fibrosus of discs in 12-week-old rats compared with 3-week-old rats. The nucleus pulposus of 3-week-old rats (Figure <a href=\"#F2\" data-ref-type=\"fig\">2A, i<\/a>) was full of notochordal cells with large vacuoles, and much of the staining was nuclear, which contrasted with the appearance in samples from 12-week-old rats (Figure <a href=\"#F2\" data-ref-type=\"fig\">2A, j<\/a>). To determine the expression of Klotho in intervertebral discs, we also measured Klotho mRNA expression both in tissue or cells and found that it was significantly higher in nucleus pulposus than in annulus fibrosus (Figure <a href=\"#F2\" data-ref-type=\"fig\">2B<\/a>). We sought to determine whether Klotho and the Wnt signal could form a direct molecular complex in nucleus pulposus cells. We demonstrated that the subcellular distribution of Klotho and \u03b2-catenin proteins within BIO-treated cells overlapped (Figure <a href=\"#F2\" data-ref-type=\"fig\">2C<\/a>).\n\n## Decreased Klotho transcriptional activity in nucleus pulposus cells treated with hypoxia\n\nWe first investigated the regulation of Klotho expression to determine whether a relation existed between Klotho and the oxemic status of the nucleus pulposus cell by analyzing the 1.83-kb promoter sequence of human Klotho and measuring the activities of different sizes of promoter constructs (Figure <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>). Sequence analysis shows that the Klotho promoter contains 12 TCF\/LEF-binding motifs (CTTTT, CTTTG, or CAAAG); that is, 12 TCF\/LEF-binding motifs in the p-Klotho Del1 (-1830\/+7) construct, but no TCF\/LEF-binding motifs in the p-Klotho Del4 (-90\/+7) construct. To analyze the promoter function further, we used luciferase reporter constructs containing -1830\/+7 bp (p-Klotho-Del1), -930\/+7 bp (p-Klotho-Del2), -415\/+7 bp (p-Klotho-Del3), or -90\/+7 bp (p-Klotho-Del4) constructs of the Klotho promoter \\[29\\]. We measured the basal activity of all four constructs in nucleus pulposus cells. Figure <a href=\"#F4\" data-ref-type=\"fig\">4A<\/a> shows that the p-Klotho-Del3 construct had maximal basal activity, and the p-Klotho-Del1 construct exhibited the lowest activity. We next examined the effect of oxygen tension on all Klotho promoter activities. Experiments using serial deletion constructs of the Klotho promoter under the hypoxic condition showed a significant decrease in the activity of the p-Klotho-Del1 and p-Klotho-Del2 promoters but not of the p-Klotho-Del3 and p-Klotho-Del4 promoters (Del1: normoxic 1.00 \u00b1 0.15, hypoxic 0.71 \u00b1 0.10; Del2: normoxic 1.71 \u00b1 0.10, hypoxic 1.53 \u00b1 0.01; Del3: normoxic 2.78 \u00b1 0.02, hypoxic 2.62 \u00b1 0.14; Del4: normoxic 2.59 \u00b1 0.05, hypoxic 2.71 \u00b1 0.12) (Figure <a href=\"#F4\" data-ref-type=\"fig\">4A<\/a>). To evaluate these results further, a p-Klotho-Del1 reporter plasmid (Klotho-luc) was transfected along with the pGL4.74 vector into nucleus pulposus cells, and the cells were stimulated with BIO under the normoxic and hypoxic conditions. Figure <a href=\"#F4\" data-ref-type=\"fig\">4B<\/a> shows that the Klotho promoter activity was increased by BIO treatment. Cells cultured under the hypoxic condition showed lower Klotho promoter activity compared with the normoxic condition. Real-time RT-PCR analysis also showed that hypoxia decreased the expression of Klotho mRNA to 0.5 to 0.7 to that in normoxia (Figure <a href=\"#F4\" data-ref-type=\"fig\">4C<\/a>). To determine whether a concomitant elevation in Klotho protein expression was associated with Wnt signaling, the cells were evaluated with immunofluorescence analysis (Figure <a href=\"#F4\" data-ref-type=\"fig\">4D<\/a>) and Western blotting (Figure <a href=\"#F4\" data-ref-type=\"fig\">4E<\/a>). As shown in Figure <a href=\"#F4\" data-ref-type=\"fig\">4D<\/a>, immunofluorescence analysis with an anti-Klotho antibody showed that BIO treatment (1.0 \u03bc*M*, 24 hours) promoted the nuclear translocation of Klotho more strongly in nucleus pulposus cells than in untreated cells. Klotho was localized to the nucleus in cells treated with BIO (arrows). Similarly, immunoblotting analysis of nucleus pulposus cells treated with BIO (1.0 \u03bc*M*, 24 hours) showed more Klotho protein than in control nucleus pulposus cells (Figure <a href=\"#F4\" data-ref-type=\"fig\">4D<\/a>).\n\n## Regulation of Klotho by Wnt signaling in nucleus pulposus cells\n\nAlthough Wnt signaling has also been shown to have a role in nucleus pulposus cells, interactions between Wnt signaling and Klotho in nucleus pulposus cells have not been previously described. Initial experiments were performed to investigate the role of Wnt signaling in the transcriptional activity of Klotho in nucleus pulposus cells. Nucleus pulposus cells were transiently transfected with plasmids encoding Klotho along with WT-\u03b2-catenin expression plasmid. Figure <a href=\"#F5\" data-ref-type=\"fig\">5A<\/a> shows that forced expression of \u03b2-catenin under both normoxic and hypoxic conditions significantly induced Klotho promoter activity (*P* \\< 0.05). To validate these findings, we performed loss-of-function experiments by using siRNA for \u03b2-catenin. Suppression of gene expression was confirmed by real-time RT-PCR (data not shown). Under the normoxic condition, Klotho promoter activity was inhibited in nucleus pulposus cells cotransfected with \u03b2-catenin siRNA (*P* \\< 0.05; 100 ng, *P* = 0.005; 300 ng, *P* = 0.002; 500 ng, *P* = 0.0001) (Figure <a href=\"#F5\" data-ref-type=\"fig\">5B<\/a>). We also examined the effect of GSK3\u03b2 treatment on Klotho promoter activity. Nucleus pulposus cells were transiently cotransfected with plasmids encoding CA-GSK3\u03b2 (Figure <a href=\"#F5\" data-ref-type=\"fig\">5C<\/a>) or DN-GSK3\u03b2 (Figure <a href=\"#F5\" data-ref-type=\"fig\">5D<\/a>). Klotho promoter activity was decreased in a dose-dependent manner by the CA-GSK3\u03b2 expression plasmid (*P* \\< 0.05) but not by the DN-GSK3\u03b2 expression plasmid. These results suggest that Klotho is downstream of \u03b2-catenin signaling in nucleus pulposus cells. To confirm whether Dkk, a secretory protein that inhibits Wnt signaling by disrupting the interactions between Wnt and Fz, regulates the transcriptional activity of Klotho, we assessed the transcriptional activity of Klotho after treatment with Dkk1, 2, 3, or 4 (500 ng) expression plasmids or backbone vector for 24 hours. As shown in Additional file <a href=\"#S1\" data-ref-type=\"supplementary-material\">1<\/a> Klotho activity was not affected by Dkk treatment.\n\n## Klotho suppression of the proliferation of nucleus pulposus cells\n\nProliferation of nucleus pulposus cells was decreased significantly by Klotho at 48 hours but not 24 hours after the treatment (*P* \\< 0.05 and *P* = 0.24, respectively) (Figure <a href=\"#F6\" data-ref-type=\"fig\">6A<\/a>). To test for cell senescence, we stained cells for the expression of the senescence-associated protein, \u03b2-gal. As shown in Figure <a href=\"#F6\" data-ref-type=\"fig\">6B<\/a>, Klotho treatment for 24 hours did not change the percentage of senescence-associated \u03b2-gal-positive nucleus pulposus cells (arrow in control, 32.8% \u00b1 6.4%; Klotho, 35.4% \u00b1 8.8%). In addition, Klotho exposure did not increase the level of apoptosis (data not shown). To analyze the methylation status of the CpG islands in the Klotho promoter region, bisulfite genomic sequencing (BGS) analysis was performed in BIO-treated nucleus pulposus cells. As shown in Figure <a href=\"#F6\" data-ref-type=\"fig\">6C<\/a>, a dense GC-rich region was identified around the -200 bp site of Klotho. To determine the methylation density of the Klotho promoter region, which encompasses 61 CpGs, we performed BGS analysis of BIO-treated and untreated nucleus pulposus cells. The methylation of the Klotho promoter region was much less extensive in the BIO-treated cells with activated Wnt signaling (that is, activation of Wnt signaling tended to be associated with unmethylated CpG islands). These data strongly suggest that the transcriptional activation of Klotho is associated with aberrant promoter demethylation by Wnt signaling in nucleus pulposus cells.\n\nWith flow cytometry, we evaluated whether the cell-cycle status was affected by Klotho (100 ng) treatment at different times after treatment. The number of cells in the G~1~ phase was significantly decreased in the presence of Klotho compared with untreated cells (24 hours, *P* = 0.035; 48 hours, *P* \\< 0.05). These results showed that activation of Klotho can suppress the proliferation of nucleus pulposus cells and may induce cells to enter a resting phase, the G~0~ phase (Figure <a href=\"#F6\" data-ref-type=\"fig\">6D<\/a>).\n\n## Functional role of Klotho and Wnt signaling crosstalk in nucleus pulposus\n\nTo investigate the possible mechanisms underlying the actions of Klotho in the induction of nucleus pulposus cell markers, we next examined the effect of Klotho treatment on expression of aggrecan (Figure <a href=\"#F7\" data-ref-type=\"fig\">7A<\/a>) and collagen type II (Figure <a href=\"#F7\" data-ref-type=\"fig\">7B<\/a>) under the normoxic condition. Cotransfection of nucleus pulposus cells with a Klotho expression plasmid significantly decreased aggrecan reporter activity (Klotho 100 ng, *P* = 0.085; Klotho 300 ng, *P* = 0.011; Klotho 500 ng, *P* = 0.001) but not collagen type-II reporter activity (Klotho 100 ng, *P* = 0.723; Klotho 300 ng, *P* = 0.308; Klotho 500 ng, *P* = 0.126). To confirm the regulatory role of Klotho in the expression of aggrecan and collagen type II, we performed real-time PCR to examine changes in the expression of aggrecan and collagen type II in nucleus pulposus cells. The results showed that treatment with 500 ng of Klotho for 24 hours also decreased aggrecan mRNA levels in nucleus pulposus cells, as shown by real-time PCR (*P* \\< 0.05; *P* = 0.004) (data not shown for collagen type II) (Figure <a href=\"#F7\" data-ref-type=\"fig\">7C<\/a>). In addition, we used immunofluorescence analysis and Western blotting to confirm the role of Klotho in aggrecan protein expression. The results demonstrated that Klotho treatment (100 ng\/ml, 24 hours) decreased aggrecan protein levels to a greater extent in nucleus pulposus cells than in control cells (data not shown). Finally, to confirm the relation between Wnt signaling and Klotho and to assess whether Klotho inhibits Wnt signaling in nucleus pulposus cells, we used a Klotho expression plasmid in the Topflash reporter assay. Topflash reporter activity was decreased by Klotho treatment in nucleus pulposus cells (normoxic: Klotho 100 ng, *P* = 0.089; Klotho 300 ng, *P* = 0.044; Klotho 500 ng, *P* = 0.0003; hypoxic: Klotho 100 ng, *P* = 0.001; Klotho 300 ng, *P* = 0.002; Klotho 500 ng, *P* = 0.0001) (Figure <a href=\"#F7\" data-ref-type=\"fig\">7D<\/a>). Similarly, Klotho treatment inhibited gene expression of \u03b2-catenin compared with untreated control cells (*P* \\< 0.05; *P* = 0.023) (Figure <a href=\"#F7\" data-ref-type=\"fig\">7C<\/a>). Real-time PCR analysis also indicated that a concomitant increase in Klotho mRNA levels occurred when nucleus pulposus cells were treated with exogenous Klotho (*P* \\< 0.05; *P* = 0.028) (Figure <a href=\"#F7\" data-ref-type=\"fig\">7C<\/a>). These findings indicate that Wnt signaling is inhibited by Klotho in nucleus pulposus cells.\n\n# Discussion\n\nIn this study, we focused on Wnt signaling to clarify the mechanism (upstream or downstream) underlying intervertebral disc degeneration. First, we examined the relation between Wnt signaling and the oxemic status of nucleus pulposus cells. Nucleus pulposus is an avascular tissue, and in some species, pO~2~ levels in large discs may be low. Oxygen tension is a powerful stimulus of nucleus pulposus cells, yet the net result of hypoxia--anabolic or catabolic--in nucleus pulposus cells is not known. In addition, the mechanism by which the nucleus pulposus cells survive in the hypoxic environment of the intervertebral disc remains largely unknown, and no evidence suggests how oxygen levels change with disc degeneration *in vivo*. However, *in vitro* studies demonstrate stimulatory effects of hypoxia on nucleus pulposus proliferation and matrix synthesis \\[35,36\\]. It is very difficult to conclude that low oxygen would be bad for discs from all of these study data, because the existence of complex signaling networks is found in disc degeneration. However, in this study, we found a hypoxia-dependent increase in the expression of \u03b2-catenin and a concomitant increase in Wnt signaling activity. We demonstrated previously that activation of Wnt signaling elevates the gene expression and activity of aggrecan by the balance of catabolic and anabolic factors in nucleus pulposus cells. This is the key to understanding the biology of intervertebral disc maintenance and degeneration \\[26\\]. Mwale *et al.* \\[37\\] reported that low oxygen levels increase aggrecan mRNA expression in nucleus pulposus cells and that disc cell metabolism is not impaired at low oxygen concentrations. Our results indicate that effect of hypoxia on matrix synthesis of the nucleus pulposus cells might be directly mediated by Wnt signaling or indirectly by Wnt stimulation of other pathways, such as the hypoxia-inducible factor (HIF) pathway. However, this study was unable to distinguish whether this is a direct or an indirect effect. Kaidi *et al.* \\[38\\] reported that Wnt signaling is mediated by the physical interaction between HIF-1\u03b1 and \u03b2-catenin. Intriguingly, the \u03b2-catenin\/HIF-1\u03b1 interaction increases HIF transcriptional activity, which might help cells adapt to severe hypoxia. Risbud *et al.* \\[39\\] reported that nucleus pulposus cells display robust and constitutive expression of both HIF-1\u03b1 and HIF-2\u03b1, suggesting that these cells reside in a hypoxic environment. Future studies will address the role of the \u03b2-catenin\/HIF pathways to determine whether regulation of Wnt signaling by hypoxic conditions is specific to nucleus pulposus cells.\n\nSecond, we investigated the expression of Klotho, a newly identified antiaging gene, and examined the relation in nucleus pulposus cells of Wnt signaling and Klotho.\n\nFinally, we investigated the role of Klotho on aggrecan or collagen type-II expression and the proliferation of nucleus pulposus cells.\n\nIt is clear that the incidence of disc degeneration increases markedly with age. Several studies have focused on Klotho and its role in the aging phenotype. The Klotho protein is composed of a large extracellular domain (130 kDa), a transmembrane domain, and a very short intracellular domain (10 amino acids). The entire extracellular domain is released into the extracellular space and is detectable in blood, urine, and cerebrospinal fluid \\[40\\]. Membrane Klotho functions as a receptor for a hormone that regulates excretion of phosphate and synthesis of active vitamin D in the kidney \\[41,42\\]. Secreted Klotho regulates the activity of multiple growth factors, including insulin\/insulin-like growth factor-1 (IGF-1) \\[11\\], Wnt \\[28\\], and transforming growth factor (TGF)-\u03b21 \\[43\\]. Liu *et al.* \\[28\\] demonstrated that Klotho acts as a Wnt antagonist and suggested that prolonged Wnt stimulation may contribute to stem cell depletion and aging. They reported that secreted Klotho inhibits Wnt signaling activity by binding directly to Wnt ligands and preventing them from binding to their receptors. In this study, we performed molecular functional analysis to study the expression of Klotho in the intervertebral disc and to elucidate the signal crosstalk between Klotho and Wnt signaling; this crosstalk is attracting attention as a potential trigger for degeneration in the intervertebral disc. Interestingly, we demonstrated for the first time that the transcriptional activation of Klotho is associated with promoter demethylation by Wnt signaling. Accordingly, these experiments strongly suggested that in nucleus pulposus cells, activation of Wnt signaling induces an increase in Klotho promoter activity and gene and protein expression by demethylation. By contrast, activation of nucleus pulposus cells by secreted Klotho seems to suppress Wnt signaling. These results might support that Klotho is not simply an antagonist of Wnt signaling but that Wnt signaling and Klotho form a negative-feedback loop in nucleus pulposus cells.\n\nWe hypothesized that Wnt signaling might control nucleus pulposus homeostasis in normal intervertebral discs. However, although we know that, in general, Klotho is an antiaging gene, activation of Wnt signaling nevertheless resulted in induction of Klotho in nucleus pulposus cells. Consequently, Klotho inhibited aggrecan synthesis and proliferation of nucleus pulposus cells. This is unexpected, because suppression of aggrecan synthesis and proliferation of nucleus pulposus cells would appear to be potentially damaging to the intervertebral disc. The reason for these discrepant effects is not clear, but several possible explanations exist.\n\nFirst, it is possible that Klotho interacts with signal pathways other than Wnt signaling. Klotho can affect the function of Wnt, TGF-\u03b21, and IGF, which can affect the expression of aggrecan and collagen type II in different ways. Doi *et al.* \\[43\\] reported that secreted Klotho suppresses TGF-\u03b21 signaling by directly binding to type-II TGF-\u03b2 receptor (TGF\u03b2R2) on the cell surface and preventing TGF-\u03b21 binding to that receptor. TGF-\u03b21 is the most potent inducer of matrix synthesis in nucleus pulposus cells. In addition, fibroblast growth factor (FGF) may act as a catabolic and antianabolic mediator in nucleus pulposus cells, stimulating matrix metalloproteinase (MMP)-13 expression and suppressing proteoglycan synthesis. Recently, Kuro-o \\[44\\] reported that the most important function of the Klotho family is to regulate the function of the endocrine FGF family. Therefore, the final effect of Klotho on the aggrecan and collagen type II gene expression may be the result of the different effects.\n\nSecond, these mechanisms may be specific to the nucleus pulposus cells, because this cell is unique both embryologically and functionally. Moreover, although regulation of Wnt signaling may be tissue specific, the signaling pathway has been shown to modulate cellular proliferation or differentiation.\n\nThird, these differences may relate to the age and species of the animal from which the cells are isolated and the environment in which the cell metabolism was studied. Other groups have shown that glycosaminoglycan production by nucleus pulposus and annulus fibrosus cells can vary, depending on the source of disc cells \\[45\\].\n\nFourth, the *Klotho* gene encodes several isoforms in mammals, which include a membrane-bound and two secreted forms. Therefore, the results for this study might be from the different isoforms through different signaling pathways.\n\nFuture studies will address the role of Wnt signaling by using human samples to determine whether the regulation of cell growth and matrix synthesis by Klotho is specific to nucleus pulposus cells. Moreover, it is necessary to examine whether, in nucleus pulposus cells, Klotho affects the activity of several signaling pathways, including those of TGF, FGF, or the MMP family, which may participate in intervertebral disc degeneration. Wnt signals are regulated in various ways in response to various extracellular and intracellular signaling. However, DKK, which regulates extracellular Wnt signaling, did not inhibit the reporter activation of Klotho in this study. The DKK family comprises four subtypes, which affect Wnt signaling by combining with the collaboration receptor LRP5\/6 of each Wnt-Fzd \\[46,47\\]. The subtypes seem to influence the functional differences and types of cells that develop, although it is generally believed that only DKK1 and DKK4 inhibit Wnt signals. Our present results suggest that the action of the regulatory factor DKK outside of cells is not dependent on the expression of Klotho in nucleus pulposus cells.\n\n# Conclusions\n\nHere, we demonstrated that (1) hypoxia stimulates Wnt signaling in nucleus pulposus cells; (2) Klotho is expressed in intervertebral discs; (3) transcriptional activation of Klotho is associated with promoter demethylation by Wnt signaling; and (4) Wnt signaling and Klotho form a negative-feedback loop in nucleus pulposus cells. In summary, our study demonstrates that in nucleus pulposus cells, the expression of Klotho is regulated by the balance between upregulation and downregulation of Wnt signaling. To elucidate the mechanism causing intervertebral disc degeneration, further studies are needed to determine how the interplay of various Wnt ligands and Klotho modulates cell growth and matrix synthesis in nucleus pulposus cells.\n\n# Abbreviations\n\nBGS: bisulfite genomic sequencing; BIO: 6-bromoindirubin-3'-oxime; CPG: cytosine-phosphate-guanosine; DKK: Dickkopf; HIF-1\u03b1: hypoxia-inducible factor 1\u03b1; TCF\/LEF: T-cell factor\/lymphoid enhancer factor.\n\n# Competing interests\n\nThe authors declare that they have no competing interests.\n\n# Authors' contributions\n\nAH and FA made substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data. AH, FA, KY, DS, and JM were involved in drafting the manuscript or revising it critically for important intellectual content. All authors read and approved the manuscript for publication.\n\n# Supplementary Material\n\n###### Additional file 1\n\n**Nucleus pulposus cells were cotransfected with the Klotho reporter plasmid along with 500 ng of Dkk1, Dkk2, Dkk3, Dkk4, or the empty backbone vector, and the reporter activity was measured (left panel)**. Nucleus pulposus cells were cotransfected with the Klotho promoter plasmid along with increasing quantities (100 to 500 ng) of the Dkk1 expression plasmid or with the empty backbone vector, and the reporter activity was measured (right panel). The results were normalized for transfection efficiency and are expressed as a relative ratio of luciferase to pGL4.74 activities (denoted as relative activity). \\**P \\< 0.05* between groups. Error bars represent SDs. NS, not significant.\n\nClick here for file\n\n## Acknowledgements\n\nWe greatly appreciate the helpful advice of Dr. Tadayuki Sato and the technical assistance of Hideki Hayashi, Masayuki Tanaka, and Mayumi Nakano. This study was supported in part by the Research and Study Program of Tokai University Educational System General Research Organization and Grant-in -Aid for Scientific Research (KAKENHI)","meta":{"dup_signals":{"dup_doc_count":134,"dup_dump_count":35,"dup_details":{"curated_sources":4,"2022-21":1,"2021-49":1,"2021-17":1,"2020-29":1,"2020-05":1,"2019-43":2,"2019-35":1,"2017-34":1,"2017-09":18,"2017-04":1,"2016-44":1,"2016-36":16,"2016-30":16,"2015-48":3,"2015-40":4,"2015-35":3,"2015-32":3,"2015-27":3,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":4,"2014-42":7,"2014-41":3,"2014-35":4,"2014-23":6,"2014-15":3,"2022-40":1,"2015-18":3,"2015-11":2,"2015-06":2,"2014-10":3,"2013-48":2,"2013-20":3,"2024-30":1}},"file":"PMC3446482"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  The 1980s marked the occasion when Geographical Information System (GIS) technology was broadly introduced into the geo-spatial community through the establishment of a strong GIS industry. This technology quickly disseminated across many countries, and has now become established as an important research, planning and commercial tool for a wider community that includes organisations in the public and private health sectors.\n  .\n  The broad acceptance of GIS technology and the nature of its functionality have meant that numerous datasets have been created over the past three decades. Most of these datasets have been created independently, and without any structured documentation systems in place. However, search and retrieval systems can only work if there is a mechanism for datasets existence to be discovered and this is where proper metadata creation and management can greatly help.\n  .\n  This situation must be addressed through support mechanisms such as Web-based portal technologies, metadata editor tools, automation, metadata standards and guidelines and collaborative efforts with relevant individuals and organisations. Engagement with data developers or administrators should also include a strategy of identifying the benefits associated with metadata creation and publication.\n  .\n  # Findings\n  .\n  The establishment of numerous Spatial Data Infrastructures (SDIs), and other Internet resources, is a testament to the recognition of the importance of supporting good data management and sharing practices across the geographic information community. These resources extend to health informatics in support of research, public services and teaching and learning.\n  .\n  This paper identifies many of these resources available to the UK academic health informatics community. It also reveals the reluctance of many spatial data creators across the wider UK academic community to use these resources to create and publish metadata, or deposit their data in repositories for sharing.\n  .\n  The Go-Geo! service is introduced as an SDI developed to provide UK academia with the necessary resources to address the concerns surrounding metadata creation and data sharing. The Go-Geo! portal, Geodoc metadata editor tool, ShareGeo spatial data repository, and a range of other support resources, are described in detail.\n  .\n  # Conclusions\n  .\n  This paper describes a variety of resources available for the health research and public health sector to use for managing and sharing their data. The Go-Geo! service is one resource which offers an SDI for the eclectic range of disciplines using GIS in UK academia, including health informatics.\n  .\n  The benefits of data management and sharing are immense, and in these times of cost restraints, these resources can be seen as solutions to find cost savings which can be reinvested in more research.\nauthor: Tony Mathys; Maged N Kamel Boulos\ndate: 2011\ninstitute: 1EDINA National Data Centre, The University of Edinburgh, Edinburgh, UK; 2Faculty of Health, The University of Plymouth, Drake Circus, Plymouth, Devon, PL4 8AA, UK\nreferences:\ntitle: Geospatial resources for supporting data standards, guidance and best practice in health informatics\n\n# Background\n\nThe 1980s marked the occasion when GIS technology was initially introduced into the geospatial community. (The 1980s mark the establishment of a strong GIS industry and new vendor products, with major influence on the discipline of Geography, though earlier non-mainstream systems also existed, e.g., Canada Geographic Information System in the mid-1960s \\[1\\].) This technology quickly disseminated across many countries, and has now become established as an important research, planning and commercial tool for a wider community that includes organisations in the public and private health sectors.\n\nThe broad acceptance of GIS technology, and the nature of its functionality, has meant that numerous datasets have been created over the past three decades. Most of these datasets have been created independently, and without any structured documentation systems in place. A 2006 spatial data audit conducted at four UK academic institutions is a testament to this. The audit yielded more than 500 spatial dataset titles, and also found hundreds more files in personal computer directories and in stored media \\[2,3\\]. These files had no provenance or descriptive documentation, but their extensions revealed that they had been created with various GIS and remote sensing software packages.\n\nEschewing metadata creation and publication is pervasive across the (Geographic Information) GI-community; the reasons being that it is perceived to be tedious and time-consuming \\[4,5\\]. A survey of archaeological organisations in the Republic of Ireland revealed that 57.1 percent of these organisations did not include metadata creation as part of their organisations' data management strategy \\[6\\]. The Open Geospatial Consortium (OGC) Working Group on Data Quality conducted a survey in 2008 which revealed that about 55.8 percent of respondents said they were not using any recognised standards for data quality work being conducted in their organisation \\[7\\]. Academia appears to represent a greater challenge based on the lack of commitment to metadata creation despite support and engagement with that community in the UK \\[8\\].\n\nUndocumented spatial datasets face a number of risks. Undocumented dataset files are likely to become redundant. If familiarity with a dataset is lost then time and costs must be assumed for any reassessments. The absence of a formal documentation system also means that datasets cannot easily be revealed to the wider geo-spatial community, therefore limiting other potential users' ability to locate both the datasets and developers. The absence of information about existing datasets can lead other organisations to expend considerable time and costs in producing data that are already in existence, but stored at an undisclosed location.\n\nThis widespread lack of proper data documentation can be improved through support mechanisms such as geospatial metadata standards and guidelines, training, metadata editor tools and metadata automation to extract information from spatial datasets. Web-based resources such as geoportal technologies must also be developed to publish metadata. A geoportal is a type of Web portal used to find and access geographic (geospatial) information and associated geographic services (display, editing, analysis, etc.) via the Internet \\[9\\]. Geoportals are important for effective use of GIS and are key to the support of Spatial Data Infrastructures (SDIs) \\[10\\].\n\nSuccessful delivery of metadata and spatial datasets via geoportals and repositories can result in the establishment of SDIs across the various GI-community sectors \\[11\\]. These can provide the infrastructure to support good data management practices, facilitate the exchange of information to advance research and teaching in academia, and to aid planners and policy makers in the public sector.\n\nWMS (Web Map Service), WFS (Web Feature Service), and WCS (Web Coverage Service) are three Web service standards from the Open Geospatial Consortium (OGC) which also provide critical SDI support. These allow Web clients to query and receive geographic information in the form of image, vector, or coverage data. The open source GeoServer application is the reference implementation of a server for the WMS, WFS, and WCS standards \\[12\\].\n\nIt is also important to engage with data developers to implement a policy of data management and sharing using these resources. The challenge lies with encouraging and sustaining these activities through identifying the benefits associated with metadata creation and publication and data sharing.\n\nUltimately, the driving force for implementation rests in the hands of individuals and organisations. Motivating people to create metadata remains the greatest challenge and requires imagination and guidance through a range of initiatives promoting metadata creation and publication in the context of the aforementioned standards, support mechanisms and identified benefits.\n\n## On metadata and metadata standards\n\nThe US Federal Geographic Data Committee (FGDC) describes geospatial metadata as follows: \"*A metadata record is a file of information, usually presented as an XML (eXtensible Markup Language) document, which captures the basic characteristics of a data or information resource. It represents the who, what, when, where, why and how of the resource. Geospatial metadata are used to document geographic digital resources such as Geographic Information System (GIS) files, geospatial databases, and earth imagery. A geospatial metadata record includes core library catalogue elements such as Title, Abstract, and Publication Data; geographic elements such as Geographic Extent and Projection Information; and database elements such as Attribute Label Definitions and Attribute Domain Values*.\" \\[13\\]\n\nGrowing attention to the critical importance of geospatial metadata throughout the past three decades sparked the development of a range of metadata collection initiatives that followed various formats within different communities of practice, agencies, and countries. The task of harmonising the plethora of overlapping formal and de facto metadata standards was initially undertaken by the FGDC with the release of the Content Standard for Digital Geospatial Metadata (CSDGM) in 1994 \\[14\\].\n\nThe FGDC CSDGM standard was implemented across the international GI-community, but from 1999 to 2002, the ISO\/TC 211 \\[15\\] worked towards publishing a new standard which culminated in the release of ISO 19115 'Geographic information - Metadata' standard in 2003 \\[16\\]. Individual countries, groups and communities of practice are in the process of rewriting their previously-used metadata standards as \"profiles\" of ISO 19115, sometimes with the inclusion of additional metadata elements as extensions to the ISO standard, e.g., the North American Profile (NAP) \\[17\\]. ISO 19139 \\[18\\] provides the XML implementation schema for ISO 19115, specifying the metadata record format to describe, validate, and exchange geospatial metadata in XML.\n\n## Implementation\n\nThe introduction of these geospatial metadata standards also required initiatives to encourage their uptake. In the late 1990s, the FGDC took the lead as part of an effort to implement a National Spatial Data Infrastructure (NSDI) for the United States and other countries \\[19\\]. The FGDC provided seed money to geospatial organisations across the US to support the development of metadata and related services and geoportals at the federal, state and local levels.\n\nThis funding also targeted a range of outreach activities in support of metadata creation. Metadata workshops, presentations and academic curriculum development have been conducted over the years. The FGDC has also published numerous documents and publicity materials \\[20\\] in support of metadata creation and producing quality records, and metadata creation and editing tools, software and utilities \\[21\\]. Business cases for the benefits of metadata creation have been provided as well to encourage uptake. Interested readers may refer to the FGDC's 'The Business Case for Metadata' section in \\[13\\].\n\nThe ratification of ISO 19115 in 2003 provided the impetus for metadata initiatives around the world. Across the Atlantic, continental Europe and the UK embraced the INSPIRE Directive \\[22\\]. In September 2001, representatives from the European Commission, the European Environment Agency (EEA) and nominated representatives of the Member States' environmental and geographic information communities convened in Brussels, Belgium to set out a legislative framework which would subsequently aim at creating a European Spatial Data Infrastructure (SDI). This SDI would be based on Member States' infrastructures, and intended to improve interoperability and delivery of environmental information across all Member States of the EU. Each Member State would be required to provide metadata catalogues to allow users to identify available data and geoservices information, and deliver online data discovery, viewing, downloading, and transformation services. The INSPIRE Directive came into force on 15 May 2007 and is being implemented in various stages, with the year 2019 targeted for full implementation \\[23\\].\n\n# Health Spatial Data Infrastructures (SDIs)\n\nHealth informatics organisations have established a range of portals and repositories to deliver metadata, spatial data and interactive mapping features to medical researchers, public health officials and the general public.\n\nThese online resources provide environmental and demographic data which are of particular benefit to epidemiology researchers. Standards, metadata and good data management practices become essential in the delivery of this information to this range of users \\[24-26\\]. Also of particular importance is the promotion of metadata creation through changing the perceptions of metadata in public health culture among public health professionals \\[26\\]. It is worth noting in this respect that ISO 19115 Topic Categories include one dedicated to health (health services, human ecology, and safety; for example, resources describing human disease and illness, factors affecting health, hygiene, mental and physical health, substance abuse, and health services) \\[16\\].\n\nThe following lists provide examples of health-related portals which deliver important information and data via interactive mapping, metadata and download services. Interactive mapping is often supported with Web Map Service (WMS), Web Feature Service (WFS), and Web Coverage Service (WCS).\n\n## Examples of data and interactive mapping portals for medical researchers and public health\n\nThe World Health Organization (WHO) delivers interactive maps, spatial data, satellite imagery and related applications through a portal \\[27\\] built with GeoNetwork opensource \\[28\\], a catalogue application to manage spatially referenced resources. It provides powerful metadata editing and search functions as well as an embedded interactive Web map viewer. The WHO Portal delivers metadata using the ISO 19115 Geographic Information Metadata Standard \\[16\\] (Figures <a href=\"#F1\" data-ref-type=\"fig\">1<\/a> and <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>). GeoNetwork opensource is becoming the primary source for most in the GI-community to use as an ISO 19115-compliant catalogue service for portals. The WHO also supports the Pan American Health Organisation's (PAHO) portal \\[29\\], which delivers health data and statistics and interactive maps for South American countries.\n\nHIVmapper \\[30\\] and the HIV Spatial Data Repository \\[31\\] provide online maps and geographically-linked HIV-related data for GIS mapping. The services are supported by USAID and the US President's Emergency Plan for AIDS Relief (PEPFAR), the US Government initiative to support partner nations around the world in responding to HIV\/AIDS. An example metadata record for Armenia from the HIV Spatial Data Repository can be found at \\[32\\].\n\nThe Earth Space Agency (ESA) and the UN Food and Agriculture Organization (FAO) have provided a joint contribution to the GEOportal \\[33\\], which provides an entry point to access Global Earth Observation System of Systems (GEOSS) information and services. It connects to a system of existing portals, addressing the GEO Societal Benefit Areas globally while also providing national and regional information to enhance understanding. GEOportal also provides the functionality to harvest metadata from portals built with GeoNetwork opensource. It offers a comprehensive series of health-related datasets which support prevention, early warning, research, health-care planning and delivery, and public alerts.\n\nThe Emergency and Disaster Information Service - EDIS \\[34\\] offers AlertMap, a map which provides global coverage and displays locations where epidemics, biological hazards and other natural and catastrophic events are taking place.\n\nThe Montreal Epidemiological and Geographical Analysis of Population Health Outcomes and Neighbourhood Effects (MEGAPHONE) catalogue \\[35\\] is a spatial data infrastructure developed at the Centre de Recherche du Centre Hospitalier de l'Universit\u00e9 de Montr\u00e9al to support research for documenting, analysing and understanding environmental influences on population health. Built with GeoNetwork opensource, MEGAPHONE provides an extensive spectrum of spatial databases enabling the characterisation of places and their contextual and compositional attributes in the Montreal region, Canada. Contextual and compositional indicators can be analysed alone or in relation to linked health outcomes data. By enabling comprehensive characterisations and analyses of exposures to physical, social and built environmental factors, MEGAPHONE contributes to advancing understanding of how the environment affects health. MEGAPHONE is currently used in a broad variety of population and public health research projects on topics including obesity, healthful aging, transmission of HIV and HCV, smoking habits, mental health, cardiometabolic diseases, adverse birth outcomes, and mortality.\n\nThe National Cancer Institute in the U.S. offers an interactive map for the Breast Cancer Relative Incidence Long Island \\[36\\]. The map shows variations in the ratios of observed to expected incidence of breast cancer by ZIP Code over a 5-year period in Long Island, New York.\n\nOneGeology Portal \\[37\\] is an example of a portal which can deliver environmental data for medical research. OneGeology is intended to provide global coverage and includes geological unit and lithological data for some countries, which can be used for radon studies. The portal is built with the latest computing technology using Geoscience Markup Language (GeoSciML) \\[38\\], a GML Application Schema that can be used to transfer information about geology, with an emphasis on the \"interpreted geology\" that is conventionally portrayed on geologic maps.\n\n## Interactive mapping portals for the public\n\nThe following interactive mapping portals offer a sample of information services for the public to assess health-related threats. Some of these allow users to enter postcode, place name or address details to retrieve information and map displays specific to their locations.\n\nThe European Environment Agency's Eye on Earth portal \\[39\\] provides users with information about air quality which is collected from 1,000 air quality monitoring stations across Europe. The portal also delivers water quality information at 22,000 bathing sites across Europe.\n\nThe Flood Maps for England and Wales \\[40\\] and Indicative River and Coastal Flood Map for Scotland \\[41\\] provide the public with flood risk information via interactive maps. Postcode, town, river and place name values can be entered to generate local maps displaying areas prone to flooding.\n\nThe Noise Mapping for England \\[42\\] and Scottish Noise Mapping \\[43\\] interactive maps display of industrial site locations and transport (roads, airports, rail) networks and estimated noise level bands based on modelling traffic volume.\n\nThe 3-D Map of Air Pollution for London \\[44\\] interactive map provides current air pollution levels for users. Postcodes can be entered or London boroughs selected from a drop-down list.\n\n# The Go-Geo! SDI Service\n\n## Go-Geo! Portal\n\nGo-Geo! \\[45\\] (Figure <a href=\"#F3\" data-ref-type=\"fig\">3<\/a>) is a Joint Information Services Committee (JISC) \\[46\\] funded UK academic spatial data portal service which is operated and maintained at EDINA \\[47\\], a JISC national data centre based at The University of Edinburgh. Go-Geo! serves as an online resource discovery tool which allows for the identification and retrieval of records describing the content, quality, condition and other characteristics of spatial data which exist within UK tertiary education and beyond. The portal offers interactive maps, grid co-ordinates and place names, as well as the more traditional topic or keyword to support users with spatial searching. Go-Geo! delivers an SDI to UK academia providing an online metadata editor tool (Geodoc), spatial data repository (ShareGeo), and a range of support resources including eLearning objects, metadata workshops and metadata quality assurance reviews.\n\nThe Go-Geo! portal started as a project in 2001 and became a fully funded service in 2008. This project commenced as a three year collaborative effort between EDINA and the UK Data Archive (UKDA) \\[48\\], at the University of Essex, with the JISC Portals Programme \\[49\\] providing the funding. Primary efforts were directed at the technical development of the Go-Geo! portal, and the collection of geographical information and metadata for content. Subsequently, Go-Geo! was enhanced and redesigned to improve functionality, appearance and ease of use. Other resources were added as well for facilitating metadata creation, supporting teaching and learning in the geo-related disciplines and publishing metadata for international spatial data.\n\nA key feature of Go-Geo! is that it allows users to find related resources, such as books, photographs, projects and maps, for their geographic area of interest. Most notable is the COPAC \\[50\\], the National, Academic, and Specialist Library Catalogue, which holds 32 million records from merged UK and Irish and national libraries. Go-Geo! also allows users to search for images from the British Geological Survey JIDI Photographic Collection \\[51\\]. These and other resources are discovered by cross searching a range of online information services within and beyond the JISC Information Environment \\[52\\]; therefore, the focus of the portal is on where a resource relates to and less on what it is about, which is the focus of other JISC -funded portals.\n\nThe Go-Geo! portal also allows users to simultaneously search across UK metadata catalogues (Figure <a href=\"#F4\" data-ref-type=\"fig\">4<\/a>) including the\n\n\u2022 National Environment Research Council's (NERC) Data Centre catalogues \\[53\\]\n\n\u2022 Arts and Humanities Data Services (AHDS) \\[54\\]\n\n\u2022 National Soils Research Institute \\[55\\] and\n\n\u2022 UK Data Archive's catalogue collections \\[56\\].\n\nThe current national, government funded GIgateway portal \\[57\\] can also be searched using the Go-Geo! portal. Reciprocally, Go-Geo! serves as the Virtual Academic Catalogue (GVAC) node for GIgateway; however, work is afoot to replace this with a metadata catalogue using UK GEMIN 2.1, an INSPIRE-compliant metadata standard for the GI-community in the UK \\[58\\]. The INSPIRE Metadata Implementing Rules are guidelines with elements derived from ISO 19115 \\[59\\]. The Go-Geo! catalogue currently holds 657 metadata records with hundreds more harvested from other UK catalogues and delivered to Go-Geo! portal users.\n\nAnother important feature for users of Go-Geo! is the geographic information resource section, which provides comprehensive information about training courses, learning materials, news, events, jobs, geographical information organisations, books and journals, online services, software, standards, data providers and more. GI-related resources are added to Go-Geo! on a daily basis with almost 3,000 added to date.\n\nGo-Geo! also includes support for place name clarification. Unlock \\[60\\], originally known as geoXwalk, is a JISC-funded middleware service which was developed in part to support geographic searching of Go-Geo! (Figure <a href=\"#F5\" data-ref-type=\"fig\">5<\/a>). Unlock can translate a place name term into a different geography that the searched target can understand. This 'crosswalk' capability of crossing different geographies to return geographical 'equivalents' significantly improves the utility of the original search target.\n\n## UK Academic Geospatial Metadata Application Profile\n\nThe UK Academic Geospatial Metadata Application Profile (UK AGMAP 2) is a template designed to comply with INSPIRE and support the documentation and discovery of spatial data, data series and geo-services within UK academia. UK AGMAP 2 contains all the elements required to generate compliant metadata records for the Go-Geo! portal. The UK AGMAP 2 profile was created to achieve the following goals:\n\n\u2022 serve as a metadata element field template for the Go-Geo! portal and metadata editor tool;\n\n\u2022 deliver support of spatial data and services documentation across an eclectic academic community, which includes extending it to support specific requirements of any discipline;\n\n\u2022 facilitate data management and sharing requirements within academia;\n\n\u2022 ensure compliance with the ISO 19115, INSPIRE and UK GEMINI 2.1, a UK standard which was ratified in 2010 and is compliant to INSPIRE; and\n\n\u2022 provide the academic user with the information necessary to assess the data, and the necessary contact details to enquire further about access and use.\n\nA guidelines document of almost 200 pages was written to serve as a reference for users of UK AGMAP 2. Use of the guidelines can also ensure that compliant metadata records are created for publication on the Go-Geo! portal site or for local data management schemes. The guidelines also represent a critical resource for improving metadata quality and currency. The guidelines also include examples to provide greater clarity for the user. Many examples are presented in a manner which can be understood across social and physical science.\n\nMetadata records displayed on the Go-Geo! portal include direct links from the individual elements within the record to the corresponding information in the guidelines. A user unfamiliar with a metadata element displayed in a record can click it and information for that element is delivered in a new window.\n\nThe UK AGMAP 2 profile has also been cross-mapped to other relevant standards. These mappings were done to assess compatibility, and address interoperability and harmonisation among standards to improve cross-searching capabilities. Cross-mapping was also done to support the export of XML metadata records into the following formats using the Geodoc metadata editor tool:\n\n\u2022 ISO 19115 Geographic Information Metadata Standard;\n\n\u2022 Federal Geographic Data Committee's (FGDC) Content Standard for Digital Geospatial Metadata (CSDGM);\n\n\u2022 UK GEMINI 2.1;\n\n\u2022 INSPIRE;\n\n\u2022 Dublin Core; and\n\n\u2022 Data Documentation Initiative (DDI) Standard.\n\nDublin Core (ISO 15836) represents a set of 15 metadata elements which can be used to provide basic descriptive information (title, abstract, date, identifier) about a resource for cataloguing purposes \\[61\\]. The descriptive information for most spatial data is too complex for Dublin Core, hence the publication of the FGDC Content Standard for Digital Geospatial Metadata in the middle 1990s in the US, with uptake across the international GI-community. Subsequently, in 2003, the ISO 19115 standard was ratified as a machine-friendly version of the FGDC standard. The ISO 19115 standard is intended to facilitate the search and harvest of metadata records from all portal catalogues. The ISO 19115 standard also allows for GI-related groups to create profiles or guidelines for their respective communities; this is the case with INSPIRE for the European GI community, and UK GEMINI 2, which is INSPIRE-compliant and serves the GI community in the UK.\n\nAs noted, the UK AGMAP 2 Profile comprises these elements to be compliant with ISO 19115, INSPIRE and UK GEMINI 2. It is also compliant with the Data Documentation Initiative (DDI) Standard. The Data Documentation Initiative (DDI) is an effort to create an international standard for describing social science data, most of which have a spatial context \\[62\\]. The assumption is that about 80 percent of all data have a spatial context including health care data \\[63\\].\n\n## Geodoc Metadata Editor Tool\n\nGeodoc (Figure <a href=\"#F6\" data-ref-type=\"fig\">6<\/a> and Figure <a href=\"#F7\" data-ref-type=\"fig\">7<\/a>) is an online Java-built metadata editor tool which delivers the UK AGMAP 2 profile to the academic community for creating discovery and descriptive level geospatial metadata records and geo services. Geodoc is accessible via the Go-Geo! portal home page \\[45\\] through an authentication service available to only those affiliated with UK academia. This restricted access allows users to create metadata with Geodoc and store records in a local and private directory, which no one else can access. This is intended to encourage metadata creation amongst those with concerns over Intellectual Property Rights (IPR), or sensitive data, but wanting to document their datasets for data management purposes. The Go-Geo! service offers support for the creation of institutional nodes, which reveal metadata to only those affiliated with an institution. This functionality can be extended to allow peers within a research group, but different academic affiliations, to create and share metadata amongst themselves for the purpose of project data management. The Go-Geo! portal provides the interface for searching their metadata, but only through the authentication log-in service, which reveals the private node created for this group or institution.\n\nGeodoc offers a range of tools in support of metadata creation, management, publication and data sharing. In terms of functionality, Geodoc delivers a map interface to capture bounding box extent coordinates, with pre-defined bounding box extents to select for the UK, Great Britain, England, Northern Ireland, Scotland and Wales; the automation of contact detail information which populates relevant fields for each new metadata record; metadata record validation; edit directory function for storing metadata records; XML export function to aforementioned standards including INSPIRE and UK GEMINI 2; direct links between Geodoc elements and supporting text in UK AGMAP 2 guidelines; and a publication function which allows user to submit records for publication on Go-Geo! or a private\/institutional node.\n\nA geospatial metadata officer reviews all submitted Geodoc metadata records to ensure they are compliant and meet quality standards for publication on Go-Geo! or an academic institutional node. The Go-Geo! service offers academic institutions the option of publishing metadata records on private nodes. This allows only those affiliated with the institution to access the metadata in their node's catalogue via an online authentication service. The creation of institutional nodes is intended to address any concerns that might arise over Intellectual Property Rights (IPR). Data developers are also encouraged to submit their datasets to the geospatial metadata officer for extracting information to create partial metadata records which can be returned to a dataset submitter for completion.\n\nGeodoc can also serve as a teaching and learning resource in the UK academic community. Academics can use Geodoc and the Go-Geo! portal to introduce metadata to students. This is critical in changing the mindset for prioritising data management and sharing amongst future GI professionals and academics in the UK.\n\n## ShareGeo Spatial Data Repository\n\nAnother important component to the Go-Geo! SDI is the ShareGeo spatial data repository which supports data sharing between creators and users of spatial data. Two versions, built using DSpace \\[64\\] exist for UK academic researchers, students and lecturers to deposit and download data. ShareGeo Open \\[65\\] (Figure <a href=\"#F8\" data-ref-type=\"fig\">8<\/a>) accepts and holds spatial data with no licensing or Intellectual Property Rights (IPR) restrictions; a closed version of ShareGeo (Figure <a href=\"#F9\" data-ref-type=\"fig\">9<\/a>) resides within the Digimap Collection services \\[66\\] and can only be accessed through an authentication service. This version is intended to hold spatial data derived from licensed data made available through the Digimap Collections, which include data products from the Ordnance Survey (OS) and others. ShareGeo Open was launched September 2010 and holds 95 datasets of various themes. The closed version of ShareGeo in Digimap Collections contains 101 datasets.\n\nThe spatial data landscape is changing with the UK Government calling for more OS data to be made free to the public and to stimulate growth in the GI industry (Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>). The availability of OS OpenData products \\[67\\] to data users should also provide the impetus for more data creation, hence more content for the ShareGeo Open repository.\n\nTable showing which OS OpenData products released to the public\n\n| 1:250,000 Scale Colour Raster                          |\n|--------------------------------------------------------|\n| MiniScale                                              |\n|                                                        |\n| Meridian                                               |\n|                                                        |\n| Strategi                                               |\n|                                                        |\n| Land-Form Panorama Contour                             |\n|                                                        |\n| Land-Form Panorama DTM                                 |\n|                                                        |\n| Boundary-Line                                          |\n|                                                        |\n| Code-Point Open (point data only; subset of Code-Point |\n|                                                        |\n| 1:50,000 Scale Gazetteer                               |\n|                                                        |\n| OS Streetview                                          |\n|                                                        |\n| OS Locator                                             |\n|                                                        |\n| OS VectorMap District                                  |\n\n## Other resources\n\nMetadata Workshops are offered as part of an effort to change the mindset and culture of the academic community. The workshops provide an overview of metadata, standards, portals, and stress the importance of creating metadata records for supporting data sharing and good data management practices. Workshop participants are also informed about the benefits associated with creating and publishing metadata on a geoportal, including protection of investments of time and cost dedicated to data development; maintenance of an inventory of datasets to reduce time required to reassess existing datasets for new and future applications; ensuring integrity of existing and new datasets using metadata as a tracking mechanism to monitor changes and edits to datasets (version control); minimisation of the disruptive effects of staff turnover; elimination or reduction of the risk of redundancy in data collection or deletion of existing datasets; compliance with the EC INSPIRE Directive that requires metadata creation at the public sector level including academia \\[14,15\\]; and how metadata publication can greatly facilitates data discoverability, which in turn encourages collaboration.\n\nWorkshop participants learn how a geoportal can serve as a repository to store and manage metadata, thus savings in cost and time; how to use metadata to announce data and applications; how to advertise (and, where applicable, sell) spatial datasets to other interested parties; how published metadata can be referenced and cited for project proposals; and how metadata can be configured as an internal resource to access and share datasets.\n\nThe INSPIRE Directive is also introduced at workshops with an explanation about its implications for the UK academic community and those students taking employment in the public sector. Moreover, the workshops offer participants the opportunity to conduct hands-on exercises for the Go-Geo! portal and Geodoc metadata editor tool to develop a familiarity with their functionality and recognise and appreciate the simplicity involved in the creation and publication of metadata records. There have been 32 workshops run over five of the past seven years at 24 UK academic institutions with an average of 14 people attending each workshop.\n\nThe Go-Geo! portal also provides eLearning objects (Figure <a href=\"#F8\" data-ref-type=\"fig\">8<\/a>) in the form of animations and interactive modules about metadata and geospatial metadata; the latter provides a scenario which shows the steps of capturing and processing data to create a spatial dataset. This serves as a bridge for transferring this information to a metadata record using the Geodoc metadata editor tool. The geospatial metadata module introduces the user to more complex information of a dataset including positional accuracy, lineage, completeness and scale.\n\nA biannual metadata newsletter is also published and disseminated to almost 200 lecturers and researchers across UK academia. Go-Geo! Metadata News provides readers with updates on Go-Geo! services and news about events and activities across the GI-community which would be of interest to the readers.\n\n# Discussion and conclusions\n\n## Challenges\n\nGood data management and sharing practices are vital to the efficient use of geospatial information. The establishment of numerous spatial information infrastructures is a testament to the recognition of this fact, but there are still significant challenges to ensure these investments are not wasted.\n\nTechnological advances and software engineering, standards and guidance are important instruments for the development of SDIs; however, reciprocal effort from the data developers still lags behind the technical developments. This must be approached through understanding their perceptions as policy implementers, then addressed with support mechanisms and a consistent policy, which does not impose obstacles and recognises the obstacles and challenges data developers face.\n\nThat the creation of metadata is seen as a tedious and time-consuming exercise is one problem which must be overcome. Software engineering can offer limited solutions through metadata automation \\[68,69\\], but considerable work remains before this comes to fruition.\n\nEven with metadata automation, and a complement of SDI services to support the delivery of metadata and spatial data, there still remain a number of concerns which must be addressed, including Intellectual Property Rights (IPR); residual licensed data rights for derived data; liability concerns; concerns over data quality (data creator and user); privacy and security issues; time and cost to anonymise data for release; time and cost for data delivery and for metadata record updates, especially descriptive level metadata; data transformation and harmonisation issues (scale, positional accuracy, projections, formats); legacy data concerns; properly dealing with VGI (Volunteered Geographic Information); issues related to performance, maintenance and enhancements of portals and repositories; data archiving requirements; data and software warehousing issues; long-term commitment and investment in the infrastructure; properly addressing revisions to standards; and solving any confusion about standards compliance and which standard to use.\n\nThere is no simple solution for confronting these issues. Intellectual Property Rights (IPR) is a primary concern especially amongst many in UK academia especially with regards to residual licensed data rights for derived data from the OS and other licensed data providers.\n\nLiability and data quality concerns are shared between data developers and users. Good metadata records can address these, but liability concerns require legal consultation, especially for spatial data which could be used for emergency responses or monitoring the spread of infectious diseases. Disclaimers would be necessary, but with regards to the distribution of altered spatial data, will a data use disclaimer protect the original data provider? Will the data quality statement in the metadata record be accurate or updated correctly to reflect changes made to the spatial dataset? In academia, are researchers willing to expose their datasets to face further scrutiny? Anonymous sources have suggested that some data developers in academia might eschew metadata creation and data sharing due to concerns over the reliability of their data?\n\nUK Health researchers face additional challenges with the creation of data which hold sensitive information and require anonymisation. The Data Protection Act 1998 \\[70\\] establishes legal requirements for health researchers to follow to ensure the confidentiality of personal information relating to research participants.\n\nThis reality imposes an expensive and time-consuming requirement on health researchers creating and using data which must be anonymised. There are also concerns about potential breaches (technological or legal) of data security and confidentiality \\[71\\].\n\nThe time and cost factors for anonymising, transforming and delivering data remains as a major obstacle for most data providers. These processes are often necessary to prepare data for deposit in a data repository. Updates made to resubmitted data must be recorded in accompanying metadata records.\n\nInfrastructure and archiving are other concerns of data developers with regards to the reliability of SDI service providers. Can data developers be assured that their data can be properly managed, stored, displayed and disseminated? Failure of a service to make data accessible to users could lead to the data provider being directly contacted for data requests. Data developers may also use SDI services for the management of their data to save on infrastructure costs, hence an incentive for them to process and deliver data to an SDI service. However, data developers raise concerns about long-term data storage and migration of their data in the event the SDI service is discontinued.\n\nChanges in data policies and revisions to standards also impose additional concerns on data developers. There are numerous standards, application profiles and competing interests across the various sectors. As noted, about 80 percent of all data have a spatial context \\[63\\]. Furthermore, privacy and security require considerable investment in the infrastructure that holds the data and metadata.\n\n## Solutions\n\n### OSGeo\n\nThe OSGeo Geodata Committee \\[72\\] is playing an important role in providing open source solutions to address a number of the concerns and challenges data developers face. These proposed remedies fall under the auspices of the Public Geospatial Data Project \\[73\\], which offers the following in its mission statement:\n\n\u2022 promote the use of open and free geospatial formats (GML, WMS, WFS-T) and metadata (Dublin Core, RDF, ISO 19115 through ISO 19139)\n\n\u2022 promote public access to state-collected spatial data\n\n\u2022 run an open spatial data repository with links to other open repositories\n\n\u2022 present and explain licences for public spatial data through the collection of licences suitable for the publication of public spatial data.\n\nThe Open Knowledge Foundation \\[74\\] also endeavours to promote 'open knowledge' through its main principles of (i) free and open access to material; (ii) freedom to redistribute the material; (iii) freedom to reuse the material; and (iv) no legal, technological or social restrictions of the above based on who someone is or their field of endeavour (e.g. commercial or non-commercial).\n\nThe OSGeo Geodata Committee and Open Knowledge Foundation have taken important steps in the investigation and challenge of key issues which dissuade data sharing, especially with regards to various restrictions and licensing concerns. Another goal of the OSGeo Public Geospatial Project is to offer, in the future, a repository of reusable public geographic data that can support open source geospatial software projects \\[75\\]. These efforts can allow various GI-communities with limited resources to use the OSGeo resources and expertise to manage and disseminate their spatial data.\n\n### Go-Geo! Services\n\nThe Go-Geo! SDI service can provide UK academics and researchers with the resources to manage and disseminate their spatial data while addressing concerns about IPR, anonymised data and residual licensed data rights for derived data.\n\nThe UK AGMAP 2 metadata application profile can be extended to support the specific requirements of a discipline. As an example, UK AGMAP 2 could include elements which support the description of anonymised health data. A compliant application profile will include the ISO 19115's core mandatory elements, then be extended to include elements which address the needs of health researchers. It is at this level where consensus must be reached within the discipline to decide which elements should be created. Health portal metadata catalogues will be built to conform to this extended health data profile, but with the core ISO 19115 elements. This will ensure that searches conducted from other thematic portals will harvest discovery-level metadata from the health portals. These metadata records should contain information which directs users from other portals to the health portals where they can access the complete metadata records.\n\nThe Geodoc Metadata Editor tool allows users concerned about IPR and data privacy to store their metadata records in private directories, or publication on institutional nodes. The private Geodoc directory can also address the concerns and needs of medical researchers using and creating data with personal, sensitive information. Geodoc's export function also allows users to export metadata records to Dublin Core, DDI, UK GEMINI 2, FGDC and INSPIRE formats. As XML files, these records can uploaded with data to repositories, or shared between data users.\n\nDerived data with residual licensing rights can be submitted to the closed version of ShareGeo which requires access via the Digimap Collections' authentication service. The ShareGeo Open version could also be extended to hold software source code and binaries, as well as software plug-ins, add-ons and extensions.\n\nThese Go-Geo! resources offer the UK academic community a no-cost and time effective solution for data management and sharing, because cost and time have been identified as primary reasons for not publishing metadata or sharing spatial data. Data developers are also invited to submit their spatial data to Go-Geo! services for extracting information to create partial metadata records, though this offer is seldom embraced.\n\n## INSPIRE\n\nThe EC INSPIRE Directive \\[22\\] has a direct effect on the European GI-community as every Member State of the EU must transpose this Directive into law. The INSPIRE Regulations 2009 No 3157 came into force on 31 December 2009 and applies to England, Northern Ireland and Wales \\[76\\]; Scotland's Parliament enacted a complementary regulation which came into force on the same date \\[77\\].\n\nThe remit of the INSPIRE Directive is to \"establish an infrastructure for spatial information in Europe to support Community environmental policies, and policies or activities which may have an impact on the environment.\" This, in effect, is a mandate for the creation of metadata and data sharing across the EU public sector in support of establishing an SDI for environmental information.\n\nThe environment has a direct impact on the daily lives of millions of EU citizens. Data modelling becomes an integral part of planning in the prevention and preparation of natural disasters which can pose serious risks to people, property and livelihoods. Experts building these models must be confident in the authenticity of the 'data' within the models, especially when situation awareness becomes critical and time is of the essence to. Inter-dependencies between environment, infrastructure and population create a situation where multi-criteria decisions become even more sensitive to accuracy of data and metadata.\n\nEnvironment and health are linked with regards to direct effects of environmental disasters on people, and environmental data used for epidemiological research.\n\nThis reality would seem to provide the impetus for data developers across the EU to embrace metadata and data sharing especially those from the health community.\n\n# Competing interests\n\nThe authors declare that they have no competing interests. TM works as the geospatial metadata co-ordinator for the Go-Geo! SDI Service.\n\n# Authors' contributions\n\nTM and MNKB conducted the literature review and wrote the manuscript. TM also provided detailed, firsthand information about the Go-Geo! SDI Service. Both authors read and approved the final version of the paper.\n\n## Acknowledgements\n\nGo-Geo! is fully funded by the Joint Information Systems Committee (JISC) under the JISC Portals Programme.","meta":{"dup_signals":{"dup_doc_count":104,"dup_dump_count":55,"dup_details":{"curated_sources":4,"2023-40":1,"2023-14":1,"2022-49":1,"2022-33":1,"2022-21":2,"2021-43":2,"2021-21":1,"2020-50":2,"2020-45":2,"2020-16":1,"2019-43":1,"2019-35":1,"2019-26":1,"2019-18":1,"2019-13":1,"2019-04":1,"2018-51":1,"2018-47":2,"2018-43":2,"2018-34":1,"2018-30":1,"2018-26":1,"2018-13":1,"2018-09":1,"2017-51":1,"2017-43":1,"2017-34":1,"2017-30":2,"2017-26":2,"2017-22":9,"2017-17":3,"2017-09":4,"2017-04":1,"2016-50":1,"2016-44":3,"2016-40":2,"2016-36":9,"2016-30":3,"2016-22":1,"2016-18":1,"2015-27":1,"2015-22":2,"2015-14":2,"2014-52":2,"2014-42":2,"2014-23":1,"2023-50":1,"2017-13":3,"2015-18":3,"2015-11":2,"2015-06":1,"2014-10":1,"2013-48":3,"2013-20":1,"2024-22":1}},"file":"PMC3224535"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  All archaeal and many bacterial genomes contain Clustered Regularly Interspaced Short Palindrome Repeats (CRISPR) and variable arrays of the CRISPR-associated (*cas*) genes that have been previously implicated in a novel form of DNA repair on the basis of comparative analysis of their protein product sequences. However, the proximity of CRISPR and *cas* genes strongly suggests that they have related functions which is hard to reconcile with the repair hypothesis.\n  .\n  # Results\n  .\n  The protein sequences of the numerous *cas* gene products were classified into \\~25 distinct protein families; several new functional and structural predictions are described. Comparative-genomic analysis of CRISPR and *cas* genes leads to the hypothesis that the CRISPR-Cas system (CASS) is a mechanism of defense against invading phages and plasmids that functions analogously to the eukaryotic RNA interference (RNAi) systems. Specific functional analogies are drawn between several components of CASS and proteins involved in eukaryotic RNAi, including the double-stranded RNA-specific helicase-nuclease (dicer), the endonuclease cleaving target mRNAs (slicer), and the RNA-dependent RNA polymerase. However, none of the CASS components is orthologous to its apparent eukaryotic functional counterpart. It is proposed that unique inserts of CRISPR, some of which are homologous to fragments of bacteriophage and plasmid genes, function as prokaryotic siRNAs (psiRNA), by base-pairing with the target mRNAs and promoting their degradation or translation shutdown. Specific hypothetical schemes are developed for the functioning of the predicted prokaryotic siRNA system and for the formation of new CRISPR units with unique inserts encoding psiRNA conferring immunity to the respective newly encountered phages or plasmids. The unique inserts in CRISPR show virtually no similarity even between closely related bacterial strains which suggests their rapid turnover, on evolutionary scale. Corollaries of this finding are that, even among closely related prokaryotes, the most commonly encountered phages and plasmids are different and\/or that the dominant phages and plasmids turn over rapidly.\n  .\n  # Conclusion\n  .\n  We proposed previously that Cas proteins comprise a novel DNA repair system. The association of the *cas* genes with CRISPR and, especially, the presence, in CRISPR units, of unique inserts homologous to phage and plasmid genes make us abandon this hypothesis. It appears most likely that CASS is a prokaryotic system of defense against phages and plasmids that functions via the RNAi mechanism. The functioning of this system seems to involve integration of fragments of foreign genes into archaeal and bacterial chromosomes yielding heritable immunity to the respective agents. However, it appears that this inheritance is extremely unstable on the evolutionary scale such that the repertoires of unique psiRNAs are completely replaced even in closely related prokaryotes, presumably, in response to rapidly changing repertoires of dominant phages and plasmids.\n  .\n  **This article was reviewed by**: Eric Bapteste, Patrick Forterre, and Martijn Huynen.\n  .\n  # Open peer review\n  .\n  Reviewed by Eric Bapteste, Patrick Forterre, and Martijn Huynen.\n  .\n  For the full reviews, please go to the Reviewers' comments section.\nauthor: Kira S Makarova; Nick V Grishin; Svetlana A Shabalina; Yuri I Wolf; Eugene V Koonin\ndate: 2006\ninstitute: 1National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA; 2Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-9050, USA\nreferences:\ntitle: A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action\n\n# Background\n\nThe discovery of the elaborate and versatile systems of RNA silencing in eukaryotes is one of the pivotal advances in biology of the last decade \\[1-6\\]. There are two major, distinct forms of regulatory small RNAs involved in eukaryotic gene silencing: small interfering (si) RNAs and micro (mi) RNAs. siRNAs are produced from double-stranded RNAs of viruses and transposable elements, which are processed by the dicer nuclease, one of the essential components of the RNA-Induced Silencing Complexes (RISCs) \\[7-9\\]. Dicer cleaves long dsRNA molecules into short, 21\u201322 nucleotide duplexes which are subsequently unwound by the RISC to yield mature siRNAs. The RISC-siRNA complex then binds to the target mRNA which is cleaved by the slicer nuclease, another crucial component of RISC, to release the RISC-siRNA which acts as a recyclable catalyst \\[9,10\\]. In addition to silencing genes of exogenous agents, a distinct class of longer, 28 nt siRNAs, the so-called repeated-associated siRNAs (rasiRNAs), silence expression of chromosomal copies of transposons and transposon-like repeats \\[11-13\\].\n\nUnlike the siRNAs, 21\u201325 nt-long miRNAs are encoded in eukaryotic genomes and are either perfectly (in plants) or imperfectly (in animals) complementary to sequences in the 3'-untranslated regions of specific endogenous mRNAs \\[6,13\\]. Base-pairing of miRNAs with the target mRNAs, which is mediated by a distinct form of RISC, results either in RNA cleavage or in down-regulation of translation \\[8\\]. Evidence is rapidly accumulating that numerous, probably, thousands of miRNAs in animals and plants are major players in development regulation and chromatin remodeling \\[6\\].\n\nProkaryotes have apparent functional counterparts to the miRNA system, i.e., regulation of bacterial gene expression by small antisense RNAs. The best characterized of these pathways employ the RNA-binding protein Hfq for small RNA presentation and RNAse E for target degradation \\[14-17\\]. *Escherichia coli* has \\~60 microRNA genes, and comparable numbers of expressed, small antisense RNAs have been detected in the archaea *Archaeoglobus fulgidus* \\[18\\] and *Sulfolobus solfataricus* \\[19\\], suggesting an important role of this regulatory mechanism in prokaryotic physiology. In addition, small antisense RNAs have been shown to regulate plasmid replication and killing of plasmid-free bacterial cells by silencing specific plasmid genes \\[20-22\\]. In contrast, counterparts to the eukaryotic siRNA mechanism so far have not been described in prokaryotes. Here, we apply comparative genomics and in-depth computational analysis of protein and RNA sequences and structures to predict a distinct prokaryotic siRNA-like system and the associated enzymatic apparatus.\n\nIn a previous comparative-genomic study, which has been originally conceived as a test case for methods for conserved gene neighborhood analysis we have developed, we characterized an extensive set of genes that included several proteins related to DNA or RNA metabolism and was, mostly, specific to thermophiles \\[23\\]. These genes comprise a complex array of overlapping neighborhoods that are partially conserved but highly diversified, in terms of both gene composition and gene order, and are represented in all archaeal and many bacterial genomes \\[23,24\\]. At the time of its discovery, we hypothesized that these genes encoded an uncharacterized, versatile repair system, largely, associated with the thermophilic lifestyle \\[23\\].\n\nIndependently and almost simultaneously, Jansen and coworkers found \\[25\\] that at least several genes from this gene neighborhood were tightly associated with the so-called Clustered Regularly Interspaced Short Palindrome Repeats (CRISPR); the acronym *cas* (for CRISPR-associated) genes was thus coined. The CRISPR are a distinct class of repetitive elements that are present in numerous prokaryotic genomes. A CRISPR element consists of a direct repeat of \\~28\u201340 base pairs (bp), with the copies separated by a unique sequence of \\~25\u201340 bp. Typically, CRISPR form tandem arrays containing from 4 to \\>100 elements. Most of the genomes contain a single array of CRISPR in which the sequences of the repeats are (nearly) identical; some, however, possess multiple CRISPR cassettes that may have substantially different sequences \\[24,25\\]. The repeats in CRISPR from different genomes show only limited similarity, but often retain distinct, conserved motifs shared even by distant species including archaea and bacteria \\[18,26\\]. There seems to be a strict link between CRISPR and cas genes, suggestive of a (nearly) mutualistic relationship: the great majority of the genomes that contain CRISPR also have at least a minimal set of cas genes, and vice versa.\n\nRecently, Mojica and coworkers reported that the unique inserts in some of the CRISPR are homologous to fragments of bacteriophage and plasmid genes \\[27\\]. This led to the hypothesis that the CRISPR might have a function in the defense of prokaryotes against invading foreign replicons and that there could be functional analogies between this putative defense system and eukaryotic RNA interference. Similar findings have been independently reported by two other groups \\[28,29\\].\n\nThe recent rapid growth of the number and diversity of sequenced prokaryotic genomes has led to a dramatic increase in the complexity of the identified cas gene arrays \\[24\\]. Here we describe the results of an exhaustive sequence analysis of the Cas protein sequences which yielded a classification of these proteins, several new functional predictions, and a reconstruction of evolutionary relationship between these genes. We propose that the cas genes encode the protein machinery of a prokaryotic siRNA-like system that performs, primarily, but perhaps, not exclusively, defense functions and is generally similar, in some respects, to eukaryotic siRNA, and in other respects, to the vertebrate immune system. The predicted enzymatic machinery of this system seems to be functionally analogous, but not homologous, to the protein apparatus involved in the eukaryotic RNA-mediated gene silencing. Finally, we outline possible molecular mechanisms of the predicted prokaryotic siRNA system. The hypothesis on the involvement of Cas proteins in an RNAi-type mechanism supplants the previous proposal that these proteins might comprise a novel DNA repair system \\[23\\] which is hardly compatible with the tight association of these proteins with CRISPR and the existence of unique CRISPR inserts homologous to phage and plasmid sequences.\n\n# Results and discussion\n\n## Identification, classification and evolutionary analysis of cas genes\n\nIn the original study of the *cas* gene neighborhoods, which was performed with \\~40 genomes, we identified \\~20 protein families that were tightly or more loosely associated with the system we now call CASS. A recent update by Haft and coworkers with \\>200 genomes yielded a diverse \"guild\" of \\~45 Cas protein families \\[24\\]. Many of the Cas proteins show very low sequence conservation which makes identification of homologous relationship between them a non-trivial task. We employed an iterative approach to the exhaustive analysis of the Cas protein sequences. The protein sequences of each Cas family were compared to the protein sequences from all available prokaryotic genomes using PSI-BLAST \\[30,31\\], the proteins encoded in the neighborhoods of all identified candidates were used as queries for further searches, and the process was iterated until convergence. The sequences of Cas proteins were, in addition, carefully compared to each other, in an attempt to identify possible traces of common origin of some of these genes that have so far eluded detection \\[for a complete list of Gene Identification (GI) numbers of the detected Cas proteins, see <a href=\"#S1\" data-ref-type=\"supplementary-material\">Additional file 1<\/a>\\].\n\n### COG1518, a universal marker of CASS\n\nIn agreement with the previous observations \\[23\\], we found that Cas1 (COG1518 in the Clusters of Orthologous Group of proteins classification system \\[32\\]) is the best marker of the CRISPR-associated systems (CASS). This gene encodes a highly conserved protein and that is represented in all cas neighborhoods, with the single exception of *Pyrococcus abyssii*. A PSI-BLAST search for COG1518 members in the completely sequenced prokaryotic genomes revealed the presence of at least one representative of this COG in 77 of the 177 analyzed genomes.\n\nGiven that COG1518 is represented in all versions of CASS, the distribution of this gene among prokaryotic lineages serves as a proxy for the distribution of CASS itself (*P. abyssii* was included as having CASS, the absence of COG1518 notwithstanding); this distribution is, obviously, non-uniform (Fig. <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>). All sequenced archaeal genomes, including, notably, the tiny genome of *Nanoarchaeum equitans*, encode this protein. In contrast, in each of the bacterial lineages with a representative set of sequenced genomes, half or more species do not have COG1518. We came across several cases of differences in the presence of CASS among closely related bacterial species and even between strains of the same species. Thus, *Corynebacterium diphtheriae* has a COG1518 gene but *Corynebacterium efficiens* does not; similarly, CASS was detected in *Mycobacterium tuberculosis* but not in *Mycobacterium bovis* or *Mycobacterium leprae*; significant differences in the composition of CASS were noticed even between strains of several bacterial species, e.g., *Thermus thermophilus* \\[33\\].\n\nPhylogenetic analysis of COG1518 combined with gene neighborhood comparisons yields a clear classification of CASS based on gene composition and (predicted) operon organization (Fig. <a href=\"#F2\" data-ref-type=\"fig\">2a<\/a>) which is, generally, in agreement with the recent study of Haft et al \\[24\\]. Altogether, we delineated 7 distinct versions of CASS which show substantial differences in gene composition and predicted operon organization. Only four CASS variants (CASS1-4) have a more or less stable operon organization. While a distinct operon organization motif is recognizable in the other CASS variants as well (Fig. <a href=\"#F2\" data-ref-type=\"fig\">2a<\/a>), these versions have undergone numerous, complex rearrangements in different prokaryotic genomes including gene duplications, fusion with other CRISPR-related operons, insertion of additional genes, and others. Thus, in line with the observation that CASS is often eliminated from genomes during evolution, this plasticity of CASS operon organization suggests that the CRISPR-associated genomic regions are \"hot spots\" of recombination and ensuing genome rearrangement. Taken together, all these observations demonstrate the extraordinary evolutionary mobility of CASS.\n\nThe recent work of Haft et al has identified a \"guild\" of over 45 protein families comprising the CASS \\[24\\]. Here we refine this classification by unifying many of the families into superfamilies and also expand the list of CASS-linked genes to include those that are found in *cas* operons less commonly and comprise the \"cloud\" surrounding the core CASS components. Only COG1518 and COG1343 (see below) are invariably present in all versions of CASS (with the exception of *P. abyssii* and several bacterial species that carry disrupted COG1518 gene). A few genomes that have a cassette of CRISPR-associated genes without COG1518 also possess another CRISPR-associated operon in a different genomic location that does include this gene. Altogether, we end up with \\~25 gene families that are tightly associated with CASS (Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>)\n\nProtein components of CASS\n\n<table>\n<thead>\n<tr>\n<th><\/th>\n<th style=\"text-align: center;\"><strong>Family<\/strong><\/th>\n<th style=\"text-align: center;\"><strong>Subfamily<\/strong><sup>A<\/sup><\/th>\n<th style=\"text-align: left;\"><strong>Phyletic distribution<\/strong><sup>B<\/sup><\/th>\n<th style=\"text-align: left;\"><strong>Comments<\/strong><\/th>\n<\/tr>\n<\/thead>\n<tbody>\n<tr>\n<td style=\"text-align: center;\">1<\/td>\n<td style=\"text-align: center;\">COG1518<\/td>\n<td style=\"text-align: center;\">COG1518 (cas1)<\/td>\n<td style=\"text-align: left;\">All<\/td>\n<td style=\"text-align: left;\">Putative novel nuclease\/integrase; Mostly \u03b1-helical protein<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">2<\/td>\n<td style=\"text-align: center;\">COG1343<\/td>\n<td style=\"text-align: center;\">COG1343 (cas2), COG3512, ygbF-like; MTH324-like; y1723_N-like;<\/td>\n<td style=\"text-align: left;\">All<\/td>\n<td style=\"text-align: left;\">Small protein related to VapD, fused to helicase (COG1203) in y1723-like proteins<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">3<\/td>\n<td style=\"text-align: center;\">COG1203<\/td>\n<td style=\"text-align: center;\">COG1203 (cas3)<\/td>\n<td style=\"text-align: left;\">All<\/td>\n<td style=\"text-align: left;\">DNA helicase; Most proteins have fusion to HD nuclease<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">4<\/td>\n<td style=\"text-align: center;\">RecB-like nuclease<\/td>\n<td style=\"text-align: center;\">COG1468 (cas4), COG4343<\/td>\n<td style=\"text-align: left;\">All<\/td>\n<td style=\"text-align: left;\">RecB-like nuclease; Contains three-cysteine C-terminal cluster<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">5<\/td>\n<td style=\"text-align: center;\">RAMP<\/td>\n<td style=\"text-align: center;\">COG1688, COG1769, COG1583, COG1567, COG1336, COG1367, COG1604, COG1337, COG1332, COG5551, BH0337-like, MJ0978-like, YgcH-like, y1726-like, y1727-like<\/td>\n<td style=\"text-align: left;\">All<\/td>\n<td style=\"text-align: left;\">Belong to \"RAMP\" superfamily, possibly RNA-binding protein, structurally related to a duplicated ferredoxin fold (PDB: <a href=\"1WJ9\">1WJ9<\/a>)<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">6<\/td>\n<td style=\"text-align: center;\">COG1857<\/td>\n<td style=\"text-align: center;\">COG1857, COG3649, YgcJ-like, y1725-like<\/td>\n<td style=\"text-align: left;\">All<\/td>\n<td style=\"text-align: left;\">\u03b1\/\u03b2 protein; probable enzymatic activity, possibly, a nuclease<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">7<\/td>\n<td style=\"text-align: center;\">HD-like nuclease<\/td>\n<td style=\"text-align: center;\">COG1203 (N-terminus), COG2254<\/td>\n<td style=\"text-align: left;\">All<\/td>\n<td style=\"text-align: left;\">HD-like nuclease<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">8<\/td>\n<td style=\"text-align: center;\">BH0338<\/td>\n<td style=\"text-align: center;\">BH0338-like MTH1090-like<\/td>\n<td style=\"text-align: left;\">All, mostly archaea and FIRM<\/td>\n<td style=\"text-align: left;\">Large Zn-finger-containing proteins, possibly, nucleases (nuclease activity has been reported for MTH1090 [75].<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">9<\/td>\n<td style=\"text-align: center;\">ygcL<\/td>\n<td style=\"text-align: center;\">ygcL<\/td>\n<td style=\"text-align: left;\">Bacteria, mostly PROTEO<\/td>\n<td style=\"text-align: left;\">Large Zn-finger containing proteins;<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">10<\/td>\n<td style=\"text-align: center;\">COG1353<\/td>\n<td style=\"text-align: center;\">COG1353, MTH326-like, alr1562, slr7011<\/td>\n<td style=\"text-align: left;\">All, mostly Archaea<\/td>\n<td style=\"text-align: left;\">Putative novel polymerase; Multidomain protein with permuted HD nuclease domain, palm domain, polymerase-thumb-like domain and Zn-ribbon; MTH326-like has inactivated polymerase catalytic domain; alr1562 and slr7011 \u2013 predicted only on the basis of size, presence of HD domain, and location with RAMPs in one operon<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">11<\/td>\n<td style=\"text-align: center;\">COG1517\/HTH<\/td>\n<td style=\"text-align: center;\">COG2462<\/td>\n<td style=\"text-align: left;\">Archaea<\/td>\n<td style=\"text-align: left;\">Former COG2462; Fusion of COG1517-like domain to HTH-type transcriptional regulator; Possible regulator of the system expression in archaea<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">12<\/td>\n<td style=\"text-align: center;\">COG1421<\/td>\n<td style=\"text-align: center;\">COG1421<\/td>\n<td style=\"text-align: left;\">All, mostly Archaea<\/td>\n<td style=\"text-align: left;\">~150 aa protein; Has a few motifs similar to ygcK-like; mostly \u03b1-helical protein<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">13<\/td>\n<td style=\"text-align: center;\">ygcK<\/td>\n<td style=\"text-align: center;\">ygcK-like<\/td>\n<td style=\"text-align: left;\">Bacteria, mostly PROTEO<\/td>\n<td style=\"text-align: left;\">~180 aa protein; has a few motifs similar to COG1421; mostly \u03b1-helical protein<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">14<\/td>\n<td style=\"text-align: center;\">COG3337<\/td>\n<td style=\"text-align: center;\">COG3337<\/td>\n<td style=\"text-align: left;\">All, mostly Archaea<\/td>\n<td style=\"text-align: left;\">~110 aa; mostly \u03b1-helical protein<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">15<\/td>\n<td style=\"text-align: center;\">COG1517<\/td>\n<td style=\"text-align: center;\">COG1517<br \/>\nCOG4006<\/td>\n<td style=\"text-align: left;\">All, mostly Archaea<\/td>\n<td style=\"text-align: left;\">Some are fused to HTH domain (see COG1517\/HTH), some proteins have the domain duplication; structure is available (1XMX); domain appears to have a Rossmann-like fold.<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">16<\/td>\n<td style=\"text-align: center;\">COG3513<\/td>\n<td style=\"text-align: center;\">COG3513<\/td>\n<td style=\"text-align: left;\">Bacteria, mostly PROTEO<\/td>\n<td style=\"text-align: left;\">Huge protein; contains McrA\/HNH-nuclease related domain and RuvC-like nuclease domain<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">17<\/td>\n<td style=\"text-align: center;\">PH0918<\/td>\n<td style=\"text-align: center;\">PH0918-like<\/td>\n<td style=\"text-align: left;\">All, mostly Archaea<\/td>\n<td style=\"text-align: left;\">Specific for <em>Pyrococcus<\/em> and <em>Thermococcus<\/em>. The pair ST0031\/SSO1401 and AF1873, most likely, belong to the same family because have similar length and located in the identical place in an operon but due to low conservation are not alignable<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">18<\/td>\n<td style=\"text-align: center;\">AF1870<\/td>\n<td style=\"text-align: center;\">AF1870-like<\/td>\n<td style=\"text-align: left;\">Archaea<\/td>\n<td style=\"text-align: left;\">Former COG3574; ~150 aa protein.<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">19<\/td>\n<td style=\"text-align: center;\">AF0070<\/td>\n<td style=\"text-align: center;\">AF0070-like<\/td>\n<td style=\"text-align: left;\">Archaea<\/td>\n<td style=\"text-align: left;\">~420 aa protein, no prediction<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">20<\/td>\n<td style=\"text-align: center;\">y1724<\/td>\n<td style=\"text-align: center;\">y1724-like<\/td>\n<td style=\"text-align: left;\">Bacteria, mostly PROTEO<\/td>\n<td style=\"text-align: left;\">~450 aa protein, no prediction<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">21<\/td>\n<td style=\"text-align: center;\">SPy1049<\/td>\n<td style=\"text-align: center;\">Spy1049-like<\/td>\n<td style=\"text-align: left;\">Bacteria, mostly FIRM<\/td>\n<td style=\"text-align: left;\">~220 aa protein, no prediction<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">22<\/td>\n<td style=\"text-align: center;\">TTE2665<\/td>\n<td style=\"text-align: center;\">TTE2665-like<\/td>\n<td style=\"text-align: left;\">Bacteria, mostly CHLOR<\/td>\n<td style=\"text-align: left;\">~130 aa protein, no prediction<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">23<\/td>\n<td style=\"text-align: center;\">LA3191<\/td>\n<td style=\"text-align: center;\">LA3191-like<\/td>\n<td style=\"text-align: left;\">Few bacteria<\/td>\n<td style=\"text-align: left;\">~650 aa, no prediction<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n^A^ \u2013 Subfamilies are named by the corresponding COG number or by a protein ID ^B^ \u2013 All indicates that the family is widespread in all major prokaryotic lineages; PROTEO \u2013 proteobacteria; FIRM \u2013 firmicutes; CHLOR \u2013 Chlorobia\n\n### COG1343 and its relatives \u2013 distant homologs of VapD\n\nCOG1343 (cas2) is another gene that is common in CASS. Typically, this gene is located immediately downstream of the COG1518 gene (Fig. <a href=\"#F2\" data-ref-type=\"fig\">2a<\/a>). Exhaustive PSI-BLAST search starting from COG1343 proteins identified proteins of COG3512 as homologs of COG1343 such that these COGs could be unified in a single superfamily. The members of this superfamily are small (80\u2013120 amino acids) proteins with distinct structural motifs, in particular, an N-terminal \u03b2-strand followed by a polar amino acid, most often, aspartate or asparagine (see <a href=\"#S2\" data-ref-type=\"supplementary-material\">Additional file 2<\/a>). In CASS2, which is typified by the *cas* operon of *E. coli*, there is an uncharacterized gene coding for a small protein immediately downstream of the COG1518 gene. Analysis of the multiple alignment of the homologs of this protein revealed motifs highly similar to those in COG1343, suggesting that these proteins actually are diverged COG1343 homologs. Only CASS3 does not seem to contain a gene for a small protein that potentially could be a COG1343 homolog. However, we found that, in CASS3, the next gene after COG1518, which codes for the CASS helicase (cas3 or COG1203) is unusually long and contains a small domain preceding the HD-hydrolase N-terminal domain present in many COG1203 proteins. We analyzed this domain separately and found that its size and motifs were consistent with a homologous relationship with COG1343 (see <a href=\"#S2\" data-ref-type=\"supplementary-material\">Additional file 2<\/a>). Thus, it appears that, either as a stand-alone protein or as part of a multidomain protein, the COG1343 domain is present in all CASS (except for a few highly degraded and, probably, non-functional ones) and, accordingly, could be essential for the CASS functions. Furthermore, searches started with the sequences of many proteins of COG3512 showed some sequence similarity to vapD (COG3309 family), a family of uncharacterized proteins that are functionally linked to the VapBC operon \\[34\\]. The VapBC operon encodes a variant of the bacterial toxin-antitoxin (TA) module which includes an HTH-containing transcription regulator and a PIN-domain nuclease. The PIN domain has been shown to possess ribonuclease activity that in eukaryotes is involved in pre-rRNA processing, nonsense-mediated decay (NMD) of aberrant mRNAs, and RNAi \\[35-37\\]. It has been proposed that there is an evolutionary connection between eukaryotic NMD and bacterial TA systems, and that the functioning of the TA module might involve mRNA degradation as well \\[38\\].Together, these observations seem to establish links between CASS and the TA system and, through the latter, between CASS and eukaryotic NMD and RNAi, but do not directly shed light on the function of the COG1343 domain. However, the COG1343 proteins and VapD show some generic similarity to the PIN domain in terms of size and the signature motifs (COG1343 proteins contain several partially conserved aspartates; see <a href=\"#S2\" data-ref-type=\"supplementary-material\">Additional file 2<\/a>) which makes it tempting to speculate that these proteins represent yet another family of nucleases.\n\n### COG1857 \u2013 another putative enzyme found in most CASS versions\n\nAnother conserved CASS family that resists straightforward functional prediction is COG1857. This gene is present in most CASS, with the exception of the \"minimal\" variants, CASS 4, 6, and 7. The COG1857 proteins consist of \\~350 aa and typically have an N-terminal \u03b2-strand followed by a loop containing a conserved asparagine (see <a href=\"#S3\" data-ref-type=\"supplementary-material\">Additional file 3<\/a>). Although no other motifs containing potential catalytic residues seem to be conserved throughout COG1857, perhaps, due to the extensive divergence between the subfamilies of this family, the observed conservation pattern suggests that this protein is an enzyme, perhaps, yet another nuclease. COG1857 proteins are typically encoded immediately upstream of another widespread CASS gene, COG1688 (named Cas5 by Haft et al. \\[24\\]). We have previously identified COG1688 as a member of the Repair-Associated Mysterious Protein (RAMP) superfamily \\[23\\] that has been renamed to Repeat-Associated Mysterious Protein (preserving the acronym) by Haft and coworkers \\[24\\]. In CASS3, COG1688 is replaced by the y1726 family which we found to include another set of RAMPs distantly related to COG1688 (Figs. <a href=\"#F2\" data-ref-type=\"fig\">2a<\/a> and <a href=\"#F3\" data-ref-type=\"fig\">3A<\/a>).\n\n### The Repeat-Associated Mysterious Proteins (RAMPs)\n\nThe RAMPs are the most diverse class of CASS genes. In addition to the previously identified 5 distinct families of RAMPs, we detected several additional ones, namely, BH0337-like, y1726-like, YgcH-like, y1727-like, and MJ0978-like families, as well as numerous diverged members of the previously described families (Fig. <a href=\"#F3\" data-ref-type=\"fig\">3A<\/a>). Despite the dramatic sequence divergence, all these protein contain the RAMP signature, the G-rich loop at the C-terminus (Fig. <a href=\"#F3\" data-ref-type=\"fig\">3A<\/a>). One family of RAMPs, COG1853\/COG5551, is often encoded outside the CASS operons or on the periphery of these. Moreover, analysis of the gene context of this gene in *Aquifex aeolicus* led us to the identification of yet another member of RAMP superfamily, COG1851. This protein family does not appear to be linked to CASS at all.\n\nWith the identification of these new families of RAMPs, it now becomes apparent that all CASS versions, with the apparent exception of the minimal CASS4, include at least one RAMP. The crystal structure of one of the RAMPs from the newly detected YgcH-like family has been solved as part of one of the structural-genomics projects (PDB: [1wj9](1wj9)). The structure of this protein from *Thermus thermophilus* reveals that the RAMP module is a duplication of a ferredoxin-like fold domain. Each domain has a two-layer \u03b1+\u03b2 architecture and is composed of four \u03b2-strands and two \u03b1-helices topologically arranged as a repeat of two \u03b2\u03b1\u03b2 units (Fig <a href=\"#F3\" data-ref-type=\"fig\">3B<\/a>, A1-A86, \u03b21 through \u03b24; and A87-A211, \u03b25 through \u03b28 for the first and the second domains, respectively). The N-terminal ferredoxin-like domain contains two additional \u03b1-helices (\u03b11' and \u03b11\", Fig <a href=\"#F3\" data-ref-type=\"fig\">3A<\/a>) inserted before and after the first \u03b1-helix. The C-terminal domain has two disordered regions and houses the conserved Gly-rich loop situated between the last \u03b1-helix and \u03b2-strand (Fig. <a href=\"#F3\" data-ref-type=\"fig\">3A<\/a>). Various structure similarity search programs detect ferredoxin fold proteins as the first hits to RAMP domains. In particular, for the N-terminal RAMP domain, DALI \\[39\\], first match is the anticodon-binding domain of Phe-tRNA synthetase (PDB Entry [1eiy](1eiy), chain B) with Z-score 4.8 and RMSD (squared Root of Mean Square Deviations) 2.9\u00c5 over 67 aligned residues. The VAST program \\[40\\] finds ribosomal protein S6 (PDB Entry [1fjg](1fjg) chain F) as the top hit for the C-terminal RMAP domain with P-value 0.039, RMSD 2.6 \u00c5 over 64 residues.\n\nThus, at least six gene (super)families seem to comprise the stable core of the CASS: COG1518 (*cas1*), COG1343 (*cas2*), COG1203 \u2013 a helicase often fused to a HD-family hydrolase (*cas3*) plus free-standing versions of the HD-hydrolase (COG2254), COG1468 (*cas4*) \u2013 a RecB family nuclease usually containing a C-terminal Zn cluster, COG1857, and RAMPs. This exact set of genes is seen only in a few genomes; most versions of CASS have substantial variations around this core \u2013 loss of some core genes in the minimal versions and addition of other genes and whole gene cassettes in others (Fig. <a href=\"#F2\" data-ref-type=\"fig\">2a,b<\/a>).\n\n### The second major module of CASS \u2013 the pol-cassette\n\nThe most notable non-core CASS module which, in a sense, constitutes a second, even if non-ubiquitous, central part of CASS may be called the \"pol-cassette\" after the predicted palm-domain RNA or DNA polymerase of COG1353. The pol-cassette also includes several distinct RAMPs and a few uncharacterized genes. The pol-cassette is strictly linked only to CASS5 and CASS7 and also, in some instances, is found in CASS1,2,4,6 (Fig. <a href=\"#F2\" data-ref-type=\"fig\">2c<\/a>), although, in some genomes, the pol-cassette is not adjacent to the CASS-core gene array. The phylogenetic tree for the predicted polymerase (COG1353), which consisted of two major branches corresponding to two distinct operon organizations (Fig. <a href=\"#F2\" data-ref-type=\"fig\">2c<\/a>), showed essentially no topological congruence with the COG1518 tree (for those CASS that have both components; compare the two trees in Fig. <a href=\"#F2\" data-ref-type=\"fig\">2a,c<\/a>). Thus, it appears that the pol-cassette comprises a distinct evolutionary unit that is often transferred horizontally independently of the CASS-core. Notably, the pol-cassette is strongly, although not strictly, linked to thermophily \u2013 the great majority of the species containing this module, typically associated with CASS5 and CASS7, are thermophiles. Additionally, several species possess a third module containing a diverged form of COG1353 with an apparently intact HD hydrolase domain but an inactivated polymerase (PALM) domain (Fig <a href=\"#F2\" data-ref-type=\"fig\">2c<\/a>).\n\n### Ancillary CASS components\n\nThe CASS-core and the pol-cassette together comprise the extended central componentry of CASS that consists of six COGs plus the RAMP superfamily (Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>). In addition, \\~20 other families were consistently found in CASS, even if in a minority of the CASS-containing species (Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>). It has to be taken into account that most of the CASS proteins have highly diverged sequences, and further unification as well as expansion and the ensuing rise in status of some of the families remains a possibility. For example, COG1517 is such a growing family. Typically, the COG1517 genes are located on the periphery of pol-cassettes and, in many cases, contain fused helix-turn-helix (HTH) domains. The core domain of COG1517 is \\~150 aa; several proteins, e.g. TM1812, contain a duplication of this domain, and several fusions, in addition to the one with HTH, were detected. The sequences of COG1517 proteins tend to be highly diverged, and some of these were detected in genomes with no CASS, indicating that the association of this gene with CASS is not as tight as it is for the extended core of CASS. For one such protein from *Vibrio cholerae*, the 3D structure has been solved, again, in one of the structural genomics projects (PDB: [1xmx](1xmx)). The structure is composed of three domains. The N-terminal domain (A-1-A137, \u03b21 through \u03b27, Fig. <a href=\"#F4\" data-ref-type=\"fig\">4<\/a>) is a modified Rossmann-like fold, with the next to the last \u03b2-strand (\u03b26) being antiparallel to the rest of the strands. The middle domain (A161-A249, \u03b16 through \u03b110) is a winged helix-turn-helix (HTH) with an additional \u03b1-helix on each terminus (\u03b16 and \u03b110, respectively). The C-terminal domain (composite of A250-A383 and A138-A156, \u03b111 through \u03b114 plus \u03b15 and \u03b28) belongs to the restriction endonuclease superfamily. In the 1xmx structure, the endonuclease domain is positioned between the HTH and the Rossmann-like domain (Fig. <a href=\"#F4\" data-ref-type=\"fig\">4<\/a>). The lowest VAST P-values of the matches between each of the 1xmx domains and the respective domains with known folds are highly statistically significant (\\<10^-5^) which is strongly suggestive of homologous relationships.\n\nThe functions of several other CASS gene families remain obscure. For instance, CASS1, 5, 7 contain genes upstream of COG1857 that encode large (500\u2013600 amino acids), homologous proteins; the best conserved family in this set of proteins is represented by BH0338 and its orthologs. Some members of the BH0338 family contain a Zn-ribbon in the middle of the sequence but otherwise have no recognizable domains or motifs. Among several conserved motifs of these proteins, are two conserved aspartates and a distal conserved glycine, a combination that resembles the motifs seen in the PALM polymerase domain (not shown). Although we were unable to obtain additional evidence of the potential connection of this family with polymerases, it is tempting to speculate that these proteins might contain an extremely diverged version of the PALM domain. Similar, albeit less pronounced, motifs are detectable in the MTH1090 family proteins which are present in CASS5 and CASS7. This similarity and the fact that the respective genes occupy the same position in the corresponding operons suggest that the BH0338-family and the MTH1090-family proteins are highly diverged homologs. CASS2 also includes a large protein (YgcL-family), some of which contain Zn-clusters; however, the conserved motifs of these proteins and their position in the respective operons are different from those of the MTH1090 and BH0338 families. CASS4 contains another huge protein (COG3513, \\~1150\u20131400 aa) with two recognizable domains, a McrA\/HNH nuclease and a RuvC-like nuclease (RNAseH fold). These observations emphasize the striking diversity of still poorly characterized CASS components, particularly, the plethora of predicted nucleases of various classes and potential novel ones.\n\n## Hypothesis: CASS is a prokaryotic defense system that functions on the RNAi principle\n\nBased on the properties of CRISPR and Cas proteins, we speculate that this system is a functional analog of the eukaryotic siRNA systems and propose possible mechanisms of the putative prokaryotic small RNA interference. The crucial observation reported independently by Mojica et al \\[27\\], Pourcel et al \\[29\\], and Bolotin et al \\[28\\] is that a certain fraction (\\~10% according to \\[27\\]) of the unique inserts in CRISPR units are homologous to fragments of viral (bacteriophage) or plasmid genomes. Only a miniscule fraction of the existing phage and plasmid sequences is currently available, whereas the total diversity of prokaryotic mobile elements is humungous and apparently exceeds the diversity of prokaryotes at least by an order of magnitude \\[41,42\\]. Thus, it is not far fetched to propose that most, if not all, CRISPR inserts are derived from mobile replicons \\[29\\].\n\nShould that be the case, it seems more or less obvious that CASS is a prokaryotic defense system against foreign replicons that functions on the antisense RNA principle. More specifically, it seems likely that the inserts are transcribed and silence the cognate phage or plasmid genes via the formation of a duplex between the prokaryotic small interfering (psiRNA) and the target mRNA followed by cleavage of the duplex or translation repression. Indeed, Mojica et al \\[27\\] mention the analogy between the CRISPR and eukaryotic RNA interference systems but propose no specific mechanisms for the action of the putative defense systems and, crucially, do not explore the connection between the putative psiRNA and the predicted activities of Cas proteins. Important supporting evidence has been independently obtained through the analysis of the small non-messenger RNA expression in the euryarchaeon *Archaeoglobus fulgidus* which showed that CRISPR are transcribed (from a leader-promoter sequence), apparently, in the form of a multiunit precursor that is subsequently cleaved into CRISPR monomers and oligomers \\[18\\]; very similar observations have been subsequently reported for the crenarchaeon *Sulfolobus solfataricus* \\[19\\]. Furthermore, as noticed by Pourcel and coworkers \\[29\\], one of the unique CRISPR inserts in the MIGAS strain of the bacterium *Streptococcus pyogenes* is homologous to a prophage present in other strains of the same bacterium that, conversely, do not carry the CRISPR. This is compatible with the possibility that the insert makes the bacterium immune to the given phage.\n\nHere, we pursue these lines in an attempt to present a coherent, even if speculative, description of the putative prokaryotic siRNA system. Circumstantial but crucial evidence in support of the psiRNA hypothesis comes from analogies between the predicted functions of CASS proteins and the protein components of eukaryotic RNA interference systems, in particular, the RISCs \\[7-9\\] (Table <a href=\"#T2\" data-ref-type=\"table\">2<\/a>). The core parts of RISCs are a helicase fused with two RNAse III domains, dicer, and the exonuclease of the argonaute family, slicer \\[9,10,43\\]. Both dicer and slicer are represented by variable numbers of paralogs in eukaryotes, and different paralogs are included into RISCs with distinct functions \\[9,10\\]. Various RISCs also contain additional RNA-binding proteins and nucleases \\[9,44\\]. Putative functional analogs of all these proteins can be gleaned among the CASS proteins. The dicer analog is immediately apparent: the COG1203 helicase that is either fused or encoded next to a predicted nuclease of the HD family (Fig. <a href=\"#F2\" data-ref-type=\"fig\">2a<\/a>); we tentatively designate this protein p-dicer (prokaryotic dicer). Identification of the slicer counterpart (p-slicer) is less straightforward because of the diversity of predicted nucleases within the CASS. One candidate is the predicted RecB-family nuclease (COG1468). Alternatively or additionally, the slicer function could be performed by a novel, still unidentified nuclease, such as COG1857. Indeed, the possibility cannot be dismissed that different or even the same version of CASS employ multiple p-slicers, in a parallel with the multiple, paralogous eukaryotic slicers of the argonaute family. We further propose that RAMP proteins, by far the most diverse components of the CASS, play a major role in the prokaryotic siRNA mechanism as RNA-binding proteins that display a degree of specificity to the psiRNAs, most likely, by specifically binding psiRNAs of different sizes. The RAMPs, diverse representatives of the same protein superfamily, can be functional analogs of the more structurally diverse RNA-binding proteins of eukaryotic RISCs (Table <a href=\"#T2\" data-ref-type=\"table\">2<\/a>). The presence of two ferredoxin-fold domains in RAMPs (Fig. <a href=\"#F3\" data-ref-type=\"fig\">3B<\/a>) is compatible with this proposal given that this fold is seen in a broad variety of RNA-binding proteins, such as the ribosomal proteins S6 and S10, several spliceosomal subunits, and others. Although the structural similarities between the two domains of RAMP and ferredoxin-like domains are not particularly strong, it might be significant that the top structural neighbors for each of the RAMP domains are different RNA-binding domains (see above).\n\nFunctional and structural parallels between CASS and eukaryotic RNAi machinery\n\n| **Eukaryotic RNAi** | **Domains\/function** | **CASS** | **Domains\/function** |\n|----|----|----|----|\n| Dicers | Helicase\/RNAseIII. Processing of long dsRNA into siRNA and pre-miRNA into miRNA, involves unwinding | Helicase (COG1203) + HD nuclease (COG2254) - fused or adjacent genes, | SFII helicase + HD nuclease |\n| Argonautes\/slicers | Ferredoxin-fold-PAZ-PIWI \u2013 endonuclease, target degradation | RecB-family nuclease (COG1468, 4343); COG1857 \u2013 a novel nuclease? | Target degradation |\n| R2D2\/RDE-4 | dsRNA-binding domain, interacts with Dicer | RAMPs | Ferredoxin-fold duplication. Size-specific psiRNA-binding, pre-psiRNA-binding, other RNA-binding functions? |\n| Fmr1\/Fxr | RGG, KH-ssRNA-binding | RAMPs | Ferredoxin-fold duplication. Size-specific psiRNA-binding, pre-psiRNA-binding, other RNA-binding functions? |\n| Tsn | Tudor, SN \u2013 RNA-binding | RAMPs | Ferredoxin-fold duplication. Size-specific psiRNA-binding, pre-psiRNA-binding, other RNA-binding functions? |\n| Vig | RGG \u2013 RNA-binding | RAMPs | Ferredoxin-fold duplication. Size-specific psiRNA-binding, pre-psiRNA-binding, other RNA-binding functions? |\n| RNA-dependent RNA polymerase | RdRp domain related to DdRp; 2^nd^-strand synthesis for siRNA production | Predicted RdRp\/RT (COG1353) | Palm polymerase domain. 2^nd^ strand synthesis for psiRNA production, reverse transcription for CRISPR formation |\n\nFigure <a href=\"#F5\" data-ref-type=\"fig\">5a<\/a> presents our current hypothesis on the basic mechanism of the functioning of the prokaryotic RNAi system. We speculate that CRISPR regions are transcribed from a promoter located in the AT-rich CRISPR leader. Transcription might be regulated with the participation of one of the Cas proteins and, conceivably, would be stimulated by stress, such as phage infection, although the results of expression analysis in *A. fulgidus* suggest some level of constitutive transcription \\[18\\]. The work of Tang and coworkers further indicates that the primary transcript is likely to encompass the entire CRISPR repeat region. This transcript would be cleaved into 70\u2013100 nt pre-psiRNA, conceivably, by the putative p-dicer, the COG1203 protein (Fig. <a href=\"#F5\" data-ref-type=\"fig\">5a<\/a>). We further postulate that p-dicer catalyzes the second, perhaps, slower (judging by the results of Tang et al) processing step that releases mature psiRNA species (Fig. <a href=\"#F5\" data-ref-type=\"fig\">5a<\/a>). The psiRNA molecules would bind RAMPs in a size-specific manner and anneal to the target mRNA. The resulting complex would recruit p-slicer, forming the minimal form of the prokaryotic analog of RISC (pRISC) that would cleave the mRNA and could be recycled to attack the next target molecule, thus, silencing the respective gene (Fig. <a href=\"#F5\" data-ref-type=\"fig\">5a<\/a>).\n\nFigure <a href=\"#F5\" data-ref-type=\"fig\">5b<\/a> shows a version of this pathway that involves the activity of the CASS polymerase, by analogy with the eukaryotic RdRp, which participates in some RNAi pathways in most eukaryotes, but apparently has been lost in arthropods and chordates \\[45-47\\]. The initial steps in this scheme are the same as in the basic one (Fig. <a href=\"#F5\" data-ref-type=\"fig\">5a<\/a>) \u2013 transcription of the CRISPR, processing of the psiRNA precursor, and annealing of psiRNA to the target mRNA mediated by the pRISC \u2013 but, at the next step, psiRNA is postulated to serve as the primer for elongation by the CASS polymerase, yielding an extended double-stranded form of the target (Fig. <a href=\"#F5\" data-ref-type=\"fig\">5b<\/a>). This form would be cleaved by p-dicer analogously to the cleavage of viral and transposon dsRNAs by the eukaryotic dicers. The p-dicer might function as a complex with the respective RAMP to form a distinct version of pRISC. This could be the endpoint of the pathway, or else, the dsRNA degradation products could be utilized as new psiRNAs, resulting in amplification of the silencing effect (Fig. <a href=\"#F5\" data-ref-type=\"fig\">5b<\/a>). The CASS polymerase is most common in thermophiles, and it is tempting to speculate that the prevalence of this form of the psiRNA pathway has to do with the instability of the psiRNA-target duplex under the high ambient temperatures of these organisms.\n\nThe most complex and uncertain aspect of the putative prokaryotic RNAi system discussed here is the formation of new CRISPR units containing unique psiRNA genes specific for new targets encountered by the organism (Fig. <a href=\"#F5\" data-ref-type=\"fig\">5c<\/a>). The path to the creation of new psiRNAs would begin just like the response pathway, i.e., with transcription of the CRISPR locus and the first processing step yielding the 70\u2013100 nt psiRNA precursors (compare Fig. <a href=\"#F5\" data-ref-type=\"fig\">5c<\/a> with Fig. <a href=\"#F5\" data-ref-type=\"fig\">5a<\/a>). At the next step, however, there must be a mechanism to replace the unique insert within the pre-psiRNA with a new fragment of foreign (e.g., phage) RNA. The nature of this mechanism remains unclear. In principle, two possibilities can be envisaged: i) reverse transcription with copy choice whereby a reverse transcriptase, most likely, the predicted CASS polymerase (COG1353) switches from using the pre-psiRNA as a template to using a phage mRNA, and then back, and ii) direct, non-homologous RNA recombination between a pre-psiRNA and a foreign mRNA, followed by reverse transcription of the resulting recombinant RNA (Fig. <a href=\"#F5\" data-ref-type=\"fig\">5c<\/a>). Both mechanisms are non-trivial in their molecular choreography and are unlikely to occur with high efficiency. Nevertheless, there are precedents for both in the molecular biology of retroviruses and other RNA viruses. In particular, reverse transcriptase switches templates in each cycle of retrovirus first-strand cDNA synthesis although, in this case, copy-choice is facilitated by the spatial juxtaposition of the two templates within the virus particle; a similar mechanism is responsible for recombination in retroviruses \\[48\\]. In addition, and probably, more relevantly to the psiRNA case, reverse transcription with copy-choice is thought to be involved in the incorporation of copies of cellular genes, such as oncogenes, into retroviral genomes \\[49,50\\]. The alternative, namely, direct recombination between RNA molecules might seem far fetched, but such a process has been demonstrated, by several groups independently, to occur in RNA viruses, apparently, via a protein-independent mechanism \\[51,52\\]. During the formation of new psiRNA species, these low-frequency processes might be facilitated by the high abundance of the phage mRNAs involved. Indeed, it has been shown that the unique inserts in CRISPR most often correspond to fragments of essential, highly conserved phage genes that are typically expressed at a high level in infected bacteria\\[27\\]. Once the dsDNA molecule consisting of a CRISPR unit with the new, unique insert is produced, by one of the mechanisms outlined here or, perhaps, via a different pathway, it must insert into the CRISPR array via homologous recombination (Fig. <a href=\"#F5\" data-ref-type=\"fig\">5c<\/a>). We suspect that this process is mediated by the COG1518 protein, the universal marker of CASS containing conserved motifs resembling those of different nucleases \\[23\\]. It seems likely that this protein functions as the CRISPR integrase\/recombinase, perhaps, in cooperation with the COG1343 protein, another universal component of CASS.\n\n## Additional lines of evidence relevant for the predicted RNAi function of CASS\n\n### Genes loosely associated with CASS\n\nIn addition to the bona fide (even if not ubiquitous) CASS components, a variety of genes were found in association with CRISPR and CASS in only one or a few closely related genomes. Thus, functional association of these genes with CASS appears uncertain. However, examination of the list of such genes clearly indicates that they are not a random set (Table <a href=\"#T3\" data-ref-type=\"table\">3<\/a>). Similarly to the common CASS components, there are several nucleases on this list. More notable is the presence of the reverse transcriptase (RT) which, on at least two independent occasions, has been fused to the COG1518 gene (Fig. <a href=\"#F2\" data-ref-type=\"fig\">2a<\/a> and Table <a href=\"#T3\" data-ref-type=\"table\">3<\/a>). This fusion suggests the intriguing possibility that, in the respective variants of CASS, the RT (probably derived from retron-type elements) takes over the proposed function of the CASS polymerase (Fig. <a href=\"#F5\" data-ref-type=\"fig\">5c<\/a>); this appears to provide indirect but substantial support for the reverse-transcription-mediated mechanism of CASS function. Perhaps, the most remarkable observation is the presence of the archaeal homolog of the eukaryotic argonaute protein (the slicer) in one of the CASS operons of the archaeon *Methanopyrus kandleri* (Table <a href=\"#T3\" data-ref-type=\"table\">3<\/a>). This suggests the possibility of a functional association of this protein with CASS, at least in some archaea, and provides the only putative (even if weak \u2013 until other archaeal genomes with such a gene arrangement are found) link between CASS and the eukaryotic RISCs at the level of homologous proteins.\n\nGenes loosely associated with CASS\n\n|  | **Family** | **Example of genes associated with CASS** | **Comments** |\n|----|----|----|----|\n| 1 | Reverse transriptase (RT) | VVA1544, PG1982, alr1468 | Fused to COG1518 in three occasions and a remnant of RT (Mbar_A1351 and MM3360) in *M. barkeri* and *mazei* genomes is located close to cas |\n| 2 | PIN-domain | alr1560, ST0017, Ava_4168 | Ribonuclease |\n| 3 | COG2442 | Ava_4167 | HTH domain, component of toxin-antitoxin system, probably targeting mRNA |\n| 4 | COG1432 | MS0983 | Large family of proteins, predicted to be a phosphatase or a nuclease on the basis of sequence motifs which is shared by all three domain of life. In multidomain proteins in plants it is associated with C2H2 Zn-finger domain |\n| 5 | PA2117-like | MS0982, MS0989 | An enzymatic domain, that is located in an operon with restriction-modification systems or in association with a diverged helicase |\n| 6 | COG3645-like | ACIAD2479 | Homologs of phage anti-repressor Ant which is known to be inhibited by an antisense RNA |\n| 7 | argonaute | MK1311 | Homolog of the eukaryotic argonaute protein, that are key player in RNA guided posttranscriptional regulation by siRNA and miRNA |\n| 8 | COG1598\/COG4226\/HicB | MCA0653, MTH321 | Probably has an RNAseH-like fold, often fused to CopG-family of transcriptional regulators; forms a conserved operon with COG1724\/hicA, which has the dsRBD-like fold; possible novel toxin-antitoxin module targeting mRNA |\n| 9 | PUA-domain | LIC10933 | RNA binding domain |\n| 10 | 3'-5' exonuclease | LcasA01001274 | Fused to COG1343 in *Lactococcus bulgaricus* and *L.casei* |\n| 11 | COG1652 | TK0459 | Regulatory ATPase of AAA family fused to RecB-family nuclease; Predicted regulator of RNA metabolism |\n| 12 | AbrB\/MazE domain | TK0457, PAE0118 | DNA-binding domain, belongs to the same fold as MazE, which involved in toxin-antitoxin system |\n| 13 | S1-domain | CaurDRAFT_2121 | Ribosomal protein S1-like RNA-binding domain, fused to RAMP domain |\n| 14 | CSP-like | Rrub02003211 | Cold shock protein-like RNA-binding domain, fused to RAMP domain |\n\n### A connection between CRISPR and RAMPs\n\nWe searched for CRISPR in the 89 complete prokaryotic genomes containing cas genes and found remarkable diversity, in both the number of repeat units and the spacer size, even among closely related species and strains, which is in agreement with previous observations \\[25,27-29\\]. Given our hypothesis that RAMPs are size-specific psiRNA-binding proteins, we examined possible connections between the number of RAMP genes, the number of CRISPR units (which is the same as the number of unique inserts) and the length heterogeneity of the unique inserts in prokaryotic genomes. The number of RAMPs showed strong positive correlations both with the number of CRISPR units (Fig. <a href=\"#F6\" data-ref-type=\"fig\">6a<\/a>) and with the variance of the insert lengths (Fig. <a href=\"#F6\" data-ref-type=\"fig\">6b<\/a>) which seems to be compatible with our hypothesis on the psiRNA-binding function of RAMPs. Significant correlations were observed also between the number of CRISPR and the variance of the insert length, and between each of these variables and the number of COG1518 proteins, COG1517 proteins or all Cas proteins minus the RAMPs. However, the correlations seen for the RAMPs and the CRISPR inserts were the strongest, suggesting that these were the biologically most relevant links (Table <a href=\"#T4\" data-ref-type=\"table\">4<\/a>). It should be emphasized that, given the extreme sequence divergence of the RAMPs, it is most likely that the correlation between the number of encoded RAMPs and the number of CRISPR units present in a genome is due to the RAMPs discriminating between inserts (by size, under our hypothesis) rather than recognition of the repeats themselves.\n\nRank correlations coefficients between CRISPR spacers and selected Cas proteins\n\n| Values selected for correlation | Rank Correlation coefficient^a^ |\n|----|----|\n| Number of spacers vs S.D. of spacer lengths | **0.398** |\n| Number of spacers vs number of COG1518 proteins | **0.475** |\n| Number of spacers vs number of RAMPs | **0.668** |\n| Number of spacers vs number of COG1517 proteins | **0.524** |\n| Number of spacers vs total number of Cas proteins minus RAMPs | **0.580** |\n| Number of spacers vs total number of Cas proteins minus RAMPs and COG1517 proteins | **0.556** |\n| Number of RAMPs vs S.D. of spacer lengths | **0.651** |\n\n^a^All correlation coefficients are highly statistically significant P \\< 10^-3^\n\n## Putative psiRNAs: relationships with phage and plasmid genes and secondary structure\n\nWe sought to reassess the relationship between the unique CRISPR inserts and bacteriophage and plasmid genes with the currently available, increased collection of prokaryotic and phage genomes. Using somewhat more stringent criteria than those adopted by Mojica et al. \\[27\\], we found that only a small fraction the unique CRISPR inserts showed significant similarity to any sequences in the current databases. Altogether, we identified 83 inserts with apparent homologs, or \\~2% of all available CRISPR sequences. Importantly, the great majority of these inserts were indeed homologous to fragments of phage or plasmid genes rather than to sequences from bacterial or archaeal chromosomes (Table <a href=\"#T5\" data-ref-type=\"table\">5<\/a>). Rather unexpectedly, it turned out that CRISPR inserts homologous to phage and plasmid genes came in both the sense and the antisense orientations (Table <a href=\"#T5\" data-ref-type=\"table\">5<\/a>). Although determination of the direction of transcription for CRISPR cassettes is ambiguous because the leader sequence thought to include the CRISPR promoter is hard to identify, it is certain that some of the CRISPR cassettes contain inserts of both orientations (Table <a href=\"#T5\" data-ref-type=\"table\">5<\/a>). This finding presents a complication to the schemes of CRISPR functioning presented in Fig. <a href=\"#F5\" data-ref-type=\"fig\">5<\/a> because sense fragments of genes, obviously cannot silence the respective targets directly. There seem to be three possible solutions to this problem, all based on the assumption that the recombination process in the scheme shown in Fig. <a href=\"#F5\" data-ref-type=\"fig\">5c<\/a> is random with respect to direction. The first possibility is that the sense inserts are non-functional by-products of indiscriminate recombination. Alternatively, it is conceivable that both sense and antisense inserts are converted to duplexes, probably, by the CASS polymerase, after which the sense strand is destroyed whereas the antisense strand becomes part of the pRISC. A very similar mechanism involving the RNA-dependent RNA polymerase has been described for one of the endogenous siRNA pathways in plants \\[53\\]. The third, more exotic possibility is that psiRNA actually function not by silencing mRNAs but by promoting degradation of the target DNA, in which case both sense and antisense psiRNAs could be active. This option is further discussed in the Conclusions section.\n\nA selection of CRISPR inserts homologous to phage, plasmid and prokaryotic genes\n\n| Species | Total spacers | Phage Plasmid | sense | antisense | Other prokaryotes. |\n|----|----|----|----|----|----|\n| *Sulfolobus solfataricus* | 413 | 22 | 9 | 12 | 4 |\n| *Sulfolobus acidocaldarius* DSM 639 | 222 | 0 |  |  | 1 |\n| *Sulfolobus tokodaii* | 449 | 4 | 4 | 0 | 2 |\n| *Xanthomonas oryzae* KACC10331 | 59 | 14 | 13 | 1 | 0 |\n| *Anabaena variabilis* ATCC 29413 | 174 | 0 |  |  | 1 |\n| *Methanobacterium thermoautotrophicum* | 169 | 10 | 4 | 4 | 0 |\n| *Methanocaldococcus jannaschii* | 185 | 0 |  |  | 1 |\n| *Bacillus halodurans* | 89 | 3 | 1 | 1 | 0 |\n| *Streptococcus thermophilus* CNRZ1066 | 41 | 20 | 15 | 5 | 0 |\n| *Streptococcus thermophilus* LMG 18311 | 37 | 10 | 5 | 5 | 0 |\n| *Streptococcus agalactiae* 2603 | 24 | 0 |  |  | 1 |\n\nImportantly, the CRISPR insert sequences from even closely related bacterial strains are unrelated to each other, with only a few exceptions among enterobacterial strains (data not shown). This suggests that the inserts are replaced rapidly on the evolutionary scale, perhaps, via the mechanism outlined in Fig. <a href=\"#F5\" data-ref-type=\"fig\">5c<\/a>. A broader implication is that the dominant phages and plasmids encountered by even closely related bacteria are different leading to the rapid generation of distinct repertoires of psiRNAs.\n\nIn terms of their ultimate function, the putative psiRNAs appear to be conceptually most similar to eukaryotic siRNAs in that they are homologous to foreign, rather than endogenous, genes and are predicted to function as part of a defense system. However, the psiRNAs also share a major feature with the miRNAs in that they are embedded in and, probably, cleaved from a specific precursor molecule, rather than from a long dsRNA like the siRNAs (Fig. <a href=\"#F5\" data-ref-type=\"fig\">5<\/a>). In an attempt to elucidate common features of the psiRNA precursors, we predicted the secondary structures of all available CRISPR units. The results were somewhat ambiguous as shown in Fig. <a href=\"#F7\" data-ref-type=\"fig\">7<\/a>. The folding free energies of GC-rich CRISPR units were substantially lower than those for the shuffled CRISPR units; notably, the CRISPR folding energies were distributed similarly to those of miRNA precursors (Fig. <a href=\"#F7\" data-ref-type=\"fig\">7a<\/a>), with numerous CRISPR units capable of folding into highly stable secondary structures. These observations suggest the possibility that psiRNA precursors possess a distinct secondary structure that could be important for recognition by p-dicer, RAMPs or other proteins. However, the folding energy distribution for AT-rich CRISPR shows only a slight shift compared to the distribution for shuffled sequences and is completely different from the distribution for miRNA precursors of the same base composition (Fig. <a href=\"#F7\" data-ref-type=\"fig\">7b<\/a>). Thus, AT-rich psiRNA precursors, which comprise approximately half of the CRISPR, on average, do not seem to be capable of folding into stable secondary structures. Thus, the existence of a functionally relevant consensus structure of psiRNA precursors remains uncertain.\n\nFigure <a href=\"#F8\" data-ref-type=\"fig\">8<\/a> shows two predicted secondary structures of putative psiRNA precursors in which the inserts are homologous to phage or plasmid genes. The relatively GC-rich sequence from *Xanthomonas axonopodis* forms a stable secondary structure, whereas the AT-rich sequence from *Streptococcus thermophilus* can fold only into a weak stem-loop structure. Curiously, in both cases, the unique insert contributes significantly to stem formation. Clearly, further analysis of the structures of putative psiRNA precursors by computational and experimental means is required to identify their functionally important features.\n\n# Conclusion\n\nObviously, the entire concept of the prokaryotic CASS defense system functioning on the RNAi principle currently remains a hypothesis. However, we believe that three lines of evidence make such a mechanism, in its broad outline, almost a logical inevitability: i) the indisputable origin of at least some of the unique CRISPR inserts from phage and plasmid genes, ii) the demonstration of transcription and processing of the CRISPR loci in *A. fulgidus* and *S. solfataricus*, and iii) the abundance of CASS components that are clearly implicated in nucleic acid degradation, processing, and possibly, recombination. The only substantial variation on the theme of RNAi could be an antisense mechanism acting on DNA. Such a mechanism is, obviously, much less common than post-transcriptional gene silencing by small RNAs, but is not without precedent. Indeed there are strong indications that elimination of intergenic DNA sequences in the macronucleus of the ciliate *Tetrahymena* occurs via a siRNA-mediated mechanism, with the participation of a specific dicer-slicer pair \\[54,55\\]. In principle, this mechanism is compatible with the finding of both sense and antisense sequences among CRISPR inserts homologous to phage and plasmid genes (Table <a href=\"#T5\" data-ref-type=\"table\">5<\/a>). Nevertheless, given the much wider prevalence of silencing pathways and, in particular, the demonstrated instances of antisense RNA regulation in bacteria, it seems most likely that CASS acts by silencing genes from invading replicons. This being said, it should be emphasized that the mechanisms depicted in Figure <a href=\"#F5\" data-ref-type=\"fig\">5<\/a> are only rough outlines of some of the ways in which this system could function. There is no doubt that experimental studies will reveal mechanisms different from these, at least, in detail. However, regardless of the specific mechanisms and even whether the predicted psiRNA systems acts on RNA or on DNA, it appears certain that its main function is RNAi-mediated defense against alien replicons invading archaea and bacteria.\n\nThe predicted psiRNA system resembles the eukaryotic counterparts not only in its functional principle but also in the general characteristics of the implicated proteins. What is most striking is the comparable complexity, diversity, and plasticity of the protein machineries involved. Both systems consist of one or more helicases, a broad spectrum of nucleases, a specific polymerase, and a variety of RNA-binding proteins. Both in CASS and in RISCs, only two or three protein subunits appear to be truly indispensable; the rest come and go, resulting in a variety of RISCs with their distinct functions, many of them still poorly understood \\[7-9\\], and, presumably, in a comparable diversity of CASS. Remarkably, however, not a single protein belonging to the bona fide CASS has an ortholog in eukaryotes, involved in RNAi or otherwise. The single direct link could be the argonaut protein which is the central active moiety of eukaryotic RISCs (slicer) and might have some functional connection to CASS in archaea as tentatively suggested by the *M. kandleri* CASS operon structure; admittedly, however, the indications of potential involvement of argonaut in the CASS functioning are currently quite weak.\n\nThe eukaryotic RNAi systems come in two basic varieties: i) siRNAs that are produced from dsRNAs of viruses and transposons and protect the host from the respective agents via perfect base-pairing with the respective target mRNAs, and ii) miRNA that regulate translation of endogenous genes via either perfect (plants) or imperfect (animals) base-pairing \\[6\\]. CASS appears to be the functional counterpart of the siRNA mechanism inasmuch as it seems to be involved in defense against infecting agents, the psiRNAs seem to be derived from the invading genome and are predicted to function via perfect base-pairing with the target. From a different viewpoint, however, this system is similar to miRNA in that the active small RNA moieties are encoded in the prokaryotic genomes rather than produced from the foreign dsRNA. The closest eukaryotic analogs of the CASS system might be the rasiRNAs that, like the putative psiRNAs, are encoded in the genome, are generated by processing of double-stranded molecules formed by symmetric transcripts of transposons (or repeats thought to be of transposon origin) and silence the latter, which contributes to heterochromatin formation \\[11-13,56\\]. In contrast, the bacterial small antisense RNA regulatory pathways that employ Hfq and RNAse E that target regular, chromosomal bacterial genes, rather than those of infectious agents or transposons \\[14-16\\], seem to be the functional counterpart of eukaryotic miRNA systems. Thus, prokaryotes seem to have at least two distinct RNAi systems none of which is operated by homologs of eukaryotic RNAi protein machinery components. Furthermore, unlike the case of eukaryotes where the siRNA, rasiRNA, and miRNA systems are operated by substantially overlapping sets of proteins \\[8\\], the prokaryotic systems seem to be completely independent from one another.\n\nClearly, our interpretation of the probable functions of CASS is based, in large part, on the analogy with the eukaryotic siRNA system. There might be danger in heavily relying on analogy as a prediction method because, if the basic premise is false, the entire scheme will fall apart. However, in the case of CASS and eukaryotic siRNA, the analogy stems from two *a priori* independent lines of evidence, namely, the discovery of CRISPR inserts homologous to phage and plasmid genes and the functional similarity between Cas proteins and components of eukaryotic RNAi systems, e.g., dicer. Should there be no *bona fide* functional analogy between CASS and RNAi, there would be no basis for this congruence. By contrast, the homology of CRISPR inserts to phage and plasmid genes and, more generally, the association of *cas* genes with CRISPR have no explanation in the context of our previous hypothesis that CASS is a repair system \\[23\\] which forces us to abandon this interpretation of the CASS function.\n\nAll the analogies notwithstanding, the predicted psiRNA system shows at least one fundamental difference from the eukaryotic counterpart: the coding segments for the putative psiRNAs are derived from genes of invading agents and incorporated into the host genome to confer heritable immunity to the respective agent. As an acquired immunity mechanism, CASS resembles more the vertebrate immune system than the eukaryotic RNAi pathways but, again, with the crucial difference that the animal immunity is not inheritable. Furthermore, the wide spread of CASS that spans a great variety of prokaryotic lineages contrasts the narrow presence of classical immunity which appears to be a mechanism specific to jawed vertebrates; the recent discovery of a dramatically different immunity system in jawless vertebrates \\[57\\] emphasizes the status of the immune system as a lineage-specific evolutionary novelty. More generally, it appears that CASS is one of the most ancient if not, indeed, the primordial biological defense system that probably emerged at an early stage of prokaryotic evolution, considering that diverse viruses, in all likelihood, have accompanied cellular life from its very beginning. Given the ubiquity of CASS in archaea and its less prominent presence in bacteria, one scenario is that CASS emerged in an ancient ancestor of archaea and spread to bacteria horizontally.\n\nInterestingly, as a mechanism of inheritance of acquired traits, CASS seems to come closest to a true Lamarckian mode of evolution among all known systems of heredity. Remarkably, however, this putative system of Lamarckian inheritance appears to be extremely volatile on the evolutionary scale as indicated by the lack of conservation of the psiRNA sequences even between closely related strains. A general implication of this aspect of CASS evolution is that the diversity of mobile replicons (phages, plasmids, transposons etc) in nature might be even more enormous than it is currently estimated \\[58,59\\] such that even closely related bacteria occupying similar niches are predominantly invaded by different agents. Additionally or alternatively, it is conceivable that, in many niches, the dominant phages and plasmids rapidly turn over with time, making existing CRISPR cassettes obsolete as defense means and triggering their exchange.\n\nFinally, a practical note. It seems that, once the psiRNA mechanism described here is investigated experimentally, it could be exploited to silence any gene in organisms that encode CASS. The simple design of such experimental gene silencing in prokaryotes will involve transfection with a plasmid containing the desired psiRNA inserted between CRISPR to facilitate homologous recombination.\n\n# Methods\n\n## Genome sequences, databases and sequence analysis\n\nThe complete bacterial and archaeal genome sequences were retrieved from National Center for Biotechnology Information (NCBI, NIH, Bethesda) FTP site. The non-redundant database of protein sequences at the National Center for Biotechnology Information (NIH, Bethesda) was iteratively searched using the PSI-BLAST program \\[31\\]. The cut-off of E \\< 0.01 was normally employed for inclusion of sequences in the position-specific weight matrices. Each retrieved sequence was used as the query for additional searches until no new sequences could be detected. For detecting subtle sequence conservation, the PSI-BLAST search results were visually examined and sequences with greater E-values, but containing signature motifs of a given protein family were included into profiles on a case by case basis \\[30,60\\]. Multiple alignments of protein sequences were constructed using the MUSCLE program \\[61\\] and corrected on the basis of PSI-BLAST results. Protein secondary structure was predicted using the JPRED program \\[62\\]. Protein structure comparisons were performed using the DALI \\[63\\] and VAST \\[40\\] programs, and ribbon diagrams of protein structures were generated made using the program BOBSCRIPT \\[64\\].\n\n## Phylogenetic analysis\n\nDistance trees were constructed from multiple protein sequence alignments after excluding poorly aligned positions, by using the least-square method as implemented in the FITCH program of the PHYLIP package \\[65,66\\]. Maximum likelihood trees were constructed using the ProtML program of the MOLPHY package, with the JTT-F model of amino acid substitutions, by optimizing the least-square trees with local rearrangements \\[67,68\\].\n\n## Identification and analysis of CRISPR repeats\n\nSearch for repeats was performed as follows: first, all exactly matching 20-mer anchor substrings were identified in the nucleotide sequence of a bacterial or archaeal genome. Alignments around these anchors were expanded in both directions, to include all adjacent positions with information content above or equal to 1.5 bits \\[69\\]. All identified high-similarity fragments were used as queries in nucleotide BLAST \\[70\\] search (word size 7; mismatch penalty -1; both gap opening and extension costs -1; E-value threshold 0.001) to detect more diverged versions of the repeats as well as the instances of the repeat in the opposite strand. To determine the repeat boundaries more precisely, sequences of all loci with short (20 nt) flanks added were collected and aligned using the MUSCLE program \\[61\\]. Alignments were trimmed from the 5' and 3' termini up to columns with information content exceeding 1.3 bits. Repeat families that shared the spatial arrangement typical of CRISPR (median repeat length of 20\u201350 nt; median spacer length of 15\u201360 nt) were identified as CRISPR candidates and further examined for chromosomal proximity to cas genes. The custom PERL scripts used for this analysis are available upon request.\n\nSequences of eukaryotic miRNAs precursors, CRISPRs, and randomly shuffled CRISPR sequences were computationally folded, and the free energy of the most stable secondary structure was calculated using a a dynamic programming algorithm that employs nearest neighbor parameters to evaluate free energy \\[71\\]. Energy minimization was performed by dynamic programming method that finds the secondary structures with the minimum free energy by summing up the contributions from stacking, loop length, and other structural features, using improved thermodynamic parameters \\[72\\].\n\n## Similarity of inter-CRISPR spacers to other sequences\n\nNucleotide sequences of inter-CRISPR spacers were used as a query in MEGABLAST \\[70\\] searches (word size 11; e-value threshold 0.01) against GenBank; hits to virus or plasmid sequences and to distantly related prokaryotes were counted separately for each source organism.\n\n# Reviewers' comments\n\n## Reviewer's report 1\n\nEric Bapteste, Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada\n\nMain comments:\n\nThe scientific quality of this paper and its methodology is certain. There has been a lot of good and interesting work done here. As indicated by its title, it thus provides multiple information and one hypothesis about RNA-interference in prokaryotes. Because of this broad scope, the manuscript is quite large. In fact, several of its parts could be read on their own, depending on the reader-specific interest, this is notably the case for the part dealing with the hypothesis of a RNAi prokaryotic immune system. I would thus suggest that a shorter version of the paper, centered around this very interesting hypothesis could be proposed online (with the first part turning into Supp. Mat.), because I feel that this part is going to receive more attention anyway, and it would be unfortunate if some readers did not consider this aspect because they are scared by the overall size of the paper. But this is simply a suggestion, and the authors are more than welcome to disregard my opinion.\n\n**Author response:** *While we fully understand the sentiment and agree that the hypothesis on prokaryotic RNAi is of greater general interest than the detailed presentation of the protein sequence-structure analysis, we strongly feel that the latter provides a badly needed foundation for the hypothesis as well as important information in its own right. Furthermore, we tend to believe that the general spirit of online publication is to present complete results of a study (of course, there are exceptions). The reader can easily navigate between sections, so the length of a paper does not represent a particularly severe problem. Furthermore, we made certain modifications to the protein analysis part in response to similar but more specific comments of Martijn Huynen, in particular, introduced additional subheadings which, hopefully, makes this part of the paper more reader-friendly*.\n\nThe seductive hypothesis of a RNAi based immune system is presented as an analogy with the eukaryotic RNAi system. The use of analogy is potentially challenging: on the one hand, it allows a powerful and elegant presentation of many complex genomic results, but on the other hand, it is questionable, since the analogy may impose an a priori model to interpret biological features, and if this model is incorrect, if the analogy does not hold, there is a risk that the genomics data receive a fairly biased interpretation. In this respect, it would be interesting if the authors discuss whether an homologous immune system could have been possible in eukaryotes and in prokaryotes, and why it is not found. Indeed, such an homologous system would be a more natural reference to interpret the data than an analogy.\n\n**Author response:** *We understand the epistemological concerns regarding the role of analogy in this study. However, as indicated by the reviewer himself, the analogy is strong. Moreover, this analogy is manifest at two different levels: i) the presence of inserts homologous to phage and plasmid genes in CRISPR units and ii) presence of predicted activities compatible with a siRNA-like system among cas gene products, in particular, the dicer analog. Had the analogy been false, there would be no reason whatever for this congruence. We briefly comment to that effect in the revised manuscript. The idea of homologous immune systems in prokaryotes and eukaryotes seems a little far-fetched. Nevertheless, this comment prompted us to incorporate a brief comparison of the evolutionary histories of RNAi and classical immune systems*.\n\nThis being said, I do not feel that the use of the analogy was a problem here, as it is convincingly presented and argued by the authors. We could eventually question more if all the so-called CASS genes are really involved in the prokaryotic immune systemand deserve their label: some may just be present in the genomic proximity of CRISP, yet having nothing to do with the RNA interference. They might just be mobile \u00abtravelling\u00bb genes. A further study of the genomic distribution of the homologs of the CASS genes in bacterial genomes may help to clarify which genes are strongly and exclusively CRISP related and which ones can be found also in alternative locations in different genomes. Then, perhaps the \u00abstriking diversity of still poorly characterized CASS components\u00bb described on page 16 would appear less striking if some CASS categories are simply not relevantly defined, and include unrelated proteins, since maybe, the use of the analogy would had led to too relaxed definitions of CASS. In another situation, by contrast, the use of the analogy could perhaps be too strict. On page 18, the authors wonder how to identify \u00abthe slicer counterpart (p-slicer)\u00bb in prokaryotes. They explain that this identification is \u00abless straightforward because of the diversity of predicted nucleases within the CASS\u00bb. But, after all, why should there be only one p-slicer, as in eukaryotes? It is possible that prokaryotes have multiple \u00abp-slicers\u00bb.\n\n**Author response:** *Yes, we agree, the possibility of multiple slicers exists, and we modified the text to acknowledge this. With regard to the rest of this comment, however, we feel that the current diversity of prokaryotic genomes is already sufficient to make conclusions on the strength of the association of individual genes with CASS, and the genes are classified here accordingly, as true CASS components and loosely associated \"satellites\". As far as the latter are concerned, inferences on involvement with CASS functions are made only for those genes whose activities appear clearly relevant, like the RT or Argonaut*.\n\nFinally, the strong suspicion about the analogous and intricate functions of COG1518 and COG1343 (cf. page 21) could be similarly toned down. Maybe these genes do play the essential analogous role of CRISPR integrase\/recombinase consistently with the analogy, but maybe they fulfill several different tasks. Perhaps the authors would like to comment more on some of these minor points.\n\n**Author response:** *Actually, this prediction is not based on analogy with eukaryotic RNAi systems but rather on the mutualistic association of these genes to CRISPR and the features of the proteins themselves. We dropped the \"strong\" suspicion but, generally, we strongly believe that this is the best possible prediction. Reference to multitasking might not be particularly productive unless there are good ideas regarding what these multiple functions might be (this is different from the above possibility that there are multiple slicers which is, indeed, compatible with the data)*.\n\nAlso, to go back to the CASS gene evolution, the authors mention, page 8, \u00ab the extraordinary evolutionary mobility of CASS\u00bb. It is unclear to me how this statement has been tested, and how the authors have established that CASS genes are more mobile than any average gene randomly picked in the same collection of prokaryotic genomes. For this reason, I am not sure if, as claimed by the authors, on page 14 \u00abthe pol-cassette comprises a distinct evolutionary unit that is often transferred horizontally independently of the CASS-core\u00bb. Does the CASS-core really have an established vertical mode of inheritanceor, as the authors stated before, a \u00abnon-uniform\u00bb distribution (cf. page 8)? This might be more strongly argued.\n\n**Author response:** *Several distinct issues are addressed here. With regard to the 'extraordinary mobility' of CASS, this is demonstrated by the trees in* Fig. <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>*(more trees have been published previously in our own 2002 paper and by Haft et al.), but even more convincingly, by the persistent pattern of presence-absence of CASS in closely related species and even strains of bacteria. We consolidated the argument such that this becomes clear the first time \"extraordinary\" mobility comes up. It is true that we did not compare the mobility of the CASS components with that of garden-variety prokaryotic genes in a rigorous, quantitative manner. While this is doable, in principle, all methods we are aware of are open to debate, and we feel that the exercise is beyond the scope of this paper. Given the above argument, we believe that, qualitatively, it is clear that CASS is unusual in this respect. With regard to the pol-casette, we believe that the discrepancy between the topologies of the two trees in* Fig. <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>*is quite sufficient for the statement on independent HGT. As for the \"vertical mode of inheritance\" of CASS, there seems to be a semantic issue here. We do not really claim vertical inheritance for CASS but neither is such a pattern necessary to detect horizontal mobility. What is required is a predominant pattern of vertical inheritance among other genes that allows us to use a species tree to detect HGT. Of course, we realize that there are substantial arguments for abandoning \"tree thinking\"*\\[73\\]*altogether but, on balance, we still believe that a species tree conceptualized as a central trend in the evolution of gene ensembles* \\[74\\]*is, at least, a useful tool for analysis of genome evolution*.\n\nThese few questions show a strength of the present work which interestingly opens perspectives and suggests that some additionnal analyses should now be conducted, because the topic deserves consideration. Maybe the authors would feel like addressing some of the points below in a revised version of the current paper, or in future analyses.\n\n**Author response:** *These are, indeed, very interesting questions, we appreciate them. Some are for future studies but we can provide certain answers now*.\n\nFurther study could iclude the following:\n\n-Do other genomic regions harboring concentrations of nucleasescomparable to the ones around CRISP exist elsewhere in the genomes?\n\n**Author response:** *Hardly. As indicated in the paper, the CRISPR neighborhood is the second most prominent (i.e., the one that ranks second in the number of genes) neighborhood in prokaryotic genomes after the ribosomal superoperons, so it is quite outstanding. However, there are other, considerably small constellations of nucleases, such as the classical recBCD operon encoding repair proteins, some restriction-modification systems, and, perhaps, others that are still poorly understood and deserve investigation*.\n\n\\- If yes, is there more than one prokaryotic immune system definable on this analogous ground? Notably, did bacteria without CRISPR evolve a totally different immune system?\n\n**Author response:** *There is no evidence of that. Furthermore, as repeatedly emphasized in this paper, CASS shows extreme evolutionary volatility, apparently being lost quite easily, in a short time, on evolutionary scale. It is hardly imaginable that these bacteria evolved a distinct immune system in the short time elapsed since the loss of CASS. Of course, purely hypothetically, one could perceive the possibility that another immune system is disseminated horizontally, like CASS, and prokaryotes having both, could differentially lose one of them. However, we are unaware of any support for such a scenario. Another prominent prokaryotic defense mechanism is restriction-modification; it would be interesting to examine the relationship between RM systems and CASS, that could be a subject for a future study*.\n\n-How did the psiRNA pathway arise in thermophiles(cf. page 20)? Does it result from a transfer? Was it ancestral?\n\n**Author response:** *Very interesting, fundamental questions, indeed. In response, we expanded the discussion of these and other aspects of evolution of CASS. The specific preponderance of CASS in thermophiles, noticed already in the 2002 paper, when we thought that this was a thermophile-specific repair system, remains a mystery. Whatever the nature of this association, it seems likely that CASS is ancestral in thermophiles (at least in hyperthermophiles)*.\n\n\\- Could we imagine that multiples promoters exist, both sense and antisense, which would activate the transcription of CRISPR, generating even more RNAi(cf.p 24)?\n\n**Author response:** *In principle, existence of multiple promoters cannot be ruled out. However, the leader sequence seems to be the only natural candidate for the promoter function. The rest of the CRISPR cassette is homogeneous (repetitive), so it is unclear where an alternative promoter would be located. Further, in the two archaeal systems that have been studied experimentally (Archaeoglobus and Sulfolobus) all transcription of CRISPR loci appear to be unidirectional*.\n\n-Finally, it might be challenging, though interesting to test in vitro on bacterial cultures if, as proposed by the authors, the presence of CRISP and CASS, has really an impact on the fitness of prokaryotes in presence of viruses.\n\n**Author response:** *We certainly hope that the computational analyses and predictions described in this paper stimulate a lot of experimentation aimed at elucidation of the biological functions of CASS and roles of its individual components*.\n\n*We greatly appreciate these insightful and stimulating comments*.\n\nMinor comments\/questions:\n\nOn page 7: \u00abfunctionally analogous\u00bb is redundant.\n\n**Author response:** *We see the point but do not really agree. The word \"functionally\" seems to add clarity*.\n\nOn page 8: the sentence \u00abthe distribution of COG1518 and, by implication, CASS among prokaryotic lineages...\u00bb is too \u00abbold\u00bb for me: even if the conclusion is correct, I am not sure one can generalize as suggested here from the case of one protein only.\n\n**Author response:** *Indeed, we can. Rephrased to clarify and emphasize this*.\n\nOn page 15: \u00abseveral other CASS gene families remain mysterious\u00bb is a mysterious sentence. I am not sure what this does really mean.\n\n**Author response:** *That there is no clue as to the possible functions of these proteins; modified to clarify*.\n\nOn page 21: I miss the idea of the sentence starting by \u00abIn addition, and probably, more relevantly etc.\u00bb to \u00abretroviral genomes\u00bb. Could you rephrase it to explicit it a little bit more?\n\n**Author response:***Rephrased \u2013 hopefully, to clarify*.\n\nOn page 23: what is the criterion retained for homology between the plasmid genes, fragments of phages and the CRISPR sequences?\n\n**Author response:** *The following quote from the Methods addresses this issue:*\n\n*\"Nucleotide sequences of inter-CRISPR spacers were used as a query in MEGABLAST* \\[70\\]*searches (word size 11; e-value threshold 0.01) against GenBank; hits to virus or plasmid sequences and to distantly related prokaryotes were counted separately for each source organism.\"*\n\nOn page 48: To me, the multiple positive correlations evoke multiple causalities and the possibility of some hidden correlations. Would you say that all the relevant combinations have been considered here?\n\n*No, we won't claim that. More complex multiple regression analysis would be required to separate correlations that reflect true causality; for the purposes of this paper, we felt it was sufficient to note the strongest correlations*.\n\n## Reviewer's report 2\n\nPatrick Forterre, Biologie Mol\u00e9culaire du G\u00e8ne chez les Extr\u00eamophiles (BMGE) Institut de G\u00e9n\u00e9tique et Microbiologie (IGM), Universit\u00e9 Paris-Sud, Centre d'Orsay, 91405 Orsay Cedex, France, and Biologie Mol\u00e9culaire du G\u00e8ne chez les Extr\u00eamophiles (BMGE), D\u00e9partement de Microbiologie Fondamentale et M\u00e9dicale, Institut Pasteur, Paris, France\n\nIn this very important paper, Makarova and coworkers propose a detailed mechanism for a putative procaryotic antiviral immunity system mediated by CRISPS sequences and their associated Cas proteins (the CAS system, CASS *sensu* the authors). Their model is based on the hypothesis that these elements represent a prokaryotic-specific antiviral mechanism analogous to the eukaryotic RNAi system. In procaryotes (Bacteria and Archaea) there is no homologs of the proteins involved in the eucaryotic RNAi system. Untill recently, it was therefore widely believed that restriction-modification mechanisms were the only defense available to procaryotes to fight viral infections. However, it has been proposed last year by several groups that procaryotic CASS could also play a significant role in fighting viral aggression in archaea and bacteria (Mojica et al. 2005, Pourcel et al., 2005, Bolotin et al., 2005). CRISPR sequences, which are transcribed but non-coding, are formed by the tandem repetition of units containing both a conserved element (similar all along a given CRISPR) and a variable element, the spacer, different from one unit to the other. The spacer sequences have strikingly no homologous sequence in databases, except for viral or plasmid sequences. Both Mojica et al. (2005) and Bolotin et al., (2005) have suggested that transcription of CRISPR sequences produce anti-sense RNA that can inhibit transcription of incoming viral (plasmid) sequences and Mojica et al. (2005) mentioned the analogy of such system with eukaryotic RNAi. However, these authors did not elaborate on the specific mechanism involved and how the cas proteins could be involved in the processing of viral RNA.\n\nIn this work Makarova and co-workers have first performed an updated analysis of cas proteins using genomic context analysis and sensitive methods (iteration approaches) to detect low level of similarity and to classify cas proteins in families and superfamilies. They were able to identify several new putative cas proteins and to define 25 superfamilies of cas proteins and 7 different types of CASS organization (named CASS1 to 7). They have also analyzed all available CRISPR repeated sequences and their putative secondary structures. More importantly, they try to predict the biological function of the cas proteins and their mechanism of action in the framework of the RNAi hypothesis. Previously, it has been suggested that cas proteins were involved in the formation and spreading of the CRISPR. For instance, Bolotin et al. Have predicted that cas proteins are acting at the DNA level by promoting cleavage, recombination and ligation. Makarova and al are the first to suggest that several cas proteins should instead interact at the RNA level, by promoting RNA degradation and RNA-RNA hybridization. They specifically suggest the existence of procaryotic homologs of eucaryotic dicer (helicase-nuclease) and splicer (nuclease). They also propose that a previously suspected DNA polymerase could be an RNA dependent RNA polymerase used to stabilize RNA\/RNA hybrid by extending iRNA hybridized to their viral mRNA target. They also suggest the involvement of a reverse transcriptase in the formation of the linker sequences from viral (plasmidic) mRNA. In my opinion, all these proposals are reasonnable and very convaincing. Another prediction is that RAMP proteins recognize linker sequences of different sizes. This is supported by a correlation between the number of linker sequences and the number of RAMPs encoding genes (Fig. <a href=\"#F6\" data-ref-type=\"fig\">6<\/a>). In that case, it's not clear to me why this could not be due to the binding of RAMPs to the repeated units, since these units exhibit conserved sequences and their number (identical to the number of linkers) should be also correlated with the number of RAMPs.\n\n**Author response:** *That RAMPs discriminate, one way or another, between CRISPR inserts, is strongly suggested by the extreme sequence divergence of RAMPs which is hardly compatible with recognition of identical repeats. To be explicit about it, we added a clarification at the end of this section*.\n\nThe search for specific secondary structure associated to the repeated units did not give convincing results and suggest for me that the dyad symmetry observed in many repeat units could be due to the binding of proteins with repeated structure (possibly the duplicated ferredoxin-like fold present in RAMPs) and not the formation of secondary structures in the transcribed repeats.\n\n**Author response:** *It is hard to see how one excludes the other: it stands to reason that CRISPR do form distinct secondary structures which bind to symmetrical proteins*.\n\nThe model proposed (including possible variation) thus implies many predictions that could be experimentally tested. Surprisingly, to my knowledge, only one cas protein has been studied at the bench up to now (ref 70 in the manuscript). This protein turns out to have DNAse activity *in vitro*, but I suspect that the authors have not tested a possible RNAse activity. This is surprising because the importance of these proteins was already highlighted in 2002 by two *in silico* papers that in one case suggested their participation to a \"mysterious DNA repair system and in the other described their association with CRISPR sequences. The present paper, with much more specfic predictions, should hopefully strongly stimulate biochemists and molecular biologists to jump onto this really exciting story. As noticed by the authors in their conclusion, if their hypothesis turned out to be correct, this prokaryotic RNAi system could be exploited to silence any gene in organisms that encode CASS. Furthermore, the experimental study of this system should help us to get new critical insights on the dynamic relationships between viruses and archaeal\/bacterial populations in nature.\n\nFinally, I would like to know if the authors have some idea about the origin of this CAS system. Why is it present in all archaeal genomes sequenced so far? Is it possible that this system originated in Archaea and was later on introduced in bacteria by LGT?\n\n**Author response:** *Given the horizontal mobility of CASS, we can only speculate on the point of its origin. We expand such speculation in the revised conclusion including the possibility of archaeal origin*.\n\n\\- In some case, the authors should be more cautious in their statement. For instance, when they talk about the pol-cassette, it might led some reader to believe that the polymerase actvity of the COG1353 protein has been experimentally validated, which is not the case.\n\n**Author response:***We added a few more \"predicted\". However, we did not want to abandon the term 'pol-cassette' as it is descriptive and succinct*.\n\n## Reviewer's report 3\n\nMartijn Huynen, Nijmegen Center for Molecular Life Sciences University Medical Center St. Radboud p\/a Center for Molecular and Biomolecular Informatics, Nijmegen, Netherlands\n\nThis paper provides a highly interesting and well documented hypothesis about a cluster of genes that E. Koonin and co-workers have discovered some time ago. By combining biological knowledge with bioinformatics methods and creative thinking the authors propose that Archaea and to a bit lesser extent Bacteria posses an RNA-interference-based immune system involving CRISPR and cas genes, that is analogous the eukaryotic RNA interference systems. Although aspects of this hypothesis have been published before, specifically with respect to CRISPR, this paper is, as far as I can tell, the first that makes the analogy between the cas genes and the RNA interference system. The idea that prokaryotic genomes would internalize pieces of foreign DNA in order to be able to defend themselves against it, thus having an immune system with a memory, would be an interesting example of Lamarckian evolution.\n\nI do have some questions and editorial comments that I think should be addressed.\n\n1\\) Do the authors have any idea why this system has the phylogenetic distribution that it does, being present in such a small genome as the nanoarchaeon, but not in e.g. the majority of Firmicutes\n\n**Author response:** *No mechanistic idea, unfortunate as this might be. We added some additional discussion of the ultimate origin of CASS (see the response to Patrick Forterre)*.\n\n2\\) concerning the feasibilty of the system proposed by the authors: Is there anything known about how many fiendly DNAs a prokaryote encounters in daily life, and how does that compare to the number of different elements in a CRISPR ?\n\n**Author response:** *Not enough for this particular comparison. However, it is well known that phages are extremely abundant, much more so than bacteria or archaea, and in the revised manuscript, we refer to this more specifically, with the corresponding references*.\n\n3\\) Regarding the Lamarckian scheme: That the unique element of the CRISPR correspond to highly conserved, essential elements of phage genes suggests that selection on genetic variation also plays role here. So the scheme would be partly Lamarckian.\n\n**Author response:** *Probably, so. The way we state it in the text \"CASS seems to come closest to a true Lamarckian mode of evolution among all known systems of heredity\" is compatible with this view*.\n\n4\\) I am not so convinced by the argument on page 3 that the results imply that even among closely related prokaryotes the most commonly encountered phages are different. First of all, it is more a corollary of the hypothesis, but second, it could also reflect the high turnover of phages over time, rather than niche.\n\n**Author response:** *This is a very good idea, we now mention this possibility both in the Abstract and in the Discussion*.\n\n5\\) I am puzzled on the involvement of more or less randomly selected pieces of DNA from foreign DNA\/RNA in exactly the same location in the secondary structure of the psiRNA (the top of the hairpin). Does this pattern occur more often?\n\n**Author response:** *The situation when the insert forms a stem of varying stability with parts of the repeats is common but not universal. The positions of the inserts are not exactly the same although they are, indeed, very similar, and the stems in which the inserts are involved are imperfect. Of course, the exciting possibility exists that the CRISPR inserts are specifically selected for their ability to base-pair with the repeats, however, we do not have enough data to make that claim*.\n\n# Authors' contributions\n\nKSM performed the protein sequence analyses, NVG performed protein structure comparisons and modeling, SAS performed RNA secondary structure predictions, YIW performed nucleotide sequence analysis, EVK developed the biological interpretations of the results and wrote the manuscript which was edited and approved by all authors.\n\n# Supplementary Material\n\n###### Additional file 1\n\nGI number for all families of Cas proteins.\n\nClick here for file\n\n###### Additional file 2\n\nmultiple protein sequence alignment for COG1343.\n\nClick here for file\n\n###### Additional file 3\n\nmultiple protein sequence alignment for COG1857.\n\nClick here for file\n\n### Acknowledgements\n\nWe thank M. Huynen and J. Van der Oost for helpful discussions, and all three reviewers for their insightful, detailed, and meticulous review that, as we believe, allowed to substantially improve the article. This work was supported in part by the Intramural Research Program of the National Library of Medicine at National Institutes of Health\/DHHS.","meta":{"dup_signals":{"dup_doc_count":124,"dup_dump_count":78,"dup_details":{"curated_sources":2,"2023-50":2,"2023-14":2,"2022-40":1,"2022-27":2,"2022-21":1,"2021-49":1,"2021-39":2,"2021-25":1,"2021-21":2,"2021-17":1,"2021-10":1,"2021-04":1,"2020-50":1,"2020-45":1,"2020-40":1,"2020-34":1,"2020-24":2,"2020-16":1,"2020-10":1,"2020-05":1,"2019-51":2,"2019-43":1,"2019-39":1,"2019-35":2,"2019-30":1,"2019-26":2,"2019-18":1,"2019-13":1,"2019-04":3,"2018-51":1,"2018-47":1,"2018-43":2,"2018-34":2,"2018-30":1,"2018-22":1,"2018-13":1,"2018-09":2,"2017-47":1,"2017-43":1,"2017-39":3,"2017-34":1,"2017-30":1,"2017-26":1,"2017-22":3,"2017-17":1,"2017-09":5,"2017-04":1,"2016-50":2,"2016-44":2,"2016-40":2,"2016-36":1,"2016-30":4,"2016-26":1,"2016-22":1,"2015-48":1,"2015-40":1,"2015-35":2,"2015-32":2,"2015-27":1,"2015-22":3,"2015-14":1,"2014-52":1,"2014-49":1,"2014-42":3,"2014-41":2,"2014-35":2,"2014-23":2,"2014-15":3,"2024-26":1,"2024-22":1,"2024-18":2,"2017-13":1,"2015-18":1,"2015-11":1,"2015-06":1,"2014-10":2,"2013-20":4,"2024-30":1}},"file":"PMC1462988"},"subset":"pubmed_central"}
{"text":"abstract: The availability of complete genomic sequences of *Saccharomyces cerevisiae* has catalyzed a cultural change in the practice of yeast biology, providing opportunities to develop high throughput techniques to define protein function, to define drug targets, and to discover and characterize drugs.\nauthor: Charles Brenner\ndate: 2000\ninstitute: 1Kimmel Cancer Center, Thomas Jefferson University, 233 South Tenth Street, Philadelphia, PA 19107, USA. E-mail: firstname.lastname@example.com\nreferences:\ntitle: A cultivated taste for yeast\n\nFor decades, yeast biology has been dually justified as an effort to understand fully the cellular processes of a group of single-celled eukaryotes and for its potential to lead to advances in human health. Sequence-based trees of life provide compelling evidence that plants, animals and fungi - once considered three primary kingdoms in a five-kingdom tree - are closely related branches within the eukaryotic domain \\[1\\]. It is well known that homologs of proteins first identified for key roles in yeast cell-cycle control \\[2\\], secretion \\[3\\] and pro-hormone processing \\[4\\] play similar roles in humans. Furthermore, similarities between fungal, animal and plant cells have created challenges in developing fungal-directed antibiotics that are not toxic to hosts. Although yeast biology has illuminated many processes in yeast and human cells by homology, the yeast cell has also become a laboratory of heterology. In the 1980s, heterologous use of yeast typically referred to protein production \\[5\\]. In the 1990s, legions of researchers were introduced to yeast two-hybrid systems to identify binding partners of proteins expressed in yeast \\[6\\]. Although it is too early to predict the leading heterologous use of yeast of the next decade, screens and selections to identify small molecules that act on proteins expressed in yeast hold tremendous promise \\[7\\]. The power of combinatorial chemistry to screen huge numbers of drugs can now be multiplied by the power of yeast genetics to assay genetic variation. At a recent Cambridge Healthtech Institute meeting in Miami entitled 'Exploiting Yeast Molecular Biology for Therapeutics' (January 20-21, 2000) \\[<http:\/\/www.healthtech.com\/conference\/yst\/yst.htm>\\], some of the latest homologous and heterologous uses of yeast biology were presented, along with new methods for studying yeast biology that became conceivable in the post-genomic era.\n\n# Given a substrate, find the enzyme\n\nThousands of yeast genes and their corresponding products were identified by classical genetic approaches that began with identification of mutant phenotype and progressed 'forward' to the gene and product. Thousands of other genes were discovered by 'reverse' genetic approaches, in which mutants were obtained last. The original reverse genetics experiments were fractionation-based; one purified a protein of interest, sequenced it, and then cloned and disrupted the corresponding gene (for example, see \\[8\\]). More recently, reverse genetic approaches have been driven by identification of homologous sequences (for example, see \\[9,10,11\\]). The availability of complete genomic information has made possible a new type of reverse genetics not driven by sequence similarity but rather based on a novel fractionation scheme.\n\nFor example, when Mark Martzen, Elizabeth Grayhack and Eric Phizicky at the University of Rochester set out to identify an enzyme, they began in the classical manner by developing an assay to screen for activity. Rather than fractionating a large, single yeast lysate to identify the enzyme by biochemical means, these researchers used a set of yeast strains carrying fusions of glutathione-S-transferase (GST) to 6,080 open reading frames (ORFs) \\[12\\]. The GST fusions, developed in the laboratory of Stanley Fields at theUniversity of Washington \\[13\\], were grown in 64 pools, each containing 95 ORF-GST fusions plus one control GST construct. To identify the gene responsible for an enzyme activity of interest, Martzen and colleagues first screened the 64 pools to identify which one contained the activity. Because each pool originated from constructs held in a 96 well plate, they then screened the 8 rows and 12 columns from the identified plate. The single point of intersection of pooled rows and pooled columns defines the single ORF responsible for an activity. The first publication from the Martzen group using this technique described three enzymes identified in this way \\[12\\], and the group's biochemical genomics scoreboard now includes a DNA-binding protein and numbers 12 additional enzymes, several of which carry out RNA splicing reactions.\n\n# Given an enzyme, validate it as a drug target\n\nIn humans, elevated serum homocysteine concentration is associated with vascular, skeletal and neurological disorders. The most common genetic basis for clinical presentation of severe hyperhomocysteinemia is cystathionine \u03b2-synthase (*CBS*) deficiency, found in 1 child in about 300,000 \\[14\\]. Moreover, levels of homocysteine vary in populations and there is strong epidemiological linkage between elevated total homocysteine levels and vascular disease and stroke \\[15\\]. Whereas 50% of *CBS*-deficient patients respond to high doses of vitamin B6, and vitamin B6 can also reduce total homocysteine in the majority of the human population, a specific drug that could boost Cbs enzyme activity in *CBS*-deficients and in those at risk for stroke might improve the quality of life for many people. Warren Kruger of the Fox Chase Cancer Center has developed a simple yeast-based assay to phenotype human *CBS* genes and has validated mutant forms of Cbs as targets that could be remedied by drugs. Wild-type human *CBS* coding sequences complement the cysteine deficiency of *S. cerevisiae cys4* deletion mutants, and loss-of-function alleles of human *CBS* fail to complement *cys4* \\[16\\]. In the course of cloning new *CBS* alleles, Kruger's laboratory amplified a patient's *CBS* genes and recovered a large number of yeast transformants that were cys^-^ and two that were CYS^+^. The two CYS^+^ isolates contained PCR-induced intragenic suppressor mutations of the patient's loss-of-function allele. Both mutations resulted in truncation of an inhibitory carboxy-terminal domain. Nearly all *CBS* patients carry missense mutations and nearly all of these alleles are suppressed by deletion of the carboxy-terminal domain \\[17\\]. At the Miami meeting, Kruger presented data showing that a variety of point mutations in the carboxy-terminus suppress the hypomorphic phenotypes of *CBS* mutations and that carboxy-terminal suppressors are not allele-specific. Thus, chemical inactivation of the carboxy-terminal domain of Cbs should be achievable and should boost the function of low-activity enzymes. Furthermore, the combinatorial chemist has a choice of primary screens: increase mutant Cbs enzyme activity *in vitro* or generate prototrophy in a *cys4* yeast strain carrying mutant human *CBS*.\n\n# Given a target, find a drug\n\nKathleen Young of Wyeth-Ayerst Inc. spoke about her group's efforts to find 'disinactivators' of a human potassium channel, Kv1.1\/Kv\u03b21. Inactivation of such channels, mediated by an interaction between the S4-S5 cytoplasmic linker domain of the \u03b1 subunit and the amino-terminal inactivation ball of the \u03b2 subunit \\[18\\] might be associated with seizures and hippocampal ischemia \\[19\\]. For this reason, small molecules that block the association of specific \u03b1 and \u03b2 subunits are considered to have therapeutic potential. As demonstrated earlier with calcium channel fragments \\[7\\], two-hybrid interactions can be constructed to produce growth inhibition such that drugs that block the interaction restore growth. The new screen for potassium channel disinactivators involved more than 176,000 compounds and has apparently identified a compound, WAY-349, with *in vitro* efficacy and specificity for the Kv1.1\/Kv\u03b21 channel.\n\n# Given a drug, find the target\n\nAlthough protein-target-directed screening has become the predominant way to identify small molecules today, the majority of approved drugs were identified on the basis of animal or cellular assays. In fact, most of the classical compounds that owe their roots to botanicals and other natural products were originally identified by human self-medication. Thus, it should come as no surprise that a great many drugs have modes of action that are not understood. John Phillips and his colleagues at Rosetta Inpharmatics Inc. use two genomic methods to clarify the mode of action of drugs. First, they have developed a complete set of promoter fusions to the green fluorescent protein of *Aequoria victoria* that allows them to monitor the effect of a small molecule on the expression of each yeast gene \\[20\\]. Administration of a drug to yeast strains transformed with the set of reporter constructs provides a fluorescent profile of the genes that become activated or repressed in response to a drug. In the best cases, such as that of *erg11*, the ergosterol-starved gene expression profile of *erg11* mutant cells is indistinguishable from that of cells treated with the antifungal drug clotrimazole. Second, they test the drug sensitivity \\[21\\] of a large and growing set of strains that are heterozygous for individual open reading frames \\[22,23\\]. Because each gene disruption strain carries two unique 20 nucleotide 'barcodes', deletion mutants can be maintained in a pool \\[22,23\\]. Upon drug administration, the relative viability of each deletion strain can be assessed by amplification of barcodes from the pooled strains using fluorescently-labeled PCR primers and array hybridization to a DNA chip containing the barcode oligonucleotide probes \\[21\\]. Particular deletion mutants that are more sensitive to a drug than the majority of deletion mutants produce a less fluorescent signal upon drug administration and are identifiable by losses of particular barcode signals. The most drug-sensitive strains are those in which the gene products targeted by the drugs have been rendered heterozygous \\[21\\]. This method is complementary to the classical method of cloning the target by virtue of multicopy-plasmid-dependent resistance \\[24\\]. The advantage of the new method is that it is highly parallel. New compounds can be profiled with ease, and can be used to identify not only primary and secondary targets via sensitivity \\[25\\] but, additionally, loss-of-function mutations that result in drug-resistance.\n\n# Given a pathogenic fungus, find a drug target\n\n*Candida* species are among the most common fungal pathogens to humans. *C. albicans*, which is asexual and diploid, is refractory to 'forward' or classical genetics though *C. glabrata* is a haploid and, thus, conditionally essential genes or genes essential for adhesion to human epithelial cells can be identified by forward mutagenesis and screening. Having developed methods for homologous and nonhomologous integrative transformation in *C. glabrata* \\[26\\], Brendan Cormack (Johns Hopkins University) selected 96 40mer oligonucleotide barcodes and integrated them into the disrupted *ura3* locus of a *C. glabrata* strain. Each of these barcoded strains was then subjected to random insertional mutagenesis with the *S. cerevisiae URA3* gene and 100 independent URA^+^ integrants were frozen away for each barcoded strain \\[27\\]. In each strain, the URA3 insertion is expected to have affected a different gene. By picking one transformant from each of the 96 barcoded transformations, Cormack was able to assemble 100 pools each containing an independent *URA3* insertion in a uniquely identifiable strain.\n\nCormack has already described a screen in which 50 of the pools, representing as many as 4,800 independent *C. glabrata* mutants, were passaged through an adherence assay on human cultured epithelial cells. By probing a DNA 'macrochip' (a nylon filter the size of a 96 well plate on which the 96 barcode oligonucleotides were spotted) with radioactively labeled barcode PCR reactions from *C. glabrata* cells that grew on human cells, Cormack identified 16 mutants that failed to adhere. Of these, 14 contained insertions in the same gene, named *EPA1* for epithelial adhesin, a calcium-dependent lectin with a consensus sequence for addition of a glycosyl-phosphatidylinositol anchor \\[27\\]. Cormack also presented data showing that pools of barcoded mutant *C. glabrata* strains can be passaged through a mouse in the primary mutant hunt: 96 mutants being screened per animal. Cormack's approach of barcoding the cells to be mutagenized rather than linking the barcode to the mutants makes his barcodes reusable and independent of specialized array hybridization equipment.\n\nThough the yeast genome has been complete for four years, the majority of yeast gene functions are poorly characterized. Many of the approaches presented in Miami have the potential to assign functions to significant portions of the yeast proteome. As knowledge of yeast cell biology expands, one can expect greater light to be reflected on all eukaryotes and further cultivation of yeast for pharmacological applications.","meta":{"dup_signals":{"dup_doc_count":102,"dup_dump_count":47,"dup_details":{"curated_sources":4,"2023-23":1,"2022-21":1,"2021-49":1,"2021-04":1,"2020-34":1,"2020-24":1,"2019-47":2,"2019-35":1,"2019-26":1,"2019-13":1,"2019-04":2,"2018-47":2,"2018-39":2,"2018-30":2,"2018-22":1,"2018-17":1,"2018-09":1,"2018-05":1,"2017-39":1,"2017-30":1,"2017-22":1,"2017-09":1,"2017-04":1,"2016-07":1,"2015-48":3,"2015-40":2,"2015-35":3,"2015-32":3,"2015-27":2,"2015-22":2,"2015-14":3,"2014-52":3,"2014-49":4,"2014-42":8,"2014-41":6,"2014-35":4,"2014-23":4,"2014-15":3,"2023-50":1,"2017-13":1,"2015-18":3,"2015-11":3,"2015-06":3,"2014-10":3,"2013-48":1,"2013-20":3,"2024-22":1}},"file":"PMC138835"},"subset":"pubmed_central"}
{"text":"abstract: Psychosocial interventions are an essential part of the treatment for people with severe mental illness such as schizophrenia. The criteria regarding what makes an intervention \"evidence-based\" along with a current list of evidence-based interventions are presented. Although many evidence-based interventions exist, implementation studies reveal that few, if any, are ever implemented in a given setting. Various theories and approaches have been developed to better understand and overcome implementation obstacles. Among these, merging two evidence-based interventions, or offering an evidence-based intervention within an evidence-based service, are increasingly being reported and studied in the literature. Five such merges are presented, along with their empirical support: cognitive behavior therapy (CBT) with skills training; CBT and family psychoeducation; supported employment (SE) and skills training; SE and cognitive remediation; and SE and CBT.\nauthor: Tania Lecomte; Marc Corbi\u00e8re; St\u00e9phanie Simard; Claude Leclerc[^1]\\*Author to whom correspondence should be addressed; E-Mail: <firstname.lastname@example.com>; Tel.: +1-514-343-6274.\ndate: 2014-11-06\ninstitute: 1Department of Psychology, University of Montreal, C-358, 90 Vincent d'Indy Street, C.P. 6128, Succ Centre-Ville, Montreal, QC H3C 3J7, Canada; E-Mail: <firstname.lastname@example.com>; 2Centre for Action in Work Disability Prevention and Rehabilitation, School of Rehabilitation, Universit\u00e9 de Sherbrooke, 150 Place Charles Le Moyne, Bureau 200, Longueuil, QC J4K 0A8, Canada; E-Mail: <firstname.lastname@example.com>; 3Universit\u00e9 du Qu\u00e9bec \u00e0 Trois-Rivi\u00e8res, 3351 Boul Des Forges, Trois-Rivi\u00e8res, QC G9A 5H7, Canada; E-Mail: <email@example.com>\nreferences:\ntitle: Merging Evidence-Based Psychosocial Interventions in Schizophrenia\n\n# 1. Introduction\n\nPsychosocial interventions are an essential part of the treatment and recovery of people with severe mental illness such as schizophrenia. It is well-accepted that medication alone is not sufficient to help with the recovery and various issues a person might encounter when attempting to lead a satisfying life in the community. Some practices have gathered sufficient empirical data to be strongly recommended by national guidelines such as the PORT (Patient outcome research team) \\[1\\], or the NICE (TheNational Institute for Health and Care Excellence) \\[2\\] guidelines in the UK, whereas others are considered promising until more studies support its effect. In order for a psychosocial practice, namely an intervention or program (an intervention is specific treatment with a targeted therapeutic aim whereas a program typically includes various interventions), to be considered evidence-based, it must not only have accumulated sufficient empirical proof of its efficacy (often in the context of at least two randomized controlled trials) but it must answer a need (such as improve functioning, overcome a deficit or help to cope with symptoms) and be standardized in order to be easily replicable \\[3\\]. The 2009 PORT report on psychosocial practices for schizophrenia highlighted eight interventions or programs that could be considered evidence-based \\[1\\]. These were in terms of interventions: family psychoeducation, cognitive behaviour therapy, social skills training, weight management, and token economy. In terms of programs, they described: intensive community treatment, supported employment and integrated dual-disorder programs (with first episode programs almost meeting criteria). Since, Mueser *et al.* \\[3\\] has added cognitive remediation, which has demonstrated in various meta-analyses its efficacy in helping individuals overcome cognitive deficits in areas such as memory, attention or problem solving. Although all of the above-mentioned interventions and programs are considered evidence-based and can truly improve the lives of people with severe mental illnesses, implementation studies reveal that few, if any, are ever implemented in a given setting \\[4\\]. For some interventions, this is understandable\u2014for instance token economy is only effective in changing problematic behaviors when used in a closed and controlled environment, like a forensic ward \\[5\\]. However, most of the other interventions and programs have demonstrated positive effects in improving symptoms, deficits, and community functioning and are under-utilized. A recent review on implementation of evidence-based psychosocial interventions and programs in psychiatry describes various strategies that have been used in order to improve implementation of one or many evidence-based practice \\[4\\]. Of the most common strategies, large demonstration projects and effectiveness trials run by researchers, broad service reforms based on new government policies and national agency-led initiatives were identified. Few of these implementation strategies have been studied over the long-term, and fewer even have considered consumer outcomes \\[4\\]. Other smaller-scale implementation strategies have also been developed, when large-scale funding was not available. For instance, supported employment programs' implementation have been facilitated by a group of researchers and clinicians \\[6\\] offering standardized training, consultation and encouraging the use of their implementation fidelity scales (The *Quality of Supported Employment Implementation Scale* (QSEIS) \\[7\\] or the *Individual Placement and Support* (IPS) fidelity scale \\[8\\]. In the UK, US and Canada, the implementation of CBT for psychosis has been improved by offering the clinical training to mental health professionals from various backgrounds (e.g., nurses, occupational therapists), offering structured manuals, and brief training sessions \\[9,10\\] rather than limiting the treatment delivery to clinical psychologists.\n\nAnother approach to improving implementation of evidence-based psychosocial interventions or programs that is being seen more and more in the literature, particularly in the last decade, is to merge two evidence-based interventions, or to offer an evidence-based intervention within an evidence-based program. Are considered merges both: (a) the simultaneous delivery of two practices (two interventions or an intervention and a program) and, (b) modified practices: interventions changed in order become integrated with another intervention or within a program. In settings with limited means, offering such merges has the advantage of targeting more than one therapeutic goal at once (e.g., cognitive distortion and work integration). Some of the merges have been developed in order to adapt an evidenced-based intervention to a specific population group or setting (such as older consumers). However, most of the merges have been developed with the hope of improving the effectiveness of an evidenced-based program by adding an empirically recognized intervention. It is important to mention that few of the merges presented here simply \"co-deliver\" interventions\u2014most are well-integrated merges or at least aim at truly integrating the interventions and programs together. The following are descriptions of such merges.\n\n# 2. Cognitive Behavior Therapy (CBT) and Social Skills Training\n\nGranholm and colleagues \\[11,12\\] have merged two evidence-based psychosocial interventions to cater to the specific needs of aging individuals with psychosis who might be struggling not only with symptoms but also with social and cognitive deficits. CBT for psychosis has been studied in over 40 randomized controlled trials and various meta-analyses since the first promising studies of the 1990s \\[13\\]. Overall, most studies have found that CBT for psychosis is effective in reducing symptoms and improving other indexes of well-being, often with stronger results compared to other interventions overtime than at post-treatment \\[14,15,16\\]. However, as in most psychotherapy studies, the most rigorous studies often reveal smaller effect sizes than the non-controlled studies \\[17\\]. CBT for psychosis aims at modifying dysfunctional beliefs by helping the person understand the link between perceptions, beliefs and emotional and behavioural reactions. CBT also helps the person question the evidence supporting his beliefs (whether they are psychotic or not). Furthermore, CBT brings the person to self-observe himself, his thoughts and behaviors, and explores various coping strategies the person might use when dealing with distressful thoughts or voices. Finally, CBT for psychosis takes into account cognitive biases a person might have and aims at modifying those biases, by seeking alternatives instead of jumping to conclusions, for instance.\n\nCBT for psychosis has been adapted for various clienteles, used with individuals at high risk of developing psychosis, individuals with early psychosis as well as older individuals with a long history of schizophrenia. This latter group was of particular interest for Granholm and colleagues \\[12\\] who wished to offer CBT for psychosis in a format that would be appealing and adapted to clients who were often isolated, and who might have difficulties grasping some CBT concepts, given their cognitive deficits. They therefore decided to include social skills training to the CBT treatment and offer the merged treatment in a group format.\n\nSocial skills training has been around since the 1980s in the USA and was considered especially useful for helping people reintegrate society after a long period of institutionalization. The goal behind skills training is to offer skills that are deemed essential to interact with others, manage one's medication and symptoms, as well as problem solve in different contexts. Skills training is based on Bandura's self-efficacy theory \\[18\\] and uses repetition and positive reinforcement to help people acquire and remember new skills. To date, over 23 randomized controlled trials have shown that skills training can help acquire skills, decrease negative symptoms, and has a moderate impact on independent living skills \\[19,20\\]. Skills training can be offered individually but works best in groups, with the use of multiple role-plays preparing for real-life interactions.\n\n## Merging CBT and Skills Training\n\nGranholm and colleagues \\[11,12\\] developed a group CBT\/skills training approach that focuses on CBT for psychosis techniques, such as checking for facts, but presents these in a skills training manner (*i.e.*, a lot of repetition, wallet cards with key words\/concepts, use of a big flag in the group to \"flag\" the beliefs without apparent facts or proof). The group included modules that were repeated over time, enabling the participants to go over the content more than once and allowing new participants to enter the group at any given moment. Although the results did not show an improvement in positive symptoms, it did show improvements in functioning and negative symptoms \\[11,12\\]. Of importance, the participants were able to remember the concepts and CBT techniques regardless of the severity of their cognitive deficits.\n\n# 3. CBT and Family Psychoeducation\n\nLeclerc and Lecomte \\[21\\] have recently published promising preliminary data on 40 family members who received a merged group CBT\/psychoeducation family intervention. Family intervention, in particular family psychoeducation, is recognized as one of the evidence-based interventions with the most empirical support, especially regarding decreasing rehospitalization rates \\[3\\]. More than 50 randomized controlled trials have been published to date supporting the effects of family psychoeducation on increased medication adherence, and decreased stress and symptoms in those receiving psychiatric care \\[15,22\\]. As for their family members, these same studies report decreased perceived burden and psychological distress. Most family interventions last an average of six to nine months and offer: information on symptoms and mental illness, recommendations for dealing with crises, emotional support, and coping skills to deal with symptoms and mental illness \\[23\\]. Family interventions can be offered to each individual family or multiple families together, with or without the family member receiving psychiatric care. Many family intervention manuals were developed in the 1990s and do not address recent concepts such as recovery and tend to focus mostly on medication, chronicity, and symptoms. During a recent trial on CBT for early psychosis, many family members asked to learn more about CBT for psychosis and how they could use the tools in their lives. We therefore developed the family psychoeducation\/CBT module entitled WITH (Wellness-Inform-Talk-Help) \\[21\\]. The module can be offered in parallel to the CBT for psychosis groups, *i.e.*, during 24, hourly multiple family sessions, or can be offered in a more intensive format: eight two-hour multiple family sessions (covering 16 activities in the module). Each multiple family group typically consists of an average of 10 parents and two co-therapists. The content of the group is psychoeducational in that it addresses concepts such as recovery, expressed emotions, parental role, personal limits, and expectations, but it is also considered CBT given that the participants learn about CBT principles and techniques and apply them to their own lives during the group and at home (homework). The parents therefore learn to not only use the skills learned with their family member with a mental illness, but also use them with themselves when they are experiencing distress for instance.\n\n## Merging CBT and Family Psychoeducation\n\nThe intensive (eight two-hour sessions) format was recently studied in a non-controlled study \\[21\\] whereby the 40 parents who participated showed significant clinical improvements in psychological distress, namely in psychoticism, depression and interpersonal sensitivity compared to their baseline scores. Qualitative data obtained revealed that parents appreciated the group, found it helpful, and they had integrated recovery as well as CBT notions and skills in order to improve their relationship with their family member receiving psychiatric services. The group format was especially appreciated, as well as the information covered in the module. Although more studies are warranted in order to compare the WITH multiple family intervention to other family interventions, social workers offering the group anecdotally mentioned that their previous multiple family psychoeducational intervention had a retention rate of only 20% of participants from the first to the last session whereas WITH had a retention rate of 80%. The merged intervention has the advantage of covering essential elements of family psychoeducation for psychosis, including updated information on recovery, and also offers concrete CBT tools that can be useful for the person with a mental illness as well for their family members.\n\n# 4. Supported Employment and Other Evidence-Based Interventions\n\nSupported employment is another evidence-based program that has attracted a few merges over the past decade. Supported employment programs help people with severe mental illness obtain real-world competitive employment, with regular wages, based on their clients' preferences \\[6\\]. Employment specialists working in supported employment programs aim at quickly finding regular paid work for their clients, and offer them unlimited support according to their needs at work. Supported employment programs are recognized as being evidence-based with more than 15 trials in various countries having demonstrated that SE programs are more efficacious in helping people with severe mental illness obtain regular jobs than other vocational or rehabilitation programs \\[6\\]. Nonetheless, there is room for improvement given that on average, in North America, between 40%\u201360% of the clients in SE programs obtain regular jobs and most jobs are only kept for three to five months. Many reasons have been suggested to explain why some individuals might struggle in finding work or in maintaining their jobs. Some have suggested lack of appropriate work-related social skills, others that cognitive deficits impede on work performance, and others still that people with mental health problems might hold irrational beliefs about themselves and the workplace.\n\n## Merging Social Skills Training and Supported Employment Programs\n\nThe first merge proposed was to offer social skills training that was work specific to people registered in a supported employment program. Charles Wallace \\[24\\] developed the *Workplace Fundamentals*, aiming at helping participants recognize the advantages of work in their lives, their potential stressors at work, how to problem solve various work-related situations, and how to avoid drugs and alcohol to maintain their jobs. The module is offered over the course of 24 sessions, typically twice a week, in groups of six to eight participants. As with most social skills training, the goal is to develop spontaneous behaviors and therefore involves multiple role-plays and repetitive behaviors. Two studies were conducted to verify its efficacy in improving job tenure. The first, including 34 participants, showed improved job tenure and better work satisfaction for those having received the skills training + SE program compared to SE program alone \\[24\\], whereas the second study did not show any differences between the two conditions on work outcomes (but reported that the sample was not typical of most studies with higher education and longer tenure, with rates close to one year for their first job) \\[25\\]. The participants receiving both conditions did show greater knowledge regarding their work setting, stressors and showed better problem solving abilities than those receiving only the SE program.\n\n# 5. Cognitive Remediation and Supported Employment\n\nAnother explanation for poor work tenure in people receiving SE programs pertains to cognitive deficits. Cognitive deficits are well documented in people with severe mental illness, namely regarding deficits in memory, attention, speed of processing and various executive functioning tasks, and can make performing at work difficult. Various cognitive remediation programs and strategies have been developed over the years with more than 40 randomized controlled trials supporting its efficacy in improving cognitive skills and overall functioning \\[26\\]. Cognitive remediation can take many forms: paper-pencil tasks, computer tasks, group training, or training in real-world tasks (using errorless learning, for instance \\[27\\]). Although some cognitive remediation can include modifying the environment to compensate for the person's most important cognitive deficit, most cognitive remediation programs aim at improving cognitive deficits to the point that they no longer interfere with work performance.\n\n## Merging Cognitive Remediation and Supported Employment Programs\n\nMcGurk and colleagues developed a cognitive remediation program called *Thinking Skills for Work* specifically for people registered in supported employment programs \\[28\\]. The program involves individual computerized training (using CogPack) for an average of 24 hours over the course of 12 weeks, along with cognitively-informed job support consultation with the employment specialist. The computerized program aims at improving attention, concentration, psychomotor speed, learning and memory as well as executive functions. The consultation aims at targeting jobs or at modifying the work environment as needed according to the person's performance and progress during the cognitive remediation training. Results at the two to three year post- cognitive remediation follow-ups revealed that those who had received the cognitive remediation program had improved on the cognitive tasks and had superior job retention rates than the control condition (registered in supported employment programs only) \\[29\\]. These results were however not found for those who presented with comorbid substance use disorders\u2014their work outcomes were poor regardless of the extra treatment added \\[30\\].\n\n# 6. CBT and Supported Employment\n\nA potential obstacle to job maintenance in people registered in supported employment is dysfunctional beliefs regarding the workplace and one's own abilities. Individuals with severe mental illness who have been away from the job market for some time can hold beliefs and act in ways that are deleterious for their work integration, and could be influenced by lack of confidence, jumping to conclusions bias, and poor coping skills, to name a few. As mentioned previously, CBT has proven efficacious in modifying beliefs and cognitive biases and helps in developing better coping strategies when dealing with stressful situations. CBT has also been modified by Davis and colleagues \\[31\\] to target work beliefs and behaviours in a transitional vocational program for veterans with severe mental illness. This program, entitled IVIP, has demonstrated improvements in work performance and job maintenance in those receiving the group IVIP compared to those participating in the vocational program alone \\[32\\]. These results were also replicated in a larger trial \\[33\\].\n\n## Merging CBT and Supported Employment Programs\n\nLecomte, Corbi\u00e8re, Titone and Lysaker \\[34\\] developed a brief CBT group intervention, inspired by the IVIP, but specifically tailored for people in supported employment programs called CBT-SE. The CBT-SE intervention is offered during 8 sessions over the course of one month, in order to ensure that the group does not impede on the rapid job search principle of supported employment programs. The content covered many essential aspects linked to the workplace, such as recognizing and managing one's stressors at work, determining and modifying dysfunctional thoughts (e.g., not jumping to conclusions, finding alternatives, seeking facts), overcoming obstacles (e.g., problem solving), improving one's self-esteem as a worker recognizing strengths and qualities), dealing with criticism, using positive assertiveness, finding coping strategies (for symptoms and stress) to use at work, negotiating work accommodations and overcoming stigma. Although the results from the trial of 160 participants are not yet available, preliminary data have been published on 24 participants \\[35\\] and suggest that the CBT-SE intervention is feasible, and acceptable, with good attendance and positive feedback regarding the group's usefulness. In terms of work outcomes, 50% of all participants in both conditions found competitive work but those in the CBT-SE condition were more likely to work more hours per week and for more consecutive weeks than those in the supported employment program alone. These preliminary results are promising, although results from the full trial are needed before concluding that CBT-SE is efficacious in improving job tenure.\n\n# 7. Conclusions\n\nEvidence-based psychosocial practices for individuals with severe mental illness can greatly improve people's lives but are unfortunately scarcely implemented. When large-scale governmental or agency supported implementation initiatives are not available, clinical or community settings who are tempted to offer one evidence-based program or intervention could also opt for a merged intervention. Merged interventions have the advantage of targeting two sets of skills at once, and could therefore generalize in other aspects of the person's life. For instance, individuals having received cognitive remediation within a supported employment program \\[28\\] could see improvements in other areas of their lives, outside of work, from their improved memory and attention skills. Similarly, the cognitive behavioural strategies used in the CBT-SE skills \\[34\\] are similar to those used in more general CBT for psychosis treatments and could be used to help the person assess situations differently at work as well as outside of work, with friends or family for instance.\n\nThis article aimed at presenting some merges of evidence-based programs but is in no way exhaustive. Other merges exist, such as social skills training with token economy for substance misuse \\[36\\] cognitive remediation with social skills training (e.g., Integrated Psychological Therapy - IPT \\[37\\]) or social cognitive training with CBT and skills training (*i.e.*, Social Cognition and Interaction Training\u2014SCIT \\[38\\]). These programs are however described as distinct and unique programs, not as merges of evidence-based interventions. Although evidence-based interventions are empirically supported, their impact on various outcomes can likely be improved by adding elements from other evidence-based interventions, or by offering them within an evidence-based program, as was demonstrated here. Although some of the proposed merged interventions have only been studied in small or uncontrolled studies so far, the strong empirical support for the non-merged evidence-based interventions from which they are derived and the preliminary data available so far is quite encouraging. Future studies on merged evidence-based interventions are warranted, particularly in terms of trials assessing the effectiveness of offering such interventions simultaneously rather than separately and at different times. Furthermore, studies should also consider measuring the level of integration of the practices in order to determine if closely-knit merges are more effective than less integrated practices. Finally, studies should also investigate if these merges increase or not generalization of the skills to other life domains.\n\n## Author Contributions\n\nThe authors contributed equally to this work.\n\n## Conflicts of Interest\n\nThe authors declare no conflict of interest.\n\n# References\n\n[^1]: These authors contributed equally to this work.","meta":{"dup_signals":{"dup_doc_count":103,"dup_dump_count":47,"dup_details":{"curated_sources":2,"2023-23":1,"2023-06":1,"2022-40":1,"2022-21":1,"2021-49":1,"2021-43":1,"2021-31":1,"2021-25":1,"2021-17":1,"2020-34":1,"2020-29":1,"2020-16":2,"2020-10":1,"2020-05":1,"2019-47":2,"2019-39":2,"2019-30":2,"2019-22":1,"2019-13":2,"2019-04":2,"2018-47":1,"2018-43":1,"2018-39":1,"2018-34":1,"2018-30":2,"2018-26":5,"2018-22":3,"2018-17":5,"2018-13":2,"2017-51":2,"2017-47":1,"2017-43":5,"2017-39":4,"2017-34":4,"2017-30":3,"2017-26":5,"2017-22":6,"2017-17":7,"2017-09":4,"2017-04":1,"2016-50":1,"2016-44":1,"2016-40":1,"2023-40":1,"2024-10":1,"2017-13":6,"2024-26":1}},"file":"PMC4287699"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  Dietary assessment tools are often too long, difficult to quantify, expensive to process, and largely used for research purposes. A rapid and accurate assessment of dietary fat intake is critically important in clinical decision-making regarding dietary advice for coronary risk reduction. We assessed the validity of the MEDFICTS (MF) questionnaire, a brief instrument developed to assess fat intake according to the American Heart Association (AHA) dietary \"steps\".\n  .\n  # Methods\n  .\n  We surveyed 164 active-duty US Army personnel without known coronary artery disease at their intake interview for a primary prevention cardiac intervention trial using the Block food frequency (FFQ) and MF questionnaires. Both surveys were completed on the same intake visit and independently scored. Correlations between each tools' assessment of fat intake, the agreement in AHA step categorization of dietary quality with each tool, and the test characteristics of the MF using the FFQ as the gold standard were assessed.\n  .\n  # Results\n  .\n  Subjects consumed a mean of 36.0 \u00b1 13.0% of their total calories as fat, which included saturated fat consumption of 13.0 \u00b1 0.4%. The majority of subjects (125\/164; 76.2%) had a high fat (worse than AHA Step 1) diet. There were significant correlations between the MF and the FFQ for the intake of total fat (r = 0.52, P \\< 0.0001) and saturated fat (r = 0.52, P \\< 0.0001). Despite these modest correlations, the currently recommended MF cutpoints correctly identified only 29 of 125 (23.3%) high fat (worse than AHA Step 1) diets. Overall agreement for the AHA diet step between the FFQ and MF (using the previously proposed MF score cutoffs of 0\u201339 \\[AHA Step 2\\], 40\u201370 \\[Step 1\\], and \\>70 \\[high fat diet\\]) was negligible (kappa statistic = 0.036). The MF was accurate at the extremes of fat intake, but could not reliably identify the 3 AHA dietary classifications. Alternative MF cutpoints of \\<30 (Step 2), 30\u201350 (Step 1), and \\>50 (high fat diet) were highly sensitive (96%), but had low specificity (46%) for a high fat diet. ROC curve analysis identified that a MF score cutoff of 38 provided optimal sensitivity 75% and specificity 72%, and had modest agreement (kappa = 0.39, P \\< 0.001) with the FFQ for the identification of subjects with a high fat diet.\n  .\n  # Conclusions\n  .\n  The MEDFICTS questionnaire is most suitable as a tool to identify high fat diets, rather than discriminate AHA Step 1 and Step 2 diets. Currently recommended MEDFICTS cutpoints are too high, leading to overestimation of dietary quality. A cutpoint of 38 appears to be providing optimal identification of patients who do not meet AHA dietary guidelines for fat intake.\nauthor: Allen J Taylor; Henry Wong; Karen Wish; Jon Carrow; Debulon Bell; Jody Bindeman; Tammy Watkins; Trudy Lehmann; Saroj Bhattarai; Patrick G O'Malley\ndate: 2003\ninstitute: 1Cardiology Service, Walter Reed Army Medical Center, Washington, DC, USA; 2Dwight D. Eisenhower Army Medical Center, Ft. Gordon, GA, USA; 3General Internal Medicine Service, Walter Reed Army Medical Center, Washington, DC, USA; 4Systems Assessment & Research, Inc., Lanham, MD, USA\nreferences:\ntitle: Validation of the MEDFICTS dietary questionnaire: A clinical tool to assess adherence to American Heart Association dietary fat intake guidelines\n\n# Background\n\nDietary fat intake is a risk factor for coronary heart disease and the modification of dietary habits is important for the prevention of cardiovascular disease. The assessment of dietary fat intake is a critically important first step in clinical decision-making regarding dietary and pharmacotherapeutic advice on coronary risk reduction. Thus, a rapid and accurate tool to assess dietary fat intake would be a clinically useful screening tool for physicians to counsel patients about diet and coronary risk reduction.\n\nCommonly used dietary assessment tools include dietary history, 24-hour recall, seven-day recall, seven-day record, and food frequency questionnaire. \\[1-7\\] Impediments to more widespread clinical use of these tools include their length, and the difficulty and expense of their analysis. Furthermore, nutrition researchers tend to focus on a method's ability to yield precise and accurate measurement of a nutrient rather than to evaluate whether a tool can simply and quickly identify an individual's distribution or pattern of food intake. We compared the accuracy of the MEDFICTS (MF) questionnaire,\\[8\\] a brief instrument developed to assess fat intake according to the American Heart Association (AHA) dietary \"steps,\"\\[9,10\\] to the standardized Block Food Frequency Questionnaire\\[11,12\\] (FFQ) in a sample of active duty US Army personnel without known coronary artery disease.\n\n# Methods\n\n## Subjects\n\nThis study contains data from 164 active-duty U.S. Army personnel who completed both the MF and Block FFQ. Subjects completed the surveys during the same intake interview for a primary prevention intervention trial \u2013 the Prospective Army Coronary Calcium (PACC) Study. The study was approved by the Department of Clinical Investigation of the Walter Reed Army Medical Center.\n\nThe methods of the PACC Study have been previously described.\\[13\\] Briefly, since October 1998, active-duty Army personnel from 39 through 45 of age who were stationed in the National Capital Area of the Walter Reed Health Care System were recruited at the time of a periodic, Army-mandated physical examination. Persons who had a history of coronary heart disease or who reported a history of angina pectoris on the Rose questionnaire were ineligible. Between October 26, 1998 and November 4, 1999, 705 eligible participants were screened and 630 provided written informed consent to undergo electron beam computed tomography (EBCT) in addition to the required physical examination procedures. A subset of these subjects also volunteered to participate in a randomized, controlled trial to assess the impact of the knowledge of EBCT results and nurse-based case management on risk factor modification. The 164 subjects included in this dietary assessment study are a consecutive sample of subjects who both consented to participate in the randomized controlled trial and who also completed the MF and Block FFQ questionnaires. Demographic characteristics of the study participants are shown in Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>.\n\nDemographic Characteristics and Select Cardiac Risk Factors of the PACC Participants (N = 164)\n\n| **Variable**                 | **Value\\*** |\n|------------------------------|-------------|\n| Demographic Characteristics: |             |\n| \u2003Male gender (%)             | 79.9        |\n| \u2003Age (yr)                    | 42 \u00b1 2      |\n| \u2003Caucasian (%)               | 65.9        |\n| \u2003College educated (%)        | 78.4        |\n| Cardiac Risk Factors:        |             |\n| \u2003Total cholesterol (mg\/dl)   | 203 \u00b1 34    |\n| \u2003LDL (mg\/dl)                 | 130 \u00b1 33    |\n| \u2003HDL (mg\/dl)                 | 52 \u00b1 14     |\n| \u2003Triglycerides (mg\/dl)       | 117 \u00b1 64    |\n| \u2003BMI                         | 27 \u00b1 4      |\n| \u2003Waist girth (cm)            | 92 \u00b1 10     |\n\n\\*Plus-minus values are means and standard deviations.\n\n## Dietary Assessment Tools\n\nEach participant filled out a series of questionnaires that included the 2 dietary assessment tools: the validated and reduced version of the Block FFQ and the full version of the MF. The reduced version of Block FFQ is a validated food survey that contains 60 food items and is intended to capture all nutrients in the diet including dietary fat intake. The survey requires approximately 15 minutes to self-administer, however, the survey requires a relatively detailed analysis, thus the results are not immediately available for patient counseling. This study focuses only on those variables from the Block FFQ that are relevant to dietary fat intake within the dietary guidelines of the American Heart Association (AHA). This includes total calories, percentage of calories from fat and saturated fat, and cholesterol intake.\n\nThe MF questionnaire was specifically designed to evaluate patient adherence to the National Cholesterol Education Program (NCEP) Step 1 and Step 2 diets adopted by the American Heart Association (AHA). The main objective of these dietary steps is incrementally reduce coronary heart disease risk through diet-induced reduction in LDL cholesterol. Both the AHA Step 1 and 2 diets focus on reducing total fat to 30% or less of daily energy, and progressively reducing saturated fat and cholesterol intake (Step 1: 7\u201310% of energy from saturated fat and \\<300 mg cholesterol; Step 2: \\<7% of energy from saturated fat and \\<200 mg cholesterol). The AHA Step 1 diet is recommended for all healthy persons for the prevention of coronary heart disease, and is recommended to precede pharmacotherapy of LDL cholesterol. The Step 2 diet is recommended to further reduce LDL cholesterol for patients that have already achieved their Step 1 dietary goals. Additionally, the Step 2 diet is the initially recommended diet for patients with either a high-risk cholesterol level (\\>240 mg\/dL) or with known coronary heart disease.\n\nThe MEDFICTS questionnaire is a brief instrument consisting of 8 food categories: **M**eats, **E**ggs, **D**airy, **F**ried foods, fat **I**n baked goods, **C**onvenience foods, fats added at the **T**able, and **S**nacks. The first column of the questionnaire addresses each of these food categories. Within each category, food items are assigned to either group 1 (desirable) or group 2 (undesirable) based upon total fat content. Numeric values are assigned to each food group, with weightings based upon weekly consumption and serving size. The questionnaire is scored using totaling the quality-adjusted intake quantity yielding a possible range of scores from 0 to 216 points. Lower MF scores indicate diets containing less dietary fat. Prior validation literature indicated that a score of \\<40 points is consistent with a Step 2 diet, a score between 40 to 69 is consistent with a Step 1 diet, and a score of \\>70 is considered as high fat diet. The MF can be self-administered in 3 to 5 minutes, and scored by the healthcare provider in approximately 2 minutes. Thus, the MF is an efficient tool enabling health care providers to quickly assess the adherence of patients to the fat components of a Step 1 or 2 diet, and identify patients consuming a diet higher in total fat, saturated fat, and cholesterol.\n\n## Statistical Analysis\n\nThe two dietary questionnaires were independently scored. The reduced version Block FFQ was coded and analyzed by the same investigator to provide consistency in scoring. The validation of MF scores with Block FFQ dietary variables (percent of fat, percentage of saturated fat, and cholesterol level) was evaluated by Spearman's rho, because both dietary scores were not normally distributed. The level of inter-test agreement between the two dietary instruments was assessed using the kappa statistic. Receiver operating characteristic (ROC) Curve analysis was applied to measure the sensitivity and specificity of the alternative MF cutpoints. All analyses were performed using SPSS for Windows (v 10.05, Chicago, IL). Data are presented as mean \u00b1 SD. A two-tailed P value of \u2264 0.05 was considered to indicate statistical significance.\n\n# Results\n\nMean daily intake values included total fat (% calories) of 35.5 \u00b1 13.0%, saturated fat 13.0 \u00b1 0.4%, and cholesterol 267 \u00b1 283 mg\/dL. These data, obtained with the Block FFQ, indicated that 76.2% of the participants had a high fat (worse than the AHA Step 1) diet. In contrast, the MF questionnaire identified only 17.7% of the group as having a high fat diet (Table <a href=\"#T2\" data-ref-type=\"table\">2<\/a>). The other subjects (82.3%) were indicated as having a low fat diet, and were approximately equally divided between AHA Step 1 and 2 diets.\n\nDietary data for the study group\n\n| **Dietary Data** | **Male (N = 131)** | **Female (N = 33)** | **Total (N = 164)** |\n|----|----|----|----|\n| MEDFICTS Data: |  |  |  |\n| \u2003Diet Group Distribution (%)\\* |  |  |  |\n| \u2003\u2003High fat diet | 19.8 | 9.1 | 17.7 |\n| \u2003\u2003Step 1 diet | 44.3 | 39.4 | 43.3 |\n| \u2003\u2003Step 2 diet | 35.9 | 51.5 | 39.0 |\n| \u2003Total MEDFICTS Score | 49 \u00b1 27\\*\\* | 41 \u00b1 27 | 48 \u00b1 27 |\n| Block Dietary Data: |  |  |  |\n| \u2003Total Calories | 1576 \u00b1 674 | 1321 \u00b1 530 | 1525 \u00b1 654 |\n| \u2003% fat | 35.8 \u00b1 14.0 | 34.4 \u00b1 1.0 | 35.5 \u00b1 13.0 |\n| \u2003% saturated fat | 12.7 \u00b1 0.4 | 12.2 \u00b1 0.4 | 12.6 \u00b1 0.4 |\n| \u2003Cholesterol (mg\/dl) | 283 \u00b1 311 | 201 \u00b1 98 | 267 \u00b1 283 |\n\n\\* MEDFICTS Diet Groups: High fat diet group: MEDFICTS score \\>70 Step 1 diet group: MEDFICTS score: 40\u201370 \\<30% fat, \\<10% saturated fat, \\<300 mg\/dl cholesterol Step 2 diet group: MEDFICTS score \\<40 \\<30% fat, \\<7% saturated fat, \\<200 mg\/dl cholesterol Data shown are means \u00b1 standard deviations.\n\nThere were significant correlations between the MF and Block FFQ for the percentage intake of fat (r = 0.52, P \\< 0.0001), saturated fat (r = 0.52, P \\< 0.0001), and cholesterol (r = 0.55, P \\< 0.0001). Subjects within the different MF diet categories did significantly differ with respect to fat intake (Table <a href=\"#T3\" data-ref-type=\"table\">3<\/a>). Despite these modest correlations, the MF (based on the currently-recommended MF score cutoffs of 0\u201339 for Step 2, 40\u201370 for Step 1, and \\>70 for high fat diets) correctly identified only 29 of 125 (23.3%) high fat (worse than AHA Step 1) diets, and 19.2% of Step 1 diets (Table <a href=\"#T4\" data-ref-type=\"table\">4<\/a>). The overall agreement for the AHA diet steps between the Block FFQ and MF was negligible (kappa statistics = 0.036). The MF was accurate at the extremes of fat intake, but could not reliably separate patient groups into 3 AHA dietary classifications.\n\nComparisons of daily caloric, fat and cholesterol intake within MEDFICTS Diet Groups\n\n| **Block Dietary** | **High Fat** | **Step 1** | **Step 2** | **ANOVA** |          |\n|-------------------|--------------|------------|------------|-----------|----------|\n| **Variable**      | **Diet**     | **Diet**   | **Diet**   | **F**     | **Sig.** |\n| Total calories    | 1980         | 1540       | 1302       | 12.3      | .0001    |\n| % fat             | 40.9         | 38         | 30.3       | 10.1      | .0001    |\n| % saturated fat   | 15.7         | 13.1       | 10.6       | 22.1      | .0001    |\n| Cholesterol       | 351          | 309        | 181        | 5.3       | .006     |\n\nMEDFICTS Diet Groups and AHA Diet Steps Crosstabulation\n\n| **MEDFICTS Diet Groups** | **AHA Diet Steps** |  |  |  |\n|----|----|----|----|----|\n|  | **High Fat** | **Step 1** | **Step 2** | **Total** |\n| High Fat Diet | 29 (100.0%)\\* (23.2%)\\*\\* |  |  | 29 (100.0%) (17.7%) |\n| Step 1 Diet | 62 (87.3%) (49.6%) | 5 (7.0%) (19.2%) | 4 (5.6%) (30.8%) | 71 (100.0%) (43.3%) |\n| Step 2 Diet | 34 (53.1%) (27.2%) | 21 (32.8%) (80.8%) | 9 (14.1%) (69.2%) | 64 (100.0%) (39.0%) |\n| Total | 125 (76.2%) (100.0%) | 26 (15.9%) (100.0%) | 13 (7.9%) (100.0%) | 164 (100.0%) (100.0%) |\n\n\\* % within MEDFICTS diet groups. \\*\\* % within AHA diet steps.\n\nExploratory analysis showed that alternative MF cutpoints of \\<30 (Step 2), 30\u201350 (Step 1), and \\>50 (high fat diet) were highly sensitive (96%), but had low specificity (46%) for a high fat diet. Receiver operating characteristic (ROC) curve analysis showed that a single MF score cutoff of 38 yielded optimal sensitivity of 75% and specificity of 72% (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>), and had modest agreement (kappa statistics = 0.39, P \\< 0.001) with the Block FFQ for the identification of patients with a high fat diet (Figure).\n\n# Discussion\n\nThe effective identification of patients requiring dietary intervention for the reduction of fat intake requires an accurate, efficient, clinically applicable dietary assessment tool. In this study, we compared the accuracy of the MEDFICTS dietary survey to the Block FFQ for the identification of compliance with the AHA dietary guidelines for fat intake. These data indicate that the MF questionnaire has moderate accuracy for the semi-quantitation of dietary fat and saturated fat intake. However, the currently-recommended MF cutpoints resulted in marked over-estimation of dietary quality, including an inability to accurately detect high fat diets. Furthermore, this tool does not appear to have adequate test characteristics to accurately discriminate Step 1 and Step 2 diets. This is likely due to the small differences in fat intake between these 2 diets, and the relatively crude nature of the MF survey. However, our data suggest that a revised cutpoint of 38 for the MF questionnaire resulted in modest accuracy for the detection of a high fat diet.\n\nBeyond ease of administration, an additional advantage of the MF survey may be its utility as a tool for dietary education and lifestyle modification. The content and design of the MF is clear and simple, but comprehensive enough to categorically provide dietary fat intake guidance for a wide variety of food goods. The survey provides rapid depiction of the greatest contributors to dietary fat intake within a given patient's diet, and suggests strategies (alternative food choices, or reduced portion size) for the reduction of dietary fat. However, whether the MF would serve as a useful tool to guide dietary modification will require further study.\n\n# Conclusions\n\nThe MF questionnaire is an efficient tool that can be used by health professionals to quickly assess the dietary fat intake. It provides immediate identification of patients consuming a diet higher in total fat, saturated fat, and cholesterol than currently recommended by the American Heart Association. However, the current recommended cutpoints of the MEDFICTS questionnaire are unable to discriminate the AHA Step 1 and Step 2 diets. Current MEDFICTS cutpoints are too high, leading to overestimation of dietary quality. A cutpoint of 38 appears to be providing optimal identification of patients who do not meet AHA dietary guidelines for fat intake.\n\nAllen J. Taylor- Project design, data analysis, manuscript preparation Henry Wong- Project design, data analysis, manuscript preparation Karen Wish- Primary data analysis, manuscript preparation Jon Carrow- Data collection, data analysis, manuscript preparation Debulon Bell- Data collection, data analysis, manuscript preparation Jody Bindeman- Data collection, data analysis, manuscript preparation Tammy Watkins- Data collection, data analysis, manuscript preparation Trudy Lehmann- Data collection, data analysis, manuscript preparation Saroj Bhattarai- Data management, data analysis, manuscript preparation Patrick G. O'Malley- Project design, data analysis, manuscript preparation","meta":{"dup_signals":{"dup_doc_count":104,"dup_dump_count":50,"dup_details":{"curated_sources":2,"2023-23":1,"2022-49":1,"2022-33":1,"2022-27":1,"2022-05":1,"2021-39":1,"2021-25":1,"2021-21":1,"2021-10":1,"2020-45":1,"2020-34":1,"2020-10":1,"2019-47":1,"2019-39":1,"2019-26":1,"2019-13":1,"2019-04":1,"2018-51":1,"2018-43":1,"2018-34":1,"2018-26":1,"2018-13":1,"2017-47":1,"2017-43":1,"2017-34":1,"2017-26":1,"2017-17":1,"2017-09":15,"2017-04":1,"2016-50":1,"2016-44":1,"2016-40":1,"2016-36":1,"2016-30":1,"2015-48":3,"2015-40":2,"2015-35":3,"2015-32":3,"2015-27":3,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":3,"2014-42":8,"2014-41":4,"2014-35":5,"2014-23":5,"2014-15":4,"2023-50":1,"2015-18":1}},"file":"PMC194867"},"subset":"pubmed_central"}
{"text":"author: JE Pope; A Stevens; K McCrea; J Ouimet\ndate: 2004\ninstitute: 1University of Western Ontario, London, Ontario, Canada\ntitle: Nonsteroidal antiinflammatory drug use may be protective to cartilage in osteoarthritis of the hip and knee\n\n# Purpose\n\nNonsteroidal antiinflammatory drugs (NSAIDs) are used to alleviate osteoarthritis (OA) symptoms and are not considered disease-modifying. We studied NSAID use in subjects with hip or knee OA to investigate the relationship between NSAIDs and OA severity.\n\n# Methods\n\nSubjects (seen over 2 years at teaching hospitals in London, Ontario) were referred to orthopedic surgeons or rheumatologists, had radiographs available, but had no inflammatory arthritis or evidence of secondary OA. Subjects were divided into mild, moderate (mod) and severe OA by X-ray (worst joint selected). A questionnaire was mailed asking about current\/ever use of specific NSAIDs (by generic and trade names) including over the counter and acetylsalicylic acid. Coxibs were not available over the study period. A total of 608 subjects were studied, having mild (71), mod (129) and severe (408) OA, with a disease duration of 10 years in each group (62% were women, who had proportionately more mild disease than men).\n\n# Results\n\nThose with severe versus mild OA were older (70 versus 61 years), so age was adjusted for. There was an inverse dose\u2013response with use of \\> 3 NSAIDs being used by mild versus severe OA (odds ratio 3.7), mild versus mod OA (odds ratio 2.5) and mod versus severe OA (odds ratio 1.5) (*P* \\< 0.0001), and the same was found with \\> 2 NSAIDs (*P* \\< 0.0002); and 7% of mild, 15% of mod and 12% of severe OA had received no NSAIDs ever. Only one NSAID (ever used) was increased in severe OA (sulindac, *P* \\< 0.008) compared with naproxen, tiaprofenic acid and diclofenac, more frequently used in mild than mod or severe OA (*P* \\< 0.01). Other NSAID use ever was not different between the groups (including indomethacin, thought to be chondrodestructive), but numbers using indomethacin were small. Limitations may include inability to study accurately the duration of NSAID use, older age in those with severe OA (which could affect NSAID treatment choice), recall bias and unknown confounders. We did not illicit body mass index or pain level in each group.\n\nThere may be biases for referring to different specialists as the rheumatologists prescribed NSAIDs more often and surgeons treated more subjects with severe OA (*P* \\< 0.0001). When analyses were stratified by specialist type, there were no differences between mild, mod and severe OA for NSAID use in subjects treated by rheumatologists; however, the trend for increased NSAID use with milder OA was evident in those seen by orthopedic surgeons. When stratified by knee and hip OA, the dose\u2013response remained (*P* \\< 0.003 and *P* \\< 0.03, respectively). Subjects were followed by general practitioners, so we assumed past NSAIDs should have been prescribed equally in all groups, but this was not the case.\n\n# Conclusions\n\nThose with more severe OA are more rapidly progressive (having similar disease duration to the other groups) and could be innate NSAID nonresponders. However, one would assume that they should be exposed to more NSAIDs (looking for one to be helpful). We conclude that NSAID use and the number of NSAIDs used was greater in mild radiographic OA and postulate that lowering prostaglandins in the joint with NSAIDs may be protective for cartilage. Prospective studies are needed to confirm observations. Some *in vitro* models of NSAIDs in OA support our observations.","meta":{"dup_signals":{"dup_doc_count":111,"dup_dump_count":45,"dup_details":{"curated_sources":2,"2022-49":1,"2022-05":1,"2021-39":1,"2021-17":1,"2021-04":1,"2020-34":1,"2020-10":1,"2019-39":1,"2019-26":1,"2018-43":1,"2018-30":1,"2018-13":1,"2017-47":1,"2017-34":1,"2017-22":1,"2017-09":7,"2016-44":1,"2016-40":1,"2016-36":6,"2016-30":5,"2016-22":1,"2016-18":1,"2016-07":7,"2015-48":3,"2015-40":2,"2015-35":3,"2015-32":3,"2015-27":3,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":4,"2014-42":7,"2014-41":5,"2014-35":3,"2014-23":3,"2014-15":2,"2023-23":1,"2015-18":3,"2015-11":2,"2015-06":3,"2014-10":3,"2013-48":2,"2013-20":3,"2024-18":1}},"file":"PMC2833518"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  The relapsing nature and varying geographical prevalence of ulcerative colitis (UC) implicates environmental factors such as diet in its aetiology.\n  .\n  # Methods\n  .\n  In order to determine which foods might be related to disease activity in UC a new method of dietary analysis was developed and applied. Eighty-one UC patients were recruited at all stages of the disease process. Following completion of a 7 d diet diary, clinical assessment including a sigmoidoscopic examination (scale 0 (normal mucosa) to 6 (very active disease)) was conducted. Food weights for each person were adjusted (divided) by the person's calorific intake for the week. Each food consumed was given a food sigmoidoscopy score (FSS) calculated by summing the products of the (adjusted) weight of food consumed and sigmoidoscopy score for each patient and occurrence of food and dividing by the total (adjusted) weight of the food consumed by all 81 patients. Thus, foods eaten in large quantities by patients with very active disease have high FSSs and vice versa. Foods consumed by \\<10 people or weighing \\<1 kg for the whole group were excluded, leaving 75 foods.\n  .\n  # Results\n  .\n  High FSS foods were characterized by high levels of the anti-thiamin additive sulfite (Mann-Whitney, p \\< 0.001), i.e. bitter, white wine, burgers, soft drinks from concentrates, sausages, lager and red wine. Caffeine also has anti-thiamin properties and decaffeinated coffee was associated with a better clinical state than the caffeine containing version. Beneficial foods (average intake per week) included pork (210 g), breakfast cereals (200 g), lettuce (110 g), apples and pears (390 g), milk (1250 ml), melon (350 g), bananas (350 g), bacon (120 g), beef and beef products (500 g), tomatoes (240 g), soup (700 g), citrus fruits (300 g), fish (290 g), yogurt (410 g), cheese (110 g), potatoes (710 g) and legumes (120 g).\n  .\n  # Conclusions\n  .\n  The dietary analysis method described provides a new tool for establishing relationships between diet and disease and indicates a potentially therapeutic diet for UC.\nauthor: Elizabeth A Magee; Laurie M Edmond; Shiona M Tasker; San Choon Kong; Richard Curno; John H Cummings\ndate: 2005\ninstitute: 1Division of Pathology and Neuroscience, Ninewells Hospital and Medical School, Dundee, DD1 9SY, Scotland, UK\nreferences:\ntitle: Associations between diet and disease activity in ulcerative colitis patients using a novel method of data analysis\n\n# Background\n\nUlcerative colitis (UC) is a chronic, relapsing mucosal disorder that extends in continuous fashion proximally from the rectum and is limited to the colon. The aetiology of UC includes a genetic component possibly involving an abnormal cell-mediated immune response to commensal enteric bacteria in the large intestine. The relapse\/remission pattern of the disorder and substrate driven nature of microbial metabolism in the large bowel implicate environmental factors such as diet.\n\nApart from nutritional repletion, dietary measures do not play a role in the management of UC. Nonetheless, attempts to link the cause of UC with specific foods date back at least 50 years\\[1\\]. Many foods or food groups have been related to UC (table 1 \u2013 see <a href=\"#S1\" data-ref-type=\"supplementary-material\">additional file 1<\/a>) \\[2-13\\] including sugar, eggs, soft drinks, fruit and vegetables, protein, carbohydrate and fat. However none have been proven to be of significant benefit or to contribute to the cause of UC. This may partly be because both the assessment of disease activity in UC and dietary intake are difficult to measure, or because the actual dietary component that is key to this relationship has not been measured.\n\nIt has been proposed that sulfide, produced in the large bowel from either amino acid fermentation or sulfate reduction, may be a triggering factor in the inflammatory process of UC \\[14-16\\]. Recently, in a prospective dietary study where foods rich in sulfur compounds were quantitated, evidence that sulfur compounds may increase the likelihood of subsequent relapse in UC was found\\[17\\].\n\nThe main source of inorganic sulfur, predominantly sulfate, in the diet are the S (IV) family of additives; the sulfiting agents. Sulfites have been used as food preservatives since the 17th century and are amongst the most widely accepted and versatile of additives. Sulfiting agents, denoted by E220\u2013228 in Europe and generally recognized as safe (GRAS) substances in the USA, include sulfur dioxide, hydrogen sulfites, sulfites and metabisulfites. Sulfiting agents are cheap, easy to use and extremely effective at preventing microbial growth and reducing spoilage\\[18\\]. They serve as antioxidants, inhibit enzymatic and non-enzymatic browning reactions and act as a texture modifier in biscuit dough. Sulfites improve color extraction from, and stabilization of grape must in winemaking and preserve lobsters and shrimps from discoloration during iced storage.\n\nHowever, there are some problems with sulfite use\\[19,20\\]. In the early 1980s ingestion or inhalation of sulfites was shown to cause bronchospasm in about 5 % of asthmatics. Sulfite sensitivity can pose a particular threat in the workplace where sulfiting agents are used, but may also occur with ingestion of sulfited foods such as potato products and wine. In addition, skin sensitivity has been reported and there are anti-nutritional effects particularly to thiamin which is readily cleaved by the sulfite ion\\[21\\]. The mechanism involves an initial nucleophilic attack to the methylene carbon activated by the positive charge on nitrogen, the reaction rate peaking between pH 5 and 6\\[18\\]. As a result of this anti-nutritional effect the GRAS status for sulfites was reviewed in the USA and in 1986 the use of sulfites in fresh and frozen fruit and vegetables revoked and a declaration on the label required\\[22,23\\]. Earlier (in the USA) their use in meat had been prohibited, because these foods are an important source of thiamin.\n\nA study of diet and disease activity in UC using a 7 d dietary diary, a full assessment of disease activity and a method of dietary data analysis that allows trends in food consumption not apparent using customary dietary software was therefore undertaken.\n\n# Methods\n\n## Subjects\n\nEighty-one UC patients were recruited and informed consent obtained. Ethical permission was granted by Tayside Committee on Medical Ethics, Dundee, UK (ref 007\/00). As it was important to have a range of disease activities present, recruitment included patients at all stages of the disease. Patients were excluded if clinical examination or histology indicated Crohn's disease or indeterminate colitis, if there was a positive stool culture for pathogens or if the patient had antibiotic treatment within 3 months preceding the start of the study.\n\n## Dietary Assessment\n\nAll the UC patients were asked to complete a 7 d diet diary\\[24\\]. The diet diary used has been validated for use in the European Prospective Investigation into Cancer study (EPIC). Following completion of the diet diary, subjects attended the research clinic and a full clinical assessment (see below) was carried out. The time interval between the first day of the diary and the clinical visit was on average 28 d. Thus the dietary data is prospective.\n\n7d diet diaries were coded and analyzed using Tinuviel, WISP v3.0 nutritional analysis software (Warrington, UK). Due to the variation in the sulfiting protocols and widespread use of sulfiting agents, current tables of food composition do not contain inorganic sulfur values and cannot be used to quantify intake. Instead of quantitating the intake of particular dietary components, foods and food groups were assessed in their entirety using the method described in the dietary data analysis section (below).\n\n## Clinical Assessment\n\nClinical assessment included history, physical examination and global clinical grading, plus full blood count, liver function tests and inflammatory markers. Patients were examined by rigid sigmoidoscopy or flexisigmoidoscopy and graded on a scale 0\u20136 (integers and half integers used) according to the macroscopic appearances of the rectal mucosa at a distance 5\u201310 cm from the anal verge\\[25\\]. The clinical assessment of disease activity was confirmed in each case by histological examination, by a single histopathologist blinded to the clinical details, of a rectal biopsy taken from the posterior rectal wall 5\u201310 cm from the anal verge\\[26\\]. A simple clinical colitis score was assigned to patients on each visit following Walmsley's scoring system\\[27\\], together with blood parameters of disease severity (Hb, plasma viscosity, CRP, serum albumin).\n\n## Dietary Data Analysis\n\nPatterns of dietary intake associated with disease activity became apparent through the study of the dietary diaries, e.g. high intakes of sulfite containing foods coupled with a modern processed, convenience diet was associated with a high sigmoidoscopy score. Traditional dietary coding (WISP) did not show any such clear associations between micro or macro nutrient intake and sigmoidoscopy score. Traditional dietary analysis was therefore thought to be missing important patterns in dietary data and a new method of dietary assessment was subsequently developed.\n\nThis new method used the following procedure. To calculate the association of a particular food with clinical score, each food or food group consumed was given a food sigmoidoscopy score (FSS) calculated by summing the products of food weight and sigmoidoscopy score for each occurrence of the food or food group and dividing by the total weight of the food or food group contained in all diaries. In order for each diary to make equal contributions to the FSSs, the weight of each food was adjusted using the calorific intake for each person. This procedure was carried out separately for every food item recorded in the 7 d diet diaries but is explained below using the example of red wine.\n\nRed wine score = (\u03a3v(i)s(i))\/\u03a3v(i) for i = 1 to 81 \u00a0\u00a0\u00a0 equation 1.\n\nWhere: -\n\ni is the 7 d dietary diary number (n = 81).\n\nv(i) is the volume (divided by calorific intake for patient (i) of red wine recorded in 7 d dietary diary i.\n\ns(i) is the sigmoidoscopy score associated with 7 d dietary diary (i).\n\nThus foods eaten in large quantities by patients with high levels of disease activity will have high scores and vice versa. The denominator in the above equation is the total volume of the food in question from all diaries (corrected for calorific intakes) so the food scores can be equated with the effect of a typical portion of the food in question on the sigmoidoscopy scores of the patients. This procedure is repeated for every food item. Foods or food groups were excluded from the analysis if 10 or fewer people consumed them or if they made up less than 1 kg of the total intake of the entire population. The decision as to where food group boundaries lay was made depending on the size of the group and whether the differences between the foods were considered important for this study.\n\n## Statistics and Data Handling\n\nDietary data was exported from WISP to Microsoft EXCEL 98 (Macintosh version, 1998). A worksheet containing the core headings; Patient ID, food description, weight and patient sigmoidoscopy score was completed. The data was then sorted by food description and each food copied to a separate EXCEL file. Equation 1 was then used to calculate food sigmoidoscopy scores for each food in a manner similar to the example in table 2 (see <a href=\"#S2\" data-ref-type=\"supplementary-material\">additional file 2<\/a>).\n\nCorrelation values for scatter plots were obtained using the linear regression function in EXCEL. The equation t = r \u221a((n-2)\/(1-r^2^)) combined with t tables provided corresponding significance levels.\n\n# Results\n\nOf the 81 patients recruited 43 were male and 38 female. The average age (range) of the males and females were respectively 53 (26\u201378) y and 47 (19\u201374). The distribution of sigmoidoscopy scores is shown in fig <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>. One third of the patients had sigmoidoscopy scores of 0, 0.5 or 1. The mean sigmoidoscopy score for all 81 patients was 2.09. The correlation between the clinical activity indexes and sigmoidoscopy scores was r^2^ = 0.25 (n = 81).\n\nTable 3 (see <a href=\"#S3\" data-ref-type=\"supplementary-material\">Additional file 3<\/a>) shows the foods and food groups with associated sigmoidoscopy scores and average portion sizes. In total 75 foods (or food groups) were given FSS scores. The higher the FSS value the greater the association with disease activity and vice versa. The total weight of foods in all diaries was 1,681 kg. The average food sigmoidoscopy score (i.e. a food sigmoidoscopy score calculated for the entire dietary intake data set was 2.127). Foods excluded from the FSS table (Table 3), by virtue of contributing \\<1 kg or being consumed by \\<10 people, made up 8 % of the total weight of all foods and had a score slightly lower (2.001) than that of an average food (2.127). Standard errors are not quoted for the food scores as the data used to generate them (weight \\* sigmoidoscopy score) was not normally distributed due to the number of sigmoidoscopy scores of 0.\n\nThe dietary diaries were assessed for completeness by comparing calorific intakes with expected values for the sexes. Expected (calculated from dietary reference tables using age and sex)\\[28\\] versus actual values for men and women were respectively 2481 kcal\/d versus 2326 kcal\/d and 1925 kcal\/d versus 1887 kcal\/d.\n\nFoods for which regulations exist in the EU permitting sulfite addition are shown in table 4 (see <a href=\"#S4\" data-ref-type=\"supplementary-material\">Additional file 4<\/a>) \\[29\\]. Typically a manufacturer will add sulfite up to the maximum permitted level in order to achieve the longest shelf life for the product. A report on sulfite usage in the UK was produced in 2001\\[30\\]. Sweet wines, langoustines (prawns), dehydrated potatoes and dried fruit were not given FSS scores because their data quantity fell below the \\<10 people or \\<1 kg rule. Soft drinks were split into those known to contain sulfite (drinks made from fruit squash concentrates and lucozade) and the rest. In terms of intake (portion size\\*sulfite concentration), for this population, the major sources of sulfite (FSS, FSS table position) were bitter beer (3.91, 75), white wine (2.87, 73), burgers (2.84, 72), soft drink concentrates (2.79, 70), sausages (2.68, 68), lager (2.47, 64) and red wine (2.00, 29). A Mann-Whitney test on the FSS positions of these foods gave a significance of p \\< 0.001. The sulfite-containing, alcoholic beverages; wines and beers, were associated with increased UC disease activity, but spirits were not, which suggests a role for sulfite rather than alcohol in the disease process. A plot of alcohol consumption from wine and beer against sigmoidoscopy score revealed a significant positive correlation (n = 81, r^2^ = 0.07, p \\< 0.02).\n\nDecaffeinated coffee appeared better for the UC patient than the caffeine-containing counterpart. Decaffeinated tea is not shown on table 3 because it was only drunk by 9 people but had a FSS of 1.71 versus 2.01 for the caffeine-containing product. Whole fruit consumption appeared better than the corresponding juice (e.g. fruit juice scored 2.43 compared to citrus fruits at 1.96 and apples at 1.67).\n\nAn average thiamin concentration (mg \/ 100 g) for each food or food group is also shown in table 3. There is a significant correlation (p \\< 0.005) between this thiamin value and the food's sigmoidoscopy score.\n\n# Discussion\n\nUlcerative colitis is considered to have a genetic component. Twin studies\\[31\\] have shown a 10% concordance of UC in monozygotic and 3% in dizygotic twins suggesting about 90% environmental and 10% genetic contributions. The pool of genetically susceptible individuals is therefore at least 10 times greater than those diagnosed with the condition. A failure to date in identifying the gene(s) responsible points to a complicated genetic component featuring multiple polymorphisms. The first acute episode of UC must disrupt either, the ecology of, or the sensitivity and selectivity of the immune system to, the commensal enteric microflora sufficiently to cause the chronic condition. More extreme versions of the environmental conditions that lead to subsequent relapses could conceivably lead to the first acute episode.\n\nOf all the dietary components studied in relation to UC risk and disease severity, milk has probably received the most attention. Andreson\\[1\\] was the first to postulate that food allergy was the cause of UC in two-thirds of his patients, and by the use of elimination diets claimed to identify the offending food and remove it. In Andreson's experience, the most common provoking antigen was cow's milk. His views were confirmed by Rowe\\[32\\] and later by Truelove\\[33\\]. They all postulated that milk protein sensitivity was an aggravating cause of disease in up to 5% of colitic patients, who benefited from a milk-free diet. While able to demonstrate circulating antibodies to milk proteins more frequently and in higher titer than in matched controls, they were unable to correlate the occurrence and titer of these antibodies with the extent, severity, or duration of colitis, or with the response to a milk-free diet. Mishkin\\[34\\] concluded, in a review of the subject, that IBD patients avoid dairy products to a much greater extent than the prevalence of lactose malabsorption and\/or milk intolerance in this population group would justify. This observation was probably due to the incorrect perceptions of patients and arbitrary advice of physicians and authors of popular diet books.\n\nIn order to ascertain whether dietary antigens may sustain the mucosal inflammatory response, two prospective controlled trials have investigated the effectiveness of bowel rest and total parenteral nutrition as primary therapy in the management of acute UC\\[35,36\\]. Neither study found any benefit over conventional corticosteroid treatment alone and so the possibility of a dietary antigen driving the chronicity of the disease seems unlikely. These results are in agreement with work demonstrating\\[37\\] that a split ileostomy is of little benefit in the management of UC, but the latter observations may have been confounded by the development of diversion colitis\\[38\\].\n\nThe dietary analysis procedure proposed here has the potential to highlight trends in dietary data that would not be apparent using traditional dietary analysis software and could be useful in the study of other diseases with dietary associations. This system would highlight any possible dietary factors both positive and negative, not just sulfite. The proposed method is less reductionist than traditional coding as it assesses the risk of each food item or group rather than the risk from the foods' (quantitated) constituents. Part of the power of this study derives from the availability of a sigmoidoscopic grading (0\u20136) of the severity and extent of the disease. This grading provides the statistical variable that is normally obtained from a non-UC control group. Other alternative systems for analysis of disease risk for dietary components are; the use of disease occurrence odds ratios between the top and bottom quartiles of intakes, and assessing the correlation coefficients between disease activity and intakes. The odds ratio method loses data and data accuracy by characterizing intakes as high, high middle, low middle and low and then discarding the middle two quartiles. The correlation method is dependent on spread. The proposed system has neither of these disadvantages. The food sigmoidoscopy score calculation does rely on the assumption that the sigmoidoscopy score is an approximately linear scale, i.e. a sigmoidoscopy score of 6 is caused by the consumption of a double portion of a harmful food item of sigmoidoscopy score 3. This could be argued to be reasonable. Both the sigmoidoscopy grading and dietary analysis method are validated methodologies. The food sigmoidoscopy score is simply a mathematical function of these two variables. As all data is transformed according to the same simple rules any statistical treatment of the results is as valid as statistical treatment of the raw data.\n\nWhilst clinical activity indices were used to generate analogous scores to the food sigmoidoscopy scores, the results from these measurements are not included in this paper. Clinical activity index involves subjective measurements such as a feeling of well being. Thus, the food orders generated by these measurements were not thought to be as accurate as those generated by the sigmoidoscopy scores.\n\nThe consensus of previous studies on diet and UC pointed to the modern, processed, highly refined, Western diets as being damaging. The results presented here linking diet with disease activity are broadly in agreement with this. Additionally they propose a new risk factor for UC, namely intake of sulfited foods.\n\nThe involvement of diet in UC is controversial. Differences in dietary intake between patients and controls could be a result of changes in diet brought on by the symptoms of the disease process\\[4\\]. While this explanation is possible it does not seem likely that patients would increase their beer and wine intake as a consequence of feeling unwell. The relationship between sulfite intake and sigmoidoscopy score in this study was extremely strong and therefore an explanation for why sulfite should be a risk factor for UC is required. Sulfite has a number of effects that may be relevant to this discussion. Sulfite may be important because it is a precursor of sulfate. Sulfate can potentially be reduced to sulfide by sulfate reducing bacteria in the colon. Sulfide is a plausible metabolic toxin in UC. Supplementing patients with sulfate decreases the microbial incorporation of hydrogen into methane (as measured by breath methane) and increases the in vitro sulfide production rate of feces\\[39\\]. The end metabolic product of both sulfite and protein is sulfate. Sulfate from both sources can be reduced to sulfide in the gut. The absence of a significant relationship between protein intake and disease activity in this study does not support a mechanism for UC that involves a common pathway for sulfite and protein.\n\nAlternatively, the relevance of sulfite to UC may be because of its ability to degrade thiamin (particularly at colonic pH). Thiamin deficiency manifests itself in the nervous and cardiovascular systems. It is unlikely that it is the status of the patient that is important, but rather the amount of thiamin available to the gut microflora. An example of the importance of thiamin to the gut microflora is the requirement of the probiotic bacteria, lactobacilli, for thiamin. Thiamin status is influenced by a number of factors. Firstly, thiamin intake; in foods such as pork, fortified cereals and legumes which are good sources of thiamin, intakes were associated with improved clinical state. Traditional dietary analysis did not reveal a significant correlation between thiamin intake and sigmoidoscopy scores though no allowance is made in dietary coding software for the reduction in thiamin content caused by sulfite usage. Secondly, carbohydrate intake; Elmadfa *et al*. demonstrated that the thiamin status of adult humans depends on carbohydrate intake\\[40\\]. Carbohydrate (and sugar) intakes have previously been associated with UC relapse (table 1). Finally, thiamin status can be affected by caffeine's anti-thiaminergic properties. For both coffee and tea intake, the decaffeinated version was associated with better clinical state.\n\nHowever, there was a sub group (n = 8) of this population who recorded an intake of either vitamin B complex or multivitamins. This sub group did not have a mean sigmoidoscopy score significantly lower that the general UC population. It is likely that vitamin B1 is a factor in the disease process but not the only nutritional one.\n\nAn additional possible interpretation for the experimentally determined food order is the carbohydrate nature and content of the foods. Carbohydrates, such as the \u03b1-amylase resistant starch (RS) and prebiotics, escape digestion in the small intestine and provide an energy substrate for the colonic microflora. Both prebiotics (found in chicory, legumes, artichokes alliums, and in small amounts in cereals) and resistant starch (potatoes, bananas, lentils and legumes) have been hypothesised to improve the colonic health of the host. For RS, resistance to digestion is a function of the morphology of the starch granules and their crystalline organisation, which is determined by the botanical source of the starch and the processing it has undergone before being eaten\\[41\\]. Prebiotics are non-digestible carbohydrates that selectively stimulate the growth of lactobacilli and bifidobacteria with benefit to health. Prebiotics are mainly fructose and galactose polymers with a degree polymerisation of between 2 and 60. Of the prebiotic sources; chicory and artichokes were not found in typical diets, legumes and cereals were seen to have probable benefits in this study and alliums were not. This study therefore provides only limited support for the use of prebiotics in UC. The foods containing RS were all found to be of benefit in this study and therefore the role of RS in UC is strongly supported.\n\nAny dietary advice provided to ulcerative colitis patients should be based on the FSS table. The table is of course imperfect because of experimental error, natural variation and the associations between foods. For example, milk and cereal are coded separately but are often consumed together. Thus the magnitude of the difference in the FSSs for these two foods is less than if they'd been independent variables. Suggestions have been made in this discussion as to the factors responsible for the FSS order and to distill these factors into the advice given in Table 5 (see <a href=\"#S5\" data-ref-type=\"supplementary-material\">Additional file 5<\/a>). This table is speculation, as this diet has not been formally tested in the UC population. It does however represent the only comprehensive dietary advice available to ulcerative colitis patients at this time.\n\nThe list of dietary risk factors for colon cancer\\[42\\] bears a similarity to the dietary risk factors presented here for UC. UC patients have an increased risk of colorectal cancer and it is probable that factors responsible for inflammation in UC patients are also responsible for neoplasia in the colon cancer population.\n\n# Conclusion\n\nA dietary analysis method is described that provides a new tool for establishing relationships between diet and disease. This method has been applied to the study of ulcerative colitis and points to sulfite and caffeine as being harmful, with thiamin and resistant starch being potentially therapeutic. For the first time, dietary guidelines for ulcerative colitis patients, including food portion sizes have been developed.\n\n# Abbreviations\n\nUlcerative colitis (UC); Food sigmoidoscopy score (FSS); European Union (EU); Odds ratio (OD) and Confidence interval (CI).\n\n# Competing interests\n\nThe author(s) declare that they have no competing interests.\n\n# Authors' contributions\n\nJHC, EAM and LME contributed to the study design and the writing of the manuscript. EAM was the co-ordinator of the study and along with ST had responsibility for managing the patients and coding the diaries. LME developed the dietary data analysis protocol assisted by RC. CK and JHC performed the sigmoidoscopic examinations of the patients. All authors read and approved the final manuscript.\n\n# Supplementary Material\n\n###### Additional File 1\n\nReview of studies of diet and ulcerative colitis (UC).\n\nClick here for file\n\n###### Additional File 2\n\nFood sigmoidoscopy score (FSS) calculation example for red wine (NB incomplete data set used).\n\nClick here for file\n\n###### Additional File 3\n\nFoods consumed in order of food sigmoidoscopy scores (FSS)\\[43\\].\n\nClick here for file\n\n###### Additional File 4\n\nPermitted levels of sulfite in the UK.\n\nClick here for file\n\n###### Additional File 5\n\nProposed dietary advice for ulcerative colitis patients\\[44\\].\n\nClick here for file\n\n### Acknowledgements\n\nCore funding for this study was provided by the National Association of Colitis and Crohn's Disease. Additional funding was provided by the Sir Halley Stewart Trust, Chief Scientist Office, Scotland and the Anonymous Trust, Dundee. We are very grateful to all these organisations.\n\nThanks go to all those who volunteered for this study. Their enthusiasm and humour throughout the study was greatly appreciated.\n\nThanks go to the endoscopy staff; Shirley McLeod, Marion Winters, Shona Pryde and Sheena McWha and to our secretary Helen Cowper without whom the study would not have run so smoothly.\n\n## Figures and Tables","meta":{"dup_signals":{"dup_doc_count":105,"dup_dump_count":42,"dup_details":{"curated_sources":4,"2022-27":1,"2021-49":1,"2021-31":1,"2020-45":1,"2020-34":1,"2020-29":1,"2020-24":1,"2020-10":1,"2019-43":1,"2019-35":1,"2018-09":1,"2017-39":1,"2017-09":14,"2017-04":1,"2016-50":1,"2016-44":1,"2016-40":1,"2016-36":1,"2016-30":1,"2016-22":1,"2016-07":17,"2015-48":2,"2015-35":2,"2015-32":3,"2015-27":2,"2015-22":3,"2015-14":2,"2014-52":2,"2014-49":3,"2014-42":5,"2014-41":3,"2014-35":3,"2014-23":4,"2014-15":3,"2023-50":1,"2017-13":1,"2015-18":3,"2015-11":2,"2015-06":2,"2014-10":2,"2013-48":2,"2024-22":1}},"file":"PMC549081"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  Recent developments in high-throughput sequencing (HTS) technologies have made it feasible to sequence the complete transcriptomes of non-model organisms or metatranscriptomes from environmental samples. The challenge after generating hundreds of millions of sequences is to annotate these transcripts and classify the transcripts based on their putative functions. Because many biological scientists lack the knowledge to install Linux-based software packages or maintain databases used for transcript annotation, we developed an automatic annotation tool with an easy-to-use interface.\n  .\n  # Methods\n  .\n  To elucidate the potential functions of gene transcripts, we integrated well-established annotation tools: Blast2GO, PRIAM and RPS BLAST in a web-based service, FastAnnotator, which can assign Gene Ontology (GO) terms, Enzyme Commission numbers (EC numbers) and functional domains to query sequences.\n  .\n  # Results\n  .\n  Using six transcriptome sequence datasets as examples, we demonstrated the ability of FastAnnotator to assign functional annotations. FastAnnotator annotated 88.1% and 81.3% of the transcripts from the well-studied organisms *Caenorhabditis elegans* and *Streptococcus parasanguinis*, respectively. Furthermore, FastAnnotator annotated 62.9%, 20.4%, 53.1% and 42.0% of the sequences from the transcriptomes of sweet potato, clam, amoeba, and *Trichomonas vaginalis*, respectively, which lack reference genomes. We demonstrated that FastAnnotator can complete the annotation process in a reasonable amount of time and is suitable for the annotation of transcriptomes from model organisms or organisms for which annotated reference genomes are not avaiable.\n  .\n  # Conclusions\n  .\n  The sequencing process no longer represents the bottleneck in the study of genomics, and automatic annotation tools have become invaluable as the annotation procedure has become the limiting step. We present FastAnnotator, which was an automated annotation web tool designed to efficiently annotate sequences with their gene functions, enzyme functions or domains. FastAnnotator is useful in transcriptome studies and especially for those focusing on non-model organisms or metatranscriptomes. FastAnnotator does not require local installation and is freely available at <http:\/\/fastannotator.cgu.edu.tw>.\nauthor: Ting-Wen Chen; Ruei-Chi Richie Gan; Timothy H Wu; Po-Jung Huang; Cheng-Yang Lee; Yi-Ywan M Chen; Che-Chun Chen; Petrus Tang\ndate: 2012\ninstitute: 1Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan; 2Bioinformatics Center, Chang Gung University, Taoyuan, Taiwan; 3Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan; 4Institute of Biomedical Informatics, National Yang-Ming University, Taipei, Taiwan; 5Graduate Institute of Biomedical Sciences, Chang Gung University, Taoyuan, Taiwan; 6Department of Microbiology and Immunology, Chang Gung University, Taoyuan, Taiwan; 7Department of Aquatic Biosciences, National Chiayi University Chiayi, Taiwan\nreferences:\ntitle: FastAnnotator- an efficient transcript annotation web tool\n\n# Background\n\nAs sequencing technologies have improved, transcriptome sequencing (RNA-Seq) or whole-genome sequencing have become faster and cheaper than ever before. In addition, sequencing projects addressing non-model organisms or environmental samples (metagenomics) have now become feasible and affordable. For example, the sequencing of relatively unexplored organisms, such as the human gut microbiome or the transcriptome of clams or butterflies, has been accomplished \\[1-3\\]. However, sequencing is only the first step in the process of understanding the biology underlying the obtained sequences. Bioinformatics analyses performed after sequencing, including assembly, functional annotation and classification, are becoming increasingly important. Several annotation pipelines have been developed, such as CycADS, PRIAM and Blast2GO \\[4-6\\], which can provide putative functions for a transcript based on sequence similarity to known genes. Although these tools are useful for understanding the newly explored sequences, they usually contain many manual steps and often are not easy to implement for biologists who are unfamiliar with the command line inputs. Web tools have been developed for the annotation of expressed sequence tags (EST), such as ESTAnnotator, ESTpass, and ESTExplorer \\[7-10\\]. These pipelines are specifically designed for EST analyses that include the cleaning, assembly and clustering and functional annotation of ESTs. However, we found that the online Uniform Resource Locators (URLs) for ESTpass and ESTAnnotator are no longer functional for ESTpass and ESTAnnotator, and ESTExplorer cannot simultaneously process more than ten thousand contigs. Thus the development of a web server that can provide an automatic annotation pipeline for contigs derived from RNA-Seq data with a user-friendly interface would be beneficial to the research community.\n\nFunctional annotation strategies are typically based on previously identified protein functions. Many protein function databases, such as BRENDA, Enzyme and Amigo, have been established as collections based on the functions of enzymes or genes \\[11-13\\]. These databases categorize protein functions into structured groups as either Enzyme Commission numbers (EC numbers) or Gene Ontology (GO) terms. EC numbers are assigned to enzymes according to the chemical reactions they catalyze \\[14\\], whereas GO terms include information on the molecular function, cellular component and biological processes of genes and can be used to describe the biological function of proteins \\[15\\]. These protein function or enzyme function databases document well-studied and well-annotated biological functions and provide resources for the annotation of newly sequenced genes. The majority of established annotation methods utilize these functional annotation systems and transfer functional annotations among sequences based on sequence similarity or pattern searches. For example, the CycADS pipeline integrates multiple annotation tools and databases \\[4\\]; PRIAM identifies enzyme functions based on profiles constructed from known enzymes \\[6\\]; and Blast2GO annotates gene functions based on a combinations of various annotation methods \\[5\\]. The strategy of annotation transfer has been shown to work well and is frequently employed.\n\nHerein, we present an automatic, efficient and easy-to-use web-based annotation tool named FastAnnotator. Given that many tools have been proposed to annotate sequences based on previously established knowledge, we chose to integrate several well-established and popular tools to construct this service. FastAnnotator utilizes Blast2GO and PRIAM to identify GO terms and EC numbers for transcript sequences. Blast2GO has a high annotation accuracy (65-70%) \\[16\\], and PRIAM provides an enzyme profile database that was updated in October 2011 \\[6\\]. As certain assembled sequences identified using RNA-Seq may not cover full-length coding regions, we also included a domain search in FastAnnotator. In addition to the possibility of identifying domains based on partial transcript sequences, domain searching also enables FastAnnotator to identify possible functions for sequences that show a significant level of divergence. This feature was included because sequences may evolve to become dissimilar, although the functional domain regions are likely to be conserved through evolution under functional constraints. It has also been shown that proteins having the same domain composition are homologs and likely have the same function \\[17-19\\]. Therefore, domain annotation in FastAnnotator can not only provide an annotation for a partially sequenced transcript but also improve the annotation performance for sequences that are highly divergent from existing database sequences. This property is particularly useful for metatranscriptomic analyses or samples from organisms that are distantly related to model organisms.\n\nIn summary, we have developed the FastAnnotator pipeline to provide automatic annotation of nucleotide sequences via a web interface. Users can begin their annotation by uploading their sequences to the FastAnnotator website. FastAnnotator then assigns possible functions and identified domains in those input query sequences based on sequence similarity and pattern searches. The output of FastAnnotator includes the best hits in the NCBI non-redundant database, GO terms, EC numbers, and domain identities \\[20,21\\]. Users can explore the output on the website, access other external functional databases (BRENDA, Amigo or Pfam) for detailed functional descriptions and simply download the output for further analysis. As a web service specifically developed for ease-of-use annotations, the use of FastAnnotator does not demand any knowledge of the command line. Moreover, FastAnnotator is free, efficient and user-friendly. Relative to existing annotation pipelines, FastAnnotator is much more convenient because it does not require installation or tedious manual steps and allows the user to analyze a large number of sequences with a single click. FastAnnotator is now available at <http:\/\/fastannotator.cgu.edu.tw\/>.\n\n# Implementation\n\n## Flowchart of the FastAnnotator pipeline\n\nThe annotation process in FastAnnotator consists of four main parts: finding the best hit in the NCBI non-redundant database, assignment of GO terms, identification of enzymes, and identification of domains. The assignment of the GO terms requires the result from LAST searching against the NCBI non-redundant database, and these four main steps can therefore be divided into three independent modules: GO term assignment, enzyme identification, and domain identification (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>). FastAnnotator runs these three steps in parallel to accelerate the annotation procedure and also calculates the basic statistics of the input sequences and provides a statistical report in the results.\n\n## The identification of GO terms\n\nLAST \\[22-24\\] and Blast2GO (B2G4Pipe) \\[5\\] were used together to identify GO annotations for the query sequences. We downloaded the non-redundant protein sequences from the NCBI database \\[21\\] and constructed a local database for Blast2GO. In FastAnnotator, LAST is used to find the similar sequences within the non-redundant protein sequence database. The output of LAST is then transformed into the equivalent BLASTX XML output format, which is the required input file format for Blast2GO. We used the statistical parameters \u03bb and \u03ba provided by the LAST output result to derive the bit score based on the following definition \\[25\\]:\n\n$$\\textsf{S'~=~}\\frac{\\lambda\\;\\textsf{S}\\;\\textsf{~-~}\\;\\textsf{In}\\;\\textsf{\u03ba}}{\\textsf{In}\\;\\;\\textsf{2}}$$\n\nThe alignment result generated by LAST is then used as an input to Blast2GO. The final assignments of GO terms are extracted and presented in a table on the website, which is made available for download.\n\n## The identification of domains\n\nWe downloaded the standalone BLAST+ (v2.2.25) program from NCBI \\[26\\] and used as database the 13,672 domain models (Pfam v26) from the Conserved Domains Database (CDD) \\[19,20\\]. FastAnnotator applies the rpstblastn to identify domains in the query nucleotide sequences by searching against the preformatted domain database with mostly default parameters except the expectation value (e-value) which is set to be less than 0.01, and the hit aligned length which is longer than 50% of the domain PSSM \\[27\\]. After the domains of the query sequences are identified, FastAnnotator calculates the length coverage and presents the percentage of coverage for the domain in the report table.\n\n## The identification of EC numbers\n\nFastAnnotator utilizes PRIAM \\[6\\] to identify potential enzyme functions. PRIAM can detect specific enzymes patterns and annotate these enzymes with EC numbers. The latest version of the enzyme profiles (released on 19 Oct, 2011) was downloaded from the PRIAM website. The nucleotide sequence inputs were translated into protein sequences in six frames using Transeq, a tool a part of the European Molecular Biology Open Software Suite (EMBOSS) \\[28\\]. These translated protein sequences are then used as inputs to search against the enzyme profile database. FastAnnotator identifies transcripts that may act as enzymes and presents the transcripts together with EC numbers in the output table.\n\n# Results and discussion\n\nThe FastAnnotator website allows users to upload *de novo* assembled sequences from RNA-Seq dataset through the upload page (shown in Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>). Only FASTA files of less than 100 megabytes are able to be uploaded to the server. After a successful upload, a unique job ID is provided as an identifier to start the annotation pipeline. All annotation results can be inspected over the Internet or downloaded in a compressed file for further analysis. The report consists of five sections. The first section is a histogram of the length distribution of the input sequences as well as a list of other statistics, including the average length, total number of bases, and the N50 value. The second section lists the best hits for all sequences against the NCBI non-redundant database. To avoid overloading the browser, only the top 100 entries are shown on the website results page as a demonstration, although users can download all of the best hits from the download link. The third section contains the results of the functional annotation (GO terms). GO term annotations are grouped according to biological processes, cellular components and molecular functions and are plotted in three separate horizontal bar charts; an example is shown in Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>. To better represent the overall distribution of functional annotations, additional spread-out or clustered views are provided based on different filtering level of GO terms. For example, when level 3 is chosen, all annotated GO terms are traced back to the root until reaching the nodes that were 2 edges away from the root (the 3^rd^ node) on the GO acyclic graph. Users can then choose among different level of GO terms to get a better biological interpretation. The fourth section presents the enzyme annotation, which lists all enzyme hits and their corresponding query sequences. The last section contains a list of domain identifications, and again, only 100 entries are shown to avoiding overloading the browser. All of these annotations can be downloaded as a compressed zip file. Annotation results for each job will be retained on the FastAnnotator server for two weeks after the job is finished, and users can retrieve the finished job using the job IDs provided by the server.\n\nTo demonstrate the performance and speed of FastAnnotator, we ran it on several transcriptome sequence datasets from different organisms. We were especially interested in how FastAnnotator could assist in the annotation of non-model organisms. The unpublished RNA-Seq dataset from a clam (*Meretrix meretrix*) was used as an example. This transcriptome included 22,129,105 cDNA sequence reads generated using an Illumina Genome Analyzer, and the average read length was 80 base pairs. A reference genome for the clam is currently not available, and thus, the reads were assembled *de novo* into 101,795 contigs using the software package CLC Genomics Workbench (CLC bio, Denmark). Using these contigs as the input, FastAnnotator required approximately 16 hours to finish the annotation. The statistical output showed that the N50 of these contigs was 390 nucleotides and the majority of these assembled contigs (73,878 contigs) ranged from 200-399 base pairs. This large number of short contigs may have resulted from a low level of coverage, and we believe that many of these contigs were actually partial transcript sequences. Of these contigs, 24,919 were found to be similar to sequences in the NCBI non-redundant protein database; 15,112 were assigned at least one Gene Ontology term; 13,015 were found to contain at least one domain; and 585 were assigned an enzyme annotation. Among all of the contigs, 20.4 % of contigs were annotated, and the annotation rate was even higher (26.4%) if we only considered contigs longer than 250 base pairs. These contig annotation rate was slightly higher than that reported in previous studies \\[1,29\\], the majority of which were based on domain identification. A further comparison of our annotation results with a recently published clam transcriptomic analysis by Huan et al. \\[1\\] showed that our annotation results for the clam transcriptome were similar.\n\nWe were interested in the performance of FastAnnotator for different organisms from different evolutionary lineages. We tested FastAnnotator using six transcriptome datasets, which includes data from plants, invertebrates, protists and prokaryotes (Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>). All of these transcriptomes were derived from the *de novo* assembly from NGS reads with the exception of the amoeba transcriptome, which was derived from EST data. Reference genomes were available for *C. elegans* and *S. parasanguinis* \\[30\\], and the highest sequence annotation percentages were obtained for these organisms. Given the high accuracy obtained using Blast2GO, which was used in the FastAnnotator annotation, these annotation results can be considered highly reliable. For the non-model organisms, FastAnnotator was able to annotate approximately half of the input sequences in three out of the four cases. We found the annotation rate was much lower than half in clam transcriptome (20.4%). Meanwhile we found the percentage of annotated contigs in our clam transcriptome was similar to those reported in previous clam studies (12.2% and 20.7% for the clam *M. meretrix* transcriptome and an Agamaki clam EST study, respectively \\[1,29\\]) and the dataset used here was composed with numerous of partial transcripts. Thus we conclude that FastAnnotator is a suitable annotation tool for non-model organisms.\n\nFastAnnotator results for five different organisms\n\n<table>\n<thead>\n<tr>\n<th style=\"text-align: center;\">Organism<\/th>\n<th style=\"text-align: center;\"># of entries (total base)<\/th>\n<th style=\"text-align: center;\">Processing time<\/th>\n<th style=\"text-align: center;\">% of sequences with best hit<\/th>\n<th style=\"text-align: center;\">% of annotated sequence<sup>*<\/sup><\/th>\n<\/tr>\n<\/thead>\n<tbody>\n<tr>\n<td style=\"text-align: center;\"><em>C. elegans<\/em><sup>+<\/sup><\/td>\n<td style=\"text-align: center;\">4,456<br \/>\n(1,339,253)<\/td>\n<td style=\"text-align: center;\">1.25 h<\/td>\n<td style=\"text-align: center;\">97.8%<\/td>\n<td style=\"text-align: center;\">88.1%<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">Bacteria example<sup>+<\/sup><\/td>\n<td style=\"text-align: center;\">1,219<br \/>\n(423,067)<\/td>\n<td style=\"text-align: center;\">21 min<\/td>\n<td style=\"text-align: center;\">94.7%<\/td>\n<td style=\"text-align: center;\">81.3%<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">Plant example<sup>+<\/sup><\/td>\n<td style=\"text-align: center;\">39,914<br \/>\n(24,168,315)<\/td>\n<td style=\"text-align: center;\">16 h<\/td>\n<td style=\"text-align: center;\">77.0%<\/td>\n<td style=\"text-align: center;\">62.9%<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">Clam<sup>+<\/sup><\/td>\n<td style=\"text-align: center;\">101,795<br \/>\n(38,886,727)<\/td>\n<td style=\"text-align: center;\">15.5 h<\/td>\n<td style=\"text-align: center;\">24.5%<\/td>\n<td style=\"text-align: center;\">20.4%<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\">Amoeba EST<\/td>\n<td style=\"text-align: center;\">13,814<br \/>\n(11,055,101)<\/td>\n<td style=\"text-align: center;\">3.5 h<\/td>\n<td style=\"text-align: center;\">68.4%<\/td>\n<td style=\"text-align: center;\">53.1%<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\"><em>Trichomonas vaginalis<\/em><sup>+<\/sup><\/td>\n<td style=\"text-align: center;\">35,989<br \/>\n(26,716,478)<\/td>\n<td style=\"text-align: center;\">11 h<\/td>\n<td style=\"text-align: center;\">93.7%<\/td>\n<td style=\"text-align: center;\">42.0%<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n^+^RNA-Seq denovo assembly result\n\n\\*Percentages of sequences had been annotated with GO, enzyme or domains.\n\nGO annotation is the most commonly used and well-established functional annotation scheme. To identify GO terms for the input sequences, FastAnnotator incorporates the Blast2GO pipeline, which is one of the most widely used annotation tools and claims to have an annotation accuracy of 65-70% \\[16\\]. However, we made a small modification to the Blast2GO pipeline by replacing BLASTX with LAST, as LAST is significantly faster and is capable of detecting evolutionarily conserved regions \\[23\\]. We also used the example file provided by the B2G4Pipe package to benchmark the computation time and comparing output results of BLASTX and LAST on the same machine (IBM X3850 with four Xeon E7540 2.0GHz CPUs and 128G RAM). We found that for this file of 10 nucleotide sequences, BLASTX took 5 times longer to search against the non-redundant database comparing to LAST. The resulting 30 annotations reported by each tool differed in only 2 cases. A closer inspection of the results revealed that the differences were minor and could be explained by the parent and child relationship within the GO acyclic graph. Therefore, we concluded that replacing BLASTX with LAST resulted in a minimal difference in the annotation while providing a substantial improvement in the computational speed.\n\nRegarding the detection of enzymes, FastAnnotator annotates enzyme functions based on the database of 2,844 EC numbers included in the most recent PRIAM release. According to Integrated relational Enzyme database (IntEnz) release 76, there were 4,812 active EC numbers \\[30\\]. Furthermore, it is well-known that more than one-third of the enzyme activities with EC numbers are so-called orphan enzyme activities, which are not associated with a protein or gene sequences \\[32-34\\]. Approximately 50% of the orphan enzyme activities are limited to only one species or closely related organisms, which implies that orphan enzyme activities may be limited to certain organisms that remain to be fully explored \\[35\\]. Due to these limitations, current annotation tools that are based on sequence similarity searches are restricted to detecting only certain enzyme activities. Consequently, FastAnnotator may overlook certain enzyme functional annotations, especially if those functions are restricted to particular organisms. In our clam transcriptome, for example, only 585 contigs were identified as enzymes. Because it has been estimated that approximately 18-29% of genes encode enzymes in eukaryotes \\[36\\], it is very likely that this level of enzyme annotation was an underestimate, which may have occurred due to the fact that the majority of the contigs are only partial transcripts in our clam example and because the enzyme database used in FastAnnotator was imcomplete. FastAnnotator may provide improved annotations in the future after additional enzyme functions and associated sequences are identified and included in the enzyme databases.\n\n# Conclusions\n\nAs sequencing technologies improve and decrease in cost, an increasing number of sequences can be generated. However, the annotation of these sequences, especially for those generated from unfamiliar biological samples, becomes an important issue. In this project, we developed FastAnnotator, an automatic annotation web tool, which integrates several well-developed annotation tools together to provide annotations for query sequences. FastAnnotator allows users to assign protein functions, cellular location, enzyme activity, and function domains to query sequences through an easy-to-use interface. By adopting a different sequence search program, LAST, and including domain identification, it is capable of efficiently annotating sequences and is suitable for annotation of sequences derived from less well-studied organisms or environmental samples. In summary, we present a web-based annotation tool, FastAnnotator, which should be helpful in transcriptome studies, particularly metatranscriptome or non-model organism studies.\n\n# Availability and requirements\n\nProject name: FastAnnotator\n\nProject home page: <http:\/\/fastannotator.cgu.edu.tw>\n\nHardware specifications: IBM X3850 with four Xeon E7540 2.0GHz CPUs and 128G RAM\n\nOperating system(s): CentOS Release 5.7 with Linux kernel 2.6.18\n\nProgramming language: Python and Perl for pipeline building, PHP for website interface.\n\nBrowser requirements: The website can be access by popular web browser with Javascript enabled include Mozilla Firefox, Google Chrome and Microsoft Internet Explorer.\n\n# Competing interests\n\nThe authors declare that they have no competing interests.\n\n# Authors' contributions\n\nTWC and THW built the pipeline. RCRG designed and constructed the website. TWC and RCRG wrote the manuscript together. PJH and CYL maintain the system. YMC and CCC performed RNA-Seq of *Streptococcus parasanguinis* and clam. PT and CCC supervised and revised the manuscript.\n\n## Acknowledgements\n\nThis work is supported by a grant (CMRPD190142) from the Chang Gung Memorial Hospital to PT. We thank Dr. Frith for his useful suggestion on parsing the output data from LAST.\n\nThis article has been published as part of *BMC Genomics* Volume 13 Supplement 7, 2012: Eleventh International Conference on Bioinformatics (InCoB2012): Computational Biology. The full contents of the supplement are available online at <http:\/\/www.biomedcentral.com\/bmcgenomics\/supplements\/13\/S7>.","meta":{"dup_signals":{"dup_doc_count":104,"dup_dump_count":68,"dup_details":{"curated_sources":4,"2023-14":1,"2022-21":1,"2022-05":1,"2021-49":1,"2021-43":2,"2021-39":1,"2021-31":1,"2021-10":1,"2021-04":1,"2020-50":1,"2020-45":1,"2020-40":1,"2020-34":1,"2020-29":1,"2020-24":1,"2020-16":1,"2020-05":2,"2019-51":1,"2019-47":1,"2019-39":2,"2019-30":1,"2019-26":1,"2019-18":1,"2019-13":1,"2019-09":1,"2019-04":1,"2018-51":2,"2018-47":1,"2018-43":1,"2018-39":1,"2018-34":2,"2018-30":2,"2018-26":1,"2018-22":2,"2018-17":2,"2018-13":1,"2018-09":1,"2018-05":1,"2017-47":1,"2017-39":1,"2017-34":1,"2017-26":1,"2017-22":7,"2017-17":5,"2017-09":3,"2017-04":1,"2016-44":1,"2016-40":1,"2016-36":3,"2016-30":4,"2016-22":1,"2016-18":1,"2015-40":1,"2015-35":1,"2015-27":1,"2015-22":2,"2014-52":3,"2014-49":1,"2014-42":1,"2014-35":1,"2014-23":1,"2023-40":1,"2017-13":2,"2015-18":1,"2015-11":1,"2014-10":1,"2013-48":4,"2024-26":1}},"file":"PMC3521244"},"subset":"pubmed_central"}
{"text":"abstract: A report on the symposium 'Genomic and Proteomic Approaches to Crustacean Biology' held as part of the Society for Integrative and Comparative Biology 2006 Annual Meeting, Orlando, USA, 4-8 January 2006.\nauthor: Timothy S McClintock; Charles D Derby\ndate: 2006\ninstitute: 1Department of Physiology, Cellular and Molecular Neuroscience of Sensory Systems Training Program, University of Kentucky, Lexington, KY 40536-0298, USA; 2Department of Biology, Brains and Behavior Program, and Center for Behavioral Neuroscience, Georgia State University, Atlanta, GA 30303, USA\ntitle: Shelling out for genomics\n\nConsistent with its goal of integrating diverse fields studying basic and applied problems in biology, the Society for Integrative and Comparative Biology showcased a symposium on the genomics and proteomics of crustaceans at its recent annual meeting. This symposium emphasized the future prospects for crustacean genomics by highlighting two themes - an explosion of sequencing efforts and the implementation of physiological genomics approaches in crustaceans.\n\n# First crustacean genome sequence\n\nCrustaceans comprise a major clade of arthropods, with thousands of extant species (see the Tree of Life web project <http:\/\/tolweb.org\/tree?group=Arthropoda>). They have significant economic, ecological, and scientific importance, but have received little attention from molecular biologists beyond efforts to assess their phylogenetic relationships. Although the list of available crustacean DNA sequences is growing exponentially, it still numbers less than 100,000 (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>), so there has been little grist for the genomics mill. But this is about to change - and change substantially. John Colbourne (Indiana University, Bloomington, USA), representing the Daphnia Genomics Consortium, reported on the sequencing of the genome of a cladoceran, the water flea *Daphnia pulex*. The selection of *D. pulex* was based on two considerations: genome size and ecology. The vast majority of crustaceans have genomes larger than that of *Homo sapiens* (see <http:\/\/www.genomesize.com> for a comparison of sizes), which is a significant source of inertia, but the *D. pulex* genome is only around 200 Mb. Ecologically, *D. pulex* populations show intriguing physiological and anatomical responses to changes in their physical environments such as shifts in pH or introduction of toxic metals, and to changes in predator abundance or type. The assembly and initial annotation of the *D. pulex* genome is expected to be completed by early 2007.\n\nThe impact of this genome will be felt far and wide as a lodestone for the comparative genomics of crustaceans and as an important outgroup for genomics studies of other arthropods. It will also invigorate ongoing cDNA sequencing efforts reported at the symposium, all of which are from the Decapoda (lobsters, crabs and shrimp). David Durica (University of Oklahoma, Norman, USA) reported the sequencing of cDNAs from blastema tissue of fiddler crab (*Celuca pugilator*) legs during limb regeneration, thereby identifying genes potentially regulated by ecdysteroids. Thomas Shafer (University of North Carolina, Wilmington, USA) has sequenced cDNAs from epidermis of the blue crab *Callinectes sapidus*. He identified at least 45 cuticular proteins, some predicted to form the flexible arthrodial cuticle in crustacean joints, others to form the calcified cuticle. David Towle (Mt Desert Island Biological Laboratory, Salisbury Cove, USA) reported cDNA sequences from the green shore crab *Carcinus maenas* and the lobster *Homarus americanus* that have helped identify enzymes, ion exchangers and pumps involved in osmoregulation, a significant challenge faced by euryhaline species (that is, species adapted to a wide range of salinity) such as *C. maenas*. cDNAs are also being sequenced from other crustaceans with the aim of producing cDNA microarrays to study gene expression, as discussed later.\n\nA common finding in these projects was that about 50-60% of new crustacean sequences lack a significant match in the public sequence databases. A major contributing factor is certainly the sequencing of ends of cDNAs, such that untranslated regions are over-represented. Another contributing factor may be the dearth of crustacean sequences combined with the large evolutionary distance of crustaceans from species whose genomes have been sequenced (the closest being insects). Plans for additional whole-genome sequencing were discussed informally, with hopes centering on the decapod with the smallest genome, *C. maenas*, and on a second *Daphnia* species.\n\n# Physiological genomics in crustaceans\n\nPhysiological ecology is the discipline currently driving crustacean genomics. *Daphnia* species are important indicators of the health of lakes and ponds, so their transcriptional responses to toxins and environmental stresses are of broad interest. Colbourne reported the analysis of cDNA libraries from populations exposed to aquatic stressors such as pH change and heavy-metal pollution as a clone-and-count method of mRNA expression profiling. The results imply that a majority of mRNAs change in abundance in response to most of these stresses - an amazing result given that expression profiling in laboratory model organisms typically detects statistically significant changes in less than 10% of the mRNAs tested. This surprising finding was supported by the report from Jonathon Stillman (San Francisco State University, California, USA) that porcelain crab (*Petrolisthes cinctipes*) cDNA microarrays reveal responses to thermal stress that are just as diverse. That two crustaceans show such broad responses raises questions as to whether environmental stresses have a greater impact in crustaceans than in other animals, whether genetic diversity is a contributing factor, and whether transcriptional regulation is especially sensitive to environmental conditions. Robert Chapman (Hollings Marine Laboratory, Charleston, USA) reported new bioinformatics approaches employing fractal mathematics to analyze microarray data on crustacean responses to environmental stress. This is an intriguing approach in which the global response and its component parts, such as clusters of related mRNAs, might both be apparent in a single integrated analysis.\n\nNuala O'Leary (Hollings Marine Laboratory, Charleston, USA) is using cDNA microarrays to investigate the effect of viral infections in the Pacific white shrimp (*Litopenaeus vannamei*) hepatopancreas. The transcriptional changes observed may help efforts to ameliorate the effects of infection on shrimp, which constitute 25% of all seafood consumed in the United States. O'Leary also revealed some success in using RNA interference, in the form of double-stranded RNAs to suppress individual mRNA expression, welcome evidence that this potent mechanism of viral defense is present in crustaceans. Even more sophisticated methods of studying gene expression, such as *in vivo* gene targeting and transgene insertion, need to be developed.\n\nOne of us (T.M.) reported the use of cDNA microarrays and differential amplification to investigate olfaction in *H. americanus* and the spiny lobster *Panulirus argus*. Specific markers were identified for the olfactory sensory neurons, their glial cells, reactive epithelial cells at sites of proliferation and regeneration, and secretory cells, the latter revealing a new exocrine gland in the olfactory organ, which has been named the aesthetasc tegumental gland. Nora Terwilliger (Oregon Institute of Marine Biology, Charleston, USA) reported analyses of the evolution of arthropod hemocyanins and related genes, including the prediction of hypoxia-response elements in their promoters. Many crustaceans experience fluctuations in oxygen tension, a problem that is exacerbated by human activity.\n\nDon Mykles (Colorado State University, Fort Collins, USA) reported the first use of proteomics in a crustacean (the red land crab *Gecarcinus lateralis*). These experiments targeted the endocrinology of molting and identified numerous proteins, including proteins that bind molt-inhibiting hormone, that are candidates for regulating molting.\n\nNone of the crustacean cDNA microarrays described above is yet sufficiently broad in its coverage of the genome and sophisticated enough in its bioinformatics to provide a systems-level view of function - that is, where activated biological processes can be identified via statistically over-represented functional groups among the affected mRNAs. Coulborne reported that coverage of the genome by the *Daphnia* microarray should soon achieve this ideal, however.\n\nCrustaceans are an ancient and highly successful clade whose members have evolved into niches that span vitally important marine and aquatic environments. Crustacean genomics is therefore poised to have a significant impact on our understanding of environmental and physiological ecology. Even though significant technological hurdles remain, this symposium was a justifiable celebration of significant achievement and the dawning of the age of crustacean genomics.\n\n## Figures and Tables","meta":{"dup_signals":{"dup_doc_count":141,"dup_dump_count":49,"dup_details":{"curated_sources":2,"2023-14":1,"2022-40":1,"2022-21":1,"2021-43":1,"2020-24":1,"2020-10":1,"2019-47":2,"2019-35":1,"2019-26":1,"2019-13":1,"2019-04":2,"2018-47":2,"2018-39":1,"2018-13":1,"2017-47":1,"2017-34":1,"2017-26":1,"2017-17":1,"2017-09":10,"2016-50":2,"2016-44":1,"2016-40":1,"2016-36":11,"2016-30":9,"2016-22":1,"2016-18":1,"2016-07":7,"2015-48":3,"2015-40":3,"2015-35":3,"2015-32":3,"2015-27":3,"2015-22":3,"2015-14":2,"2014-52":3,"2014-49":3,"2014-42":9,"2014-41":5,"2014-35":6,"2014-23":4,"2014-15":4,"2023-40":1,"2015-18":3,"2015-11":2,"2015-06":3,"2014-10":4,"2013-48":4,"2013-20":3,"2024-22":1}},"file":"PMC1557997"},"subset":"pubmed_central"}
{"text":"abstract: A report on the Keystone symposium 'Non-coding RNAs' held at Snowbird, Utah, USA, 31 March to 5 April 2012.\nauthor: Ezgi Hacisuleyman; Moran N Cabili; John L Rinn\ndate: 2012\ninstitute: 1Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA; 2Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA 02142, USA; 3Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; 4Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA\ntitle: A Keystone for ncRNA\n\nOnce upon a time RNA fit cleanly into the central dogma as a messenger between DNA and protein. Over the past 50 years, RNA molecules have continually emerged as dynamic and versatile regulators of the genome. Our modern understanding of non-coding RNAs (ncRNAs) may look like an intertwined mess of molecules, but collectively they exhibit architecture and coordination, leading to elegantly choreographed regulation of DNA and protein by RNA. The emerging role of RNA as an orchestrator resonated throughout the inaugural Keystone symposium on ncRNAs, with many examples of long ncRNAs (lncRNAs) having critical roles across numerous biological pathways. After the meeting, it was clear that more surprises would emerge from the 'dark matter' of the genome.\n\nThe keynote address by Nick Proudfoot (University of Oxford) set the stage by describing how the most classically studied regions of the genome, such as the \u03b2-globin locus, are now emerging as being exquisitely regulated by RNA. Specifically, Proudfoot summarized years of work showing how the act of transcription through non-coding regions, and importantly where transcriptional termination occurs, regulates the epigenetic dynamics of the locus. Intriguingly, convergent transcription by RNA polymerase II (RNA pol II) may serve as a substrate to recruit Dicer and other factors of the RNA interference (RNAi) machinery. Similarly, Robert Martienssen (Cold Spring Harbor Laboratory) presented an interplay between RNA\/DNA polymerase activity and RNAi in establishing heterochromatic domains. The dependence on co-transcriptional RNAi allows the release of RNA polymerase and prevents collision with the centromeric DNA replication machinery. Together these studies demonstrate the need for not only identifying lncRNAs involved in epigenetic establishment but also for understanding many simultaneous intertwined layers of regulation.\n\n# The human noncoding transcriptome reveals a map of 'noncodarnia'\n\nThomas Gingeras (Cold Spring Harbor Laboratory) provided an overview of the complexity of the human transcriptome resulting from the efforts of the ENCODE consortium. The transcriptomic map has gained an unprecedented resolution, revealing that 76% of our genome is transcribed. With an average of approximately eight transcripts per genic region, the wealth of ENCODE has redefined the 'one gene - one function' hypothesis into 'many transcripts - one function', or possibly many. Using complementary datasets and approaches, Piero Carninci and the Riken OMICs Center have provided new insights into lncRNA promoter regulation. By fine mapping of the 5' 7-methyl guanosine caps on RNA, the group have found that 6 to 30% of 5' start sites of mouse and human transcripts initiate within repetitive elements. Remarkably, over 250,000 retrotransposon-derived transcription start sites show tissue- and cell-compartment-specific expression.\n\nLeonard Lipovich (Wayne State University) and colleagues added 6,000 lncRNAs to this catalog by examining unclassified human cDNA clones and their expression profiles to determine whether these lncRNAs contribute to neurological disease phenotypes. They found that certain primate-specific and non-conserved lncRNAs are differentially expressed in brain regions that show high levels of activity. Some of these lncRNAs, antisense to protein-coding genes, can regulate their neighbors' expression. Weaving the intricacy of the transcriptome with the complexity of the mammalian body development and cognition, John Mattick (Garvan Institute of Medical Research) presented examples that emphasized the need to further understand the diversity of lncRNAs. Digging into the depths of the 'dark matter in the genome' using capture enrichment methods revealed not only numerous novel lncRNAs and their isoforms but also isoforms of well-studied protein-coding mRNAs such as p53. Hundreds of lncRNAs were shown to change during stem cell differentiation and to have similar transcript stability to mRNAs, and many are associated with epigenetic complexes, suggesting that this complexity cannot be dismissed *en masse* as transcriptional noise.\n\n# RNA-RNA interactions\n\nAn ever emerging theme is the importance of RNA-RNA interactions and gene regulation. Kevin Morris (The Scripps Research Institute) described new findings on lncRNA-directed epigenetic regulation through RNA-RNA interactions. Morris and colleagues observed an antisense transcript from the *PTEN* pseudogene (*PTENpg1* asRNA), which is transcribed in the opposite direction to the previously reported *PTENpg1* sense transcript (which can sequester microRNAs and affects *PTEN* translation rates). The *PTENpg1* asRNA seems to direct transcriptional gene silencing of *PTEN* by interacting with the DNA methyltransferase Dnmt3a and the histone-lysine N-methyltransferase Ezh2 and affecting their localization to the *PTEN* promoter. Moreover, the *PTENpg1* asRNA (containing a poly(A) tail) seems to facilitate the cellular localization of *PTENpg1* sense transcript (lacking a poly(A) tail). Thus, this pseudogene node seems to control *PTEN* at both the translational and the transcriptional level.\n\n# ncRNAs in cellular and developmental biology\n\nDavid Spector (Cold Spring Harbor Laboratory) and Shinichi Nakagawa (RIKEN Advanced Science Institute) presented a comprehensive investigation of the physiological roles of lncRNAs in mouse model systems. Using knockout mice, Spector and colleagues concluded that the lncRNA Malat1 was physiologically dispensable under laboratory conditions. This lack of phenotype may be attributed to genetic redundancy or stress conditions that were not examined. Indeed, Nakagawa examined the physiological role of the nuclear lncRNA *Neat1* (which is essential for the formation of nuclear structures involved in splicing termed paraspeckles) using knockout mice and observed a phenotype only in adult homozygote females, which became infertile at an earlier age. Seth Blackshaw (John Hopkins University School of Medicine) presented the *Six3OS* RNA as another example of a lncRNA required in developmental processes. Loss and gain of function of *Six3OS*, which is transcribed in the opposite direction from the Six3 transcription factor (TF) mRNA, showed that *Six3OS* regulates retinal cell specification. Several elegant follow-up experiments suggest that *Six3OS* regulates Six3-dependent transcription by recruiting the Polycomb repressive complex 2 (PRC2) to Six3 target genes.\n\nMartin Walsh (Mount Sinai School of Medicine) presented his recent work on the complex interplay between RNA pol II and pol III, whereby RNA pol III can affect transcription by accentuating the function of RNA pol II. Ablation of the components of RNA pol III caused a significant depletion of an enhancer ncRNA in the *Sox2* locus followed by changes in chromatin\n\nsignatures. Further analysis of the three-dimensional spacing of RNA pol III sites suggest that RNA pol III-regulated transcription could have a role in chromatin architecture, especially in the context of embryonic stem cell differentiation.\n\nRachel Duffi\u00e9 (Institut Curie) demonstrated how lncRNAs could control differential methylation patterns and imprinting during germ layer specification. Duffi\u00e9 presented a biallelically expressed lncRNA that was sufficient to induce DNA methylation *in* *cis*. Carla Klattenhoff (Massachusetts Institute of Technology) presented *braveheart*, a lncRNA required for stem cell differentiation into cardiomyocytes. Loss of *braveheart* causes disorganization and lack of beating in myofibrils and shorter sarcomeres. Collectively, these studies demonstrate the importance of lncRNAs in establishing cellular identity.\n\n# RNA and chromatin\n\nThe intersection of RNA with chromatin was introduced by the well-studied example of *Xist* by Neil Brockdorff (University of Oxford) and Jeannie Lee (Massachusetts General Hospital). Brockdorff showed that *Xist* RNA associates directly with additional proteins such as heterogeneous nuclear ribonucleoprotein U (hnRNPU) and PRC1. Brockdorff and colleagues have also identified a distinct form of PRC1, in which RING1 and YY1 binding protein (Rybp) replaces the catalytic Cbx subunit and associates with *Xist* RNA, suggesting that in addition to PRC2, hnRNPU and PRC1 are also involved in localizing *Xist* RNA to the inactive X. Lee further elucidated the mechanism by which the *Xist* lncRNA controls the silencing on the inactive X. Allele-specific localization profiles of PRC2 during \u00d7 inactivation suggest a highly selective localization: first, PRC2 localizes to limited number of nucleation spots and, as *Xist* RNAs spreads *in cis*, PRC2 is recruited to additional sites in a gradient, unlike recruitment on autosomes.\n\nAs an attempt to generalize the observation that ncRNAs such as *Xist* bind the PRC2 and recruit it to its target loci, Richard Jenner (University College London) and colleagues observed short RNAs that are transcribed from CpG islands surrounding PRC2-bound promoters. Jenner's group showed that these short RNAs are required for efficient methylation of histone H3 on lysine 27 (H3K27me3) through interaction with a newly characterized amino-terminal domain of Suppressor of zeste 12 (Suz12). Collectively, these studies clearly demonstrate the interplay between DNA, lncRNAs, and proteins to establish proper epigenetic states and cellular physiology.\n\nWith the goal of identifying where RNA localizes on chromatin, Matthew Simon (Massachusetts General Hospital) and Jason West (Massachusetts General Hospital) introduced a new method (CHART) to purify regions directly bound by lncRNAs. CHART successfully identified the protein and chromatin targets of the paraspeckle-associated lncRNA *NEAT1* in human cells. Initial analysis of CHART coupled with sequencing revealed the chromosomal binding sites of *NEAT1* and suggested that *NEAT1* facilitates tertiary DNA interactions involved in nuclear architecture.\n\nDeepening our understanding of recruitment of chromatin modifiers by lncRNAs, Howard Chang (Stanford University) and colleagues discovered the lncRNA *HOTTIP*, which activates the expression of its neighboring genes *in cis* by recruiting the activating modifier WDR5. Using crystallography and site-specific mutagenesis, the group identified the RNA-binding domain of WDR5. Moreover, binding to *HOTTIP* regulates the stability of WDR5, suggesting a model in which a RNA-based molecular clock regulates protein complex formation and stability. Similar to *HOTTIP*, Ramin Shiekhattar (Wistar Institute) presented a set of lncRNAs with enhancer-like functions. Shiekhattar described a specific example, in which an lncRNA enhances the expression of genes *in cis* through physical interactions with the Mediator complex. The lncRNA-Mediator interaction facilitates DNA looping to activate distal genes. Together, these suggest that lncRNAs emanating from enhancer regions may have RNA-based functions.\n\nUsing a detailed map of large intergenic ncRNAs (lincRNAs), one of us (JR) and colleagues used a computational 'guilt-by-association approach' to identify lincRNAs involved in specific pathways. One such example led to the discovery of a lncRNA, Required for adipogenesis 1 (*RAP1*). *RAP1* contains a striking feature: multiple repetitive exonic sequences that physically associate with hnRNPU. Single-molecule RNA localization methods showed that *RAP1* shows nuclear localization indicative of large chromosomal domains. Using a similar guilt-by-association approach, Keith Vance (University of Oxford) from Chris Ponting's group used computational and evolutionary genomics to identify mouse lincRNAs close to genes encoding developmental TFs. One such lincRNA is *Plinc*, which can act *in cis* to repress its bidirectionally promoted neighbor Pax6, as well as *in trans* to repress other targets involved in cell cycle and synaptic function. *Plinc* is enriched on chromatin and associates with the transcriptional cofactors Kap1 and Erh.\n\n# Non-mammalian ncRNA\n\nncRNAs also have critical regulatory roles in prokaryotes. Gisela Storz (National Institute of Child Health and Human Development) gave several examples from bacteria, in which small RNAs act in parallel to TFs to fine-tune the TFs' target gene levels through a base-pairing mechanism. In parallel, Jennifer Doudna (University of California, Berkeley) gave new insights into the CRISPR adaptive immune system in bacteria, in which viral DNA segments are integrated within bacterial DNA repeat clusters and are later transcribed to short RNAs that act in a viral defense pathway. Doudna highlighted some similarities between eukaryotic RNAi and prokaryotic CRISPR target recognition mechanisms based on molecular structures of these machineries.\n\n# RNA and disease\n\nAndrew Feinberg (John Hopkins University School of Medicine) presented a comprehensive analysis of the epigenetic changes in cancer. Surveying differential DNA methylation in cancer revealed the loss of DNA methylation of the lncRNA termed *HOTS* (H19 opposite tumor suppressor) encoded antisense to the maternally imprinted *H19* gene, which encodes an untranslated RNA of unknown function. Feinberg described large lamin-associated DNA domains that are also lost in cancer. Similar to *HOTS*, almost all genes have naturally occurring antisense transcripts (NATs) that are ripe for RNA-RNA interactions and are often misregulated in disease states. Claes Wahlestedt (University of Miami) and colleagues have developed single-stranded oligonucleotides termed antagonists of NATs (antagoNATs) that upregulate specific endogenous genes *in vitro* and *in vivo* by interfering with the function of NATs. As NATs are widely expressed, the approach holds promise for potential RNA-based therapeutics. Together these studies highlight the need to understand the link between epigenetic regulation and lncRNAs in the etiology of disease.\n\n# Trans-generational RNA for the future\n\nParamutation is a form of non-Mendelian heredity in which the phenotypic expression of an allele is mutated while its genotype is intact. The meeting concluded with exciting updates on the current understanding of this phenomenon from worms to mammals. Andrew Spence (University of Toronto) described how a maternal transcript of the sex-determining gene *fem-1* does not require any coding capacity to trigger *fem-1* expression, which it does by preventing RNA-dependent epigenetic silencing of this gene. Oded Rechavi (Columbia University Medical Center) demonstrated how virus-derived small interfering RNAs can be trans-generationally transmitted in a non-Mendelian manner. Finally, Minoo Rassoulzadegan (University of Nice) showed evidence for RNA-induced hereditary transmission of variation affecting the phenotype of three different genes in mouse animals. By electroporating an inducer small RNA homologous to the gene of interest to embryonic stem cells, Rassoulzadegan and colleagues were able to obtain a paramutable effect over many passages in cell culture. Collectively, these studies have provided exciting new evidence for the potential role of RNA in trans-generational inheritance in mammals.\n\nThis meeting was a resounding success and clearly demonstrated the versatility, diversity, and importance of ncRNAs in gene regulatory programs. John Mattick ended the meeting with inspiring words, highlighting the imminence of many groundbreaking discoveries to come from the dark matter of the genome. Kevin Morris reinforced the need for a collaborative spirit among the ncRNA community, which will rapidly drive the field to a greater success. One thing was clear: the transcriptome has revealed many mysterious creatures that comprise the mysterious world of 'Noncodarnia'.","meta":{"dup_signals":{"dup_doc_count":157,"dup_dump_count":52,"dup_details":{"curated_sources":2,"2023-14":1,"2022-27":1,"2021-43":1,"2020-40":1,"2019-22":1,"2019-09":1,"2018-51":2,"2018-43":2,"2018-34":1,"2018-13":2,"2018-09":1,"2018-05":2,"2017-51":3,"2017-47":1,"2017-39":1,"2017-34":1,"2017-30":1,"2017-26":1,"2017-22":1,"2017-17":1,"2017-09":8,"2017-04":1,"2016-50":1,"2016-44":1,"2016-40":1,"2016-36":9,"2016-30":9,"2016-22":1,"2016-18":1,"2016-07":6,"2015-48":4,"2015-40":2,"2015-35":4,"2015-32":4,"2015-27":4,"2015-22":5,"2015-14":4,"2014-52":4,"2014-49":6,"2014-42":12,"2014-41":6,"2014-35":4,"2014-23":6,"2014-15":4,"2023-50":1,"2015-18":4,"2015-11":4,"2015-06":4,"2014-10":5,"2013-48":2,"2013-20":1,"2017-13":1}},"file":"PMC3446285"},"subset":"pubmed_central"}
{"text":"date: 2019\ntitle: ADOPTING A HEALTHY LIFESTYLE HELPS REDUCE THE RISK OF DEMENTIA\n\n**14 May 2019** - People can reduce their risk of dementia by getting regular exercise, not smoking, avoiding harmful use of alcohol, controlling their weight, eating a healthy diet, and maintaining healthy blood pressure, cholesterol and blood sugar levels, according to new guidelines issued by the World Health Organization (WHO) today.\n\n\"In the next 30 years, the number of people with dementia is expected to triple,\" said WHO Director-General Dr Tedros Adhanom Ghebreyesus. \"We need to do everything we can to reduce our risk of dementia. The scientific evidence gathered for these Guidelines confirm what we have suspected for some time, that what is good for our heart, is also good for our brain.\"\n\nThe Guidelines provide the knowledge base for health-care providers to advise patients on what they can do to help prevent cognitive decline and dementia. They will also be useful for governments, policy-makers and planning authorities to guide them in developing policy and designing programmes that encourage healthy lifestyles.\n\nThe reduction of risk factors for dementia is one of several areas of action included in WHO's Global action plan for the public health response to dementia. Other areas include: strengthening information systems for dementia; diagnosis, treatment and care; supporting carers of people with dementia; and research and innovation.\n\nWHO's Global Dementia Observatory, launched in December 2017, is a compilation of information about country activities and resources for dementia, such as national plans, dementia-friendly initiatives, awareness campaigns and facilities for care. Data from 21 countries, including Bangladesh, Chile, France, Japan, Jordan and Togo, have already been included, with a total of 80 countries now engaged in providing data.\n\nCreating national policies and plans for dementia are among WHO's key recommendations for countries in their efforts to manage this growing health challenge. During 2018, WHO provided support to countries such as Bosnia and Herzegovina, Croatia, Qatar, Slovenia and Sri Lanka to help them develop a comprehensive, multi-sectoral public health response to dementia.\n\nAn essential element of every national dementia plan is support for carers of people with dementia, said Dr D\u00e9vora Kestel, Director of the Department of Mental Health and Substance Abuse at WHO. \"Dementia carers are very often family members who need to make considerable adjustments to their family and professional lives to care for their loved ones. This is why WHO created iSupport. iSupport is an online training programme providing carers of people with dementia with advice on overall management of care, dealing with behaviour changes and how to look after their own health.\"\n\niSupport is currently being used in eight countries, with more expected to follow.\n\nDementia: a rapidly growing public health problem\n\nDementia is an illness characterized by a deterioration in cognitive function beyond what might be expected from normal ageing. It affects memory, thinking, orientation, comprehension, calculation, learning capacity, language and judgement. Dementia results from a variety of diseases and injuries that affect the brain, such as Alzheimer disease or stroke.\n\nDementia is a rapidly growing public health problem affecting around 50 million people globally. There are nearly 10 million new cases every year. Dementia is a major cause of disability and dependency among older people. Additionally, the disease inflicts a heavy economic burden on societies as a whole, with the costs of caring for people with dementia estimated to rise to US\\$ 2 trillion annually by 2030.\n\nAvailable from: <https:\/\/www.who.int\/news-room\/detail\/14-05-2019-adopting-a-healthy-lifestyle-helps-reduce-the-risk-of-dementia>","meta":{"dup_signals":{"dup_doc_count":157,"dup_dump_count":41,"dup_details":{"curated_sources":2,"2023-50":5,"2023-40":1,"2023-23":4,"2023-14":2,"2023-06":7,"2022-40":7,"2022-33":2,"2022-27":3,"2022-21":4,"2022-05":4,"2021-49":1,"2021-43":5,"2021-39":3,"2021-31":10,"2021-25":1,"2021-21":5,"2021-17":4,"2021-10":4,"2021-04":3,"2020-50":4,"2020-45":1,"2020-40":4,"2020-34":2,"2020-29":4,"2020-24":6,"2020-16":5,"2020-10":2,"2020-05":3,"2019-51":1,"2019-47":2,"2019-43":2,"2019-39":4,"2019-35":4,"2019-30":9,"2019-26":2,"2019-22":11,"2024-30":2,"2024-26":3,"2024-22":4,"2024-18":4,"2024-10":1}},"file":"PMC6757200"},"subset":"pubmed_central"}
{"text":"abstract: A report on the FASEB meeting 'Nuclear Structure and Cancer', Saxtons River, USA, 16-21 June 2007.\nauthor: David M Gilbert; Daniele Zink\ndate: 2007\ninstitute: 1Department of Biological Science, Florida State University, Tallahassee, FL 32306, USA; 2Ludwig-Maximilians-University Munich, Department of Biology II, Grosshaderner Str., 82152 Planegg-Martinsried, Germany\ntitle: Intranuclear changes in cancer cells\n\nThis year's Federation of American Societies for Experimental Biology (FASEB) conference on nuclear structure and cancer opened with keynote speaker Donald Coffey (Johns Hopkins University, Baltimore, USA), who together with Ronald Berezney (University at Buffalo, Buffalo, USA) is credited with the discovery of the nuclear matrix. Coffey pointed out that changes in nuclear structure are diagnostic hallmarks of many cancers that are frequently utilized by the pathologist but are poorly understood from a molecular standpoint. This set the tone for a stimulating meeting, where talks on the fundamental organization of the nucleus were intermingled with reports of direct links to cancer and perspectives from pathologists. There were many interesting talks and here we report a few of the highlights.\n\n# Chromosomal organization and aberrations\n\nHow universal are the 'rules' of chromosome organization within the nucleus that are so frequently abrogated in cancer cells? Thomas Cremer (Ludwig-Maximilians-University, Munich, Germany) reported that in rod cells of the retina of nocturnal animals, the heterochromatin is in the center of the nucleus and euchromatin at the periphery, which is the inverse of nearly all other cell types examined to date. He speculates that this arrangement may be less diffractive to light and might, therefore, provide an advantage for nocturnal animals.\n\nLive-cell imaging is now a standard tool in the analysis of nuclear structure and function. Andrew Belmont (University of Illinois, Urbana, USA) addressed the large-scale chromatin organization of actively transcribing genes in somatic diploid nuclei of living cells. In contrast to models proposing the transition from loops of 30-nm chromatin fibers to 10-nm fibers during transcriptional activation, Belmont finds that transcribed chromatin remains several-fold more compact than even the 30-nm fiber. How do transcription factors find their cognate sites in these thick fibers? Gordon Hager (National Cancer Institute, Bethesda, USA) emphasized the highly dynamic nature of transcription factor binding as illustrated by the implication of chromatin remodeling as a key mechanism in nuclear receptor 'hit-and-run'. He described recent work addressing the question of whether chromatin remodeling is always required for interactions of the glucocorticoid receptor (GR) with its binding sites and whether these are always dependent on Brg1, a component of the chromatin-remodeling SWI\/SNF complex. Genome-wide analyses revealed that chromatin remodeling takes place at all GR-binding sites, but is not always dependent on Brg1. The results suggest that GR searches chromatin randomly, but that chromatin remodeling distinguishes functional and non-functional interactions. Hager also pointed out that mapping of DNase I hypersensitive sites is an important tool in the identification of functional regulatory sites where chromatin remodeling takes place.\n\nA common characteristic of tumor cells is genomic instability leading to chromosomal aberrations. For example, aggregates of telomeres are frequently observed in the nuclei of tumor cells but not in those of normal cells. Sabine Mai (University of Manitoba, Winnipeg, Canada) reported that expression of the proto-oncogene *c-Myc* leads to the formation of telomeric aggregates and telomeric fusions. She has found that the normal intranuclear positioning of centromeres and chromosomes was disturbed in cells over-expressing c-Myc, and that this was associated with the formation of chromosomal aberrations. These nuclear changes required the Myc box II domain of c-Myc, which associates with the histone acetyltransferase (HAT) Tip60; c-Myc overexpression in combination with Tip60 heterozygosity led to enhanced lymphoma formation in mice. These findings suggest that c-Myc-induced changes in nuclear organization play an important role in the genomic instability associated with tumorigenesis.\n\nJeffrey Salisbury (Mayo Clinic, Rochester, USA) presented an elegant study on the regulation of centrosome duplication. Centrosome amplification is often observed in tumor cells and can lead to aberrant spindle formation and chromosomal instability. Normally, centrosome duplication is coordinated with the cell cycle. During the cell-cycle arrest that follows DNA damage the protein xeroderma pigmentosum C (XPC) becomes upregulated. XPC binds to centrin 2, which is essential for centrosome duplication, and the complex moves into the nucleus - a movement dependent on the presence of the tumor suppressor protein p53. This depletes the cytoplasmic pool of centrin 2, and the resulting low levels are not sufficient to support centrosome duplication. Salisbury has found that in cells, such as tumor cells, that are defective in p53 function, or following post-transcriptional silencing of XPC, the cytoplasmic pool of centrin 2 remains high enough to support aberrant duplication, resulting in spindle abnormalities and chromosome instability.\n\nThe relation between cell-cycle stage and vulnerability to carcinogens was addressed by David Kaufman (University of North Carolina, Chapel Hill, USA), who has shown previously that cells are most susceptible to transformation by chemical carcinogens when treated at the beginning of S phase. He now reported that in two different human cell types (normal fibroblasts and lymphoblastoid cells), genes replicated at the beginning of S phase include the majority of apoptosis genes and Wnt genes, both of which are frequently associated with cancer. This suggests the existence of a class of important cancer-related genes, located on several different chromosomes, which are replicated simultaneously during the cell cycle and are most vulnerable to cancer-causing mutations during replication.\n\nThe organization of the sites at which DNA replication and transcription take place within the nucleus and their potential roles in the generation of chromosomal aberrations associated with cancer were addressed in several presentations. James Davie (University of Manitoba, Winnipeg, Canada) outlined work showing that transcription factors Sp1 and Sp3, which are ubiquitously expressed in mammalian cells, form distinct unrelated nuclear foci and associate with histone deacetylase (HDAC) 1 and phosphorylated HDAC2. Using chromatin immunoprecipitation (ChIP), he studied the binding of the estrogen receptor alpha, Sp1 and Sp3 to the estrogen-responsive trefoil factor 1 promoter in MCF-7 breast cancer cells. The findings suggested that Sp1 and Sp3 aid in recruitment of HDACs and HATs. Spectral karyotype analyses revealed high chromosomal instability in MCF-7 cells and Davie pointed out that the question of cell-to-cell variation in the karyotype of cancer cell lines must be carefully addressed when performing ChIP analyses.\n\nThe importance of the organization of the transcriptional regulatory machinery in nuclear microenvironments in cancer was underlined by work presented by Gary Stein (University of Massachusetts, Worcester, USA). He reported that the NMTS domain of the Runx transcription factors is necessary and sufficient for subnuclear targeting and trafficking. Perturbations in subnuclear targeting of Runx1 (also known as AML1) are associated with altered competence for myeloid differentiation and with expression of the transformed\/leukemia phenotype in myeloid progenitors. Impaired intranuclear trafficking of Runx2 (also known as AML3) in metastatic breast cancer cells interferes with the formation of osteolytic lesions *in vivo*.\n\nCameron Osborne (Babraham Institute, Cambridge, UK) reported that immediate early genes, including *c-Myc*, are recruited within minutes into transcription factories - complexes of transcribing genes - upon B-cell stimulation. He has found that 25% of transcribing *c-Myc* alleles colocalize with the *IgH* locus. Other active loci did not show a correspondingly high degree of association. Intriguingly, *c-Myc* and *IgH* are the most frequent translocation partners in mouse plasmacytoma and Burkitt's lymphoma, suggesting that intermingling of chromatin from the *c-Myc* and *IgH* loci during co-transcription makes it more likely that these loci will undergo translocations.\n\n# Epigenetic changes in cancer cells\n\nIn addition to genetic lesions, cancer cells display characteristic epigenetic changes associated with altered gene-expression patterns, which include CpG island hyper- and hypo-methylation. CpG island hypermethylation leads to epigenetic silencing by recruiting methyl-CpG-binding domain (MBD) family proteins. William Nelson (Sidney Kimmel Comprehensive Cancer Center, Baltimore, USA) reported that hypermethylation leads to epigenetic silencing of the *GSTP1* locus (encoding the \u03c0-class glutathione S-transferase) in nearly all prostate cancers, and most breast and liver cancers. High-throughput chemical screening has yielded a number of compounds capable of activating transcription from hypermethylated *GSTP1* promoters, and several of these compounds antagonized the binding of MBDs to methyl-CpG-containing DNA. Nelson described a strategy for characterizing methyl-CpG patterns based on the binding of DNA to the DNA-binding domain of MBD2 and subsequent hybridization to whole-genome tiled arrays. This approach has potential applications in cancer screening, detection, diagnosis and prognosis.\n\nA direct link between oncogene activation and diagnostic nuclear changes in human papillary thyroid carcinoma cells was reported by Andrew Fischer (University of Massachusetts, Worcester, USA). Changes diagnostic of this type of cancer are the dispersal of heterochromatin and irregularity of the nuclear lamina. Activation of either of the tyrosine kinases Ret or Trk is sufficient to induce the diagnostic changes *in vitro*. Fischer reported that dispersal of heterochromatin is associated with a decrease in monomethylation of lysine 4 of histone H3, and that Trk is sufficient to induce this decrease.\n\nPrevious work from the laboratory of Donald Tindall (Mayo Clinic, Rochester, USA) showed that the transcriptional co-activator p300 is involved in the progression of prostate cancer. At the meeting, Tindall reported that transfection of p300 into prostate cancer cells induces quantifiable nuclear alterations associated with prostate cancer. Furthermore, he proposed the provocative idea that androgen ablation, a routine treatment for prostate cancer, might initiate an androgen-independent cycle of activity. This suggestion is based on the findings that p300 mediates androgen-independent activation of the androgen receptor and that androgens downregulate p300 in cancer cell lines. Thus, androgen ablation might increase p300 expression in prostate cancer cells, which might initiate aberrant androgen-independent activation of the androgen receptor. Androgen-independent activation of the androgen receptor is usually observed in refractory prostate cancer after androgen-deprivation therapy.\n\nThe molecular basis by which alterations in nuclear structure and function lead to cancer is unquestionably an exciting new area for young researchers. Exchange between clinicians and basic researchers in this field is still limited, but is an inevitable prerequisite for investigating and understanding nuclear changes in cancer. This biennial meeting will continue to provide an exceptional forum for such discussions between clinicians, pathologists and basic researchers. The conference also provided a forum for investigators from academia and the biotech and pharmaceutical industries to explore cancer-related changes in the organization and localization of regulatory machinery within the cell nucleus as a platform for new diagnostic and therapeutic strategies.","meta":{"dup_signals":{"dup_doc_count":116,"dup_dump_count":41,"dup_details":{"curated_sources":2,"2020-29":1,"2019-18":1,"2019-04":1,"2018-47":1,"2018-39":1,"2018-30":1,"2018-22":1,"2018-13":1,"2017-47":1,"2017-34":1,"2017-22":1,"2017-09":9,"2016-44":1,"2016-40":1,"2016-36":9,"2016-30":7,"2016-22":1,"2016-18":1,"2016-07":6,"2015-48":3,"2015-40":2,"2015-35":3,"2015-32":3,"2015-27":2,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":3,"2014-42":6,"2014-41":5,"2014-35":3,"2014-23":4,"2014-15":4,"2022-27":1,"2015-18":3,"2015-11":3,"2015-06":3,"2014-10":4,"2013-48":2,"2013-20":4,"2024-18":1}},"file":"PMC2374982"},"subset":"pubmed_central"}
{"text":"abstract: The American focus on the sexual purpose of breasts, rather than the physiological function of breasts, has serious public health consequences. Discomfort with breastfeeding in public lowers breastfeeding rates, which in turn negatively affects women's and children's short- and long-term health.\nauthor: Jacqueline H Wolf\ndate: 2008\ninstitute: 1Department of Social Medicine, College of Osteopathic Medicine, Ohio University, Athens, Ohio, USA\nreferences:\ntitle: Got milk? Not in public!\n\n# Debate\n\nWhen I was growing up in the 1960s my best friend's father was an obstetrician. This was the heyday of formula feeding and my friend's father, even with his vast experience in women's reproductive health, had virtually no experience with breastfeeding. His expertise continued to be so negligible, in fact, that when my friend nursed her newborn son in front of him in the early 1980s \u2013 this is his first grandchild being nursed \u2013 he watched for a minute and then he said to his daughter, \"That will ruin your breasts, you know.\" When my friend called to report this comment to me sometime later, she was still bewildered. She asked me: \"What does he think breasts are *for*?\"\n\nHer story captures the essence of why breastfeeding in public is such a controversial topic in American culture. Americans think breasts are primarily for enhancing sexual activity, which results in widespread discomfort when they are reminded that breasts go into babies' mouths. While *our* culture defines breasts primarily as enhancing sexual appeal, other cultures emphasize the \"sexiness,\" if you will, of *other* body parts: buttocks, hips, shoulders, feet. It varies according to culture, since sexual attractiveness is always culturally defined.\n\nYet the American obsession with breasts is so unique that it is often puzzling to people from other cultures. One of my graduate students who conducted field work in Samoa reported to me that when she told a Samoan woman that American men like to suck on women's breasts, the Samoan was amazed. She asked my student, apparently with genuine interest and concern, \"Do American men like to pretend they're babies?\" That is how strictly Samoans define the purpose of women's breasts. In many countries, breastfeeding in public is as mundane an activity as public conversation; no one is concerned when women use their breasts in public for their primary biological function: to feed babies.\n\nMy purpose today is not to argue that we should think more like Samoans, but to point out that the American obsession with breasts has consequences most Americans fail to consider: ready access to human milk is vital to babies' short- and long-term health. We are all affected by our culture's sexual emphasis on breasts and our consequent discomfort with breastfeeding in public. While people from other cultures often find this controversy inexplicable, the reasons for the controversy are obvious to Americans \u2013 even those of us who fully support breastfeeding in public. We understand that many equate public breastfeeding with lewd behavior. However, equating breastfeeding with vulgarity has dire consequences; this attitude lowers the country's breastfeeding rates which in turn affects women's and children's short and long-term health. If women are made to feel uncomfortable with public breastfeeding, breastfeeding becomes difficult, if not impossible, to sustain. Women who have successfully breastfed for long periods of time know that unless women can feed their babies anytime, anywhere, they're going to end up housebound. And it's the rare American woman who is willing to be housebound for months on end. So, many women give up breastfeeding early on and opt for the bottle.\n\nHere are the statistics: 70% of new mothers initiate breastfeeding. But all initiation means is breastfeeding once before hospital discharge. Of far greater meaning is the breastfeeding exclusivity rate, which is terrible. By the time American babies are six weeks old, 53% of breastfeeding mothers have introduced at least some infant formula to their babies' diet and by six months, 90% \\[1\\]. Fewer than 18% of mothers who initiate breastfeeding are giving their babies *any breast milk at all* at the end of the minimal year recommended by the American Academy of Pediatrics.\n\nOne reason that rates are so high *initially* and then plummet almost immediately when it comes to exclusivity and duration is because most women are *not c*omfortable breastfeeding in public because the public is not comfortable seeing them breastfeed. That is a fundamental problem because human milk is low in fat and it is especially low in protein, much lower, for example, than cows' milk. That means human babies are almost constant feeders by design \u2013 which is why women in so many cultures literally wear their babies on their bodies, for easy access so they can feed often. In the U.S., people who oppose breastfeeding in public often argue, \"What's the problem? Feed the baby before you leave the house. There's no reason, with a little planning, to breastfeed in a restaurant or at the mall.\" This insistence that babies should only be breastfed behind closed doors demonstrates a fundamental lack of understanding of both the composition of human milk and babies' needs. Babies have to nurse while they are out and about due to the nature of human milk.\n\nThis insistence that babies should only be breastfed behind closed doors exposes our split personality on breastfeeding. We insist breastfeeding is a good thing to do. And, we insist it is an offensive thing to do.\n\nOur squeamishness with breastfeeding in public has consequences we refuse to acknowledge. Hurricane Katrina is a prime example of an unacknowledged consequence of our culture's revulsion at breastfeeding in public. We all watched with horror as New Orleans drowned two years ago. For days, Katrina and its aftermath were the only items in the news. And, whenever there is a news story of that magnitude there are always a lot of sidebars to the story. Remember? Katrina exposed America's class and racial divisions. We heard stories about inefficient government agencies, abandoned pets, lethal mold.\n\nBut do you know what story I kept looking for and never found? What happens to formula fed babies during a disaster when mothers cannot buy infant formula and they do not even have access to water? And there was ample opportunity to have a sidebar that pondered those awful questions. Some of the most memorable film clips coming out of New Orleans in 2005 pictured frantic mothers clutching their barely conscious, dehydrated babies.\n\nDiscussing our culture's attitude toward breastfeeding in relation to the Katrina disaster would have been a tremendous public service. Think about how much our discomfort with breastfeeding would have been mitigated if one of the Katrina reports contrasted the convenience and dependability of breastfeeding with the difficulty and unreliability of formula feeding. Imagine: images of mothers' breasts saving babies' lives. That would have been a national revelation. We talk about the importance of breastfeeding, yet we're a formula feeding culture. It seemed perfectly natural to all the reporters and much of the viewing audience during Katrina that mothers were hysterical and babies were dehydrated because there was not enough infant formula available. The entire nation seemed to be saying, \"Of course that's a consequence of a hurricane.\" No reporter thought to ask, \"Why aren't these women breastfeeding?\" No reporter thought to ask, \"What roadblocks have we constructed as a nation that would dissuade women from breastfeeding and put their babies through this completely avoidable horror?\"\n\nTo those of us who work on breastfeeding, the \"issue\" of breastfeeding in public is a periodic amusing and frustrating annoyance. However, we have to start treating it as more than that. The negative attitude toward public breastfeeding is a cornerstone of low breastfeeding rates and a basis of our persistently formula feeding culture. Aside from all the mothers who quickly learn to use infant formula because they are embarrassed by their hungry babies when there is no private space to breastfeed, women in the U.S. often fail at breastfeeding because they do not have adequate opportunity to observe other women breastfeeding. Breastfeeding is not intuitive, it is a learned behavior. In other words: Breastfeeding in our culture is deemed a private bodily function when \u2013 for many reasons, all having to do with infant and maternal health \u2013 it *should* be a public one.\n\n# Competing interests\n\nThe author declares that they have no competing interests.\n\n### Acknowledgements\n\n\"Got milk?\" is an American advertising campaign marketing cow's milk.","meta":{"dup_signals":{"dup_doc_count":110,"dup_dump_count":54,"dup_details":{"curated_sources":2,"2021-43":1,"2021-25":1,"2020-34":1,"2020-10":1,"2019-47":1,"2019-30":1,"2019-22":1,"2019-18":1,"2019-13":1,"2019-04":1,"2018-51":3,"2018-43":1,"2018-34":2,"2018-26":1,"2018-13":2,"2018-05":1,"2017-39":1,"2017-30":1,"2017-22":2,"2017-17":1,"2017-09":3,"2017-04":2,"2016-50":1,"2016-44":2,"2016-40":1,"2016-36":1,"2016-30":2,"2016-22":1,"2016-18":1,"2016-07":6,"2015-48":6,"2015-40":1,"2015-35":2,"2015-32":2,"2015-27":2,"2015-22":2,"2015-14":2,"2014-52":2,"2014-49":5,"2014-42":5,"2014-41":3,"2014-35":4,"2014-23":5,"2014-15":4,"2023-40":1,"2024-18":1,"2017-13":1,"2015-18":2,"2015-11":3,"2015-06":2,"2014-10":2,"2013-48":2,"2013-20":3,"2024-22":1}},"file":"PMC2518137"},"subset":"pubmed_central"}
{"text":"date: 2009-01\ntitle: Journals under Threat: A Joint Response from History of Science, Technology and Medicine Editors\n\nWe live in an age of metrics. All around us, things are being standardized, quantified, measured. Scholars concerned with the work of science and technology must regard this as a fascinating and crucial practical, cultural and intellectual phenomenon. Analysis of the roots and meaning of metrics and metrology has been a preoccupation of much of the best work in our field for the past quarter century at least. As practitioners of the interconnected disciplines that make up the field of science studies we understand how significant, contingent and uncertain can be the process of rendering nature and society in grades, classes and numbers.\n\nWe now confront a situation in which our own research work is being subjected to putatively precise accountancy by arbitrary and unaccountable agencies. Some may already be aware of the proposed European Reference Index for the Humanities (ERIH), an initiative originating with the European Science Foundation. The ERIH is an attempt to grade journals in the humanities\u2014including \"history and philosophy of science\". The initiative proposes a league table of academic journals, with premier, second and third divisions. According to the European Science Foundation, ERIH \"aims initially to identify, and gain more visibility for, top-quality European Humanities research published in academic journals in, potentially, all European languages\". It is hoped \"that ERIH will form the backbone of a fully-fledged research information system for the Humanities\". What is meant, however, is that ERIH will provide funding bodies and other agencies in Europe and elsewhere with an allegedly exact measure of research quality. In short, if research is published in a premier league journal it will be recognized as first rate; if it appears somewhere in the lower divisions, it will be rated (and not funded) accordingly.\n\nThis initiative is entirely defective in conception and execution. Consider the major issues of accountability and transparency. The process of producing the graded list of journals in science studies was overseen by a committee of four (the membership is currently listed at http:\/\/www.esf.org\/research-areas\/humanities\/research-infrastructures-including-erih\/erih-governance-and-panels\/erih-expert-panels.html). This committee cannot be considered representative. It was not selected in consultation with any of the various disciplinary organizations that currently represent our field such as the European Association for the History of Medicine and Health, the Society for the Social History of Medicine, the British Society for the History of Science, the History of Science Society, the Philosophy of Science Association, the Society for the History of Technology or the Society for Social Studies of Science. Journal editors were only belatedly informed of the process and its relevant criteria or asked to provide any information regarding their publications. No indication has been given of the means through which the list was compiled; nor how it might be maintained in the future.\n\nThe ERIH depends on a fundamental misunderstanding of conduct and publication of research in our field, and in the humanities in general. Journals' quality cannot be separated from their contents and their review processes. Great research may be published anywhere and in any language. Truly ground-breaking work may be more likely to appear from marginal, dissident or unexpected sources, rather than from a well-established and entrenched mainstream. Our journals are various, heterogeneous and distinct. Some are aimed at a broad, general and international readership, others are more specialized in their content and implied audience. Their scope and readership say nothing about the quality of their intellectual content. The ERIH, on the other hand, confuses internationality with quality in a way that is particularly prejudicial to specialist and non-English language journals. In a recent report, the British Academy, with judicious understatement, concludes that \"the European Reference Index for the Humanities as presently conceived does not represent a reliable way in which metrics of peer-reviewed publications can be constructed\" (*Peer Review: the Challenges for the Humanities and Social Sciences*, September 2007: http:\/\/www.britac.ac.uk\/reports\/peer-review). Such exercises as ERIH can become self-fulfilling prophecies. If such measures as ERIH are adopted as metrics by funding and other agencies, then many in our field will conclude that they have little choice other than to limit their publications to journals in the premier division. We will sustain fewer journals, much less diversity and impoverish our discipline.\n\nAlong with many others in our field, this Journal has concluded that we want no part of this dangerous and misguided exercise. This joint Editorial is being published in journals across the fields of history of science and science studies as an expression of our collective dissent and our refusal to allow our field to be managed and appraised in this fashion. We, among others, have asked the compilers of the ERIH to remove our journals' titles from their lists.\n\nHal Cook and Anne Hardy (*Medical History*)\n\nHanne Andersen (*Centaurus*)\n\nRoger Ariew and Moti Feingold (*Perspectives on Science*)\n\nA K Bag (*Indian Journal of History of Science*)\n\nJune Barrow-Green and Benno van Dalen (*Historia Mathematica*)\n\nKeith Benson (*History and Philosophy of the Life Sciences*)\n\nMarco Beretta (*Nuncius*)\n\nMichel Blay (*Revue d'Histoire des Sciences*)\n\nJohanna Bleker (*Medizinhistorisches Journal*)\n\nCornelius Borck (*Berichte zur Wissenschaftsgeschichte*)\n\nGeof Bowker and Susan Leigh Star (*Science, Technology and Human Values*)\n\nMassimo Bucciantini and Michele Camerota (*Galilaeana: Journal of Galilean Studies*)\n\nJed Buchwald and Jeremy Gray (*Archive for History of Exact Sciences*)\n\nVincenzo Cappelletti and Guido Cimino (*Physis*)\n\nCathryn Carson (*Historical Studies in the Natural Sciences*)\n\nMark Clark and Alex Keller (*ICON*)\n\nRoger Cline (*International Journal for the History of Engineering & Technology*)\n\nStephen Clucas and Stephen Gaukroger (*Intellectual History Review*)\n\nLeo Corry, Alexandre M\u00e9traux and J\u00fcrgen Renn (*Science in Context*)\n\nBrian Dolan and Bill Luckin (*Social History of Medicine*)\n\nHilmar Duerbeck and Wayne Orchiston (*Journal of Astronomical History and Heritage*)\n\nMoritz Epple, Mikael H\u00e5rd, Hans-J\u00f6rg Rheinberger and Volker Roelcke (*NTM: Zeitschrift f\u00fcr Geschichte der Wissenschaften, Technik und Medizin)*\n\nPaul Farber (*Journal of the History of Biology*)\n\nMary Fissell and Randall Packard (*Bulletin of the History of Medicine*)\n\nRobert Fox (*Notes & Records of the Royal Society*)\n\nMarina Frasca-Spada (*Studies in History and Philosophy of Science*)\n\nSteven French (*Metascience*)\n\nJim Good (*History of the Human Sciences*)\n\nWillem Hackmann (*Bulletin of the Scientific Instrument Society*)\n\nRobert Halleux (*Archives Internationales d'Histoire des Sciences*)\n\nBosse Holmqvist (*Lychnos*)\n\nRod Home (*Historical Records of Australian Science*)\n\nMichael Hoskin (*Journal for the History of Astronomy*)\n\nIan Inkster (*History of Technology*)\n\nNick Jardine (*Studies in History and Philosophy of Biological and Biomedical Sciences*)\n\nTrevor Levere (*Annals of Science*)\n\nBernard Lightman (*Isis*)\n\nChristoph L\u00fcthy (*Early Science and Medicine*)\n\nMichael Lynch (*Social Studies of Science*)\n\nStephen McCluskey and Clive Ruggles (*Archaeastronomy: The Journal of Astronomy in Culture*)\n\nPeter Morris (*Ambix*)\n\nIwan Rhys Morus (*History of Science*)\n\nE Charles Nelson (*Archives of Natural History*)\n\nIan Nicholson (*Journal of the History of the Behavioral Sciences*)\n\nKathy Olesko (*Osiris*)\n\nLiliane P\u00e9rez (*Documents pour l'Histoire des Techniques*)\n\nJohn Rigden and Roger H Stuewer (*Physics in Perspective*)\n\nJulio Sams\u00f3 (*Suhayl: Journal for the History of the Exact and Natural Sciences in Islamic Civilisation*)\n\nSimon Schaffer (*British Journal for the History of Science*)\n\nNorbert Schappacher (*Revue d'Histoire des Math\u00e9matiques*)\n\nJohn Staudenmaier SJ (*Technology and Culture*)\n\nClaire Strom (*Agricultural History*)\n\nPaul Unschuld (*Sudhoffs Archiv*)\n\nPeter Weingart (*Minerva*)\n\nStefan Zamecki (*Kwartalnik Historii Nauki i Techniki*)\n\nHuib Zuidervaart (*Studium: Tijdschrift voor Wetenschaps- en Universiteitgeschiedenis* \/*Revue de l'Histoire des Sciences et des Universit\u00e9s*)","meta":{"dup_signals":{"dup_doc_count":138,"dup_dump_count":77,"dup_details":{"curated_sources":2,"2023-50":5,"2023-14":2,"2023-06":2,"2022-49":3,"2022-40":2,"2022-33":1,"2022-27":3,"2022-21":2,"2022-05":1,"2021-49":1,"2021-43":2,"2021-39":1,"2021-25":1,"2021-21":2,"2021-17":2,"2021-10":2,"2020-45":2,"2020-29":1,"2020-10":1,"2019-47":1,"2019-39":2,"2019-30":2,"2019-22":2,"2019-18":3,"2019-09":3,"2018-51":3,"2018-47":1,"2018-39":1,"2018-30":2,"2018-26":4,"2018-13":3,"2018-09":1,"2018-05":2,"2017-51":3,"2017-47":3,"2017-43":3,"2017-34":3,"2017-30":2,"2017-26":4,"2017-17":4,"2017-09":1,"2017-04":2,"2016-50":2,"2016-44":1,"2016-40":1,"2016-36":1,"2016-30":1,"2016-26":1,"2016-22":1,"2016-18":1,"2016-07":1,"2015-48":1,"2015-40":1,"2015-35":1,"2015-32":1,"2015-27":1,"2015-22":1,"2015-14":1,"2014-52":1,"2014-49":2,"2014-42":3,"2014-41":1,"2014-35":1,"2014-23":2,"2014-15":2,"2024-26":2,"2024-22":1,"2024-18":1,"2024-10":1,"2017-13":4,"2015-18":1,"2015-11":1,"2015-06":1,"2014-10":1,"2013-48":1,"2013-20":1,"2024-30":1}},"file":"PMC2629173"},"subset":"pubmed_central"}
{"text":"abstract: A report on the 10th annual Research in Computational Molecular Biology (RECOMB) Conference, Venice, Italy, 2-5 April 2006.\nauthor: Balaji S Srinivasan; Chuong B Do; Serafim Batzoglou\ndate: 2006\ninstitute: 1Department of Electrical Engineering, Stanford University, Stanford CA 94305, USA; 2Department of Developmental Biology, Stanford University, Stanford CA 94305, USA; 3Department of Computer Science, Stanford University, Stanford CA 94305, USA\ntitle: Evidence for intelligent (algorithm) design\n\nMore than 700 computational biologists convened in beautiful Venice in early April for RECOMB 2006, the 10th annual Conference on Research in Computational Molecular Biology. After 40 talks, 6 keynote lectures, 180 posters, and at least two cameos by the Riemann zeta function, several emerging trends in computational biology are apparent.\n\nFirst, there has been a strong shift towards empirical studies of molecular evolution and variation, with approximately 25% of the papers in this broad area. We expect that this number can only increase in the near future, given the ENCODE project <http:\/\/www.genome.gov\/10005107> and the forthcoming release of several new eukaryotic genomes.\n\nSecond, there is a resurgence of interest in two of the oldest problems in computational biology: RNA folding and protein sequence alignment. The interest in noncoding RNAs (ncRNAs) is driven by experiment: recent work on RNA interference (RNAi), microRNAs, ribozymes, and the rest of the 'modern RNA world' has once again stimulated interest in the classical problems of ncRNA identification and fold prediction. Advances in protein sequence alignment draw on the development of new algorithmic and machine-learning techniques for principled estimation of gap penalties (the penalty for inserting a gap in the alignment to improve it) and the rigorous incorporation of non-local similarity measures that move beyond residue-residue similarity.\n\nInterest in classic areas such as protein structure and folding remains strong, with several papers tacitly or explicitly motivated by the coming flood of data promised by structural genomics. Many of the other mainstays of computational biology were also represented at the conference, including old favorites such as expression analysis and genome evolution, as well as the newer areas of data integration and network alignment. Notable by their absence were papers on genome assembly and human single-nucleotide polymorphism (SNP) variation; this is likely to be a fluke rather than a trend, however, given the impending deluge of data from high-throughput sequencing and resequencing projects. We have selected a few of the talks that particularly caught our eye out of the many excellent ones given at the conference.\n\n# Focus on ncRNA folding\n\nOne of the highlights of the conference was the demonstration by Ydo Wexler (Technion-Israel Institute of Technology, Haifa, Israel) of a quadratic time algorithm for RNA folding, a result that deservedly won a special mention award. For several decades, RNA folding algorithms had running times that scaled with at least the cube of the length of the RNA sequence. This O(L^3^) time complexity worsens further if pseudoknots are involved in the folding model. By combining a simple 'triangle inequality' heuristic with empirical validation of the 'polymer zeta' behavior of RNA folding, Wexler and colleagues developed an O(L^2^) average time algorithm for RNA folding, a result which makes high-throughput ncRNA prediction far more feasible.\n\nThe pseudoknot, a fold comprising two or more helical segments connected by single-stranded loops, was the subject of a talk by Banu Dost (University of California, San Diego, USA), who presented a new algorithm for aligning a subset of ncRNAs with computationally tractable pseudoknots to a database of known ncRNA sequences. Sequences that interact to serve a structural or biochemical function often show coevolution. In this regard, Jeremy Darot (University of Cambridge, UK) presented a general probabilistic graphical model for detecting interdependent evolution between sites in nucleic acid and protein sequences, which he applied to the problem of identifying secondary and tertiary structure interactions in tRNA.\n\n# Interaction networks and microarray analysis\n\nThe broad area of functional genomics encompasses methods for the prediction of gene function and interaction. Talks covered the inference and comparison of protein-interaction networks, and the statistical issues associated with the detection of functional enrichment in microarray data. One of us (B.S.S) described an algorithm for integrating a number of different predictors of protein interaction without making assumptions about statistical dependence. He showed that this approach revealed hidden interactions that would not have been found without data integration, and used the method to produce probabilistic protein-interaction networks for 11 microbes. Benny Chor (Tel-Aviv University, Tel-Aviv, Israel) presented work on graphs of metabolic reactions from different species, showing that a taxonomy inferred from network-based characters corresponded fairly well to the known consensus phylogeny.\n\nThe problem of comparing large collections of networks from different species motivates work on network alignment, whose goal is to detect conserved modules between networks. By analogy with the existing theory for sequence alignment, Mehmet Koyut\u00fcrk (Purdue University, West Lafayette, USA) presented an asymptotic theory for estimating the statistical significance of network alignments, with respect to certain classes of large random networks. Developing a version of this theory applicable to alignments of few proteins, which are more common in practice, is an open problem.\n\nSteffen Grossmann (Max Planck Institute for Molecular Genetics, Berlin, Germany) presented an improved statistic for estimating the functional enrichment of gene sets based on Gene Ontology (GO) that takes account of the complex parent-child dependencies in the GO hierarchy (this statistic is implemented in the Ontologizer package available at <http:\/\/www.charite.de\/ch\/medgen\/ontologizer>). Stefanie Scheid (Max Planck Institute for Molecular Genetics) presented a novel permutation-filtering technique for the detection of differentially expressed genes in microarray analysis. Her method filters the results of a naive data permutation to estimate a more accurate null distribution, and her work is implemented in the Twilight package available online <http:\/\/www.bioconductor.org>.\n\n# Parameter estimation in protein sequence alignment\n\nTwo speakers addressed the issue of estimating parameters such as substitution scores and gap penalties for protein sequence alignment. John Kececioglu (University of Arizona, Tucson, USA) provided a solution to the 'inverse sequence alignment' problem, where one estimates parameter values from a training set of alignments. He described a linear programming algorithm for determining a set of alignment parameters under which every example alignment in a given training set is guaranteed to be nearly optimal with respect to that parameter set. The algorithm can learn both residue substitution and gap scores simultaneously, and it will be interesting to see how the resulting parameters perform when used to make new alignments.\n\nOne of us (C.B.D) introduced pair-conditional random fields for incorporating non-local sequence similarities (such as hydropathy) into the alignment scoring framework. As such similarities are functions of peptide windows of variable length rather than of individual residues, they cannot easily be incorporated into standard methods based on hidden Markov models (HMMs) for sequence alignment without heuristics. The resulting algorithm, CONTRAlign (source code available online <http:\/\/contra.stanford.edu\/contralign>), achieves the highest cross-validated pairwise protein alignment accuracies to date.\n\n# Protein structure, dynamics and identification\n\nPerhaps the biggest obstacle to deriving insights from protein structure is the sheer size and complexity of a typical polypeptide. Addressing the problem of protein structure alignment, Wei Xie (University of Illinois at Urbana-Champaign, USA) and Jinbo Xu (Massachusetts Institute of Technology, Cambridge, USA) manage this complexity by focusing on maps of intra-protein contacts. Xie presented work on aligning structures by overlapping their contact maps, by developing a brand-and-reduce algorithm that allows rapid superposition of structurally homologous proteins. By analogy with sequence alignment, Xu presented a polynomial time-parametric algorithm for aligning a protein represented by a contact map to another protein represented by a contact map or an interatomic distance matrix.\n\nMany scientists are interested not just in alignments of stable protein structures, but also in the dynamics of the folding process. Chakra Chennubhotla (University of Pittsburgh, Pittsburgh, USA) reduced the complexity of an all-atom protein simulation by calculating a low-rank, eigenmode-based approximation to the molecular dynamics that is designed to preserve certain stochastic properties of the original protein. Shawna Thomas (Texas A&M University, College Station, USA) took a technically different but conceptually similar approach by approximating a protein as a chain of rigid bodies and then sampling its conformation space with a probabilistic roadmap method imported from motion planning for robotics (for further details, see the parasol website <http:\/\/parasol.tamu.edu\/foldingserver>). The 'roadmap' in the protein context contains thousands of feasible folding pathways.\n\nThe ultimate purpose of protein-folding simulation (as distinct from protein-structure prediction) is to use the observed dynamics to yield insight into aspects of protein biochemistry, such as cooperativity or macromolecular assembly. To this end, Tsung-Han Chiang (National University of Singapore, Singapore) showed that the probabilistic roadmap framework can be used to calculate the probability of proper folding from any given protein conformation, and then to estimate protein-folding rates.\n\nTwo speakers addressed problems of fast protein identification by clever hashing methods. Brian Chen in collaboration with Viacheslav Fofanov (both from Rice University, Houston, Texas, USA) showed that one can use geometric hashing techniques to speed up the identification of three-dimensional structural motifs in functionally uncharacterized proteins of known structure. In a different problem domain, Nuno Bandeira (University of California, San Diego, USA) demonstrated a rapid hashing algorithm for identifying proteins from tandem mass spectrometry (MS\/MS) spectra. The input protein sample is split into two groups, chemical modifications are applied to one group, and spectra are obtained for both groups. Bandeira showed how using correlations between the two spectra greatly reduces the noise of protein identification.\n\n# Reconstructing the past\n\nTalks on evolution and phylogenetics included richer models of sequence evolution, new methods for tree building, and applications of molecular evolution to questions in functional genomics. On the topic of richer models for deducing phylogeny from sequences, Yun Song (University of California, Davis, USA) described a method for including gene conversion in reconstructions of SNP phylogenies (software available online <http:\/\/www.cs.ucdavis.edu\/~gusfield>); existing methods typically incorporate only point mutation and recombination as possible events. Sagi Snir (University of California, Berkeley, USA) presented work on the inference of micro-indel events (insertions and\/or deletions) from multiple sequence alignments; the method has a time-complexity that is exponential in the number of species, but is linear in terms of sequence length. Mikl\u00f3s Cs\u00fcr\u00f6s (Universit\u00e9 de Montr\u00e9al, Montreal, Canada) dealt with gene evolution. He described a parametric model for gene family evolution that models gene duplication, gene loss, and (most significantly) horizontal gene transfer.\n\nWith respect to the general problem of building trees from data, Constantinos Daskalakis (University of California, Berkeley, USA) described an algorithm for calculating phylogenies from distance matrices (compiled from the differences between sequences), which compares favorably to neighbor-joining on specific examples, without requiring strong assumptions about possible model tree topologies. Adam Siepel (University of California, Santa Cruz, USA) addressed the problem of using molecular evolution to detect functional elements in genomic sequences. He has extended the phastCons program, a phylogenetic HMM model for segmenting a genomic sequence into conserved and nonconserved regions, by introducing lineage-specific models which allow for simple gains or losses of constraint along specific branches of the evolutionary tree relating the sequences. The output of the program, called DLESS, is available as a track on the University of California Santa Cruz genome browser <http:\/\/genome.ucsc.edu\/encode>.\n\nEvolution also figured prominently in the only talk at the conference given by a non-scientist. In his keynote address, author and journalist Carl Zimmer warned of the perils of 'genomic myopia' and challenged computational molecular biologists to create a model of life's evolution that was consistent with the wealth of knowledge from paleontology and the fossil record. Given the rapid advance of bioinformatics apparent at RECOMB 2006, we have no doubt that our community is up to the challenge.","meta":{"dup_signals":{"dup_doc_count":125,"dup_dump_count":46,"dup_details":{"curated_sources":4,"2020-40":1,"2020-24":1,"2019-30":1,"2019-22":1,"2019-09":1,"2018-51":1,"2018-47":1,"2018-39":1,"2018-30":1,"2018-13":1,"2017-47":1,"2017-34":1,"2017-26":1,"2017-17":2,"2017-09":10,"2017-04":1,"2016-50":1,"2016-44":1,"2016-40":1,"2016-36":10,"2016-30":8,"2016-22":1,"2016-18":1,"2015-48":3,"2015-40":2,"2015-35":3,"2015-32":3,"2015-27":3,"2015-22":3,"2015-14":2,"2014-52":3,"2014-49":3,"2014-42":9,"2014-41":6,"2014-35":4,"2014-23":4,"2014-15":3,"2021-39":1,"2017-13":1,"2015-18":3,"2015-11":3,"2015-06":3,"2014-10":3,"2013-48":2,"2013-20":4,"2024-22":1}},"file":"PMC1779572"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  Self-management is rarely studied 'in the wild'. We sought to produce a richer understanding of how people live with diabetes and why self-management is challenging for some.\n  .\n  # Method\n  .\n  Ethnographic study supplemented with background documents on social context. We studied a socio-economically and ethnically diverse UK population. We sampled 30 people with diabetes (15 type 1, 15 type 2) by snowballing from patient groups, community contacts and NHS clinics. Participants (aged 5-88, from a range of ethnic and socio-economic groups) were shadowed at home and in the community for 2-4 periods of several hours (total 88 visits, 230 hours); interviewed (sometimes with a family member or carer) about their self-management efforts and support needs; and taken out for a meal. Detailed field notes were made and annotated. Data analysis was informed by structuration theory, which assumes that individuals' actions and choices depend on their dispositions and capabilities, which in turn are shaped and constrained (though not entirely determined) by wider social structures.\n  .\n  # Results\n  .\n  Self-management comprised both practical and cognitive tasks (e.g. self-monitoring, menu planning, medication adjustment) and socio-emotional ones (e.g. coping with illness, managing relatives' input, negotiating access to services or resources). Self-management was hard work, and was enabled or constrained by economic, material and socio-cultural conditions within the family, workplace and community. Some people managed their diabetes skilfully and flexibly, drawing on personal capabilities, family and social networks and the healthcare system. For others, capacity to self-manage (including overcoming economic and socio-cultural constraints) was limited by co-morbidity, cognitive ability, psychological factors (e.g. under-confidence, denial) and social capital. The consequences of self-management efforts strongly influenced people's capacity and motivation to continue them.\n  .\n  # Conclusion\n  .\n  Self-management of diabetes is physically, intellectually, emotionally and socially demanding. Non-engagement with self-management may make sense in the context of low personal resources (e.g. health literacy, resilience) and overwhelming personal, family and social circumstances. Success of self-management as a policy solution will be affected by interacting influences at three levels: \\[a\\] at micro level by individuals' dispositions and capabilities; \\[b\\] at meso level by roles, relationships and material conditions within the family and in the workplace, school and healthcare organisation; and \\[c\\] at macro level by prevailing economic conditions, cultural norms and expectations, and the underpinning logic of the healthcare system. We propose that the research agenda on living with diabetes be extended and the political economy of self-management systematically studied.\nauthor: Susan Hinder; Trisha Greenhalgh\ndate: 2012\ninstitute: 1Centre for Primary Care and Public Health, Barts and The London School of Medicine and Dentistry, London E1 2AB, UK\nreferences:\ntitle: \"This does my head in\". Ethnographic study of self-management by people with diabetes\n\n# Background\n\nPrevalence of diabetes is rising rapidly in most countries. Epidemiologists predict that within a generation, demand will outstrip supply for essential medical services \\[1\\]. Based on economic evaluations of randomised controlled trials, some researchers have estimated that educating people to self-manage their diabetes will significantly increase capacity in the healthcare system \\[2,3\\]. Neither self-management nor self-care has a universally agreed definition. The former is sometimes viewed as a complex construct which, in addition to the biomedical tasks of self care (defined as the various behaviours linked to managing illness and promoting and restoring health \\[4\\]), includes personal, cultural, social and political dimensions such as making sense of illness, rebuilding what sociologists have called \"spoiled identity\" after the diagnosis of a potentially stigmatising condition and lobbying for disability rights \\[5-10\\]. Lorig and Holman define self management as the full range of tasks undertaken by a person with chronic illness, including medical management, role management and emotional management \\[11\\].\n\nThe centrepiece of self-management policy in UK has been the Expert Patient Programme, which aims to increase independence and reduce use of health services \\[12\\]. Whilst self-efficacy has improved in people completing the Expert Patient Programme, improvements in disease outcomes have been modest or absent, especially in lower socio-economic and minority ethnic groups \\[13\\]. The programme had zero impact, for example, on type 2 diabetes in Hackney's multi-ethnic, inner-city population (Griffiths C, personal communication, unpublished results of MEDEA trial). Diabetes-specific structured education programmes include DAFNE ('dose adjustment for normal eating') and DESMOND ('diabetes education and self management for ongoing and newly diagnosed') for type 1 and type 2 diabetes respectively, which have led to significant improvements in disease outcomes compared to usual-care controls in controlled trials \\[14,15\\]. But they have been criticised for focusing too heavily on a relatively narrow biomedical skill set, and the transferability of such programmes to people with low health literacy and\/or complex personal or social circumstances has been questioned \\[5,16,17\\].\n\nQuestionnaire studies have shown a positive association between psychological constructs (such as self-efficacy and locus of control) and effective diabetes self-management \\[6,18,19\\]. Researchers who invited people with diabetes to describe their experiences have concluded that self-management is culturally embedded, dependent on core knowledge and understanding, and improves with family and social support (see Discussion) \\[20-28\\]. Given the high research and policy interest in self-management, it is surprising that there are almost no recent research studies of self-management in individuals going about their daily lives. A classic sociological study of \"living at home with chronic illness\" was published in 1988, based on interviews rather than direct observation \\[29\\]. More recently, Korean researchers observed eating practices in 15 people with diabetes and concluded that cultural meanings were important in food choices \\[30\\]. An ethnographic study in Thailand of 33 people with diabetes found no evidence of \"spoiled identity\", perhaps because diabetes did not have visible manifestations in most people; but participants spent much time and effort in self-management work (diet control, exercise, clinic attendance, self-monitoring) in order to achieve what they described as \"normality\" \\[31\\].\n\nPeople with diabetes spend around 1% of their time in contact with health professionals; the other 99% is virtually a closed book to clinicians and researchers. Our study sought to fill this evidence gap by directly observing how people live with diabetes in the home, family and community - and especially to identify the positive and negative influences on effective self-management.\n\n# Methods\n\n## Theoretical and methodological approach\n\nWe adopted a sociological perspective drawn from structuration theory. In its original formulation, Giddens proposed a two-part (structure-agency) relationship: social structures (such things as social norms, moral codes, meaning-systems and political-economic institutions) both shape and constrain individual agency (people's actions and choices), which in turn serve to reproduce and shape social structures \\[32\\]. More recently, Stones has refined and extended structuration theory to accommodate the complexity and inherent contradictions of the social order in today's globalised, multicultural, networked and technology-dominated society \\[33\\]. He invites researchers to consider four aspects of the structure-agency relationship as it plays out in social situations: (a) external structures (the physical, social or economic environment in which actions are contemplated and take place, which are strongly influenced by an individual's position in society and which may be either enabling or constraining); (b) internal structures (what individuals 'know', including general life experience, educational and cultural background, and personal morals; and also the beliefs, assumptions, skills and capabilities that are relevant to a specific, here-and-now action or choice); (c) active agency (in which individuals draw on their stock of knowledge and experience and their assessment of a social situation to inform particular actions or choices); and (d) outcomes (which may feed back on both external and internal structures, reproducing and perhaps changing these). These are shown diagrammatically in Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>.\n\nIn this model, 'what individuals know' is not limited to their cognitive awareness of facts; it also includes perspectival or partial understandings (such as lay epidemiology), unconscious emotional urges (such as the desire to please an authority figure), and past experiences (such as a previous confrontation over 'bad' test results). In any social experience, the individual draws not merely on his or her own internal structures (what he or she 'knows' about the social world), but on their knowledge of the internal structures of other agents (i.e. what A thinks B 'knows'). For example, a patient from a particular socio-cultural background makes assumptions about what their clinician would expect, and tailors their own action accordingly.\n\nStones' adaptation of structuration theory thus combines an etic perspective (considering *external* social structures independently of individuals' understanding and perception) and an emic one (considering *internal* social structures via a hermeneutic study of what individuals 'know'), along with actions and outcomes. It is well suited to studying how people's actions and choices are situated within a particular meso- (home, family, organisation) and macro- (socio-cultural, political-economic) context. The study design recommended by Stones is ethnographic observation, supplemented by analysis of background data on the wider social context. The ethnographer immerses him or herself in a social situation and collects naturalistic data (i.e. real-world observations and naturally-occurring talk rather than 'under experimental conditions') in a pragmatic, reflexive and emergent way \\[34\\]. Ethnographic data are rich in qualitative description, allowing the researcher to interpret what is \"really going on\". Significantly, ethnography allows the researcher to capture data on what people actually do, rather than what they say they do. In this study, we sought to use an ethnographic approach to explain why individuals in different situations and settings took particular actions and choices relating to diabetes (including choosing *not* to undertake what a health professional would call 'self-management').\n\n## Sampling and recruitment\n\nIn order to capture as wide a range of illness experience as possible, we sought a sample of 30 people with diabetes representing maximum variety by age, gender, ethnicity, health literacy, IT literacy, stage and severity of diabetes, level of family support and socio-economic status. We recruited from a range of settings including a local diabetes service spanning primary and secondary care, patient groups, community contacts and ethnic organisations in four counties in northern England. We used snowball sampling from our initial participants (i.e. we asked them to nominate another person with diabetes known to them). All participants gave written informed consent or assent. For the three participants under 13 and one adult with a mild learning disability, we used a specially designed assent procedure and form, and included the parents or designated carer in the consent process and the ethnography. Two participants were limited English speakers; they chose to have a lay (family member) interpreter rather than a professional interpreter.\n\n## Data collection\n\nData collection occurred between 2008 and 2011. Our dataset was designed to inform the four analytic levels shown in Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>. In particular, we sought to inform both an etic analysis (from the researchers' perspective) of the wider economic, social and material context in which self-management took place or was contemplated, as well as an emic analysis (from the participants' perspective) of what individuals 'knew', including their cultural and educational background and the overall interpretive schema through which they made sense of their world, as well as the particular knowledge, abilities, practical skills, assumptions and desires which were brought to bear (or not) on the management of their diabetes. The data sources for the different levels of analysis are shown in Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>.\n\nData sources and analytic methods for structuration analysis of diabetes self-management\n\n<table>\n<thead>\n<tr>\n<th style=\"text-align: left;\">Level of analysis<\/th>\n<th style=\"text-align: left;\">Data sources<\/th>\n<th style=\"text-align: left;\">Analytic approach<\/th>\n<th style=\"text-align: left;\">Output<\/th>\n<\/tr>\n<\/thead>\n<tbody>\n<tr>\n<td style=\"text-align: left;\">External structures<\/td>\n<td style=\"text-align: left;\">National policy and policy-related documents e.g. public health strategy, NICE guidance, agreements with the<br \/>\nfood industry<br \/>\nPublicly accessible data on local demographics, disease patterns (from local public health reports) and built environment (food<br \/>\noutlets, leisure centres)<br \/>\nIndex of multiple deprivation<br \/>\nfor locality<br \/>\nField notes on neighbourhoods, homes, workplaces, schools,<br \/>\nfood outlets<\/td>\n<td style=\"text-align: left;\">Thematic analysis of documents<br \/>\nin historical context and texts. Findings drawn together by narrative synthesis to identify overall themes and key changes over time.<\/td>\n<td style=\"text-align: left;\">'Etic' understanding (from the researchers' perspective) of the wider structural context in which 'self-management' is contemplated and takes place<\/td>\n<\/tr>\n<tr>\n<td colspan=\"4\"><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Internal structures<\/td>\n<td style=\"text-align: left;\">Ethnographic observations and naturalistic interviews with participant, including:<br \/>\nParticipant's explanations of<br \/>\nwhat they were doing and why<br \/>\nParticipant's drawing of 'my diabetes'<br \/>\nParticipant's spontaneously disclosed beliefs, values and assumptions<\/td>\n<td style=\"text-align: left;\">Phenomenological analysis.<br \/>\nWhere a series of interviews<br \/>\nwas obtained from one agent,<br \/>\nthese were analysed<br \/>\nlongitudinally for change<br \/>\nover time<\/td>\n<td style=\"text-align: left;\">Hermeneutic understanding of dispositions, perceptions and understandings of the index case and other relevant actors<\/td>\n<\/tr>\n<tr>\n<td><\/td>\n<td><\/td>\n<td><\/td>\n<td><\/td>\n<\/tr>\n<tr>\n<td><\/td>\n<td style=\"text-align: left;\">Ethnographic observations and naturalistic interviews with other relevant actors e.g.<br \/>\nParent and teacher (if a child)<br \/>\nPartner or carer<br \/>\nFriends<br \/>\nAdult children<\/td>\n<td><\/td>\n<td><\/td>\n<\/tr>\n<tr>\n<td colspan=\"4\"><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Actions<\/td>\n<td style=\"text-align: left;\">Ethnographic observation of<br \/>\nparticipant in activities of daily living at home, work, school<br \/>\nMeal in a caf\u00e9<br \/>\nTaking exercise (if this occurred)<br \/>\nVisiting a health professional<br \/>\n(if this occurred)<\/td>\n<td style=\"text-align: left;\">Interpretive analysis of actions in context, drawing on theories of symbolic interactionism and ethnomethodology<\/td>\n<td style=\"text-align: left;\">Understanding of why the agent-in-focus acted in particular ways in particular situations<\/td>\n<\/tr>\n<tr>\n<td colspan=\"4\"><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Outcomes<\/td>\n<td style=\"text-align: left;\">Study of immediate consequences<br \/>\nof action e.g. via direct ethnographic observation<\/td>\n<td style=\"text-align: left;\">Interpretive analysis of actions in context<\/td>\n<td style=\"text-align: left;\">Understanding of the short-term intended and unintended impact of social action<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\nMany potential data sources were relevant to our etic study of context, but for practical reasons we limited the dataset to sources we considered to have the most direct bearing on the social, cultural and political context of self-management decisions and actions: \\[a\\] policy and policy-related documents on economic conditions, diabetes management, food labelling and promotion of positive behaviour choices; \\[b\\] our ethnographic observations in the home, neighbourhood and, where accessible, the participant's work or school environment (see details below); and \\[c\\] the Index of Multiple Deprivation (IMD) of the participant's home postcode. IMD is a composite score for a small neighbourhood, derived from aggregated responses to the ten-yearly national census and with some but not all components updated annually using local statistics. It consists of 38 indicators of deprivation grouped in 7 domains: household income, employment, health status and disability, crime, skills and training (including but not limited to formal education), barriers to housing and services, and living environment (including access to open spaces and leisure facilities). IMD scores for localities across the UK range from 0.4 to 84 (median 17.1); higher scores indicate more severe deprivation.\n\nFor the emic component of the study, we used three data sources, described in detail below: \\[a\\] ethnographic observation of the participant; \\[b\\] naturalistic interviews; and \\[c\\] a drawing of \"my diabetes\". We undertook between two and four periods of detailed ethnographic observation on each participant (230 person-hours of observation in all). We accompanied them for periods of 2-5 hours as they went about their daily life, noting issues to do with their condition as these arose in conversation, actions or events. We were interested in how participants managed their diabetes; what their information, communication and support needs were in relation to it and how they addressed them (or why they didn't). We noted any individuals (e.g. partner, other relatives, friends, peers, GP, specialist nurse) who helped the participant manage their condition and the technologies (e.g. blood glucose meter, insulin pump, telephone) which they or their carers made use of.\n\nIn half the cases, we observed the participant making a meal or snack (or one being made for them) and eating it in the home environment. We also took all but four participants to a local caf\u00e9 or restaurant (selected by them) where they were invited to order a meal and a drink. This provided an opportunity to observe how diabetes affected their food choices and how (if relevant) they coped with measuring blood sugar and injecting insulin. Four participants invited the researcher to accompany them on visits to a health professional in relation to their diabetes (nurse, podiatrist, optometrist, pharmacist); in these visits, verbal informed consent was sought, and in all cases obtained, from the health professional for the researcher to be present. We went on walks with two participants and to the gym with one.\n\nDuring the ethnography, we conducted naturalistic interviews (i.e. we interviewed the participant and any relatives or friends they chose to have with them, as they went about daily activities, and asked them to explain their perspective on things which came up spontaneously - for example why they chose a particular meal in a caf\u00e9, or why they were upset in a situation). Whist we did not use a formal topic list, we were particularly interested in knowledge needs for managing diabetes and information sources used to meet those needs. Rather, we used a conversational format, taking cues from things that interested or surprised us. To help engage the participant in the research, and to add to the diversity of data sources, we invited them to draw a picture of \"my diabetes\", showing themselves in the centre plus the people and technologies involved in managing the illness. We used the drawing activity mainly to engage participants (because people talked about their diabetes while considering what to draw), and some people preferred to describe rather than draw their illness. We made brief contemporaneous notes and spent several hours immediately afterwards annotating these and adding further recollections and reflections. This approach had the advantage of being minimally intrusive to participants but did not allow us to capture verbatim talk.\n\n## Data interpretation and analysis\n\nThe different analytic approaches used for our large and diverse dataset are summarised in Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>. Following Atkinson and Hammersley, who emphasise the value of observing naturally occurring talk and action and interpreting these in context \\[35\\], we placed greater significance on what people did, and how we observed them to react to events in real-life situations, than on what they said they believed, \"would do\" or \"had done\". To analyse our ethnographic notes, we used a traditional approach of immersion in the text through repeated reading (and studying any drawings in a similar way), thereby developing provisional analytic categories and iteratively refining these categories by the constant comparative method (comparing our analysis to date with new data as these emerged) \\[36\\]. To help achieve this, we used an Excel spreadsheet to aid initial data management and support a preliminary framework analysis to gain familiarity with the data, and Atlas Ti qualitative software for a more detailed thematic analysis, again chiefly to gain familiarity with the data before going on to apply the strong structuration theory approach shown in Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>. Each researcher prepared an initial interpretation of several cases before meeting to combine these and resolve discrepancies through discussion; and this process was repeated as more cases were added.\n\nTo achieve the four-level analytic approach shown in Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>, we first produced a description of the macro context, then considered the individual backgrounds and world views represented in our sample. Next, we considered particular actions or incidents relating to self-management in detail to explore how the possibilities available to the person were influenced by the macro economic, social, cultural and material context, how this context shaped and constrained their actions and choices in this incident, and how the outcomes of action (or inaction) fed back into the system at both micro and macro level. This involved both individual reflection by both researchers and discussion between us. We offered participants the opportunity to hear our interpretation of their case verbally, or see it in writing, and feed back comments or factual corrections We anonymised cases for publication using ethnically appropriate pseudonyms and an adaptation of the critical fiction technique (i.e. we systematically fictionalised identifying details in a way which retained essential themes of the clinical case) \\[37\\].\n\n# Results and discussion\n\n## Description of study participants\n\nOur 30 participants ranged in age from 5 to 88 (median 48.5); 22 were white British, 7 British South Asian (two of whom were limited English speakers) and one African. 22 were taking insulin, of whom 15 described themselves as 'type 1'. Duration of diabetes ranged from 1 to 48 years (median 9). Only 7 of the 30, including the five aged 16 or under, felt they had no other illnesses apart from diabetes; 17 described complications of diabetes. The index of multiple deprivation (IMD) score of their home postcode ranged from 8.6 to 76.1 (median 40.6). Of the 25 participants over 16, 8 were in paid work, 4 retired and 11 drawing benefits; three preferred not to say. Three were educated to degree level and four more had A-levels; the others had secondary education and\/or diploma certificates or preferred not to say. In sum, this sample broadly reflected the demographics of people with diabetes, including the higher prevalence of this condition in Asians and in lower socio-economic and less educated groups. It was deliberately skewed towards people treated with insulin (since self-management raises additional challenges in this group).\n\n## The wider context for self-management\n\nOur etic analysis revealed a number of overlapping external social structures (political\/policy, economic, socio-cultural, health system) within which the opportunities for, and constraints on, self-management were nested. We briefly outline those relevant to this study of self-management. Our field work occurred at a time (2008-11) of negative economic growth, including the worst recession since the second world war in 2009-10 (Office of National Statistics data). Unemployment levels rose over this period, as did job uncertainty, food and fuel prices, public transport costs, and personal debt - and these disproportionately affected the poorest tenth of society \\[38\\]. However, this was also a period of relatively generous funding for the National Health Service, when general practitioners were being offered strong financial incentives to prevent and manage diabetes; significant improvement in diabetes outcomes nationally occurred in this period \\[39\\].\n\nUK health policy throughout our data collection period emphasised individual responsibility for lifestyle choices rather than social determinants of health. For example, whilst the 2010 white paper *Healthy Lives, Healthy People* promised a \"radical new approach\" to public health based on life course epidemiology and talked in somewhat vague terms about \"empowering local communities\" \\[40\\], actual policies were based largely on behavioural economics and emphasised strategies to \"nudge\" individual behaviour through incentives, prompts and health education \\[41\\]. In the Public Health Responsibility Deal, for example, the food industry was encouraged to provide healthier eating options on a voluntary basis \\[40\\].\n\n## Individual dispositions and capacities for self-management\n\nOur ethnographic observations illustrated how the wider social, economic and cultural context described above influenced self-management via individuals' social position and via the cultural and material resources available to them. Importantly, however, these external structures did not wholly determine self-management behaviour, since our 30 participants varied considerably in the type of people they were and in the personal resources (abilities, skills and traits) they were able to bring to bear on self-management in any given context. For example, they differed widely in education, personality, ambition, motivation, cultural perceptions and beliefs, religiosity, self-confidence, health and information literacy, and how they perceived and framed their condition. Seven (including one with a diagnosed mild learning disability) described themselves as \"slow\" or having \"memory\" or \"understanding\" difficulties. Five had evident and severe disabilities such as paralysis, blindness, or heart failure, which made them dependent on others for basic care needs. Four more were largely housebound through a combination of non-specific disability, obesity, depression or low mood, generalised tiredness, sleep apnoea and lack of fitness. All these differences affected people's capacity and motivation to self-manage and how they went about this.\n\n## The work of self-management\n\nList: What people did when they 'self-managed' their diabetes\n\nPRACTICAL AND COGNITIVE TASKS\n\nMonitoring bodily symptoms (indicative of high or low blood glucose, high blood pressure, \"stress\") and taking steps to feel normal again\n\nShopping\n\nPreparing meals (sometimes separately from the rest of the family)\n\nExercising (usually, walking or attending gym)\n\nInjecting insulin or taking tablets\n\nCalculating variable insulin dosage by estimating nutritional content of food\n\nMeasuring and recording blood glucose levels\n\nCoping with 'hypos' or preventing them (by ensuring that they took food and drink when going out)\n\nMaintaining equipment e.g. replacing needles on insulin pen\n\nSeeking general information on diabetes from the Internet or libraries\n\nSeeking specific information relating to own diabetes e.g. from health professionals\n\nSOCIO-EMOTIONAL TASKS\n\nCoping with feelings about diabetes (including anger and frustration)\n\nMaking sense of information or advice from health professionals and\/or seeking confirmation of this from peers or other professionals\n\nMaintaining smoking cessation e.g. avoiding situations where they might be tempted to smoke\n\nExplaining to relatives, friends and work colleagues what diabetes is and how it is managed\n\nTraining relatives or work colleagues how to manage diabetic emergencies\n\nSeeking and\/or offering advice and support in diabetes peer support groups (e.g. Facebook)\n\nNegotiating access to health care (e.g. seeking appointments or referrals)\n\nObtaining resources (medication, blood testing strips, blood glucose meter)\n\nNegotiating the diagnosis, symptoms or management of diabetes with health professionals (including persuading doctors and nurses to engage with their self-management efforts)\n\nParents persuading children (and family members persuading the adult with diabetes) to consume recommended food\/drink and discouraging them from consuming 'bad' foods\n\nParents managing wider family dynamics (e.g. siblings feeling cheated when the family diet is restricted to align with the diabetic child)\n\nA strong over-arching theme in our data was the perception that self-management involved physical, cognitive or emotional *work*. By 'work' we mean an ongoing need to put in effort in order to achieve physical wellbeing and a sense of autonomy and social worth. A few expressed this directly, using metaphors like \"hassle\", \"grief\" or \"ball ache\" (see quote below). The list above shows examples of self-management tasks we observed directly, which correspond broadly to Corbin and Strauss's categories of 'illness work', 'identity work' and 'everyday life work' \\[29\\]. Much time was spent on practical tasks such as blood glucose testing (which some found painful and stressful), planning and preparing meals, matching food intake and energy expenditure with medication, and caring for feet, as well as on socio-emotional tasks including negotiating access to health services; obtaining resources for their diabetes care; and managing (i.e. encouraging, resisting or moderating) the input of family, friends and colleagues.\n\nA few insulin-treated participants drew confidently on an extensive knowledge base to make adjustments in their medication so as to accommodate variations in diet and activity.\n\n'Tina orders a panini. \\[..\\] She says she'll get her plate of food and look at it and decide the size of dose \\[for her insulin pump\\]. She informs me that there are 2 different ways of giving a bolus - all at once, for something very sugary such as fruit juice, which will cause blood sugar to increase straight away. Something like a white bread panini needs a half an hour split dose - 30% of the dose straightaway and the remainder over the following half an hour. Depending on protein, fat or fibre, she needs to increase the length of time the bolus is given over.'\n\nField notes and interview from caf\u00e9 visit with Tina, age 26, type 1 diabetes for 20 years\n\nTina had had little formal education and lived in a deprived locality (IMD score 32); she worked as a healthcare assistant. But she had recently engaged enthusiastically with an intensive diabetes education course and considered her life \"transformed\" by the pump. When asked for an image to depict her diabetes, she suggested \"a picture of me with diabetes always there, but in the background\". In short, she was competent in the work of self-management and this work was effective in achieving her goal of dietary freedom.\n\nInsulin-treated participants with limited knowledge and\/or confidence sometimes achieved 'self-management' by maintaining a strict and invariant dietary routine.\n\n'He said his mum used to give him spinach and ricotta cannelloni (microwaveable) and half a loaf of bread to take for his lunch. But after a year he got fed up of spinach and ricotta cannelloni butties every day. He asked his mum to get him something else so she got him beef lasagne (microwaveable) for the next six months.'\n\nNotes from naturalistic interview on visit to Callum, age 28, type 1 diabetes for 17 years\n\nWhilst Callum had subsequently discovered greater dietary freedom following a DAFNE course, his metaphor for diabetes was still \"a vice\" (meaning, we assume, that he had to put in considerable effort to achieve this freedom). Between these extremes, many insulin-treated participants had developed a simplified dietary regimen driven by a few rules of thumb.\n\nMany participants talked of \"balance\". But in contrast to the biomedical meaning of this term (physiological homeostasis), they saw self-management as relating to balance in their wider lives, including controlling personal stress levels, nurturing family and social relationships and achieving work-life balance. One aspect of \"balance\", for example, was between the immediate physical pleasure and social significance of food treats and the deferred benefits of a strict dietary regimen. Another was the balance between being open and transparent about the work of managing their diabetes and not burdening their friends and relatives with these issues.\n\n## The 'meso' environment of the home, family and social group\n\nOur 88 visits to participants' homes revealed striking differences in the material and social context for self-management. A few had comfortable houses which were well-equipped with working technologies, including broadband Internet and landline telephone, and located in settings close to fresh food sources and open spaces. But many of our participants enjoyed few or none of these benefits. There was a wide range of domestic arrangements from living alone (single, divorced or widowed); shared occupancy; nuclear, single-parent or 'blended' families; and multi-generational extended families. Participants' homes differed greatly in the availability of food - and in the symbolic meaning which food held within the family.\n\n'Very large bowl of sweets on the bookcase. Large bottle of Coke plus large jar of toffees on a small table at the side of Wendy's armchair. Terry's Chocolate Orange on small table at the other side of the armchair plus a bottle of tomato sauce. \\[..\\] Wendy explains that her mother (aged 79) buys her loads of \"goodies\" e.g. recently brought back 3 carrier bags full of biscuits, sweets, cakes, joints of meat, burgers, etc. She has tackled her Mum before about giving her and her children so much sweet stuff but her response is \"well, kids love goodies\".'\n\nField notes from visit to Wendy, age 58, type 2 diabetes for 18 years, is obese and has had two strokes\n\nFor many participants, the prevailing material and social conditions within the home and the immediate neighbourhood provided challenges to effective self-management (Table <a href=\"#T2\" data-ref-type=\"table\">2<\/a>). The impacts of poverty and family disruption were sometimes multiple and mutually reinforcing. Karl, for example, lived with his mother in a 2-bedroomed council house (IMD score 58); his brother lived with his father.\n\nChallenges to self-management in the home environment\n\n| Feature | Potential impact on self-management |\n|----|----|\n| Presence of sweets, sugared drinks and other health-negative foods, perhaps supplied by relatives as \"gifts\" | Difficulty following diabetes diet |\n|  |  |\n| Cramped housing | No dedicated place to keep diabetes monitoring equipment |\n|  |  |\n| Multiple occupants in the home, sometimes from several generations | Psychosocial stress from limited privacy and intergenerational conflict, with \\[perceived\\] impact on blood glucose control, blood pressure and lifestyle choices e.g. alcohol, smoking |\n|  |  |\n| Social problems within family (e.g. family member involved in drugs or crime; unemployment; domestic strife) | Psychosocial stress as above; family members less able to support the person with diabetes |\n|  |  |\n| Computer not working | More difficult to access health information and advice |\n|  |  |\n| Financial pressures | Food choices made primarily on the grounds of cost rather than nutritional value |\n|  |  |\n| Conflict with neighbours e.g. noise through walls | Psychosocial stress as above |\n|  |  |\n| Crime or fear of crime | Reluctance to exercise outside the home |\n\n'I ask if Karl goes to any activities outside school. He says he used to go to boxing but his dad lost his driving licence and they had to stop for three months and didn't start again.'\n\nField notes from visit to Karl, age 11, type 1 diabetes for 4 years, parents divorced\n\nWhilst only a minority of participants were in paid work, many had domestic and\/or carer duties, did voluntary work or were studying for a qualification. The various self-management tasks had to be fitted in with these wider commitments. Below, we describe a case example to illustrate the complexity of this:\n\nBeryl, aged 50, has had type 2 diabetes for 15 years. She lives on a council estate (IMD score 58) and is on state benefits. She has hypertension, hypercholesterolaemia, retinopathy, neuropathy and had a gastric bypass for morbid obesity two years ago. Beryl left school with few qualifications and can barely use a computer. She signed up for a further education course two years ago but had to give this up to care for a sick relative. She recently resumed the course following the relative's death. Beryl's adult children have various social and financial problems (including fear of crime on another local estate) and depend heavily on her for childcare and financial and emotional support.\n\nBeryl views diabetes as one of numerous interacting stressors in her life. During a home visit, Beryl takes a piece of paper and writes \"Things that are doing my head in today\". Prompted by the researcher, she writes a list of current issues in her life down the left hand side of the page and scores them where 1 = worst impact and 10 = no impact. \"\\[adult daughter\\] - 3\/10, \\[adult son\\] - 4\/10; new puppy: 2\/10; college course - 5\/10; diabetes - 2\/10; losing eyesight - 1\/10; pains in feet - 1\/10; shopping - 3\/10; cleaning - 7\/10; looking after grandkids - 1\/10.\"\n\nBefore her gastric bypass operation, Beryl had too little energy to take exercise. She had been on escalating doses of insulin and her attempts to lose weight had failed; she had abandoned her diet and described herself at the time as a \"chocoholic\". At that time she did not even have the motivation to bath regularly or clean the house. Following bypass surgery, she was delighted to have lost a third of her body weight. She has begun to follow a much stricter diet, which involves cooking separately from her live-in relative (whom she says now has to \"fend for himself\"). She has also given up smoking and manages to fit regular exercise around her family and college commitments, even recently competing in a 3-mile charity walk ('Race for Life').\n\nPartners played a very variable role in the management of participants' diabetes. Some took lead responsibility for the work of measuring and monitoring blood glucose, administering medication and supporting the individual in following their diet. At the other extreme, some partners appeared to demarcate diabetes management work, and the knowledge and skills needed to undertake it, as beyond the bounds of their relationship. Some partners viewed self-management of diabetes in moral terms (because neglect of one's own care had implications for the couple or family). In such circumstances, self-management was a potential source of conflict.\n\n'Steve has not done his blood sugar today - he's just done \"guesstimates\". \\[...\\] Steve orders a pint of beer and (later) a pint of shandy. Steve and Lucia have an intense discussion about how she feels about him not managing his condition properly, because she sees it as an insult to her that he's not prepared to do all he can to be well and stay around for her. Lucia says people close to them have warned her off having a relationship with a person with diabetes.'\n\nField notes from home visit and restaurant outing with Steve (age 32, type 1 diabetes for 26 years, microvascular complications) and his partner Lucia\n\nA key socio-emotional self-management task was managing partners' and other relatives' level of involvement with their diabetes, and in particular dealing with relatives who either refused to engage with the recommended restrictions on diet and lifestyle or sought to enforce them rigidly.\n\n'He only gets Flora \\[margarine\\] on the toast if his wife makes it, he gives himself butter if he makes it. \\[...\\] Tonight his wife is away; he will have chunky soup with some sausage leftovers from last night sliced into it.'\n\nNotes from naturalistic interview on visit to Vic, age 61, type 2 diabetes for 6 years, hypercholesterolaemia\n\nInterestingly, in this example and that of Wendy (see above), the participant chose a 'workaround' (i.e. they accepted an implicit dietary arrangement when the relative was present but did not adhere to this in their absence) rather than continue to confront them about it. Others preferred to stand their ground and resist family pressure. Saleem, for example, was the patriarch of a traditional South Asian family; in this particular meso environment his relatives were less able to enforce their views on what he should eat.\n\n'Saleem's son comments that he's on insulin because of his bad habits. I ask what the bad habits might be. Sweet, sugary things. Saleem says \"they can't tell me I can't have them\". \\[...\\] He says he has something sweet every day. His son says he's \"zithy\", the Urdu word for stubborn.'\n\nField notes from visit to Saleem, age 88, type 2 diabetes for 15 years\n\nSome participants drew considerable practical or emotional support from friends, neighbours and extended family. Even when this support was limited, participants' self-management actions and choices were strongly influenced by their desire to act appropriately and keep face within their social group. This was particularly evident in teenagers and young adults.\n\n'He tells me he plays football and goes to the gym. He doesn't make any special preparation for doing sports. Mum says he takes Lucozade with him. Asghar insists he doesn't and then Mum says he drank a whole bottle before football. She gets frustrated with him \"What about the time I chased after you because you'd taken four bottles!\" \"I was taking them for my mates\" Mum looks disgruntled - \"They're too expensive to give them to your mates\".'\n\nField notes from home visit to Asghar, age 16, type 1 diabetes for 7 years, IMD score 67.1\n\nLucozade is a commercial carbonated carbohydrate drink which many participants used to treat hypoglycaemic attacks, but which is also marketed as a sports drink. By handing out bottles to his friends, Asghar may have successfully de-medicalised his treatment and achieved social gain, but this trade-off had a very different social meaning for his mother, who was struggling to feed a family of six on state benefits.\n\nA minority of participants were members of diabetes patient groups and\/or online peer support networks where they exchanged experiences and tacit knowledge with other people with diabetes. Below, we reproduce examples of postings on a diabetes patient organisation public-access bulletin board used by one of our participants (identifying details have been fictionalised).\n\n\"Hi\n\nthe best way to get out of a low, is sugar and milk. trust me... and also quick question... can anyone tell when there blood levels are high, cus its obvious when there low but my doctor said i couldnt tell when they are high,, but i can: D: D... can anyone else?????\"\n\n-------------------------------------------------------------\n\n\"Hi All, i was wondering if some one could give me some advice about the pumps. i am 32 and a type one. i have tried everything under the sun to control my levels. i eat well, work out and take good care of myself AND still i cant get control myself. i feel rather alone and scared esp after reading some things online ref...\"\n\n\"Hi i'm in the same position as you, i just cant get my sugars under control (its been an uphill struggle for 22 yrs). I have asked my consultant about a pump for several yrs but she always just ignored me... but finally last aug she said she would consider it !! In nov i was told that i have been accepted, but that there is a 2 yr waiting list. so i'm waiting. I've read that it is very hard work at first but the benifits are well worth it. My advise to you would be to first of all speak to your G.P\/consultant\/D.L.S \\[Diabetes Liaison Sister\\] and see what they say. If you meet all the NICE requirements (which it sounds like you do), keep on asking your consultant. Once you have got your consultants backing, your local PCT have to fund it! but in some areas they drag their feet because they dont want to part with the money. If this happens speak to your local MP (that's what I'm doing) and just keep on nagging. try looking at these websites: -<http:\/\/www.nice.org.uk\/TA151><http:\/\/www.input.me.uk>...\"\n\n\"Try our online diary - record your glucose levels and the carbs you are eating online so they are all in one place <http:\/\/diabeticfriend.co.uk\/> I found it really useful to understand my levels while I was pregnant. Xxx\"\n\n-------------------------------------------------------------\n\n\"After my last check at hospital clinic they were concerned about my blood preesure as it was a tad on the high side and am already on medication for it. They asked my GP to keep an eye on it. First reading 155\/70, 2 weeks later 152\/70. I told receptionist that was too high as a person with diabetes should not have a reading that high, not above 130\"\n\n\"Maybe if you brought a blood pressure machine. Take it daily and show your GP the results when you see them again. Personally I dont always rely on the receptionists. Good luck and hope you are settled soon.\"\n\n---------------------------------------------------------------------------\n\n\"I just gave my 4 year old daughter 8 units of novorapid instead of 8 units of lantus can any1 let me know wot 2 do.\"\n\n\"Been there! If you have any milkshake or juice or junky food I'm sure she can manage something... x\"\n\n\"I gave my son 28 units of novorapid by mistake 1 night, it was a very long nite, making sure he was ok, won't make that mistake again\"\n\n## Self-management outside the home\n\nA recurring theme in our data was how much easier people found it to manage diabetes when they were in a familiar environment (usually but not always their own home) and following a regular routine. Settings outside the home were inherently more challenging and experienced as harder work. Some participants commented that their diabetes was more difficult to control at weekends because life was less predictable then. Several led restricted social lives and were reticent to go out to places with which they were unfamiliar.\n\nWe had hoped to follow people with diabetes in the work environment, but only one (a semi-retired administrator who worked part time for a small company) was prepared to be shadowed at work. The reason for this seemed to be that having a researcher accompanying them would have made the diabetes more visible in the workplace whereas the individual preferred not to draw attention to it. Several participants said they had given up a job because the \"stress\" had interfered with their diabetes control. Three issues seemed to contribute to this problem: intensity and unpredictability of workload, inability to control timing of meals, and shift work. Three participants described coping with a particular job until a 'squeeze' occurred (reduction in staffing or increase in expected performance). Two had given up nursing or nurse training after developing diabetes.\n\nIn contrast, the one school we visited appeared keen to accommodate and support the diabetic pupil. Staff were knowledgeable; a formal policy on the condition was evident; and the child's parents had made great efforts to engage staff in the strict routine needed for optimal diabetes control.\n\n'At 10 am she gives William the biscuit his mum has brought in. Mum always wraps it in silver foil so it's very noticeable and puts it in the middle of the teacher's desk.'\n\nNotes from naturalistic interview with class teacher of William, age 5, type 1 diabetes for 1 year\n\nMany participants found eating out a strain because it was impossible to predict what the nutritional content of the meal would be or assess this accurately once the meal arrived. Several insulin-treated participants chose to eat out at corporate chain outlets such as McDonalds because these offered a detailed breakdown of nutritional content on their website (enabling the insulin dose for a particular menu choice to be worked out in advance). In the light of these findings, it is not surprising that several participants abandoned their otherwise tight dietary regimen when we took them out for a meal. Some but not all were reluctant to inject in caf\u00e9s or restaurants either in the public areas or in the lavatory, since neither space was considered sufficiently \"private\". Injecting insulin was not a value-neutral medical procedure but a social practice which people with diabetes deemed appropriate or inappropriate in different contexts.\n\nNone of the food outlets we visited in this study offered a 'diabetic' option, and even when some nutritional information was given, it was inadequate to inform a titration of insulin dose. When questioned about a menu item, waiting staff were typically able to explain what a meal would look or taste like and whether it would contain nuts, but could give no indication of its total energy, fat or carbohydrate content.\n\nParticipants offered examples of limited understanding of diabetes by staff in other organisations. One person with an insulin pump, for example, described a humiliating episode at an airport where he had been strip-searched by security staff. Since then he has decided not to take his pump on holiday.\n\n## The healthcare system and the self-managing patient\n\nThe main hospital diabetes service covering the localities in our sample was delivered by a multidisciplinary team and included free structured diabetes education and a telephone 'Care Call' support service.\n\n'The Care Call system is that someone (non-clinical) phones her once a month. Four days before the call, people have to take blood sugars 4 times a day. The figures are then passed on to the diabetes nurse who will then phone to say whether to increase, decrease or stay the same with the insulin. Pauline can phone the service with any concerns. She says she phones them all the time.'\n\nNotes from naturalistic interview on visit to Pauline, age 62, insulin-treated type 2 diabetes for 6 years\n\nPauline appeared particularly to value the responsive nature of the Care Call support service. For example, when she was booked for a hospital admission for an abdominal operation, the surgeon had dismissed with vague reassurance her questions about what would happen with her diabetes. The Care Call nurse had explained in detail how an insulin-treated patient would be managed on the surgical ward.\n\nAlmost all general practitioners in the locality received the maximum incentive payments for diabetes care. But whilst services appeared to be of a high standard overall, some participants experienced difficulties with individual practice staff whom they felt did not recognise or value their knowledge, showed little interest in their self-management efforts and sometimes seemed actively to discourage such efforts. One participant was in the process of changing his general practitioner because the practice nurse \"did not like his questions\". Such accounts were sometimes borne out by our own observations.\n\nOptometrist: \"Are you diabetic?\"\n\nHarry: \"Yes\".\n\nOptometrist: \"On tablets or insulin?\"\n\nHarry: \"I'm on tablets and insulin.\"\n\nOptometrist: \"You're type 1 then.\"\n\nHarry: \"No, I'm type 2.\"\n\nOptometrist: \"Well you must have become type 1 now\"\n\nField notes on accompanying Harry (age 73, insulin-treated type 2 diabetes for 2 years) to optometrist\n\nParticipants who sought actively to self-manage greatly valued continuity of care with a particular doctor or nurse. and saw them as pivotal in aiding the self-management. From their perspective, effective conversations about self-management could occur only when the health professional acknowledged their knowledge and expertise and trusted their account - and this was rarely the case in the absence of an established relationship.\n\n## Observations of 'non self-management'\n\nThe ethnographic method allowed us to observe what we interpreted as 'non self-management' - that is, situations in which the self-management practices listed above might have been employed but were not. We were not surprised to observe some of our participants eating energy-dense, nutrient-poor ('junk') foods; snacking (e.g. on glucose tablets when not hypoglycaemic); not taking account of portion size; missing meals; not testing blood glucose levels even when they suspected them of being very high or low; managing (presumed) hypoglycaemic attacks by drinking large amounts of Lucozade from the bottle; smoking; consuming sugary foods and drinks to counteract the known hypoglycaemic effect of alcohol; and not recognising or addressing what we viewed as self-management needs which emerged in daily life. Many ordered high-sugar drinks when out for a meal even though 'diet' options were available. Few took regular physical exercise beyond household duties. Some omitted their prescribed medication (because of side effects) or insulin (to control their weight or reduce the risk of hypoglycaemic attacks when going out).\n\nThe underlying reasons for episodes of 'non self-management' were varied and complex. An important influence was the numerous other facets of people's daily lives (such as childcare, domestic duties or the restrictions and demands of paid employment); competing demands on a limited family budget; the constraints of co-morbidity; and the material and social contexts in which particular self-management tasks were (perceived as) more or less achievable and socially acceptable. Some participants found the requirement to plan menus, restrict dietary content, adjust medication dosage and keep written records of their progress difficult and dispiriting, especially in the context of psychosocial stress, complications (e.g. visual impairment, cognitive impairment) or co-morbidity.\n\n'I ask if she counts carbohydrates. She says yes but says \"it doesn't sit comfortably in my head\". Melissa's husband tells me \"it's a ball ache\" to keep the log of blood sugars and food. He says it's doing his head in having to write down everything in the log. He's been doing it for the last 6 months.'\n\nField notes from home visit to Melissa, age 38, type 1 diabetes for 28 years, pregnant with multiple diabetes complications including severe visual impairment\n\nIn one participant, a pattern of insulin omission was linked to denial of diabetes in the context of high psychosocial stress and low health literacy. The individual had lost weight dramatically recently and insisted that she was not \"dependent on\" insulin any more. (With her consent, we supplied her with a letter of concern to take to her GP, and also informed the practice).\n\nSome participants viewed self-management mainly or exclusively in terms of responding to bodily symptoms (a finding others have described previously \\[42\\]). Some considered routine diabetes check-ups \"pointless\" if there was no direct symptomatic improvement as a result. Participants who smoked generally justified this in terms of coping with \"stress\" and\/or by denying the risks. Health-negative food choices were often described as \"treats\" and taken in what participants considered to be small quantities. In many cases, these were justified in terms of their social significance - for example at a social event or as part of a positive relationship with a family member or friend.\n\nSome participants who had not attended self-management education told us that their current self-management regimen had been approved by a health professional. This affirmation was typically depicted in vague and non-specific terms.\n\n'I ask was he given any information on diet. He says there was \"a slight mention\". Don't have greasy foods, don't have too many sweets. He saw the dietician who apparently told him that he wasn't doing anything wrong.'\n\nField notes on visit to Kamlesh, age 61, type 2 diabetes for 2 years, overweight with heart disease\n\n## The consequences of self-management (and non self-management)\n\nStructuration theory proposes that the outcomes of human action feed back to reinforce or change internal and external structures (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>). One of the most powerful influences on participants' attitudes towards diabetes and their consequent self-management activity was the extent to which their efforts were effective.\n\nThe case example of Beryl (see above) illustrates how the consequences of self-management can feed back either positively or negatively to change internal structures. Before her gastric bypass operation, Beryl's attempts to lose weight had failed, creating a vicious circle of low motivation, poor eating, weight gain and increasing insulin dose. But with an altered metabolic profile, her self-management efforts had begun to bear fruit and the circle became positively reinforcing; she took increasing control of her diet, exercise and smoking despite adverse contextual influences.\n\nA number of other participants had reversed a previous \"chaotic\" pattern by moving to an insulin pump or by going on a DAFNE or DESMOND course. As their efforts to control their diabetes became more successful, they described greater motivation and effort in self-management activities. In contrast, both Mary, aged 58, with poorly controlled insulin-treated type 2 diabetes, obesity and multiple complications (who described her diabetes as a \"monster\") and Priya, aged 14, with recurrent hypoglycaemic attacks with fits (whose parent described her diabetes as \"a box of scary things\") had made several unsuccessful attempts in the past to adjust their diet and medication.\n\n## Summary of findings\n\nThis ethnographic study, in which a total of 88 extended visits were made to 30 people with diabetes in their homes and community, has shown that self-management comprised both practical and cognitive tasks (e.g. self-monitoring, menu planning, medication adjustment) and socio-emotional ones (e.g. coping with illness, managing relatives' input, negotiating access to healthcare or self-management resources, and making evaluative judgements about immediate versus deferred benefits). All this was hard work and time-consuming for people with diabetes and their families. Some managed their diabetes skilfully and flexibly, drawing on personal capabilities and family and social networks. For others, capacity to self-manage (including overcoming contextual barriers) was limited by co-morbidity, cognitive ability, psychological factors (e.g. under-confidence, denial) and social capital. As predicted by structuration theory, economic, material and socio-cultural conditions within the family, workplace and community sometimes created an enabling context for self-management and at other times provided significant barriers to achieving it.\n\n'Non self-management' tended to occur in contexts where people's material, intellectual or emotional resources were stretched, including poverty, low health literacy, a demanding family or social context, or multiple co-morbidity - and especially when all these factors were present and interacting. Some healthcare staff appeared reluctant to accommodate advanced knowledge and\/or self-managing attitudes and activities in their patients, especially when they did not have an ongoing care relationship with them. Food labelling in caf\u00e9s and restaurants was inadequate to allow people with diabetes to apply advanced insulin dosage algorithms even when they were confident to use these.\n\n## Strengths and limitations of this study\n\nThe main strength of this study is that it is, to our knowledge, the first detailed observational study of all aspects of self-management, including the context in which it occurs, in a sample of people with diabetes selected for maximum variety in both medical and socio-economic variables. The ethnographic design enabled us not merely to see self-management activities in family and social context, but also to study the multiple interacting influences on the under-researched phenomenon of *non* self-management.\n\nSignificant limitations of the study are that whilst we gained extensive access to people's homes, there were many aspects of personal and family life which we did not witness; some of our observations may have been influenced by the presence of the researcher; and we had limited access to the workplace. Furthermore, people may have behaved differently while being observed, though they might be expected to amend their behaviour towards what they considered to be compliance with professional expectations of self-management (e.g. to try to be seen to be following a diabetes diet). Whilst we still collected multiple examples of participants choosing not to self-manage, our findings should be interpreted in the light of this possible bias.\n\n## Links to previous research\n\nOur findings support and extend those from previous studies (most based on narrative interviews of people with diabetes, and a single ethnographic study) which highlighted the need to align the self-management agenda with the social demands of people's everyday lives; their need to maintain a coherent identity and a \"normal\" social life; and the finding that poverty and the physical and social environment may impact on self-management \\[21,25,31,43\\]. In particular, our findings resonate strongly with those of a previous study by our team which highlighted a number of 'storylines' within which the practical tasks of self-management acquire social meaning and moral worth, including rebuilding spoiled identity, living a disciplined and balanced life, mobilising a care network, navigating and negotiating in the health care system, and making ethical choices (such as allocating a limited family budget) \\[25\\]. They also align with work by an Australian team who identified eight dimensions for chronic disease self-management, including positive and active engagement in life, health-directed behaviour (e.g. dietary choices), self-management skills, health services navigation and social integration and support \\[44\\].\n\nIt is worth comparing our findings with those of qualitative interview studies linked to trials of self-management education. In the DAFNE trial, three qualitative interviews were conducted with each of 30 participants over a 12-month period \\[27,45\\]. These suggested that opportunities presented by more sophisticated self-management regimens for greater dietary freedom were counterbalanced by new challenges and burdens (e.g. having to simplify food choices). Researchers who interviewed 36 people recruited from a DESMOND course classified them into four 'types' depending on the degree of personal responsibility they took for their self-management activity \\[46\\].\n\nIt is perhaps unsurprising that the study reported here, which sought to study self-management in all its messy and idiosyncratic detail and take account of its multi-layered context has failed to explain participants' behaviour solely in terms of psychological states or traits. Our findings resonate more closely with the social ecology model offered by Glass and McAttee, in which psychology is just one of many dynamically interacting influences \\[47\\]. Whilst our work supports a conclusion that psychological constructs such as low motivation, low self-efficacy and external locus of control are significant mediators and moderators of diabetes self-management efforts, it also supports a more sophisticated, 'nested determinants' model in which these traits are in large part the product of prevailing social, economic and material circumstances.\n\n# Conclusion\n\n## Implications for policy and practice\n\nOur findings suggest that the success of self-management as a policy solution will be affected by interacting influences at three levels. At the micro level, self-management depends crucially on individuals' dispositions and capabilities, and will have relatively little purchase in those with low health literacy and other relevant capabilities. At the meso level, self-management depends on key roles, relationships and material conditions within the family and also on the presence of a supportive infrastructure in the workplace, school and healthcare organisation. And at the macro level, self-management is likely to be influenced by prevailing economic conditions, cultural norms and expectations, and the underpinning logic of the healthcare system (e.g. the extent to which support for self-care is an expected and adequately remunerated aspect of the service). People's self-management efforts, in turn, will influence the meso environment of the family and (more indirectly) the organisational and wider social structures which support this practice (or not).\n\nMany authors have argued that structured education for self-management should be supplemented with attention to the wider environment \\[5,13,17,48-50\\]. The Robert Wood Johnson Foundation in the USA, for example, has proposed a multi-level set of resources and support for self-management which include individual components (self-management education, continuity of clinical care, collaborative goal-setting, personalised support and troubleshooting) as well as community development (e.g. food outlets, safe exercise spaces), community health support workers, and health system development. Instruments to evaluate such multi-level resource packages have been developed and validated \\[51,52\\].\n\nIn contrast, and notwithstanding a small but politically high-profile demonstration project to encourage shops in low-income areas to sell more fresh fruit \\[53\\], self-management policy in the UK remains relatively narrowly focused on the person with chronic illness and assumes that the key focus for change is that individual's knowledge, attitudes and behaviour. The findings reported here suggest that a much broader approach should be taken. We need to develop ways of educating and developing health professionals (and allied staff such as healthcare assistants) so that they recognise, value and effectively support the efforts of the self-managing patient. At the very least, clinicians need to explore the details of their patients' home, work\/school and community environments more, so that consideration of these can be factored into any 'goal setting' and management plans. We also need to work on removing barriers to effective diabetes self-management in wider society. Voluntary agreements with the food industry might be less effective than legislative changes, but in the absence of the latter, higher standards for nutritional information in caf\u00e9s and restaurants should be strongly encouraged.\n\n## Suggestions for further research\n\nWhilst we have claimed some theoretical generalisability (we have shown, for example, that there is a recursive, mutually reinforcing, relationship between wider social determinants of health and individual action in relation to self-management), we do not claim that the findings from this small study are empirically generalisable. A number of themes from our findings should be explored further in empirical studies. A larger sample of participants, perhaps broken down by ethnic and demographic subgroups, studied more intensively for a longer period, would almost certainly reveal important additional insights into the challenges of self-management, though the acceptability and resource implications of an extended ethnographic approach may be prohibitive. The use of design tools such as 'cultural probes', which engage research participants in collecting ethnographic data in the absence of the researcher \\[54\\], could take self-management research into fruitful new directions. Such studies might inform the development and evaluation of multi-level intervention programmes which address the complex needs of individuals who find self-management challenging. Ethnographic studies of diabetes in the workplace might be undertaken in partnership with occupational health departments.\n\nThere is also a need for further theoretical and methodological work on what might be termed the political economy of self-management. Tudor Hart's inverse care law states that because of pervasive economic and socio-cultural forces, people most in need of health care are least likely to seek it or receive it \\[55\\]. The findings from this preliminary study suggest that because of the pervasive impact of economic and socio-cultural forces on the opportunities for health-related actions, people who would benefit most from self-management of their diabetes may be least likely to achieve it. Social ecology models of risk and chronic illness \\[47\\], perhaps combined with a 'critical theory' application of ethnographic methods \\[56\\], could help take this agenda forward. A bold and innovative approach to researching self-management is needed not least because without it, the social determinants of poor diabetes outcome are likely to exert their impact in the next generation as well as the present one.\n\n## Funding, governance and ethical approval\n\nThe study was initially conceptualised (and data collection commenced) as a component of a study funded by the Department of Health under the Connecting for Health Evaluation Programme and reported previously \\[57\\]. We subsequently extended our sample and analysis using funding from a Senior Investigator Award for TG from the National Institute of Health Research. The work was overseen by an External Advisory Group chaired by a layperson with representation from key stakeholders including patients, professional bodies and external academics. Ethical approval was obtained from two Multi-centre Research Ethics Committees: Thames Valley in January 2007 (06\/MRE12\/81 and subsequent amendments) and North West 8 in September 2009 (09\/H1013\/36 and subsequent amendments). The sponsor had no role in the preparation or submission of the paper.\n\n# Competing interests\n\nAll authors have completed the Unified Competing Interest form at <http:\/\/www.icmje.org\/coi_disclosure.pdf> (available on request from the corresponding author) and declare: no support from any organisation for the submitted work \\[or describe if any\\]; no financial relationships with any organisations that might have an interest in the submitted work in the previous three years \\[or describe if any\\], no other relationships or activities that could appear to have influenced the submitted work.\n\n# Authors' contributions\n\nTG conceptualised the study. SH did the fieldwork. Both authors analysed the data. TG led on writing the paper with input from SH. Both authors read and approved the final manuscript.\n\n# Pre-publication history\n\nThe pre-publication history for this paper can be accessed here:\n\n<http:\/\/www.biomedcentral.com\/1472-6963\/12\/83\/prepub>\n\n## Acknowledgements\n\nWe thank the study participants for their time and commitment to this research; health professionals and others who supported the study and helped recruit participants; the project steering group for their contribution to overseeing the work; Clive Seale for sociological advice; and two reviewers of an earlier draft for their helpful constructive comments.\n\n### Data sharing statement\n\nData not available for further sharing because this possibility was not included in information to participants.","meta":{"dup_signals":{"dup_doc_count":104,"dup_dump_count":57,"dup_details":{"curated_sources":4,"2023-23":1,"2022-40":1,"2022-27":1,"2022-05":1,"2021-43":2,"2021-25":1,"2021-17":2,"2020-50":2,"2020-40":1,"2020-29":1,"2020-16":1,"2020-05":1,"2019-51":1,"2019-47":1,"2019-39":1,"2019-35":1,"2019-22":2,"2019-13":3,"2019-09":1,"2019-04":2,"2018-51":1,"2018-47":3,"2018-43":2,"2018-39":1,"2018-34":2,"2018-30":2,"2018-26":1,"2018-22":3,"2018-17":1,"2018-13":1,"2018-09":2,"2018-05":2,"2017-51":2,"2017-47":4,"2017-43":2,"2017-39":3,"2017-34":1,"2017-30":2,"2017-26":1,"2017-22":5,"2017-17":4,"2017-09":1,"2017-04":2,"2016-50":2,"2016-44":2,"2016-40":1,"2016-36":2,"2016-30":6,"2023-50":1,"2024-30":2,"2024-18":1,"2024-10":1,"2017-13":3,"2015-18":1,"2015-06":1,"2013-48":1,"2013-20":1}},"file":"PMC3349490"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  During the first wave of pandemic H1N1 influenza in 2009, most cases outside North America occurred in the UK. The clinical characteristics of UK patients hospitalised with pandemic H1N1 infection and risk factors for severe outcome are described.\n  .\n  # Methods\n  .\n  A case note-based investigation was performed of patients admitted with confirmed pandemic H1N1 infection.\n  .\n  # Results\n  .\n  From 27 April to 30 September 2009, 631 cases from 55 hospitals were investigated. 13% were admitted to a high dependency or intensive care unit and 5% died; 36% were aged \\<16\u2005years and 5% were aged \u226565\u2005years. Non-white and pregnant patients were over-represented. 45% of patients had at least one underlying condition, mainly asthma, and 13% received antiviral drugs before admission. Of 349 with documented chest x-rays on admission, 29% had evidence of pneumonia, but bacterial co-infection was uncommon. Multivariate analyses showed that physician-recorded obesity on admission and pulmonary conditions other than asthma or chronic obstructive pulmonary disease (COPD) were associated with a severe outcome, as were radiologically-confirmed pneumonia and a raised C-reactive protein (CRP) level (\u2265100\u2005mg\/l). 59% of all in-hospital deaths occurred in previously healthy people.\n  .\n  # Conclusions\n  .\n  Pandemic H1N1 infection causes disease requiring hospitalisation of previously fit individuals as well as those with underlying conditions. An abnormal chest x-ray or a raised CRP level, especially in patients who are recorded as obese or who have pulmonary conditions other than asthma or COPD, indicate a potentially serious outcome. These findings support the use of pandemic vaccine in pregnant women, children \\<5\u2005years of age and those with chronic lung disease.\nauthor: J S Nguyen-Van-Tam; P J M Openshaw; A Hashim; E M Gadd; W S Lim; M G Semple; R C Read; B L Taylor; S J Brett; J McMenamin; J E Enstone; C Armstrong; K G Nicholson**Correspondence to** Professor J Nguyen-Van-Tam, Division of Epidemiology and Public Health, University of Nottingham, Clinical Sciences Building, City Hospital, Hucknall Road, Nottingham NG5 1PB, UK; <email@example.com>\ndate: 2010-07-13\ninstitute: 1Division of Epidemiology and Public Health, University of Nottingham, Nottingham, UK; 2Centre for Respiratory Infections, National Heart and Lung Institute, Imperial College, London, UK; 3Department of Health, Skipton House, London, UK; 4Department of Respiratory Medicine, Nottingham University Hospitals NHS Trust, Nottingham, UK; 5School of Reproductive and Developmental Medicine, University of Liverpool, Liverpool, UK; 6Department of Infection and Immunity, University of Sheffield, Royal Hallamshire Hospital, Sheffield, UK; 7Department of Critical Care, Portsmouth Hospitals NHS Trust, Portsmouth, UK; 8Centre for Peri-operative Medicine and Critical Care Research, Imperial College Healthcare NHS Trust, London, UK; 9Health Protection Scotland, NHS National Services, Glasgow, UK; 10Infectious Diseases Unit, University Hospitals of Leicester NHS Trust, Leicester Royal Infirmary, Leicester, UK\nreferences:\ntitle: Risk factors for hospitalisation and poor outcome with pandemic A\/H1N1 influenza: United Kingdom first wave (May\u2013September 2009)\n\n# Introduction\n\nOn 11 June 2009 the World Health Organization announced the first influenza pandemic of the 21st century.1 2 While most pandemic H1N1 infections were mild or subclinical, the case fatality rate was 0.1\u20130.7%.3 4 Early reports suggested a case hospitalisation rate of 2\u20138%,5\u20139 later moderated to approximately 1% in view of under-ascertainment of mild cases.10 Although atypical, patients with severe disease imposed a considerable burden on hospital systems. Hospitalisation due to pandemic H1N1 infection was most common in children, teenagers and younger adults. At least half had underlying medical conditions.1 2 8\u201312 Relatively few studies have examined the risk factors associated with a severe outcome.7 13\n\n# Methods\n\n## Data collection\n\nThe Influenza Clinical Information Network (FLU-CIN) surveillance network was established by the Department of Health in England on 11 May 2009. Clinical data were collected from 55 hospitals in 20 cities or towns (see figure 1 in online supplement). Trained FLU-CIN data collectors gathered information from the case notes of patients hospitalised with pandemic H1N1 infection without pre-selection. All patients had acute respiratory illness and pandemic H1N1 infection confirmed by real-time reverse-transcriptase PCR. Diagnostic tests were performed as dictated by clinical management.\n\nData were extracted using a standard form that included demographic characteristics, past medical history, prehospital medication, clinical presentation, care timelines, initial assessment (emergency department and\/or acute medical unit), investigations and care escalation from levels 0 to 3,[^1] discharge and death. Since height and weight are not uniformly recorded in UK hospital notes, obesity based on physicians' observations was captured when recorded on admission.\n\n## Analysis of data\n\nAnonymised data were analysed using STATA Version 10 (StataCorp). \u03c7^2^ or Fisher exact tests were used to assess differences in proportions and the Mann\u2013Whitney test was used for continuous variables. The demographic data were compared with those of the combined populations of London, East Midlands and Northern Ireland because these areas provided \\>75% of the FLU-CIN cases. As paediatric normal reference ranges for respiratory rate and blood pressure vary with age, and heart rate also varies with body temperature, abnormal values in children (aged \\<16\u2005years) were defined after appropriate adjustment.14\u201317\n\nUnivariate analysis was performed using logistic regression to identify factors affecting the risk of requiring enhanced care (levels 2 or 3) or death in hospital (or both) as a combined measure of severe outcome. Unadjusted odds ratios (Wald test) and 95% CIs were computed. A multivariable regression technique was applied using all statistically significant variables identified during univariate analysis, including exploration for robustness and interactions using likelihood ratio tests. Age (continuous variable) was forced into each model, being an a priori confounder for comorbid conditions and case fatality. Two separate models were constructed\u2014one for demography, comorbidities and clinical characteristics and one for selected investigations and other prognostic indicators. Where continuous variables (eg, serum C-reactive protein (CRP)) were found to be significant, these were converted into categorical variables to facilitate clinical interpretation. A peripheral oxygen saturation threshold of 94% (breathing air) was selected based on British Thoracic Society recommendations for the instigation of supplemental oxygen therapy.18\n\n# Results\n\nA total of 631 hospitalised cases with H1N1 infection (226 children, 405 adults; median age 23\u2005years; range 3\u2005months\u201390\u2005years) admitted between 27 April and 30 September 2009 are described. The onset of illness, available for 522 patients, occurred from April 25 to September 29 (see figure 2 in online supplement). Most cases were non-white (<a href=\"#tbl1\" data-ref-type=\"table\">table 1<\/a>) and the distribution of age and ethnicity differed substantially from the source population. Of the 631 patients, 284 (45% overall; 34% children and 51% adults) had one or more underlying medical conditions (<a href=\"#tbl2\" data-ref-type=\"table\">table 2<\/a>), of which asthma was most common in children (16%) and adults (31%). Of 159 subjects with asthma, 88 (55%) regularly used oral or inhaled steroids. Twenty-seven patients were pregnant, representing 4% of total admissions and 18% of admissions among women aged 16\u201344\u2005years (<a href=\"#tbl2\" data-ref-type=\"table\">table 2<\/a>). Two, 13 and 8 pregnancies were in the first, second and third trimesters, respectively (missing data, n=4), and 8 (30%) had one or more underlying medical conditions. Obesity was recorded on admission in 8 (2%) of 405 adults; no child was noted to be obese.\n\nDemographic characteristics of 631 UK patients hospitalised with pandemic H1N1 infection during the first pandemic wave compared with source population data\n\n|  | n (%) | Population comparison<a href=\"#table-fn1\" data-ref-type=\"table-fn\">*<\/a> (%) |\n|----|----|----|\n| Sex: female | 320 (51) | 51.3 |\n| Age (years) |  |  |\n| \u2003\\<1 | 42 (7) | 1.2 |\n| \u20031\u20134 | 59 (9) | 5.2 |\n| \u20035\u201315 | 125 (20) | 14.2 |\n| \u200316\u201324 | 114 (18) | 11.7 |\n| \u200325\u201334 | 83 (13) | 16.9 |\n| \u200335\u201344 | 75 (12) | 15.4 |\n| \u200345\u201354 | 68 (11) | 12.3 |\n| \u200355\u201364 | 32 (5) | 9.4 |\n| \u200365\u201374 | 22 (3) | 7.3 |\n| \u2003\\>75 | 11 (2) | 6.4 |\n| Ethnicity<a href=\"#table-fn2\" data-ref-type=\"table-fn\">\u2020<\/a> |  |  |\n| \u2003White | 202 (38) | 81.9 |\n| \u2003Mixed | 7 (1) | 2.1 |\n| \u2003Asian\/Asian British | 169 (31) | 8.0 |\n| \u2003Black\/Black British | 100 (19) | 6.3 |\n| \u2003Chinese and other | 59 (11) | 1.7 |\n\nData are number (%) unless otherwise indicated.\n\nData for comparison of sex, age structure and ethnicity (London, East Midlands and Northern Ireland combined) were obtained from the Office for National Statistics ([www.statistics.gov.uk](http:\/\/www.statistics.gov.uk)) and Northern Ireland Statistics and Research Agency ([www.nisra.gov.uk](http:\/\/www.nisra.gov.uk)): National Census 2001 data: KS01 sex, KS02 age structure, KS06 ethnicity and corresponding census area statistics (CAS) data: UV03, UV04 and UV09 (available on Neighbourhood Statistics and [www.nomis.co.uk](http:\/\/www.nomis.co.uk)) for further subdivisional data.\n\nInformation on ethnicity was unavailable for 94 cases; assessments of ethnicity are based on 537 cases.\n\nPrehospital comorbidity in 631 patients hospitalised with pandemic H1N1 infection during the first pandemic wave compared with national prevalence data\n\n|  | Children (n=226) | Adults (n=405) | All admissions (n=631) | Background prevalence<a href=\"#table-fn3\" data-ref-type=\"table-fn\">*<\/a> (%) |\n|----|----|----|----|----|\n| No. of comorbidities<a href=\"#table-fn4\" data-ref-type=\"table-fn\">\u2020<\/a> |  |  |  |  |\n| \u20030 | 149 (66) | 198 (49) | 347 (55) | \u2013 |\n| \u20031 | 66 (29) | 138 (34) | 204 (32) | \u2013 |\n| \u2003\u22652 | 11 (5) | 69 (17) | 80 (13) | \u2013 |\n| Comorbidity |  |  |  |  |\n| \u2003Cardiovascular disease | 5 (2) | 64 (16) | 69 (11) | 3.5 |\n| \u2003Pulmonary disease |  |  |  |  |\n| \u2003COPD | 0 (0) | 25 (6) | 25 (4) | 1.5 |\n| \u2003Asthma | 35 (16) | 124 (31) | 159 (25) | 5.9 |\n| \u2003Other pulmonary disease | 8 (3) | 13 (3) | 21 (3) | \\- |\n| \u2003Diabetes | 3 (1) | 48 (12) | 51 (8) | 4.1 |\n| \u2003Other metabolic disease | 4 (2) | 2 (\\<1) | 6 (1) | \\- |\n| \u2003Neurological disease | 9 (4) | 10 (3) | 19 (3) | \\- |\n| \u2003Cerebrovascular disease | 0 (0) | 3 (\\<1) | 3 (\\<1) | 1.7 |\n| \u2003Obesity recorded on admission | 0 (0) | 8 (2) | 8 (1) | 8.1<a href=\"#table-fn5\" data-ref-type=\"table-fn\">\u2021<\/a> |\n| \u2003Pregnancy | 0 (0) | 27 (7)<a href=\"#table-fn6\" data-ref-type=\"table-fn\">\u00a7<\/a> | 27 (4) | 6.2<a href=\"#table-fn7\" data-ref-type=\"table-fn\">\u00b6<\/a> |\n\nData are number (%) unless otherwise indicated.\n\nNational prevalence data on comorbidity were obtained from the Quality and Outcomes Framework (QOF) primary care data for 2009 ([www.qof.ic.nhs.uk](http:\/\/www.qof.ic.nhs.uk)) and are based on all ages except for pregnancy.\n\nRecorded obesity and pregnancy are excluded from the number of comorbidities in the upper portion of the table.\n\nNational prevalence data on obesity based on QOF obesity registers defined as body mass index \u226530\u2005kg\/m^2^.\n\n27 pregnancies represent 7% of all adults but 18% of women aged 16\u201344\u2005years in the study.\n\n6.2% of females aged 15\u201344\u2005years estimated to be pregnant in source population: in addition to 207\u2009474 live births in London, East Midlands and Northern Ireland in 2008, we assumed that 4% of females aged 15\u201344\u2005years experienced miscarriage or abortion in the same time period (119\u2009668). To calculate the prevalence of pregnancy we took 9\/12 of annual live births (assuming 9-month duration of pregnancy) and 3\/12 of miscarriages\/abortions (assuming 3-month duration), divided by the total female population aged 15\u201344\u2005years.\n\nCOPD, chronic obstructive pulmonary disease.\n\n## Preadmission care\n\nThe median interval between onset of illness and admission recorded for 522 cases was 2\u2005days (range 0\u201333; interquartile range 0\u201324). It was shorter in children than in adults (1\u2005day vs 2\u2005days, p\\<0.001) but was unaffected by comorbid conditions (median 2 vs 2\u2005days; p=0.42). Twenty children (9%) and 62 adults (15%) received antiviral drugs before admission (p=0.02). Preadmission antiviral drugs were given to 35 patients (12%) with comorbidity and to 47 patients (14%) without comorbidity (p=0.80).\n\n## Clinical presentation, treatment and outcome\n\nPatients presented most frequently with fever (71%), cough (68%), breathlessness (36%), headache (27%), sore throat (23%) and nausea or vomiting (22%) in adults; and fever (74%), cough (50%) and nausea or vomiting (27%) in children. Overall, 11% presented with diarrhoea, which was almost twice as common in white ethnic groups (15.4% vs 8.1%; p=0.008) in both adults (16% vs 9%) and children (14% vs 7%).\n\nUpon presentation, 63% of children and 87% of adults had tachycardia and 30% of children and 33% of adults had tachypnoea (<a href=\"#tbl3\" data-ref-type=\"table\">table 3<\/a>). About half had fever (\u226538\u00b0C); 16% of children and 24% of adults had a peripheral oxygen saturation of \\<94% breathing air, and 13% of children and 24% of adults had systolic hypotension. Alanine aminotransferase was raised in 19% of children and 34% of adults. Increased levels of CRP of \\<100\u2005mg\/l occurred in 19% of children and 31% of adults; increases of \u2265100\u2005mg\/l occurred in 8% of children and 18% of adults. Eighteen patients (3%) presented with symptoms of encephalitis (new behavioural change, meningism, focal signs, seizures or confusion), one of whom died. Twenty-nine patients (5%) presented with muscle weakness or tenderness suggestive of myositis, of whom one died.\n\nPresenting signs and initial investigations and the percentage that were abnormal in patients hospitalised with pandemic H1N1 infection during the first pandemic wave\n\n| Observations | n | Mean | Range | Interquartile range | Level of abnormality | % |\n|----|----|----|----|----|----|----|\n| Temperature (\u00b0C) |  |  |  |  |  |  |\n| Children | 183 | 38.0 | 35.3\u201341.4 | 36.0\u201340.1 | 37.1\u201337.9 | 22 |\n|  |  |  |  |  | \u226538.0 | 50 |\n| Adults | 344 | 39.0 | 35.1\u201340.2 | 35.9\u201340.3 | 37.1\u201337.9 | 24 |\n|  |  |  |  |  | \u226538.0 | 53 |\n| Pulse (rate\/min) |  |  |  |  |  |  |\n| Children | 97 | 137 | 54\u2013240 | 89\u2013184 | \\> Normal<a href=\"#table-fn8\" data-ref-type=\"table-fn\">*<\/a> | 63 |\n| Adults | 218 | 107 | 54\u2013184 | 62\u2013155 | \\>85 | 87 |\n| Pulse (respirations\/min) |  |  |  |  |  |  |\n| Children | 94 | 37 | 18\u201398 | 20\u201366 | \\> Normal | 30 |\n| Adults | 205 | 23 | 10\u201344 | 14\u201340 | \\>30 | 33 |\n| Systolic BP (mmHg) |  |  |  |  |  |  |\n| Children | 32 | 106 | 83\u2013126 | 93\u2013119 | \\< Normal | 13 |\n| Adults | 216 | 125 | 68\u2013191 | 84\u2013185 | \\<110 | 24 |\n| Diastolic BP (mmHg) |  |  |  |  |  |  |\n| Children | 33 | 60 | 39\u201390 | 42\u201377 | \\< Normal | 36 |\n| Adults | 217 | 71 | 32\u2013121 | 43\u2013100 | \\<65 | 32 |\n| C-reactive protein (mg\/l) |  |  |  |  |  |  |\n| Children | 98 | 60 | 1\u2013322 | 4\u2013146 | 31\u201399 | 19 |\n|  |  |  |  |  | \u2265100 | 8 |\n| Adults | 208 | 57 | 1\u2013322 | 1.5\u2013274 | 31\u201399 | 31 |\n|  |  |  |  |  | \u2265100 | 18 |\n| White cell count (\u00d710^9^\/l) |  |  |  |  |  |  |\n| Adults and children | 425 | 9.0 | 1.1\u201338.0 | 1.1\u201332.9 | \\>11.0 | 17 |\n| Children | 102 | 44 | 3\u2013163 | 19\u201382 | \\>195 | 0 |\n| Adults | 285 | 91 | 5\u2013800 | 7\u2013611 | \\>200 | 3 |\n| Urea (\u03bcmol\/l) |  |  |  |  |  |  |\n| Children | 104 | 7.2 | 0.6\u2013106 | 1.1\u201340 | \\>6.4 | 14 |\n| Adults | 286 | 7.3 | 0.9\u2013130 | 1.5\u201356 | \\>6.7 | 18 |\n| Alanine transaminase (IU\/l) |  |  |  |  |  |  |\n| Children | 70 | 42.7 | 2\u2013393 | 7\u2013177 | \\>40 | 19 |\n| Adults | 179 | 36.3 | 8\u2013167 | 10\u2013127 | \\>35 | 34 |\n| Sp<span class=\"smallcaps\">o<\/span>~2~ (%, breathing room air) |  |  |  |  |  |  |\n| Children | 175 | 95.9 | 54\u2013100 | 80\u2013100 | \\<94% | 16 |\n| Adults | 334 | 95.1 | 66\u2013100 | 60\u2013100 | \\<94% | 24 |\n\nSee Methods for derivation of abnormal values in children.\n\nSp<span class=\"smallcaps\">o<\/span>~2~, peripheral oxygen saturation.\n\n## Pneumonia\n\nChest radiographic results were recorded in the notes in 349 cases (106 children, 243 adults), of which 37 children (35%) and 65 adults (27%) had findings consistent with pneumonia. Thirty-seven of 65 adults had sufficient data to derive a CURB65 score of 0, 1 and 2 in 15 (41%), 17 (46%) and 5 (14%) cases, respectively. The median age of patients with pneumonia was 26\u2005years; 46 (45%) had one or more underlying medical conditions, 4 were pregnant and 2 were obese. Initial arterial blood gas analysis readings (while breathing room air) were available from 34 patients with pneumonia; 1\/23 (4%) with an arterial oxygen tension \u22658\u2005kPa died compared with 3\/11 (27%) with an initial reading \\<8\u2005kPa (p=0.085).\n\nThe occurrence of radiological pneumonia was unrelated to the presence of underlying medical conditions (p=0.09). Of the 102 cases with radiological pneumonia, 14 (6 children) had received preadmission antiviral drugs (14%) compared with 28\/247 (11%) patients (2 children) without pneumonia on the chest x-ray (p=0.53). The median length of stay for patients with pneumonia was 6\u2005days compared with 3\u2005days for patients without pneumonia (p=0.0001). Thirty-seven of 102 patients (36%) required level 2 (n=6) or level 3 (n=31) care, 21 (21%) underwent mechanical ventilation (intubated) and 12 (12%) died, of whom 11 were ventilated. Mortality in cases with radiographic pneumonia was significantly higher than in cases without (OR 4.57, 95% CI 1.71 to 12.18; p=0.0008). Four cases of pneumonia (4%) had positive bacteriological findings. Two children with severe developmental delay and extreme prematurity, respectively, grew methicillin-resistant *Staphylococcus aureus* (MRSA) and one adult grew *Streptococcus pneumoniae* in sputum; all three died. One adult had *S aureus* bacteraemia and survived.\n\n## Inpatient treatment\n\nAfter admission, antiviral drugs were prescribed to 474 patients (75%) and 366 (58%) received antibiotics. One hundred and forty-seven of 157 cases (93%) who did not receive an antiviral drug in hospital did not receive an antiviral agent before hospitalisation. One hundred and fourteen patients (18%) received steroids as an acute intervention; of these, 71 had underlying asthma, 57 of whom were previously maintained on steroids. The 43 patients without asthma treated in hospital with steroids included 13 with COPD or other chronic lung disease on long-term steroids; 7 on steroids for other long-term conditions (eg, myeloma); 8 with sudden deterioration (of whom 4 were pregnant or recently post-partum); 5 with wheeze on admission; 2 obese patients; 2 with suspected new asthma; and 1 suspected allergic reaction (the reason for steroid treatment was unclear in 5 cases).\n\n## Length of stay\n\nThe median length of hospital stay was 3\u2005days in children (range 1\u201332; interquartile range 1\u201328) and 4\u2005days in adults (range 1\u201341; interquartile range 1\u201329; p=0.004); it was unaffected by comorbidity or treatment with antiviral drugs before hospitalisation.\n\n## Deaths, severity criteria and requirement for critical care\n\nOverall, 85 of the 631 cases (14%) had a severe outcome. Eighty patients received level 2 (n=27) or level 3 (n=53) care and 29 died. The recorded case fatality rate was 4.6%, 3.5% in children and 5.2% in adults (likelihood ratio=1.5, p=0.34). Seventeen of the 29 fatalities (59%) were previously healthy. The median age of those who died was 49\u2005years in adults and 7\u2005years in children. There were no significant differences in use of level 2 or level 3 care with age; however, the in-hospital case fatality rate increased with age (\\<5\u2005years (3%), 5\u201315\u2005years (4%), 16\u201344\u2005years (3.7%), 45\u201364\u2005years (9%), \u226565\u2005years (6.0%)) and was significantly higher in patients aged \u226545\u2005years (3.6% vs 8.3%; p=0.02). Twenty-five patients (31%) died while in level 2 or level 3 care. A further two deaths occurred on standard wards (a patient with metastatic carcinoma and another with severe chronic lung disease), one in the emergency room (after 4\u2005h resuscitation) and one after transfer to another hospital (no subsequent information). Of cases receiving level 2 or level 3 care, those who died received a median of 10\u2005days care at this level whereas survivors received 3\u2005days of care (p=0.001). No patients were declined level 2 or level 3 care for non-clinical reasons during the study period.\n\nRecipients of antiviral drugs before admission were less likely to require level 2 or level 3 care (8\/82 (9.8%) vs 72\/549 (13%); likelihood ratio=0.72, p=0.394) or to die (2\/82 (2.4%) vs 27\/549 (4.9%); likelihood ratio=0.48, p=0.31) than non-recipients, but both trends were non-significant. Two of 27 pregnant women (7%) died in hospital while in level 3 care; four others (15%) received level 2 (n=1) or level 3 (n=3) care and survived.\n\n<a href=\"#tbl4\" data-ref-type=\"table\">Table 4<\/a> shows the association between admission variables and severe outcome. Altered conscious level, dyspnoea, requirements for intravenous fluids or supplementary oxygen, radiologically-confirmed pneumonia and CRP levels \u2265100\u2005mg\/l were each associated with a severe outcome, as were obesity recorded on admission and chronic pulmonary disease other than asthma or COPD (eg, cystic fibrosis, fibrosing alveolitis and congenital lung defects). In a multivariable model of demography, comorbidities and clinical characteristics, obesity recorded on admission and pulmonary conditions other than asthma or COPD were found to be associated with a severe outcome (<a href=\"#tbl5\" data-ref-type=\"table\">table 5<\/a>). An additional multivariable analysis of selected investigations and possible prognostic indicators showed that radiologically-confirmed pneumonia and a CRP level \u2265100\u2005mg\/l were independently significant (<a href=\"#tbl5\" data-ref-type=\"table\">table 5<\/a>). The addition of further variables did not significantly alter either model.\n\nAnalysis of comorbidity, demography, clinical characteristics and selected investigations as risk factors for severe outcome in patients hospitalised with pandemic H1N1 infection during the first pandemic wave\n\n|  | People affected by condition or feature (number with severe outcome) | Likelihood ratio (95% CI) | p Value |\n|----|----|----|----|\n| Age (years) | \u2013 | 1.01<a href=\"#table-fn9\" data-ref-type=\"table-fn\">*<\/a> (0.99 to 1.02) | 0.089 |\n| Asthma | 159 (18) | 0.77 (0.44 to 1.34) | 0.360 |\n| COPD | 25 (6) | 2.10 (0.81 to 5.43) | 0.123 |\n| Chronic pulmonary conditions, excluding asthma or COPD | 21 (7) | 3.41 (1.33 to 8.71) | 0.010 |\n| Cardiovascular disease | 69 (12) | 1.41 (0.72 to 2.75) | 0.314 |\n| Diabetes | 51 (7) | 1.02 (0.44 to 2.35) | 0.956 |\n| Neurological disorders | 19 (3) | 1.21 (0.34 to 4.25) | 0.764 |\n| Hepatic disease | 7 (2) | 2.60 (0.49 to 13.65) | 0.257 |\n| Obesity recorded on admission | 8 (4) | 6.96 (1.46 to 27.28) | 0.008 |\n| Smoker (current and former)<a href=\"#table-fn11\" data-ref-type=\"table-fn\">\u2021<\/a> | 103 (16) | 1.01 (0.53 to 1.91) | 0.972 |\n| Pregnancy | 27 (6) | 1.87 (0.73 to 4.77) | 0.190 |\n| White ethnicity<a href=\"#table-fn10\" data-ref-type=\"table-fn\">\u2020<\/a><a href=\"#table-fn11\" data-ref-type=\"table-fn\">\u2021<\/a> | 202 (29) | 1.08 (0.65 to 1.78) | 0.764 |\n| Altered conscious level | 10 (6) | 1.11 (1.04 to 1.17) | 0.001 |\n| Dyspnoea | 181 (44) | 1.32 (1.81 to 1.49) | 0.001 |\n| Heart rate (abnormal)<a href=\"#table-fn10\" data-ref-type=\"table-fn\">\u2020<\/a> | 198 (31) | 1.62 (0.80 to 3.30) | 0.180 |\n| Respiratory rate (abnormal)<a href=\"#table-fn10\" data-ref-type=\"table-fn\">\u2020<\/a> | 89 (16) | 1.78 (0.89 to 3.56) | 0.102 |\n| Required supplemental oxygen on admission | 99 (33) | 4.51 (2.72 to 7.40) | 0.001 |\n| Intravenous fluid replacement on admission | 145 (28) | 1.76 (1.07 to 2.89) | 0.005 |\n| Radiologically-confirmed pneumonia | 102 (37) | 5.28 (2.95 to 9.47) | 0.001 |\n| CRP \u2265100\u2005mg\/l<a href=\"#table-fn10\" data-ref-type=\"table-fn\">\u2020<\/a> | 46 (16) | 4.41 (2.14 to 9.10) | 0.001 |\n| Sp<span class=\"smallcaps\">o~2~<\/span> \\<94% on air<a href=\"#table-fn10\" data-ref-type=\"table-fn\">\u2020<\/a> | 107 (31) | 3.60 (2.17 to 6.27) | 0.001 |\n\nReflects a 1% increase in risk of severe outcome for each additional year of age.\n\nInformation about smoking history was documented for 216 cases; ethnicity was recorded for 537 cases; heart rate, with corrections for temperature, was available for 512 cases; respiratory rate was recorded for 470 cases; chest radiography findings were recorded for 349 cases; CRP levels were recorded for 306 cases; Sp<span class=\"smallcaps\">o~2~<\/span> data were recorded for 252 cases.\n\n45 severe outcomes among 335 non-white patients.\n\nCOPD, chronic obstructive pulmonary disease; CRP, C-reactive protein; Sp<span class=\"smallcaps\">o<\/span>~2~, peripheral oxygen saturation.\n\nResults of logistic regression analyses of (A) demography, comorbidities and clinical characteristics and (B) selected investigations and other possible prognostic indicators for critical care requirement or death in hospital\n\n|  | Odds ratio (95% CI) | p Value |\n|----|----|----|\n| \\(A\\) Clinical conditions<a href=\"#table-fn12\" data-ref-type=\"table-fn\">*<\/a> |  |  |\n| Obesity recorded on admission | 6.08 (1.45 to 25.37) | 0.013 |\n| Chronic respiratory disease other than asthma or COPD | 3.17 (1.22 to 8.24) | 0.018 |\n|  |  |  |\n| \\(B\\) Other clinical parameters<a href=\"#table-fn13\" data-ref-type=\"table-fn\">\u2020<\/a> |  |  |\n| Radiologically-confirmed pneumonia | 4.97 (2.09 to 11.81) | 0.001 |\n| C-reactive protein \\>100\u2005mg\/l | 3.06 (1.20 to 7.81) | 0.019 |\n\nMultivariable analysis of demography, comorbidities and clinical characteristics was constructed based on observations from 631 cases.\n\nA separate multivariable analysis was constructed for selected investigations and other possible prognostic indicators based on observations from 193 cases.\n\nCOPD, chronic obstructive pulmonary disease.\n\n# Discussion\n\nThe strengths of this study include confirmation by standardised PCR criteria, relatively few missing data and a setting in which hospitalisation and management of cases is driven by national guidelines.19 Reported cases were followed up without selection. Except in Scotland, the acquisition of cases closely mirrored the national epidemic curve geographically and temporally, with most occurring in Greater London, the English Midlands and Northern Ireland (see figures 1 and 2 in the online supplement). Overall, 12% of patients required high dependency or intensive care and 4.6% died; the mortality among those requiring enhanced care was 31%.\n\nThe median age of cases was 23\u2005years and 46% had risk factors for seasonal influenza complications. In relation to the source population, hospitalisations were highest in those aged \\<5\u2005years and lowest in those aged \u226555\u2005years, consistent with the age-specific prevalence of cross-reacting antibodies.20 Similar to other reports,13 mortality was significantly higher above 44\u2005years of age.\n\nOver one-half of all admissions and 59% of all in-hospital deaths occurred in previously healthy people. In contrast, Donaldson *et al* found that 36% of patients who died had no (19%) or only mild (17%) underlying illnesses.21 The principal comorbidity was asthma (in adults and children). About 45% of hospitalised patients with asthma with pandemic H1N1 infection did not routinely use inhaled or oral steroids, suggesting that pandemic influenza vaccine (H1N1) might be beneficial for all patients with asthma rather than just those with more severe disease.\n\nPregnancy substantially increases the risks for severe respiratory illness and excess deaths during pandemics and seasonal influenza,22 and in our series pregnant women comprised 18% of admissions among women aged 16\u201344\u2005years compared with an expected prevalence of 6% in the source population. These findings suggest that pregnant women are about three times more likely to be admitted to hospital with H1N1 infection than non-pregnant women of similar age and confirm the importance of vaccinating pregnant women, which may also protect their newborn infant.23\n\nObesity has been previously identified as a risk factor for severe pandemic H1N1 infection.13 24 25 Obesity recorded on admission was identified as an independent risk factor for a severe outcome. This observation possibly reflects that gross (as opposed to mild) obesity is more likely to be recorded by physicians in case notes and the lack of reserve respiratory capacity in such individuals.\n\nUnivariate analyses showed that patients with a severe outcome were more likely to be recorded as obese and to have pulmonary disease other than asthma or COPD (eg, cystic fibrosis, fibrosing alveolitis and congenital lung defects), altered consciousness level, shortness of breath, radiologically-confirmed pneumonia, CRP level \u2265100\u2005mg\/l, peripheral oxygen saturation of \\<94% on air or to have required supplemental oxygen or intravenous fluids on admission than those managed on standard wards. These findings are similar to those of previous studies7 13 and highlight the importance of regular monitoring for dyspnoea and peripheral oxygen saturation. Multivariable analysis of clinical features revealed that obesity recorded on admission and pulmonary conditions other than asthma or COPD remained significant clinical risk factors for a severe outcome. In addition, radiologically-confirmed pneumonia and CRP levels \u2265100\u2005mg\/l were independently associated with a severe outcome. In general, CRP levels are higher in patients with bacterial infections than in those with non-bacterial infections, and high levels are associated with mortality.26 Our findings should alert physicians to the possibility of very high CRP levels in patients with pandemic H1N1 infection and its potential seriousness. In our study, 29% of patients with chest x-rays had findings consistent with pneumonia. The median duration of stay of these patients was twice as long as those without pneumonia, and mortality in cases with radiographic pneumonia was several fold higher than for the whole case series (12% vs 4.6%). Bacterial co-infections were less commonly reported than in other case series,27 28 although we did not access autopsy data.29\n\nHigher rates of severe pandemic H1N1 infection have been reported in indigenous or disadvantaged populations,24 30 31 as also occurred during 1918\u201319.32 In our study, the number of Asian and black people who were admitted exceeded population estimates of ethnic profile 3\u20134-fold. The reasons for this excess are unclear, but could include language barriers affecting consulting behaviour or treatment access, overcrowding, household size and genetic susceptibility.\n\nFewer than one-sixth of all children and adults in this case series, including those with underlying medical risk factors for complications, received antiviral drugs before admission; this requires further investigation. In this case series, 25% were not prescribed antiviral drugs during the admission for reasons that are unclear; in most cases (93%) they did not receive an antiviral drug before hospital admission either. While the median interval between illness onset and hospital admission was just 1\u2005day in children and 2\u2005days in adults, 28% of children and 23% of adults in our study were apyrexial on admission, while just over half had a fever of \u226538\u00b0C. Such findings question the appropriateness of specifying fever of at least 38\u00b0C as part of the clinical case definition in current diagnostic, treatment and infection control algorithms.\n\nThe median length of stay in hospital was 3\u2005days in children and 4\u2005days in adults, which was unaffected by comorbidity or the use of antiviral drugs before admission. While cases that received antiviral drugs before admission were less likely to require high dependency or intensive care and were 50% less likely to die in hospital, neither trend was significant. A number of other studies now suggest that early treatment with oseltamivir may reduce the likelihood of hospitalisation and death due to pandemic H1N1 influenza.33\u201335\n\n## Conclusions\n\nWhile most patients with pandemic H1N1 influenza experience mild disease, 12% of those admitted to hospital require high dependency or intensive care, about 30% have radiographic pneumonia and 5% die. Pandemic H1N1 influenza should be considered in the differential diagnosis of any respiratory illness while the pandemic virus is circulating in the community; fever \u226538\u00b0C is a poor discriminator. Patients admitted to hospital with illness compatible with influenza should have a chest x-ray on admission and should be actively monitored for altered level of consciousness, dyspnoea and low peripheral oxygen saturation. An abnormal chest x-ray or raised CRP level\u2014especially in patients who are observed to be obese, have pulmonary conditions other than asthma or COPD or are pregnant\u2014may suggest a potentially serious outcome. Our findings support the use of H1N1 pandemic vaccine in pregnant women, children aged \\<5\u2005years and those with chronic lung disease as a priority, including patients with asthma, regardless of severity.\n\nThe authors gratefully acknowledge individuals who helped identify cases and collated clinical data: Alison Booth, Margaret Charlesworth, Sarah Rodenhurst, Angela Ballard and Alison Holmes at Imperial College Healthcare NHS Trust, London, UK; Sally Batham, Phayre Parkinson, Tracy Kumar and Aiden Dunphy at the University Hospitals of Leicester NHS Trust, Leicester, UK; Anne Tunbridge, Patty Hempsall, Joyce Linskill, Aimee Turner and Eric Moulds at Sheffield University Hospitals NHS Trust, Sheffield, UK; Elvina White, Elaine Scott, Jennifer Cater, Erica Sergi and Helen Hill at Alder Hey Children's Hospital NHS Foundation Trust, Liverpool, UK; Deborah Fleetwood, Lorna Roche, Sarah Dyas and Maria Boswell at the Royal Liverpool and Broadgreen University Hospital's Trust, Liverpool, UK; Gillian Vernon, Gillian Houghton, Heather Longworth and Angela Kerrigan at Liverpool Women's Hospital, Liverpool, UK; Sonia Greenwood, Gemma Thompson, Emily Jarvis, Tom Bewick and Charlotte Minter at the Nottingham University Hospitals NHS Trust, Nottingham, UK; Kristina Lum Kin, Jacqueline Daglish, Sam Hayton and Gemma Slinn at Birmingham Children's Hospital, Birmingham, UK; Michelle Lacey at Quality Improvement Scotland; Karen Duffy, Anne Gordon and Kevin Rooney at Greater Glasgow and Clyde NHS Hospitals, Scotland; Brian Smyth and Cathriona Kearns from the National Public Health Agency, Northern Ireland for identifying cases and facilitating data collection; Teresa Cunningham at the Southern Trust and Leslie Boydell at the Belfast Trust for facilitating data collection; Alemayehu Amberbir, Safaa Al-Badri, Baraa Mahgoob and Nachi Arunachalam at the University of Nottingham for data entry and obtaining background population data; also Graham Watson for database development and support. They also thank Professor Sir Gordon Duff, Co-Chair of the Scientific Advisory Group for Emergencies, and Professor Dame Sally Davies and Professor Janet Darbyshire, who Co-Chair the Influenza Clinical Information Network Strategy Group, for their support and constructive remarks; Dr Shona Kelly of the University of Nottingham for help in developing the initial data collection tool; and Dr Barbara A Bannister of the Department of Health, England and Dr Patrick O'Brien of University College London Hospitals NHS Foundation Trust for assistance in further developing the data set. The authors thank the Chief Executive Officers, clinicians, virologists and managers, too numerous to mention, who were active in notifying cases to FLU-CIN. SJB and PJMO wish to acknowledge the support of the UK NIHR Biomedical Research Centre scheme.\n\n# References\n\n[^1]: Level 0: Patients whose care needs can be met through normal ward care. Level 1: Patients at risk of deteriorating or recently relocated from higher levels of care whose needs can be met on an acute ward with additional advice and support from the critical care team. Level 2: Patients requiring more detailed observation or intervention including support for a single failing organ system and those 'stepping down' from higher levels of care (high dependency unit). Level 3: Patients requiring advanced respiratory support alone or basic respiratory support together with support of at least two organ systems. This includes all complex patients requiring support for multiorgan failure (intensive care unit).","meta":{"dup_signals":{"dup_doc_count":103,"dup_dump_count":58,"dup_details":{"curated_sources":2,"2023-06":1,"2022-33":2,"2022-27":1,"2022-21":1,"2021-39":1,"2021-31":1,"2021-21":1,"2021-17":1,"2020-45":1,"2019-43":1,"2019-39":1,"2019-30":1,"2018-43":1,"2018-39":1,"2018-26":1,"2018-17":1,"2018-09":2,"2017-51":1,"2017-47":1,"2017-43":1,"2017-39":2,"2017-34":1,"2017-30":2,"2017-22":1,"2017-17":2,"2017-09":3,"2017-04":3,"2016-50":2,"2016-44":2,"2016-40":1,"2016-36":2,"2016-30":2,"2016-26":1,"2016-22":2,"2016-18":1,"2016-07":2,"2015-48":2,"2015-40":2,"2015-35":1,"2015-32":2,"2015-27":2,"2015-22":2,"2015-14":2,"2014-52":2,"2014-49":2,"2014-42":7,"2014-41":3,"2014-35":3,"2014-23":1,"2014-15":4,"2023-14":1,"2017-13":2,"2015-18":2,"2015-11":1,"2015-06":2,"2014-10":3,"2013-48":2,"2013-20":2}},"file":"PMC2921287"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  Compared to more general networks, biochemical networks have some special features: while generally sparse, there are a small number of highly connected metabolite nodes; and metabolite nodes can also be divided into two classes: internal nodes with associated mass balance constraints and external ones without. Based on these features, reclassifying selected internal nodes (separators) to external ones can be used to divide a large complex metabolic network into simpler subnetworks. Selection of separators based on node connectivity is commonly used but affords little detailed control and tends to produce excessive fragmentation.\n  .\n  The method proposed here (Netsplitter) allows the user to control separator selection. It combines local connection degree partitioning with global connectivity derived from random walks on the network, to produce a more even distribution of subnetwork sizes. Partitioning is performed progressively and the interactive visual matrix presentation used allows the user considerable control over the process, while incorporating special strategies to maintain the network integrity and minimise the information loss due to partitioning.\n  .\n  # Results\n  .\n  Partitioning of a genome scale network of 1348 metabolites and 1468 reactions for *Arabidopsis thaliana* encapsulates 66% of the network into 10 medium sized subnets. Applied to the flavonoid subnetwork extracted in this way, it is shown that Netsplitter separates this naturally into four subnets with recognisable functionality, namely synthesis of lignin precursors, flavonoids, coumarin and benzenoids. A quantitative quality measure called *efficacy* is constructed and shows that the new method gives improved partitioning for several metabolic networks, including bacterial, plant and mammal species.\n  .\n  # Conclusions\n  .\n  For the examples studied the Netsplitter method is a considerable improvement on the performance of connection degree partitioning, giving a better balance of subnet sizes with the removal of fewer mass balance constraints. In addition, the user can interactively control which metabolite nodes are selected for cutting and when to stop further partitioning as the desired granularity has been reached. Finally, the blocking transformation at the heart of the procedure provides a powerful visual display of network structure that may be useful for its exploration independent of whether partitioning is required.\nauthor: Wynand S Verwoerd\ndate: 2011\ninstitute: 1Centre for Advanced Computational Solutions, Dept WF & Molecular Bioscience, Lincoln University, Ellesmere Junction Road, Christchurch, New Zealand\nreferences:\ntitle: A new computational method to split large biochemical networks into coherent subnets\n\n# Background\n\nThe genome scale metabolic network of small molecule reactions for cells (particularly eukaryotic cells) is sufficiently complex that it is hard to visualize, let alone interpret. Using conventional biochemical pathways is a bottom-up approach that helps to bridge the complexity gap between individual reactions and the complete network. But this still leaves scope for an intermediate level of granularity, namely subnets. A subnet allows the study of the interplay between pathways and reactions in a broader context, while still focussing attention on a limited biological functionality of interest.\n\nThis line of thought has been pursued by many authors in the recent literature, together with algorithms that use a top-down approach utilising the inherent structure of the complete network to determine its natural subdivision points. In addition to the conceptual argument, there are also practical considerations that motivate this endeavour in particular contexts. The use of structural analysis tools such as elementary modes and extreme pathways \\[1\\], suffers from the problem of a combinatorial explosion \\[2\\] of the number of such modes in a complex network. In essence the problem is that if two small networks are joined together sequentially, each pathway in one can be joined to each pathway in the other. So reversing this and partitioning a large network into subnets is a useful strategy to keep mode numbers manageable. Alternatively, significant advances have also been made in large scale mode calculations in genome scale networks \\[3\\] and analyzing the results by sorting \\[4\\] or pattern matching \\[5\\] techniques. Whether such methods or partitioning is preferable depends on the goals of a particular project.\n\nAnother significant context is flux balance analysis (FBA). There, knowledge of at least some measured fluxes is needed in order to calculate others by applying stoichiometric and other constraints. Current technology allows simultaneous measurement of about a dozen flux values or several hundred metabolite concentrations \\[6\\]. Optimization of an objective function such as biomass production has been used successfully to supplement the constraints for metabolic modelling of unicellular organisms, but the choice of objective for multicellular organisms is problematic and even for unicellular systems, maximising biomass is not always appropriate \\[7\\]. So in a study that focuses on a particular aspect of metabolism, it would be helpful if a way can be found to limit the FBA calculation to a \"relevant\" section of the network and avoid needing boundary conditions that only affect other metabolic aspects.\n\nDepending on the priority allocated to these three sets of considerations, different approaches have been advocated, and a recent review including the application of more general network theory approaches to biological networks, can be found in a recent article by Nayak and De \\[8\\].\n\nThe conceptual network simplification problem is typically addressed by clustering- or community finding algorithms. A typical example is the Markov clustering (MCL) algorithm\\[9\\]. There, the focus is on identifying groups of nodes that are closely connected to each other, while intergroup connections are weaker by comparison. A clustering method somewhat similar to MCL has also been applied to metabolic networks \\[10\\]. This approach uses simulated annealing to process connectivity information to find modules with high connectivity within and minimal links between modules, but takes no account of mass balance constraints.\n\nHowever, clustering of this kind is not really appropriate for metabolic subnetworks. The most highly connected metabolites are commodity or currency compounds such as H~2~O and NADH, but generally (depending on the context) they are of least interest in terms of function. Conversely, the conventional pathways of biochemistry that should form the core of a functionally oriented partitioning are typically linear or circular and only weakly connected in terms of graph structure.\n\nAn alternative approach to the conceptual clarification of biochemical network structure is as hierarchy trees, an approach advocated in the work of Holme, Huss and Jeong \\[11\\] and of Gagneur, Jackson and Casari \\[12\\]. However, such hierarchies are not very amenable to isolating a particular subnetwork for FBA or mode analysis.\n\nAn approach that prioritises the appropriateness of a biochemical subnet for use in practical applications, was demonstrated by Schuster et al \\[13\\]. The approach is based on the observation that metabolite nodes in a biochemical network are of two distinct types: Internal nodes that have associated stoichiometric mass balance constraints, and external nodes that represent metabolite inflows and outflows from the environment and have no associated mass conservation. The external nodes define the periphery of the network, and so a new boundary that separates a subset of nodes from the rest can be created by reclassifying some of the internal nodes as external. Changing the status of a node from internal to external, can in a graphical network representation be seen as splitting it into two: one copy becomes a sink in one subnet, and the other a source in another subnet. This demonstrates another difference from clustering, where networks are usually partitioned by deleting links rather than splitting nodes. The selection criterion used \\[13\\], is that all internal metabolites that participate in more reactions than a chosen threshold value are made external. One rationale behind this criterion is that the many reactions that contribute to mass balance of such a highly connected metabolite are reasonably represented by considering it as buffered, in a subnet not containing all those reactions. Another is that this choice is particularly effective at avoiding the combinatorial explosion problem. And finally, for a high enough threshold, mainly carrier and commodity metabolites are selected that are not the focus of interest in a typical subnet.\n\nUsing a threshold connectivity of 5, the metabolic network of *Mycoplasma pneumoniae* was found \\[13\\] to divide into 19 subnetworks, with identifiable biological functions. Similarly the human redox metabolism was found \\[14\\] to split into 7 subnetworks for the threshold value of 5. Although successful in these relatively small metabolic networks, a criticism expressed by several authors \\[11,12,15\\] is that it relies totally on a local property, the degree of node connectivity, and takes no account of the global network structure. Compared to their method that uses the global \"bow-tie\" structure, Ma et al \\[15\\] found that while both methods classify most currency metabolites similarly, there are examples in their method of both internal metabolites with high connection degree and external metabolites with a low degree. It was also acknowledged in the original article \\[13\\] that the fully automated selection on connection degree alone can be improved by minor editing based on biochemical knowledge. Despite these reservations, connectivity selection is still implemented in the current version of the network analysis software application YANAsquare \\[16\\].\n\nThe network splitting procedure presented in this article aims to incorporate the insights outlined above. In addition it provides flexibility to interactively guide how the splitting proceeds, based on the purpose and biochemical knowledge of the user, within the limits set by the inherent network structure.\n\nThe formulation adopts internal\/external reassignment as the splitting paradigm, but only uses the connectivity degree as a preliminary coarse filter to identify the most obvious external metabolites. This is optionally supplemented or refined by an explicit listing of metabolites that are\/are not taken as external. The main algorithm uses random walks to explore long range network structure, in a similar way as MCL clustering \\[9\\]. However instead of the rigid automated cluster delineation produced by the \"inflation\" step of the MCL, the results are displayed to the user as a matrix that summarises network structure even for large networks in a powerful visual form. At the heart of the visualisation is a blocking transformation designed to express subtleties of the status of each node in relation to an underlying hierarchical clustering, in a way that resembles fuzzy clustering algorithms. Optimisation using linear programming delivers a small set of candidate externals, which the user can accept or reject and this process is repeated until acceptably small subnets are produced. In a final postprocessing step, externals that are not essential for the partitioning achieved, are reincorporated to ensure that the inevitable loss of mass balance information is kept to a minimum. The procedure described here has been implemented in a software application called Netsplitter, and is subsequently referred to as the \"netsplitter algorithm\".\n\n# Methods\n\n## General overview\n\nProcessing of a metabolic network consisting of an unordered list of chemical reactions specified in the standard way by a matrix of stoichiometric coefficients, proceeds through four computational stages:\n\n1\\. Generating a matrix representation of the network connectivity structure from random walks, which expresses each internal metabolite as a distinct source or sink node in an associated directed acyclic graph ( DAG).\n\n2\\. Using hierarchical clustering and a blocking transformation to rearrange the DAG matrix into latent blocks that express the underlying partially separated subnets.\n\n3\\. Proposing prospective separator nodes for approval to the user, implementing the decision and recalculating the DAG with improved blocking, leading to the next round of separator selection.\n\n4\\. Post-processing to consolidate subnets by reincorporating superfluous externals and to reconstitute a stoichiometry matrix specification of each subnet from the DAG matrix blocks.\n\nEach of these stages is described in more detail in the following subsections, followed by introduction of a quantitative measure of effectiveness. Fuller justifications for some of the steps are supplied in a separate subsection at the end of Methods.\n\n## Matrix representation of biochemical networks\n\n### Random walks and probability matrices\n\nThe procedure is based on representing the network as a matrix of probabilities that reflect random walks on a simple graph, similar to that used in the well-known Markov Clustering (MCL) algorithm \\[9\\]. However, since a metabolic network contains nodes of two distinct types (metabolite and reaction), the first task is to reduce the stoichiometry matrix (S-matrix) used conventionally to specify a metabolic network, to a probability matrix for a simple graph containing metabolite nodes only. For this step it suffices to treat the metabolic network as a bipartite graph, although it has been pointed out that metabolic networks are best considered hypergraphs \\[17\\].\n\nFor a simple graph, one starts from a probability matrix **P**~1~ where the elements in row *i* are the probabilities that a random walk starting from node *i* in the network will reach node *j* in a single step. For simplicity, equal probabilities are assigned to all links emanating from a particular node. To achieve this, one merely needs to normalise each row of the adjacency matrix **C** of the graph, by dividing each element by the sum of all elements in the row. The probability matrix **P**~N~ for a random walk of *N* steps, is calculated by raising **P**~1~ to the *N*-th power. Walks of *n* \u2264 *N* steps, will be included in this provided that there is a non-zero probability for a random walk step to \"stay put\" on a node; in other words self-loops are included in the adjacency matrix, or in matrix terms a multiple of the identity matrix is added before the row normalisation is carried out.\n\nIf we start from a state where there is a single \"random walker\" on each node of the network at step 0, the probability associated with each walker has the value 1 for being localised on its starting node. Then **P**~1~ represents propagation of this probability to nearest neighbour nodes in step 1, and generally the potentiating of the matrix can be visualised as the flow of probability through the network after increasing numbers of steps. This is expressed in MCL terminology by referring to potentiating as the \"expansion\" operation.\n\nAs constructed, the matrix **P**~1~ has non-negative elements with a row sum = 1, which makes it an example of a *right stochastic matrix*. From the theory of stochastic matrices \\[18\\], it is known that raising a matrix of this type to consecutive powers converges to a matrix denoted as **P**~\u221e~. In practice, for metabolic networks numerical convergence to an approximation of **P**~\u221e~ is obtained for values of *N* in the low tens. A typical feature of this matrix is that many of its columns are zero vectors, implying that after a sufficient number of steps, the probability of finding a random walker on the node corresponding to that column approaches zero, regardless of the node from which the walker started at step 0. Such nodes therefore act as *sources*, while the remainder with non-zero columns are *sink* nodes. In a thought experiment with one walker starting from each node of the network at time 0, all walkers will congregate on the sink nodes after the number of steps needed for convergence. A binary version of **P**~\u221e~ , obtained by replacing all non-zero elements by 1, can be interpreted as the adjacency matrix of a new graph, containing the same nodes as the original network, but in which all links connect sources directly to sinks in a star-like configuration. This is formally described as a directed acyclic graph, and is directed irrespective of whether any links in the original network were directed. In what follows, either **P**~\u221e~ or its binary version is referred to as the DAG-matrix. Qualitatively the features described above are quite similar to those in the MCL, but note that in MCL terminology only the \"expansion\" operation (raising **P**~1~ to a power) is applied while the \"inflation\" operation that is key to the MCL, is not used. Consequently the DAG obtained here does not usually separate into disconnected clusters, and needs to be further manipulated by the algorithm to extract subnetworks.\n\nThe generalisation of the procedure outlined above to the bipartite metabolic network case, starts by defining two separate adjacency matrices **CR** and **RC**. For a network of *m* reactions and *n* metabolites, the S-matrix is (*n* \u00d7 *m*) with rows representing metabolites and columns reactions. **CR** is similarly (*n* \u00d7 *m*) and each row contains the adjacencies of a metabolite node that act as a substrate to each reaction column, while **RC** is (*m* \u00d7 *n*) and gives the adjacencies of reaction products but with the roles of rows and columns reversed. Formally, these are constructed from **S** by the relations\n\n$$\\mathbf{C}\\mathbf{R} = \\frac{1}{2}\\left( {\\left| {Sign(\\mathbf{S})} \\right| - Sign(\\mathbf{S})} \\right)\\,;\\,\\mathbf{R}\\mathbf{C} = \\frac{1}{2}Transpose\\left( {\\left| {Sign(\\mathbf{S})} \\right| + Sign(\\mathbf{S})} \\right)$$\n\nHere the Sign function takes the values -1, 0 or 1 and serves to ensure that **CR** and **RC** are nonnegative binary matrices. From these the probability matrix for the reduced metabolites-only network is calculated by\n\n$$\\mathbf{P}_{1} = RowNorm\\left( {RowNorm\\left( {\\mathbf{C}R} \\right) \\bullet RowNorm\\left( {\\mathbf{R}\\mathbf{C}} \\right) + 0.25\\,\\mathbf{I}} \\right)$$\n\nHere the function RowNorm normalises each matrix row by a simple row sum, and converts the adjacency matrices to probability matrices. The summation implied in the matrix multiplication accumulates the probabilities for a random walk jump from metabolite node *i* to metabolite node *j*, over all reactions that connect them. Note that links in the metabolites-only network are directed in accordance with underlying reaction directions, whether these are reversible or not. In the added term, **I** represents an (*n* \u00d7 *n*) identity matrix to account for selfloops, and its coefficient is chosen to scale their probabilities in accordance with typical values in a sparse network. The results for the DAG are found to be insensitive to the actual coefficient value. Unlike for a simple graph, the selfloops could not be introduced into the adjacency matrices because they are non-square and even if not, it would have destroyed the bipartite nature.\n\nCalculation of the DAG matrix proceeds by straightforward iterative potentiation of **P**~1~, using convergence of the Frobenius norm of the matrix to within an absolute value of 10^-10^ as the criterion.\n\n### Matrix implementation of partitioning\n\nReclassifying an internal node as external to produce network partitioning, is implemented by deleting the corresponding row from **CR** and column from **RC**. This implies that at any stage the DAG matrix only represents internal metabolites, and as this changes during the course of the partitioning the DAG is regularly updated. A detailed account of this implementation is given in the justifications section further below.\n\n### Preprocessing the DAG matrix\n\nThe first step in processing the DAG matrix is to sort its columns so all non-zero columns are collected on the left and rows sorted in the same order, then deleting the zero columns. In this way, by definition only sink nodes remain in the column sequence and rows are sorted with all sinks appearing first, followed by all source nodes.\n\nTo demonstrate the method, an example network consisting of 137 metabolites and 117 reactions is used in what follows. This network happens to be a subnet for flavonoid metabolism in *Arabidopsis thaliana* extracted from the Aracyc \\[19\\] database, but serves here merely as a realistic example of a small metabolic network. The specification of this network in SBML format is available as Additional File <a href=\"#S1\" data-ref-type=\"supplementary-material\">1<\/a>.\n\nFigure <a href=\"#F1\" data-ref-type=\"fig\">1(a)<\/a> shows the DAG matrix for this network using a colour scale to represent numerical values of the matrix elements. Of the original 137 metabolites, 66 have been identified as a structural external (i.e. it acts uniquely as either a substrate or a product in all reactions in which it participates) and eliminated. The remaining 71 internals are found to separate into 16 sinks and 55 source nodes.\n\nThe top square 16 \u00d7 16 submatrix is seen to be (block) diagonal. For the majority of single diagonal elements, this merely indicates the finite probability that a random walk starting from a sink node will end there as a result of a selfloop, while it will not terminate at any other sink. There are also a few small blocks; they represent small clusters of nodes that are fully connected and hence jointly act as a \"supersink\". This top square does not reflect much of the overall network structure and further manipulation centers on the lower part, i.e. the DAG matrix is further truncated to contain just the (*nsources* \u00d7 *nsinks*) lower left submatrix of the original.\n\nInspection of the lower 55 \u00d7 16 submatrix in Figure <a href=\"#F1\" data-ref-type=\"fig\">1(a)<\/a> shows very little internal structure on which to base network partitioning; almost all source nodes are connected to almost all sinks. The reason is that in a metabolic network there are typically a small number of ubiquitous metabolites such as carrier molecules that participate in many reactions. Sacrificing mass balance for these, is arguably not a serious loss of information, as an excess or lack of the molecule in the subnet can be assumed to be made up by other subnets. The common convention of presenting metabolic pathways as a backbone structure while suppressing secondary metabolites, is an implicit recognition that such crosslinking tends to obscure the underlying connectivity structure that is important to understand functional units in a metabolic network.\n\nReclassifying just the four highest connectivity internal metabolites (Water, Coenzyme A, NADP and NADPH) in the demonstration network produces the drastic change shown by Figure <a href=\"#F1\" data-ref-type=\"fig\">1(b)<\/a>. The truncated DAG now exhibits clear structure, creating scope for further manipulation as described in the next section.\n\nA useful strategy to determine such a set of a priori ubiquitous metabolites is to simply choose a fixed threshold value and reclassify all internal metabolites with connectivities higher than the threshold, in order to reveal the connection structure. A threshold of 8 was found to work well for networks over a wide range of sizes, from about 100 metabolites upwards. Manual adjustment of the threshold can also be done as its effect is easily monitored by visual inspection of the truncated DAG as in Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>.\n\nAlternatively, an explicit list of commonly occurring ubiquitous metabolites can be used instead of a threshold, to avoid inadvertent reclassifications. The most efficient strategy was found to be a combination, using a threshold to automatically reclassify the most \"obvious\" carrier metabolites automatically, and supplementing this with an explicit list of less obvious ones.\n\n## Rearranging the DAG matrix to identify subnets\n\n### Subnetworks and matrix blocks\n\nThe key insight needed to use the mathematical infrastructure described so far for network partitioning, is that separated subnets can be made to appear in the truncated DAG matrix as non-overlapping blocks.\n\nA block is defined as a rectangular submatrix, formed by the intersection of a horizontal band of rows and a vertical band of columns, and where any non-zero matrix elements in either band occur only inside the intersection (so elements in the bands outside of the block are all zero). It follows that the row and column ranges of a block does not overlap with those of any other block. So if it exhibits a non-trivial block structure, the full set of rows in the truncated DAG matrix will be partitioned with no overlap into two or more bands, and similarly the columns into the same number of bands. This definition does not require that blocks are arranged diagonally.\n\nThe connection with disjoint subnets is established by noting that a non-zero element (*i*,*j*) in a particular block of the truncated DAG means that there is a finite probability, and hence a path through the network from source node *i* to sink node *j* in the same block. Conversely, the zero elements in the bands belonging to a particular block but outside that block, means that probability does not flow from source nodes in one block, to nodes in any other block; nor are sink nodes in one block fed by sources in any other block. So the collection of sources and sinks of each block, specifies the internal nodes of an isolated subnet.\n\nThe truncated DAG as constructed so far will not show such block structure, but two operations are available to produce the block structure:\n\n\u2022 Rows and columns may be reordered. There is no penalty to this, as the ordering of internal metabolites inherited from the S-matrix is arbitrary.\n\n\u2022 Internal metabolites may be reclassified as external and deleted from the adjacency matrices. This carries a penalty, as information is lost - the mass balance of the metabolite is not enforced any more. The DAG matrix needs to be recalculated in this case and usually has a different allocation of sinks and sources.\n\n### Rearrangement of rows and columns\n\nThe first step is to rearrange rows and columns so that metabolites belonging to a block are grouped together. For computational efficiency, operations described here are performed on a binary version of the truncated DAG matrix, on the grounds that it is the connectivity of the network that is relevant rather than detailed probabilities from the random walk. In a binary matrix with simple rectangular blocks, all rows\/columns in a particular block are identical but are orthogonal to those in any other block. However, the definition of a block given previously allows zero elements inside a block as well, so this is relaxed to say that rows\/columns belonging to a block needs to be similar to each other but dissimilar from those in other blocks - i.e., it reduces to a vector clustering problem. The Sokal-Sneath vector dissimilarity is used to quantify this, as discussed in more detail in the justifications section.\n\nUsing this measure the rearrangement problem reduces to one of finding row and column sequences that give optimal clustering. Of various standard clustering methods that were considered the hierarchical clustering method \\[20\\] was found to be most suitable.\n\nHierarchical clustering, as expressed in a dendrogram representation, has the advantage that - unlike most other clustering methods - it gives a definite sequence (the ordering of leaves in the dendrogram) while not committing to a fixed number or size of clusters. These can be subsequently determined by choosing a cutoff level in the dendrogram, a property exploited in the next stage of the procedure.\n\nFigure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a> shows the result of reordering rows and columns in the truncated binary version of the matrix of Figure <a href=\"#F1\" data-ref-type=\"fig\">1(b)<\/a> according to an agglomerative hierarchical clustering using single linkage and the Sokal-Sneath dissimilarity criterion. The figure shows a far better organised structure with black regions identifying associations between metabolites groups, but no separated blocks. This is expected - reordering alone can only isolate blocks if the underlying network (after removal of externals) was already divided into disjoint subnets.\n\n### Blocking transformation\n\nThe next challenge is to identify latent blocks that can be separated in further processing. A crucial decision to be made is the optimal number, size and shape of blocks. Reordering alone as in Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>, does not give any obvious clues to whether many small blocks or fewer large ones will best represent the network.\n\nThe decision is facilitated by introducing a *blocking transformation* that expresses the extent to which the hierarchical clusters succeed in defining blocks. The transformation proceeds in five steps:\n\n1\\. Truncating the column dendrogram at a particular chosen level, defines a collection of consecutive column clusters such as *C* = (1-3,4-8,9,10-11,...). In the matrix, these clusters define vertical bands. In row *i* of the rearranged, truncated DAG matrix with elements *p*~ij~, we make the replacement\n\n$$\\left. p_{ij}\\rightarrow r_{ij} = \\frac{\\sum\\limits_{j \\in C_{j}}b_{ij}}{\\sum\\limits_{j}b_{ij}} \\right.$$\n\nHere *b*~ij~ are the binary matrix elements and *C*~j~ is the column cluster to which column *j* belongs.\n\nIn the case of a perfectly blocked matrix, all non-zero elements in a row will belong to the same unique cluster and their values are left unchanged at 1. Any zero element in the same cluster is replaced by 1, i.e. any gaps inside the cluster are filled in. All row elements in the remaining clusters will be, and remain, zero.\n\nHowever, for an imperfectly blocked matrix, any non-zero element outside the range of a particular cluster will serve to dilute the common value of elements inside the cluster to a fractional value. Hence in a gray-scale representation the row appears as a sequence of bands in different shades of grey; the darkest grey identifies the cluster containing the largest fraction of non-zero elements.\n\nApplying this transformation to all rows of the matrix in Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a> produces the row blocking matrix shown in Figure <a href=\"#F3\" data-ref-type=\"fig\">3(a)<\/a>. Analogously, columns are blocked next:\n\n2\\. Truncating the row dendrogram at a particular chosen level, defines a collection of consecutive row clusters *C*~i~. In column *j* of the rearranged, truncated DAG matrix, we make the replacement\n\n$$\\left. p_{ij}\\rightarrow c_{ij} = \\frac{\\sum\\limits_{i \\in C_{i}}b_{ij}}{\\sum\\limits_{i}b_{ij}} \\right.$$\n\nApplication of this transformation to all columns similarly gives the column blocking matrix shown in Figure <a href=\"#F3\" data-ref-type=\"fig\">3(b)<\/a>.\n\nIn a perfectly blocked matrix, blocks based on grouping rows or columns are identical, but the demonstration example shows that for imperfect blocking the row and column blocking matrices are similar but not identical. The next step superimposes the information from the two separate hierarchies.\n\n3\\. Combine row and column blocking matrices by elementwise averaging:\n\n$$s_{ij} = \\frac{1}{2}(r_{ij} + c_{ij})$$\n\nThe combined blocking matrix obtained in this way is shown in Figure <a href=\"#F3\" data-ref-type=\"fig\">3(c)<\/a>. In this matrix, grey shades again indicate deviations from perfect blocking - whether by leakage of amplitude from hierarchical clusters or by discrepancies between row and column blocking. Black areas, on the other hand, identify areas where all evidence from the grouping procedures agrees and that can plausibly be taken as cores for blocks still to be further delineated. Moreover, the gap filling effect mentioned above serves to highlight the intrinsically rectangular shape of blocks.\n\nAn important aspect of the algorithm has been glossed over above. The dendrograms used in steps 1 and 2 define hierarchical lists of distances between subclusters. It is by choice of a particular cutoff value in each list (defining the minimal distance for subclusters to be recognised as separate) that one can choose between many smaller clusters or fewer larger ones.\n\nTo exploit that, a quantitative criterion *Q* for the optimal blocking has been defined as detailed in the justifications section, leading to the next step:\n\n4\\. Calculate *Q* from equation (1.8) after repeating steps 1-3 for each of the trial list of cutoff values, and select the one that maximises Q.\n\nThe blocking matrices shown in Figure <a href=\"#F3\" data-ref-type=\"fig\">3(a)-(c)<\/a> are those that maximise Q for the demonstration network and represent the visual structure of Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a> quite well.\n\nHowever, there is still one noticeable deficiency in Figure <a href=\"#F3\" data-ref-type=\"fig\">3(c)<\/a>. Some blocks appear split - for example, there are two medium grey horizontal lines in the lower half of Figure <a href=\"#F3\" data-ref-type=\"fig\">3(c)<\/a> that each clearly belongs with the large dark areas directly above it. The reason for this is a conflict between similarity at the fine-grained (element) level used when grouping, and the coarse-grained (block) level that applies after blocking. This is rectified by the final step of the blocking transformation.\n\n5\\. Consolidate blocks by reordering rows and columns according to hierarchical clustering now applied to the combined blocking matrix.\n\nAs Figure <a href=\"#F3\" data-ref-type=\"fig\">3(d)<\/a> shows, the final result of the blocking transformation gives a succinct and visually appealing overview of the size, shape and location of blocks, as well as indicating areas that produce block overlap.\n\nFinally, in order to computationally process individual subnets, automated recognition of separated blocks is required. This is a fairly straightforward image processing problem, and a heuristic procedure based on the block definition given above is described in Additional file <a href=\"#S2\" data-ref-type=\"supplementary-material\">2<\/a>. A side effect of the heuristic is that matrix rows and columns are rearranged once more, aligning recognised blocks along the matrix diagonal.\n\n## Selection of separation nodes\n\nHaving prepared the DAG matrix to express any underlying partial block structure, the procedure now enters an iterative loop in which in each round, a small number of nodes are identified that when \"cut\" (i.e., the corresponding metabolite is reclassified as external), will lead to separation into subnets. The goal is to keep this set of separation nodes as small as possible, both to minimise the loss of mass balance information and to preserve as far as possible the local structure of the full network.\n\nFor example, applying block recognition scanning to the matrix of Figure <a href=\"#F3\" data-ref-type=\"fig\">3(d)<\/a>, recognises only the full matrix as a single block. That is also visually apparent - there are four or more latent blocks highlighted in black, but none are fully separated as signified by grey areas.\n\nFor a number of reasons, it is postulated that the lighter grey cells in the figure are the most promising candidates for removal to induce separation. One rationale is that by construction they reflect a status as exceptions while the majority of cells in their row or column belong to the same group and end up as dark grey. Also, they tend to result from cases where there is already separation from the perspective of the row grouping and only weak overlap from the column grouping, or vice versa. Middle grey, on the other hand reflects either strong evidence from one of the groupings, or moderate consensus that may solidify once the weakest overlaps are removed. Also, there is some analogy to the effects of the \"inflation\" step of the MCL. In that method, \"inflation\" is produced by taking the Hadamard power of a probability matrix; that tends to suppress low probabilities and leads to the \"weakest\" links between node clusters to be removed first. These arguments can be made more elaborate, but in the final analysis the justification lies in the result obtained. As detailed in the justifications section, linear programming is used to select a small number of metabolites that optimally cover the lighter grey cells in the blocking matrix and propose these to the user as candidate externals.\n\nAn example is illustrated in Figure <a href=\"#F4\" data-ref-type=\"fig\">4(a)<\/a> where this is applied to the case of the consolidated blocking matrix in Figure <a href=\"#F3\" data-ref-type=\"fig\">3(d)<\/a>. The picture shows that two columns, i.e. metabolites that produce block overlaps (separation nodes), are identified by solving equation (1.9). These metabolites are made external, i.e. they are deleted from the adjacency matrices **RC** and **CR**, the DAG matrix recalculated and the blocking transformation repeated to identify further candidates in a second round, and this iteration is continued until either sufficiently fine-grained splitting has been achieved, or no more separation nodes are found.\n\nFigure <a href=\"#F4\" data-ref-type=\"fig\">4(b)<\/a> shows the result in selection round 5. The blocking matrix still shows only one block, but has decreased in size, firstly because of the removal of separation nodes. Also, at each round the algorithm inspects the blocking matrix for any non-overlapping block that consists of a single row or column, like the two middle blocks shown in Figure <a href=\"#F4\" data-ref-type=\"fig\">4(c)<\/a>. Such blocks cannot be split further, and are therefore removed from the blocking process to be restored later.\n\nAnother case of such irreducible blocks that is usually encountered, is the appearance in the DAG matrix of isolated sinks or \"orphans\". These appear as entries in the top, diagonal section of the DAG matrix with no accompanying source node entries in the corresponding column of the truncated matrix used for blocking. Such an orphan metabolite node signifies the simplest possible subnet, with only a single internal metabolite, and typically containing only two reactions. As these can obviously not be further split and the presence of an empty column complicates block recognition, they are best eliminated in each round from the adjacency matrices along with single row\/column irreducible blocks.\n\n## Postprocessing and reconstruction of subnetworks\n\nOnce the iterative process of progressively selecting separation nodes has terminated, the main outcome is a list of internal metabolites, partitioned into disjoint subsets that belong to each block. The remaining metabolites constitute a list of external metabolites. This list may contain entries that are not, in fact, essential for block separation. For example, a metabolite may have been made external during initialisation on the grounds that it participates in a large number of reactions, but if all of those reactions belong to the same subnet it should be reinstated as an internal metabolite in this subnet. Also, it can happen that the effects of a metabolite selected early on in the progressive selection process, are superseded by one selected later. In the interests of maintaining maximal network integrity compatible with the separation, all such superfluous externals need to be reincorporated before finalising the subnets.\n\nThis is done in a loop that inspects the stoichiometry matrix for each external metabolite on the list, to determine all internal metabolites to which it connects by reaction links in either direction in the bipartite representation. If all those belong to a single block, the external metabolite is reincorporated into that block. If they belong to a single block, except for a connection to one or more orphan metabolite nodes, those orphan nodes are also reincorporated into the block as detailed below. As this reincorporation loop changes the composition of the lists of internal and external metabolites, the loop is repeated iteratively until there is no further change in the composition of the lists.\n\nThe approach that was chosen to select separation nodes progressively, a few at a time, has the advantage that it allows the user to steer the network splitting by accepting or rejecting proposed separation nodes and terminating the process at the desired level of granularity. However, a disadvantage is that the results may become dependent on the order in which separation nodes are identified. That is counteracted by performing a one-off blocking step in which the full list of external metabolites are applied simultaneously. This step is performed as part of the post-processing done after the selection process is finished; but the question arises whether it should be done before or after the reincorporation step. Each choice has some advantages, and the most robust result is in fact achieved by repeating the reincorporation step. So the full post-processing procedure consists of the 3-step sequence: a first reincorporation step, then the one-off blocking, followed by a second reincorporation. Figure <a href=\"#F4\" data-ref-type=\"fig\">4(c)<\/a> shows the effects of the reincorporation for the demonstration network.\n\nOnce the partitioned list of internal metabolites is finalised by this post-processing, the individual subnets can be reconstructed in a straightforward way from the original stoichiometry matrix **S**. For each subnet, all reactions in which its internal metabolites participate are extracted from **S** and allocated to this subnet. All metabolites that participate in these reactions are collected; those not appearing on the list of internals for the subnet, are by definition the external metabolites of the subnet. The submatrix of **S** pertaining to the reactions and metabolites so identified is extracted and saved in appropriate format as a full specification of the subnetwork, which can be further analysed by standard network analysis or FBA software tools.\n\nBy construction, the internal metabolites of different subnets are mutually exclusive sets. External metabolites, on the other hand, are often shared between subnets. In the vast majority of cases, there is also no overlap between external metabolites of any subnet and the internals of any other.\n\nThere are, however, rare exceptions where an external of one subnet is in fact an internal of another. This phenomenon can be considered an artefact of the way that the algorithm mainly operates on a reduced metabolites-only simple graph. At this level where the blocking procedure is carried out, there is a strict distinction between internal and external metabolites; they form non-overlapping sets. However, when translated back to the underlying bipartite graph representation, cutting all metabolite nodes that were identified as external, can sometimes still leave subnets connected by a shared reaction node.\n\nA typical case is shown in Figure <a href=\"#F5\" data-ref-type=\"fig\">5(a)<\/a>, where subnets A and B are connected by reaction node R1 which has input reactants *a* and *b* respectively from each subnet. Figure <a href=\"#F5\" data-ref-type=\"fig\">5(b)<\/a> shows the metabolites-only representation, where splitting node *c* by making metabolite *c* external will separate the subnets. But then, after the blocking procedure, the external metabolites of each subnet are found by collecting metabolites that participate in all the reactions in which each internal metabolite is involved. In the case of subnet A, both nodes *b* and *c* are connected to its internal node *a* by reaction R1 and will be added to its list of externals; and conversely, for subnet B, metabolite *b* is internal while *a* and *c* become externals.\n\nThe existence of this kind of limited overlap between two subnets does not compromise the integrity of either as a coherent subnet: it remains true that for all internal metabolites in a subnet, all reactions in which they participate are included in the subnet, and so the mass conservation constraints of all internal metabolites are identical in the subnet and in the full network. However, it does uniquely create the complication that the same reaction is present in both subnets, which can lead to conflicting values for the flux through this reaction in separate FBA calculations for each subnet. To avoid that, it may be preferred to merge the two subnets into a larger one when this exceptional case arises.\n\nIt should also be noted that for a similar reason the reincorporation of orphan metabolite nodes is slightly more complicated than outlined above. By definition, an orphan node is isolated from all other internal nodes in terms of probability flow, but it could still be connected by a unidirectional link towards the orphan. Consequently, incorporation of an orphan takes place in two steps. When an external connected to an orphan metabolite node is incorporated into a block, the orphan is first promoted to an external of that block. In the next round of the incorporation loop, it is then tested for links to internals of other blocks and only incorporated as an internal if no such links in either direction is found.\n\n## Detailed justifications\n\n### Internal and external metabolites and network partitioning\n\nConventionally, external nodes are placed on the periphery when drawing a network to indicate that they form the interface between the metabolic system that the network represents and its environment. However, the distinction between nodes that are associated with mass balance constraints (internal metabolites), and those that are not (external metabolites) is not apparent when the network topology is simply specified as a list of reactions. Most external metabolites can be recognised computationally by the fact that an external metabolite is either taken up or delivered to the environment so that all network links impinging on an external node are directed away from or towards the node; but in cases where the metabolite is exchanged with the environment that distinction is lost.\n\nA convention commonly used in FBA of metabolic networks \\[1\\] to keep track of externals, is to order the rows of the S-matrix so that internal metabolites appear first and externals last. Then the lack of stoichiometry constraints for externals is easily implemented by using only the top (internals) section of the matrix for FBA calculations.\n\nAnother feature of representing a chemical network by a bipartite graph, is that as reaction nodes represent a chemical transformation of one or more reactants, reaction nodes can never be external.\n\nThese issues become relevant for partitioning a network, because in isolating a subnetwork a new periphery is created for it. Severing the connection between the subnet and the rest of the network, some metabolites are received from or\/and delivered to the rest of the network. Their mass balance can no longer be guaranteed by the subnet alone; in other words, the status of these metabolites is changed from internal to external. From a graph theory perspective, partitioning corresponds most naturally to deleting a link of a graph. However, that will not do for the biochemical network; in the bipartite representation, that would make a reaction node external, and it makes even less sense in the metabolites-only simple graph representation where a link represents a sum over several reactions. In clustering methods such as MCL, each node is allocated to a particular cluster, but that would not make sense here either as a metabolite that is made external by partitioning belongs to both subnets - as a product of one, and substrate of the other subnet. Clearly the appropriate way to represent partitioning is to split the metabolite node into two, each becoming an external node in either subnet. This leaves all reaction nodes as internal and uniquely assigned to a subnet. The effect of splitting a node is to stop probability flow through the node, and the simplest way to implement that in the matrix representation, is to delete the corresponding row from **P**~1~ and hence ultimately from the DAG.\n\nThe problem of partitioning the network hence reduces to finding a suitable (by criteria to be formulated) subset of internal metabolites such that when deleted, the network divides into self-contained subnets with no probability flow between them.\n\nRecognising that the algorithmically found externals are due to be deleted in this way, it follows that metabolites that are already external in the full network should similarly be deleted from **P**~1~ even before the partitioning starts. This step in fact corresponds to the restriction of FBA calculations to the internal rows of the S-matrix as mentioned before. However, in the procedure presented here rows are deleted from the adjacency matrices **CR** and **RC** while **S** is left intact, so that the reaction stoichiometries can be used to restore the externals to each subnet once partitioning is complete.\n\n### Vector clustering\n\nA quantitative measure of dissimilarity *d* for binary vectors can be based on the formula\n\n$$d = \\frac{n_{10} + n_{01}}{n_{10} + n_{01} + a\\, n_{11} + b\\, n_{00}}$$\n\nHere, corresponding elements in two equal length binary vectors are paired, and *n*~ij~ is the number of pairs with value (*i*,*j*). Commonly used such measures are known as Matching (*a* = 1, *b* = 1), Jaccard (*a* = 1, *b* = 0), Sokal-Sneath (*a* = 1\/2, *b* = 0), Rogers-Tanimoto (*a* = 1\/2, *b* = 1\/2) and Dice (*a* = 2, *b* = 0) dissimilarities. Experimentation with all of these in the present context has shown that Matching and Rogers-Tanimoto give low contrast. Dice, Jaccard and Sokal-Sneath are similar but the best contrast and hence block identification is obtained with Sokal-Sneath dissimilarity.\n\nHierarchical clustering in addition needs a measure for the distance between clusters (a \"linkage criterion\"), and again several common measures were tried: single linkage (minimum intercluster dissimilarity), complete (maximum intercluster dissimilarity), average dissimilarity, dissimilarity of cluster centroids or medians, and finally the Ward minimum variance criterion. Single linkage was found to be both fast to calculate and gives good contrast; average, centroid and median are slower but give similar results, while complete and Ward lead to excessive fragmentation of the network.\n\nClustering of the combined blocking matrix in step 5 of the blocking transformation is performed broadly as described for the DAG. However since the combined blocking matrix can contain fractional values, the binary dissimilarity measure described by equation (1.6) is replaced by a generalisation of the Dice dissimilarity to real values and known as the Bray-Curtis distance between vectors *a* and *b*:\n\n$$d = \\frac{\\sum\\left| {a_{i} - b_{i}} \\right|}{\\sum\\left| {a_{i} + b_{i}} \\right|}$$\n\n### Blocking quality\n\nExperimentation with various possibilities yielded the following scoring formula for the blocking quality *Q* of an (I\u00d7J) combined blocking matrix:\n\n$$Q = \\left( \\frac{\\sum\\limits_{i,j}c_{ij}}{I\\, J - W} \\right)^{2}\\frac{W}{I\\, J}$$\n\nHere, W is the total number of zero (white) elements in the matrix. The two factors in this formula express distinct features that seem qualitatively reasonable to judge the quality of the blocking matrix. The first, squared, factor would be 1 if all non-zero elements are 1 (black) and decreases when there are more and lighter grey cells; so it is a measure of \"how black\" the block parts of the matrix is. On its own, however, maximising this tends to favour a small number of large blocks because that makes it easier to capture all the non-zero values inside blocks. To counteract that, the second factor represents the fraction of cells that are white, so this tends to be maximised by keeping blocks as compact as possible. It clear that for a perfectly blocked matrix, a maximum *Q* value *Q*~max~ \\< 1 will be achieved if the cutoff produces clustering that coincides exactly with the blocks.\n\nIn an imperfectly blocked matrix, adjusting the dendrogram cutoff gives unpredictable fluctuations in the *Q* value and so a search method is necessary to maximise *Q*. Three strategies are employed to keep this manageable. First, as the number of recognised clusters only changes when the cutoff rises above an actual intercluster distance value in the dendrogram, the search space is restricted to a trial list of discrete cutoffs falling midway between values in the ordered intercluster distance list. Second, the same cutoff value is applied to both the row and column dendrograms. Although it constrains the flexibility of the search, this helps to avoid large disparities in the number of clusters for rows and columns (which ideally should be equal) despite the fact that the truncated DAG matrix generally has far more rows than columns. To implement that, the intercluster distance lists for rows and columns are merged before selecting midpoints. Finally, bearing in mind that distance values from equation (1.6) fall between 0 and 1, the discrete list of trial cutoffs is truncated to values in the range \\[0.2,0.7\\]. This avoids either very few or very many blocks that would not be desirable for subnet partitioning and rarely produce high *Q* scores anyway.\n\n### Optimal selection of separator nodes\n\nTo implement the recognition of light grey cells in the blocking matrix as most promising for eliminating block overlap while keeping the metabolites taken as external to a minimum, the strategy is to select the smallest set of rows and columns that together cover all matrix cells with values below a chosen threshold. The threshold is determined as the value that selects a total number of light grey cells, no more than a low multiple of the column dimension of the matrix. This gives a flexible threshold value adapted to the size and nature of the matrix, which will lead to only a few metabolites eliminated at a time before checking for adequate subnet separation.\n\nAs any light grey cell could be eliminated by taking either its row or column metabolite external, the optimal selection from both sets is determined by reformulating this as an integer linear programming (ILP) problem. To set that up mathematically, introduce a binary column vector **x** of dimension = number of internal metabolites. Each vector element is 1 or 0 according to whether the corresponding metabolite is selected. The total number selected is obtained by premultiplying **x** with a row vector **b** of the same dimension with all elements equal to 1. The constraints are that for each light grey element *s*~ij~ included, either its row or column or both needs to be present in **x**. That is codified by a constraints matrix **A** in which each row corresponds to a light grey cell, and in such a row the only non-zero elements are 1's for the columns corresponding to *i* and *j*. Then the ILP problem is:\n\n$$\\text{Minimize}\\quad\\mathbf{b} \\cdot \\mathbf{x}\\quad\\text{subject~to}\\quad\\mathbf{A} \\cdot \\mathbf{x} \\geq \\mathbf{b}$$\n\nThis problem is to be solved in the domain of binary vectors, and is guaranteed to be feasible, since all constraints are satisfied by **x** = **b**. Solution by standard methods typically yields small sets of selected metabolites.\n\n## A quantitative measure of overall splitting effectiveness\n\nThe goal of subnet splitting is to reduce the complexity of interpretation (mentally or by further computation) by reducing the size of networks that need to be considered. It is shown here that a robust quantitative measure of how effective a particular splitting procedure is in achieving this goal can be developed under quite general assumptions.\n\nThe original network constitutes the obvious lower limit of simplification. In the opposite extreme where the network is fragmented into subnets consisting of a single node each, no overall simplification has been achieved either: while the subnets are simple, their interconnections reconstitute exactly the original network. This suggests that to judge overall effectiveness, the subnets should be considered together with a \"metanetwork\" which is derived from the original network by contracting the internal nodes of each subnetwork to a single meta-node. It should then be possible to construct a measure that evaluates to zero at the two extremes, and reaches at least one maximum at a suitable intermediate network partitioning.\n\nTo quantify the concept of simplification, it useful to introduce a monotonically increasing function *f*(*n*) that represents the effort to interpret a network with *n* internal nodes. Having split a network with *N* internal nodes into *k* subnets, each with *n~i~* internal nodes, the metanet has *k* nodes and the total effort *F* to interpret the metanetwork and all the subnets is then given by\n\n$$F(\\overset{\\rightarrow}{n},k) = f(k) + W\\sum\\limits_{i = 1}^{k}\\, f(n_{i})$$\n\nand this is subject to the constraint $\\sum{n_{i} = N}$. With a weight *W* = 1, equation (1.10) represents the effort to interpret the metanet and all subnets; choosing *W* = 1\/*k* is relevant to the case where a single subnet and its interconnections to other subnets are of interest. The latter case is algebraically simplest so is considered first.\n\nThe term \"effort\" is used to emphasize that this is not about network complexity as such. Many sophisticated measures of network or graph complexity have been defined by various authors, and network size usually does not play an important part in this - for example, both a square lattice and a fully connected network are conceptually simple, irrespective of size. Also, biochemical networks are known to be scale-free (having a power law distribution of node connectivity) and so complexity measures should give a similar value when applied to the full network and its subnetworks.\n\nFor a given *k*, it follows by straightforward differentiation of equation (1.10) that the constrained minimisation of *F* is achieved by choosing all subnets of equal size, i.e. *n*~i~ = *N*\/*k* , provided that *f*(*n*) is concave up, i.e. increases monotonically faster than *n*. This is quite reasonable, considering that the number of reaction links in a metabolic network rises approximately quadratically with the number of internal metabolites and the number of pathways to be interpreted much faster than that.\n\nMoreover, setting *n*~i~ = *N*\/*k* into (1.10) shows that the minimum is achieved at the optimal number of subnets *k* = $\\sqrt{N}$, irrespective of the functional form of *f*. This result makes intuitive sense, as it implies that all subnets and the metanet have the same number *n~i~* = $\\sqrt{N}$ of internal nodes, so the interpretative effort is spread equally across them all. The value of *F* at its minimum is given by the simple formula\n\n$$F_{l}(N) = 2f\\left( \\sqrt{N} \\right)$$\n\nWhen $\\sqrt{N}$ is not integer, this minimum cannot actually be reached, but it still serves as a lower limit to the range of *F*, while the upper limit is obviously *F*~u~(*N*) = *f*(*N*). In these terms the goal of partitioning can be formulated as finding a vector $\\overset{\\rightarrow}{n} = (n_{1},n_{2},...n_{k})$ that comes as close as possible to *F~l~*, having started from *F*~u~. This leads to defining a performance measure (designated as the *efficacy*) as\n\n$$E = 100\\frac{Log\\left\\lbrack {f(N)} \\right\\rbrack - Log\\left\\lbrack {F\\left( {\\overset{\\rightarrow}{n},k} \\right)} \\right\\rbrack}{Log\\left\\lbrack {f(N)} \\right\\rbrack - Log\\left\\lbrack {2f\\left( \\sqrt{N} \\right)} \\right\\rbrack}$$\n\nThis may be interpreted as the percentage of the distance between the upper and lower limits that has been achieved by a given partitioning, as measured on a logarithmic plot. Use of logarithmic scaling is not conceptually essential but helps to smooth the distribution of efficacy values when *f* is a fast-rising function.\n\nIt is easily checked that *E* evaluates to zero for the original network and could even reach a slightly negative value for the case of complete fragmentation, while it gives 100% if the optimum *k* = *n*~i~ = $\\sqrt{N}$ is reached.\n\nTo get concrete values, a power law assumption produces the required concave up behaviour while still allowing the actual rate of increase to be adjusted:\n\n$$f(N) = \\alpha N^{p}\\quad;\\quad p > 1$$\n\nA value *p* = 2 to reflect the size of the adjacency matrix that fully specifies the network seems reasonable, but the choice is left open for now. The proportionality constant *\u03b1* cancels out in constructing (1.12) so is suppressed in what follows.\n\nThe efficacy curves calculated from equations (1.12) and (1.13) for equal-sized subnets, at subnet counts *k* ranging from 1 to *N*, are shown in Figure <a href=\"#F6\" data-ref-type=\"fig\">6<\/a> for *p* = 1.5 and *p* = 10. With a smooth variation and quite a steep gradient, in particular near the optimal subnet count, *E* gives a sensitive measure of how well a particular network split performs in terms of its subnet count. The figure also shows that this behaviour is almost independent of the *p*-value, with higher values merely sharpening the maximum slightly.\n\nThe main significance of the *p*-value is in determining the discrimination between partitionings with the same *k*, but homogeneous versus distributed subnet sizes. Raising the power value increases the dominance of larger subnets over smaller ones in the summation term of equation (1.10), which tends to lower the efficacy value for a split with a large spectrum of subnet sizes.. Experimenting with various networks has shown that for large networks *p* values in the range 6 to 8 are required to adequately discriminate between network splits that are dominated by large subnets, and those with more even size distributions. For small networks the available range of subnet sizes is correspondingly small and the value of *p* less important. The best results are obtained by adjusting *p* to the network size, and the following empirical formula performs this adequately:\n\n$$p = 0.25\\sqrt{N}$$\n\nThis formula is merely a calibration of the efficacy scale and has no fundamental significance. The results below illustrate its effects.\n\nIt is finally noted that the efficacy measure is constructed quite independently of the Netsplitter method; as its only required input is a list of subnet sizes, it can equally well be applied to diverse partitioning algorithms.\n\n# Results\n\nThe results obtained from the Netsplitter procedure are illustrated by considering the problem of investigating the flavonoid metabolism of the model plant *Arabidopsis thaliana*. The complete network was obtained by extracting all stoichiometrically balanced reactions from the Aracyc 4.5 database as curated by the Arabidopsis Information Resource (TAIR) \\[19\\] and contains 1468 reactions and 1348 metabolites. Reaction directions and reversibilities were assigned in accordance with the pathways and enzymes tables of the database. It is not intended as a definitive metabolic network for the organism (no further curation e.g. using flux balance calculations was done) but for the demonstration purpose it is considered adequately representative of genome scale metabolic networks. The specification of this network in SBML format is available as Additional File <a href=\"#S3\" data-ref-type=\"supplementary-material\">3<\/a>.\n\nFor comparison, Figure <a href=\"#F7\" data-ref-type=\"fig\">7<\/a> first shows the performance of the simple connection degree partitioning \\[13\\] for various threshold values. Visual representation of the DAG matrix is as in Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>, but elaborated here by the addition of a blue background that displays automatically recognised block limits.\n\nFor a large threshold value of 20, Figure <a href=\"#F7\" data-ref-type=\"fig\">7(a)<\/a> shows that only a few small blocks are split off from the main block that still contains more than 90% of all internal metabolites. As observed in the demonstration example, internal structure in this large block is not resolved. Reducing the cutoff threshold to a value of 5 used in previous work \\[13,14\\], the internal structure is well resolved but the main block still contains 20% of all metabolites. Reducing the threshold still further to 4 finally avoids the domination of a single large block, but at the cost of fragmenting the network into 164 very small blocks containing only 4 internal metabolites on average.\n\nFigure <a href=\"#F8\" data-ref-type=\"fig\">8<\/a> shows that the netsplitter algorithm achieves a much more even distribution of subnet sizes. Using the same initial threshold value of 10 shown in Figure <a href=\"#F7\" data-ref-type=\"fig\">7(b)<\/a> but supplementing this with a targeted list of general external metabolites as specified in Additional File <a href=\"#S4\" data-ref-type=\"supplementary-material\">4<\/a>, has the effect shown in Figure <a href=\"#F8\" data-ref-type=\"fig\">8(a)<\/a> of resolving the internal structure of the initial main block. The netsplitter algorithm exploits this internal structure to split it progressively. In its first round, a medium sized block which turns out to contain all flavonoid compounds is split off as seen in Figure <a href=\"#F8\" data-ref-type=\"fig\">8(b)<\/a>. For the purpose outlined above the procedure can be terminated at this point, and this subnet extracted for further study as was done to create the flavonoid demonstration network used above. However, if the procedure is allowed to run its course, the final result is shown in Figure <a href=\"#F8\" data-ref-type=\"fig\">8(c)<\/a>. There is a substantial improvement, in that rather than a single large block, there are several medium sized ones. Quantitatively, 66% of all internal metabolites are captured in the 10 largest blocks which range in size between 20 and 76 internal metabolites. There is still some fragmentation, with the remaining 34% spread over 68 small blocks containing 3.6 metabolites on average. Some fragmentation is probably inevitable when making cuts to a network, but future work will be aimed at reducing this to a minimum.\n\nThe reincorporation step is important to keep the number of externals as low as possible. For example, in the full *Arabidopsis* network, 17 of the 57 high connectivity externals and 12 of the 22 orphan metabolite nodes created by taking a connectivity degree threshold of 10, can be reincorporated without disturbing the block structure in Figure <a href=\"#F8\" data-ref-type=\"fig\">8(a)<\/a>. When the netsplitter algorithm is subsequently applied, these numbers are reduced because of the increased partitioning achieved, but in turn 112 of the 150 externals proposed during the course of externals selection rounds are in fact reincorporated in the final stage. Even in the case of the small flavonoid network where only a single separation round is needed producing 8 proposed externals, 6 of these get reincorporated leaving only two separation nodes. The overall effect is that in both cases less than 5% of the significant mass balance constraints in the full network are sacrificed to decompose it into subnetworks.\n\nIt is also instructive to see the action of the netsplitter procedure in an explicit network diagram. The actual layout of the flavonoid demonstration network, for which stages in the procedure were traced out in matrix form in Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a> to <a href=\"#F4\" data-ref-type=\"fig\">4<\/a>, is shown in Figure <a href=\"#F9\" data-ref-type=\"fig\">9<\/a>. A larger version of this figure identifying the metabolites is available as Additional File <a href=\"#S5\" data-ref-type=\"supplementary-material\">5<\/a>.\n\nThe algorithm identifies two separation nodes in this case - the metabolites trans-cinnamate and coumaroyl-CoA (shown in red); cutting these, the network falls apart into four natural subnets, plus two small fragments or \"orphans\". By inspection of the metabolite names (not shown in the figure) the subnets can be identified as synthesis of flavonoids (purple), lignin precursors (green), benzenoids (blue) and coumarin (yellow). While in this relatively small network it may have been possible (although not easy) to identify these separators by inspection, it should be borne in mind that much of the work to group nodes coherently has already been done in the manual construction of the two-dimensional layout displayed. In a realistic example, the input to the algorithm is merely arbitrarily ordered lists of metabolites and reactions, making the task much harder.\n\nTo place the results in a more general perspective, Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a> compares efficacy values for various cutoff values in simple connectivity-based partitioning \\[13,14,16\\], with Netsplitter results applied to four different networks of increasing size. In addition to the flavonoid demonstration network and the genome scale model plant *Arabidopsis* discussed in detail above, genome scale networks for a simple bacterial species and a mammal are included. The first of these is a metabolic model for *M. pneumoniae* recently published by Yus et al \\[21\\]. Details of the application of Netsplitter to this model has been published elsewhere \\[22\\] and is shown there to give a partitioning that is virtually identical to the assignment by Yus et al of pathways to functional blocks, based on biochemical knowledge. The second is a model for *Mus musculus* validated by FBA \\[23\\].\n\nEfficacy values\n\n|  | Flavonoid | *M. pneumon* | *Arabid. t*. | *M. musculus* |\n|----|----|----|----|----|\n| S-matrix size | 137 \u00d7 117 | 189 \u00d7 229 | 1468 \u00d7 1348 | 2016 \u00d7 2158 |\n|  |  |  |  |  |\n| *p*-value | 2.1 | 2.8 | 7.2 | 7.6 |\n|  |  |  |  |  |\n| Threshold | *High connectivity cutoff method* |  |  |  |\n| 20 | 0 | 33 | 18 | 25 |\n| 10 | 19 | 33 | 24 | 32 |\n| 8 | 19 | 33 | 27 | 37 |\n| 6 | 31 | 37 | 49 | **41** |\n| 5 | **80** | 45 | **54** | 33 |\n| 4 | 76 | **60** | 43 | 27 |\n| 3 | 29 | 32 | 32 | 21 |\n|  |  |  |  |  |\n| Netsplitter | *Default automated externals and reincorporation* |  |  |  |\n|  | 88 | 85 | 70 | 48 |\n\nValues are shown as percentages, and peak values highlighted in bold. The p-value increases with network size as described in the text.\n\nConsidering first the connectivity based splitting, Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a> illustrates that as expected, lower efficacies are calculated for large connectivity cutoffs where a large part of the network remains undivided, and also for small cutoffs where the network is fragmented. This produces an intermediate maximal efficacy, at cutoff values close to 5, in accordance with the values chosen empirically \\[13,14\\] on similar small networks. The p-values assigned by equation (1.14) fall well within the range between the curves in Figure <a href=\"#F6\" data-ref-type=\"fig\">6<\/a> in all cases.\n\nIn judging efficacy percentages, its increased sensitivity near the optimum as illustrated in Figure <a href=\"#F6\" data-ref-type=\"fig\">6<\/a> should be borne in mind - e.g., a partitioning with an efficacy *E* = 80% is about six times closer to the optimal subnet size than one with *E* = 40%.\n\nWhile a single numerical score can hardly be expected to capture all the varied considerations (some subjective) of what constitutes the best partitioning, the more detailed graphical representation in Figure <a href=\"#F10\" data-ref-type=\"fig\">10<\/a> of the most interesting cases, suggests that *E* as calculated here does give a good overall quality indication.\n\nIn that figure, each bar segment corresponds to a subnet and the total height of each bar represents the total number of internal metabolites for that partitioning. Thus the height difference from the reference bar on the right, indicates the total number of internal metabolite mass balance constraints that have been sacrificed to achieve a particular split. The reference bar also shows the theoretical maximal efficacy $\\sqrt{N}$ size partitioning of the original network. As this totally ignores the network topology, no actual partitioning can be expected to achieve that. But a reasonable aim would be for subnets to have this size on average without too large deviations on either side.\n\nFigure <a href=\"#F10\" data-ref-type=\"fig\">10(a)<\/a> illustrates that a large connectivity cutoff for the bacterial network (the first bar) gives the opposite effect, with one large block and a few very small ones, and this has a correspondingly low efficacy value of 33%. The cutoff *C* = 4 in this case splits the large block into medium sized ones approximating the reference value, but at the price of considerably increasing both the number of small blocks and the number of lost constraints, while achieving an efficacy of 60%. The Netsplitter result improves on both aspects and gives *E* = 85%.\n\nIn the case of *Arabidopsis*, the *C* = 5 value that maximises *E* gives a couple of rather large blocks and many small ones, including fragments too small to be resolved graphically and appearing as black or grey areas at the bottom. Decreasing the cutoff to 4 worsens the fragmentation, and this is reflected in the *E*-values of 54% and 43% respectively. It is again visually obvious that the Netsplitter result improves on both of these by spanning the reference subnet size more effectively, and has a moderately high score of *E* = 70%. It has less fragmentation than either of the cutoff results, and it also retains more constraints than either.\n\nA rather similar situation is shown in Figure <a href=\"#F10\" data-ref-type=\"fig\">10(c)<\/a> for the case of *M. musculus*, The best cutoff value increases slightly to *C* = 6 for this larger network, but already shows large fragmentation reflected in *E* = 41% and again this worsens both visually and in terms of the calculated efficacy to *E* = 33% if *C* is reduced to 5. Visually, the Netsplitter result has a slightly better spread of sizes at the top and somewhat better but still significant fragmentation at the bottom, giving an only moderately improved efficacy score of 48%.\n\nIn all cases, the efficacy score based on equation (1.14) accords quite well with observations from the more detailed graphical display.\n\nThe analysis above of the performance of the netsplitter algorithm for the larger networks, shows that there is a decline with size but that this is not due to its efficiency in splitting, but rather that fragmentation becomes an increasing problem as networks grow. A direct approach to solve that is to introduce controlled merging of subnets and this will be further explored in a subsequent article.\n\n# Discussion\n\nPrevious work \\[24\\] has shown that graph theory algorithms to trace pathways through a network can give results in conflict with stoichiometric constraints. Netsplitter is not of this kind; graph analysis is merely used to identify a list of external metabolites, but network splitting is done directly on the stoichiometry matrix and (apart from the reclassifications) all the constraints that it expresses are maintained intact. Nevertheless, the concern may exist that e.g. elementary modes may be lost through the partitioning.\n\nA general counterargument is that removal of constraints cannot reduce the number of solutions to a problem. More specifically, consider for example a single mode that in the full network traverses two of the subsequent subnets. When the subnets are separated by reclassifying the metabolite node on their interface as external, the mode is correspondingly split into two parts. Since the original mode satisfied all constraints set by mass balances of internal compounds along its path, the two parts must separately continue to be viable. One part will now belong to the first subnet and terminate at the boundary node, which has become an unconstrained external sink and cannot affect its viability. The other will start at the corresponding unconstrained boundary source node in the second subnet and similarly remain viable because by construction the network context of internal metabolites nodes in each subnet is identical to that in the full network.\n\nA direct demonstration of this is obtained from a comparison of the null space of the internal stoichiometry matrix in the full network and in the subnetworks. As the flux vector of a mode lies in the null space, a reaction can only be active (i.e., participate in any of the modes) if there is a non-zero entry in at least one basis vector of the null space. For example, in the flavonoid demonstration network shown in Figure <a href=\"#F9\" data-ref-type=\"fig\">9<\/a>, there are 117 reactions, and calculation of the null space basis shows that 116 of those are active. Repeating this calculation for the internal stoichiometry matrix of each of the six subnets gives (42, 17, 5, 47, 2, 2) active reactions respectively in each subnet. As reactions are uniquely allocated to subnets in this case, the counts can be added to give a total of 115 reactions that are active collectively in the subnets. That leaves a discrepancy of one reaction, the one indicated by an \"X\" at the top of Figure <a href=\"#F9\" data-ref-type=\"fig\">9<\/a>. This reaction becomes excluded from the flux space because reclassification of the separator metabolite trans-cinnamate (indicated as the red oval node nearest the top) implies that after partitioning the reaction becomes irrelevant as it then only involves external metabolites. The single inactive reaction in the full network, ends up in subnet 4 and remains inactive there.\n\nPerforming this null space analysis for genome scale networks such as those shown in Figure <a href=\"#F10\" data-ref-type=\"fig\">10<\/a> reveals the same behaviour in all cases. Reclassification can result in some reactions involving only external metabolites and consequent reduction of the flux space; but in the remainder, all reactions that are active in the full network remain active in the subnets, and inactive ones remain inactive in subnets as well. This confirms that subnets collectively support the same set of modes as the full network.\n\nThe reduction of the flux space is another perspective on the desirability of keeping the set of external metabolites as small as possible, as is implemented in Netsplitter. Nevertheless, it is observed that the reactions eliminated are (like the one in Figure <a href=\"#F9\" data-ref-type=\"fig\">9<\/a>) mostly those at the periphery that only involve a single internal metabolite in the full network, and that their removal has a minimal effect on the structure of modes.\n\nComputing efficiency has been taken into account in several aspects of the Netsplitter procedure. Performing the main computation on a metabolites only simple graph rather than the bipartite representation, reduces matrix dimensions roughly by a factor of two, since the numbers of metabolites and reactions are usually similar. As about half of the metabolites are typically external, including only internals gives a further dimension reduction by a factor of about two. Focussing on the (*nsources* x *nsinks*) submatrix of the DAG gives a further reduction by a similar factor, enhanced also by using binary representations for clustering. Bearing in mind that matrix manipulations usually scale quadratically or worse, the overall reduction in complexity could approach two orders of magnitude.\n\nThis is borne out by moderate computing times. The total running time observed for the demonstration network of 117 reactions \u00d7 137 metabolites is 1.25 seconds, while for a genome scale network of 2037 reactions and 2179 metabolites this increased to 59 seconds, on a Core 2 Duo PC with 4 Gb of memory running at 2.66 GHz. These values appear quite acceptable and indicate that the algorithm scales better than quadratic with network dimension. It may be possible to achieve better performance by rewriting the code in a compiled language, but as extensive use is made of sophisticated graph theory and user interface functions built into *Mathematica*, this would be a major undertaking. Experimentation with *Mathematica* options to compile the most computing-intensive sections of the code did not produce significant performance improvements, suggesting that the coding of these functions is already quite efficient.\n\nThe procedure as presented is quite elaborate and requires considerable programming for its implementation. To facilitate its practical use, a software application \"Netsplitter\" has been developed as a *Mathematica* \\[25\\] notebook which is available for download \\[26\\]. This application provides an interactive interface that displays the progress of the subnet separation in the way illustrated by Figure <a href=\"#F4\" data-ref-type=\"fig\">4<\/a>, and offers additional facilities not discussed here such as the merging of selected subnets and display of subnet layouts. A more complete description of the software aspects and illustrations of its application to large scale networks, will be presented in a subsequent article.\n\nAn intriguing observation made in applying Netsplitter, is the radical change in resolving network structure that results from excluding high connectivity metabolites. An example is seen by comparing Figure <a href=\"#F1\" data-ref-type=\"fig\">1(a)<\/a> and <a href=\"#F1\" data-ref-type=\"fig\">1(b)<\/a>.\n\nIt is surmised that the reason for this behaviour can be understood from percolation theory \\[27\\]. In networks where only a random subset of the potential links in an infinite regular lattice are occupied (i.e., present in the actual network), it is found that there is a critical occupancy of potential links, termed the percolation threshold *p*~c~. Long range paths that penetrate the entire network only exist for occupancies greater than *p*~c~ . Values \\[28\\] of *p*~c~ for a variety of lattices have been derived mathematically or numerically; for a simple infinite 2-dimensional square lattice this is 0.5 and typical values range between 0.3 and 0.6, including some non-regular or randomized lattices, while lower values are obtained in higher dimensions. The values also depend strongly on the coordination number *z* (i.e. the number of links impinging on a node). While a metabolic network is not a regular lattice nor necessarily 2-dimensional, a rough estimate of the occupancy can be made from the adjacency matrices by the formula\n\n$$p = \\frac{\\sum{\\mathbf{C}\\mathbf{R} \\cdot \\mathbf{R}\\mathbf{C}}}{\\, z\\, N}$$\n\nwhere a simple sum over all matrix elements is taken, and N is the total number of nodes. Applying this to the example network, it is found that the removal of the four high connectivity metabolites produces a sharp drop in the value of the occupancy from a value of 0.630 for the network in Figure <a href=\"#F1\" data-ref-type=\"fig\">1(a)<\/a> to 0.295 for Figure <a href=\"#F1\" data-ref-type=\"fig\">1(b)<\/a>. This is consistent with the interpretation that the disappearance of long-range connectivity displayed by the figure, is caused by a drop of the occupancy number below an unknown percolation threshold. The same phenomenon has been observed in several other networks including those of genome-scale size discussed above.\n\nBased on this understanding, an automated strategy could be pursued to progressively reclassify the highest connectivity internal metabolites as external until there is a sharp drop in the *p* value calculated from equation (1.15). In practice, however, it works equally well to simply choose a fixed threshold value and reclassify all internal metabolites with connectivities higher than the threshold.\n\nRegarding the proposed efficacy measure, it was indicated above that it relies mainly on a general framework and even where a particular functional form such as the power law in equations (1.13) and (1.14) was postulated, its parameters merely readjust relative sensitivities to detailed features of the partitioning. This was further tested by experimenting with different functional forms such as an exponential dependence instead of a power law, taking *W* = 1 as might seem more plausible in equation (1.10), and even replacing the arithmetic mean term in that equation by a geometric mean expression. These changes were found to have marked effects on the complexity of the analysis, some of which could as a result only be done numerically. In the end the results were quite similar (but in some cases inferior in terms of smoothness and stability) and the simple option presented is deemed adequate.\n\nThe efficacy score measures the degree of simplification achieved by a given network partitioning. As shown in its derivation this is mathematically maximised for equal sized subnets. That does not mean that equal sized subnets is the ideal partitioning outcome; simplification is not the only criterion by which to judge success. Clearly there would be no special functional or biological relevance to an equal sized partitioning. On the other hand the low efficacy opposite extreme (towards which simple degree-based partitioning tends for large networks) of a large monolithic block and small fragments, or even complete fragmentation, is also functionally meaningless. As the *M. Pneumoniae* example illustrates, good agreement with conventional pathway assignments accompanies a moderately high efficacy value. In this sense, despite the inherent limitations of a single number score to represent varied considerations for judging the success of splitting a network for a particular purpose, efficacy values are useful as an overall guideline. It is noted that the efficacy measure is only used after the fact, optimising it does not form any part of the Netsplitter algorithm.\n\n# Conclusions\n\nThe modularization of a large, complex biochemical network into subnets that can be associated with recognisable biological functions, can be helpful both in the conceptual understanding and interpretation of the network, and to reduce practical problems that arise in the application of analysis methods such as constraint-based modelling. The challenge in constructing an algorithm for this task is to accommodate both the objective structural properties of the network, and more subjective requirements such as the desire for a manageable number of subnets of roughly similar sizes. Also, while it is inevitable that some information will be lost when a subnet is isolated from its larger context, it is desirable to restrict this loss to information that is not subjectively relevant for a particular study.\n\nIn the procedure proposed here, dealing with the information aspect is facilitated by selecting metabolite node cutting as the partitioning operation, since this pinpoints the nature of the information loss as removal of a mass balance constraint. Then the subjective requirements are met by allowing the flexibility to veto the selection of particular nodes to be cut, or to terminate partitioning at a suitable subnet size. Both local and long range network structure is taken into account by the use of random walks and clustering strategies, and finally information loss is minimised by using optimisation techniques in selecting candidate separation nodes and by explicit reincorporation of nodes not essential for the separation.\n\nThe combination of these strategies succeeds in moderating the extremes of the subnet size distribution that results from partitioning based simply on connectivity degree.\n\nThis point is illustrated by considering Figure <a href=\"#F7\" data-ref-type=\"fig\">7(c)<\/a> and <a href=\"#F8\" data-ref-type=\"fig\">8(c)<\/a> as alternative outcomes for somewhat similar levels of intervention with Figure <a href=\"#F7\" data-ref-type=\"fig\">7(b)<\/a> as a common starting point. Figure <a href=\"#F7\" data-ref-type=\"fig\">7(c)<\/a> shows how simply decreasing the connection degree threshold from 10 to 5, introduces 61 new externals and increases the block count by 70, but most of these are very small and a large block encompassing 20% of the network remains. In Figure <a href=\"#F8\" data-ref-type=\"fig\">8(c)<\/a> the netsplitter algorithm incurs a similar but smaller information loss of 52 new externals, but only forms 30 additional blocks and encapsulates 66% of the network in 10 medium sized blocks. This is clearly a much better cost:benefit ratio, although it is recognised that parts of the network are still dispersed into small fragments leaving room for future improvements.\n\nThe efficacy measure *E* that was introduced encapsulates these considerations into a single quantitative quality score.\n\nAt present, it seems that the most promising applications of subnet splitting would be to studies and interpretation of network structure, such as those based on elementary mode analysis, rather than for the more quantitative FBA. In this context, subnetworks can play an important role in reducing the often very large number of elementary modes in a large network. The use of subnets for FBA would similarly simplify the problem and allow the elimination of extraneous detail not relevant for study of a particular aspect of metabolism. However, the obstacle that arises is that it would usually be more difficult to fix the boundary conditions ( i.e. flux values for metabolite exchange with the environment) for a subnet than for the full network. At least for a single cell organism, full network boundary fluxes reflect overall nutrient uptake or waste elimination rates that are relatively easy to measure. Externals of a subnet are likely to include metabolites shared with another subnet and measuring the associated fluxes may require much more detailed metabolic measurements. In special cases, such as when the subnet is spatially localised e.g. to a particular cellular organelle, this might present less of a problem.\n\nA by-product of the matrix oriented approach used by the netsplitter algorithm, is the visually powerful display of network structure. Even for large networks for which a network layout diagram is totally unintelligible, features of network connectivity can be recognised at a glance from the colourscale plot of the truncated DAG matrix.\n\nEven more striking is the characterisation of fully and partially resolved subnetworks afforded by grayscale plots of the blocking matrices. The blocking transformation that was introduced as the basis for computational recognition and optimisation of blocks and their overlaps, serves this second purpose to visualise rather subtle structural network properties. Quite apart from the purpose to separate subnets, this visualisation should be a useful tool e.g. to explore the structure of large networks or to compare how related networks differ from one another.\n\n# Competing interests\n\nThe author declares that they have no competing interests.\n\n# Supplementary Material\n\n###### Additional file 1\n\n**Demonstration model**. Specification of the network model used for demonstration in the Methods section, as an SBML file.\n\nClick here for file\n\n###### Additional file 2\n\n**Heuristic for block recognition**. A description of the heuristic employed by Netsplitter for automated recognition of non-overlapping matrix blocks as defined in the text.\n\nClick here for file\n\n###### Additional file 3\n\n**Genome scale Arabidopsis model**. Specification of the network model extracted from Aracyc 4.5 and used for demonstration in the Methods section, as an SBML file.\n\nClick here for file\n\n###### Additional file 4\n\n**External Metabolites**. Listing of default external metabolites, specified as Biocyc compound ID's.\n\nClick here for file\n\n###### Additional file 5\n\n**Demonstration network layout**. The network layout shown in Figure <a href=\"#F8\" data-ref-type=\"fig\">8<\/a>, with all metabolite and reaction nodes labelled with their Biocyc ID's and names.\n\nClick here for file\n\n## Acknowledgements\n\nThe author gratefully acknowledges the hospitality of the University of Queensland, Brisbane where part of this work was completed.","meta":{"dup_signals":{"dup_doc_count":104,"dup_dump_count":46,"dup_details":{"curated_sources":3,"2022-40":1,"2022-21":1,"2021-43":2,"2021-25":1,"2021-10":1,"2020-40":1,"2020-24":1,"2020-10":1,"2019-43":1,"2019-39":1,"2019-26":1,"2019-22":1,"2019-18":1,"2019-13":1,"2019-09":1,"2019-04":1,"2018-51":1,"2018-43":1,"2018-34":1,"2018-26":1,"2018-22":1,"2018-09":1,"2017-47":1,"2017-39":1,"2017-30":1,"2017-26":7,"2017-22":12,"2017-17":10,"2017-09":5,"2017-04":1,"2016-44":2,"2016-40":2,"2016-36":9,"2016-30":10,"2016-22":1,"2016-18":1,"2015-40":1,"2015-22":2,"2014-49":1,"2014-42":2,"2014-35":1,"2023-14":1,"2017-13":4,"2015-06":1,"2013-48":1,"2024-10":1}},"file":"PMC3045323"},"subset":"pubmed_central"}
{"text":"author:\ndate: 2009-01\nreferences:\ntitle: Standards of Medical Care in Diabetes\u20142009\n\nDiabetes is a chronic illness that requires continuing medical care and patient self-management education to prevent acute complications and to reduce the risk of long-term complications. Diabetes care is complex and requires that many issues, beyond glycemic control, be addressed. A large body of evidence exists that supports a range of interventions to improve diabetes outcomes.\n\nThese standards of care are intended to provide clinicians, patients, researchers, payors, and other interested individuals with the components of diabetes care, treatment goals, and tools to evaluate the quality of care. While individual preferences, comorbidities, and other patient factors may require modification of goals, targets that are desirable for most patients with diabetes are provided. These standards are not intended to preclude more extensive evaluation and management of the patient by other specialists as needed. For more detailed information, refer to references 1\u20133.\n\nThe recommendations included are screening, diagnostic, and therapeutic actions that are known or believed to favorably affect health outcomes of patients with diabetes. A grading system (<a href=\"#t1\" data-ref-type=\"table\">Table 1<\/a>), developed by the American Diabetes Association (ADA) and modeled after existing methods, was utilized to clarify and codify the evidence that forms the basis for the recommendations. The level of evidence that supports each recommendation is listed after each recommendation using the letters A, B, C, or E.\n\n# I. CLASSIFICATION AND DIAGNOSIS\n\n## A. Classification\n\nIn 1997, ADA issued new diagnostic and classification criteria (4); in 2003, modifications were made regarding the diagnosis of impaired fasting glucose (5). The classification of diabetes includes four clinical classes:\n\n-   type 1 diabetes (results from \u03b2-cell destruction, usually leading to absolute insulin deficiency)\n\n-   type 2 diabetes (results from a progressive insulin secretory defect on the background of insulin resistance)\n\n-   other specific types of diabetes due to other causes, e.g., genetic defects in \u03b2-cell function, genetic defects in insulin action, diseases of the exocrine pancreas (such as cystic fibrosis), and drug- or chemical-induced (such as in the treatment of AIDS or after organ transplantation)\n\n-   gestational diabetes mellitus (GDM) (diabetes diagnosed during pregnancy)\n\nSome patients cannot be clearly classified as type 1 or type 2 diabetes. Clinical presentation and disease progression vary considerably in both types of diabetes. Occasionally, patients who otherwise have type 2 diabetes may present with ketoacidosis. Similarly, patients with type 1 may have a late onset and slow (but relentless) progression of disease despite having features of autoimmune disease. Such difficulties in diagnosis may occur in children, adolescents, and adults. The true diagnosis may become more obvious over time.\n\n## B. Diagnosis of diabetes\n\nCurrent criteria for the diagnosis of diabetes in nonpregnant adults are shown in <a href=\"#t2\" data-ref-type=\"table\">Table 2<\/a>. Three ways to diagnose diabetes are recommended at the time of this statement, and each must be confirmed on a subsequent day unless unequivocal symptoms of hyperglycemia are present. Although the 75-g oral glucose tolerance test (OGTT) is more sensitive and modestly more specific than the fasting plasma glucose (FPG) to diagnose diabetes, it is poorly reproducible and difficult to perform in practice. Because of ease of use, acceptability to patients, and lower cost, the FPG has been the preferred diagnostic test. Though FPG is less sensitive than the OGTT, the vast majority of people who do not meet diagnostic criteria for diabetes by FPG but would by OGTT will have an A1C value well under 7.0% (6).\n\nThough the OGTT is not recommended for routine clinical use, it may be useful for further evaluation of patients in whom diabetes is still strongly suspected but who have normal FPG or IFG (see Section I.C).\n\nThe use of the A1C for the diagnosis of diabetes has previously not been recommended due to lack of global standardization and uncertainty about diagnostic thresholds. However, with a world-wide move toward a standardized assay and with increasing observational evidence about the prognostic significance of A1C, an Expert Committee on the Diagnosis of Diabetes was convened in 2008. This joint committee of ADA, the European Association for the Study of Diabetes, and the International Diabetes Federation will likely recommend that the A1C become the preferred diagnostic test for diabetes. Diagnostic cut-points are being discussed at the time of publication of this statement. Updated recommendations will be published in *Diabetes Care* and will be available at diabetes.org.\n\n## C. Diagnosis of pre-diabetes\n\nHyperglycemia not sufficient to meet the diagnostic criteria for diabetes is categorized as either impaired fasting glucose (IFG) or impaired glucose tolerance (IGT), depending on whether it is identified through the FPG or the OGTT:\n\n-   IFG = FPG 100 mg\/dl (5.6 mmol\/l) to 125 mg\/dl (6.9 mmol\/l)\n\n-   IGT = 2-h plasma glucose 140 mg\/dl (7.8 mmol\/l) to 199 mg\/dl (11.0 mmol\/l)\n\nIFG and IGT have been officially termed \"pre-diabetes.\" Both categories of pre-diabetes are risk factors for future diabetes and for cardiovascular disease (CVD) (7).\n\n# II. TESTING FOR PRE-DIABETES AND DIABETES IN ASYMPTOMATIC PATIENTS\n\n## Recommendations\n\n-   Testing to detect pre-diabetes and type 2 diabetes in asymptomatic people should be considered in adults of any age who are overweight or obese (BMI \u226525 kg\/m^2^) and who have one or more additional risk factors for diabetes (<a href=\"#t3\" data-ref-type=\"table\">Table 3<\/a>). In those without these risk factors, testing should begin at age 45 years. (B)\n\n-   If tests are normal, repeat testing should be carried out at least at 3-year intervals. (E)\n\n-   To test for pre-diabetes or diabetes, an FPG test or 2-h OGTT (75-g glucose load) or both are appropriate. (B)\n\n-   An OGTT may be considered in patients with IFG to better define the risk of diabetes. (E)\n\n-   In those identified with pre-diabetes, identify and, if appropriate, treat other CVD risk factors. (B)\n\nFor many illnesses, there is a major distinction between screening and diagnostic testing. However, for diabetes, the same tests would be used for \"screening\" as for diagnosis. Type 2 diabetes has a long asymptomatic phase and significant clinical risk markers. Diabetes may be identified anywhere along a spectrum of clinical scenarios ranging from a seemingly low-risk individual who happens to have glucose testing, to a higher-risk individual whom the provider tests because of high suspicion of diabetes, to the symptomatic patient. The discussion herein is primarily framed as testing for diabetes in those without symptoms. Testing for diabetes will also detect individuals with pre-diabetes.\n\n## A. Testing for pre-diabetes and type 2 diabetes in adults\n\nType 2 diabetes is frequently not diagnosed until complications appear, and approximately one-third of all people with diabetes may be undiagnosed. Although the effectiveness of early identification of pre-diabetes and diabetes through mass testing of asymptomatic individuals has not been definitively proven (and rigorous trials to provide such proof are unlikely to occur), pre-diabetes and diabetes meet established criteria for conditions in which early detection is appropriate. Both conditions are common, increasing in prevalence, and impose significant public health burdens. There is a long presymptomatic phase before the diagnosis of type 2 diabetes is usually made. Relatively simple tests are available to detect preclinical disease (8). Additionally, the duration of glycemic burden is a strong predictor of adverse outcomes, and effective interventions exist to prevent progression of pre-diabetes to diabetes (see Section IV) and to reduce risk of complications of diabetes (see Section VI).\n\nRecommendations for testing for pre-diabetes and diabetes in asymptomatic, undiagnosed adults are listed in <a href=\"#t3\" data-ref-type=\"table\">Table 3<\/a>. Testing should be considered in adults of any age with BMI \u226525 kg\/m^2^ and one or more risk factors for diabetes. Because age is a major risk factor for diabetes, testing of those without other risk factors should begin no later than age 45 years.\n\nEither FPG testing or the 2-h OGTT is appropriate for testing. The 2-h OGTT identifies people with either IFG or IGT, and thus, more pre-diabetic people at increased risk for the development of diabetes and CVD. It should be noted that the two tests do not necessarily detect the same pre-diabetic individuals (9). The efficacy of interventions for primary prevention of type 2 diabetes (10\u201316) has primarily been demonstrated among individuals with IGT, not individuals with IFG (who do not also have IGT). As noted in the diagnosis section (Section I.B), the FPG test is more convenient, more reproducible, less costly, and easier to administer than the 2-h OGTT (4,5). An OGTT may be useful in patients with IFG to better define the risk of diabetes.\n\nThe appropriate interval between tests is not known (17). The rationale for the 3-year interval is that false-negatives will be repeated before substantial time elapses, and there is little likelihood that an individual will develop significant complications of diabetes within 3 years of a negative test result.\n\nBecause of the need for follow-up and discussion of abnormal results, testing should be carried out within the health care setting. Community screening outside a health care setting is not recommended because people with positive tests may not seek, or have access to, appropriate follow-up testing and care. Conversely, there may be failure to ensure appropriate repeat testing for individuals who test negative. Community screening may also be poorly targeted, i.e., it may fail to reach the groups most at risk and inappropriately test those at low risk (the worried well) or even those already diagnosed (18,19).\n\n## B. Testing for type 2 diabetes in children\n\nThe incidence of type 2 diabetes in adolescents has increased dramatically in the last decade, especially in minority populations (20), although the disease remains rare in the general adolescent population (21). Consistent with recommendations for adults, children and youth at increased risk for the presence or the development of type 2 diabetes should be tested within the health care setting (22). The recommendations of the ADA consensus statement on type 2 diabetes in children and youth, with some modifications, are summarized in <a href=\"#t4\" data-ref-type=\"table\">Table 4<\/a>.\n\n## C. Screening for type 1 diabetes\n\nGenerally, people with type 1 diabetes present with acute symptoms of diabetes and markedly elevated blood glucose levels, and most cases are diagnosed soon after the onset of hyperglycemia. However, evidence from type 1 prevention studies suggests that measurement of islet autoantibodies identifies individuals who are at risk for developing type 1 diabetes. Such testing may be appropriate in high-risk individuals, such as those with prior transient hyperglycemia or those who have relatives with type 1 diabetes, in the context of clinical research studies (see, for example, <http:\/\/www2.diabetestrialnet.org>). Widespread clinical testing of asymptomatic low-risk individuals cannot currently be recommended, as it would identify very few individuals in the general population who are at risk. Individuals who screen positive should be counseled about their risk of developing diabetes. Clinical studies are being conducted to test various methods of preventing type 1 diabetes, or reversing early type 1 diabetes, in those with evidence of autoimmunity.\n\n# III. DETECTION AND DIAGNOSIS OF GDM\n\n## Recommendations\n\n-   Screen for GDM using risk factor analysis and, if appropriate, use of an OGTT. (C)\n\n-   Women with GDM should be screened for diabetes 6\u201312 weeks postpartum and should be followed up with subsequent screening for the development of diabetes or pre-diabetes. (E)\n\nGDM is defined as any degree of glucose intolerance with onset or first recognition during pregnancy (4). Although most cases resolve with delivery, the definition applies whether or not the condition persists after pregnancy and does not exclude the possibility that unrecognized glucose intolerance may have antedated or begun concomitantly with the pregnancy. Approximately 7% of all pregnancies (ranging from 1 to 14% depending on the population studied and the diagnostic tests employed) are complicated by GDM, resulting in more than 200,000 cases annually.\n\nBecause of the risks of GDM to the mother and neonate, screening and diagnosis are warranted. The screening and diagnostic strategies, based on the 2004 ADA position statement on gestational diabetes mellitus (23), are outlined in <a href=\"#t5\" data-ref-type=\"table\">Table 5<\/a>.\n\nResults of the Hyperglycemia and Adverse Pregnancy Outcomes study (24), a large-scale (including \u223c25,000 pregnant women) multinational epidemiologic study, demonstrated that risk of adverse maternal, fetal, and neonatal outcomes continuously increased as a function of maternal glycemia at 24\u201328 weeks, even within ranges previously considered normal for pregnancy. For most complications, there was no threshold for risk. These results have led to careful reconsideration of the diagnostic criteria for GDM. An international group representing multiple obstetrical and diabetes organizations, including ADA, is currently working on consensus toward *1*) a world-wide standard for which diagnostic test to use for GDM and *2*) rational diagnostic cut points.\n\nBecause women with a history of GDM have a greatly increased subsequent risk for diabetes (25), they should be screened for diabetes 6\u201312 weeks postpartum, using nonpregnant OGTT criteria, and should be followed up with subsequent screening for the development of diabetes or pre-diabetes, as outlined in Section II. For information on the National Diabetes Education Program (NDEP) campaign to prevent type 2 diabetes in women with GDM, go to [www.ndep.nih.gov\/diabetes\/pubs\/NeverTooEarly_Tipsheet.pdf](www.ndep.nih.gov\/diabetes\/pubs\/NeverTooEarly_Tipsheet.pdf).\n\n# IV. PREVENTION\/DELAY OF TYPE 2 DIABETES\n\n## Recommendations\n\n-   Patients with IGT (A) or IFG (E) should be referred to an effective ongoing support program for weight loss of 5\u201310% of body weight and for increasing physical activity to at least 150 min per week of moderate activity such as walking.\n\n-   Follow-up counseling appears to be important for success. (B)\n\n-   Based on potential cost savings of diabetes prevention, such counseling should be covered by third-party payors. (E)\n\n-   In addition to lifestyle counseling, metformin may be considered in those who are at very high risk for developing diabetes (combined IFG and IGT plus other risk factors such as A1C \\>6%, hypertension, low HDL cholesterol, elevated triglycerides, or family history of diabetes in a first-degree relative) and who are obese and under 60 years of age. (E)\n\n-   Monitoring for the development of diabetes in those with pre-diabetes should be performed every year. (E)\n\nRandomized controlled trials have shown that individuals at high risk for developing diabetes (those with IFG, IGT, or both) can be given interventions that significantly decrease the rate of onset of diabetes (10\u201316). These interventions include intensive lifestyle modification programs that have been shown to be very effective (\u226558% reduction after 3 years) and use of the pharmacologic agents metformin, acarbose, orlistat, and thiazolidinediones (TZDs), each of which has been shown to decrease incident diabetes to various degrees. A summary of major diabetes prevention trials is shown in <a href=\"#t6\" data-ref-type=\"table\">Table 6<\/a>.\n\nTwo studies of lifestyle intervention have shown persistent reduction in the rate of conversion to type 2 diabetes with 3 (26) to 14 years (27) of postintervention follow-up.\n\nBased on the results of clinical trials and the known risks of progression of pre-diabetes to diabetes, an ADA Consensus Development Panel (7) concluded that persons with pre-diabetes (IGT and\/or IFG) should be counseled on lifestyle changes with goals similar to those of the Diabetes Prevention Program (DPP) (5\u201310% weight loss and moderate physical activity of \u223c30 min per day). Regarding the more difficult issue of drug therapy for diabetes prevention, the consensus panel felt that metformin should be the only drug considered for use in diabetes prevention. For other drugs, the issues of cost, side effects, and lack of persistence of effect in some studies led the panel to not recommend their use for diabetes prevention. Metformin use was recommended only for very-high-risk individuals (those with combined IGT and IFG who are obese and under 60 years of age with at least one other risk factor for diabetes). In addition, the panel highlighted the evidence that in the DPP, metformin was most effective compared to lifestyle in those with BMI of at least 35 kg\/m^2^ and those under age 60 years.\n\n# V. DIABETES CARE\n\n## A. Initial evaluation\n\nA complete medical evaluation should be performed to classify the diabetes, detect the presence of diabetes complications, review previous treatment and glycemic control in patients with established diabetes, assist in formulating a management plan, and provide a basis for continuing care. Laboratory tests appropriate to the evaluation of each patient's medical condition should be performed. A focus on the components of comprehensive care (<a href=\"#t7\" data-ref-type=\"table\">Table 7<\/a>) will assist the health care team to ensure optimal management of the patient with diabetes.\n\n## B. Management\n\nPeople with diabetes should receive medical care from a physician-coordinated team. Such teams may include, but are not limited to, physicians, nurse practitioners, physician's assistants, nurses, dietitians, pharmacists, and mental health professionals with expertise and a special interest in diabetes. It is essential in this collaborative and integrated team approach that individuals with diabetes assume an active role in their care.\n\nThe management plan should be formulated as an individualized therapeutic alliance among the patient and family, the physician, and other members of the health care team. A variety of strategies and techniques should be used to provide adequate education and development of problem-solving skills in the various aspects of diabetes management. Implementation of the management plan requires that each aspect is understood and agreed on by the patient and the care providers and that the goals and treatment plan are reasonable. Any plan should recognize diabetes self-management education (DSME) as an integral component of care. In developing the plan, consideration should be given to the patient's age, school or work schedule and conditions, physical activity, eating patterns, social situation and personality, cultural factors, and presence of complications of diabetes or other medical conditions.\n\n## C. Glycemic control\n\n### 1. Assessment of glycemic control\n\nTwo primary techniques are available for health providers and patients to assess the effectiveness of the management plan on glycemic control: patient self-monitoring of blood glucose (SMBG) or of interstitial glucose and measurement of A1C.\n\n#### a. Glucose monitoring\n\n##### Recommendations\n\n-   SMBG should be carried out three or more times daily for patients using multiple insulin injections or insulin pump therapy. (A)\n\n-   For patients using less frequent insulin injections, noninsulin therapies, or medical nutrition therapy (MNT) and physical activity alone, SMBG may be useful as a guide to the success of therapy. (E)\n\n-   To achieve postprandial glucose targets, postprandial SMBG may be appropriate. (E)\n\n-   When prescribing SMBG, ensure that patients receive initial instruction in, and routine follow-up evaluation of, SMBG technique and their ability to use data to adjust therapy. (E)\n\n-   Continuous glucose monitoring (CGM) in conjunction with intensive insulin regimens can be a useful tool to lower A1C in selected adults (age \u226525 years) with type 1 diabetes (A).\n\n-   Although the evidence for A1C lowering is less strong in children, teens, and younger adults, CGM may be helpful in these groups. Success correlates with adherence to ongoing use of the device. (C)\n\n-   CGM may be a supplemental tool to SMBG in those with hypoglycemia unawareness and\/or frequent hypoglycemic episodes. (E)\n\nThe ADA's consensus and position statements on SMBG provide a comprehensive review of the subject (31,32). Major clinical trials of insulin-treated patients that demonstrated the benefits of intensive glycemic control on diabetes complications have included SMBG as part of multifactorial interventions, suggesting that SMBG is a component of effective therapy. SMBG allows patients to evaluate their individual response to therapy and assess whether glycemic targets are being achieved. Results of SMBG can be useful in preventing hypoglycemia and adjusting medications (particularly prandial insulin doses), MNT, and physical activity.\n\nThe frequency and timing of SMBG should be dictated by the particular needs and goals of the patients. SMBG is especially important for patients treated with insulin to monitor for and prevent asymptomatic hypoglycemia and hyperglycemia. For most patients with type 1 diabetes and pregnant women taking insulin, SMBG is recommended three or more times daily. For this population, significantly more frequent testing may be required to reach A1C targets safely without hypoglycemia. The optimal frequency and timing of SMBG for patients with type 2 diabetes on noninsulin therapy is unclear. A meta-analysis of SMBG in non\u2013insulin-treated patients with type 2 diabetes concluded that some regimen of SMBG was associated with a reduction in A1C of \u22650.4%. However, many of the studies in this analysis also included patient education with diet and exercise counseling and, in some cases, pharmacologic intervention, making it difficult to assess the contribution of SMBG alone to improved control (33). Several recent trials have called into question the clinical utility and cost-effectiveness of routine SMBG in non\u2013insulin-treated patients (34\u201336).\n\nBecause the accuracy of SMBG is instrument and user dependent (37), it is important to evaluate each patient's monitoring technique, both initially and at regular intervals thereafter. In addition, optimal use of SMBG requires proper interpretation of the data. Patients should be taught how to use the data to adjust food intake, exercise, or pharmacological therapy to achieve specific glycemic goals, and these skills should be reevaluated periodically.\n\nCGM through the measurement of interstitial glucose (which correlates well with plasma glucose) is available. These sensors require calibration with SMBG, and the latter are still recommended for making acute treatment decisions. CGM devices also have alarms for hypo- and hyperglycemic excursions. Small studies in selected patients with type 1 diabetes have suggested that CGM use reduces the time spent in hypo- and hyperglycemic ranges and may modestly improve glycemic control. A larger 26-week randomized trial of 322 type 1 patients showed that adults age 25 years and older using intensive insulin therapy and CGM experienced a 0.5% reduction in A1C (from \u223c7.6 to 7.1%) compared with usual intensive insulin therapy with SMBG (38). Sensor use in children, teens, and adults to age 24 years did not result in significant A1C lowering, and there was no significant difference in hypoglycemia in any group. Importantly, the greatest predictor of A1C lowering in this study for all age-groups was frequency of sensor use, which was lower in younger age-groups. Although CGM is an evolving technology, emerging data suggest that, in appropriately selected patients who are motivated to wear it most of the time, it may offer benefit. CGM may be particularly useful in those with hypoglycemia unawareness and\/or frequent episodes of hypoglycemia, and studies in this area are ongoing.\n\n#### b. A1C\n\n##### Recommendations\n\n-   Perform the A1C test at least two times a year in patients who are meeting treatment goals (and who have stable glycemic control). (E)\n\n-   Perform the A1C test quarterly in patients whose therapy has changed or who are not meeting glycemic goals. (E)\n\n-   Use of point-of-care testing for A1C allows for timely decisions on therapy changes, when needed. (E)\n\nBecause A1C is thought to reflect average glycemia over several months (37), and has strong predictive value for diabetes complications (10,39), A1C testing should be performed routinely in all patients with diabetes at initial assessment and then as part of continuing care. Measurement approximately every 3 months determines whether a patient's glycemic targets have been reached and maintained. For any individual patient, the frequency of A1C testing should be dependent on the clinical situation, the treatment regimen used, and the judgment of the clinician. Some patients with stable glycemia well within target may do well with testing only twice per year, while unstable or highly intensively managed patients (e.g., pregnant type 1 women) may be tested more frequently than every 3 months. The availability of the A1C result at the time that the patient is seen (point-of-care testing) has been reported to result in increased intensification of therapy and improvement in glycemic control (40,41).\n\nThe A1C test is subject to certain limitations. Conditions that affect erythrocyte turnover (hemolysis, blood loss) and hemoglobin variants must be considered, particularly when the A1C result does not correlate with the patient's clinical situation (37). In addition, A1C does not provide a measure of glycemic variability or hypoglycemia. For patients prone to glycemic variability (especially type 1 patients, or type 2 patients with severe insulin deficiency), glycemic control is best judged by the combination of results of SMBG testing and the A1C. The A1C may also serve as a check on the accuracy of the patient's meter (or the patient's reported SMBG results) and the adequacy of the SMBG testing schedule.\n\n<a href=\"#t8\" data-ref-type=\"table\">Table 8<\/a> contains the correlation between A1C levels and mean plasma glucose levels based on data from the international A1C-Derived Average Glucose (ADAG) trial utilizing frequent SMBG and continuous glucose monitoring in 507 adults (83% Caucasian) with type 1, type 2, and no diabetes (49) The ADA and American Association of Clinical Chemists have determined that the correlation (*r* = 0.92) is strong enough to justify reporting both an A1C result and an estimated average glucose (eAG) result when a clinician orders the A1C test. The table in previous versions of the Standards of Medical Care in Diabetes describing the correlation between A1C and mean glucose was derived from relatively sparse data (one seven-point profile over 1 day per A1C reading) in the primarily Caucasian type 1 participants in the Diabetes Control and Complications Trial (DCCT) trial (43). Clinicians should note that the numbers in the table are now different, as they are based on \u223c2,800 readings per A1C in the ADAG trial.\n\nIn the ADAG study, there were no significant differences among racial and ethnic groups in the regression lines between A1C and mean glucose, although there was a trend toward a difference between African\/African-American and Caucasian participants' regression lines that might have been significant had more African\/African-American participants been studied. A recent study comparing A1C to CGM data in 48 type 1 children found a highly statistically significant correlation between A1C and mean blood glucose, although the correlation (*r* = 0.7) was significantly lower than in the ADAG trial (44). Whether there are significant differences in how A1C relates to average glucose in children or in African-American patients is an area for further study. For the time being, the question has not led to different recommendations about testing A1C or to different interpretations of the clinical meaning of given levels of A1C in those populations.\n\nFor patients in whom A1C\/eAG and measured blood glucose appear discrepant, clinicians should consider the possibilities of hemoglobinopathy or altered red cell turnover and the options of more frequent and\/or different timing of SMBG or use of CGM. Other measures of chronic glycemia such as fructosamine are available, but their linkage to average glucose and their prognostic significance are not as clear as is the case for A1C.\n\n### 2. Glycemic goals in adults\n\n-   Lowering A1C to below or around 7% has been shown to reduce microvascular and neuropathic complications of type 1 and type 2 diabetes. Therefore, for microvascular disease prevention, the A1C goal for nonpregnant adults in general is \\<7%. (A)\n\n-   In type 1 and type 2 diabetes, randomized controlled trials of intensive versus standard glycemic control have not shown a significant reduction in CVD outcomes during the randomized portion of the trials. Long-term follow-up of the DCCT and UK Prospective Diabetes Study (UKPDS) cohorts suggests that treatment to A1C targets below or around 7% in the years soon after the diagnosis of diabetes is associated with long-term reduction in risk of macrovascular disease. Until more evidence becomes available, the general goal of \\<7% appears reasonable for many adults for macrovascular risk reduction. (B)\n\n-   Subgroup analyses of clinical trials such as the DCCT and UKPDS and the microvascular evidence from the Action in Diabetes and Vascular Disease: Preterax and Diamicron MR Controlled Evaluation (ADVANCE) trial suggest a small but incremental benefit in microvascular outcomes with A1C values closer to normal. Therefore, for selected individual patients, providers might reasonably suggest even lower A1C goals than the general goal of \\<7%, if this can be achieved without significant hypoglycemia or other adverse effects of treatment. Such patients might include those with short duration of diabetes, long life expectancy, and no significant CVD. (B)\n\n-   Conversely, less stringent A1C goals than the general goal of \\<7% may be appropriate for patients with a history of severe hypoglycemia, limited life expectancy, advanced microvascular or macrovascular complications, extensive comorbid conditions, and those with longstanding diabetes in whom the general goal is difficult to attain despite DSME, appropriate glucose monitoring, and effective doses of multiple glucose-lowering agents including insulin. (C)\n\nGlycemic control is fundamental to the management of diabetes. The DCCT, a prospective, randomized, controlled trial of intensive versus standard glycemic control in patients with relatively recently diagnosed type 1 diabetes, showed definitively that improved glycemic control is associated with significantly decreased rates of microvascular (retinopathy and nephropathy) as well as neuropathic complications (45). Follow-up of the DCCT cohorts in the Epidemiology of Diabetes Interventions and Complications (EDIC) study has shown persistence of this effect in previously intensively treated subjects, even though their glycemic control has been equivalent to that of previous standard arm subjects during follow-up (46,47).\n\nIn type 2 diabetes, the Kumamoto study (48) and the UKPDS (49,50) demonstrated significant reductions in microvascular and neuropathic complications with intensive therapy. Similar to the DCCT-EDIC findings, long-term follow-up of the UKPDS cohort has recently demonstrated a \"legacy effect\" of early intensive glycemic control on long-term rates of microvascular complications, even with loss of glycemic separation between the intensive and standard cohorts after the end of the randomized controlled (51).\n\nIn each of these large randomized prospective clinical trials, treatment regimens that reduced average A1C to \u22657% (\u22651% above the upper limits of normal) were associated with fewer long-term microvascular complications; however, intensive control was found to increase the risk of severe hypoglycemia, most notably in the DCCT, and led to weight gain (39,52).\n\nEpidemiological analyses of the DCCT and UKPDS (39,45) demonstrate a curvilinear relationship between A1C and microvascular complications. Such analyses suggest that, on a population level, the greatest number of complications will be averted by taking patients from very poor control to fair or good control. These analyses also suggest that further lowering of A1C from 7 to 6% is associated with further reduction in the risk of microvascular complications, albeit the absolute risk reductions become much smaller. Given the substantially increased risk of hypoglycemia (particularly in those with type 1 diabetes) and the relatively much greater effort required to achieve near-normoglycemia, the risks of lower targets may outweigh the potential benefits on microvascular complications on a population level. However, selected individual patients, especially those with little comorbidity and long life expectancy (who may reap the benefits of further lowering of glycemia below 7%) may, at patient and provider judgment, adopt glycemic targets as close to normal as possible as long as significant hypoglycemia does not become a barrier.\n\nWhereas many epidemiologic studies and meta-analyses (53,54) have clearly shown a direct relationship between A1C and CVD, the potential of intensive glycemic control to reduce CVD has been less clearly defined. In the DCCT, there was a trend toward lower risk of CVD events with intensive control (risk reduction 41%, 95% CI 10\u201368%), but the number of events was small. However, 9-year post-DCCT follow-up of the cohort has shown that participants previously randomized to the intensive arm had a 42% reduction (*P* = 0.02) in CVD outcomes and a 57% reduction (*P* = 0.02) in the risk of nonfatal myocardial infarction (MI), stroke, or CVD death compared with those previously in the standard arm (55).\n\nThe UKPDS trial of type 2 diabetes observed a 16% reduction in cardiovascular complications (combined fatal or nonfatal MI and sudden death) in the intensive glycemic control arm, although this difference was not statistically significant (*P* = 0.052), and there was no suggestion of benefit on other CVD outcomes such as stroke. In an epidemiologic analysis of the study cohort, a continuous association was observed, such that for every percentage point lower median on-study A1C (e.g., 8 to 7%) there was a statistically significant 18% reduction in CVD events, again with no glycemic threshold. A recent report of 10 years of follow-up of the UKPDS cohort describes, for the participants originally randomized to intensive glycemic control compared with those randomized to conventional glycemic control, long-term reductions in MI (15% with sulfonylurea or insulin as initial pharmacotherapy, 33% with metformin as initial pharmacotherapy, both statistically significant) and in all-cause mortality (13 and 27%, respectively, both statistically significant) (51).\n\nBecause of ongoing uncertainty regarding whether intensive glycemic control can reduce the increased risk of CVD events in people with type 2 diabetes, several large long-term trials were launched in the past decade to compare the effects of intensive versus standard glycemic control on CVD outcomes in relatively high-risk participants with established type 2 diabetes.\n\nThe Action to Control Cardiovascular Risk in Diabetes (ACCORD) study randomized 10,251 participants with either history of a CVD event (ages 40\u201379 years) or significant CVD risk (ages 55\u201379) to a strategy of intensive glycemic control (target A1C \\<6.0%) or standard glycemic control (A1C target 7.0\u20137.9%). Investigators used multiple glycemic medications in both arms. ACCORD participants were on average 62 years old and had a mean duration of diabetes of 10 years, with 35% already treated with insulin at baseline. From a baseline median A1C of 8.1%, the intensive arm reached a median A1C of 6.4% within 12 months of randomization, while the standard group reached a median A1C of 7.5%. Other risk factors were treated aggressively and equally in both groups. The intensive glycemic control group had more use of insulin in combination with multiple oral agents, significantly more weight gain, and more episodes of severe hypoglycemia than the standard group.\n\nIn February 2008, the glycemic control study of ACCORD was halted on the recommendation of the study's data safety monitoring board due to the finding of an increased rate of mortality in the intensive arm compared with the standard arm (1.41%\/year vs. 1.14%\/year; HR 1.22 \\[95% CI 1.01\u20131.46\\]), with a similar increase in cardiovascular deaths. The primary outcome of ACCORD (MI, stroke, or cardiovascular death) was lower in the intensive glycemic control group due to a reduction in nonfatal MI, although this finding was not statistically significant when the study was terminated (HR 0.90 \\[95% CI 0.78\u20131.04\\]; *P* = 0.16) (56).\n\nExploratory analyses of the mortality findings of ACCORD (evaluating variables including weight gain, use of any specific drug or drug combination, and hypoglycemia) were reportedly unable to identify an explanation for the excess mortality in the intensive arm. Prespecified subset analyses showed that participants with no previous CVD event and those who had a baseline A1C \\<8% had a statistically significant reduction in the primary CVD outcome.\n\nThe ADVANCE study randomized 11,140 participants to a strategy of intensive glycemic control (with primary therapy being the sulfonylurea gliclizide and additional medications as needed to achieve a target A1C of \u22646.5%) or to standard therapy (in which any medication but gliclizide could be used and the glycemic target was according to \"local guidelines\"). ADVANCE participants (who had to be at least 55 years of age with either known vascular disease or at least one other vascular risk factor) were slightly older and of similar high CVD risk as those in ACCORD. However, they had an average duration of diabetes 2 years shorter, lower baseline A1C (median 7.2%), and almost no use of insulin at enrollment. The median A1C levels achieved in the intensive and standard arms were 6.3 and 7.0%, respectively, and maximal separation between the arms took several years to achieve. Use of other drugs that favorably impact CVD risk (aspirin, statins, ACE inhibitors) was lower in ADVANCE than in the ACCORD or Veterans Affairs Diabetes Trial (VADT).\n\nThe primary outcome of ADVANCE was a combination of microvascular events (nephropathy and retinopathy) and major adverse cardiovascular events (MI, stroke, and cardiovascular death). Intensive glycemic control significantly reduced the primary endpoint (HR 0.90 \\[95% CI 0.82\u20130.98\\]; *P* = 0.01), although this was due to a significant reduction in the microvascular outcome (0.86 \\[0.77\u20130.97\\], *P* = 0.01), primarily development of macroalbuminuria, with no significant reduction in the macrovascular outcome (0.94 \\[0.84\u20131.06\\]; *P* = 0.32). There was no difference in overall or cardiovascular mortality between the intensive and the standard glycemic control arms (57).\n\nThe VADT randomized 1,791 participants with type 2 diabetes uncontrolled on insulin or maximal dose oral agents (median entry A1C 9.4%) to a strategy of intensive glycemic control (goal A1C \\<6.0%) or standard glycemic control, with a planned A1C separation of at least 1.5%. Medication treatment algorithms were used to achieve the specified glycemic goals, with a goal of using similar medications in both groups. Median A1C levels of 6.9 and 8.4% were achieved in the intensive and standard arms, respectively, within the first year of the study. Other CVD risk factors were treated aggressively and equally in both groups.\n\nThe primary outcome of the VADT was a composite of CVD events (MI, stroke, cardiovascular death, revascularization, hospitalization for heart failure, and amputation for ischemia). During a mean 6-year follow-up period, the cumulative primary outcome was nonsignificantly lower in the intensive arm (HR 0.87 \\[95% CI 0.73\u20131.04\\]; *P* = 0.12). There were more CVD deaths in the intensive arm than in the standard arm (40 vs. 33; sudden deaths 11 vs. 4), but the difference was not statistically significant. Post hoc subgroup analyses suggested that duration of diabetes interacted with randomization such that participants with duration of diabetes less than about 12 years appeared to have a CVD benefit of intensive glycemic control while those with longer duration of disease before study entry had a neutral or even adverse effect of intensive glycemic control. Other exploratory analyses suggested that severe hypoglycemia within the past 90 days was a strong predictor of the primary outcome and of CVD mortality (58).\n\nThe cause of the excess deaths in the intensive glycemic control arm of ACCORD compared with the standard arm has been difficult to pinpoint. By design of the trial, randomization to the intensive arm was associated with or led to many downstream effects, such as higher rates of severe hypoglycemia; more frequent use of insulin, TZDs, other drugs, and drug combinations; and greater weight gain. Such factors may be associated statistically with the higher mortality rate in the intensive arm but may not be causative. It is biologically plausible that severe hypoglycemia could increase the risk of cardiovascular death in participants with high underlying CVD risk. Other plausible mechanisms for the increase in mortality in ACCORD include weight gain, unmeasured drug effects or interactions, or the overall \"intensity\" of the ACCORD intervention (use of multiple oral glucose-lowering drugs along with multiple doses of insulin, frequent therapy adjustments to push A1C and self-monitored blood glucose to very low targets, and an intense effort to aggressively reduce A1C by \u223c2% in participants entering the trial with advanced diabetes and multiple comorbidities).\n\nSince the ADVANCE trial did not show any increase in mortality in the intensive glycemic control arm, examining the differences between ADVANCE and ACCORD supports additional hypotheses. ADVANCE participants on average appeared to have earlier or less advanced diabetes, with shorter duration by 2\u20133 years and lower A1C at entry despite very little use of insulin at baseline. A1C was also lowered less and more gradually in the ADVANCE trial, and there was no significant weight gain with intensive glycemic therapy. Although severe hypoglycemia was defined somewhat differently in the three trials, it appears that this occurred in fewer than 3% of intensively treated ADVANCE participants for the entire study duration (median 5 years) compared with \u223c16% of intensively treated subjects in ACCORD and 21% in VADT.\n\nIt is likely that the increase in mortality in ACCORD was related to the overall treatment strategies for intensifying glycemic control in the study population, not the achieved A1C per se. The ADVANCE study achieved a median A1C in its intensive arm similar to that in the ACCORD study, with no increased mortality hazard. Thus, the ACCORD mortality findings do not imply that patients with type 2 diabetes who can easily achieve or maintain low A1C levels with lifestyle modifications with or without pharmacotherapy are at risk and need to \"raise\" their A1C.\n\nThe three trials compared treatments to A1C levels in the \"flatter\" part of the observational glycemia-CVD risk curves (median A1C of 6.4\u20136.9% in the intensive arms compared with 7.0\u20138.4% in the standard arms). Importantly, their results should not be extrapolated to imply that there would be no cardiovascular benefit of glucose lowering from very poor control (e.g., A1C \\>9%) to good control (e.g., A1C \\<7%).\n\nAll three trials were carried out in participants with established diabetes (mean duration 8\u201311 years) and either known CVD or multiple risk factors suggesting the presence of established atherosclerosis. Subset analyses of the three trials suggested a significant benefit of intensive glycemic control on CVD in participants with shorter duration of diabetes, lower A1C at entry, and\/or or absence of known CVD. The DCCT-EDIC study and the long-term follow-up of the UKPDS cohort both suggest that intensive glycemic control initiated soon after diagnosis of diabetes in patients with a lower level of CVD risk may impart long-term protection from CVD events. As is the case with microvascular complications, it may be that glycemic control plays a greater role before macrovascular disease is well developed and minimal or no role when it is advanced.\n\nThe benefits of intensive glycemic control on microvascular and neuropathic complications are well established for both type 1 and type 2 diabetes. The ADVANCE trial has added to that evidence base by demonstrating a significant reduction in the risk of new or worsening albuminuria when A1C was lowered to 6.3% compared with standard glycemic control achieving an A1C of 7.0%. The lack of significant reduction in CVD events with intensive glycemic control in ACCORD, ADVANCE, and VADT should not lead clinicians to abandon the general target of an A1C \\<7.0% and thereby discount the benefit of good control on what are serious and debilitating microvascular complications.\n\nThe evidence for a cardiovascular benefit of intensive glycemic control primarily rests on long-term follow-up of study cohorts treated early in the course of type 1 and type 2 diabetes and subset analyses of ACCORD, ADVANCE, and VADT. Conversely, the mortality findings in ACCORD suggest that the potential risks of very intensive glycemic control may outweigh its benefits in some patients, such as those with very long duration of diabetes, known history of severe hypoglycemia, advanced atherosclerosis, and advanced age\/frailty. Certainly, providers should be vigilant in preventing severe hypoglycemia in patients with advanced disease and should not aggressively attempt to achieve near-normal A1C levels in patients in whom such a target cannot be reasonably easily and safely achieved.\n\nRecommended glycemic goals for nonpregnant adults are shown in <a href=\"#t9\" data-ref-type=\"table\">Table 9<\/a>. The recommendations are based on those for A1C, with listed blood glucose levels that appear to correlate with achievement of an A1C of \\<7%. The issue of pre- versus postprandial SMBG targets is complex (59). Elevated postchallenge (2-h OGTT) glucose values have been associated with increased cardiovascular risk independent of FPG in some epidemiological studies. In diabetic subjects, some surrogate measures of vascular pathology, such as endothelial dysfunction, are negatively affected by postprandial hyperglycemia (60). It is clear that postprandial hyperglycemia, like preprandial hyperglycemia, contributes to elevated A1C levels, with its relative contribution being higher at A1C levels that are closer to 7%. However, outcome studies have clearly shown A1C to be the primary predictor of complications, and landmark glycemic control trials such as the DCCT and UKPDS relied overwhelmingly on preprandial SMBG. Additionally, a randomized controlled trial presented at the 68th Scientific Sessions of the American Diabetes Association in June 2008 found no CVD benefit of insulin regimens targeting postprandial glucose compared with those targeting preprandial glucose. A reasonable recommendation for postprandial testing and targets is that for individuals who have premeal glucose values within target but have A1C values above target, monitoring postprandial plasma glucose (PPG) 1\u20132 h after the start of the meal and treatment aimed at reducing PPG values to \\<180 mg\/dl may help lower A1C.\n\nAs noted above, less stringent treatment goals may be appropriate for adults with limited life expectancies or advanced vascular disease. Glycemic goals for children are provided in Section VII.A.1.a. Severe or frequent hypoglycemia is an absolute indication for the modification of treatment regimens, including setting higher glycemic goals.\n\nRegarding goals for glycemic control for women with GDM, recommendations from the Fifth International Workshop-Conference on Gestational Diabetes Mellitus (61) were to target the following maternal capillary glucose concentrations:\n\n-   preprandial: \u226495 mg\/dl (5.3 mmol\/l) and either\n\n    -   1-h postmeal: \u2264140 mg\/dl (7.8 mmol\/l) or\n\n    -   2-h postmeal: \u2264120 mg\/dl (6.7 mmol\/l)\n\nFor women with preexisting type 1 or type 2 diabetes who become pregnant, a recent consensus statement (62) recommended the following as optimal glycemic goals, if they can be achieved without excessive hypoglycemia:\n\n-   premeal, bedtime, and overnight glucose 60\u201399 mg\/dl\n\n-   peak postprandial glucose 100\u2013129 mg\/dl\n\n-   A1C \\<6.0%\n\n### 3. Approach to treatment\n\n#### a. Therapy for type 1 diabetes.\n\nThe DCCT clearly showed that intensive insulin therapy (three or more injections per day of insulin or continuous subcutaneous insulin infusion (CSII, or insulin pump therapy) was a key part of improved glycemia and better outcomes (45). At the time of the study, therapy was carried out with short- and intermediate-acting human insulins. Despite better microvascular outcomes, intensive insulin therapy was associated with a marked increase in severe hypoglycemia (62 episodes per 100 patient-years of therapy). Since the time of the DCCT, a number of rapid-acting and long-acting insulin analogs have been developed. These analogs were designed to be more \"physiological\" in their pharmacokinetics and pharmacodynamics and are associated with less hypoglycemia with equal A1C lowering in type 1 diabetes (63,64).\n\nTherefore, recommended therapy for type 1 diabetes consists of the following components: *1*) use of multiple dose insulin injections (3\u20134 injections per day of basal and prandial insulin) or CSII therapy; *2*) matching of prandial insulin to carbohydrate intake, premeal blood glucose, and anticipated activity; and *3*) for many patients (especially if hypoglycemia is a problem), use of insulin analogs. There are excellent reviews available that guide the initiation and management of insulin therapy to achieve desired glycemic goals (3,63,65).\n\n#### b. Therapy for type 2 diabetes.\n\nThe ADA and the European Association for the Study of Diabetes published a consensus statement on the approach to management of hyperglycemia in individuals with type 2 diabetes (66) and recently published an update (67). Highlights of this approach are: intervention at the time of diagnosis with metformin in combination with lifestyle changes (MNT and exercise) and continuing timely augmentation of therapy with additional agents (including early initiation of insulin therapy) as a means of achieving and maintaining recommended levels of glycemic control (i.e., A1C \\<7% for most patients). The overall objective is to achieve and maintain glycemic control and to change interventions when therapeutic goals are not being met.\n\nThe algorithm took into account the evidence for A1C-lowering of the individual interventions, their additive effects, and their expense. The precise drugs used and their exact sequence may not be as important as achieving and maintaining glycemic targets safely. Medications not included in the consensus algorithm, owing to less glucose-lowering effectiveness, limited clinical data, and\/or relative expense, still may be appropriate choices in individual patients to achieve glycemic goals. Initiation of insulin at time of diagnosis is recommended for individuals presenting with weight loss or other severe hyperglycemic symptoms or signs. For a list of currently approved diabetes medications, see <http:\/\/ndep.nih.gov\/diabetes\/pubs\/Drug_tables_supplement.pdf>.\n\n## D. MNT\n\n### General recommendations\n\n-   Individuals who have pre-diabetes or diabetes should receive individualized MNT as needed to achieve treatment goals, preferably provided by a registered dietitian familiar with the components of diabetes MNT. (B)\n\n-   MNT should be covered by insurance and other payors. (E)\n\n### Energy balance, overweight, and obesity\n\n-   In overweight and obese insulin-resistant individuals, modest weight loss has been shown to reduce insulin resistance. Thus, weight loss is recommended for all overweight or obese individuals who have or are at risk for diabetes. (A)\n\n-   For weight loss, either low-carbohydrate or low-fat calorie restricted diets may be effective in the short-term (up to 1 year). (A)\n\n-   For patients on low-carbohydrate diets, monitor lipid profiles, renal function, and protein intake (in those with nephropathy) and adjust hypoglycemic therapy as needed. (E)\n\n-   Physical activity and behavior modification are important components of weight loss programs and are most helpful in maintenance of weight loss. (B)\n\n### Primary prevention of diabetes\n\n-   Among individuals at high risk for developing type 2 diabetes, structured programs that emphasize lifestyle changes that include moderate weight loss (7% body weight) and regular physical activity (150 min\/week), with dietary strategies including reduced calories and reduced intake of dietary fat, can reduce the risk for developing diabetes and are therefore recommended. (A)\n\n-   Individuals at high risk for type 2 diabetes should be encouraged to achieve the U.S. Department of Agriculture recommendation for dietary fiber (14 g fiber\/1,000 kcal) and foods containing whole grains (one-half of grain intake). (B)\n\n### Dietary fat intake in diabetes management\n\n-   Saturated fat intake should be \\<7% of total calories. (A)\n\n-   Intake of *trans* fat should be minimized. (B)\n\n### Carbohydrate intake in diabetes management\n\n-   Monitoring carbohydrate, whether by carbohydrate counting, exchanges, or experience-based estimation, remains a key strategy in achieving glycemic control. (A)\n\n-   For individuals with diabetes, the use of the glycemic index and glycemic load may provide a modest additional benefit for glycemic control over that observed when total carbohydrate is considered alone. (B)\n\n### Other nutrition recommendations\n\n-   Sugar alcohols and nonnutritive sweeteners are safe when consumed within the acceptable daily intake levels established by the Food and Drug Administration (FDA). (A)\n\n-   If adults with diabetes choose to use alcohol, daily intake should be limited to a moderate amount (one drink per day or less for adult women and two drinks per day or less for adult men). (E)\n\n-   Routine supplementation with antioxidants, such as vitamins E and C and carotene, is not advised because of lack of evidence of efficacy and concern related to long-term safety. (A)\n\n-   Benefit from chromium supplementation in people with diabetes or obesity has not been conclusively demonstrated and, therefore, cannot be recommended. (E)\n\nMNT is an integral component of diabetes prevention, management, and self-management education. In addition to its role in preventing and controlling diabetes, ADA recognizes the importance of nutrition as an essential component of an overall healthy lifestyle. A full review of the evidence regarding nutrition in preventing and controlling diabetes and its complications and additional nutrition-related recommendations can be found in the ADA position statement \"Nutrition Recommendations and Interventions for Diabetes,\" published in 2007 and updated for 2008 (68). Achieving nutrition-related goals requires a coordinated team effort that includes the active involvement of the person with pre-diabetes or diabetes. Because of the complexity of nutrition issues, it is recommended that a registered dietitian who is knowledgeable and skilled in implementing nutrition therapy into diabetes management and education be the team member who provides MNT.\n\nClinical trials\/outcome studies of MNT have reported decreases in A1C at 3\u20136 months ranging from 0.25 to 2.9% with higher reductions seen in type 2 diabetes of shorter duration. Multiple studies have demonstrated sustained improvements in A1C at 12 months and longer when a registered dietician provided follow-up visits ranging from monthly to three sessions per year (69\u201376). Meta-analyses of studies in nondiabetic, free-living subjects report that MNT reduces LDL cholesterol by 15\u201325 mg\/dl (77) or can lower LDL cholesterol by up to 16% (78), while clinical trials support a role for lifestyle modification in treating hypertension (78,79).\n\nBecause of the effects of obesity on insulin resistance, weight loss is an important therapeutic objective for overweight or obese individuals with pre-diabetes or diabetes (80). Short-term studies have demonstrated that moderate weight loss (5% of body weight) in subjects with type 2 diabetes is associated with decreased insulin resistance, improved measures of glycemia and lipemia, and reduced blood pressure (81); longer-term studies (52 weeks) showed mixed effects on A1C in adults with type 2 diabetes (82\u201385), and results were confounded by pharmacologic weight loss therapy. A systematic review of 80 weight loss studies of \u22651 year duration demonstrated that moderate weight loss achieved through diet alone, diet and exercise, and meal replacements can be achieved and maintained over the long term (4.8\u20138% weight loss at 12 months) (86). The multifactorial intensive lifestyle intervention employed in the DPP, which included reduced intake of fat and calories, led to weight loss averaging 7% at 6 months and maintenance of 5% weight loss at 3 years, associated with a 58% reduction in incidence of type 2 diabetes (10). Look AHEAD (Action for Health in Diabetes) is a large clinical trial designed to determine whether long-term weight loss will improve glycemia and prevent cardiovascular events in subjects with type 2 diabetes. One-year results of the intensive lifestyle intervention in this trial show an average of 8.6% weight loss, significant reduction of A1C, and reduction in several CVD risk factors (87). When completed, the Look AHEAD trial should provide insight into the effects of long-term weight loss on important clinical outcomes.\n\nThe optimal macronutrient distribution of weight loss diets has not been established. Although low-fat diets have traditionally been promoted for weight loss, several randomized controlled trials found that subjects on low-carbohydrate diets (\\<130 g\/day of carbohydrate) lost more weight at 6 months than subjects on low-fat diets (88,89); however, at 1 year, the difference in weight loss between the low-carbohydrate and low-fat diets was not significant, and weight loss was modest with both diets. Another study of overweight women randomized to one of four diets showed significantly more weight loss at 12 months with the Atkins low-carbohydrate diet than with higher-carbohydrate diets (90). Changes in serum triglyceride and HDL cholesterol were more favorable with the low-carbohydrate diets. In one study, those subjects with type 2 diabetes demonstrated a greater decrease in A1C with a low-carbohydrate diet than with a low-fat diet (89). A recent meta-analysis showed that at 6 months, low-carbohydrate diets were associated with greater improvements in triglyceride and HDL cholesterol concentrations than low-fat diets; however, LDL cholesterol was significantly higher on the low-carbohydrate diets (91). In a 2-year dietary intervention study, Mediterranean and low-carbohydrate diets were found to be effective and safe alternatives to a low-fat diet for weight reduction in moderately obese participants (85).\n\nThe recommended dietary allowance for digestible carbohydrate is 130 g\/day and is based on providing adequate glucose as the required fuel for the central nervous system without reliance on glucose production from ingested protein or fat. Although brain fuel needs can be met on lower carbohydrate diets, long-term metabolic effects of very-low-carbohydrate diets are unclear, and such diets eliminate many foods that are important sources of energy, fiber, vitamins, and minerals that are important in dietary palatability (92).\n\nAlthough numerous studies have attempted to identify the optimal mix of macronutrients for meal plans of people with diabetes, it is unlikely that one such combination of macronutrients exists. The best mix of carbohydrate, protein, and fat appears to vary depending on individual circumstances. For those individuals seeking guidance as to macronutrient distribution in healthy adults, the Dietary Reference Intake (DRI) system may be helpful (92). It must be clearly recognized that regardless of the macronutrient mix, total caloric intake must be appropriate for the weight management goal. Further, individualization of the macronutrient composition will depend on the metabolic status of the patient (e.g., lipid profile, renal function) and\/or food preferences. Individuals who choose to consume plant-based diets that are well planned and nutritionally adequate (i.e., vegetarian) may continue, as this can be done without being deleterious to metabolic control (93,94).\n\nThe primary goal with respect to dietary fat in individuals with diabetes is to limit saturated fatty acids, *trans* fatty acids, and cholesterol intake so as to reduce risk for CVD. Saturated and *trans* fatty acids are the principal dietary determinants of plasma LDL cholesterol. There is a lack of evidence on the effects of specific fatty acids on people with diabetes, so the recommended goals are consistent with those for individuals with CVD (78,95).\n\nThe FDA has approved five nonnutritive sweeteners for use in the U.S.: acesulfame potassium, aspartame, neotame, saccharin, and sucralose. Before being allowed on the market, all underwent rigorous scrutiny and were shown to be safe when consumed by the public, including people with diabetes and women during pregnancy. Reduced calorie sweeteners approved by the FDA include sugar alcohols (polyols) such as erythritol, isomalt, lactitol, maltitol, mannitol, sorbitol, xylitol, tagatose, and hydrogenated starch hydrolysates. The use of sugar alcohols appears to be safe; however, they may cause diarrhea, especially in children.\n\n### Reimbursement for MNT\n\nMNT, when delivered by a registered dietitian according to nutrition practice guidelines, is reimbursed as part of the Medicare program as overseen by the Centers for Medicare and Medicaid Services (CMS) ([www.cms.hhs.gov\/medicalnutritiontherapy](www.cms.hhs.gov\/medicalnutritiontherapy))\n\n## E. Bariatric surgery\n\n### Recommendations\n\n-   Bariatric surgery should be considered for adults with BMI \u226535 kg\/m^2^ and type 2 diabetes, especially if the diabetes is difficult to control with lifestyle and pharmacologic therapy. (B)\n\n-   Patients with type 2 diabetes who have undergone bariatric surgery need life-long lifestyle support and medical monitoring. (E)\n\n-   Although small trials have shown glycemic benefit of bariatric surgery in patients with type 2 diabetes and BMI of 30\u201335 kg\/m^2^, there is currently insufficient evidence to generally recommend surgery in patients with BMI \\<35 kg\/m^2^ outside of a research protocol. (E)\n\n-   The long-term benefits, cost-effectiveness, and risks of bariatric surgery in individuals with type 2 diabetes should be studied in well-designed randomized controlled trials with optimal medical and lifestyle therapy as the comparator. (E)\n\nGastric reduction surgery, either gastric banding or procedures that involve bypassing or transposing sections of the small intestine, when part of a comprehensive team approach, can be an effective weight loss treatment for severe obesity, and national guidelines support its consideration for people with type 2 diabetes who have BMI at or exceeding 35 kg\/m^2^. Bariatric surgery has been shown to lead to near or complete normalization of glycemia in \u223c55\u201395% of patients with type 2 diabetes, depending on the surgical procedure. A meta-analysis of studies of bariatric surgery reported that 78% of individuals with type 2 diabetes had complete \"resolution\" of diabetes (normalization of blood glucose levels in the absence of medications), and that the resolution rates were sustained in studies that had follow-up exceeding 2 years (96). Resolution rates are lowest with procedures that only constrict the stomach and higher with those that bypass portions of the small intestine. Additionally, there is increasing evidence that intestinal bypass procedures may have glycemic effects that are independent of, and additive to, their effects on weight.\n\nA recent randomized controlled trial compared adjustable gastric banding to \"best available\" medical and lifestyle therapy in subjects with type 2 diabetes diagnosed less than 2 years before randomization and BMI 30\u201340 kg\/m^2^ (97). In this trial, 73% of surgically treated patients achieved \"remission\" of their diabetes, compared with 13% of those treated medically. The latter group lost only 1.7% of body weight, suggesting that their therapy was not optimal. Overall, the trial had 60 subjects, and only 13 had a BMI under 35 kg\/m^2^, making it difficult to generalize these results widely to diabetic patients who are less severely obese or with longer duration of diabetes.\n\nBariatric surgery is costly in the short term and has some risks. Rates of morbidity and mortality directly related to the surgery have been reduced considerably in recent years, with 30-day mortality rates now 0.28%, similar to those of laparoscopic cholecystectomy (98). Longer-term concerns include vitamin and mineral deficiencies, osteoporosis, and rare but often severe hypoglycemia from insulin hypersecretion. Cohort studies attempting to match subjects suggest that the procedure may reduce longer-term mortality rates (99), and it is reasonable to postulate that there may be recouping of costs over the long run. However, studies of the mechanisms of glycemic improvement, long-term benefits and risks, and cost-effectiveness of bariatric surgery in individuals with type 2 diabetes will require well-designed randomized clinical trials, with optimal medical and lifestyle therapy of diabetes and cardiovascular risk factors as the comparitor.\n\n## F. DSME\n\n### Recommendations\n\n-   People with diabetes should receive DSME according to national standards when their diabetes is diagnosed and as needed thereafter. (B)\n\n-   Self-management behavior change is the key outcome of DSME and should be measured and monitored as part of care. (E)\n\n-   DSME should address psychosocial issues, since emotional well-being is strongly associated with positive diabetes outcomes. (C)\n\n-   DSME should be reimbursed by third-party payors. (E)\n\nDSME is an essential element of diabetes care (100\u2013106), and National Standards for DSME (107) are based on evidence for its benefits. Education helps people with diabetes initiate effective self-care when they are first diagnosed. Ongoing DSME also helps people with diabetes maintain effective self-management as their diabetes presents new challenges and treatment advances become available. DSME helps patients optimize metabolic control, prevent and manage complications, and maximize quality of life in a cost-effective manner (108).\n\nSince the 1990s, there has been a shift from a didactic approach, with DSME focusing on providing information, to a skill-based approach that focuses on helping those with diabetes make informed self-management choices. Care of diabetes has shifted to an approach that is more patient centered and that places the person with diabetes, and joint decision-making with heath care professionals, at the center of the care model. Patient-centered care is respectful of and responsive to individual patient preferences, needs, and values and ensures that patient values guide all decision making (109).\n\n### Evidence for the benefits of DSME\n\nSeveral studies have found that DSME is associated with improved diabetes knowledge and improved self-care behavior (101), improved clinical outcomes such as lower A1C (102,103,105,106,110), lower self-reported weight (101), and improved quality of life (104). Better outcomes were reported for DSME interventions that were longer and included follow-up support (101), that were tailored to individual needs and preferences (100), and that addressed psychosocial issues (100,101,105). Both individual and group approaches have been found effective ((111,112). There is increasing evidence for the role of a community health worker in delivering diabetes education in addition to the core team (113).\n\n### National standards for DSME\n\nADA-recognized DSME programs have staff that must be certified diabetes educators or have recent experience in diabetes education and management. The curriculum of ADA-recognized DSME programs must cover all nine areas of diabetes management, with the assessed needs of the individual determining which areas are addressed. The ADA Education Recognition Program (ERP) is a mechanism to ensure diabetes education programs meet the national standards and provide quality diabetes care.\n\n### Reimbursement for DSME\n\nDSME, when provided by a program that meets ADA ERP standards, is reimbursed as part of the Medicare program as overseen by the Centers for Medicare and Medicaid Services (CMS) ([www.cms.hhs.gov\/DiabetesSelfManagement](www.cms.hhs.gov\/DiabetesSelfManagement)).\n\n## G. Physical activity\n\n### Recommendations\n\n-   People with diabetes should be advised to perform at least 150 min\/week of moderate-intensity aerobic physical activity (50\u201370% of maximum heart rate). (A)\n\n-   In the absence of contraindications, people with type 2 diabetes should be encouraged to perform resistance training three times per week. (A)\n\nADA technical reviews on exercise in patients with diabetes have summarized the value of exercise in the diabetes management plan (114,115). Regular exercise has been shown to improve blood glucose control, reduce cardiovascular risk factors, contribute to weight loss, and improve well being. Furthermore, regular exercise may prevent type 2 diabetes in high-risk individuals (10\u201312). Structured exercise interventions of at least 8 weeks' duration have been shown to lower A1C by an average of 0.66% in people with type 2 diabetes, even with no significant change in BMI (116). Higher levels of exercise intensity are associated with greater improvements in A1C and in fitness (117).\n\n### Frequency and type of exercise\n\nThe U.S. Surgeon General's report (118) recommended that most adults accumulate at least 30 min of moderate-intensity activity on most, ideally all, days of the week. The studies included in the meta-analysis of effects of exercise interventions on glycemic control (116) had a mean number of sessions per week of 3.4, with a mean of 49 min per session. The DPP lifestyle intervention, which included 150 min per week of moderate intensity exercise, had a beneficial effect on glycemia in those with pre-diabetes. Therefore, it seems reasonable to recommend \u223c150 min of exercise per week for people with diabetes.\n\nResistance exercise improves insulin sensitivity to about the same extent as aerobic exercise (119). Clinical trials have provided strong evidence for the A1C-lowering value of resistance training in older adults with type 2 diabetes (120,121) and for an additive benefit of combined aerobic and resistance exercise in adults with type 2 diabetes (122).\n\n### Evaluation of the diabetic patient before recommending an exercise program\n\nPrior guidelines suggested that before recommending a program of physical activity, the provider should assess patients with multiple cardiovascular risk factors for coronary artery disease (CAD). As discussed more fully in Section VI.A.5, the area of screening asymptomatic diabetic patients for CAD remains unclear, and a recent ADA consensus statement on this issue concluded that routine screening is not recommended (123). Providers should use clinical judgment in this area. Certainly, high-risk patients should be encouraged to start with short periods of low-intensity exercise and increase the intensity and duration slowly.\n\nProviders should assess patients for conditions that might contraindicate certain types of exercise or predispose to injury, such as uncontrolled hypertension, severe autonomic neuropathy, severe peripheral neuropathy or history of foot lesions, and advanced retinopathy. The patient's age and previous physical activity level should be considered.\n\n### Exercise in the presence of nonoptimal glycemic control\n\n#### Hyperglycemia.\n\nWhen people with type 1 diabetes are deprived of insulin for 12\u201348 h and are ketotic, exercise can worsen hyperglycemia and ketosis (124); therefore, vigorous activity should be avoided in the presence of ketosis. However, it is not necessary to postpone exercise based simply on hyperglycemia, provided the patient feels well and urine and\/or blood ketones are negative.\n\n#### Hypoglycemia.\n\nIn individuals taking insulin and\/or insulin secretagogues, physical activity can cause hypoglycemia if medication dose or carbohydrate consumption is not altered. For individuals on these therapies, added carbohydrate should be ingested if pre-exercise glucose levels are \\<100 mg\/dl (5.6 mmol\/l) (125,126). Hypoglycemia is rare in diabetic individuals who are not treated with insulin or insulin secretagogues, and no preventive measures for hypoglycemia are usually advised in these cases.\n\n### Exercise in the presence of specific long-term complications of diabetes\n\n#### Retinopathy.\n\nIn the presence of proliferative diabetic retinopathy (PDR) or severe nonproliferative diabetic retinopathy (NPDR), vigorous aerobic or resistance exercise may be contraindicated because of the risk of triggering vitreous hemorrhage or retinal detachment (127).\n\n#### Peripheral neuropathy.\n\nDecreased pain sensation in the extremities results in increased risk of skin breakdown and infection and of Charcot joint destruction. Therefore, in the presence of severe peripheral neuropathy, it may be best to encourage non\u2013weight-bearing activities such as swimming, bicycling, or arm exercises (128,129).\n\n#### Autonomic neuropathy.\n\nAutonomic neuropathy can increase the risk of exercise-induced injury or adverse events through decreased cardiac responsiveness to exercise, postural hypotension, impaired thermoregulation, impaired night vision due to impaired papillary reaction, and unpredictable carbohydrate delivery from gastroparesis predisposing to hypoglycemia (128). Autonomic neuropathy is also strongly associated with CVD in people with diabetes (130,131). People with diabetic autonomic neuropathy should undergo cardiac investigation before beginning physical activity more intense than that to which they are accustomed.\n\n#### Albuminuria and nephropathy.\n\nPhysical activity can acutely increase urinary protein excretion. However, there is no evidence that vigorous exercise increases the rate of progression of diabetic kidney disease and likely no need for any specific exercise restrictions for people with diabetic kidney disease (132).\n\n## H. Psychosocial assessment and care\n\n### Recommendations\n\n-   Assessment of psychological and social situation should be included as an ongoing part of the medical management of diabetes. (E)\n\n-   Psychosocial screening and follow-up should include, but is not limited to, attitudes about the illness, expectations for medical management and outcomes, affect\/mood, general and diabetes-related quality of life, resources (financial, social, and emotional), and psychiatric history. (E)\n\n-   Screen for psychosocial problems such as depression, anxiety, eating disorders, and cognitive impairment when adherence to the medical regimen is poor. (E)\n\nPsychological and social problems can impair the individual's (133\u2013138) or family's (139) ability to carry out diabetes care tasks and therefore compromise health status. There are opportunities for the clinician to assess psychosocial status in a timely and efficient manner so that referral for appropriate services can be accomplished.\n\nKey opportunities for screening of psychosocial status occur at diagnosis, during regularly scheduled management visits, during hospitalizations, at discovery of complications, or when problems with glucose control, quality of life, or adherence are identified (140). Patients are likely to exhibit psychological vulnerability at diagnosis and when their medical status changes, i.e., the end of the honeymoon period, when the need for intensified treatment is evident, and when complications are discovered (135,137).\n\nIssues known to impact self-management and health outcomes include but are not limited to attitudes about the illness, expectations for medical management and outcomes, affect\/mood, general and diabetes-related quality of life, resources (financial, social, and emotional) (136), and psychiatric history (137,140,141). Screening tools are available for a number of these areas (142). Indications for referral to a mental health specialist familiar with diabetes management may include gross noncompliance with medical regimen (by self or others) (141), depression with the possibility of self-harm (134,143), debilitating anxiety (alone or with depression), indications of an eating disorder (144), or cognitive functioning that significantly impairs judgment (143). It is preferable to incorporate psychological assessment and treatment into routine care rather than waiting for identification of a specific problem or deterioration in psychological status (142). Although the clinician may not feel qualified to treat psychological problems, utilizing the patient-provider relationship as a foundation for further treatment can increase the likelihood that the patient will accept referral for other services. It is important to establish that emotional well-being is part of diabetes management (140).\n\n## I. When treatment goals are not met\n\nFor a variety of reasons, some people with diabetes and their health care providers do not achieve the desired goals of treatment (<a href=\"#t9\" data-ref-type=\"table\">Table 9<\/a>). Re-thinking the treatment regimen may require assessment of barriers to adherence including income, educational attainment, and competing demands, including those related to family responsibilities and family dynamics. Other strategies may include culturally appropriate and enhanced DSME, co-management with a diabetes team, referral to a medical social worker for assistance with insurance coverage or change in pharmacological therapy. Initiation of or increase in SMBG, utilization of continuous glucose monitoring, frequent contact with the patient, or referral to an endocrinologist may be useful.\n\n## J. Intercurrent illness\n\nThe stress of illness, trauma, and\/or surgery frequently aggravates glycemic control and may precipitate diabetic ketoacidosis (DKA) or nonketotic hyperosmolar state, life-threatening conditions that require immediate medical care to prevent complications and death (145). Any condition leading to deterioration in glycemic control necessitates more frequent monitoring of blood glucose and (in ketosis-prone patients) urine or blood ketones. Marked hyperglycemia requires temporary adjustment of the treatment program and, if accompanied by ketosis, vomiting, or alteration in level of consciousness, immediate interaction with the diabetes care team. The patient treated with noninsulin therapies or MNT alone may temporarily require insulin. Adequate fluid and caloric intake must be assured. Infection or dehydration are more likely to necessitate hospitalization of the person with diabetes than the person without diabetes.\n\nThe hospitalized patient should be treated by a physician with expertise in the management of diabetes. For further information on management of patients with hyperglycemia in the hospital, see Section VIII.A. For further information on management of DKA or nonketotic hyperosmolar state, refer to the ADA position statement on hyperglycemic crises (145).\n\n## K. Hypoglycemia\n\n### Recommendations\n\n-   Glucose (15\u201320 g) is the preferred treatment for the conscious individual with hypoglycemia, although any form of carbohydrate that contains glucose may be used. If SMBG 15 min after treatment shows continued hypoglycemia, the treatment should be repeated. Once SMBG glucose returns to normal, the individual should consume a meal or snack to prevent recurrence of hypoglycemia. (E)\n\n-   Glucagon should be prescribed for all individuals at significant risk of severe hypoglycemia, and caregivers or family members of these individuals should be instructed in its administration. Glucagon administration is not limited to health care professionals. (E)\n\n-   Individuals with hypoglycemia unawareness or one or more episodes of severe hypoglycemia should be advised to raise their glycemic targets to strictly avoid further hypoglycemia for at least several weeks to partially reverse hypoglycemia unawareness and reduce risk of future episodes. (B)\n\nHypoglycemia is the leading limiting factor in the glycemic management of type 1 and insulin-treated type 2 diabetes (146). Treatment of hypoglycemia (plasma glucose \\<70 mg\/dl) requires ingestion of glucose- or carbohydrate-containing foods. The acute glycemic response correlates better with the glucose content than with the carbohydrate content of the food. Although pure glucose is the preferred treatment, any form of carbohydrate that contains glucose will raise blood glucose. Added fat may retard and then prolong the acute glycemic response (147). Ongoing activity of insulin or insulin secretagogues may lead to recurrence of hypoglycemia unless further food is ingested after recovery.\n\nSevere hypoglycemia (where the individual requires the assistance of another person and cannot be treated with oral carbohydrate due to confusion or unconsciousness) should be treated using emergency glucagon kits, which require a prescription. Those in close contact with, or having custodial care of, people with hypoglycemia-prone diabetes (family members, roommates, school personnel, child care providers, correctional institution staff, or coworkers) should be instructed in use of such kits. An individual does not need to be a health care professional to safely administer glucagon. Care should be taken to ensure that unexpired glucagon kits are available.\n\nPrevention of hypoglycemia is a critical component of diabetes management. Teaching people with diabetes to balance insulin use, carbohydrate intake, and exercise is a necessary but not always sufficient strategy. In type 1 diabetes and severely insulin-deficient type 2 diabetes, the syndrome of hypoglycemia unawareness, or hypoglycemia-associated autonomic failure, can severely compromise stringent diabetes control and quality of life. The deficient counter-regulatory hormone release and autonomic responses in this syndrome are both risk factors for, and caused by, hypoglycemia. A corollary to this \"vicious cycle\" is that several weeks of avoidance of hypoglycemia has been demonstrated to improve counter-regulation and awareness to some extent in many patients (146,148,149). Hence, patients with one or more episodes of severe hypoglycemia may benefit from at least short-term relaxation of glycemic targets.\n\n## L. Immunization\n\n### Recommendations\n\n-   Annually provide an influenza vaccine to all diabetic patients \u22656 months of age. (C)\n\n-   Administer pneumococcal polysaccharide vaccine to all diabetic patients \u22652 years of age. A one-time revaccination is recommended for individuals \\>64 years of age previously immunized when they were \\<65 years of age if the vaccine was administered \\>5 years ago. Other indications for repeat vaccination include nephrotic syndrome, chronic renal disease, and other immunocompromised states, such as after transplantation. (C)\n\nInfluenza and pneumonia are common, preventable infectious diseases associated with high mortality and morbidity in the elderly and in people with chronic diseases. Though there are limited studies reporting the morbidity and mortality of influenza and pneumococcal pneumonia specifically in people with diabetes, observational studies of patients with a variety of chronic illnesses, including diabetes, show that these conditions are associated with an increase in hospitalizations for influenza and its complications. People with diabetes may be at increased risk of the bacteremic form of pneumococcal infection and have been reported to have a high risk of nosocomial bacteremia, which has a mortality rate as high as 50% (150).\n\nSafe and effective vaccines are available that can greatly reduce the risk of serious complications from these diseases (151,152). In a case-control series, influenza vaccine was shown to reduce diabetes-related hospital admission by as much as 79% during flu epidemics (151). There is sufficient evidence to support that people with diabetes have appropriate serologic and clinical responses to these vaccinations. The Centers for Disease Control and Prevention's Advisory Committee on Immunization Practices recommends influenza and pneumococcal vaccines for all individuals with diabetes (<http:\/\/www.cdc.gov\/vaccines\/recs\/>). For a complete discussion on the prevention of influenza and pneumococcal disease in people with diabetes, consult the technical review and position statement on this subject (150,153).\n\n# VI. PREVENTION AND MANAGEMENT OF DIABETES COMPLICATIONS\n\n## A. CVD\n\nCVD is the major cause of morbidity and mortality for individuals with diabetes and the largest contributor to the direct and indirect costs of diabetes. The common conditions coexisting with type 2 diabetes (e.g., hypertension and dyslipidemia) are clear risk factors for CVD, and diabetes itself confers independent risk. Numerous studies have shown the efficacy of controlling individual cardiovascular risk factors in preventing or slowing CVD in people with diabetes. Large benefits are seen when multiple risk factors are addressed globally (154). Evidence is summarized in the following sections and reviewed in detail in the ADA technical reviews on hypertension (155), dyslipidemia (156), aspirin therapy (157), and smoking cessation (158) and in the American Heart Association (AHA)\/ADA scientific statement on prevention of CVD in people with diabetes (159).\n\n### 1. Hypertension\/blood pressure control\n\n#### Recommendations\n\n##### Screening and diagnosis\n\n-   Blood pressure should be measured at every routine diabetes visit. Patients found to have a systolic blood pressure of \u2265130 mmHg or a diastolic blood pressure of \u226580 mmHg should have blood pressure confirmed on a separate day. Repeat systolic blood pressure of \u2265130 mmHg or diastolic blood pressure of \u226580 mmHg confirms a diagnosis of hypertension. (C)\n\n##### Goals\n\n-   Patients with diabetes should be treated to a systolic blood pressure \\<130 mmHg. (C)\n\n-   Patients with diabetes should be treated to a diastolic blood pressure \\<80 mmHg. (B)\n\n##### Treatment\n\n-   Patients with a systolic blood pressure of 130\u2013139 mmHg or a diastolic blood pressure of 80\u201389 mmHg may be given lifestyle therapy alone for a maximum of 3 months and then, if targets are not achieved, be treated with the addition of pharmacological agents. (E)\n\n-   Patients with more severe hypertension (systolic blood pressure \u2265140 mmHg or diastolic blood pressure \u226590 mmHg) at diagnosis or follow-up should receive pharmacologic therapy in addition to lifestyle therapy. (A)\n\n-   Pharmacologic therapy for patients with diabetes and hypertension should be with a regimen that includes either an ACE inhibitor or an angiotensin receptor blocker (ARB). If one class is not tolerated, the other should be substituted. If needed to achieve blood pressure targets, a thiazide diuretic should be added to those with an estimated GFR (see below) \u226530 ml\/min per 1.73 m^2^ and a loop diuretic for those with an estimated GFR \\<30 ml\/min per 1.73 m^2^. (C)\n\n-   Multiple drug therapy (two or more agents at maximal doses) is generally required to achieve blood pressure targets. (B)\n\n-   If ACE inhibitors, ARBs, or diuretics are used, kidney function and serum potassium levels should be closely monitored. (E)\n\n-   In pregnant patients with diabetes and chronic hypertension, blood pressure target goals of 110\u2013129\/65\u201379 mmHg are suggested in the interest of long-term maternal health and minimizing impaired fetal growth. ACE inhibitors and ARBs are contraindicated during pregnancy. (E)\n\nHypertension is a common comorbidity of diabetes, affecting the majority of patients, with prevalence depending on type of diabetes, age, obesity, and ethnicity. Hypertension is a major risk factor for both CVD and microvascular complications. In type 1 diabetes, hypertension is often the result of underlying nephropathy, while in type 2 it usually coexists with other cardiometabolic risk factors.\n\n#### Screening and diagnosis\n\nMeasurement of blood pressure in the office should be done by a trained individual and follow the guidelines established for nondiabetic individuals: measurement in the seated position, with feet on the floor and arm supported at heart level, after 5 min of rest. Cuff size should be appropriate for the upper arm circumference. Elevated values should be confirmed on a separate day. Because of the clear synergistic risks of hypertension and diabetes, the diagnostic cutoff for a diagnosis of hypertension is lower in people with diabetes (blood pressure \u2265130\/80) than in those without diabetes (blood pressure \u2265140\/90 mmHg) (160).\n\nHome blood pressure self-monitoring and 24-h ambulatory blood pressure monitoring may provide additional evidence of \"white coat\" and masked hypertension and other discrepancies between office and \"true\" blood pressure, and studies in nondiabetic populations show that home measurements may better correlate with CVD risk than office measurements (161,162). However, the preponderance of the clear evidence of benefits of treatment of hypertension in people with diabetes is based on office measurements.\n\n#### Treatment goals\n\nRandomized clinical trials have demonstrated the benefit (reduction of CHD events, stroke, and nephropathy) of lowering blood pressure to \\<140 mmHg systolic and \\<80 mmHg diastolic in individuals with diabetes (160,163\u2013165). Epidemiologic analyses show that blood pressure \\>115\/75 mmHg is associated with increased cardiovascular event rates and mortality in individuals with diabetes (160,166,167). Therefore, a target blood pressure goal of \\<130\/80 mmHg is reasonable if it can be safely achieved. The ongoing ACCORD trial is designed to determine whether blood pressure lowering to systolic blood pressure \\<120 mmHg provides greater cardiovascular protection than a systolic blood pressure level of \\<140 mmHg in patients with type 2 diabetes ([www.accord.org](www.accord.org)).\n\n#### Treatment strategies\n\nAlthough there are no well-controlled studies of diet and exercise in the treatment of hypertension in individuals with diabetes, studies in nondiabetic individuals have shown anti-hypertensive effects similar to pharmacologic monotherapy of reducing sodium intake and excess body weight; increasing consumption of fruits, vegetables, and low-fat dairy products; avoiding excessive alcohol consumption; and increasing activity levels (160,168). These nonpharmacological strategies may also positively affect glycemia and lipid control. Their effects on cardiovascular events have not been established. An initial trial of nonpharmacologic therapy may be reasonable in diabetic individuals with mild hypertension (systolic blood pressure 130\u2013139 mmHg or diastolic blood pressure 80\u201389 mmHg). If the blood pressure is \u2265140 mmHg systolic and\/or \u226590 mmHg diastolic at the time of diagnosis, pharmacologic therapy should be initiated along with nonpharmacologic therapy (160).\n\nLowering of blood pressure with regimens based on a variety of antihypertensive drugs, including ACE inhibitors, ARBs, \u03b2-blockers, diuretics, and calcium channel blockers, has been shown to be effective in reducing cardiovascular events. Several studies have suggested that ACE inhibitors may be superior to dihydropyridine calcium channel blockers in reducing cardiovascular events (169\u2013171). However, a variety of other studies have shown no specific advantage to ACE inhibitors as initial treatment of hypertension in the general hypertensive population, but rather an advantage on cardiovascular outcomes of initial therapy with low-dose thiazide diuretics (160,172,173).\n\nIn people with diabetes, inhibitors of the renin-angiotensin system (RAS) may have unique advantages for initial or early therapy of hypertension. In a nonhypertension trial of high-risk individuals, including a large subset with diabetes, an ACE inhibitor reduced CVD outcomes (174). In patients with congestive heart failure (CHF), including diabetic subgroups, ARBs have been shown to reduce major CVD outcomes (175\u2013178), and in type 2 patients with significant nephropathy, ARBs were superior to calcium channel blockers for reducing heart failure (179\u2013181). Though evidence for distinct advantages of RAS inhibitors on CVD outcomes in diabetes remains conflicting (163,182), the high CVD risks associated with diabetes, and the high prevalence of undiagnosed CVD, may still favor recommendations for their use as first-line hypertension therapy in people with diabetes (160). Recently, the blood pressure arm of the ADVANCE trial demonstrated that routine administration of a fixed combination of the ACE inhibitor perindopril and the diuretic indapamide significantly reduced combined microvascular and macrovascular outcomes, as well as CVD and total mortality. The improved outcomes could also have been due to lower achieved blood pressure in the perindopril-indapamide arm (183). The compelling benefits of RAS inhibitors in diabetic patients with albuminuria or renal insufficiency provide additional rationale for use of these agents (see section VI.B below).\n\nAn important caveat is that most patients with hypertension require multi-drug therapy to reach treatment goals, especially diabetic patients whose targets are lower. Many patients will require three or more drugs to reach target goals (160). If blood pressure is refractory to multiple agents, clinicians should consider an evaluation for secondary forms of hypertension.\n\nDuring pregnancy in diabetic women with chronic hypertension, target blood pressure goals of systolic blood pressure 110\u2013129 mmHg and diastolic blood pressure 65\u201379 mmHg are reasonable, as they contribute to long-term maternal health. Lower blood pressure levels may be associated with impaired fetal growth. During pregnancy, treatment with ACE inhibitors and ARBs is contraindicated, since they are likely to cause fetal damage. Antihypertensive drugs known to be effective and safe in pregnancy include methyldopa, labetalol, diltiazem, clonidine, and prazosin. Chronic diuretic use during pregnancy has been associated with restricted maternal plasma volume, which might reduce uteroplacental perfusion (184).\n\n### 2. Dyslipidemia\/lipid management\n\n#### Recommendations\n\n##### Screening\n\n-   In most adult patients, measure fasting lipid profile at least annually. In adults with low-risk lipid values (LDL cholesterol \\<100 mg\/dl, HDL cholesterol \\>50 mg\/dl, and triglycerides \\<150 mg\/dl), lipid assessments may be repeated every 2 years. (E)\n\n##### Treatment recommendations and goals\n\n-   Lifestyle modification focusing on the reduction of saturated fat, *trans* fat, and cholesterol intake; weight loss (if indicated); and increased physical activity should be recommended to improve the lipid profile in patients with diabetes. (A)\n\n-   Statin therapy should be added to lifestyle therapy, regardless of baseline lipid levels, for diabetic patients:\n\n    -   with overt CVD (A)\n\n    -   without CVD who are over the age of 40 and have one or more other CVD risk factors. (A)\n\n-   For lower-risk patients than the above (e.g., without overt CVD and under the age of 40), statin therapy should be considered in addition to lifestyle therapy if LDL cholesterol remains above 100 mg\/dl or in those with multiple CVD risk factors. (E)\n\n-   In individuals without overt CVD, the primary goal is an LDL cholesterol \\<100 mg\/dl (2.6 mmol\/l). (A)\n\n-   In individuals with overt CVD, a lower LDL cholesterol goal of \\<70 mg\/dl (1.8 mmol\/l), using a high dose of a statin, is an option. (B)\n\n-   If drug-treated patients do not reach the above targets on maximal tolerated statin therapy, a reduction in LDL cholesterol of \u223c30\u201340% from baseline is an alternative therapeutic goal. (A)\n\n-   Triglycerides levels \\<150 mg\/dl (1.7 mmol\/l) and HDL cholesterol \\>40 mg\/dl (1.0 mmol\/l) in men and \\>50 mg\/dl (1.3 mmol\/l) in women are desirable. However, LDL cholesterol\u2013targeted statin therapy remains the preferred strategy. (C)\n\n-   If targets are not reached on maximally tolerated doses of statins, combination therapy using statins and other lipid-lowering agents may be considered to achieve lipid targets but has not been evaluated in outcome studies for either CVD outcomes or safety. (E)\n\n-   Statin therapy is contraindicated in pregnancy. (E)\n\n#### Evidence for benefits of lipid-lowering therapy\n\nPatients with type 2 diabetes have an increased prevalence of lipid abnormalities, contributing to their high risk of CVD. For the past decade or more, multiple clinical trials demonstrated significant effects of pharmacologic (primarily statin) therapy on CVD outcomes in subjects with CHD and for primary CVD prevention (185). Subanalyses of diabetic subgroups of larger trials (186\u2013190) and trials specifically in subjects with diabetes (191,192) showed significant primary and secondary prevention of CVD events \u00b1 CHD deaths in diabetic populations. As shown in <a href=\"#t10\" data-ref-type=\"table\">Table 10<\/a>, and similar to findings in nondiabetic subjects, reduction in \"hard\" CVD outcomes (CHD death and nonfatal MI) can be more clearly seen in diabetic subjects with high baseline CVD risk (known CVD and\/or very high LDL cholesterol levels), but overall the benefits of statin therapy in people with diabetes at moderate or high risk for CVD are convincing.\n\nLow levels of HDL cholesterol, often associated with elevated triglyceride levels, are the most prevalent pattern of dyslipidemia in persons with type 2 diabetes. However, the evidence base for drugs that target these lipid fractions is significantly less robust than that for statin therapy (193). Nicotinic acid has been shown to reduce CVD outcomes (194), although the study was done in a nondiabetic cohort. Gemfibrozil has been shown to decrease rates of CVD events in subjects without diabetes (195,196) and in the diabetic subgroup in one of the larger trials (195). However, in a large trial specific to diabetic patients, fenofibrate failed to reduce overall cardiovascular outcomes (197).\n\n#### Dyslipidemia treatment and target lipid levels\n\nFor most patients with diabetes, the first priority of dyslipidemia therapy (unless severe hypertriglyceridemia is the immediate issue) is to lower LDL cholesterol to a target goal of \\<100 mg\/dl (2.60 mmol\/l) (198). Lifestyle intervention, including MNT, increased physical activity, weight loss, and smoking cessation, may allow some patients to reach lipid goals. Nutrition intervention should be tailored according to each patient's age, type of diabetes, pharmacological treatment, lipid levels, and other medical conditions and should focus on the reduction of saturated fat, cholesterol, and *trans* unsaturated fat intake. Glycemic control can also beneficially modify plasma lipid levels, particularly in patients with very high triglycerides and poor glycemic control.\n\nIn those with clinical CVD or over age 40 with other CVD risk factors, pharmacological treatment should be added to lifestyle therapy regardless of baseline lipid levels. Statins are the drugs of choice for LDL cholesterol lowering.\n\nIn patients other than those described above, statin treatment should be considered if there is an inadequate LDL cholesterol response to lifestyle modifications and improved glucose control, or if the patient has increased cardiovascular risk (e.g., multiple cardiovascular risk factors or long duration of diabetes). Very little clinical trial evidence exists for type 2 patients under the age of 40, or for type 1 patients of any age. In the Heart Protection Study, the subgroup of 600 patients with type 1 diabetes (lower age limit 40 years) had a proportionately similar reduction in risk as patients with type 2 diabetes, although not statistically significant (187). Although the data are not definitive, consideration should be given to similar lipid-lowering goals in type 1 diabetic patients as those in type 2 diabetic patients, particularly if they have other cardiovascular risk factors.\n\n#### Alternative LDL cholesterol goals\n\nVirtually all trials of statins and CVD outcomes have tested specific doses of statins against placebo, other doses of statin, or other statins, rather than aiming for specific LDL cholesterol goals (199). As can be seen in <a href=\"#t10\" data-ref-type=\"table\">Table 10<\/a>, placebo-controlled trials generally achieved LDL cholesterol reductions of 30\u201340% from baseline. Hence, LDL cholesterol lowering of this magnitude is an acceptable outcome for patients who cannot reach LDL cholesterol goals due to severe baseline elevations in LDL cholesterol and\/or intolerance of maximal, or any, statin doses. Additionally, for those with baseline LDL cholesterol minimally above 100 mg\/dl, prescribing statin therapy to lower LDL cholesterol about 30\u201340% from baseline is probably more effective than prescribing just enough to get LDL cholesterol slightly below 100 mg\/dl.\n\nRecent clinical trials in high-risk patients, such as those with acute coronary syndromes or previous cardiovascular events (200\u2013202), have demonstrated that more aggressive therapy with high doses of statins to achieve an LDL cholesterol of \\<70 mg\/dl led to a significant reduction in further events. Therefore, a reduction in LDL cholesterol to a goal of \\<70 mg\/dl is an option in very-high-risk diabetic patients with overt CVD (203).\n\nIn individual patients, LDL cholesterol lowering with statins is highly variable, and this variable response is poorly understood (204). Reduction of CVD events with statins correlates very closely with LDL cholesterol lowering (185). When maximally tolerated doses of statins fail to significantly lower LDL cholesterol (\\<30% reduction from patients baseline), the primary aim of combination therapy should be to achieve additional LDL cholesterol lowering. Niacin, fenofibrate, ezetimibe, and bile acid sequestrants all offer additional LDL cholesterol lowering. The evidence that combination therapy provides a significant increment in CVD risk reduction over statin therapy alone is still elusive.\n\n#### Treatment of other lipoprotein fractions or targets\n\nSevere hypertriglyceridemia may warrant immediate therapy of this abnormality with lifestyle and usually pharmacologic therapy (fibric acid derivative or niacin) to reduce the risk of acute pancreatitis. In the absence of severe hypertriglyceridemia, therapy targeting HDL cholesterol or triglycerides has intuitive appeal but lacks the evidence base of statin therapy (162). If the HDL cholesterol is \\<40 mg\/dl and the LDL cholesterol is between 100 and 129 mg\/dl, gemfibrozil or niacin might be used, especially if a patient is intolerant to statins. Niacin is the most effective drug for raising HDL cholesterol. It can significantly increase blood glucose at high doses, but recent studies demonstrate that at modest doses (750\u20132,000 mg\/day), significant improvements in LDL cholesterol, HDL cholesterol, and triglyceride levels are accompanied by only modest changes in glucose that are generally amenable to adjustment of diabetes therapy (205,206).\n\nCombination therapy, with a statin and a fibrate or a statin and niacin, may be efficacious for treatment for all three lipid fractions, but this combination is associated with an increased risk for abnormal transaminase levels, myositis, or rhabdomyolysis. The risk of rhabdomyolysis is higher with higher doses of statins and with renal insufficiency and seems to be lower when statins are combined with fenofibrate than gemfibrozil (207). Several ongoing trials may provide much-needed evidence for the effects of combination therapy on cardiovascular outcomes.\n\nIn 2008, a consensus panel convened by ADA and the American College of Cardiology recommended a greater focus on non-HDL cholesterol and apolipoprotein B (apo B) in patients who are likely to have small LDL particles, such as people with diabetes (208). The consensus panel suggested that for statin-treated patients in whom the LDL cholesterol goal would be \\<70 mg\/dl (non-HDL cholesterol \\<100 mg\/dl), apo B should be measured and treated to \\<80 mg\/dl. For patients on statins with an LDL cholesterol goal of \\<100 mg\/dl (non-HDL cholesterol \\<130 mg\/dl), apo B should be measured and treated to below 90 mg\/dl.\n\n<a href=\"#t11\" data-ref-type=\"table\">Table 11<\/a> summarizes the general recommendations for glycemic, blood pressure, and lipid control for adults with diabetes.\n\n### 3. Antiplatelet agents\n\n#### Recommendations\n\n-   Use aspirin therapy (75\u2013162 mg\/day) as a primary prevention strategy in those with type 1 or type 2 diabetes at increased cardiovascular risk, including those who are \\>40 years of age or who have additional risk factors (family history of CVD, hypertension, smoking, dyslipidemia, or albuminuria). (C)\n\n-   Use aspirin therapy (75\u2013162 mg\/day) as a secondary prevention strategy in those with diabetes with a history of CVD. (A)\n\n-   For patients with CVD and documented aspirin allergy, clopidogrel (75 mg\/day) should be used. (B)\n\n-   Combination therapy with ASA (75\u2013162 mg\/day) and clopidogrel (75 mg\/day) is reasonable for up to a year after an acute coronary syndrome. (B)\n\n-   Aspirin therapy is not recommended in people under 30 years of age due to lack of evidence of benefit and is contraindicated in patients under the age of 21 years because of the associated risk of Reye's syndrome. (E)\n\nThe use of aspirin in diabetes is reviewed in detail in the ADA technical review (157) and position statement (210) on this topic. Aspirin has been recommended for primary (211,212) and secondary (213,214) prevention of cardiovascular events in high-risk diabetic and nondiabetic individuals. One large meta-analysis and several clinical trials demonstrate the efficacy of using aspirin as a preventive measure for cardiovascular events, including stroke and myocardial infarction. Many trials have shown an \u223c30% decrease in myocardial infarction and a 20% decrease in stroke in a wide range of patients, including young and middle-aged patients, patients with and without a history of CVD, men and women, and patients with hypertension.\n\nDosages used in most clinical trials ranged from 75 to 325 mg\/day. There is little evidence to support any specific dose, but using the lowest possible dosage may help reduce side effects (215). Conversely, a randomized trial of 100 mg of aspirin daily showed less of a primary prevention effect, without statistical significance, in the large diabetic subgroup in contrast to significant benefit in those without diabetes (216), raising the issue of aspirin resistance in those with diabetes.\n\nThe systematic review of evidence for the U.S. Preventive Services Task Force (USPSTF) estimated that aspirin reduced the risk for nonfatal and fatal MI (odds ratio 0.72 \\[95% CI 0.60\u20130.87\\]). The review acknowledged the low numbers of diabetic subjects in most trials but concluded that subset analyses and a single trial in diabetic patients suggested that the estimates extended to those with diabetes (211). The USPSTF stated that the risk-to-benefit ratio favors aspirin use when 5-year CHD risk equals or exceeds 3% and suggested aspirin therapy be considered for men \\>40 years of age, postmenopausal women, and younger persons with CHD risk factors (including diabetes) (212).\n\nThere is no evidence for a specific age at which to start aspirin, but aspirin has not been studied at ages \\<30 years.\n\nClopidogrel has been demonstrated to reduce CVD events in diabetic individuals (217). Adjunctive therapy in the first year after acute coronary syndrome in very-high-risk patients, or as alternative therapy in aspirin-intolerant patients, should be considered.\n\n### 4. Smoking cessation\n\n#### Recommendations\n\n-   Advise all patients not to smoke. (A)\n\n-   Include smoking cessation counseling and other forms of treatment as a routine component of diabetes care. (B)\n\nIssues of smoking in diabetes are reviewed in detail in the ADA technical review (158) and position statement (218) on this topic. A large body of evidence from epidemiological, case-control, and cohort studies provides convincing documentation of the causal link between cigarette smoking and health risks. Cigarette smoking contributes to one of every five deaths in the U.S. and is the most important modifiable cause of premature death. Much of the prior work documenting the impact of smoking on health did not separately discuss results on subsets of individuals with diabetes, suggesting that the identified risks are at least equivalent to those found in the general population. Other studies of individuals with diabetes consistently found a heightened risk of CVD and premature death among smokers. Smoking is also related to the premature development of microvascular complications of diabetes and may have a role in the development of type 2 diabetes.\n\nA number of large randomized clinical trials have demonstrated the efficacy and cost-effectiveness of smoking cessation counseling in changing smoking behavior and reducing tobacco use. The routine and thorough assessment of tobacco use is important as a means of preventing smoking or encouraging cessation. Special considerations should include assessment of level of nicotine dependence, which is associated with difficulty in quitting and relapse (219,220). Free telephone quit lines are available in each state (see [www.naquitline.org](www.naquitline.org)).\n\n### 5. CHD screening and treatment\n\n#### Recommendations\n\n##### Screening\n\n-   In asymptomatic patients, evaluate risk factors to stratify patients by 10-year risk, and treat risk factors accordingly. (B)\n\n##### Treatment\n\n-   In patients with known CVD, ACE inhibitor (C), aspirin (A), and statin therapy (A) (if not contraindicated) should be used to reduce the risk of cardiovascular events.\n\n-   In patients with a prior myocardial infarction, add \u03b2-blockers (if not contraindicated) to reduce mortality. (A)\n\n-   In patients \\>40 years of age with another cardiovascular risk factor (hypertension, family history, dyslipidemia, microalbuminuria, cardiac autonomic neuropathy, or smoking), aspirin and statin therapy (if not contraindicated) should be used to reduce the risk of cardiovascular events. (B)\n\n-   In patients with CHF, TZD use is contraindicated. (C)\n\n-   Metformin may be used in patients with stable CHF if renal function is normal. It should be avoided in unstable or hospitalized patients with CHF. (C)\n\nScreening for CAD is reviewed in a recently updated consensus statement (123). To identify the presence of CAD in diabetic patients without clear or suggestive symptoms, a risk factor\u2013based approach to the initial diagnostic evaluation and subsequent follow-up has intuitive appeal. However, recent studies concluded that using this approach fails to identify which patients will have silent ischemia on screening tests (130,221).\n\nCandidates for cardiac testing include those with *1*) typical or atypical cardiac symptoms and *2*) an abnormal resting electrocardiogram (ECG). The screening of asymptomatic patients remains controversial, especially as intensive medical therapy indicated in diabetic patients at high risk for CVD has an increasing evidence base for providing equal outcomes to invasive revascularization, including in diabetic patients (222). There is also recent preliminary evidence that silent myocardial ischemia may reverse over time, adding to the controversy concerning aggressive screening strategies (223). Finally, a recent randomized observational trial presented at the ADA's Scientific Sessions in June 2008 demonstrated no clinical benefit to routine screening of asymptomatic patients with type 2 diabetes and normal ECGs. Despite abnormal myocardial perfusion imaging in more than one in five patients, cardiac outcomes were essentially equal (and very low) in screened versus unscreened patients.\n\nIn all patients with diabetes, cardiovascular risk factors should be assessed at least annually. These risk factors include dyslipidemia, hypertension, smoking, a positive family history of premature coronary disease, and the presence of micro- or macroalbuminuria. Abnormal risk factors should be treated as described elsewhere in these guidelines. Patients at increased CHD risk should receive aspirin and a statin and ACE inhibitor or ARB therapy if hypertensive, unless there are contraindications to a particular drug class. While clear benefit exists for ACE inhibitor and ARB therapy in patients with nephropathy or hypertension, the benefits in patients with CVD in the absence of these conditions is less clear, especially when LDL cholesterol is concomitantly controlled (224,225)\n\n## B. Nephropathy screening and treatment\n\n### Recommendations\n\n#### General recommendations\n\n-   To reduce the risk or slow the progression of nephropathy, optimize glucose control. (A)\n\n-   To reduce the risk or slow the progression of nephropathy, optimize blood pressure control. (A)\n\n#### Screening\n\n-   Perform an annual test to assess urine albumin excretion in type 1 diabetic patients with diabetes duration of \u22655 years and in all type 2 diabetic patients, starting at diagnosis. (E)\n\n-   Measure serum creatinine at least annually in all adults with diabetes regardless of the degree of urine albumin excretion. The serum creatinine should be used to estimate GFR and stage the level of chronic kidney disease (CKD), if present. (E)\n\n#### Treatment\n\n-   In the treatment of the nonpregnant patient with micro- or macroalbuminuria, either ACE inhibitors or ARBs should be used. (A)\n\n-   While there are no adequate head-to-head comparisons of ACE inhibitors and ARBs, there is clinical trial support for each of the following statements:\n\n    -   In patients with type 1 diabetes, hypertension, and any degree of albuminuria, ACE inhibitors have been shown to delay the progression of nephropathy. (A)\n\n    -   In patients with type 2 diabetes, hypertension, and microalbuminuria, both ACE inhibitors and ARBs have been shown to delay the progression to macroalbuminuria. (A)\n\n    -   In patients with type 2 diabetes, hypertension, macroalbuminuria, and renal insufficiency (serum creatinine \\>1.5 mg\/dl), ARBs have been shown to delay the progression of nephropathy. (A)\n\n    -   If one class is not tolerated, the other should be substituted. (E)\n\n-   Reduction of protein intake to 0.8\u20131.0 g \u00b7 kg body wt^\u22121^ \u00b7 day^\u22121^ in individuals with diabetes and the earlier stages of CKD and to 0.8 g \u00b7 kg body wt^\u22121^ \u00b7 day^\u22121^ in the later stages of CKD may improve measures of renal function (urine albumin excretion rate, GFR) and is recommended. (B)\n\n-   When ACE inhibitors, ARBs, or diuretics are used, monitor serum creatinine and potassium levels for the development of acute kidney disease and hyperkalemia. (E)\n\n-   Continued monitoring of urine albumin excretion to assess both response to therapy and progression of disease is recommended. (E)\n\n-   Consider referral to a physician experienced in the care of kidney disease when there is uncertainty about the etiology of kidney disease (active urine sediment, absence of retinopathy, rapid decline in GFR), difficult management issues, or advanced kidney disease. (B)\n\nDiabetic nephropathy occurs in 20\u201340% of patients with diabetes and is the single leading cause of end-stage renal disease (ESRD). Persistent albuminuria in the range of 30\u2013299 mg\/24 h (microalbuminuria) has been shown to be the earliest stage of diabetic nephropathy in type 1 diabetes and a marker for development of nephropathy in type 2 diabetes. Microalbuminuria is also a well-established marker of increased CVD risk (226,227). Patients with microalbuminuria who progress to macroalbuminuria (300 mg\/24 h) are likely to progress to ESRD (228,229). However, a number of interventions have been demonstrated to reduce the risk and slow the progression of renal disease.\n\nIntensive diabetes management with the goal of achieving near normoglycemia has been shown in large prospective randomized studies to delay the onset of microalbuminuria and the progression of micro- to macroalbuminuria in patients with type 1 (230,231) and type 2 (49,50) diabetes. The UKPDS provided strong evidence that control of blood pressure can reduce the development of nephropathy (163). In addition, large prospective randomized studies in patients with type 1 diabetes have demonstrated that achievement of lower levels of systolic blood pressure (\\<140 mmHg) resulting from treatment using ACE inhibitors provides a selective benefit over other antihypertensive drug classes in delaying the progression from micro- to macroalbuminuria and can slow the decline in GFR in patients with macroalbuminuria (180,181,232). In type 2 diabetes with hypertension and normoalbuminuria, ACE inhibition has been demonstrated to delay progression to microalbuminuria (233).\n\nIn addition, ACE inhibitors have been shown to reduce major CVD outcomes (i.e., myocardial infarction, stroke, death) in patients with diabetes (174), thus further supporting the use of these agents in patients with microalbuminuria, a CVD risk factor. ARBs have also been shown to reduce the rate of progression from micro- to macroalbuminuria as well as ESRD in patients with type 2 diabetes (234\u2013236). Some evidence suggests that ARBs have a smaller magnitude of rise in potassium compared with ACE inhibitors in people with nephropathy (237,238). It is important to note that the benefits of both ACE inhibitors and ARBs in those with diabetic nephropathy are strongly associated with the reduction in albuminuria. Combinations of drugs that block the rennin-angiotensin-aldosterone system (e.g., an ACE inhibitor plus an ARB, a mineralocorticoid antagonist, or a direct renin inhibitor) have been shown to provide additional lowering of albuminuria (239\u2013242). However, the long-term effects of such combinations on renal or cardiovascular outcomes have not yet been evaluated in clinical trials.\n\nOther drugs, such as diuretics, calcium channel blockers, and \u03b2-blockers, should be used as additional therapy to further lower blood pressure in patients already treated with ACE inhibitors or ARBs (179) or as alternate therapy in the rare individual unable to tolerate ACE inhibitors or ARBs.\n\nStudies in patients with varying stages of nephropathy have shown that protein restriction helps slow the progression of albuminuria, GFR decline, and occurrence of ESRD (243\u2013246). Protein restriction should be considered particularly in patients whose nephropathy seems to be progressing despite optimal glucose and blood pressure control and use of ACE inhibitor and\/or ARBs (246).\n\n### Assessment of albuminuria status and renal function\n\nScreening for microalbuminuria can be performed by measurement of the albumin-to-creatinine ratio in a random spot collection (preferred method); 24-h or timed collections are more burdensome and add little to prediction or accuracy (247,248). Measurement of a spot urine for albumin only, whether by immunoassay or by using a dipstick test specific for microalbumin, without simultaneously measuring urine creatinine, is somewhat less expensive but susceptible to false-negative and -positive determinations as a result of variation in urine concentration due to hydration and other factors.\n\nAbnormalities of albumin excretion are defined in <a href=\"#t12\" data-ref-type=\"table\">Table 12<\/a>. Because of variability in urinary albumin excretion, two of three specimens collected within a 3- to 6-month period should be abnormal before considering a patient to have crossed one of these diagnostic thresholds. Exercise within 24 h, infection, fever, CHF, marked hyperglycemia, and marked hypertension may elevate urinary albumin excretion over baseline values.\n\nInformation on presence of abnormal urine albumin excretion in addition to level of GFR may be used to stage CKD. The National Kidney Foundation classification (<a href=\"#t13\" data-ref-type=\"table\">Table 13<\/a>) is primarily based on GFR levels and therefore differs from other systems, in which staging is based primarily on urinary albumin excretion (249). Studies have found decreased GFR in the absence of increased urine albumin excretion in a substantial percentage of adults with diabetes (250,251). Epidemiologic evidence suggests that a substantial fraction of those with CKD in the setting of diabetes have little or no detectable albuminuria (250). Serum creatinine should therefore be measured at least annually in all adults with diabetes, regardless of the degree of urine albumin excretion.\n\nSerum creatinine should be used to estimate GFR and to stage the level of CKD, if present. GFR can be estimated using formulae such as the Cockroft-Gault equation or a prediction formula using data from the Modification of Diet and Renal Disease study (252). GFR calculators are available at <http:\/\/www.nkdep.nih.gov>. Many clinical laboratories now report estimated GFR in addition to serum creatinine.\n\nThe role of continued annual quantitative assessment of albumin excretion after diagnosis of microalbuminuria and institution of ACE inhibitor or ARB therapy and blood pressure control is unclear. Continued surveillance can assess both response to therapy and progression of disease. Some suggest that reducing abnormal albuminuria (\\>30 mg\/g) to the normal or near-normal range may improve renal and cardiovascular prognosis, but this approach has not been formally evaluated in prospective trials.\n\nComplications of kidney disease correlate with level of kidney function. When the estimated GFR is \\<60 ml\/min per 1.73 m^2^, screening for anemia, malnutrition, and metabolic bone disease is indicated. Early vaccination against hepatitis B is indicated in patients likely to progress to end-stage kidney disease.\n\nConsider referral to a physician experienced in the care of kidney disease when there is uncertainty about the etiology of kidney disease (active urine sediment, absence of retinopathy, rapid decline in GFR), difficult management issues, or advanced kidney disease. The threshold for referral may vary depending on the frequency with which a provider encounters diabetic patients with significant kidney disease. Consultation with a nephrologist when stage 4 CKD develops has been found to reduce cost, improve quality of care, and keep people off dialysis longer (253,254). However, nonrenal specialists should not delay educating their patients about the progressive nature of diabetic kidney disease, the renal preservation benefits of aggressive treatment of blood pressure, blood glucose, and hyperlipidemia, and the potential need for renal replacement therapy.\n\n## C. Retinopathy screening and treatment\n\n### Recommendations\n\n#### General recommendations\n\n-   To reduce the risk or slow the progression of retinopathy, optimize glycemic control. (A)\n\n-   To reduce the risk or slow the progression of retinopathy, optimize blood pressure control. (A)\n\n#### Screening\n\n-   Adults and children aged 10 years or older with type 1 diabetes should have an initial dilated and comprehensive eye examination by an ophthalmologist or optometrist within 5 years after the onset of diabetes. (B)\n\n-   Patients with type 2 diabetes should have an initial dilated and comprehensive eye examination by an ophthalmologist or optometrist shortly after the diagnosis of diabetes. (B)\n\n-   Subsequent examinations for type 1 and type 2 diabetic patients should be repeated annually by an ophthalmologist or optometrist. Less frequent exams (every 2\u20133 years) may be considered following one or more normal eye exams. Examinations will be required more frequently if retinopathy is progressing. (B)\n\n-   Women with preexisting diabetes who are planning pregnancy or who have become pregnant should have a comprehensive eye examination and be counseled on the risk of development and\/or progression of diabetic retinopathy. Eye examination should occur in the first trimester with close follow-up throughout pregnancy and 1 year postpartum. (B)\n\n#### Treatment\n\n-   Promptly refer patients with any level of macular edema, severe NPDR, or any PDR to an ophthalmologist who is knowledgeable and experienced in the management and treatment of diabetic retinopathy. (A)\n\n-   Laser photocoagulation therapy is indicated to reduce the risk of vision loss in patients with high-risk PDR and clinically significant macular edema and in some cases of severe NPDR. (A)\n\n-   The presence of retinopathy is not a contraindication to aspirin therapy for cardioprotection, as this therapy does not increase the risk of retinal hemorrhage. (A)\n\nDiabetic retinopathy is a highly specific vascular complication of both type 1 and type 2 diabetes, with prevalence strongly related to the duration of diabetes. Diabetic retinopathy is the most frequent cause of new cases of blindness among adults aged 20\u201374 years. Glaucoma, cataracts, and other disorders of the eye occur earlier and more frequently in people with diabetes.\n\nIn addition to duration of diabetes, other factors that increase the risk of, or are associated with, retinopathy include chronic hyperglycemia (255), the presence of nephropathy (256), and hypertension (257). Intensive diabetes management with the goal of achieving near normoglycemia has been shown in large prospective randomized studies to prevent and\/or delay the onset and progression of diabetic retinopathy (45,49,50). Lowering blood pressure has been shown to decrease the progression of retinopathy (163). Several case series and a controlled prospective study suggest that pregnancy in type 1 diabetic patients may aggravate retinopathy (258,259); laser photocoagulation surgery can minimize this risk (259).\n\nOne of the main motivations for screening for diabetic retinopathy is the established efficacy of laser photocoagulation surgery in preventing vision loss. Two large trials, the Diabetic Retinopathy Study (DRS) and the Early Treatment Diabetic Retinopathy Study (ETDRS), provide the strongest support for the therapeutic benefits of photocoagulation surgery.\n\nThe DRS (260) showed that panretinal photocoagulation surgery reduced the risk of severe vision loss from PDR from 15.9% in untreated eyes to 6.4% in treated eyes. The benefit was greatest among patients whose baseline evaluation revealed high-risk characteristics (chiefly disc neovascularization or vitreous hemorrhage). Given the risks of modest loss of visual acuity and contraction of the visual field from panretinal laser surgery, such therapy is primarily recommended for eyes with PDR approaching or having high-risk characteristics.\n\nThe ETDRS (261) established the benefit of focal laser photocoagulation surgery in eyes with macular edema, particularly those with clinically significant macular edema, with reduction of doubling of the visual angle (e.g., 20\/50 to 20\/100) from 20% in untreated eyes to 8% in treated eyes. The ETDRS also verified the benefits of panretinal photocoagulation for high-risk PDR, but not for mild or moderate NPDR. In older-onset patients with severe NPDR or less-than-high-risk PDR, the risk of severe vision loss or vitrectomy was reduced \u223c50% by early laser photocoagulation surgery at these stages.\n\nLaser photocoagulation surgery in both trials was beneficial in reducing the risk of further vision loss, but generally not beneficial in reversing already diminished acuity. This preventive effect and the fact that patients with PDR or macular edema may be asymptomatic provide strong support for a screening program to detect diabetic retinopathy.\n\nAs retinopathy is estimated to take at least 5 years to develop after the onset of hyperglycemia (262), patients with type 1 diabetes should have an initial dilated and comprehensive eye examination within 5 years after the onset of diabetes. Patients with type 2 diabetes, who generally have had years of undiagnosed diabetes (263) and who have a significant risk of prevalent diabetic retinopathy at time of diabetes diagnosis, should have an initial dilated and comprehensive eye examination soon after diagnosis. Examinations should be performed by an ophthalmologist or optometrist who is knowledgeable and experienced in diagnosing the presence of diabetic retinopathy and is aware of its management. Subsequent examinations for type 1 and type 2 diabetic patients are generally repeated annually. Less frequent exams (every 2\u20133 years) may be cost effective after one or more normal eye exams (264\u2013266), while examinations will be required more frequently if retinopathy is progressing.\n\nExaminations can also be done with retinal photographs (with or without dilation of the pupil) read by experienced experts. In-person exams are still necessary when the photos are unacceptable and for follow-up of abnormalities detected. This technology has great potential in areas where qualified eye care professionals are not available and may enhance efficiency and reduce costs when the expertise of ophthalmologists can be utilized for more complex examinations and for therapy (267).\n\nResults of eye examinations should be documented and transmitted to the referring health care professional. For a detailed review of the evidence and further discussion of diabetic retinopathy, see the ADA's technical review and position statement on this subject (268,269).\n\n## D. Neuropathy screening and treatment (270)\n\n### Recommendations\n\n-   All patients should be screened for distal symmetric polyneuropathy (DPN) at diagnosis and at least annually thereafter using simple clinical tests. (B)\n\n-   Electrophysiological testing is rarely needed, except in situations where the clinical features are atypical. (E)\n\n-   Screening for signs and symptoms of cardiovascular autonomic neuropathy should be instituted at diagnosis of type 2 diabetes and 5 years after the diagnosis of type 1 diabetes. Special testing is rarely needed and may not affect management or outcomes. (E)\n\n-   Medications for the relief of specific symptoms related to DPN and autonomic neuropathy are recommended, as they improve the quality of life of the patient. (E)\n\nThe diabetic neuropathies are heterogeneous with diverse clinical manifestations. They may be focal or diffuse. Most common among the neuropathies are chronic sensorimotor DPN and autonomic neuropathy. Although DPN is a diagnosis of exclusion, complex investigations to exclude other conditions are rarely needed.\n\nThe early recognition and appropriate management of neuropathy in the patient with diabetes is important for a number of reasons: *1*) nondiabetic neuropathies may be present in patients with diabetes and may be treatable; *2*) a number of treatment options exist for symptomatic diabetic neuropathy; *3*) up to 50% of DPN may be asymptomatic, and patients are at risk of insensate injury to their feet; *4*) autonomic neuropathy may involve every system in the body; and *5*) cardiovascular autonomic neuropathy causes substantial morbidity and mortality. Specific treatment for the underlying nerve damage is currently not available, other than improved glycemic control, which may slow progression but not reverse neuronal loss. Effective symptomatic treatments are available for some manifestations of DPN and autonomic neuropathy.\n\n### Diagnosis of neuropathy\n\n#### Distal symmetric polyneuropathy\n\nPatients with diabetes should be screened annually for DPN using tests such as pinprick sensation, vibration perception (using a 128-Hz tuning fork), 10-g monofilament pressure sensation at the distal plantar aspect of both great toes and metatarsal joints, and assessment of ankle reflexes. Combinations of more than one test have \\>87% sensitivity in detecting DPN. Loss of 10-g monofilament perception and reduced vibration perception predict foot ulcers (270).\n\n#### Diabetic autonomic neuropathy (271)\n\nThe symptoms and signs of autonomic dysfunction should be elicited carefully during the history and physical examination. Major clinical manifestations of diabetic autonomic neuropathy include resting tachycardia, exercise intolerance, orthostatic hypotension, constipation, gastroparesis, erectile dysfunction, sudomotor dysfunction, impaired neurovascular function, \"brittle diabetes,\" and hypoglycemic autonomic failure.\n\nCardiovascular autonomic neuropathy, a CVD risk factor (93), is the most studied and clinically important form of diabetic autonomic neuropathy. Cardiovascular autonomic neuropathy may be indicated by resting tachycardia (\\>100 bpm), orthostasis (a fall in systolic blood pressure \\>20 mmHg upon standing without an appropriate heart rate response), or other disturbances in autonomic nervous system function involving the skin, pupils, or gastrointestinal and genitourinary systems.\n\nGastrointestinal neuropathies (e.g., esophageal enteropathy, gastroparesis, constipation, diarrhea, fecal incontinence) are common, and any section of the gastrointestinal tract may be affected. Gastroparesis should be suspected in individuals with erratic glucose control or with upper gastrointestinal symptoms without other identified cause. Evaluation of solid-phase gastric emptying using double-isotope scintigraphy may be done if symptoms are suggestive, but test results often correlate poorly with symptoms. Constipation is the most common lower-gastrointestinal symptom but can alternate with episodes of diarrhea.\n\nDiabetic autonomic neuropathy is also associated with genitourinary tract disturbances. In men, diabetic autonomic neuropathy may cause erectile dysfunction and\/or retrograde ejaculation. Evaluation of bladder dysfunction should be performed for individuals with diabetes who have recurrent urinary tract infections, pyelonephritis, incontinence, or a palpable bladder.\n\n### Symptomatic treatments\n\n#### DPN\n\nThe first step in management of patients with DPN should be to aim for stable and optimal glycemic control. Although controlled trial evidence is lacking, several observational studies suggest that neuropathic symptoms improve not only with optimization of control, but also with the avoidance of extreme blood glucose fluctuations. Patients with painful DPN may benefit from pharmacological treatment of their symptoms: many agents have efficacy confirmed in published randomized controlled trials, with several FDA-approved for the management of painful DPN. See <a href=\"#t14\" data-ref-type=\"table\">Table 14<\/a> for examples of agents to treat DPN pain.\n\n### Treatment of autonomic neuropathy\n\nGastroparesis symptoms may improve with dietary changes and prokinetic agents such as metoclopramide or erythromycin. Treatments for erectile dysfunction may include phosphodiesterase type 5 inhibitors, intracorporeal or intraurethral prostaglandins, vacuum devices, or penile prostheses. Interventions for other manifestations of autonomic neuropathy are described in the ADA statement on neuropathy (270). As with DPN treatments, these interventions do not change the underlying pathology and natural history of the disease process but may have a positive impact on the quality of life of the patient.\n\n## E. Foot care\n\n### Recommendations\n\n-   For all patients with diabetes, perform an annual comprehensive foot examination to identify risk factors predictive of ulcers and amputations. The foot examination should include inspection, assessment of foot pulses, and testing for loss of protective sensation (10-g monofilament plus testing any one of the following: vibration using 128-Hz tuning fork, pinprick sensation, ankle reflexes, or vibration perception threshold). (B)\n\n-   Provide general foot self-care education to all patients with diabetes. (B)\n\n-   A multidisciplinary approach is recommended for individuals with foot ulcers and high-risk feet, especially those with a history of prior ulcer or amputation. (B)\n\n-   Refer patients who smoke, have loss of protective sensation and structural abnormalities, or have history of prior lower-extremity complications to foot care specialists for ongoing preventive care and life-long surveillance. (C)\n\n-   Initial screening for peripheral arterial disease (PAD) should include a history for claudication and an assessment of the pedal pulses. Consider obtaining an ankle-brachial index (ABI), as many patients with PAD are asymptomatic. (C)\n\n-   Refer patients with significant claudication or a positive ABI for further vascular assessment and consider exercise, medications, and surgical options. (C)\n\nAmputation and foot ulceration, consequences of diabetic neuropathy and\/or PAD, are common and major causes of morbidity and disability in people with diabetes. Early recognition and management of risk factors can prevent or delay adverse outcomes.\n\nThe risk of ulcers or amputations is increased in people who have the following risk factors:\n\n-   previous amputation\n\n-   past foot ulcer history\n\n-   peripheral neuropathy\n\n-   foot deformity\n\n-   peripheral vascular disease\n\n-   vision impairment\n\n-   diabetic nephropathy (especially patients on dialysis)\n\n-   poor glycemic control\n\n-   cigarette smoking\n\nMany studies have been published proposing a range of tests that might usefully identify patients at risk of foot ulceration, creating confusion among practitioners as to which screening tests should be adopted in clinical practice. An ADA task force was therefore assembled in 2008 to concisely summarize recent literature in this area and then recommend what should be included in the comprehensive foot exam for adult patients with diabetes. Their recommendations are summarized below, but clinicians should refer to the task force report (272) for further details and practical descriptions of how to perform components of the comprehensive foot examination.\n\nAt least annually, all adults with diabetes should undergo a comprehensive foot examination to identify high-risk conditions. Clinicians should ask about history of previous foot ulceration or amputation, neuropathic or peripheral vascular symptoms, impaired vision, tobacco use, and foot care practices. A general inspection of skin integrity and musculoskeletal deformities should be done in a well-lit room. Vascular assessment should include inspection and assessment of pedal pulses.\n\nThe neurologic exam recommended is designed to identify loss of protective sensation (LOPS) rather than early neuropathy. The clinical examination to identify LOPS is simple and requires no expensive equipment. Five simple clinical tests (use of a 10-g monofilament, vibration testing using a 128-Hz tuning fork, tests of pinprick sensation, ankle reflex assessment, and testing vibration perception threshold with a biothesiometer), each with evidence from well-conducted prospective clinical cohort studies, are considered useful in the diagnosis of LOPS in the diabetic foot. The task force agrees that any of the five tests listed could be used by clinicians to identify LOPS, although ideally two of these should be regularly performed during the screening exam\u2014normally the 10-g monofilament and one other test. One or more abnormal tests would suggest LOPS, while at least two normal tests (and no abnormal test) would rule out LOPS. The last test listed, vibration assessment using a biothesiometer or similar instrument, is widely used in the U.S.; however, identification of the patient with LOPS can easily be carried out without this or other expensive equipment.\n\nInitial screening for PAD should include a history for claudication and an assessment of the pedal pulses. A diagnostic ABI should be performed in any patient with symptoms of PAD. Due to the high estimated prevalence of PAD in patients with diabetes and the fact that many patients with PAD are asymptomatic, an ADA consensus statement on PAD (273) suggested that a screening ABI be performed in patients over 50 years of age and be considered in patients under 50 years of age who have other PAD risk factors (e.g., smoking, hypertension, hyperlipidemia, or duration of diabetes \\>10 years). Refer patients with significant symptoms or a positive ABI for further vascular assessment and consider exercise, medications, and surgical options (273).\n\nPatients with diabetes and high-risk foot conditions should be educated regarding their risk factors and appropriate management. Patients at risk should understand the implications of the loss of protective sensation, the importance of foot monitoring on a daily basis, the proper care of the foot, including nail and skin care, and the selection of appropriate footwear. Patients with loss of protective sensation should be educated on ways to substitute other sensory modalities (hand palpation, visual inspection) for surveillance of early foot problems. The patient's understanding of these issues and their physical ability to conduct proper foot surveillance and care should be assessed. Patients with visual difficulties, physical constraints preventing movement, or cognitive problems that impair their ability to assess the condition of the foot and to institute appropriate responses will need other people, such as family members, to assist in their care.\n\nPeople with neuropathy or evidence of increased plantar pressure (e.g., erythema, warmth, callus, or measured pressure) may be adequately managed with well-fitted walking shoes or athletic shoes that cushion the feet and redistribute pressure. Callus can be debrided with a scalpel by a foot care specialist or other health professional with experience and training in foot care. People with bony deformities (e.g., hammertoes, prominent metatarsal heads, bunions) may need extra-wide or -depth shoes. People with extreme bony deformities (e.g., Charcot foot) who cannot be accommodated with commercial therapeutic footwear may need custom-molded shoes.\n\nFoot ulcers and wound care may require care by a podiatrist, orthopedic or vascular surgeon, or rehabilitation specialist experienced in the management of individuals with diabetes. For a complete discussion, see the ADA's consensus statement on diabetic foot wound care (274).\n\n# VII. DIABETES CARE IN SPECIFIC POPULATIONS\n\n## A. Children and adolescents\n\n### 1. Type 1 diabetes\n\nThree-quarters of all cases of type 1 diabetes are diagnosed in individuals \\<18 years of age. Because children are not simply \"small adults,\" it is appropriate to consider the unique aspects of care and management of children and adolescents with type 1 diabetes. Children with diabetes differ from adults in many respects, including changes in insulin sensitivity related to sexual maturity and physical growth, ability to provide self-care, supervision in child care and school, and unique neurologic vulnerability to hypoglycemia and DKA. Attention to such issues as family dynamics, developmental stages, and physiologic differences related to sexual maturity are all essential in developing and implementing an optimal diabetes regimen. Although recommendations for children and adolescents are less likely to be based on clinical trial evidence, because of current and historical restraints placed on conducting research in children, expert opinion and a review of available and relevant experimental data are summarized in the ADA statement on care of children and adolescents with type 1 diabetes (275).\n\nIdeally, the care of a child or adolescent with type 1 diabetes should be provided by a multidisciplinary team of specialists trained in the care of children with pediatric diabetes. At the very least, education of the child and family should be provided by health care providers trained and experienced in childhood diabetes and sensitive to the challenges posed by diabetes in this age-group. At the time of initial diagnosis, it is essential that diabetes education be provided in a timely fashion, with the expectation that the balance between adult supervision and self-care should be defined by, and will evolve according to, physical, psychological, and emotional maturity. MNT should be provided at diagnosis, and at least annually thereafter, by an individual experienced with the nutritional needs of the growing child and the behavioral issues that have an impact on adolescent diets, including risk for disordered eating.\n\n#### a. Glycemic control\n\n##### Recommendations\n\n-   Consider age when setting glycemic goals in children and adolescents with type 1 diabetes, with less stringent goals for younger children. (E)\n\nWhile current standards for diabetes management reflect the need to maintain glucose control as near to normal as safely possible, special consideration must be given to the unique risks of hypoglycemia in young children. Glycemic goals need to be modified to take into account the fact that most children \\<6 or 7 years of age have a form of hypoglycemic unawareness. Their counterregulatory mechanisms are immature and they may lack the cognitive capacity to recognize and respond to hypoglycemic symptoms, placing them at greater risk for severe hypoglycemia and its sequelae. In addition, and unlike the case in adults, young children below the age of 5 years are at risk for permanent cognitive impairment after episodes of severe hypoglycemia (276\u2013278). Extensive evidence indicates that near normalization of blood glucose levels is seldom attainable in children and adolescents after the honeymoon (remission) period. The A1C level achieved in the \"intensive\" adolescent cohort of the DCCT group was \\>1% higher than that achieved by adult DCCT subjects and above current ADA recommendations for patients in general. However, the increased frequency of use of basal bolus regimens (including insulin pumps) in youth from infancy through adolescence has been associated with more children reaching ADA blood glucose targets (279,280) in those families in which both parents and the child with diabetes are motivated to perform the required diabetes-related tasks.\n\nIn selecting glycemic goals, the benefits on long-term health outcomes of achieving a lower A1C must be weighed against the unique risks of hypoglycemia and the difficulties achieving near normoglycemia in children and youth. Age-specific glycemic and A1C goals are presented in <a href=\"#t15\" data-ref-type=\"table\">Table 15<\/a>.\n\n#### b. Screening and management of chronic complications in children and adolescents with type 1 diabetes\n\n##### i. Nephropathy\n\n###### Recommendations\n\n-   Annual screening for microalbuminuria, with a random spot urine sample for microalbumin-to-creatinine ratio, should be initiated once the child is 10 years of age and has had diabetes for 5 years. (E)\n\n-   Confirmed, persistently elevated microalbumin levels on two additional urine specimens should be treated with an ACE inhibitor, titrated to normalization of microalbumin excretion if possible. (E)\n\n##### ii. Hypertension\n\n###### Recommendations\n\n-   Treatment of high-normal blood pressure (systolic or diastolic blood pressure consistently between the 90\u201395th percentile for age, sex, and height) should include dietary intervention and exercise aimed at weight control and increased physical activity, if appropriate. If target blood pressure is not reached with 6\u201312 months of lifestyle intervention, pharmacologic treatment should be initiated. (E)\n\n-   Pharmacologic treatment of high blood pressure (systolic or diastolic blood pressure consistently above the 95th percentile for age, sex, and height or consistently \\>130\/80 mmHg for adolescents) should be initiated along with lifestyle intervention as soon as the diagnosis is confirmed. (E)\n\n-   ACE inhibitors should be considered for the initial treatment of hypertension. (E)\n\n-   The goal of treatment is a blood pressure consistently \\<130\/80 or below the 90th percentile for age, sex, and height, whichever is lower. (E)\n\nHypertension in childhood is defined as an average systolic or diastolic blood pressure 95th percentile for age, sex, and height percentile measured on at least 3 separate days. \"High-normal\" blood pressure is defined as an average systolic or diastolic blood pressure 90th but \\<95th percentile for age, sex, and height percentile measured on at least 3 separate days. Normal blood pressure levels for age, sex, and height and appropriate methods for determinations are available online at [www.nhlbi.nih.gov\/health\/prof\/heart\/hbp\/hbp_ped.pdf](www.nhlbi.nih.gov\/health\/prof\/heart\/hbp\/hbp_ped.pdf).\n\n##### iii. Dyslipidemia\n\n###### Recommendations\n\n####### Screening\n\n-   If there is a family history of hypercholesterolemia (total cholesterol \\>240 mg\/dl) or a cardiovascular event before age 55 years, or if family history is unknown, then a fasting lipid profile should be performed on children \\>2 years of age soon after diagnosis (after glucose control has been established). If family history is not of concern, then the first lipid screening should be performed at puberty (\u226510 years). All children diagnosed with diabetes at or after puberty should have a fasting lipid profile performed soon after diagnosis (after glucose control has been established). (E)\n\n-   For both age-groups, if lipids are abnormal, annual monitoring is recommended. If LDL cholesterol values are within the accepted risk levels (\\<100 mg\/dl \\[2.6 mmol\/l\\]), a lipid profile should be repeated every 5 years. (E)\n\n###### Treatment\n\n-   Initial therapy should consist of optimization of glucose control and MNT using a Step 2 AHA diet aimed at a decrease in the amount of saturated fat in the diet. (E)\n\n-   After the age of 10, the addition of a statin is recommended in patients who, after MNT and lifestyle changes, have LDL \\>160 mg\/dl (4.1 mmol\/l) or LDL cholesterol \\>130 mg\/dl (3.4 mmol\/l) and one or more CVD risk factors. (E)\n\n-   The goal of therapy is an LDL cholesterol value \\<100 mg\/dl (2.6 mmol\/l). (E)\n\nPeople diagnosed with type 1 diabetes in childhood have a high risk of early subclinical (281\u2013283) and clinical (284) CVD. Although intervention data are lacking, the AHA categorizes type 1 children in the highest tier for cardiovascular risk and recommends both lifestyle and pharmacologic treatment for those with elevated LDL cholesterol levels (285,286). Initial therapy should be with a Step 2 AHA diet, which restricts saturated fat to 7% of total calories and restricts dietary cholesterol to 200 mg per day. Data from randomized clinical trials in children as young as 7 months of age indicate that this diet is safe and does not interfere with normal growth and development (287,288).\n\nFor children over the age of 10 with persistent elevation of LDL cholesterol despite lifestyle therapy, statins should be considered. Neither long-term safety nor cardiovascular outcome efficacy has been established for children. However, recent studies have shown short-term safety equivalent to that seen in adults and efficacy in lowering LDL cholesterol levels, improving endothelial function, and causing regression of carotid intimal thickening (289\u2013291). No statin is approved for use under the age of 10, and statin treatment should generally not be used in type 1 children before this age.\n\n##### iv. Retinopathy\n\n###### Recommendations\n\n-   The first ophthalmologic examination should be obtained once the child is \u226510 years of age and has had diabetes for 3\u20135 years. (E)\n\n-   After the initial examination, annual routine follow-up is generally recommended. Less frequent examinations may be acceptable on the advice of an eye care professional. (E)\n\nAlthough retinopathy most commonly occurs after the onset of puberty and after 5\u201310 years of diabetes duration, it has been reported in prepubertal children and with diabetes duration of only 1\u20132 years. Referrals should be made to eye care professionals with expertise in diabetic retinopathy, an understanding of the risk for retinopathy in the pediatric population, and experience in counseling the pediatric patient and family on the importance of early prevention\/intervention.\n\n##### v. Celiac disease\n\n###### Recommendations\n\n-   Patients with type 1 diabetes should be screened for celiac disease by measuring tissue transglutaminase or anti-endomysial antibodies, with documentation of normal serum IgA levels, soon after the diagnosis of diabetes. (E)\n\n-   Testing should be repeated if growth failure, failure to gain weight, weight loss, or gastroenterologic symptoms occur. (E)\n\n-   Consideration should be given to periodic re-screening of asymptomatic individuals. (E)\n\n-   Children with positive antibodies should be referred to a gastroenterologist for evaluation. (E)\n\n-   Children with confirmed celiac disease should have consultation with a dietitian and placed on a gluten-free diet. (E)\n\nCeliac disease is an immune-mediated disorder that occurs with increased frequency in patients with type 1 diabetes (1\u201316% of individuals compared with 0.3\u20131% in the general population) (292,293). Symptoms of celiac disease include diarrhea, weight loss or poor weight gain, growth failure, abdominal pain, chronic fatigue, malnutrition due to malabsorption, and other gastrointestinal problems and unexplained hypoglycemia or erratic blood glucose concentrations.\n\n##### vi. Hypothyroidism\n\n###### Recommendations\n\n-   Patients with type 1 diabetes should be screened for thyroid peroxidase and thyroglobulin antibodies at diagnosis. (E)\n\n-   TSH concentrations should be measured after metabolic control has been established. If normal, they should be rechecked every 1\u20132 years, or if the patient develops symptoms of thyroid dysfunction, thyromegaly, or an abnormal growth rate. Free T4 should be measured if TSH is abnormal. (E)\n\nAutoimmune thyroid disease is the most common autoimmune disorder associated with diabetes, occurring in 17\u201330% of patients with type 1 diabetes (294). The presence of thyroid autoantibodies is predictive of thyroid dysfunction, generally hypothyroidism but less commonly hyperthyroidism (295). Subclinical hypothyroidism may be associated with increased risk of symptomatic hypoglycemia (296) and with reduced linear growth (297). Hyperthyroidism alters glucose metabolism, potentially resulting in deterioration of metabolic control.\n\n#### c. \"Adherence.\"\n\nNo matter how sound the medical regimen, it can only be as good as the ability of the family and\/or individual to implement it. Family involvement in diabetes remains an important component of optimal diabetes management throughout childhood and into adolescence. Health care providers who care for children and adolescents, therefore, must be capable of evaluating the behavioral, emotional, and psychosocial factors that interfere with implementation and then must work with the individual and family to resolve problems that occur and\/or to modify goals as appropriate.\n\n#### d. School and day care.\n\nSince a sizable portion of a child's day is spent in school, close communication with school or day care personnel is essential for optimal diabetes management, safety, and maximal academic opportunities. See Section V.III.B, Diabetes Care in the School and Day Care Setting, for further discussion.\n\n### 2. Type 2 diabetes\n\nThe incidence of type 2 diabetes in adolescents is increasing, especially in ethnic minority populations (20). Distinction between type 1 and type 2 diabetes in children can be difficult, since the prevalence of overweight in children continues to rise and since autoantigens and ketosis may be present in a substantial number of patients with features of type 2 diabetes (including obesity and acanthosis nigricans). Such a distinction at the time of diagnosis is critical because treatment regimens, educational approaches, and dietary counsel will differ markedly between the two diagnoses. Because type 2 diabetes has a significant incidence of hypertension, dyslipidemia, and microalbuminuria at diagnosis (298), it is recommended that screening for the comorbidities and complications of diabetes, including fasting lipid profile, microalbuminuria assessment, and dilated eye examinations, begin at the time of diagnosis. The ADA consensus statement on this subject (22) provides guidance on the prevention, screening, and treatment of type 2 diabetes and its comorbidities in young people.\n\n## B. Preconception care\n\n### Recommendations\n\n-   A1C levels should be as close to normal as possible (\\<7%) in an individual patient before conception is attempted. (B)\n\n-   Starting at puberty, preconception counseling should be incorporated in the routine diabetes clinic visit for all women of child-bearing potential. (C)\n\n-   Women with diabetes who are contemplating pregnancy should be evaluated and, if indicated, treated for diabetic retinopathy, nephropathy, neuropathy, and CVD. (E)\n\n-   Medications used by such women should be evaluated before conception, since drugs commonly used to treat diabetes and its complications may be contraindicated or not recommended in pregnancy, including statins, ACE inhibitors, ARBs, and most noninsulin therapies. (E)\n\nMajor congenital malformations remain the leading cause of mortality and serious morbidity in infants of mothers with type 1 and type 2 diabetes. Observational studies indicate that the risk of malformations increases continuously with increasing maternal glycemia during the first 6\u20138 weeks of gestation, as defined by first-trimester A1C concentrations. There is no threshold for A1C values below which risk disappears entirely. However, malformation rates above the 1\u20132% background rate of nondiabetic pregnancies appear to be limited to pregnancies in which first-trimester A1C concentrations are \\>1% above the normal range for a nondiabetic pregnant woman.\n\nPreconception care of diabetes appears to reduce the risk of congenital malformations. Five nonrandomized studies compared rates of major malformations in infants between women who participated in preconception diabetes care programs and women who initiated intensive diabetes management after they were already pregnant. The preconception care programs were multidisciplinary and designed to train patients in diabetes self-management with diet, intensified insulin therapy, and SMBG. Goals were set to achieve normal blood glucose concentrations, and \\>80% of subjects achieved normal A1C concentrations before they became pregnant (299\u2013303). In all five studies, the incidence of major congenital malformations in women who participated in preconception care (range 1.0\u20131.7% of infants) was much lower than the incidence in women who did not participate (range 1.4\u201310.9% of infants). One limitation of these studies is that participation in preconception care was self-selected rather than randomized. Thus, it is impossible to be certain that the lower malformation rates resulted fully from improved diabetes care. Nonetheless, the evidence supports the concept that malformations can be reduced or prevented by careful management of diabetes before pregnancy.\n\nPlanned pregnancies greatly facilitate preconception diabetes care. Unfortunately, nearly two-thirds of pregnancies in women with diabetes are unplanned, leading to a persistent excess of malformations in infants of diabetic mothers. To minimize the occurrence of these devastating malformations, standard care for all women with diabetes who have child-bearing potential, beginning at the onset of puberty or at diagnosis, should include *1*) education about the risk of malformations associated with unplanned pregnancies and poor metabolic control and *2*) use of effective contraception at all times, unless the patient has good metabolic control and is actively trying to conceive.\n\nWomen contemplating pregnancy need to be seen frequently by a multidisciplinary team experienced in the management of diabetes before and during pregnancy. The goals of preconception care are to *1*) involve and empower the patient in the management of her diabetes, *2*) achieve the lowest A1C test results possible without excessive hypoglycemia, *3*) assure effective contraception until stable and acceptable glycemia is achieved, and *4*) identify, evaluate, and treat long-term diabetic complications such as retinopathy, nephropathy, neuropathy, hypertension, and CHD.\n\nAmong the drugs commonly used in the treatment of patients with diabetes, a number may be relatively or absolutely contraindicated during pregnancy. Statins are category X (contraindicated for use in pregnancy) and should be discontinued before conception, as should ACE inhibitors (304). ARBs are category C (risk cannot be ruled out) in the first trimester but category D (positive evidence of risk) in later pregnancy and should generally be discontinued before pregnancy. Among the oral antidiabetic agents, metformin and acarbose are classified as category B (no evidence of risk in humans) and all others as category C. Potential risks and benefits of oral antidiabetic agents in the preconception period must be carefully weighed, recognizing that data are insufficient to establish the safety of these agents in pregnancy.\n\nFor further discussion of preconception care, see the ADA's technical review (305) and position statement (306) on this subject.\n\n## C. Older adults\n\n### Recommendations\n\n-   Older adults who are functional, cognitively intact, and have significant life expectancy should receive diabetes treatment using goals developed for younger adults. (E)\n\n-   Glycemic goals for older adults not meeting the above criteria may be relaxed using individual criteria, but hyperglycemia leading to symptoms or risk of acute hyperglycemic complications should be avoided in all patients. (E)\n\n-   Other cardiovascular risk factors should be treated in older adults with consideration of the time frame of benefit and the individual patient. Treatment of hypertension is indicated in virtually all older adults, and lipid and aspirin therapy may benefit those with life expectancy at least equal to the time frame of primary or secondary prevention trials. (E)\n\n-   Screening for diabetes complications should be individualized in older adults, but particular attention should be paid to complications that would lead to functional impairment. (E)\n\nDiabetes is an important health condition for the aging population; at least 20% of patients over the age of 65 years have diabetes, and this number can be expected to grow rapidly in the coming decades. Older individuals with diabetes have higher rates of premature death, functional disability, and coexisting illnesses such as hypertension, CHD, and stroke than those without diabetes. Older adults with diabetes are also at greater risk than other older adults for several common geriatric syndromes, such as polypharmacy, depression, cognitive impairment, urinary incontinence, injurious falls, and persistent pain.\n\nThe American Geriatric Society's guidelines for improving the care of the older person with diabetes (307) have influenced the following discussion and recommendations. The care of older adults with diabetes is complicated by their clinical and functional heterogeneity. Some older individuals developed diabetes years earlier and may have significant complications; others who are newly diagnosed may have had years of undiagnosed diabetes with resultant complications or may have few complications from the disease. Some older adults with diabetes are frail and have other underlying chronic conditions, substantial diabetes-related comorbidity, or limited physical or cognitive functioning. Other older individuals with diabetes have little comorbidity and are active. Life expectancies are highly variable for this population, but often longer than clinicians realize. Providers caring for older adults with diabetes must take this heterogeneity into consideration when setting and prioritizing treatment goals.\n\nThere are few long-term studies in older adults demonstrating the benefits of intensive glycemic, blood pressure, and lipid control. Patients who can be expected to live long enough to reap the benefits of long-term intensive diabetes management and who are active, have good cognitive function, and are willing to undertake the responsibility of self-management should be encouraged to do so and be treated using the goals for younger adults with diabetes.\n\nFor patients with advanced diabetes complications, life-limiting comorbid illness, or substantial cognitive or functional impairment, it is reasonable to set less intensive glycemic target goals. These patients are less likely to benefit from reducing the risk of microvascular complications and more likely to suffer serious adverse effects from hypoglycemia. However, patients with poorly controlled diabetes may be subject to acute complications of diabetes, including dehydration, poor wound healing, and hyperglycemic hyperosmolar coma. Glycemic goals at a minimum should avoid these consequences.\n\nAlthough control of hyperglycemia may be important in older individuals with diabetes, greater reductions in morbidity and mortality may result from control of other cardiovascular risk factors rather than from tight glycemic control alone. There is strong evidence from clinical trials of the value of treating hypertension in the elderly (308,309). There is less evidence for lipid-lowering and aspirin therapy, although the benefits of these interventions for primary and secondary prevention are likely to apply to older adults whose life expectancies equal or exceed the time frames seen in clinical trials.\n\nSpecial care is required in prescribing and monitoring pharmacologic therapy in older adults. Metformin is often contraindicated because of renal insufficiency or significant heart failure. TZDs can cause fluid retention, which may exacerbate or lead to heart failure. They are contraindicated in patients with CHF (New York Heart Association class III and IV) and if used at all should be used very cautiously in those with, or at risk for, milder degrees of CHF. Sulfonylureas, other insulin secretagogues, and insulin can cause hypoglycemia. Insulin use requires that patients or caregivers have good visual and motor skills and cognitive ability. Drugs should be started at the lowest dose and titrated up gradually until targets are reached or side effects develop.\n\nScreening for diabetes complications in older adults also should be individualized. Particular attention should be paid to complications that can develop over short periods of time and\/or that would significantly impair functional status, such as visual and lower-extremity complications.\n\n# VIII. DIABETES CARE IN SPECIFIC SETTINGS\n\n## A. Diabetes care in the hospital\n\n### Recommendations\n\n-   All patients with diabetes admitted to the hospital should have their diabetes clearly identified in the medical record. (E)\n\n-   All patients with diabetes should have an order for blood glucose monitoring, with results available to all members of the health care team. (E)\n\n-   Goals for blood glucose levels:\n\n    -   Critically ill surgical patients' blood glucose levels should be kept as close to 110 mg\/dl (6.1 mmol\/l) as possible and generally \\<140 mg\/dl (7.8 mmol\/l). (A) These patients require an intravenous insulin protocol that has demonstrated efficacy and safety in achieving the desired glucose range without increasing risk for severe hypoglycemia. (E)\n\n    -   Critically ill nonsurgical patients' glycemic targets are less well defined. Intravenous insulin infusion protocols targeting blood glucose levels \\<110\u2013140 mg\/dl have been shown to reduced morbidity and mortality in some, but not all studies. Intravenous insulin infusion protocols that effectively and safely keep blood glucose \\<140 mg\/dl are recommended. (C)\n\n    -   For non\u2013critically ill patients, there is no clear evidence for specific blood glucose goals. Since cohort data suggest that outcomes are better in hospitalized patients with fasting glucose \\<126 mg\/dl and all random glucoses \\<180\u2013200, these goals are reasonable if they can be safely achieved. Insulin is the preferred drug to treat hyperglycemia in most cases. (E)\n\n    -   Due to concerns regarding the risk of hypoglycemia, some institutions may consider these blood glucose levels to be overly aggressive for initial targets. Through quality improvement, glycemic goals should systematically be reduced to the recommended levels. (E)\n\n-   Scheduled prandial insulin doses should be appropriately timed in relation to meals and should be adjusted according to point-of-care glucose levels. The traditional sliding-scale insulin regimens are ineffective as monotherapy and are generally not recommended. (C)\n\n-   Using correction dose or \"supplemental\" insulin to correct premeal hyperglycemia in addition to scheduled prandial and basal insulin is recommended. (E)\n\n-   Glucose monitoring with orders for correction insulin should be initiated in any patient not known to be diabetic who receives therapy associated with high risk for hyperglycemia, including high-dose glucocorticoids therapy, initiation of enteral or parenteral nutrition, or other medications such as octreotide or immunosuppressive medications. (B) If hyperglycemia is documented and persistent, initiation of basal\/bolus insulin therapy may be necessary. Such patients should be treated to the same glycemic goals as patients with known diabetes. (E)\n\n-   A plan for treating hypoglycemia should be established for each patient. Episodes of hypoglycemia in the hospital should be tracked. (E)\n\n-   All patients with diabetes admitted to the hospital should have an A1C obtained if the result of testing in the previous 2\u20133 months is not available. (E)\n\n-   A diabetes education plan including \"survival skills education\" and follow-up should be developed for each patient. (E)\n\n-   Patients with hyperglycemia in the hospital who do not have a diagnosis of diabetes should have appropriate plans for follow-up testing and care documented at discharge. (E)\n\nThe management of diabetes in the hospital is extensively reviewed in an ADA technical review (310). This review, as well as a consensus statement by the American Association of Clinical Endocrinologists (AACE) with co-sponsorship by ADA (311,312) and a report of a joint ADA-AACE task force on the topic (313), form the basis for the discussion and guidelines in this section.\n\nThe literature on hospitalized patients with hyperglycemia typically describes three categories:\n\n-   Medical history of diabetes: diabetes previously diagnosed and acknowledged by the patient's treating physician.\n\n-   Unrecognized diabetes: hyperglycemia (fasting blood glucose 126 mg\/dl or random blood glucose 200 mg\/dl) occurring during hospitalization and confirmed as diabetes after hospitalization by standard diagnostic criteria but unrecognized as diabetes by the treating physician during hospitalization.\n\n-   Hospital-related hyperglycemia: hyperglycemia (fasting blood glucose 126 mg\/dl or random blood glucose \u2265200 mg\/dl) occurring during the hospitalization that reverts to normal after hospital discharge.\n\nThe prevalence of diabetes in hospitalized adult patients is not precisely known. In the year 2000, 12.4% of hospital discharges in the U.S. listed diabetes as a diagnosis, but this is likely an underestimate. The prevalence of diabetes in hospitalized adults is conservatively estimated at 12\u201325%, depending on the thoroughness used in identifying patients. In the year 2003, there were 5.1 million hospitalizations with diabetes as a listed diagnosis, a 2.3-fold increase over 1980 rates (314).\n\nThe management of hyperglycemia in the hospital was traditionally considered secondary in importance to the condition that prompted admission (313).\n\nA rapidly growing body of literature supports targeted glucose control in the hospital setting for potential improved mortality, morbidity, and health economic outcomes. Hyperglycemia in the hospital may result from stress, decompensation of type 1 or type 2 or other forms of diabetes, and\/or may be iatrogenic due to withholding of antihyperglycemic medications or administration of hyperglycemia-provoking agents such as glucocorticoids or vasopressors.\n\n### 1. In-hospital hyperglycemia and outcomes\n\n#### a. General medicine and surgery.\n\nObservational studies suggest an association between hyperglycemia and increased mortality. Surgical patients with at least one blood glucose value \\>220 mg\/dl (12.2 mmol\/l) on the first postoperative day have significantly higher infection rates (315).\n\nWhen admissions on general medicine and surgery units were studied, patients with new hyperglycemia had significantly increased in-hospital mortality, as did patients with known diabetes. In addition, length of stay was higher for the new hyperglycemic group, and patients in either hyperglycemic group were more likely to require intensive care unit (ICU) care and transitional or nursing home care. Better outcomes were demonstrated in patients with fasting and admission blood glucose \\<126 mg\/dl (7 mmol\/l) and all random blood glucose levels \\<200 mg\/dl (11.1 mmol\/l) (316).\n\n#### b. CVD and critical care.\n\nA significant relationship exists between blood glucose levels and mortality in the setting of acute myocardial infarction. A meta-analysis of 15 studies compared in-hospital mortality in both hyper- and normoglycemic patients with and without diabetes. In subjects without known diabetes whose admission blood glucose averaged 109.8 mg\/dl (6.1 mmol\/l), the relative risk for in-hospital mortality was increased significantly. When diabetes was present and admission glucose averaged 180 mg\/dl (10 mmol\/l), risk of death was moderately increased compared with patients who had diabetes but less hyperglycemia on admission (317). Another study (318) demonstrated a strong independent relationship between admission blood glucose values and both in-hospital and 1-year mortality; rates were significantly lower in subjects with admission plasma glucose \\<100.8 mg\/dl (5.6 mmol\/l) than in those with plasma glucose 199.8 mg\/dl (11 mmol\/l).\n\nThese studies focused on admission blood glucose as a predictor of outcomes, rather than inpatient glycemic management per se. Higher admission plasma glucose levels in patients with a prior history of diabetes could reflect the degree of glycemic control in the outpatient setting, thus linking outpatient glycemic control to outcomes in the inpatient population. In patients without a prior history of diabetes, admission hyperglycemia could represent case finding of patients with previously undiagnosed diabetes, an unmasking of risk in a population at high risk for diabetes or more severe illness at admission.\n\nIn the initial Diabetes and Insulin-Glucose Infusion in Acute Myocardial Infarction (DIGAMI) study (319,320), insulin-glucose infusion followed by at least 3 months of subcutaneous insulin treatment in diabetic patients with acute myocardial infarction improved long-term survival. Mean blood glucose in the intensive insulin intervention arm was 172.8 mg\/dl (9.6 mmol\/l), compared with 210.6 mg\/dl (11.7 mmol\/l) in the \"conventional\" group. The broad range of blood glucose levels within each arm limits the ability to define specific blood glucose target thresholds.\n\nThree more recent studies (321\u2013323) using an insulin-glucose infusion did not show a reduction in mortality in the intervention groups. However, in each of these studies, blood glucose levels were positively correlated with mortality. In the Hyperglycemia: Intensive Insulin Infusion In Infarction (HI-5) study, a decrease in both CHF and reinfarction was observed in the group receiving intensive insulin therapy for at least 24 h.\n\n#### c. Cardiac surgery.\n\nAttainment of targeted glucose control in patients with diabetes undergoing cardiac surgery is associated with reduced mortality and risk of deep sternal wound infections (324,325). Although these studies used historical controls and were not randomized, they support the concept that perioperative hyperglycemia is an independent predictor of infection in patients with diabetes (326), with the lowest mortality in patients with blood glucose \\<150 mg\/dl (8.3 mmol\/l) (327).\n\n#### d. Critical care.\n\nA mixed group of patients with and without diabetes admitted to a surgical ICU were randomized to receive intensive insulin therapy (target blood glucose 80\u2013110 mg\/dl \\[4.4\u20136.1 mmol\/l\\]) or conventional therapy. Intensive insulin therapy achieved a mean blood glucose of 103 mg\/dl (5.7 mmol\/l) and was associated with reduced mortality during the ICU stay and decreased overall in-hospital mortality (328). Hospital and ICU survival were linearly associated with ICU glucose levels, with the highest survival rates occurring in patients achieving an average blood glucose \\<110 mg\/dl (6.1 mmol\/l) (329).\n\nA subsequent study of a similar intervention in patients in a medical ICU (330) showed that the group receiving intensive insulin therapy had reduced morbidity but no difference in mortality overall. Death rates were significantly lower in those patients who were treated for longer than 3 days; but these patients could not be identified before therapy. In another study using a similar intervention targeting a blood glucose range of 4.4\u20136.1 mmol in patients admitted with sepsis, no difference in mortality from the conventionally treated group was observed. There were more episodes of hypoglycemia, defined as a blood glucose \\<40 mg\/dl (2.2 mmol\/l), and more serious adverse events in the group receiving intensive insulin therapy (331).\n\nWhile an earlier meta-analysis concluded that insulin therapy in critically ill patients had a beneficial effect on short-term mortality in different clinical settings (332), a more recent meta-analysis, involving 29 studies and over 8,000 patients, failed to show any mortality benefit from intensive glucose control. Tight glucose control did reduce the relative risk of septicemia by 26% (333). While this latter meta-analysis investigated strategies with target blood glucose levels of 80\u2013110 mg\/dl (4.4\u20136.1 mmol\/l), studies with less stringent glucose targets were also included. Stratification by glucose target did not demonstrate any heterogeneity. The authors of this analysis as well as an accompanying editorial both recommend that glycemic targets in critically ill patients be revisited (328\u2013331).\n\nWhile results from ongoing clinical trials are still pending, it is clear that uncontrolled hyperglycemia is associated with adverse outcomes in critically ill patients and that achieving levels of glucose control below 140 mg\/dl are reasonable, provided that protocols that minimize risk for hypoglycemia are utilized and that personnel are well educated in the direct application of these protocols.\n\n### 2. Glycemic targets in hospitalized patients\n\nThere is relatively strong evidence from randomized controlled trials for a glycemic target of blood glucose \\<110 mg\/dl (6.1 mmol\/l) in surgical patients in critical care units (328\u2013330). However, in several studies of critically ill medical patients (330,331,333), the incidence of severe hypoglycemia (blood glucose \\<40 mg\/dl) was approximately threefold greater in intensively treated patients. The identification of hypoglycemia as an independent risk factor for death in the medical ICU population (334) may merit caution in widely promoting the 80\u2013110 mg\/dl target range for all critically ill populations.\n\nFor patients in general medical-surgical units, the evidence for specific glycemic goals is less definitive. Epidemiologic and physiologic data suggest that higher blood glucose levels are associated with worse outcomes, but whether glucose is simply a marker of the severity of underlying illness or a mediator of adverse outcomes is unclear. Glycemic targets similar to those of outpatients may be difficult to achieve in the hospital due to the effects of stress hyperglycemia, altered nutritional intake, and multiple interruptions to medical care. Blood glucose levels shown to be associated with improved outcomes in these patients (fasting glucose \\<126 mg\/dl and all blood glucose readings \\<180\u2013200 mg\/dl) would appear reasonable, if they can be safely achieved.\n\nIn both the critical care and noncritical care venue, glycemic goals must take into account the individual patient's situation as well as hospital system support for achieving these goals. A continuous quality improvement strategy may facilitate gradual improvement in mean glycemia hospital wide.\n\n### 3. Treatment options in hospitalized patients\n\n#### a. Noninsulin glucose-lowering agents.\n\nNo large studies have investigated the potential roles of various noninsulin glucose-lowering agents on outcomes of hospitalized patients with diabetes. Use of the various noninsulin classes in the inpatient setting presents some specific issues.\n\nThe long action of sulfonylureas and their predisposition to hypoglycemia in patients not consuming their normal nutrition serve as relative contraindications to routine use of these agents in the hospital (335). While the meglitinides, repaglinide and neteglinide, theoretically would produce less hypoglycemia than sulfonylureas, lack of clinical trial data for these agents, and the fact that they are primarily prandial in effect, would preclude their use. The major limitation to metformin use in the hospital is a number of specific contraindications to its use, related to risk of lactic acidosis, many of which occur in the hospital. The most common risk factors for lactic acidosis in metformin-treated patients are cardiac disease, including decompensated CHF, hypoperfusion, renal insufficiency, old age, and chronic pulmonary disease (336). Lactic acidosis is a rare complication in the outpatient setting (337), despite the relative frequency of risk factors (338). However, in the hospital the risks of hypoxia, hypoperfusion, and renal insufficiency are much higher, and it is prudent to avoid the use of metformin in most patients.\n\nTZDs are not suitable for initiation in the hospital because of their delayed onset of effect. In addition, they increase intravascular volume, a particular problem in those predisposed to CHF and potentially a problem for patients with hemodynamic changes related to admission diagnoses (e.g., acute coronary ischemia) or interventions common in hospitalized patients. Pramlintide and exenatide work mainly by reducing postprandial hyperglycemia and would therefore not be appropriate for patients not eating (nil per os, NPO) or with reduced caloric consumption. Furthermore, initiation of these drugs in the inpatient setting would be problematic due to alterations in normal food intake and their propensity to induce nausea initially. There is limited experience, and no published data, on the DPP-IV inhibitors in the hospital setting, although there are no specific safety concerns. They are mainly effective on postprandial glucose and therefore would have limited effect in patients who are not eating.\n\nIn summary, each of the major classes of noninsulin glucose-lowering drugs has significant limitations for inpatient use. Additionally, they provide little flexibility or opportunity for titration in a setting where acute changes often demand these characteristics. Therefore, insulin, when used properly, is preferred for the majority of hyperglycemic patients in the hospital setting.\n\n#### b. Insulin\n\n##### i. Subcutaneous insulin therapy.\n\nSubcutaneous insulin therapy may be used to attain glucose control in most hospitalized patients with diabetes outside of the critical care arena. The components of the daily insulin dose requirement can be met by a variety of insulins, depending on the particular hospital situation. Subcutaneous insulin therapy should cover both basal and nutritional needs and is subdivided into scheduled insulin and supplemental, or correction-dose, insulin. Correction-dose insulin therapy is an important adjunct to scheduled insulin, both as a dose-finding strategy and as a supplement when rapid changes in insulin requirements lead to hyperglycemia. If correction doses are frequently required, the appropriate scheduled insulin doses should be increased to accommodate the increased insulin needs. There are currently no published studies comparing human regular insulin with rapid-acting analogs for use as correction-dose insulin.\n\nThe traditional \"sliding-scale\" insulin regimens, usually consisting of regular insulin without any intermediate or long-acting insulins, have been shown to be ineffective when used as monotherapy in patients with an established insulin requirement (339\u2013341). Problems with sliding-scale insulin regimens include the fact that the sliding-scale regimen prescribed on admission is likely to be used throughout the hospital stay without modification, even when control remains poor. Additionally, sliding-scale insulin therapy treats hyperglycemia after it has already occurred, instead of preventing the occurrence of hyperglycemia. This \"reactive\" approach can lead to rapid changes in blood glucose levels, which may exacerbate both hyper- and hypoglycemia.\n\nA recent study demonstrated the safety and efficacy of using basal-bolus insulin therapy utilizing weight-based dosing in insulin-na\u00efve hospitalized patients with type 2 diabetes (342). Glycemic control, defined as a mean blood glucose \\<140 mg\/dl, was achieved in 68% of patients receiving basal-bolus insulin versus only 38% of those receiving sliding-scale insulin alone. There were no differences in hypoglycemia between the two groups. It is important to note that the patients in this study were obese, and the doses used in this study (0.4 to 0.5 units \u00b7 kg \u00b7 day^\u2212*1*^) are higher than what may be required in patients who are more sensitive to insulin, such as those who are lean or who have type 1 diabetes.\n\n##### ii. Intravenous insulin infusion.\n\nThe only method of insulin delivery specifically developed for use in the hospital is continuous intravenous infusion, using regular crystalline insulin. There is no advantage to using rapid acting analogs, whose structural modifications increase the rate of absorption from subcutaneous depots, in an intravenous insulin infusion. The medical literature supports the use of intravenous insulin infusion in preference to the subcutaneous route of insulin administration for several clinical indications among nonpregnant adults. These include DKA and nonketotic hyperosmolar state; general preoperative, intraoperative, and postoperative care; the postoperative period following heart surgery; following organ transplantation; with cardiogenic shock; exacerbated hyperglycemia during high-dose glucocorticoid therapy; type 1 patients who are NPO; or in critical care illness in general. It may be used as a dose-finding strategy in anticipation of initiation or reinitiation of subcutaneous insulin therapy in type 1 or type 2 diabetes.\n\nMany institutions use insulin infusion algorithms that can be implemented by nursing staff. Although numerous algorithms have been published, there have been no head-to-head comparisons between insulin infusion strategies. Algorithms should incorporate the concepts that maintenance requirements differ between patients and change over the course of treatment. Ideally, intravenous insulin algorithms should consider both current and previous glucose levels, the rate of change of plasma glucose, and the current intravenous insulin infusion rate. For all algorithms, frequent (Q 1\u20132 h) bedside glucose testing is required.\n\n##### iii. Transition from intravenous to subcutaneous insulin therapy.\n\nFor those who will require subcutaneous insulin, the very short half-life of intravenous insulin necessitates administering the first dose of subcutaneous insulin before discontinuation of the intravenous insulin infusion. If short or rapid-acting insulin is used, it should be injected 1\u20132 h before stopping the infusion. If intermediate- or long-acting insulin is used alone, it should be injected 2\u20133 h before. A combination of short\/rapid- and intermediate\/long-acting insulin is usually preferred. Basal insulin therapy can be initiated at any time of the day and should not be withheld to await a specific dosing time, such as bedtime. A recent clinical trial demonstrated that a regimen using 80% of the intravenous insulin requirement over the preceding 24 h, divided into basal and bolus insulin components, was effective at achieving blood glucose levels between 80 and 150 mg\/dl following discontinuation of the intravenous insulin (343).\n\n### 4. Self-management in the hospital\n\nSelf-management of diabetes in the hospital may be appropriate for competent adult patients who have a stable level of consciousness, have reasonably stable daily insulin requirements, successfully conduct self-management of diabetes at home, have physical skills needed to successfully self-administer insulin and perform SMBG, have adequate oral intake, and are proficient in carbohydrate counting, use of multiple daily insulin injections or insulin pump therapy, and sick-day management. The patient and physician, in consultation with nursing staff, must agree that patient self-management is appropriate under the conditions of hospitalization. For patients conducting self-management in the hospital, it is imperative that basal, prandial, and correction doses of insulin and results of bedside glucose monitoring be recorded as part of the patient's hospital medical record. While many institutions allow patients on insulin pumps to continue these devices in the hospital, others express concern regarding use of a device unfamiliar to staff, particularly in patients who are not able to manage their own pump therapy. If a patient is too ill to self-manage either multiple daily injections or CSII, then appropriate subcutaneous doses can be calculated on the basis of their basal and bolus insulin needs during hospitalization, with adjustments for changes in nutritional or metabolic status.\n\n### 5. Preventing hypoglycemia\n\nHypoglycemia, especially in insulin-treated patients, is the leading limiting factor in the glycemic management of type 1 and type 2 diabetes (146). In the hospital, multiple additional risk factors for hypoglycemia are present, even among patients who are neither \"brittle\" nor tightly controlled. Patients with or without diabetes may experience hypoglycemia in the hospital in association with altered nutritional state, heart failure, renal or liver disease, malignancy, infection, or sepsis (344,345). Additional triggering events leading to iatrogenic hypoglycemia include sudden reduction of corticosteroid dose, altered ability of the patient to self-report symptoms, reduction of oral intake, emesis, new NPO status, inappropriate timing of short- or rapid-acting insulin in relation to meals, reduction of rate of administration of intravenous dextrose, and unexpected interruption of enteral feedings or parenteral nutrition.\n\nDespite the preventable nature of many inpatient episodes of hypoglycemia, institutions are more likely to have nursing protocols for the treatment of hypoglycemia than for its prevention. Tracking such episodes and analyzing their causes are important quality improvement activities.\n\n### 6. Diabetes care providers in the hospital\n\nInpatient diabetes management may be effectively provided by primary care physicians, endocrinologists, or hospitalists, but involvement of appropriately trained specialists or specialty teams may reduce length of stay, improve glycemic control, and improve outcomes (346\u2013349). In the care of diabetes, implementation of standardized order sets for scheduled and correction-dose insulin may reduce reliance on sliding-scale management. A team approach is needed to establish hospital pathways. To achieve glycemic targets associated with improved hospital outcomes, hospitals will need multidisciplinary support for using insulin infusion therapy outside of critical care units or will need to develop protocols for subcutaneous insulin therapy that effectively and safely achieve glycemic targets (350).\n\n### 7. DSME in the hospital\n\nTeaching diabetes self-management to patients in hospitals is a challenging task. Patients are ill, under increased stress related to their hospitalization and diagnosis, and in an environment not conducive to learning. Ideally, people with diabetes should be taught at a time and place conducive to learning, as outpatients in a recognized program of diabetes education.\n\nFor the hospitalized patient, diabetes \"survival skills\" education is generally a feasible approach. Patients and\/or family members receive sufficient information and training to enable safe care at home. Those newly diagnosed with diabetes or who are new to insulin and\/or blood glucose monitoring need to be instructed before discharge. Those patients hospitalized because of a crisis related to diabetes management or poor care at home need education to prevent subsequent episodes of hospitalization. An assessment of the need for a home health referral or referral to an outpatient diabetes education program should be part of discharge planning for all patients.\n\n### 8. MNT in the hospital\n\nHospital diets continue to be ordered by calorie levels based on the \"ADA diet.\" However, since 1994 the ADA has not endorsed any single meal plan or specified percentages of macronutrients, and the term \"ADA diet\" should no longer be used. Current nutrition recommendations advise individualization based on treatment goals, physiologic parameters, and medication usage. Because of the complexity of nutrition issues in the hospital, a registered dietitian, knowledgeable and skilled in MNT, should serve as an inpatient team member. The dietitian is responsible for integrating information about the patient's clinical condition, eating, and lifestyle habits and for establishing treatment goals in order to determine a realistic plan for nutrition therapy (351,352).\n\n### 9. Bedside blood glucose monitoring\n\nImplementing intensive diabetes therapy in the hospital setting requires frequent and accurate blood glucose data. This measure is analogous to an additional \"vital sign\" for hospitalized patients with diabetes. Bedside glucose monitoring using capillary blood has advantages over laboratory venous glucose testing because the results can be obtained rapidly at the \"point of care,\" where therapeutic decisions are made.\n\nBedside blood glucose testing is usually performed with portable meters that are similar or identical to devices for home SMBG. Staff training and ongoing quality control activities are important components of ensuring accuracy of the results. Ability to track the occurrence of hypo- and hyperglycemia is necessary. Results of bedside glucose tests should be readily available to all members of the care team.\n\nFor patients who are eating, commonly recommended testing frequencies are premeal and at bedtime. For patients not eating, testing every 4\u20136 h is usually sufficient for determining correction insulin doses. Patients on continuous intravenous insulin typically require hourly blood glucose testing until the blood glucose levels are stable, then every 2 h.\n\n### 10. Discharge planning\n\nIt is important to anticipate the postdischarge antihyperglycemic regimen in all patients with diabetes or newly discovered hyperglycemia. The optimal program will need to consider the type and severity of diabetes, the effects of the patient's illness on blood glucose levels, and the capacities and desires of the patient. Smooth transition to outpatient care should be ensured, especially in those new to insulin therapy or in whom the diabetes regimen has been substantially altered during the hospitalization. All patients in whom the diagnosis of diabetes is new should have, at minimum, \"survival skills\" training before discharge. More expanded diabetes education can be arranged in the community. For those with hyperglycemia who do not require treatment upon discharge, follow-up testing through their primary care physician should be arranged, since many of these individuals are found to have diabetes when tested after discharge.\n\n## B. Diabetes care in the school and day care setting (353)\n\n### Recommendations\n\n-   An individualized Diabetes Medical Management Plan (DMMP) should be developed by the parent\/guardian and the student's personal diabetes health care team with input from the parent\/guardian. (E)\n\n-   All school staff members who have responsibility for a student with diabetes should receive training that provides a basic understanding of diabetes and a student's needs. (E)\n\n-   While the school nurse is the coordinator and primary provider of diabetes care, a small number of school personnel should be trained in routine and emergency diabetes procedures (including monitoring of blood glucose levels and administration of insulin and glucagon) and in the appropriate response to high and low blood glucose levels and should perform these diabetes care tasks when the school nurse is not available to do so. These school personnel need not be health care professionals. (E)\n\n-   As specified in the DMMP and as developmentally appropriate, the student with diabetes should have immediate access to diabetes supplies at all times and should be permitted to self-manage his or her diabetes in the classroom or anywhere the student may be in conjunction with a school activity. Such self-management should include blood glucose monitoring and responding to blood glucose levels with needed food and medication. (E)\n\nThere are \u223c186,300 individuals \\<20 years of age with diabetes in the U.S., most of whom attend school and\/or some type of day care and need knowledgeable staff to provide a safe environment. Despite legal protections, including coverage of children with diabetes under Section 504 of the Rehabilitation Act of 1973, the Individuals with Disabilities Education Act, and the Americans with Disabilities Act, children in the school and day care setting still face discrimination. The ADA position statement on Diabetes Care in the School and Day Care Setting (353) provides the legal and medical justifications for the recommendations provided herein.\n\nAppropriate diabetes care in the school and day care setting is necessary for the child's immediate safety, long-term well-being, and optimal academic performance. Parents and the health care team should provide school systems and day care providers with the information necessary for children with diabetes to participate fully and safely in the school\/day care experience by developing an individualized DMMP.\n\nThe school nurse should be the key coordinator and provider of care and should coordinate the training of an adequate number of school personnel and ensure that if the school nurse is not present at least one adult is present who is trained to perform the necessary diabetes procedures (e.g., blood glucose monitoring and insulin and glucagon administration) and provide the appropriate response to high and low blood glucose levels in a timely manner while the student is at school, on field trips, participating in school-sponsored extracurricular activities, and on transportation provided by the school or day care facility. These school personnel need not be health care professionals.\n\nThe student with diabetes should have immediate access to diabetes supplies at all times, with supervision as needed. The student should be able to obtain a blood glucose level and respond to the results as quickly and conveniently as possible in the classroom or wherever the child is in conjunction with a school-related activity, minimizing the need for missing instruction in the classroom and avoiding the risk of worsening hypoglycemia or hyperglycemia if the child must go somewhere else for treatment. The student's desire for privacy during blood glucose monitoring and insulin administration should also be accommodated.\n\nThe ADA and partner organizations have developed tools for school personnel to provide a safe and nondiscriminatory educational environment for all students with diabetes (354,355).\n\n## C. Diabetes care at diabetes camps (356)\n\n### Recommendations\n\n-   Each camper should have a standardized medical form completed by his\/her family and the physician managing the diabetes. (E)\n\n-   Camp medical staff should be led by with a physician with expertise in managing type 1 and type 2 diabetes and include nurses (including diabetes educators and diabetes clinical nurse specialists) and registered dietitians with expertise in diabetes. (E)\n\n-   All camp staff, including physicians, nurses, dietitians, and volunteers, should undergo background testing to ensure appropriateness in working with children. (E)\n\nThe concept of specialized residential and day camps for children with diabetes has become widespread throughout the U.S. and many other parts of the world. The mission of diabetes camps is to provide a camping experience in a safe environment. An equally important goal is to enable children with diabetes to meet and share their experiences with one another while they learn to be more personally responsible for their disease. For this to occur, a skilled medical and camping staff must be available to ensure optimal safety and an integrated camping\/educational experience.\n\nEach camper should have a standardized medical form completed by his\/her family and the physician managing the diabetes that details the camper's past medical history, immunization record, and diabetes regimen. The home insulin dosage should be recorded for each camper, including type(s) of insulin used, number and timing of injections, and the correction factor and carbohydrate ratios used for determining bolus dosages for basal-bolus regimens. Campers using CSII should also have their basal rates specified. Because camp is often associated with more physical activity than experienced at home, the insulin dose may have to be decreased during camp.\n\nThe diabetes camping experience is short term, with food and activity different than the home environment. Thus, goals of glycemic control at camp are to avoid extremes in blood glucose levels rather than attempting optimization of intensive glycemic control.\n\nDuring camp, a daily record of the camper's progress should be made, including all blood glucose levels and insulin dosages. To ensure safety and optimal diabetes management, multiple blood glucose determinations should be made throughout each 24-h period: before meals, at bedtime, after or during prolonged and strenuous activity, and in the middle of the night when indicated for prior hypoglycemia. If major alterations of a camper's regimen appear to be indicated, it is important to discuss this with the camper and the family in addition to the child's local physician. The record of what transpired during camp should be discussed with the family at the end of the camp session and a copy sent to the child's physician.\n\nEach camp should secure a formal relationship with a nearby medical facility so that camp medical staff can refer to this facility for prompt treatment of medical emergencies. ADA requires that the camp medical director be a physician with expertise in managing type 1 and type 2 diabetes. Nursing staff should include diabetes educators and diabetes clinical nurse specialists. Registered dietitians with expertise in diabetes should have input into the design of the menu and the education program. All camp staff, including medical, nursing, nutrition, and volunteer, should undergo background testing to ensure appropriateness in working with children.\n\n## D. Diabetes management in correctional institutions (357)\n\n### Recommendations\n\n-   Correctional staff should be trained in the recognition, treatment, and appropriate referral for hypo- and hyperglycemia, including serious metabolic decompensation. (E)\n\n-   Patients with a diagnosis of diabetes should have a complete medical history and physical examination by a licensed health care provider with prescriptive authority in a timely manner upon entry. Insulin-treated patients should have a capillary blood glucose (CBG) determination within 1\u20132 h of arrival. Staff should identify patients with type 1 diabetes who are at high risk for DKA with omission of insulin. (E)\n\n-   Medications and MNT should be continued without interruption upon entry into the correctional environment. (E)\n\n-   In the correctional setting, policies and procedures should enable CBG monitoring to occur at the frequency necessitated by the patient's glycemic control and diabetes regimen and should require staff to notify a physician of all CBG results outside of a specified range, as determined by the treating physician. (E)\n\n-   For all inter-institutional transfers, a medical transfer summary should be transferred with the patient, and diabetes supplies and medication should accompany the patient. (E)\n\n-   Correctional staff should begin discharge planning with adequate lead time to ensure continuity of care and facilitate entry into community diabetes care. (E)\n\nAt any given time, over 2 million people are incarcerated in prisons and jails in the U.S., and it is estimated that nearly 80,000 of these inmates have diabetes. In addition, many more people with diabetes pass through the corrections system in a given year.\n\nPeople with diabetes in correctional facilities should receive care that meets national standards. Correctional institutions have unique circumstances that need to be considered so that all standards of care may be achieved. Correctional institutions should have written policies and procedures for the management of diabetes and for training of medical and correctional staff in diabetes care practices.\n\nReception screening should emphasize patient safety. In particular, rapid identification of all insulin-treated individuals with diabetes is essential in order to identify those at highest risk for hypo- and hyperglycemia and DKA. All insulin-treated patients should have a CBG determination within 1\u20132 h of arrival. Patients with a diagnosis of diabetes should have a complete medical history and physical examination by a licensed health care provider with prescriptive authority in a timely manner. It is essential that medication and MNT be continued without interruption upon entry into the correctional system, as a hiatus in either medication or appropriate nutrition may lead to either severe hyper- or hypoglycemia.\n\nPatients must have access to prompt treatment of hypo- and hyperglycemia. Correctional staff should be trained in the recognition and treatment of these conditions, and appropriate staff should be trained to administer glucagon. Institutions should implement a policy requiring staff to notify a physician of all CBG results outside of a specified range, as determined by the treating physician.\n\nCorrectional institutions should have systems in place to ensure that insulin administration and meals are coordinated to prevent hypo- and hyperglycemia, taking into consideration the transport of residents off site and the possibility of emergency schedule changes. The frequency of CBG monitoring will vary by patients' glycemic control and diabetes regimens. Policies and procedures should ensure that the health care staff has adequate knowledge and skills to direct the management and education of individuals with diabetes.\n\nPatients in jails may be housed for a short period of time before being transferred or released, and patients in prison may be transferred within the system several times during their incarceration. Transferring a patient with diabetes from one correctional facility to another requires a coordinated effort, as does planning for discharge. The ADA position statement on Diabetes Management in Correctional Institutions (357) should be consulted for more information on this topic.\n\n## E. Emergency and disaster preparedness (358)\n\n### Recommendations\n\n-   People with diabetes should maintain a disaster kit that includes items important to their diabetes self-management and continuing medical care. (E)\n\n-   The kit should be reviewed and replenished at least twice yearly. (E)\n\nThe difficulties encountered by people with diabetes and their health care providers in the wake of Hurricane Katrina (359) highlight the need for people with diabetes to be prepared for emergencies, whether natural or otherwise, affecting a region or just their household. Such preparedness will lessen the impact an emergency may have on their condition. It is recommended that people with diabetes keep a waterproof and insulated disaster kit ready with items critically important to their self-management. These may include glucose testing strips, lancets, and a glucose-testing meter; medications including insulin in a cool bag; syringes; glucose tabs or gels; antibiotic ointments\/creams for external use; glucagon emergency kits; and photocopies of relevant medical information, particularly medication lists and recent lab tests\/procedures if available. If possible, prescription numbers should be noted, since many chain pharmacies throughout the country will refill medications based on the prescription number alone. In addition, it may be important to carry a list of contacts for national organizations, such as the American Red Cross and ADA. This disaster kit should be reviewed and replenished at least twice yearly.\n\n# IX. DIABETES AND EMPLOYMENT (360)\n\n## Recommendations\n\n-   When questions arise about the medical fitness of a person with diabetes for a particular job, a health care professional with expertise in treating diabetes should perform an individualized assessment; input from the treating physician should always be included. (E)\n\n-   Proper safety assessments for employment should include review of blood glucose test results, history of severe hypoglycemia, presence of hypoglycemia unawareness, and presence of diabetes-related complications but should not include urine glucose or A1C\/eAG tests or be based on a general assessment of level of control. (E)\n\nAny person with diabetes, whether insulin treated or noninsulin treated, should be eligible for any employment for which he\/she is otherwise qualified. Questions are sometimes raised by employers about the safety and effectiveness of individuals with diabetes in a given job. When such questions are legitimately raised, a person with diabetes should be individually assessed by a health care professional with expertise in diabetes to determine whether or not that person can safely and effectively perform the particular duties of the job in question.\n\nEmployment decisions should never be based on generalizations or stereotypes regarding the effects of diabetes. \"Blanket bans\" that restrict individuals with diabetes from certain jobs or classes of employment solely because of the diagnosis of diabetes or the use of insulin are medically and legally inappropriate and ignore the many advancements in diabetes management that range from the types of medications used to the tools used to administer them and to monitor blood glucose levels. For most types of employment, there is no reason to believe that the individual's diabetes will put employees or the public at risk. In certain safety-sensitive positions the safety concern is whether the employee will become suddenly disoriented or incapacitated. Episodes of severe hypoglycemia should be examined by a health care professional with expertise in diabetes to determine any impact on safe performance of the job. Hyperglycemia is not typically a barrier to employment unless long-term complications are present that interfere with the performance of the job.\n\nMost accommodations that help an individual with diabetes do his or her job may be provided easily and with little or no cost to the employer. Typical accommodations include breaks to test blood glucose, administer insulin, or access food and beverages. Some individuals may need leave or a flexible work schedule or accommodations for diabetes-related complications.\n\nThe ADA position statement on Diabetes and Employment should be consulted for more information on this topic.\n\n# X. THIRD-PARTY REIMBURSEMENT FOR DIABETES CARE, SELF-MANAGEMENT EDUCATION, AND SUPPLIES (361)\n\n## Recommendations\n\n-   Patients and practitioners should have access to all classes of antidiabetic medications, equipment, and supplies without undue controls. (E)\n\n-   MNT and DSME should be covered by insurance and other payors. (E)\n\nTo achieve optimal glucose control, the person with diabetes must be able to access health care providers who have expertise in the field of diabetes. Treatments and therapies that improve glycemic control and reduce the complications of diabetes will also significantly reduce health care costs. Access to the integral components of diabetes care, such as health care visits, diabetes supplies and medications, and self-management education, is essential. All medications and supplies, such as syringes, strips, and meters, related to the daily care of diabetes must also be reimbursed by third-party payors.\n\nIt is recognized that the use of formularies, prior authorization, and provisions such as competitive bidding can manage provider practices as well as costs to the potential benefit of payors and patients. However, any controls should ensure that all classes of anti-diabetic agents with unique mechanisms of action and all classes of equipment and supplies designed for use with such equipment are available to facilitate achieving glycemic goals and to reduce the risk of complications. Without appropriate safeguards, undue controls could constitute an obstruction of effective care.\n\nMedicare and many other third-party payors cover DSME (Centers for Medicare and Medicaid Services \\[CMS\\] term is diabetes self-management training \\[DSMT\\]) that meets the national standards for DSME (107) and MNT. The qualified beneficiary, with referral from the provider managing his or her diabetes, can receive an initial benefit of 10 h of DSMT and 3 h of MNT, with a potential total of 13 h of initial. More information on Medicare policy, including follow-up benefits, is available at [www.diabetes.org\/for-health-professionals-and-scientists\/recognition.jsp](www.diabetes.org\/for-health-professionals-and-scientists\/recognition.jsp) or on the CMS Web sites: DSME, [www.cms.hhs.gov\/DiabetesSelfManagement](www.cms.hhs.gov\/DiabetesSelfManagement); and diabetes MNT, [www.cms.hhs.gov\/MedicalNutritionTherapy](www.cms.hhs.gov\/MedicalNutritionTherapy), reimbursement.\n\n# XI. STRATEGIES FOR IMPROVING DIABETES CARE\n\nThe implementation of the standards of care for diabetes has been suboptimal in most clinical settings. A recent report (362) indicated that only 37% of adults with diagnosed diabetes achieved an A1C of \\<7%, only 36% had a blood pressure \\<130\/80 mmHg, and just 48% had a total cholesterol \\<200 mg\/dl. Most distressing was that only 7.3% of people with diabetes achieved all three treatment goals.\n\nWhile numerous interventions to improve adherence to the recommended standards have been implemented, the challenge of providing uniformly effective diabetes care has thus far defied a simple solution. A major contributor to suboptimal care is a delivery system that too often is fragmented, lacks clinical information capabilities, often duplicates services, and is poorly designed for the delivery of chronic care. The Institute of Medicine has called for changes so that delivery systems provide care that is evidence based, patient centered, and systems oriented and takes advantage of information technologies that foster continuous quality improvement. Collaborative, multidisciplinary teams should be best suited to provide such care for people with chronic conditions like diabetes and to empower patients' performance of appropriate self-management. Alterations in reimbursement that reward the provision of quality care, as defined by the attainment of quality measures developed by such programs as the ADA\/National Committee for Quality Assurance Diabetes Provider Recognition Program will also be required to achieve desired outcome goals.\n\nThe NDEP recently launched a new online resource to help health care professionals better organize their diabetes care. The [www.betterdiabetescare.nih.gov](www.betterdiabetescare.nih.gov) Web site should help users design and implement more effective health care delivery systems for those with diabetes.\n\nIn recent years, numerous health care organizations, ranging from large health care systems such as the U.S. Veterans Administration to small private practices, have implemented strategies to improve diabetes care. Successful programs have published results showing improvement in process measures such as measurement of A1C, lipids, and blood pressure. Effects on important intermediate outcomes, such as mean A1C for populations, have been more difficult to demonstrate (363\u2013365), although examples do exist (366\u2013370). Successful interventions have been focused at the level of health care professionals, delivery systems, and patients. Features of successful programs reported in the literature include:\n\n-   Improving health care professional education regarding the standards of care through formal and informal education programs.\n\n-   Delivery of DSME, which has been shown to increase adherence to standard of care.\n\n-   Adoption of practice guidelines, with participation of health care professionals in the process. Guidelines should be readily accessible at the point of service, such as on patient charts, in examining rooms, in \"wallet or pocket cards,\" on PDAs, or on office computer systems. Guidelines should begin with a summary of their major recommendations instructing health care professionals what to do and how to do it.\n\n-   Use of checklists that mirror guidelines have been successful at improving adherence to standards of care.\n\n-   Systems changes, such as provision of automated reminders to health care professionals and patients, reporting of process and outcome data to providers, and especially identification of patients at risk because of failure to achieve target values or a lack of reported values.\n\n-   Quality improvement programs combining continuous quality improvement or other cycles of analysis and intervention with provider performance data.\n\n-   Practice changes, such as clustering of dedicated diabetes visits into specific times within a primary care practice schedule and\/or visits with multiple health care professionals on a single day and group visits.\n\n-   Tracking systems with either an electronic medical record or patient registry have been helpful at increasing adherence to standards of care by prospectively identifying those requiring assessments and\/or treatment modifications. They likely could have greater efficacy if they suggested specific therapeutic interventions to be considered for a particular patient at a particular point in time (371).\n\n-   A variety of nonautomated systems, such as mailing reminders to patients, chart stickers, and flow sheets, have been useful to prompt both providers and patients.\n\n-   Availability of case or (preferably) care management services, usually by a nurse (372). Nurses, pharmacists, and other nonphysician health care professionals using detailed algorithms working under the supervision of physicians and\/or nurse education calls have also been helpful. Similarly, dietitians using MNT guidelines have been demonstrated to improve glycemic control.\n\n-   Availability and involvement of expert consultants, such as endocrinologists and diabetes educators.\n\nEvidence suggests that these individual initiatives work best when provided as components of a multifactorial intervention. Therefore, it is difficult to assess the contribution of each component; however, it is clear that optimal diabetes management requires an organized, systematic approach and involvement of a coordinated team of health care professionals.\n\n# References\n\nADA evidence grading system for clinical practice recommendations\n\n<table>\n<thead>\n<tr>\n<th style=\"text-align: center;\">Level of evidence<\/th>\n<th style=\"text-align: center;\">Description<\/th>\n<\/tr>\n<\/thead>\n<tbody>\n<tr>\n<td style=\"text-align: left;\">A<\/td>\n<td style=\"text-align: left;\">Clear evidence from well-conducted, generalizable, randomized controlled trials that are adequately powered, including:\n<ul>\n<li><p>Evidence from a well-conducted multicenter trial<\/p><\/li>\n<li><p>Evidence from a meta-analysis that incorporated quality ratings in the analysis<\/p><\/li>\n<\/ul><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: left;\">Compelling nonexperimental evidence, i.e., \"all or none\" rule developed by the Centre for Evidence-Based Medicine at Oxford<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: left;\">Supportive evidence from well-conducted randomized controlled trials that are adequately powered, including:\n<ul>\n<li><p>Evidence from a well-conducted trial at one or more institutions<\/p><\/li>\n<li><p>Evidence from a meta-analysis that incorporated quality ratings in the analysis<\/p><\/li>\n<\/ul><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">B<\/td>\n<td style=\"text-align: left;\">Supportive evidence from well-conducted cohort studies, including:\n<ul>\n<li><p>Evidence from a well-conducted prospective cohort study or registry<\/p><\/li>\n<li><p>Evidence from a well-conducted meta-analysis of cohort studies<\/p><\/li>\n<\/ul><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: left;\">Supportive evidence from a well-conducted case-control study<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">C<\/td>\n<td style=\"text-align: left;\">Supportive evidence from poorly controlled or uncontrolled studies\n<ul>\n<li><p>Evidence from randomized clinical trials with one or more major or three or more minor methodological flaws that could invalidate the results<\/p><\/li>\n<li><p>Evidence from observational studies with high potential for bias (such as case series with comparison to historical controls)<\/p><\/li>\n<li><p>Evidence from case series or case reports<\/p><\/li>\n<\/ul><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: left;\">Conflicting evidence with the weight of evidence supporting the recommendation<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">E<\/td>\n<td style=\"text-align: left;\">Expert consensus or clinical experience<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\nCriteria for the diagnosis of diabetes\n\n|  |  |\n|----|----|\n| 1\\. | FPG \u2265126 mg\/dl (7.0 mmol\/l). Fasting is defined as no caloric intake for at least 8 h.<a href=\"#t2fn1\" data-ref-type=\"table-fn\">*<\/a> |\n| OR |  |\n| 2\\. | Symptoms of hyperglycemia and a casual (random) plasma glucose \u2265200 mg\/dl (11.1 mmol\/l). Casual (random) is defined as any time of day without regard to time since last meal. The classic symptoms of hyperglycemia include polyuria, polydipsia, and unexplained weight loss. |\n| OR |  |\n| 3\\. | 2-h plasma glucose \u2265200 mg\/dl (11.1 mmol\/l) during an OGTT. The test should be performed as described by the World Health Organization using a glucose load containing the equivalent of 75-g anhydrous glucose dissolved in water.<a href=\"#t2fn1\" data-ref-type=\"table-fn\">*<\/a> |\n\nIn the absence of unequivocal hyperglycemia, these criteria should be confirmed by repeat testing on a different day (5).\n\nCriteria for testing for pre-diabetes and diabetes in asymptomatic adult individuals\n\n<table>\n<tbody>\n<tr>\n<td style=\"text-align: left;\">1.<\/td>\n<td style=\"text-align: left;\">Testing should be considered in all adults who are overweight (BMI \u226525 kg\/m<sup>2<\/sup><a href=\"#t3fn1\" data-ref-type=\"table-fn\">*<\/a>) and have additional risk factors:\n<ul>\n<li><p>physical inactivity<\/p><\/li>\n<li><p>first-degree relative with diabetes<\/p><\/li>\n<li><p>members of a high-risk ethnic population (e.g., African American, Latino, Native American, Asian American, Pacific Islander)<\/p><\/li>\n<li><p>women who delivered a baby weighing &gt;9 lb or were diagnosed with GDM<\/p><\/li>\n<li><p>hypertension (\u2265140\/90 mmHg or on therapy for hypertension)<\/p><\/li>\n<li><p>HDL cholesterol level &lt;35 mg\/dl (0.90 mmol\/l) and\/or a triglyceride level &gt;250 mg\/dl (2.82 mmol\/l)<\/p><\/li>\n<li><p>women with polycystic ovarian syndrome (PCOS)<\/p><\/li>\n<li><p>IGT or IFG on previous testing<\/p><\/li>\n<li><p>other clinical conditions associated with insulin resistance (e.g., severe obesity, acanthosis nigricans)<\/p><\/li>\n<li><p>history of CVD<\/p><\/li>\n<\/ul><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">2.<\/td>\n<td style=\"text-align: left;\">In the absence of the above criteria, testing for pre-diabetes and diabetes should begin at age 45 years<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">3.<\/td>\n<td style=\"text-align: left;\">If results are normal, testing should be repeated at least at 3-year intervals, with consideration of more frequent testing depending on initial results and risk status.<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\nAt-risk BMI may be lower in some ethnic groups.\n\nTesting for type 2 diabetes in asymptomatic children\n\n<table>\n<tbody>\n<tr>\n<td style=\"text-align: left;\">Criteria:\n<ul>\n<li><p>Overweight (BMI &gt;85th percentile for age and sex, weight for height &gt;85th percentile, or weight &gt;120% of ideal for height)<\/p><\/li>\n<\/ul><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Plus any two of the following risk factors:\n<ul>\n<li><p>Family history of type 2 diabetes in first- or second-degree relative<\/p><\/li>\n<li><p>Race\/ethnicity (Native American, African American, Latino, Asian American, Pacific Islander)<\/p><\/li>\n<li><p>Signs of insulin resistance or conditions associated with insulin resistance (acanthosis nigricans, hypertension, dyslipidemia, PCOS, or small-for-gestational-age birthweight)<\/p><\/li>\n<li><p>Maternal history of diabetes or GDM during the child's gestation<\/p><\/li>\n<\/ul><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Age of initiation: age 10 years or at onset of puberty, if puberty occurs at a younger age<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Frequency: every 3 years<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Test: FPG preferred<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\nScreening for and diagnosis of GDM\n\n<table>\n<tbody>\n<tr>\n<td style=\"text-align: left;\">Carry out GDM risk assessment at the first prenatal visit.<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Women at very high risk for GDM should be screened for diabetes as soon as possible after the confirmation of pregnancy. Criteria for very high risk are:\n<ul>\n<li><p>severe obesity<\/p><\/li>\n<li><p>prior history of GDM or delivery of large-for-gestational-age infant<\/p><\/li>\n<li><p>presence of glycosuria<\/p><\/li>\n<li><p>diagnosis of PCOS<\/p><\/li>\n<li><p>strong family history of type 2 diabetes<\/p><\/li>\n<\/ul><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Screening\/diagnosis at this stage of pregnancy should use standard diagnostic testing (<a href=\"#t2\" data-ref-type=\"table\">Table 2<\/a>).<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">All women of greater than low risk of GDM, including those above not found to have diabetes early in pregnancy, should undergo GDM testing at 24\u201328 weeks of gestation. Low risk status, which does not require GDM screening, is defined as women with ALL of the following characteristics:\n<ul>\n<li><p>age &lt;25 years<\/p><\/li>\n<li><p>weight normal before pregnancy<\/p><\/li>\n<li><p>member of an ethnic group with a low prevalence of diabetes<\/p><\/li>\n<li><p>no known diabetes in first-degree relatives<\/p><\/li>\n<li><p>no history of abnormal glucose tolerance<\/p><\/li>\n<li><p>no history of poor obstetrical outcome<\/p><\/li>\n<\/ul><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Two approaches may be followed for GDM screening at 24\u201328 weeks:\n<ol>\n<li><p>Two-step approach:<\/p>\n<ol type=\"A\">\n<li><p>Perform initial screening by measuring plasma or serum glucose 1 h after a 50-g oral glucose load. A glucose threshold after 50-g load of \u2265140 mg\/dl identifies \u223c80% of women with GDM, while the sensitivity is further increased to \u223c90% by a threshold of \u2265130 mg\/dl.<\/p><\/li>\n<li><p>Perform a diagnostic 100-g OGTT on a separate day in women who exceed the chosen threshold on 50-g screening.<\/p><\/li>\n<\/ol><\/li>\n<li><p>One-step approach (may be preferred in clinics with high prevalence of GDM): Perform a diagnostic 100-g OGTT in all women to be tested at 24\u201328 weeks.<\/p><\/li>\n<\/ol><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">The 100-g OGTT should be performed in the morning after an overnight fast of at least 8 h.<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">To make a diagnosis of GDM, at least two of the following plasma glucose values must be found:\n<ol>\n<li><p>Fasting: \u226595 mg\/dl<\/p><\/li>\n<li><p>1 h: \u2265180 mg\/dl<\/p><\/li>\n<li><p>2 h: \u2265155 mg\/dl<\/p><\/li>\n<li><p>3 h: \u2265140 mg\/dl<\/p><\/li>\n<\/ol><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\nTherapies proven effective in diabetes prevention trials\n\n| Study (ref.) | *n* | Population | Mean age (years) | Duration (years) | Intervention (daily dose) | Conversion in control subjects (%\/year) | Relative risk |\n|----|----|----|----|----|----|----|----|\n| Lifestyle |  |  |  |  |  |  |  |\n| \u2003\u2003\u2003\u2003Finnish DPS (11) | 522 | IGT, BMI \u226525 kg\/m^2^ | 55 | 3.2 | Individual diet\/exercise | 6 | 0.42 (0.30\u20130.70) |\n| \u2003\u2003\u2003\u2003DPP (10) | 2,161<a href=\"#t6fn1\" data-ref-type=\"table-fn\">*<\/a> | IGT, BMI \u226524 kg\/m^2^, FPG \\>5.3 mmol\/l | 51 | 3 | Individual diet\/exercise | 10 | 0.42 (0.34\u20130.52) |\n| \u2003\u2003\u2003\u2003Da Qing (12) | 259<a href=\"#t6fn1\" data-ref-type=\"table-fn\">*<\/a> | IGT (randomized groups) | 45 | 6 | Group diet\/exercise | 16 | 0.62 (0.44\u20130.86) |\n| \u2003\u2003\u2003\u2003Toranomon study (28) | 458 | IGT (men), BMI = 24 kg\/m^2^ | 55 | 4 | Individual diet\/exercise | 2 | 0.33 (0.10\u20131.0)<a href=\"#t6fn2\" data-ref-type=\"table-fn\">\u2020<\/a> |\n| \u2003\u2003\u2003\u2003Indian DPP (16) | 269<a href=\"#t6fn1\" data-ref-type=\"table-fn\">*<\/a> | IGT | 46 | 2.5 | Individual diet\/exercise | 22 | 0.71 (0.63\u20130.79) |\n| Medications |  |  |  |  |  |  |  |\n| \u2003\u2003\u2003\u2003DPP (10) | 2,155<a href=\"#t6fn1\" data-ref-type=\"table-fn\">*<\/a> | IGT, BMI \\>24 kg\/m^2^, FPG \\>5.3 mmol\/l | 51 | 2.8 | Metformin (1,700 mg) | 10 | 0.69 (0.57\u20130.83) |\n| \u2003\u2003\u2003\u2003Indian DPP (16) | 269<a href=\"#t6fn1\" data-ref-type=\"table-fn\">*<\/a> | IGT | 46 | 2.5 | Metformin (500 mg) | 22 | 0.74 (0.65\u20130.81) |\n| \u2003\u2003\u2003\u2003STOP NIDDM (14) | 1,419 | IGT, FPG \\>5.6 mmol\/l | 54 | 3.2 | Acarbose (300 mg) | 13 | 0.75 (0.63\u20130.90) |\n| \u2003\u2003\u2003\u2003XENDOS (29) | 3,277 | BMI \\>30 kg\/m^2^ | 43 | 4 | Orlistat (360 mg) | 2 | 0.63 (0.46\u20130.86) |\n| \u2003\u2003\u2003\u2003DREAM (15) | 5,269 | IGT or IFG | 55 | 3.0 | Rosiglitazone (8 mg) | 9 | 0.40 (0.35\u20130.46) |\n\nNumber of participants in the indicated comparisons, not necessarily in the entire study.\n\nCalculated from information in the article. DPP, Diabetes Prevention Program; DREAM, Diabetes REduction Assessment with ramipril and rosiglitazone Medication; DPS, Diabetes Prevention Study; STOP NIDDM, Study to Prevent Non-Insulin Dependent Diabetes; XENDOS, Xenical in the prevention of Diabetes in Obese Subjects. This table has been reprinted with permission (30) with some modification.\n\nComponents of the comprehensive diabetes evaluation\n\n<table>\n<tbody>\n<tr>\n<td style=\"text-align: left;\">Medical history\n<ul>\n<li><p>age and characteristics of onset of diabetes (e.g., DKA, asymptomatic laboratory finding)<\/p><\/li>\n<li><p>eating patterns, physical activity habits, nutritional status, and weight history; growth and development in children and adolescents<\/p><\/li>\n<li><p>diabetes education history<\/p><\/li>\n<li><p>review of previous treatment regimens and response to therapy (A1C records)<\/p><\/li>\n<li><p>current treatment of diabetes, including medications, meal plan, physical activity patterns, and results of glucose monitoring and patient's use of data<\/p><\/li>\n<li><p>DKA frequency, severity, and cause<\/p><\/li>\n<li><p>hypoglycemic episodes<\/p>\n<ul>\n<li><p>hypoglycemia awareness<\/p><\/li>\n<li><p>any severe hypoglycemia: frequency and cause<\/p><\/li>\n<\/ul><\/li>\n<li><p>history of diabetes-related complications<\/p>\n<ul>\n<li><p>microvascular: retinopathy, nephropathy, neuropathy (sensory, including history of foot lesions; autonomic, including sexual dysfunction and gastroparesis)<\/p><\/li>\n<li><p>macrovascular: CHD, cerebrovascular disease, PAD<\/p><\/li>\n<li><p>other: psychosocial problems,<a href=\"#t7fn1\" data-ref-type=\"table-fn\">*<\/a> dental disease<a href=\"#t7fn1\" data-ref-type=\"table-fn\">*<\/a><\/p><\/li>\n<\/ul><\/li>\n<\/ul><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Physical examination\n<ul>\n<li><p>height, weight, BMI<\/p><\/li>\n<li><p>blood pressure determination, including orthostatic measurements when indicated<\/p><\/li>\n<li><p>fundoscopic examination<a href=\"#t7fn1\" data-ref-type=\"table-fn\">*<\/a><\/p><\/li>\n<li><p>thyroid palpation<\/p><\/li>\n<li><p>skin examination (for acanthosis nigricans and insulin injection sites)<\/p><\/li>\n<li><p>comprehensive foot examination:<\/p>\n<ul>\n<li><p>inspection<\/p><\/li>\n<li><p>palpation of dorsalis pedis and posterior tibial pulses<\/p><\/li>\n<li><p>presence\/absence of patellar and Achilles reflexes<\/p><\/li>\n<li><p>determination of proprioception, vibration, and monofilament sensation<\/p><\/li>\n<\/ul><\/li>\n<\/ul><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Laboratory evaluation\n<ul>\n<li><p>A1C, if results not available within past 2\u20133 months<\/p><\/li>\n<\/ul><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">If not performed\/available within past year:\n<ul>\n<li><p>fasting lipid profile, including total, LDL- and HDL-cholesterol and triglycerides<\/p><\/li>\n<li><p>liver function tests<\/p><\/li>\n<li><p>test for urine albumin excretion with spot urine albumin\/creatinine ratio<\/p><\/li>\n<li><p>serum creatinine and calculated GFR<\/p><\/li>\n<li><p>thyroid-stimulating hormone in type 1 diabetes, dyslipidemia or women over age 50<\/p><\/li>\n<\/ul><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Referrals\n<ul>\n<li><p>annual dilated eye exam<\/p><\/li>\n<li><p>family planning for women of reproductive age<\/p><\/li>\n<li><p>registered dietitian for MNT<\/p><\/li>\n<li><p>diabetes self-management education<\/p><\/li>\n<li><p>dental examination<\/p><\/li>\n<li><p>mental Health professional, if needed<\/p><\/li>\n<\/ul><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\nSee appropriate referrals for these categories.\n\nCorrelation of A1C with average glucose\n\n| A1C (%) | Mean plasma glucose |        |\n|---------|---------------------|--------|\n|         | mg\/dl               | mmol\/l |\n| 6       | 126                 | 7.0    |\n| 7       | 154                 | 8.6    |\n| 8       | 183                 | 10.2   |\n| 9       | 212                 | 11.8   |\n| 10      | 240                 | 13.4   |\n| 11      | 269                 | 14.9   |\n| 12      | 298                 | 16.5   |\n\nEstimates based on ADAG data of \u223c2,700 glucose measurements over 3 months per A1C measurement in 507 adults with type 1, type 2, and no diabetes. Correlation between A1C and average glucose: 0.92 (42). A calculator for converting A1C results into eAG, in either mg\/dl or mmol\/l, is available at <http:\/\/professional.diabetes.org\/eAG>.\n\nSummary of glycemic recommendations for non-pregnant adults with diabetes\n\n<table>\n<tbody>\n<tr>\n<td style=\"text-align: left;\">A1C<\/td>\n<td style=\"text-align: left;\">&lt;7.0%<a href=\"#t9fn1\" data-ref-type=\"table-fn\">*<\/a><\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Preprandial capillary plasma glucose<\/td>\n<td style=\"text-align: left;\">70\u2013130 mg\/dl (3.9\u20137.2 mmol\/l)<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Peak postprandial capillary plasma glucose<\/td>\n<td style=\"text-align: left;\">&lt;180 mg\/dl (&lt;10.0 mmol\/l)<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Key concepts in setting glycemic goals:\n<ul>\n<li><p>A1C is the primary target for glycemic control.<\/p><\/li>\n<li><p>Goals should be individualized based on:<\/p>\n<ul>\n<li><p>duration of diabetes<\/p><\/li>\n<li><p>age\/life expectancy<\/p><\/li>\n<li><p>comorbid conditions<\/p><\/li>\n<li><p>known CVD or advanced microvascular complications<\/p><\/li>\n<li><p>hypoglycemia unawareness<\/p><\/li>\n<li><p>individual patient considerations<\/p><\/li>\n<\/ul><\/li>\n<li><p>More or less stringent glycemic goals may be appropriate for individual patients.<\/p><\/li>\n<li><p>Postprandial glucose may be targeted if A1C goals are not met despite reaching preprandial glucose goals.<\/p><\/li>\n<\/ul><\/td>\n<td><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\nReferenced to a nondiabetic range of 4.0\u20136.0% using a DCCT-based assay. Postprandial glucose measurements should be made 1\u20132 h after the beginning of the meal, generally peak levels in patients with diabetes.\n\nReduction in 10-year risk of major CVD end points (CHD death\/non-fatal MI) in major statin trials, or substudies of major trials, in diabetic subjects (*n* = 16,032)\n\n| Study (ref.) | CVD prevention | Statin dose and comparator | Risk reduction (%) | Relative risk reduction (%) | Absolute risk reduction (%) | LDL cholesterol reduction |\n|----|----|----|----|----|----|----|\n| 4S-DM (186) | 2\u00b0 | Simvastatin 20\u201340 mg vs. placebo | 85.7 to 43.2 | 50 | 42.5 | 186 to 119 mg\/dl (36%) |\n| ASPEN 2\u00b0 (191) | 2\u00b0 | Atorvastatin 10 mg vs. placebo | 39.5 to 24.5 | 34 | 12.7 | 112 to 79 mg\/dl (29%) |\n| HPS-DM (187) | 2\u00b0 | Simvastatin 40 mg vs. placebo | 43.8 to 36.3 | 17 | 7.5 | 123 to 84 mg\/dl (31%) |\n| CARE-DM (188) | 2\u00b0 | Pravastatin 40 mg vs. placebo | 40.8 to 35.4 | 13 | 5.4 | 136 to 99 mg\/dl (27%) |\n| TNT-DM (189) | 2\u00b0 | Atorvastatin 80 mg vs. 10 mg | 26.3 to 21.6 | 18 | 4.7 | 99 to 77 mg\/dl (22%) |\n| HPS-DM (187) | 1\u00b0 | Simvastatin 40 mg vs. placebo | 17.5 to 11.5 | 34 | 6.0 | 124 to 86 mg\/dl (31%) |\n| CARDS (209) | 1\u00b0 | Atorvastatin 10 mg vs. placebo | 11.5 to 7.5 | 35 | 4 | 118 to 71 mg\/dl (40%) |\n| ASPEN (191) | 1\u00b0 | Atorvastatin 10 mg vs. placebo | 9.8 to 7.9 | 19 | 1.9 | 114 to 80 mg\/dl (30%) |\n| ASCOT-DM (190) | 1\u00b0 | Atorvastatin 10 mg vs. placebo | 11.1 to 10.2 | 8 | 0.9 | 125 to 82 mg\/dl (34%) |\n\nStudies were of differing lengths (3.3\u20135.4 years) and used somewhat different outcomes, but all reported rates of CVD death and non-fatal MI. In this tabulation, results of the statin on 10-year risk of major CVD end points (CHD death\/non-fatal MI) are listed for comparison between studies. Correlation between 10-year CVD risk of the control group and the absolute risk reduction with statin therapy is highly significant (*P* = 0.0007). Analyses provided by Craig Williams, Pharm.D., Oregon Health & Science University, 2007.\n\nSummary of recommendations for glycemic, blood pressure, and lipid control for adults with diabetes\n\n|  |  |\n|----|----|\n| A1C | \\<7.0%<a href=\"#t11fn1\" data-ref-type=\"table-fn\">*<\/a> |\n| Blood pressure | \\<130\/80 mmHg |\n| Lipids |  |\n| \u2003\u2003\u2003\u2003LDL cholesterol | \\<100 mg\/dl (\\<2.6 mmol\/l)<a href=\"#t11fn2\" data-ref-type=\"table-fn\">\u2020<\/a> |\n\nReferenced to a nondiabetic range of 4.0\u20136.0% using a DCCT-based assay.\n\nIn individuals with overt CVD, a lower LDL cholesterol goal of \\<70 mg\/dl (1.8 mmol\/l), using a high dose of a statin, is an option.\n\nDefinitions of abnormalities in albumin excretion\n\n| Category                     | Spot collection (\u03bcg\/mg creatinine) |\n|------------------------------|------------------------------------|\n| Normal                       | \\<30                               |\n| Microalbuminuria             | 30\u2013299                             |\n| Macro (clinical)-albuminuria | \u2265300                               |\n\nStages of CKD\n\n| Stage | Description | GFR (ml\/min per 1.73 m^2^ body surface area) |\n|----|----|----|\n| 1 | Kidney damage<a href=\"#t13fn1\" data-ref-type=\"table-fn\">*<\/a> with normal or increased GFR | \u226590 |\n| 2 | Kidney damage<a href=\"#t13fn1\" data-ref-type=\"table-fn\">*<\/a> with mildly decreased GFR | 60\u201389 |\n| 3 | Moderately decreased GFR | 30\u201359 |\n| 4 | Severely decreased GFR | 15\u201329 |\n| 5 | Kidney failure | \\<15 or dialysis |\n\nKidney damage defined as abnormalities on pathologic, urine, blood, or imaging tests. Adapted and reprinted with permission (248).\n\nTable of drugs to treat symptomatic DPN\n\n| Class | Examples | Typical doses<a href=\"#t14fn1\" data-ref-type=\"table-fn\">*<\/a> |\n|----|----|----|\n| Tricyclic drugs | Amitriptyline | 10\u201375 mg at bedtime |\n|  | Nortriptyline | 25\u201375 mg at bedtime |\n|  | Imipramine | 25\u201375 mg at bedtime |\n| Anticonvulsants | Gabapentin | 300\u20131,200 mg t.i.d. |\n|  | Carbamazepine | 200\u2013400 mg t.i.d. |\n|  | Pregabalin<a href=\"#t14fn2\" data-ref-type=\"table-fn\">\u2020<\/a> | 100 mg t.i.d. |\n| 5-hydroxytryptamine and norepinephrine uptake inhibitor | Duloxetine<a href=\"#t14fn2\" data-ref-type=\"table-fn\">\u2020<\/a> | 60\u2013120 mg daily |\n| Substance P inhibitor | Capsaicin cream | 0.025\u20130.075% applied t.i.d. or q.i.d. |\n\nDose response may vary; initial doses should be low and titrated up.\n\nHas FDA indication for treatment of painful diabetic neuropathy.\n\nPlasma blood glucose and A1C goals for type 1 diabetes by age-group\n\n<table>\n<thead>\n<tr>\n<th rowspan=\"2\" style=\"text-align: center;\">Values by age (years)<\/th>\n<th colspan=\"2\" style=\"text-align: center;\">Plasma blood glucose goal range (mg\/dl)<\/th>\n<th style=\"text-align: center;\"><\/th>\n<th rowspan=\"2\" style=\"text-align: center;\">A 1C<\/th>\n<\/tr>\n<tr>\n<th style=\"text-align: center;\"><\/th>\n<th style=\"text-align: center;\">Before meals<\/th>\n<th style=\"text-align: center;\">Bedtime\/overnight<\/th>\n<\/tr>\n<\/thead>\n<tbody>\n<tr>\n<td style=\"text-align: left;\">Toddlers and preschoolers (0\u20136)<\/td>\n<td style=\"text-align: center;\">100\u2013180<\/td>\n<td style=\"text-align: center;\">110\u2013200<\/td>\n<td style=\"text-align: left;\">&lt;8.5% (but &gt;7.5%)<\/td>\n<td style=\"text-align: left;\">High risk and vulnerability to hypoglycemia<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">School age (6\u201312)<\/td>\n<td style=\"text-align: center;\">90\u2013180<\/td>\n<td style=\"text-align: center;\">100\u2013180<\/td>\n<td style=\"text-align: left;\">&lt;8%<\/td>\n<td style=\"text-align: left;\">Risks of hypoglycemia and relatively low risk of complications prior to puberty<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Adolescents and young adults (13\u201319)<\/td>\n<td style=\"text-align: center;\">90\u2013130<\/td>\n<td style=\"text-align: center;\">90\u2013150<\/td>\n<td style=\"text-align: left;\">&lt;7.5%<\/td>\n<td style=\"text-align: left;\">\u2022Risk of severe hypoglycemia<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: left;\">\u2022Developmental and psychological issues<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: center;\"><\/td>\n<td style=\"text-align: left;\">\u2022A lower goal (&lt;7.0%) is reasonable if it can be achieved without excessive hypoglycemia<\/td>\n<\/tr>\n<tr>\n<td style=\"text-align: left;\">Key concepts in setting glycemic goals:\n<ul>\n<li><p>Goals should be individualized and lower goals may be reasonable based on benefit-risk assessment.<\/p><\/li>\n<li><p>Blood glucose goals should be higher than those listed above in children with frequent hypoglycemia or hypoglycemia unawareness.<\/p><\/li>\n<li><p>Postprandial blood glucose values should be measured when there is a discrepancy between pre-prandial blood glucose values and A1C levels and to help assess glycemia in those on basal\/bolus regimens.<\/p><\/li>\n<\/ul><\/td>\n<td><\/td>\n<td><\/td>\n<td><\/td>\n<td><\/td>\n<\/tr>\n<\/tbody>\n<\/table>","meta":{"dup_signals":{"dup_doc_count":117,"dup_dump_count":47,"dup_details":{"curated_sources":2,"2023-23":1,"2022-40":1,"2022-27":2,"2022-21":4,"2021-49":4,"2021-43":4,"2021-39":5,"2021-31":4,"2021-25":6,"2021-21":3,"2021-17":3,"2021-10":1,"2021-04":4,"2020-50":1,"2020-45":3,"2020-40":3,"2020-34":3,"2020-29":1,"2020-16":4,"2020-10":1,"2020-05":4,"2019-51":2,"2019-47":2,"2019-43":6,"2019-39":2,"2019-26":3,"2019-18":2,"2019-13":2,"2019-09":1,"2019-04":2,"2018-51":1,"2018-47":3,"2018-43":3,"2018-39":1,"2018-34":4,"2018-30":2,"2018-26":2,"2018-22":2,"2018-17":1,"2018-13":1,"2018-09":2,"2018-05":2,"2017-51":1,"2017-47":2,"2023-50":1,"2024-26":2,"2024-22":1}},"file":"PMC2613589"},"subset":"pubmed_central"}
{"text":"abstract: Identifying tradeoffs between hunting and scavenging in an ecological context is important for understanding predatory guilds. In the past century, the feeding strategy of one of the largest and best-known terrestrial carnivores, *Tyrannosaurus rex*, has been the subject of much debate: was it an active predator or an obligate scavenger? Here we look at the feasibility of an adult *T. rex* being an obligate scavenger in the environmental conditions of Late Cretaceous North America, given the size distributions of sympatric herbivorous dinosaurs and likely competition with more abundant small-bodied theropods. We predict that nearly 50 per cent of herbivores would have been within a 55\u201385 kg range, and calculate based on expected encounter rates that carcasses from these individuals would have been quickly consumed by smaller theropods. Larger carcasses would have been very rare and heavily competed for, making them an unreliable food source. The potential carcass search rates of smaller theropods are predicted to be 14\u201360 times that of an adult *T. rex*. Our results suggest that *T. rex* and other extremely large carnivorous dinosaurs would have been unable to compete as obligate scavengers and would have primarily hunted large vertebrate prey, similar to many large mammalian carnivores in modern-day ecosystems.\nauthor: Chris Carbone; Samuel T. Turvey; Jon Bielby\\*Author for correspondence (<email@example.com>).\ndate: 2011-09-07\ninstitute: 1Institute of Zoology, Zoological Society of London, Regent's Park, London NW1 4RY, UK; 2Department of Infectious Disease Epidemiology, Imperial College London, St. Mary's Hospital, Norfolk Place, London W2 1PG, UK\nreferences:\ntitle: Intra-guild competition and its implications for one of the biggest terrestrial predators, *Tyrannosaurus rex*\n\n# Introduction\n\nScavenging behaviour is associated with a diverse range of important ecological and behavioural processes including kleptoparasitism \\[1\\], disease transmission \\[2\\], species distributions \\[3\u20135\\] and nutrient cycling \\[6\\], but despite its importance it remains a poorly understood phenomenon \\[7\\]. Most carnivorous species today rely on a combination of hunting and scavenging \\[7,8\\], although some groups of species subsist almost exclusively on scavenging \\[9\\]. Effective scavengers must possess high movement speeds but low locomotion costs, and the ability to detect carcasses over long distances in order to outcompete other scavengers and predators \\[10\\]. These factors explain why vultures are such highly successful obligate scavengers, while sit-and-wait predators (e.g. viperid snakes) are reliant almost exclusively on hunting \\[7\\]. However, many physical features that help species scavenge successfully (e.g. powerful build; sharp teeth; fast speed; good vision and sense of smell) are also useful for hunting. As a result, it can be difficult to diagnose feeding strategies of species from their physical characteristics alone, especially for extinct carnivores known only from the fossil record \\[11\u201313\\].\n\nIn the past century, the feeding strategy of one of the best known and largest terrestrial carnivores, *Tyrannosaurus rex*, has been the subject of considerable debate and attention (e.g. \\[14\u201320\\]). Proposed support for the species being a primary or obligate scavenger includes its disproportionately tiny forelimbs and apparently reduced eyes \\[16\\], enlarged olfactory bulbs \\[21\\], and large body size and upright stance as a possible adaptation for finding carcasses, although the latter two traits may also have been an advantage for locating live prey \\[15\\]. However, enlarged olfactory bulbs may be associated with behaviours unrelated to food acquisition \\[22,23\\], and the eyes of *T. rex* are in fact large in both relative and absolute terms \\[24\\]. The binocular vision, bite force and impact-resistant teeth of *T. rex* suggest instead that it may have been better adapted for an active predatory lifestyle \\[18,20,25,26\\].\n\nNearly all of the debate over the feeding ecology of *T. rex* has focused on the interpretation of functional morphology rather than on energetic or ecosystem-level considerations. A recent attempt to determine the feasibility of *T. rex* being an obligate scavenger addressed the energetic costs of movement and the amount of meat available from carcasses \\[17\\]. Based on reasonable assumptions of energetic rates, travel speeds and carcass detection ability, this study concluded that *T. rex* could feasibly have survived as an obligate scavenger, although energetically it might have been a marginal strategy. However, as yet no analyses of the feeding ecology of *T. rex* have investigated wider patterns of theropod and other dinosaurian diversity in Late Cretaceous faunas \\[27\\], in particular, the impacts of competition from other sympatric carnivorous theropods, or the size distribution of carcasses available to potential scavengers of that period.\n\nMost extant carnivore guilds contain a number of competing predatory\/scavenging species \\[28\\], and the Late Cretaceous North American dinosaur fauna also consisted of several small-bodied non-avian carnivorous theropods in the Tyrannosauridae, Oviraptorosauria (Caenagnathidae), Deinonychosauria (Dromaeosauridae and Troodontidae) and Coelurosauria incertae sedis \\[27\\]. Although oviraptorosaurs and troodontids may not have been obligate carnivores \\[29,30\\], many of these sympatric theropods were largely or completely carnivorous, and there is evidence for both active predation and late-stage carcass consumption (probable scavenging) by dromaeosaurs \\[31\\]. In modern-day ecosystems, carcasses are typically a rare, widely dispersed and unpredictable resource, and competition among scavengers is often seen as a form of scramble competition \\[7\\]. If the same conditions applied in Late Cretaceous ecosystems, *T. rex* could have been in competition for resources with a range of different-sized theropods, also including juvenile and sub-adult *T. rex* individuals, which may have been relatively abundant compared with adults \\[32\\]. In our analysis, we focus on Late Cretaceous theropod species weighing 16\u201320 kg and up to the size of *T. rex*. Our lower size range was based on evidence from terrestrial mammalian carnivore guilds, because species of this weight range (e.g. wild dogs, jackals, hyenas) are extremely effective hunter\/scavengers \\[8\\] (<a href=\"#RSPB20102497TB1\" data-ref-type=\"table\">table\u00a01<\/a>). If the smaller, more abundant theropod species and *T. rex* age classes were therefore also primarily hunters that scavenged opportunistically, this might have placed extreme pressure on the resources available from scavenging in the Late Cretaceous theropod community.\n\nSpecies and body masses of carnivorous non-avian theropod dinosaurs of Late Cretaceous North America. Midpoint taken when a range of body mass estimates is given in the literature. *Richardoestesia* is only known from jaws and teeth; since maximum tooth size is slightly smaller than that of *Saurornitholestes* \\[33\\], we estimate a body mass of *ca* 20 kg for this genus based on *Saurornitholestes* body mass estimates. Body mass estimates for *Albertosaurus sarcophagus* and *T. rex* taken as mean of all measurements for adult individuals that had reached somatic maturity given in Erickson *et al*. \\[34\\]. *Nanotyrannus* has been interpreted as a possible juvenile *Tyrannosaurus* by some authors \\[35\\], but we provisionally retain it here as a valid taxon following \\[27\\]. Mass categories and the estimated percentage that each size category contributes to the total carnivore guild are also presented (<a href=\"#RSPB20102497F1\" data-ref-type=\"fig\">figure\u00a01<\/a>).\n\n| species | family | mass (kg) | mass categories, kg (with estimated %) | reference |\n|:---|:---|----|:---|:---|\n| *Dromaeosaurus albertensis* | Dromaeosauridae | 16 | 20.6 (79.9%) | \\[36\\] |\n| *Richardoestesia gilmorei* | Coelurosauria incertae sedis | 20 |  | see legend |\n| *Richardoestesia isosceles* | Coelurosauria incertae sedis | 20 |  | see legend |\n| *Saurornitholestes langstoni* | Dromaeosauridae | 23 |  | \\[37\\] |\n| *Velociraptor* sp. | Dromaeosauridae | 24 |  | \\[38\\] |\n| *Troodon formosus* | Troodontidae | 50 | 58.3 (19.0%) | \\[39\\] |\n| *Chirostenotes elegans* | Caenagnathidae | 63 |  | \\[40\\] |\n| *Chirostenotes pergracilis* | Caenagnathidae | 63 |  | \\[40\\] |\n| *Nanotyrannus lancensis* | Tyrannosauridae | 1100 | 1123 (0.9%) | \\[39\\] |\n| *Albertosaurus sarcophagus* | Tyrannosauridae | 1146 |  | \\[34\\] |\n| *Tyrannosaurus rex* | Tyrannosauridae | 5347 | 5347 (0.1%) | \\[34\\] |\n\nInterpreting patterns of relative abundance is notoriously difficult from the fossil record, due to taphonomic biases typically favouring the preservation of large-bodied species \\[41\\]. Skeletons of small-bodied Late Cretaceous dinosaurs are very rare and new species continue to be discovered, whereas the remains of large-bodied dinosaurs typically dominate Late Cretaceous fossil deposits \\[38,42\\]. However, even though they are recognized to have a much lower preservation potential, the observed fossil abundance of small putatively carnivorous theropods (dromaeosaurs and troodontids) is similar to that of tyrannosaurids in North American Late Cretaceous deposits \\[42\\], and many of these deposits show a relatively high abundance of both small theropods and small herbivorous dinosaurs (hypsilophodontids and pachycephalosaurs) \\[43\\]. This suggests that smaller carnivores and herbivores were proportionally much more abundant than tyrannosaurids in Late Cretaceous ecosystems. The abundance of small species within dinosaur guilds would be supplemented by the presence of earlier life stages of larger species. Body mass abundance patterns in fishes, another vertebrate group where individuals are independent before adulthood, are similar to those found in mammals when one considers the overall size classes rather than species weight classes \\[44\\]; assumptions about relationships between abundance and size at a faunal level are therefore likely to be robust to changes in the relative abundance of some of the smaller species.\n\nIn this paper, we explore the potential competitive ability of *T. rex* as an obligate scavenger. We take into account the presence of other carnivorous theropod individuals in Late Cretaceous ecosystems, and the estimated spatial distribution of carcasses of different herbivorous dinosaur species that would be available for scavengers. We make the assumption that, by analogy with vertebrate assemblages in modern-day terrestrial ecosystems, many predators are also opportunistic scavengers, so that there are more potential consumers of carcasses than carcasses available for these predatory guilds. We assess the likelihood that adult *T. rex* could have been an obligate scavenger based on expected carcass encounter rates for this species compared with those for sympatric smaller theropod dinosaurs. We follow Ruxton & Houston \\[17\\] in drawing explicitly on modern ecological analogues, by basing our analysis on the production rate of large carcasses estimated from the terrestrial large mammal community of the well-studied modern-day Serengeti ecosystem \\[9\\]. We also base our predictions of relative abundance and travel speed on well-established body size abundance and speed relationships found in terrestrial mammals \\[45\u201347\\].\n\n# Methods\n\nLists of non-avian dinosaur species that were stratigraphically coeval and geographically sympatric with *T. rex* and which could potentially act as competitors or prey were collected from records of Late Cretaceous (late Campanian to late Maastrichtian) fossil formations in North America \\[27\\]. Only those dinosaur taxa that were positively identified to species or genus level from formations that also contained tyrannosaurid fossils identified as *T. rex* were categorized in our study as being sympatric; all provisional or tentative species or genus identifications were interpreted as being definite identifications for the purposes of analysis. Body mass estimates for these dinosaurs were obtained from the scientific literature (tables\u00a0<a href=\"#RSPB20102497TB1\" data-ref-type=\"table\">1<\/a> and <a href=\"#RSPB20102497TB2\" data-ref-type=\"table\">2<\/a>). We exclude the herbivorous ornithomimosaurs \\[54\\] from our analyses of carnivorous theropods but include caenagnathid oviraptorosaurs and troodontids, following earlier studies \\[55\u201357\\]. Although there may have been minor variations in dinosaur faunal composition across the geographical range of *T. rex* during the Late Cretaceous (e.g. titanosaurid sauropods were restricted to the southwestern United States \\[27\\]), our species list is treated as representing a consistent sympatric faunal unit across this region for the purposes of analysis. A slightly different species list is also available on the Paleobiology Database (<http:\/\/paleodb.org\/cgi-bin\/bridge.pl>). However, as our analysis of theropod performance focuses on a comparison between the largest and smallest theropod size classes (see below), it is robust to such minor changes in faunal composition.\n\nSpecies and body masses of herbivorous dinosaurs of Late Cretaceous North America. Ornithischian and sauropod body masses taken from the midpoint of genus-level estimates in Peczkis \\[48\\]; ornithomimosaur body masses taken from Christiansen \\[37\\]. Species diversity from Weishampel *et al*. \\[27\\] revised following \\[49\u201352\\]; *Dyslocosaurus polyonychius* also excluded as the suggested Late Cretaceous occurrence of this species is probably erroneous \\[53\\]. Mass categories and the estimated percentage each category contributes to the total herbivore guild are based on herbivore mass\u2013abundance relationships found in extant mammalian herbivores (see also <a href=\"#RSPB20102497F1\" data-ref-type=\"fig\">figure\u00a01<\/a>).\n\n| species | family | body mass (kg) | mass categories, kg (with estimated %) |\n|:---|:---|----|:---|\n| *Parksosaurus warreni* | Hypsilophodontidae | 55 | 75 (49.3%) |\n| *Prenocephale edmontonensis* | Pachycephalosauridae | 85 |  |\n| *Ornithomimus velox* | Ornithomimosauridae | 155 | 216 (36.8%) |\n| *Struthiomimus* sp. | Ornithomimosauridae | 175 |  |\n| *Thescelosaurus garbanii* | Hypsilophodontidae | 250 |  |\n| *Thescelosaurus neglectus* | Hypsilophodontidae | 250 |  |\n| *Leptoceratops gracilis* | Leptoceratopsidae | 250 |  |\n| *Montanoceratops* sp. | Leptoceratopsidae | 550 | 700 (6.0%) |\n| *Pachycephalosaurus wyomingensis* | Pachycephalosauridae | 850 |  |\n| *Edmontosaurus annectens* | Hadrosauridae | 2500 | 2500 (6.7%) |\n| *Edmontosaurus regalis* | Hadrosauridae | 2500 |  |\n| *Edmontosaurus saskatchewanensis* | Hadrosauridae | 2500 |  |\n| *Lambeosaurus* sp. | Hadrosauridae | 2500 |  |\n| *Parasaurolophus walkeri* | Hadrosauridae | 2500 |  |\n| *Edmontonia rugosidens* | Nodosauridae | 2500 |  |\n| *Ankylosaurus magniventris* | Ankylosauridae | 5500 | 5000 (0.6%) |\n| *Triceratops horridus* | Ceratopsidae | 8500 | 8500 (0.4%) |\n| *Alamosaurus sanjuanensis* | Saltasauridae | 25 000 | 25 000 (0.2%) |\n\nWe based our estimates of carcass production on data from the Serengeti ecosystem \\[17\\]. We assume that this is a valid ecological comparison, because although ecosystems in the Late Cretaceous may have been more productive and contained more large-bodied species than terrestrial mammalian systems today, they would also be expected to contain a higher abundance of predators, and the fossil record of large-bodied species contains similar predator\u2013prey ratios to those recorded in modern-day ecosystems \\[58\\].\n\nOur approach relies on the use of allometric equations to estimate how factors such as abundance and search capacity vary with body mass. Many biological characteristics of animals are related to body mass and can be described with power equations taking the form of *Y* = *a* \u00d7 *M*^*b*^, where *a* is a constant, *M* is mass (kg) and *b* represents the allometric exponent, which influences the relative change in the value *Y* with changes in body mass. These equations are commonly used in multi-species studies on the influence of body mass on species characteristics \\[59\\].\n\nWe assume that population densities of herbivorous and theropod dinosaurs, *N*~h~ and *N*~t~ (number of individuals km^\u22122^), are related to the following power equations:\n\n![](rspb20102497-e1)\n\nand\n\n![](rspb20102497-e2)\n\nwhere *M*~h~ and *M*~t~ are in kilograms, *c* and *e* are constants corresponding to the number of 1 kg animals km^\u22122^, and *d* and *f* are scaling factors.\n\nTo estimate the relative numbers of Late Cretaceous herbivorous dinosaurs, we used values of *c* = 100 and *d* = \u22120.75, similar to the values estimated from data on herbivorous mammal abundance \\[45\\]. Estimates of carnivorous theropod dinosaur abundances were derived from mammalian carnivore data \\[47\\], where *e* = 1.97 and *f* = \u22120.88. For the purposes of illustration, herbivorous and carnivorous dinosaur species, including different age classes of the larger species, were then grouped into seven and four size classes, respectively, corresponding to the natural body size groupings shown by the data (<a href=\"#RSPB20102497TB1\" data-ref-type=\"table\">tables\u00a01<\/a>, <a href=\"#RSPB20102497TB2\" data-ref-type=\"table\">2<\/a> and <a href=\"#RSPB20102497F1\" data-ref-type=\"fig\">figure\u00a01<\/a>).\n\nTo explore how the presence of sympatric competing theropods affects the likelihood of adult *T. rex* being an obligate scavenger, we used estimates of carcass encounter rate following the model proposed by Ruxton & Houston \\[17\\]. Our approach, however, differs from Ruxton & Houston in that we focus on estimating relative encounter rates of theropod scavengers accounting for size-dependent differences in estimated population density, travel speed and search capacity in relation to the size and likely distribution of available carcasses. Also unlike Ruxton & Houston, we do not explicitly attempt to estimate intake rates of a scavenging *T. rex* but focus our analysis on the relative search areas of an adult *T. rex* compared with other competing species and age classes. Following Ruxton & Houston, we assumed that the forager moves along a path where the search area is defined by the speed *S*~t~ (m s^\u22121^) and detection distance *D*~t~. Because we also need to estimate how these factors vary with body mass, we used the following allometric equations:\n\n![](rspb20102497-e3)\n\nwhere *g* and *h* represent the constant and exponent for speed, respectively, and:\n\n![](rspb20102497-e4)\n\nwhere *j* and *k* represent the constant and exponent for detection range, respectively.\n\nIn order to explore the potential impact of scavenging on carcasses, we calculated the total potential impact of the populations of different theropod species on carcass feeding by multiplying estimated population sizes by individual search rates (see above).\n\nTo estimate the population search rate *NA*~tot~ (km^2^ d^\u22121^) would be:\n\n![](rspb20102497-e5)\n\nwhich expands to:\n\n![](rspb20102497-e6)\n\nThe constants *e*, *g* and *j* in this equation do not affect the relative impacts of scavenging by different theropods, but the scaling exponents have a critical influence on these relative values. Therefore, in order to compare these scanning rates we do not need to know the absolute values of the constants, which is useful as we do not have a good understanding of the absolute values of abundance, speed and detection range for any dinosaur species.\n\nWe initially chose variables for the constants and exponents to match Ruxton & Houston's \\[17\\] predictions for *T. rex* (<a href=\"#RSPB20102497TB3\" data-ref-type=\"table\">table\u00a03<\/a>, scenario 1), but also used alternative values based on more extreme assumptions about relative differences between theropod species (<a href=\"#RSPB20102497TB3\" data-ref-type=\"table\">table\u00a03<\/a>, scenarios 2\u20135). In scenario 1, we set the speed constant *g* = 11.9 and the speed scaling factor *h* = 0.05. We set the detection range constant *j* = 0.034 and the detection range scaling factor *k* = 0.1. These values predict a daily distance for an individual *T. rex* of 18.3 km and a detection distance of 80 m (approximately the values estimated in \\[17\\]), compared with 13.8 km d^\u22121^ and a 50 m detection range for a 20 kg theropod. We increased the exponents for the scaling of both speed and\/or detection range with mass (scenarios 2\u20134) to increase the relative speed and detection distance for *T. rex*. Here we used combinations of values of 0.16 and 0.25 for the speed and detection range scaling factors *h* and *k*, respectively (broadly similar to values described in scaling studies on mammals \\[46,60\\]; <a href=\"#RSPB20102497TB3\" data-ref-type=\"table\">table\u00a03<\/a>). For the purposes of illustration, we calculated an extreme case (scenario 5) where *T. rex* performs at the same level as smaller theropod species, with scaling factor values *h* = 0.35 and *k* = 0.54.\n\nSensitivity analysis of the impact of varying speed and detection range for 'small' (a hypothetical 20 kg species) and 'large' (*T. rex*) theropod dinosaur populations. Scenario 5 shows extreme scaling where *T. rex*-sized theropods would be predicted to be able to cover the same areas as all other theropod species. In this case the constant *sc* was set to 6.6 to reduce the km d^\u22121^ estimate for *T. rex*.\n\n|  | speed |  | detection |  | theropod mass (kg) | km d^\u22121^ | *S*~t~ (km h^\u22121^) | *D*~t~ (km) | ratio^a^ |\n|:---|----|----|:---|:---|:---|----|----|----|----|\n| scenario | const. *g* | exp. *h* | const. *j* | exp. *k* |  |  |  |  |  |\n| 1 | 11.9 | 0.05 | 0.034 | 0.1 | 20 | 13.8 | 1.2 | 0.05 | 60.1 |\n|  |  |  |  |  | 5347 | 18.3 | 1.5 | 0.08 |  |\n| 2 | 11.9 | 0.16 | 0.034 | 0.1 | 20 | 19.2 | 1.6 | 0.05 | 32.5 |\n|  |  |  |  |  | 5347 | 47.0 | 3.9 | 0.08 |  |\n| 3 | 11.9 | 0.05 | 0.034 | 0.25 | 20 | 13.8 | 1.2 | 0.07 | 26 |\n|  |  |  |  |  | 5347 | 18.3 | 1.5 | 0.29 |  |\n| 4 | 11.9 | 0.16 | 0.034 | 0.25 | 20 | 19.2 | 1.6 | 0.07 | 14 |\n|  |  |  |  |  | 5347 | 47.0 | 3.9 | 0.29 |  |\n| 5 | 6.6 | 0.35 | 0.034 | 0.54 | 20 | 18.8 | 1.6 | 0.17 | 1.0 |\n|  |  |  |  |  | 5347 | 133.2 | 11.1 | 3.50 |  |\n\n^a^Ratio of the daily area covered by members of a species of 20 kg over that predicted for the *T. rex* population, based on an estimated density of 177.4 per 1000 km^2^ for the small species and 2.9 per 1000 km^2^ for *T. rex* (see text for details).\n\n# Results\n\nBased on size\u2013abundance relationships found in modern-day herbivorous mammals \\[45\\], we predict that just under 50 per cent of herbivorous carcasses in the *T. rex* community would have been in the 55\u201385 kg size range (<a href=\"#RSPB20102497F1\" data-ref-type=\"fig\">figure\u00a01<\/a>). A productive modern-day ecosystem such as the Serengeti generates 4.38 kg of carcasses km^\u22122^ d^\u22121^ \\[9\\]. If we can assume that levels of carcass abundance were similar in Mesozoic terrestrial ecosystems, a substantial number of dinosaur carcasses would therefore have been present in Late Cretaceous North America, enough to sustain an individual adult *T. rex*. However, it becomes clear that finding these carcasses becomes a problem if we estimate the distributions of different-sized carcasses given this predicted rate of supply. Using the simplified assumption that all size classes of species produce carcasses at the same rate \\[47\\], we can easily calculate an expected distribution based on the Serengeti production rate as an even fraction of the number of herbivorous dinosaur size classes. If every carcass lasted 7 days before becoming consumed (or 14 days at 50% of the original body mass), most carcasses would be very widely dispersed indeed: on average there would be just one 75 kg carcass every 17 km^2^, one 700 kg carcass every 160 km^2^, and one 5 tonne carcass every 1000 km^2^, with a 25 tonne carcass every 5000 km^2^. Clearly, these carcasses would be difficult for individual scavengers to find without travelling great distances.\n\nBased on Ruxton & Houston's calculations \\[17\\], we expect an adult *T. rex* to have covered just under 3 km^2^ a day (18.3 km daily distance \u00d7 2 \u00d7 0.08 km detection distance). Using our estimates of carcass distributions described above, we predict that *T. rex* would have been able to find a 75 kg carcass on average in just under 6 days, and a 700 kg carcass in approximately 55 days; on average, it would have had to search for over a year to find a 5 tonne carcass and five times that to find a 25 ton carcass (<a href=\"#RSPB20102497F2\" data-ref-type=\"fig\">figure\u00a02<\/a>). The potential absence of titanosaurid sauropods, the largest size class of herbivorous dinosaurs, from part of the geographical range of *T. rex* \\[27\\] therefore has little bearing on the availability of carcasses in Late Cretaceous ecosystems.\n\nHow might populations of other smaller carnivorous theropod dinosaurs have impacted these carcasses during the time *T. rex* spent searching for them? Given the much higher predicted relative abundances of smaller theropod species and age classes compared with an adult *T. rex*, one might expect individuals of these species to have had a considerable effect on the amount of meat supplied by carcasses. In <a href=\"#RSPB20102497F2\" data-ref-type=\"fig\">figure\u00a02<\/a>, we show the average expected numbers of theropod dinosaurs that would arrive at a carcass before an adult *T. rex* could reach it. The smaller, more abundant species would be expected to take less time to locate and arrive at a carcass. For a 75 kg carcass, we predict that on average five *Dromaeosaurus*-sized individuals and one *Troodon*-sized individual would already have arrived first, and would be expected to quickly consume most or all of the carcass. Larger carcasses would have attracted more theropods, and again would probably have been fully consumed before the arrival of *T. rex*. Overall for the *T. rex* community, we predict that the smallest carnivorous theropods collectively would have had carcass search rates over 60 times higher than adult *T. rex* under a model of modest scaling exponents for travel speed and detection range (i.e. small differences between theropods), and between 26\u201333 times higher given moderate scaling exponents (<a href=\"#RSPB20102497TB3\" data-ref-type=\"table\">table\u00a03<\/a>). Only if we assume extreme scaling exponents, where an adult *T. rex* travelled over 130 km d^\u22121^ and had a detection range of around 3.5 km, could it have had the same search rate as smaller theropod species (<a href=\"#RSPB20102497TB3\" data-ref-type=\"table\">table\u00a03<\/a>). However, even under these extreme and implausible parameters, competition would have been intense across the Late Cretaceous theropod guild. Although we do not show these calculations, if an adult *T. rex* had been even larger than our body mass estimate and instead nearer to 7 tonnes \\[34\\] following alternative estimates, we would expect approximately 30\u201373-fold lower search rates in this species given conditions in scenarios 1\u20133.\n\n# Discussion\n\nOur results highlight the importance of taking both intraspecific and interspecific competition and the distribution of food resources into account when investigating the ecological viability of alternative carnivore foraging strategies. Ultimately, the nutritional ecology of a carnivore will be determined by the costs and benefits of different available strategies. Most terrestrial mammalian carnivores are primarily hunters, but scavenge opportunistically when carcasses become available \\[7\\]. A heavy reliance on scavenging will only be viable if this strategy can provide a reliable source of food. While our analysis does not examine these costs\/benefits in relation to hunting at an individual level, we clearly show that given the distribution of carcasses and the potential for competition with other members of the carnivorous theropod guild, it is extremely unlikely that an adult *T. rex* could use scavenging as a long-term sustainable foraging strategy. The simple reason for this lies in the predicted overwhelming abundance of smaller-bodied competing theropods, with the smallest of these in the 16\u201325 kg range estimated to represent approximately 80 per cent of individuals in the guild (<a href=\"#RSPB20102497TB2\" data-ref-type=\"table\">table\u00a02<\/a>).\n\nIn modern-day carnivore guilds, groups of African wild dogs (*Lycaon pictus*), wolves (*Canis lupus*) and spotted hyena (*Crocuta crocuta*) weighing 20\u201360 kg can reduce a 70+ kg carcass to scraps of skin and bone extremely rapidly, certainly in under an hour \\[61,62\\]. If similar-sized carnivorous theropods had the same ecological impact in the Late Cretaceous, this would have severely reduced the availability of the most common carcass size available to larger carnivorous species. Although a fully grown *T. rex*, being such a large carnivore, may have dominated carcasses once it had arrived \\[63,64\\], there would have been a limited number of carcasses persisting in the environment to support such a giant obligate scavenger. Even without the impact of small theropods, predicted travel speeds and detection ranges indicate that carcasses would have rotted by the time an adult *T. rex* was typically able to find them. Furthermore, whereas our analysis has only considered potential dinosaur competitors, *T. rex* may also have competed with other species such as giant azhdarchid pterosaurs for access to carcasses \\[12,65\\], suggesting that Late Cretaceous North America would have been even less likely to support a giant obligate terrestrial scavenger than we have predicted. Decomposers would also have played their part in further reducing the amount of meat available to vertebrate scavengers \\[7\\]. In addition, our analyses made the conservative assumption that theropod speed increases with size. Recent studies on theropod locomotion, however, suggest that smaller species may in fact have been faster and more mobile \\[66,67\\]. This is consistent with patterns of home range size in modern carnivores, where the largest home ranges are found in the intermediate body size range \\[68\\]. If movement rates of smaller species were in fact higher than we have assumed, the difference in search rates would be even more extreme.\n\nThe question of whether dinosaurs were endothermic or ectothermic remains one of the great unresolved controversies of palaeontology, and has important implications for reconstructing aspects of dinosaurian trophic ecology such as metabolic requirements and predator : prey ratios \\[69\u201371\\]. Our comparison of the search rates of theropod dinosaurs is sensitive to changes in relative abundance, speed of movement and detection distances. While these variables would be affected by metabolic rate, our main conclusions regarding competition between theropods would not be affected by their thermoregulatory status if we assume that all theropods in the *T. rex* community had similar metabolic rates, a plausible assumption following several lines of evidence (e.g. phylogeny, shared possession of feathers or protofeathers, bipedal stance \\[27,70\\]). If theropods were ectotherms, we would expect *T. rex* to be able to tolerate longer periods of fasting, but we might also expect it to move more slowly and have a lower search rate \\[17\\]. We might expect ectothermic theropods to obtain higher densities for a given density of prey\/carcasses, but this would also lead to higher levels of competition for each carcass. These tradeoffs therefore suggest that our conclusions about the trophic status of *T. rex* should hold true whether it was endothermic or ectothermic.\n\nIn a wider ecological context, it should not be surprising that a fully grown *T. rex* cannot be realistically interpreted as an obligate scavenger. Large-bodied theropods (e.g. neoceratosaurs, carnosaurs, megalosaurs, tyrannosauroids) are characteristic members of all well-studied diverse Jurassic and Cretaceous dinosaur assemblages, and almost all of these species other than *T. rex* have been interpreted uncontroversially as active terrestrial predators \\[24\\]. The only large-bodied theropods that probably had a markedly different nutritional ecology are the fish-eating spinosaurs, which unlike *T. rex* have been reconstructed on the basis of several independent lines of evidence (functional morphology, isotope signatures and stomach contents \\[72\u201374\\]). Several Jurassic and Cretaceous predator guilds (e.g. Morrison Formation, Bahariya Formation) contained multiple lineages of extremely large sympatric predatory theropods \\[24,75\\], for which direct competition for prey must have been mitigated by morphologically or ecologically mediated niche partitioning \\[56\\]. In contrast, tyrannosaurids are the only large-bodied theropods in Late Cretaceous North America, with *T. rex* as the only giant carnivore (see \\[34\\] for tyrannosaurid body masses and growth rates). Since the *T. rex* community also contained a wide range of large-bodied potential prey species, it can be considered extremely unlikely on comparative ecological grounds that this system for some reason lacked a similar theropod 'super-predator'.\n\nMany extant mammalian carnivores feed in groups, especially in open and highly competitive ecosystems, and recent work on Pleistocene carnivores suggests that sociality may make species more effective at defending scavenged carcasses from competitors \\[64,76\\]. If intra-guild competition was similarly high in Late Cretaceous terrestrial ecosystems, we might also expect selection for social grouping behaviour in carnivorous theropods. Interestingly, although reconstructing social behaviour for extinct species in the fossil record is an extremely difficult process, dromaeosaurs have often been interpreted as social carnivores ('pack hunters') on the basis of *Deinonychus*\u2013*Tenontosaurus* fossil assemblages \\[77\\] (although see \\[78\\]), and there is also some taphonomic evidence to suggest gregarious behaviour in the small tyrannosaurid *Albertosaurus* \\[79\\].\n\nWhat can our analysis tell us about other aspects of the predatory behaviour of *T. rex* and other extremely large theropods? Clearly, *T. rex* would have been able to dominate almost all other terrestrial predators at any time or any place. However, given its large mass and the fact that it would have been vastly outnumbered by smaller theropods, it was probably a poor competitor in indirect scramble competition. We have evaluated the potential impact of intra- and interspecific competition between sympatric theropods on scavenging opportunities for *T. rex*, but similar arguments could also be applied to its ability to find hatchling or juvenile dinosaurs or other sources of food if these had an unpredictable and ephemeral distribution, even though these are sometimes interpreted as possible specialist tyrannosaurid prey items \\[80\\]. Given its physical dominance, being one of the largest known theropods \\[36\\], *T. rex* would have been better able to compete for more predictable food resources, and as a large prey specialist it would have had a unique position in its guild at being able to effectively hunt herbivorous dinosaurs that were far larger than would be available to other theropod species. Indeed, although pack hunting may have allowed dromaeosaurs to target larger prey species, taphonomic evidence suggests that small Cretaceous deinonychosaurian theropods probably hunted relatively small-bodied herbivorous dinosaurs such as basal neoceratopsians or hypsilophodontids, or dinosaur hatchlings \\[31,57,77\\], rather than the larger hadrosaurs, neoceratopsians or titanosaurs. As an active large prey specialist, feeding on herbivores of similar or greater mass, *T. rex* would have had feeding habits consistent with prey size selection patterns found in extant mammalian carnivores \\[81,82\\]. We propose that this is the most likely feeding strategy for *T. rex*. Future research into the energetic and behavioural constraints on this extreme giant predator might also help to better understand the evolutionary constraints on carnivory.\n\n## Acknowledgements\n\nWe thank Marcus Clauss, James Farlow and John R. Hutchinson for their helpful comments on previous versions of the manuscript and thank Tom Kennedy for help with the figures.\n\nC.C. acknowledges funding provided by the Deutsche Forschungsgemeinschaft (DFG) for publishing this manuscript. This is contribution no. 104 of the DFG Research Unit 533 'Biology of the sauropod dinosaurs: the evolution of gigantism'.\n\n# References","meta":{"dup_signals":{"dup_doc_count":138,"dup_dump_count":44,"dup_details":{"curated_sources":2,"2018-43":1,"2018-39":1,"2018-34":1,"2018-30":2,"2018-26":2,"2018-22":1,"2018-17":3,"2018-13":1,"2018-09":2,"2018-05":1,"2017-51":3,"2017-47":1,"2017-43":3,"2017-39":1,"2017-34":3,"2017-30":5,"2017-26":4,"2017-22":2,"2017-17":4,"2017-09":3,"2017-04":5,"2016-50":5,"2016-44":7,"2016-40":5,"2016-36":5,"2016-30":5,"2016-26":3,"2016-22":4,"2016-18":3,"2016-07":4,"2015-48":4,"2015-40":3,"2015-35":3,"2015-32":3,"2015-27":3,"2015-22":4,"2015-14":3,"2014-52":3,"2014-49":5,"2018-47":1,"2017-13":4,"2015-18":3,"2015-11":3,"2015-06":4}},"file":"PMC3136829"},"subset":"pubmed_central"}
{"text":"abstract: Speculations on the potential impact of climate change on human health frequently focus on malaria. Predictions are common that in the coming decades, tens \u2013 even hundreds \u2013 of millions more cases will occur in regions where the disease is already present, and that transmission will extend to higher latitudes and altitudes. Such predictions, sometimes supported by simple models, are persuasive because they are intuitive, but they sidestep factors that are key to the transmission and epidemiology of the disease: the ecology and behaviour of both humans and vectors, and the immunity of the human population. A holistic view of the natural history of the disease, in the context of these factors and in the precise setting where it is transmitted, is the only valid starting point for assessing the likely significance of future changes in climate.\nauthor: Paul Reiter\ndate: 2008\ninstitute: 1Insects and Infectious Disease Unit, Institut Pasteur, 25-28 rue du Dr Roux, 75724 Paris, France\nreferences:\ntitle: Global warming and malaria: knowing the horse before hitching the cart\n\n*Malaria is ... a thousand different diseases and epidemiological puzzles. Like chess, it is played with a few pieces, but is capable of an infinite variety of situations*.\n\n*Malaria in Europe, An Ecological Study* \\[1\\]\n\nMan-made climate change has become a defining moral and political issue of our age. Speculations on its potential impact often focus on infectious diseases, and on malaria in particular. Predictions are common that in the coming decades, tens \u2013 even hundreds \u2013 of millions more cases will occur in regions where the disease is already present, and that the vectors and the pathogens will move to higher latitudes and altitudes.\n\nNumerous review publications \\[2-34\\] and substantial media attention have had a major impact on public perceptions of the issue. In most cases, these publications make brief mention of where malaria occurs and how it is transmitted, followed by a succession of statements on the action of temperature, rainfall and other climate variables on specific components of the transmission cycle. These statements \u2013 often valid in themselves \u2013 are used to justify disquieting predictions that are persuasive because they are intuitive. Some are based on mathematical models that select a climate variable (usually temperature), propose a direct interaction with a transmission parameter (e.g. multiplication of the parasite, survival of the vector), and inevitably arrive at the same conclusions. Many focus on the vulnerability of people in poorer countries and place the blame squarely on the activities of the industrial nations. A deplorable trend in the scientific press is the inclusion of a political message, much as in the popular media.\n\nThe great majority of these publications sidestep four factors that are key to the transmission and epidemiology of the disease: the ecology and behaviour of humans, and the ecology and behaviour of the vectors. There is rarely any mention of a fifth, the immunity of the host and of the host population. These factors interact with each other, and are themselves made up of an intricate network of highly variable parameters. The true dynamics of transmission can only be assessed by taking this daunting complexity into account. Moreover, the key climate variables \u2013 temperature, rainfall and humidity \u2013 cannot be viewed independently; the effects of temperature are modified by humidity, the daily range of each may be more significant than the daily mean, brief periods of atypical heat or cold can be more significant than long-term averages, heavy storms can have a different impact than light prolonged rainfall, one year's events may have a significant impact on subsequent years, and so on.\n\nIn summary, a holistic view of the complex natural history of malaria in the precise setting where it is transmitted is mandatory for any speculation on the role of climate variables; the significance of these variables, and their putative role in future climates, can only be assessed in the perspective of this complexity.\n\n# Mathematical models\n\nThe mathematical modelling of malaria began with Ronald Ross, soon after he had demonstrated the transmission cycle of the parasite. Fifty years later, Macdonald modified his approach to build a more comprehensive model that proved useful in developing strategies for control. Both men were aware that their approach was limited; they recognized that factors such as immunity, behaviour and seasonality were not included, mainly because they are hard to quantify. Nevertheless, many of the claims that are made in the climate change debate blithely present Macdonald's simple deterministic model, list its component variables, point out that the extrinsic incubation period is sensitive to temperature and thereby reason that higher temperatures will mean higher rates of transmission. To the layman, this na\u00efve approach may be persuasive because it is intuitive, but Ronald Ross and Macdonald would certainly not have approved.\n\nThe advent of low-cost computers has propelled mathematical modelling into a major role in the description of complex systems, including ecology, epidemiology and public health \\[35\\]. It is now relatively simple to run stochastic models, built of a selected set of variables, with interactions driven by sets of differential equations. Complex systems imply the need for a large number of variables and operators, but as these numbers increase, so does the variance \u2013 and the uncertainty \u2013 of the models. In a sense, therefore, they remain an extension of the intuitive approach because the selection of variables, the assumptions of the frequency distributions that are involved, the mathematical descriptors of the operators, and the constraints on both are made by the modeller. This is not to say that such models are inappropriate. On the contrary, they have become a central component of infectious disease epidemiology, mathematical representations that can be used to explore the dynamics of transmission and the fit of such models to field data. Nevertheless, an inescapable constraint in the case of vector-borne diseases is that the natural history and ecology, of both vector and host, are so complex that it is facile to predict future prevalence and incidence on the basis of temperature.\n\n# Common misconceptions\n\nThere is a widespread misconception that mosquito-borne diseases require tropical temperatures, or at least the temperatures of the warmer temperate regions. A glance at a map of global isotherms reveals that summer temperatures in many temperate regions are at least as high as in the warmest seasons of many regions in the tropics. The crucial difference is that the tropics do not have cold winters. Moreover, if tropical mosquito-borne pathogens are introduced to temperate regions in the right season, they can be transmitted, if suitable vectors are present \\[34\\].\n\nThere is also a misconception that mosquitoes die in winter, and that more die in colder winters, but it is obvious that mosquitoes \u2013 and indeed all life-forms that are native to temperate regions \u2013 have evolved strategies to survive low temperatures. In the tropics, comparable adaptations are necessary for survival during unfavorable dry periods that may last for several years. In both cases, such adaptations merely impose seasonality on transmission. In southern Europe, for example, *Plasmodium falciparum* (the most pathogenic species of malarial pathogen) was once transmitted from July to September \\[36\\]. In Mali, where the disease is still endemic, it is limited to the same three months, the wet season \\[37\\].\n\nThe physical environment is an important modifier of local climate. *Anopheles arabiensis*, an important vector of malaria in Africa, can survive in the Sudan when outdoor temperatures are above 55\u00b0C by hiding in the thatch of buildings in the daytime, feeding after mid-night, and ovipositing at dawn or dusk \\[38\\]. In Lapland, in the past, anopheline species survived the winter in houses and stables, feeding occasionally, and transmitting malaria when outdoor temperatures were below -40\u00b0C \\[39\\].\n\nThese examples underline the limited value of meteorological variables as a guide to the behaviour and geographic range of vector species, and of the pathogens they transmit.\n\n# Malaria in temperate regions\n\nFew people are aware that it is less than forty years since the final eradication of malaria in Europe and the United States. Indeed, the disease was common in the period from the 16^th^ to 18^th^ centuries that climatologists term the Little Ice Age \\[33\\], and data from burial records around the Thames estuary reveal that mortality in \"marsh parishes\" of England was comparable to that in areas of transmission in sub-Saharan Africa today \\[40,41\\].\n\nUntil the mid-19^th^ century, the northern limit of transmission was roughly defined by the present 15\u00b0C July isotherm. Denmark and parts of Sweden suffered devastating epidemics until the 1860s. Incidence diminished thereafter and the disease had essentially disappeared around the turn of the 20^th^ Century. The same was true in Finland, except for a brief recrudescence in 1941, during the Russo-Finnish war. Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a> shows the distribution of malaria cases in Norway between 1860 and 1920. In England, there was a gradual decrease in transmission until the 1880s, after which it dropped precipitously and became relatively rare, except in a short period following World War I. In Germany, transmission also diminished rapidly. After World War I it was mainly confined to a few marshy localities \\[36\\].\n\nThis regional decline is attributed to a combination of factors:\n\n## Ecological change\n\nImproved drainage, reclamation of swampy land for cultivation and the adoption of new farming methods all served to eliminate mosquito habitat (there is an old Italian saying: \"malaria flees before the plough\").\n\n## New farm crops\n\nNew root crops such as turnips and mangel-wurzels were adopted as winter fodder. These enabled farmers to maintain larger numbers of animals, particularly cattle, throughout the year. European vectors of malaria readily bite cattle, so the increasing size of herds diverted mosquitoes from feeding on humans. Human malaria parasites cannot infect cattle, so this diversion reduced the number of infected mosquitoes and infective bites.\n\n## New rearing practices\n\nSelective breeding of cattle, and new introductions (e.g. the Chinese domestic pig), in combination with the new fodder crops, enabled farmers to keep large populations of stock in farm buildings rather than in open fields and woodland. These buildings provided attractive sites for adult mosquitoes to rest and feed, diverting them from human habitation.\n\n## Urbanization and mechanization\n\nRural populations declined as industrialization drew people to urban areas. The increased ratio of cattle to people further reduced the attack rates of mosquitoes on humans.\n\n## Human living conditions\n\nNew building materials and improvements in construction methods made houses more mosquito-proof, especially in winter, another factor that reduced contact with the vector.\n\n## Medical care\n\nGreater access to medical care, and wider use of quinine (in part due to a major reduction in price) reduced the survival rate of the malaria parasite in its human host, thus limiting the infection rate of mosquitoes.\n\nThese factors are a classic illustration of the role of ecology and behaviour in the prevalence and incidence of a mosquito-borne disease. Moreover, the decline cannot be attributed to climate change, for it occurred at the start of the current warming phase. Nor can it be attributed, as is often stated, to deliberate mosquito control, for it came before recognition of the role of the vector.\n\nIn contrast, in countries that lagged in these changes, malaria did not decline \"spontaneously\" \\[36\\]. In the Soviet-block countries, for example, from Poland to eastern Siberia, major epidemics occurred throughout the 19^th^ century and the disease remained one of the principal public health problems for the entire first half of the 20^th^ century. Indeed, in the 1920s, in the wake of massive social and economic disruption, a pandemic swept through the entire Soviet Union. Official figures for 1923\u201325 listed 16.5 million cases, of which not less than 600,000 were fatal \\[36\\]. Tens of thousands of infections, many caused by *P. falciparum*, occurred as far north as the Arctic seaport of Arkhangelsk (61\u00b0 30^'^N).\n\nThe advent of DDT revolutionized malaria control. Cheap, safe, effective applications of the chemical could be targeted at the site where most infections occur \u2013 in the home. Initial efforts in Italy, Cyprus and Greece were so successful that a decision was made to eradicate the disease from all of Europe. The entire continent was finally declared free of endemic malaria in 1975. One of the last countries affected was Holland.\n\nSeveral efficient malaria vectors are indigenous to North America, and are common in many areas. The pathogen probably arrived in the hemisphere in infected European colonists and African slaves. The history of its decline is similar to that of Europe. In the 1880s, it was widespread in nearly all states east of the Rocky Mountains, from the semitropical Gulf Coast states to the northern border and into Canada. It was also present west of the Rocky Mountains, particularly in areas where rainfall is abundant. As living conditions improved, and anti-malarial drugs became more widely available, the incidence of the disease declined. During World War II, the US Military created an Office of Malaria Control in War Areas, responsible for military bases throughout the country. In a symposium published in 1941 \\[42\\], the disease was described as \"of moderate endemic intensity in the sixteen south-eastern states...with scattered epidemics at intervals of approximately seven years.\" In 1946 the United States Congress established a new agency, the Communicable Disease Center. This was the forerunner of the U.S. Centers for Disease Control and Prevention (CDC), and its principal mission was to eradicate malaria from the entire country. Its headquarters were in Atlanta, Georgia, because the southern states were the main region still affected by the disease. When operations began, foci of transmission were already hard to find, but the disease was not totally eliminated until the late 1950s. Today, as in Europe, transmission cycles have been disrupted and the pathogens are absent.\n\nMalaria vectors are, of course, still widespread in Europe and North America, and there are occasional cases of autochthonous transmission when an infected traveler infects local mosquitoes. Given the efficacy of anti-malarial therapy, however, and the increasing sophistication of disease surveillance, it is safe to say that there is no chance of significant transmission arising from such cases, and certainly no chance of the disease becoming endemic, at least under current economic circumstances.\n\n# Malaria in the tropics\n\nNinety percent of the estimated 300\u2013500 million cases of malaria worldwide occur each year in sub-Saharan Africa. Statements on climate change and human health often focus on this region, with predictions that, by the mid-21^st^ century, tens of millions more cases will occur there as a direct result of increasing temperatures (see for example \\[2\\]).\n\nA critical aspect of malaria epidemiology is the concept of *stability*. In much of sub-Saharan Africa, parts of northern India, Indonesia, South America and elsewhere, transmission is termed *stable* because it is fairly constant from year to year; the disease is endemic, but epidemics are uncommon. In other regions, including parts of India, Southeast Asia, Central and South America, the disease is also endemic but is termed *unstable* because transmission can vary greatly from year to year, and the potential for epidemics is high. These terms are, of course, a simplification; there is a wide range of degrees of stability, depending on complex factors in local circumstances.\n\n## Stable endemic malaria\n\nIn regions where the principal malaria vectors are anthropophilic (i.e. prefer to feed on humans) and have a high survival rate, transmission is usually stable. The disease is hard to control because transmission is efficient and transmission rates are so high that most people experience many infective bites per year. Severe illness and mortality occurs mainly among \"new arrivals,\" i.e. children and non-immune immigrants. Older inhabitants have survived multiple infections and maintain a degree of immunity by repeated re-infection. They can have bouts of illness that may be life-threatening, but are usually relatively mild.\n\n## Unstable endemic malaria\n\nThis generally occurs in regions where the anophelines are zoophilic (i.e. bite animals as well as humans), or their survival rates are low, or where both apply. Transmission can vary greatly from year to year, with epidemics separated by many years of relatively low activity during which the overall immunity of the population declines. The factors that precipitate such epidemics are often difficult to identify. The disease may appear suddenly, for no apparent reason, only to disappear again without obvious cause. In general, transmission is relatively inefficient and, therefore, requires high mosquito populations. In theory this implies that the disease is easier to control. In practice, it can be catastrophic, not least because the attack rate is not restricted to \"new arrivals\".\n\nThe vast majority of people in sub-Saharan Africa live in regions of stable transmission. In other words, throughout their lives, people living in the shaded areas of the map (Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>) are regularly exposed to multiple bites from infective mosquitoes, as many as 300 infective bites per year in some regions. Under such circumstances, just as it is impossible to pour more water into a glass that is already full, it is illogical to suggest that increased temperatures will result in an increased incidence of infections. On the other hand, changes in rainfall patterns could alter stability and distribution.\n\nThus, as in temperate regions, the behaviour and ecology of vector and host are the dominant factors in transmission. In many cases, these are attributable to explosive population growth and poverty.\n\n## Birth rate\n\nThe world's population has grown from 2.5 billion in 1950 to 6.7 billion at the time of writing (September 2008). In sub-Saharan Africa, there are now nearly five times as many people (ca. 750 million) as there were in 1955. In some countries, more than half the population is under 15 years of age. High birth rates often give rise to larger communities with higher densities of people, which can lead to a higher attack rate; clinical studies in some parts of Africa quote 998 infections per 1,000 infants.\n\n## Forest clearance\n\nMany malaria vectors breed in open, sunlit pools. Forest clearance provides abundant new habitat for these species, a classic cause of the emergence of malaria problems \\[43\\].\n\n## Agriculture\n\nIrrigation creates an ideal habitat for mass-production of mosquitoes, as can construction of dams for hydroelectric power. Rice cultivation provides an environment for many of the most efficient malaria vectors. Conversely, the cultivation of ground depressions can suppress such vectors and thereby reduce transmission \\[\\].\n\n## Movement of people\n\nInfected people in pursuit of work can introduce malaria to areas where it is rare. Non-immune people are at high risk if they move to areas of transmission. Extensive road building and modern transportation have greatly exacerbated this factor.\n\n## Urbanization\n\nWater storage and inadequate water disposal can provide habitat for mosquitoes, particularly in rapidly expanding urban areas. The absence of cattle can promote stable transmission by forcing zoophilic species to feed on people. Moreover, many tropical cities are surrounded by densely-populated, satellite settlements that are essentially rural in nature.\n\n## Insecticide resistance\n\n*Physiological resistance* to insecticides is common in many regions. *Behavioural resistance* can also be a major problem: species that prefer to feed and rest indoors (*endophilic*) can switch to outdoor (*exophilic*) activity in response to treatment of indoor surfaces with insecticides.\n\n## Drug resistance\n\nIn many parts of the world, the malaria parasite has evolved resistance to commonly used anti-malarial drugs. Substitutes are available, but are much more expensive.\n\n## Degradation of the health infrastructure\n\nLack of funding, institutional difficulties, rapid urbanization and other problems associated with rapid development have eroded the public health sector of many countries. In addition, the AIDS pandemic has overwhelmed the ability of authorities to deal with other diseases.\n\n## War and civil strife\n\nIn times of conflict, mass movements of people, e.g. soldiers and refugees, often promote malaria transmission. The breakdown of public health services, damage to water distribution and drainage systems, and the destruction of homes often exacerbate the situation. High concentrations of people in camps for displaced persons can also be disastrous.\n\n# Highland malaria in the tropics\n\nA topic that is repeatedly cited in the climate change debate, both in the scientific and the popular press, is that warmer temperatures will drive malaria transmission to higher altitudes in the tropical highlands, particularly East Africa. Indeed, environmental activists frequently state that this is already happening.\n\nIt is certainly true that the incidence of malaria has increased in some highland areas, and it is perfectly acceptable to cite temperature as a limiting factor in transmission; vectors such as *Anopheles gambiae* have been reported at altitudes up to 3,000 m above sea level, but endemic malaria disappears above 1,800\u20132,000 m. What is rarely mentioned is that less than two percent of the African continent (including North Africa) is above 2,000 m, and that much of this is so arid that it offers little opportunity for cultivation. Moreover, the history of malaria in highland areas is a compelling example of the dominant role of human behaviour and human ecology, not climate, as the driving factors in the dynamics of transmission.\n\nTwo examples illustrate this point: the Kenya Highlands and the New Guinea Highlands.\n\n# Kenya Highlands\n\nNairobi, capital of Kenya, was founded in 1899 during the construction of a railway from Mombassa, on the coast, to Lake Victoria. The site was chosen because it was on the last stretch of level ground before the steep descent into the Rift Valley. It was a swampy area, and had always been known as an unhealthy locality \"swarming with mosquitoes\" \\[45\\]. Indeed, in 1904, when the town had already grown substantially, a committee of doctors petitioned the colonial administration that the entire municipality be relocated because it was a spawning ground for disease. At that time it marked the upper limits (1,068 m) of malaria transmission in the region, but the disease began to appear at higher altitudes after the clearance of forests for the development of tea estates and the importation of infected labourers \\[46\\]. The first sizeable epidemic, shortly after World War I, was attributed to the return of local soldiers from Tanzania. A major epidemic in 1926 led to recognition that economic development was a key factor in the proliferation of mosquito breeding sites, and hence the source of the increasingly serious problem:\n\n*That there have been no notable general alterations in the domestic environment of the natives of these reserves during recent years is true, but on the other hand it is to be remembered that in every direction roads, and to a lesser extent, railways, have been carried into and through these areas, and always where there are roads, artificial and un-drained excavations are to be found* \\[47\\] quoted by Snow *et al* \\[48\\]\n\nIn the following year, the colonial administration and the Municipal Corporation of Nairobi agreed to spend \u00a340,000 (the equivalent of nearly \u00a3800,000 (US\\$ 1,400,000) in current currency) for eradication of anopheline breeding sites in the Nairobi area. Nevertheless, there were six major epidemics in the city between the two World Wars, with serious rates of transmission extending to the Londiani district (2,250\u20132,490 m) and even at a farm near Mount Timboroa, at 2,490\u20132,550 m \\[49\\].\n\nThe fundamental cause of the upward advance of the disease was widespread deforestation and development as the areas were opened up for large farming ventures. As in the quote above, the construction of roads and railways created innumerable flooded \"borrow pits\" (depressions left by excavation for materials) and also contributed to the dispersal of the mosquito. The introduction of the ox-wagon caused a proliferation of rough cart roads; water in the wheel ruts provided a prolific breeding site for vectors. Mill-dams on rivers interfered with natural drainage \\[49\\].\n\nThese and many other factors were components of a drastic ecological change, and it was this change that brought transmission to the Highlands. The disease continued to be a serious public health problem until the 1950s, when the administration organized an extensive control programme, mainly based on DDT, after which the area was essentially malaria-free until the 1970s.\n\nThe tea-growing estates in the Kericho district (1,780\u20132,225 m) have an extensive medical service for employees and their dependents that was initiated in 1925. Health care at the central hospital of Unilever Kenya Ltd. is extended to some 100,000 inhabitants of the region. However, there is no attempt at mosquito control, and malaria has re-emerged as a serious problem. Epidemics occurred almost every year from 1990 to 1997, with a mean annual attack rate of around 50 percent \\[50\\]. Peak transmission was from May to July, after the principal rainy season and before mean monthly temperatures drop below 18\u00b0C. A questionnaire survey (June 1997) indicated that only 8 percent of patients had travelled to areas with known malaria transmission in the previous 30 days.\n\nThe main factor in this recrudescence may be increased resistance to anti-malarial drugs, as well as the unsupervised use of ineffective medications, but the picture is not entirely clear \\[51\\]. Whatever the cause, the history of multiple epidemics in the earlier part of the century, including many at higher altitudes, makes it unnecessary to infer climate change as a contributory factor. Moreover, a set of well-maintained meteorological records shows no significant change in temperature over recent decades \\[52\\]. Indeed, in a detailed report to the World Health Organization, a group of malaria specialists based in Nairobi dismissed those who claim a global warming link as \"scientific Nostradamuses\" \\[48\\].\n\n# New Guinea Highlands\n\nIn the early 1930s, a human population estimated at 1,000,000, previously unknown to outsiders, was contacted in the mountains of New Guinea. It appeared that these so-called \"Stone Age people\" were malaria-free, and this was attributed to their unique state of isolation. By contrast, the lowland coastal regions were highly malarious.\n\nAt first, the highlanders became a new source of labour for the coastal plantations, but after World War II there was a rapid increase in the number labourers from the lowlands employed by landowners growing *Arabica* coffee and other crops in the mountains. In the late 1940s, government scientists warned that the increasing contact between the regions could bring disaster, for epidemic malaria had already appeared in several highland areas at around 1,500 m \\[53\\]. By the mid-1950s several alarming outbreaks lead to the enforcement of a law that required employers of highlanders working on the coast to supply them with anti-malarials, and to ensure that the medications were actually taken. On repatriation, highlanders were held by the government in compulsory quarantine for two weeks and given curative malaria therapy \\[54,55\\].\n\nThese regulations failed to stop the emergence of the disease and its spread to many isolated valleys. As in Kenya, the increase in prevalence was clearly attributable to a rapid increase of anopheline populations after forest clearance, the influx of people from malarious areas, and the construction of roads, airstrips, plantations, mines, water impoundmentsand other human artifacts. Several vectors were involved, including *Anopheles farauti* and *Anopheles punctulatus* \\[56\\]. These species breed in open sunlit pools and are essentially the local equivalent of *An. gambiae* and *Anopheles funestus* in sub-Saharan Africa.\n\nSubsequent studies suggest that the parasite may have arrived in the highlands as early as the 1940s but did not become evident until forest clearance and development were more widespread. Whatever the chronology, the history of the disease is clearly attributable to the introduction of the parasite to non-immune populations and the proliferation of its vectors as a result of large-scale ecological change.\n\n# Conclusion\n\nSimplistic reasoning on the future prevalence of malaria is ill-founded; malaria is not limited by climate in most temperate regions, nor in the tropics, and in nearly all cases, \"new\" malaria at high altitudes is well below the maximum altitudinal limits for transmission. Future changes in climate may alter the prevalence and incidence of the disease, but obsessive emphasis on \"global warming\" as a dominant parameter is indefensible; the principal determinants are linked to ecological and societal change, politics and economics. There is a critical need for cheap, effective control campaigns, as were implemented during the DDT era. A creative and organized search for new strategies, perhaps based on new technologies, is urgently required, irrespective of future climate change.\n\n# Competing interests\n\nThe authors declare that they have no competing interests.\n\n### Acknowledgements\n\nThis article has been published as part of *Malaria Journal* Volume 7 Supplement 1, 2008: Towards a research agenda for global malaria elimination. The full contents of the supplement are available online at <http:\/\/www.malariajournal.com\/supplements\/7\/S1>","meta":{"dup_signals":{"dup_doc_count":126,"dup_dump_count":55,"dup_details":{"curated_sources":2,"2022-49":1,"2022-33":2,"2021-49":1,"2021-39":1,"2020-50":1,"2020-34":1,"2020-10":2,"2019-51":1,"2019-47":1,"2019-43":1,"2019-35":1,"2019-30":1,"2019-22":1,"2019-13":1,"2019-09":2,"2018-51":2,"2018-43":3,"2018-26":1,"2018-09":1,"2018-05":1,"2017-39":1,"2017-30":1,"2017-26":1,"2017-17":1,"2017-09":5,"2017-04":1,"2016-50":1,"2016-44":2,"2016-40":2,"2016-36":1,"2016-30":2,"2016-22":1,"2016-18":1,"2016-07":6,"2015-48":3,"2015-40":3,"2015-35":2,"2015-32":3,"2015-27":3,"2015-22":3,"2015-14":2,"2014-52":3,"2014-49":2,"2014-42":10,"2014-41":4,"2014-35":6,"2014-23":3,"2014-15":5,"2023-23":1,"2015-18":4,"2015-11":3,"2015-06":3,"2014-10":3,"2013-48":3,"2013-20":3}},"file":"PMC2604878"},"subset":"pubmed_central"}
{"text":"author: David S Pisetsky\ndate: 2012\ninstitute: 1Duke University Medical Center, 151G, Durham VA Medical Center, Durham, NC 27705, USA\ntitle: The LE cell: crime scene or crime stopper?\n\nI have often said that the lupus erythematosus (LE) cell is the most important cell in all of medicine. This judgment does not reflect the cell's putative function in auto-immunity but rather the insights it has provided into the mechanisms of autoimmunity. Discovered fortuitously by Hargraves and colleagues (see Suggested reading below) at the Mayo Clinic, the test to detect this cell is very simple. A sample of peripheral blood is disrupted, incubated, and then spread onto a slide for inspection, after Wright's staining, under an old-fashioned light microscope. If the patient has lupus, then, *voil\u00e0*, the LE cell stands out among the mundane and ordinary red and white cells that populate the blood.\n\nNo doubt, my view of the significance of the LE cell is parochial and relates to my identity as a rheumatologist who has spent a career trying to solve the enigmas of anti-DNA antibodies. The competition for the LE cell is also not that stiff. 'Tart' cells are now forgotten and the Reed-Sternberg cell, though valuable for the diagnosis of Hodgkin's disease, has a very confusing phenotype, representing a B cell of some kind. Truly, what has the Reed-Sternberg cell revealed about the basis of malignancy? In contrast, the study of the LE cell has been a bonanza for autoimmunity.\n\nAs is now recognized, the LE cell phenomenon involves, in the presence of anti-nuclear antibodies (ANAs) and complement, the phagocytosis of a cell nucleus. In peripheral blood, the cell doing the engulfing is likely a neutrophil. For many years, the focus on the LE cell phenomenon was the ANA component and specificities that can bind nuclear molecules; this focus spawned simpler and more widely used tests such as the fluorescence anti-nuclear antibody (FANA) test. Other elements of the LE cell phenomenon raise salient issues that remain prime topics for investigation: the role of complement in the binding and clearance of nuclear material; the activation of neutrophils by nuclear molecules; and, perhaps most important, the existence of nuclear material outside cells.\n\nOf events key to the creation of an LE cell, the translocation of an intact nucleus from the inside to the outside of a cell is perhaps the most mysterious and, for many years, attracted little investigative interest. Physiological enucleation is thought to occur essentially only in the bone marrow during red cell development and in the eye during the formation of the lens. Otherwise, cells quite reasonably hold onto their nuclei with their treasures of genetic information. As suggested by the apparent supply of nuclei available for the LE cell phenomenon, enucleation of hematopoietic cells perhaps occurs readily in the blood, a feature of a cell death process yet to be described and defined. If such a process exists among blood cells, it differs from apoptosis, in which nuclei condense and fragment for safe disposal in the form of apoptotic bodies and microparticles.\n\nThe enucleation central to the LE phenomenon also must differ from the extrusion of nuclear material from neutrophils as neutrophil extracellular traps (NETs). NETs are a unique form of DNA designed to entrap and ensnare bacteria and fungi. The process of the formation of NETs is called NETosis. During NETosis, which can be triggered by a variety of stimuli (including cytokines and Toll-like receptor (TLR) ligands), the nuclear membrane breaks down to allow mixing of DNA with cytoplasmic and granule contents which emerge from cells as strands or mesh. A NET should differ in appearance from the seemingly intact nucleus inside the LE cell. The striking images of NETs by fluorescence microscopy may not be the true story, however. Perhaps, during NETosis, the neutrophil ejects a more intact structure that unravels and disassembles over time once outside the cell to produce the now-classic image of a NET.\n\nWhatever the mechanism for the generation of an LE cell, its close association with lupus, especially the more severe manifestations, has enforced the idea that it represents a pathological process linked to inflammation. Furthermore, the demonstration of LE cells in pleural effusions, for example, places the LE cell directly at the site of pathology. Even if the LE cell is an *in vitro* finding, the evidence for its involvement in pathogenicity, though circumstantial, is compelling. In other words, the LE cell has the tell-tale signs of a crime scene. All that is missing is the yellow tape the police use to keep gawkers away.\n\nI do not watch the *CSI* shows, nor am I an expert in forensics. But I am not ready to declare the case closed and the LE cell guilty as charged. I think that another interpretation is possible, and the LE cell as shown under the microscope signifies something quite different. Let me reconstruct the crime scene in a different way, which I hope will encourage a more general examination of the role of ANAs in lupus. This interpretation stems from exciting studies on the treatment of immune-mediated disease by monoclonal antibodies to nuclear molecules.\n\nAs shown now in many studies, the boundary between foreign (non-self) and self in the induction of innate immunity is not that sharp, and many self molecules can acquire immunological activity. This acquisition occurs during the exposure of these molecules to the immune system during cell injury and death, and the death processes often alter their structure by cleavage, denaturation, or post-translational modification. Such self molecules go by a number of terms; 'danger' molecules is the most dramatic and vivid description of their ability to signal that the organism is in dire straits. These molecules can also alert the immune system to trouble, hence the term alarmin. Finally, these molecules, by analogy to PAMPs, or pathogen-associated molecular patterns, can be called DAMPs, or damage-associated (or death-associated) molecular patterns. Indeed, DAMPs and PAMPs can trigger the same set of sensors, such as the TLRs. The interaction of DAMPs and PAMPs with the same receptors indicates that the immune system does not really discriminate self from foreign or non-self but rather recognizes patterns that are structurally similar despite disparate origins.\n\nAmong the most prominent and potent DAMPs are nuclear molecules, and HMGB1 (high-mobility group box 1 protein) is at the top of the list. HMGB1 is a nonhistone nuclear protein that can bind DNA and serves as the prototype alarmin. HMGB1 leaves cells during cell death (apoptosis and necrosis), activation (especially of macrophages), and NETosis, and its extracellular activity is determined in part by its redox state. In addition, HMGB1 can partner with other molecules, such as lipopolysaccharide or cytokines like interleukin-1, to create new immunostimulatory moieties. Relevant to this discussion are observations that antibodies to HMGB1 are highly effective therapies in animal models of sepsis and arthritis. Coupled with the observations that HMGB1 is elevated in the blood or tissue during inflammatory disease, these findings have made HMGB1 a compelling target in new treatments.\n\nOther nuclear components with immunological activity are nucleosomes, histones, and DNA, although DNA may need a partner like HMGB1 or LL37 for its immunostimulatory activity. Interestingly, histones can directly inflict tissue injury, and monoclonal anti-histone (H1 and H3) antibodies are effective in blocking models of thrombosis, shock, and liver injury in mice. Together, these considerations suggest that nuclear material can be pathogenic and that ANAs (anti-HMGB1 and anti-histones) can be therapeutic. Thus, the binding of an ANA can prevent disease as well as cause it.\n\nIn light of these data, the LE cell can be reconceptualized as a beneficial response, in which the binding of the nucleus facilitates neutralization or removal of a source of DAMPs. This process would be directly analogous to the opsonization and phagocytosis of a bacterium to remove a bundle of PAMPs before they cause harm, such as shock. Another way to look at this is that, if the target antigen in the LE cell were a bacterium instead of nucleus, you would say, 'Way to go, immune system. Get that critter.' Because the target is a nucleus, however, you say, 'Omigod, it's lupus. Get steroids'.\n\nAlthough this line of thought sounds reasonable (at least to me), one can argue that there is an obvious flaw since the LE cell is associated with a more serious set of disease manifestations. If LE cells were preventing inflammation and damage, the opposite relationship could be expected. Thus, owing to a robust protective response, individuals with high numbers of LE cells (a sign of anti-nuclear defense) should display disease that is less severe, not more severe. I would counter this argument by saying that, in those individuals with active disease with LE cells, the system is working in overdrive but has been overwhelmed with nuclear material. This situation may be exacerbated in lupus by inadequate handling of the burden of dangerous molecules. Clearance defects abound in patients with lupus, providing plausibility for this model. In this scenario, the presence of LE cells at sites of inflammation does not mean that the cells are committing a crime but that they are trying to stop or prevent one.\n\nA further issue in conceptualizing the LE cell relates to the heterogeneity in the serological response of patients with lupus. These responses are highly diverse, targeting a multitude of nuclear antigens. Importantly, many ANAs have never been definitively associated with clinical disease manifestations, and some patients can remain 'serologically positive, clinically negative' for many years, seemingly spared active disease despite abundant ANA production. In such patients, this discordancy suggests that the ANAs, though pathological, are neither pathogenic nor nephritogenic. Mouse transfer models show clearly that, although some anti-DNAs can deposit in the kidney, many cannot despite *bona fide* DNA binding. Clearly, interaction with a nuclear antigen does not necessitate a role in promoting disease, or exclude a role in preventing disease. It is of interest, therefore, that between 15% and 25% of otherwise healthy individuals can express an ANA when blood is assayed by sensitive techniques. A high frequency of ANA expression in normal individuals could signify a propensity to autoreactivity in the general population but could also indicate the ordinary operation of immunoregulatory mechanisms to constrain immune responses to nuclear molecules. Unfortunately, the specificity of the ANAs in normal individuals is usually not known, although the characterization of these ANAs could provide additional insights into the way that the immune system protects against the activity of nuclear molecules. Therefore, it is of great interest that, in a study by Li and colleagues (see Suggested reading below), healthy controls without ANA reactivity showed more evidence of upregulated gene expression by array analysis of peripheral blood cells than healthy controls with ANA positivity; those with ANA positivity, however, showed evidence of upregulation of genes associated with the interferon signature. Therefore, further genetic and genomic studies of patients and controls should be informative in elucidating immune disturbances underlying lupus, especially if studies allow for the possibilities that ANAs can act defensively as well as offensively and that serological disturbances can indicate a response to impending danger and not necessarily its cause.\n\nThe remarkable success of anti-HMGB1 antibodies in animal models of disease and the equally impressive but more limited studies on anti-histone antibodies indicate that some ANAs are protective. This protection could result from increased clearance of nuclear antigens or blockade of TLR binding. Since many nuclear molecules with DAMP activity function in the context of a nanostructure or microstructure (for example, microparticles or microvesicles), protective ANAs could indeed pro-mote their phagocystosis after opsonization, effectively reproducing the LE cell phenomenon. This discussion is not just for intellectual amusement but rather to suggest new lines of research into the serology of lupus and other diseases characterized by ANA production. Given the possibility that ANAs can be protective and not just pathogenic (or non-pathogenic), the relationship between serology and clinical disease events may be clarified. Furthermore, the possibility that some ANAs are protective could spur inquiry into new biological therapies based on the paradigm, now so well established with anti-HMGB1 antibodies, that ANAs can block disease by eliminating deleterious nuclear material. If the LE cell phenomenon provided evidence for the prevention of disease and not its induction, then a venerable but clunky assay could have a revival as a screen for agents to stop the immunological crime known as lupus.\n\n# Abbreviations\n\nANA: anti-nuclear antibody; DAMP: damage-associated (or death-associated) molecular pattern; HMGB1: high-mobility group box 1 protein; LE: lupus erythematosus; NET: neutrophil extracellular trap; PAMP: pathogen-associated molecular pattern; TLR: Toll-like receptor.\n\n# Suggested reading\n\nAndersson U, Tracey KJ: **HMGB1 is a therapeutic target for sterile inflammation and infection**. *Annu Rev Immunol* 2011, **29:**139-162.\n\nFarrell K, Jarrett RF: **The molecular pathogenesis of Hodgkin lymphoma**. *Histopathology* 2011, **58:**15-25.\n\nGriffith TS, Ferguson TA: **Cell death in the maintenance and abrogation of tolerance: the five Ws of dying cells**. *Immunity* 2011, **35:**456-466.\n\nGulhane S, Gangane N: **Detection of lupus erythematosus cells in pleural effusion: an unusual presentation of systemic lupus erythematosus**. *J Cytol* 2012, **29:**77-79.\n\nHargraves M, Richmond H, Morton R: **Presentation of two bone marrow elements; the tart**. *Proc Staff Meet Mayo Clin* 1948, **23:**25-28.\n\nHarris HE, Andersson U, Pisetsky DS: **HMGB1: a multifunctional alarmin driving autoimmune and inflammatory disease**. *Nat Rev Rheumatol* 2012, **8:**195-202.\n\nHebederen historical series: **The LE cell**. *Rheumatology* 2001, **40:**826-827.\n\nHreggvidsdottir HS, Lundberg AM, Aveberger AC, Klevenvall L, Andersson U, Harris HE: **High mobility group box protein 1 (HMGB1)-partner molecule complexes enhance cytokine production by signaling through the partner molecule receptor**. *Mol Med* 2012, **18:**224-230.\n\nKeerthivasan G, Wickrema A, Crispino JD: **Erythroblast enucleation**. *Stem Cells Intl* 2011, 139851.\n\nKruse K, Janko C, Urbonaviciute V, Mierke CT, Winkler TH, Voll RE, Schett G, Munoz LE, Hermann M: **Inefficient clearance of dying cells in patients with SLE; anti-dsDNA autoantibodies, MFG-E8, HMGB-1 and other players**. *Apoptosis* 2010, **15:**1098-1113.\n\nLi Q-Z, Zhou J, Lian Y, Zhang B, Branch VK, Carr-Johnson F, Karp DR, Mohan C, Wakeland EK, Olsen NJ: **Interferon signature gene expression is correlated with autoantibody profiles in patients with incomplete lupus syndromes**. *Clin Exp Immunol* 2009, **159:**281-291.\n\nMortensen ES, Rekvig OP: **Nephritogenic potential of anti-DNA antibodies against necrotic nucleosomes**. *J Am Soc Nephrol* 2009, **20:**696-704.\n\nNakano T, Goto S, Lai C-Y, Hsu L-W, Takaoka Y, Kawamoto S, Chiang K-C, Shimada Y, Ohmori N, Goto T, Sato S, Ono K, Cheng Y-F, Chen C-L: **Immunological aspects and therapeutic significance of an autoantibody against histone H1 in a rat model of concanavalin A-induced hepatitis**. *Immunology* 2010, **129:**547-555.\n\nQuan-Zhen L, Karp DR, Quan J, Branch VK, Zhou J, Lian Y, Chong BF, Wakeland EK, Olsen NJ: **Risk factors for ANA positivity in healthy persons**. *Arthritis Res Ther* 2011, **13:**1-11.\n\nPisetsky D: **Cell death in the pathogenesis of immune-mediated diseases: the role of HMGB1 and DAMP-PAMP complexes**. *Swiss Med Wkly* 2011, **141:**w13256.\n\nPark JY, Malik A, Dumoff KL, Gupta PK: **Case report and review of lupus erythematosus cells in cytology fluids**. *Cytopathol* 2007, **35:**806-809.\n\nRamijsen Q, Kuijpers TW, Wirawan E, Lippens S, vandenabeele P, Vanden-Berghe T: **Dying for a cause: NETosis, mechanisms behind an antimicrobial cell death modality**. *Cell Death Differ* 2011, **18:**581-588.\n\nXu J, Zhang X, Pelayo R, Monestier M, Ammollo CT, Semeraro F, Taylor FB, Esmon NL, Lupu F, Esmon CT: **Extracellular histones are major mediators of death in sepsis**. *Nature Med* 2009, **15:**1318-1322.\n\nXu J, Zhang X, Monestier M, Esmon NL, Esmon CT: **Extracellular histones are mediators of death through TLR2 and TLR4 in mouse fatal liver injury**. *J Immunol* 2011, **187:**2626-2631.","meta":{"dup_signals":{"dup_doc_count":123,"dup_dump_count":46,"dup_details":{"curated_sources":2,"2021-39":1,"2021-17":1,"2020-45":1,"2020-40":1,"2020-34":1,"2020-29":1,"2020-24":1,"2020-16":1,"2020-10":1,"2020-05":1,"2019-51":1,"2019-47":1,"2019-43":1,"2019-39":1,"2019-35":1,"2017-17":2,"2017-09":9,"2017-04":1,"2016-50":1,"2016-44":1,"2016-36":9,"2016-30":8,"2016-22":1,"2016-18":1,"2015-48":3,"2015-40":3,"2015-35":3,"2015-32":3,"2015-27":3,"2015-22":3,"2015-14":3,"2014-52":3,"2014-49":3,"2014-42":7,"2014-41":5,"2014-35":5,"2014-23":6,"2014-15":3,"2023-40":1,"2015-18":3,"2015-11":3,"2015-06":3,"2014-10":3,"2013-48":3,"2013-20":3,"2017-13":1}},"file":"PMC3446528"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  The reported health benefits of Korean red ginseng (KRG) include antioxidant, antitumor, antimutagenic, and immunomodulatory activities; however, the effects on oxidative stress have not yet been evaluated. Therefore, we assessed the effect of KRG on antioxidant enzymes and oxidative stress markers in humans.\n  .\n  # Methods\n  .\n  We conducted a randomized, double-blind, placebo-controlled study with three groups, including placebo, low-dose (3\u2009g\/day), and high-dose (6\u2009g\/day), which were randomly assigned to healthy subjects aged 20\u201365\u2009years. Lymphocyte DNA damage, antioxidative enzyme activity, and lipid peroxidation were assessed before and after the 8-week supplementation.\n  .\n  # Results\n  .\n  Fifty-seven subjects completed the protocol. Plasma superoxide dismutase (SOD) activity after the 8-week KRG supplementation was significantly higher in the low-and high-dose groups compared to baseline. Plasma glutathione peroxidase (GPx) and catalase activities were also increased after the high-dose supplementation. Furthermore, the DNA tail length and tail moment were significantly reduced after the supplementation (low-dose and high-dose), and plasma oxidized low-density lipoprotein (LDL) levels were reduced in low-dose and high-dose groups, but increased in the placebo group. The net changes in oxidized LDL after the supplementation differed significantly between both KRG supplementation groups and the placebo group. Net changes in GPx, SOD and catalase activities, and DNA tail length and tail moment were significantly different between the high-dose group and the placebo group. Additionally, the net changes in urinary 8-epi-PGF~2\u03b1~ were significantly different between the KRG supplementation groups and the placebo group.\n  .\n  # Conclusions\n  .\n  KRG supplementation may attenuate lymphocyte DNA damage and LDL oxidation by upregulating antioxidant enzyme activity.\nauthor: Ji Young Kim; Ju Yeon Park; Hee Jung Kang; Oh Yoen Kim; Jong Ho Lee\ndate: 2012\ninstitute: 1Department of Culinary Nutrition, Woosong University, Daejeon, Korea; 2Division of Metabolic Diseases, Center for Biomedical Science, Korea National Institute of Health, Chungbuk, Korea; 3Department of Food and Nutrition, Research Laboratory for Clinical Nutrigenetics\/Nutrigenomics, Yonsei University, Seoul, Korea; 4Department of Food Science and Nutrition, Dong-A University, Busan, Korea; 5Department of Food and Nutrition, Brain Korea 21 Project, College of Human Ecology, Yonsei University, Seoul, Korea; 6Department of Food and Nutrition, Yonsei University, 134 Shinchon-Dong, Sudaemun-Gu, Seoul, 120-749, Korea\nreferences:\ntitle: Beneficial effects of Korean red ginseng on lymphocyte DNA damage, antioxidant enzyme activity, and LDL oxidation in healthy participants: a randomized, double-blind, placebo-controlled trial\n\n# Background\n\nOxidative stress describes a set of intracellular or extracellular conditions that lead to the chemical or metabolic generation of reactive oxygen\/nitrogen species, including superoxide radicals, hydrogen peroxide (H~2~O~2~), hydroxyl radicals, singlet oxygen, lipid hydroperoxides, peroxynitrite, and related species \\[1\\]. Reactive oxygen\/nitrogen species can cause oxidative damage to essential cellular constituents, such as membrane lipids, proteins, and DNA, which may ultimately result in cell death \\[2\\]). Aerobic organisms attempt to protect themselves against oxidative damage with exogenous antioxidants (e.g., vitamin E, ascorbic acid, and carotenoids) obtained through the diet as well as endogenous antioxidants (e.g., glutathione, glutathione peroxidase (GPx), catalase, and superoxide dismutase (SOD)). Furthermore, oxidative damage mediated by the reactive oxygen species listed above is known to contribute to the aging process and the pathogenesis of cancer, cardiovascular disease, and other degenerative diseases \\[3\\].\n\nGinseng (*Panax ginseng* C.A. Meyer) is a perennial herb belonging to the *Panax* family (Araliaceae). Its roots are used as a general tonic in traditional oriental medicine to increase health, longevity, and vitality, especially in the elderly \\[4\\]. White Ginseng is known to exert a number of effects, such as suppressing the growth and metastasis of melanoma (B16) \\[5\\] and activating GPx and SOD to protect against free radical damage \\[6\\]. Interest in Korean red ginseng (KRG) has grown recently, as it is one of the foods that can easily be consumed daily in Korea. Red ginseng is produced by steaming and drying fresh ginseng, a process that chemically transforms the components and alters their biological properties \\[7\\]. The non-saponin components of red ginseng are thought to improve learning and memory \\[8\\], while acid polysaccharides activate natural killer cells and stimulate interferon production \\[9\\]. Mochizuki et al. \\[10\\] reported that 20(R)- and 20(S)-ginsenoside-Rg3, the ginseng saponins, possess the ability to inhibit the lung metastasis of cancer by inhibiting the invasion and adhesion of tumor cells. According to Kubo and Tong \\[11\\], red ginseng extract influenced tumor cell lysosomes by promoting the uptake of mitomycin C, which has cytocidal effects. Kim et al. \\[12\\] also found that ginseng extracts scavenged 40% of hydroxyl radicals at 0.1\u2009mg\/ml and completely scavenged superoxide radicals at 2\u2009mg\/ml. In addition, KRG was also shown to affect immunological markers in individuals with human immunodeficiency virus \\[13\\]. Although a number of pre-clinical studies have reported medicinal benefits of KRG including antioxidant, antitumor, antimutagenic \\[10-12\\] and a clinical study has reported immunomodulatory actions \\[13\\], the effects of KRG on oxidative stress and lipid peroxidation have not been properly evaluated in healthy subjects. Thus, we conducted a randomized, double-blind, placebo-controlled clinical trial in healthy subjects to evaluate the effects of KRG supplementation on biomarkers of oxidative stress and lipid peroxidation, which included lymphocyte DNA damage, urinary 8-epi-prostaglandin F~2\u03b1~ (PGF~2\u03b1~), plasma oxidized low-density lipoprotein (LDL), and antioxidant enzymes.\n\n# Methods\n\n## Subjects\n\nSixty-nine healthy subjects (aged 20\u201365\u2009years) were recruited during routine check-ups at a health promotion center at Yonsei University Hospital from May to November in 2009. Participants were asked to visit four times (1\u2009week before and at 0, 4, and 8\u2009weeks). Blood samples were taken at weeks 0 and 8, and diet and capsule intakes were assessed at weeks 0, 4, and 8. Healthy subjects who either used cigarettes or consumed alcohol (or both) were enrolled in this study at the Laboratory of Clinical Nutrigenetics\/Nutrigenomics at Yonsei University. The exclusion criteria were the consumption of more than three servings of vegetables or fruit per day, antioxidant or vitamin\/mineral supplementation, history of chronic disease (e.g., diabetes, heart disease, renal disease), or the use of medication (e.g., lipid-lowering or antihypertensive medications, anti-platelets, or disease-related medications). The participants were interviewed to determine their smoking and drinking habits. Smoking status was categorized as \"current smoker\" or \"nonsmoker,\" and alcohol consumption was categorized as \"current drinker\" or \"nondrinker.\" Nonsmokers or nondrinkers included both ex-consumers and those who had never consumed. Ex-smokers and ex-drinkers were defined as subjects who stopped smoking or drinking at least 1\u2009year prior to participating in the study. Current smokers were asked the number of cigarettes smoked per day. Current drinkers were asked about the type and amount of alcohol consumed per day, and the amount of alcohol was then calculated as grams per day.\n\nThe study subjects number calculation assumed a two-tailed alpha\u2009=\u20090.05 and 1-\u03b2\u2009=\u200990% to detect a 10% difference with a standard error of 0.28 in the comet assay which is primary outcome and an attrition rate (20%). Biomarkers for testing antioxidant\/oxidative effects and the number of subjects were determined by following the criteria recommended by the Korea Food and Drug Administration. Of the 69 study subjects, 12 subjects (placebo group, *n*\u2009=\u20094; low-dose group \\[3\u2009g KRG\/day\\], *n*\u2009=\u20094; high-dose group, \\[6\u2009g KRG\/day\\], *n*\u2009=\u20094) discontinued the study for personal reasons. No adverse reactions (e.g., fever, hot flush, nausea, vomiting, diarrhea) due to KRG supplementation were observed among the 57 subjects who completed the study.\n\n## Test capsule and study design\n\nIdentical-looking capsules contained red ginseng (low dose, 300\u2009mg; high dose, 600\u2009mg) or placebo (300\u2009mg of KRG-flavored capsule containing corn starch). Red ginseng and placebo capsules were provided by the Korea Ginseng Cooperation (KGC, Daejeon, Korea). The red ginseng contained 16.58\u2009mg\/g total ginsenosides, and the ratio of protopanaxadiol ginsenosides (Rb1, Rb2, Rc, Rd, and Rg3) to protopanaxatriol ginsenosides (Rg1, Re, and Rf) was 1.65. Analyses of common ginsenosides were performed in quadruplicate using standard HPLC-UV techniques \\[14\\] at the Korean Ginseng Research Institute in Daejeon, Korea.\n\nThis study design was the randomized double-blind, placebo-controlled intervention trial for 8\u2009week and was approved by the Institutional Review Board of Yonsei University (#RA-2009-650). After written informed consent was obtained, subjects were randomly assigned to placebo (n\u2009=\u200923) or KRG (low-dose \\[n\u2009=\u200924\\] or high-dose \\[n\u2009=\u200922\\]) groups. All subjects were asked to take 10 capsules in total per day immediately after any main meals. For example, three capsules after breakfast, three capsules after lunch, and four capsules after dinner. Compliance of KRG consumption was assessed at the end of the study by counting capsules remaining and self-recording. If compliance was beneath 75%, the subject dropped out. All participants were encouraged to maintain their usual lifestyle and dietary habits.\n\n## Assessment of dietary intake and level of physical activity\n\nThe subjects' usual diet information was obtained using both a 24-hour recall method and a semi-quantitative food frequency questionnaire (SQFFQ), which has been tested previously for validity \\[15\\]. We used the former to carry out analyses and the latter to check if the data collected by the 24-hour recall method was representative of the usual dietary pattern.\n\nAll the subjects were given written and verbal instructions by a registered dietitian for the completion of a 3-day (two weekdays and one weekend) dietary record every 4\u2009weeks. On the sheet, the subjects were instructed to record the amount of food before ingestion and any remaining after ingestion by weighing the foods. One week before the start of the study, all the participants were advised to continue their usual diet for a week and were instructed to complete a 3-day dietary record as the baseline measurement, which was compared with the record obtained by 24-hour recall methods at a previous visit to check their records. The participants were also instructed to record their physical activity in a diary for 24 hours every 4\u2009weeks. To check participants' compliance during the whole study period, the dietitian interviewed them biweekly by telephone. During the study period, all participants were also encouraged to maintain their usual lifestyle.\n\nDietary energy values and nutrient content from the 3-day food records were calculated using the Computer Aided Nutritional analysis program (CAN-pro 2.0, Korean Nutrition Society, Seoul, Korea). Total energy expenditure (kcal\/day) was calculated from activity patterns, including basal metabolic rate, physical activity for 24 hours \\[16\\], and specific dynamic action of food. Basal metabolic rates for each subject were calculated with the Harris-Benedict equation \\[17\\].\n\n## Anthropometric parameters, blood pressure, and blood and urine collection\n\nBody weight and height were measured without clothes or shoes in the morning to determine the body mass index (BMI; kg\/m^2^). Blood pressure was measured with an automatic blood pressure monitor (TM-2654, A&D, Tokyo, Japan) in triplicate using the left arm of a seated subject after a 20-min rest. After a 12\u2009h fasting period, venous blood specimens were collected in EDTA-treated and plain tubes, centrifuged to produce plasma or serum, and stored at \u221270\u00b0C until analysis. Urine was collected in polyethylene bottles containing 1% butylated hydroxytoluene after 12\u2009h of fasting. The tubes were immediately covered with aluminum foil and stored at \u221270\u00b0C until analysis.\n\n## Serum lipid profile and fasting glucose\n\nFasting total cholesterol and triglycerides were measured using commercially available kits on a Hitachi 7150 Autoanalyzer (Hitachi Ltd., Tokyo, Japan). After precipitation of chylomicrons with dextran sulfate magnesium, concentrations of LDL and high-density lipoprotein (HDL) cholesterol in the supernatants were determined enzymatically. In participants with serum triglyceride concentrations below 400\u2009mg\/mL, LDL cholesterol was indirectly measured using the Friedewald formula. Fasting glucose levels were determined by the glucose oxidase method using a Beckman glucose analyzer (Beckman Instruments, Irvine, CA, USA).\n\n## Safety parameters\n\nSerum Blood Urea Nitrogen (BUN) and creatinine concentrations were measured by a colorimetric method using commercially available kits on a Hitachi 7180 Autoanalyzer (Hitachi Ltd., Tokyo, Japan). Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured by a kinetic UV method based on recommendations by the International Federation of Clinical Chemistry (IFCC) using commercially available kits on a Hitachi 7180 Autoanalyzer (Hitachi Ltd., Tokyo, Japan). A complete blood count (CBC) test was determined using the HOROBA ABX diagnostic (HORIBA ABX SAS, Parc Euromedicine, France).\n\n## Alkaline comet assay for DNA damage\n\nAs a primary outcome measure, DNA damage was assessed using the comet method described by Green et al. \\[18\\]. Whole blood was mixed with phosphate buffered saline and poured gently over a lymphocyte separation solution (Histopaque-1077, Sigma Chemical Co., Korea). After centrifugation at 1,450\u2009rpm for 4\u2009min, lymphocytes were transferred to another tube, mixed with 0.7% low-melting agarose, and then added to slides pre-coated with 0.5% agarose. The slides were immersed in freshly prepared cold lysing solution and subjected to electrophoresis. The slides were then washed with neutralizing buffer and treated with ethanol. Tail length (\u03bcm) and tail moment (% of DNA in the tail \u00d7 tail length) were evaluated using image analysis software (Komet 5.0, Kinetic Imaging, UK) and fluorescence microscopy (Leica, Germany) equipment with filters. Images from 50 cells from each slide were analyzed. All steps were performed in dim light, and the electrophoresis tank was covered with black paper to avoid additional light-induced DNA damage.\n\n## Antioxidative enzyme activities\n\nPlasma and erythrocyte antioxidative enzyme activities were measured as secondary outcomes. Plasma and erythrocyte SOD activity was determined based on the generation of superoxide radicals produced by xanthine and hypoxanthine. Superoxide radicals react with tetrazolium chloride salts to produce a red formazan dye. SOD activity was estimated by the inhibition of this colorimetric reaction using an assay kit (Cayman Chemical, Ann Arbor, MI, USA), and the absorbance at 450\u2009nm was measured with a Wallac Victor^2^ multilabel counter (Perkin Elmer Life Sciences, Turka, Finland). The intra-assay and inter-assay coefficients of variance were 4.43% and 8.26%, respectively. Plasma GPx activity was indirectly measured using an assay kit that involved a coupled reaction with glutathione reductase (Cayman Chemical). Oxidized glutathione, generated by the reduction of hydroperoxide by GPx, was restored to its reduced state by glutathione reductase and nicotinamide adenine dinucleotide phosphate (NADPH). NADPH oxidation was monitored at a lower 340\u2009nm absorbance, which was measured with the Wallac Victor^2^ multilabel counter. The intra-assay and inter-assay coefficients of variance were 6.01% and 7.68%, respectively. Plasma and erythrocyte catalase activity was measured based on the generation of formaldehyde from methanol in the presence of H~2~O~2~ using an assay kit (Cayman Chemical). Formaldehyde forms a bicyclic heterocycle with the chromogen Purpald, which changes from colorless to a purple color when oxidized. The absorbance was measured at 540\u2009nm with the Wallac Victor^2^ multilabel counter. The intra-assay and inter-assay coefficients of variance were 5.52% and 6.78%, respectively.\n\n## Plasma oxidized LDL concentration and Urinary 8-epi-PGF~2\u03b1~\n\nAs one of the secondary outcome measures, plasma oxidized LDL concentration and urinary 8-epi-PGF~2\u03b1~ excretion. Plasma oxidized LDL was determined with an enzyme immunoassay (Mercodia, Uppsala, Sweden), and the color reaction was measured at 450\u2009nm with the Wallac Victor^2^ multilabel counter. The intra-assay and inter-assay coefficients of variance were 6.52% and 8.15%, respectively. Urinary 8-epi-PGF~2\u03b1~ was measured using a colorimetric enzyme immunoassay (Bioxytech urinary 8-epi-PGF~2\u03b1~ assay kit; OXIS International Inc., OR, USA). The resulting color was measured at 650\u2009nm using the Wallac Victor^2^ multilabel counter. Urinary creatinine was determined using a Hitachi 7180 Autoanalyzer (Hitachi Ltd., Tokyo, Japan) by the alkaline picrated (Jeffe) reaction. Urinary 8-epi-PGF~2\u03b1~ concentrations were expressed as pmol\/mmol of creatinine. The intra-assay and inter-assay coefficients of variance were 4.32% and 9.57%, respectively.\n\n## Statistical analysis\n\nStatistical analyses were performed with SPSS version 12.0 for Windows (Statistical Package for the Social Sciences (SPSS) Inc., Chicago, IL, USA). We used the Wilcoxon signed-rank test to determine within-group differences of biochemical parameters before (baseline) and after the intervention. The nonparametric Kruskal-Wallis test and analysis of covariance (ANCOVA) using the general linear model (GLM) with adjustments for age, sex, BMI, smoking, drinking, systolic BP, diastolic BP, and baseline values were used to compare the three groups. Spearman's correlation coefficients were used to determine relationships between variables. After determining whether data was normally distributed by the Kolmogorov-Smirnov and Shapiro-Wilk W tests, a logarithmic transformation was applied to non-normal data before statistical analysis. For descriptive purposes, the results are expressed as untransformed and unadjusted mean values. Continuous variables are expressed as mean \u00b1 SEM, and categorical variables are expressed as absolute numbers and percentages. A two-tailed value of *P* less than 0.05 was considered significant.\n\n# Results\n\n## Baseline clinical characteristics and dietary intake\n\nTable <a href=\"#T1\" data-ref-type=\"table\">1<\/a> shows the baseline clinical characteristics of the study subjects. Anthropometric and biochemical parameters before treatment were not significantly different among the groups. In addition, KRG supplementation had no significant effect on BMI and blood pressure. After treatment, serum lipid profiles (e.g., total cholesterol, triglyceride, HDL- and LDL-cholesterol) and safety parameters (e.g., liver and kidney function tests, complete blood count) did not differ among groups (data not shown). In addition, no significant differences were observed for daily nutrient intake before and after the intervention in all the groups (Table <a href=\"#T2\" data-ref-type=\"table\">2<\/a>).\n\nAnthropometric parameters and blood pressure before and after intervention\n\n| \u00a0 | **Placebo (n\u2009=\u200919)** | **Low-dose (n\u2009=\u200919)** | **High-dose (n\u2009=\u200919)** | ***P***^***a)***^ | \u00a0 | \u00a0 | \u00a0 |\n|:---|----|----|----|----|----|----|----|\n| Age (yr) | 37.0 \u00b1 2.24 | 36.4 \u00b1 1.73 | 35.3 \u00b1 1.68 | 0.910 | \u00a0 | \u00a0 | \u00a0 |\n| Male \/ Female (n) | 7\/12 | 7\/12 | 9\/10 | 0.747 | \u00a0 | \u00a0 | \u00a0 |\n| Body mass index (BMI; kg\/m^2^) | \u00a0 | \u00a0 | \u00a0 | \u00a0 | \u00a0 | \u00a0 | \u00a0 |\n| \u2003Pre-treatment | 23.4 \u00b1 0.60 | 24.0\u00b10.93 | 23.5\u00b10.70 | \u00a0 | \u00a0 | \u00a0 | \u00a0 |\n| \u2003Post-treatment | 23.6 \u00b1 0.67 | 24.3\u00b10.93 | 23.6\u00b10.65 | \u00a0 | \u00a0 | \u00a0 | \u00a0 |\n| \u2003Change | 0.27\u00b10.16 | 0.26\u00b10.15 | 0.11\u00b10.14 | 0.850 | \u00a0 | \u00a0 | \u00a0 |\n| Cigarette smoker, n (%) | 8 (42.1) | 6 (31.6) | 8 (42.1) | 0.948 | \u00a0 | \u00a0 | \u00a0 |\n| Alcohol drinker, n (%) | 15 (78.9) | 13 (68.4) | 13 (68.4) | 0.706 | \u00a0 | \u00a0 | \u00a0 |\n| Blood pressure (mmHg) | \u00a0 | \u00a0 | \u00a0 | \u00a0 | \u00a0 | \u00a0 | \u00a0 |\n| Systolic blood pressure (mmHg) |  |  |  |  | \u00a0 | \u00a0 | \u00a0 |\n| \u2003Pre-treatment | 115.3\u00b12.92 | 118.8\u00b13.22 | 117.5\u00b12.32 | \u00a0 | \u00a0 | \u00a0 | \u00a0 |\n| \u2003Post-treatment | 114.7\u00b12.08 | 118.8\u00b13.10 | 118.2\u00b11.94 | \u00a0 | \u00a0 | \u00a0 | \u00a0 |\n| \u2003Change | \u22120.55\u00b11.97 | \u22120.03\u00b10.19 | 0.74\u00b11.65 | 0.740 | \u00a0 | \u00a0 | \u00a0 |\n| Diastolic blood pressure (mmHg) |  |  |  |  | \u00a0 | \u00a0 | \u00a0 |\n| \u2003Pre-treatment | 76.6\u00b11.83 | 80.6\u00b11.98 | 78.4\u00b11.81 | \u00a0 | \u00a0 | \u00a0 | \u00a0 |\n| \u2003Post-treatment | 75.6\u00b11.93 | 77.3\u00b12.14^\\*^ | 76.2\u00b11.71 | \u00a0 | \u00a0 | \u00a0 | \u00a0 |\n| \u2003Change | \u22120.92\u00b11.88 | \u22123.29\u00b11.28 | \u22122.16\u00b11.75 | 0.599 | \u00a0 | \u00a0 | \u00a0 |\n\nResults are expressed as mean\u2009\u00b1\u2009standard error of the mean (SEM) or n (%).\n\nBaseline values for the placebo and test groups were not significantly different (nonparametric Kruskal-Wallis test).\n\n^\\*^*P*\u2009\\<\u20090.05 compared with the baseline in each group (Wilcoxon signed-rank test).\n\n^a)^ Evaluated by nonparametric Kruskal-Wallis test.\n\nDaily energy expenditure and nutrient intake before and after intervention\n\n| \u00a0 | **Placebo (n\u2009=\u200919)** | **Low-dose (n\u2009=\u200919)** | **High-dose (n\u2009=\u200919)** | ***P***^***a)***^ |\n|:---|----|----|----|----|\n| Total energy expenditure (kcal) | \u00a0 | \u00a0 | \u00a0 | \u00a0 |\n| \u2003Pre-treatment | 2329.3\u00b178.9 | 2331.8\u00b190.5 | 2299.5\u00b157.6 | \u00a0 |\n| \u2003Post-treatment | 2268.3\u00b176.7 | 2273.2\u00b198.1 | 2300.0\u00b168.1 | \u00a0 |\n| \u2003Change | \u221261.0\u00b136.9 | \u221258.6\u00b133.6 | 0.50\u00b132.9 | 0.317 |\n| Estimates of dietary nutrient intake | \u00a0 | \u00a0 | \u00a0 | \u00a0 |\n| Total calorie intake (kcal) | \u00a0 | \u00a0 | \u00a0 | \u00a0 |\n| \u2003Pre-treatment | 1789.2\u00b179.6 | 1726.4\u00b1104.4 | 1847.9\u00b1114.2 | \u00a0 |\n| \u2003Post-treatment | 1892.9\u00b172.9 | 1820.1\u00b181.9 | 1966.3\u00b197.8 | \u00a0 |\n| \u2003Change | 103.7\u00b179.0 | 93.7\u00b169.1 | 118.4\u00b175.1 | 0.991 |\n| Carbohydrate (%) | \u00a0 | \u00a0 | \u00a0 | \u00a0 |\n| \u2003Pre-treatment | 60.0\u00b11.57 | 60.8\u00b11.09 | 62.4\u00b10.92 | \u00a0 |\n| \u2003Post-treatment | 61.2\u00b11.66 | 59.9\u00b11.72 | 61.1\u00b11.34 | \u00a0 |\n| \u2003Change | 1.16\u00b11.78 | \u22120.94\u00b11.78 | \u22121.28\u00b11.44 | 0.767 |\n| Protein (%) | \u00a0 | \u00a0 | \u00a0 | \u00a0 |\n| \u2003Pre-treatment | 16.0\u00b10.61 | 16.3\u00b10.62 | 15.8\u00b10.42 | \u00a0 |\n| \u2003Post-treatment | 16.5\u00b10.75 | 16.0\u00b10.61 | 15.4\u00b10.53 | \u00a0 |\n| \u2003Change | 0.56\u00b10.86 | \u22120.36\u00b10.75 | \u22120.38\u00b10.67 | 0.280 |\n| Fat (%) | \u00a0 | \u00a0 | \u00a0 | \u00a0 |\n| \u2003Pre-treatment | 24.4\u00b11.47 | 23.9\u00b11.05 | 22.4\u00b11.07 | \u00a0 |\n| \u2003Post-treatment | 22.7\u00b11.79 | 24.3\u00b11.55 | 22.9\u00b11.14 | \u00a0 |\n| \u2003Change | \u22121.67\u00b11.65 | 0.43\u00b11.22 | 0.50\u00b11.22 | 0.733 |\n| TEE\/TCI | \u00a0 | \u00a0 | \u00a0 | \u00a0 |\n| \u2003Pre-treatment | 1.37\u00b10.11 | 1.43\u00b10.09 | 1.34\u00b10.09 | \u00a0 |\n| \u2003Post-treatment | 1.23\u00b10.06 | 1.29\u00b10.08 | 1.23\u00b10.07 | \u00a0 |\n| \u2003Change | \u22120.14\u00b10.10 | \u22120.14\u00b10.07 | \u22120.12\u00b10.07 | 0.990 |\n\nResults are expressed as standard error of the mean (SEM).\n\nNo significant differences in the week 0 value between the placebo and test groups were found by nonparametric Kruskal-Wallis test.\n\n^a)^ Evaluated by nonparametric Kruskal-Wallis test.\n\n## Antioxidant enzyme activities\n\nPlasma SOD activity after the 8-week KRG supplementation was significantly higher than baseline activity (low-dose; *P*\u2009=\u20090.038, high-dose; *P*\u2009=\u20090.016), and the net changes were significantly different between the high-dose group and the placebo group (*P*\u2009=\u20090.021) after adjusting for age, sex, BMI, smoking, drinking, systolic BP, diastolic BP, and baseline values (Table <a href=\"#T3\" data-ref-type=\"table\">3<\/a>). The plasma GPx and catalase activities after the 8-week high-dose supplementation were higher than baseline (*P*\u2009=\u20090.006 and 0.016, respectively), and the net changes also were significantly different between the high-dose group and the placebo group (*P*\u2009=\u20090.032 and 0.019, respectively) after adjustment. Erythrocyte SOD and catalase activities were also measured. No significant differences were observed in the baseline activities of both enzymes among the three groups (SOD: placebo, 44.1\u2009\u00b1\u20093.14; low-dose, 46.0\u2009\u00b1\u20092.93; high-dose; 42.3\u2009\u00b1\u20092.99; Catalase: placebo, 231.1\u2009\u00b1\u20091.91; low-dose, 224.3\u2009\u00b1\u20093.48; high-dose; 223.7\u2009\u00b1\u20092.78). After the eight weeks, the activities of both enzymes were significantly increased in the high-dose group (p\u2009\\<\u20090.05). In particular, the net changed values for erythrocyte SOD activities were significantly different among the three groups after the adjustment (SOD: placebo, -3.58\u2009\u00b1\u20092.39; low-dose, -0.07\u2009\u00b1\u20091.81; high-dose; 3.50\u2009\u00b1\u20091.95, p\u2009=\u20090.030; Catalase: placebo, -6.74\u2009\u00b1\u20092.94; low-dose, 1.21\u2009\u00b1\u20093.45; high-dose; 4.46\u2009\u00b1\u20092.55, p\u2009=\u20090.120).\n\nAntioxidant enzyme activity and lipid peroxidation before and after intervention\n\n| \u00a0 | **Placebo (n\u2009=\u200919)** | **Low-dose (n\u2009=\u200919)** | **High-dose (n\u2009=\u200919)** | \u00a0 | \u00a0 | \u00a0 |\n|:---|:---|:---|:---|:---|:---|:---|\n| SOD activity (U\/ml) | \u00a0 | \u00a0 | \u00a0 | \u00a0 | \u00a0 | \u00a0 |\n| \u2003Pre-treatment | 12.6\u00b10.97 | 10.1\u00b10.68 | 11.9\u00b10.98 | \u00a0 | \u00a0 |  |\n| \u2003Post-treatment | 12.2\u00b10.96 | 12.1\u00b11.03^\\*^ | 14.2\u00b11.15^\\*^ | \u00a0 | \u00a0 | \u00a0 |\n| \u2003Change | \u22120.46\u00b10.57 | 1.95\u00b10.80 | 2.24\u00b10.86^\u2020^ | \u00a0 | \u00a0 | \u00a0 |\n| GPx activity (nmol\/min\/ml) | \u00a0 | \u00a0 | \u00a0 | \u00a0 | \u00a0 | \u00a0 |\n| \u2003Pre-treatment | 41.1\u00b13.71 | 34.3\u00b11.88 | 34.9\u00b12.53 | \u00a0 | \u00a0 | \u00a0 |\n| \u2003Post-treatment | 39.1\u00b13.72 | 37.4\u00b12.27 | 43.5\u00b12.53^\\*\\*^ | \u00a0 | \u00a0 | \u00a0 |\n| \u2003Change | \u22122.00\u00b12.08 | 3.05\u00b12.15 | 8.66\u00b12.63^\u2020^ | \u00a0 | \u00a0 | \u00a0 |\n| Catalase (nmol\/min\/ml) | \u00a0 | \u00a0 | \u00a0 | \u00a0 | \u00a0 | \u00a0 |\n| \u2003Pre-treatment | 103.2\u00b111.5 | 94.5\u00b18.56 | 86.9\u00b111.7 | \u00a0 | \u00a0 | \u00a0 |\n| \u2003Post-treatment | 99.0\u00b113.2 | 118.3\u00b111.8 | 135.5\u00b112.3^\\*\\*^ | \u00a0 | \u00a0 | \u00a0 |\n| \u2003Change | \u22124.17\u00b112.4 | 23.8\u00b112.4 | 48.5\u00b112.3^\u2020^ | \u00a0 | \u00a0 | \u00a0 |\n| 8-epi-PGF~2\u03b1~ (pg\/mg creatinine)^a)^ |  | \u00a0 | \u00a0 | \u00a0 | \u00a0 | \u00a0 |\n| \u2003Pre-treatment | 1450.3\u00b1103.7 | 1519.0\u00b1222.3 | 1516.3\u00b1105.9 | \u00a0 | \u00a0 | \u00a0 |\n| \u2003Post-treatment | 1641.6\u00b1136.5 | 1323.4\u00b1106.6 | 1383.5\u00b1120.2 | \u00a0 | \u00a0 | \u00a0 |\n| \u2003Change | 191.3\u00b1107.7 | \u2212195.6\u00b1147.6^\u2020^ | \u2212132.8\u00b1127.9^\u2020^ | \u00a0 | \u00a0 | \u00a0 |\n\nSOD, superoxide dismutase; GPx, glutathione peroxidase; 8-epi-PGF~2\u03b1~, 8-epi-prostaglandin F~2\u03b1~.\n\n^a)^Tested after logarithmic transformation.\n\nResults are expressed as standard error of the mean (SEM).\n\nTested by Wilcoxon signed-rank test or analysis of covariance (ANCOVA) using a general linear model (GLM) with adjustments for age, sex, BMI, smoking, drinking, systolic BP, diastolic BP, and baseline values.\n\n^\\*^*P* \\<0.05,^\\*\\*^*P* \\<0.01 compared with the baseline in each group; ^\u2020^*P* \\<0.05, ^\u2020\u2020^*P* \\<0.01 compared with the placebo group.\n\n## Lymphocyte DNA damage\n\nThe DNA tail length significantly decreased after KRG supplementation compared to baseline (low-dose; *P*\u2009=\u20090.004, high-dose; *P*\u2009=\u20090.003; Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>). The tail moment was also significantly reduced by KRG supplementation (low-dose; *P*\u2009=\u20090.012, high-dose; *P*\u2009=\u20090.001). The net changes in tail length and tail moment differed significantly between the high-dose group and the placebo group (*P*\u2009=\u20090.032 and *P*\u2009=\u20090.019, respectively) after adjustment. The images showing lymphocyte DNA damage, as performed by comet assay before and after the treatment, between the placebo and high-dose groups are presented in Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>.\n\n## Oxidative stress biomarkers, oxidized LDL, and urinary 8-epi-PGF~2\u03b1~\n\nAfter 8\u2009weeks, the plasma oxidized LDL levels decreased in the low-dose (*P*\u2009=\u20090.003) and high-dose (*P*\u2009=\u20090.001) groups, but increased in the placebo group (*P*\u2009=\u20090.035). The net changes differed significantly between the KRG supplementation groups and the placebo group (vs. low-dose, *P*\u2009=\u20090.001; vs. high-dose, *P \\<*0.001) after adjustment (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>). We also found significant differences in the net changes of urinary excretion of 8-epi-PGF~2\u03b1~ between the KRG supplementation groups and the placebo group (vs. low-dose, *P*\u2009=\u20090.033; vs. high-dose, *P\u2009=*\u20090.049) after adjustment (Table <a href=\"#T3\" data-ref-type=\"table\">3<\/a>).\n\n## Relationship between changes in oxidative stress markers and catalase activity\n\nAmong all subjects, changes in oxidized LDL from baseline correlated positively with changes in urinary 8-epi-PGF~2\u03b1~ (*r*\u2009=\u20090.305, *P*\u2009=\u20090.030), negatively with changes in catalase activity (*r*\u2009=\u2009\u22120.419, *P*\u2009=\u20090.002), and appeared to correlate positively with changes in DNA tail moment (*r*\u2009=\u20090.231, *P*\u2009=\u20090.096); however, this correlation was not significant. After adjusting for sex and age, changes in oxidized LDL correlated positively with both changes in urinary 8-epi-PGF~2\u03b1~ (*r*\u2009=\u20090.332, *P*\u2009=\u20090.026) and DNA tail moment (*r*\u2009=\u20090.295, *P*\u2009=\u20090.049) and correlated negatively with changes in catalase activity (*r*\u2009=\u2009\u22120.401, *P*\u2009=\u20090.006).\n\n# Discussion\n\nIt has been reported that about 200 substances, such as ginsenosides, polysaccharides, polyacetylenes, peptides, and amino acids etc, have been isolated from Korean ginseng, and more than 100 substances have also been isolated from American ginseng and Notoginseng \\[19,20\\]. Among the substances isolated from ginseng, ginsenosides are the primary active components of ginseng \\[21\\]. According the recent report, more than 60 ginsenosides have been found in the American ginseng \\[22\\]. These ginsenosides have been discovered continuously in the Korean ginseng, and it has been assumed that Korean ginseng contains over 60 ginsenosides \\[20\\]. Furthermore, Korean ginseng contains more primary non-saponin compounds, phenol compounds, acid polysaccharides, and polyethylene compounds than American ginseng or Sanchi ginseng \\[23\\]. KRG is produced by a special process of steaming and drying fresh Korean ginseng. The steaming process hydrolyzes and converts ginsenosides into other types of ginsenosides, such as red ginseng-specific compounds with anticancer properties (ginsenoside-Rh2, -Rh4, -Rs3, -Rs4, -Rg5) and a compound with anti-metastasis and vasodilation properties (ginsenoside-Rg3). The anti-oxidative and anti-cancer activities of KRG appear to be superior to those of white ginseng \\[21,24\\].\n\nThe reduction in oxidative DNA damage after KRG supplementation observed in the present study is consistent with findings from a previous study using a rat model. Park et al. \\[25\\] reported that red ginseng extract inhibited the breaking of *Escherichia coli* ColE1 plasmid DNA as well as the nuclear DNA of rat hepatocytes damaged by oxidative stress. In the present study, 8\u2009weeks of KRG supplementation (6\u2009g\/day) significantly decreased DNA tail length (\u03bcm) and tail moment, indicators of lymphocyte DNA damage, compared to placebo. Zhang et al. \\[26\\] also reported in mice that a 2-day oral administration of 20(S)-ginsenoside Rg3 (20\u2009mg\/kg per day) significantly inhibited cyclophosphamide-induced DNA damage in bone marrow cells and peripheral lymphocytes.\n\nOxidative stress results from an imbalance between antioxidants and reactive oxygen species, such as superoxide anions, H~2~O~2~, and hydroxyl radicals \\[27\\]. High levels of reactive oxygen species damage biomolecules (nucleic acids, proteins, and lipids) and generate oxidized LDL, which upregulates lectin-like oxidized low-density lipoprotein receptor-1 in endothelial cells, thereby inducing the generation of more reactive oxygen species through an unclear intracellular feedback mechanism \\[28\\]. The antioxidant enzyme SOD eliminates superoxide anions by producing H~2~O~2~ and is the first line of defense against reactive oxygen species toxicity. Furthermore, the enzymes catalase and GPx convert harmful H~2~O~2~ to harmless H~2~O \\[3,29\\]. Thus, boosting the activity of these three main antioxidant enzymes by KRG supplementation may decrease oxidative stress.\n\nIn the present study, plasma SOD, catalase, GPx, and erythrocyte SOD activities were significantly higher in subjects who received KRG supplementation than in subjects who received the placebo. In the past, the antioxidative effects of KRG have primarily been demonstrated in animal models or *in vitro* studies. Kim et al. \\[30\\] reported that, in mice subjected to paraquat-mediated oxidative stress, SOD, catalase, and GPx levels in liver tissue were significantly increased by red ginseng saponin supplementation. In a separate publication, Kim et al. \\[31\\] demonstrated that ginsenoside Rh2, a characteristic ginsenoside of red ginseng, is a particularly important active compound that significantly boosts catalase activity. Similarly, Chang et al. \\[32\\] reported that the Rb2 subfraction of panaxadiol ginsenosides appeared to strongly induce SOD1 and catalase, which maintain cell viability by lowering the level of oxygen radicals generated by the intracellular metabolism.\n\nThe limitations of our study include a small sample size, which renders us unable to generalize our study findings to other populations. We were also limited by the short study duration, and the mechanisms of action were not addressed in the study design. The important findings presented in this study will require further investigation in larger trials that will be carefully designed to include the optimal dose and form of KRG intervention, the study duration, and subject characteristics. In addition, enzyme mass and gene and protein expression levels should be measured to determine whether the increased enzyme activity is due to an increase in protein mass or through the elevation of the specific activities of the enzyme.\n\n# Conclusions\n\nIn this randomized, double-blind, placebo-controlled study, KRG supplementation improved biomarkers of oxidative stress*,* as evidenced by decreased plasma oxidized LDL, attenuated lymphocyte DNA damage and increased plasma antioxidant enzyme activity in healthy participants (20\u201365\u2009years old). In addition, changes in plasma oxidized LDL correlated positively with the decreases in oxidative DNA damage and urinary 8-epi-PGF~2\u03b1~ and negatively with changes in catalase activity. Larger prospective studies with a longer intervention period (3 or 6\u2009months) will be necessary to further characterize the beneficial antioxidative activity of KRG and its mechanism of action.\n\n# Abbreviations\n\nGPx, Glutathione peroxidase; SOD, Superoxide dismutase; KRG, Korean red ginseng; PGF2\u03b1, 8-epi-prostaglandin F2\u03b1; LDL, Low-density lipoprotein; HDL, High-density lipoprotein; AST, Serum aspartate aminotransferase; ALT, Alanine aminotransferase; BUN, Blood urea nitrogen; CBC, Complete blood count; NADPH, Nicotinamide adenine dinucleotide phosphate.\n\n# Competing interests\n\nThe authors declare that they have no competing interests.\n\n# Authors' contributions\n\nJYK and JHL conceptualized this study, and JHL was the Principal Investigator. JYP and HJK also participated in the design of the study and performed the analyses. JYK and OYK contributed to the writing of the manuscript. All authors have read and approved the final manuscript.\n\n## Acknowledgements\n\nWe sincerely thank the research subjects who participated in the studies described in this report. This work was supported by a 2009 grant from the Korean Society of Ginseng funded by the Korea Ginseng Cooperation and National Research Foundation, Ministry of Education, Science and Technology (Mid-Career Researcher Program: 2010\u20130015017), Seoul, Korea.","meta":{"dup_signals":{"dup_doc_count":161,"dup_dump_count":38,"dup_details":{"curated_sources":3,"2022-33":1,"2021-43":1,"2020-05":2,"2019-39":1,"2017-17":1,"2017-09":19,"2017-04":1,"2016-50":1,"2016-44":1,"2016-40":1,"2016-36":1,"2016-30":1,"2016-22":1,"2016-18":1,"2015-48":5,"2015-40":4,"2015-35":5,"2015-32":3,"2015-27":5,"2015-22":5,"2015-14":5,"2014-52":5,"2014-49":7,"2014-42":14,"2014-41":8,"2014-35":8,"2014-23":8,"2014-15":9,"2023-23":1,"2024-26":1,"2024-10":1,"2015-18":5,"2015-11":5,"2015-06":5,"2014-10":5,"2013-48":5,"2013-20":5,"2024-30":1}},"file":"PMC3426460"},"subset":"pubmed_central"}
{"text":"abstract: A^\u2212^\u03b2^+^ ketosis-prone diabetes (KPD) is an emerging syndrome of obesity, unprovoked ketoacidosis, reversible \u03b2-cell dysfunction, and near-normoglycemic remission. We combined metabolomics with targeted kinetic measurements to investigate its pathophysiology. Fasting plasma fatty acids, acylcarnitines, and amino acids were quantified in 20 KPD patients compared with 19 nondiabetic control subjects. Unique signatures in KPD\u2014higher glutamate but lower glutamine and citrulline concentrations, increased \u03b2-hydroxybutyryl-carnitine, decreased isovaleryl-carnitine (a leucine catabolite), and decreased tricarboxylic acid (TCA) cycle intermediates\u2014generated hypotheses that were tested through stable isotope\/mass spectrometry protocols in nine new-onset, stable KPD patients compared with seven nondiabetic control subjects. Free fatty acid flux and acetyl CoA flux and oxidation were similar, but KPD had slower acetyl CoA conversion to \u03b2-hydroxybutyrate; higher fasting \u03b2-hydroxybutyrate concentration; slower \u03b2-hydroxybutyrate oxidation; faster leucine oxidative decarboxylation; accelerated glutamine conversion to glutamate without increase in glutamate carbon oxidation; and slower citrulline flux, with diminished glutamine amide\u2013nitrogen transfer to citrulline. The confluence of metabolomic and kinetic data indicate a distinctive pathogenic sequence: impaired ketone oxidation and fatty acid utilization for energy, leading to accelerated leucine catabolism and transamination of \u03b1-ketoglutarate to glutamate, with impaired TCA anaplerosis of glutamate carbon. They highlight a novel process of defective energy production and ketosis in A^\u2212^\u03b2^+^ KPD.\nauthor: Sanjeet G. Patel; Jean W. Hsu; Farook Jahoor; Ivonne Coraza; James R. Bain; Robert D. Stevens; Dinakar Iyer; Ramaswami Nalini; Kerem Ozer; Christiane S. Hampe; Christopher B. Newgard; Ashok BalasubramanyamCorresponding author: Ashok Balasubramanyam, <email@example.com>.[^1]\ndate: 2013-03\nreferences:\ntitle: Pathogenesis of A^\u2212^\u03b2^+^ Ketosis-Prone Diabetes\n\nKetosis-prone diabetes (KPD) is characterized by presentation with diabetic ketoacidosis (DKA) in persons who do not fit traditional categories of types 1 or 2 diabetes (1\u20135). We have defined four subgroups of KPD based on presence or absence of \u03b2-cell autoantibodies (A^+^ or A^\u2212^), and recovery or lack of recovery of \u03b2-cell functional reserve following the index episode of DKA (\u03b2^+^ or \u03b2^\u2212^) (1,6,7).\n\nThe A^\u2212^\u03b2^+^ KPD subgroup represents a novel syndrome of severe but reversible \u03b2-cell dysfunction (1,3,5,8,9). Approximately 50% of these patients develop DKA without a precipitating factor at diagnosis of diabetes. These new-onset, unprovoked A^\u2212^\u03b2^+^ KPD patients display male predominance (10) and low frequencies of human leukocyte antigen class II susceptibility alleles for autoimmune diabetes (11). \u03b2-Cell function increases markedly within 1\u20133 months after the index DKA, with sustained glycemic improvement and insulin independence (1,9,11,12). The cause of the unprovoked ketoacidosis is unknown. Over 5\u201310 years, patients may relapse to unprovoked ketosis (3,5).\n\nThis syndrome provides a model to identify novel mechanisms of obesity, ketosis, and reversible \u03b2-cell dysfunction. We used a metabolomics approach to identify unique alterations in A^\u2212^\u03b2^+^ KPD patients, with a kinetics approach to specify the pathophysiology.\n\n# RESEARCH DESIGN AND METHODS\n\n## Metabolomic analysis\n\n### Human subjects.\n\nProtocols were approved by the Human Studies Institutional Review Boards of Baylor College of Medicine and Duke University (13). Written informed consent was obtained. New-onset, unprovoked male A^\u2212^\u03b2^+^ KPD patients (*n* = 20) were identified by absence of GAD65\/67, IA-2, or ZnT8 autoantibodies and presence of \u03b2-cell functional reserve 4\u20138 weeks after the index DKA (1,6). Men (*n* = 19) from the Study of the Effects of Diet on Metabolism and Nutrition study (13) were nondiabetic obese control subjects. KPD patients were selected from a longitudinal database using the MatchIT program (15) to minimize intragroup differences in initial HbA~1c~ and intergroup differences in age, ethnicity, BMI, waist circumference, fasting C-peptide, glucose, and lipids. At the time of blood sampling, all patients were clinically stable, taking twice-daily neutral protamine Hagedorn insulin (\u00b1 short-acting insulin); three patients were also taking metformin (1 g daily). Five additional, newly diagnosed, unprovoked A^\u2212^\u03b2^+^ KPD patients were subsequently recruited for measurements of glutamine, glutamate, isoleucine, and leucine.\n\n### Plasma collection.\n\nThe KPD samples were collected in acid citrate dextrose (#364606, BD Biosciences) after an overnight fast before the morning dose of insulin, 20 days (median) after the index DKA (5). Blood was kept on ice for 2\u20134 h, centrifuged at 4\u00b0C, and the plasma stored at \u221220\u00b0C. Control subjects' samples were collected after an overnight fast in serum separator tubes (#366510, BD Biosciences), centrifuged, and the serum stored at \u221280\u00b0C. Blood samples were also taken from five fresh A^\u2212^\u03b2^+^ KPD patients 1 month after their index DKA, collected in serum separator tubes, processed immediately, and stored.\n\n### Metabolite analysis.\n\nTo measure 15 amino acids and 45 acylcarnitines, samples were deproteinized by methanol precipitation, esterified with hot, acidic methanol or *n*-butanol, and then analyzed by tandem mass spectrometry (MS\/MS) with a Quattro Micro instrument (Waters Corporation, Milford, MA) (13). Leucine and isoleucine are reported as a single analyte (Leu\/Ile) because they were not resolved by this method. These conditions partially hydrolyze glutamine to glutamate and asparagine to aspartate, so \"glx\" (Glu\/Gln) or \"asx\" (Asp\/Asn) signify glutamate or aspartate with contributions from hydrolysis reactions of glutamine and asparagine. Final concentrations of isoleucine, leucine, glutamine, and glutamate were measured by reverse-phase high-performance liquid chromatography (HPLC; Hewlett-Packard 1090; Hewlett-Packard, Avondale, PA); frozen samples were thawed on ice prior to HPLC and glutamine standards prepared fresh in ice-cold water.\n\nTo measure seven nonesterified free fatty acids (FFAs), samples were methylated and purified by solid-phase extraction (13). Derivatized fatty acids were analyzed by gas chromatography\/mass spectrometry (GC\/MS; Trace DSQ; Thermo Electron Corporation, Austin, TX).\n\nAll MS analyses used stable isotope dilution to quantify metabolites (13). Because acid citrate dextrose tubes were used for the original KPD patients, a dilution correction factor (1.31) was used for analytes measured in those samples (16).\n\nPlasma insulin, C-peptide, glucagon, cortisol, and free metanephrines were measured by radioimmunoassay (Linco, St. Louis, MO), and \u03b2-hydroxybutyrate (BOHB) by spectrophotometry (Wako, Richmond, VA).\n\n### Statistical analysis.\n\nData were inspected for normality using the Shapiro-Wilk tests. The *t* tests were used to compare levels of hormones, baseline clinical parameters, and metabolites. BOHB values were not normally distributed, so median levels were compared using the Mann-Whitney U test. The Fisher exact test was used to assess deviations from the order of metabolites as an indicator of substrate preference due to altered consumption or production. Significance was defined at *P* \\< 0.05.\n\n## Kinetic analyses\n\n### Human subjects.\n\nThe study was approved by the Baylor institutional review board and written informed consent obtained. Nine new-onset, unprovoked A^\u2212^\u03b2^+^ KPD patients (six men, three women), all clinically stable, were recruited 6\u20138 weeks after the index DKA episode. Seven nondiabetic control subjects (six men, one woman) were matched for BMI, age, and ethnicity, with the six men and one woman among the KPD patients. Exclusions were: serum creatinine \\>1.2 mg\/day, retinopathy, neuropathy, or heart failure; history of alcohol abuse; alanine aminotransferase\/aspartate aminotransferase (AST)\/alkaline phosphatase \\>2\u00d7 upper limit of normal; medications likely to affect fat, glucose, or protein metabolism; and other chronic illnesses. Fasting plasma C-peptide and glucose, peak and area under the curve of C-peptide response to glucagon, fasting lipids, and liver and thyroid functions were measured. Details of the glucagon stimulation test and cutoffs to classify \u03b2-cell reserve have been reported (1).\n\nSubjects underwent four stable isotope protocols in the General Clinical Research Center. For 2 days prior to each study, they consumed an isocaloric, standardized diet (30% calories from fat, 65% from carbohydrates, 1 g\/kg protein) prepared by the General Clinical Research Center kitchen. KPD patients were treated with low doses of neutral protamine Hagedorn insulin for 8 weeks prior to the studies. The last dose was administered at 8\u201310 <span class=\"smallcaps\">p.m.<\/span>, and each study commenced the following morning after a 10\u201314-h fast, with no insulin administered until study completion. One venous catheter was placed for infusions, with another in the opposite hand, heated for arterialized blood sampling.\n\n### Protocol 1.\n\nAfter baseline blood and breath sampling, an oral 50 mg\/kg bolus of ^2^H~2~O was administered. After an intravenous priming dose of NaH^13^CO~3~ (1.2 \u00b5mol\/kg), ^2^H~5~-glycerol (prime 4.5 \u00b5mol\/kg, constant 9.0 \u00b5mol\/kg\/h), ^2^H~2~-potassium palmitate (prime 3 \u00b5mol\/kg, constant 4 \u00b5mol\/kg\/h), and ^13^C~1~-acetate (prime 62.5 \u00b5mol\/kg, constant 150 \u00b5mol\/kg\/h) were infused for 4 h. Blood was collected hourly for the first 3 h and every 15 min during the fourth hour. Indirect calorimetry was performed during the last 30 min of the fourth hour. At hour 4, glycerol and palmitate infusions ceased, but the acetate infusion continued for another 2 h. Blood and breath samples were drawn every 15 min between hours 5 and 6.\n\nTracer-to-tracee ratios of plasma free palmitate, acetate, and BOHB were determined by negative chemical ionization GC\/MS of its pentafluorobenzyl derivative (17). Plasma palmitate and BOHB concentrations were measured by in vitro isotope dilution with U-^13^C-palmitate or ^13^C~2~-BOHB (Cambridge Isotope Laboratories, Andover, MA) as internal standards, using negative chemical ionization-GC\/MS (17).\n\nTracer-to-tracee ratio of plasma glycerol was determined by electron impact ionization GC\/MS of its tripropionate derivative (18). Breath ^13^CO~2~ content was determined by GC\/gas isotope ratio MS. Zinc was used to reduce water in 10-\u00b5l plasma in quartz vessels and ^2^H~2~ abundance of the resulting hydrogen determined by GC\/gas isotope ratio MS.\n\n### Protocol 2.\n\nAfter baseline blood sampling, ^2^H~2~-citrulline (prime 1.0 \u00b5mol\/kg, constant 1.0 \u00b5mol\/kg\/h), ^15^N~1~-amide-glutamine (prime 13.6 \u00b5mol\/kg, constant 13.6 \u00b5mol\/kg\/h), ^2^H~8~-valine (prime 1 \u00b5mol\/kg, constant 1 \u00b5mol\/kg\/h), and NaH^13^CO~3~ (prime 4 \u00b5mol\/kg, constant 4 \u00b5mol\/kg\/h) were infused. A 0.5-\u00b5mol\/kg prime of ^15^N~1~-citrulline was also given. Citrulline, glutamine, and valine were infused for 6 h. NaH^13^CO~3~ infusion was infused for 2 h; at the second hour, ^13^C~1~-leucine (prime 4 \u00b5mol\/kg, constant 4 \u00b5mol\/kg\/h) was started and infused until hour 6. Blood was collected hourly during the first 4 h and every 15 min during the fifth hour. Breath was collected at baseline and every 15 min between hours 1 and 2 and 5 and 6.\n\nTracer-to-tracee ratio of plasma \u03b1-ketoisocaproic acid (\u03b1-KICA) from leucine was measured by negative chemical ionization-GC\/MS of its pentafluorobenzyl derivative (17).\n\nPlasma citrulline, glutamine, and valine isotopic enrichment (IE) were measured after conversion to their 5-(dimethylamino)-1-napthalene sulfonamide derivatives by liquid chromatography-MS\/MS (Synergi MAX-RP column; Phenomenex, Torrance, CA) (19).\n\n### Protocol 3.\n\nAfter baseline blood and breath sampling, ^13^C~1~-glutamine (prime 13.6 \u00b5mol\/kg, constant 13.6 \u00b5mol\/kg\/h) was infused for 6 h. A priming dose of NaH^13^CO~3~ (4 \u00b5mol\/kg) was given at hour 0. Indirect calorimetry was performed during the last 30 min of the fourth hour. Blood and breath were collected hourly for 5 h and then every 15 min between hours 5 and 6.\n\nPlasma glutamine and glutamate IE were measured by liquid chromatography-MS\/MS as in protocol 2.\n\n### Protocol 4.\n\nAfter baseline blood and breath sampling, a priming dose of NaH^13^CO~3~ (4 \u00b5mol\/kg) was given, and then 2,4-^13^C-\u03b2-hydroxybutyrate (prime 20 \u00b5mol\/kg, constant 12 \u00b5mol\/kg\/h) was infused for 3 h. Blood and breath were collected hourly for the first 2 h and every 15 min during the third hour. Indirect calorimetry was performed during the last 30 min of the second hour. Blood was collected in K-EDTA tubes and spun immediately and one aliquot deproteinized with perchloric acid and frozen in liquid nitrogen.\n\nIE of BOHB was measured as in protocol 1.\n\nPlasma glucose was measured with a glucose analyzer (YSI, Yellow Springs, OH), insulin by radioimmunoassay (Linco) and FFAs by spectrophotometry (Wako). Amino acids were measured by HPLC.\n\n### Calculations.\n\nRa (palmitate, glycerol, acetate, or BOHB) (in \u00b5mol\/kg\/h):\n\nwhere IE~inf~ is the IE (mole %) in the infusate, IE~p~ is IE (mole %) in the plasma at steady state, and *i* is the tracer infusion rate.\n\nRate of appearance of FFAs (\u00b5mol \u00b7 kg^\u22121^ \u00b7 h^\u22121^):\n\nwhere Ra palmitate is the rate of appearance of palmitate (Eq. <a href=\"#e1\" data-ref-type=\"disp-formula\">1<\/a>), \\[palmitate\\]~p~ is the concentration of plasma palmitate, and \\[FFA\\]~p~ is the concentration of plasma FFA.\n\nWhole-body fatty acid oxidation is calculated from RQ, VO~2~, and (20).\n\nRate of acetate oxidation (\u00b5mol \u00b7 kg^\u22121^ \u00b7 h^\u22121^) in the tricarboxylic acid (TCA) cycle:\n\nwhere is the CO~2~ excretion rate measured by indirect calorimetry, IE~CO2~ is the IE of CO~2~ in the breath, IE~acetate~ is the IE of plasma acetate, and 0.56 is the acetate retention factor (21).\n\nAcetate oxidized in the TCA cycle (%):\n\nBOHB from acetate (\u00b5mol\/kg\/h):\n\nwhere Ra~BOHB~ is the value from Eq. <a href=\"#e1\" data-ref-type=\"disp-formula\">1<\/a>, IE~BOHB~ is the M+1 isotopomer enrichment of BOHB derived from the ^13^C~1~-acetate infusate, and IE~acetate~ is the IE of acetate.\n\nTotal body water (TBW) (from oral deuterated water):\n\nwhere is the enrichment of deuterated water consumed, is the enrichment of plasma H~2~O at steady state, and the constant 1.04 corrects for deuterium's volume of distribution. Fat-free mass (FFM) is TBW\/0.72, where 0.72 is the hydration constant of fat-free tissue. Fat mass (kg) = body weight (kg) \u2212 FFM.\n\nRa (citrulline or glutamine amide-N) (\u03bcmol\/kg\/h) was calculated as in Eq. (<a href=\"#e1\" data-ref-type=\"disp-formula\">1<\/a>).\n\nRate of transfer of glutamine amide ^15^N to citrulline:\n\nwhere Ra citrulline is the value from Eq. <a href=\"#e7\" data-ref-type=\"disp-formula\">7<\/a>, IE^15^~N-citrulline~ is the isotopic (M+1) enrichment of citrulline from ^15^N-glutamine, and IE^15^~N-glutamine~ is the isotopic (M+1) enrichment of glutamine.\n\nFlux of glutamine carbon is calculated as in Eq. (<a href=\"#e7\" data-ref-type=\"disp-formula\">7<\/a>).\n\n^13^C-glutamine reaches steady state quickly, but ^13^C-glutamate formed from the glutamine does not, because of the large whole-body free glutamate pool. Hence, the fraction of the glutamate pool derived from glutamine (fractional synthesis rate \\[FSR\\] of glutamate from glutamine) is calculated using the precursor-product equation:\n\nwhere IEglu*~t6~* \u2212 IEglu*~t3~* is the increase in glutamate IE from 3\u20136 h of the infusion, and IEgln*~pl~* is the plateau IE of glutamine.\n\nAbsolute synthesis rate (ASR) of glutamate from glutamine:\n\nThe rate of formation of ^13^CO~2~ from ^13^C~1~-glutamine over the 6-h infusion, an index of glutamate dehydrogenase activity, is calculated as follows:\n\n^13^C~1~-glutamine oxidation (mmol\/6 h) = area under the curve of breath ^13^CO~2~ = AUC~0\u20136\\ h~ of ^13^CO~2~:\n\nwhere V~CO2~\/0.78 is the production rate of CO~2~, assuming 22% of CO~2~ is retained in the bicarbonate pool (22), and IE~CO2~ is the IE of CO~2~ (atom percent excess).\n\na relative measure of glutamate entry in the TCA cycle at \u03b1-ketoglutarate and an estimate of glutaminase plus glutamate dehydrogenase activity.\n\n# RESULTS\n\n## Metabolomics\n\n### Clinical and biochemical parameters.\n\nBMI and waist circumference were similar in KPD and control subjects (<a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>). KPD subjects had elevated initial HbA~1c~ (13.5 \u00b1 0.5 %) and fasting plasma glucose levels 4\u201312 weeks after the index DKA episode. Characteristically, HbA~1c~ was markedly improved 3\u20136 months after the index DKA (7.2 \u00b1 0.5), when the patients were on low doses of insulin (*N* = 13) or off insulin (*N* = 7). Fasting C-peptide was higher in control subjects. The C-peptide\/glucose ratio was one-third of the control level in the KPD group. Fasting insulin levels were not different, and quantitative insulin sensitivity check index (QUICKI), an index of insulin sensitivity, demonstrated similar levels of insulin resistance in the two groups. Triglycerides showed a trend to be higher, and HDL-cholesterol (HDL-C) was \u223c30% lower in KPD. The mean BOHB level was twice as high in KPD as in control subjects, but the difference did not attain statistical significance due to individual variability. KPD patients had normal fasting metanephrines and cortisol and normal thyroid, renal, and liver functions. The KPD group included 1 white, 7 African American, and 11 Hispanic subjects, whereas the control subjects included 14 white, 4 African American, and no Hispanic subjects; ethnicity was not correlated with any inter- or intragroup differences in metabolite or hormone levels.\n\nMetabolomic survey: demographic and biochemical data and fasting plasma concentrations of amino acids, acylcarnitines, and FFAs\n\n![](912tbl1)\n\n### Amino acids and related acylcarnitines.\n\n\"Asx\" was 2.3-fold (*P* \\< 0.01) and \"glx\" 4.8-fold (*P* \\< 0.0001) higher in the KPD group (<a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>). Smaller elevations were observed in KPD for glycine (1.4-fold; *P* \\< 0.001), serine (1.4-fold; *P* \\< 0.001), alanine (1.2-fold; *P* = 0.03), proline (1.2-fold; *P* = 0.02), and histidine (1.2-fold; *P* \\< 0.001). Methionine (0.85-fold; *P* = 0.02) and citrulline (0.75-fold; *P* = 0.02) were lower in KPD.\n\nThe unusually high levels of glx in KPD suggested partial ex vivo conversion of glutamine to glutamate or pyroglutamine (23\u201325) in those samples. HPLC also demonstrated very low glutamine and very high glutamate levels in the KPD samples. To avoid alterations of glutamine and glutamate due to collection methods or storage, five additional, newly diagnosed A^\u2212^\u03b2^+^ KPD patients were recruited and their plasma collected using stringent collection and processing methods (26). The mean glutamine concentration in these new KPD samples was 207.20 \u00b1 10.29 \u03bcmol\/L (0.56-fold less than control subjects; *P* = 0.0001) and the mean glutamate concentration 192.03 \u00b1 7.98 \u03bcmol\/L (1.75-fold more than control subjects; *P* = 0.02).\n\nConcentrations of total branched chain amino acids (BCAA) were similar in KPD and control subjects, with no significant differences in valine or isoleucine\/leucine. Because MS\/MS cannot distinguish isoleucine from leucine, HPLC was used to separate them in samples from 11 KPD patients and 16 control subjects. Leucine and isoleucine concentrations were not different between KPD and control subjects (128.8 \u00b1 11.6 vs. 137.3 \u00b1 7.7 and 59.6 \u00b1 7.0 vs. 62.2 \u00b1 5.3 \u03bcmol\/L, respectively; *P* = NS for both). This was surprising because fasting plasma concentrations of leucine and isoleucine are usually elevated in diabetic patients with imperfect glycemic control (27\u201330). Hence, we tracked the catabolic pathways of BCAA through acylcarnitine esters of their distinct ketoacid metabolites (<a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>). Isobutyryl-carnitine (C4-AC), from decarboxylation of the valine ketoacid, was 1.5-fold higher in KPD (*P* \\< 0.01). In contrast, C5-AC, representing acylcarnitine esters of both isovaleryl-CoA and \u03b1-isomethylbutyryl-CoA (from decarboxylation of the leucine and isoleucine ketoacids, respectively), was 1.3-fold lower in KPD (*P* \\< 0.01), but tiglyl-carnitine (C5:1-AC), an isoleucine-specific catabolite, was 1.2-fold higher in KPD (*P* = 0.1). In sum, these data indicated that the leucine ketoacid metabolite was decreased in KPD patients.\n\nBCAA are deaminated by branch chain aminotransferase (BCAT) to produce KICA from leucine, \u03b1-keto-\u03b2-methyvaleric acid (KMVA) from isoleucine and \u03b1-ketoisovaleric acid (KIVA) from valine, and the NH~2~ group is transferred to \u03b1-ketoglutarate to generate glutamate. The ketoacids are processed by branch chain ketoacid dehydrogenase (BCKDH) and subsequently follow distinct pathways to either generate ketone bodies (KICA, KMVA) or enter the TCA cycle for oxidation (KIVA, KMVA). Glutamate is exported from mitochondria with aspartate and the remaining ketoacids. Glutamate, aspartate, KMVA, and KIVA are precursors for gluconeogenesis; in contrast, KICA is a primary amino acid source of ketones. The metabolomic data suggested acceleration of the initial steps of BCAA catabolism in muscle as an explanation for key metabolic signatures in KPD patients: lack of elevated BCAA, low C5-AC, elevated glutamate, and elevated ketones.\n\n### TCA\/anaplerotic metabolites.\n\nAcetylcarnitine (C2, acylcarnitine ester of acetyl CoA) was lower in KPD (1.3-fold; *P* = 0.001), suggesting either decreased production of acetyl CoA or increased shunting of acetyl CoA into oxidative or synthetic pathways (<a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>). \u03b2-hydroxybutyryl-carnitine (C4-OH), representing intracellular BOHB, was threefold higher in KPD (*P* = 0.01), suggesting increased ketone synthesis or diminished ketone oxidation. Other short-chain acylcarnitines (C4-DC, C5-DC), which include metabolites arising distal to \u03b1-ketoglutarate in the TCA cycle, such as methylmalonyl\/succinyl-carnitine and glutaryl-carnitine, were diminished in KPD.\n\n### Fatty acids and ketones.\n\nThe molar sum of the seven most abundant fatty acids was similar in KPD and control subjects (<a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>). Persons consuming American diets have the following rank order of fasting fatty acid concentrations: oleate \\> palmitate \u2265 linoleate (31); this order was preserved in both groups, and levels of palmitate and oleate were similar, making it unlikely that there were group differences in habitual dietary fat composition. These data suggested that the proclivity for ketosis in KPD patients is not due to excessive fatty acid supply.\n\n## Kinetics.\n\nIn the kinetic study cohort, age, weight, and BMI were similar in the two groups (<a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a>). HbA~1c~ was very high in the KPD patients at the time of the index DKA; 3 to 4 months later, mean HbA~1c~ and fasting glucose were markedly improved, but still higher than in the control subjects. At that time, fasting and glucagon-stimulated peak C-peptide and C-peptide\/glucose ratio were similar in KPD and control subjects. Fasting insulin levels were higher in KPD; however, they were very similar to those of the KPD patients in the metabolomics study (<a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>), and QUICKI demonstrated a significantly higher degree of insulin resistance in KPD. Thyroid-stimulating hormone, blood urea nitrogen, creatinine, and AST were normal and similar in both groups. Triglycerides (163 \u00b1 9 mg\/dL) were elevated, whereas HDL-C (40.6 \u00b1 3.9 mg\/dL) was low, and LDL-cholesterol level was 85 \u00b1 9 mg\/dL in the KPD group.\n\nKinetic study: demographic, hormonal, and biochemical data\n\n![](912tbl2)\n\n1\\. *Fasting rates of FFA release and acetyl CoA flux are similar in KPD and control subjects. BOHB oxidation and fatty acid disposal are slowed, leading to elevated plasma concentrations of BOHB and FFA*.\n\nRa FFA (total FFA flux) was not different, whereas Ra glycerol (index of triglyceride hydrolysis) was significantly lower, and Ra palmitate (palmitate flux) trended lower in KPD (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<\/a>). Furthermore, Ra acetate (index of whole-body acetyl CoA flux) was similar in KPD and control subjects (1,344 \u00b1 260 vs. 1,570 \u00b1 141 \u00b5mol\/kgFM\/h; *P* = 0.50), and acetate oxidation (breath ^13^CO~2~ liberated by isocitrate dehydrogenase activity, reflecting acetyl CoA flux through the TCA cycle) was also similar (861 \u00b1 175 vs. 1,007 \u00b1 87 \u00b5mol\/kgFM\/h; *P* = 0.49). Despite the similar FFA flux, plasma concentration of FFA was higher in KPD (587 \u00b1 51 vs. 412 \u00b1 8 \u00b5mol\/L; *P* = 0.04) (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<\/a>), suggesting slower FFA disposal (corroborated by the slower palmitate clearance). Plasma concentration of BOHB was also higher in the KPD patients (97 \u00b1 17 vs. 43 \u00b1 10 \u03bcmol\/L; *P* = 0.02) in the face of slower conversion of acetyl CoA to BOHB (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<\/a>). Collectively, these findings indicated defective BOHB and fatty acid disposal, which, after a 10\u201314-h fast, is predominantly oxidative. To test BOHB oxidation directly, protocol 4 was performed in three control subjects and seven KPD patients. KPD had slower BOHB flux (330 \u00b1 80 vs. 696 \u00b1 103 \u00b5mol\/kgFM\/h; *P* = 0.03) but also slower oxidative BOHB disposal (191 \u00b1 47 vs. 427 \u00b1 84 \u03bcmol\/kgFM\/h; *P* = 0.03), a combination that would increase BOHB concentration. (Of note, plasma BOHB enrichment was used in this study as a proxy for intracellular acetate or acetyl CoA enrichment; in reality, it is acetyl CoA that is oxidized. The IE of the acetyl CoA would be lower than the plasma BOHB enrichment, because there are additional sources of unlabeled acetyl CoA in the intracellular pool. Because the more highly enriched BOHB is the denominator used in the calculations, the actual oxidation rate of BOHB \\[which must first be converted to acetyl CoA to be oxidized via intermediates in the TCA cycle\\] is probably underestimated in all subjects.)\n\n2\\. *Oxidative decarboxylation of leucine is accelerated in KPD.*\n\nThere was no difference between KPD and control subjects in leucine flux and a trend toward higher valine flux in KPD (<a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<\/a>). The rate of oxidative decarboxylation, or removal of the \\#1 carbon of leucine by BCKDH, showed a strong trend to be faster in KPD (60.0 \u00b1 2.8 vs. 51.7 \u00b1 2.9 \u03bcmol\/kgFFM\/h; *P* = 0.06). Increased BCKDH activity requires increased flux through the earlier BCAT-catalyzed step that drives transfer of NH~2~ from leucine to \u03b1-ketoglutarate to generate glutamate (32). Unless matched by acceleration of the opposing reaction that oxidatively deaminates glutamate to \u03b1-ketoglutarate, this would promote accumulation of glutamate.\n\n3\\. *Conversion of glutamine to glutamate is accelerated in KPD, without a parallel increase in glutamate carbon oxidation.*\n\nThere was no group difference in glutamine flux, but plasma glutamine concentration trended lower in KPD (557 \u00b1 29 vs. 655 \u00b1 36 \u03bcmol\/L; *P* = 0.06) (<a href=\"#F4\" data-ref-type=\"fig\">Fig. 4<em>A<\/em><\/a>). The rate of glutamine carbon transfer to glutamate was faster in KPD by several measures: slope of change, absolute synthesis rate of glutamate from glutamine (<a href=\"#F4\" data-ref-type=\"fig\">Fig. 4<em>A<\/em><\/a>), and fractional synthesis rate of glutamate from glutamine (<a href=\"#F4\" data-ref-type=\"fig\">Fig. 4<em>B<\/em><\/a>). However, the rate of oxidation of the same glutamate carbon in the TCA cycle was not different between the groups (<a href=\"#F4\" data-ref-type=\"fig\">Fig. 4<em>B<\/em><\/a>). Thus, increased glutamate production from glutamine in KPD is not matched by a parallel increase in glutamate carbon anaplerosis into the TCA cycle, as predicted from the results of the leucine infusion study.\n\n4\\. *Transfer of amide nitrogen from glutamine to citrulline is slower in KPD.*\n\nCitrulline flux was slower in KPD than in control subjects (<a href=\"#F5\" data-ref-type=\"fig\">Fig. 5<\/a>), with a trend toward slower transfer of glutamine amide nitrogen to ornithine (7.8 \u00b1 0.4 vs. 8.6 \u00b1 0.3 \u03bcmol\/kgFFM\/h; *P* = 0.1) for citrulline synthesis.\n\n# DISCUSSION\n\nIn this study, the pathophysiology of A^\u2212^\u03b2^+^ KPD was investigated comprehensively using a two-step discovery method. First, fasting plasma metabolomics compared KPD patients to nondiabetic obese subjects. Hypotheses generated from key differences were tested by kinetic tracer studies targeting the suspected metabolic pathways in a fresh cohort of patients and control subjects.\n\nThe metabolomic analysis revealed signals of altered BCAA catabolism in KPD associated with changes in glutamine\/glutamate. We hypothesized that the lack of elevated leucine in KPD was due to accelerated catabolism of this amino acid, indicated by decreased C5-AC and elevated glutamate (33,34). Evidence that accelerated leucine catabolism extended beyond isovaleryl CoA to \u03b2-methylcrotonyl CoA and ketogenesis was provided by the lower level of 3-hydroxyisovaleryl-carnitine (C5-OH). Concomitantly, glutamine was 50% lower, whereas glutamate was 175% higher, in KPD. Glutamine and glutamate are nitrogen sinks for catabolism of other amino acids, including BCAA, in the fasted state. Elevated glutamate and decreased glutamine levels could reflect absence of upregulation of glutamate oxidation rates in KPD subjects in the face of faster conversion of glutamine to glutamate and accelerated leucine deamidation.\n\nChanges in small-chain acylcarnitines reflecting TCA cycle intermediates and anaplerosis broadened the pathophysiologic hypothesis. Acetylcarnitine was significantly lower in KPD, suggesting decreased production or increased consumption of acetyl CoA. Acylcarnitines that include glutaryl CoA and succinyl CoA were lower and \u03b2-hydroxybutyryl-carnitine was threefold higher in KPD, suggesting diversion of acetyl CoA disposal toward ketogenesis. Finally, fasting FFA levels and composition were similar in KPD and control subjects, suggesting that excessive fatty acid flux from lipolysis is not the driver of ketogenesis in KPD.\n\nIn sum, the metabolomic data suggested that patients with unprovoked A^\u2212^\u03b2^+^ KPD have accelerated leucine catabolism toward ketogenesis. This would expand the nitrogen sinks of transamination reactions, resulting in elevated levels of glutamate and aspartate. The data also indicated that KPD patients have defects in transferring carbon from glutamine\/glutamate to the TCA cycle and nitrogen from glutamine\/glutamate to the urea cycle.\n\nThe results of the kinetic studies confirmed the hypothesis and revealed also that KPD patients have altered ketone oxidation. Clinically stable KPD patients with excellent glycemic control have elevated plasma BOHB. The cause is not excessive fatty acid\u2013derived ketone production\u2014the conversion of acetyl CoA to BOHB is slower, and rates of acetyl CoA flux and isocitrate decarboxylation are unaltered. However, the rate of ketone oxidation is significantly slowed in KPD, leading to elevated concentrations of BOHB and its intracellular metabolites, and this likely plays a prominent role in the most unique characteristic of the phenotype, proclivity for ketosis. Fatty acid disposal is also impaired.\n\nUnable to maximize rates of oxidation of fat-derived sources for energy, KPD patients after an overnight fast draw upon amino acids for energy. There is increased flux through the leucine catabolic pathway toward ketone production. Acceleration of leucine catabolism also leads, through reductive deamination of \u03b1-ketoglutarate, to glutamate accumulation (exacerbated by increased conversion from glutamine) and blunted glucose carbon anaplerosis in the TCA cycle as manifested by an unchanged rate of glutamate carbon oxidation. A similar cascade occurs in McArdle syndrome, in which accelerated BCAA catabolism eventually reduces flux into the TCA cycle through increased conversion of \u03b1-ketoglutarate to glutamate (35).\n\nPeripheral oxidation of ketone bodies is a TCA cycle-dependent process; in this regard, an apparent conflict arises between the finding of slowed BOHB oxidation in KPD and the finding that there is no difference in the rate of oxidation in the TCA cycle when the groups are challenged with a similar flux of oxidative substrate (^13^C~1~-acetate). This apparent paradox may be explained because our measurements specifically demonstrate that KPD show no defect in the rate of removal of CO~2~ from isocitrate (at the step of isocitrate dehydrogenase) in the TCA cycle, and this leaves open the possibility of kinetic defects distal to that step in the TCA cycle and therefore in the rates at which reduced energy intermediates are delivered for oxidative phosphorylation. The data suggest that there may be such a kinetic defect at the level of \u03b1-ketoglutarate dehydrogenase associated with more rapid decarboxylation of leucine, as evidenced by the accumulation of glutamate and reduced levels of acylcarnitines of TCA intermediates beyond that step.\n\nThis study combines for the first time an unbiased metabolomics approach and a targeted kinetics approach to specify the pathophysiology of a novel diabetic syndrome (<a href=\"#F6\" data-ref-type=\"fig\">Fig. 6<\/a>). Umpierrez et al. (36) previously explored the pathophysiology of ketosis-prone type 2 diabetes, a condition identical to unprovoked A^\u2212^\u03b2^+^ KPD. They reported no ketosis or \u03b2-cell decompensation following prolonged infusion of fatty acids, supporting the concept that proneness to ketosis in KPD is not due to excessive fatty acid flux to the liver.\n\nDefective oxidation of ketones and accelerated leucine catabolism are key defects of fasting energy metabolism that underlie the distinctive syndrome of unprovoked A^\u2212^\u03b2^+^ KPD. The findings indicate the need to investigate mitochondrial function and BCAT and BCKDH regulation in the skeletal muscle of KPD patients. These defects are associated with pronounced changes in glutamine\/glutamate metabolism; interestingly, elevated glutamate predicted type 1 diabetes in a longitudinal metabolomics study (37). Clinically and metabolically, patients with A^\u2212^\u03b2^+^ KPD manifest features of both type 1 and type 2 diabetes. The present data explain how overweight patients with a type 2 diabetes phenotype and adequate \u03b2-cell reserve are prone to developing unprovoked ketoacidosis. The coupled metabolomics-kinetics approach should be useful in elucidating the pathophysiology of other phenotypes of diabetes.\n\n## ACKNOWLEDGMENTS\n\nThis study was supported by National Institutes of Health (NIH) R21-DK-082827 (to A.B.), the Diabetes and Endocrinology Research Center (P30-DK-079638) at Baylor College of Medicine, NIH RO1-DK-056689 (to F.J.), funds from the U.S. Department of Agriculture, Agricultural Research Service under Cooperative Agreement Number 58-6250-6001, and NIH PO1-DK-58398 (to C.B.N.).\n\nNo potential conflicts of interest relevant to this article were reported.\n\nS.G.P. and F.J. helped to design the kinetic studies, analyzed data, and edited the manuscript. J.W.H. performed mass spectrometric analyses and analyzed data. I.C. recruited subjects and implemented the stable isotope protocols. J.R.B. and R.D.S. performed metabolomic analyses and reviewed the manuscript. D.I. performed biochemical assays. R.N. and K.O. maintained clinical care of the study subjects and edited the manuscript. C.S.H. performed autoantibody testing of the study subjects and reviewed the manuscript. C.B.N. helped with study design, supervised the metabolomic analyses, and edited the manuscript. A.B. designed and obtained funding for the study, implemented the kinetic protocols, analyzed data, and wrote the manuscript. A.B. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.\n\nParts of this study were presented in poster form at the 71st Scientific Sessions of the American Diabetes Association, San Diego, California, 24\u201328 June 2011.\n\nThe authors thank Elizabeth Fraser, Vy Pham, and Toni Oplt (all from Baylor College of Medicine) for technical assistance; Resa Labbe-Morris, RN, and the nursing staff of the Baylor College of Medicine General Clinical Research Center for meticulous attention to protocol and care of the study subjects; Varsha Patel, RPh, and the Investigational Pharmacy (The Methodist Hospital Research Institute, Houston, TX) for stable isotope preparation; Drs. Heinrich Taegtmeyer (University of Texas Health Sciences Center, Houston, TX), William Mitch, and Arun Sreekumar (both from Baylor College of Medicine) for helpful discussions; and all of the study subjects.\n\n# REFERENCES\n\n[^1]: S.G.P. and J.W.H. are joint first authors.","meta":{"dup_signals":{"dup_doc_count":134,"dup_dump_count":49,"dup_details":{"curated_sources":4,"2022-05":1,"2020-50":1,"2019-43":2,"2019-30":1,"2019-09":1,"2018-39":1,"2018-26":2,"2018-22":1,"2018-17":2,"2018-13":1,"2018-09":2,"2018-05":1,"2017-51":2,"2017-47":1,"2017-43":8,"2017-39":1,"2017-34":2,"2017-30":7,"2017-26":3,"2017-22":1,"2017-17":7,"2017-09":3,"2017-04":10,"2016-50":4,"2016-44":4,"2016-40":4,"2016-36":4,"2016-30":3,"2016-26":3,"2016-22":4,"2016-07":3,"2015-48":3,"2015-40":1,"2015-35":2,"2015-32":1,"2015-27":3,"2015-22":2,"2015-14":2,"2014-52":3,"2014-49":2,"2014-42":2,"2014-41":4,"2014-35":4,"2014-23":2,"2023-06":1,"2017-13":4,"2015-18":2,"2015-06":1,"2024-22":1}},"file":"PMC3581228"},"subset":"pubmed_central"}
{"text":"abstract: A report on the Conference on Systems Biology of Mammalian Cells, Dresden, Germany, 22-24 May 2008.\nauthor: Hanspeter Herzel; Nils Bl\u00fcthgen\ndate: 2008\ninstitute: 1Institute for Theoretical Biology, Humboldt University, Invalidenstrasse, 10115 Berlin, Germany; 2Institute of Pathology, Charit\u00e9, Schumannstr., D-10117 Berlin, Germany\ntitle: Mathematical models in mammalian cell biology\n\nSystems biology has been defined in many ways, emphasizing various aspects of interdisciplinary research. At the recent symposium in Dresden on systems biology of mammalian cells, this emerging field was presented as a fruitful symbiosis of genomics, imaging, and high- and medium-throughput technologies with data analysis and mathematical modeling. Here we focus on selected applications of mathematical modeling in mammalian cell biology - a challenging but promising branch of systems biology.\n\n# Mathematically modeling the circadian clock\n\nOne application of mathematical models is in analyzing the workings of the mammalian circadian clock. About 20,000 synchronized neurons in the suprachiasmatic nucleus (SCN) control daily rhythms of physiology, metabolism and behavior. In addition, almost all peripheral tissues of mammals and even cell lines contain cellular clocks, in which endogenous oscillations of mRNA and protein abundance rhythms with a period of about 24 hours are driven by intracellular feedback loops involving clock genes such as *Per1-2*, *Cry1-2*, *Clock* and *Bmal1*. Post-translational events such as phosphorylation of clock proteins contribute to the delay in negative feedback and thus are crucial for the dynamics of circadian rhythms.\n\nOne of us (HH) reported that a combination of experiments in the lab of Achim Kramer and mathematical modeling led to a deeper understanding of the molecular mechanisms underlying human circadian behavior. In the first example, it was reported that the molecular explanation for a human behavioral disorder called familial advanced sleep phase syndrome (FASPS), which leads to a 4-hour advance in sleep and wakefulness, can be attributed to a point mutation in the circadian *Per2* gene. This mutation leads to a phosphorylation defect of the PER2 protein, changing its stability and subcellular localization in a cell culture model for FASPS. This cell culture model nicely recapitulates the 4-hour phase advance of human behavior by showing advanced rhythms of clock gene expression. Other phosphorylations of PER2, however, have partly opposite effects. Mathematical modeling integrated these experimental data and proposed a dynamical model with differential roles of PER2 phosphorylation sites for circadian dynamics.\n\nIn the second example, human skin fibroblasts from extreme chronotypes (that is, either 'night owls' or 'morning larks') have been used to characterize intrinsic circadian properties of these cells. Although for a large part of the subjects a good correlation between behavioral phase (that is, 'morningness' or 'eveningness' assessed by a questionnaire) and period of clock gene rhythms in skin fibroblasts (assayed by live-cell imaging using luciferase-based reporters) could be found, some subjects have normal circadian periods in their cells, but do display extreme behavioral phases. Computer models here helped to explain these phenotypes by suggesting that the amplitude and input sensitivity of the cellular oscillators should be experimentally investigated.\n\n# Quantifying apoptosis and signaling cascades\n\nAnother impressive example of mathematical modeling and quantitative experimentation going hand-in-hand is the analysis of apoptotic pathways. Heinrich Huber (Royal College of Surgeons, Dublin, Ireland) reported the monitoring of cytochrome *c* release during apoptosis at a resolution of seconds, using confocal and FRET-based imaging techniques. In this way the onset of mitochondrial outer membrane permeabilization in individual HeLa cells was monitored. Combining this imaging approach with mathematical modeling allowed the identification of two separate kinetic phases: an 'ignition phase' during which the mitochondria were not yet fully permeabilized, and a second phase of cytochrome *c* redistribution.\n\nA highlight of the conference was the opening lecture by Douglas Lauffenburger (Massachusetts Institute of Technology, Cambridge, USA). He asked how information about extracellular cues is encoded in the intracellular signaling network and causes a specific cellular response - in this case apoptosis. His group stimulated cells with different levels of tumor necrosis factor \u03b1, insulin, and epidermal growth factor (EGF), and measured phosphoprotein levels distributed across five kinase pathways as well as four apoptotic outputs. This impressive dataset showed that the response is not encoded in a single pathway, but that the information is distributed over the signaling network. It also enabled a comparison of different modeling strategies, including principal component analysis (PCA), fuzzy logic, and differential equations. The combination of these approaches led to interesting insights into the time-dependent role of the kinase IKK in the NF-\u03baB pathway in inducing the apoptotic response and cross-talk mediated via autocrine loops involving transforming growth factor \u03b1 and interleukin 1.\n\nThe question posed by Lauffenburger as to how information about extracellular cues is encoded in intracellular signaling pathways was addressed throughout the conference. Takashi Naka (RIKEN, Yokohama, Japan) presented data from breast cancer cell lines showing that EGF and heregulin induce broadly overlapping immediate-early gene expression patterns despite triggering different cellular responses. Naka speculated that the specificity of response is generated during late-phase signaling by feed-forward loop duration decoding through transcription factors such as c-Fos, which generate waves of expression of secondary, more specific transcriptional regulators depending on the length of the signal. In his closing talk, Hans Westerhoff (Free University, Amsterdam, The Netherlands) pointed out that the length of the signal in a pathway, and thereby the biological response, can be selectively regulated by the expression of phosphatases. He illustrated this with experimental data from growth-factor stimulated mammalian cells, where inhibition of the kinase in the MAP kinase (MAPK) pathway leads to smaller amplitudes of the signal, whereas inhibition of the phosphatases leads to an increase in the durations. This behavior has been previously postulated by Reinhard Heinrich using mathematical models of kinase cascades.\n\nLeonidas Alexopoulos (Massachusetts Institute of Technology, Cambridge, USA) presented data from large-scale experiments on the response of the signaling network of primary and transformed hepatocytes following treatment with various cytokines and small-molecule inhibitors, alone and in combination. Using linear regression, he could work out alterations in the signaling network that were caused by oncogenic transformation. Again, these alterations were distributed in the network and not confined to a single pathway. As a consequence of the distributed information in the network, conventional single-target therapies could have limited efficacy. In this regard, Westerhoff pointed out that targeting the signaling network would require novel therapeutic agendas, such as targeting multiple nodes in the network, and that these targets might not necessarily be close to the original mutation in the network. Responses to such combinatorial inhibitor treatment can also be used to reverse-engineer the network, as demonstrated by Sven Nelander (Memorial Sloan-Kettering Cancer Center, New York, USA). He and colleagues applied combinations of pharmacological inhibitors to perturb mitogenic and pro-apoptotic signaling pathways in breast cancer cells and measured the activity of the pathways' components. From these data they were able to infer feedbacks within the pathway, and the use of combinatorial inhibition allowed for the inference of nonlinear interactions ('synergies') in the pathways. The method may be applicable to the design of targeted combination therapies for cancer.\n\n# Mathematical models in pattern formation\n\nThere is a long history of applying partial differential equations to spatio-temporal pattern formation in morphogenesis, as pioneered by Alan Turing and Hans Meinhardt. Advanced imaging techniques such as fluorescence recovery after photobleaching (FRAP) now allow the quantification of diffusion-reaction dynamics. Frank J\u00fclicher (Max Planck Institute of the Physics of Complex Systems, Dresden, Germany) has combined modeling with experimental data to describe morphogen gradients in the wing imaginal disc of the fruit fly. He reported that quantitative studies reveal that morphogen transport in the tissue is coupled to cellular kinetic processes such as ligand-receptor binding, endocytosis of ligand-receptor pairs and the recycling of ligands to the cell surface. The resulting nonlinear transport equations guarantee a robust gradient profile to regulate the expression of genes in a manner that depends on the distance to the source of the morphogen.\n\nThe classical 'clock and wavefront model' proposed by Erik Christopher Zeeman in 1976 has been applied by Oliver Pourqui\u00e9 (Howard Hughes Medical Institute, Kansas City, USA) to study the vertebrate segmentation clock. This periodic pattern is established in the embryo during the formation of the somites. Somitogenesis involves an oscillator driving the dynamic expression of genes in the presomitic mesoderm from which the somites are derived. Microarray data indicated that the mutually exclusive activation of Notch\/FGF and Wnt pathways coordinate the oscillator. The FGF pathway controls the positioning of the wavefront of gene expression and couples the spatio-temporal activation of segmentation to the posterior elongation of the embryo.\n\n# How useful are mathematical models?\n\nFor small subsystems, dynamical models can be fitted directly to experimental time-course data. But despite extensive microarray, proteomics and imaging data, larger systems such as the mammalian cell-cycle machinery cannot yet be described in quantitative detail by mathematical models. In these cases, however, statistical approaches such as PCA, Bayesian networks and fuzzy logic help to extract useful information.\n\nPresentations at the conference impressively illustrated that complex processes such as apoptosis or morphogenesis can be tackled with a combination of quantitative spatio-temporal data and modeling, thus giving hope that we might be able to make dynamic models of other more complex processes in future as quantitative data becomes available. The conference nicely illustrated that the collaboration of experimentalists and theoreticians helps to design appropriate experimental strategies for the analysis of mammalian cells. Therefore, we believe that the time is ripe to apply systems biology more widely in mammalian cells. Although many models will be semi-quantitative for the near future, they will help guide our experimental design and integrate our data.\n\n### Acknowledgements\n\nWe thank Kieran Smallbone, Stefan Legewie and Achim Kramer for commenting on this report. Attendance of NB at the conference was made possible by an award from the MTZ Foundation.","meta":{"dup_signals":{"dup_doc_count":119,"dup_dump_count":47,"dup_details":{"curated_sources":2,"2022-21":1,"2021-43":1,"2020-24":2,"2019-47":1,"2019-26":1,"2019-09":1,"2018-51":1,"2018-43":1,"2018-39":1,"2018-30":1,"2018-26":2,"2018-22":1,"2018-05":1,"2017-47":1,"2017-34":1,"2017-26":1,"2017-22":1,"2017-09":8,"2016-44":1,"2016-40":1,"2016-36":8,"2016-30":6,"2016-22":1,"2016-18":1,"2016-07":5,"2015-48":3,"2015-40":2,"2015-35":3,"2015-32":3,"2015-27":2,"2015-22":3,"2015-14":1,"2014-52":2,"2014-49":5,"2014-42":7,"2014-41":5,"2014-35":5,"2014-23":5,"2014-15":2,"2023-50":1,"2015-18":3,"2015-11":3,"2015-06":2,"2014-10":3,"2013-48":3,"2013-20":3,"2024-26":1}},"file":"PMC2530880"},"subset":"pubmed_central"}
{"text":"abstract: A recently departed genome biologist discusses the impact factor of his life with St Peter at the Pearly Gates.\nauthor: Gregory A Petsko\ndate: 2008\ninstitute: 1Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02454-9110, USA\ntitle: Having an impact (factor)\n\n*The time: Some time in the not-too-distant future*.\n\n*The place: The entrance to The Pearly Gates. There are fluffy clouds everywhere. In the center is a podium with an enormous open book. A tall figure in white robes with white hair and beard stands at the podium. Approaching is a thin, middle-aged man with glasses and a bewildered expression. He is the soul of a recently deceased genome biologist*.\n\n**GB**: My gosh is this...? Are you...? Am I really...?\n\n**St Peter**: Yes, I'm St Peter. And yes, this is where souls such as yours enter heaven.\n\n**GB**: Wow. I mean, I didn't expect to live forever, but still, this is something of a shock. (*Pauses*.) OK, I guess I can live with it. Uh, I mean ...\n\n**St Peter**: I know.\n\n**GB**: Well, at least I'm here. I'm not thrilled to be dead, but it's a relief to know I'm going to heaven.\n\n**St Peter**: I'm afraid it's not that simple. We have to check.\n\n**GB**: Check what?\n\n**St Peter**: Your life history. (*He leafs through the enormous book*.) It's all here, you know.\n\n**GB**: I'm sure it is. I can imagine you guys keep records that make PubMed seem like a stack of index cards. I'm a little surprised you don't use something more up-to-date, though.\n\n**St Peter**: If you mean a personal computer, no - we don't. After all, they were invented elsewhere.\n\n**GB**: You mean on earth?\n\n**St Peter**: No, somewhere a lot warmer. (*He stops at a page*.) Here you are.\n\n**GB**: Hey, I'm not worried. I was a good scientist, a good citizen, a good family man, I think, too. I never...\n\n**St Peter**: Yes, yes, I'm sure, but you see, none of that matters. The only thing that matters is your IF.\n\n**GB**: IF?\n\n**St Peter**: Your impact factor. That's all we use now. If your IF is above 10, then you enter here. If it's lower, well...\n\n**GB**: My impact factor? What the hell - oops, sorry - is that?\n\n**St Peter**: It's something we borrowed from you science chaps on earth. Oh, we used to do it the hard way: send a fledgling angel down to check on your deeds; look at how your life affected your friends and family, consider your intentions versus your actions. All that sort of thing. It was tedious and required huge numbers of new angels, who have become somewhat scarce since free-market capitalism became all the rage down there. Then we noticed that you scientists never bothered to do anything like that. If you had to evaluate someone, all you did was look at this number called the impact factor. So we did the same thing. Now when anyone comes here, all we do is look up their number.\n\n**GB**: A single number? Are you nuts? You can't sum up someone's whole life in a single number!\n\n**St Peter**:You do. At least, you sum up their career that way, when you decide if they've published in the best journals or done the best work. It's how you work out who gets promoted and who's a star and who gets funded and...\n\n**GB**: Yes, but it's a terrible idea! We should never have done it. It ruined European science in a matter of a few years, and then it spread to Australia, China and Japan, and finally to Canada and the US; and before too long, science was totally controlled by unimaginative bureaucrats who just used that number for everything. It was a disaster!\n\n**St Peter**: That's not what St Garfield thinks.\n\n**GB**: St who?\n\n**St Peter**: St Eugene Garfield, PhD. He invented citation analysis, remember? He thought using the IF was a great idea - really, a logical extension of his own work creating the Citation Index. So we set it up: for example, I see here that you contributed regularly to several local charities.\n\n**GB**: Of course. They do good work. I never did it because I thought it would get me into heaven, but...\n\n**St Peter**: Just as well, because it won't. Local charities, you know. Small impact factor. Doesn't really add much to your total. Besides, how bad could the idea be? Why, the journal *Genome Biology* advertises its impact factor right at the top of their website. Didn't you use to write a column for them? (*He looks at the ledger again*.) Oh my, I see that won't add much to your total either.\n\n**GB**: But that's all ridiculous! It's the whole problem I was trying to explain to you. That's like saying that a paper only has significant impact if it's published in *Nature*, *Science*, or *Cell*. Once you do that, then the impact factor of where you publish becomes a surrogate for the use of your own judgment. No one bothers to read anyone's papers when they're up for a fellowship or being considered for a job or for a promotion or having their grant proposal evaluated; all you do is look to see how many papers they've published in high-impact journals. No one considers whether the work was better suited to a more specialized journal or a journal where other work that puts it in context was published previously; no one considers whether those handful of high impact-factor journals have the best referees or whether they in fact may have a disproportionate number of incorrect papers because of the pressure to publish there. And look, over reliance on one stupid number gave a small bunch of editors enormous power over the careers of people who, for the most part, they never met or heard speak, and whose body of work they never read. It was probably the worst idea since General Custer thought he could surround the whole Sioux Nation with a couple of hundred troops.\n\n**St Peter**: Ah, yes. St Sitting Bull still talks about that.\n\n**GB**: Huh? (*Shakes himself*.) Look, once the impact factor dominated scientific judgments, creative people were doomed. Bureaucrats didn't need to know anything or have any wisdom; all they had to do was rely on arbitrary numbers. And now you're telling me you're doing that to determine who gets into heaven?\n\n**St Peter**: Yes; it's a lot simpler. It doesn't matter if you were kind or tried hard or did good work or were pious or modest or generous. The only thing that matters is how big an impact we calculate you had.\n\n**GB**: But that's just wrong! Look, maybe I could talk to the people who thought up that idea and pushed for its use. If I can just get in for a minute...\n\n**St Peter**: Oh, they're not here. (*He waves his hand and an image appears on a cloud. It shows a huge pit of boiling sulfur and brimstone. In it, up almost to their necks, are a bunch of men in business suits*.) As you can see, they're in a warmer climate.\n\n**GB**: Well, at least, that seems fair somehow. Wait a minute - is that George W Bush?\n\n**St Peter**: Yes.\n\n**GB**: But his impact factor should have been huge.\n\n**St Peter**: Oh, the absolute value was off the charts. But we do take the sign into consideration...\n\n**GB**: Then why is he only in brimstone up to his knees?\n\n**St Peter**: Oh. He's standing on Dick Cheney's shoulders. (*The image vanishes*.) Now let's get back to you...\n\n**GB**: But don't you see, the idea that you can determine someone's impact in the future from where they publish today is totally absurd. On that basis, God would have an impact factor of zero. I mean, He did his best work a long time ago; it has never been repeated by anyone; and all His ideas were published in a book, not in a peer-reviewed journal!\n\n**St Peter**: Very funny. Go to hell.","meta":{"dup_signals":{"dup_doc_count":124,"dup_dump_count":66,"dup_details":{"curated_sources":2,"2023-14":1,"2022-40":1,"2022-21":1,"2022-05":1,"2021-39":1,"2021-25":2,"2021-17":1,"2021-04":1,"2020-50":1,"2020-40":1,"2020-29":1,"2020-16":1,"2020-10":1,"2019-51":1,"2019-43":1,"2019-35":1,"2019-26":2,"2019-18":2,"2019-09":1,"2018-51":2,"2018-43":1,"2018-34":2,"2018-26":1,"2018-13":1,"2017-51":1,"2017-43":2,"2017-39":1,"2017-34":1,"2017-30":1,"2017-26":1,"2017-22":2,"2017-17":1,"2017-09":8,"2017-04":1,"2016-50":1,"2016-44":1,"2016-40":1,"2016-36":8,"2016-30":7,"2016-22":1,"2016-18":1,"2016-07":3,"2015-48":2,"2015-40":1,"2015-35":2,"2015-32":2,"2015-27":3,"2015-22":2,"2015-14":1,"2014-52":2,"2014-49":3,"2014-42":4,"2014-41":3,"2014-35":4,"2014-23":3,"2014-15":2,"2023-40":1,"2024-22":2,"2024-10":1,"2017-13":1,"2015-18":2,"2015-11":2,"2015-06":2,"2014-10":2,"2013-48":2,"2013-20":2}},"file":"PMC2530861"},"subset":"pubmed_central"}
{"text":"author: Emil R. Unanue; Fumihiko UranoCorresponding author: Fumihiko Urano, <firstname.lastname@example.com>.\ndate: 2014-01\nreferences:\ntitle: Endoplasmic Reticulum: An Interface Between the Immune System and Metabolism\n\nEndoplasmic reticulum (ER) is a busy cell organelle that participates in many important cellular tasks. It has been established that ER is involved in protein and lipid biosynthesis, calcium regulation, redox regulation, cell signaling, and cell death. Given the many vital and complex functions of ER, there is little wonder that its failure can trigger a range of diseases. Recent genetic and clinical evidence indicates that inherited or acquired dysregulation of ER homeostasis can give rise to genetic diseases, including Wolfram syndrome (which is characterized by juvenile-onset diabetes and neurodegeneration) and a number of common metabolic diseases including diabetes and atherosclerosis. Accelerating interest in the role of ER in metabolic disease has been fueled by recent reports showing pathways that link ER to inflammation. The role of ER as an interface between the immune system and metabolism is an emerging concept (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<\/a>). However, currently there is no treatment targeting ER for combating immunometabolic diseases. To fulfill this unmet medical need, we need to identify pathways and molecules that link the immune system to metabolism at ER.\n\nER dysfunction has been a suspect as a major pathogenic component of human chronic diseases, such as diabetes, atherosclerosis, and Wolfram syndrome (1\u201310). However, the precise role of ER in the etiology of these diseases is not clear. It has been recognized that inflammation plays a central role in chronic metabolic diseases, raising the possibility that ER is at the intersection of inflammation and metabolism (7,11\u201314). Over the past several years, this concept has been supported by genetic, experimental, and clinical evidence (10,15). One of the unmet scientific needs in this emerging field is to identify the pathways linking ER to production of inflammatory cytokines. Two molecular pathways linking ER to production of interleukin (IL)-1\u03b2, a major player in inflammation, have been recently identified. These are activating transcription factor (ATF) 5 and miR-17 (16,17). Both molecules are regulated by key regulators of the ER stress response, Perk and Ire1\u03b1.\n\nIn this issue, Iwasaki et al. (18) describe the molecular pathway linking ER to IL-6 production. Using DNA microarray and network analyses of macrophages, they show compelling evidence that ATF4, which is involved in the ER stress response, plays an essential role in IL-6 expression induced by various metabolic stresses, including ER stress. Furthermore, they reveal that the ATF4 pathway has a synergistic effect on the Toll-like receptor-4 signaling pathway, enhancing IL-6 expression. IL-6 has been shown to play crucial roles in insulin resistance and type 2 diabetes (19), raising the possibility that ATF4 signaling is a novel target for the treatment of metabolic diseases. The new findings from Iwasaki et al. (18) also suggest that ER-stressed macrophages may trigger autoimmune diseases through IL-6 production.\n\nCompelling evidence indicates that ER is at the intersection of inflammation and metabolism and is therefore an attractive target for immunometabolic diseases. For example, recent evidence strongly suggests that ER dysfunction in antigen-presenting macrophages and \u03b2-cells triggers autoimmunity during the onset and progression of type 1 diabetes (20,21). Despite the underlying importance of ER dysfunction in these diseases, no current therapies target ER. The unmet scientific and medical need in the field of ER immunometabolism is to target the common molecular processes that are altered in ER diseases as a novel therapeutic discovery strategy. The strategy of performing clinical studies using drugs previously known to target ER, such as glucagon-like peptide 1 agonists and vitamin D, on patients with immunometabolic diseases should be explored (22,23).\n\n## Article Information\n\n**Funding.** F.U. is supported by grants from the National Institutes of Health (DK-067493, DK-020579, and UL1 TR000448), JDRF (47-2012-760, 17-2013-512), American Diabetes Association (1-12-CT-61), the Ellie White Foundation for Rare Genetic Disorders, and the Jack and J.T. Snow Scientific Research Foundation.\n\n**Duality of Interest.** No potential conflicts of interest relevant to this article were reported.\n\n# References","meta":{"dup_signals":{"dup_doc_count":133,"dup_dump_count":50,"dup_details":{"curated_sources":2,"2023-06":1,"2022-05":1,"2019-43":1,"2019-18":1,"2018-43":2,"2018-30":2,"2018-22":1,"2018-17":3,"2018-13":2,"2018-09":2,"2018-05":1,"2017-51":3,"2017-47":3,"2017-43":6,"2017-39":2,"2017-34":2,"2017-30":7,"2017-26":3,"2017-22":2,"2017-17":9,"2017-09":3,"2017-04":9,"2016-50":3,"2016-44":4,"2016-40":3,"2016-36":3,"2016-30":2,"2016-26":3,"2016-22":3,"2016-18":3,"2016-07":3,"2015-48":3,"2015-40":1,"2015-35":1,"2015-32":1,"2015-27":3,"2015-22":3,"2015-14":1,"2014-52":3,"2014-42":4,"2014-41":1,"2014-35":1,"2014-23":2,"2023-50":1,"2017-13":4,"2015-18":3,"2015-11":2,"2015-06":2,"2014-10":1,"2024-30":1}},"file":"PMC3968434"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  In May 2000, AstraZeneca launched a Web service for asthma patients and health-care providers called LinkMedica, which includes an asthma diary for monitoring and self-management. In the diary, the patient enters his or her peak flow, number of doses of rescue medication, and if there have been any asthma symptoms during the previous 24 hours. The patient receives an immediate response from LinkMedica, telling him or her if the asthma is under control and what to do if not, eg, increase the dose of inhaled steroid. Health-care providers have access to the patient diary.\n  .\n  # Objectives\n  .\n  The primary objective of the study was to describe patients' and health-care providers' use of LinkMedica. Secondary objectives were to evaluate their perception of the system and how the users' interaction with the system is influenced by their everyday lives.\n  .\n  # Methods\n  .\n  Site statistics regarding number of registered users and diary usage were analyzed. An online survey among users (85 respondents), a mailed questionnaire to health-care providers (131 respondents; response rate 26.8%), as well as in-depth interviews with 10 patients and 5 general practitioners, elicited further quantitative and qualitative data on users' perceptions.\n  .\n  # Results\n  .\n  In February 2003, a total of 7653 users had registered. During 2002, the growth in registered users averaged 50 per month. In the same period, the number of unique diary users per month decreased from 307 to 138. Patients usually stopped using the diary after a short time; the doctors were reluctant to introduce the diary to patients because of time constraints. Several user subtypes were identified among patients and their relatives.\n  .\n  # Conclusion\n  .\n  The self-selected survey responses and in-depth interviews indicated that LinkMedica is generally considered a trustworthy and reliable site by both patients and doctors. However, there was a contrast between users' positive perception of LinkMedica and their unwillingness to use the site for more than short periods. The primary reason for this was that LinkMedica did not fit into their everyday lives because of technical and psychological aspects. A number of recommendations to improve LinkMedica are suggested.\nauthor: Jacob Anh\u00f8j; Lene Nielsen\ndate: 2004\ninstitute: ^1^AstraZeneca A\/SBusiness CommunicationAlbertslundDenmark; ^2^Copenhagen Business SchoolDepartment of InformaticsFrederiksbergDenmark\nreferences:\ntitle: Quantitative and Qualitative Usage Data of an Internet-Based Asthma Monitoring Tool\n\n# Introduction\n\nThe cornerstone of modern asthma care is self-management, allowing the patient to monitor his or her disease severity continuously and to adjust the dose of inhaled corticosteroid based on symptoms, lung function, and use of rescue medication \\[1\\]. A recent Cochrane Review concluded that self-management might improve asthma outcomes significantly \\[2\\]. Several strategies have been developed, including patient education and written actions plans.\n\nWith the appearance of the World Wide Web, new opportunities for communication and interaction between patients and health-care providers have emerged. The Internet has been suggested as a tool for monitoring and for self-management of a number of chronic diseases, eg, diabetes, hypertension, and asthma, and a small number of studies has been reported \\[3-8\\].\n\nLinkMedica DK was launched in May 2000 as a Web service for asthma patients and health- care providers. The service enables asthma patients to monitor their condition using an electronic asthma diary, and allows health-care providers to access their patients' diary data. LinkMedica was sponsored and designed by AstraZeneca Denmark in cooperation with the Danish Asthma and Allergy Association and an independent advisory board of asthma specialists.\n\nTo our knowledge, LinkMedica was one of the first publicly available services taking advantage of the Internet for self-management of asthma and allowing health-care providers to access patient diary data online, thus improving and facilitating the cooperation between health-care providers and patients.\n\nThe LinkMedica Web site is currently available in Denmark \\[9\\]. Until 31 March 2004 LinkMedica was also available in the UK \\[10\\]. Besides different languages, the main difference between the two sites was different algorithms controlling feedback messages to patientsin the diary. This reflects the fact that LinkMedica is prepared for localized set-ups in different countries with different clinical guidelines and treatment practices.\n\nBelow, LinkMedica is briefly described. Readers are encouraged to visit www.linkmedica.dk (Danish) for personal study. Fig. 1 shows a screenshot from linkmedica.co.uk\n\n## System Description\n\nLinkMedica has three main sections: Asthma Management Centre (AMC), Knowledge Centre (KC), and Forum. KC and Forum are immediately available to everyone, whereas AMC requires the user to register and create a user name and a password. Patients are able to create their own accounts online, while health-care providers are required to contact AstraZeneca to get registered. This is to confirm the identity of doctors and nurses available to patients on LinkMedica. When a patient has registered, he or she may select one or more doctors or nurses from the list of available health-care providers. This grants the health-care providers access to the patient's asthma diary if he or she accepts the patient. This procedure ensures that both parties have accepted their collaboration via LinkMedica. After the doctor or nurse has accepted or rejected a patient, the patient receives a notification about this at next login.\n\n### LinkMedica Asthma Management Centre\n\nAMC contains the asthma diary. The intention is that the patients log on every day and enter their asthma values: morning peak flow, number of doses of rescue medication, and whether they have had asthma symptoms at night. After submitting diary values, the patient receives an immediate response saying whether or not his or her asthma is under control and, if it is not, providing detailed instructions on what to do. For example, the user may be instructed to double the dose of inhaled corticosteroids for a period of two weeks if he or she has reported asthma symptoms on two consecutive nights. AMC also has graphics that show trends in peak flow and symptoms coupled with environmental factors such as pollen counts and air pollution.\n\nWhen a health-care provider logs on to LinkMedica, he or she is shown the list of patients who have permitted him or her to access their diary data. By clicking on a patient's name, the health-care provider can see that patient's diary data and graphs.\n\n### LinkMedica Knowledge Centre\n\nIn KC, users can find a large number (\\>100) of articles and news about asthma and allergies. The article section contains summaries of evidence-based scientific papers from peer-reviewed journals. These summaries are presented in two formats: 1) \"In summary,\" a user-friendly summary of scientific papers, written in consumer language, and 2) \"In detail,\" a fuller version of the scientific paper and link to the published paper abstract.\n\nThe following process is used to select papers: A project coordinator appointed by a subcontractor (Foresight Links Corporation) oversees the selection process. The coordinator, who has professional expertise in evidence-based decision-making, is the main liaison between Foresight Links Corporation, the LinkMedica team and the advisory board.\n\nFirst, the coordinator conducts a search of the databases of \"distilled\" evidence using \"asthma\" as the only keyword, and produces a list of the citations yielded by the search.\n\nDatabases used include [The Cochrane Database of Systmatic Reviews](http:\/\/www.cochrane.org\/index2.htm), [Best Evidence](http:\/\/www.bestbets.org\/home\/dbintro.html), [Database of Abstracts or Reviews of Evidence (DARE)](http:\/\/www.ovid.com\/site\/products\/ovidguide\/daredb.htm), [Clinical Evidence](http:\/\/www.clinicalevidence.com\/ceweb\/conditions\/index.jsp), and [Bandolier](http:\/\/www.jr2.ox.ac.uk\/bandolier\/).\n\nThese databases have been built up over the last 10 years by internationally recognized initiatives (including professional and governmental organizations such as the American College of Physicians, the British Medical Association, and the Cochrane Collaboration), engaged in the collection, appraisal, and synthesis of the best available evidence from clinical research.\n\nAs a second step, this list of citations is sent to the advisory board members, together with the list of topics identified as relevant to patients and health professionals. The members of the advisory board are asked to select articles covering as many of the topics on this list as possible. When there are no articles on the list to address topics, members of the advisory board are invited to identify \"classic\" or recent articles published in peer-reviewed journals, based on their own content knowledge and expertise. These articles are retrieved from the Cochrane Controlled Trials Database and PubMed, and are included only if they meet the selection criteria used by [ACP Journal Club](http:\/\/www.acpjc.org\/). The full article selection process was done before launch of LinkMedica UK in summer 2001. Every four or five years, articles will be reviewed again by the advisory board members who are responsible for ensuring the content is up-to-date.\n\nIn addition, news from Danish and international media of interest to asthma and allergy patients is being added on a regular basis from Observer Denmark \\[11\\].\n\n### LinkMedica Forum\n\nIn the Forum section, users may participate in unmoderated discussion groups and ask questions, which are dealt with by experts. The experts are advisers from the Danish Asthma and Allergy Association, a specialist in environmental medicine, a pediatrician, a dermatologist, and a general practitioner \\[12\\]. AstraZeneca and the Danish Asthma and Allergy Association selected the experts.\n\n### Objectives of This Study\n\nThe primary objective of this study was to describe patients' and health-care providers' use of LinkMedica. Secondary objectives were to evaluate users' perception of LinkMedica and how their everyday lives interact with the system.\n\n# Methods\n\nA total of four user studies (two surveys, two interview rounds) were launched in summer\/fall 2002. Site statistics were evaluated in February 2003.\n\n## Site Statistics\n\nSite statistics are available to the site administrator online from LinkMedica's back end based on Web site log files. Total number of registered users at the end of each month and number of unique diary users each month (number of users entering diary values at least once per month) were extracted and plotted against time for visual inspection.\n\n## Surveys\n\nTwo user surveys were carried out. The first was an online pop-up survey targeted at all users visiting the site from April 29 to May 30, 2002. When visiting LinkMedica during this period, the user was presented with a pop-up window asking the user if he or she was willing to participate in an online survey. Except for the introductory text, a yes and a no button were the only elements on the pop-up form. If the user pressed the no button, the window was closed. If the user pressed the yes button, he or she was redirected to the survey form. Regardless of the button pressed, a cookie was set on the user's hard disk to prevent the pop-up window appearing on subsequent visit. If the pop-up window was closed by other means-eg, by clicking the cross in the upper right corner-no cookie was set. No attempts were made to prevent users from submitting more than one survey form by filling in the form on different computers or by deleting the cookie from their own hard disk. We assumed, however, that the risk of a significant number of users doing this was negligible.\n\nThe response rate was not monitored, but the number of submissions was compared with the number of unique visitors (by IP-address) in May 2002.\n\nThe survey questionnaire contained 17 questions. In this article we present the results of 7 selected questions: \"Your age?\"; \"Your gender?\" (male, female); \"What is your background?\" (patient, relative, health care professional); \"What is your primary reason for visiting LinkMedica?\" (seeking information, seeking advice, asthma diary); \"How often do you visit LinkMedica at present?\" (daily, weekly, monthly, less than monthly); \"How often do you intend to visit LinkMedica in the future?\" (daily, weekly, monthly, less than monthly); \"How do you rate the quality of LinkMedica?\" (very good, good, poor, don't know). The last question was asked for each subsection of LinkMedica (diary, knowledge centre, forum).\n\nComplete results from the full questionnaire are available in an internal AstraZeneca report, which is available free (in Danish) to anyone interested.\n\nMales and females were compared with respect to age distribution, reason for visiting LinkMedica (information, advice, diary), and user's background (patient, relative, health-care provider).\n\nThe second survey, a mailed questionnaire, was sent to all health-care providers that- according to AstraZeneca's customer database-had received a user name and password for LinkMedica. The questionnaire was in two sections. The first section of 4 questions was intended for all respondents. The second section of 15 questions was intended for those who, in their own opinion, had ample experience in using LinkMedica. Only the results from the first section are presented in this article. The full report (in Danish) is available free to anyone interested. The questions from the first section were: \"Your profession?\" (physician, nurse, secretary, other); \"Have you heard of LinkMedica?\" (yes; no); \"Do you think that there is a need for Internet tools like LinkMedica in medical practice?\" (yes, no); \"Do you ever use LinkMedica in collaboration with your patients?\" (Yes-frequently, Yes-sometimes, I have looked at it-but did not find it useful, No, No-but I would like to try).\n\nThe questionnaire results allowed us to select persons representing different types of LinkMedica users for interviews; respondents of both surveys were asked to provide name, address, and phone number if they were interested in being interviewed.\n\n## Semi-structured Interviews\n\n> To seek to understand the social world as it is for those people whose social world it is, is possible only if one practices the art of listening to them in their own terms and attends to the social world they construct for themselves. (Zaner \\[13\\])\n\nIn-depth qualitative interviews were conducted with users in order to get an understanding of the patients' approach to their illness and their use of the Internet and the doctors' approach to the use of Web-based monitoring systems.\n\nA total of 15 users were selected from respondents who were willing to be interviewed for semi-structured interviews: 8 patients, 2 mothers of children with asthma, and 5 GPs. They were selected to represent: 1) male and female users, 2) users of AMC and of KC and Forum, 3) frequent visitors and occasional visitors, and 4) health-care providers, patients, and relatives.\n\nThe interviews were designed to address three issues:\n\n-   Who are the users?\n\n-   How do the users use LinkMedica?\n\n-   How do the users' everyday lives interact with LinkMedica?\n\nThe perspective of the qualitative method is to understand the world as inter-subjective-to understand the world from the point of view of those who live in the world. The purpose of this interpretative approach is to understand social phenomena-to understand the lived experience and the complex world this experience takes place in \\[14,15\\].\n\nAll interviews were conducted as semi-structured qualitative interviews according to Kvale's criteria for conducting and analyzing qualitative interviews \\[16\\]. They were taped and transcribed. Each interview was broken down into thematic units and these were compared across interviews. As the qualitative method provides insight into the inter-subjective world, it is not possible to quantify the data: they are interpreted as themes from the lived experience of those interviewed.\n\nThe starting point of the interviews was the patients' relation to their disease, and both doctors' and patients' strategies for information seeking and use of the Internet. Themes that appeared during the first interviews were pursued in later interviews.\n\n# Results\n\n## Trends in Number of Registered Users and Diary Users\n\nIn February 2003, a total of 7653 diary users were registered on LinkMedica. The number of unique diary users at that time was 138 per month.\n\n<a href=\"#figure2\" data-ref-type=\"fig\">Figure 2<\/a> shows that the growth rate of user numbers falls into three phases: In the first 3 months after launch, more than 2000 new users registered. During the next year and a half approximately 4000 users registered, and during the last year there was a growth of approximately 50 new users per month.\n\nThe trend in unique diary users is more uneven. After a peak of 100 diary users in May 2000, the number decreased until November 2000, when only 9 users kept an online diary. During the next year the number of diary users increased (with a decrease during holiday seasons) to a maximum of 307 in January 2002. This increase occurred at the same time as AstraZeneca started marketing LinkMedica. After this, the number of unique diary users was steady for a period of about 4 months. However, in February 2003, the diary user number had declined to 138 per month.\n\n## User Survey\n\nBetween April 29 and May 30, 2002, 85 users responded to the online pop-up survey. Compared to a total of 3689 unique visitors in May 2002, this gives an estimated response rate of 2.3%. Of these 59 (69%) were patients, 12 (14%) were mothers of children with asthma and 8 (9%) were health-care providers (see <a href=\"#table1\" data-ref-type=\"table\">Table 1<\/a>). Inter-quartile age range was 29 to 43 years.\n\nBecause of a programming error in the survey, gender was not recorded for 13 users. Of those remaining, two thirds were female. Mean age was 36 years for females and 41 years for males. This difference was not statistically significant (p=0.12). The age distribution for males and females respectively is shown in <a href=\"#figure3\" data-ref-type=\"fig\">Figure 3<\/a>.\n\nUser background and reason for visiting LinkMedica differed somewhat between males and females. Most females were seeking information and advice (49%), while the majority of males gave the asthma diary as primary reason (56%) (<a href=\"#table2\" data-ref-type=\"table\">Table 2<\/a>). Only 1 of 7 health-care providers was female. This difference between male and female users with respect to their background (patient, relative, health-care provider) and their reason for visiting LinkMedica (information, advice, diary) was statistically significant (p\\<0.05, chi square test).\n\nSeventy-two percent reported that they visited LinkMedica at least once a month, and 92% reported that they expected to visit LinkMedica at least monthly in the future.\n\nWhen asked how they perceived the quality of LinkMedica, the majority of users answered that the quality was good or very good. However, for each main section a rather large percentage of users answered that they did not know (<a href=\"#table3\" data-ref-type=\"table\">Table 3<\/a>).\n\nDemographics of respondents to pop-up online survey\n\n| Background | All | Males<a href=\"#table1fn2\" data-ref-type=\"table-fn\">**<\/a> | Females<a href=\"#table1fn2\" data-ref-type=\"table-fn\">**<\/a> |\n|----|----|----|----|\n| Patient | 59 (69%) | 17 (68%) | 32 (68%) |\n| Relative | 12 (14%) | 0 (0%) | 11 (23%) |\n| Health care professional | 8 (9%) | 6 (24%) | 1 (2%) |\n| Other | 6 (7%) | 2 (8%) | 3 (6%) |\n| Total | 85 (99%)<a href=\"#table1fn1\" data-ref-type=\"table-fn\">*<\/a> | 25 (100%) | 47 (99%) |\n\n^\\*^ Because of a programming error in the survey, gender could not be accounted for in 13 users\n\n^\\*\\*^ p = 0.04, chi square test\n\nResponse to the online pop-up survey question \"What is your primary reason for visiting LinkMedica?\"\n\n| Reason | All | Males<a href=\"#table2fn1\" data-ref-type=\"table-fn\">*<\/a> | Females<a href=\"#table2fn1\" data-ref-type=\"table-fn\">*<\/a> |\n|----|----|----|----|\n| Information (Knowledge Centre) | 23 (27%) | 4 (16%) | 18 (38%) |\n| Advice (Forum) | 6 (7%) | 0 (0%) | 5 (11%) |\n| Asthma diary (Management Centre) | 39 (46%) | 14 (56%) | 18 (38%) |\n| Other | 17 (20%) | 7 (28%) | 6 (13%) |\n| **Total** | 85 (100%) | 25 (100%) | 47 (100%) |\n\n^\\*^ Because of a programming error in the survey, gender could not be accounted for in 13 users\n\nUsers' responses to the online pop-up survey question: \"How do you rate the quality of Asthma Management Centre, Knowledge Centre, and Forum respectively?\"\n\n| Assessed quality | Asthma Management Centre | Knowledge Centre | Forum      |\n|------------------|--------------------------|------------------|------------|\n| Very good        | 27 (33.7%)               | 15 (18.7%)       | 8 (10.3%)  |\n| Good             | 28 (35%)                 | 33 (41.2%)       | 24 (30.8%) |\n| Poor             | 2 (2.5%)                 | 2 (2.5%)         | 2 (2.6%)   |\n| Don't know       | 23 (28.7%)               | 30 (37.5%)       | 44 (56.4%) |\n\n## Health Care Providers Survey\n\nOut of 489 questionnaires mailed to health-care providers that-according to AstraZeneca's customer database-had been given user name and password for LinkMedica, 131 were returned (response rate 26.8%). Among the respondents there were 127 (97%) physicians and 4 (3%) nurses. Fifty-one (39%) used LinkMedica intermittently or frequently for their asthma patients. Questionnaire results are summarized in <a href=\"#table4\" data-ref-type=\"table\">Table 4<\/a> and <a href=\"#table5\" data-ref-type=\"table\">Table 5<\/a>.\n\nHealth-care providers' answers to two questions from the mailed questionnaire\n\n| **Question** | **Yes** | **No** | **Total** |\n|----|----|----|----|\n| Have you heard of LinkMedica? | 113 (86%) | 18 (14%) | 131 (100%) |\n| Do you think that there is a need for Internet tools like LinkMedica in medical practice? | 96 (73%) | 35 (27%) | 131 (100%) |\n\nHealth-care providers' answers to the question: \"Do you ever use LinkMedica in collaboration with your patients?\" (from mailed questionnaire)\n\n| **Answer**                                     | **Frequency** |\n|------------------------------------------------|---------------|\n| Frequently                                     | 4 (3%)        |\n| Sometimes                                      | 47 (36%)      |\n| I have looked at it-but did not find it useful | 4 (3%)        |\n| No                                             | 46 (35%)      |\n| No-but I would like to try                     | 29 (22%)      |\n| Total                                          | 130 (99%)     |\n\n## Interviews\n\nA total of 15 one-to-one in-depth interviews, each lasting approximately 60 minutes, were conducted.\n\n### Who Are the Users?\n\nFive thematic types of users were identified among the interviewees. Characteristics are summarized and compared in <a href=\"#table6\" data-ref-type=\"table\">Table 6<\/a>.\n\nInterviewees\n\n| User | Demography |\n|----|----|\n| Patient 1 | Male, 28, severe asthma, uses the diary. Has posed questions to expert. |\n| Patient 2 | Female, 37, asthma, diary user, mother of two, one has asthma. |\n| Patient 3 | Female 33, asthma, mother of a child with asthma, does not use the site. |\n| Patient 4 | Female 53, severe asthma and allergy, diary user. |\n| Patient 5 | Female 43, asthma and allergy, has used the site for information. |\n| Patient 6 | Male, 40, asthma, diary user. |\n| Patient 7 | Female, 35, asthma, diary user, pregnant with first child. |\n| Patient 8 | Female, 38, daughter with allergy, has posed questions to expert. |\n| Patient 9 | Female, 38, son with severe asthma, uses the whole site. |\n| Patient 10 | Male 48, asthma, diary user. |\n| GP 1 | Female 40, been a GP for 3 years, uses the diary. |\n| GP 2 | Male 53, been a GP for 18 years, uses the diary. |\n| GP 3 | Male 52, been a GP for 26 years, uses the diary. |\n| GP 4 | Male 52, been a GP for 20 years, does not use the diary. |\n| GP 5 | Male 58, been a GP for 20 years, working part time, uses the diary. |\n\n### Patients\n\nWe identified a thematic difference amongst the patients. We labeled the two distinctly different types of patients as *controllers* and *neglecters*.\n\nThe **controllers** wish to gain control of their disease. They establish daily routines to control and monitor the disease so they do not have to worry about it. Their homes are designed to prevent asthma attacks. They use AMC to monitor their condition.\n\nThe following is an excerpt from a patient interview. Interviewees are listed and characterized in <a href=\"#table7\" data-ref-type=\"table\">Table 7<\/a>.\n\n> Interviewer: \"Almost everybody in the family suffers from asthma. Does it influence your everyday life?\"\n\n> Patient 2: \"No, not really. It influences us in that way-as you can see-that we do not have carpets, just the bare floor. And our son cannot have the pets that he would like to have. And we have installed a ventilating system in order to try and reduce the humidity. So in that way it has influence on our surroundings. We have chosen to hire a cleaner to clean the house because we realize that we cannot do it properly ourselves. So it does influence our lives, but we do not think about it on a daily basis.\"\n\nThe **neglecters** do not want to think about their disease. By not focusing on it, they feel better. In this way they do not use mental energy on the disease, and they consider this to be good for their health. To feel secure, they just need to carry their rescue medication with them. This excerpt from an interview reflects this attitude.\n\n> Patient 4: \"Then I must figure out myself what is good for me.\n\n> Interviewer: \"Instead of exploring and reading?\"\n\n> Patient 4: \"Yes-you can get so focussed on it at times. Sometimes it is better to pretend nothing is wrong. It's a balance, you know.\"\n\nThere was no distinction between the sexes in these attitudes.\n\nWe also observed that different user types might very well be expressed within the same person at different times and that most users possess traces of both the controller and the neglecter types. Thus, a person could say that he or she did not pay attention to the disease and at the same time talk about refurbishing the entire house or about being highly aware of things that might provoke an attack.\n\n### Mothers\n\nThe two mothers of children with asthma or allergy interviewed were different in their needs and response patterns to information. One motherexpressed an urgent need for information and responded emotionally to the information. She was mainly interested in guidelines that could help her in her present situation. She was not interested in abstract knowledge such as research results or scientific information.\n\nThe other mother used all her energy to control her child's disease. She subscribed to news and was active in the Forum. She was empowered by the use of AMC and strived to control her child's disease. She did not think this behavior influenced her family life. The following excerpt is an example of this.\n\n> Interviewer:\" I can see you spend a lot of time looking for information. How does that influence your everyday life?\"\n\n> Patient 9: \"It does not-in any negative way. I think it is good that I don't have to spend all day reading newspapers. Now I can use the Internet if there is anything I need to know.\"\n\nThese two mothers were comparable to the neglecter and the controller types. They will be referred to as the **emotional** and the **professional** mother respectively.\n\n### General Practitioners\n\nThe typical GP user is a male around fifty years old who works at a GP clinic with a small number of other doctors. Two thematic subgroups were identified: the *user*, who had experience in monitoring asthma patients with LinkMedica and the *interested*, who was considering using LinkMedica in the future.\n\nThe **user** was introduced to AMC through participation in a clinical trial. He finds the system of great value for the patients, but he doesn't use it himself now the trial has ended. He finds that AMC has too many functions-more than he needs. He finds the system to be complex with a complicated login procedure.\n\nThe **interested** has no experience in using LinkMedica but has heard about it. He believes that both he and his patients might benefit from using the system.\n\nThe typical GP user is not a confident user of either the Internet or a PC. The GP knows his own electronic patient record system, but he doesn't use the PC for anything else. He is connected to the Internet through an integrated services digital network (ISDN) connection. This creates obstacles for a smooth login procedure and prevents him from being online all the time.\n\nThematic user segmentation\n\n| User segmentation | Patient: The disease controller | Patient: The disease neglecter | Relative: the emotional mother | Relative: the professional mother | The GP |\n|----|----|----|----|----|----|\n| Cause & action | Causal (it is possible to find the cause for an attack\/rash) | Deterministic (the causes can be found but they are not important to find) | Causal (it is possible to find the cause for an attack\/rash) | Causal (it is possible to find the cause for an attack\/rash) | No focus on the causal relationship |\n| Relation to disease | Instrumental | Ad hoc information seeking according to need | Instrumental | Ad hoc information seeking according to need | Patient must learn to accept their asthma |\n| Knowledge | Disseminated information.Active search for information | Experienced information.Information is sought when acute needs appear. | Disseminated information.Active search for information | Experienced information.Information is sought when acute needs appear. | Courses arranged by the Danish Medical Association or the medical industry |\n| Information type | Research articles, news, etc. | Funny information, clarifying information. | Instructive information | Research articles, news, etc. | Medical journals, easy read articles |\n| Relation between GP and patient | The GP is perceived as ignorant on subjects concerning asthma and allergy | The GP is perceived as ignorant on subjects concerning asthma and allergy | The GP is perceived as ignorant on subjects concerning asthma and allergy | The GP is perceived as ignorant on subjects concerning asthma and allergy | The GP is a consultant for the patient |\n\n### How Do Users Use LinkMedica?\n\nFrom the interviews we found that the user's perspective plays an important role in how the system is perceived and used.\n\nIn the **outside-in** perspective, the users have a problem that arises in the outside world and expect to find answers in LinkMedica. These users have an acute need for information and browse the site to fulfill this need. They ask questions, and they expect a quick reply. It is mainly women who have the outside-in perspective.\n\nIn the **inside-out** perspective, the users focus on the use of the diary. They do not read or look for articles and news. But once in a while, an interesting headline may catch their attention. They expect the system to operate as smoothly and as quickly as possible. They have high expectations to the usability of the system. Most of the users who used the diary participated in another research project. It is more often males that have the inside-out perspective. This also applies to GPs.\n\n### How Does the User's Everyday Life Interact With LinkMedica?\n\nMost patients interviewed found it easy to use LinkMedica and to enter diary data. In spite of this, none of them used the diary as intended, ie, entering diary values immediately after measuring morning peak flow. All patients wrote the values on a piece of paper and entered the values in LinkMedica whenever it was convenient. Some had access to the Internet at work while others used their home computer in the evenings or during the weekends.\n\nIn general, we found that the users were satisfied with LinkMedica; some expressed a will to continue using the site to monitor their asthma over time and to identify asthma triggers. But even though they were motivated, most of the users believed that they would not continue using the site. This excerpt shows a typical behavior:\n\n> Patient 2: \"I do not enter diary values every day. I do it in batches. On the other hand, you should enter values every day in order to benefit from the system. I really want to, but I never get it done.\"\n\nAs a system, AMC is seen as a reliable tool. However, when the patient receives an unexpected message, one that contradicts his or her previous experience, the patient reacts with disbelief. The patients that had experienced a red alert instructing them to increase the dose of inhaled steroid did not understand why this was important, and none of them took the prescribed action.\n\nIn general, the patients were reluctant to use medication on a regular basis. It was difficult for them to accept the fact that a daily dose of medicine is better than using medicine only when experiencing symptoms, as this excerpt shows:\n\n> Patient 6: \"I thought it was a high dose. I did follow the instructions on the Internet-but not the dose.\"\n\n> Interviewer: \"Why not?\"\n\n> Patient 6: \"I might have had a low peak flow for a couple of days. But it kept instructing me to increase the dose, and I did not think it was necessary.\n\n> Interviewer: How much would be reasonable?\"\n\n> Patient 6: It should have said a little bit more.... I do not know the side effects \\[talking to the microphone\\]-do I?\n\nWhen asked to consider the ideal patient for the system, the GPs generally described a young man who does sports and has an interest in computers, as expressed here:\n\n> GP 4: \"I imagine a young man around 20 who is troubled by his asthma and knows about computers. So I don't have to explain everything to him. I think it is complicated and I would rather not explain it.\"\n\nThe GPs said that their relations with the patients are currently in transition. From being considered experts, doctors are now more like consultants who identify problems and cures in collaboration with the patients**.** The GPs find that this is a positive development. But resources are limited, and the consultations still have to fit the 10-minute slots GPs can make available for appointments. This puts a strain on the GPs. Furthermore, as most GPs themselves are not confident PC users, they find it very difficult to instruct the patients in the system. The consultant role requires confidence as well as technical insight, and most GPs do not feel they have this insight.\n\nFrom the GP's point of view, the patients benefit from using LinkMedica. The system helps patients understand their disease, improves compliance, and reduces symptoms. Furthermore, LinkMedica stresses the patient's own responsibility for his or her disease, as this GP says:\n\n> GP 5: \"It is really motivating. The first of my patients who used the system came back to me and said: I'm so happy I tried this. I went hill-walking in Norway, and you know what? I went all the way to the top and back down again. I haven't been able to do that for many years.\"\n\nNone of the GPs reported that the patients had difficulties in using the system. The GPs themselves, however, found that using LinkMedica was difficult. The login procedure, especially, was perceived as an obstacle. A GP expressed it like this:\n\n> \"It is an obstacle. I think it is important that the computer logs you in automatically by remembering your login information. All that about changing your password: Forget it! People don't do it.\"\n\nFrom the GP's perspective, AMC is a useful tool, especially for things that computer systems do well: record keeping and performing calculations. This is the major advantage of the system. The disadvantage is that it takes time to log in and to instruct the patients. Also, the GPs found that the system has more functions than necessary.\n\nEven though the GPs have a positive attitude towards the system, their use of it is influenced by external factors such as time and economy. As the GPs put emphasis on these factors, they do not use the system.\n\n# Discussion\n\nSurvey and interview data indicate that users are happy with LinkMedica in general. Patients find that the asthma diary helps them manage their disease, and doctors find that the diary improves asthma control in patients using it. This observation is supported by preliminary data from a clinical trial. These data, which are currently under evaluation and have been published in abstract form**,** suggest that LinkMedica improves lung function, asthma severity score, and bronchial hyperreactivity compared to traditional treatment regimens initiated by either a GP or a pulmonologist \\[17\\].\n\nLinkMedica as a whole is considered a reliable system that offers information of high quality about asthma and allergy. Furthermore, doctors and patients haveexpressed a need for improved tools for asthma monitoring and management. In this respect, the users consider the Internet a medium of high interest.\n\nInterestingly, however, we also found that despite their positive attitude and readiness, both doctors and patients usually stop using LinkMedica after a short period of time. From site statistics and informal user contacts we were already aware of this problem before the project started. The project, however, has given us insight into possible reasons forthis evident paradox:\n\n-   Users are inexperienced with the Internet and computers.\n\n-   Access to the Internet is limited and cumbersome.\n\n-   Users' everyday lives interact with LinkMedica in unpredictable ways.\n\n-   Different user types have conflicting needs.\n\n-   Internet information may support but not change users' inherent attitudes.\n\n-   The benefits of using an asthma diary are not recognized immediately.\n\n## Users Are Inexperienced With Internet and Computers\n\nSurprisingly, we found that the users having most difficulties with LinkMedica were the doctors, whom we expected to be confident computer and Internet users. None of the patient users expressed any difficulties whatsoever using LinkMedica. The main complaint from doctors was that the login procedure is complicated and time consuming. The doctors have difficulties managing different user names and passwords for different services. Even though changing user name and password to something easy to remember is straightforward in LinkMedica, none of the doctors did this.\n\nFrom training sessions with GPs, it is our personal experience that doctors find it difficult to use more than one application at a time. In general, doctors are satisfied with their electronic patient record system available from their desktop, but they do not feel confident in \"windows juggling.\" This prevents them from having LinkMedica at hand whenever an asthma patient comes to the clinic.\n\nIn this sense, lack of practice and confidence hinders the use of LinkMedica. But surprisingly, only doctors seem to have this problem. This observation could be the result of a selection bias, leaving only computer literate patients for survey and interview. Although this may be partially true, we do not believe this fully explains why patients apparently have fewer problems than doctors accessing LinkMedica. It may be that doctors have higher demands and are more critical because of time constraints in their work. The fact that doctors on average are older, have less experience with the Internet, and seem to use the Internet less than patients for general information seeking may also play a role.\n\n## Access to Internet Is Limited and Cumbersome\n\nEasy access to the Internet is critical for users' experience with LinkMedica. In 2002, 76% of Danes had access to the Internet either from work or home, and 56% used the Internet at least weekly (38% daily) \\[18\\]. Thus, in Denmark, Internet availability is hardly a barrier. But speed of connection may be. In the surveys, we did not ask how the users connected to the Internet. But in the interviews, some users complained that the connection and logon time through an analog modem was an obstacle. Although our impression is that LinkMedica loads faster than many other Web sites with comparable content, booting the computer and dialing up with a modem may take several minutes. In comparison, it usually takes less than 20 seconds to fill in the diary and receive the feedback message. As broadband connections become more available and affordable in the future, we expect these problems to diminish.\n\n## Users' Everyday Lives Interact With LinkMedica in Unpredictable Ways\n\nIn our opinion, an interesting finding from this study is how much users' everyday lives interact with LinkMedica. As an example, take the doctors' time schedules. They have about 10 minutes per patient. This, together with the fact that most doctors do not feel confident in using the Internet, or even their computers, has an enormous impact on how the doctors look at LinkMedica. The end result is that not a single one of the doctors that we interviewed used LinkMedica on a regular basis, despite their positive attitude. Furthermore, the doctors were mistaken in their views of who would use LinkMedica. According to the doctors, the typical LinkMedica user would be a young, sporty man, when in fact, the typical user is a mother around 40 years of age.\n\nThe following is another example of how everyday life influences the use of LinkMedica: Typically, the diary users measure their peak flow in the morning and write down the value. They collect the values for a week and type them in later, during the weekend. This way, feedback messages are received days after the condition that triggered them, and the whole idea of immediate dose adjustments due to changes in symptoms or peak flow is lost.\n\nWe are convinced that this problem has something to do with the Internet still being separate from the rest of people's everyday lives. The Internet is something you actively connect to, not something that is just there like the telephone or the television. In this sense, LinkMedica and other Internet-based disease management systems are ahead of their time, and we would expect these programs to gain popularity and usability as the Internet gets more integrated into our everyday lives. Currently, we are investigating other means of connecting to LinkMedica, eg, short messaging system (SMS) and general packet radio service (GPRS). These technologies have the advantage of being closely integrated into people's everyday lives and are immediately available to anyone, anywhere without the inconvenience of having to start the computer or wait for slow dial-up connections.\n\n## Different User Types Have Conflicting Needs\n\nA number of patient subtypes were identified: controllers, neglecters, professional mothers and emotional mothers. The doctors came in two groups: users (or more correctly, former users) and potential users, who had no prior experience with LinkMedica but who were interested in trying it. During the interviews, it became obvious that these highly different user types have very different requirements and expectations of LinkMedica.\n\nDifferent parts of LinkMedica (AMC, KC, Forum) are considered important depending on the user's perspective: inside-out or outside-in. The doctor and the controller, together with the professional mother, expect the diary (AMC) to function without a hitch. News, discussion forums, and ask-the-expert sections are merely distracting elements preventing them from having fast access to the diary. If they ask for information, they want it to be as complete as possible, enabling them to decide for themselves how to act. They prefer evidence-based articles and expert opinions to news and advice from other users. The neglecter and the emotional mother, on the other hand, seek information only when they need it. They expect concise information and concrete advice to help them in their current situation. They usually avoid scientific articles and expert opinions unless they are directly applicable to their current needs. They are not interested in monitoring their disease using the diary.\n\nCreating a Web site that seeks to satisfy such conflicting needs and user perspectives may not be a good idea. For future Web projects like LinkMedica, we suggest that the target users be defined clearly from the very beginning. For existing Web sites of complex nature like LinkMedica, it may be worthwhile considering a split into several more focused sites, which may, of course, be interlinked.\n\n## Internet Information May Support but Not Change Users' Inherent Attitudes\n\nIt is an interesting observation that diary users (controllers) were the ones least likely to follow the advice in the feedback messages. As mentioned previously, not a single interviewee who received an alert message instructing him or her to increase the dose of steroid followed this advice. This observation calls into question the whole idea of the diary design as it is today. If the users most likely to use the diary are also the users least likely to follow specific advice from feedback messages, it might be worthwhile reconsidering the format and the content of the feedback messages.\n\nThe idea of having an electronic asthma diary with an \"intelligent\" feedback system was to support and educate asthma patients in self-management. In this respect, the diary may serve as a daily consultation with a virtual asthma expert. This study taught us, however, that an important difference between virtual and real experts is that patients do not readily accept advice from a virtual expert if this advice conflicts with the patient's own previous experience and attitudes in general, eg, that they are opposed to the use of steroids. The lesson learned is that no matter how intelligent and how well supported by acknowledged experts, a computer system cannot replace real face-to-face contact between doctor and patient. In future versions of LinkMedica and similar systems, we suggest that the very detailed (and complicated) feedback system be replaced by a simple \"traffic light\" approach. For example: \"If your asthma diary says green, all is well. If it says yellow two days in a row or more, or red on a single day, contact your asthma doctor.\"\n\n## The Benefits of Using an Asthma Diary Are Not Recognized Immediately\n\nFrom classical behavioral psychology we know that in order to reinforce certain behavior, the latency time between behavior and reward must be short. This phenomenon has great implications for treatment of asthma.\n\nIn general, it takes time to achieve asthma control. The effect of inhaled steroids on symptoms and exacerbations resulting from previously inadequate treatment may be delayed weeks or months after start of treatment. Furthermore, relapse after cessation of treatment may also be delayed. Since the reward (improved health) is delayed, it can be difficult for the patient to understand why taking regular medication is important. This latency problem is probably one of the reasons that inhaled steroids are being used much less than recommended \\[19\\] and also a main reason why patients tend to stop using LinkMedica after a short time.\n\nAs with medical treatment, self-management using an electronic asthma diary may take some time to prove its value to the patient. The immediate advantage of measuring peak flow, turning on the computer, connecting to the Internet, logging on LinkMedica, and entering diary values is simply not big enough for the patient to continue doing this for longer periods. Even if a patient has experienced improved asthma control from using LinkMedica, the advantage of continuing use may not (from the patient's viewpoint) justify the inconvenience.\n\nIn principle, there are two ways to solve this problem: by improving the accessibility of the diary, which could be achieved by, for example, improved Internet connections, mobile phones, or wireless devices (peak flow meters, electronic dispensing devices, etc.); or by increasing the immediate advantage of diary entry. There may be several ways to achieve the latter. Further studies need to be carried out to reveal whether it is the technology or the lack of immediate advantages that creates obstacles. User studies and explorative design methods \\[20\\] can help to clarify the users' needs.\n\nAs an example, teenagers would probably adhere more to the diary if they were permitted a small number of free SMS messages for their mobile phones after each diary entry, while others would be encouraged by reimbursement of a portion of their medicine costs linked to how often they entered their diary values. The decrease in unscheduled doctor visits, days off work, etc. resulting from the improved health of users would probably offset the cost of such reimbursement programs. Rewards may also be \"virtual\": small games where points earned from filling in the diary give access to new game levels might encourage children (and some adults) to adhere to the diary. General information on the user's health-eg, weekly or monthly messages with overall information about the user's asthma, whether it has improved or worsened, and what to do about it-might also prove useful.\n\n## What Can Be Done to Support the Use of LinkMedica and Similar Web Sites?\n\nGiven 3 years of experience developing and marketing LinkMedica together with the results of this study, we are able to suggest improvements that would make new users more likely to hold onto their Web-based asthma diaries for longer periods:\n\n-   Split LinkMedica into two sites, one for the diary and one for information, discussion groups and ask-the-expert sections.\n\n-   The diary should be developed after the \"lean-mean-machine\" principle, completely free of distracting elements like news, flashy graphics, opinion polls, etc. Fields for login and diary values should preferably be available on the front page, allowing for one-click access and diary entry.\n\n-   Redesign the feedback messages to merely *inform* about asthma status, rather than give concrete *advice* regarding dosage etc.\n\n-   Remove peak flow from the diary. The value of peak flow monitoring in asthma care is debatable \\[21\\], and many users find peak flow measurements cumbersome, which hinders the use of the diary. Alternatively, recording peak flow should be optional.\n\n-   Explore other methods for data entry and feedback than conventional Web forms. A combination of daily data entry via mobile phone and occasional Web access for diary overview and graphing facilities could prove valuable.\n\n-   Consider how to \"reward\" users immediately after diary entry. This is essential. Users of the diary must have some sort of immediate reward after dairy entry in order to continue using it for longer periods.\n\n## Conclusion\n\nIn general, LinkMedica is regarded as a very reliable and advantageous system by both patients and doctors. However, only a few users are using LinkMedica as intended, and most users, patients as well as doctors, stop using the diary after a short time. There are several reasons for this, the main reason being that the Internet in general and LinkMedica in particular are still not integrated into people's everyday lives. Consequently, if LinkMedica is to become more popular, it needs to be adapted to the conditions of the users, so it becomes a natural and integrated part of their everyday lives.\n\nAstraZeneca A\/S, Roskildevej 22, 2620 Albertslund, Denmark sponsored this study.\n\nThe Danish Asthma-Allergy Association, Astma-Allergiforbundet, which was involved in the design of the website, is a private, independent, non-profit organization whose aim it is to promote better health for people with asthma and allergy in Denmark. Astma-Allergiforbundet is a member of the European Federation of Asthma and Allergy Associations ([www.efanet.org](http:\/\/www.efanet.org\/)).\n\nThe members of the International and the Danish advisory board are: Martyn Partridge, UK; Paul O'Byrne, Canada; S\u00f8ren Pedersen, Denmark; David Price, Scotland; John Haughney, UK; Lone Agertoft, Denmark; Klaus Phanareth, Denmark; Carsten Vrang, Head of the Department of Pediatrics, Storstr\u00f8ms Sygehus, N\u00e6stved, Denmark; J\u00f8rgen Vestbo, UK; Asger Dirksen, Denmark.\n\nJacob Lenau, e-Business Manager, AstraZeneca, reviewed and approved the manuscript.\n\nKirsten Broe Pedersen, AstraZeneca, did proofreading and translated citations of the interviewees.\n\nTorkild Clemmensen, TCPsych, and Claus Bornemann, Kamene Research, participated in planning and evaluation of the online user survey.\n\nThomas Dunk, frontAvenue A\/S, programmed the online survey.\n\n## Abbreviations\n\nAMC\n\n:   Asthma Management Centre\n\nGP\n\n:   General Practitioner\n\nISDN\n\n:   Integrated Services Digital Network\n\nKC\n\n:   Knowledge Centre\n\nSMS\n\n:   Short Messaging System\n\nGPRS\n\n:   General Packet Radio Service","meta":{"dup_signals":{"dup_doc_count":102,"dup_dump_count":54,"dup_details":{"curated_sources":2,"2021-39":1,"2021-21":1,"2021-17":1,"2020-10":3,"2019-39":1,"2019-22":1,"2019-18":1,"2018-51":2,"2018-43":1,"2018-30":1,"2018-17":1,"2018-13":1,"2018-05":1,"2017-47":1,"2017-39":2,"2017-30":1,"2017-22":2,"2017-17":1,"2017-09":2,"2017-04":2,"2016-50":2,"2016-44":1,"2016-40":2,"2016-36":2,"2016-30":2,"2016-26":2,"2016-22":2,"2016-18":2,"2016-07":2,"2015-48":2,"2015-40":2,"2015-35":2,"2015-32":1,"2015-27":2,"2015-22":2,"2015-14":2,"2014-52":2,"2014-49":2,"2014-42":7,"2014-41":3,"2014-35":3,"2014-23":2,"2014-15":3,"2022-40":1,"2024-30":3,"2024-26":1,"2024-10":3,"2017-13":1,"2015-18":2,"2015-11":2,"2015-06":2,"2014-10":2,"2013-48":2,"2013-20":2}},"file":"PMC1550611"},"subset":"pubmed_central"}
{"text":"abstract: Type 2 diabetes is characterized by poor glucose uptake in metabolic tissues and manifests when insulin secretion fails to cope with worsening insulin resistance. In addition to its effects on skeletal muscle, liver, and adipose tissue metabolism, it is evident that insulin resistance also affects pancreatic \u03b2-cells. To directly examine the alterations that occur in islet morphology as part of an adaptive mechanism to insulin resistance, we evaluated pancreas samples obtained during pancreatoduodenectomy from nondiabetic subjects who were insulin-resistant or insulin-sensitive. We also compared insulin sensitivity, insulin secretion, and incretin levels between the two groups. We report an increased islet size and an elevated number of \u03b2- and \u03b1-cells that resulted in an altered \u03b2-cell\u2013to\u2013\u03b1-cell area in the insulin- resistant group. Our data in this series of studies suggest that neogenesis from duct cells and transdifferentiation of \u03b1-cells are potential contributors to the \u03b2-cell compensatory response to insulin resistance in the absence of overt diabetes.\nauthor: Teresa Mezza; Giovanna Muscogiuri; Gian Pio Sorice; Gennaro Clemente; Jiang Hu; Alfredo Pontecorvi; Jens J. Holst; Andrea Giaccari; Rohit N. KulkarniCorresponding authors: Andrea Giaccari, <firstname.lastname@example.com>, and Rohit N. Kulkarni, <firstname.lastname@example.com>.\ndate: 2014-03\nreferences:\ntitle: Insulin Resistance Alters Islet Morphology in Nondiabetic Humans\n\n# Introduction\n\nInsulin resistance, along with \u03b2-cell inadequacy, represent the key features in the pathogenesis of type 2 diabetes, and that both are essential for the full manifestation of the disease is generally accepted (1).\n\nA feature that has been recognized in rodents (2,3) and humans (4\u20136) is the ability of the pancreas to compensate for insulin resistance by an increase in \u03b2-cell mass and insulin secretion. Indeed, \u03b2-cell mass is dynamic and capable of adapting to physiological and pathological conditions to maintain normoglycemia (7\u20139). Studies in humans suggest that the number and mass of \u03b2-cells increase in response to obesity; however, the time of onset of the increase and the precise origin of such new \u03b2-cells are still unknown (7). It is also evident that a failure of this ability of the \u03b2-cells to compensate for insulin resistance leads to progressive hyperglycemia and glucose toxicity (10) and to overt diabetes (11). A challenge to identifying the pathways and investigating the mechanisms that underlie compensatory changes in islets is the lack of longitudinal access to human tissue samples of appropriate quality for analyses coupled with accurate metabolic and hormonal profiling.\n\nWe took advantage of the unique opportunity to collect pancreas samples from patients undergoing surgical removal of a tumor of the ampulla of Vater to explore the hypothesis that insulin resistance directly contributes to adaptive changes in \u03b2-cell mass and function. To this end, we measured insulin sensitivity, insulin secretion, and incretin levels in nondiabetic, nonobese subjects before and after pancreatoduodenectomy. We also evaluated markers of \u03b2-cell proliferation, apoptosis, hypertrophy, and islet neogenesis, as well as ductal cell markers. Our data indicate that alterations in insulin sensitivity are linked to markers of compensation in humans and suggest ductal cells and \u03b1-cell transdifferentation as sources for new \u03b2-cells.\n\n# Research Design and Methods\n\n## Selection and Description of Participants\n\nThe study recruited 18 patients (9 males and 9 females) scheduled to undergo pylorus-preserving pancreatoduodenectomy from the Hepato-Biliary Surgery Unit of the Department of Surgery (Agostino Gemelli University Hospital, Rome, Italy). The local ethics committee approved the study protocol, and all participants provided written informed consent, followed by a comprehensive medical evaluation.\n\nIndication for surgery was tumor of the ampulla of Vater. None of the patients had a family history of diabetes, and all were classified as nondiabetic as determined by a 75-g oral glucose tolerance test and HbA~1c~ according to the American Diabetes Association criteria (12). Only patients with normal cardiopulmonary and kidney functions, as determined by medical history, physical examination, electrocardiography, creatinine clearance, and urinalysis were included in the study. Altered serum lipase and amylase levels before surgery, as well as morphologic criteria for pancreatitis, were considered exclusion criteria. Potential patients who had severe obesity (BMI \\>40 kg\/m^2^), uncontrolled hypertension, and\/or hypercholesterolemia were excluded.\n\nTo assess differences in islet morphology in response to insulin-resistant versus insulin-sensitive states, patients were divided into insulin-resistant and insulin-sensitive groups according to their insulin sensitivity, as measured with the euglycemic hyperinsulinemic clamp procedure before surgery. As previously described (13), the cutoff for insulin sensitivity was the median value of glucose uptake in the overall cohort (4.9 mg \u22c5 kg^\u22121^ \u22c5 min^\u22121^); therefore, subjects whose glucose uptake exceeded the median value were classified as \"more insulin sensitive\" than subjects whose glucose uptake was less than the median; for ease of comprehension, the two groups were defined \"insulin sensitive\" or \"insulin resistant.\" Clinical and metabolic characteristics of the two groups are summarized in <a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a>.\n\n## Study Design and Experimental Procedures\n\nAnthropometric parameters were determined according to standard procedures (14). BMI was calculated as weight in kilograms divided by height in meters squared (kg\/m^2^). Blood samples were drawn from all patients for serum lipid assays (total cholesterol and HDL and LDL) in the morning after an overnight (8-h) fast. All procedures were performed with subjects supine throughout the experiments. Each subject underwent a hyperinsulinemic euglycemic clamp, a hyperglycemic clamp, and a mixed-meal test 1 week before the surgical procedure and after a variable period of recovery from the operation. A sufficient recovery period was judged on normalization of inflammatory parameters, such as C-reactive protein, erythrocyte sedimentation rate, stability of weight, and absence of symptoms of abnormal intestinal motility or exocrine pancreatic deficiency. During the clamp procedures, an intravenous catheter was inserted into each arm, one for infusions and the other for blood sampling.\n\n### Oral Glucose Tolerance Test\n\nNormal glucose metabolism was confirmed by a standard 75-g oral glucose tolerance test measuring glycemia, insulin, and C-peptide at 0, 30, 60, 90, 120 min after the glucose load.\n\n### Hyperinsulinemic Euglycemic Clamp Procedure\n\nThe hyperinsulinemic euglycemic clamp test was performed after a 12-h overnight fast using insulin 40 mIU\u22c5m^\u22122^\u22c5min^\u22121^ of body surface according to DeFronzo et al. (15) A primed constant infusion of insulin was administered (Actrapid HM, 40 mIU\u22c5m^\u22122^\u22c5min^\u22121^; Novo Nordisk, Copenhagen, Denmark). The constant rate for the insulin infusion was reached within 10 min to achieve steady-state insulin levels. In the meantime, a variable infusion of 20% glucose was started with a separate infusion pump, and the rate was adjusted, on the basis of plasma glucose samples drawn every 5 min, to maintain the plasma glucose concentration at each participant's fasting plasma glucose level. During the last 20 min of the clamp procedure, plasma samples from blood drawn at 5- to 10-min intervals were used to determine glucose and insulin concentrations. Whole-body peripheral glucose utilization was calculated during the last 30-min period of the steady-state insulin infusion and was measured as the mean glucose infusion rate (mg\u22c5kg^\u22121^\u22c5min^\u22121^).\n\n### Hyperglycemic Clamp Procedure\n\nThe plasma glucose was clamped at a stable level of 125 mg\/dL above the fasting blood glucose concentration. The hyperglycemic clamp was started with a 200 mg\/mL bolus dose of dextrose (150 mg\/kg) administered into the antecubital vein. Blood was drawn from a cannulated dorsal hand vein on the opposite arm. Venous plasma glucose was analyzed every 5 min with a glucose analyzer, and the infusion of 20% glucose was adjusted to achieve a stable glucose level of 125 mg\/dL above the fasting value. Serum samples for insulin and C-peptide were drawn at 0, 2.5, 5, 7.5, 10, 15, 30, 60, 90, 120, 130, 140, and 150 min.\n\nThe first-phase insulin release, reflecting the early insulin peak secreted from the pancreatic \u03b2-cell in response to glucose stimulation, was calculated as the area under the curve (AUC) during the first 10 min of the clamp by using the trapezium rule. The second-phase insulin release, reflecting \u03b2-cell function under sustained elevated glucose levels, was calculated as the AUC from 10 to 120 min. Subsequently a 5-g arginine bolus was administered to measure maximum C-peptide secretory capacity at a steady-state blood glucose concentration of 250 mg\/dL. Combined hyperglycemia- and arginine-stimulated \u03b2-cell secretory capacity was calculated as the insulin AUC during the 30 min after the arginine bolus (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>A<\/em><\/a>).\n\n### Mixed-Meal Test\n\nPatients were instructed to consume a meal of 830 kcal (107 kcal from protein, 353 kcal from fat, and 360 kcal from carbohydrates) within 15 min. Blood samples were drawn twice in the fasting state and at 30-min intervals over the following 240 min (sample time 0, 30, 60, 90, 120, 150, 180, 210, and 240 min) for the measurement of plasma glucose, insulin, C-peptide, glucagon, and glucagon-like peptide 1 (GLP-1) or glucose-dependent insulinotropic polypeptide (GIP) concentrations. Blood samples for glucagon, total GLP-1, or intact GIP were sampled in tubes containing EDTA and a dipeptidyl peptidase-4 inhibitor (Millipore, Billerica, MA); after centrifugation (1,000 rpm for 10 min at 4\u00b0C), samples were stored at \u221280\u00b0C until analyses. Insulin levels were determined using a commercial radioimmunoassay kit (Medical System, Immulite DPC, Los Angeles, CA). Plasma glucose concentrations were determined by the glucose oxidase technique, using a glucose analyzer (Beckman Instruments, Palo Alto, CA). Plasma C-peptide was measured by AutoDELPHIA automatic fluoroimmunoassay (Wallac, Turku, Finland), with a detection limit of 17 pmol\/L. Immunoreactive glucagon was measured in ethanol-extracted plasma by radioimmunoassay using antibody code no. 4305, which is directed against the C-terminus of glucagon and reacts specifically with pancreatic glucagon (16). Total GLP-1 concentrations were measured using antiserum no. 89390, reacting equally with intact GLP-1 (7-36) amide and its primary N-terminally truncated metabolite GLP-1 (9-36) amide. Intact GIP was measured using antiserum no. 98171, reacting with the N-terminus of GIP, but not with the metabolite, GIP 3-42 (17).\n\n## Surgical Procedures\n\nPancreatoduodenectomy was performed according to the pylorus-preserving technique (18,19). Briefly, the pancreatic head, the entire duodenum, common bile duct, and gallbladder were removed en bloc, leaving a functioning pylorus intact at the gastric outlet. All adjacent lymph nodes were carefully removed. The continuity of the gastrointestinal tract was restored by an end-to-side invaginated pancreatojejunostomy. Further downstream, an end-to-side hepaticojejunostomy and side-to side gastroenterostomy or an end-to-side pylorus-jejunostomy was made. The removed volume of pancreas during the surgery was constant (\u223c50%), as previously reported by Schrader et al. (20). A pancreas sample was collected during the surgery from the downstream edge of the surgical cut.\n\n## Immunohistochemical Analysis of Pancreas Samples\n\n### Pancreatic Tissue Processing\n\nPancreas samples were fixed in formaldehyde and embedded in paraffin for subsequent analysis. Five-micrometer sections were stained with hematoxylin and eosin or by immunohistochemistry for islet hormones using a cocktail of antibodies to insulin, glucagon, or somatostatin (21). In addition, sections were immunostained for insulin, Ki67, or DAPI (nucleus) to assess proliferation, TUNEL for apoptosis, and for duct marker using anti-CK19 antibodies, and GLP-1 to identify incretin immunoreactivity. The hematoxylin and eosin slides were examined in all cases by two pathologists to exclude those with pancreatitis, autolysis, and tumor infiltration.\n\nPrimary antibodies included insulin (guinea pig antibody, 1:200; Abcam), glucagon (mouse mono, 1:500; Sigma-Aldrich), somatostatin (rabbit poly, 1:500; Abcam), or Ki67 (mouse mono antibody, 1:50; BD Biosciences), GLP-1 (rabbit antibody, 1:1000; J.F. Habener, MD, Massachusetts General Hospital, Boston, MA), and CK19 (rabbit poly, 1:100; Abcam). For TUNEL we used an Apoptag Fluorescein in situ apoptosis detection kit (Roche) and PDX1 (rabbit poly, 1:200; Cell Signaling).\n\nSecondary antibodies were donkey anti\u2013GP-594, donkey anti\u2013mouse-AMCA, donkey anti\u2013rabbit-488, donkey anti\u2013mouse-AMCA, and biotinylated donkey anti-rabbit (all from Jackson ImmunoResearch, West Grove, PA), and peroxidase labeled polymer (Dako).\n\n### Morphometric Analysis\n\nAnalysis of \u03b2-, \u03b1-, and \u03b4-cell area was done as described previously (22). Each section was analyzed separately by measuring total insulin-, glucagon-, or somatostatin-positive areas, as well as the total pancreas section area, using Image Pro Plus 4.5.1 software (Media Cybernetics, Silver Springs, MD). The \u03b2-, \u03b1-, or \u03b4-cell areas were expressed as a percentage of the total pancreas section area. The islet size was calculated as the sum of the individual \u03b2-, \u03b1-, and \u03b4-cell areas divided by the number of islets counted in each pancreas section. Islet density was quantified by measuring the total area of the pancreas using Image Pro Plus and then counting the number of islets contained within that pancreas area, the results being expressed as islets per mm^2^. Islet size distribution was determined using the insulin-stained sections of pancreas counterstained with DAPI. At least 100 islets per section were examined and classified according to the number of insulin-positive cells (i.e., 1\u20138 cells, 9\u201319 cells, 20\u201349 cells, and 50 or more \u03b2-cells) and the data expressed as a percentage of islets. The ratio of the \u03b2-cell to \u03b1-cell area was evaluated for each section, dividing the individual percentage of \u03b2-cell area by the \u03b1-cell area. To evaluate \u03b2-cell size and nuclear area, five randomly selected islets per case were immunostained for insulin or DAPI and imaged at original magnification \u00d7400 (\u00d740 objective). The insulin-positive area for each islet was measured, and the number of nuclei present in the insulin-stained area (\u03bcm^2^) was manually counted to calculate the individual \u03b2-cell cross-sectional area (\u03bcm^2^). The number of \u03b2-cells was manually counted for each section and expressed as the ratio of \u03b2-cells per total pancreas section. To measure the \u03b2-cell nuclear area, insulin-stained sections of pancreas counterstained with DAPI were used; five randomly selected islets per case were photographed at original magnification \u00d7400. Then, five representative \u03b2-cell nuclei were identified in each islet. Selection criteria included clear presence of the nucleus within a \u03b2-cell, the ability to clearly visualize nuclear boundaries, circular shape (similar dimensions in all directions), and the appearance to the observer that the nucleus had been sectioned through its maximum diameter. Once the identified, nucleus was encircled, and the nuclear area (\u03bcm^2^) was measured using Image Pro Plus software. The number of insulin and glucagon double-positive cells was manually counted in sections costained for insulin or glucagon. A mean \u00b1 SE of 1,172 \u00b1 269 endocrine cells was evaluated per subject, and the resulting data were expressed as percentage of endocrine cells. The double-positive cells were confirmed in randomly selected islets by confocal microscopy. All data were expressed as the mean \u00b1 SE for each group.\n\n### Quantification of Scattered Islet and Exocrine Duct Cells Positive for Insulin\n\nAs previously described (23), clusters of less than eight endocrine cells were considered as new islets (neogenesis). On sections stained specifically for insulin, clusters with less than eight insulin-positive cells were manually counted and considered as scattered islets and then expressed as the ratio of the number of scattered islets per total pancreas area. Sections costained for pancreatic ductal marker CK19 or insulin were imaged by confocal microscopy, and the number of CK19- and insulin-positive cells was manually counted. A mean \u00b1 SE of 1,107 \u00b1 475 duct cells were evaluated for each section. The resulting data were expressed as a percentage of duct cells positive for insulin in each pancreas. All data were expressed as the mean \u00b1 SE for each group.\n\n### Proliferation and Apoptosis\n\nTo determine replication in \u03b2-cells, the number of \u03b2-cells costaining with Ki67 was counted and expressed as the percentage of the total number of \u03b2-cells (at least 2,000 \u03b2-cells were counted in each case). For the evaluation of apoptosis, the number of \u03b2-cells costaining with TUNEL was counted and expressed as percentage of the total number of \u03b2-cells (at least 2,000 \u03b2-cells for each case). The entire analysis was performed by a single observer in a blinded fashion.\n\n## Statistics\n\nAll data are expressed as mean \u00b1 SE, unless indicated otherwise. Because samples were normally distributed, differences in means were tested by the two-tailed Student *t* test. The relationship between variables was derived with linear regression analysis using SPSS 9 software (SPSS, Chicago, IL). A *P* value of \\<0.05 was considered statistically significant.\n\n## Study Approval\n\nThis study was approved by the ethical committee of the Catholic University of the Sacred Heart, Rome, Italy. All study subjects provided written informed consent before screening and participating in the study.\n\n# Results\n\nThe current study included 18 patients (9 female and 9 male; mean age, 53 \u00b1 15 years) undergoing pylorus-preserving pancreatoduodenectomy for a tumor of the ampulla of Vater. Clinical and metabolic characteristics of study subjects are provided in <a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>.\n\nClinical and metabolic characteristics of patients before and after surgery\n\n![](994tbl1)\n\n## Hemipancreatectomy Induced a Marked Reduction in Insulin Secretion, Without Affecting Insulin Sensitivity, and Resulted in the Onset of Diabetes Only in Insulin-Resistant Subjects\n\nSubjects were evaluated 1 week before surgery and at 40 \u00b1 7 days (range 34\u201348) after surgery. To evaluate the insulin secretory capacity, we performed hyperglycemic clamps over 2 h, followed by an acute stimulation with <span class=\"smallcaps\">l<\/span>-arginine (5 g). As expected, insulin secretion was significantly reduced after surgery (*P* \\< 0.001, <a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>B<\/em><\/a>). The response to arginine (121 to 150 min after glucose infusion) revealed an even higher (76%) reduction of insulin secretion (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>C<\/em><\/a>). Conversely, insulin sensitivity, as assessed by the hyperinsulinemic euglycemic clamp (15), did not change significantly after surgery (<a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>D<\/em><\/a>).\n\nEvaluation of glucose homeostasis by standard oral glucose tolerance tests (75 g) in the overall cohort revealed worsening of glucose tolerance after surgery (<a href=\"#T1\" data-ref-type=\"table\">Table 1<\/a>).\n\nTo further characterize changes in glucose tolerance after removal of \u223c50% of the pancreas and to assess differences in islet morphology, patients were divided into insulin-resistant and insulin-sensitive groups (<a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a>). Despite the removal of the head of the pancreas, which includes \u223c50% of the \u03b2-cell mass, patients identified as insulin-sensitive before surgery preserved their glucose tolerance, whereas seven of nine (77.7%) insulin-resistant patients developed diabetes, as confirmed by a 75-g oral glucose tolerance test and an HbA~1c~ higher than 7% (53 mmol\/mol; <a href=\"#T2\" data-ref-type=\"table\">Table 2<\/a>).\n\nClinical and metabolic characteristics of insulin-sensitive and insulin-resistant patients before and after surgery\n\n![](994tbl2)\n\nGLP-1 secretion in response to a mixed meal significantly increased (AUC GLP-1 before vs. after surgery: 10.4 \u00b1 3.2 nmol \u22c5 L^\u22121^ \u22c5 min vs. 15.6 \u00b1 4.3 nmol \u22c5 L^\u22121^ \u22c5 min, *P* = 0.01; <a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>E<\/em><\/a>), whereas GIP response was significantly reduced (AUC GIP before vs. after surgery: 19.1 \u00b1 8.1 nmol \u22c5 L^\u22121^ \u22c5 min vs. 7.7 \u00b1 2.8 nmol \u22c5 L^\u22121^ \u22c5 min, *P* \\< 0.001; <a href=\"#F1\" data-ref-type=\"fig\">Fig. 1<em>E<\/em><\/a>) after surgery.\n\nAlthough 50% pancreatectomy led to a decrease in insulin secretion in insulin-sensitive and insulin-resistant patients (<a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<em>A<\/em><\/a>), the latter exhibited a greater attenuation in all of the phases of insulin secretion (insulin-sensitive 924 \u00b1 900 \u00b5IU \u22c5 mL^\u22121^ \u22c5 min vs. insulin-resistant 6,952 \u00b1 1,951 \u00b5IU \u22c5 mL^\u22121^ \u22c5 min; \u0394AUC insulin secretion, *P* = 0.04; <a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<em>C<\/em><\/a>). Furthermore, the increase in glucagon secretion during the mixed-meal test after surgery was higher in insulin-resistant patients (insulin-sensitive 112 \u00b1 101 nmol \u22c5 L^\u22121^ \u22c5 min vs. insulin-resistant 1,812 \u00b1 326.3 nmol \u22c5 L^\u22121^ \u22c5 min; \u0394AUC glucagon secretion, *P* = 0.02; <a href=\"#F2\" data-ref-type=\"fig\">Fig. 2<em>B<\/em> and <em>D<\/em><\/a>).\n\n## Insulin-Resistant Individuals Exhibit Increased Islet Size\n\nTo evaluate changes in islet morphology, we performed immunohistochemical analyses of sections of pancreas removed during surgery. Compared with insulin-sensitive individuals, insulin-resistant subjects exhibited an increased percentage of insulin area (insulin-sensitive 0.58 \u00b1 0.17% vs. insulin-resistant 1.10 \u00b1 0.23%, *P* = 0.05), percentage of glucagon area (insulin-sensitive 0.04 \u00b1 0.01% vs. insulin-resistant 0.23 \u00b1 0.06%, *P* \\< 0.01) and percentage of somatostatin area (insulin-sensitive 0.01 \u00b1 0.00% vs. insulin resistant 0.03 \u00b1 0.01%, *P* = 0.01; <a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<em>A<\/em><\/a>). Overall, these differences resulted in an increased mean islet size in insulin-resistant subjects compared with the insulin-sensitive ones (mean islet size: insulin sensitive 2,456 \u00b1 332 \u00b5m^2^ vs. insulin resistant 5,156 \u00b1 944 \u00b5m^2^, *P* \\< 0.001; <a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<em>B<\/em><\/a>). Further, we observed a strong inverse correlation between islet size and glucose uptake in the entire cohort (*r* = \u22120.74, *P* \\< 0.01; <a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<em>C<\/em><\/a>), which suggests that changes in islet morphology are influenced by insulin sensitivity.\n\n## Increased Islet Size Is Likely Caused by \u03b2-Cell Hyperplasia\n\nTo identify potential mechanism(s) underlying the increased islet size in insulin resistance, we examined \u03b2-cell replication, apoptosis, and cell size. Among a total of 37,845 \u03b2-cells counted in the entire cohort, replication was undetectable, as determined by Ki67 immunostaining. \u03b2-Cell apoptosis was also infrequent among the 39,600 cells examined in the entire cohort. Although positive cells were undetectable in the insulin-sensitive group, we detected 24 positive apoptotic cells in three insulin-resistant patients (mean percentage of \u03b2-cell apoptosis among the insulin-resistant group, 0.1%); however, the differences between groups were not significant (*P* = 0.20). By measuring the mean individual \u03b2-cell cross-sectional area in the two groups, we also ruled out a possible contribution of cell hypertrophy to the increase in islet size (insulin-sensitive 127 \u00b1 9 \u00b5m^2^ vs. insulin-resistant 129 \u00b1 17 \u00b5m^2^, *P* = 0.79; <a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<em>D<\/em><\/a>). Insulin-resistant patients showed an increased number of \u03b2-cells per mm^2^ of pancreas area (insulin-sensitive 22.7 \u00b1 2.7 \u03b2-cells\/mm^2^ vs. insulin-resistant 80.9 \u00b1 15.8 \u03b2-cells\/mm^2^, *P* \\< 0.01). These data suggest that the increase in islet size is due to increased number of cells (i.e., hyperplasia) rather than altered \u03b2-cell volume. Further, \u03b2-cell nuclear size was increased in the insulin-resistant group (insulin-sensitive 34.5 \u00b1 1.1 \u00b5m^2^ vs. insulin resistant 42.4 \u00b1 6.2 \u00b5m^2^, *P* = 0.03; <a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<em>E<\/em><\/a>), suggesting that secretory \u03b2-cells are relatively young (24,25).\n\n## Insulin Resistance Is Associated With Increased Islet Neogenesis\n\nOn the basis of the results discussed above and the finding of increased islet density (insulin-sensitive 3.5 \u00b1 0.5 islets\/mm^2^ vs. insulin resistant 7.5 \u00b1 1.4 islets\/mm^2^, *P* \\< 0.01; <a href=\"#F3\" data-ref-type=\"fig\">Fig. 3<em>F<\/em><\/a>), we hypothesized that neogenesis, rather than proliferation, contributes to \u03b2-cell hyperplasia in insulin-resistant patients. To explore whether the pancreas shows evidence of neogenesis, we quantified the number of scattered islets with less than eight nuclei (23) and observed an increase of such islets in insulin-resistant subjects (insulin-sensitive 1.80 \u00b1 0.18 nuclei\/mm^2^ vs. insulin resistant 4.65 \u00b1 1.16 nuclei\/mm^2^, *P* = 0.04; <a href=\"#F4\" data-ref-type=\"fig\">Fig. 4<em>A<\/em><\/a>). Although these scattered islets were distributed within the exocrine tissue, we cannot ascertain whether these cells arise directly from acinar cells without further detailed investigation.\n\nFurther, because previous studies have reported potential formation of new islets from duct cells, we evaluated the number of cells that were double-positive for the duct marker CK19 and insulin. The mean percentage of CK19\/insulin double-positive cells was increased in insulin-resistant subjects (insulin-sensitive 0.28 \u00b1 0.12% vs. insulin-resistant 1.47 \u00b1 0.26%, *P* \\< 0.001; <a href=\"#F4\" data-ref-type=\"fig\">Fig. 4<em>B<\/em> and <em>D<\/em><\/a>). As shown in <a href=\"#F4\" data-ref-type=\"fig\">Fig. 4<em>C<\/em><\/a>, insulin-resistant subjects displayed a greater number of small clusters and islets with more than 50 cells.\n\n## Insulin Resistance and Alterations in \u03b1-Cells\n\nThe fractional \u03b1-cell area was greater in the insulin-resistant compared with the insulin-sensitive group and was inversely correlated with glucose uptake (*r* = \u22120.65, *P* = 0.03; <a href=\"#F5\" data-ref-type=\"fig\">Fig. 5<em>A<\/em><\/a>). The mean ratio of \u03b2-cell to \u03b1-cell areas was lower in the insulin-resistant subjects (insulin-sensitive 0.13 \u00b1 0.01 vs. insulin-resistant 0.08 \u00b1 0.01, *P* = 0.05; <a href=\"#F5\" data-ref-type=\"fig\">Fig. 5<em>B<\/em><\/a>), suggesting a relative increase in the \u03b1-cell area.\n\nBecause previous reports have suggested transdifferentiation of \u03b1-cells as a mechanism that contributes to alterations in \u03b2-cell mass, we immunostained pancreas sections to identify insulin and glucagon coexpressing cells. Interestingly, we detected an increased number of double-positive cells in insulin-resistant subjects compared with insulin-sensitive subjects (mean percentage of double-positive cells: insulin sensitive 4.51 \u00b1 1.07% vs. insulin resistant 10.86 \u00b1 2.17%, *P* = 0.02; <a href=\"#F5\" data-ref-type=\"fig\">Fig. 5<em>C<\/em> and <em>D<\/em><\/a>) ([Supplementary Figs. 1](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db13-1013\/-\/DC1) and [2](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db13-1013\/-\/DC1)). We also examined PDX1 immunoreactivity in the insulin and glucagon double-positive cells and detected PDX1-positive cells in insulin-sensitive and insulin-resistant subjects ([Supplementary Fig. 3](http:\/\/diabetes.diabetesjournals.org\/lookup\/suppl\/doi:10.2337\/db13-1013\/-\/DC1)).\n\nFurther immunohistochemical analyses using a specific anti-GLP-1 antibody, which is highly selective for processed amidated GLP-1 directed to the COOH-terminal, revealed that glucagon colocalizes with GLP-1 in both groups (<a href=\"#F6\" data-ref-type=\"fig\">Fig. 6<em>A<\/em><\/a>). Interestingly, the \u03b1-cell area correlated with GLP-1 (*r* = 0.63, *P* = 0.04; <a href=\"#F6\" data-ref-type=\"fig\">Fig. 6<em>B<\/em><\/a>) but not GIP secretion (*r* = 0.08, *P* = 0.79). The AUC of GLP-1 secretion during the mixed-meal test also correlated with glucose uptake in the entire cohort (*r* = \u22120.57, *P* \\< 0.01). Whether these correlations indicate a link between circulating GLP-1 and \u03b1-cell biology requires further study.\n\nThe relative increase in \u03b1-cell area could lead to an increase in \u03b2-cells by transdifferentiation and also to an increase in intraislet GLP-1 production. Furthermore, it is possible that this change in the relative proportion of \u03b1-cells could be a first step toward hyperglucagonemia, a hallmark of type 2 diabetes.\n\n# Discussion\n\nIn the current study, we evaluated pancreas samples obtained from nondiabetic subjects to investigate the effects of altered insulin sensitivity on islet morphology. The major finding of our study is that insulin-resistant subjects exhibited increased islet size, which was strongly inversely correlated with insulin sensitivity (*r* = \u22120.74, *P* \\< 0.001). This suggests that insulin resistance directly impacts islet biology in nondiabetic humans by inducing an increase in \u03b2-cell area to compensate for the increased insulin demand. These findings are consistent with reports in humans and mouse models (26,27) in which defects in insulin-signaling pathways in \u03b2-cells have been suggested to be responsible for a decrease in mass and reduced secretory function. Indeed, impaired \u03b2-cell responsiveness to insulin has been shown in insulin-resistant patients (28). Consistent with previous reports in insulin-resistant obese patients (7), who exhibit an increase in islet size due to increased cell number, \u03b2-cells from insulin-resistant individuals in our study also exhibited an increased number. In addition, the \u03b2-cells showed an increased nuclear area, suggesting that cells were relatively young and with increased secretory capacity (29).\n\nSeveral studies (7,30,31) have investigated proliferation of \u03b2-cells in humans mostly in pancreata from autopsy samples. Although some variability is evident, most reports agree that the rate of \u03b2-cell proliferation is extremely low in the adult human pancreas. Consistently, we noted virtually undetectable \u03b2-cell proliferation by Ki67 immunostaining in pancreas sections from the entire group. We also failed to detect \u03b2-cell apoptosis, in contrast to the increase seen in patients with type 2 diabetes (5). Our data showing the lack of alterations between groups in Ki67 and TUNEL staining suggest that neither proliferation nor apoptosis contributes significantly to the \u03b2-cell adaptive response to insulin resistance in this cohort of patients.\n\nRecent reports have suggested that plasticity of the adult \u03b2-cell mass is linked to neogenesis during different periods of life (early postnatal life, pregnancy, and aging) as well as in obesity, impaired glucose tolerance, and in individuals with newly diagnosed diabetes (32,33). Other reports suggest that cells lining the ducts or acinar cells may serve as a source of new \u03b2-cells (34). Indeed, our findings of a higher number of islet clusters and the presence of insulin-positive duct cells suggest that these pathways underlie the alteration in islet size associated with insulin resistance.\n\nBecause inappropriate glucagon secretion is a feature of patients with diabetes (4,35) and previous reports indicate a role for insulin signaling in the regulation of \u03b1 cell function (36), we explored the link between \u03b1-cells and insulin sensitivity. Analyses of islet morphology revealed that the ratio between \u03b2- and \u03b1-cell area was lower in insulin-resistant subjects due to a relatively greater increase in \u03b1-cell area. The relative low \u03b1-cell number in the insulin-sensitive group is a feature of the significantly higher insulin sensitivity, whereas the increased number of \u03b1-cells in insulin-resistant subjects occurs as compensation for insulin resistance. Similar changes are also evident in diabetic patients (37) and insulin-resistant primates (38). Furthermore, we found a strong inverse correlation between \u03b1-cell area and insulin sensitivity (*r* = \u22120.65, *P* = 0.003). These observations raise several questions. For example, do alterations in \u03b1-cell biology precede changes in \u03b2-cell mass? Could transdifferentiation of \u03b1-cells contribute to an increase in \u03b2-cells? Does the imbalance between \u03b2- and \u03b1-cells result from dedifferentiation (39)? Despite the cross-sectional nature of our study, it is worth noting that our data are timely and highly relevant to islet biology because an ideal longitudinal study on human pancreata is extremely difficult to undertake due to ethical limitations.\n\nA second link between insulin sensitivity and \u03b1-cell mass was evident from the correlation with GLP-1 secretion. As previously reported by our group (19), patients who underwent pancreatoduodenectomy and a mixed-meal test exhibited a significant increase in GLP-1 secretion in contrast to a significant decrease in GIP. Although the latter is likely due to removal of the duodenum, a major site of production of GIP (40), the mechanism contributing to greater GLP-1 secretion is not fully understood and suggests hypersecretion by existing intestinal L-cells and\/or other potential sources of the incretin hormone.\n\nOur observations on GLP-1 gain significance in light of previous reports (41) suggesting \u03b1-cells are a potential source of the incretin hormone, which, in turn, can exert a local paracrine effect on islet function, as previously suggested by Marchetti et al. (41). Indeed, the presence of GLP-1 in the islet has been suggested to have multiple effects, including differentiation of progenitor cells into \u03b2-cells in the pancreatic duct epithelium (42,43) and direct stimulation of \u03b2-cell proliferation and inhibition of apoptosis (44,45). In addition, pancreas extracts from glucagon receptor knockout mice (46) exhibit an increase in GLP-1 that is associated with an up to 10-fold increase in circulating GLP-1 amide, the active form of the incretin hormone. The lack of change in GLP-1 in intestinal extracts suggests that the pancreas is one of the sources contributing to circulating GLP-1.\n\nIn our study, none of the insulin-sensitive patients developed diabetes after surgery, whereas 77.7% of insulin-resistant patients became diabetic. The latter displayed a greater reduction in all phases of insulin secretion and a higher increase in glucagon secretion in response to a mixed-meal test after surgery. It is tempting to speculate that these alterations are secondary to insulin resistance in \u03b1-cells and, as a consequence, to an inability of ambient insulin to adequately suppress glucagon secretion (34,47).\n\nIn conclusion, our findings suggest that neogenesis from duct cells and\/or transdifferentiation from \u03b1-cells are likely explanations for the alterations in \u03b2-cell mass observed in insulin-resistant subjects. Our study provides an example of a unique approach in the investigation of islet morphology in nondiabetic patients. A strength of this approach is the comprehensive evaluation of metabolic parameters in conjunction with analyses of pancreatic tissue from living donors, which allows comparison of in vivo and ex vivo studies that would otherwise not be possible unless sequential biopsies throughout life are performed. These findings provide a platform to plan studies to directly identify the source of new \u03b2-cells and determine the molecular mechanisms responsible for the dynamic changes that impact \u03b2-cell mass over the time course of progression of type 2 diabetes, with the long-term goal of enhancing islet compensation to insulin resistance.\n\n# Supplementary Material\n\n###### Supplementary Data\n\n## Article Information\n\n**Acknowledgments.** The authors thank S. Bonner-Weir, PhD, (Joslin Diabetes Center) for advice and assistance with histological examination of the samples; J.F. Habener, MD, (Massachusetts General Hospital) for the gift of the anti-GLP-1 antibody; C. Cahill and S. Sioletic, MD, PhD, for expert technical assistance; and A. Molven, PhD, A. El Ouaamari, PhD, and C. Conte, MD, for constructive comments.\n\n**Funding.** This study was supported by grants to A.G. from Universit\u00e0 Cattolica del Sacro Cuore (Fondi Ateneo Linea D.3.2 Sindrome Metabolica), from the Italian Ministry of Education, University and Research (PRIN 2010JS3PMZ_011) and from Fondazione Don Gnocchi. T.M. is the recipient of the Albert Reynolds Travel Fellowship by the European Association for the Study of Diabetes and the Fellowship Prize by the Societ\u00e0 Italiana di Diabetologia. G.P.S. is the recipient of a fellowship from Laboratori Guidotti. Some of the reagents used in the study were supported by National Institutes of Health grant R01-DK-67536 to R.N.K.\n\n**Duality of Interest.** No potential conflicts of interest relevant to this article were reported.\n\n**Author Contributions.** T.M. generated the data and wrote the manuscript. G.M. and A.P. contributed to discussion and reviewed and edited the manuscript. G.P.S., G.C., and J.H. researched data. J.J.H. generated data. A.G. generated the protocol and reviewed and edited the manuscript. R.N.K. reviewed and edited the manuscript and supervised the work at the Joslin Diabetes Center. T.M., A.G., and R.N.K. are the guarantors of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analyses.\n\n# References","meta":{"dup_signals":{"dup_doc_count":160,"dup_dump_count":52,"dup_details":{"curated_sources":2,"2023-06":1,"2021-21":1,"2021-10":2,"2021-04":2,"2020-05":1,"2019-51":3,"2019-47":2,"2019-13":1,"2019-09":1,"2018-47":1,"2018-39":1,"2018-30":1,"2018-22":3,"2018-17":1,"2018-13":2,"2018-09":1,"2018-05":3,"2017-51":1,"2017-47":3,"2017-43":6,"2017-39":4,"2017-34":1,"2017-30":9,"2017-26":3,"2017-22":2,"2017-17":11,"2017-09":6,"2017-04":13,"2016-50":4,"2016-44":5,"2016-40":5,"2016-36":4,"2016-30":6,"2016-26":4,"2016-22":4,"2016-18":2,"2016-07":2,"2015-48":3,"2015-40":4,"2015-35":3,"2015-32":1,"2015-27":2,"2015-22":4,"2014-52":4,"2014-49":1,"2014-42":1,"2014-41":2,"2014-35":1,"2023-40":1,"2017-13":4,"2015-18":4,"2015-06":1}},"file":"PMC3931397"},"subset":"pubmed_central"}
{"text":"abstract: Why do we have so much trouble distinguishing life from non-life, and distinguishing our biology from another? When in doubt, one should count the \"bits\" of heritable information.\nauthor: Gerald F. Joyce\\* E-mail: <firstname.lastname@example.com>[^1]\ndate: 2012-05\ninstitute: 1Department of Molecular Biology, Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, United States of America; 2Department of Chemistry, The Scripps Research Institute, La Jolla, California, United States of America\nreferences:\ntitle: Bit by Bit: The Darwinian Basis of Life\n\nThanks to a combination of ground- and space-based astronomical observations, the number of confirmed extrasolar planets will soon exceed 1,000. An increasing number of these will be said to lie within the \"habitable zone\" and even be pronounced as \"Earth-like.\" Within a decade there will be observational data regarding the atmospheric composition of some of those planets, and just maybe those data will indicate something funny going on\u2014something well outside the state of chemical equilibrium\u2014on a potentially hospitable planet. Perhaps our astronomy colleagues should be forgiven for their enthusiasm in declaring that humanity is on the brink of discovering alien life.\n\nBut haven't we heard this before? Didn't President Clinton announce in 1996 that a Martian meteorite recovered in Antarctica \\[1\\] \"speaks of the possibility of life\" on Mars? (No, it turned out to be mineralic artifacts.) Wasn't some \"alien\" arsenic-based life discovered recently in Mono Lake, California \\[2\\]? (No, it's a familiar proteobacterium struggling to survive in a toxic environment.) Didn't Craig Venter and his colleagues recently create a synthetic bacterial cell \\[3\\], \"the first self-replicating species we've had on the planet whose parent is a computer\"? (No, its parent is *Mycoplasma mycoides* and its genome was dutifully reconstructed through DNA synthesis and PCR amplification.)\n\nWhy are we so confused (or so lonely) that we have such trouble distinguishing life from non-life and distinguishing our biology from another? A key limitation is that we know of only one life form, causing us to regard life from that singular perspective (<a href=\"#pbio-1001323-g001\" data-ref-type=\"fig\">Figure 1<\/a>). We see life as cellular, with a nucleic acid genome that is translated to a protein machinery. Life self-reproduces, transmits heritable information to its progeny, and undergoes Darwinian evolution based on natural selection. Life captures high-energy starting materials and converts them to lower-energy products to drive metabolic processes. Life exists on at least one temperate, rocky planet, where it has persisted for about four billion years. There are likely to be tens of thousands of \"habitable\" planets within a thousand light years of Earth, and more than a billion such planets in our galaxy, so *surely* (say the astronomers) we are not alone.\n\n# Rolling the Dice\n\nWhat, in fact, is the probability that a temperate, rocky planet will generate life? Science cannot say. That is because, based on the one known example of obscure origins, even a Bayesian would not want to assign a probability to such an event. The probability assessment would be more meaningful if there were even one more genuine example of life, whether discovered in space, on Earth, or in a test tube. If that entity had all of the properties of terrestrial life described above, then one would conclude that, indeed, we are not alone. But what if the entity had only some of those properties? What if it could self-reproduce, directing the assembly of progeny of identical composition, but could not evolve new functions? What if it consisted of complex chemical processes within a cellular compartment but had no basis for maintaining heritable genetic information? What if it had all of the properties of life but was descended from our own life form rather than derived from an independent origin?\n\nWhen faced with such real or hypothetical situations regarding alternative life, it is useful to frame the question in terms of information: How many heritable bits are involved, and where did they come from? (<a href=\"#pbio-1001323-box001\" data-ref-type=\"boxed-text\">Box 1<\/a>) Biological systems are distinguishable from chemical systems because they contain components that have many potential alternative compositions but adopt a particular composition based on the history of the system. In this sense biological systems have a molecular memory (genotype), which is shaped by experience (selection) and maintained by self-reproduction. One can count the number of bits in this molecular memory, for example, up to two bits per base pair for a nucleic acid genome. The bits accrue as potential alternative compositions are excluded and specific compositions are adopted. More formally, the number of bits is calculated as log~2~ of the number of potential compositions divided by the number of realized compositions. One must count only those bits that accrue within the system, not those that were evolved elsewhere and bestowed upon the system for free.\n\n## Box 1. Counting Bits\n\nWhen evaluating potential examples of alternative life, one should look at the genetic material and ask the following questions regarding its information content. The aim is to determine how many heritable bits are involved, excluding those that did not arise within the genetic system.\n\n**Q1.** How many different types of subunits (x) does the genetic material contain? For a binary polymer, x\u200a=\u200a2; for a nucleic acid polymer, x\u200a=\u200a4 (A, T\/U, G, C).\n\n**Q2.** What is the length (n) of the genetic material? Count only those positions that can potentially be occupied by different types of subunits.\n\n**Q3.** Do all of the possible compositions have the same prior probability of occurrence? Some combinations may be less probable if one of the subunits is less abundant or less frequently incorporated into the genetic material. Some combinations may be disfavored for chemical or biochemical reasons (e.g., long runs of G residues in DNA).\n\nThe number of possible compositions is x^n^. If these are all equally probable, then each has a prior probability of occurrence of x^\u2212n^, and the information content (number of bits) associated with a particular realized composition is log~2~(x^n^). This can also be expressed as 2^\\#bits^\u200a=\u200ax^n^. For a binary polymer, \\#bits\u200a=\u200an; for a nucleic acid polymer, \\#bits\u200a=\u200a2n.\n\nIf the various compositions do *not* have the same prior probability of occurrence, then the information content associated with a particular realized composition must be calculated based on its prior probability of occurrence (*p* ~k~), which ranges from 0 to 1. The information content (number of bits) associated with a particular realized composition is \u2212log~2~(*p* ~k~).\n\n**Q4.** Was a portion of the composition of the genetic material determined outside the genetic system? Some of the composition may have evolved in a different system and was merely available for the taking. Do not include preexisting information when counting bits because its prior probability of occurrence is 1, and therefore its information content with respect to the genetic system being considered is \u2212log~2~(1)\u200a=\u200a0.\n\nSuppose one has a molecule that self-duplicates indefinitely, directing the ordered assembly of building blocks to produce additional copies of itself. That would be an interesting chemical process, but unless there is the opportunity for alternative compositions to arise and similarly reproduce\u2014that is, for Darwinian evolution to occur\u2014the bit content would be zero. Suppose one has a complex reaction cascade, perhaps even an autocatalytic cycle, contained within a growing and dividing physical compartment. That too would be an interesting chemical process, but it would not involve any heritable bits. Suppose one uses the genetic information from a preexisting biological organism to construct a facsimile that, going forward, can evolve alternative compositions. New bits could accrue within such a system, but all of the bits that were provided at the outset would have derived from the preexisting organism. To be considered a new life form, the majority of bits must be self-derived.\n\n# Pathways to Life\n\nIn principle, there are two pathways by which a new life form can arise: either directly from chemistry or spun off from some other biology. If life arises from chemistry, as is thought to have occurred on the primitive Earth (<a href=\"#pbio-1001323-g002\" data-ref-type=\"fig\">Figure 2<\/a>), then it begins with zero heritable bits and organizes into a bit-generating system. Following an era of prebiotic chemistry, perhaps reaching a high level of chemical complexity, molecular memory arises. Molecules of variable composition begin to replicate, mutate, and evolve in a Darwinian manner. If, instead, life arises from another life form, then it may have a privileged beginning, benefitting from a chemical environment that has been shaped by preexisting life. A new life form may grow on the spoils of prior life with no carryover of bits, or it may descend from prior life with some carryover of bits into a different genetic system. The latter type of transition is thought to have occurred during the early history of life on Earth when RNA-based life gave way to DNA\/protein-based life \\[4\\]. That transition likely involved a substantial transfer of bits that had accrued within RNA and were ported over to DNA through what might be termed The Great Reverse Transcription. We see echoes of those ancestral bits in the sequences of ribosomal RNA, tRNA, and possibly other contemporary RNAs that are present across all three kingdoms of life.\n\nIt has been suggested that RNA-based life was preceded by yet another life form \\[5\\], perhaps based on a nucleic-acid-like genetic molecule such as threose nucleic acid (TNA) \\[6\\] or glycol nucleic acid (GNA) \\[7\\]. There does not appear to be any trace of pre-RNA life in contemporary biology, although maybe we do not know how to look. It is conceivable, for example, that some of the deeply conserved bits in contemporary RNAs are the double echo of information that was carried from pre-RNA life to RNA life and then to DNA\/protein-based life. It also is possible that there was life on Earth prior to RNA but that it had no chemical resemblance to RNA. Information from such a pre-RNA genetic molecule could have been translated into RNA through a machinery analogous to the ribosome, but with RNA as the output rather than the input. Alternatively, pre-RNA life may have facilitated the emergence of RNA life, but with no transfer of information. Pre-RNA life may have generated RNA molecules as a metabolic product, and eventually the RNA molecules began replicating and evolving on their own.\n\n# Getting Started\n\nThe above discussion emphasizes that there are many possible routes by which one life form can give rise to another, but it does not address the question of how an initial life form arises in the first place. A key question to ask is: What is the minimum number of bits it takes to provide a replicating, evolving system that has the ongoing capacity to accrue more bits? That will depend on the chemical nature of the first self-replicating molecules and the resources that are available in its environment. When astronomers speak of habitable zones they refer to a planetary orbit that is of appropriate distance from a star to maintain liquid water on the planetary surface. This may be too restrictive or too generous a definition, depending on your point of view \\[8\\]. Perhaps life can exist in a non-aqueous environment, although there is little data to support this conjecture. Conversely, even a temperate little pond of water, salts, and dilute organics may be insufficient for life to originate. The pond would need to accumulate heteropolymers of variable composition, including some that could replicate and provide the basis for molecular memory, and it is not clear whether this is a common or exceedingly rare occurrence.\n\nIn terms of bits, a binary heteropolymer that contains 10 subunits would have 1,024 potential compositions, and any particular composition would represent up to 10 bits of heritable information. It seems doubtful that there is any heteropolymer for which 10 bits is enough to get started. Now suppose that it is a quaternary heteropolymer (like RNA) that is assembled into chains of 40 subunits. Then there would be 10^24^ possible compositions, and any particular composition would represent up to 80 bits of heritable information. An RNA biochemist might argue (see below) that this is just enough to get started. However, to represent all of these combinations as even a single copy would require that the little pond contain 27 kilograms of RNA, which seems highly implausible. Finally, suppose there are millions of winning compositions of 40 subunits, any of which could self-replicate and initiate Darwinian evolution. Then even a lucky milligram of RNA might contain the seed of life.\n\nIn the laboratory one can prepare milligram quantities of random-sequence RNA molecules containing 40 or more subunits. One can provide an endless supply of activated nucleotide building blocks and control all aspects of the reaction conditions. Stacking the deck in this way, why haven't we witnessed the origin of RNA life experimentally? Because even a lucky milligram of RNA is insufficient. In order for a seed copy of replicating RNA to germinate, it must produce additional copies of itself faster than the existing copies become degraded, and it must operate with sufficient fidelity that the accurate copies are not overwhelmed by error copies \\[9\\]. The requisite rate and fidelity of replication might reasonably be achieved in a pure reaction system that contains only the replicator and its corresponding building blocks. However, in a complex mixture of almost entirely mismatched parts, what process singles out the rare matching components? Darwinian evolution can enrich one molecule in a milligram, but before the onset of Darwinian evolution there would be only chemistry, and the chemistry of complex mixtures of cross-interacting molecules is very messy.\n\nOne way to avoid complex mixtures would be for each replicating RNA, together with its corresponding building blocks, to be spatially isolated within a separate compartment. These compartments need not be \"cells,\" but at least separate locales to avoid crosstalk among the replicators and to allow the fruits of their labor to accumulate locally for their own benefit. The need for compartments places additional restrictions on the nature of the environment, although at a minimum a \"compartment\" could be as simple as a non-covalent assemblage of the replicator and its components. Again it comes down to bits. How many bits of specific chemical organization are required to exclude the molecular riff-raff and allow a fledgling replicator to begin evolving? If 80 bits are required and there are a million such 80-bit combinations, then there would need to be 10^18^ separate locales of suitable composition to provide a reasonable opportunity for life to begin. If each suitable locale is a bubble in the sea, then the origin of life may not be a rare event, but if each is a different habitable planet in our galaxy, then even among a billion such planets, we may be very much alone.\n\n# Second Life?\n\nMy laboratory recently described an example, outside of biology, of a chemical system that can undergo Darwinian evolution in a self-sustained manner \\[10\\]. \"Self-sustained\" in this context means that all of the bits necessary for the system to undergo Darwinian evolution are part of the system that is evolving. The chemical system involves pairs of RNA enzymes that catalyze each other's synthesis by joining together two oligonucleotide building blocks (<a href=\"#pbio-1001323-g003\" data-ref-type=\"fig\">Figure 3A<\/a>). Each of the two building blocks can adopt thousands of potential alternative compositions and thus can be joined to form millions of different combinations. Each of the many possible combinations can self-replicate and transmit compositional information to its progeny. Variants that replicate most efficiently grow to dominate the population until new, more advantageous variants arise to supplant their predecessors in a never-ending Darwinian battle for survival.\n\nThe population of evolving RNA enzymes constitutes a synthetic genetic system, but it is limited in two important respects. First, the molecules contain only 24 bits (12 base pairs) of heritable information to encode function. Second, replication depends on 60 bits (30 defined nucleotides) that are provided at the outset and are not subject to mutation and selection (<a href=\"#pbio-1001323-g003\" data-ref-type=\"fig\">Figure 3B<\/a>). Thus of the 84 total bits required for the system to replicate and evolve, only about one-fourth can be counted as part of the system's molecular memory. The synthetic genetic system is not a new life form because it operates mostly on borrowed bits.\n\n# Prospects\n\nSomeday the threshold may be crossed in which an alternative genetic system contains more heritable bits than the number of bits required to initiate its operation. Crossing that threshold is a reasonable criterion for what would constitute a new form of life. A life form that arises directly from bit-free chemistry would be considered \"new\" from the outset, while one that derives from a biological cell would have a long way to go before reaching the threshold. Between these two extremes lie the possibilities of starting with a modest number of bits, whether by the luck of combinatorial chemistry or derived from preexisting life, then accruing enough bits within the system to be regarded as new life. Perhaps the first true alternative to terrestrial biology will be found on an extrasolar planet, in a rock from Mars, or within an extreme environment on Earth. More likely, it will be the handiwork of an intelligent species that has discovered the principles of Darwinian evolution and learned to devise chemical systems that have the capacity to generate bits on their own.\n\n# References\n\n## Abbreviations\n\nGNA\n\n:   glycol nucleic acid\n\nTNA\n\n:   threose nucleic acid\n\n[^1]: Essays articulate a specific perspective on a topic of broad interest to scientists.","meta":{"dup_signals":{"dup_doc_count":112,"dup_dump_count":48,"dup_details":{"curated_sources":4,"2023-40":1,"2023-23":1,"2023-06":1,"2022-40":1,"2022-33":1,"2021-49":1,"2021-39":1,"2021-25":1,"2021-10":1,"2020-45":1,"2019-22":1,"2019-18":1,"2019-04":2,"2018-47":2,"2018-26":1,"2017-09":6,"2017-04":3,"2016-50":1,"2016-44":1,"2016-40":3,"2016-36":4,"2016-30":6,"2016-26":1,"2016-22":1,"2016-18":1,"2016-07":2,"2015-48":4,"2015-40":3,"2015-35":2,"2015-32":4,"2015-27":2,"2015-22":2,"2015-14":3,"2014-52":3,"2014-49":1,"2014-42":6,"2014-41":4,"2014-35":6,"2014-23":8,"2014-15":3,"2023-50":1,"2024-10":1,"2017-13":2,"2015-18":2,"2015-11":2,"2014-10":1,"2013-48":1,"2024-30":1}},"file":"PMC3348159"},"subset":"pubmed_central"}
{"text":"author: Ursula Neubauer; Gregor Kastl\\*To whom correspondence should be addressed: Ursula Neubauer, Michael-Gaismair Strasse 11, 6020 Innsbruck, Austria, E-mail: <email@example.com>\ndate: 2010-04-22\ninstitute: 1University of Innsbruck, student in medicine, Innsbruck, Austria\ntitle: A student's perspective from the Medical University of Innsbruck\n\n# Report\n\nInnsbruck is known for its skiing resorts, the 'olden Dacherl'(golden roof) and the medical school, which recently has become a refuge for foreign students desperately seeking admission to medical education. However, the Medical University of Innsbruck is much more than a place of last resort for students; it is first and foremost an outstanding medical school which is held in high esteem internationally. It is well-renowned for its medical research and practice and its international reputation transcends the small size of Austria as a country. Numerous scientists such as Ignaz Semmelweis, Karl Landsteiner, Sigmund Freud and Konrad Lorenz gained international recognition for their significant contributions to the field of medicine.\n\nInnsbruck therefore combines for students the spirit of a young city dominated by students and situated in the midst of famous skiing resorts with the heritage of a great tradition and the professional environment of a high ranking institution for education and training.\n\nToday Austria has four medical universities: public universities at Vienna, Graz and Innsbruck and the private Paracelsus University at Salzburg.\n\nAs in most other countries, it is not easy to get into medical school as the number of applicants far exceeds the number of available places. Medical Universities have therefore adopted various admission procedures. In Vienna and Innsbruck, applicants take a general admissions test, the 'Eignungstest fuer das Medizinstudium' (EMS) (ability test for medical education), which tests skills and competencies like stereoscopic vision, concentration, knowledge of maths and the ability to understand scientific texts. The test is designed to be very exhausting and stressful. The tension is palpable when one enters the large examination hall where approximately 1800 applicants are gathered to compete for a mere 400 places at the medical school. During the test the level of pressure rises because the test is designed to be too long to complete in the short time allowed. Accordingly, there is a strong sense of discouragement among students after the test. Six weeks after the test, the results are published together with the list of the 400 candidates that are invited to start their studies in the upcoming semester.\n\nFrom the very start the curriculum is quite challenging. To help students cope with their new situation they are guided during the first four weeks before the first exam by senior more experienced students acting as tutors.\n\nThe basic structure of the curricula of the three public medical universities in Austria is similar. The curriculum is divided into three parts: 'pre-clinical', 'middle-clinical' and 'clinical', consisting of modules, such as 'musculoskeletal system' or 'components of life'.\n\nDespite the basic similarity, the curricula differ significantly because medical schools are free to design their own programme and determine their own concept and standards of academic excellence. Unfortunately, these differences are a barrier to students moving between universities and most students complete their studies at one medical school.\n\nHowever, in order to offer students international experience, there are many scholarships enabling students to work in hospitals abroad during semester breaks.\n\nThe medical programme at Innsbruck Medical University comprises twelve semesters. From the first year to the tenth semester, students have to take an exam consisting of 800 multiple choice questions every second semester.\n\nIt is therefore important that these questions test students' basic medical knowledge. However, as teachers are required to produce new questions for the same topics every year, in practice questions tend to ask about details that may be overly specific and not adequately reflect basic medical knowledge. This system has come under strong criticism from both students and teachers.\n\nInnsbruck has a long and established history in medical education and it is the seat of one of the oldest faculties of anatomy in Europe. Unlike other medical schools where students often do not have sufficient access to practical courses in anatomy, Innsbruck Medical University upholds its longstanding tradition of intensive dissection courses in the first two years. Groups of eight students are provided with one human body for anatomical observation and medical procedures. This offers students extensive practice in gross anatomy and gives them ample opportunity to develop their anatomical skills and knowledge.\n\nThe 11^th^ and 12^th^ semesters are designed as clinical clerkships ('Klinisch-Praktisches Jahr') in six disciplines. During this year students can also gain international experience. Because this practical year was only established fairly recently with the introduction of a new curriculum, there was no previous experience with this type of training and there has not been enough time for improvement. An important benefit of the new curriculum is the reduction in the size of student groups to maximize their work experience and enhance their learning.\n\nMedical studies in Austria are a diploma study, which means that students have to write a diploma thesis to complete their studies. Strangely, this diploma thesis is the basis for the title of Medicinae Universalis Doctor (Dr. med. univ., Doctor of Medicine). However, it is not equivalent to a PhD degree. Consequently, students additionally have to earn a PhD degree in order to obtain a postgraduate lecture qualification.\n\nAfter completing undergraduate medical studies students in Austria have to work as a 'Turnusarzt' in different medical disciplines for a minimum period of 36 months. Having successfully completed this period, graduates are awarded the *ius practicandi* (licence to practice). The 'Turnusarzt' system is subject to strong criticism because it is considered too time consuming and to delay specialty training until very late in a trainee's development. As a consequence, plans are in the offing to change this system fundamentally, or at least to shorten the required 36 months of working as a 'Turnusarzt'.\n\nAnother issue that should be mentioned is the relationship between German and Austrian students at Innsbruck. The population of Germany is ten times the size of the Austrian population and approximately 20% of medical students in Austria are from Germany. In Innsbruck, which is located just 30 km from the German border, this percentage is even higher. Unable to secure a place at a German medical school because of the \"numerus clausus\" (limited admission), many young Germans try to gain entrance to an Austrian Medical University by participating in the EMS test. This situation has aroused much heated debate in Austria. Presently, Austria is attempting to control the number of German students by allocating a maximum number places for foreign students from countries within the European Union. According to current regulations, at least 75% of students have to be Austrian citizens.\n\nOverall there are several advantages to studying medicine at Innsbruck Medical University. From the first semester onwards, students benefit from a good education in clinical practical subjects. As early as the first weeks of the first semester students can take part in clinical training in the hospital where they can join nurses and doctors. Moreover, there are lectures by general practitioners in addition to lectures by professors specialized in anatomy or biochemistry. This enhances learning, since students are brought into contact with \"real medical practice\" from the very beginning of their training. The strong clinical orientation is the main reason for the good reputation that medical students from Innsbruck have in numerous hospitals across Europe. Many of our students undertaking clinical traineeships elsewhere have been complimented by doctors on the standard of their clinical knowledge, which even in the early semesters is very high compared to that of medical students from other universities.\n\nThe early clinical orientation makes it easy for students to stay highly motivated even during the preclinical phase. Obviously, there are also theoretical lectures but students additionally gain more in-depth experience by practical training in small student groups.\n\nAnother important aspect is that students are involved in almost every decision of the university concerning their education and take an important and proactive role in improving the curriculum.\n\nThe experience we have gained over the last seven semesters has convinced us that education at Innsbruck Medical University is highly competitive. Looking back we have not once regretted our decision to study at Innsbruck and we can definitely recommend it to all those considering Innsbruck Medical University for their medical studies.\n\n# The authors\n\n1\\. Ursula Neubauer, 3th year student in medicine, University of Innsbruck.\n\n2\\. Gregor Kastl. 3th year student in medicine, University of Innsbruck.","meta":{"dup_signals":{"dup_doc_count":109,"dup_dump_count":40,"dup_details":{"curated_sources":2,"2018-51":1,"2018-47":1,"2018-30":1,"2018-26":1,"2018-05":2,"2017-30":3,"2017-22":2,"2017-09":2,"2017-04":2,"2016-50":2,"2016-44":3,"2016-40":2,"2016-36":3,"2016-30":3,"2016-26":2,"2016-22":2,"2016-18":2,"2016-07":3,"2015-48":3,"2015-40":1,"2015-35":3,"2015-32":3,"2015-27":2,"2015-22":3,"2015-14":2,"2014-52":3,"2014-49":2,"2014-42":9,"2014-41":4,"2014-35":6,"2014-23":5,"2014-15":6,"2019-09":1,"2015-18":3,"2015-11":3,"2015-06":3,"2014-10":2,"2013-48":3,"2013-20":2,"2024-26":1}},"file":"PMC3140351"},"subset":"pubmed_central"}
{"text":"abstract: The number of neuroimaging studies has grown exponentially in recent years and their results are not always consistent. Meta-analyses are helpful to summarize this vast literature and also offer insights that are not apparent from the individual studies. In this review, we describe the main methods used for meta-analyzing neuroimaging data, with special emphasis on their relative advantages and disadvantages. We describe and discuss meta-analytical methods for global brain volumes, methods based on regions of interest, label-based reviews, voxel-based meta-analytic methods and online databases. Regions of interest-based methods allow for optimal statistical analyses but are affected by a limited and potentially biased inclusion of brain regions, whilst voxel-based methods benefit from a more exhaustive and unbiased inclusion of studies but are statistically more limited. There are also relevant differences between the different available voxel-based meta-analytic methods, and the field is rapidly evolving to develop more accurate and robust methods. We suggest that in any meta-analysis of neuroimaging data, authors should aim to: only include studies exploring the whole brain; ensure that the same threshold throughout the whole brain is used within each included study; and explore the robustness of the findings via complementary analyses to minimize the risk of false positives.\nauthor: Joaquim Radua; David Mataix-Cols\ndate: 2012\ninstitute: 1Institute of Psychiatry, King's College London, De Crespigny Park, London, UK; 2Research Unit, FIDMAG-CIBERSAM, Sant Boi de Llobregat, Barcelona, Spain\nreferences:\ntitle: Meta-analytic methods for neuroimaging data explained\n\n# Introduction\n\nThe number of neuroimaging studies has grown exponentially in recent years. However, findings from different studies may sometimes be difficult to integrate into a coherent picture. Inconsistent results are not uncommon. Furthermore, a few influential studies might often eclipse robust findings from other studies. In other words, we may at times not see the forest for the trees. In this context, meta-analyses are helpful to combine and summarize the data of interest and potentially offer insights that are not immediately apparent from the individual studies.\n\nThe present paper aims to describe the main methods which have been used for the meta-analysis of neuroimaging data, as well as their advantages and drawbacks, with some examples of application to mood and anxiety disorders. The first section of the paper introduces how a standard meta-analysis is conducted, that is, when there is only one variable of interest, with an example from a meta-analysis of global brain volumes. This is important for a better appreciation of the pros and cons of the meta-analytic methods that we review later. The second section of the paper describes the meta-analyses of neuroimaging studies based on regions of interest (ROI) and their particular issues. The third section introduces the various available voxel-based meta-analytic methods, which aim to overcome some of the limitations of the ROI-based methods but have, in turn, their own limitations. The similarities and differences between the various voxel-based methods are also discussed in depth. Finally, we describe the available online databases of neuroimaging studies. This paper is meant to be accessible for the applied researcher in the fields of psychiatry, neurology and allied disciplines. Other excellent, more technical, reviews of meta-analytical methods can be found elsewhere \\[1,2\\].\n\n## Standard meta-analyses\n\nPrior to any meta-analytic calculation, researchers conduct an exhaustive and critical literature search, often including contact with the authors of the original studies in order to retrieve important pieces of missing information. Then, researchers conduct a mathematical summary of the findings of the included studies (that is, the meta-analysis proper). Finally, researchers apply a series of tests, plots and subgroup analyses to assess the heterogeneity and robustness of the results. The latter step, along with the exhaustive and critical inclusion of studies, is of utmost importance in order to obtain unbiased meta-analytic conclusions.\n\nWith the aim of introducing the reader to the logics of a standard meta-analysis, in this section we will use as an example a meta-analysis of global gray matter volumes in patients with obsessive-compulsive disorder (OCD) (see Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>). The included studies correspond to seven publications reporting global gray matter volume, which were included in a published meta-analysis of voxel-based morphometry studies in OCD \\[3\\].\n\nGlobal gray matter volumes reported in seven studies on obsessive-compulsive disorder\n\n|  | Reference | Patients |  | Controls |  | Patients + controls |  | Difference |  | Effect size |  |\n|----|----|----|----|----|----|----|----|----|----|----|----|\n|  |  |  |  |  |  |  |  |  |  |  |  |\n|  |  | Number | Volume \u00b1 SD | Number | Volume \u00b1 SD | N | Variance | Estimate | Variance | Estimate | Variance |\n| Study 1 | \\[46\\] | 18 | 773 \u00b1 56 | 18 | 822 \u00b1 56 | 36 | 3,114 | -49 | 346 | -0.854 | 0.122 |\n| Study 2 | \\[47\\] | 55 | 685 \u00b1 74 | 50 | 708 \u00b1 72 | 105 | 5,323 | -23 | 203 | -0.313 | 0.039 |\n| Study 3 | \\[48\\] | 25 | 850 \u00b1 83 | 25 | 834 \u00b1 71 | 50 | 5,997 | +16 | 480 | 0.196 | 0.080 |\n| Study 4 | \\[49\\] | 72 | 739 \u00b1 82 | 72 | 763 \u00b1 78 | 144 | 6,404 | -24 | 178 | -0.298 | 0.028 |\n| Study 5 | \\[50\\] | 37 | 776 \u00b1 69 | 26 | 747 \u00b1 68 | 63 | 4,680 | +29 | 307 | 0.418 | 0.067 |\n| Study 6 | \\[51\\] | 19 | 827 \u00b1 44 | 15 | 836 \u00b1 63 | 34 | 3,041 | -9 | 363 | -0.179 | 0.120 |\n| Study 7 | \\[52\\] | 71 | 740 \u00b1 66 | 71 | 738 \u00b1 63 | 142 | 4,119 | +2 | 116 | 0.035 | 0.028 |\n\nAll volumes are in milliliters. SD: standard deviation.\n\n### Weighting of the studies\n\nIn order to summarize these seven studies, a simple meta-analysis could consist of calculating the mean difference in global gray matter volume between patients and controls as reported in the original studies \\[4\\]. Thus, we could summarize Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a> by saying that the mean global gray matter volume is 8.4 mL inferior in patients than in healthy controls-this number is just the arithmetic mean of the differences shown in the table.\n\nThe use of the arithmetic mean, however, may be too simplistic, because the different studies should have different weights. For example, the number of patients in study 4 is four times larger than the number of patients in study 1. Clearly, we cannot give the same weight to both studies and should give more weight to study 4. Probably, we should give it about four times more weight, as it includes four times as many patients.\n\nIncluding all the studies in Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a> and weighting the mean difference by the sample sizes of the studies, we would conclude now that the mean global gray matter volume is 8.8 mL inferior in patients than in controls. Note that when we previously calculated the mean difference as the simple arithmetic mean, we were indeed assuming that all the studies had the same sample size. This erroneous assumption had only a minor effect here (we thought that the difference was about 5% smaller than what we think now), but it could have important effects in other meta-analyses, especially if studies with smaller sample sizes inexplicably find more differences than studies with larger sample sizes-we will introduce how to detect this kind of bias later.\n\nUnfortunately, weighting the calculations only by sample size would still be too simplistic, because the weight of a study should also include its precision. For example, study 1 included fewer patients than study 4, but its volume estimates seem much more precise, as its sample variance is approximately the half than that in study 4 (see Patients + controls column in Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>). We do not know the reason for this higher precision (maybe the sample was more homogenous; maybe the technical procedures were cleaner; maybe it was just chance); however, we must take this precision into account by weighting by the inverse of the variance of the difference-which also includes the sample size.\n\nIncluding all the studies in Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a> and weighting by the inverse of the variance of the difference, we would conclude that the mean global gray matter volume is 8.9 mL inferior in patients than in controls (z-value = -1.55, *P* = 0.121). When previously we did not weight by sample variance we were assuming that all the studies had the same variance, though in this case this assumption was acceptable because the variance of the studies is rather homogeneous.\n\nHowever, as explained in the next section, weighting the calculations only by the inverse of the variance of the difference may still be too simplistic.\n\n### Heterogeneity in the studies\n\nHealthy individuals have different global gray matter volumes, that is, some have larger brains, some have thicker cortices, and so on. When conducting an analysis with original data, we are usually able to explain or model a part of this variability, but there is also a part of this variability that remains unexplained. This residual error may be due to unobserved variables, etiological heterogeneity within particular diagnoses, poor model fitting, or maybe just pure chance. This individual-based within-study variability cause the sample means to be variable, so that different studies obtain different results.\n\nHowever, within-study variability is not the only source of the between-study variability or heterogeneity. Given the relatively small amount of robust findings in neuroimaging, it would be highly desirable that all researchers conducted their studies using the exact same inclusion criteria and methods so that all between-study variability was only related to the within-study variability. However, the fact is that clinical and methodological differences between studies are often substantial.\n\nOn the one hand, patients included in the individual studies may have been sampled from clinically different populations; for example, one study of major depressive disorder may include outpatients with mild reactive depressive episodes while another study may be focused on inpatients suffering from severe endogenous depressions with melancholic symptoms. Similarly, patients in different studies may be receiving different treatments, or be in different phases of the disorder (for example, having a first episode or having a history of multiple episodes).\n\nOn the other hand, researchers may have been investigated similar but still different aspects of a disorder; for example, one study may have described the blood oxygen level-dependent (BOLD) brain response to a task involving a high memory load, while another study may be interest in the BOLD response to a task related to decision-making. Or even if studying the same particular cognitive function, each study may employ a particular statistical package, and its large set of associated assumptions.\n\nFinally, there may be a relevant part of the between-study heterogeneity which can be neither related to the within-study variability, nor explained by clinical or methodological differences between studies. This is called residual heterogeneity.\n\nIt is highly recommended to study the between-study variability or heterogeneity in any meta-analysis. For example, if the main analysis detects differences between patients and controls, it may be of interest to explore whether these differences depend on the severity of the disorder, or if they are related to special subtypes of the disorder. These questions may be assessed with meta-regressions. But even if the meta-analysis does not aim to explore the modulating effects of clinical variables on the main outcomes, heterogeneity should still be taken into account.\n\nIndeed, there is agreement on always including the residual heterogeneity in the weighting of the calculations \\[5-8\\]. Meta-analyses conducted this way are said to follow random-effects models, in opposition to the fixed-effects models that we saw in the previous point, which did not include heterogeneity. In the example of Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a> the use of a random-effects model would lead us to conclude that the mean global gray matter volume is 9.0 mL inferior in patients than in controls (z-value = -0.98, *P* = 0.328; see Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>, left). Note the increase of *P*-value in the random-effects model (from 0.121 to 0.328), thus better controlling the false positive rate.\n\n### Other complementary analyses\n\nThe above meta-analysis of global gray matter volumes in OCD was only aimed to help interpret the findings of a regional voxel-based meta-analysis, for which no other tests beyond a correctly weighted random-effects model would probably be required. However, if global gray matter volumes were the main outcome of a meta-analysis, some complementary plots and tests would be recommended to help the reader assess the reliability and robustness of the findings \\[9\\].\n\nOn the one hand, the meta-regressions described above may be useful for assessing if the findings are predominantly (or only) present in one group of patients, for example, in those with more severe forms of OCD. In this regard, specific meta-analyses of subgroups of patients may further confirm these hypotheses or, also important, may state that the abnormalities are present in all subgroups of patients, increasing the robustness of the findings. Similarly, sensitivity analyses consisting of repeating the meta-analysis many times, each time with a different combination of studies, may be useful to assess whether the findings are driven by one or few studies. Finally, funnel plots (see Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>, right) may be useful for appraising whether studies with small samples report more statistically significant findings than studies with larger samples. This is typical of subjects with publication bias, where studies with small samples are only published if their results match *a priori* hypotheses.\n\nIt is important to note that these kinds of tests and graphical aids are necessary but do not provide conclusive information, and should only be interpreted in the context of the field under investigation. A symmetrical funnel plot, for example, is not an amulet against publication bias, especially in some types of meta-analysis. ROI-based studies, for instance, may be more prone to be affected by publication biases, as the authors may decide which brain regions are reported and which are not. Conversely, an asymmetrical funnel plot would not necessarily invalidate a meta-analysis if publication bias appears unlikely. This may be the case of voxel-based studies, where the whole brain is included in the analysis.\n\n### Use of effect sizes\n\nMost meta-analyses do not use the raw volume differences as we exemplified in the previous points, but rather, they use standardized volume differences, that is, effect sizes \\[10\\]. Briefly, a raw difference is the difference in milliliters between patients' and controls' global gray matter volume, while a standardized difference is the difference is standard deviations-usually corrected for small sample size bias.\n\nThis subtle difference has a series of consequences. First, the unit of measure (milliliters, in this case) is lost, which makes the interpretation of the findings less straightforward but indeed more comparable with other measures, for example, an effect size of d = 0.5 is considered 'medium' independently of whether it refers to a difference in gray matter volume, in BOLD response or in a questionnaire score. Using the data from Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>, the effect size of the difference in global gray matter volume between patients and controls is d = -0.122 (z-value = -0.93, *P* = 0.354; Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>), which is below the conventional range of 'small' effect (0.2 to 0.3) \\[11\\]. Second, a study reporting a larger difference may be found to have a smaller effect size, or vice versa, depending on the sample variance. For instance in Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>, the raw difference is slightly larger in study 4 than in study 3, whilst the effect size is slightly larger in study 3 than in study 4. Third, and very important, effect size can be directly derived from many statistics like a *t*-value or a *P*-value, which are much more often reported than sample statistics; that is, we can often know the effect size but not the raw difference. This advantage usually allows a much more exhaustive inclusion of studies, thus clearly justifying the use of effect sizes in many meta-analyses.\n\n## Meta-analyses based on regions of interest\n\nA ROI is a part of the brain that the authors of the study wish to investigate, usually based on *a priori* hypotheses. ROI-based studies usually select a set of few ROIs and manually delimitate them on the raw images. Researchers then analyze the volume of these ROIs, their mean BOLD response to a stimulus, their positron emission tomography ligand volume of distribution, or any other measure of interest.\n\n### Region of interest-based meta-analyses\n\nA typical ROI-based meta-analysis can be viewed as a set of different meta-analyses, each of them applied to a different ROI. These meta-analyses can usually be optimally conducted with all appropriate weightings and complementary analyses, as seen for example in the meta-analysis of regional brain volumes in OCD conducted by Rotge *et al.* \\[12\\], in which the analyses are based on effect sizes and random-effects models and complemented with explicit assessments of the heterogeneity, several sensitivity analyses, funnel plots and meta-regressions. Unfortunately, each original study included in this meta-analysis only investigated a small set of brain regions, causing the meta-analyses to include only a very small number of studies for each brain region. Indeed, only three or four studies could be included for highly relevant regions such as the putamen or the anterior cingulate cortex, both of which were found as abnormal in subsequent voxel-based meta-analyses \\[3,13\\]. Other brain regions could not be meta-analyzed because they had been investigated by too few or no studies. Needless to say, this would not be the case for those ROI studies reporting whole brain results in online supplements or similar, but this is seldom the case.\n\nMoreover, it must be noted that some brain regions are more frequently studied than others, which causes the statistical power to differ depending on the brain region under study. In the example, while data from five studies or more were available for the orbitofrontal cortex, the thalamus and the caudate nuclei, some brain regions could not be meta-analyzed at all.\n\nUltimately, the authors of the original studies have a set of *a priori* hypotheses which influence their decision to investigate differences in a given brain region at the expense of other regions. These decisions determine the number of studies investigating that brain region, and thus the statistical power to detect that brain region as significantly different between patients and controls in a ROI-based meta-analysis. Publication bias is also a problem as studies failing to report statistically significant differences on hypothesized ROIs may be less likely to ever be become publicly available. A recent analysis of more than 450 ROI-based neuroimaging studies in psychiatry illustrates this point well \\[14\\]. The author demonstrated that the number of studies reporting significant results was nearly the double than expected, suggesting strong publication biases in the ROI literature.\n\nAnother consideration is the heterogeneous definition or the boundaries of the ROIs, which may differ from one study to the other \\[12\\]. However, this variability might have a relatively small impact on effect sizes, as boundary definitions are the same for the patients and controls included in a study. Furthermore, the spatial error may probably be counteracted by the higher anatomical accuracy achieved by the manual delimitation of the ROIs in the original studies \\[15,16\\].\n\n### Label-based reviews\n\nSome authors have used a simplified type of ROI-based meta-analysis, consisting of just counting how many times a particular ROI is detected as significantly abnormal in patients versus healthy controls. This procedure has been called label-based review \\[17\\]. For example, in their functional neuroimaging meta-analysis of the brain's response to emotional tasks, Phan *et al.* \\[18\\] represented each activation peak as a dot in an atlas of the brain. They then divided the brain into 20 ROIs and counted how many studies had one or more activation peak in each ROI.\n\nA fictitious example of such approach is shown in Figure <a href=\"#F3\" data-ref-type=\"fig\">3A<\/a>. Here, the studies would have reported that the patients with mood or anxiety disorders have increases of gray matter volume in the basal ganglia, extending to the anterior part of the right insula, as well as decreases of gray matter volume in the anterior cingulate and insular cortices. The authors of a label-based review would have first plotted the peaks of the clusters of significant increase (red) or decrease (blue) of gray matter volume in a brain template. Then, they would divide the brain into several regions, for example, anterior cingulate gyrus, left and right inferior frontal gyri, insulas, superior temporal gyri, caudate nuclei, putamen nuclei, and so on. Finally, they would have counted how many peaks lay within each of these regions.\n\nThis method may be useful when other approaches are not feasible, for example when not enough information is available for conducting a ROI-based or a voxel-based meta-analysis. Its simplicity, however, may conceal a series of important drawbacks which must be taken into account. First, no weighting of the studies is performed, which means that all studies are assumed to have the same sample size and precision. This is a strong and unrealistic assumption which may be violated in most meta-analysis. Fortunately, sample size information is always available, and so label-based meta-analyses should at least be weighted by sample size. Second, the findings of the studies are binarized (significant versus not-significant), leading to a loss of information on the magnitude of the raw differences or on the effect sizes. Third, it is not clear whether studies reporting opposite findings in a particular ROI (for example, volume decrease in some studies and volume increase in others) are adequately dealt with. Finally, they may be also affected by the particular issues of ROI-based meta-analyses described above.\n\n## Voxel-based meta-analyses\n\nScanner three-dimensional images are composed of thousands of tiny cubes (or rectangular cuboids) called voxels, in the same way that digital photographs are composed of thousands of tiny squares called pixels. Voxel-based methods consist of conducting the meta-analytic calculations separately in each voxel of the brain, thus freeing the meta-analysis from aprioristic anatomical definitions. There are different types of voxel-based meta-analyses, including image-based, coordinate-based and mixed image- and coordinate-based meta-analyses.\n\n### Image-based meta-analyses\n\nAn image-based meta-analysis should be understood as a voxel-based version of the standard meta-analysis, that is, it consists of thousands of standard meta-analyses, each of them applied to a different voxel \\[2,19\\]. The data of each study is retrieved from its statistical parametric maps (the three-dimensional images resulting from the comparison between patients and controls), and thus include the whole brain. This technique shares some limitations with any voxel-based analysis, mainly relating to the massive number of statistical tests (that is, one test for each voxel). The correction of multiple comparisons is an unsolved issue, with current methods being either too liberal or too conservative \\[19\\]. For this reason, thresholds based on uncorrected *P*-values and cluster-size are usually preferred \\[1,19,20\\]. Also, such massive-scale testing prevents a careful visual inspection of the analyses (for example, to describe relevant non-significant trends).\n\nHowever, the biggest drawback of image-based meta-analyses is that the statistical parametric maps of the original studies are seldom available, therefore seriously limiting the inclusion of studies.\n\n### Coordinate-based meta-analyses\n\nGiven the poor availability of statistical parametric maps, early meta-analyses of voxel-based studies consisted of label-based (rather than image-based) reviews, as discussed earlier. These methods quickly evolved to coordinate-based meta-analyses, which in their simplest form consisted of counting, for each voxel, how many activation peaks had been reported within its surroundings \\[21\\]. In the fictitious example, the dots of the label-based review (Figure <a href=\"#F3\" data-ref-type=\"fig\">3A<\/a>) would be replaced with spheres (Figure <a href=\"#F3\" data-ref-type=\"fig\">3B, C<\/a>), and the brain would not be divided into conventional regions but rather the number of spheres surrounding each voxel would be counted, thus obtaining a count for each voxel. It must be noted that calculations in activation likelihood estimation (ALE) \\[22\\] are not exactly based on counting the number of spheres but on computing the probability of a union, though in practice, the latter behaves like the former.\n\nThe use of voxels rather than conventional divisions of the brain improved the anatomical localization of the findings. However, the first available methods, namely ALE and kernel density analysis (KDA) \\[21\\], had some additional issues which enlarged the list of drawbacks of label-based reviews. Specifically, they only counted the total number of peaks, independently of whether they came from the same or different studies, and thus the analysis could not be weighted by sample size and a single study reporting many peaks in close proximity could drive the findings of the whole analysis.\n\nThese drawbacks led to the creation of a second generation of coordinate-based meta-analytic methods, mainly evolved versions of KDA, such as multilevel KDA \\[23\\] and parametric voxel-based meta-analysis \\[24\\]; evolved versions of ALE \\[25,26\\]; and signed differential mapping (SDM) \\[3,27-30\\] (Figure <a href=\"#F3\" data-ref-type=\"fig\">3D, E<\/a>), which overcame these limitations by separating the peaks of each study. Moreover, some of these new methods weighted the studies by their sample size and included a series of complementary analyses to assess the reliability and robustness of the findings. One of the methods, SDM, also addressed between-study heterogeneity by reconstructing positive and negative maps in the same image, thus counteracting the effects of studies reporting findings in opposite directions \\[31\\] and incorporating meta-regression methods. Finally, SDM included templates for white matter \\[32\\], allowing multimodal meta-analyses which were not possible with previous methods \\[33\\]. However, these methods still did not employ the standard statistical methods of the ROI-based meta-analyses, for example, they did not weight by the precision of each study.\n\nSeveral of these methods have recently been applied to mood and anxiety disorders. One such was a meta-analysis of studies investigating bipolar disorder \\[34\\] using SDM, which found patients to have gray matter reductions in the left medial frontal and\/or anterior cingulate cortex (MF\/ACC) and bilateral anterior insula, with complementary analyses showing findings in the left ACC and right insula to be robust, and left MF\/ACC volume to be higher in samples where patients were being treated with lithium. An SDM meta-analysis of studies investigating major depressive disorder also found patients to have gray matter reductions in the MF\/ACC \\[35\\], and complementary analyses showed this finding to be robust and more severe in multiple-episode samples. Recent SDM meta-analyses of studies investigating anxiety disorders have also found patients to have gray matter reduction in the MF\/ACC \\[13\\], along with abnormalities in the basal ganglia. Specifically, patients with OCD were found to have increased gray matter volume in the bilateral putamen and caudate nuclei \\[3\\], with complementary analyses showing findings in the MF\/ACC and left basal ganglia to be robust, and bilateral increases of basal ganglia volume to be higher in samples including patients with higher symptom severity. Conversely, patients with anxiety disorders other than OCD were found to have decreased gray matter volume in the left putamen nucleus \\[13\\].\n\nAn example of the use of these methods in functional neuroimaging is the recent study by Delvecchio *et al.* \\[36\\], which meta-analyzed the functional brain response to emotional faces using ALE. They found that both patients with bipolar disorder and patients with unipolar depression displayed limbic hyperactivations. However, only patients with bipolar disorder showed a set of hypoactivations in the prefrontal cortex and hyperactivations in the thalamus and basal ganglia. Conversely, only patients with major depressive disorder showed hypoactivations in the sensorimotor cortices. Another example from the anxiety disorders literature is a multilevel KDA meta-analysis, which found that patients showed hyperactivations in the amygdala and insula \\[37\\]. Interestingly, these hyperactivations were more observed in phobias, whilst patients with post-traumatic stress disorder displayed a hypoactivation of the MF\/ACC. Finally, an ALE meta-analysis of functional differences in patients with OCD detected that symptom provocation was possibly associated with activation of the orbitofrontal cortex, prefrontal cortex, anterior cingulate cortex, precuneus, premotor cortex, superior temporal gyrus, basal ganglia, hippocampus and uncus \\[38\\].\n\nThese methods may also be applied to functional connectivity studies \\[39,40\\]. Laird *et al*., for instance, studied the default mode network by first creating ALE maps, and then deriving meta-analytic co-activation maps \\[40\\]. With this approach, they could identify an affective sub-network component. This approach is promising, given the increasing interest in functional connectivity studies in various mood and anxiety disorders.\n\n### Mixed image- and coordinate-based meta-analyses\n\nRecently, effect size SDM (ES-SDM) was designed to allow the combination of studies from which images (statistical parametric maps) are available with studies from which only peak coordinates are reported, thus allowing a more exhaustive inclusion of studies, as well as more accurate estimations \\[19\\].\n\nThis is achieved by first using peak coordinates and their statistical values to recreate the statistical parametric maps, and then conducting an image-based meta-analysis. Thus, this method has some statistical advantages as compared to previous coordinate-based methods, namely the use of standard statistical methods (for example, weighting the calculations by both sample size and study precision, use of effect sizes, inclusion and assessment of residual heterogeneity, and so on).\n\nIn a meta-analysis of the BOLD response to emotional facial stimuli \\[19\\], the sensitivity to detect real activations (that is, the number of actually activated voxels appearing as significant in the meta-analysis, divided by the total number of actually activated voxels) was similar between ES-SDM (55%) and SDM (51%) when only using peak coordinates. However, the inclusion of the statistical parametric maps led to a gradual and substantial increase of the sensitivity of ES-SDM (73% when the statistical parametric map of one study was included, 87% when the statistical parametric maps of two studies were included, 93% when the statistical parametric maps of three studies were included, and so on). Therefore, given the potential of this new method, we would encourage authors to make their statistical parametric maps widely available to the community on their laboratory websites or via other means.\n\n## Online databases\n\nIn parallel with the development of new meta-analytical methods, several freely-available website-based databases of neuroimaging data have been made available. These online databases may be classified in three groups, namely: sets of original data (for example, the raw scanner images from several samples of individuals); summary statistics from the studies included in one meta-analysis (for example, the mean \u00b1 standard deviation ROI volumes); and sets of summary statistics of virtually all published studies.\n\nThe online sets of original data are composed of the raw and\/or pre-processed brain images, along with the demographic and clinical characteristics of each of the many anonymous participants. These databases may be used by researchers to conduct their studies, thus being a useful resource for highly accurate data analyses. It must be noted, however, that analyses derived from these datasets should not be strictly considered meta-analyses, as they do not necessarily exhaustively include all available data. Examples of these datasets are BRAINNet (<http:\/\/www.brainnet.net>), the fMRI Data Center (<http:\/\/www.fmridc.org>) and OpenfMRI (<http:\/\/www.openfmri.org>).\n\nOnline databases containing the summary statistics from the studies included in particular meta-analyses represent a more interactive (and often complete) alternative to the traditional 'supplementary materials' that accompany published meta-analyses. Importantly, these online data may be used by other researchers to conduct updated or secondary analyses. Examples of this type of databases are the Bipolar Disorder Neuroimaging Database (<http:\/\/www.bipolardatabase.org>) and the Major Depressive Disorder Neuroimaging Database (<http:\/\/www.depressiondatabase.org>) by Kempton and colleagues \\[41,42\\], as well as the series of peak-coordinate databases from SDM meta-analyses (<http:\/\/www.sdmproject.com\/database>).\n\nFinally, many sets of summary statistics of virtually all published neuroimaging studies exist, allowing a rapid retrieval of specific data in order to facilitate the meta-analytic process. The developers of BrainMap (<http:\/\/www.brainmap.org>), for instance, have been building and updating an impressive database of neuroimaging findings since 1987 \\[43\\]. Other available databases are the AMAT toolbox (<http:\/\/www.antoniahamilton.com\/amat.html>), the Brede Database (<http:\/\/neuro.imm.dtu.dk\/services\/brededatabase>) \\[44\\], the Internet Brain Volume Database (<http:\/\/www.cma.mgh.harvard.edu\/ibvd>) and the Surface Management System Database (<http:\/\/sumsdb.wustl.edu\/sums\/index.jsp>).\n\nAnother recent and promising online development called NeuroSynth (<http:\/\/www.neurosynth.org>) \\[45\\] deserves mentioning. NeuroSynth contains a set of summary statistics together with online functions aimed to conduct real-time meta-analyses online. Unfortunately, extraction of coordinates from publications is not manually verified, which may bias the results towards those regions that the authors of the original articles wanted to emphasize in the tables of the manuscripts. However, when the researcher's goal is to obtain a very fast and preliminary meta-analysis of the literature, NeuroSynth may be one of the first options.\n\n# Conclusions\n\nIn this paper we have reviewed the main types of meta-analytic methods available for neuroimaging studies, using examples from the mood and anxiety disorder literature to illustrate these methods.\n\nROI- and voxel-based methods each have advantages and disadvantages, which are summarized in Table <a href=\"#T2\" data-ref-type=\"table\">2<\/a>. Specifically, ROI-based meta-analyses use optimal statistical methods, but they usually have a limited and likely biased inclusion of studies \\[14\\]. Conversely, voxel-based meta-analyses usually have a more exhaustive and unbiased inclusion of studies, but their statistical methods are less accurate. There are also relevant differences between the different available voxel-based meta-analytic methods. Fortunately, the field is rapidly evolving to develop more accurate and robust methods.\n\nComparison of the main meta-analytic methods for neuroimaging studies comparing patients and controls\n\n|  |  |  |  |  |  |\n|----|----|----|----|----|----|\n|  | **ROI-based meta-analyses** | **Voxel-based meta-analyses** |  |  |  |\n|  |  |  |  |  |  |\n|  |  | **KDA\/old ALE** | **Multilevel KDA\/new ALE** | **SDM** | **ES-SDM** |\n|  |  |  |  |  |  |\n| **Selection of studies** |  |  |  |  |  |\n| \u2003Exhaustive inclusion of studies | Limited, as information for a given brain region is present in few or no studies | Probable, as far as the included studies investigate the whole brain and not only some ROIs (in which case should be discarded) |  |  | More probable, because statistical parametric maps can also be included |\n| \u2003Unbiased inclusion of studies | Limited, as information is only available for regions hypothesized *a priori*, ignoring the rest of the brain | Probable, as far as the included studies do not use different statistical thresholds for different parts of the brain (this is a strict inclusion criterion in SDM and ES-SDM) |  |  |  |\n| **Statistical analyses** |  |  |  |  |  |\n| \u2003Weighting of the studies | Complete (sample size and study precision) | None | Partial (only sample size) |  | Complete (sample size and study precision) |\n| \u2003Control of the heterogeneity | Residual heterogeneity is correctly included in the analyses | Residual heterogeneity is not controlled, and increases and decreases are not counteracted, potentially leading to voxels being detected as increased and decreased at the same time |  | Residual heterogeneity is not included in the weightings, but increases and decreases are counteracted | Residual heterogeneity is correctly included in the weightings |\n| \u2003Study of the heterogeneity | Possible, by means of meta-regressions and subgroup analyses | Limited to subgroup analyses |  | Possible, by means of meta-regressions and subgroup analyses |  |\n| \u2003Correction for multiple comparisons | Possible | Not possible, questionable or limited to conventional voxel-thresholded cluster-based methods |  |  |  |\n| \u2003Description of the effect sizes | Possible | Not possible | Possible though limited to pseudo-effect sizes based on the proportion of studies reporting significant findings |  | Possible |\n| \u2003Description of relevant non-significant trends | Possible, as the number of ROIs is manageable | Not possible, or limited to the visual inspection of liberally thresholded maps, as the number of voxels is too massive for a more accurate individual inspection |  |  |  |\n\nPlease see text for further details. ALE: activation likelihood estimation; ES: effect size; KDA: kernel density analysis; ROI: region of interest; SDM: signed differential mapping.\n\nAlthough voxel-based meta-analyses minimize the effects of selectively reporting certain ROI, they are not totally immune to publication biases, as negative results may still be less likely to be published (what is known as the file drawer problem). Authors of the original papers are strongly encouraged to publish their results even if they perceive them as being disappointing or they do not find differences between patients and controls.\n\nFinally, we suggest that in any meta-analysis of neuroimaging data, independently of the chosen method, authors should aim to: only include studies which explored the whole brain; ensure that the same threshold throughout the whole brain was used within each included study; and explore the robustness of the findings with several complementary analyses, for example, sensitivity analyses, quantification of the ROI- or voxel-based between-study heterogeneity \\[19\\], funnel plots of the values extracted from the meta-analytic clusters or their peaks, and so on, just like in any standard meta-analysis.\n\n# Competing interests\n\nThe authors declare that they have no competing interests.\n\n# Authors' contributions\n\nAll authors drafted, read and approved the final manuscript.","meta":{"dup_signals":{"dup_doc_count":160,"dup_dump_count":44,"dup_details":{"curated_sources":3,"2019-13":1,"2019-04":1,"2018-51":1,"2018-43":2,"2018-39":1,"2018-30":2,"2018-17":1,"2018-05":1,"2017-39":1,"2017-30":1,"2017-22":1,"2017-17":1,"2017-09":18,"2017-04":1,"2016-50":1,"2016-44":1,"2016-40":1,"2016-36":1,"2016-30":1,"2016-22":1,"2016-18":1,"2015-48":5,"2015-40":5,"2015-35":5,"2015-32":5,"2015-27":4,"2015-22":5,"2015-14":4,"2014-52":6,"2014-49":4,"2014-42":14,"2014-41":8,"2014-35":8,"2014-23":7,"2014-15":6,"2019-22":1,"2024-18":1,"2015-18":5,"2015-11":5,"2015-06":5,"2014-10":4,"2013-48":5,"2013-20":4,"2024-30":1}},"file":"PMC3384225"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  The recent emergence of hypervirulent subtypes of avian influenza has underlined the potentially devastating effects of pandemic influenza. Were such a virus to acquire the ability to spread efficiently between humans, control would almost certainly be hampered by limited vaccine supplies unless global spread could be substantially delayed. Moreover, the large increases that have occurred in international air travel might be expected to lead to more rapid global dissemination than in previous pandemics.\n  .\n  # Methods and Findings\n  .\n  To evaluate the potential of local control measures and travel restrictions to impede global dissemination, we developed stochastic models of the international spread of influenza based on extensions of coupled epidemic transmission models. These models have been shown to be capable of accurately forecasting local and global spread of epidemic and pandemic influenza. We show that under most scenarios restrictions on air travel are likely to be of surprisingly little value in delaying epidemics, unless almost all travel ceases very soon after epidemics are detected.\n  .\n  # Conclusions\n  .\n  Interventions to reduce local transmission of influenza are likely to be more effective at reducing the rate of global spread and less vulnerable to implementation delays than air travel restrictions. Nevertheless, under the most plausible scenarios, achievable delays are small compared with the time needed to accumulate substantial vaccine stocks.\nauthor: Ben S Cooper; Richard J Pitman; W. John Edmunds; Nigel J Gay\\* To whom correspondence should be addressed. E-mail: <email@example.com>[^1]\ndate: 2006-06\ninstitute: **1**Statistics, Modelling, and Bioinformatics Department, Centre for Infections, Health Protection Agency, London, United Kingdom; Mexican National Institutes of HealthMexico\nreferences:\ntitle: Delaying the International Spread of Pandemic Influenza\n\n# Introduction\n\nThe scale of threat posed by hypervirulent avian influenza subtypes \\[1,2\\], and the memory of the 20\u2013100 million who died in the 1918 pandemic \\[3,4\\], warrant consideration of large-scale, concerted, and potentially highly disruptive control measures \\[5\\]. Were such a virus to acquire the ability to spread efficiently between humans, control would almost certainly be hampered by limited vaccine supplies \\[6\\]. Interventions able to substantially impede global spread, by providing time for vaccine stocks to accumulate, could have profound public health benefits.\n\nBorder controls and World Health Organization travel advisories formed central and sometimes controversial components of the control efforts during the severe acute respiratory syndrome (SARS) epidemic \\[7,8\\], and travel restriction is thought likely to occur during an influenza pandemic (although enforcement is currently considered by the World Health Organization to be impractical in most countries) \\[9\\]. In the absence of sufficient vaccine stocks, other control measures such as the use of antiviral agents could also be used \\[10,11\\]. Ideally, such measures would reduce the average number of secondary cases caused by each primary case (the effective reproduction number, *R* ~t~) to below one, making sustained transmission impossible. This happened during the SARS epidemic, where isolation, quarantine, and behaviour change were able to bring about control \\[12\\]. The much shorter serial interval for influenza makes the chances for early epidemic termination much lower \\[13\\]. The main value of interventions is more likely to be in reducing the incidence and slowing the rate of spread of the virus.\n\nTo evaluate the potential of travel restriction and local control measures to impede global dissemination we developed a stochastic (i.e., probabilistic) model of the international spread of influenza based on extensions of coupled deterministic epidemic transmission models \\[14\u201319\\]. This class of models has been shown to be capable of accurately forecasting local and global spread of epidemic and pandemic influenza \\[14\u201319\\] and accounting for the global distribution of other pathogens \\[20,21\\], but has not previously been used to assess the impact of travel restrictions or other control options for pandemic influenza.\n\n# Methods\n\nWe used a metapopulation model that consists of a set of coupled dynamic epidemic transmission models (<a href=\"#pmed-0030212-g001\" data-ref-type=\"fig\">Figure 1<\/a>). Each component model represents one city and tracks the progression of individuals through four classes: susceptible to infection (S); exposed to the virus but not yet infectious (E); infectious (I); and recovered and no longer susceptible (R). We assumed that infectiousness coincides with disease onset and that infectious cases do not travel.\n\nPrevious work has used deterministic approximations to study the evolution of this system \\[14\u201319\\]. With this approach the first case in each city (except the originating city) was assumed to occur only when the average number of incubating cases arriving from other cities exceeded one, an approximation that will artificially slow the rate of spread between cities. Our stochastic model, which has a similar underlying structure to its deterministic counterpart, avoids this distortion and uses probabilistic transitions to capture the inherent uncertainty in the course of the epidemic. This is more appropriate than the deterministic approaches previously adopted, because chance effects dominate in the early stages of the epidemic in each city and in the seeding of each city's epidemic. As well as providing greater realism, this approach allowed us to quantify the uncertainty in model predictions due to demographic stochasticity. Thus, rather than assuming that each city's epidemic starts at a determined time, we assumed that the initiation of an epidemic depends on the timing of a sequence of chance events: a person incubating the virus must board a plane; that person must infect others in the destination city; some of those others must cause further transmission, and so on. Each time the model is run, even with identical starting conditions and parameter values, a different answer is obtained. This stochastic model was used to estimate key parameters using data from the 1968\u20131969 (1968\/9) influenza pandemic and to evaluate the impact of interventions using contemporary demographic and transport data. Mathematical details of the model are provided in <a href=\"#sd001\" data-ref-type=\"supplementary-material\">Protocol S1<\/a>.\n\nCoupling between cities was estimated using data from the International Air Transport Association for 2002 that gives the number of seats on flights between 105 cities, including the 100 with the highest number of international scheduled passengers and all 52 used in the 1968\/9 data. City sizes were taken from the United Nations urban agglomeration data (available at: <http:\/\/unstats.un.org\/unsd\/demographic\/sconcerns\/densurb\/urban.aspx>). When fitting models to 1968\/9 data, air transport data, sizes of urban agglomerations, and influenza data were taken from a previous study \\[14,15\\].\n\nTo select from variants of the basic model, we compared deterministic and stochastic model fits to data from the 1968\/9 pandemic by choosing parameters to minimize the sum of squared deviations (SSQ) between times of observed and predicted peaks (this contrasts with previous work that aimed to forecast the pandemic, and therefore based parameter estimates only on data from the first affected city \\[14\\]). A major strength of our approach is that the observed time of an epidemic peak should be unaffected even by large between-country variation in influenza reporting rates. The predicted epidemic peak for a given city was defined as the day on which the highest number of infected people first developed symptoms. When a city had more than one observed peak in the 1968\/9 data, only the first peak was used. We used the statistic SSQ(*m*)\/(*n* \u2212 2*m*) to compare model fits \\[22\\], where *m* is the number of fitted parameters (between one and four), and *n* the number of fitted data points (the number of cities with observed and predicted epidemic peak times). This formula selects models that are parsimonious and fit the data well. The full stochastic model was then used to estimate parameters for the best-fitting model by choosing parameters that minimised the mean SSQ from ten simulation runs for each combination of parameter values.\n\nWe evaluated models with sine wave, square wave, and no seasonal variation in transmission parameters for cities outside the tropics. In the sine wave formulation, the peak transmissibility occurred on the shortest day in each hemisphere, while in the square wave formulation, peak transmissibility lasted 6 mo, also centred on the winter solstice. We also considered model formulations where the transmission parameter in the tropics was taken as the maximum, minimum, and mean over 1 y of that outside the tropics. For some parameter values, the model predicted no epidemic peaks in some cities for which an epidemic peak was in fact recorded. When fitting the models we penalized these regions of parameter space by arbitrarily assigning a deviation between model and data of 500 d.\n\n## Interventions\n\nWe used the stochastic model to consider the effects of (i) reducing local transmission (this simulates the effects of isolation, behaviour changes, antiviral use, or other measures that may reduce the average number of secondary cases produced by one primary case); and (ii) restricting travel to and from affected cities. We assessed the ability of these measures to delay epidemics in individual cities. We considered only major epidemics, which we defined as those peaking with at least one case per 10,000 people per day. We assumed that measures were introduced only after the first 100 symptomatic cases in each city except the originating city, for which 1,000 cases were required, although we also evaluated the sensitivity of the results to these assumptions.\n\nWe considered a number of other scenarios to assess the sensitivity of the results to the most important unknowns: patterns of seasonal variation in influenza transmission; variation in transmissibility between tropical and temperate regions; the proportion of individuals initially susceptible to the virus; the basic reproduction number, *R* ~0~ (defined as the mean number of secondary cases in a local and susceptible population caused by the introduction of one primary case); the distribution of the infectious period; the city in which the pandemic begins; and the date on which the virus first begins to spread.\n\n# Results\n\nAmongst the model variants considered, the best fit to data from the 1968\/9 pandemic was achieved when transmissibility varied sinusoidally in temperate regions and was constant and equal to the north\/south maximum in the tropics. We used this model to estimate key parameters using 1968\/9 data, and to evaluate the impact of interventions. Models without seasonal forcing terms gave poor fits to data and could not account for the large differences in epidemic timing between cities in the north and south temperate regions. Models in which transmissibility in the tropics was set to the north\/south mean also performed surprisingly poorly, with best-fit SSQs approximately three times greater than those obtained when transmissibility in the tropics was set to the north\/south maximum (<a href=\"#pmed-0030212-g002\" data-ref-type=\"fig\">Figure 2<\/a>A and <a href=\"#pmed-0030212-g002\" data-ref-type=\"fig\">2<\/a>B). Less surprisingly, models that assumed all cities were equally connected by air travel (but with the same total volume of air traffic) also performed poorly, with best-fit SSQs about twice as large as those from the models that used the air travel data. Previous work with the deterministic version of the model has assumed a square wave variation in transmissibility, assigning transmission outside the influenza season to be one-tenth of the value during the season \\[14\\]. We found the fit to data under this assumption to be substantially poorer compared with models in which maximum and minimum seasonal transmission parameters were both estimated.\n\nAn exploration of the parameter space for the best-fit model showed that, assuming 60% of the population to be initially susceptible (the approximate value estimated previously \\[14\\]), maximum *R* ~0~ values (*R* ~0,max~) ranging from about 2.5 to 3.5 gave the best fits to data, while minimum *R* ~0~ values (*R* ~0,min~) between about 0.5 and 1.5 had the most support (<a href=\"#pmed-0030212-g002\" data-ref-type=\"fig\">Figure 2<\/a>C). The maximum *R* ~0~ value and the fraction initially susceptible could not be identified simultaneously: A high value of one implied a low value of the other (<a href=\"#pmed-0030212-g002\" data-ref-type=\"fig\">Figure 2<\/a>D). However, the initial maximum *effective* reproduction number, *R* ~max~ (equal to the product of the two and giving the average number of secondary cases produced by one primary case in an actual population, accounting for immunity) was well defined, with only a narrow range of values between about 1.5 and 2.2 supported by the data. This result is consistent with other estimates from influenza pandemics \\[14,23\\]. We therefore took as our baseline scenario an *R* ~0,max~ value of 3 and an *R* ~0,min~ of 1.2, assumed 60% of the population to be initially susceptible, and used a model in which the *R* ~0~ value varied sinusoidally and peaked in midwinter, and in which the pandemic originated in Hong Kong on 1 June.\n\nThe model showed good agreement with data from the 1968\/9 pandemic, with observed epidemic peaks almost always occurring at times when the model predicted a very high probability of influenza activity (<a href=\"#pmed-0030212-g003\" data-ref-type=\"fig\">Figure 3<\/a>). Observed and predicted times of epidemic peaks differed, on average, by 31 d. There were, however, some anomalies: the first epidemic peaks occurred much later than might have been expected in London and Tokyo, and somewhat earlier than predicted in Manila and Madras.\n\nDespite large variation in the timing of predicted epidemic peaks in individual cities between simulation runs, the overall course of the pandemic was quite predictable (<a href=\"#pmed-0030212-g003\" data-ref-type=\"fig\">Figure 3<\/a>A), although there was markedly more between-run variability in the tropics and the south than in the north. The roughly ten-fold increase in air traffic since 1968 causes epidemics in most cities to peak between 1 and 2 mo earlier than they would have done in 1968 (in some southern hemisphere cities the epidemic peaks 1 y earlier) and substantially reduces variation between simulation runs (<a href=\"#pmed-0030212-g003\" data-ref-type=\"fig\">Figure 3<\/a>B). The model reproduced another interesting aspect of influenza epidemiology: the tendency for peak periods of influenza activity in the tropics to shift with latitude, so that in the northern tropics they are closer to countries north of the tropics, while the southern tropics tend to be more closely aligned with countries south of the tropics \\[24\\]. This occurs despite the fact that the model has no explicit assumptions about seasonality for cities in the tropics; the behaviour arises only as a result of the strength of transport connections between different regions. It is also notable that the pandemic starts early enough to allow some probability of influenza activity in the south during the end of the flu season in 1968. Despite this, predicted epidemic peaks (the weeks with the greatest number of reported cases in each location) still occur in 1969 in the south.\n\nWhen we used the model to evaluate interventions using contemporary air travel and demographic data, we found that travel restrictions to and from affected cities would slow epidemic spread, but unless almost all air travel from affected cities (i.e., greater than 99%) was suspended, the potential for delaying the pandemic was limited (<a href=\"#pmed-0030212-g004\" data-ref-type=\"fig\">Figures 4<\/a>\u2013<a href=\"#pmed-0030212-g006\" data-ref-type=\"fig\">6<\/a> and <a href=\"#pmed-0030212-t001\" data-ref-type=\"table\">Table 1<\/a>). Even when 99.9% of air traffic was suspended, most cities had a low probability of ultimately escaping the pandemic (<a href=\"#pmed-0030212-g004\" data-ref-type=\"fig\">Figure 4<\/a>), and delays large enough to be of clinical significance (6 mo or more) were common only if interventions were made after the first few cases (<a href=\"#pmed-0030212-g005\" data-ref-type=\"fig\">Figure 5<\/a>). Interventions that reduced transmission could typically lead to more pronounced delays (<a href=\"#pmed-0030212-g005\" data-ref-type=\"fig\">Figures 5<\/a> and <a href=\"#pmed-0030212-g006\" data-ref-type=\"fig\">6<\/a> and <a href=\"#pmed-0030212-t001\" data-ref-type=\"table\">Table 1<\/a>), although only when *R* ~t~ was reduced to slightly above one were these sufficient to delay epidemics until the next influenza season. These findings were not highly sensitive to assumptions about initial susceptibility and transmissibility (<a href=\"#pmed-0030212-t001\" data-ref-type=\"table\">Table 1<\/a>).\n\nMedian Delays in Epidemic Peak for Diverse Interventions and Percentage of Cities Experiencing Major Epidemics\n\n![](pmed.0030212.t001)\n\nDecreasing the number initially susceptible (while holding *R* ~max~ constant) has two opposing effects (<a href=\"#pmed-0030212-t001\" data-ref-type=\"table\">Table 1<\/a>). First, within cities the time between seeding with influenza cases and the epidemic peak decreases. This is because the initial epidemic growth rate is unaffected, but each new case represents a greater proportional reduction in the susceptibles and causes a greater reduction in *R* ~t~ (the epidemic peaks when *R* ~t~ = 1). Conversely, between-city dynamics are slowed because there are fewer infectious people to spread the disease. Which effect dominates varies between cities; those affected at the start of the pandemic tend to experience peak activity earlier when there are fewer initial susceptibles; for the rest it usually occurs later. Although travel restriction always reduces the rate of spread between cities, under most scenarios so many people become infected that even near-total restriction has remarkably little effect. However, for a given *R* ~max~, the smaller the number of susceptibles the greater the impact of this intervention. For example, when 90% of the population are initially immune, the most extreme travel restrictions can be quite effective in preventing international spread. Conversely, reducing transmission has the greatest effect on impeding international spread when (for a given *R* ~max~) more people are susceptible. The large delays and reductions in the number of affected cities result from two effects acting in the same direction: The reduced *R* ~t~ slows the epidemic within each city (delaying epidemic peaks), and the reduced total number of cases reduces the rate of spread between cities. Larger reductions in transmission led, in extreme cases, to smaller delays in epidemic peaks (<a href=\"#pmed-0030212-t001\" data-ref-type=\"table\">Table 1<\/a>). This happened only when *R* ~t~ was reduced to below one, causing the epidemic decline to begin immediately; the peak therefore occurred at the time of the intervention, earlier than it would have done with a less effective intervention. Under such circumstances the time of the epidemic peak is not a good measure for fully evaluating local control measures.\n\nPrevious influenza modelling work has used both square and sine wave seasonal forcing terms \\[14,25\\]. We found that the outcomes of interventions were not highly sensitive to the precise assumptions made. The delays in the timing of epidemic peaks depended only to a limited extent on the city in which the pandemic started and to a somewhat greater extent on the date of release (<a href=\"#pmed-0030212-t002\" data-ref-type=\"table\">Table 2<\/a>), with larger delays more likely when the first cases occurred towards the end of the influenza season in the place of origin. Results were, however, highly sensitive to the timing of the intervention (<a href=\"#pmed-0030212-g005\" data-ref-type=\"fig\">Figure 5<\/a>). Large delays in the timing of epidemic peaks and the prevention of epidemics in a large number of locations could be achieved with the most extreme interventions, but only when they were made sufficiently early. However, making the interventions after fewer than 1,000 cases in the place of origin had minimal additional benefit in slowing pandemic spread. Similarly, preemptive travel restrictions had no advantage over interventions made after one case in affected cities (<a href=\"#pmed-0030212-g005\" data-ref-type=\"fig\">Figure 5<\/a>A and <a href=\"#pmed-0030212-g005\" data-ref-type=\"fig\">5<\/a>B).\n\nMedian Delays in Timing of Epidemic Peaks with Different Dates and Locations for the Start of the Pandemic\n\n![](pmed.0030212.t002)\n\nThe course of infection with a future pandemic influenza virus might differ in important ways from our baseline assumptions, and could be quite unlike typical interpandemic influenza. We therefore assessed the robustness of our conclusions to the assumed latent and infectious periods. We found that assuming a greater degree of infectiousness early in the course of infection (reducing the serial interval from 4.2 to 2.6 d, as suggested by recent analysis of household influenza transmission data \\[11,26\\]) did not substantially alter the conclusions about the value of the interventions (<a href=\"#pmed-0030212-g006\" data-ref-type=\"fig\">Figure 6<\/a>A\u2013<a href=\"#pmed-0030212-g006\" data-ref-type=\"fig\">6<\/a>C) compared with the baseline scenario (<a href=\"#pmed-0030212-g005\" data-ref-type=\"fig\">Figure 5<\/a>C and <a href=\"#pmed-0030212-g005\" data-ref-type=\"fig\">5<\/a>D), although if this assumption was used when fitting the model to the 1968\/9 data the estimated value of *R* ~max~ was reduced from about 1.8 to 1.5. Conclusions were also robust to moderate variation in the distribution of the latent period (<a href=\"#pmed-0030212-g006\" data-ref-type=\"fig\">Figure 6<\/a>D\u2013<a href=\"#pmed-0030212-g006\" data-ref-type=\"fig\">6<\/a>F). If, however, the virus behaved more like the SARS coronavirus, with extended latent and infectious periods (<a href=\"#pmed-0030212-g006\" data-ref-type=\"fig\">Figure 6<\/a>G\u2013<a href=\"#pmed-0030212-g006\" data-ref-type=\"fig\">6<\/a>I), a greatly delayed rate of global spread could be expected, giving more chance of delaying epidemics until the next influenza season. In this case, smaller reductions in travel and transmission can achieve clinically significant delays (6 mo or more) in epidemic take-off in many cities. Assuming reduced transmission in the tropics (<a href=\"#pmed-0030212-g006\" data-ref-type=\"fig\">Figure 6<\/a>J\u2013<a href=\"#pmed-0030212-g006\" data-ref-type=\"fig\">6<\/a>L) also led to a substantial reduction in the rate of global dissemination. Under this scenario much smaller reductions in transmission would be sufficient to greatly reduce the chance of a pandemic; this happens because the lower transmission in the tropics (where the virus is assumed to originate) means that a further transmission reduction of just 21% would be sufficient to make sustained spread impossible in this region.\n\n# Discussion\n\nThe relative ineffectiveness of travel restrictions for controlling pandemic influenza is a consequence of the rapid initial rate of growth of the epidemic in each city and the large number of people infected. For example, with a serial interval of 3 d, ignoring depletion of susceptibles, an *R* ~t~ of two would cause a 128-fold increase in new cases within 21 d (128 = 2^21\/3^). This means that if travel from the first affected city was restricted to 1\/128 of its former value on (and after) day 1, there would be approximately the same number of influenza cases leaving the city on day 21 + *t* as there would have been on day *t* had there been no intervention; even such an extreme intervention would therefore buy only about 3 wk. The highly connected nature of the air travel network prevents such minor delays between pairs of cities combining into substantial delays over the whole network.\n\nHufnagel et al. \\[20\\] used a related model to study the global spread of SARS. Although this model differed in important respects from the one used here (the implicit assumptions that air travel frequency varies with neither infection state nor country would not be tenable in the context of pandemic influenza), the conclusion that \"remarkable success \\[in SARS epidemic control\\] is guaranteed if the largest cities are isolated in response to an outbreak\" might, at first sight, be thought to apply equally to influenza. In fact, pandemic influenza is expected to have a much shorter serial interval than SARS, and delays in international spread that could be achieved by restricting almost all travel would be far more modest. Even if 99.9% of all travel could be stopped, epidemics in most cities would be delayed by no more than 4 mo. Moreover, the conclusion that a policy of isolating only the largest cities would guarantee success implicitly assumes that closing major airports would cause infected individuals who would have travelled through them to abandon their journeys rather than seek alternative routes, and that disease spread by routes other than air travel can be ignored without substantially altering the conclusions. This seems rather implausible, and for these reasons we think that the conclusions of Hufnagel and colleagues, while of undoubted theoretical interest, would be misleading if taken too literally.\n\nLarge and important uncertainties abound in influenza epidemiology: We do not know whether or not a significant proportion of transmission occurs before the onset of symptoms or whether subclinical infections are an important source of transmission, and we know very little about the determinants of seasonality \\[24,27,28\\]. In evaluating the potential to delay the spread of influenza by restricting travel and reducing transmission, we have systematically adopted optimistic assumptions, chosen to give the interventions the greatest chance of success. Thus we have assumed that seasonal effects are important (delaying the rate of spread outside the influenza season), and that asymptomatic cases do not contribute to transmission (minimizing the numbers capable of spreading the virus, and maximising the chance of detecting them); we have ignored travel that is not by air and not between major airports; and we have ignored the possibility of transmission during flights themselves. Despite these optimistic assumptions we found that even large and widely enforced travel restrictions would usually delay epidemic peaks by only a few days; to have a major impact, restrictions would have to be almost total and almost instantaneous. Only if a pandemic strain were considerably less transmissible, or had a considerably longer serial interval than influenza strains seen in the past, or if very few people were initially susceptible, would such measures be likely to have an important impact on the rate of pandemic spread. Local control measures able to reduce influenza transmission were found to have greater potential for reducing the rate of global spread (they could also substantially reduce the total number of cases, although an evaluation of this benefit is beyond the scope of this paper). Under most plausible scenarios, however, delays would still fall far short of those required to produce large quantities of vaccine unless they were implemented early and able to reduce *R* ~t~ to close to one. Elsewhere it has been shown that airport entry screening would be unlikely to detect more than 10% of passengers latently infected with influenza when boarding \\[29\\]. The results in this paper show that such an intervention would have a negligible impact on the course of a pandemic once it was underway.\n\nThe results also raise interesting questions about the importance of seasonality in influenza transmission. The evidence for strong seasonal effects in temperate regions found here with 1968\/9 data is supported by a recent analysis of interpandemic influenza \\[30\\]. However, it is not clear how important such seasonal effects have been in previous pandemics, nor is it clear why a much better model fit should be obtained when transmission in the tropics is assumed to be the maximum (rather than the mean) of that in temperate regions. Indeed, a fuller understanding of the determinants of seasonal effects and their variation with latitude remains one of the outstanding problems of influenza epidemiology \\[24,27,28\\].\n\nRecent models of pandemic influenza have accounted for household and social contact patterns \\[10,11\\]. While such details are needed for evaluating the possibility of containment at source, they would not be expected to affect the broad conclusions presented here. However, for a given *R* ~0~, assuming nonhomogeneous local mixing patterns would result in a somewhat reduced attack rate and rate of spread within each city, causing a slight decrease in the rate of global spread. For this reason, estimates of *R* ~max~ based on fitting models that assume homogeneous local mixing to pandemic data may underestimate the true value.\n\nA new pandemic strain might not show the same pattern of seasonality as in 1968\/9 and could potentially have greater transmissibility than strains seen previously. Both SARS and smallpox transmission can be greatly amplified by nosocomial spread \\[31,32\\]; a similar amplification effect could occur with an unusually virulent influenza virus that led to many hospitalisations. In these more pessimistic scenarios, even more heroic efforts would be required to have any chance of significantly delaying the virus's spread by restricting travel. The results here suggest that resources might be better directed at reducing transmission locally and at attempting to control outbreaks during the earliest stages of sustained human-to-human spread, when movement restrictions are likely to be a more valuable containment measure \\[10,11\\]\n\n# Supporting Information\n\n###### Detailed Description of the Model\n\n(65 KB DOC)\n\nClick here for additional data file.\n\n## Editors' Summary\n\n### Background.\n\nMost people who get influenza (flu) recover quickly, although it can cause serious illness and death, most often in the elderly. Sometimes a new type of flu virus appears that is much more likely to kill. This happened, for example, in 1918, when a worldwide flu pandemic killed between 20 million and 100 million people. Recently, there have been concerns about a flu virus that affects birds, and often kills them. At present the virus does not pass easily from birds to humans, and it does not seem to pass from one human to another. However, the fear is that this virus might change and that human-to-human infection could then be possible. Should all this happen, the changed virus would be a major threat to human health. With current technology, it would take several months to produce enough vaccine for even a small proportion of the world's population. By that time, it would probably be too late; the virus would already have spread to most parts of the world. It is therefore important for health authorities to consider all the methods that might control the spread of the virus. With the increase in international travel that has taken place, the virus could spread more quickly than in previous worldwide pandemics. Restrictions on international travel might, therefore, be considered necessary, particularly travel by air.\n\n### Why Was This Study Done?\n\nIt is important to estimate how useful restrictions on air travel might be in controlling the spread of a flu virus. Travel restrictions are usually unpopular and could themselves be harmful, and, if they are not effective, resources could be wasted on enforcing them.\n\n### What Did the Researchers Do and Find?\n\nThis research involved mathematical modelling. In other words, complex calculations were done using information that is already available about how flu viruses spread, particularly information recorded during a worldwide flu outbreak in 1968\u20131969. Using this information, virtual experiments were carried out by simulating worldwide outbreaks on a computer. The researchers looked at how the virus might spread from one city to another and how travel restrictions might reduce the rate of spread. Their calculations allowed for such factors as the time of the year, the number of air passengers who might travel between the cities, and the fact that some people are more resistant to infection than others. From the use of their mathematical model, the researchers concluded that restrictions on air travel would achieve very little. This is probably because, compared with some other viruses, the flu virus is transmitted from one person to another very quickly and affects many people. Once a major outbreak was under way, banning flights from affected cities would be effective at significantly delaying worldwide spread only if almost all travel between cities could be stopped almost as soon as an outbreak was detected in each city. It would be more effective to take other measures that would control the spread of the virus locally. These measures could include use of vaccines and antiviral drugs if they were available and effective against the virus.\n\n### Additional Information\n\nPlease access these Web sites via the online version of this summary at <http:\/\/dx.doi.org\/10.1371\/journal.pmed.0030212>.\n\n\u2022\u2009 Fact sheets are available about various aspects of flu from the Web site of the World Health Organization, which takes a global overview of the impact of the infection\n\nMany health Web sites aimed at patients provide basic information about flu.\n\n\u2022\u2009US National Institute of Allergy and Infectious Diseases page about flu\n\n\u2022\u2009National Institute of Allergy and Infectious Diseases fact sheet about cold and flu symptoms\n\n\u2022\u2009US Centers for Disease Control and Prevention page about flu\n\n\u2022\u2009The Journal of the American Medical Association's patient page about influenza\n\n\u2022\u2009Page on flu from BBC Health\n\n\u2022\u2009Information about pandemic influenza from The Health Protection Agency\n\nWe thank Emilia Vynnycky and Maria Zambon (Health Protection Agency) for helpful discussions; the International Air Transport Association for supplying data; and Professor Neil Ferguson (Imperial College) for helpful suggestions that led to significant improvements in the manuscript.\n\n**Author contributions.** BSC, WJE, and NJG conceived the study. RJP obtained and processed the travel data. BSC implemented and analysed the model. All authors contributed to writing the manuscript.\n\n# References\n\n## Abbreviations\n\nIQR\n\n:   interquartile range\n\nSARS\n\n:   severe acute respiratory syndrome\n\nSSQ\n\n:   sum of squared deviations\n\n[^1]: **Competing Interests:** The authors have declared that no competing interests exist.","meta":{"dup_signals":{"dup_doc_count":136,"dup_dump_count":37,"dup_details":{"curated_sources":2,"2022-49":1,"2022-27":1,"2022-21":2,"2021-10":1,"2020-50":1,"2020-24":1,"2019-26":1,"2019-22":1,"2018-47":1,"2018-34":1,"2017-09":10,"2017-04":1,"2016-36":2,"2016-30":7,"2016-26":1,"2016-18":1,"2016-07":4,"2015-48":3,"2015-40":4,"2015-35":3,"2015-32":5,"2015-27":5,"2015-22":2,"2015-14":5,"2014-52":7,"2014-49":3,"2014-42":8,"2014-41":10,"2014-35":8,"2014-23":10,"2014-15":8,"2023-40":1,"2024-10":1,"2015-18":6,"2015-11":3,"2014-10":4,"2024-18":1}},"file":"PMC1450020"},"subset":"pubmed_central"}
{"text":"abstract: Impaired physical performance is a common but not obligate result of a low carbohydrate diet. Lessons from traditional Inuit culture indicate that time for adaptation, optimized sodium and potassium nutriture, and constraint of protein to 15\u201325 % of daily energy expenditure allow unimpaired endurance performance despite nutritional ketosis.\nauthor: Stephen D Phinney\ndate: 2004\ninstitute: 16108 Boothbay Court, Elk Grove, CA 95758, United States of America\nreferences:\ntitle: Ketogenic diets and physical performance\n\n# Introduction\n\nIn the opinion of most physicians and nutrition scientists, carbohydrate must constitute a major component of one's daily energy intake if optimum physical performance is to be maintained \\[1\\]. This consensus view is based upon a long list of published studies performed over the last century that links muscle glycogen stores to high intensity exercise. It has also been reinforced by the clinical experience of many physicians, whose patients following low carbohydrate formula or food diets frequently complain of lightheadedness, weakness, and ease of fatigue.\n\nDuring the time that this consensus view of the necessity of carbohydrate for vigorous exercise was forming, the last pure hunting cultures among the peoples of North America finally lost out in competition with expanding European cultural influences. Between 1850 and 1930, the routine consumption of carbohydrates spread north from the U.S. Plains States through central Canada, where the indigenous peoples had heretofore made at most seasonal use of this nutrient class. However the last of these groups to practice their traditional diet, the Inuit people of the Canadian and Alaskan Arctic regions, were luckily observed by modern scientists before their traditional dietary practices were substantially altered. The reports of these early scientists imply that the Inuit people were physically unhampered despite consuming a diet that was essentially free of identifiable carbohydrate.\n\nGiven this juxtaposition of clinical research results favoring carbohydrate against observed functional well-being in traditional cultures consuming none, it is an interesting challenge to understand how these opposing perspectives can be explained. This paper will review the observations of early explorer scientists among the Inuit, track the controversy that they stimulated among nutritionists in the last century, and utilize some of the forgotten lessons from the Inuit culture to explain how well-being and physical performance can be maintained in the absence of significant dietary carbohydrate.\n\n# The origins of carbohydrate supremacy\n\nUntil the development of agriculture over last few millennia, our human ancestors' consumption of dietary carbohydrate was opportunistic. As some groups adapted to hunting and fishing for their sustenance, they were able to move into temperate and then arctic regions, where limited access to wild grain, nuts, and fruit dictated sustained dependence upon fat and protein as primary sources of dietary energy.\n\nWith the development of agriculture came the ability to grow and store grain, allowing societies to remain in a stable physical location, build permanent dwellings, and potentially stimulating the development of written language (those early stone tablets would have been difficult to transport from camp to camp on a dog sled). Starting from locations in the Middle East and Asia, cultures based upon agricultural wheat and rice spread over 5 millennia to dominate Europe, Africa, and the Americas.\n\nWith its ability to support a non-nomadic life style, greater population density, and permanent communities; there were clear advantages of agriculture-based societies over those based upon hunting and fishing, particularly as agricultural communities built the infrastructure to support trade and transport. Given its success in this competition of cultures (and by implication, the competition of their diets), it is an easy assumption that a grain-based diet is functionally superior to one based upon the meat and fish (fat and protein) of the hunting societies that they superseded.\n\nAs the science of nutrition developed in the early 20^th^ Century, numerous comparative studies were undertaken to assess differences between diets. Although there were some advocates of low carbohydrate diets (eg, the Banting diet of the 19^th^ Century, promoted for weight loss and diabetes control), the prevailing premise for these studies was that carbohydrate was a necessary nutrient for optimum human health and function. Among studies confirming this view, a classic was the 1939 study by two Danish scientists, Christensen and Hansen \\[2\\]. They did a crossover study of low carbohydrate, moderate carbohydrate, and high carbohydrate diets, each lasting one week. At the end of each diet, the subjects' endurance time to exhaustion on a stationary bicycle was assessed. Compared to the mean endurance time on the low carb diet of 81 minutes, the subjects were able to ride for 206 minutes after the high carb diet.\n\nDuring the Second World War, another oft-cited study was performed, this time examining the practicality of pemmican (a mixture of dried meat and fat) as a light-weight emergency ration for soldiers. This experiment by Kark et al \\[3\\] involved abruptly switching soldiers in winter training in the Canadian Arctic from standard carbohydrate-containing rations to pemmican. This study only lasted 3 days, as the soldiers rapidly became unable to complete their assigned tasks, which included pulling loaded sleds 25-miles per day through deep snow.\n\nWith the resurgence of biomedical science in the 1960's came development of the percutaneous needle biopsy, facilitating assessment of intra-muscular fuel stores and metabolism. This led to the concept of muscle glycogen as the limiting fuel for high intensity exercise \\[4\\] and to the nutritional strategy of carbohydrate loading \\[5\\]. The clear consensus that developed from this research was that fat had limited utility as a fuel for vigorous exercise, and that humans are physically impaired if given a low carbohydrate diet.\n\n# The hunter's counterpoint \u2013 practical observations on ketogenic diets\n\nAlthough high-carbohydrate diets might be more effective in short-term tests of high-intensity exercise, there are multiple clues in the published literature that the debilitating effects of ketogenic diets are overstated. Not only is there the demographic evidence that whole populations of people lived for millennia as hunters, but there are many reports of Europeans crossing over to live within the cultures of these hunting societies without apparent impediment.\n\nOne of the earliest documented demonstrations of physical stamina during a ketogenic diet was the Schwatka 1878\u201380 expedition in search of the lost Royal Navy Franklin expedition. The Schwatka expedition, sponsored by the *New York Herald* and the American Geographical Society, departed from the west coast of Hudson's Bay in April of 1879 with 4 Caucasians, 3 families of Inuits, and 3 heavily laden dog sleds. Totaling 18 people, they started out with a month's supply of food (mostly walrus blubber) and a prodigious supply of ammunition for their hunting rifles. After covering over 3000 miles on foot over ice, snow and tundra, all 18 members of the original party plus their 44 dogs returned to Hudson's Bay in March of 1880. Once their initial provisions were depleted, the expedition's only source of additional food was hunting and fishing, as there were no other sources of supply along their route.\n\nThe leader of this expedition, Lt. Frederick Schwatka, was a graduate of both West Point and Bellevue Hospital Medical College. His summary of the expedition was published as a news article in the *New York Herald* in the Fall of 1880, but his written diary was lost for 85 years until its discovery and publication by the Marine Historical Association of Mystic CT in 1965 \\[6\\]. This fascinating 117-page saga describes how Schwatka, a frontiersman and U.S. Army surgeon, collaborated with his Inuit guides to accomplish a remarkable feat of physical endurance.\n\nIn one notation, Schwatka provides an interesting insight into his weaning from their initial supply of carbohydrate-containing food.\n\n\"*When first thrown wholly upon a diet of reindeer meat, it seems inadequate to properly nourish the system, and there is an apparent weakness and inability to perform severe exertive fatiguing journeys. But this soon passes away in the course of two or three weeks.\"*\n\nThis observation, written a century before the current author first came to grips with the issue of \"keto-adaptation\", offers an early clue to resolve the dichotomy between impaired performance with low carbohydrate diets in the laboratory and their lack of debilitating effects when taken among people practiced in their use. That Schwatka was not impaired by his prolonged experience eating meat and fat is evidenced by his diary entry for the period 12\u201314 March 1880, during which he and an Inuit companion walked the last 65 miles in less than 48 hours to make a scheduled rendezvous with a whaling ship and complete his journey home.\n\nTwenty-six years later, a Harvard-trained anthropologist named Vilhjalmur Stefansson entered the Arctic with the purpose of studying the Inuit language and culture. Having been born in 1879 in Manitoba and grown up in North Dakota, it is unlikely that Stefansson was aware of the Schwatka expedition or its reported technique of extended dogsled travel while living by hunting. However when separated from his expedition and thus his source of supply over the winter of 1906\u20137, Stefansson was taken in by a group of Inuit on the Canadian Arctic coast. With the arrival of spring in June of 1907, he both spoke their language and had acquired their skill of living and traveling by dogsled on a hunter's diet.\n\nFor the next decade, Stefansson traveled extensively over the arctic mainland and among the islands to the north. During this period, he was away from the outposts of European settlement for periods of up to 18 months at a time, and in the remote regions of the Canadian Arctic he lived with groups of Inuit for whom he was the first European they had met.\n\nStefansson wrote extensively about these experiences in both the scientific literature and in books for the lay public \\[7\\]. One of the main themes of his writing was the adaptation of the Inuit culture to survive as nomadic groups in the arctic on a diet consisting solely of the products of hunting and fishing. Coming as it did in the same time period that the science of nutrition was blossoming with the discovery and characterization of vitamins (eg, the first vitamin to be chemically defined was thiamin by Funk in 1911), Stefansson's claim that one could live and function well on the products of just one food group caused tremendous controversy \\[8\\].\n\nSubjected to great criticism and even scorn, Stefansson agreed to recreate the Inuit diet under scientific observation. Therefore, for the calendar year of 1929 he and a colleague from his arctic explorations ate a diet consisting of meat and fat for 12 months. This experiment, supervised by Dr. Eugene DuBois, was conducted at Bellevue Hospital in New York. For the first 3 months of this study, the two explorers were under constant observation to guarantee dietary compliance, after which they were allowed more freedom of movement but with frequent tests to document that they remained in ketosis. This study was reported in multiple peer-reviewed publications, the primary reports being published in the Journal of Biological Chemistry in 1930 \\[9,10\\], As noted by DuBois \\[8\\], the study results were essentially \"negative\", in that both subjects survived the 12 months in apparent good health, having no signs of scurvy (which was predicted to occur within the first 3 months) or other deficiency diseases.\n\nIt is interesting to note from the careful observations published from the Bellevue study that Stafansson ate relatively modestly of protein, deriving between 80\u201385% of his dietary energy from fat and only 15\u201320% from protein \\[9\\]. This was, and still remains, at odds with the popular conception that the Inuit ate a high protein diet, whereas in reality it appears to have been a high fat diet with a moderate intake of protein. In his writings, Stefansson notes that the Inuit were careful to limit their intake of lean meat, giving excess lean meat to their dogs and reserving the higher fat portions for human consumption \\[11\\].\n\nIt is also interesting to conjecture that the vigorous defense of his arctic observations by Stefansson may have led indirectly to the development of the carbohydrate loading hypothesis. Stefansson was a polarizing influence in the field of nutrition, and his advocacy of pemmican as an emergency ration for troops during the Second World War led directly to the Kark study quoted above, which in turn was a predecessor to many comparative dietary trials performed in Europe and the U.S. in later decades.\n\n# Modern ketogenic diet performance studies\n\nThere was a resurgence of interest in very low calorie ketogenic diets for weight loss in the 1970's, followed closely by the complications (including sudden death) associated with the Liquid Protein diet popularized in 1976. However, the fatigue and apparent cardiac dysfunction caused by this collagen-based fad diet stood in stark contrast to the published experience of arctic explorers such as Schwatka and Stefansson. In addition, physicians who monitored patients following very low calorie diets observed wide variations between the exercise-tolerance of these individuals.\n\nGiven that the elegant research on the metabolism of total fasting by Dr. George Cahill and colleagues had demonstrated that full adaptation of nitrogen, fat, and carbohydrate metabolism required a number of weeks \\[12\\], it seemed reasonable to hypothesize that exercise tolerance would take more than a week to recover after removal of carbohydrate from the diet. This view was supported by the subsequent discovery of the prescient adaptation quote from Schwatka's diary \\[6\\] noted above.\n\nTo test this hypothesis, the current author (under the mentorship of Drs. Ethan Sims and Edward Horton at the University of Vermont) undertook a study of subjects given a very low calorie ketogenic diet for 6 weeks in a metabolic research ward \\[13\\]. The protein for this diet, along with a modicum of inherent fat, was provided by lean meat, fish, and poultry providing 1.2 grams of protein per kg of reference (\"ideal\") body weight daily. In addition, mindful that the natriuresis of fasting could reduce circulating blood volume and cause secondary renal potassium wasting, the subjects were prescribed 3 grams of supplemental sodium as bouillion and 25 mEq (1 g) of potassium as bicarbonate daily.\n\nTreadmill performance testing of these subjects included determinations of peak aerobic power (VO~2~max) after a 2-week weight maintenance baseline diet, and again after 6 weeks of the ketogenic weight loss diet. Endurance time to exhaustion was quantitated at 75% of the baseline VO~2~max. This endurance test was repeated again after one week of weight loss and finally after 6 weeks of weight loss. Other than these tests, the subjects did no training exercise during their participation in this study. To compensate for the fact that the average subject had lost over10 kg, the final endurance treadmill test was performed with the subject carrying a backpack equivalent in weight to the amount lost.\n\nThe energy expenditure data (expressed as oxygen consumption) and exercise times across this 8-week inpatient study are shown in Table <a href=\"#T1\" data-ref-type=\"table\">1<\/a>. That these subjects'peak aerobic power did not decline despite 6 weeks of a carbohydrate-free, severely hypocaloric diet implies that the protein and mineral contents of the diet were adequate to preserve functional tissue. As can be noted, endurance time to exhaustion was reduced after one week of the ketogenic diet, but it was significantly increased over the baseline value by the 6-week time point. However the interpretation of this endurance test is confounded by the fact that the oxygen cost (ie, energy cost) of the treadmill exercise had significantly decreased following the weight loss, and this occurred despite the subjects being made to carry a backpack loaded to bring them back to their initial exercise test weight.\n\nExercise parameters of Vermont study \\[13\\]\n\n|                          | **Baseline** | **Week 1** | **Week 6** |\n|--------------------------|--------------|------------|------------|\n| **VO~2~max** (LPM)       | 2.49         | --         | 2.49       |\n| **Exercise VO~2~**(LPM)  | 1.88\\*       | 1.71       | 1.50\\*     |\n| **Endurance time** (min) | 168^+^       | 130        | 249^+^     |\n\nLPM, liter per minute \\*week 6 \\< baseline, P \\< 0.05 ^+^week 6 \\> baseline, P \\< 0.01\n\nThis question of improved efficiency notwithstanding, it is clear that our subjects experienced a delayed adaptation to the ketogenic diet, having reduced endurance performance after one week followed by a recovery to or above baseline in the period between one and six weeks. Given the reduced energy cost of the exercise despite the backpack, the extent of this adaptation cannot be determined from this study. To explain this improved exercise efficiency, we can speculate that humans are more efficient carrying weight in a modern backpack than under their skin as excess body fat. It is also possible that these untrained subjects became more comfortable with prolonged treadmill walking by their third test, and therefore improving their overall efficiency.\n\nGiven the uncertainties of this study caused by the subject's weight loss and potential for improved technique with multiple tests, the current author undertook a second study under the mentorship of Dr. Bruce Bistrian at MIT in Cambridge MA \\[14,15\\]. The diet employed in this followup study was patterned after that consumed by Stefansson during his year in the Bellevue study (and thus presumably close to that traditionally consumed by the Inuit) with the intention that the subjects would be in ketosis without weight loss.\n\nThis second study utilized competitive bicycle racers as subjects, confined to a metabolic ward for 5 weeks. In the first week, subjects ate a weight maintenance (eucaloric) diet providing 67% of non-protein energy as carbohydrate, during which time baseline performance studies were performed. This was followed by 4 weeks of a eucaloric ketogenic diet (EKD) providing 83% of energy as fat, 15% as protein, and less than 3% as carbohydrate. The meat, fish, and poultry that provided this diets protein, also provided 1.5 g\/d of potassium and was prepared to contain 2 g\/d of sodium. These inherent minerals were supplemented daily with an additional 1 g of potassium as bicarbonate, 3 grams of sodium as bouillon, 600 mg of calcium, 300 mg of magnesium, and a standard multivitamin.\n\nThe bicyclist subjects of this study noted a modest decline in their energy level while on training rides during the first week of the Inuit diet, after which subjective performance was reasonably restored except for their sprint capability, which remained constrained during the period of carbohydrate restriction. On average, subjects lost 0.7 kg in the first week of the EKD, after which their weight remained stable. Total body potassium (by ^40^K counting) revealed a 2% reduction in the first 2 weeks (commensurate with the muscle glycogen depletion documented by biopsy), after which it remained stable in the 4^th^ week of the EKD. These results are consistent with the observed reduction in body glycogen stores but otherwise excellent preservation of lean body mass during the EKD.\n\nThe results of physical performance testing are presented in Table <a href=\"#T2\" data-ref-type=\"table\">2<\/a>. What is remarkable about these data is the lack of change in aerobic performance parameters across the 4-week adaptation period of the EKD. The endurance exercise test on the cycle ergometer was performed at 65% of VO~2~max, which translates in these highly trained athletes into a rate of energy expenditure of 960 kcal\/hr. At this high level of energy expenditure, it is notable that the second test was performed at a mean respiratory quotient of 0.72, indicating that virtually all of the substrate for this high energy output was coming from fat. This is consistent with measures before and after exercise of muscle glycogen and blood glucose oxidation (data not shown), which revealed marked reductions in the use of these carbohydrate-derived substrates after adaptation to the EKD.\n\nExercise parameters of MIT EKD study \\[15\\]\n\n|  | **VO~2~max** (LPM) | **Exercise VO~2~**(LPM) | **Exercise RQ** | **Endurance time** (min) |\n|----|----|----|----|----|\n| **Baseline** | 5.1 | 3.18 | 0.83\\* | 147 |\n| **EKD-4** | 5.0 | 3.21 | 0.72\\* | 151 |\n\nLPM, liter per minute \\* P \\< 0.01\n\nExamining the results of these two ketogenic diet performance studies together indicates that both groups experienced a lag in performance across the first week or two of carbohydrate restriction, after which both peak aerobic power and sub-maximal (60\u201370% of VO~2~max) endurance performance were fully restored. In both studies, one with untrained subjects and the other with highly trained athletes who maintained their training throughout the study, there was no loss of VO~2~max despite the virtual absence of dietary carbohydrate for 4\u20136 weeks. This whole-body measure of oxidative metabolism could not be maintained unless there was excellent preservation of the full complement of functional tissues including skeletal muscle (and mitochondrial) mass, circulating red cell mass, and cardiopulmonary functions.\n\nThe possibility raised by the first study of improved endurance time after keto-adaptation was not substantiated by the second study employing highly trained athletes without the complicating variable of major weight loss. It is thus likely that the increased endurance time in the Vermont study was due to improved efficiency (ie, less hobbling from a backpack than from an equal weight of internal body fat) and\/or improved acclimation to the endurance test procedure. Such acclimation would not be expected in the second study, as the highly trained bicycle racers were well conditioned to the stationary ergometer at the start of the study. It is also worth noting that the bicycle racers remained weight stable (excepting the half kilogram of reduced muscle glycogen) across the 4 weeks of the EKD, which was equi-caloric with the baseline diet. Although 4 weeks is a relatively short period to assess small differences in energy efficiency between diets, this observation implies that there was no great reduction in the efficiency of energy metabolism after keto-adaptation.\n\nAs a final note in this section, neither the Vermont study nor the MIT study has been refuted in the 2 decades since their publication. Understandably given the expense of human metabolic ward studies and the orthogonal conclusions of these two studies, neither study has been corroborated by a similar human study. However two subsequent animal studies examining physical performance after keto-adaptation have yielded results consistent with those presented above \\[16,17\\].\n\n# Resolving the performance paradox\n\nThere are three factors that can help us explain the paradox presented by studies showing superior performance with high carbohydrate diets versus the present author's two studies noted above.\n\n## Adaptation\n\nThe most obvious of these is the time allotted (or not) for keto-adaptation. In this context, the prescient observation of Schwatka (that adaptation to \"a diet of reindeer meat\" takes 2\u20133 weeks) says it all. None of the comparative low-carbohydrate versus high-carbohydrate studies done in support of the carbohydrate loading hypothesis sustained the low carbohydrate diet for more than 2 weeks \\[5\\], and most (including the classic report of Christensen and Hansen \\[2\\]) maintained their low-carbohydrate diets for 7 days or less.\n\nThere are to date no studies that carefully examine the optimum length of this keto-adapataion period, but it is clearly longer than one week and likely well advanced within 3\u20134 weeks. The process does not appear to happen any faster in highly trained athletes than in overweight or untrained individuals. This adaptation process also appears to require consistent adherence to carbohydrate restriction, as people who intermittently consume carbohydrates while attempting a ketogenic diet report subjectively reduced exercise tolerance.\n\n## Sodium and potassium\n\nThe second factor differentiating the author's studies from many others is optimized mineral nutriture, which has benefits for both cardiovascular reserve in the short term and preservation of lean body mass and function over longer time periods. The Inuit people lived much of the year on coastal ice (which is partially desalinated sea water), and much of their food consisted of soup made with meat in a broth from this brackish source of water. When they went inland to hunt, they traditionally added caribou blood (also a rich source of sodium) to their soup. With these empirically derived techniques, the Inuit culture had adapted the available resources to optimize their intakes of both sodium and potassium.\n\nWhen meat is baked, roasted, or broiled; or when it is boiled but the broth discarded, potassium initially present in the meat is lost, making it more difficult to maintain potassium balance in the absence of fruits and vegetables. Because our research subjects were accustomed to eating meat, fish, and poultry prepared as something other than soup, we chose to give them most of their sodium separately as bouillon and a modest additional supplement of potassium as potassium bicarbonate. With these supplements maintaining daily intakes for sodium at 3\u20135 g\/d and total potassium at 2\u20133 g\/d, our adult subjects were able to effectively maintain their circulatory reserve (ie, allowing vasodilatation during submaximal exercise) and effective nitrogen balance with functional tissue preservation.\n\nAn example of what happens when these mineral considerations are not heeded can be found in a study prominently published in 1980 \\[18\\]. This was a study designed to evaluate the relative value of \"protein only\" versus \"protein plus carbohydrate\" in the preservation of lean tissue during a weight loss diet. The protein only diet consisted solely of boiled turkey (taken without the broth), whereas the protein plus carbohydrate consisted of an equal number of calories provided as turkey plus grape juice. Monitored for 4 weeks in a metabolic ward, the subjects taking the protein plus carbohydrate did fairly well at maintaining lean body mass (measured by nitrogen balance), whereas those taking the protein only experienced a progressive loss of body nitrogen.\n\nA clue to what was happening in this \"Turkey Study\" could be found in the potassium balance data provided in this report. Normally, nitrogen and potassium gains or losses are closely correlated, as they both are contained in lean tissue. Interestingly, the authors noted that the protein only diet subjects were losing nitrogen but <u>gaining<\/u> potassium. As noted in a rebuttal letter published soon after this report \\[19\\], this anomaly occurred because the authors assumed the potassium intake of their subjects based upon handbook values for raw turkey, not recognizing that half of this potassium was being discarded in the unconsumed broth. Deprived of this potassium (and also limited in their salt intake), these subjects were unable to benefit from the dietary protein provided and lost lean tissue. Also worthy of note, although this study was effectively refuted by a well-designed metabolic ward study published 3 years later \\[20\\], this \"Turkey Study\" continues to be quoted as an example of the limitations of low carbohydrate weight loss diets.\n\n## Protein dose\n\nThe third dietary factor potentially affecting physical performance is adjusting protein intake to bring it within the optimum therapeutic window for human metabolism. The studies noted herein \\[13-15,20\\] demonstrate effective preservation of lean body mass and physical performance when protein is in the range of 1.2 \u2013 1.7 g\/kg reference body weight daily, provided in the context of adequate minerals. Picking the mid-range value of 1.5 g\/kg-d, for adults with reference weights ranging from 60\u201380 kg, this translates into total daily protein intakes 90 to 120 g\/d. This number is also consistent with the protein intake reported in the Bellevue study \\[9\\]. When expressed in the context of total daily energy expenditures of 2000\u20133000 kcal\/d, about 15% of ones daily energy expenditure (or intake if the diet is eucaloric) needs to be provided as protein.\n\nThe effects of reducing daily protein intake to below 1.2 g\/kg reference weight during a ketogenic diet include progressive loss of functional lean tissue and thus loss of physical performance, as demonstrated by Davis et al \\[21\\]. In this study, subjects given protein at 1.1 g\/kg-d experienced a significant reduction in VO~2~max over a 3 month period on a ketogenic diet, whereas subjects given 1.5 g\/kg-d maintained VO~2~max.\n\nAt the other end of the spectrum, higher protein intakes have the potential for negative side-effects if intake of this nutrient exceeds 25% of daily energy expenditure. One concern with higher levels of protein intake is the suppression of ketogenesis relative to an equi-caloric amount of fat (assuming that ketones are a beneficial adaptation to whole body fuel homeostasis). In addition, Stefansson describes a malady known by the Inuit as rabbit malaise \\[8\\]. This problem would occur in the early spring when very lean rabbits were the only available game, when people might be tempted to eat too much protein in the absence of an alternative source of dietary fat. The symptoms were reported to occur within a week, and included headache and lassitude. Such symptoms are not uncommon among people who casually undertake a \"low carbohydrate, high protein\" diet.\n\n# Conclusions\n\nBoth observational and prospectively designed studies support the conclusion that submaximal endurance performance can be sustained despite the virtual exclusion of carbohydrate from the human diet. Clearly this result does not automatically follow the casual implementation of dietary carbohydrate restriction, however, as careful attention to time for keto-adaptation, mineral nutriture, and constraint of the daily protein dose is required. Contradictory results in the scientific literature can be explained by the lack of attention to these lessons learned (and for the most part now forgotten) by the cultures that traditionally lived by hunting. Therapeutic use of ketogenic diets should not require constraint of most forms of physical labor or recreational activity, with the one caveat that anaerobic (ie, weight lifting or sprint) performance is limited by the low muscle glycogen levels induced by a ketogenic diet, and this would strongly discourage its use under most conditions of competitive athletics.\n\n# List of abbreviations\n\nVO~2~max \u2013 maximum aerobic capacity\n\nRQ \u2013 respiratory quotient\n\nEKD \u2013 eucaloric ketogenic diet\n\n# Competing interests\n\nNone declared.","meta":{"dup_signals":{"dup_doc_count":135,"dup_dump_count":69,"dup_details":{"curated_sources":8,"2023-40":2,"2023-23":1,"2023-14":1,"2022-49":2,"2022-33":1,"2022-27":1,"2022-05":1,"2021-39":2,"2021-25":2,"2021-17":1,"2021-04":1,"2020-50":1,"2020-40":1,"2020-29":1,"2020-16":1,"2020-10":2,"2020-05":1,"2019-51":1,"2019-47":1,"2019-43":1,"2019-35":1,"2019-13":1,"2018-47":1,"2018-39":1,"2018-30":1,"2018-22":1,"2018-05":1,"2017-47":1,"2017-43":1,"2017-34":1,"2017-30":1,"2017-26":1,"2017-22":1,"2017-17":3,"2017-09":3,"2017-04":2,"2016-50":2,"2016-44":3,"2016-40":2,"2016-36":3,"2016-30":3,"2016-26":1,"2016-22":1,"2016-18":1,"2016-07":1,"2015-48":3,"2015-40":2,"2015-35":2,"2015-32":2,"2015-27":2,"2015-22":3,"2015-14":2,"2014-52":2,"2014-49":4,"2014-42":6,"2014-41":4,"2014-35":3,"2014-23":4,"2014-15":4,"2024-30":2,"2024-26":1,"2024-10":2,"2017-13":4,"2015-18":2,"2015-11":2,"2015-06":2,"2014-10":2,"2013-48":1,"2013-20":3}},"file":"PMC524027"},"subset":"pubmed_central"}
{"text":"abstract: # Background\n  .\n  The South African health care delivery system has shifted focus to primary health care since 1994. For this purpose the Batho Pele principles were introduced. Nurses claim, however, that since the introduction of these principles patients and their families have been making unnecessary and sometimes impossible demands of nursing staff. This article presents the perceptions of the professional nurses regarding the introduction of the Batho Pele principles in their workplace.\n  .\n  # Objectives\n  .\n  To describe the perceptions of professional nurses regarding introduction of the Batho Pele principles and to recommend guidelines to facilitate measures to realise the objects of these principles.\n  .\n  # Method\n  .\n  A qualitative, exploratory, descriptive and contexual research design was used. Six audio-taped focus group discussions and field notes were used to collect data from purposively sampled participants who have worked in the outpatient departments of hospitals in the Port Elizabeth Hospital Complex. Guba's model of trustworthiness was used to confirm integrity of the study, whilst the participants were kept anonymous, protected from harm and participated voluntarily. Data analysis was done using Tesch's data analysis spiral and with the involvement of an independent-coder.\n  .\n  # Results\n  .\n  Three themes emerged, revealing that the professional nurses perceived the objectives of the Batho Pele principles as difficult to uphold due to the inadequate planning prior to their implementation. Inadequacy of human and material resources aggravated this perception.\n  .\n  # Conclusion\n  .\n  Professional nurses are not happy with how things are in terms of introduction of the Batho Pele principles, but are optimistic of a positive change in the near future.\nauthor: Sindiwe James; Thenjiwe M. Miza**Correspondence to:** Sindiwe James **Email:**<email@example.com>, **Postal address:** Department of Nursing Science, Nelson Mandela Metropolitan University, 2nd Floor, R-Block, North Campus, Port Elizabeth 6031, South Africa[^1]\ndate: 2015-03-09\ninstitute: 1Department of Nursing Science, North Campus, Nelson Mandela Metropolitan University, Port Elizabeth, South Africa; 2Livingstone Hospital, Port Elizabeth, South Africa\nreferences:\ntitle: Perceptions of professional nurses regarding introduction of the Batho Pele principles in State hospitals\n\n# Introduction and background\n\nThe quality of care rendered to patients has always been the main focus of health care, and yet this still remains a challenge to State hospitals in South Africa (SA). Quality of care refers to the attributes or characteristics of excellence, with an emphasis on the principles of applicability and acceptability (Muller 2009). If these principles are adhered to, quality healthcare delivery can be achieved. Patients associate 'quality of care' with the quality of care that they receive as well as the amount of empathy shown towards them by nursing staff (Booyens 2008).\n\nIn recent years the rendering of quality care in South African State hospitals has been compounded by the resignations of senior and experienced nurses in all sectors, including outpatient departments of different hospital units. This brain and skill 'drain' happened at the same time as inception of primary healthcareopportunities for all in SA. Primary health care was seen as the key element in the Government's plan to transform the health services in SA and to bridge healthcare service delivery gaps where possible (Muller 2009). In order to accomplish this a White Paper on the Batho Pele principles was adopted and published in 1997 as a framework for provision of service delivery by the Department of Health (DoH) (DoH 2001) The core value of these principles, as stated in the English translation, is that people are made a priority. The aim is to ensure that healthcare providers are sensitive to patients' healthcare needs. These principles, which guide healthcare providers to observe standards of consultation, service standards, access, courtesy, information, openness and transparency, redress and value for money (Muller 2009) are discussed next.\n\n## The Batho Pele principles\n\nThese prinicples are almost synonymous and therefore difficult to view separately. For the purposes of optimal discussion they will therefore be grouped accordingly.\n\n### Standards of consultation, service, access and courtesy\n\nThe above principles insist that citizens should be *consulted* regarding the level and quality of the public service they receive. Should the need arise, they should be further referred to gain *access* to the correct healthcare facility. Here the emphasis is that patients know how healthcare services are classified to ensure that they receive prompt care. As those who have easy access to this information, healthcare workers (for the purposes of this article, professional nurses) should frequently inform the patients in this regard, be tolerant to their inquiries and be respectful of patients as individuals. Such actions will enhance the realisation of effective access to healthcare services.\n\nThe principle of access in terms of the Batho Pele principles means that patients should have equal opportunities in terms of resources and facilities (DoH1997, in Muller 2009). In this regard the DoH has vowed to promote and improve access to healthcare facilities through provision of comprehensive healthcare services within a 5 km radius of catchment areas at all times (DoH 2001). However, access may go beyond the designated catchment areas, and hence the need at times for a further referral.\n\n### Information, openness and transparency, redress and value for money\n\nBesides consultation, access and courtesy, the Batho Pele principles also make reference to the provision of fully accurate information to citizens about the public health services to which they are entitled. The public have faith that the professional nurse will give them accurate information regarding health issues; therefore the nurse has the responsibility to ensure that (s)he is aware of all changes and new developments in the healthcare sector. Whilst sharing information with patients, healthcare workers (professional nurses) should observe the culture of openness and transparency. Furthermore, the Batho Pele principles determine that public services should be provided economically and efficiently in order to provide citizens with the best possible service for the value of their money.\n\nHaving mentioned the various principles that comprise the notion of Batho Pele, one must also remember that these are informed by three sets of beliefs: care, teamwork and service.\n\n## Care, teamwork and service\n\nCaring for patients in outpatient departments cannot be done in isolation of other healthcare providers and relevant stakeholders. Teamwork and support is of even more importance in an outpatient department for the sake of interdependence and good-quality service. Covey (1989) states that interdependence opens up worlds of possibilities for deep, rich, meaningful associations and increased productivity. As mentioned earlier, some patients will access the healthcare service as referrals from other service centres or will need to be referred to other services. The procedure relating to referral of patients requires consultation of expert knowledge as well as coordination of all processes involved, hence the need for efficient teamwork.\n\nThe concept of teamwork becomes more appreciated in health care as it brings with it the experience of interdependence, complementary skills, information sharing and making decisions together towards the set goals of the unit (Muller, Bezuidenhout & Jooste 2011:334). In most hospitals a multidisciplinary team is formed whereby all the health care disciplines meet to discuss the care and referral needs of their patients. Booyens (2008) states that the aim of this teamwork is to determine priorities and clarify roles and responsibilities which, in the context of this article, are significant in promoting quality healthcare service.\n\nIn some instances, as is the case in the hospital where the co-author of this article works, the same outpatient department's patients are referred from clinic to clinic to satisfy their healthcare needs. This action may be confusing and annoying to some patients. Explaining the process of referral in a caring manner to patients is therefore a crucial role of professional nurses in identifying patient concerns, problems related to referral systems, possible conflict situations and improvement of communication between team members and community.\n\nNurses as messengers of either bad or good healthcare-related information are sometimes challenged with being non-caring, especially by patients who do not want to hear the bad news or results related to their health (Huber 2014:120). Patients usually become confused when they do not understand medical instructions, and thus come into conflict with the healthcare person (Huber 2014:120), as in the case of the referral system brought about by introduction of the Batho Pele principles. Doctors and professional nurses should be mindful of the consequences of miscommunication and always try to be as clear in their instructions as possible. Muller (2009) asserts that caring is integral to the ethical and philosophical foundation of nursing. Nurses join the nursing profession with a vision to serve society, this vision being driven by the values of passion and caring which could be enhanced through practising the Batho Pele principles.\n\nSearle (2006) states that when nurses are admitted to the professional register they declare publicly that they are competent and willing to meet the standards of nursing required from them, and that they will dedicate themselves to serving people through meeting their nursing needs. The eight Batho Pele principles and belief sets can easily be applied within patient nursing care by professional nurses working in outpatient departments, as these are also entrenched in the message of caring as translated through the Pledge of Service. Fundamental to adequately serving the patient are the principles of responsibility and accountability, as well as a willingness to go beyond the call of duty, for which the Batho Pele principles chiefly stand. Professional nurses are held responsible for their actions in their day-to-day activities, and despite the conditions under which they serve, their duties include being held accountable for caring for their patients.\n\n## Problem statement\n\nThe researcher is exposed to nurses who almost daily express concern that, since introduction of the Batho Pele principles, patients have become abusive towards them, making their work unbearable. According to these nurses, patients seem to have unrealistic demands due to their expectations having been raised by these principles. Some of these nurses feel that it is not easy to uphold the principle of access, for example when there is a shortage of staff, especially skilled staff, and a shortage of medications.\n\nOn average, one professional nurse takes care of 20 patients a day in an outpatients department. These patients, as reported by the professional nurses working in the outpatient departments, present themselves to the department as early as 06h00 and might leave between 14h00 and 16h00. This delay is caused mainly by limited material and human resources.\n\nLack of material resources may at times include a lack of medicines to be given to patients, as well as a lack of diagnostic equipment. Nurses are confronted with the anger of patients who are exposed to such supply shortages. As the nursing category that carries the bulk of patient care in the outpatient departments, Professional nurses bear the brunt of the bad behaviour of patients and feel that the authorities do not listen when they report abusive tendencies or behaviour of patients.\n\nFurthermore, there have been many negative reports in the media in recent years regarding the 'bad attitude' of nurses towards patients and their relatives, as well as about the substandard housekeeping and nursing care in State hospitals. Professional nurses expressed concern about these reports.\n\nThis study was designed to answer the following research question: What is the perception of professional nurses regarding the introduction of the Batho Pele principles?\n\n### Research objectives\n\nThe objectives of the study were to: describe the perceptions of professional nurses regarding the introduction of the Batho Pele principles; and to recommend guidelines to facilitate measures for all parties involved in realisation of the objects of the Batho Pele principles.\n\n# Research design\n\nThe research design for the study was qualitative, exploratory, descriptive and contextual in nature. The design suited the study as it allowed the researcher to engage with and to fully understand the lived experiences of the participants (Johnston & Onwuegbuzie 2004:20).\n\nThe population for the study was professional nurses who had been working in the outpatient departments of the three hospitals in the Port Elizabeth Hospital Complex within the Nelson Mandela Metropolitan Municipality for at least five years. Specific inclusion criteria were at least five years' experience of working in the outpatients department in a State hospital; work in either the adult or children outpatients department; and knowledge and practical skills which meant that they should possess a better understanding of what nursing stands for, and enhanced the purposive sampling of participants for this study (Parahoo 2006). Forty professional nurses were sampled; only 32 participated as the remaining 8 were either sick or not on duty on the day of data collection.\n\n## Data collection methods\n\nData were collected through six audio-taped focus group discussions (FGDs) with no less than four participants each, as well as the taking of field notes. Of these six FGDs, two were for the purposes of a pilot study. No errors were identified in the data from the pilot study, and therefore data collected from these two groups were also included in the data analysis and results. Following the discussion with regard to the pilot study, with no major concerns identified, the researcher continued with data collection (Hornby 2005).\n\nThe FGDs were scheduled to last no longer than 45 uninterrupted minutes, but at times this schedule was not easy to maintain due to the nature of engagement with the participants, and they would be extended by 5\u201310 minutes. The interviews were based on one central question, with subsequent questions developing from the responses of the group members, and a set of two predetermined questions.\n\nThe central question was: Tell me, how do you feel about the introduction of Batho Pele principles in your institution? The predetermined questions were: What would you tell me about the implementation of the Batho Pele principles?; and What do you think could assist with the implementation of the Batho Pele principles?\n\nThe FGDs were conducted over a period of seven days during March 2010. As the researcher had no previous experience in conducting research interviews, the supervisor of the study assisted with facilitation of the FGD with one group for the pilot study and two of the main study FGDs. This facilitation was mainly to teach the researcher how to prepare participants before commencing the interview and to direct a question to a specific participant using the name code before or after the question has been asked, so as to allow individual responses. The researcher was also guided in keeping the focus to the research questions. Data saturation was the indication to discontinue data collection and complete the data analysis process (Burns & Grove 2011; Green & Thorogood 2009).\n\n# Ethical considerations\n\nThe researchers were mindful of meeting all ethical considerations (Silverman 2011). A complete research proposal was presented to the research committee of the Nursing Science Department for approval. Written permission to conduct research was obtained from the Research, Technology and Innovation Committee of the Faculty of Health Sciences, with ethical clearance from the Ethical Committee of Nelson Mandela Metropolitan University. Furthermore, the DoH via the Chief Executive Officer of the Port Elizabeth Hospital Complex gave permission to use the hospitals as the site for conducting the study. Informed consent was obtained from the individual participants, as well as permission to identify them using codes such as P1 or P5.\n\nOther ethical issues that were adhered to included preventing harm, no deception of participants, confidentiality and anonymity (De Vos *et al*. 2013). Participants were further assured of anonymity and confidentiality of their information and their privacy. Name codes were used to identify the tapes and transcripts and none of the participants' particulars were divulged. In order not to cause harm, each FGD was conducted in a sensitive manner (Babbie 2010). Participants maintained their right to withdraw at any stage of the study without any penalties.\n\n## Trustworthiness\n\nBabbie and Mouton (1998) state that the eminence of any qualitative study is determined by the level of trustworthiness it 'portrays'. To ensure valid results and trustworthiness, the researcher used Guba's model as described by Krefting (1991). The focus of the model is truth value, applicability, consistency and neutrality. Credibility was assured through member checking and the use of peer examination reflexivity, whilst confirmability was maintained through an audit trail.\n\nTransferability refers to the ability to apply the findings of the study in another context or to other participants. The activities used to enhance transferability include a thick description, achieved by collecting and providing sufficient detailed data within the context of the study, purposive sampling, and data saturation. Dependability was maintained by use of a code-recode procedure and peer examination.\n\n# Results and discussion\n\nParticipants were between the ages of 35 and 50years. The participants were all black people with most being Xhosa-speaking, whilst a few were mixed-race. Two groups had one male participant and two groups had one mixed-race participant. The male participants represented 100% of the male professional nurses in the department chosen as the research site.\n\nThere were no white employees at these research sites, and hence no white participants. In SA most townships \u2013 the environment in which the study was conducted \u2013 are predominantly populated by Xhosa families and individuals, whilst the nursing profession is a predominantly female-dominated profession.\n\nData were analysed using Tesch's method as stated in Creswell (2008:154) and by making use of an independent-coder. Three main themes and several sub-themes and categories emerged from the data analysis.\n\n## Theme 1: Lack of planning with the introduction of Batho Pele\n\nParticipants indicated positive and negative emotions regarding introduction of the Batho Pele principles. The participants felt strongly that introduction of the Batho Pele principles lacked a sound foundation to their implementation. Said one participant: 'My experience with Batho Pele principles is that it was designed to transform the health services, the aim was good, but the government fails to do the ground work.'\n\nAs a result, the Batho Pele principles could not meet the set positive objectives of providing quality service delivery or of developing competent, committed and professional health workers for whom people or patients come first (DoH 2008).\n\nAccording to the participants a lack of planning was the chief shortcoming with introduction of the Batho Pele principles: 'No planning was done before implementing the Batho Pele principles'. The participants felt that proper planning to implement the principles would have assisted the authorities to anticipate challenges and devise strategies to deal with such challenges. The experience of limited planning before implementing the principles was expressed as follows: '\u2026 without checking on the consequences the patients were informed of the Batho Pele principles. Now the patients want to hear what they want to hear'. Adequate planning would also have afforded well thought-through processes to correctly inform the public of the introduction of the Batho Pele principles.\n\nThe Batho Pele principles were meant to bring about a change in the usual manner of healthcare service delivery in the country. The environmental dynamics related to the change process, namely emotional involvement and resistance, were therefore to be observed (Jooste 2009; Huber 2010). Planning allows involvement of all stakeholders, which according to this study are the professional nurses and the community, thus encouraging ownership (Jooste 2009). Planned change enhances productivity and lessens the amount of individual stress resulting from the stakeholders' efforts to cope (Huber 2010).\n\nAccording to the participants, the patients and the public are largely misinformed about the Batho Pele principles. As one put it: 'Some of these patients are not educated. These principles were read to them then they picked up what they want to hear and interpret it wrongly.'\n\nAs a result, they are inclined to make inappropriate demands, such as self-referring to an institution of a higher level of care instead of being referred from a nearby clinic or private doctor. Healthcare services were not rendered previously in terms of different levels; this resulted in duplication of services being the order of the day. Hospitals were providing primary to secondary services simultaneously \u2013 thus patients could present for a consultation and then would either be admitted and managed further or referred from one service to another. The professional nurses felt that the newly introduced referral system is not easy for patients and their families to understand.\n\nAvailability of medicines and prescriptions are currently categorised according to the level of care of that particular institution, and at times are out of stock. These are demanded by the patients from the professional nurses, although it is a managerial responsibility to order medicines. When the participants explained the impossibility of certain patient demands and informed patients of the correct procedure to be followed, the patients and their relatives became angry \u2013 shouting and accusing them of being uncaring and selfish. In this regard one of the participants expressed herself as follows:\n\n> 'Patients shout and threaten us. You become scared and humiliated because patients shout at you in front of colleagues and other patients.'\n\nSome even threatened to channel their dissatisfaction through the media, which would cause even worse conflict between the professional nurses and their patients.\n\nInforming and explaining the merits of change to subordinates and followers enhances the process of organisational change (Jooste 2009). Here the 'followers' \u2013 the community members \u2013 seemed to misunderstand the practical meaning of the 'right to access of health care' due to a lack of adequate explanation regarding the full scope and meaning of this 'right'. Below are examples of expressions bythe participants with regard to limited understanding of the 'right to access of health care' by the community:\n\n> 'Patients are not aware that other things are beyond the control of a nurse.'\n>\n> 'Patients are rude. They have taken this the wrong way. They think when they shout at a nurse it is their right. They think that these rights are to disrespect and abuse nurses.'\n\nFurther mixed emotions were experienced by study participants because they felt that implementation of the policy was further grossly affected by a shortage of human and material resources:\n\n> 'Nurses would like to work and produce good results. When there are no resources I feel disgruntled, fed up. I feel like taking my bag and resign\\[*ing*\\] and find\\[*ing*\\] work where there are resources and I can give optimal care.'\n>\n> 'Another thing that makes us bad to the public is shortage of resources \u2026'\n>\n> 'You feel embarrassed when you must re-book a patient because of no availability of resources. Some of these patients are poor. At times for most of these patients they have kept that money to come to the hospital; some have borrowed the money, and they must go back without any help because there are no resources.'\n\nBoth proper planning and adequate availability of resources are needed to influence achievement of the principles' goals. Despite existing disparities between policy and implementation, healthcare professionals cannot refuse to practice, because they are bound by professional ethics and the Pledge of Service that charges South African nurses to protect the dignity of their patients and families and the community at large, and to protect and maintain a safe and therapeutic health care environment, regardless of colour and creed, based on the human needs of the patient.\n\n### Sub-theme 1.1: Difficulty in upholding the policy of the Batho Pele principles\n\nParticipants expressed concern that because of a lack of planning in implementing and incorporating the Batho Pele principles into policy, it is sometimes difficult for them to act in an ethical manner in response to the way in which patients and community members address or treat them:\n\n> 'I can say Batho Pele principles is something good introduced by the government but sometimes it is difficult to uphold.'\n>\n> 'It is a good tool, but patients use it the wrong way.'\n\nEthics are vital in nursing practice and any occupation dealing with human life needs, both to regulate the practitioner so as to protect the good name of the profession, and to protect the public (Huber 2010). As aforementioned, at the core of provision of nursing is the value of and respect for human life. Professional nurses found it difficult to uphold this value as introduction of the principles did not fully accommodate issues related to human and material supplies, resulting in negative responses from the patients and community.\n\nBurns and Grove (2009) define ethics as a means of endeavouring towards realistic outcomes where others are involved. Ethics determine that which is 'good' and 'bad', 'right' or 'wrong', or a moral duty and obligation (Pope & May 2005).\n\nIn this regard Burns and Grove (2009) maintain that problems of ethics relate to conscience, justice and responsibility, amongst others. The BathoPele principles were formulated to ensure that patients are treated with respect and consideration, and that the healthcare providers show courtesy and respect when interacting with patients. With this understanding in mind, professional nurses often have to keep quiet and ignore abusive behaviour from their patients, despite feeling heartbroken.\n\n### Sub-theme 1.2: Batho Pele principles not planned properly\n\nChange is complicated and should therefore be planned and implemented properly. If proper planning is not done, it leads to frustration, confusion and resistance amongst employees (Tomey 2009). Planning, according to Booyens (2008), is the cornerstone of good management and is grounded in the vision, mission, goals, philosophy and objectives of the organisation. Therefore, when planning for change the manager should not settle on a change approach before first examining the problem, the personal environment, time constraints, resources and goals to be achieved (Hellriegel *et al*. 2002). In the opinion of the participants this approach should have been adopted by the health authorities before their implementation of the Batho Pele principles:\n\n> 'No planning was done before implementing the Batho Pele principles.'\n>\n> '\u2026 without checking on the consequences the patients were informed of the Batho Pele principles. Now the patients want to hear what they want to hear.'\n\nPlanning affords an organisation a formal structure which will lead to better coordination of activities and, in the context of this article, better service delivery. In a study of cross-sectional surveys of nurses and patients to explore patient safety, satisfaction and quality hospital care in 12 countries of Europe and the United States of America, conducted and published by Aiken *et al*. (2012), results show that an organised healthcare worker environment is closely assosciated with quality care and patient and nurse satisfaction. For this reason the participants felt that the Batho Pele principles needed to be revisited.\n\n### Sub-theme 1.3: Introduction of Batho Pele principles affected by shortage of human and material resources\n\nThe participants indicated that besides the lack of planning by the relevant authorities, there were also human and material resource constraints which made it difficult for them to achieve the goals of the Batho Pele principles. Inadequate resources can have a negative impact on provision of care, leading to delays and deaths of patients. When there are no resources nurses are unable to render quality care and give of their best (Yoder\u2013Wise 2007). The participants reported that they often have to rely on crisis management. In the patients' eyes this is often perceived as non-caring inefficiency. A lack of resources, such as staff shortages, means patients might not always get their treatment on time or are sent home to return to collect outstanding medication later.\n\nAiken *et al*. (2012) argue that provision of resources (staffing and materials) improves patient outcomes as there is less occurence of staff burnout and patient complaints. Furthermore, Hellriegel *et al*. 2002) state that managers need to identify and commit the organisation's resources to achieving its goals. In view of Hellriegel's statement, Benjamin (2008) recommends that managers develop project management Gantt charts to help them focus on tracking tasks and allocating resources, thus identifying shortcomings and providing the necessary help. The following are some of the statements made by participants in relation to inadequacy of material resources:\n\n> 'Most of the time we nurses we improvise or go round borrowing from other departments or hospitals because there are no resources.'\n>\n> 'Nurses would like to work and produce good results. When there are no resources I feel disgruntled, fed up. I feel like taking my bag and resign\\[*ing*\\] and find\\[*ing*\\] work where there are resources and I can give optimal care.'\n\nCaring promotes patient satisfaction. For patients, caring denotes positive connectedness, being instructed and taught, and having a nurse spending time with them and being patient with them (Palese *et al*. 2011:341). In the current study participants perceived themselves as not fulfilling this expectation due to limited materials, resulting in conflict between themselves and their patients.\n\n## Theme 2: Owing to introduction of the Batho Pele principles, nurse-managers are letting the professional nurses down\n\nParticipants felt that they were not receiving enough support from their managers, expressed in statements such as that below:\n\n> 'To report a patient or even a staff member who threatens you, you will be wasting time. Nothing happens.'\n\nDessler (2004) argues that a manager is a person who plans, organises, leads and controls the work of others so that the organisation can achieve its goals. If the manager fails to adhere to these attributes the department will be chaotic and nursing care will be compromised (Dessler 2004).\n\nIn their recent study Aiken *et al*. (2012) found that managerial support of nursing care, nurse participation in decision-making and organisational priorities on care quality were directly associated with nurse-workforce outcomes and nurses who were intent on leaving or remaining in their nursing jobs. This is in keeping with the perceptions of the participants in this study. The finding of that study can be applied in this current study, as the participants perceived the nurse-managers as not supportive of their actions, and at times were embarrassed in front of the patients as the nurse-managers took sides with the patients.\n\n### Sub-theme 2.2: Batho Pele principles cause the nurse managers to lack discipline in their leadership\n\nIn the participants' opinion challenges to introduction of the Batho Pele principles were further aggravated by inconsistencies in the application of disciplinary measures by nurse managers \u2013 yet there were appropriate policies and guidelines in place. The participants reported that such policies and guidelines were not adhered to:\n\n> 'The disciplinary policies are there but they are not implemented, especially if that person is a shop steward nothing is done. They do as they please. They are rewarded for their behaviour, like they will be promoted or sent for courses.'\n>\n> 'There is no discipline in our hospitals, everyone does what he\/she likes, and even if you report that person nothing happens.'\n\nOwing to this lack of disciplinary consistency, everyone could do as they pleased. This finding is similar to what was found in a similar study conducted in Ekurhuleni District of Gauteng Province, SA. That study revealed that nurse managers are to be blamed at times for non-supervision of staff performance, lack of role-modelling, and ineffective planning and control of their nursing units, all of which negatively affects implementation of the Batho Pele principles (Khoza, Du Toit & Roos 2010). However, ill-discipline was not only related to inadequately upheld disciplinary measures but also to matters concerning professionalism.\n\n## Theme 3: Doctors are not committed to implementation of the Batho Pele principles\n\nDoctors have the same responsibility as the nurses with regards to upholding the Batho Pele principles; however participants felt that sometimes their actions belied this.\n\n**Sub-theme 3.1:** Doctors perceived as being impolite to patients and nurses.\n\nMost of the participants felt that some doctors were extremely impolite and sarcastic. Participants had experienced doctors shouting at them in front of patients when they queried their behaviour or prescription. Participants felt that some doctors ignore the fact that a nurse is part of the health team:\n\n> 'Batho Pele is for all categories, not only for nurses.'\n>\n> 'Doctors must get lectures on the upholding of the Batho Pele principles.'\n\nSearle (2006:172) contends that nurses act in the interests of the patient and have a joint responsibility with the doctor for ensuring that the patient receives the correct prescribed diagnostic and therapeutic treatment. It is therefore important for doctors to familiarise themselves with the contents of this policy.\n\n## Reaching the research goals\n\nThe research conducted was guided by two objectives, which were accomplished at the end of the study. Participants shared their perceptions relating to introduction of the Batho Pele principles. Through the use of exploratory questions, participants were able to provide in-depth information related to their perceptions, that could be both described and analysed. Findings revealed that professional nurses are overwhelmed by challenges resulting from the introduction of these principles. According to the participants, the objective of introduction of these principles was good, but implementation of the introduction of the Batho Pele principles lacked proper planning.\n\nData were analysed and the findings were used to develop guidelines that will be recommended to hospital managers as a means to facilitate realisation of the objectives of the Batho Pele principles within State hospitals.\n\n# Limitations of the study\n\nThe concept of the Batho Pele principles is fairly new in the South African healthcare system and, like any other change introduced in a system, it has been confronted with some challenges. The researcher experienced it as being limiting to having to only interview professional nurses who work in outpatients' departments, excluding professional nurses from other departments and wards of the hospitals in the study.\n\n# Recommendations\n\nA second objective of this study was to recommend measures that could be used for facilitation of all the parties involved in the realisation of the objectives of the Batho Pele principles. Based on the findings of the study, the guidelines outlined below are recommended to facilitate effective implementation of the Batho Pele principles.\n\n## Guideline 1: Planning of health services\n\nThe researcher believes that the staff would be eager supporters of the Batho Pele principles if:\n\n-   they could be assured of their access to the necessary human and material resources, as well as to an adequate and reliable public and emergency services transport system for the purpose of speedy referrals;\n\n-   the hospitals have fully functional Accident and Emergency Departments to allow for direct referrals;\n\n-   all the hospitals are provided with the necessary security measures to safeguard the environment for both patients and staff; and\n\n-   all stakeholders are provided with educational programmes and other formal means to inform them about the standards of care they will receive.\n\n## Guideline 2: Effective leadership and management\n\nThe hospital and nurse managers should and are to:\n\n-   be sensitive to employees' problems and show genuine concern;\n\n-   create an environment in which employees are able to render optimal care, a non-threatening environment characterised by safety and security, where discipline is the order of the day;\n\n-   ensure orientation of new personnel to the policies of the institution, basing all punitive discipline on accepted legal prescriptions and remaining uninfluenced by other staff members or unions in consistency in implementing discipline; and\n\n-   be exemplary and keep to issues related to punctuality and work attendance, in keeping with the necessary institutional policies.\n\n## Guideline 3: Availability of human and material resources\n\nThere are never enough resources for the work that needs to be done. In order to overcome this gap managers need to be:\n\n-   well-versed in financial management and budgetary control; and\n\n-   able to prioritise and be cost-effective without compromising quality care.\n\nTo achieve the clinical-related recommendations the researchers provide a further guide, based on the principles informed by the article 'Healthcare in a land called People Power: nothing about me without me' (Delbanco *et al*. 2001:144). A summary of those principles is listed below:\n\n-   Collaborative partnerships with relevant stakeholders.\n\n-   Approach the groups identified and inform them of the intentions of the collaboration.\n\n-   The groups should be represented by non-governmental organisations, lay persons, patients, doctors, multi-disciplinary teams, nurse managers and, where possible, legal staff.\n\n-   Allow involvement at decision-making level, as this allows for stakeholder commitment and ownership.\n\n-   Share information with interested groups.\n\n-   Identified groups are to be informed of the changes and interpretation of the changes so as to allow them to take that information to the masses.\n\n-   Information should also relate to the objectives of (in this instance) the Batho Pele principles.\n\n-   Collaborations with community-based leaders.\n\n-   Lay, retired and other well-respected, experienced people in the community could be approached for assistance in dealing with misinformation and incorrect perceptions of the introduction of the Batho Pele principles.\n\n-   In-service education.\n\n-   Nurse managers or persons in these positions should be continuously guided in their roles.\n\n-   Nurse managers need also to be guided as to how to motivate surbordinates, for example by using incentives.\n\n-   Nurse managers should be trained on how to be adequate role models.\n\n# Conclusion\n\nBatho Pele is a well-founded strategy towards a quality healthcare service, but is posing a challenge in the South African healthcare context. A revised strategy is needed to encourage ownership and uptake of these principles by key stakeholders, namely professional nurses, managers and the community.\n\nThe Batho Pele principles are intended for empowerement of the community through developing responsible attitudes towards health care and usage of healthcare services. From the findings of this study it is evident that this objective has not yet been achieved, owing to the diverse factors discussed in this article. The implication is that funds are still not being adequately channelled or used correctly. It is possible that because of the funds not being used correctly, some patients are still not getting the care they need and, as a result, are prone to complications that could lead to either chronic conditions or longer stays in hospital. Both of the latter ultimately lead to the hospital losing more funds.\n\nNotwithstanding the perceptions of participants with regard to introduction of the Batho Pele principles, the researcher saw it necessary to recommend measures that could facilitate enhancement of implementation of these principles. Three guidelines were developed from the study, in the hope of ultimately developing permanent policies to guide implementation of the Batho Pele principles.\n\n## Acknowledgements\n\nWe thank Jean Morrissey for assisting with the editorial requirements of this article.\n\n### Competing interests\n\nThe authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article.\n\n### Authors' contributions\n\nS.J. (Nelson Mandela Metropolitan University) was supervisor and co-researcher for this study, as well as being responsible for conceptualisation, responding to the reviewers' comments and finalising the manuscript. T.M.M. (Livingstone Hospital) conducted the research and approved the initial draft article.\n\n# References\n\n[^1]: **How to cite this article:** James, S. & Miza, T.M., 2015, 'Perceptions of professional nurses regarding introduction of the Batho Pele principles in State hospitals', *Curationis* 38(1), Art. \\#1128, 9 pages. <http:\/\/dx.doi.org\/10.4102\/curationis.v38i1.1128>","meta":{"dup_signals":{"dup_doc_count":119,"dup_dump_count":24,"dup_details":{"curated_sources":2,"2018-22":5,"2018-17":2,"2018-13":5,"2018-09":2,"2017-51":5,"2017-47":2,"2017-43":5,"2017-39":2,"2017-34":5,"2017-30":2,"2017-26":5,"2017-22":5,"2017-17":4,"2017-09":6,"2017-04":6,"2016-50":7,"2016-44":6,"2016-40":7,"2016-36":7,"2016-30":7,"2016-26":8,"2016-22":8,"2018-26":1,"2017-13":5}},"file":"PMC6091678"},"subset":"pubmed_central"}
{"text":"abstract: Three tools for simulating genome evolution are presented: for neutrally evolving DNA, for phylogenetic context-free grammars and for richly structured syntenic blocks of genome sequence.\nauthor: Avinash Varadarajan; Robert K Bradley; Ian H Holmes\ndate: 2008\ninstitute: 1Computer Science Division, University of California, Berkeley, CA 94720-1776, USA; 2Biophysics Graduate Group, University of California, Berkeley, CA 94720-3200, USA; 3Department of Bioengineering, University of California, Berkeley, CA 94720-1762, USA\nreferences:\ntitle: Tools for simulating evolution of aligned genomic regions with integrated parameter estimation\n\n# Rationale\n\nAlmost every kind of genome annotation tool has, at some point in its lifecycle, been benchmarked on simulated data. Exceptions occur when the data are too poorly understood to be simulated, or so well understood that simulation is unnecessary. For the regime in between, generation of synthetic datasets is a nontrivial task. To accurately predict the strengths and weaknesses of a tool, one needs to generate simulated data with the same underlying statistics as the real data on which the tool will eventually be used. Relevant statistics include compositional bias (including local deviations from randomness like microsatellites or inverted repeats), phylogenetic correlations (including tree topology and branch lengths), hetergeneous substitution and indel rates, structured and conserved features (such as genes or binding sites) and context-dependence of mutational parameters. All the above factors lead to fluctuations in information density that can reduce the effectiveness of an annotation tool in practice.\n\nA few concrete examples of applications that have been critically evaluated using simulated data are protein-coding genefinding \\[1\\], identification of transcription factor binding sites \\[2\\] and other functional non-coding DNA \\[3\\], phylogenetic reconstruction using distances based on substitutions \\[4,5\\] or indels \\[6\\], protein homology detection \\[7\\] and protein multiple alignment \\[8\\]. This list is a gross under-sampling from recent years, based on a citation search using the simulators described below; since simulation is almost a prerequisite for any new tool, the full list is much longer than this.\n\nA number of tools address different aspects of the sequence simulation problem over long evolutionary timescales. Some of these tools are primarily protein-coding simulators; some handle DNA, and some do both. ROSE \\[9\\] is a canonical model for neutral sequence that implements substitution and indel events. DAWG \\[10\\] implements similar mutations to ROSE but is based on a deeper theoretical analysis of the underlying model, which is somewhat similar to the 'long indel' model of statistical alignment \\[11\\]. Several programs simulate substitution processes without indels; a recent example, which generates data of a given dinucleotide content (with a somewhat constrained choice of substitution model), is SISSIz \\[12\\]. Protein simulators include SimProt \\[13\\], Indel-Seq-Gen \\[14\\] and EvolveAGene \\[15\\]; of these, Indel-Seq-Gen offers the most realism, with rate heterogeneity and a diverse set of mutations. Simulators for genomic sequence (including conserved features) include EvolSimulator \\[16\\], which simulates gene family dynamics, and PSPE \\[17\\], which simulates turnover in promoter regions.\n\nMany of these model-based simulation tools share a fundamental deficiency in their treatment of missing parameters and data. Specifically, they often lack robust functionality for: direct measurement of the model parameters from data; or integrated inference of missing information (gene boundaries, evolutionary histories, and so on) via the underlying model. The first aspect is important because the properties of the synthetic dataset may be parameter-dependent; the second aspect is important because even with direct measurement, the parameters measured may be strongly biased by the annotation of the training data.\n\nEqually seriously, few of these models capture even a fraction of the true variety of genome features. At the mutational level, context-dependent substitution and indel rates (within which category we include microsatellite expansion and contraction) can have a significant impact on prediction accuracy \\[18\\]. At the level of selection for particular genomic features, a realistic simulator should model all the commonly encountered features of genomes, including protein-coding genes, non-coding RNA genes, conserved elements (such as binding sites), pseudogenes and transposons. All of these can bias different predictors in different ways.\n\nThere are notable exceptions to the above sweeping critique. DAWG does include a Perl script to estimate indel rates directly from data. SISSIz offers some heuristics for estimating parameters, without proof of the convergence or accuracy of these estimates. EvolSimulator does combine evolution of gene families and intergenic regions, but does not model the other genomic features mentioned above; nor does it provide tools to measure rate parameters. Indel-Seq-Gen is restricted to protein sequences; it offers domain-specific mutational parameters and includes events such as indels and domain shuffling but, again, does not offer a way to estimate these parameters. PSPE simulates promoter turnover, including loss-of-function mutations; the PSPE paper also describes ways to estimate parameters for promoter turnover directly from data, though it does not provide software to do this.\n\nWe have developed three related simulation engines: GSIMULATOR, SIMGRAM and SIMGENOME. The first, GSIMULATOR, models substitutions and indels in neutrally evolving DNA, with context-dependent rates for those events. The second, SIMGRAM, samples paths from a generic class of models called 'phylo-grammars', which include a broad class of models for proteins, coding DNA sequences, RNA genes, promoters and other features. The third, SIMGENOME, combines the previous tools with a rich diversity of genomic features (proteins, RNAs, pseudogenes, promoters and transposons) backed by a comprehensive repository of empirically measured substitution and indel rates, drawn from previously published analyses of large datasets such as PANDIT \\[19\\] and the 12 sequenced *Drosophila* genomes \\[20\\], and collected for the first time in a single place.\n\nAll three tools are based on generative models that offer efficient algorithms for parameter inference, annotation and\/or ancestral reconstruction. The particular generative models used are transducers \\[21\\] and phylo-grammars \\[22\\], which both may be viewed as subsets of the set of mutational grammars. The theoretical advantages are several. One can learn parameters directly from data: for phylo-grammars we use the previously published XRATE program \\[22\\], while GSIMULATOR has a transducer estimator built-in. Generative grammars also offer parametric modularity: one can isolate important or repeated parameters, and place these conveniently at the top of the grammar file so that they can easily be edited or replaced with re-estimated parameters. Equally significantly, our grammars offer structural modularity. All the tools described here use documented file formats for representing their models. One can easily break down the grammar into sub-models, add new features or modify existing ones, without the need for directly modifying or re-compiling the underlying program code.\n\nThe present work is addressed at the problem of simulating syntenic blocks of genome sequence. We did not attempt to model genome rearrangements or duplications in this first release of our simulator, despite a clear need for benchmarking of genome rearrangement algorithms \\[23-30\\]. This was primarily because (at the time of writing) we lacked a unified framework for measurement in such models, or for dealing with missing data. In contrast, each simulation tool that we describe here is consistently complemented by robust measurement and reconstruction tools \\[22,31\\]. Adding rearrangement or duplication events would, however, be a natural future extension to our work. Another related but distinct area that we have omitted is evolution at the population level. Theoretical frameworks for populations exist, such as the coalescent \\[32\\] and super-processes \\[33\\], but exact inference in those frameworks is a significantly more challenging problem. Several recent simulators have addressed evolution at the shorter timescales associated with population genetics, incorporating effects like recombination, geographic migration, demography and ecology \\[34-36\\].\n\n# Results\n\nThe fundamental component models of our simulator each have applications in training, simulation and inference. We propose that a single coherent evolutionary theory, encompassing and unifying these three aspects, is an extremely desirable feature for a simulation framework. There can be no simulation without parameters, which must be measured using some model; the parameters are typically measured from data that must have been aligned or annotated, using some model; and the alignment or annotation tools must be benchmarked using simulations, also using some model. The common feature of all the approaches described here, which we seek to emphasize, is that all three of these models are the same: parameter estimation, annotation and simulation can all be conducted using the same probabilistic model.\n\nWe now describe three tools. GSIMULATOR randomly generates alignments of neutrally evolving DNA; SIMGRAM randomly generates alignments of generically structured features under selection; and SIMGENOME combines these to randomly generate alignments of syntenic regions in genomes, using a reasonably detailed model of the genomic feature landscape. All three of these programs, together with examples of input\/output files and command-line usage, may be accessed via the webpage \\[37\\].\n\n## GSIMULATOR: a transducer-based simulator for neutrally evolving DNA\n\nThe GSIMULATOR tool simulates the neutral evolution of DNA on a phylogenetic tree. Along each branch, substitution and insertion-deletion mutations are modeled, using a context-dependent transducer. The theory of transducers is described in previous work \\[21\\] and summarized in the Materials and methods (see 'Sampling from lexicalized transducers'). Essentially, a transducer is a finite-state machine, similar to a Pair hidden Markov model ('Pair-HMM'), that mutates a sequence by introducing random substitutions and indels.\n\nThe GSIMULATOR transducer is context-dependent, meaning that the substitution and indel 'rates' are dependent on the past *K* absorbed and emitted symbols, where *K* is a parameter that can be configured. The purpose of allowing context-dependence is to model local sequence-dependent fluctuations in substitution and indel rates, such as methylation-dependent CpG deamination, microsatellite expansion and contraction, and 'micro-duplications' or 'micro-inversions' corresponding to *K* nucleotides or fewer.\n\nGSIMULATOR also permits flexible modeling of gap length distributions, by allowing *N* multiple degenerate insertion and deletion states, where *N* is a configurable parameter. In the absence of context-dependence (that is, when *K* = 0), this yields a mixture of geometric distributions for the lengths of gaps. The distribution is more complicated for context-dependent transducers (*K* \u2265 1), since the gap length depends on the inserted (or deleted) sequence.\n\nAs with all the methods described in this paper, GSIMULATOR is a trainable simulator, meaning that the parameters can be directly estimated from pairwise alignment data (and need not be 'guesstimated' by a user). Compositional biases of gene predictors, aligners, motif finders and other annotation tools can be exquisitely sensitive to the underlying evolutionary statistics, so this feature is extremely important for a robust simulator.\n\n## SIMGRAM: a phylo-grammar simulation tool\n\nThe SIMGRAM tool generates sample alignments from a user-specified phylogenetic context-free grammar, or 'phylo-grammar'. In contrast to the transducers used by GSIMULATOR, the phylo-grammars of SIMGRAM can model genome features under finely structured selection, including co-ordination of the relative layout of these features (analogously to the way in which a human-language grammar specifies the layout of the various parts of speech). Examples of features that can be so modeled include protein-coding genes \\[1,18\\], non-coding RNA genes \\[22,38,39\\], protein binding sites \\[40\\], protein domains \\[41,42\\] and protein secondary structure \\[22,43\\]. The full theory of phylo-grammars has been widely described (see the above-cited applications by ourselves and others); a brief introduction can be found in the Materials and methods (see 'Sampling from phylo-grammars').\n\nThe phylo-grammar format used by SIMGRAM is the same as that of the XRATE program, a previously described tool that uses the Expectation Maximization algorithm to estimate the rate and probability parameters of any custom phylo-grammar \\[22\\]. Therefore, XRATE can be used to estimate simulation parameters directly from training data, then SIMGRAM can be used to generate synthetic data with similar properties, but no direct homology (excluding cases where the phylo-grammar itself encodes homology information \\[41,42\\]). This represents a new application of XRATE: the SIMGRAM program has not been previously described, and previous uses of XRATE have involved using the phylo-grammar to annotate sequence, or to measure substitution rates that are themselves of direct interest. (Thus, as with all the methods here, the generative model underpinning SIMGRAM can readily be trained on data and used for annotation and inference.)\n\nAn appealing feature of phylo-grammars for simulating an evolving and feature-rich genome sequence is that it is extremely easy to combine several sub-models into an all-encompassing model. Furthermore, the XRATE format allows several features that are useful for simulation. One such feature is parametric models, where the rates and probabilities are constrained to have a particular functional form that depends on a smaller parameter set. This is useful to construct models that have desired symmetry properties, such as strand-symmetric substitution processes, or *K*~*a*~\/*K*~*s*~ codon models. The XRATE format can also approximate context-dependent substitution rate models, using the technique of \\[44\\]. Finally, a powerful macro language exists that can be used to compactly describe grammars with many states, or to model lineage-specific parameterizations.\n\nThe DART (DNA, Amino Acid and RNA Tests) package, in which SIMGRAM is distributed, includes reproductions of several previously published phylo-grammars that can be simulated using SIMGRAM. Examples include models of secondary structure for proteins \\[43\\] and RNA \\[38\\], as well as a number of point substitution models for protein and nucleic acid sequences. The phylo-grammar format is described in full online \\[45\\].\n\n## SIMGENOME: a feature-rich phylo-grammar for genome alignments\n\nSo far, we have described the GSIMULATOR program for context-dependent mutation of neutral DNA, and the SIMGRAM program for mutation of structured features under selection. Each model has its strengths and weaknesses: GSIMULATOR richly models neutral DNA, but not features under selection, whereas SIMGRAM has better models for such features but lacks GSIMULATOR's context-dependent rates or sophisticated indel model. We now describe a program that combines these approaches, using a modular framework that can easily be extended to incorporate future, specialized feature-simulators.\n\nThe combined simulator, SIMGENOME, starts by generating a multiple alignment from a template phylo-grammar that includes a rich array of genome features. The features are described below in more detail, together with outlines of how the template can be extended by an expert user to incorporate new features.\n\nIn the generated alignment, certain columns are flagged as intergenic. The SIMGENOME program then repeatedly calls GSIMULATOR to generate alignments of neutral DNA corresponding to these intergenic regions, and splices them into the main alignment. This process is extensible: the template phylo-grammar can be edited to add new features or change the underlying parameters of the model. Furthermore, other external feature simulators can be specified in the template grammar, and their output alignments will be spliced into the main alignment in exactly the same way as GSIMULATOR's output is.\n\nThe features modeled by the template phylo-grammar include protein-coding genes (with a rough approximation to exon-intron-untranslated region (UTR) structure that includes exon length distributions), non-coding RNA genes, conserved elements (such as transcription factor binding sites), pseudogenes and DNA transposons with terminal inverted repeats. Features can appear on forward or reverse strands. All features are annotated in the generated alignment, so that their recovery in automated benchmarks can be assessed.\n\nThe sub-models that generate these features use substitution rate parameters that all were estimated directly from the following experimental datasets, using the XRATE program (and could be re-estimated from alternative datasets).\n\n### The protein-coding gene model\n\nThe protein-coding gene model uses an empirical, fully reversible and otherwise unconstrained 61\u00d761 rate matrix over codons, estimated in previous work \\[46\\]. Frame-preserving deletions are allowed. The training set for this model was the PANDIT database consisting of DNA-level alignments of protein domain families \\[19\\].\n\n### The non-coding RNA gene model\n\nThe non-coding RNA gene model treats gaps as a fifth character and therefore uses (4 + 1) \u00d7 (4 + 1) single-stranded nucleotide and (4 + 1)^2^ \u00d7 (4 + 1)^2^ double-stranded base-pair rate matrices that are fully reversible and otherwise unconstrained, and were estimated separately. The training set for this model consisted of alignments provided with the program CONSAN \\[47\\], which in turn were derived from the European large subunit rRNA database \\[48\\]. The initial probability distribution over base-pairs in double-stranded regions was also used to generate the terminal inverted repeats in simulated DNA transposons (although these features subsequently evolve under a neutral model, so that they do not display the compensatory mutations characteristic of non-coding RNA (ncRNA) genes under selection).\n\n### The strand-symmetric neutral substitution model\n\nThe strand-symmetric neutral substitution model that underpins the pseudogene and transposon models treats gaps as a fifth character and was trained on a random 1% of alignments of 12 *Drosophila* genomes \\[20,49\\]. The alignments themselves were made using the PECAN program \\[50\\]. The model was constrained to be strand-symmetric and reversible using XRATE's parameterization functionality. A slower, ungapped version of this substitution rate matrix is also used to model conserved features.\n\n### The transducer model\n\nThe transducer model used by GSIMULATOR to simulate intergenic sequence evolving under a neutral context-dependent model was trained on a set of pairwise alignments drawn from a subset of twelve-species *Drosophila* alignments, which were made using the PECAN program \\[50\\]. The subset was drawn from approximately 5% of the original multiple alignment data, to which a 95% minimum percentage identity threshold was applied.\n\n### The frequencies and length distributions\n\nThe frequencies and length distributions of genomic features were estimated from the *Drosophila* genome literature \\[20,51,52\\] using *Minos* as a model for DNA transposons \\[53,54\\].\n\nThe underlying template phylo-grammar is written using the publicly documented XRATE format and can be readily edited. High-level parameters, such as the frequencies with which genes or other features appear, are declared at the top level of the grammar and may be easily changed. We invite users to try adding sub-models representing new features of relevance to benchmarking (or to consult with us in this process). New sub-models can be parameterized directly from data using XRATE, and the parameters copied and pasted into SIMGENOME's grammar; this also applies to the existing models, which can be re-trained and\/or re-parameterized (for example, to reflect differing codon usage or GC content). Even greater extensibility is afforded by the modular plug-in architecture that allows the use of third-party programs to generate features that cannot currently be simulated by SIMGENOME's phylo-grammars or transducers; such as tandem arrays, long-range duplications, and so on.\n\nThe following are examples of features that are not currently included in the SIMGENOME model, but are possible using SIMGRAM and could be incorporated by modifying SIMGENOME's grammar file.\n\n### Codon frequencies\n\nThe PANDIT dataset that was used to estimate the SIMGENOME codon model spans a wide spectrum of compositional and codon usage biases. In terms of overall patterns of conservation and suppressed mutation rates, we consider it an acceptable general model of codon substitution. For example, if one is benchmarking a motif-finding tool, the coding regions generated by SIMGENOME will result in more false positives than non-coding regions (because the level of conservation is higher) and this may be adequate for the purposes of that benchmark. However, for other purposes, one might conceivably want a richer parametric model that takes account of genome-specific effects such as compositional bias, codon bias, transition-transversion ratios or CpG methylation-induced deamination. Using such a parametric model is a simple case of swapping out the relevant rate matrix in the SIMGENOME grammar file. The model can be fit to data using XRATE in the normal way. We are preparing a manuscript describing a direct comparison between XRATE and PAML for these purposes, including Perl code for generating such richer parametric models (Heger A, Ponting C and Holmes IH, in preparation).\n\n### Lineage-specific parameterizations\n\nThe SIMGRAM macro language allows for different parameters on different branches of the tree. We have not made use of this feature in the SIMGENOME grammar, since its usage is somewhat dependent on the phylogenetic clade under investigation: one may (for example) want to use different parameters on every branch, on a single internal branch, or within a specific clade. With reference to the XRATE documentation, it is quite possible to design grammars that make use of this feature, so that the model may (for example) use different codon frequencies or compositional biases in different parts of the tree.\n\n### Loss-of-function mutations\n\nThe SIMGRAM format also allows for lineage-specific evolution of whole features, in the manner of the DLESS program by Pollard *et al.* \\[55\\]. Internally, we have developed phylo-grammars modeling loss-of-function mutations in protein-coding genes, for investigations of pseudogene evolution. We have not included loss-of-function mutations in this first release of SIMGENOME, but it would be quite feasible to extend it without needing to write new code.\n\n### Splice sites, initial methionines, UTRs and other aspects of protein-coding gene structure\n\nThe grammar of SIMGENOME currently includes a crude semblance of exon and intron structure, in order to reproduce the broad compositional fluctuations associated with protein-coding genes. This currently includes slow-evolving sequence at the exon-intron boundaries, as a mock-up of splice site conservation. The current release does not, however, model protein-coding genes at a sufficient level of detail to be used as positive examples for a protein-coding gene predictor. One could readily modify the simulator to do this, modeling splice sites as GT-AG and perhaps even AT-AC donor-acceptor pairs, and including other features such as poly-A signals, initial ATGs, TATA boxes and UTRs. These sorts of feature are straightforward to add to a phylo-grammar framework.\n\n### Higher-order correlations between codons\n\nThe SIMGRAM distribution includes example phylo-HMMs demonstrating higher-order correlations between amino acids in protein alignments. For example, there is a replica of the Thorne-Goldman-Jones 3-state phylo-HMM for modeling secondary structure \\[43\\]. Given a suitable parameterization to map from amino-acid substitution rate matrices to codon matrices, it would be straightforward to use something like this to model higher-level correlations between codons in SIMGENOME's coding-DNA regions. One may reasonably ask why we have not included such higher-order dependencies in the first release of SIMGENOME, when we have included higher-order context-dependencies in the intergenic regions. The answer is that intergenic features (such as microsatellites or conserved regions) may include strong compositional biases extending over tens of bases and so generally contribute more to fluctuations in information density than correlations between codons, which are typically weak, local and often detectable only at the level of the encoded amino acids \\[43\\].\n\n### Detailed length distributions\n\nMost of the length distributions of features modeled in SIMGENOME are geometric (the simplest kind of distribution that one can model with an HMM). By chaining together duplicate states, it is possible to generate more complex (realistic) length distributions. For example, the SIMGENOME grammar file includes functions for generating a peaked 'negative binomial' distribution (also known as the 'Pascal' distribution) for exon lengths; so there are examples that can be used to do this. However, a straightforward geometric distribution was empirically found to be a better fit to exon length data in *Drosophila*.\n\n## Evaluation: benchmarking a non-coding RNA predictor\n\nEvaluating a simulation tool is a slightly different problem from evaluating an annotation tool, such as a gene predictor. When benchmarking a predictor, one is typically interested in minimizing the number of false positives that the predictor finds in a null dataset at a given threshold of the prediction score cutoff. One can eliminate all false positives by setting the score threshold arbitrarily high, but at the cost of also missing all the real genes in the dataset. To get a true picture of the predictor's performance, one must therefore consider how the number of false positives varies as a function of this threshold; or (more meaningfully) as a function of the sensitivity of the predictor, that is, the number of real genes that it correctly detects at a particular score threshold.\n\nA common practice is to use a set of real genes to evaluate the sensitivity of the predictor, but to use simulated null data to evaluate the false positive rate. The reason for using simulated null data, instead of a real DNA sequence that does not contain any genes, is the paucity of negative annotations: it is difficult to demonstrate experimentally that a particular sequence of DNA definitively does not contain any genes. This is particularly true of hard-to-identify genes, such as RNA genes or short open reading frames.\n\nA good simulator, therefore, is one that closely reproduces the statistics of real DNA, and generates a similar number of false positives to real DNA. In practice, the second criterion rests on the choice of statistics for the first criterion In general, if the statistical model is too simplistic (for example, omitting low-complexity regions), then the complexity of the simulated DNA will be more than it should be, leading to fewer false positives. To take a specific example: a DNA simulator that randomly emits a sequence of independent, identically distributed symbols will match the nucleotide-level composition of real DNA, but will not reproduce the short-range fluctuations in information content that can be found in real DNA, and so will generate fewer false positives in a motif-finding benchmark than a simulator that includes short-range complexity fluctuation phenomena (such as microsatellites).\n\nOn this basis, we argue that a measure of a good probabilistic simulator is that its model (after being fit to real DNA) should maximize the number of false positives for a given predictor at a given sensitivity. Another way of putting this is that the simulator should provide the most stringent possible benchmark for the gene predictor, by minimizing the area under the receiver operating characteristic (ROC) curve over the range of interest (see below).\n\nTo evaluate the simulation engines described here, we used them to estimate the false positive rate (FPR) for a computational whole-genome screen for conserved structural RNA genes, conducted using XRATE \\[22\\]. We chose RNA gene prediction as a test case because it has an extremely high FPR, whose actual extent is still unknown \\[39,56,57\\]; and because the estimated FPR for this screen is highly sensitive to the underlying properties of the simulation engine, making it a good motivator of increased realism \\[12,58\\].\n\nSpecifically, the FPR was estimated by sliding a window across the simulated alignments, and running XRATE on each window using an RNA gene-prediction grammar. This grammar models the distinct patterns of nucleotide substitution in RNA genes, including covariation of base-paired nucleotides, and is closely related to the EVOFOLD program for comparative RNA gene prediction \\[39\\] and the PFOLD program for comparative RNA folding \\[59\\]. The grammar itself, along with detailed instructions for reproducing the screen, can be found online \\[37\\]. The full rationale underlying the development of the grammar, and its critical evaluation (as a genefinder) and comparison to related grammars, will be described elsewhere (Bradley RK, Uzilov AV, Skinner M, Benda\u00f1a YR, Barquist L and Holmes I, submitted).\n\nThe plots show ROC curves where the FPR is plotted against the sensitivity of the screen (as measured using annotations of known ncRNAs in *Drosophila melanogaster*), as a parametric function of the score threshold for the screen. Since ncRNAs represent positive results for this screen, and we already have a set of known curated ncRNAs for *D. melanogaster*, we omitted the ncRNA submodel from the SIMGENOME grammar for these tests.\n\nThe results are shown in Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>. The general conclusion is that increased realism makes for a higher FPR. In the case of GSIMULATOR, increasing either *N* or *K* radically increases the FPR; in the case of SIMGENOME, inclusion of conserved genomic features with slower evolutionary rates markedly increases the FPR relative to both a pure point substitution model as well as the most realistic GSIMULATOR model.\n\nWe also compared our simulation methods, GSIMULATOR and SIMGENOME, to DAWG \\[10\\], a widely cited program for simulation of neutral substitution and indel events. We chose DAWG because it most closely exemplifies the goals we have identified here: it is clearly based on an underlying evolutionary model and provides tools for estimating the parameters of the indel model directly from sequence data. It appears to be the leading general-purpose simulator at the time of writing. Other simulators (such as PSPE) are richer, but do not provide the parameter-estimation functionality that DAWG does.\n\nThe parameters for DAWG were as follows. We used the same general-time reversible substitution model (REV) that we estimated from PECAN alignments of *Drosophila* genomes. DAWG's 'geometric' indel model (that is, geometrically distributed indel lengths) was parameterized using the script provided with the program. Although the 'power-law' model for indel lengths gave a better fit, it produced alignments that were mostly gaps. DAWG allows for heterogeneous rates and invariant sites using the \u0393 + *I* model for rate heterogeneity \\[60\\], also providing some example parameters for this model (\u03b3 = 1, \u03b9 = 0.1), which we used for these simulations.\n\nFigure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a> compares DAWG with the most realistic GSIMULATOR and richest SIMGENOME models. The figure shows that the context-dependence modeled by GSIMULATOR and the genomic features modeled by SIMGENOME result in much tighter false-positive estimates than DAWG produced.\n\nSince DAWG's example \u03b3 and \u03b9 parameters (which determine the rate heterogeneity and conserved-site density) were manually adjusted for human-chimp alignments by the program's author, they may be an underestimate for *Drosophila* (where conserved elements are more closely spaced than in primates, due to smaller genomes and a higher deletion rate of nonfunctional DNA). However, there is no automated method for setting these parameters in DAWG; nor does the DAWG package or paper provide explicit guidance on how to relate these parameters to straightforward statistics on genomic feature density. (The SIMGENOME grammar file, in contrast, includes comments that outline the derivation of the feature distribution parameters from published *Drosophila* annotations.) Further, the GSIMULATOR program includes a fully automated training procedure, has no model for conserved sites or rate heterogeneity (other than context-dependent substitution) and nevertheless generates a higher false prediction rate than DAWG, even when these features are enabled in DAWG (Figure <a href=\"#F2\" data-ref-type=\"fig\">2<\/a>). Since SIMGENOME, in turn, generates a higher FPR than GSIMULATOR (Figure <a href=\"#F1\" data-ref-type=\"fig\">1<\/a>), we reason transitively that SIMGENOME is also a more realistic simulator than DAWG. Correspondingly, we note that this argument strongly motivates an automated tool (or even a simple heuristic) for estimating the \u03b3 and \u03b9 parameters of DAWG's heterogeneous-rate model. There is considerable analysis of such models \\[60\\], so this may be a reasonable goal of future study.\n\n# Discussion\n\nWe have described an extensible system for genome simulation that includes a rich array of features and parameters, together with integrated methods for estimating those parameters, and comes with a comprehensive repository of mutation rate measurements for genomic features and intergenic DNA. Features modeled by our simulator include protein-coding and non-coding RNA genes, pseudogenes, transposons, conserved elements, microsatellite expansion\/contraction and other context-sensitive substitutions and indels in neutrally evolving regions.\n\nThere are several obvious extensions to this work. We did not include large duplications or rearrangements in our simulator because, at the time of writing, we lacked up-to-date methods for measuring the frequencies of these events in evolution (although see \\[61\\]). However, such events would be an obvious extra layer to add to our system. A variant on this would be to simulate gene family dynamics using a birth-death model \\[62,63\\] for benchmarking of orthology\/paralogy prediction methods. This might also be achieved by constructing gene features from explicit models of protein domains, for example, based on HMMs from the PFAM database. Likewise, we could generate ncRNA features based on explicit families (such as tRNAs).\n\nThe power of efficient generative probabilistic models is that they provide a common framework for measuring parameters, simulating from those parameters, and reconstructing missing information such as ancestral genotypes or gene boundaries. Another strong advantage, which we have used for this work, is the ease with which such models can be extended to incorporate new features or variations on existing features. The technologies that we have used to build our simulator (phylo-grammars and transducers) can, in fact, be viewed as overlapping subsets of a larger set of mutational grammars. With the inevitable trend of richer prediction tools whose dependence on the intricate structure of sequence evolution becomes ever harder to predict, the future of such generative grammatical models is as bright in computational biology as it is in linguistics.\n\n# Materials and methods\n\n## Sampling from phylo-grammars\n\nA phylo-grammar is a stochastic context-free grammar whose every state generates alignment columns (or groups of alignment columns). The residues in each row of such a column are related by an underlying phylogenetic tree (whose topology remains constant), using a continuous-time Markov chain subsitution model (which is allowed to vary from state to state). Each state in the grammar is allowed to generate several columns simultaneously; these sites then evolve co-ordinately, so that a state can (for example) emit three columns that evolve together as a codon triplet, or two distantly separated columns that evolve as a base-pair in a non-coding RNA alignment.\n\nOne can vary the evolutionary rate throughout the tree, and for this behavior to be state-dependent, so that different states in the grammar can have trees with different branch lengths. However, SIMGRAM requires that the tree topology stay constant throughout the alignment. (While it is mathematically straightforward to construct phylo-grammars with alternative tree topologies in the various states, such models are not implemented by SIMGRAM.)\n\nSampling from the phylo-grammar proceeds in two stages. First, a parse tree is sampled by repeated application of randomized grammar rules. Next, a Gillespie algorithm is used to simulate continuous-time Markov chain trajectories for each set of co-evolving columns \\[64\\].\n\n## Sampling from lexicalized transducers\n\nA transducer is a finite state machine, similar to a 'Pair-HMM', but conditionally normalized; thus, instead of emitting two sequences (as does a Pair-HMM), the transducer absorbs one sequence (*X*) as an input and emits the second sequence (*Y*) as an output. Practically, a transducer looks very similar to a Pair-HMM: it has match, delete and insert states, and the training and alignment algorithms are almost identical. The key difference is the probabilistic normalization: a transducer's emission and transition probabilities are normalized such that the likelihood of any state path \u03c0 corresponds to the conditional probability *P*(\u03c0,*Y*\\|*X*), whereas in a Pair-HMM the state path likelihood corresponds to the joint probability *P*(\u03c0,*X*,*Y*). Conceptually, a transducer absorbs a symbol on the input, then decides (randomly) what state to go into, then emits a symbol (or symbols) on the output; in contrast, a Pair-HMM first changes state, then emits symbol(s) on either or both outputs.\n\nA transducer models random changes in a sequence, as might occur on an individual branch of a phylogenetic tree. It is also convenient to introduce a special type of transducer, located at the root of the tree, that only emits sequence (that is, it has only 'insert' states, and lacks 'match' or 'delete' states to absorb an input sequence). Such a transducer is referred to as a 'singleton' transducer and is exactly equivalent to a single sequence-emitting HMM. The composition of an (emitting) singleton transducer with an (absorbing-and-emitting) branch transducer yields a machine that is exactly equivalent to a Pair-HMM.\n\nA lexicalized transducer is one in which the probability parameters for making transitions between states, and for absorbing or emitting various symbols within states, are dependent on the last few symbols that were emitted or absorbed. This dependence allows the root transducer to generate higher-order compositional statistics of DNA (such as dinucleotide or trinucleotide frequencies). It also allows the branch transducers to model phenomena such as increased rates of indels and substitutions in low-complexity regions, such as microsatellites.\n\nThe lexicalized transducer underlying GSIMULATOR is a discrete mutator that introduces substitutions and indels at a frequency corresponding to a finite span \u03c4 of evolutionary time, corresponding (typically) to a short branch on a phylogenetic tree. For example, the pre-trained transducers that are distributed with GSIMULATOR, and parameterized from *Drosophila* alignments, correspond to a branch length of \u03c4 \u2248 0.07 substitutions per site. Longer branches of the tree, with length *T* \\> \u03c4, are simulated by iterated application of the transducer (for example, a branch of length *T* = 0.35 corresponds to five repeated applications of an evolutionary transducer with branch length \u03c4 \u2248 0.07); branches of length *T* \\< \u03c4 are rounded up (if *T* \u2265 \u03c4\/2) or down (if *T* \\< \u03c4\/2).\n\nIn other words, evolutionary time *T* is treated as a discrete parameter, corresponding to some integer multiple of \u03c4. This contrasts with many point substitution models that are used in molecular evolution (and in previous evolutionary simulators), which are based on continuous-time Markov chains. This behavior is deliberate and justified on the grounds of realism, as follows: it is extremely difficult to obtain closed-form matrix exponential solutions to the transition probabilities for context-dependent substitution and indel models of the kind used by GSIMULATOR. Indeed, only approximate solutions currently exist for context-dependent substitution models \\[44,65\\], and there is currently no good theory at all for context-dependent indels. We argue that the increased realism of context-dependence is easily worth a small reduction in granularity of the evolutionary time parameter. In cases where fine-grained time distinctions are critical, the SIMGRAM phylo-grammar simulator described here can be used instead.\n\nThe training procedure for GSIMULATOR is straightforward. The algorithm for estimating the parameters is essentially the same as the Baum-Welch algorithm for training a Pair-HMM from a pairwise sequence alignment \\[66\\]. The corresponding inference algorithms are not detailed here, but there exist a range of dynamic programming algorithms, from exhaustive inference to Markov Chain Monte Carlo algorithms \\[31,67-69\\] that can be used to impute alignments and evolutionary histories, to reconstruct ancestral sequences, or to annotate sequences in alignment-free (as well as alignment-dependent) ways. The particular algorithm used by GSIMULATOR to infer parameters is a variant on the Baum-Welch algorithm \\[66\\]. During training, GSIMULATOR finds the branch length \u03c4 from the underlying data using a Jukes-Cantor distance estimate \\[70\\].\n\nFor the four symbols of the DNA alphabet, order-*K* context-dependence multiplies the number of transducer parameters by a factor of 4^2*K*^. In practice, the quantity of training data (and potentially runtime and\/or memory considerations) will limit the value of *K*. We find that a value of *K* = 3 yields a transducer that can reasonably be trained from eukaryotic genome alignments of the approximate size of *Drosophila*, but that this transducer takes a long time to train (especially if the degeneracy *N* of the indel states is greater than 1).\n\nWe do not currently have a specialized ancestral reconstruction tool for lexicalized transducers; instead, we have a more general tool. The previously published Handel program \\[68\\] is a Gibbs sampler for multiple alignments and ancestral sequence reconstructions under the TKF91 model \\[71\\]. More recently, it has been extended to model affine indel-length distributions (unpublished). Handel can also be coupled to any external program that implements a probabilistic scoring function for alignments; that is, given any ancestral reconstruction *A* in Stockholm alignment format \\[72\\], the program must compute and then output *P*(*A*,*S*\\|*M*) for some model *M*, where *S* represents the observed sequences at leaf nodes. (GSIMULATOR is itself one such program.) Handel then accepts the Markov Chain Monte Carlo move from *A* \u2192 *A'* with probability:\n\n$$\\min\\left( {1,\\frac{\\left. P(A^{\\prime},S \\middle| M)P(A,S \\middle| T^{\\prime}) \\right.}{\\left. P(A,S \\middle| M)P(A^{\\prime},S \\middle| T) \\right.}} \\right)$$\n\nwhere *T* represents the TKF91 model; this is a Hastings ratio \\[73\\]. Asymptotically, this yields a direct sample from *P*(*A*\\|*S*,*M*).\n\n## Generative model features\n\nMost features of the SIMGENOME phylo-grammar are self-explanatory, either from this paper or by inspection of the commented grammar file (located in the subdirectory `dart\/grammars\/SIMGENOME.eg`). The length distribution of exons is a negative binomial distribution, or Pascal distribution, obtained by serial chaining of left-regular states, each of which emits a geometrically distributed number of codons. (The Pascal distribution is that of the sum of independent, geometrically distributed random variables.) This results in far fewer unrealistically shorter exons than would be generated by a geometric distribution, while allowing easy parameterization of the mean exon length.\n\nThe default GSIMULATOR model used by SIMGENOME has two insert states and two delete states, and uses three nucleotides of both input and output context. Roughly speaking, this corresponds to a mixture of geometric distributions over gap lengths; however, due to the input and output context, the precise length of inserted or deleted sequences will also depend on the sequence content of those indels.\n\n# Abbreviations\n\nDART, DNA, Amino Acid and RNA Tests; FPR, false positive rate; HMM, hidden Markov model; ncRNA, non-coding RNA; ROC, receiver operating characteristic; UTR, untranslated region.\n\n# Authors' contributions\n\nAV wrote GSIMULATOR. RB wrote the SIMGENOME phylo-grammar, ran the gene-prediction benchmarks and prepared the ROC curve figures. IH wrote SIMGRAM (and related programs), the SIMGENOME Perl script, and the manuscript. All authors read and approved the final manuscript.\n\n### Acknowledgements\n\nThis work was funded by NIH\/NHGRI grant GM076705. RKB was partially supported by an NSF Graduate Research Fellowship.","meta":{"dup_signals":{"dup_doc_count":108,"dup_dump_count":36,"dup_details":{"curated_sources":3,"2020-45":1,"2020-24":1,"2019-47":1,"2018-30":1,"2018-09":1,"2017-17":1,"2017-09":9,"2017-04":1,"2016-50":1,"2016-44":1,"2016-40":1,"2016-36":11,"2016-30":6,"2015-48":3,"2015-40":2,"2015-35":3,"2015-32":2,"2015-27":2,"2015-22":3,"2015-14":1,"2014-52":3,"2014-49":6,"2014-42":8,"2014-41":5,"2014-35":3,"2014-23":4,"2014-15":3,"2021-39":1,"2017-13":2,"2015-18":3,"2015-11":3,"2015-06":3,"2014-10":2,"2013-48":3,"2013-20":3,"2024-18":1}},"file":"PMC2760874"},"subset":"pubmed_central"}